[go: up one dir, main page]

WO2025096730A1 - Nouveaux lymphocytes t car autorégulés et leurs utilisations - Google Patents

Nouveaux lymphocytes t car autorégulés et leurs utilisations Download PDF

Info

Publication number
WO2025096730A1
WO2025096730A1 PCT/US2024/053822 US2024053822W WO2025096730A1 WO 2025096730 A1 WO2025096730 A1 WO 2025096730A1 US 2024053822 W US2024053822 W US 2024053822W WO 2025096730 A1 WO2025096730 A1 WO 2025096730A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
car
cir
cells
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/053822
Other languages
English (en)
Inventor
Daniel W. Lee
Dustin A. Cobb
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UVA Licensing and Ventures Group
Original Assignee
University of Virginia Patent Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Virginia Patent Foundation filed Critical University of Virginia Patent Foundation
Publication of WO2025096730A1 publication Critical patent/WO2025096730A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/36—Immune checkpoint inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/23—On/off switch
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • This disclosure relates to engineered autoregulated chimeric antigen receptor (CAR) T cells and uses thereof.
  • Chimeric antigen receptor (CAR)-T cell therapies targeting CD 19 or CD22 have been highly successful at salvaging children and adults with multiply relapsed or refractory acute lymphoblastic leukemia (r/rALL) and adults with B-cell lymphomas.
  • B-cell maturation antigen (BCMA) targeted CAR-T cell therapies have been successfully used in adults with multiple myeloma. However, they have significant toxicities for which few solutions exist.
  • Cytokine release syndrome (CRS), immune effector cell associated neurotoxicity syndrome (ICANS) and hemophagocytic lymphohistiocytosis/macrophage activation-like syndrome (HLH/MAS) can be life threatening and difficult to manage even with tocilizumab (anti-IL-6R antibody) and corticosteroids.
  • CRS Cytokine release syndrome
  • ICANS immune effector cell associated neurotoxicity syndrome
  • HHLH/MAS hemophagocytic lymphohistiocytosis/macrophage activation-like syndrome
  • tocilizumab anti-IL-6R antibody
  • corticosteroids for r/rALL patients, as many as 77% develop Grade 3-5 CRS and 40% Grade 3-5 ICANS.
  • the underlying cause of these toxicities is the exponential and uncontrollable nature of CAR-T cell expansion and activation leading to an as of yet mechanistically poorly characterized supra-physiologic and generalized immune system activation. None of the FDA-approved CAR-T
  • Severe CRS occurs when hyperactivated CAR-T cells secrete copious inflammatory cytokines that hyperactivate the rest of the immune system. Left unchecked, sCRS can cause multi-organ failure and even death or morph into carHLH/MAS, which is often refractory to aggressive interventions. Many times, sCRS and carHLH/MAS do not respond to multiple doses of tocilizumab, the only FDA approved drug for sCRS, or high-dose steroids suggesting additional undiscovered mechanisms of toxicity exist. The pathophysiology of ICANS is also poorly understood but is related to uncontrolled CAR-T cell activity. There is presently no proven therapy for mitigating or treating ICANS, though steroids are often used.
  • T cell receptor (TCR) engagement by its cognate peptide-MHC complex in the context of co-stimulatory signals results in productive activation.
  • immune checkpoint receptors such as PD-1 and TIGIT (T cell immunoreceptor with Ig and ITIM domains) are upregulated to control the T cell response and maintain immune homeostasis.
  • CAR-T cell engagement at least in B cell malignancies, results in a supraphysiologic and uncontrolled activation of the T cells and many other components of innate immunity such as macrophages and monocytes.
  • CAR constructs lack regulatory mechanisms to counterbalance CAR activation.
  • This disclosure relates to novel chimeric inhibitory receptors (CIRs) for attenuating and mitigating systemic toxicity in a subject, and methods of making and using thereof.
  • CIRs chimeric inhibitory receptors
  • a mammalian cell comprising a nucleic acid encoding a CIR domain, the CIR domain comprising a ligand-sensing polypeptide able to sense, recognize, or interact with, an extracellular ligand, operably linked to an inhibitory signaling molecule, such that binding of the ligand to the ligand- sensing polypeptide delivers inhibitory signaling to automatically regulate and titrate ligand expression or production.
  • a nucleic acid sequence encoding a CIR domain the nucleic acid identified in any one of SEQ ID No. 1-6, or a nucleic acid with at least 75% homology to said sequence.
  • an amino acid sequence encoding a CIR domain the amino acid sequence identified in any one of SEQ ID NO: 7-12, or an amino acid with at least 75% identity to said sequence.
  • a mammalian cell comprising a nucleic acid encoding a CIR domain of the invention and a chimeric antigen receptor (CAR) domain, a CIR/CAR.
  • the mammalian cell is a T-cell transduced with a nucleic acid encoding a CIR/CAR of the invention, a CIR/CAR T-cell.
  • an amino acid sequence encoding a CIR/CAR, the amino acid identified in any of SEQ ID No. 25-33, or an amino acid with at least 75% identity to said sequence is also considered to be identical to said sequence.
  • a method for producing a CIR/CAR T-cell comprising transducing a T cell with a nucleic acid encoding a CIR/CAR (or CAR/CIR) described herein.
  • a method for controlling CAR T cell activation and cytokine production comprising transducing a mammalian cell or an immune effector cell with a nucleic acid encoding CIR/CAR described herein.
