WO2022066894A1 - Humanized mouse models for assessing immune cell therapy - Google Patents

Humanized mouse models for assessing immune cell therapy Download PDF

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Publication number
WO2022066894A1
WO2022066894A1 PCT/US2021/051734 US2021051734W WO2022066894A1 WO 2022066894 A1 WO2022066894 A1 WO 2022066894A1 US 2021051734 W US2021051734 W US 2021051734W WO 2022066894 A1 WO2022066894 A1 WO 2022066894A1
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hours
cells
cell
mouse
human
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French (fr)
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James Keck
Jing JIAO
Chunting YE
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Jackson Laboratory
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Jackson Laboratory
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Priority to CA3196823A priority Critical patent/CA3196823A1/en
Priority to IL301634A priority patent/IL301634A/en
Priority to CN202180076671.4A priority patent/CN116916748A/zh
Priority to EP21873419.2A priority patent/EP4216971A4/en
Priority to KR1020237013626A priority patent/KR20230074758A/ko
Priority to AU2021347320A priority patent/AU2021347320A1/en
Application filed by Jackson Laboratory filed Critical Jackson Laboratory
Priority to JP2023518849A priority patent/JP2023542539A/ja
Priority to US17/705,037 priority patent/US12196744B2/en
Publication of WO2022066894A1 publication Critical patent/WO2022066894A1/en
Anticipated expiration legal-status Critical
Priority to US18/972,072 priority patent/US20250164467A1/en
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2207/00—Modified animals
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • A01K2207/15—Humanized animals
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    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • A01K2227/10—Mammal
    • A01K2227/105—Murine
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
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    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
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Definitions

  • Adoptive cell therapy such as chimeric antigen receptor (CAR) immune cell therapy (e.g., CAR T cell therapy or CAR-natural killer cell (CAR-NK) therapy) has become a revolutionary new cancer treatment. It has proven to be an effective new treatment for hematological malignancies and is currently being developed to treat solid tumor cancers.
  • ACT utilizes gene transfer to reprogram immune cells expressing an engineered antigen receptor, which enables immune cells (e.g., T cells, B cells, and/or natural killer (NK) cells) to recognize and target (bind to) cell surface antigens specific to a diseased cell, such as a tumor cell, further eliminating diseased cells carrying the antigen.
  • CAR chimeric antigen receptor
  • CAR-NK CAR-natural killer cell
  • FDA Food and Drug Administration
  • CAR T cell products for example: two for the treatment B-cell lymphoma, one for the treatment of advanced mantle cell lymphoma (MCL).
  • MCL mantle cell lymphoma
  • NK cells play a pivotal role as the body's first-line defense against vitally infected and malignant cells.
  • CRS cytokine release syndrome
  • encephalopathy syndrome neurotoxicity
  • CRS is a cytokine-mediated systemic inflammatory response caused by multiple cytokines following in vivo immune cell (e.g., T cell, B cell, NK cell) activation and expansion.
  • Immune cells comprising an engineered antigen receptor (i.e., engineered immune cells), diseased cells (e.g., tumor cells), and other immune cells can release cytokines and contribute to the induction of CRS.
  • the main cytokines associated with pathogenesis of CRS include interleukin (IL) 6, IL10, interferon (IFN)-y, monocyte chemoattractant protein 1 (MCP-1), and granulocyte-macrophage colony- stimulating factor (GM-CSF).
  • IL interleukin
  • IFN interferon
  • MCP-1 monocyte chemoattractant protein 1
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • TNF tumor necrosis factor
  • IL1 IL1
  • IL2Ra IL2 receptor alpha
  • IL8 granulocyte-macrophage colony- stimulating factor
  • T cell, B cell, NK cell infusion dose
  • other patient-specific factors such as pre-existent state of inflammation and baseline endothelial activation.
  • tocilizumab IL6 antagonist
  • tocilizumab is approved by the FDA for the treatment of severe or life-threatening engineered immune cell (e.g., T cell, B cell, NK cell) induced CRS.
  • Preclinical models of CRS are useful for identifying agents effective for CRS treatment that do not interfere with the cytokine-mediated anti-tumor effects of engineered immune cells (e.g., CAR T cells, CAR B cells, or CAR NK cells).
  • engineered immune cells e.g., CAR T cells, CAR B cells, or CAR NK cells.
  • preclinical models of CRS are helpful for evaluating which engineered immune cells (e.g., T cells, B cells, NK cells) (e.g., which specific CAR construct) induce the least CRS and remain therapeutically effective.
  • engineered immune cell refers to any immune cell (e.g., T cell, B cell, or NK cell) that comprises (e.g., expresses) an engineered antigen receptor, i.e., a non-naturally-occurring receptor that specifically binds to a cell surface antigen of interest.
  • a “CAR immune cell” such as a “CAR T cell” is considered an “engineered immune cell.”
  • engineered immune cells include T cells with an engineered T cell receptor (TCR), engineered (e.g., edited) tumor infiltrating lymphocytes (eTIL) and engineered regulatory T cells (eTregs).
  • TCR engineered T cell receptor
  • eTIL tumor infiltrating lymphocytes
  • eTregs engineered regulatory T cells
  • Some aspects of the present disclosure provide a method comprising: administering human immune cells and human peripheral blood mononuclear cells (PBMCs) to an immunodeficient mouse, wherein the human immune cells comprise an engineered receptor that specifically binds to a cell surface antigen on the diseased human cells, and the immunodeficient mouse has been engrafted with diseased human cells; and assaying the immunodeficient mouse for symptoms of CRS and/or efficacy of the human immune cells.
  • PBMCs peripheral blood mononuclear cells
  • the method further comprises administering the diseased human cells to an immunodeficient mouse.
  • the human immune cells are selected from T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells, and neutrophils.
  • NK natural killer
  • the human immune cells are genomically-modified immune cells.
  • the engineered receptor is a CAR.
  • the engineered receptor is a T cell receptor (TCR).
  • TCR T cell receptor
  • the human immune cell is a T cell with an engineered TCR.
  • the human immune cells are regulatory T cells (Tregs).
  • the diseased human cells are selected from blood cells, muscle cells, and neuronal cells.
  • the diseased human cells are tumor cells.
  • the PBMCs and the human immune cells are autologous.
  • the diseased human cells, the PBMCs and the human immune cells are autologous.
  • the PBMCs and the human immune cells are allogeneic.
  • the method further comprises irradiating the immunodeficient mouse prior to administering the human immune cells and the human PBMCs to an immunodeficient mouse.
  • the human immune cells and the human PBMCs are administered simultaneously.
  • the method further comprises administering to the immunodeficient mouse a candidate agent for treating CRS prior to the assaying.
  • the mouse is a non-obese diabetic (NOD) mouse.
  • the mouse comprises a null mutation in a Prkdc gene and a null mutation in an Il2rg gene.
  • the mouse has a ⁇ OV)-Cg.-Prkdc sad IL2rg tmlwJl ISz] genotype.
  • the mouse lacks functional major histocompatibility complex I (MHC I) and major histocompatibility complex II (MHC II).
  • the mouse comprises a null H2-Abl gene.
  • the mouse is a NOD .Cg-Prkdc scld H2-Kl tmlBpe H2-Abl emlMvw H2-Dl tmlBpe Il2rg tmlWjl/ SzJ mouse (NSG-(£ fo D /; ) IIU " (IA nul1 ) mouse).
  • the method further comprises determining that the likelihood of CRS induction is high when: a human IFN-y level in the mouse is > 1,800 pg/ml ⁇ 10%; a human IL-10 level in the mouse is > 120 pg/ml ⁇ 10%; a human IL-6 level in the mouse is > 25 pg/ml ⁇ 10%; a human IL-2 level in the mouse is > 80 pg/ml ⁇ 10%; a human IL-4 level in the mouse is > 120 pg/ml ⁇ 10%; a human TNFa level in the mouse is > 120 pg/ml ⁇ 10%; a human IL-8 level in the mouse is > 15 pg/ml ⁇ 10%; a human MCP-1 level in the mouse is > 120 pg/ml ⁇ 10%; and/or a human GM-CSF level in the mouse is > 600 pg/ml ⁇ 10%.
  • the method further comprises assaying the mouse for macrophage activation syndrome (MAS).
  • MAS macrophage activation syndrome
  • the likelihood of MAS is determined by measuring the circulating levels of IL-6, IL-1, and/or IFN-y.
  • the method further comprises assaying the mouse for neurotoxicity.
  • the likelihood of neurotoxicity is determined by measuring the circulating levels of IFN-y, IL-6, and/or TNF-a.
  • the method further comprises performing a serum biochemical analysis of liver-kidney function.
  • the serum biochemical analysis comprises measuring the levels of at least one of the following markers: aspartate transaminase (AST), albumin, total bilirubin, creatinine, or blood urea nitrogen.
  • AST aspartate transaminase
  • the method further comprises determining whether the candidate agent reduces the level of one or more circulating cytokines.
  • the diseased human cells are human tumor cells
  • the assaying comprises measuring growth of the human tumor cells.
  • the growth of the human tumor cells is measured over time.
  • a reduction in tumor volume of 20% or more relative to a control mouse that was not administered the human immune cells is indicative of efficacy.
  • a reduction in tumor burden of 20% or more relative to a control mouse that was not administered the human immune cells is indicative of efficacy.
  • the growth of the human tumor cells is used to determine progression-free survival, tumor volume doubling time, relative tumor volume, tumor growth inhibition, or tumor growth rate.
  • FIG. 1A provides data demonstrating that irradiated MHC class VII knockout (KO) NSGTM mice had increased cytokine release compared to control unirradiated mice after CD 19 CAR T cell treatment.
  • FIG. IB provides data demonstrating that unirradiated mice (D10) and irradiated mice (D9) had similar tumor burden upon CAR T cell treatment.
  • FIG. 2A provides data demonstrating that CD22 CAR T cell treatment in mice with low tumor burden efficiently blocked Raji-Luc tumor growth in both NSGTM and Raji-Luc NSG Class I/II KO (DKO).
  • FIG. 2B provides data showing very low levels of human cytokine induction after CD22 CAR T cell infusion in both the Raji-Luc NSGTM and Raji- Luc NSGTM VII KO models.
  • FIG. 3A provides data demonstrating that increased cytokine levels were correlated with high tumor burden following CD 19 CAR T cell treatment in the Raji-Luc model.
  • FIG. 3B provides data demonstrating that CD 19 CAR T cell treatment in mice with a high tumor burden was poorly effective, while CD 19 CAR T cell treatment in mice with a lower tumor burden can prevent tumor progression.
  • FIGs. 7A-7B are graphs showing the effect of CART dosage on tumor burden (FIG. 7A, left), mouse body weight change (FIG. 7A, right), and cytokine release (FIG. 7B).
  • FIGs. 8A-8C are graphs showing data from CAR T cell therapy in PBMC humanized mice compared to control mice.
  • Tumor burden (FIG. 8A), body weight (FIG. 8B), and levels of human interferon (IFN), tumor necrosis factor (TNF), interleukin- 10 (IL- 10), and IL-6 (FIG. 8C) are shown.
  • IFN human interferon
  • TNF tumor necrosis factor
  • IL- 10 interleukin- 10
  • IL-6 FIGs. 8C
  • FIGs. 9A-9C are graphs showing the efficacy of allogeneic CD 19 CART from different PBMC humanized mice having a Raji_Luc tumor.
