Methods of using immune organoids to predict drug safety

Abstract

The present disclosure relates to methods of assessing therapeutic efficacy using immune organoids. The disclosure also provides compositions useful for in such methods. In some embodiments, the present disclosure demonstrates the development of a new method for assessing the safety of therapeutic using 3D immune organoids. The method includes (a) contacting a three dimensional immune organoid comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells with a therapeutic and (b) measuring the response of the three dimensional immune organoid to the therapeutic after said contacting.

Description

METHODS OF USING IMMUNE ORGANOIDS TO PREDICT DRUG SAFETYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 621,937, filed January 17, 2024, the disclosure of which is incorporated by reference herein in its entirety, including any drawings.FIELD

[0002] The present disclosure relates to methods of using immune organoids to predict the safety of a therapeutic. The disclosure also provides compositions of immune organoids and a therapeutic for use in the methods.BACKGROUND

[0003] A main concern in assessing drug safety of immunotherapeutics is analysis of the production of anti-drug antibodies, cytokine / chemokine storm, cytotoxicity, and cell death. Thus, it is important to have a suitable model in which to test potential drugs and their safety in patients. Drugs entering clinical development have a high level of attrition (about 95%) despite the rising cost of new drug development (about 800 million). Such a high rate of attrition has been attributed to the current approaches used for drug discovery, safety testing, and drug development in two-dimensional (2D) cell-culture assays and in vivo animal models. Currently mice & non-human primates (NHP) are used, but there is considerable evidence of drugs that are shown to be safe in mice but are dangerous in human and vice versa. Reasons for this include that mouse and NHP physiology, tissue structure, proteins, etc. are significantly different from human. Not to mention that mice also inbred, single sex, and kept in sterile conditions. As such, there is an urgent and unmet need for improved tools and techniques for immunotherapy safety testing.SUMMARY

[0004] The present disclosure demonstrates the development of a new method for assessing the safety of therapeutic using 3D immune organoids.

[0005] Provided herein is a method of assessing the safety of a therapeutic. The method includes(a) contacting a three dimensional immune organoid comprising a plurality of self-assembled primary immune cells obtained from one or more secondary' lymphoid organs and a plurality' of stem cells with a therapeutic and (b) measuring the response of the three dimensional immune organoid to the therapeutic after said contacting.

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

[0007] In some embodiments, the one or more secondary ly mphoid organs are from spleen, lymph node, Peyer’s patch, and / or MALT.

[0008] In some embodiments, the immune organoid is a human immune organoid.

[0009] In some embodiments, the immune organoid further comprises peripheral blood mononuclear cells.

[0010] In some embodiments, the secondary' lymphoid organs are obtained from living patients, surgical resections, fine needle aspirates, biopsy, and deceased patients.

[0011] In some embodiments, the plurality of primary immune cells comprises B cells, T cells, plasmablasts, plasma cells, ILCs, granulocytes, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof. In some embodiments, the B cells comprise one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, plasmablasts, plasma cells, or a combination thereof. In some embodiments, 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, gamma delta T cells, CD4 effector memory' T cells, CD8 effector memory' T cells, or a combination thereof. In some embodiments, the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or a combination thereof.

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

[0013] In some embodiments, the immune organoid is 8000 pm or less in diameter.

[0014] In some embodiments, the immune organoid comprises germinal centers and / or B / T cell zones.

[0015] In some embodiments, the immune organoid produces antibodies. In some embodiments, the antibodies are IgG, IgM, or IgA, antibodies. In some embodiments, the antibodies have full humoral functionality. In some embodiments, the antibodies bind human and non-human targets. In some embodiments, the human targets comprise proteins, sugars, and nucleic acid. In someembodiments, the non-human targets comprise infectious disease antigens, venoms, poisons, small molecules.

[0016] In some embodiments, the therapeutic is an immunotherapeutic. In some embodiments, the immunotherapeutic is selected from the group consisting of a vaccine, an antibody, a gene therapy, a cell therapy, a small molecule, and a nanobody.

[0017] In some embodiments, measuring the response comprises measuring (i) the presence of anti-drug antibodies, (ii) cytokine and chemokine production, (iii) cell viability, and / or (iv) cytotoxicity’.

[0018] Also provided herein is a method of assessing the ability of a therapeutic to induce AD As. The method includes (a) contacting a three dimensional immune organoid comprising a plurality7of self-assembled primary7immune cells obtained from one or more secondary7lymphoid organs and a plurality of stem cells with a therapeutic and (b) measuring the presence or absence of AD As produced by the organoid after said contacting.

[0019] Also provided herein is a method of assessing the ability of a therapeutic to induce a cytokine / chemokine storm. The method includes (a) contacting a three dimensional immune organoid comprising a plurality7of self-assembled primary7immune cells obtained from one or more secondary lymphoid organs and a plurality7of stem cells with a therapeutic and (b) measuring the levels of cytokines / chemo kines produced by the organoid after said contacting.

[0020] Also provided herein is a method of assessing the cytotoxicity7of a therapeutic. The method includes (a) contacting a three dimensional immune organoid comprising a plurality of self-assembled primary7immune cells obtained from one or more secondary7lymphoid organs and a plurality of stem cells with a therapeutic and (b) measuring the levels of cytotoxic T lymphocytes produced by the organoid after said contacting.

[0021] Also provided herein is a method of assessing the ability' of a therapeutic to induce cell death. The method includes (a) contacting a three dimensional immune organoid comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells with a therapeutic and (b) measuring the cell viability' the organoid after said contacting.

[0022] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0023] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination.Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.DESCRIPTION OF THE DRAWINGS

[0024] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0025] FIG. 1 shows morphological changes in immune organoids after therapeutic treatment.

[0026] FIGs. 2A-2C show the stem cell population represented in immune organoids after being treated with an experimental influenza subunit vaccine. FIG. 2A and 2B show quantification of the stem cell types in representative immune organoids in day 7 cultures of total live cells. Cellular frequencies were determined by flow cytometry. Values plotted are across two different donors. FIG. 2C shows representative flow cytometry' analysis of the stem cell populations contained in representative immune organoids in day 7 cultures. Cells shown are pre-gated on live, single, CD45+CD34+CD45RA- cells. Data shown across two donors.

[0027] FIGs. 3A-3F show the diversity of immune cell populations in human immune organoids is maintained following immunotherapy treatment. FIG. 3A shows representative flow cytometry analysis of B (CD 19+) and T cells (CD3+) contained in representative immune organoids in day 7 cultures following stimulation with an experimental influenza subunit vaccine at day 0. Cells shown are pre-gated on live, single, CD45+ cells. Data shown across two donors. FIG. 3B shows representative flow' cytometry analysis of plasmablasts (CD27+CD38++) following stimulation with Imovax Rabies Vaccine to which the donors were naive. Cells shown are pre-gated on live, single, CD45+. CD19+ cells. FIG. 3C shows quantification of CD138+ plasma cell populations in immune organoids following exposure to five developmental influenza mRNA vaccine conditions and one unstimulated control. Analysis was performed using samples from three independent donors, with data presented as mean ± standard deviation (n=4 technical replicates per condition). FIG. 3D shows representative flow' cytometry analysis of NK cells in an unstimulated control immune organoid (left) and an immune organoid 7 days following treatment with an experimental antibody therapeutic designed to activate the immune system. Cells shown are pre-gated on live, single, CD45+ cells. NK cells are characterized by those that areCD56+CD16+. FIG. 3E shows representative flow cytometry analysis of the granulocyte population in an immune organoid following vaccination with a subunit influenza vaccine. Cells were gated based on high side scatter (SSChlgh) and CD 15 expression and plots demonstrate the presence of SSChlghCD 15+ granulocyte populations. FIG. 3F shows representative flow cytometry analysis of immune organoid-derived myeloid populations following treatment with a subunit influenza vaccine. Flow cytometry plots show dendritic cells (CD1 lb+CD45+), monocytes (CD14+CD1 lb+), and macrophages (visualized as SSC versus CD14+). Distinct populations were identified using sequential gating strategies for each myeloid subset.

[0028] FIGs. 4A-4C show immune organoids undergo B cell differentiation upon treatment. FIG. 4A shows representative images of flow cytometry analysis demonstrating representative immune organoid B cell differentiation at days 0, 7, 14, and 21 following treatment. All cells shown were previously gated on total B cells (CD3- CD19+ CD45+). Phenotypes shown: CD38- CD27- naive B cell, CD38-CD27+ memory B cell, CD38+CD27- pre-GC B cell. CD38+CD27+ GC B cell. Plots from one representative donor shown. FIG. 4B shows quantification of B cells represented in day 0, day 7, day 14, and day 21 immune organoids after being immunized with hemagglutinin protein. Cellular frequencies were determined by flow cytometry. Cells were previously gated on total B cells (CD3- CD 19+ CD45+). FIG. 4C shows quantification of CD 138+ plasma cell populations in immune organoids following exposure to five developmental mRNA influenza vaccination conditions and one unstimulated control. Analysis was performed using samples from three independent donors, with data presented as mean ± standard deviation (n=4 technical replicates per condition).

[0029] FIGs. 5A-5C show immune organoids are composed of T cell subtypes following immunotherapy treatment. FIG. 5A shows representative flow cytometry analysis of T cell populations in day 7 immune organoids following therapeutic treatment with an experimental adjuvanted influenza vaccine. Analysis was performed on gated T cells (CD19-CD3+), showing 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 is shown from two representative donors with relevant markers indicated. FIGs. 5B-5C show representative flow' cytometry analysis of T cell subsets in immune organoids following treatment at day 0 with an experimental adjuvanted influenza vaccine. Analysis was performed on gated T cells (CD19-CD3+) with subsequent subset-specific gating. Populations shown include naive CD8+ T cells (CD3+CD8+CD45RA+), memory CD8+T cells (CD3+CD8+CD45RO+), CD8+ effector memory cells (CD3+CD8+CD45RO+CCR7-), regulator)- T cells (CD3+CD4+CD25+), gamma delta T cells (CD3+CD27+), and T follicular helper cells (CD3+CD25+CXCR5+). Data is shown from two representative donors with relevant markers indicated.

[0030] FIGs. 6A-6C show immune organoids are composed of other immune cell types including myeloid dendritic cells, plasmacytoid dendritic cells, and dendritic cells. Representative flow cytometry analysis of dendritic cell populations in immune organoids following treatment with an experimental subunit influenza vaccine are shown. FIG. 6A shows flow cytometry plots showing CD14+CD1 lc+ myeloid dendritic cells from two independent donors. FIG. 6B shows analysis of plasmacytoid dendritic cells (CD123+) pre-gated on CD45RA+CDl lc+ populations. FIG. 6C shows identification of CD1 lb+CD45+ dendritic cells. Data shown are representative plots from independent experiments.

[0031] FIGs. 7A-7C show immune organoids are composed of other immune cell types including stromal cells, fibroblastic reticular cells, and follicular dendritic cells after treatment with an experimental subunit influenza vaccine. Representative flow cytometry analysis of stromal populations in immune organoids following treatment is shown. FIG. 7A show s flow cytometry plots demonstrating CD45- stromal cell populations. FIG. 7B shows identification of fibroblastic reticular cells (PDPN+CD31+). FIG. 7C shows analysis of follicular dendritic cells (CD45+PDPN+CD35+). Data showTi are representative plots from independent donors.

[0032] FIG. 8 shows size and morphology' of immune organoids can be a critical indicator of therapeutic effect. Representative brightfield images of day 5 and 21 immune organoids are shown after being vaccinated with developmental mRNA influenza vaccines on day 0. Blue line indicates the area of a subset of representative organoids, w ith which diameter for each organoid was calculated. Rows A through E represent different vaccine formulations and columns 1-5 represent decreasing concentrations of mRNA.

[0033] FIGs. 9A-9C show immune organoids form germinal centers in response to specific treatments designed to drive plasmablast and plasma cell differentiation consistent with human lymph node function. FIG. 9A shows representative brightfield images of day 14 immune organoids vaccinated with an inactivated Hep A vaccine. Lighter structures in the organoids outlined in broken circles are consistent with germinal center morphology. FIG. 9B shows representative images of flow cytometry staining demonstrating immune organoids consisting of B (CD 19+) and T cell (CD3+) zones. All cells were previously gated on live, single, CD45+cells. Plot is from a representative day 7 immune organoid treated with an experimental influenza subunit vaccine. FIG. 9C shows representative images of flow cytometry analysis demonstrating immune organoids consisting of germinal center B cells. Cells shown are gated on live, single, CD45+, CD3-, CD19+ cells and germinal center B cells are CD27+CD38+. Plots are from four representative day 14 immune organoids treated with hemagglutinin protein.

[0034] FIGs. 10A-10E show immune organoids form antigen-specific antibodies in response to vaccination. FIG. 10A shows antigen-specific IgG antibodies from day 4, day 8, day 11, day 15 immune organoids across 12 different doses and formulations of an experimental influenza subunit vaccine. Data shown is from one representative donor. FIG. 10B shows representative images of flow cytometry analysis demonstrating immune organoids undergoing plasmablast differentiation upon vaccination with an adjuvanted influenza subunit vaccine. Plots are from representative DO and D 14 vaccinated immune organoids. Cells shown were previously gated on live, single, and total B cells (CD 19+ CD3- CD45+). Phenotypes shown: CD38-CD27- naive B cell, CD38-CD27+ memory B cell, CD38+CD27- pre-GC B cell, CD38+CD27+ GC B cell, CD38++CD27+ Plasmablasts. Plots from one representative donor are shown. FIG. 10C shows antigen-specific immunoglobulin production profiles following vaccination in two independent donors. Concentrations of IgGl-4 subtypes, IgM, and IgA were measured in response to Treatment A and Treatment B, where treatment A and B are two different doses of rabies vaccine. Analysis was performed across four technical replicates per condition, with concentrations reported in pg / mL. Data are presented as mean ± standard deviation (n=4). FIG. 10D shows antigen-specific IgM antibodies from day 4. day 8, day 11, day 14 immune organoids stimulated tw ice with 6 different formulations of an experimental adjuvanted influenza subunit vaccine. Plots from one representative donor are shown. FIG. 10E shows antigen-specific IgG antibodies from day 4, day 8, day 11, day 14 immune organoids stimulated twice w ith 6 different formulations of an experimental adjuvanted influenza subunit vaccine. Plots from one representative donor are shown.

[0035] FIGs. 11A-11D show immune organoids are able to respond to foreign antigens and can also be stimulated to break tolerance to generate antibodies against human targets. FIG. 11A shows influenza-specific IgM and IgG antibodies from day 11 immune organoids from unstimulated control organoids and 12 different stimulation conditions consisting of influenza hemagglutinin protein and different experimental adjuvants. Plots from one representative donor are shown. FIG. 11B show's SARS-CoV-2-specific IgM and IgG antibodies from day 11 immuneorganoids from unstimulated control organoids and 12 different stimulation conditions consisting of SARS-CoV-2 spike protein and different experimental adjuvants. Plots from one representative donor are shown. FIG. 11C shows myelin-specific IgM and IgG antibodies from day 11 immune organoids from unstimulated control organoids and 8 different stimulation conditions consisting of myelin protein and different experimental adjuvants. Plots from one representative donor are shown. FIG. 11D shows NK cell-specific IgM and IgG antibodies from day 11 immune organoids from unstimulated control organoids and 12 different stimulation conditions consisting of an NK cell protein and different experimental adjuvants. Plots from one representative donor are shown.

[0036] FIGs. 12A-12E show immune organoids enable comprehensive safety assessment of immunotherapies through multiparameter analysis of cellular responses. FIG. 12A shows quantification of anti-drug antibodies (ADA) in response to 10 ng / ml of OKT3 antibody. ADA levels were measured in untreated controls and following administration of therapeutic antibody at 10 ng / mL. Individual data points represent independent measurements, with bars showing mean ± standard deviation (n=10). Treatment induced variable ADA responses across samples, with levels ranging from baseline to 0.28 ng / mL ADA. FIG. 12B shows analysis of IFN-y and IL- 10 production quantified by LegendPlex™ immunoassay in immune organoid supernatants from three independent donors across an untreated control condition and treatment with an experimental therapeutic antibody. Data are presented as mean ± standard deviation (n=3). FIG. 12C shows CXCL10 concentrations measured by LegendPlex™ immunoassay in immune organoid supernatants from five independent donors (A-E) across an untreated control condition and an experimental therapeutic antibody. Data are presented as mean ± standard deviation (n=2). FIG. 12D shows cell viability analysis by flow cytometry following therapeutic antibody treatment at vary ing dilutions over 6 days. Data are presented as mean ± standard deviation, with viability determined by single-cell gating. FIG. 12E shows analysis of cytotoxic T cell function through GZMB (Granzyme B) and Perforin expression via flow cytometry analysis over a 6-day period following treatment with an experimental therapeutic antibody. Single replicates were performed across 2 donors.DETAILED DESCRIPTION

[0037] To address the problem of a lack of a sufficient model for assessing safety of immunotherapeutics, the disclosure provided herein provides 3D in vitro immune organoidscultured in the presence of an immunotherapeutic that that fully recapitulate the cellular complexity and critical functions of an in vivo secondary lymphoid organ from a subject being treated with such an immunotherapeutic.

[0038] The disclosure provides methods for assessing the safety of a therapeutic, such as an immunotherapeutic, in a manner that ensures the drug is safe when it goes on to be tested in patients. No other system is able to capture real patient heterogeneity to ensure drugs are safe across age, sex, race, genetic background, and exposure history.Definition

[0039] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”. and “A and B.”

[0040] It is understood that aspects and embodiments of the disclosure described herein include "comprising", "consisting", and "consisting essentially of aspects and embodiments. As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0041] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0042] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so forth. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently- broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art. a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0043] It is appreciated that certain features of the disclosure, which are, for clarity , described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every- such sub-combination was individually and explicitly disclosed herein.

[0044] Although features of the disclosures may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the disclosures may be described herein in the context of separate embodiments for clarity, the disclosures may also be implemented in a single embodiment. Any published patent applications and any other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference for any purpose. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.METHODS OF THE DISCLOSURE

[0045] The present disclosure provides, among others, a method of assessing the safety of a therapeutic. The method includes (a) contacting a three dimensional immune organoid comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality’ of stem cells with a therapeutic and (b) measuring the response of the three dimensional immune organoid to the therapeutic after the contacting. Immune organoids

[0046] As described above, one aspect of the present disclosure relates to a method of assessing the safety of a therapeutic. The method includes contacting a three dimensional immune organoid comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality' of stem cells with a therapeutic.

[0047] As used herein, a “three dimensional immune organoid” can be a composition of live immune cells, arranged in a three-dimensional or multi-layered configuration (as opposed to a monolayer).