  • composition comprising a mammalian cell described herein and a pharmaceutically acceptable carrier.
  • a vector comprising one or more of the nucleic acids as described herein.
  • a cell comprising the vector as described herein.
  • the cell is an immune effector cell.
  • a cell comprising one or more of the nucleic acids as described herein. In another aspect, a cell comprising two of the nucleic acids as described herein.
  • a method of treating or reducing cytokine toxicity in a subject receiving CAR T-cell therapy comprising administering a therapeutically effective amount of a composition comprising a mammalian cell comprising a nucleic acid encoding a CIR domain described herein to the subject.
  • the mammalian cell comprises a nucleic acid encoding both a CIR domain and a CAR domain, or a CIR/CAR (or CAR/CIR) nucleic acid.
  • Fig. 1 shows proposed model of chimeric inhibitory receptor (CIR) -mediated attenuation of CD19 CAR activity.
  • CIR chimeric inhibitory receptor
  • CIRs Upon binding to a specific pro-inflammatory cytokine or chemokine in the extracellular environment, CIRs deliver an intracellular inhibitory signal to counter activating signals of the CAR and deliver inhibitory signaling to automatically regulate and titrate CAR-T cell activity and the resultant global immune hyperactivation, which together mitigate severe CAR-T cell toxicities in a “hands-free” approach.
  • FIG. 2 shows a schematic representative model of a chimeric inhibitory receptor (CIR) composed of an anti-IFN-gamma binding scFv coupled to the intracellular inhibitory signaling domain of the TIGIT receptor.
  • CIR chimeric inhibitory receptor
  • the schematic shows interchangeability of extracellular cytokine-binding domain and intracellular inhibitory signaling domain.
  • FIG. 3 shows construct design of CD19.28z CAR-T cell co-expressing a representative chimeric inhibitory receptor (CIR).
  • CIR chimeric inhibitory receptor
  • scFv single-chain variable fragment
  • T2A “self-cleaving” peptide sequence
  • EGFRt truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs
  • GM-CSFR granulocyte-monocyte-colony stimulating factor receptor
  • 32M Beta 2 microglobulin
  • LTR long terminal repeat.
  • Figs. 4A and 4B show normal expansion and proliferation of CD19.28z CAR- T cells co-expressing CIR domain during in vitro manufacture.
  • 4A CAR-T cells were enumerated at indicated timepoints post-activation during in vitro expansion following retroviral transduction of CAR construct.
  • 4B Fold expansion of CAR-T cells relative to Day 7 postactivation during in vitro production. Results shown are representative of at least six individual experiments using T cells from 3 different normal human donors.
  • Figs. 5A and 5B show CD19.28z.CIR CAR-T cells exhibit attenuated activation following exposure to NALM-6 CD19 + tumor cells.
  • CD19.28z CAR-T cells, CD19.28z.CIR CAR-T cells, or non-transduced (NT) T cells were cultured with (dark-shaded histograms) or without (light- shaded histograms) NALM-6 for 24 hours then CAR-T cell activation was evaluated by staining for CD3, CD4, CD8, CD69, CD25, CD38, PD-1, CD107a and analyzed by flow cytometry. Representative flow plots for expression of T cell activation markers CD69 and CD38 are shown for CD4 + and CD8 + CAR-T cells.
  • Bar graphs represent the percentage of CAR-T cells expressing the indicated number of activation markers per cell.
  • Fig. 6 shows in vitro cytotoxicity of CD19.28z CAR-T cells expressing CIR receptor against CD19 + leukemia cell line NALM-6.
  • Nontransduced T cells were co-cultured overnight with NALM-6 tumor cell line expressing firefly luciferase, at various effector to target (E:T) cell ratios.
  • Anti-tumor cytotoxicity was then evaluated by measuring bioluminescent activity of remaining tumor cells following addition of luciferin. Culture plates were subsequently read in a Lago X instrument (Spectral Instruments Imaging).
  • percent cytotoxicity 100 - (((average signal of CAR-T cell-treated wells) I (average signal from untreated tumor cell only wells)) x 100).
  • the graphs above are representative individual experiments performed with CARs generated from multiple donors.
  • Figs. 7A - 7C show CD19.28z.CIR/CAR-T cells exhibit attenuated cytotoxicity kinetics which is dependent upon CIR-mediated binding to IFN-gamma.
  • NALM-6 were co-incubated with NT, CD19.28z CAR, or CD19.28z CAR/CIR-T cells. Sequential NALM-6 stimulations were carried out as denoted by adding fresh target cells.
  • IFN-gamma binding by CIR scFv domain is required for attenuated cytotoxic activity during in vitro stimulation by tumor cells.
  • CD19.28z.CIR CAR-T cells require IFN-y-scFv binding domain and are responsive to IFN-y.
  • NALM-6 cells were co-incubated with CD19.28z CAR, CD19.28z.CIR, or CD19.28z.CIR (scFv-less, cannot bind IFN-y) T cells.
  • CD19.28z.CIR (scFv-less) T cells mimic the anti-tumor killing of control CD19.28z CAR-T cells, demonstrating the necessity for IFN-y recognition to drive TIGIT signaling.
  • Figs. 8A and 8B show CIR signaling in CD19.28z CAR-T cells results in attenuated production of cytokines and chemokines.
  • 8A Attenuated pro-inflammatory cytokine production by CD19.28z.CIR CAR-T cells relative to CD19.28z CAR-T cells following stimulation by tumor cells.