  • Tumor burden imaging FIG. 9A
  • flow analysis of the CD3-CD19+ cell population FIG. 9B
  • body weight loss FIG. 9C
  • FIGs. 10A-10C are graphs showing levels of IFN (FIG. 10A), IL- 10 (FIG. 10B), and IL-6 (FIG. 10C) in different PBMC humanized mice with Raji_Luc tumor following allogeneic CD 19 CART treatment at two time points.
  • FIGs. 11A-11D show the variation of cytokine release and toxicity from different PBMC humanized mice following autologous CD 19 CART treatment. The protocol is shown schematically in FIG. 11A.
  • FIG. 11B shows the percentage of CD3-CD19+ cells after treatment.
  • FIG. 11C shows body weight over time and FIG. 11D depicts cytokine levels following treatment.
  • Engineered immune cell therapies such as chimeric antigen receptor (CAR) immune cell therapies and other engineered immune cell therapies, use gene transfer to reprogram immune cells (e.g., T cells, B cells, NK cells) so that they express at least one engineered antigen receptor (e.g., CAR or TCR), enabling the resulting immune cells to recognize and target cell surface antigens specific to a particular disease (e.g., cancer) or cell type.
  • CAR T cells eliminate malignant cells after recognizing and binding to them.
  • engineered immune cell (e.g., T cell, B cell, or NK cell) therapy is used to treat hematological malignancies and is currently being developed to treat solid tumor cancers.
  • engineered immune cells can be used to target (e.g., bind to) cell surface antigens specific to diseased cells (e.g., those associated with cardiovascular disease, metabolic disease, or other pathological states).
  • Engineered immune cell therapies such as engineered T cell therapies, have several known side effects, such as cytokine release syndrome (CRS) and T cell-related encephalopathy syndrome (neurotoxicity). Either or both complications can lead to significant morbidity and mortality.
  • CRS cytokine release syndrome
  • NGS T cell-related encephalopathy syndrome
  • CAR natural killer cell (CAR NK) therapy can be an off-the- shelf t e.g., universal) therapy, as NK cells do not require strict human leukocyte antigen (HLA) matching or carry the risk of graft-versus-host disease.
  • CAR NK therapy is developing, as primary NK cell isolation, expansion, and transduction are still being refined.
  • immune cells such as B cells, dendritic cells, monocytes/macrophages, and neutrophils, may also be reprogramed to express at least one engineered antigen receptor (e.g., CAR or TCR).
  • engineered antigen receptor e.g., CAR or TCR
  • the mouse models described herein may be used to assess whether a particular engineered immune cell (e.g., CAR or TCR immune cell (e.g., T cell, B cell, NK cell)) therapy is likely to be associated with CRS or other side effects.
  • the mouse models described herein are humanized and therefore include human immune cells (e.g., T cells, monocytes, and NK cells) which may contribute to cytokine release. In this way, the mouse models described herein more precisely represent in vivo CRS induction and enable a more accurate assessment of human cytokine release.
  • the mouse models described herein are also useful for identifying agents effective for treating CRS without interfering with the therapeutic efficacy of the engineered immune cells, such as immune cells expressing a CAR (the therapeutic effects of which are often mediated by the release of cytokines).
  • the mouse models describe herein may be used to identify engineered immune cells (e.g., CAR immune cells, e.g., which CAR constructs, are effective for treating certain diseases (e.g., cancers) without inducing CRS.
  • Engineered Immune Cell Therapies e.g., the human immune cell comprises an engineered receptor that specifically binds to a cell surface antigen on a diseased human cell.
  • the human immune cell is one that may be used in adoptive cell therapy (ACT).
  • ACT refers to a cell-based immunotherapy that relates to the transfusion of autologous or allogenic immune cells, genetically modified or not, that have been expanded ex vivo prior to the transfusion.
  • the human immune cells in some embodiments, are engineered immune cells.
  • the engineered receptor is an engineered T cell receptor (eTCR).
  • eTCR refers to a dimeric heterologous cell surface signaling protein forming an alpha-beta or gamma-delta receptor typically involved in recognizing an antigen presented by a major histocompatibility complex (MHC) molecule (i.e., antigen recognition in the context of an MHC molecule).
  • MHC major histocompatibility complex
  • eTCRs are modified to target or recognize histocompatibility antigen 1 (HA1), Wilms tumor 1 (WT1), cytomegalovirus (CMV), melanoma antigen (MAGE), glycoprotein 100 (gp100), MAR-1, human papillomavirus-16 E6 protein (HPV-16 E6), New York esophageal squamous cell carcinoma (NY-ESO-1), hepatitis B virus (HBV), protein 53 (P53), carcinoembryonic antigen (CEA), HPV E7, HIVgag-specific peptide SLYNTVATL (SL9), transforming growth factor-beta 2 (TGF ⁇ 2), monocyte chemotactic protein (MCP ⁇ V), TNF-related apoptosis-inducing ligand (TRAIL), preferentially expressed antigen in melanom
  • H1 histocompatibility antigen 1
  • WT1 Wilms tumor 1
  • CMV cytomegalovirus
  • the human immune cell is a T cell with an eTCR.
  • the human immune cell comprises an engineered tumorinfiltrating lymphocyte (TIL).
  • TIL therapy or engineered TIL therapy, eTIL therapy
  • TILs are removed from a subject’s tumor (e.g., during a biopsy or surgical resection) and grown and expanded ex vivo with interleukin-2 (IL-2) and/or other cytokines.
  • IL-2 interleukin-2
  • the TILs which are naturally present in some tumors and are capable of recognizing and killing cancer cells, are then administered to the subject (e.g., by infusion).
  • the TILs are engineered TILs (eTILs), which have been modified to increase tumor homing ability, cytotoxicity and/or to improve longevity (prevent exhaustion) (Jimenez-Reinoso et al., Front. Oncol., 16 February 2021).
  • eTILs engineered TILs
  • the eTILs may be transfected with TRAIL, IL- 12, CXCL8, and/or CXCR2.
  • the human immune cell is a regulatory T cell (Treg).
  • a "regulatory T cell” also known as a suppressor T cell, is a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease.
  • Tregs are CD4 + CD25 + FoxP3 + , immunosuppressive and generally suppress or downregulate induction and proliferation of T effector cells.
  • administration of the Tregs may treat or prevent cancer. For example, it has been found that administration of Tregs downregulates inflammation, blocking the development of bacteria-triggered colitis and colorectal cancer (Poutahidis et al., Carcinogenesis.
  • the Tregs comprise a chimeric antigen receptor (CAR) as described below (Mohseni et al., Front. Immunol., 24 July 2020). It should be understood that while many embodiments describe herein are directed to assessing the effects of cell therapies for treating cancer, the disclosure is not so limited. The mouse models described herein may be used to assess a myriad of engineered immune cell therapies, particularly those associated with the induction of CRS.
  • the human immune cells are human T cells.
  • the human immune cells are human B cells.
  • a CAR is designed for a T cell and is a chimera of a signaling domain of the T cell receptor (TcR) complex and an antigen-recognizing domain (e.g., a single chain fragment (scFv) of an antibody or other antibody fragment) (Enblad et al., Human Gene Therapy. 2015; 26(8):498-505).
  • TcR T cell receptor
  • scFv single chain fragment
  • a T cell that expresses a CAR is referred to as a “CAR T cell.”
  • the T cell is a Treg (CD4 + CD25 + FoxP3 + ) and resulting CAR T cell is referred to as a “CAR Treg cell.”
  • CAR Treg cell There are five generations of CARs, each of which contains different components.
  • First generation CARs join an antibody-derived scFv to the CD3zeta ( ⁇ or z) intracellular signaling domain of the T cell receptor through hinge and transmembrane domains.
  • Second generation CARs incorporate an additional domain, e.g., CD28, 4-1BB (41BB), or ICOS, to supply a costimulatory signal.
  • Third-generation CARs contain two costimulatory domains fused with the TcR CD3- ⁇ chain.
  • Third-generation costimulatory domains may include, e.g., a combination of CD3z, CD27, CD28, 4-1BB, ICOS, or OX40.
  • CARs in some embodiments, contain an ectodomain (e.g., CD3 ⁇ ), commonly derived from a single chain variable fragment (scFv), a hinge, a transmembrane domain, and an endodomain with one (first generation), two (second generation), or three (third generation) signaling domains derived from CD3 ⁇ and/or co-stimulatory molecules (Maude et al., Blood.2015; 125(26):4017-4023; Kakarla and Gottschalk, Cancer J.2014; 20(2):151-155).
  • the chimeric antigen receptor (CAR) is a T cell redirected for universal cytokine killing (TRUCK), also known as a fourth generation CAR.
  • TRUCKs are CAR-redirected T cells used as vehicles to produce and release a transgenic cytokine, IL-12, that accumulates in the targeted tissue, e.g., a targeted tumor tissue.
  • the transgenic cytokine is released upon CAR engagement of the target. This may result in therapeutic concentrations at the targeted site and avoid systemic toxicity.
  • the CAR T cell is a fifth generation CAR or next-generation CAR.
  • Fifth generation CAR T cells are based on second generation CARs, having additional intracellular domains of cytokine receptors.
  • the additional intracellular domain is a cytoplasmic IL-2 receptor (e.g., IL-2R ⁇ having a STAT3/5 binding motif), which is a binding site for STAT3/5, a transcription factor (Tokarew et al., British Journal of Cancer.2019; 120: 26-37).
  • the CAR is capable of producing all three synergistic signals necessary to physiologically to drive full T cell activation and proliferation: TCR (through the CD3 ⁇ domains), co-stimulatory (CD28 domain), and cytokine (JAK–STAT3/5) signaling.
  • CARs typically differ in their functional properties.
  • the CD3 ⁇ signaling domain of the T cell receptor when engaged, will activate and induce proliferation of T cells but can lead to anergy (a lack of reaction by the body's defense mechanisms, resulting in direct induction of peripheral lymphocyte tolerance). Lymphocytes are considered anergic when they fail to respond to a specific antigen.
  • the addition of a costimulatory domain in second- generation CARs improved replicative capacity and persistence of modified T cells. Similar antitumor effects are observed in vitro with CD28 or 4-1BB CARs, but preclinical in vivo studies suggest that 4-1BB CARs may produce superior proliferation and/or persistence.
  • Second-generation CARs are capable of inducing substantial T cell proliferation in vivo, but CARs containing the 4-1BB costimulatory domain appear to persist longer.
  • Third generation CARs combine multiple signaling domains (costimulatory) to augment potency.
  • Fourth generation CARs are additionally modified with a constitutive or inducible expression cassette for a transgenic cytokine, which is released by the CAR T cell to modulate the T cell response. See, for example, Enblad et al., Human Gene Therapy.2015; 26(8):498-505; Chmielewski and Hinrich, Expert Opinion on Biological Therapy.2015;15(8): 1145-1154.
  • fifth generation CARs further comprise cytokine receptor domains and are able to trigger cytokine signaling, further enhancing T cell proliferation and maintenance (Tokarew et al., British Journal of Cancer.2019; 120: 26-37).
  • Other immune cells may be reprogramed using CAR technology.
  • NK cells, B cells, dendritic cells, monocytes/macrophages, and neutrophils may also be reprogramed to expression at least one CAR.
