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WO2025155727A1 - Procédés d'utilisation d'organoïdes immunitaires pour prédire la sécurité d'un médicament - Google Patents

Procédés d'utilisation d'organoïdes immunitaires pour prédire la sécurité d'un médicament

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Publication number
WO2025155727A1
WO2025155727A1 PCT/US2025/011889 US2025011889W WO2025155727A1 WO 2025155727 A1 WO2025155727 A1 WO 2025155727A1 US 2025011889 W US2025011889 W US 2025011889W WO 2025155727 A1 WO2025155727 A1 WO 2025155727A1
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Prior art keywords
cells
immune
organoid
therapeutic
organoids
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Robert M. DIFAZIO
Juliana L. HILLIARD
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Parallel Bio
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Parallel Bio
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/0634—Cells from the blood or the immune system
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    • C12N5/0602—Vertebrate cells
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    • C12N5/0602—Vertebrate cells
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
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    • C12N2513/00—3D culture

Definitions

  • 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.
  • 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.
  • 2D two-dimensional
  • mice & non-human primates 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.
  • 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.
  • the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+.
  • the one or more secondary ly mphoid organs are from spleen, lymph node, Peyer’s patch, and/or MALT.
  • the immune organoid is a human immune organoid.
  • the immune organoid further comprises peripheral blood mononuclear cells.
  • the secondary' lymphoid organs are obtained from living patients, surgical resections, fine needle aspirates, biopsy, and deceased patients.
  • 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.
  • 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.
  • 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.
  • the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or a combination thereof.
  • the plurality of primary cells further comprises one or more stromal cells, follicular dendritic cells, and fibroblastic reticular cells.
  • the immune organoid is 8000 pm or less in diameter.
  • the immune organoid comprises germinal centers and/or B/T cell zones.
  • the immune organoid produces antibodies.
  • the antibodies are IgG, IgM, or IgA, antibodies.
  • the antibodies have full humoral functionality.
  • the antibodies bind human and non-human targets.
  • the human targets comprise proteins, sugars, and nucleic acid.
  • the non-human targets comprise infectious disease antigens, venoms, poisons, small molecules.
  • the method includes (a) contacting a three dimensional immune organoid comprising a plurality 7 of self-assembled primary 7 immune cells obtained from one or more secondary 7 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.
  • 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 plurality 7 of self-assembled primary 7 immune cells obtained from one or more secondary lymphoid organs and a plurality 7 of stem cells with a therapeutic and (b) measuring the levels of cytokines/ chemo kines produced by the organoid after said contacting.
  • Also provided herein is a method of assessing the cytotoxicity 7 of a therapeutic.
  • the method includes (a) contacting a three dimensional immune organoid comprising a plurality of self-assembled primary 7 immune cells obtained from one or more secondary 7 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.
  • 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.
  • 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. 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'
  • 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. 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
  • 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.
  • 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+).
  • 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.
  • FIGs. 12A-12E show immune organoids enable comprehensive safety assessment of immunotherapies through multiparameter analysis of cellular responses.
  • FIG. 12B shows analysis of IFN-y and IL- 10 production quantified by LegendPlexTM immunoassay in immune organoid supernatants from three independent donors across an untreated control condition and treatment with an experimental therapeutic antibody.
  • 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.
  • the disclosure provided herein provides 3D in vitro immune organoids cultured 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.
  • 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.
  • aspects and embodiments of the disclosure described herein include “comprising”, “consisting”, and “consisting essentially of aspects and embodiments.
  • “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.
  • “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 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.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so forth.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the immune cells comprising the three dimensional immune organoid of the present disclosure are derived from one or more secondary lymphoid organs.
  • “derived from” generally refers to the source of primary cells that form the organoid.
  • “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.
  • “derived from one or more secondary lymphoid organs” can mean that the organoids are formed after 1 passage of the primary cells.
  • “derived from one or more secondary lymphoid organs” can mean that the organoids are formed after more than 1 passage of the primary cells.
  • 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.
  • the secondary lymphoid tissue is obtained from a human and thereby results in a human immune organoid.
  • 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.
  • the naive CD4 T cells are CD4+CCR7+CD45RA+.
  • the naive CD8 T cells are CD8+CD45RA-CD27+.
  • the memory CD4 T cells are CD4+ CCR7+ CD45RA+, CD4+ CD45RA- CD45RO+.
  • the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or a combination thereof.
  • the dendritic cells are CD45+CDl lb+ dendritic cells.