  • Fig. 9 shows overall polyfunctionality of CD19.28z CAR-T cells co-expressing CIR is not compromised by CIR signaling.
  • CD19.28z CAR-T cells or CD19.28z.CIR CAR-T cells were cultured in the presence or absence of NALM-6 cells for 22 hrs followed by singlecell secretome analysis using the Isolight® platform (Bruker ⁇ Cellular Analysis).
  • FIGs. 10A - 10C show CD19.28z CAR-T cells attenuated by CIR signaling results in diminished pro-inflammatory cytokine/chemokine secretion by bystander autologous macrophages and DCs.
  • 10A Schematic diagram of an in vitro co-culture assay consisting of NALM-6, CD19.28z, or CD19.28z.CIR CAR-T cells, and either dendritic cells (DC) or macrophages (Mac) for the production of CRS-associated cytokines and chemokines.
  • DC dendritic cells
  • Mac macrophages
  • Fig. 11 shows Nanostring® nCounter gene expression profiling demonstrates significant regulation of CAR-T cell pathways by the presence of CIR signaling.
  • CD19.28z CAR-T cells or CD19.28z.CIR CAR-T cells were cultured in the presence or absence of NALM- 6 cells for 24 hrs followed by total cell lysis and RNA extraction.
  • RNA was then subjected to Nanostring® nCounter analysis using the CAR-T Characterization Panel, a 780-plex gene expression panel (Nanostring® Technologies). Standard quality control procedures and data normalization were performed using the ROSALIND online platform.
  • RNA Samples from each condition were analyzed in triplicate.
  • Figs. 12A-12E show CIR-regulated CD19.28z CAR-T cells exhibit similar anti-tumor activity and survival in vivo relative to CD19.28z controls.
  • 12A On day -2, NOD.Cg-Prkdc sc ’ d I12rg tmlw j
  • BBI Bioluminescent imaging
  • 12C Bioluminescent tumor growth curves. Statistical significance was calculated using Two-way ANOVA with Tukey’s multiple comparisons test. ****p ⁇ 0.0001.
  • 12D Kaplan- Meier survival curve analysis.
  • Figs. 13A-13E show CIR-mediated attenuation of CD19 CAR-T cell responses is observed in vivo in tumor-bearing mice.
  • 13A On day -2, NSG mice were injected with 0.5xl0 6 NALM-6. Two days later, mice were treated i.v. with CD19.28z or CD19.28z.CIR CAR-T cells (10xl0 6 ).
  • 13B Bioluminescent tumor growth curves.
  • Fig. 14 shows Chimeric Inhibitory Receptors (CIRs) can be applied to any CAR product, such as CD19.BBz CAR-T cells.
  • CD19.BBz.CIR anti- IFN-y/TIGIT
  • scFv single-chain variable fragment
  • T2A “selfcleaving” peptide sequence
  • EGFRt truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs
  • GM-CSFR granulocyte-monocyte-colony stimulating factor receptor
  • 32M Beta-2 microglobulin
  • LTR long terminal repeat.
  • Figs. 15A and 15B show CD19.BBz CAR/CIR-T cells exhibit attenuated activation following exposure to NALM-6 CD19 + tumor cells.
  • 15A CD19.BBz CAR-T cells, CD19.BBz.CIR CAR-T cells, or non-transduced (NT) T cells were cultured with (dotted line histograms) or without (full line histograms) NALM-6 for 24 hours then CAR-T cell activation was evaluated by staining for CD3, CD4, CD8, CD69, PD-1, and CD25 and analyzed by flow cytometry.
  • Fig. 16 shows in vitro cytotoxicity of CD19.4-lBBz CAR T cells expressing CIR receptor against leukemia cell line NALM-6 demonstrates similar leukemia control.
  • CD19.BBz CAR T cells, CD19.BBz.CIR CAR T cells, or control Nontransduced T cells were co-cultured overnight with NALM-6 tumor cell line expressing firefly luciferase, at various effector to target (E:T) cell ratios.
  • Anti-tumor cytotoxicity was then evaluated by measuring bioluminescent activity of remaining tumor cells following addition of luciferin. Culture plates were subsequently read in a Lago X instrument (Spectral Instruments Imaging).
  • percent cytotoxicity 100 - (((average signal of CAR T cell-treated wells) I (average signal from untreated tumor cell only wells)) x 100).
  • the graphs above are representative individual experiments performed with CARs generated from multiple donors.
  • FIGs. 17A and 17B show diminished pro-inflammatory cytokine/chemokine secretion by bystander autologous macrophages and DCs during in vitro CRS-assay.
  • 17A Schematic diagram of an in vitro co-culture assay consisting of NALM-6, CD19.BBz, or CD19.BBz.CIR CAR-T cells, and macrophages (Mac) for the production of CRS- associated cytokines/chemokines.
  • CD19.BBz CAR-T cells attenuated by CIR signaling results in decreased release of innate pro-inflammatory CRS-associated cytokines/chemokines by monocyte-derived macrophages (MoMac). All groups shown include NALM-6 tumor cells. Cocultures were incubated for 24 Hrs. and the supernatants were collected for analysis by Luminex®. Error bars represent standard deviation. The above data are representative of two individual experiments.
  • Figs. 18A-18D show self-regulating CD19.BBz CAR-T cells expressing anti- IFN-y/TIGIT CIR exhibit similar anti-tumor activity and survival but improved persistence in vivo.