  • NK cells are derived from the bone marrow and defend against viruses and prevent cancer. These cells can kill cells (e.g., virus-infected cells) by injecting a combination of chemicals lethal to the cell.
  • B cells are immune cells that develop in the bone marrow from hematopoietic stem cells and produce antibodies. B cells are “trained” so that they do not produce antibodies against healthy tissue, and when they encounter foreign (non-self) material, they mature into plasma cells or memory cells.
  • Dendritic cells are antigen-presenting cells that process antigen material and present it on their respective cell surfaces to T cells. In this way, they act as a liaison between the innate and adaptive immune systems.
  • CAR dendritic cells CAR-DC
  • Monocytes are phagocytic cells of the immune system found in all tissues. They play a role in both adaptive and innate immunity, and in some instances, work with T cells to kill microorganisms.
  • the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy is autologous; that is, for example, a subject’s T cells are collected and used to generate the CAR T cells that are later used to treat the subject.
  • the engineered immune cells are universal allogeneic engineered immune cells (e.g., “off-the-shelf” engineered immune cells). Allogeneic engineered immune cells use donor immune cells; that is, immune cells from a source other than the subject (recipient) who undergoes the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • the donor immune cells are from a healthy human (e.g., adult or child).
  • Allogeneic engineered immune cells e.g., CAR T cells
  • GVHD graft- versus-host disease
  • the T cell ⁇ receptor (TCR ⁇ ) of the CAR T cell may be knocked out using gene editing tools (e.g., zinc finger nucleases, transcription activator like effector nucleases, or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)) (Kim et al., Biomolecules.2020; 10(2):263).
  • gene editing tools e.g., zinc finger nucleases, transcription activator like effector nucleases, or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas9 CRISPR-associated protein 9
  • a chimeric antigen receptor (CAR) comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and a cytoplasmic domain.
  • a CAR is fully human.
  • the antigen binding domain of a CAR is specific for one or more antigens.
  • a “spacer” domain or “hinge” domain is located between an extracellular domain (comprising the antigen binding domain) and a transmembrane domain of a CAR, or between a cytoplasmic domain and a transmembrane domain of the CAR.
  • a “spacer domain” refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the cytoplasmic domain in the polypeptide chain.
  • a CAR of the disclosure comprises an antigen binding domain, such as a single chain Fv (scFv) specific for an antigen (e.g., a tumor antigen).
  • an antigen e.g., a tumor antigen.
  • the choice of binding domain depends upon the type and number of ligands that define the surface of a target cell.
  • the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state, such as cancer, cardiovascular disease, metabolic disease, neurobiological disease, or an autoimmune disease.
  • examples of cell surface markers that may act as ligands for the antigen binding domain in the CAR of the present disclosure include those associated with cancer cells and/or other forms of diseased cells.
  • immune cells expressing a CAR are genetically modified to recognize multiple targets or antigens, which permits the recognition of unique target or antigen expression patterns on diseased cells (e.g., tumor cells).
  • diseasesd cells e.g., tumor cells.
  • CARs that can bind multiple targets include: “split signal CARs,” which limit complete immune cell activation to tumors expressing multiple antigens; “tandem CARs” (TanCARs), which contain ectodomains having two scFvs; and “universal ectodomain CARs,” which incorporate avidin or a fluorescein isothiocyanate (FITC)-specific scFv to recognize tumor cells that have been incubated with tagged monoclonal antibodies (mAbs).
  • FITC fluorescein isothiocyanate
  • a CAR is considered “bispecific” if it recognizes two distinct antigens (has two distinct antigen recognition domains).
  • a bispecific CAR is comprised of two distinct antigen recognition domains present in tandem on a single transgenic receptor (referred to as a TanCAR; see, e.g., Grada Z et al. Molecular Therapy Nucleic Acids 2013;2:el05, incorporated herein by reference).
  • these iCARs block T cell responses from T cells activated by either their endogenous T cell receptor or an activating CAR.
  • CARs are engineered for use in adoptive cell transfer, wherein immune cells are removed from a subject and modified so that they express receptors specific to an antigen, e.g., a tumor- specific antigen. The modified immune cells, which may then recognize and kill the cancer cells, are reintroduced into the subject (Pule, et al., Cytotherapy. 2003; 5(3): 211-226; Maude et al., Blood. 2015; 125(26): 4017-4023, each of which is incorporated herein by reference).
  • Cytokine release syndrome occurs with activation of T cells and Natural Killer (NK) cells as well as other immune cell populations (e.g., macrophages).
  • engineered immune cells e.g., T cells, B cells, or NK cells
  • T cells, B cells, or NK cells the activation of immune cells can lead to the release of high levels of cytokines and downstream injury and possibly death.
  • Both T cells and NK cells have been found to be sources of CRS in response to certain immunomodulators (Wing M.G. et al. (1995) Ther. Immunol. 2:183-190; Carson W.E., (1999) J Immunol 162;4943-4951).
  • CRS can manifest with high levels of cytokine release that can vary with the various activated immune cell populations.
  • CAR T cell-induced CRS treatments for CAR T cell-induced CRS include anti- IL-6 antibodies (e.g., siltuximab), corticosteroids (e.g., methylprednisone), anti-TNF-a drugs (e.g., etanercept), IL-1R inhibitors (e.g., anakinra), GM-CSF inhibitors, and small molecule inhibitors (e.g., ruxolitinib (JAK 1/2 inhibitor) and Bruton’s tyrosine kinase inhibitor).
  • anti- IL-6 antibodies e.g., siltuximab
  • corticosteroids e.g., methylprednisone
  • anti-TNF-a drugs e.g., etanercept
  • IL-1R inhibitors e.g., anakinra
  • GM-CSF inhibitors e.g., GM-CSF inhibitors
  • Immunodeficiency mouse models include the single-gene mutation models such as nude- mice (nu) strains and the severe combined immunodeficiency (scid) strains, non-obese diabetic (NOD) strain, RAG (recombination activating gene) strains with targeted gene deletion and a variety of hybrids originated by crossing doubly and triple mutation mice strains with additional defects in innate and adaptive immunity.
  • Non-limiting examples of spontaneous and transgenic immunodeficient mouse models include the following mouse strains: • Nude (nu) [Flanagan SP. Genet Res 1966; 8: 295-309; and Nehls M et al. Nature 1994; 372: 103-7]; • Scid (scid) [Bosma GC et al.
  • an immunodeficient mouse has a NOD (non-obese diabetic) genotype.
  • the NOD mouse (e.g., the NOD/ShiLtJ mouse, Jackson Labs Stock #001976) is a polygenic model for autoimmune type 1 diabetes, characterized by hyperglycemia and insulitis, a leukocytic infiltration of the pancreatic islet cells.
  • the NOD mice are hypoinsulinemic and hyperglucagonemic, indicating a selective destruction of pancreatic islet beta cells.
  • the major component of diabetes susceptibility in NOD mice is the unique MHC haplotype.
  • NOD mice also exhibit multiple aberrant immunophenotypes including defective antigen presenting cell immunoregulatory functions, defects in the regulation of the T lymphocyte repertoire, defective NK cell function, defective cytokine production from macrophages (Fan et al., 2004) and impaired wound healing. They also lack hemolytic complement, C5. NOD mice also are severely hard-of-hearing. A variety of mutations causing immunodeficiencies, targeted mutations in cytokine genes, as well as transgenes affecting immune functions, have been backcrossed into the NOD inbred strain background.
  • an immunodeficient mouse provided herein based on the NOD background may have a genotype selected from NOD-Cg.- Prkdc scid IL2rg tm1wJl /SzJ (NSG), a NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl /SzJ (NRG), and NOD.Cg- Prkdc scid Il2rg tm1Sug /ShiJic.
  • the mouse may have a NOD-Cg.- Prkdc scid IL2rg tm1wJl /SzJ (NOG) genotype.
  • an immunodeficient mouse has an NSGTM genotype.
  • the NSGTM mouse e.g., Jackson Labs Stock No: #005557
  • NK natural killer
  • the NS mouse derived from the NOD mouse strain NOD/ShiLtJ (see, e.g., (Makino et al., 1980), which is incorporated herein by reference), include the Prkdc scid mutation (also referred to as the “severe combined immunodeficiency” mutation or the “scid” mutation) and the Il2rg tm1Wjl targeted mutation.
  • Prkdc scid mutation is a loss-of-function (null) mutation in the mouse homolog of the human PRKDC gene – this mutation essentially eliminates adaptive immunity (see, e.g., (Blunt et al., 1995; Greiner, Hesselton, & Shultz, 1998), each of which is incorporated herein by reference).
  • the Il2rg tm1Wjl mutation is a null mutation in the gene encoding the interleukin 2 receptor gamma chain (IL2R ⁇ , homologous to IL2RG in humans), which blocks NK cell differentiation, thereby removing an obstacle that prevents the efficient engraftment of primary human cells (Cao et al., 1995; Greiner et al., 1998; Shultz et al., 2005), each of which is incorporated herein by reference).
  • an immunodeficient mouse has an NRG genotype.
  • the NRG mouse e.g., Jackson Labs Stock #007799
  • This mouse two mutations on the NOD/ShiLtJ genetic background a targeted knockout mutation in recombination activating gene 1 (Rag1) and a complete null allele of the IL2 receptor common gamma chain (IL2rg null ).
  • the Rag1 null mutation renders the mice B and T cell deficient and the IL2rg null mutation prevents cytokine signaling through multiple receptors, leading to a deficiency in functional NK cells.
  • the severe immunodeficiency allows the mice to be humanized by engraftment of human CD34+ hematopoietic stem cells (HSC) and patient derived xenografts (PDX) at high efficiency.
  • HSC hematopoietic stem cells
  • PDX patient derived xenografts
  • an immunodeficient mouse has an NOG genotype.
  • the NOG mouse (Ito M et al., Blood 2002) is an extremely severe combined immunodeficient mouse established by combining the NOD/scid mouse and the IL-2 receptor- ⁇ chain knockout (IL2r ⁇ KO) mouse (Ohbo K. et al., Blood 1996).
  • the NOG mouse lacks T and B cells, lacks natural killer (NK) cells, exhibits reduced dendritic cell function and reduced macrophage function, and lacks complement activity.
  • an immunodeficient mouse has an NCG genotype.
  • the NCG mouse (e.g., Charles River Stock #572) was created by sequential CRISPR/Cas9 editing of the Prkdc and Il2rg loci in the NOD/Nju mouse, generating a mouse coisogenic to the NOD/Nju.
  • the NOD/Nju carries a mutation in the Sirpa (SIRP ⁇ ) gene that allows for engrafting of foreign hematopoietic stem cells.
  • SIRP ⁇ Sirpa
  • the Prkdc knockout generates a SCID-like phenotype lacking proper T cell and B-cell formation.
  • the knockout of the Il2rg gene further exacerbates the SCID-like phenotype while additionally resulting in a decrease of NK cell production.
  • strain symbol conveys basic information about the type of strain or stock used and the genetic content of that strain.
  • Rules for symbolizing strains and stocks have been promulgated by the International Committee on Standardized Genetic Nomenclature for Mice. The rules are available on-line from the Mouse Genome Database (MGD; informatics.jax.org) and were published in print copy (Lyon et al. 1996).
  • Strain symbols typically include a Laboratory Registration Code (Lab Code). The registry is maintained at the Institute for Laboratory Animal Research (ILAR) at the National Academy of Sciences, Washington, D.C.