[0048] In some embodiments, the immune organoid is produced ex vivo. A person of ordinary skill in the art would readily appreciate that the immune organoids described herein are also non- naturally occurring.

[0049] An organoid, in general, is an artificial construct created in vitro to mimic or resemble the functionality and / or histological structure of an organ, tissue, or a portion thereof. An organoid, as used herein, can be a cellular structure obtained by' expansion of immune cells and stem cells, and consisting of tissue-specific cell ty pes that self-organize. In the present disclosure, the term “organoid” can be used to refer to normal (e.g., non-tumour) organoids. An organoid can comprise one or more (e.g., 1, 2. 3, 4, or more) differentiated cell type(s) depending upon the particular tissue and / or organ being modeled or emulated.Immune cells

[0050] The immune cells comprising the three dimensional immune organoid of the present disclosure are derived from one or more secondary lymphoid organs. As used herein, “derived from” generally refers to the source of primary cells that form the organoid. In some embodiments, “derived from one or more secondary lymphoid organs” can mean that the organoids are formed without any passage of the primary cells from the secondary lymphoid organs. In some embodiments, “derived from one or more secondary lymphoid organs” can mean that the organoids are formed after 1 passage of the primary cells. In some embodiments, “derived from one or more secondary lymphoid organs” can mean that the organoids are formedafter more than 1 passage of the primary cells. A secondary lymphoid organ is a site where an adaptive immune response is initiated, and lymphocytes are maintained. Exemplary secondary' lymphoid organs include, lymph nodes (LNs), spleen, Peyer's patches (PPs), and mucosal associated lymphoid tissue (MALT), adenoids, and tonsils. In some embodiments, the primary immune cells are obtained from spleen, lymph node, Peyer’s patch, and / or MALT.

[0051] The secondary lymphoid tissue can be obtained from a mammal (z.e., donor or patient) such as human, dog, cat, rabbit, monkey, chimpanzee, cow, pig, or goat. The secondary' lymphoid tissue can be taken directly from living patients, surgical resections, fine needle aspirates, or biopsy or deceased patients. In some embodiments, the secondary lymphoid tissue is obtained from a human and thereby results in a human immune organoid.

[0052] In some embodiments, the secondary' lymphoid tissue is obtained and dissociated mechanically, enzymatically, or both. In some embodiments, the secondary lymphoid tissue is dissociated with a proteolytic and / or collagenolytic enzyme. In some embodiments, the secondary lymphoid tissue is enzymatically dissociated with Accutase (StemCell Technologies), Accumax (StemCell Technologies), trypsin, trypsin / EDTA, collagenase, dispase, TrypLE Express (Thermo Fisher), TrypLE Select (Thermo Fisher), or any combination thereof. In some embodiments, the secondary lymphoid tissue is mechanically dissociated by trituration, for example, with a pipette. In some embodiments, the single cell suspension of resulting cells is filtered to remove any non-dissociated cell masses.

[0053] The primary immune cells derived from the secondary7lymphoid tissue can be any cell of hematopoietic origin that is functionally involved in the initiation and / or execution of innate and / or adaptive immune response, such as typically CD3 or CD4 positive cells. Exemplary types of primary immune cells include, without limitation, a dendritic cell, a mast cell, aNK-cell, a plasmablast, a macrophage, a B-celL a T-cell, a plasma cell, 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.

[0054] In some embodiments, the B cells comprise one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, plasmablasts, plasma cells or a combination thereof. In some embodiments, the B cells comprise primarily naive B cells. In some embodiments, the B cells undergo B cell differentiation upon stimulation to yield pre-GC B cells, GC B cells, memory' B cells, or a combination thereof. In some embodiments, the naive B cells are CD38-CD27-. Insome embodiments, the memory B cells are CD38-CD27+. In some embodiments, the pre-GC B cells are CD38+CD27-. In some embodiments, the GC B cells are CD38+CD27+.

[0055] In some embodiments, 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, gamma delta T cells, CD4 effector memory cells, CD8 effector memory cells, or a combination thereof. In some embodiments, the naive CD4 T cells are CD4+CCR7+CD45RA+. In some embodiments, the naive CD8 T cells are CD8+CD45RA-CD27+. In some embodiments, the memory CD4 T cells are CD4+ CCR7+ CD45RA+, CD4+ CD45RA- CD45RO+. In some embodiments, the memory CD8 T cells are CD8+ CD45RA- CD45RO+. In some embodiments, the T regulatory cells are CD3+CD4+CD25+. In some embodiments, the T follicular helper cells are CD3+CXCR5+CD25+. In some embodiments, the gamma delta T cells are CD3+CD27+gdTCR+.

[0056] In some embodiments, the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or a combination thereof. In some embodiments, the dendritic cells are CD45+CDl lb+ dendritic cells. In some embodiments, the dendritic cells are CD14+CDl lc+ myeloid dendritic cells. In some embodiments, the dendritic cells are CD 123+ plasmacytoid dendritic cells.

[0057] In some embodiments, the monocytes are CD14+CD1 lb+ monocytes.

[0058] In some embodiments, the macrophages are CD68+ macrophages. In some embodiments, the macrophages are CD14+ macrophages.

[0059] In some embodiments, the plasmablasts are CD38++CD27++ plasmablasts.

[0060] In some embodiments, the NK cells are CD56+ NK cells. In some embodiments, the NK cells are CD56+CD16+ NK cells.

[0061] In some embodiments, the granulocytes are SSChlghCD15+ granulocytes.

[0062] In some embodiments, the immune organoid as described herein produces and / or modifies T cells. By way of example, new T cell phenotype changes can occur in the immune organoid such as, for example, an increase in T helper cells. In some embodiments, new cytotoxic T cells and / or memory' T cells can be produced in the immune organoid described herein. Such phenotypic changes can be identified using assays including, without limitation, flow cytometry to detect identifying markers on the T cells. Cytotoxic T cells can be further identified, for example, using a T cell Cytotoxicity Assay.

[0063] In some embodiments, the plurality of immune cells present in an immune organoid of the present disclosure can be an amount of about 1%, 5%, 10%, 25%, 50% to about 55%, 60%, 75%, 80%, 90%, or 95%, of the total number of cells present in the organoid. In some embodiments, an organoid of the present disclosure comprises 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 organoid. If an organoid of the present disclosure comprises primary' immune cells in an amount less than 100% of the total number of cells present in the organoid, then stem cells, stromal cells, fibroblastic reticular cells, and follicular dendritic cells, in any suitable amount, can make up the remaining number / percentage of cells.

[0064] In some embodiments, B cells are present in an immune organoid of the present disclosure in an amount of about 1%, 5%, 10%, 15%, or 20%. In some embodiments, T cells are present in an immune organoid of the present disclosure in an amount of about 1%, 5%, 10% or 15%. In some embodiments, NK cells are present in an immune organoid of the present disclosure in an amount of about 1%, 5%, 10% or 15%. In some embodiments, macrophages are present in an immune organoid of the present disclosure in an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, monocytes are present in an immune organoid of the present disclosure in an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, dendritic cells are present in an immune organoid of the present disclosure in an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, plasmablasts are present in an immune organoid of the present disclosure in an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, plasma cells are present in an immune organoid of the present disclosure in an amount of about 1%. 2%, 3%. 4%, or 5%. In some embodiments, ILCs are present in an immune organoid of the present disclosure in an amount of about 1%, 2%, 3%, 4%, or 5%. In some embodiments, granulocytes are present in an immune organoid of the present disclosure in an amount of about 1%, 2%, 3%, 4%, or 5%.Stem Cells

[0065] As described above, the immune organoid also includes a plurality of stem cells. Stem cells may be characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers as identified by the lack of binding of specific antibodies. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiatedprogeny. The stem cells of the immune organoid described herein are identified as CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+. Accordingly, the stem cells can include hematopoietic stem cells. Hematopoietic stem cells refer to a subset of multipotent stem cells that give rise to all the blood or immune cell types, including myeloid (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 retain the ability to renew themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types. The stem cells can also include mesenchymal stem cells. Mesenchymal stem cells include stem cells that can be obtained, for example, from the bone marrow, peripheral blood, skin, hair root, muscle tissue, uterine endometrium, blood, cord blood, and primary cultures of various tissues. Mesenchymal stem cells can differentiate into all or several of osteocytes. chondrocytes, and adipocy tes.

[0066] Stem cells can be identified using flow cytometry and immunofluorescence as described in the Examples below. These methods are known in the art 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).Other cell types

[0067] In some embodiments, the immune organoid further includes 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 provides for an increase in the size of the immune repertoire of the organoid. By way of example, in some embodiments, the PBMCs are isolated from the same donor as the secondary lymphoid tissue. In some embodiments, the PBMCs are isolated from a different donor.

[0068] In other embodiments, the immune organoid further includes one or more stromal cells, fibroblastic reticular cells, and follicular dendritic cells. A stromal cell is a type of cell that makes up certain types on connective tissue in the body. Fibroblastic reticular cells are stromal cells found in the secondary lymphoid organs. Follicular dendritic cells are non-haematopoietic cells that are of stromal origin. They are integrated into the continuous stromal network within lymphoid organs. The presence of these types of cells in the immune organoid described herein contribute to the deposition of extracellular matrix and the overall architecture of the three- dimensional immune organoid. By way of 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 methods described in the Examples herein.

[0069] In some embodiments, stromal cells are present in an immune organoid of the present disclosure in an amount of about 0.5% 1%, 1.5%, or 2%. In some embodiments, fibroblastic reticular cells are present in an immune organoid of the present disclosure in an amount of about 1%, 2%. 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some embodiments, follicular dendritic cells are present in an immune organoid of the present disclosure in an amount of about 1%, 2%. 3%, 4%, 5%, 6%. 7%, 8%, 9%. or 10%.Structure

[0070] In some embodiments, the immune organoid of the present disclosure has a diameter and / or largest dimension of about 8000 pm, 7500 pm, 7000 pm, 7000 pm, 6500 pm, 6000 pm, 5500 pm, 5000 pm, 4500 pm, 4000 pm, 3500 pm, 3000 pm, 2500 pm, 2000 pm, 1500 pm, 1000 pm, 500 pm. 250 pm, 100 pm. 50 pm or less. In some embodiments, the organoid may comprise about 1 ,500, 2,000, or 5,000 to about 10,000, 25,000, or 50,000 cells in total or 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 total. In some embodiments, an immune organoid of the present disclosure may comprise about 1, 2, or 5 million to about 10. 25. 50. or 100 million cells per mL. In some embodiments, an organoid of the present disclosure may comprise about 10 million cells per mL or 20 million cells per mL. In some embodiments, an organoid of the present disclosure may comprise about 5 or 10 million cells per mL to about 15 or 20 million cells per mL. An organoid of the present disclosure may be in any suitable three-dimensional shape or multi-layered shape. In some embodiments, an organoid of the present disclosure is in the form of a spheroid. In some embodiments, an organoid of the present disclosure may be selforganized in a suspension or medium.

[0071] In some embodiments, the immune organoid contains germinal centers and / or B / T cell zones. A germinal center (GC) is a sub-anatomical structure that programs B cell conversion into antibody producing cells. Within the GC, B cells undergo somatic mutation of the genes encoding their B cell receptors which, following successful selection, can lead to the emergence of B cell clones that bind antigen with high affinity. As described in Stebegg et al., “Regulation of the Germinal Center Response,” Front. Immunol. 9 (2018), the GC is divided into two distinct compartments: the light zone and the dark zone. In some embodiments, the immune organoid of the present disclosure contains both a light zone and a dark zone. The dark zone (DZ) contains anetwork of CXCL12-producing reticular cells and is the site of GC B cell proliferation and somatic hypermutation (SHM). Centroblasts then follow a CXCL13 gradient to enter the light zone (LZ) as centrocytes through their expression of CXCR5. In the LZ, centrocytes capture antigen presented on follicular dendritic cells which they internalize, process and subsequently present to T follicular helper cells in order to undergo selection. This process is regulated by T follicular regulatory cells which are also present in the LZ. Upon receiving survival signals from Tfh cells, centrocytes re-enter the DZ for further rounds of proliferation and SHM after which they exit the GC as memory B cells or high-affinity antibody-secreting plasma cells. Thus, the presence of both the dark zone and the light zone in an immune organoid of the present disclosure can be identified using techniques including, without limitation, immunofluorescence as described in the Examples. In some embodiments, the germinal centers in the immune organoid are characterized as CXCR4+, CD83+, Ki67+, and IgD+. In some embodiments, the B and T cell zones in the germinal centers of the immune organoid are characterized as CD31and CD20+.Antibody Production

[0072] In some embodiments, the immune organoid of the present disclosure can be capable of producing antibodies. Antibodies are proteins used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen. Antibodies can come in different varieties known as isotypes or classes. In placental mammals there are five antibody classes known as IgA, IgD, IgE, IgG, and IgM, which are further subdivided into subclasses such as IgAl. IgA2. Accordingly, the immune organoid of the present disclosure can be capable of producing IgA. IgD, IgE, IgG, IgM, and combinations thereof. The prefix "Ig" stands for immunoglobulin, while the suffix denotes the type of heavy chain the antibody contains: the heavy chain types a (alpha), y (gamma). 5 (delta), 8 (epsilon), p (mu) give rise to IgA, IgG, IgD, IgE, IgM, respectively. In some embodiments, the immune organoid of the present disclosure produces IgG antibodies. In some embodiments, the immune organoid of the present disclosure produces IgA antibodies. In some embodiments, the immune organoid of the present disclosure produces IgM antibodies.

[0073] The antibody isotype of a B cell changes during cell development and activation. Immature B cells, which have never been exposed to an antigen, express only the IgM and IgD isotype in a cell surface bound form. The B lymphocyte, in this ready-to-respond form, is known as a "naive B lymphocyte." The naive B lymphocyte expresses both surface IgM and IgD. Theco-expression of both of these immunoglobulin isotypes renders the B cell ready to respond to antigen. B cell activation follows engagement of the cell-bound antibody molecule with an antigen, causing the cell to divide and differentiate into an antibody-producing cell called a plasma cell. In this activated form, the B cell starts to produce antibody in a secreted form rather than a membrane-bound form. Some daughter cells of the activated B cells undergo isotype switching, a mechanism that causes the production of antibodies to change from IgM or IgD to the other antibody isotypes. IgE, IgA, or IgG, that have defined roles in the immune sy stem. Accordingly, the immune cells of the immune organoid of the present disclosure can also encompass those that have undergone or will undergo the process of isotype switching.

[0074] Antibodies are critical for development of a humoral immune response in which antibodies are produced by B cells and are secreted into the blood and / or lymph in response to an antigenic stimulus. In a properly functioning immune response, the antibody binds specifically to antigens on the surface of cells (e.g.. a pathogen), marking the cell for destruction by phagocytic cells and / or complement-mediated mechanisms. Briefly, antibodies participate in several important functions including antibody dependent cellular cytotoxicity (ADCC), phagocytosis (opsonization), and complement-dependent cytotoxicity (CDC), upon binding to antibody -bound target cell.

[0075] Thus, in some embodiments, the immune organoid has full humoral functionality. Specifically, in some embodiments, antibodies produced by the immune organoid of the present disclosure can function in ADCC. ADCC is an in vitro or in vivo process where an antibody can bind to an antigen on a surface of a cell then engage with immune-effector cells via sequences within the antibody’s Fc domain that in turn results in their release of toxins that can kill bound cell. ADCC activity can be measured using methods known in the art including, without limitation, using in vitro methods as known in the art.

[0076] In some embodiments, antibodies produced by the immune organoid of the present disclosure can function in CDC. CDC refers to an in vitro or in vivo process where an antibody can bind to an antigen on a surface of a eukaryotic or prokaryotic cell then engage with the Clq protein via sequences within the antibody’s Fc domain that in turn results in initiation of classical complement cascade that can kill bound cell. CDC activity can be measured using methods known in the art including, without limitation, using in vitro methods as known in the art.

[0077] In some embodiments, antibodies produced by the immune organoid of the present disclosure can function in opsonization. Opsonization is a process where an antibody can bind toan antigen on a surface of a cell then engage with immune cells via sequences within its Fc domain that in turn results in immune cells engulfing, consuming and ultimately killing antibody bound cell. Opsonization activity' can be measured using methods known in the art including, without limitation, using in vitro methods as known in the art.

[0078] In some embodiments, the immune organoid as described herein exhibits at least one of ADCC, CDC, and opsonization activity. In some embodiments, the immune organoid as described herein exhibits at least two of ADCC, CDC, and opsonization activity7. In some embodiments, the immune organoid as described herein exhibits all three of ADCC, CDC, and opsonization activity.

[0079] In some embodiments, the antibodies produced by7the immune organoid of the present disclosure can also undergo the process of somatic hypermutation. Somatic hypermutation refers to a process of enhanced mutation of a gene, thought to require activation-induced cytidine deaminase (AID), an error-prone DNA repair enzyme. SHM was initially described from observations of the increased mutation of immunoglobulin gene regions encoding variable regions of the light and heavy chains in B lymphocytes following antigen stimulation. AID is discussed, for example, in Smith et al., Trends Genet. 20:224-227 (2004). The presence of Somatic hypermutation can be identified using methods including, without limitation, measurement of AID upregulation by, for example, quantitative PCR methods. B cell receptor sequencing can also be used to identify collections of mutations.

[0080] In some embodiments, the immune organoids described herein produce plasmablasts and antigen-specific antibodies against targets to which patient donors have been exposed (recall response) and to which patients are naive.

[0081] In some embodiments, the immune organoids described herein produce autoimmune responses against self-antigen.

[0082] In some embodiments, the antibodies produced by the immune organoids as described herein bind human and non-human targets. Exemplary human targets include, without limitation, proteins, sugars, and nucleic acid. Exemplary non-human targets can include, without limitation, infectious disease antigens, venoms, poisons, small molecules.Therapeutics

[0083] As described above, the method includes the step of contacting the three dimensional immune organoid described above with a therapeutic.

[0084] As used herein, a therapeutic can refer to a molecule or composition that has an effect on a cell. The therapeutic used in the methods of the disclosure can be selected from one or more of the following therapeutic classes: immunotherapeutic, self-antigen, tumour-specific peptides, checkpoint inhibitors, alkylating agent, antimetabolite, metabolic agonist, metabolic antagonist, plant alkaloid, mitotic inhibitor, antitumour, antibiotic, topoisomerase inhibitor, radiotherapeutics, chemotherapeutics, antibodies, nanobodies, photosensitizing agent, stem cell transplant, vaccine, cytotoxic agent, cytostatic agent, tyrosine kinase inhibitor, proteasome inhibitor, cytokine, interferon, interleukin, intercalating agent, targeted therapy agent, gene therapy, small-molecule drug, hormone, steroid, cellular therapeutic, viral vector, and nucleic acid therapeutic.