  • 18A On day -2, NOD.Cg-Prkdc sc ‘ d H2rg tmlw
  • Fig. 19 shows generic CIR constructs for “sensing” various inflammatory cytokines/chemokines coupled to various inhibitory signaling domains.
  • CD 19 CAR-T cells could also express CIRs engineered to bind additional extracellular cytokines/chemokines and incorporate different inhibitory signaling domains for attenuating CAR-T activation and mitigating toxicity.
  • scFv single-chain variable fragment
  • T2A “self-cleaving” peptide sequence
  • EGFRt truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs
  • GM-CSFR granulocyte-monocyte-colony stimulating factor receptor
  • 32M Beta-2 microglobulin
  • LTR long terminal repeat.
  • Fig. 20 shows alternative CIR constructs for “sensing” various inflammatory cytokines/chemokines coupled to various inhibitory signaling domains.
  • CIR domain is designed to be modular so alternative inhibitory signaling domains can be utilized and combined with alternative cytokine -binding domains.
  • scFv single-chain variable fragment
  • T2A “selfcleaving” peptide sequence
  • EGFRt truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs
  • GM-CSFR granulocyte-monocyte-colony stimulating factor receptor
  • 2M Beta-2 microglobulin
  • LTR long terminal repeat.
  • Fig. 21 shows potential incorporation of CIR domains into other CAR-T cell constructs.
  • CIR domain could also be co-expressed with other CAR-T cell domains for attenuating activation and mitigating toxicity.
  • scFv single-chain variable fragment
  • T2A “self-cleaving” peptide sequence
  • EGFRt truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs
  • GM- CSFR granulocyte-monocyte-colony stimulating factor receptor
  • 32M Beta-2 microglobulin
  • LTR long terminal repeat.
  • CIR chimeric inhibitory receptor
  • a chimeric inhibitory receptor comprising a ligand-sensing polypeptide, or a sensor, that when engaged with its ligand, will trigger inhibitory signaling within the cell.
  • CIRs upon binding to a specific ligand, e.g. a pro-inflammatory cytokine or chemokine, in the extracellular environment, CIRs deliver inhibitory signaling to counter and automatically titrate cell activation signals, for example from an activated chimeric antigen receptor (CAR) or other immune cell producing the signal, resulting in regulation of the amount of ligand produced.
  • CAR activated chimeric antigen receptor
  • CIR can mitigate severe CAR-T cell toxicities in a “hands-free” approach.
  • a mammalian cell comprising a nucleic acid encoding a CIR domain, the CIR domain comprising a ligand-sensing polypeptide able to recognize an extracellular ligand, operably linked to an inhibitory signaling molecule, such that sensing of the ligand by the ligand- sensing polypeptide delivers inhibitory signaling to automatically regulate and titrate ligand expression or production by the cell.
  • ligand-sensing is meant that the polypeptide is able to detect, bind, internalize or react, or a combination of any of these functions, to the presence of the ligand.
  • inhibitory signaling molecule is meant a molecule able to inhibit or reduce the amount of ligand at the level of transcription or translation, or reduce the function of the ligand, or modify the ligand to reduce its activity, or divert the ligand’s intended effect into a different effect on the cell as designed, or bind the internalized ligand to prevent its activity, or prevent entry of the ligand to the cell.
  • the ligand-sensing polypeptide interacts with the ligand.
  • the ligand-sensing polypeptide recognizes and binds the ligand.
  • the ligand- sensing polypeptide recognizes, binds, and internalizes the ligand.
  • extracellular ligand-sensing polypeptide and the intracellular inhibitory signaling molecule are modular and exchangeable, i.e. can be selected or chosen as needed.
  • the ligand is a circulating signal, a hormone, a neurotransmitter, a lipid, a peptide, a cytokine or a chemokine.
  • the ligand is toxic to the cell.
  • the ligand is a soluble mediator of toxicity in a cell.
  • the ligand causes systemic toxicity in a subject.
  • the ligand is a cytokine or chemokine produced by direct or indirect chimeric antigen receptor (CAR) activation.
  • CAR chimeric antigen receptor
  • the ligand is interferon gamma (IFNy), interleukin 6 (IL- 6), interleukin 1 (IL-1), monocyte chemoattractant protein 1 (MCP-1), granulocyte macrophage colony stimulating factor (GM-CSF) or a combination thereof.
  • IFNy interferon gamma
  • IL- 6 interleukin 6
  • IL-1 interleukin 1
  • MCP-1 monocyte chemoattractant protein 1
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the ligand- sensing polypeptide is able to sense the ligand.
  • sensing the ligand is meant, sensing the presence of, detecting, interacting with, binding, internalizing, or any combination thereof, of the ligand.
  • the ligand- sensing polypeptide is a naturally occurring or modified ligand receptor.
  • the ligand-sensing polypeptide is an antibody or antigen-binding fragment that recognizes the ligand.
  • the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) of an antibody able to recognize the ligand.
  • the scFv recognizes a cytokine or chemokine.
  • the scFv recognizes and interacts with a cytokine or chemokine resulting from direct or indirect CAR activation.
  • the inhibitory signaling molecule is any naturally occurring, or naturally or artificially modified, inhibitory or regulatory signal molecule.
  • the inhibitory molecule is an inhibitory motif derived from a signaling molecule, the inhibitory motif comprising part or whole of an inhibitory molecule.