  • Lab Codes may be obtained electronically at ILAR's web site (nas.edu/cls/ilarhome.nsf). See also Davisson MT, Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutes an Acceptable Genetic- Phenotypic Definition, National Research Council (US) International Committee of the Institute for Laboratory Animal Research. Washington (DC): National Academys Press (US); 1999.
  • a genetically modified immunodeficient mouse (e.g., NSG, NRG, or NOG mouse) includes a genomic modification, wherein the genomic modification renders the immunodeficient mouse deficient in major histocompatibility complex class I (MHC I) and major histocompatibility complex class II (MHC II), such that the genetically modified immunodeficient mouse lacks functional MHC I and lacks functional MHC II.
  • MHC I major histocompatibility complex class I
  • MHC II major histocompatibility complex class II
  • a genetically modified immunodeficient mouse deficient in MHC class I and MHC class II is a NOD.Cg-Prkdc scid H2-K1 tm1Bpe H2-Ab1 em1Mvw H2-D1 tm1Bpe Il2rg tm1Wjl /SzJ (abbreviated as NSG-(K b D b ) null (IA null ) mouse, e.g., Jackson Labs Stock #025216).
  • the NSG-(K b D b ) null (IA null ) mouse lacks functional MHC I due to a homozygous null mutation of H2-K and H2-D MHC I ⁇ protein subclasses (abbreviated (K b D b ) null ) and lacks functional MHC II due to a homozygous null mutation of H-2A subclass of MHC II (abbreviated as IA null ).
  • a genetically modified immunodeficient mouse deficient in MHC class I and MHC class II is a NO D.Cg-B2m tm1Unc Prkdc scid H2 dlAb1-Ea Il2rg tm1Wjl /SzJ (abbreviated as NSG-B2M null (IA IE) null , e.g., Jackson Labs Stock #030547) mouse.
  • the NSG- B2M null (IA IE) null mouse lacks functional MHC I due to a homozygous null mutation of ⁇ 2 microglobulin (abbreviated B2M null ).
  • the NSG-B2M null (IA IE) null mouse lacks functional MHC II due to a homozygous null mutation of H-2A and H-2E subclasses of MHC II (abbreviated as (IA IE) null ).
  • a genetically modified immunodeficient mouse deficient in MHC class I and MHC class II is a NOD.Cg-Prkdc scid H2-K1 tm1Bpe H2-Ab1 em1Mvw H2-D1 tm1Bpe Il2rg tm1Wjl Tg(Ins2-HBEGF)6832Ugfm/Sz transgenic mouse (abbreviated as NSG-RIP-DTR (K b D b ) null (IA null ), e.g., Jackson Labs Stock # 027976), which expresses the diphtheria toxin receptor under the control of the rat insulin promoter on an NSGTM background.
  • NSG-RIP-DTR K b D b
  • IA null e.g., Jackson Labs Stock # 027976
  • a humanized immunodeficient mouse model is used to screen an engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • an engineered immune cell e.g., T cell, B cell, or NK cell
  • humanized mouse As used herein, the terms “humanized mouse”, “humanized immune deficient mouse”, “humanized immunodeficient mouse”, and the plural versions thereof are used interchangeably to refer to an immunodeficient mouse humanized by engraftment with human peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Humanized mice are generated by starting with an immunodeficient mouse and, if necessary, depleting and/or suppressing any remaining murine immune cells (e.g., chemically or with radiation). That is, successful survival of the human immune system in the immunodeficient mice may require suppression of the mouse’s immune system to prevent GVHD (graft-versus-host disease) rejections.
  • GVHD graft-versus-host disease
  • the mouse is engrafted with human cells (e.g., PBMCs).
  • human cells e.g., PBMCs
  • engraft refers to the process of the human cells migrating to, and incorporating into, an existing tissue of interest in vivo.
  • the engrafted human PBMCs provide functional mature human cells (e.g., immune cells, such as T cells or NK cells).
  • the model has a specific time window of 4-5 weeks after engraftment before GVHD sets in.
  • DKO double-knockout mice lacking functional MHC I and MHC II, as described above, may be used. Irradiation
  • the immunodeficient mice are irradiated prior to engraftment with PBMCs. It is thought that irradiation of an immunodeficient mouse destroys mouse immune cells in peripheral blood, spleen, and bone marrow, which facilitates engraftment of human cells, such as human PBMCs (e.g., by increasing human PBMC survival factors), as well as expansion of immune cells, and ultimately, engineered immune cells (e.g., T cells, B cells, or NK cells). Irradiation also shortens the time it takes to accumulate the required number of human immune cells to “humanize” the mouse models.
  • human PBMCs e.g., by increasing human PBMC survival factors
  • engineered immune cells e.g., T cells, B cells, or NK cells
  • mice For immunodeficient mice (e.g., NSGTM mice), this preparation is commonly accomplished through whole-body gamma irradiation.
  • Irradiators may vary in size depending on their intended use. Animals are generally irradiated for short periods of time (less than 15 min). The amount of time spent inside the irradiator varies depending on the radioisotope decay charts, amount of irradiation needed, and source of ionizing energy (that is, X-rays versus gamma rays, for which a cesium or cobalt source is needed).
  • a myeloablative irradiation dose is usually 700 to 1300 cGy, though in some embodiments, lower doses such as 1-100 cGy (e.g., about 2, 5, or 10 cGy), or 300-700 cGy may be used.
  • the mouse is irradiated with 100 cGy X-ray (or 75 cGy - 125 cGy X- ray).
  • the dose is about 1, 2, 3, 4, 5, 10, 20, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 cGy, or between any of the two recited doses herein, such as 100-300 cGy, 200-500 cGy, 600-1000 cGy, or 700-1300 cGy.
  • the immunodeficient mouse is irradiated about 15 minutes, 30 minutes, 45 minutes, 1 hour, or more before engraftment with PBMCs and diseased cells (e.g., from a cell line or from a patient-derived xenograft).
  • the immunodeficient mouse is engrafted with PBMCs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days after irradiation.
  • the irradiated immunodeficient mice are engrafted with PBMCs, humanizing the mice.
  • the PBMCs may be engrafted after irradiation and before engraftment with diseased cells (e.g., tumor cells), after irradiation and engraftment with diseased cells (e.g., tumor cells) and before the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy is administered, or concurrently with the administration of the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • PBMCs peripheral blood mononuclear cells
  • lymphocytes typically include T cells, B cells and NK cells.
  • PBMCs may be isolated from whole blood samples, for example (e.g., Ficoll gradient).
  • PBMCs from a subject e.g., a human subject
  • an engineered immune cell e.g., T cell, B cell, or NK cell
  • mice with PBMCs to yield a humanized mouse model
  • Methods of engrafting immunodeficient mice with PBMCs to yield a humanized mouse model include, but are not limited to, intraperitoneal or intravenous injection (Shultz et al., J Immunol, 2015, 174:6477-6489; Pearson et al., Curr Protoc Immunol.2008; 15-21; Kim et al., AIDS Res Hum Retrovirus, 2016, 32(2): 194-2020; Yaguchi et al., Cell & Mol Immunol, 2018, 15:953-962).
  • the mouse is engrafted with 0.5-3.0x10 7 PBMCs.
  • the mouse is engrafted with 0.5 x10 7 , 0.6 x10 7 , 0.7 x10 7 , 0.8 x10 7 , 0.9 x10 7 , 1.0 x10 7 , 1.1 x10 7 , 1.2 x10 7 , 1.3 x10 7 , 1.4 x10 7 , 1.5 x10 7 , 1.6 x10 7 , 1.7 x10 7 , 1.8 x10 7 , 1.9 x10 7 , 2.0 x10 7 , 2.5 x10 7 , 3.0 x10 7 or more PBMCs.
  • the mouse is engrafted with 0.5-0.75 x10 7 , 0.5-1.0 x10 7 , 0.5-1.1 x10 7 , 0.5-1.2 x10 7 , 0.5-1.3 x10 7 , 0.5-1.4 x10 7 , 0.5-1.5 x10 7 , 0.5-1.6 x10 7 , 0.5-1.7 x10 7 , 0.5-1.8 x10 7 , 0.5- 1.9 x10 7 , 0.5-2.0 x10 7 , 0.5-2.25 x10 7 , 0.5-2.5 x10 7 , 0.5-3.0 x10 7 , 0.75-1.0 x10 7 , 0.75-1.1 x10 7 , 0.75-1.2 x10 7 , 0.75-1.3 x10 7 , 0.75-1.4 x10 7 , 0.75-1.5 x10 7 , 0.75-1.6 x10 7 , 0.75-1.7 x10 7 , 0.75- 1.8 x10 7 , 0.75-
  • the mouse is engrafted with 2x10 7 PBMCs.
  • the mouse is engrafted with 4.5-5.5x10 7 (4.5-5.0x10 7 , 5.0-5.5x10 7 ) PBMCs.
  • the human PBMCs are engrafted 5 minutes, 10 minute, 15 minute, 20 minutes, 25 minutes, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or more after irradiation.
  • the human PBMCs are engrafted 1-2 hours, 1-3 hours, 1-4 hours, 1-5 hours, 1-6 hours, 1-7 hours, 1-8 hours, 1-9 hours, 1-10 hours, 1-12 hours, 1-14 hours, 1-16 hours, 1-18 hours, 1-20 hours, 1-22 hours, 1-24 hours, 2-3 hours, 2-4 hours, 2-5 hours, 2-6 hours, 2-7 hours, 2-8 hours, 2-9 hours, 2-10 hours, 2-12 hours, 2-14 hours, 2-16 hours, 2-18 hours, 2-20 hours, 2-22 hours, 2-24 hours, 3-4 hours, 3-5 hours, 3-6 hours, 3-7 hours, 3-8 hours, 3-9 hours, 3-10 hours, 3-12 hours, 3-14 hours, 3-16 hours, 3-18 hours, 3-20 hours, 3-22 hours, 3-24 hours, 4-5 hours, 4-6 hours, 4-7 hours, 4-8 hours, 4-9 hours, 4-10 hours, 4-12 hours, 4-14 hours, 4-16 hours, 4-18 hours, 4-20 hours, 4- 22 hours, 4-24 hours, 5-6 hours, 5-7 hours,
  • the immunodeficient mouse is administered/engrafted with diseased cells (e.g., from a cell line or a patient-derived xenograft) after irradiation.
  • diseased cell refers to a cell which is found in a diseased subject (e.g., an individual suffering from a disease or pathological condition, including cancer) and which is abnormal in terms of its structure and/or functioning and/or metabolism and/or genome compared to a cell having a structure, function, metabolism, and genome that are characteristic of a physiological cell found in a healthy subject (e.g., an individual not suffering from a disease or condition).
  • diseased cells include, but are not limited to, cancer or tumor cells (discussed below), diseased vascular smooth muscle cells, diseased endothelial cells (e.g., in the case of atherosclerosis), diseased cells infected by a pathogen such as a virus (e.g., in the case of infectious diseases), and diseased cells undergoing fibrosis (e.g., in the case of fibrotic diseases).
  • cancer or tumor cells discussed below
  • diseased vascular smooth muscle cells e.g., in the case of atherosclerosis
  • diseased endothelial cells e.g., in the case of atherosclerosis
  • diseased cells infected by a pathogen such as a virus
  • diseased cells undergoing fibrosis e.g., in the case of fibrotic diseases.