  • the dendritic cells are CD14+CDl lc+ myeloid dendritic cells.
  • the dendritic cells are CD 123+ plasmacytoid dendritic cells.
  • the monocytes are CD14+CD1 lb+ monocytes.
  • the macrophages are CD68+ macrophages. In some embodiments, the macrophages are CD14+ macrophages.
  • the plasmablasts are CD38++CD27++ plasmablasts.
  • the dark zone (DZ) contains a network 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.
  • LZ light zone
  • 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.
  • centrocytes 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.
  • 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.
  • the germinal centers in the immune organoid are characterized as CXCR4 + , CD83 + , Ki67 + , and IgD + .
  • the B and T cell zones in the germinal centers of the immune organoid are characterized as CD3 1 and CD20 + .
  • 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.
  • Ig immunoglobulin
  • 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.
  • the immune organoid of the present disclosure produces IgG antibodies.
  • the immune organoid of the present disclosure produces IgA antibodies.
  • the immune organoid of the present disclosure produces IgM antibodies.
  • 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.
  • the co-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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ADCC antibody dependent cellular cytotoxicity
  • opsonization phagocytosis
  • CDC complement-dependent cytotoxicity
  • the immune organoid has full humoral functionality.
  • 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.
  • 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.
  • antibodies produced by the immune organoid of the present disclosure can function in opsonization.
  • Opsonization is a process where an antibody can bind to an 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.
  • 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 activity 7 . In some embodiments, the immune organoid as described herein exhibits all three of ADCC, CDC, and opsonization activity.
  • 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
  • MAG myelin-associated glycoprotein
  • AQP4 aquaporin-4
  • nAChR nicotinic acetylcholine receptor
  • DSG1 desmoglein-1
  • DSG2 desoglein-2
  • TSHR thyroid-stimulating hormone receptor
  • the self-antigen is MOG and the autoimmune disorder is multiple sclerosis (MS).
  • 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
  • the vaccine can be a vaccine for an infectious disease.
  • the vaccine for an infectious diseases comprises an antigen or immunogen selected from 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).
  • the antigen or immunogen can be an infectious agent, or a product of an infectious agent.
  • the antigen or immunogen comprises an inactivated infectious agent, e.g., that has been killed or otherwise attenuated.
  • the antigen or immunogen comprises a live infectious agent.
  • the infectious agent is a virus, for example and without limitation, a pox virus (e g., vaccinia virus), smallpox virus, marburg virus, flaviviruses (e.g.
  • 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 e.g., rubeola virus
  • human immunodeficiency virus 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 or 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.
  • filovirus e.g., ebola virus
  • coxsackievirus equine encephalitis virus
  • alphavirus e.g., Chikungunyavirus. Sindbis virus
  • hepatitis A virus hepatitis B virus
  • 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.
  • 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.
  • 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.
  • the infectious agent is a parasite, or a parasite derived product.
  • suitable parasite (or parasite derived products) for use in the vaccines and/or methods of the invention include Plasmodium species, including P. falciparum;
  • 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.
  • 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.
  • 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.
  • an alloantigen a self-antigen
  • a protein or peptide 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.
  • influenza vaccines can include, without limitation, influenza subunit vaccines, influenza mRNA vaccines, and adjuvanted influenza vaccines.
  • influenza vaccine is an influenza subunit vaccine.
  • influenza subunit comprises the hemagglutinin protein (HA).
  • influenza vaccine is an influenza mRNA vaccine.
  • influenza vaccine is an adjuvanted influenza vaccine.
  • the vaccine is a SARS-CoV-2 vaccine.
  • 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.
  • 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.
  • immunoglobulins can be assigned to different classes.
  • the antibody used in the present disclosure can be a monoclonal antibody.
  • the term "monoclonal antibody” 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.
  • a monoclonal antibody exists in a homogeneous or substantially homogeneous population.
  • 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.
  • the monoclonal antibody’ contemplated for use herein can encompass murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies.
  • 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.
  • 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’.
  • CDR complementary determining region
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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)
  • HCAbs Heavy-chain only 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.
  • 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.
  • 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.
  • HER2 Humanssack G, Raphael S, Lowden MJ, Henry KA. Isolation and characterization of camelid single-domain antibodies against HER2.
  • CAIX Araste F, Ebrahimizadeh W, Rasooli I, Rajabibazl M, Mousavi Gargari SL.
  • any method available in the art for gene therapy may be used in accordance with the present disclosure.
  • 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).
  • 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.
  • 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.