  • the inhibitory signals molecule is from an immune checkpoint receptor.
  • the signaling molecule is from a TIGIT (a T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine -based inhibitory motif or ITIM), from a PD-1 (programmed cell death protein 1), positive programmed death-ligand 1 (PD-L1), from a cytotoxic T-lymphocyte protein 4 (CTLA-4), from a lymphocyte activation gene 3 (LAG-3), from a T-cell immunoglobulin and mucin domain 3 (TIM-3).
  • TIGIT a T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine -based inhibitory motif or ITIM
  • PD-1 programmed cell death protein 1
  • CTLA-4 cytotoxic T-lymphocyte protein 4
  • LAG-3 lymphocyte activation gene 3
  • the inhibitory signaling molecule is TIGIT, comprising the transmembrane and intracellular signaling domain thereof.
  • the inhibitory molecule TIGIT has a nucleic acid sequence set forth in SEQ ID NO:37, a polypeptide encoded by SEQ ID NO: 37 set for in SEQ ID NO: 38, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;
  • SEQ ID NO: 38 LLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSC VQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG
  • the inhibitory signaling molecule is PD-1, comprising the transmembrane and intracellular signaling domain of PD-1.
  • the inhibitory molecule PD-1 has a nucleic acid sequence set forth in SEQ ID NO:39, a polypeptide encoded by SEQ ID NO: 39 set forth in SEQ ID NO: 40, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence having a nucleic acid sequence;
  • a nucleic acid encoding a CIR domain wherein the ligandsensing polypeptide is a single-chain variable fragment (scFv) (a single-chain variable fragment heavy chain, a single-chain variable domain light chain, an IgGl hinge sequence) capable of sensing the ligand.
  • scFv single-chain variable fragment heavy chain, a single-chain variable domain light chain, an IgGl hinge sequence
  • the scFv is an anti-interferon gamma single-chain variable fragment.
  • the anti-interferon gamma single-chain variable fragment has a sequence set forth in SEQ ID NO: 41, a polypeptide encoded by SEQ ID NO 41 set forth in SEQ ID NO: 42, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence having a nucleic acid sequence;
  • the ligand- sensing polypeptide is an anti-interleukin-6 (IL-6) single chain variable fragment (scFv).
  • the anti-IL-6 scFv has a nucleic acid sequence set forth in SEQ ID NO: 43, a polypeptide encoded by SEQ ID NO: 43 set forth in SEQ ID NO: 44, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence having a nucleic acid sequence;
  • the ligand-sensing polypeptide is an anti-MCP- 1 single chain variable fragment having a nucleic acid sequence.
  • the anti-MCP- 1 scFv has a nucleic acid sequence set forth in SEQ ID NO: 45, a polypeptide encoded by SEQ ID NO:45 set forth in SEQ ID NO: 46, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence having a nucleic acid sequence;
  • a nucleic acid encoding a CIR domain wherein the ligand-sensing polypeptide is anti-IL-6 scFv and the inhibitory signaling molecule is from a TIGIT
  • the nucleic acid set forth in SEQ ID NO:2 below an amino acid encoded by SEQ ID NO: 2 set forth in SEQ ID NO: 8, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;
  • a nucleic acid encoding a CIR domain wherein the CIR domain comprises an scFv recognizing MCP-1 and the inhibitory signaling molecule is from a TIGIT, set forth in SEQ ID NOG, an amino acid sequence encoded by SEQ ID NOG set forth in SEQ ID NO: 9, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;
  • a nucleic acid encoding a CIR domain wherein the CIR domain comprises an scFv recognizing IFN gamma and the inhibitory signaling molecule is from a PD-1, set forth in SEQ ID NO:4, an amino acid sequence encoded by SEQ ID NO:4 set forth in SEQ ID NO: 10, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;
  • a nucleic acid encoding a CIR domain wherein the CIR domain comprises an scFv recognizing IL-6 and the inhibitory signaling molecule is from a PD-1, set forth in SEQ ID NO:5, an amino acid sequence encoded by SEQ ID NO:5 set forth in SEQ ID NO: 11, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;
  • a nucleic acid encoding a CIR domain wherein the CIR domain comprises an scFv recognizing MCP-1 and the inhibitory signaling molecule is from a PD-1, set forth in SEQ ID NO:6; an amino acid sequence encoded by SEQ ID NO:6 set forth in SEQ ID NO: 12, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.
  • Sequences required for cloning a nucleic acid, for proper expression of a nucleic acid, for proper expression of an encoded polypeptide, for proper localization of the encoded peptide, for detection of the encoded peptide, or for transformation or transduction of cells, are known in the art and can be substituted with the ones described herein, such as for example, linkers, signal sequences, His Tags, 5’ETR, 3’ETR, transmembrane localization signals antibiotic resistance, etc. and methods for producing a nucleic acid for its desired function, to name a few.