[0085] In some embodiments, the therapeutic is a self-antigen. In some embodiments, the selfantigen is associated with an autoimmune disorder. Exemplary autoimmune disorders include, without limitation, multiple sclerosis (MS), peripheral neuritis, Sjogren's syndrome, rheumatoid arthritis, alopecia, autoimmune pancreatitis, Behcet's disease, Bullous pemphigoid, Celiac disease, Devic's disease (neuromyelitis optica), Glomerulonephritis, IgA nephropathy, assorted vasculitides, scleroderma, diabetes, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, systemic lupus erythematosus, Graves' disease, myasthenia gravis (MG), pemphigus vulgaris, anti-glomerular basement membrane disease (Goodpasture syndrome), Hashimoto's thyroiditis, autoimmune hepatitis, or combinations thereof. In some aspects, the selfantigen comprises beta-cell proteins, insulin, islet antigen 2 (IA-2), glutamic acid decarboxylase (GAD65), zinc transporter 8 (ZNT8), myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP). proteolipid protein (PLP). myelin-associated glycoprotein (MAG), citrullinated antigens, synovial proteins, aquaporin-4 (AQP4), nicotinic acetylcholine receptor (nAChR), desmoglein-1 (DSG1), desoglein-2 (DSG2), thyrotropin receptor, type IV collagen, thyroglobulin, thyroid peroxidase, thyroid-stimulating hormone receptor (TSHR), or combinations thereof. In some embodiments, the self-antigen is MOG and the autoimmune disorder is multiple sclerosis (MS).

[0086] In some embodiments, the self-antigen is an induced self-antigen. In some embodiments, the induced self-antigen is an NK cell receptor protein. In some embodiments, the NK cell receptor protein is NKG2D.

[0087] The self-antigen can be contacted with the immune organoid as described herein at any dose at which the self-antigen is sought to be tested for efficacy. In some embodiments, the self-antigen is contacted with the immune organoid at a dose of about 1 .0 pg / mL, 2.0 pg / mL, 3.0 pg / mL, 4.0 pg / mL, 5.0 pg / mL, 6.0 pg / mL, 7.0 pg / mL, 8.0 pg / mL, 9.0 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL, 45 pg / mL, or 50 pg / mL.

[0088] In some embodiments, the therapeutic is an immunotherapeutic. An “immunotherapeutic” can refer to any medical intervention that induces, suppresses or enhances the immune system of a patient for the treatment of a disease. In some embodiments, immunotherapies activate a patient's innate and / or adaptive immune responses (e.g., T cells) to more effectively target and remove a pathogen or cure a disease, such as cancer or an immune disease.

[0089] In some embodiments, the immunotherapeutic is selected from the group consisting of a vaccine, an antibody, a gene therapy, a cell therapy, a small molecule, and a nanobody.

[0090] Any of the therapeutics used in the methods of the present disclosure can also be combined with one or more adjuvants which can function to enhance the immune responses (humoral and / or cellular). Adjuvants can include, without limitation, Pam3CSK4 (0.1-1 pg / mL), MALP-2 (50-500 ng / mL), Poly I:C (1-50 pg / mL), Poly ICLC (1-25 pg / mL), MPL (0.1-10 pg / mL), GLA (0.5-5 pg / mL), Flagellin (0.1-2 pg / mL), Imiquimod (1-10 pM), Resiquimod (0.1-5 pM), Gardiquimod (0.5-5 pM), CpG ODN (0.1-10 pM), MDP (1-20 pg / mL), M-Tri-DAP (5-50 pg / mL), cGAMP (1-20 pg / mL), ADU-S 100 (0.5-10 pM), Aluminum hydroxide (50-250 pg / mL), Aluminum phosphate (50-250 pg / mL), Calcium phosphate (100-500 pg / mL), MF59 (2-20% v / v), AS03 (2-20% v / v), AS04 (5-50 pg / 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 IFNs (100-1000 U / mL), TSLP (5-50 ng / mL), QS-21 (1-20 pg / mL), ISCOM (1-25 pg / mL), Matnx-M (1-25 pg / mL), AS01 (1-20 pg / mL), AS02 (1-20 pg / mL), Cyclic dinucleotides (0.1-10 pg / mL), R848 (0.1-5 pM), Glucopyranosyl lipid A (0.1-10 pg / mL), a-GalCer (50-500 ng / mL), Muramyl dipeptide (0.5-10 pg / mL), |3-glucans (10-100 pg / mL). Inulin (50-500 pg / mL), Chitosan (1-50 pg / mL), Dextran (10-100 pg / mL). Mannose derivatives (1-50 pg / mL), Montanide (5-20% v / v), Complete Freund's (1: 1 ratio), Incomplete Freund's (1: 1 ratio), Squalene-based (2-20% v / v), PLGA (10-200 pg / mL), PLA (10-200 pg / mL), PCL (10-200 pg / mL), Gold nanoparticles (1-50 pg / mL), Liposomes (10-200 pg / mL), CD40 agonists (0.1-10 pg / mL), 0X40 agonists (0.1-10 pg / mL), 4-1BB agonists (0.1-10 pg / mL), GITR agonists (0.1-10 pg / mL), Quillaja saponins (1-50 pg / mL), Essential oils (0.01-0.1% v / v), Plant extracts (1-100 pg / mL), Bacterial toxins (modified) (0.1-1 pg / mL), AS01 (MPL 1-10 pg / mL + QS-21 1-20 pg / mL), AS04 (MPL 1-10 pg / mL +Alum 50-250 pg / mL), CAF01 (DDA 250-500 pM + TDB 50-100 pM), and IC31 (KLK 100-500 pM + ODNla 0.1-10 pM).Vaccines

[0091] In some embodiments, the immunotherapeutic is a vaccine. As used herein, vaccines can include any material administered to raise either humoral and / or cell mediated immune response, such as live or attenuated viral and bacterial immunogens and inactivated viral, tumor- derived, protozoal, 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. Any vaccine is contemplated for use in the methods described herein. Exemplary vaccines include, without limitation, vaccines for cancer, BCG, cholera, plague, typhoid, hepatitis A, B, and C, influenza A and B, parainfluenza, polio, rabies, measles, mumps, rubella, yellow fever, tetanus, diphtheria, haemophilus influenza b, tuberculosis, meningococcal and pneumococcal vaccines, adenovirus, HIV, chicken pox, cytomegalovirus, dengue, feline leukemia, fowl plague, HSV-1 and HSV-2, hog cholera, Japanese encephalitis, respiratory syncytial virus, rotavirus, papilloma virus, severe acute respiratory syndrome (SARS), SARS-CoV-2, anthrax, and yellow fever.

[0092] The antigens or immunogens used to prepare the vaccines may be derived from a wide variety of sources. For example, suitable antigens or immunogens may include an infectious agent (e.g., bacterial, fungal, protozoan, parasitic, or viral), an infectious agent-derived product, e.g., protein, peptide, nucleic acid, polysaccharide, glycoprotein, glycolipid, antigen or antigenic preparations, a degenerative disease antigen, an atopic disease antigen, an autoimmune disease antigen, an alloantigen, a xenoantigen. a metabolic disease enzyme or enzymatic product, a recombinantly produced protein or peptide, a chimeric fusion protein, and / or a small molecule.

[0093] Suitable antigens or immunogens may be in the form of whole cells or purified or partially purified antigens or antigenic preparations. Suitable antigens or immunogens may be used without modification, in galenic form, or in combination with vehicles or carriers such as e.g. microspheres, liposomes, nanospheres, and other antigen delivery systems familiar to one of ordinary skill in the art.

[0094] The vaccine can be based on an antigen that is prepared or derived from natural sources or produced through recombinant technologies.

[0095] In some embodiments, the vaccine can be a vaccine for an infectious disease. In some embodiments, the vaccine for an infectious diseases comprises an antigen or immunogen selectedfrom microbial structures (cell walls, capsules, flagella, pili, viral capsids, envelope-associated glycoproteins); microbial toxins (Allergens: dust, pollen, hair, foods, dander, bee venom, drugs, and other agents causing allergic reactions; Foreign tissues and cells (from transplants and transfusions); and the body's own cells that the body fails to recognize as “normal self (cancer cells, infected cells, cells involved in autoimmune diseases).

[0096] In one embodiment, the antigen or immunogen can be an infectious agent, or a product of an infectious agent. In one embodiment, the antigen or immunogen comprises an inactivated infectious agent, e.g., that has been killed or otherwise attenuated. In another embodiment, the antigen or immunogen comprises a live infectious agent.

[0097] In one embodiment, the infectious agent (or infectious agent product) is a virus, for example and without limitation, a pox virus (e g., vaccinia virus), smallpox virus, marburg virus, flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus), influenza virus (or antigens, such as F and G proteins or derivatives thereof), e.g., influenza A; or purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof), parainfluenza virus (e.g., sendai virus), respiratory syncytial virus, rubeola virus, human immunodeficiency virus (or antigens, e g., such as tat, nef, gpl20 or gp!60), 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, e.g., such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2), cytomegalovirus (or antigens such as gB or derivatives thereof), Epstein-Barr virus (or antigens, such as gp350 or derivatives thereof), JC virus, rhabdovirus, rotavirus, rhinovirus. adenovirus, papillomavirus, parvovirus, picomavirus. poliovirus, virus that causes mumps, virus that causes rabies, reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus, hantavirus, junin virion, filovirus (e.g., ebola virus), coxsackievirus, equine encephalitis virus, Rift Valley fever virus, alphavirus (e.g., Chikungunyavirus. sindbis virus), hepatitis A virus, hepatitis B virus (or antigens thereof, for example Hepatitis B Surface antigen or a derivative thereof), hepatitis C virus, hepatitis D virus, or hepatitis E virus.

[0098] In one embodiment, the infectious agent is a bacterium. Non-limiting examples of suitable bacteria (or bacterially derived products) for use in the vaccines and / or methods of the disclosure include Neisseria species, including N. gonorrhea and N. meningitidis (or antigens, such as, for example, capsular polysaccharides and conjugates thereof, transferrin-bindingproteins, lactoferrin binding proteins, Pi l C, adhesins); Haemophilus species, e.g., H. influenzae,' S. pyogenes (or antigens, such as, for example, M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans; H. ducreyi: Moraxella spp, including M catarrhalis, also known as Branhamella catarrhalis (or antigens, such as, for example, high and low molecular weight adhesins and invasins); Bordetella spp, including B. pertussis (or antigens, such as, for example, pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica;Mycobacterium species, including M. tuberculosis (or antigens, such as, for example, ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium. M. paratuberculosis. M. smegmatis: Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (or antigens, such as, for example, colonization factors, heat-labile toxin or derivatives thereof, heatstable toxin or derivatives thereof), enterohemorragic E. coli, enteropathogenic E. coli (or antigens, such as, for example, shiga toxin-like toxin or derivatives thereof); Vibrio spp, including V. cholera (or antigens, such as, for example, cholera toxin or derivatives thereof); Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y enterocolitica (or antigens, such as, for example, a Yop protein), Y pestis. Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (or antigens, such as, for example, toxins, adhesins and invasins) and C. coli; Salmonella sp\ including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis, S. typhimurium, and S. dysenteriae; Listeria species, including L. monocytogenes; Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus species, including S. aureus, S. epidermidis; Proteus species, e.g., P. mirabilis; Enterococcus species, including E. faecalis, E. faecium; Clostridium species, including C. tetani (or antigens, such as, for example, tetanus toxin and derivative thereof), C. botulinum (or antigens, such as, for example, botulinum toxin and derivative thereof). C. difficile (or antigens, such as. for example, Clostridium toxins A or B and derivatives thereof), and C. perfringens; Bacillus species, including / ?, anthracis (or antigens, such as, for example, botulinum toxin and derivatives thereof), B. cereus, B. circulans and B. megaterium; Corynebacterium species, including C. diphtheriae (or antigens, such as, for example, diphtheria toxin and derivatives thereof); Borrelia species, including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (or antigens, such as, for example. OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (or antigens, such as, for example, OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia species, including E.equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia species, including C. trachomatis (or antigens, such as, for example, MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP, heparin-binding proteins), C. psittaci; Leptospira species, including L. interrogans,' Streptococcus species, such as S. pyogenes, S. agalactiae, S. pneumonia; Treponema species, including T. pallidum (or antigens, such as, for example, the rare outer membrane proteins), T denticola, and T. hyodysenteriae.

[0099] In one embodiment, the infectious agent is a parasite, or a parasite derived product. Non-limiting examples of suitable parasite (or parasite derived products) for use in the vaccines and / or methods of the invention include Plasmodium species, including P. falciparum;Toxoplasma species, including T. gondii (or antigens, such as, for example SAG2, SAGS, Tg34); Entamoeba species, including E. histolytica; Babesia species, including B. microti; Trypanosoma species, including T cruzi; Giardia species, including G. lamblia; Leshmania species, including L. major; Pneumocystis species, including P. carinii; Trichomonas species, including T. vaginalis; and Schisostoma species, including S. mansoni.

[0100] In another embodiment, the infectious agent is a fungus, or a fungal derived product. Suitable fungi (or fungal derived products) for use in the vaccines and / or methods of the invention include, without limitation, Candida species, including C. albicans and parapsilosis; Cryptococcus species, including C. neoformans; Aspergillus fumigates and niger. Fusarium spp, Trychophyton spp, Absidia species, e.g., Absidia corymbifera, Ajellomyces spp, e.g., Ajellomyces capsulatus, Arthroderma species, e.g., Arthroderma benhamiae, Blastomyces species, e.g., Blastomyces dermatitidis, Cladophialophor a species, e.g., Cladophialophora carrionii, Coccidioides spp, e.g., Coccidioides immitis. Cryptococcus spp, e.g., Cryptococcus neoformans, Cunninghamella species, Epidermophyton species, e.g., Epidermophyton floccosum, Exophiala spp, e.g., Exophiala dermatitidis, Filobasidiella spp, e.g., Filobasidiella neoformans. Fonsecaea spp, e.g., Fonsecaea pedrosoi, Fusarium spp, e.g.. Fusarium solani, Geotrichum spp, e.g., Geotrichum candidum, Histoplasma spp. e.g., Histoplasma capsulatum, Hortaea spp, e.g., Hortaea werneckii, Issatschenkia spp, e.g., Issatschenkia orientalis, Madurella spp, e.g., Madurella grisae, Malassezia spp, e.g, Maias sezia furfur , Microsporum spp, e.g., Microsporum canis, Mucor spp, e.g., Mucor circinelloides, Nectria spp, e.g., Nectria haematococca, Paecilomyces spp, e.g., Paecilomyces variotii, Paracoccidioides spp, e.g., Paracoccidioides brasiliensis, Penicillium spp, e.g., Penicillium marneffei, Pichia spp, e.g., Pichia guilliermondii,Pneumocystis spp, e.g., Pneumocystis carinii, Pseudallescheria spp, e.g., Pseudallescheria boydii, Rhizopus spp, e.g., Rhizopus oryzae, Rhodotorula spp, e.g., Rhodotorula rubra, Scedosporium spp, e.g., Scedosporium apiospermum, Schizophyllum spp, e.g., Schizophyllum commune, Sporothrix spp, e.g., Sporothrix schenckli, Trichophyton spp, e.g., Trichophyton violaceum, and Trichosporon spp, e.g., Trichosporon mucoides.

[0101] In another embodiment, the infectious agent is a protozoan, or a protozoan derived product. Suitable protozoans (or protozoan derived products) for use in the vaccines and / or methods of the invention include, without limitation, protests (unicellular or multicellular), e.g, Plasmodium falciparum, and helminths, e.g., cestodes, nematodes, and trematodes.

[0102] In one embodiment, a suitable antigen or immunogen for use in the vaccines and methods of the invention is an alloantigen (a self-antigen), such as a protein or peptide, lipoprotein, lipid, carbohydrate, a nucleic acid, an enzyme, a structural protein, a secreted protein, a cell surface receptor, and a cytokine, e.g, TNF, IFN-y, IL-1, or IL-6.

[0103] In some embodiments, the vaccine is an influenza vaccine. Influenza vaccines can include, without limitation, influenza subunit vaccines, influenza mRNA vaccines, and adjuvanted influenza vaccines. In some embodiments, the influenza vaccine is an influenza subunit vaccine. In some embodiments, the influenza subunit comprises the 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.

[0104] In some embodiments, the vaccine is a rabies vaccine.

[0105] In some embodiments, the vaccine is a hepatitis A vaccine.

[0106] In some embodiments, the vaccine is a SARS-CoV-2 vaccine.Antibodies

[0107] In some embodiments, the immunotherapeutic is an antibody. An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen binding fragment (z.e., "antigen-binding portion") or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site including, for example without limitation, scFv, single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies,triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1 126-1 136). An antibody can include an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy’ chains, immunoglobulins can be assigned to different classes.

[0108] There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0109] The antibody used in the present disclosure can be a monoclonal antibody. The term "monoclonal antibody" (mAb) refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokary otic, or phage clone, and not the method by which it is produced. Preferably, a monoclonal antibody exists in a homogeneous or substantially homogeneous population. In immunotherapy, monoclonal antibodies (mAbs) are manufactured in vitro to recognize specific targeted antigens. They are used to treat, for example, solid and hematopoietic tumors, inflammatory disorders, and infections. Most mAbs in clinical use target a single antigen, but a few are engineered to be bispecific. The monoclonal antibody’ contemplated for use herein can encompass murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies.

[0110] "Humanized" antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity’, affinity, and capacity’. The term "chimeric antibody" can refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0111] Antibodies of the invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, S.D.. Clin. Chem.. 45: 1628-50 (1999) and Fellouse, F.A., et al., J. Mol. Biol., 373(4):924-40 (2007)).

[0112] Immunotherapeutic antibodies are w ell known in the art, and include, without limitation, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, ustekinumab, etanercept, gemtuzumab. alemtuzumab, rituximab, trastuzumab, nimotuzumab, palivizumab, daratumumab. denosumab, dinutuximab. elotuzumab, isatuximab, margetuximab, mogamulizumab, naxitamab, necitumab, obintuzumab, and abeiximab.

[0113] The antibody can be contacted with the immune organoid as described herein at any dose at which the antibody is sought to be tested for efficacy. In some embodiments, the antibody is contacted with the immune organoid at a dose of about 0.1 pg / mL, 0.2 pg / mL, 0.3 pg / mL, 0.4 pg / mL, 0.5 pg / mL, 0.6 pg / mL, 0.7 pg / mL, 0.8 pg / mL, 0.9 pg / mL, 1.0 pg / mL, 2.0 pg / mL, 3.0 pg / mL, 4.0 pg / mL, 5.0 pg / mL, 6.0 pg / mL, 7.0 pg / mL, 8.0 pg / mL, 9.0 pg / mL, 10 pg / mL, 15 pg / mL. or 20 pg / mL.