  • the cell therapy can include pluripotent stem cell therapy, adult stem cell therapy 7 , 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.
  • pluripotent stem cell therapy e.g., pluripotent stem cell therapy, adult stem cell therapy 7 , 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.
  • ty pes are described in more detail in, for example, El-Kadiry et al., Front Med 8 (2021).
  • 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.
  • T cells 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.
  • 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.
  • 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.
  • 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.
  • the immunotherapeutic is a small molecule.
  • 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.
  • 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).
  • small molecule immunomodulators are p38 kinase inhibitors such as VX 702 (Vertex Pharmaceuticals), SCIO 469 (Scios), doramapimod (Boeringer Ingelheim), RO 30201195 (Roche) and SCIO 323 (Scios), TACE inhibitors such as DPC 333 (Bristol Myers Squibb), ICE inhibitors such as pranalkasan (Vertex Pharmaceuticals) and IMPDH inhibitors such as my cophenolate (Roche) and merimepodib (Vertex Pharmaceuticals).
  • VX 702 Verytex Pharmaceuticals
  • SCIO 469 Scios
  • doramapimod Boeringer Ingelheim
  • RO 30201195 Roche
  • 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).
  • 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.
  • 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.
  • 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.
  • the therapeutic is contacted with the organoid for about 5 days.
  • the therapeutic is contacted with the organoid for about 7 days.
  • the therapeutic is contacted with the organoid for about 11 days.
  • 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.
  • the therapeutic as described herein can also be contacted with the immune organoid at various time points in immune organoid formation.
  • 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.
  • the therapeutic is contacted with the immune organoid at day 0.
  • the therapeutic is contacted with the immune organoid at day 1.
  • the therapeutic is contacted with the immune organoid at day 2.
  • the therapeutic is contacted with the immune organoid at day 3.
  • the therapeutic is contacted with the immune organoid at day 3.
  • 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 some embodiments, 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.
  • the contacting step involves a prime-boost regimen.
  • 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.
  • 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.
  • 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 p
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cytotoxic T-lymphocytes that are able to induce apoptosis in body 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.
  • 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.
  • 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.
  • the humoral immune response is measured by detection of germinal center formation.
  • the humoral immune response is measured by detecting antibody class switching.
  • the humoral immune response is measured by detecting memory B cell induction.
  • the immune response is measured by measuring both the innate and adaptive immune response.
  • control refers to a sample or standard used for comparison with a test sample.
  • control is a three dimensional organoid obtained from a healthy subject that is not exposed to any therapeutic.
  • control is a historical control or standard reference value or range of values (such as a previously tested control sample).
  • immune cell proliferation is measured.
  • Immune cell proliferation can be measured using any suitable method known in the art.
  • lymphocyte proliferation can be measured using a carboxy fluorescein diacetate succinimidyl diester (CFSE) dilution assay or by [ 31 -l]-thymidine incorporation.
  • CFSE carboxy fluorescein diacetate succinimidyl diester
  • 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.
  • 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.
  • 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.
  • no change 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 not efficacious.
  • 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.
  • control 7 refers to a sample or standard used for comparison with a test sample.
  • the control is a three dimensional organoid obtained from a healthy subject that is not exposed to any therapeutic.
  • the control is a historical control or standard reference value or range of values (such as a previously tested control sample).
  • 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.
  • 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.
  • 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.
  • immune organoids treated with a therapeutic can exhibit time dependent changes in B cell phenotype.
  • 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.
  • 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.
  • 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.
  • cytokines including, for example, IL10, CXCL10, IFN-g, IL-17, TNF, IL-4, and IL-2.
  • IL10 production is measured.
  • CXCL10 production is measured.
  • IFN-g production is measured.
  • cytokines and chemokines are known to one skilled in the art.
  • levels of cytokines produced by immune organoids can be measured using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • levels of cytokines can be measured using the EMDmillipore LUMINEX® xMAP® multiplex assay.
  • cytokine and chemokine production are measured using a bead-based multiplex assay.
  • the bead-based multiplex assay is LEGENDplexTM.
  • 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 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.
  • Antibody production can be measured using methods known in the art.
  • antibody production is measured using an ELISA assay.
  • antibody production is measured using a bead-based multiplex assay.
  • the bead-based multiplex assay is LEGENDplexTM.
  • specific IgG, IgM, and IgA antibody production by the immune organoids is measured using ELISA as described in the Examples provided herein.
  • IgG antibody production is measured.
  • IgGl antibody production is measured.
  • IgG2 antibody production is measured.