  • the cell is a mammalian cell. In another aspect, the cell is an immune effector cell:
  • -a T cell selected from the group consisting of a helper CD4 + T cell, a CD4 + stem cell memory T cell, a cytotoxic CD8 + T cell, a CD8 + stem cell memory T cell, a memory T cell, a naive and/or central memory T cell, a regulatory CD4 + T cell, an innate-like T cell, a natural killer T cell, a mucosal associated invariant T cell, and a Gamma Delta T cell;
  • -a monocyte selected from the group consisting of a CD14++CD16 monocyte, a CD14+CD16++ monocyte, and a CD14++CD16+ monocyte
  • -a B cell selected from the group consisting of a plasmablast, a plasma cell, a lymphoplasmacytoid cell, a memory B cell, a B-2 cell, a B-l cell, and a regulatory B cell (Breg);
  • -a NK cell selected from the group consisting of a CD56 BRIGHT NK cell and a CD56 DM NK cell;
  • a mononuclear phagocytic cell selected from the group consisting of an adipose tissue macrophage, a monocyte, a Kupffer cell, a sinus histiocyte, an alveolar macrophage (dust cell), a tissue macrophage (histiocyte), a microglial cell, a Hofbauer cell, an intraglomerular mesangial cell, an osteoclast, a Langerhans cell, an epithelioid cell, a red pulp macrophage (sinusoidal lining cell), a peritoneal macrophage, a lysomac, and a perivascular macrophage.
  • a mammalian cell is a T-cell.
  • a mammalian cell is a CAR T-cell.
  • CARs are artificial receptors that redirect antigen specificity, activate T cells, and further enhance T cell function through their costimulatory component.
  • CAR T-cells are genetically modified with targeted CARs, CARs targeted to a cell surface molecule, or antigen, specific to a disease cell.
  • autologous T-cells are transduced with a nucleic acid encoding a CAR which targets the modified T-cell against a specified antigen, i.e. not expressed in normal non-disease cells
  • the fundamental structure of a CAR involves 3 components: an extracellular antigen recognition domain, a transmembrane domain, and a cytoplasmic signaling domain.
  • the extracellular antigen-binding domain is usually a singlechain variable fragment (scFv) derived from an antigen -reactive antibody.
  • the scFv comprises of the variable regions of heavy (VH) and light (VL) chains of the antibody joined together by a flexible peptide linker.
  • VH variable heavy
  • VL light
  • This ectodomain scFv provides the target antigen specificity for CAR T cells.
  • the scFv of CAR does not need antigen processing and presentation of peptide epitope in the context of MHC molecules.
  • CAR T-cell targeting molecules include a native protein, an antibody or an antigen-binding fragment thereof, a T cell receptor (TCR), an integrin, or a cell adhesion molecule; an integrin selected from the group consisting of integrin beta-1 (ITG
  • the first-generation CAR had only one intracellular signaling domain, most commonly a CD3 ⁇ chain, which provided signal 1 for activation of CAR T-cells.
  • second- and third-generation CARs were developed by adding one or more costimulatory domains, respectively to provide signal 2 for CAR T-cell activation.
  • the costimulatory domains that are most commonly used are CD28 and CD137 (4-1BB). Costimulatory domains with significant therapeutic potential include CD28, CD137 (4-1BB), 0X40, CD27, and ICOS. Any of these CAR T-cells can be used for expressing a CIR as described herein.
  • a method of producing a CAR T-cell with a CIR domain comprising transducing a CAR T-cell with a nucleic acid encoding a CIR domain.
  • the T-cell can be transduced with a nucleic acid encoding a CIR domain and a nucleic acid encoding a CAR domain targeted to a specific antigen.
  • a T-cell cell can be transduced with two nucleic acids, one encoding a CIR domain as described herein, and one encoding a CAR domain.
  • the T cell can be transduced with one nucleic acid encoding both a CIR domain and a CAR domain as is exemplified herein.
  • a nucleic acid encoding both a CIR domain and a CAR domain, or co-expressing a CIR domain and a CAR domain is a CIR/CAR nucleic acid (or a CAR/CIR nucleic acid).
  • the cell expressing both a CIR domain and a CAR domain is a CIR/CAR cell or a CAR/CIR cell.
  • the cell is a T-cell, it is referred to as a CIR/CAR T-cell.
  • a nucleic acid comprising a CIR/CAR nucleic acid described herein.
  • the CAR contains at least one signaling domain, in a non-limiting example, the CAR signaling domain is CD28, CD3z or 4- 1BB, OX-40, CD27, CD30, GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein), herpes virus entry mediator (HVEM), ICOS (inducible T cell co stimulator).
  • the nucleic acid when designing a CIR/CAR nucleic acid for proper expression, may encode molecules for detection, isolation, cleavage, integration in the genome, transmembrane or surface localization, secretion, endoplasmic retention, and others as found necessary for the proper expression of the CIR/CAR.
  • the nucleic acid encodes a molecule for detection and isolation of cells transformed with the nucleic acid.
  • an inert receptor EGFRt a truncated form of EGFR is used in the detection/isolate or depletion of CARs.
  • Other detection/isolation molecules will be evident to those with ordinary skill in the art.
  • a nucleic acid encoding a self-cleaving peptide inserted into the nucleic acid so that the CIR domain is cleaved from the CAR domain after translation.
  • the self-cleaving peptide is a 2A peptide which can induce ribosomal skipping during translation of the protein. Examples of 2A peptides include P2A, T2A, F2A.
  • the nucleic acid encodes a signal sequence for surface expression of the peptide.
  • the signal sequence is a CD8a signal sequence and/or B2M (beta-2-microglobulin) signal sequence, a GM-CSFR signal sequence for surface localization.
  • the nucleic acid may be inserted into a retroviral vector or a lenti viral vector by including viral 5’ LTR (long terminal repeat) and 3’ LTR as promoter and terminator regions for transcription and integration into the host cell genome.