[0114] In some embodiments, the immunotherapeutic antibody is a nanobody. Nanobodies in immunotherapy are known in the art and described in, for example, Maali et al., “Nanobodies in Cell-Mediated Immunotherapy: On the Road to Fight Cancer,” Front Immunol. 2023; 14: 1012841. Nanobodies or VHHs (Variable domain of Heavy chain from Heavy-chain only antibodies (HCAbs)), are derived from camelid heavy-chain antibodies. Nanobodies, as the smallest natural antigen binding domains, can have dimensions in the range of about 2.5 nm in diameter and about 4 nm in height and a molecular w eight of about 15 kD. High-affinity nanobodies against different targets, including tumor markers, can be selected from phage- displayed libraries through the biopanning process.

[0115] Nanobodies can be produced easily in microorganisms, mammalian cells, or plants. Nanobody expression yield is high, whether in the periplasm of Escherichia coli or the cytoplasm of eukaryotic cells.

[0116] Nanobodies have been used in different applications, including biosensing, affinitycapturing of proteins, and protein crystallization. They have been especially used for cancer therapeutics by targeting surface receptors of tumor cells 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 / sl3104-018-3955-8), CAIX (Araste F, Ebrahimizadeh W, Rasooli I, Rajabibazl M, Mousavi Gargari SL. A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis. Biotechnol left (2014) 36( 1 ):21 — 8. doi: 10. 1007 / sl0529-013-1340-l), 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. Mol Biotechnol (2013) 54(2): 590- 601. doi: 10.1007 / S12033-012-9601-0), DR5 (Huet HA, 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. mAbs (2014) 6(6): 1560-70. doi: 10.4161 / 19420862.2014.975099), c-Met (Slordahl TS, Denayer T, Moen SH, Standal T, Borset M, Ververken C. et al., Anti-c-MET nanobody - a new potential drug in multiple myeloma treatment. Eur J haematol (2013) 91(5):399-410. doi: 10.1 11 1 / ejh. 12185), EGFR (Chen T, Liu X, Hong H, Wei H. Novel single-domain antibodies against the EGFR domain III epitope exhibit the anti-tumor effect. J Trans Med (2020) 18(1):376. doi: 10. 1186 / sl 2967-020-02538-y), mesothelin (Tang Z, Feng M, Gao W, Phung Y. Chen W, Chaudhary A, et al., A human singledomain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface. Mol Cancer Ther (2013) 12(4):416-26. doi: 10.1158 / 1535-7163. MCT- 12-0731), AgSKl (Rashidi SK, Mousavi Gargari SL, Ebrahimizadeh W. Targeting colorectal cancer cell lines using nanobodies; AgSKl as a potential target. Iranian J Biotechnol (2017) 15(2):78-86. doi: 10. 15171 / ijb. 1472) and CD33 (Romao E. Krasniqi A, Maes L. Vandenbrande C, Sterckx YG, Stijlemans B, et al., Identification of nanobodies against the acute myeloid leukemia marker CD33. IntJMol Sci (2020) 21(l):310. doi: 10.3390 / ijms21010310).

[0117] Now, about 16 therapeutic nanobodies have entered clinical trials for various disease types (Arbabi-Ghahroudi M. Camelid single-domain antibodies: Promises and challenges as lifesaving treatments. IntJMol Sci (2022) 23(9): 5009. doi: 10.3390 / ijms23095009).Gene Therapy

[0118] In some embodiments, the immunotherapeutic is a gene therapy . The term “gene therapy” is given its ordinary meaning in the art. Briefly, “gene therapy” refers to the transfer of genetic material (e.g., a DNA or RNA polynucleotide) of interest into a host cell and / or tissue to treat or prevent a disease condition. The target genetic material encodes a productthat is desired to be produced in vivo (e.g., a protein, polypeptide, or functional RNA). For example, the target genetic material may encode a therapeutically valuable enzyme, hormone, receptor, or polypeptide.

[0119] Any method available in the art for gene therapy may be used in accordance with the present disclosure. For example, gene therapy techniques are described in, for example, 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). Generally known recombinant DNA techniques used for gene therapy are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology7, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0120] The main immunological targets for gene therapy include, without limitation, cytokine / chemokine genes, tumor-associated antigens, fusion proteins, including tumor antigens, genetically modified tumor cells, or immune cells. These are described in detail in the art, e.g., Akbulut, “Immune Gene Therapy of Cancer,’' Turk J Med Sci 202 50(7): 1679-1690.Cell Therapy

[0121] In some embodiments, the immunotherapeutic is a cell therapy. Cell therapy refers to a method of treatment in which normal cells or biotechnologically modified cells are expanded in vitro and then transplanted or transfused into a patient. The newly input cells can replace damaged cells to reconstruct tissue structure and function (stem cell therapy technology) or have stronger immune killing function (immune cell therapy technology) to achieve the aim of treating diseases.

[0122] For the purposes of the methods disclosed herein, any type of cell therapy is contemplated. For example, the cell therapy can include pluripotent stem cell therapy, adult stem cell therapy7, cancer stem cell therapy, fibroblast cell therapy, chondrocyte cell therapy keratinocyte cell therapy, hepatocyte cell therapy, pancreatic islet cell therapy, T cell therapy, dendritic cell therapy, Natural killer cell therapy, and macrophage cell therapy. Such ty pes are described in more detail in, for example, El-Kadiry et al., Front Med 8 (2021).

[0123] In some embodiments, the cell therapy is an adoptive cell therapy. "Adoptive cell therapy" refers to immunotherapy in which immune cells are administered to a subject to help the subject fight a disease such as cancer or viral infection. In cancer therapy, for example, Tcells are harvested from a subject's own blood (or from a donor's blood) or tumor tissue, grown in large numbers, and then given back to the subject to help the subject fight cancer. Types of adoptive cell therapy include tumor-infiltrating lymphocyte (“TIL”) therapy, T-cell receptor (“TCR”) therapy, and chimeric antigen receptor T-cell (CAR-T-cell) therapy.

[0124] “Tumour-infiltrating lymphocyte (“TIL”) therapy” or “TIL therapy” refers to immunotherapy, which is adoptive cell therapy using lymphocytes that are in or near a tumor and have the ability to recognize the tumor. In TIL therapy, ly mphocytes such as T-cells in or near a tumor are isolated and then treated with substances that cause them to grow rapidly in large numbers. These lymphocytes are then given back to the subject.

[0125] “T-cell receptor therapy” or “TCR therapy” refers to a type of adoptive cell therapy that involves engineering a subject's or donor's T or immune cells to express a special or specific T- cell receptor or TCR.

[0126] “Chimeric antigen receptor T cell therapy” or “CAR-T therapy” refers to adoptive cell therapy in which one or more portions of a T cell receptor are changed to an extracellular binding moiety such as an antibody or antibody fragment. Extracellular binding moieties such as antibodies or antibody fragments can be targeted with tumor-associated antigens (TAAs) or tumor-specific antigens.Small Molecules

[0127] In some embodiments, the immunotherapeutic is a small molecule. A"small molecule" refers to a composition that has a molecular w eight 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 can comprise nucleic acids, peptides, polypeptides. peptidomimetics, peptoids, carbohydrates, lipids, components of these or other organic or inorganic molecules. In some embodiments, the small molecule is a small molecule-based immunomodulator. Such small molecules include those targeting innate immune system, adaptive immune system, and tumor microenvironment. A small molecule-based immunomodulator refers to a nonsteroidal agent that reduces the production or secretion of a proinflammatory cytokine, causes a reduction in the proinflammatory response, or otherwise modulates the immune system. Such small molecules are known in the art and are 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 are p38 kinase inhibitors such as VX702 (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 my cophenolate (Roche) and merimepodib (Vertex Pharmaceuticals).Contacting

[0128] The therapeutics as described herein can be contacted with the immune organoid in a manner that involves exposing the organoid to therapeutic levels of a known or unknown therapeutic. Typically, an agent will be dissolved in solution to a (predicted) therapeutically effective concentration and administered to the culture into a vessel in which the culture is maintained. The therapeutic dose range will vary depending on the particular composition and therapeutic agent contacted with the organoid.

[0129] In some embodiments, the therapeutic is contacted with the organoid for 1 day. 2 days. 3 days. 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days. 27 days, 28 days, 29 days, or 30 days. In some embodiments, the therapeutic is contacted with the organoid for about 5 days. In some embodiments, the therapeutic is contacted with the organoid for about 7 days. In some embodiments, the therapeutic is contacted with the organoid for about 11 days. In some embodiments, the therapeutic is contacted with the organoid for about 14 days. In some embodiments, the therapeutic is contacted with the organoid for about 21 days. In some embodiments, the therapeutic is contacted with the organoid for 25 days. In some embodiments, the therapeutic is contacted with the organoid for about 28 days.

[0130] The therapeutic as described herein can also be contacted with the immune organoid at various time points in immune organoid formation. In some embodiments, the therapeutic is contacted with the immune organoid at day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11. day 12. In some embodiments, the therapeutic is contacted with the immune organoid at day 0. In some embodiments, the therapeutic is contacted with the immune organoid at day 1. In some embodiments, the therapeutic is contacted with the immune organoid at day 2. In some embodiments, the therapeutic is contacted with the immune organoid at day 3. In some embodiments, the therapeutic is contacted with the immune organoid at day 3. In some embodiments, the therapeutic is contacted with the immune organoid at day 4. In some embodiments, the therapeutic is contacted with the immune organoid at day 5. In someembodiments, the therapeutic is contacted with the immune organoid at day 6. In some embodiments, the therapeutic is contacted with the immune organoid at day 7. In some embodiments, the therapeutic is contacted with the immune organoid at day 8. In some embodiments, the therapeutic is contacted with the immune organoid at day 9. In some embodiments, the therapeutic is contacted with the immune organoid at day 10. In some embodiments, the therapeutic is contacted with the immune organoid at day 11. In some embodiments, the therapeutic is contacted with the immune organoid at day 12. In some embodiments, the contacting step involves a prime-boost regimen. For example, in some embodiments, the therapeutic is contacted with the immune organoid at day 0, day 1. day 2, day 3, day 4, day 5, and day 6 and then contacted again with the immune organoid at day 14, day 15, day 16, day 17, day 18, day 19, and day 20.

[0131] The vaccine can be contacted with the immune organoid as described herein at any dose at which the vaccine is sought to be tested for safety. In some embodiments, the vaccine is contacted with the immune organoid at a dose of about 0. 1 pg / mL, 0.2 pg / mL, 0.3 pg / mL, 0.4 pg / mL, 0.5 pg / mL, 0.6 pg / mL, 0.7 pg / mL, 0.8 pg / mL, 0.9 pg / mL, 1.0 pg / mL, 2.0 pg / mL, 3.0 pg / mL, 4.0 pg / mL, 5.0 pg / mL, 6.0 pg / mL, 7.0 pg / mL, 8.0 pg / mL, 9.0 pg / mL, 10 pg / mL, 15 pg / mL. 20 pg / mL, 25 pg / mL, 30 pg / mL, 35 pg / mL, 40 pg / mL. 45 pg / mL, or 50 pg / mL.

[0132] In some embodiments, the vaccine is contacted with the immune organoid at a dose of about 0.1 lU / mL, 0.5 lU / mL, 1 lU / mL, 1.5 lU / mL, 2 lU / mL, 2.5 IU / m , 5 lU / mL, 10 lU / mL, 15 lU / mL, 20 lU / mL, 25 lU / mL, 30 lU / mL, 35 lU / mL, 40 lU / mL, 45 lU / mL, 50 lU / mL, 55 lU / mL, 60 lU / mL, 65 lU / mL, 70 lU / mL, 75 lU / mL, 80 lU / mL, 85 lU / mL, 90 lU / mL, 95 lU / mL. 100 lU / mL. 1 10 lU / mL, 120 lU / mL. 130 lU / mL, 140 lU / mL, 150 lU / mL, or 160 lU / mL.

[0133] In some embodiments, the vaccine is an influenza subunit vaccine contacted with the immune organoid at a dose of about 0.5 pg / mL, 0.6 pg / mL, 0.7 pg / mL, 0.8 pg / mL, 0.9 pg / mL, 1.0 pg / mL, 2.0 pg / mL, 3.0 pg / mL, 4.0 pg / mL, 5.0 pg / mL, 6.0 pg / mL, 7.0 pg / mL, 8.0 pg / mL, 9.0 pg / mL. or 10 pg / mL. In some embodiments, the vaccine is an influenza mRNA vaccine contacted with the immune organoid at a dose of about 0.1 pg / mL, 0.2 pg / mL, 0.3 pg / mL, 0.4 pg / mL, 0.5 pg / mL, 0.6 pg / mL, 0.7 pg / mL, 0.8 pg / mL, 0.9 pg / mL, 1.0 pg / mL, 2.0 pg / mL, 3.0 pg / mL, 4.0 pg / mL, 5.0 pg / mL, 6.0 pg / mL, 7.0 pg / mL, 8.0 pg / mL, 9.0 pg / mL, 10 pg / mL, 15 pg / mL. 20 pg / mL, 25 pg / mL, 30 pg / mL, or 35 pg / mL.

[0134] In some embodiments, the vaccine is a rabies vaccine contacted with the immune organoid at a dose of about 2.5 IU / mL, 5 lU / mL, 10 lU / mL, 15 lU / mL, 20 lU / mL, or 25 lU / mL.

[0135] In some embodiments, the vaccine is a hepatitis A vaccine contacted with the immune organoid at a dose of about 20 ZU / mL, 25 lU / mL, 30 lU / mL, 35 lU / mL, 40 lU / mL, 45 ZU / mL, 50 lU / mL, 55 lU / mL, 60 lU / mL, 65 lU / mL, 70 lU / mL, 75 lU / mL, 80 lU / mL, 85 lU / mL, 90 lU / mL, 95 lU / mL, 100 lU / mL. 110 lU / mL, 120 lU / mL. 130 lU / mL, 140 lU / mL, 150 lU / mL, or 160 lU / mL.Measuring Immune Organoid Response

[0136] The methods described herein further involve measuring the response of the three dimensional immune organoid to the therapeutic after the contacting step. One important indication of drug efficacy is how a drug acts on the immune system. Measuring the immune response can include measuring any aspect of the immune response, such as e.g. induction of the innate or adaptive immune response. In some embodiments, the method described herein involves measuring the innate immune response. In some embodiments, the method described herein involves measuring the adaptive immune response.

[0137] The innate immune response refers to non-antigen specific responses, for example the release of soluble effector compounds, non-specific phagocytosis performed by dendritic cells, macrophages, etc. It also refers to cellular changes that affect the abilities of cells to act as antigen presenting cells and / or modulate the antigen-specific adaptive immune response. Monocyte lineage cells mediate the initiation and progression of inflammation and other early immune responses by direct cytotoxicity , the secretion of soluble factors, and / or by regulating the adaptive immune response. These include the expression of adhesion molecules on monocyte derived cells and underlying vascular endothelium and the release of cytokines, chemokines. tissue-destructive metalloproteases, and reactive oxygen species.

[0138] The adaptive immune response can involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The adaptive immune response includes both a cellular immune response and humoral immune response.

[0139] In some embodiments, the method described herein involves measuring the cellular immune response. Cellular immunity relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to a stimulus. In a more general way, cellular immunity is not related to antibodies but to the activation of cells of the immune system. A cellular immune response is characterized e.g. by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis inbody cells displaying epitopes of an antigen on their surface, such as virus -infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; activating macrophages and natural killer cells, enabling them to destroy pathogens: and stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses. Specifically, the cellular immune response involves the presentation of polypeptide epitopes in conjunction with class II or class I MHC molecules to activate antigen specific CD4+T helper cells and / or CD8+cytotoxic T cells, respectively. This response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined, for example, by a proliferation assay (CD4+T cells) or a CTL (cytotoxic T lymphocyte) assay.

[0140] In some embodiments, the method described herein involves measuring the humoral immune response. Humoral immunity refers typically to antibody production and the accessory processes that may accompany it. A humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination. In some embodiments, the humoral immune response is measured by detection of germinal center formation. In some embodiments, the humoral immune response is measured by detecting antibody class switching. In some embodiments, the humoral immune response is measured by detecting memory B cell induction.

[0141] In some embodiments, the immune response is measured by measuring both the innate and adaptive immune response.

[0142] In some embodiments, the immune response is measured by measuring immune cell number, 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 immunological memory.

[0143] In some embodiments, the immune response is measured by measuring immune cell number, immune cell proliferation, immune cell phenotype, immune cell polarization, antibodyproduction, cytokine production, chemokine production, changes in B cell receptors, changes in T cell receptors, and / or immunological memory7.

[0144] Another important indication of drug safety is whether the drugs induce anti-drug antibodies (AD As), cytokine / chemokine storm, cytotoxicity, and / or cell death. In some embodiments, the method described herein involves measuring levels of AD As. In some embodiments, the method described herein involves measuring the presence or absence of cytokine storm. In some embodiments, the method described herein involves measuring the presence or absence of cytotoxicity. In some embodiments, the method described herein involves measuring the presence or absence of cell death.

[0145] In the methods described herein, the response is measured with reference to a control. A “control’' refers to a sample or standard used for comparison with a test sample. In some embodiments, the control is a three dimensional organoid obtained from a healthy subject that is not exposed to any therapeutic. In some embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample).Immune Cell Proliferation / Number

[0146] In some embodiments, immune cell proliferation is measured. Immune cell proliferation can be measured using any suitable method known in the art. For example, lymphocyte proliferation can be measured using a carboxy fluorescein diacetate succinimidyl diester (CFSE) dilution assay or by [31-l]-thymidine incorporation. Further, immune cell number can be measured using techniques such a flow cytometry, as described in the Examples provided herein, that can identify immune cell types based on markers such as CD3+ for T cells, CD 19+ for B cells. CD56+ and / or CD16+ for NK cells. CD27+CD8++ for plasmablasts. CD 138+ for plasma cells, CD15+ for granulocytes, CD1 lb+CD45+ for dendritic cells, CD14+CDl lc+ for myeloid dendritic cells, CD123+ for plasmacytoid dendritic cells, CD45- for stromal cells, PDPN+CD31+ for fibroblastic reticular cells, CD45+PDPN+CD35+ for follicular dendritic cells , CD27+CD38+ for germinal center B cells, CD14+CD1 lb+ for monocytes, and CD68+ or CD14+ for macrophages.

[0147] In one embodiment, an increase in immune cell proliferation and / or immune cell number in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. In one embodiment, a decrease in immune cell proliferation and / or immune cell number in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. In one embodiment, no change in immunecell proliferation and / or immune cell number in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.