  • the phenotype, physical aspects or characteristics of the diseased cells will vary depending on the disease or condition (e.g., cancer, atherosclerosis, fibrotic disease and infectious disease, etc.) and standard techniques and knowledge (e.g., using disease-specific markers) can be used to distinguish a diseased cell from a non-diseased or healthy cell depending on the disease or condition.
  • Diseased cells may be administered using any method known in the art, for example, intravenous (e.g., tail vein injection), subcutaneous, intrafemoral, intraventricular, intracardial, intraperitoneal routes of administration, and the like.
  • the route of administration is intravenous infusion.
  • PDX Patient-Derived Xenograft
  • the immunodeficient mouse is administered/engrafted with a patient-derived xenograft (PDX) comprising diseased cells (e.g., tumor cells).
  • PDXs are tissues that have been removed from a human.
  • the PDX comprises tumor cells.
  • the tumor is from a hematological malignancy, such as adult acute myeloid leukemia (AML).
  • AML adult acute myeloid leukemia
  • the appropriate amount of PDX cells is 0.1-0.2 ⁇ 10 6 , 0.1-0.3 ⁇ 10 6 , 0.1-0.4 ⁇ 10 6 , 0.1-0.5 ⁇ 10 6 , 0.1-0.6 ⁇ 10 6 , 0.1-0.7 ⁇ 10 6 , 0.1- 0.8 ⁇ 10 6 , 0.1-0.9 ⁇ 10 6 , 0.1-1.0 ⁇ 10 6 , 0.1-1.25 ⁇ 10 6 , 0.1-1.5 ⁇ 10 6 , 0.1-1.75 ⁇ 10 6 , 0.1-2.0 ⁇ 10 6 , 0.2- 0.3 ⁇ 10 6 , 0.2-0.4 ⁇ 10 6 , 0.2-0.5 ⁇ 10 6 , 0.2-0.6 ⁇ 10 6 , 0.2-0.7 ⁇ 10 6 , 0.2-0.8 ⁇ 10 6 , 0.2-0.9 ⁇ 10 6 , 0.2- 1.0 ⁇ 10 6 , 0.2-1.25 ⁇ 10 6 , 0.2-1.5 ⁇ 10 6 , 0.2-1.75 ⁇ 10 6 , 0.2-2.0 ⁇ 10 6 , 0.3-0.4 ⁇ 10 6 , 0.3-10 6
  • the PDX is introduced into the mice before the PBMCs are engrafted and the human immune cells (e.g., human B- or T cells or NK cells) appear.
  • a PDX is introduced into a mouse immediately after irradiation.
  • the PDX is introduced 5 minutes, 10 minute, 15 minute, 20 minutes, 25 minutes, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or more after irradiation.
  • the tumor cells are introduced 1-2 hours, 1-3 hours, 1-4 hours, 1-5 hours, 1-6 hours, 1-7 hours, 1-8 hours, 1-9 hours, 1-10 hours, 1-12 hours, 1-14 hours, 1-16 hours, 1-18 hours, 1-20 hours, 1-22 hours, 1-24 hours, 2-3 hours, 2-4 hours, 2-5 hours, 2-6 hours, 2-7 hours, 2-8 hours, 2-9 hours, 2-10 hours, 2-12 hours, 2-14 hours, 2-16 hours, 2-18 hours, 2-20 hours, 2-22 hours, 2-24 hours, 3-4 hours, 3-5 hours, 3-6 hours, 3-7 hours, 3-8 hours, 3-9 hours, 3-10 hours, 3-12 hours, 3-14 hours, 3-16 hours, 3-18 hours, 3-20 hours, 3-22 hours, 3-24 hours, 4-5 hours, 4-6 hours, 4-7 hours, 4-8 hours, 4-9 hours, 4-10 hours, 4-12 hours, 4-14 hours, 4-16 hours, 4-18 hours, 4-20 hours, 4-22 hours, 4-24 hours, 5- 6 hours, 5-7 hours, 5-8 hours, 5-9
  • the immunodeficient mice are engrafted with primary cells.
  • Human primary cells are isolated directly from tissues and retain the morphological and functional characteristics of their tissue of origin.
  • a primary cell is a cancer cell.
  • a primary cell is a neuronal cell.
  • a primary cell is a metabolic cell.
  • a primary cell is a cardiac cell.
  • Other primary cells are contemplated herein.
  • the immunodeficient mice are engrafted with stem cells, such as induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • iPSCs are a type of pluripotent stem cell that can be generated directly from a somatic cell (see, e.g., Takahashi K et al. Cell.2006; 126 (4): 663–76).
  • the immunodeficient mice are engrafted with immortalized cells (immortalized cell lines).
  • Immortalized cell lines are cells that have been manipulated to proliferate indefinitely and can thus be cultured for long periods of time.
  • Non-limiting examples of commonly used immortalized cell lines include 3T3 cells, HeLa cells, COS cells, 293/293T/HEK-293T cells, MDCK cells, CHO cells, S2 cells, PC12 cells, Neuro-2a/N2a cells, and SH-SY5Y cells.
  • the immunodeficient mice are engrafted with tumor cells from tumor cell lines.
  • Tumor cell lines are known in the art and are publicly accessible, for example, through ATCC or other collections.
  • the cell line is from a human tumor.
  • Raji a cell line associated with human B cell lymphoma is used.
  • Jeko-1 a cell line associated with human mantle cell lymphoma is used.
  • tumor cell lines include, but are not limited to, human lung carcinoma cell lines, such as A549 (SRCC768), Calu-1 (SRCC769), Calu-6 (SRCC770), H157 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774), SW900 (SRCC775), H522 (SRCC832), and H810 (SRCC833).
  • human lung carcinoma cell lines such as A549 (SRCC768), Calu-1 (SRCC769), Calu-6 (SRCC770), H157 (SRCC771), H441 (SRCC772), H460 (SRCC773), SKMES-1 (SRCC774), SW900 (SRCC775), H522 (SRCC832), and H810 (SRCC833).
  • the cell line is associated with human lung tumors, such as SRCC724 (adenocarcinoma, abbreviated as “AdenoCa”) (LT1), SRCC725 (squamous cell carcinoma, abbreviated as “SqCCa) (LTla), SRCC726 (adenocarcinoma) (LT2), SRCC727 (adenocarcinoma) (LT3), SRCC728 (adenocarcinoma) (LT4), SRCC729 (squamous cell carcinoma) (LT6), SRCC730 (adeno/squamous cell carcinoma) (LT7), SRCC731 (adenocarcinoma) (LT9), SRCC732 (squamous cell carcinoma) (LT10), SRCC733 (squamous cell carcinoma) (LT11), SRCC734 (adenocarcinoma) (LT12), SRCC735 (adeno/squamous cell carcinoma)
  • human lung tumors designated SRCC1125 [HF-000631], SRCC1127 [HF-000641], SRCC1129 [HF-000643], SRCC1133 [HF-000840], SRCC1135 [HF-000842], SRCC1227 [HF-001291], SRCC1229 [HF-001293], SRCC1230 [HF-001294], SRCC1231 [HF-001295], SRCC1232 [HF-001296], SRCC1233 [HF-001297], SRCC1235 [HF-001299], and SRCC1236 [HF-001300],
  • the cell line is associated with human colon cancers.
  • colon cancer cell lines include, but are not limited to, SW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), Colo320 (carcinoma, SRCC778), HT29 (adenocarcinoma, SRCC779), HM7 (a high mucin producing variant of ATCC colon adenocarcinoma cell line, SRCC780), CaWiDr (adenocarcinoma, SRCC781), HCT116 (carcinoma, SRCC782), SKCO1 (adenocarcinoma, SRCC783), SW403 (adenocarcinoma, SRCC784), LS174T (carcinoma, SRCC785), Colo205 (carcinoma, SRCC828), HCT15 (carcinoma, SRCC829)
  • SW480
  • Primary colon tumors include colon adenocarcinomas designated CT2 (SRCC742), CT3 (SRCC743), CT8 (SRCC744), CT10 (SRCC745), CT12 (SRCC746), CT14 (SRCC747), CT15 (SRCC748), CT16 (SRCC749), CT17 (SRCC750), CT1 (SRCC751), CT4 (SRCC752), CT5 (SRCC753), CT6 (SRCC754), CT7 (SRCC755), CT9 (SRCC756), CT11 (SRCC757), CT18 (SRCC758), CT19 (adenocarcinoma, SRCC906), CT20 (adenocarcinoma, SRCC907), CT21 (adenocarcinoma, SRCC908), CT22 (adenocarcinoma, SRCC909), CT23 (adenocarcinoma, SRCC910), CT24 (adenocarcinoma, SRCC911), CT25 (
  • human colon tumors designated SRCC1051 [HF-000499], SRCC1052 [HF-000539], SRCC1053 [HF-000575], SRCC1054 [HF-000698], SRCC1142 [HF-000762], SRCC1144 [HF-000789], SRCC1146 [HF-000795] and SRCC1148[HF-000811].
  • the cell line is associated with human breast cancers.
  • human breast carcinoma cell lines include, for example, HBL100 (SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468 (SRCC762), MB175 (SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), and SKBR3 (SRCC767), and human breast tumor center designated SRCC1057 [HF-000545]. Also included are human breast tumors designated SRCC1094, SRCC1095, SRCC1096, SRCC1097, SRCC1098, SRCC1099, SRCC1100, SRCC1101, and human breast-met-lung-NS tumor designated SRCC893 [LT 32].
  • the cell line is associated with human kidney cancers.
  • human kidney tumor cell lines include SRCC989 [HF-000611] and SRCC1014 [HF-000613].
  • the cell line is associated with human testicular cancers. Examples of human testis tumor center includes SRCC1001 [HF-000733] and testis tumor margin SRCC999 [HF-000716].
  • the cell line is associated with human parathyroid cancers. Examples of human parathyroid tumor cell lines include SRCC1002 [HF-000831] and SRCC1003 [HF-000832]. Other human tumor cell lines are contemplated herein.
  • the immunodeficient mice are injected with an appropriate amount of tumor cells from a cancer cell line. In some embodiments, the immunodeficient mice are injected with 0.25 ⁇ 10 6 cancer cells. In some embodiments, the appropriate amount of tumor cells is 0.1-0.2 ⁇ 10 6 , 0.1-0.3 ⁇ 10 6 , 0.1-0.4 ⁇ 10 6 , 0.1-0.5 ⁇ 10 6 , 0.1-0.6 ⁇ 10 6 , 0.1- 0.7 ⁇ 10 6 , 0.1-0.8 ⁇ 10 6 , 0.1-0.9 ⁇ 10 6 , 0.1-1.0 ⁇ 10 6 , 0.1-1.25 ⁇ 10 6 , 0.1-1.5 ⁇ 10 6 , 0.1-1.75 ⁇ 10 6 , 0.1- 2.0 ⁇ 10 6 , 0.2-0.3 ⁇ 10 6 , 0.2-0.4 ⁇ 10 6 , 0.2-0.5 ⁇ 10 6 , 0.2-0.6 ⁇ 10 6 , 0.2-0.7 ⁇ 10 6 , 0.2-0.8 ⁇ 10 6 , 0.2- 0.9 ⁇ 10 6 , 0.2-1.0 ⁇ 10 6 ,
  • the cancer cells are introduced into the mice before the PBMCs are engrafted and the human immune cells (e.g., human B cells or T cells or NK cells) appear.