  • viral 5’ LTR long terminal repeat
  • 3’ LTR 3’ LTR
  • Other sequences and methods for integration and transcription of the recombinant CAR/CIR nucleic acid sequence into the cell genome, such as CRISPR technology, are known in the art. It is evident that the position of the CIR and CAR domains in the nucleic acid can be interchanged, or the CIR can be positioned upstream or downstream of the nucleic acid encoding the CAR domain.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-interferon-gamma scFv for recognizing the ligand IFNy, and an inhibitory signal from TIGIT comprising the transmembrane and intracellular signaling domain of TIGIT receptor; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain.
  • a nucleic acid is identified in SEQ ID NO: 13, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:25 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-interferon-gamma scFv, and an inhibitory signal from TIGIT comprising the transmembrane and intracellular signaling domain of TIGIT receptor; and encoding a CAR domain targeted to CD19 with a CD19.BBz stimulatory domain and a CD3z costimulatory domain.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-interferon-gamma scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain.
  • a nucleic acid identified in SEQ ID NO: 15, and the encoded an amino acid sequence is set forth in SEQ IDNO:27 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-interferon-gamma scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.BBz antigen and a CD3z costimulatory domain.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-IL-6 scFv, and an inhibitory signal from TIGIT; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.29z antigen and a CD3z costimulatory domain.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-IL-6 scFv, and an inhibitory signal from TIGIT; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.BBz antigen and a CD3z costimulatory domain.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-IL-6 scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-IL-6 scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.BBz antigen and a CD3z costimulatory domain.
  • a nucleic acid identified in SEQ ID NO:20 or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:32, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-MCPl scFv, and an inhibitory signal from TIGIT; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain.
  • a nucleic acid identified in SEQ ID NO:21 or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO: 33 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-MCPl scFv, and an inhibitory signal from TIGIT; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.BBz antigen and a CD3z costimulatory domain.
  • a nucleic acid identified in SEQ ID NO:22 or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:34, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-MCP-1 scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain.
  • a nucleic acid identified in SEQ ID NO:23 or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:35 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.
  • a CIR/CAR nucleic acid encoding a CIR domain comprising an anti-MCP-1 scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.41BBz antigen and a CD3z costimulatory domain.
  • a nucleic acid identified in SEQ ID NO:24 or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:36 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.
  • a polynucleotide encoding a CIR or CIR/CAR is introduced and expressed in an immune effector cell using routine methods and readily available reagents.
  • a polynucleotide encoding a CIR or CIR/CAR is synthesized by conventional techniques, including automated DNA synthesizers.
  • PCR amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments that can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992).
  • a vector comprising one or more of the nucleic acids described herein.
  • a polynucleotide or a vector encoding a CIR or CIR/CAR is introduced into a cell (e.g., an immune effector cell or a mammalian cell) using a physical or chemical method, for example, by transfection, transformation, or transduction.
  • a cell e.g., an immune effector cell or a mammalian cell
  • Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J. (ed.), Methods in Molecular Biology, Vol.
  • Phage or viral vectors can be introduced into cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available.
  • a retrovirus is used to deliver a polynucleotide encoding a CIR or a CIR/CAR into a cell (e.g., an immune effector cell or a mammalian cell).
  • a cell e.g., an immune effector cell or a mammalian cell.
  • Retroviruses are a common tool for gene delivery (Miller, 2000, Nature 357: 455-60).
  • Non-limiting examples of retroviruses suitable for use in particular embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV), Rous Sarcoma Virus (RSV), and lentivirus.
  • M-MuLV Moloney murine leukemia virus
  • MoMSV Moloney murine sarcoma virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • RSV Rous Sarcoma Virus
  • Non-limiting examples of lentiviruses include human immunodeficiency virus (e.g., HIV type 1 and HIV type 2), visna-macdi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV).
  • human immunodeficiency virus e.g., HIV type 1 and HIV type 2
  • VMV visna-macdi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anemia virus
  • FV feline immunodeficiency virus
  • BIV bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • a polynucleotide encoding an CIR or a CIR/CAR is integrated into the genome of a cell (e.g., an immune effector cell or a mammalian cell). In some embodiments, a polynucleotide encoding an CIR or a CIR/CAR is inserted into a gene. In some embodiments, a polynucleotide encoding CIR or CIR/CAR is extrachromosomal in a cell (e.g., an immune effector cell or a mammalian cell).
  • a mammalian cell or an immune effector cell expresses a CIR or a CIR/CAR disclosed herein.
  • a mammalian cell or immune effector cell expressing a CAR domain is transduced with a nucleic acid encoding a CIR resulting in a CIR/CAR cell as described herein.
  • the immune effector cell is a CIR/CAR T-cell.
  • the level of a CIR or a CIR/CAR in an immune effector cell or a mammalian cell is conditionally (e.g., tunable, reversible, spatial control, and/or temporal control) regulatable (e.g., inducible or suppressible), for example, at the level of transcription, protein synthesis, protein degradation, or a combination thereof.
  • the level of a CIR or a CIR/CAR in an immune effector cell or a mammalian cell is conditionally inducible.
  • the level of a CIR or CIR/CAR in an immune effector cell or a mammalian cell is conditionally suppressible.
  • the level of a CIR or CIR/CAR in an immune effector cell or a mammalian cell is conditionally regulated by controlling gene expression (e.g., reversibly or irreversibly).