[0148] In some embodiments, the increase in immune cell proliferation and / or immune cell number is increased 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% in immune organoids cultured with a therapeutic as compared to a control.

[0149] In some embodiments, the decrease in immune cell proliferation and / or immune cell number is increased 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% in immune organoids cultured with a therapeutic as compared to a control.

[0150] A “control’7refers to a sample or standard used for comparison with a test sample. In some embodiments, the control is a three dimensional organoid obtained from a healthy subject that is not exposed to any therapeutic. In some embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample).Immune Cell Phenotype

[0151] In some embodiments, immune cell phenotype is measured. The phenotypic characterization of a cell population by surface markers can be performed either by individual staining of the cells (flow cytometry as described in the Examples provided herein) or by making histological cuts of the population in situ, done in accordance with normal methods. The determination of the profile of expression of surface markers by antibodies, immunophenotype characterization, may be direct, using a labeled antibody or indirect, using a second labeled antibody against the primary specific antibody of the cell marker, thus achieving signal amplification. On the other hand, the presence or absence of binding to the antibody can be determined by different methods that include but are not limited to immunofluorescence microscopy and radiography. Similarly, it is possible to carry out the monitoring of the levels of binding of the antibody by flow cytometry', a technique that allows the levels of fluorochrome to be correlated with the quantity of antigens present on the cell surface bound specifically to thelabeled antibodies. The differential expression of a series of surface markers on a cell population provides a method for identification and isolation of said population.

[0152] In some embodiments, changes in immune cell phenotype in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. In some embodiments, no changes in immune cell phenotype in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.

[0153] By way of example, in some embodiments, immune organoids treated with a therapeutic can exhibit time dependent changes in B cell phenotype. As shown in the Examples herein, distinct populations can emerge at different timepoints. For example, there can be a progression from predominantly naive B cells (CD38-CD27-) at day 0 to significant populations of germinal center B cells (CD38+CD27+) by day 7-14, and ultimately to plasmablasts (CD38+++CD27+) by day 21, which demonstrates successful B cell activation and maturation within the organoid system.

[0154] Similarly, in some embodiments, immune organoids treated with a therapeutic can exhibit time dependent plasmablast differentiation. As show n in the Examples herein, organoids can exhibit an increase in CD38++CD27+ plasmablasts in response to vaccination.Immune Cell Polarization

[0155] In some embodiments, immune cell polarization is measured. Immune cell polarization is the process in which immune cells adopt distinct programs and perform specialized functions in response to specific signals. Immune cell polarization can be measured using a variety of methods known in the art including, but not limited to, intracellular cytokine staining, flow cytometry, ELISA. ELISpot. MSD, and / or Luminex methods.

[0156] In some embodiments, immune cell polarization is measured by measuring the production of cytokines including, for example, IL10, CXCL10, IFN-g, 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-g production is measured.

[0157] Techniques for measuring cytokines and chemokines are known to one skilled in the art. For example, levels of cytokines produced by immune organoids can be measured using an enzyme-linked immunosorbent assay (ELISA). In one embodiment, levels of cytokines can be measured using the EMDmillipore LUMINEX® xMAP® multiplex assay. In someembodiments, cytokine and chemokine production are measured using a bead-based multiplex assay. In some embodiments, the bead-based multiplex assay is LEGENDplex™.

[0158] In some embodiments, changes in immune cell polarization in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. In some embodiments, no changes in immune cell polarization in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.Antibody Production

[0159] In some embodiments, antibody production is measured. Production of antibodies can indicate the immune system’s reaction to a certain therapeutic and is thus an indicator of efficacy or, in some cases, a lack of efficacy. This is particularly relevant to vaccines for infectious diseases as well as cancer vaccines, where production of antibodies against antigens of interest is a desired outcome. For other therapies, antibodies can be produced against the treatment itself, leading to potential resistance to the therapy.

[0160] Antibody production can be measured using methods known in the art. 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, specific IgG, IgM, and IgA antibody production by the immune organoids is measured using ELISA as described in the Examples provided herein. In some embodiments, IgG antibody production is measured. In some embodiments, IgGl 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.

[0161] In some embodiments, an increase in the production of antibodies from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. Alternatively, in some embodiments, an increase in the production of antibodies from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious. In some embodiments, a decrease, or no change in the production of antibodies from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.Cytokine / Chemokine Production

[0162] In some embodiments, in the methods disclosed herein, the immune response is measured by measuring cytokine and / or chemokine production. Assessing cytokine / chemokine responses in response to immunotherapy treatment is highly relevant as these signaling molecules play a critical role activating and regulating the immune response, a primary goal of immunotherapy. Immunotherapies that induce an appropriate cytokine response is a good indicator of drug efficacy.

[0163] Cytokines are 8-30 kDa proteins and glycoproteins, which are produced by many cell types and operate as signals in cell-cell communication. They play a central role in the immune system and are involved in a variety of immunological, inflammatory, and infectious diseases. Exemplary cytokines that can be measured include, without limitation, 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. IL37, IL38, ILIA, IL1B, IL1RN, IFNA, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA14, IFNA16, IFNA17, IFNA21 , IFNB, IFNg, IL-la, IL-lb, IL-6, IL-13, IL-17a, TNFA, TNFB, TRAIL, TL1, BAFF, APRIL, RANKL, CD40LG, EDA, FASLG, CD70. In some embodiments, production of IL10 is measured. In some embodiments, production of IFNy is measured.

[0164] Chemokines are small chemoattractant secreted molecules regulating cell positioning and cell recruitment into tissues, playing a pivotal role in embryogenesis, tissue development and immune response. Approximately 50 chemokines and 20 chemokine receptors have been discovered so far. 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. Int J Mol Sci. 2018 Dec 27;20(l):96. Chemokines can be widely divided into two major groups based on their prominent functions: inflammatory' and homeostatic chemokines. Among inflammatory’ chemokines. which are induced by inflammation, some nonlimiting examples that can be measured in the methods of the disclosure include CXCL1, CXCL2, CXCL3, CXCL5, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, and CXCL14. On the other hand, homeostatic chemokines such as, without being limited to, CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12 and CXCL13 are constitutively expressed and are involved in homeostatic leukocyte trafficking. In some embodiments, the chemokine comprises a CXC chemokine, or an isoform or a derivative capable of binding thereof. In certain non-limitingexemplary embodiments, the chemokine can 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 vCXCl. In some embodiments, the chemokine includes 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 chemokine includes CXCL12, CXCL11. CXCL1, CXCL2, CXCL3. CXCL4. CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2. In some embodiments the chemokine includes vMIPII, U83, or vCXCl. In some embodiments, the chemokine is CXCL10.

[0165] Techniques for measuring cytokines and chemokines are known to one skilled in the art. For example, levels of cytokines produced by immune organoids can be measured using an enzyme-linked immunosorbent assay (ELISA). In one embodiment, levels of cytokines can be measured using the EMDmillipore LUMINEX® xMAP® multiplex assay. In some embodiments, cytokine and chemokine production are measured using a bead-based multiplex assay. In some embodiments, the bead-based multiplex assay is LEGENDplex™

[0166] In some embodiments, an increase in the production of cytokines and / or chemokines from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. Alternatively, in some embodiments, an increase in the production of cytokines and / or chemokines from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious. In some embodiments, a decrease, or no change in the production of cytokines and / or chemokines from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.BCR / TCR Changes

[0167] In other embodiments, the immune response is measured by analyzing changes in BCRs and / or TCRs. In one embodiment, BCR changes are measured. In one embodiment, TCR changes are measured. In one embodiment, both BCR and TCR changes are measured.

[0168] Both BCR and TCR changes can be measuring using sequencing techniques, such as with the use of the 10X Genomics platform and 5’ kits to obtain BCR and TCR libraries, whichwill be sequenced using NGS technology and analyzed for changes in the BCR and TCR repertoires.

[0169] BCR changes that can be measured via sequencing in the methods of the present disclosure include, without limitation, antibody isotype switching, clonal expansion, and somatic hypermutation. Isotype switching and somatic hypermutation are described in more detail above. Clonal expansion is the process by which daughter cells arise from a parent cell. During B cell clonal expansion (and T cell clonal expansion), many copies of that B cell are produced that share affinity with and specificity of the same antigen. This can be identified by sequencing as described above.

[0170] In some embodiments, changes in the BCR will lead to memory B cells which can measured. For example, in some embodiments, immune organoids that have been treated with a therapeutic will show an increase in the number of memory B cells in response to the therapeutic. Memory B cells can be measured using methods known in the art, including, but not limited to, flow cytometry for the detection of CD27+CD19+ cells. In some embodiments, memory B cells are measured using flow cytometry for detection of CD19+CD27+CD38- cells.

[0171] In some embodiments, an increase in the clonal expansion of B and / or T cells from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. Alternatively, in some embodiments, an increase in the clonal expansion of B and / or T cells from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious. In some embodiments, a decrease, or no change in the clonal expansion of B and / or T cells from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.

[0172] In some embodiments, an increase in memory B cells from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. In some embodiments, a decrease, or no change in memory B cells from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.

[0173] In some embodiments, an increase in isotype switching from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. In some embodiments, a decrease or no change in the isotype switching from animmune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.In some embodiments, an increase in somatic hypermutation from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is efficacious. In some embodiments, a decrease, or no change in somatic hypermutation from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.Immunological Memory

[0174] In other embodiments, the immune response is measured by measuring the formation of immunological memory. Immunological memory represents an important aspect of the immune response in mammals. Memory7responses form the basis for the effectiveness of many therapeutics, such as vaccines in particular, due to increased response to subsequent exposures, long-term protection created by the immunotherapy, and a strong induction of immunological memory is indicative of a better drug. Thus, the formation of immunological memory is a key indicator of therapeutic efficacy. As used herein, “immunological memory" refers to a state in which long-lived antigen-specific lymphocytes are available and are capable of rapidly mounting responses upon repeat exposure to a particular antigen. Memory7B cells are long lived B cells that express BCR on the surface, but they do not secrete large amounts of antibodies. Memory B cells recirculate throughout the body and respond rapidly to initiate a neyv immune response upon antigen re-encounter. The memory B cell has already undergone clonal expansion and differentiation and affinity maturation, so it is able to divide multiple times faster and produce antibodies with much higher affinity. Memory T cells can be both CD4+ and CD8+.These memory T cells have a lower threshold for activation via MHC, so they need less antigen to become activated compared to naive T cells.

[0175] In some embodiments, immunological memory is measured by the identification and quantification of both memory T cell subsets and memory B cell subsets using techniques known in the art such as flow cytometry as described in the Examples herein. In some embodiments, identification of the memory T cell subsets comprises identification of CD45RO, CCR7, CD62L, and CD127 on T cells. In some embodiments, identification of the memory B cell subsets comprises identification of CD27 on B cells and CD138 on plasma cells.

[0176] In some embodiments, the formation of immunological memory7from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic ofinterest is efficacious. In some embodiments, a decrease or no change immunological memory from an immune organoid cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not efficacious.Anti-Drug Antibodies (ADAs)

[0177] In some embodiments, the method described herein involves measuring levels of ADAs. The term "ADA" or "Anti -Drug Antibody" refers to an antibody that binds to a therapeutic antibody in a subject. ADAs can bind to various epitopes in therapeutic antibodies, including variable or constant regions.

[0178] It is well known that many therapeutics have immunogenic potential, and administration of therapeutic proteins to a patient sometimes results in the production of antibodies against the therapeutic protein. Such anti-drug antibodies (ADA) may reduce the effectiveness of the therapeutic protein, for example they may bind to or / and neutralize the therapeutic protein, resulting in changes of drug pharmacokinetics or pharmacodynamics that alters drug efficacy. ADA may cause serious side effects, including allergic reactions, crossreactivity against endogenous proteins, and complement activation. A life-threatening deficiency syndrome can result if ADA cross-reacts with and neutralizes a critical endogenous protein.

[0179] To screen for immunogenic activity of biologic drugs, assays for antibodies specific for potential therapeutic proteins, or components thereof, are often used during clinical drug development. Drug interference is regarded as one of the toughest challenges in such immunogenicity testing. For a drug with a long half-life and / or one administered at a high dose or a repeated dose, such as an antibody-based therapy, the ADA usually complexes with the drug, typically making the ADA unavailable for detection. In this situation, acid dissociation is often employed in an attempt to break up the ADA / drug immune complex so as to make the ADA available for detection. When a drug is present at high levels in the blood, it has proven extremely difficult to detect the ADA. even with acid dissociation, and it is often impossible to do so in a reproducible fashion.

[0180] Screening test subjects for the production of ADA is an important step in ensuring the safety and efficacy of many therapeutic proteins, and immunogenicity assessment of many- therapeutic biologies (drugs comprising proteins) is required by regulatory agencies as part of the pre-clinical and clinical phases of drug development, as well as in the post-market phase to ensure their safety. See, e.g., US Food and Drug Administration (FDA) Draft Guidance forIndustry: Assay Development for immunogenicity testing of therapeutic proteins, December 2009.

[0181] Immunodetection of ADA can be carried out by different types or platforms of immuno-assays including, without limitation, ELISA, immunocapture, microarrays (protein chips), flow cytometry (including, e.g, Fidis), or multiplex dot. The enzyme linked immunosorbent sandwich assay (ELISA) in a bridging format represents the state-of-the-art assay format for immunogenicity' testing due to its high throughput and sensitivity7and its easy applicability to different projects (Mikulskis, A., et al., J. Immunol. Meth. 365 (2011) 38-49).

[0182] Standard solid-phase anti-drug antibody immunoassays with monoclonal antibodies involve the formation of a complex between the drug antibody adsorbed on or bound to a solid phase (capture antibody), the anti-drug antibody, and the drug antibody conjugated to a detectable label, e.g. an enzyme (tracer antibody). Thus, a sandwich is formed: solid phasecapture antibody-anti-drug antibody-tracer antibody. In the reaction catalyzed by the sandwich, the activity of the antibody-conjugated enzyme is proportional to the anti-drug antibody concentration. The standard sandwich method is also called bridging immunoassay because the anti-drug antibody bridges between the capture and tracer antibodies, i.e,, the drug antibody. Immunoassays such as the bridging ELISA are common assay types in the investigation of an immunogenic answer of a patient to an antibody drug. Mire-Sluis, A.R., et al., J. Immunol. Methods 289 (2004) 1-16, summarized the recommendations for the design and optimization of immunoassays using detection of host antibodies against biotechnology products. Wadhwa, M., et al.. J. Immunol. Methods 278 (2003) 1-17, reported strategies for the detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals. The principles of different immunoassays are described, for example, by Hage, D.S., Anal. Chem. 71 (1999) 294R- 304R. Lu, B., et al., Analyst. 121 (1996) 29R-32R, reported the orientated immobilization of antibodies for the use in immunoassays. Avidin-biotin-mediated immunoassays are reported, for example, by Wilchek, M., and Bayer, E.A., Methods Enzymol. 184 (1990) 467-469. In WO 2017 / 072210 anti-variant Fc-region antibodies and methods of use thereof have been reported. Wessels, U., et al., Bioanal. 9 (2017) 849-859, reported novel drug and soluble target tolerant antidrug antibody assay for therapeutic antibodies bearing the P329G mutation. In US 2008 / 0118939 a conjugate and its use as a standard in an immunoassay has been reported.

[0183] In some embodiments, production of ADA is measured by ELISA as described in the Examples herein.

[0184] In one embodiment, detection of ADA in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest may be rendered ineffective due to the presence of antibodies or will cause adverse responses for patients. In one embodiment, no detection of ADA in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is likely safe.Cytokine / Chemokine Storm

[0185] The term "cytokine storm" refers to the dysregulated, abnormal systemic release of inflammatory cytokines that leads to disease and has also been called "cytokine release syndrome" or "inflammatory cascade." A cytokine storm or cascade is often referred to as part of a sequence because one cytokine typically leads to the production of multiple other cytokines that can enhance and amplify the immune response.

[0186] Cytokines are 8-30 kDa proteins and glycoproteins, which are produced by many cell types and operate as signals in cell-cell communication. They play a central role in the immune system and are involved in a variety of immunological, inflammatory and infectious diseases. Exemplary cytokines that can be measured include, without limitation, 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, IL37, IL38, ILIA, IL1B, IL1RN, IFNA, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA14, IFNA16, IFNA17, IFNA21, IFNB, IFNg, IL-la, IL-lb, IL-6, IL-13, IL-17a. TNFA. TNFB. TRAIL, TL1, BAFF, APRIL, RANKL, CD40LG, EDA, FASLG, CD70 GM-CSF, VEGF. In some embodiments, production of IL 10 is measured. In some embodiments, production of IFNy is measured.

[0187] Chemokines are small chemoattractant secreted molecules regulating cell positioning and cell recruitment into tissues, playing a pivotal role in embry ogenesis, tissue development and immune response. Approximately 50 chemokines and 20 chemokine receptors have been discovered so far. 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. Int J Mol Sci. 2018 Dec 27;20(l): 96. Chemokines can be widely divided into two major groups based on their prominent functions: inflammatory and homeostatic chemokines. Among inflammatory chemokines, which are induced by inflammation, some non-limiting examples that can be measured in the methods of the disclosure include CXCL1, CXCL2, CXCL3, CXCL5, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, and CXCL14. On the other hand, homeostatic chemokines such as, without being limited to, CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12 and CXCL13 are constitutively expressed and are involved in homeostatic leukocyte trafficking. In some embodiments, the chemokine comprises a CXC chemokine, or an isoform or a derivative capable of binding thereof. In certain non-limiting exemplary7embodiments, the chemokine can 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 vCXCl. In some embodiments, the chemokine is CXCL10.

[0188] Methods of detecting cytokines / chemokines involved in cytokine storm are known in the art and include, without limitation, enzyme-linked immunosorbent spot (ELISpot) and enzyme-1 inked immunosorbent assay (ELISA), flow cytometry, Luminex bead-based assays, and the electrochemiluminescent multiplex immunoassays (Meso Scale Discovery, MSD). In some embodiments, cytokine and chemokine production are measured using a bead-based multiplex assay. In some embodiments, the bead-based multiplex assay is LEGENDplex™

[0189] In some embodiments, the presence or absence of cytokine storm is measured by' measuring one or more levels of IL-2, IL-6, IL-8, IL-9, IL-10, IL-12, IL-17, IL-18, IFNg, TNF, GM-CSF, VEGF, MCP-1, MIP-la, and / or MIP-lb. In some embodiments, the one or more levels of IL-2, IL-6, IL-8. IL-9. IL-10, IL-12, IL-17. IL-18, IFNg. TNF. GM-CSF, VEGF, MCP-1. CXCL10, MIP-la, and / or MIP-lb is measured using Luminex. In some embodiments, the beadbased multiplex assay is LEGENDplex™.