  • the tumor cells are introduced immediately after irradiation.
  • the tumor cells are introduced 5 minutes, 10 minute, 15 minute, 20 minutes, 25 minutes, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or more after irradiation.
  • sufficient time it is meant that the tumor is the size or has the number of tumor cells needed to study the effects of the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy, or in the case of a non-cancer disease mouse model, “sufficient time” refers to the amount of time needed to obtain the number of diseased cells necessary to study the effects of the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • the engineered immune cell e.g., T cell, B cell, or NK cell
  • Moderate tumor burdens are 1 ⁇ 5% of the body weight of the mouse (e.g., 1%, 1.25%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 1.95%, 1.96%, 1.97%, 1.98%, 1.99%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25% 4.5%, 4.75%).
  • the moderate tumor burden is 1.5-1.6%, 1.5-1.7%, 1.5-1.8%, 1.5-1.9%, 1.6-1.7%, 1.6-1.8%, 1.6-1.9%, 1.7-1.8%, 1.7-1.9%, or 1.8- 1.9%.
  • the low tumor burden is 0.1-0.2%, 0.1-0.3%, 0.1- 0.4%, 0.2-0.3%, 0.2-0.4%, or 0.3-0.4%
  • the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the mouse was injected with the diseased cells (e.g., tumor cells) (e.g., 1-2 days, 1-3 days, 1-4 days, 1-5 days, 1-6 days, 1-7 days, 1-8 days, 1-9 days, 1-10 days, 1-11 days, 1-12 days, 1-13 days, 1-14 days, 2-3 days, 2-4 days, 2-5 days, 2-6 days, 2-7 days, 2-8 days, 2-9 days, 2-10 days, 2
  • the diseased cells e.g., tumor cells
  • the mouse is administered a dose of 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 10 ⁇ 10 6 , 11 ⁇ 10 6 , 12 ⁇ 10 6 , 13 ⁇ 10 6 , 14 ⁇ 10 6 , 15 ⁇ 10 6 , 16 ⁇ 10 6 , 17 ⁇ 10 6 , 18 ⁇ 10 6 , 19 ⁇ 10 6 , 20 ⁇ 10 6 , or more engineered immune cells.
  • the mouse is administered one dose of engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • engineered immune cell e.g., T cell, B cell, or NK cell
  • the mouse is administered 1-2, 1-3,1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 doses (e.g., 2, 3, 4, 5, or more doses) of engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • the initial dose is divided into two or more smaller doses to mitigate the risk of side effects (e.g., instead of administering one initial dose, half the initial dose is administered twice).
  • the time between administrations can be, for example, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more.
  • the time between administrations is 1-2 days, 1-3 days, 1-4 days, 1-5 days, 1-6, days, 1-7 days, 1-8 days, 1-9 days, 1-10 days, 2-3 days, 2-4 days, 2-5 days, 2-6 days, 2-7 days, 2-8 days, 2-9 days, 2-10 days, 3-4 days, 3-5 days, 3-6 days, 3-7 days, 3-8 days, 3-9 days, 3-10 days, 4-5 days, 4-6 days, 4-7 days, 4-8 days, 4-9 days, 4-10 days, 5-6 days, 5-7 days, 5-8 days, 5-9 days, 5-10 days, 6-7 days, 6-8 days, 6-9 days, 6-10 days, 7-8 days, 7-9 days, 7-10 days, 8-9 days, 8-10 days, or 9-10 days.
  • the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy can be administered to PBMC humanized mice using any applicable route of administration.
  • routes of administration include, but not limited to, intravenous (e.g., via tail vein), subcutaneous, intrafemoral, intraventricular, intracardial, intraperitoneal routes of administration.
  • the route of administration is intravenous injection via tail vein.
  • the PBMCs and the immune cells for engineered immune cell (e.g., T cell, B cell, or NK cell) therapy are from the same subject (the two cell types are autologous). In other embodiments, the PBMCs and the immune cells are from different subjects (the two cell types are allogeneic). For example, the models described herein may be used to test a universal allogeneic engineered immune cell (e.g., T cell, B cell, or NK cell) therapy. In some embodiments, the PBMCs, the immune cells (e.g., T cells, B cells, NK cells), and the tumor cells are from the same subject.
  • the PBMCs, immune cells (e.g., T cells, B cells, NK cells), and tumor cells are from two or more subjects (e.g., the PBMCs and immune cells are from one subject and the tumor cells are from a different subject; the PBMCs and the tumor cells are from one subject and the immune cells are from a different subject; or the immune cells and the tumor cells are from one subject and the PBMCs are from a different subject).
  • a subject from which the PBMCs and/or immune cells (e.g., T cells, B cells, NK cells) are obtained is a human subject. Other mammals are contemplated herein.
  • a candidate agent effective for the treatment of CRS treatment may be administered (e.g., to prevent or reduce the effects of CRS).
  • the mouse models are used to determine whether a candidate CRS treatment will eliminate or reduce CRS in response to a specific engineered immune cell (e.g., T cell, B cell, or NK cell) therapy, as described herein.
  • the CRS treatment is administered simultaneously with the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • the CRS treatment is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or more after the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy has been administered.
  • the engineered immune cell e.g., T cell, B cell, or NK cell
  • the CRS treatment is administered 1-5 minutes, 1-10 minutes, 1-15 minutes, 1-20 minutes, 1-30 minutes, 1-45 minutes, 1-60 minutes, 2-5 minutes, 2-10 minutes, 2-15 minutes, 2-20 minutes, 2-30 minutes, 2-45 minutes, 2-60 minutes, 3-5 minutes, 3-10 minutes, 3-15 minutes, 3-20 minutes, 3-30 minutes, 3-45 minutes, 3-60 minutes, 4-5 minutes, 4-10 minutes, 4-15 minutes, 4-20 minutes, 4-30 minutes, 4-45 minutes, 4-60 minutes, 5-10 minutes, 5-20 minutes, 5-30 minutes, 5-45 minutes, 5-60 minutes, 10-20 minutes, 10-30 minutes, 10-40 minutes, 10-50 minutes, 10-60 minutes, 15-30 minutes, 15-45 minutes, 15-60 minutes, 30-45 minutes, 30-60 minutes, 1-2 hours, 1-3 hours, 1-4 hours, 1-5 hours, 1-6 hours, 1-7 hours, 1-8 hours, 1-9 hours, 1-10 hours, 1-11 hours, 1-12 hours, 2-3 hours, 2-4 hours, 2-5 hours, 2-6 hours, 2-7 hours, 2-8 hours, 2-9 hours,
  • the CRS treatment is administered prophylactically, such as 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, or 4 days before the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy is administered.
  • the engineered immune cell e.g., T cell, B cell, or NK cell
  • the CRS treatment is administered 1-5 minutes, 1-10 minutes, 1-15 minutes, 1- 20 minutes, 1-30 minutes, 1-45 minutes, 1-60 minutes, 2-5 minutes, 2-10 minutes, 2-15 minutes, 2-20 minutes, 2-30 minutes, 2-45 minutes, 2-60 minutes, 3-5 minutes, 3-10 minutes, 3-15 minutes, 3-20 minutes, 3-30 minutes, 3-45 minutes, 3-60 minutes, 4-5 minutes, 4-10 minutes, 4-15 minutes, 4-20 minutes, 4-30 minutes, 4-45 minutes, 4-60 minutes, 5-10 minutes, 5-20 minutes, 5-30 minutes, 5-45 minutes, 5-60 minutes, 10-20 minutes, 10-30 minutes, 10-40 minutes, 10-50 minutes, 10-60 minutes, 15-30 minutes, 15-45 minutes, 15-60 minutes, 30-45 minutes, 30-60 minutes, 1-2 hours, 1-3 hours, 1-4 hours, 1-5 hours, 1-6 hours, 1-7 hours, 1-8 hours, 1-9 hours, 1-10 hours, 1-11 hours, 1-12 hours, 2-3 hours, 2-4 hours, 2-5 hours, 2-6 hours, 2-7 hours, 2-8 hours, 2-9 hours,
  • the mouse is observed to assess the efficacy of the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • efficacy refers to the ability of the therapy administered to a subject to produce a therapeutic effect in the subject.
  • the therapy comprises engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • the therapy comprises engineered immune cell (e.g., T cell, B cell, or NK cell) therapy and anticytokine therapy.
  • the mouse models are used to determine whether a candidate CRS treatment will eliminate or reduce CRS in response to a specific engineered immune cell (e.g., T cell, B cell, or NK cell) therapy, as described herein.
  • a specific engineered immune cell e.g., T cell, B cell, or NK cell
  • the CRS treatment reduces the level of circulating cytokines following engineered immune cell (e.g., T cell, B cell, or NK cell) therapy in the mouse to the level of circulating cytokines present in the mouse prior to administration of the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy or to the level of circulating cytokines in a control mouse that did not receive the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • engineered immune cell e.g., T cell, B cell, or NK cell
  • the CRS treatment reduces the level of circulating cytokines following engineered immune cell (e.g., T cell, B cell, or NK cell) therapy in the mouse to a level that is lower than would be found in a mouse administered the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy without the CRS treatment.
  • the CRS treatment reduces the circulating cytokine level (e.g. , the cytokine level of one or more cytokines) 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.
  • the circulating cytokine level (e.g., one or more cytokines) in the mouse is reduced 10-20%, 10- 30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 20-30%, 20-40%, 20- 50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30- 80%, 30-90%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, 60-70%, 60-80%, 60-90%, 60-100%, 70-80%, 70-90%, 70-100%, 80-90%, 80-100%, or 90-100%.
  • the circulating cytokine level e.g., one or more cytokines
  • tumor growth can be monitored, for example, with in vivo bioluminescence imaging (BLI) as described herein, genomic studies, histology studies, or with any other method of measuring or approximating the volume of a tumor.
  • Efficacy may be determined by using the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, the 3-category method, the 4-response mRECIST criterion, and the 5-category method (Eisenhauer et al., Eur J Cancer, 2009, 45(2): 228-247; Bertotti et al., Nature.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • tumor volume includes, but are not limited to, progression-free survival, tumor volume doubling time, relative tumor volume (RTV), tumor growth inhibition (changes in tumor volume relative to initial tumor volume), and tumor growth rate.
  • progression-free survival is the length of time during and following treatment when the subject has the disease, but it does not get worse (e.g., the amount of time, during and after engineered immune cell (e.g., T cell, B cell, or NK cell) therapy, that the tumor does not grow).
  • engineered immune cell e.g., T cell, B cell, or NK cell
  • Tumor volume doubling time is the amount of time it takes the tumor volume to double (faster doubling times indicate a more malignant tumor) and typically determined from two volume estimations with measurement time intervals comparable with or shorter than DT.
  • Relative tumor volume is the relative in tumor volume over time and is calculated as: (absolute tumor volume on day X) x (100/absolute tumor volume on day 0).
  • Day 0 is the day the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy begins.
  • tumor growth inhibition which is expressed as a percentage, examines the changes in tumor volume relative to the initial tumor volume using the formula: (1 - (mean volume of treated tumors)/(mean volume of control tumors)) x 100%.