  • IgG3 antibody production is measured.
  • IgG4 antibody production is measured.
  • IgM antibody production is measured.
  • IgA antibody production is measured.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • production of IL10 is measured.
  • production of IFNy is measured.
  • 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.
  • 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.
  • 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.
  • the chemokine comprises a CXC chemokine, or an isoform or a derivative capable of binding thereof.
  • the chemokine includes CXCL12, CXCL11. CXCL1, CXCL2, CXCL3. CXCL4. CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL13, CXCL14, CXCL16, CXCL17, CX3CL1, XCL1, XCL2.
  • the chemokine includes vMIPII, U83, or vCXCl.
  • the chemokine is CXCL10.
  • cytokines and chemokines are known to one skilled in the art.
  • levels of cytokines produced by immune organoids can be measured using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • levels of cytokines can be measured using the EMDmillipore LUMINEX® xMAP® multiplex assay.
  • cytokine and chemokine production are measured using a bead-based multiplex assay.
  • the bead-based multiplex assay is LEGENDplexTM
  • the immune response is measured by analyzing changes in BCRs and/or TCRs.
  • BCR changes are measured.
  • TCR changes are measured.
  • both BCR and TCR changes are measured.
  • 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, which will be sequenced using NGS technology and analyzed for changes in the BCR and TCR repertoires.
  • 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.
  • 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.
  • 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.
  • the method described herein involves measuring levels of ADAs.
  • 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.
  • ADA anti-drug antibodies
  • 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.
  • ELISA enzyme linked immunosorbent sandwich assay
  • 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 sensitivity 7 and its easy applicability to different projects (Mikulskis, A., et al., J. Immunol. Meth. 365 (2011) 38-49).
  • 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).
  • a sandwich is formed: solid phasecapture antibody-anti-drug antibody-tracer antibody.
  • 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.
  • production of ADA is measured by ELISA as described in the Examples herein.
  • 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.
  • 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 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.
  • 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.
  • TNFA TNFB.
  • TRAIL TL1
  • BAFF APRIL
  • RANKL CD40LG
  • EDA FASLG
  • CD70 GM-CSF VEGF.
  • production of IL 10 is measured.
  • production of IFNy is measured.
  • 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.
  • 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.
  • 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.
  • the chemokine comprises a CXC chemokine, or an isoform or a derivative capable of binding thereof.
  • 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.
  • cytokine and chemokines production are measured using a bead-based multiplex assay.
  • the bead-based multiplex assay is LEGENDplexTM
  • 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.
  • 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.
  • an increase in cytotoxicity e.g., the presence of CD8+ killer T cells or Granzyme B+Perforin+ cells
  • cytotoxicity e.g., the presence of CD8+ killer T cells or Granzyme B+Perforin+ cells
  • no change in cytotoxicity e.g., the presence of CD8+ killer T cells
  • organoids cultured with a therapeutic as compared to a control indicate that the therapeutic of interest is not cytotoxic and is likely safe.
  • cell viability is measured.
  • Cell viability can be measured using any suitable method known in the art.
  • 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.
  • Cell death can also be measured by analyzing the total number of viable cells following culture with a particular therapeutic as described herein.
  • 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.
  • 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.
  • 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 plurality 7 of stem cells from a diverse pool of donors.
  • Exemplary 7 genetically diverse donors include diverse populations, such as donors of both genders and donors of multiple races.
  • 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.
  • 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).
  • 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.
  • ROCK inhibitor e.g. , Y-27632
  • Complete medium 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).
  • 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.
  • 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.
  • 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.
  • 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. [00213] 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.
  • Blocking Buffer 1% BSA in lx PBS w/o +Mg and +Ca
  • Assay Diluent is stored at 4°C for short term storage and -20°C for long term storage. Wash Buffer (IX
  • 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.
  • 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. [00217] 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.
  • 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.
  • Sandwich Antigen-Specific IgM/IgA ELISA ELISA.
  • 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.
  • 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.
  • 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.
  • 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 prepared antigen/ antibody solution to the plate. The plate is covered and incubated on a shaker at room temperature for 1 hour.
  • 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.
  • 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. 5xl0 6 stock 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.
  • 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.
  • PE phycoerythrin
  • 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 pL Drug Conjugate is added to each well.
  • Wells are washed 1 time with 200 pL IX Wash Buffer per well.
  • 25 pL of Detection Antibodies is added and incubated for 1 hour at room temperature, shaking -800 rpm.
  • 25 pL SA-PE is added.