[0190] In one embodiment, detection of increases in one or more levels of IL-2, IL-6, IL-8, IL- 9, IL-10, IL-12, IL-17. IL-18, IFNg. TNF. GM-CSF, VEGF, MCP-1. CXCL10, MIP-la. and / or MIP-lb in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest will cause adverse responses for patients. In one embodiment, no detection change in in one or more levels of IL-2, IL-6, IL-8, IL-9, IL-10, IL-12, IL-17, IL-18, IFNg, TNF, GM-CSF, VEGF, MCP-1, CXCL10, MIP-la, and / or MIP-lb in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is likely safe.Cytotoxicity

[0191] In some embodiments, the method described herein involves measuring cytotoxicity. Cytotoxicity can include the presence of cytotoxic cells, like CTL (CD8+ killer T cells) and NK cell. In some embodiments, cytotoxicity is measured by measuring the numbers of expanded CD8+ T cells with the ability to kill. In some embodiments, this is measured using flow cytometry and markers for cytotoxic T cells including, CD3, CD8, CD69, Granzyme B, FasL, IFN-g, and TNF. Cytotoxic T cells can be further identified, for example, using a T cell Cytotoxicity Assay as described in the Examples below.

[0192] In some embodiments, cytotoxicity is measured by analyzing cytotoxic T cell function via Granzyme B and Perforin expression as described in the Examples herein.

[0193] In one embodiment, an increase in cytotoxicity (e.g., the presence of CD8+ killer T cells or Granzyme B+Perforin+ cells) in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is cytotoxic and will cause adverse responses for patients. In one embodiment, no change in cytotoxicity (e.g., the presence of CD8+ killer T cells) in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not cytotoxic and is likely safe.

[0194] Cytotoxicity can also be measured by analyzing the total number of viable cells following culture with a particular therapeutic as described herein. In some embodiments, a decrease in the total number of viable cells in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is cytotoxic and will cause adverse responses for patients. In one embodiment, no change in cytotoxicity (e.g., the total number of viable cells) in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not cytotoxic and is likely safe.Cell Death

[0195] In some embodiments, cell viability is measured. Cell viability can be measured using any suitable method known in the art. For example, cell viability can be measured using flow cytometry and markers including, without limitation, live / dead, CD3, CD19, CD14, CDl lc, CD45 to look for populations of immune cells from the organoid that have potentially died as a result of a therapeutic.

[0196] In one embodiment, a decrease in cell viability' in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest induces cell death and will causeadverse responses for patients. Tn one embodiment, no change in the cell viability in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest does not induce cell death and is likely safe.

[0197] Cell death can also be measured by analyzing the total number of viable cells following culture with a particular therapeutic as described herein. In some embodiments, a decrease in the total number of viable cells in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest induces cell death and will cause adverse responses for patients. In one embodiment, no change in cell death (e.g., the total number of viable cells) in organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest does not induce cell death and is likely safe.Other Methods

[0198] Also provided herein are methods of assessing the ability of a therapeutic to induce one or more of ADA, cytokine / chemokine storm, cytotoxicity, and / or cell death. The methods involve contacting a three dimensional immune organoid comprising a plurality7of selfassembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells with a therapeutic and measuring (i) the presence or absence of AD As, (ii) the levels of cytokines / chemokines produced, (iii) the levels of cytotoxic T lymphocytes produced and / or cell viability by the organoid after the contacting.

[0199] Also provided herein is a method to predict the range of outcomes of a drug across a diverse group of patients. This method includes generating three dimensional immune organoids comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality7of stem cells from a diverse pool of donors.Exemplary7genetically diverse donors include diverse populations, such as donors of both genders and donors of multiple races.

[0200] The various responses of immune organoids from a large number of donors to a therapeutic are analyzed, and the differential responses among the tested organoids are analyzed, to provide a variety' of useful data, including, but not limited to, the total percentage of the representative human population likely to experience an adverse reaction, or the key phenoty pic characteristics, for example, gender or race, of those individuals within the represented human population that are most likely to experience an adverse reaction.

[0201] Immune organoids as well as therapeutics useful in the methods and compositions of the present disclosure are described above.COMPOSITIONS

[0202] As described in greater detail below, one aspect of the present disclosure relates to a composition of a three dimensional immune organoid 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 CD 105+ and a therapeutic.

[0203] Immune organoids as well as therapeutics useful in the methods and compositions of the present disclosure are described above.EXAMPLES

[0204] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.EXAMPLE 1: METHODS

[0205] Tissue Collection and freezing. Whole spleens and whole lymph nodes from healthy donors, e.g., deceased donors (of all ages) or live donors (of all ages) are collected and sliced into segments by accredited clinicians, placed in hypothermosol + 2x Pen / Strep, lx Normocin and stored at 4C for shipment and until further processing. Hypothermosol medium is prepared, and workspace is prepared. Cryovials are labeled with date, tissue type, batch ID number, name [link to cryovial sticker template]. Upon arrival in the lab, tissues are fully immersed in fresh hypothermosol + 2x Pen / Strep, lx Normocin and unprocessed segments are kept on ice (4°C). Using a disposable scalpel, tissue is carefully dissected into 5 mm x 5 mm x 5 mm pieces and placed into 24 well plates for dissociation. Tissue is rinsed with 1ml 0.5 mM EDTA and then incubated at 37°C in 1 ml 0.5 mM EDTA for 1 hour (modulate and record change depending on results). Tissue is incubated with 1 ml Accumax at 37°C for 1 hour, the reaction is stopped with 1 ml Complete Medium. The cells are counted using the NC-200 and live cell count and viability is recorded. Cells are centrifuged at 250 x g for 10 minutes. Cells are aspirated and resuspended in1 ml Cryostore at 10 x 106cells / mL per vial. Cells are then transferred to pre-barcoded cryovials. Vials are transferred to Mr. Frosty and stored at -80°C overnight and transferred to LN2 the next day. The barcode is recorded in LN2 inventory' (LN2 number, hotel number, box number, and placement in box).

[0206] Organoid formation. Upon thaw, ROCK inhibitor (e.g. , Y-27632) is added to Complete medium. Cryovials are partially submerged in a 37°C bath and thawed until a sliver of ice remains. Cells are transferred from the cryovial to a 50 ml conical tube. 1 ml Complete medium is added dropwise, swirling between each drop until 9 ml Complete medium has been added. Spin at 200xg for 5 minutes. Supernatant is discarded and cells are resuspended in 2 ml of complete medium. The cells are counted using the NC-200 and the tubes are spun again while counting (200xg for 5 minutes). Resuspension volume is calculated to achieve 100 pl per well in the upper chamber (IxlO6to 2xl06cells total). The complete medium is supplemented with 1 pg / ml BAFF and 200ul supplemented medium is added per well of e.g., transwells, ULA plates, flat-bottom plates, V-bottom plates, or a pedestal. Stimulation is added directly to the culture prior to incubation 37°C overnight. Organoids are supplemented with BAFF supplemented complete medium every 3 days.

[0207] Addition ofPBMCs. Once formed, organoids are supplemented with adaptive cells from PBMCs.

[0208] Flow cytometry. Staining buffer is prepared (lx DPBS +0.5% BSA, or lx DPBS + 1%FBS) and a Flow Panel is designed using the Biolegend Spectra Analyzer tool (https: / / www.biolegend.com / en-us / spectra-analyzer). The organoid containing plate is removed out of the incubator and placed in a precleaned biosafety cabinet. The organoid is disrupted to create a single cell suspension. Cells are counted and placed into flow tubes. 2 mL of staining buffer is added to each tube. Cells are centrifuged at 500g for 5 minutes and resuspended in about 50 pL of staining buffer. The appropriate amount of primary antibody is added per manufacturer's directions and incubated for 30 minutes at 4°C, protected from light. The panel of antibodies includes CD3, CD4, CD8, CD19, CD38, CD27, CD127, CD138, PDPN, CDl lc, HLA-DR, CD123, live / dead, CD45, CD45RO, CD14, CD56, CD68, CD25, CXCR5, PD-1, CD35, CD20, CDl lb, CFSE). The sample is washed 2x with 2 mL of Flow' Staining buffer, the supernatant is aspirated, and cells are resuspended in residual buffer. Secondary antibody is added per manufacturer’s instructions and incubated for 30 at 4°C, protected from light. Sample is washed 2x with 2 mL of Flow' Staining buffer, supernatant is aspirated, and testing sample isresuspended in 250 pL of staining buffer and unstained control in 350 pL. If needed, cells are be fixed at this point using 1% paraformaldehyde. Single-color compensation controls are prepared, and sample is analyzed.

[0209] ELISA. First the assay diluent (1% BSA in lx PBS w / o +Mg and +Ca) and wash buffer (IX PBS + 0.05% Tween-20) is prepared. The ELISA plate is coated using a coating buffer diluted from 5x ELISA coating buffer to lx working solution in DI water. Columns “1” and “2” are coated with coating buffer mixed with IgG capture Ab at a dilution of 1:300 (100 pL / well dilution mix capture antibody). The remaining wells of plate are coated with coating buffer mixed with influenza A recombinant protein at a concentration of 0.1 pg / well (Seed 100 pL per well). The plate is covered with film and placed in shaker for 1 hour at 20-25°C or covered with film and incubated overnight at 4°C. The ELISA plate is blocked by washing with 300 pL of prepared wash buffer in each well. This is repeated until it is completed for a total of 4 times. 200 pL of Assay Diluent is added to each well of plate and placed on shaker at room temperature for a total of 1 hour. While plates are blocking, all standards are prepared. The standard is diluted with assay diluent to a concentration of 1000 ng / mL. 500 pL of assay diluent is added to each tube. From the initial prepared standard (200 ng / mL) 500 pL of AD is added. This is now tube 1 and final concentration is 100 ng / mL. 2x serial dilutions are continued down to the final tube. One tube will contain just assay diluent.

[0210] The plate is removed from shaker washed for a total of 4 times. Prepared standards are added to wells in 100 pL duplicates, and sample dilution is determined. Assay diluent is added to all sample wells of plate based on sample dilution for a total volume of 100 pL / well. The plate is placed on shaker for 2 hours at room temperature.

[0211] For the addition of detection antibody-HRP, detection antibody dilution is prepared at a 1: 100,000 dilution. The plate is removed from shaker and washed for a total of 4 times. 100 pL of prepared detection antibody solution is added to all wells of plate. The plate is covered with film and placed on shaker for 1 hour.

[0212] The next step is a 2TMB Substrate Incubation and Reaction STOP. TMB substrate is prepared and kept away from light. For one plate 5.5 mL of each solution is added. The plate is removed from the shaker w ashed with 300 pL of prepared wash buffer per each well for a total of 5 times. 100 pL of prepared TMB solution is added to each well of the plate. The plate is placed in the dark and monitored for color change. 100 pL of ELISA STOP solution or (2N H2SO4) is added to each well when color in 4th standard is developed.

[0213] Data is acquired on plate reader. For data analysis, duplicate absorbance values should be within 10% of each other. A standard curve is plotted for the IgG standard (known concentration) samples, and the average absorbance value minus (-) the blank value for each standard for each standard concentration is plotted on the vertical (Y) axis. The corresponding human IgG concentration is plotted the horizontal axis (X) that correlates with the absorbance values. This is used to extrapolate the concentration for the unknown sample.

[0214] Automated Image Segmentation for Area and Diameter. Brightfield image is opened, and its histogram equalized. The portion of saturated pixels is set to 0%. Canny edge detection is performed with Gaussian kernel radius pixel resolution set according to setup. Maximum filter is run with radius set to value according to setup. This filter creates a running window that replaces the central pixel with the maximum value of the neighboring pixels. Morphological operation Closing is performed with number of iterations set to 10 and count set to 3. This fills the remaining small holes in the image. Morphological Opening is run with number of iterations set to 10 and count set to 3. This eliminates small structures like debris localized outside the organoids. The outline of segmented areas is overlayed with the original brightfield image, and the precision of segmentation is checked. If necessary, the parameters of procedure are adjusted, focused especially on High threshold setting of Canny edge detection. Area is calculated using Analyze > Measure. Diameter is calculated using the formula d = 2v / ( A / JC).

[0215] Sandwich Antigen-Specific IgG ELISA. On the day of sample transfer, the plates containing the supernatant are thaw ed by placing them on ice for a couple of hours. Assay Diluent is made which is also the Blocking Buffer (1% BSA in lx PBS w / o +Mg and +Ca). 4 ml of 7.5% BSA is added to 26 ml of DPBS (total = 30 ml). Assay Diluent is stored at 4°C for short term storage and -20°C for long term storage. Wash Buffer (IX PBS + 0.05% Tween-20) is made. 10X wash buffer (lOx PBS + 0.5% Tween-20) is prepared by adding 2.5 ml of Tw een-20 into 500 ml of lOx PBS. 900 ml of MilliQ water is added to 100 ml of 10X buffer to prepare 1 1 of IX Wash Buffer. Any remaining 10X wash buffer is stored at 4°C for later use.

[0216] Coat ELISA Plate. Coating solution (1 pg / ml antibody in lx Coating Buffer) is prepared as follows, ensuring a total volume of 100 pl / well. 5x ELISA coating buffer is diluted to lx working solution in DI w ater. Capture antibody is added to a final concentration of 1 pg / ml. 100 pl / well is added. The plate is covered with film and incubated on shaker with gentle shaking for 1 hour at 20-25°C or overnight at 4°C.

[0217] Block ELISA Plate. The plate is washed as follows. Wash buffer is loaded into the plate washer. The plate is washed 4 times with 250 pl / well of wash buffer. The plate is blocked with 200 pl / well of Assay Diluent. The plate is covered with film and incubated on shaker at room temperature for 1 hour or overnight at 4°C.

[0218] Capture antigen / antibody. The plate is washed 4 times with 250 pl / well of wash buffer. The capture antigen / antibody solution is prepared as follows. Enough solution is prepared to ensure a total volume of 100 pl per well. The capture antigen / antibody is added in assay diluent to achieve a final concentration of 1 pg / ml. The plate is coated with 100 pl / well of the prepared antigen / antibody solution to the plate. The plate is covered and incubated on a shaker at room temperature for 1 hour.

[0219] Sandwich Antigen-Specific IgM / IgA ELISA. ELISA. On the day of sample transfer, the plates containing the supernatant are thawed by placing them on ice for a couple of hours. Assay Diluent is made which is also the Blocking Buffer (1% BSA in lx PBS w / o +Mg and +Ca). 4 ml of 7.5% BSA is added to 26 ml of DPBS (total = 30 ml). Assay Diluent is stored at 4°C for short term storage and -20°C for long term storage. Wash Buffer (IX PBS + 0.05% Tween-20) is then made. 10X wash buffer (lOx PBS + 0.5% Tween-20) is prepared by adding 2.5 ml of Tween-20 into 500 ml of lOx PBS. 900 ml of MilliQ water is added to 100 ml of 10X buffer to prepare 1 1 of IX Wash Buffer. Any remaining 10X wash buffer is stored at 4°C for later use.

[0220] Coat ELISA Plate. Coating solution (1 pg / ml antibody in lx Coating Buffer) is prepared as follows, ensuring a total volume of 100 pl / well. 5x ELISA coating buffer is diluted to lx working solution in DI water. Capture antibody is added to a final concentration of 1 pg / ml and 100 pl / well was added. The plate is covered with film and incubated on shaker with gentle shaking for 1 hour at 20-25 °C or overnight at 4°C.

[0221] Block ELISA Plate. Wash buffer is loaded into the plate washer, and the plate is washed 4 times with 250 pl / well of wash buffer. The plate is blocked with 200 pl / well of Assay Diluent. The plate is covered with film and incubated on shaker at room temperature for 1 hour or overnight at 4°C.

[0222] The plate is then washed 4 times with 250 pl / well of wash buffer. The capture antigen / antibody solution is prepared as follows. Enough solution is prepared to ensure a total volume of 100 pl per well. The capture antigen / antibody is added in assay diluent to achieve a final concentration of 1 pg / ml. The plate is coated with 100 pl / well of the preparedantigen / antibody solution to the plate. The plate is covered and incubated on a shaker at room temperature for 1 hour.

[0223] Capture antigen / antibody. The plate is washed 4 times with 250 pl / well of wash buffer. The capture antigen / antibody solution is prepared as follows. Enough solution is prepared to ensure a total volume of 100 pl per well. The capture antigen / antibody is added in assay diluent to achieve a final concentration of 1 pg / ml. The plate is coated with 100 pl / well of the prepared antigen / antibody solution to the plate. The plate is covered and incubated on a shaker at room temperature for 1 hour.

[0224] Luminex. Cell culture supernatants are harvested at time points of interest. It is ensured that no cells are collected when removing supernatants from organoid cultures. Aliquots are stored at -80°C. Carboxylated Luminex beads are coated with monoclonal antibodies required for study following the two-step carbodiimide procedure described by the manufacturer. 5xl06stock microspheres are washed in 100 pl of distilled water. The microspheres are pelleted by centrifuging at 8000 x g for 2 minutes, and the supernatants are removed. The beads are resuspended in 80 pl of 100 mM monobasic sodium phosphate pH 6.2. 10 pl of 50 mg / mL Sulfo- NHS (diluted in dH20) and 10 pl of 50 mg / mL EDC (diluted in dH20) is added to the microspheres and gently vortexed. Beads are incubated for 20 minutes at room temperature with gentle mixing at 1000 rpm in a Thermomixer. Microspheres are washed three times in 250 pl of 50 mM MES pH 5.0. Beads are resuspended in 100 pl 50 mM MES pH 5.0 and mixed with 25 pg monoclonal antibody for a final volume of 500 pl in the same buffer. The coupling reaction is incubated for 2 hours with mixing at 1000 rpm at room temperature. The beads are washed and resuspended with PBS containing 1% bovine serum albumin and 0.05% sodium azide. The efficiency of coupling is determined by incubating 2000 coated beads with 50 pl of 1 pg / ml R- phycoerythrin (PE) goat anti-mouse IgG (H + L) antibody. Beads are washed two times with 500 pl PBS - 0.05% Tween 20 (PBS-T) and resuspended in 125 pl PBS-T and analyzed on the Luminex instrument.

[0225] ADA ELISA. All reagents are brought to room temperature prior to use. Plates and standards are prepared as follows. Briefly, excess microplate strips are removed if the entire plate is not being used. Next, 250 ng / mL top standard is prepared by diluting 50 pl standard stock in 150 pl Assay Buffer A. Six two-fold serial dilutions are then performed (205 to 3.9 ng / mL) using Assay Buffer A. Assay Buffer A is used as zero standard (0 ng / mL). Next, the plate is washed 4 times with >300 pL IX Wash Buffer per well, blotting residual buffer on absorbent paper. 50 pLDrug Conjugate is added to each well. Next 25 pL reconstituted Matrix A is added to standard and QC wells, and 25 pL Assay Buffer A is added to sample wells. The plate is sealed and incubated at room temperature for 2 hours while shaking at 200 rpm. The plate is then washed 5 times with IX Wash Buffer, soaking wells 30-60 seconds per wash. 100 pL Substrate Solution C is then added to each well and incubated for 15 minutes in the dark. 100 pL Stop Solution is then added to each well. Absorbance is read at 450 nm within 30 minutes (subtract 570 nm readings if available).