  • Tumor growth rate is estimated using a variety of different models. For an exponentially growing tumor, the growth rate is proportional to its volume: (l/V)x( ⁇ 7VZ7/), where V is the volume of the tumor, and dV and dl are the change in volume and time, respectively.
  • the tumor volume may be measured over time, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days after engraftment with the tumor cells.
  • the tumor volume is measured 1-2 days, 1-3 days, 1-4 days, 1-5 days, 1-6 days, 1-7 days, 1-8 days, 1-9 days, 1-10 days, 1-11 days, 1-12 days, 2-3 days, 2-4 days, 2-5 days, 2-6 days, 2-7 days, 2-8 days, 2-9 days, 2-10 days, 2-11 days, 2-12 days, 3-4 days, 3-5 days, 3-6 days, 3-7 days, 3-8 days, 3-9 days, 3-10 days, 3-11 days, 3-12 days, 4-5 days, 4-6 days, 4-7 days, 4-8 days, 4-9 days, 4-10 days, 4-11 days, 4-12 days, 5-6 days, 5-7 days, 5-8 days, 5-9 days, 5-10 days, 5-11 days, 5-12 days, 6-7 days, 6-8 days, 6-9 days, 6-10 days, 6-11 days,
  • the change in tumor volume is indicative of the efficacy of the human engineered immune cell (e.g., T cell, B cell, or NK cell) therapy (and, optionally, the CRS treatment).
  • the tumor volume in a mouse treated with human engineered immune cell (e.g., T cell, B cell, or NK cell) therapy may be compared to the tumor volume in a mouse that was not treated with the human engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • the tumor volume is reduced 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more relative to a mouse that did not receive the human engineered immune cell (e.g., T cell, B cell, or NK cell) therapy or relative to an earlier time point.
  • human engineered immune cell e.g., T cell, B cell, or NK cell
  • the tumor volume is reduced 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10- 70%, 10-80%, 10-90%, 10-100%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20- 90%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, 60-70%, 60-80%, 60-90%, 60-100%, 70-80%, 70-90%, 70-100%, 80-90%, 80-100%, or 90- 100% relative to a mouse that did not receive the human engineered immune cell (e.g., T cell, B cell, or NK cell) therapy or relative to an earlier time point.
  • human engineered immune cell e.g., T cell, B cell, or NK cell
  • CRS induction may also be monitored through body weight measurement, as acute toxicity relates to significant mouse body weight loss. Further, clinical observations may be indicative of CRS. Examples of clinical observations relevant to CRS include: a hunched posture with tiptoe/abnormal gait, reduced activity (e.g., not moving unless being stimulated), and/or non-responsiveness to touch. Survival rate (and duration) may also be used to evaluate the efficacy of a human engineered immune cell (e.g., T cell, B cell, or NK cell) therapy (and/or anti-CRS treatment).
  • a human engineered immune cell e.g., T cell, B cell, or NK cell
  • Efficacy may also be evaluated by examining cytokine-induced liver and kidney damage. This may be determined, for example, by a serum biochemical analysis of liverkidney function, such as measuring levels of aspartate transaminase (AST), albumin, total bilirubin, creatinine and blood urea nitrogen.
  • AST aspartate transaminase
  • the change in liver weight of preclinical mouse model is indicative of the efficacy of the human engineered immune cell (e.g., T cell, B cell, or NK cell) therapy (and, optionally, the CRS treatment).
  • a healthy mouse’s liver weight is approximately 5% of its body weight. Injection of tumor cells (e.g., Raji_Luc cells) leads to dissemination of the tumor cells to the liver, increasing the liver weight.
  • engineered immune cell e.g., T cell, B cell, or NK cell
  • anti-CRS treatment one may determine whether the treatment effectively eliminated or reduced tumor cell accumulation in the liver.
  • the methods described herein may be used to assess the possible side effects of engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • Possible side effects include but are not limited to cytokine release syndrome (CRS), macrophage activation syndrome (MAS), neurotoxicity (encephalopathy syndrome), tumor lysis syndrome (TLS), anaphylaxis, on-target, off-tumor toxicity, and B cell aplasia.
  • certain cytokines can be measured in a blood sample from the PBMC humanized mouse model following administration of the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy and/or CRS treatment.
  • the cytokine may be selected from the group consisting of IFN-y, IL-10, IL-6, IL-2, IL-4, and TNFa.
  • the level of cytokine measured is indicative of the severity of immunotoxicity of the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy.
  • the method further comprises determining that the severity of immunotoxicity of the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy is high (e.g., the likelihood of CRS induction is high) when: an IFN-y level in the mouse is > 1,800 pg/ml ⁇ 10%; an IL- 10 level in the mouse is > 120 pg/ml ⁇ 10%; an IL-6 level in the mouse is > 25 pg/ml ⁇ 10%; an IL-2 level in the mouse is > 80 pg/ml ⁇ 10%; an IL-4 level in the mouse is > 120 pg/ml ⁇ 10%; TNFa level in the mouse is > 120 pg/ml ⁇ 10%; MCP-1 level in the mouse is > 120 pg/ml ⁇ 10%; GM-CSF level in the mouse is > 600 pg/ml ⁇ 10%; and IL8 level in the mouse is > 15 pg/m
  • Macrophage activation syndrome or hemophagocytic lymphohistiocytosis (HLH), which clinically manifests as liver dysfunction, increased ferritin levels and, in some cases, decreased fibrinogen levels, may also be examined using the mouse models described herein. Macrophages mediate the major production of cytokines including IL-6, IL-1, and IFN- y, and their activation (MAS) is thought to play a role in CRS (Hao et al., Experimental Hematology & Oncology, 2020, 9:15). Therefore, elevated levels of the three cytokines in the mouse model may indicate that that the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy may result in MAS.
  • the engineered immune cell e.g., T cell, B cell, or NK cell
  • the mouse may be administered a MAS treatment to determine whether the treatment would prevent or reduce MAS in the human subject.
  • MAS treatments include but are not limited to, glucocorticoids (e.g., methylprednisone, dexamethasone), cyclosporin A, etoposide, immunoglobulins, and cyclophosphamide.
  • Neurotoxicity can develop approximately 5-17 days after engineered immune cell (e.g., T cell, B cell, or NK cell) therapy in humans (Herlopian et al., Neurology, 2018, 91(5): 227-229). It is characterized by global encephalopathy, aphasia, seizure/seizure- like activity, obtundation, tremor/myoclonus, and hallucinations. Subjects with neurotoxicity also have high levels of IFN- y, IL-6, and TNF-a.
  • Elevated levels of the three cytokines in the mouse model may indicate that the engineered immune cell (e.g., T cell, B cell, or NK cell) therapy may result in neurotoxicity. Therefore, in some embodiments, the mouse may be administered a neurotoxicity treatment to determine whether the treatment would prevent or reduce neurotoxicity in the human subject.
  • neurotoxicity treatments include but are not limited to corticosteroids (e.g., dexamethasone, prednisone), anti-IL-6 antibodies (e.g., siltuximab), and platelet hypertransfusion.
  • a method comprising: engrafting an immunodeficient mouse with tumor cells; engrafting the mouse with human peripheral blood mononuclear cells (PBMCs); administering to the mouse human immune cells engineered to express a chimeric antigen receptor (CAR) that specifically targets a cell surface antigen on tumor cells (human CAR immune cells); and assaying the mouse for induction of cytokine release syndrome (CRS) and/or efficacy of the human CAR immune cells for treating the tumor cells.
  • PBMCs peripheral blood mononuclear cells
  • CAR chimeric antigen receptor
  • CRS cytokine release syndrome
  • tumor cells are from a tumor cell line or a patient-derived xenograft (PDX).
  • PDX patient-derived xenograft
  • mice is administered the CAR immune cell (e.g., T cell, B cell, NK cell) therapy 6-12 days after tumor cell engraftment.
  • the CAR immune cell e.g., T cell, B cell, NK cell
  • the candidate agent is selected from the group consisting of: IL-6 antagonists, anti-IL-6 antibodies, corticosteroids, anti- TNF-a drugs, IL-1R inhibitors, GM-CSF inhibitors, and small molecule inhibitors.
  • mice are a NOD.Cg-Prkdcscid H2- KltmlBpe H2-AblemlMvw H2-DltmlBpe I12rgtmlWjl/SzJ mouse (NSG-(Kb Db)null (lAnull) mouse).
  • assaying the mouse for induction of CRS comprises measuring a circulating level of a cytokine selected from the group consisting of: interleukin (IL)-6, IL10, interferon (IFN)-y, monocyte chemoattractant protein 1 (MCP-1), granulocyte-macrophage colony- stimulating factor (GM-CSF), tumor necrosis factor (TNF), IL-1, IL-2, IL-2-receptor alpha (IL-2Ra), IL-8, IL-4, IL- 18, and macrophage inflammatory protein (MIP) 4.
  • a cytokine selected from the group consisting of: interleukin (IL)-6, IL10, interferon (IFN)-y, monocyte chemoattractant protein 1 (MCP-1), granulocyte-macrophage colony- stimulating factor (GM-CSF), tumor necrosis factor (TNF), IL-1, IL-2, IL-2-receptor alpha (IL-2Ra
  • serum biochemical analysis comprises measuring the levels of at least one of the following markers: aspartate transaminase (AST), albumin, total bilirubin, creatinine, or blood urea nitrogen.
  • AST aspartate transaminase
  • the method of paragraph 43 comprising determining that the candidate agent effective for treatment of CRS does reduce the level of one or more circulating cytokines when the circulating level of the one or more cytokines is reduced 30-100% in a mouse administered the human CAR immune cell (e.g., T cell, B cell, NK cell) therapy and the candidate agent, relative to a mouse administered the human CAR immune cell (e.g., T cell, B cell, NK cell) therapy without the candidate agent.
  • the human CAR immune cell e.g., T cell, B cell, NK cell
  • a method comprising: irradiating an immunodeficient mouse; engrafting the mouse with tumor cells; engrafting the mouse with human peripheral blood mononuclear cells (PBMCs); administering to the mouse universal allogeneic human immune cells engineered to express a chimeric antigen receptor (CAR) that specifically targets a cell surface antigen on tumor cells (universal allogeneic human CAR immune cells); and assaying the mouse for induction of cytokine release syndrome (CRS) and/or efficacy of the universal allogeneic human CAR immune cells for treating the tumor cells.
  • CRS cytokine release syndrome
  • human immune cells comprise at least two of the following: T cells, natural killer cells, B cells, monocytes, dendritic cells, and neutrophils.
  • CD19 CAR T and the PBMCs were derived from the same donor. Mice sera were collected were at different days (D1, D2, D3) following CAR T/PBMC treatment. Circulating cytokine concentrations were measured by the BD CBA Th1/Th2 II kit. CD19 CAR T batch 1 and PBMC were from the same donor. There were 4 mice per group and the data are presented as mean ⁇ SEM. PBMC/CAR T co-treated mice had increased human IFN- ⁇ , TNF and IL6 compared to mice receiving CD19 CAR T alone (FIG.4). This data demonstrated that CAR T cell therapy induced higher levels of human cytokines in the PBMC humanized mice compared to the control mice that did not receive PBMCs.
  • mice were bled 2 days after CAR T/Mock treatment and circulating cytokine concentrations were measured by the BD CBA Th1/Th2 II kit. Daily body weight and clinical observation were performed after CAR T treatment.