  • the plate is incubated for 30 minutes at room temperature, shaking -800 rpm.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 memory 7 cell formation and maintenance. They can also be an indicator of successful immune activation as well as potential long-term durability of the treatment.
  • organoids incubated with immunotherapies do not display differences in organoid integrity.
  • 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.
  • 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.
  • 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 suppression can have differing effects on organoid size, with significant growth seen after successful stimulation due to cellular proliferation.
  • EXAMPLE 6 IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES FORM WITHIN 24 HOURS AND REMAIN VIABLE FOR AT LEAST 30 DAYS
  • 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.
  • 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
  • This example demonstrates the ability of the immune organoids treated with immunotherapies to form their essential structures and begin function after one day.
  • 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 at day 0 and organoids were visualized using brightfield microscopy after 24 hours and then through day 28.
  • EXAMPLE 8 DIVERSE IMMUNE CELL POPULATIONS ARE CONTAINED IN IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES, INCLUDING BOTH INNATE AND ADAPTIVE CELLS
  • 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.
  • 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.
  • 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. [00242] 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.
  • 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.
  • NK cells 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.
  • EXAMPLE 10 IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES ARE COMPOSED OF OTHER IMMUNE CELL TYPES [00248]
  • 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.
  • 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+).
  • myeloid DCs CD1 lc+
  • plasmacytoid DCs CD123+
  • conventional DCs CDl lb+ CD45+
  • CD14+ DCs CD14+ CDl lc+
  • stromal cells CD45-
  • fibroblastic reticular cells CD31+ PDPN+
  • 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.
  • 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.
  • myeloid DCs CDl lc+
  • plasmacytoid DCs CD123+
  • conventional DCs CDl lb+ CD45+
  • CD14+ DCs CD14+ CDl lc+
  • stromal cells CD45-
  • fibroblastic reticular cells CD31+ PDPN+
  • 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
  • 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
  • EXAMPLE 14 IMMUNE ORGANOIDS TREATED WITH IMMUNOTHERAPIES FORM GERMINAL CENTERS AND HAVE CELLS CONSISTENT WITH GERMINAL CENTER FUNCTION
  • 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.
  • 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.
  • 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. [00262] 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.
  • 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
  • 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.
  • immune organoids are composed of T cells, including T cell subtypes that are consistent with those found in human lymph nodes.
  • 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+).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 for therapeutic 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.
  • myelin self-antigen
  • 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
  • EXAMPLE 22 IMMUNE ORGANOIDS ARE ABLE TO MODEL DRUG INDUCED CYTOKINE/CHEMOKINE STORM
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • ADA anti-drug antibodies
  • immune organoids were generated using the methods described in Example 1. Some organoids were left untreated, and some organoids were treated with 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.
  • cytokine and chemokine production were examined in immune organoids in response to treatment with an experimental therapeutic antibody.
  • 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.
  • 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.
  • 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 cytotoxicity 7 , while untreated controls maintained higher viability (-70%).
  • 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.
  • GZMB Granzyme B
  • 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.
  • 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
  • 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.
  • 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.

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Abstract

La présente divulgation se rapporte à des procédés d'évaluation de l'efficacité thérapeutique à l'aide d'organoïdes immunitaires. La divulgation concerne également des compositions utiles dans de tels procédés. Dans certains modes de réalisation, la présente divulgation démontre le développement d'un nouveau procédé d'évaluation de la sécurité d'un agent thérapeutique à l'aide d'organoïdes immunitaires 3D. Le procédé comprend (a) la mise en contact d'un organoïde immunitaire tridimensionnel comprenant une pluralité de cellules immunitaires primaires autoassemblées obtenues à partir d'un ou de plusieurs organes lymphoïdes secondaires et une pluralité de cellules souches avec un agent thérapeutique et (b) la mesure de la réponse de l'organoïde immunitaire tridimensionnel à l'agent thérapeutique après ladite mise en contact.
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WO2023114785A2 (fr) * 2021-12-14 2023-06-22 Prellis Biologics, Inc. Organes et organoïdes lymphoïdes biomodifiés tridimensionnels pour thérapies à base de cellules
WO2024016001A2 (fr) * 2022-07-15 2024-01-18 Parallel Bio Compositions et procédés pour produire des organoïdes immunitaires générant des anticorps

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WO2023114785A2 (fr) * 2021-12-14 2023-06-22 Prellis Biologics, Inc. Organes et organoïdes lymphoïdes biomodifiés tridimensionnels pour thérapies à base de cellules
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