[0226] LegendPlex. All reagents are brought to room temperature prior to use. Multiplexed standards are reconstituted with Assay Buffer. Next, standards are serially diluted in Assay Buffer according to the specific kit protocol. Supernatants are then appropriately in Assay Buffer. Next, capture beads are vortexed for 1 minute. Then 25 pL Assay Buffer, 25 pL diluted samples or standards, 25 pL mixed beads were added to each well of a 96- well V-bottom plate. The plate is incubated for 2 hours at room temperature, shaking -800 rpm. Next, the beads are spun down, and supernatant is removed without disturbing bead pellets. Wells are washed 1 time with 200 pL IX Wash Buffer per well. Next, 25 pL of Detection Antibodies is added and incubated for 1 hour at room temperature, shaking -800 rpm. Next, without washing. 25 pL SA-PE is added. The plate is incubated for 30 minutes at room temperature, shaking -800 rpm. Next, the beads are spun down and supernatant is again removed. The wells are washed IX, and the beads are resuspended in 150 pL of IX Wash Buffer. Samples are read on a flow cytometer.EXAMPLE 2: THE STEM CELL POPULATION IS REPRESENTED IN IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES

[0227] This example demonstrates that, similar to control immune organoids, drugged immune organoids contain stem cell populations only found in secondary lymphoid organs and not blood. These stem cells proliferate and play a role in lymph node support and function as well as immune organoid support and function.

[0228] Briefly, immune organoids are generated using the methods described in Example 1. Some organoids are left untreated, and others are incubated with various immunotherapies across numerous doses for 25 days. Immunotherapies are added at day 1 only. Day 25 cultures are stained for stem cell markers, and flow cytometry is performed using antibodies specific to CD45, CD34, CD45RA. CD201 (EPCR), CD49c (ITGA3). CD105, and CD73.EXAMPLE 3: THE STEM CELL POPULATION IS REPRESENTED IN IMMUNE ORGANOIDS

[0229] This example demonstrates that immune organoids contain stem cell populations only found in secondary lymphoid organs and not blood. These stem cells proliferate and play a role in lymph node support and function as well as immune organoid support and function.

[0230] Briefly, immune organoids were generated using the methods described in Example 1. Vaccinated Day 7 cultures were stained for stem cell markers, and flow cytometry was performed using antibodies specific to CD45, CD34, CD45RA, CD201 (EPCR), CD49c (ITGA3). CD105, and CD73. As shown in FIGs. 2A-2C, characterizing stem cell population modulation following therapeutic treatment is possible. These cells are important to consider during immunotherapy treatment as they play an important role in maintaining structure in lymph nodes and lymphoid tissues and support memory7cell formation and maintenance. They can also be an indicator of successful immune activation as well as potential long-term durability of the treatment.EXAMPLE 4: SIZE OF IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES

[0231] This example demonstrates that organoids incubated with immunotherapies (vaccines, antibodies, gene therapies, cell therapies, small molecules) do not display differences in organoid integrity. Thus, the immunotherapies do not alter the organoids to a point where they render the organoids useless or nonfunctional, and the organoids can be used to screen immunotherapies.

[0232] Briefly, immune organoids are generated using the methods described in Example 1. Some organoids are left untreated, and others are incubated with various immunotherapies across numerous doses for 25 days. Immunotherapies are added at day 1 only. Day 25 cultures are imaged with brightfield microscopy and diameter is measured as well as shape, integrity, and opacity.EXAMPLE 5: SIZE OF IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES

[0233] This example demonstrates that immunotherapy treatment can have significant effects on immune organoid size, structure, and morphology depending on successful activation of the immune system as well as successful dosing. While the immunotherapies do not alter the organoids to a point where they render the organoids useless or nonfunctional, and thus the organoids can be used to screen immunotherapies, suboptimal formulations or concentrations can lead to no activation and proliferation of cells or they can lead to toxicity and cell death. However, immune organoid size and morphology is highly dependent on the type of treatment and how it is designed to impact the immune system. Immune cell activation versus suppressioncan have differing effects on organoid size, with significant growth seen after successful stimulation due to cellular proliferation.

[0234] Briefly, immune organoids were generated using the methods described in Example 1. Rows A-E represent different influenza mRNA vaccine formulations and columns 1-5 represent decreasing concentrations of mRNA. Immunotherapies are added at day 0 only. As shown in FIG. 8, Day 5 and Day 21 cultures were imaged with brightfield microscopy and diameter was measured. All organoids exhibited morphological changes in response to vaccination however, higher concentrations of mRNA exhibited larger morphological changes compared to lower doses, with larger structures forming around the organoids. In this example, different mRNA concentrations have a larger effect on organoid morphology than vaccine formulation. By 21 these morphological differences disappear, and diameters are similar across conditions. These early morphological differences led to accelerated antibody and cellular responses in earlier timepoints, but by day 21 all organoids had exhibited the same responses across all concentrations and all vaccine formulations (data not shown).EXAMPLE 6: IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES FORM WITHIN 24 HOURS AND REMAIN VIABLE FOR AT LEAST 30 DAYS

[0235] This example demonstrates the ability of the immune organoids treated with immunotherapies to form their essential structures and begin function after one day. These organoids currently remain viable in culture for 30 or more days.

[0236] Briefly, immune organoids are generated using the methods described in Example 1. Some organoids are left untreated, and others are incubated with various immunotherapies across numerous doses for 30 days. Organoids are visualized using brightfield microscopy after 24 hours and then through day 30. Cell viability is also assessed using flow cytometry. The data is quantified and compared across conditions.EXAMPLE 7: IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES FORM WITHIN 24 HOURS AND REMAIN VIABLE FOR AT LEAST 30 DAYS

[0237] This example demonstrates the ability of the immune organoids treated with immunotherapies to form their essential structures and begin function after one day. Briefly, immune organoids were generated using the methods described in Example 1. Some organoids were left untreated, and others were incubated with an experimental subunit vaccine atday 0 and organoids were visualized using brightfield microscopy after 24 hours and then through day 28.

[0238] As shown in FIG. 1, representative longitudinal brightfield images of immune organoids following therapeutic treatment exhibit morphological changes over time. Immune organoids matured within 24 hours. Initial transcriptional responses and cellular expansion occurred over Day 3 to Day 4. Initial germinal center responses and antibody responses occurred on Day 11 through Day 14. Peak antibody responses occurred on Day 21.EXAMPLE 8: DIVERSE IMMUNE CELL POPULATIONS ARE CONTAINED IN IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES, INCLUDING BOTH INNATE AND ADAPTIVE CELLS

[0239] This example demonstrates that, similar to untreated immune organoids, immune organoids treated with immunotherapies are composed of the same diverse immune cell types as those found in a human lymph node. These cells w ork together to recapitulate lymph node structure and function.

[0240] Briefly, immune organoids are generated using the methods described in Example 1. Some organoids are left untreated, and others are incubated with various immunotherapies across numerous doses for 25 days. Cultures are stained for markers of 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, CDl lc, HLA-DR. CD123, live / dead, CD45, CD45RO, CD14, CD56, CD68, CD25, CXCR5, PD-1, CD35, CD20, CD1 lb, and CFSE. The cells are analyzed by flow cytometry. The data is quantified and analyzed and compared across all conditions.EXAMPLE 9: DIVERSE IMMUNE CELL POPULATIONS ARE CONTAINED IN IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES, INCLUDING BOTH INNATE AND ADAPTIVE CELLS.

[0241] This example demonstrates that immune organoids are composed of the same diverse immune cell types as those found in a human lymph node and the diversity of immune cells are maintained following treatment. Modulation of specific immune cells is an important readout for assessing treatment safety, depending on the target product profile. For example, plasmablast or plasma cell differentiation is an important cellular consideration for the safety of the vaccine. Alternatively, disrupting the immune response by for example disrupting germinal center B cells or depleting B, T, and other immune cells in the case of assessing safety of many antibody-based therapeutic strategies.

[0242] Briefly, immune organoids were generated using the methods described in Example 1 . Some organoids were left untreated, and others were incubated with various immunotherapies across numerous doses for 7 days. Cultures were stained for markers of B cells, T cells, NK cells, macrophages, monocytes, dendritic cells, plasma cells, granulocytes, and plasmablasts. The cells were analyzed by flow cytometry. The data was quantified and analyzed using flowjo and compared across all conditions.

[0243] As shown in FIGs. 3A-3B, organoids treated with an influenza subunit vaccine where the ratios of CD19+ B cells and CD3+ T cells were assessed, while induction of CD27+CD38++ plasmablasts following rabies vaccination were assessed.

[0244] Next, immune organoids from three independent donors were treated with different formulations of an experimental influenza mRNA vaccine. As show n in FIG. 3C, all five vaccine conditions resulted in the presence of CD138+ plasma cells in the immune organoids suggesting possible safety of the vaccines.

[0245] The presence of NK cells was then examined. Immune organoids from different donors were subjected to either no stimulation or stimulation with an experimental antibody therapeutic for 7 days. As shown in FIG. 3D, stimulation with the antibody 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 demonstrates successful immune activation and successful pathway targeting. The antibody can effectively promote NK cell expansion and potentially enhance innate immune responses, highlighting the immune organoid's ability to provide valuable insights into the antibody’s mechanism of action.

[0246] Next the granulocyte population was studied. Immune organoids from a single donor were subjected to vaccination with an influenza subunit vaccine for 7 days. As shown in FIG. 3E, vaccination resulted in a large SSChlghCD 15+ granulocyte population within the immune organoid, consistent with assumptions of an early granulocyte response following successful vaccination priming the initial inflammatory response.

[0247] Finally, immune organoids from a single donor were treated an influenza subunit vaccine for 7 days. As shown in FIG. 3F, distinct dendritic cell (CD1 lb+CD45+), monocyte (CD14+CDl lb+), and macrophage (visualized as SSC versus CD14+) populations w ere identified using sequential gating strategies for each myeloid subset to assess how these immune cells were impacted following vaccination.EXAMPLE 10: IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES ARE COMPOSED OF OTHER IMMUNE CELL TYPES

[0248] This example demonstrates similar to untreated immune organoids, immune organoids treated with immunotherapies are composed of rarer immune cell types critical for human lymph node.

[0249] Briefly, immune organoids are generated using the methods described in Example 1. Some organoids are left untreated, and others are incubated with various immunotherapies across numerous doses for 25 days. Cultures are stained for myeloid DCs (CD1 lc+), plasmacytoid DCs (CD123+), conventional DCs (CDl lb+ CD45+), and CD14+ DCs (CD14+ CDl lc+), stromal cells (CD45-) and fibroblastic reticular cells (CD31+ PDPN+).EXAMPLE 11: IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES ARE COMPOSED OF OTHER IMMUNE CELL TYPES

[0250] This example demonstrates immune organoids are composed of rarer immune cell types critical for human lymph nodes that can be assessed for population effects following immunotherapy treatment.

[0251] Briefly, immune organoids were generated using the methods described in Example 1. Some organoids were left untreated, and others were treated with an experimental influenza subunit vaccine for 7 days. Cultures were stained for myeloid DCs (CDl lc+), plasmacytoid DCs (CD123+), conventional DCs (CDl lb+ CD45+), and CD14+ DCs (CD14+ CDl lc+), stromal cells (CD45-) and fibroblastic reticular cells (CD31+ PDPN+), and flow cytometry was performed.

[0252] As shown in FIGs. 6A-6C and FIGs. 7A-7C, immune organoids treated with the vaccine contained CD14+CDl lc+ myeloid dendritic cells (FIG. 6A), plasmacytoid dendritic cells (CD123+) (FIG. 6B), CDl lb+CD45+ dendritic cells (FIG. 6C), CD45- stromal cells (FIG. 7A), fibroblastic reticular cells (PDPN+CD31+) (FIG. 7B). and follicular dendritic cells (CD45+PDPN+CD35+) (FIG. 7C).EXAMPLE 12: IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES FORM GERMINAL CENTERS

[0253] This example demonstrates that germinal centers with light zones and dark zones are required to recapitulate lymph node biology and are unable to be found in current in vitro technologies. The organoid platform described herein uniquely produces germinal centers and thus reproduces a critical feature of human biology.

[0254] Briefly, immune organoids were generated using the methods described in Example 1 . Some organoids are left untreated, and others are incubated with various immunotherapies across numerous doses for 25 days. Day 14 cultures are stimulated with Hepatitis B vaccines and organoids were visualized under brightfield microscopy.

[0255] Confocal microscopy is then performed to image the germinal centers in the day 14 immune organoids with B (CD20) and T cell (CD3) organization, plasmablasts (CD138), BCL6+ cells, and PD1+ cells.EXAMPLE 13: IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES HAVE CELLS CONSISTENT WITH GERMINAL CENTER FUNCTION

[0256] This example demonstrates that germinal centers need to be present as well as functional. These data indicate that these germinal centers are fully functional and reproduce all essential aspects of lymph node function, being the first organoid technology capable of doing so.

[0257] Flow cytometry staining demonstrates immune organoids consisting of B and T cell zones. Flow cytometry is also performed to identify germinal center B cells.EXAMPLE 14: IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES FORM GERMINAL CENTERS AND HAVE CELLS CONSISTENT WITH GERMINAL CENTER FUNCTION

[0258] This example demonstrates that immune organoids treated with immunotherapies designed to stimulate the immune system, in this case vaccines, form germinal centers and contain B and T cell zones.

[0259] Briefly, immune organoids were generated using the methods described in Example 1. Some organoids were left untreated, and others vaccinated with a Hepatitis A vaccine on Day 0. On Day 14 organoids were visualized under brightfield microscopy.

[0260] As shown in FIG. 9A, the immune organoids form germinal centers in response to vaccination with Hepatitis A vaccine. Lighter structures in the organoids outlined in red are consistent with germinal center morphology.

[0261] Next, immune organoids were examined for the presence of B and T cell zones following vaccination with an experimental influenza vaccine. Flow cytometry was performed to analyze both CD 19+ B cells and CD3+ T cells in Day 14 organoids. As shown in FIG. 9B, immune organoids contain both CD 19+ B cells and CD3+ T cells, which are consistent with the presence of both B and T cell zones.

[0262] Finally, the presence of germinal center B cells within the organoids was confirmed following stimulation with hemagglutinin protein. Flow cy tometry was performed to detect CD27+CD38+ germinal center B cells as shown in FIG. 9C.

[0263] Therefore, the immune organoids reproduce all essential aspects of lymph node function, being the first organoid technology capable of doing so.EXAMPLE 15: IMMUNE ORGANOIDS UNDERGO B CELL DIFFERENTIATION UPON TREATMENT

[0264] This example demonstrates that the immune organoid platform of the disclosure uniquely produces germinal centers and thus reproduces a critical feature of human biology.

[0265] First, it is shown that immune organoids undergo a variety of different responses upon stimulation, including B cell differentiation during which immune organoids stimulated at day 0 are primarily naive B cells and then differentiate into pre-GC B cells. GC B cells, memory B cells, plasmablasts, and plasma cells. These B cell phenotypes are consistent with human B cell responses to infection or vaccination or other stimulation in a lymph node.

[0266] Briefly, immune organoids were generated using the methods described in Example 1. As shown in FIG. 4A, B cell differentiation was monitored by flow cytometry over a 21 -day period following treatment with hemagglutinin protein, with distinct populations emerging at different timepoints. The progression from predominantly naive B cells (CD38-CD27-) at day 0 to significant populations of germinal center B cells (CD38+CD27+) by day 7-14, and ultimately to plasmablasts (CD38+++CD27+) by day 21 demonstrates successful B cell activation and maturation within the organoid system.

[0267] Briefly, immune organoids were generated using the methods described in Example 1 . Some organoids were left untreated, and others were immunized with hemagglutinin protein on Day 0. Flow cytometry' was performed on Day 0, Day 7, Day 14. and Day 21 using markers for CD38-CD27- naive B cells, CD38-CD27+ memory B cells. CD38+CD27- pre-GC B cells, and CD38+CD27+ GC B cells. As shown in FIG. 4B, over time, the amount of pre-GC and GC B cells increased in organoids treated with hemagglutinin protein, demonstrating successful B cell differentiation.

[0268] Finally, plasma cell populations were examined in immune organoids following vaccination. Briefly, immune organoids were generated using the methods described in Example 1 , and immune organoids from three independent donors were treated with a panel of 5 different formulations of experimental mRNA influenza vaccines. As shown in FIG. 4C, all five vaccineconditions induced CD138+ plasma cells compared to unstimulated controls in the immune organoids suggesting possible safety of the vaccines. This temporal analysis of B cell differentiation validates the ability of immune organoids to recapitulate key aspects of the germinal center reaction and subsequent B cell differentiation. Additionally, the organoids provide more insight into the temporal nature of these responses in assessing vaccine safety.EX MPLE 16: IMMUNE ORGANOIDS ARE COMPOSED OF T CELL SUBTYPES FOLLOWING IMMUNOTHERAPY TREATMENT

[0269] This example demonstrates that immune organoids are composed of T cells, including T cell subtypes that are consistent with those found in human lymph nodes.Briefly, immune organoids were generated using the methods described in Example 1. Some organoids were left untreated, and others were treated with an experimental adjuvanted influenza vaccine on Day 0. Flow cytometry was performed on Day 7 using markers for various T cell populations. As shown in FIGs. 5A-5B, immune organoids treated with the vaccine exhibit CD4+ naive T cells (CD3+CD4+CD45RA+), CD4+ memory T cells (CD3+CD4+CD45RO+). and 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+), gamma delta T cells (CD3+CD27+). and T follicular helper cells (CD3+CD25+CXCR5+).The results demonstrate that immune organoids can respond to immunotherapy in a manner consistent with human lymph nodes.EXAMPLE 17: IMMUNE ORGANOIDS ARE COMPOSED OF BOTH B AND T CELLS THAT CAN BE MODULATED UPON VARIOUS STIMULATION CONDITIONS.