  • in vivo Bioluminescence Imaging (BLI) was plotted using average radiance to quantitatively measure tumor burden.
  • the bottom portion of FIG. 5A provides dorsal (D) and ventral (V) images for each mouse. There were 4-5 mice per group and data are presented as mean ⁇ SEM. CAR T efficacy was determined over that seen with the PBS or mock CAR T treatment.
  • the data demonstrates that one may predict toxicity on an individual, in some instances, prior to the treatment so that the subject can be pretreated with a drug to reduce cytokine induction.
  • Example 6 The PBMC humanized DKO mice model was used to examine efficacy and cytokine release. PBMC humanized mice or control (no PBMC) mice were treated IV with PBS or 5 x10 6 CD19 CAR T cells or 5 x10 6 Mock T cells 6 days after Raji_Luc (0.25x10 6 per mouse). The CD19 CAR T cells and PBMCs were derived from the same donor. Sera were collected 2 days after CAR T/PBMC treatment and cytokine level (D8) were analyzed by the BD CBA Th1/Th2 II kit.
  • FIG.6A shows in vivo Bioluminescence Imaging (BLI) plotted using average radiance to quantitatively measure tumor burden.
  • CAR T efficacy was seen with and without PBMC humanization but a complete response was evident with PBMC humanization at day 12.
  • FIG.6C The bioluminescence images of these mice at different experiment days are shown in FIG.6C. All mice were imaged with identical camera settings. Dorsal (D) and ventral (V) images are shown for each.
  • mice were treated intravenously beginning on day 8 with PBS or 15 x10 6 PBMC or 1 x10 6 CD19 CAR T plus 15 x10 6 PBMC, 3 x10 6 CD19 CAR T plus 15 x10 6 PBMC or 5 x10 6 CD19 CAR T plus 15 x10 6 PBMC.
  • CD19 CAR T and the PBMCs were derived from the same donor.
  • Mice sera were collected 2 days following CAR T/PBMC treatment. Circulating cytokine concentrations were measured by the BD CBA Th1/Th2 II kit. There were 5 mice per group and the data are presented as mean ⁇ SEM in FIGs.7A-7B.
  • PBMC/CAR T co-treated mice were found to have dose-dependent increased levels of human IFN- ⁇ (FIG.7B).
  • the high CART dose had improved efficacy (tumor burden) compared to lower CART dose treatments (FIG.7A, left graph), while both CART doses were observed to rescue mice body weight loss due to Raji_Luc tumor growth. (FIG.7A, right graph).
  • This data demonstrated that CAR T cell therapy induced higher levels of human cytokines in the PBMC humanized mice in a dose-dependent manner.
  • Example 8 The effects of CAR T cell therapy on human cytokines levels in PBMC humanized mice compared to the control mice that did not receive PBMCs following autologous PBMC/CD19 CAR T treatment were examined.
  • CD19 CART cells were generated using a new CD19 CAR construct and a new PBMC donor (donor 9534).
  • DKO NSGTM mice were engrafted with an intravenous injection of 0.25x10 6 /mouse after irradiation (D0).
  • Raji_Luc tumor growth was monitored by in vivo Bioluminescence Imaging (BLI).
  • Mice were treated intravenously beginning on day 8 with PBS or 15 x10 6 PBMC or 5x10 6 CD19 CAR T alone, or 5 x 10 6 CD19 CAR T plus 15 x 10 6 PBMC.
  • CD19 CAR T and the PBMCs were derived from the same donor. Mice sera were collected 2 days following CAR T/PBMC treatment.
  • Circulating cytokine concentrations were measured by the BD CBA Th1/Th2 II kit. There were 5 mice per group and the data are presented as mean ⁇ SEM. PBMC/CAR T co-treated mice and CART alone treated mice had decreased tumor burden (FIG.8A) and did not lose significant body weight (FIG.8B). Moreover, PBMC/CART co-treated mice had high levels of human IFN and TNF release compared to mice treated with CART alone (FIG.8C).
  • Example 9 The efficacy of allogeneic CD19 CART treatment from different PBMC humanized mice with Raji_Luc tumor was examined. Allogeneic CD19 CART cells were generated using a modified CD19 CAR construct and a new PBMC donor.
  • DKO NSGTM mice were engrafted with an intravenous injection of 0.25x10 6 /mouse after irradiation (D0).
  • Raji_Luc tumor growth was monitored by in vivo Bioluminescence Imaging (BLI).
  • Mice were treated intravenously beginning on day 8 with PBS or 15 x10 6 PBMC or 5x10 6 CD19 CAR T alone, or 5 x10 6 CD19 CAR T plus 15 x10 6 PBMC.
  • Two different donors’ PBMCs PBMC 8058 or PBMC 9601 were used for the humanization.
  • Allogeneic CART showed some level of efficacy through tumor burden imaging (FIG.9A) and flow analysis CD3-CD19+ cell population (FIG.9B).
  • Allogeneic CD19 CART treatment blocked body weight loss induced by Raji-Luc tumor development (FIG.9C).
  • Circulating cytokine concentrations were measured by the BD CBA Thl/Th2 II kit.
  • Six days after CART treatment whole blood and spleen were collected from all mice for flow analysis of the CD3-CD19+ population.
  • Both autologous CD19 CART samples showed good efficacy and decreased the CD3-CD19+ population compared to the corresponding PBS control and Mock T treated mice (FIG. 11B).
  • the PBMC 9534-humanized mice had significant body weight loss (FIG. 11C) and significantly higher levels of IL- 10 and IL-4 compared to the PBMC 9531 -humanized mice after autologous CART treatment (FIG. 11D), demonstrating a donor- specific response.
  • All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024029561A1 (ja) * 2022-08-02 2024-02-08 学校法人東海大学 担がん－免疫不全非ヒト動物、これを用いた供試物質に関するがん免疫応答評価方法
CN117686676A (zh) * 2023-12-15 2024-03-12 扬州大学 一种基于多水平生物标志物响应法评价水中污染物转代生物效应的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2937797T3 (es) 2017-04-17 2023-03-31 Jackson Lab Método de determinación de la toxicidad de un fármaco inmunomodulador para el uso en seres humanos

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130316326A1 (en) * 2010-08-27 2013-11-28 Technologie Integrale Ltd. Animal model for the evaluation of the efficacy of an hiv vaccine
US20180187210A1 (en) * 2015-06-23 2018-07-05 The Jackson Laboratory Non-hla matched humanized nsg mouse model with patient-derived xenograft
WO2019141251A1 (en) * 2018-01-19 2019-07-25 Beijing Biocytogen Co., Ltd Immunodeficient non-human animal

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9100481D0 (en) 1991-01-10 1991-02-20 Inst Nat Sante Rech Med Genetically engineered mice
US7105158B1 (en) 1992-11-16 2006-09-12 The Corporation Of Mercer University Methods of administering microencapsulated materials for immune modulated diseases
FR2827302B1 (fr) 2001-07-13 2003-10-10 Genoway Cellule et animal transgenique modelisant la presentation antigenique humaine et leurs utilisations
US20060116321A1 (en) 2004-07-01 2006-06-01 Robbins Paul D Immunosuppressive exosomes
JP2007244268A (ja) 2006-03-15 2007-09-27 Osaka Prefecture Univ イヌの造血前駆細胞を認識するモノクローナル抗体の製造方法
EP1878342A1 (en) 2006-07-13 2008-01-16 Institut Pasteur Immunodeficient mice transgenic for HLA class I and HLA class II molecules and their uses
US20080299091A1 (en) 2007-04-06 2008-12-04 International Stem Cell Corporation Patient-specific stem cell lines derived from human parthenogenetic blastocysts
US20110082091A1 (en) 2009-09-28 2011-04-07 Theramab Gmbh Method for Preclinical Testing of Immunomodulatory Drugs
EP4502152A3 (en) 2009-10-06 2025-04-30 Regeneron Pharmaceuticals, Inc. Genetically modified mice and engraftment
EP2558576B1 (en) 2010-04-14 2016-10-19 Envigo RMS, Inc. Hairless nod scid mouse
KR20140004174A (ko) 2011-01-18 2014-01-10 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 암 치료를 위한 조성물 및 방법
SG10202008003VA (en) 2011-02-15 2020-10-29 Regeneron Pharma Humanized m-csf mice
CN104160272A (zh) 2011-12-06 2014-11-19 麻省理工学院 利用人源化的小鼠来确定毒性
CN103304669A (zh) 2012-03-12 2013-09-18 复旦大学 人源化肿瘤免疫细胞因子tntil2及其制备方法和用途
NZ630363A (en) 2012-07-25 2018-09-28 Celldex Therapeutics Inc Anti-kit antibodies and uses thereof
MX377561B (es) 2012-11-05 2025-03-10 Regeneron Pharma Animales no humanos genéticamente modificados y métodos de uso de los mismos.
ES2664352T3 (es) 2013-03-22 2018-04-19 Imperial Innovations Ltd Método in vitro para evaluar respuestas de tormenta de citoquinas
WO2017040930A2 (en) 2015-09-03 2017-03-09 The Trustees Of The University Of Pennsylvania Biomarkers predictive of cytokine release syndrome
HUE066460T2 (hu) * 2016-10-26 2024-08-28 Iovance Biotherapeutics Inc A kriokonzervált tumor infiltráló limfociták restimulációja
CN110740641A (zh) 2016-11-30 2020-01-31 杰克逊实验室 具有改善的人先天免疫细胞发育的人源化小鼠模型
ES2937797T3 (es) 2017-04-17 2023-03-31 Jackson Lab Método de determinación de la toxicidad de un fármaco inmunomodulador para el uso en seres humanos
RU2019140867A (ru) * 2017-05-12 2021-06-15 Зе Джексон Лаборатори Nsg мыши, не имеющие mhc класса i и класса ii
US20190091310A1 (en) * 2017-09-26 2019-03-28 Stephen E. Wright Nonreleased il-12 for therapy of cancer
US11712026B2 (en) 2017-10-18 2023-08-01 The Jackson Laboratory Murine-MHC-deficient HLA-transgenic nod-mouse models for T1D therapy development
US12496338B2 (en) * 2017-10-23 2025-12-16 City Of Hope CAR for treatment of HIV infection
JP7411578B2 (ja) * 2018-06-01 2024-01-11 マヨ ファウンデーション フォー メディカル エデュケーション アンド リサーチ 癌を治療するための物質及び方法
US20220389092A1 (en) * 2019-04-05 2022-12-08 The Regents Of The University Of California Methods and compositions involving chimeric binding polypeptides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130316326A1 (en) * 2010-08-27 2013-11-28 Technologie Integrale Ltd. Animal model for the evaluation of the efficacy of an hiv vaccine
US20180187210A1 (en) * 2015-06-23 2018-07-05 The Jackson Laboratory Non-hla matched humanized nsg mouse model with patient-derived xenograft
WO2019141251A1 (en) * 2018-01-19 2019-07-25 Beijing Biocytogen Co., Ltd Immunodeficient non-human animal

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4216971A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024029561A1 (ja) * 2022-08-02 2024-02-08 学校法人東海大学 担がん－免疫不全非ヒト動物、これを用いた供試物質に関するがん免疫応答評価方法
CN117686676A (zh) * 2023-12-15 2024-03-12 扬州大学 一种基于多水平生物标志物响应法评价水中污染物转代生物效应的方法

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