[0270] This example demonstrates that immune organoids treated with immunotherapies are composed of a large mixture of different immune cells that are activated and can proliferate upon stimulation. B and T cells are one example of dramatic differences in cell abundance after stimulating the immune organoids with six different stimulation conditions, where some stimulation conditions preferentially expanded B cell populations and some expanded T cell populations.EXAMPLE 18: IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES UNDERGO A FULL ADAPTIVE IMMUNE RESPONSE

[0271] This example demonstrates that organoids produce plasmablasts and antigen-specific antibodies against targets to which patient donors have been exposed (recall response) and to which patients are naive. The antibodies produced by the organoids have the abi li ty to class switch, for example IgM to IgG antibodies, consistent with an adaptive immune response.EXAMPLE 19: IMMUNE ORGANOIDS FORM ANTIGEN-SPECIFIC ANTIBODIES IN RESPONSE TO VACCINATION

[0272] This example demonstrates that, upon stimulation, immune organoids undergo a full immune response including a full adaptive immune response. This includes full B cell differentiation from naive B cells into germinal center B cells and mature cells like memory B cells, plasmablasts, and plasma cells. Broad antibodies are produced throughout but antigenspecific and higher affinity antibodies are produced once germinal centers are formed and the more mature B cells differentiate. IgM production occurs first in a temporal manner and then class-switch to IgG and IgA. IgG subtypes are also seen in the immune organoids.

[0273] First, antigen-specific IgG antibodies were measured over time across different stimulation conditions. Briefly, immune organoids were generated using the methods described in Example 1. Organoids were stimulated with 12 different formulations for an experimental influenza subunit vaccine. Day 4, Day 8, Day 11, and Day 15 organoids were analyzed by ELISA for the presence of antigen-specific IgG antibodies.

[0274] As shown in FIG. 10A, antigen-specific IgG responses were measured across twelve different experimental influenza vaccine formulations in immune organoids over a 15-day period. The ELISA data reveals significant variation in anti-HA IgG production between different stimulation conditions, with several formulations showing distinct temporal patterns. Notably, Stimulations 7 and 11 demonstrated sustained antibody production reaching higher concentrations (>400 ng / mL) at later timepoints (day 11-15), while others like Stimulations 1-4 showed minimal IgG production (<100 ng / mL) throughout the observation period. Stimulation 6 exhibited a gradual increase in antibody concentration over time, suggesting a more delayed but progressive immune response.

[0275] Next, the ability of immune organoids to undergo plasmablast differentiation upon vaccination was analyzed. Briefly, immune organoids were generated using the methods described in Example 1. Organoids were vaccinated with an experimental adj uv anted influenza vaccine, and flow cytometry was performed on either Day 0 or Day 14 to assess CD38 and CD27expression on individual cells. As shown in FIG. 10B, organoids exhibited an increase in CD38++CD27+ plasmablasts in response to vaccination.

[0276] Antigen-specific immunoglobulin profiles were then examined in two different donors. Briefly, immune organoids were generated using the methods described in Example 1, and organoids were treated with either Treatment A or Treatment B, two different doses of rabies vaccine. On Day 7 supernatants were collected and LEGENDplex Human Immunoglobulin Isotyping Panel was performed to assess concentrations of IgGl-4 subty pes, IgM, and IgA in response to Treatment A and Treatment B. As shown in FIG. 10C, both donors exhibited similar patterns of immunoglobulin production, with notable levels of IgGl, IgM, and IgA across both treatment conditions.

[0277] Finally, as shown in FIGs. 10D and 10E, immune organoids were used to evaluate anti- HA antibody responses following vaccination with different formulations and doses of experimental influenza vaccines. The data demonstrates successful immunoglobulin class switching, progressing from early IgM responses to later IgG production. All formulations w ere tested at two doses (1 being higher than 2), with Stimulations 3 and 4 emerging as the most immunogenic, showing both sustained IgM responses (OD 0.8- 1.0) and robust IgG production (70-80 ng / mL) by day 15. While most formulations exhibited early IgM responses by day 4-8. there was considerable variation in the magnitude and kinetics of antibody production across different conditions. Higher doses generally elicited stronger responses, though this dosedependency varied among formulations. Notably, Stimulations 5 and 6 showed relatively weak IgG responses despite initial IgM production, suggesting these formulations may be less effective at promoting class switching or sustaining the antibody response.EXAMPLE 20: IMMUNE ORGANOIDS CAN BREAK TOLERANCE TO GENERATE ANTIBODIES AGAINST HUMAN TARGETS

[0278] This example demonstrates that immune organoids produce both IgM and IgG antibodies against foreign antigens, examples here include influenza and SARS-CoV-2.

[0279] Immunotolerance is a key attribute of systemic immunity, in which immune organoids replicate this feature by not responding to stimulation. Another key attribute of systemic immunity' is the ability' to break tolerance (z.e. autoimmune disease and allergic disease). Under certain stimulation conditions the immune organoids are able to be induced to model breaking tolerance, a key feature for the immune organoid to produce antibodies against human targets fortherapeutic purposes (i.e. cancer and autoimmune disease) and the organoid has the ability to produce autoimmune responses against self-antigen (myelin) and are key to being able to model autoimmune and allergic diseases for the first time.

[0280] Briefly, immune organoids were generated using the methods described in Example 1. Some organoids were left untreated, and others were stimulated with either influenza hemagglutinin protein, SARS-CoV-spike protein, myelin protein, or NK cell protein combined with different experimental adjuvants. On Day 11, ELISA was performed to detect IgM and IgG antibodies.

[0281] As shown in FIGs. 11A-11D, the organoids have versatility in modeling both conventional vaccine responses and autoimmune phenomena. While stimulations with influenza HA and SARS-CoV-2 spike proteins elicited expected antigen-specific antibody responses, notably, the organoid also successfully generated antibodies against self-antigens (myelin and NK. cell receptor proteins) when combined with specific adjuvant formulations. Particularly. Stimulation 2 consistently produced the highest IgG responses across all antigens tested (absorbance ~0.3-0.5), while IgM responses showed more variable patterns among different conditions. The ability to break immunological tolerance in this controlled system is particularly significant for immunotherapy development, as it demonstrates the potential to overcome selftolerance mechanisms that often limit anti-tumor immune responses. This feature could be especially valuable for cancer immunotherapy development, where generating effective immune responses against self-antigens (tumor-associated antigens) is crucial but challenging due to natural tolerance mechanisms. The immune organoids therefore provide a unique platform for screening and optimizing stimulation formulations that can effectively break tolerance.EXAMPLE 21: IMMUNE ORGANOIDS MODEL PRODUCTION OF ANTI-DRUG ANTIBODIES

[0282] This example demonstrates that organoids treated with immunotherapies known to induce anti-drug antibodies (ADA) in patients are able to produce AD As specific to the immunotherapy used. This is to demonstrate that immune organoids can be used to screen drugs for ADA production and predict rate of AD As for a given drug in the population. ADA production is a major concern for immunotherapies, especially for antibodies, where the impact on the patient can be either that the drugs are rendered ineffective due to the presence of antibodies, or this can cause adverse responses for the patients.

[0283] Briefly, immune organoids are generated using the methods described in Example 1. Organoids are set up across 3 different donors. Organoids are treated with immunotherapy knownto induce ADA in some patients (e.g., Humira), ADA in all patients (e.g., foreign IgG) or ADA in no patients (e.g., Avastin) on day 0 and day 7. Supernatants are collected across different timepoints, and ELISAs are performed to assess ADA production. Data is quantified and ELISA data is graphed. No IgG is seen for no ADA conditions (e.g. Avastin), and increasing IgG levels is seen across the controls accordingly which match the clinical published data.EXAMPLE 22: IMMUNE ORGANOIDS ARE ABLE TO MODEL DRUG INDUCED CYTOKINE / CHEMOKINE STORM

[0284] This example demonstrates that organoids are able to readout cytokine / chemokine production following immunotherapy treatment. Assessing cytokine / chemokine responses in response to immunotherapy treatment is highly relevant to safety some drugs can induce a cytokine / chemokine storm. Immunotherapies that induce an appropriate (or inappropriate) cytokine response is a good indicator of drug safety.

[0285] Briefly, immune organoids are generated using the methods described in Example 1. Organoids are set up across 3 different donors. Organoids are left untreated, treated with immunotherapy known to induce cytokine storm in patients (e.g., Muromonab), or known not to induce cytokine storm in patients (e.g., rituximab). Organoids go out to 25 days, and supernatants are collected across different time-points. Luminex is performed to assess cytokine and chemokine production.EXAMPLE 23: IMMUNE ORGANOIDS ARE ABLE TO MODEL DRUG CYTOTOXICITY

[0286] This example demonstrates that organoids can predict safety for cytotoxicity.

[0287] Briefly, immune organoids are generated using the methods described in Example 1. Organoids are set up across 3 different donors. Some organoids are left untreated, and others are incubated with drugs known to cause / not cause cytotoxicity, as evidenced by expanded CD8+ cells with killing capacity. Organoids go out to 25 days. Organoids are harvested and disaggregated into single cell suspensions, cells are stained with antibodies and / or dyes indicative of CTL phenotypes and viability (live / dead, CD3, CD8. CD69, FasL. granzyme B, IFN-g, TNF), read on cytometer, quantified and plotted.

[0288] Controls are compared to drugs known to induce cytotoxicity and show' that known drugs wdth cytotoxicity’ have an increase in these cells over control.EXAMPLE 24: IMMUNE ORGANOIDS ARE ABLE TO MODEL DRUG INDUCED CELL DEATH

[0289] This example demonstrates that organoids can predict safety for cell death. Similar to cytotoxicity, safety issues can lead to major changes in cell viability mediated either by cells or by cytokine production and induced cell death.

[0290] Briefly, immune organoids are generated using the methods described in Example 1. Organoids are set up across 3 different donors. Some organoids are left untreated, and others are incubated with drugs known to cause / not cause changes in cell vi ability. Organoids go out to 25 days. Organoids are harvested and disaggregated into single cell suspensions, cells are stained with antibodies and / or dyes indicative of viability (Live / dead, CD3, CD19, CD14, CDl lc, CD45). read on cytometer, quantified and plotted.EXAMPLE 25: IMMUNE ORGANOIDS ENABLE COMPREHENSIVE SAFETY ASSESSMENT OF IMMUNOTHERAPIES THROUGH MULTIPARAMETER ANALYSIS OF CELLULAR RESPONSES

[0291] This Example demonstrates the immune organoid enables comprehensive evaluation of immunotherapeutic safety through multiple complementary readouts within a physiologically relevant human immune microenvironment. The system captures key parameters including antidrug antibodies, cytotoxic responses, and cell viability. Current drug development workflows lack predictive preclinical models that effectively mirror human immune responses. Traditional in vitro systems fail to capture the complex cellular interactions and tissue architecture necessary for proper immune function, while animal models often poorly predict human immune responses due to species-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.

[0292] The ability to assess multiple aspects of immune response within a single platform, using donor-matched human cells, positions immune organoids as a valuable tool for therapeutic screening and optimization. This system enables more informed decision-making in drug development by providing comprehensive immunological profiling before advancing candidates to clinical trials, potentially improving success rates and reducing development costs in immunotherapy development.

[0293] First, the development of anti-drug antibodies (ADA) in immune organoids in response to OKT3 antibody was examined Briefly, immune organoids were generated using the methods described in Example 1. Some organoids were left untreated, and some organoids were treatedwith 10 ng / mL of OKT3 antibody. As shown in FIG. 12A, treatment induced variable ADA responses across samples, with levels ranging from baseline to 0.28 ng / mL ADA.

[0294] Next, cytokine and chemokine production were examined in immune organoids in response to treatment with an experimental therapeutic antibody. Briefly, immune organoids from two donors were generated using the methods described in Example 1. The organoids were treated with an experimental therapeutic antibody on Day 0, and LegendPlex immunoassay was performed. As shown in FIGs 12B and 12C, there was substantial donor-specific variation in IFN-y and CXCL10 production, highlight the importance of understanding individual immune responses for safety assessment. Particularly noteworthy is the range of CXCL10 responses across five donors (500-15,000 pg / mL) and the dramatic differences in IFN-y production (ranging from 15,000 to 200,000 pg / mL), suggesting potential risks for cytokine-mediated adverse events in certain patient populations.

[0295] Next, cell viability was examined in immune organoids in response to therapeutic antibody treatment. Briefly, immune organoids were generated using the methods described in Example 1. Cell viability was measured in untreated conditions and following treated with a therapeutic antibody. As shown in FIG. 12D, initial viability at day 0 was consistent across all conditions (-75% viable cells). By day 6, a dose-dependent effect on cell viability was observed, with higher concentrations of immunotherapy (1 :8 and 1: 16 dilutions) inducing greater cytotoxicity (approximately 15-20% viable cells) compared to more dilute treatments. Lower concentrations (1:64 and 1: 128) showed moderate cytotoxicity7, while untreated controls maintained higher viability (-70%).

[0296] Next, cytotoxic T cell function was measured in immune organoids following treatment with an experimental therapeutic antibody. Briefly, immune organoids were generated using the methods described in Example 1. Organoids were either left untreated or were treated with an experimental therapeutic antibody. Flow cytometry was performed over a 6-day period to assess Granzyme B (GZMB) and Perforin expression. As shown in FIG. 12E, Negative control (untreated) organoids showed minimal increase in cytotoxic activity, while positive control (bead stimulation) organoids demonstrated robust T cell activation, reaching 25-35% GZMB+Perforin+ cells by day 6. The antibody treatment showed moderate but consistent activation reaching -15- 18% GZMB+Perforin+ cells suggesting that the antibody effectively activates cytotoxic T cell responses, though not as strongly as maximal stimulation with the beads. The cytotoxic response develops over time, with major differences emerging by day 6.

[0297] The multi-parameter analysis reveals critical aspects of immunotherapy safety evaluation. The dose-dependent cytotoxicity data demonstrates that higher concentrations of immunotherapy can significantly impact cell viability, suggesting a potential therapeutic window that balances efficacy with safety. The proliferation of cytotoxic T cells across two donors demonstrate that the immune organoid platform can effectively model the kinetics of CTL activation and expansion. The cytokine profiling results, showing substantial donor-specific variation in IFN-y and CXCL10 production, highlight the importance of understanding individual immune responses for safety assessment. Particularly noteworthy is the range of CXCL10 responses across five donors (500-15,000 pg / mL) and the dramatic differences in IFN-y production (ranging from 15,000 to 200,000 pg / mL), suggesting potential risks for cytokine- mediated adverse events in certain patient populations. The detection of anti-drug antibodies (ADA) further emphasizes the complexity of safety considerations, indicating potential immunogenicity that could affect both safety and efficacy. Immune organoids have a unique ability to predict patient-specific responses and identify potential safety' concerns before clinical implementation, particularly for cytokine release syndrome and immunogenicity risks.EXAMPLE 26: IMMUNE ORGANOIDS CAN PREDICT THE RANGE OF OUTCOMES OF DRUG ACROSS A Diverse Group of Patients

[0298] This example demonstrates that immune organoids can predict the range of outcomes that would exist across a real, diverse group of patients.

[0299] Organoids are co-cultured with drugs known to have differences in safety profiles across patient backgrounds (e.g.. sex, age, etc). These donor-to-donor dependent effects are recapitulated in the organoids by generating organoids from a diverse pool of donors.

[0300] For example, a drug is used that exhibits different ADA in women versus men. Organoids are made from both sexes and are stimulated with drug. ADA is quantified in supernatants and plotted. The same difference in women versus men is seen in the organoids as is seen in the literature.

[0301] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0302] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the rightto challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0303] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure and are to be included within the spirit and purview of this application.

Claims

WHAT IS CLAIMED IS:

1. A method of assessing the safety of a therapeutic, the method comprising:(a) contacting a three dimensional immune organoid comprising a plurality of selfassembled primary' immune cells obtained from one or more secondary' lymphoid organs and a plurality of stem cells with a therapeutic and(b) measuring the response of the three dimensional immune organoid to the therapeutic after said contacting.

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

3. The method of any preceding claim, wherein the one or more secondary lymphoid organs are from spleen, lymph node, Peyer's patch, and MALT.

4. The method of any preceding claim, wherein the immune organoid is a human immune organoid.

5. The method of any preceding claim, wherein the immune organoid further comprises peripheral blood mononuclear cells.

6. The method of any preceding claim, wherein the secondary lymphoid organs are obtained from living patients, surgical resections, fine needle aspirates, biopsy, and deceased patients.

7. The method of any preceding claim, wherein the plurality of primary immune cells comprises B cells. T cells, plasmablasts, plasma cells, ILCs, granulocytes, NK cells, monocytes, dendritic cells, macrophages and combinations thereof.

8. The method of claim 7, wherein the B cells comprise one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, plasmablasts, plasma cells, or a combination 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, gamma delta T cells, CD4 effector memory T cells, CD8 effector memory' T cells, or a combination thereof.

10. The method of claim 7, wherein the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or a combination thereof.

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

12. The method of any preceding claim, wherein the immune organoid is 8000 pm or less in diameter.

13. The method of any preceding claim, wherein the immune organoid comprises germinal centers and / or B / T cell zones.

14. The method of any preceding claim, wherein the immune organoid produces antibodies.

15. The method of claim 14, wherein the antibodies are IgG, IgM, or IgA, antibodies.

16. The method of claim 15, wherein the antibodies have full humoral functionality.

17. The method of claim 16, wherein the antibodies bind human and non-human targets.

18. The method of claim 17, wherein the human targets comprise proteins, sugars, and nucleic acid.

19. The method of claim 18, wherein the non-human targets comprise infectious disease antigens, venoms, poisons, small molecules.

20. The method of any preceding claim, wherein the therapeutic is an immunotherapeutic.

21. The method of claim 20, wherein the immunotherapeutic is selected from the group consisting of a vaccine, an antibody, a gene therapy, a cell therapy, a small molecule, and a nanobody.

22. The method of any preceding claim, wherein measuring the response comprises measuring (i) the presence of anti-drug antibodies, (ii) cytokine and chemokine production, (iii) cell viability, and / or (iv) cytotoxicity.

23. A method of assessing the ability of a therapeutic to induce AD As, the method comprising:(a) contacting a three dimensional immune organoid comprising a plurality of selfassembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells with a therapeutic and(b) measuring the presence or absence of AD As produced by the organoid after said contacting.

24. A method of assessing the ability of a therapeutic to induce a cytokine / chemokine storm, the method comprising:(a) contacting a three dimensional immune organoid comprising a plurality of selfassembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells with a therapeutic and(b) measuring the levels of cytokines / chemokines produced by the organoid after said contacting.

25. A method of assessing the cytotoxicity’ of a therapeutic, the method comprising:(a) contacting a three dimensional immune organoid comprising a plurality of selfassembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells with a therapeutic and(b) measuring the levels of cytotoxic T lymphocytes produced by the organoid after said contacting.

26. A method of assessing the ability of a therapeutic to induce cell death, the method comprising:(a) contacting a three dimensional immune organoid comprising a plurality of selfassembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells with a therapeutic and(b) measuring the cell viability the organoid after said contacting.

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