  • the level (e.g., transcription level) of a CIR or a CIR/CAR in an immune effector cell or a mammalian cell is conditionally regulatable (e.g., inducible or suppressible) by a small molecule, a light-switchable system, a ligand-switchable system, or a combination thereof.
  • the level (e.g., transcription level) of a CIR or CIR/CAR in an immune effector cell or a mammalian cell is conditionally regulatable (e.g., inducible or suppressible) by a small molecule.
  • small molecules include tetracycline (TET), doxycycline (DOX), caffeine, 4-hydroxytamoxifen, estrogen, ecdysone, abscisic acid, mifepristone, xylose, FKBP12-rapamycin, and HCV NS3/4A protease inhibitors.
  • TET tetracycline
  • DOX doxycycline
  • caffeine 4-hydroxytamoxifen
  • estrogen ecdysone
  • abscisic acid mifepristone
  • xylose xylose
  • FKBP12-rapamycin xylose
  • HCV NS3/4A protease inhibitors HCV NS3/4A protease inhibitors.
  • the level (e.g., transcription level) a CIR or CIR/CAR in an immune effector cell or a mammalian cell is conditionally regulatable (e.g., inducible or suppressible) by a light-switchable system.
  • a light-switchable system responds to light in the range of about 450-700 nm, for example, in the range of about 450-500 nm (e.g., about 450 nm) or about 620-700 nm (e.g., about 660 nm).
  • an immune effector cell comprises: (a) a tetracycline operator (tetO) motif operably linked to a nucleic acid encoding a CIR or CAR/CIR, (b) a phytochrome-interacting factor 6 (PIF6) sequence (e.g., amino acids 1-100 of PIF6 from Arabidopsis lhaliana) fused to tetracycline repressor (TetR), and (c) A. thaliana red- and far-red light receptor phytochrome B (PhyB) (e.g., amino acids 1-650 of PhyB) linked to VP16 transactivation domain and a nuclear localization sequence (NLS).
  • PIF6 phytochrome-interacting factor 6
  • an immune effector cell comprises a transcriptional control element responsive to a small molecule described herein operably linked to a nucleic acid encoding a CIR or CIR/CAR.
  • a composition e.g., a pharmaceutical composition
  • a serum-free (without serum or substantially free of serum) cry opreservation medium Any suitable cryopreservation media and/or excipients may be used.
  • a cryopreservation medium comprises 5% DMSO and Dextran 40.
  • kits comprising a container and optionally an instruction for use, wherein the container comprises any one or more compositions of pharmaceutical compositions disclosed herein.
  • a method of treating or reducing cytokine toxicity in a subject receiving CAR T-cell therapy comprising administering a therapeutically effective amount of a composition comprising a mammalian cell comprising a nucleic acid encoding a CIR domain described herein to the subject.
  • the mammalian cell is a CIR/CAR T cell comprising a nucleic acid encoding both a CIR domain and a CAR domain.
  • the cytokine toxicity is one of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), immune effector cell- associated hematotoxicity (ICAHT), immune effector cell- associated hemophagocytic lymphohistiocytosis-like syndrome (CAR-HLH), cerebral edema associated with CAR therapy, parkinsonism-like syndrome associated with CAR therapy.
  • CRS cytokine release syndrome
  • ICANS immune effector cell-associated neurotoxicity syndrome
  • ICAHT immune effector cell- associated hematotoxicity
  • CAR-HLH immune effector cell- associated hemophagocytic lymphohistiocytosis-like syndrome
  • cerebral edema associated with CAR therapy parkinsonism-like syndrome associated with CAR therapy.
  • the subject is receiving CAR T therapy for treating a disease.
  • the disease is cancer.
  • cancers include: acute lymphoblastic leukemia (ALL); Acquired immunodeficiency syndrome (AIDS)-related cancer (e.g., Kaposi Sarcoma, AIDS-related lymphoma, primary central nervous system (CNS) lymphoma); acute myeloid leukemia (AML); adrenocortical carcinoma; anal cancer; appendix cancer; basal cell carcinoma of the skin; bile duct cancer; bladder cancer; bone cancer (including Ewing sarcoma, osteosarcoma and malignant fibrous histiocytoma); brain tumors/cancer; breast cancer; Burkitt lymphoma; carcinoid tumor (gastrointestinal); cervical cancer; childhood adrenocortical carcinoma; childhood astrocytoma; childhood atypical CNS teratoid/rhabdoid tumor; childhood bladder cancer; childhood carcinoid tumor
  • ALL acute lymphoblastic leukemia
  • DCIS Ductal carcinoma in situ
  • endometrial cancer uterine cancer
  • esophageal cancer esthesioneuroblastoma
  • extragonadal germ cell tumor extragonadal germ cell tumor
  • eye (ocular) cancer fallopian tube cancer
  • gallbladder cancer gastric (stomach) cancer
  • gastrointestinal carcinoid tumor gastrointestinal stromal tumors (GIST)
  • germ cell tumors gestational trophoblastic disease
  • hairy cell leukemia head and neck cancer
  • hepatocellular (liver) cancer Hodgkin lymphoma; intraocular (eye) melanoma; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia; liver cancer; lung cancer (non-small cell and small cell); lymphoma; male breast cancer; malignant mesothelioma; melanoma; Merkel cell carcinoma; metastatic cancer; metastatic squamous neck cancer
  • childhood rhabdomyosarcoma childhood vascular tumors, Ewing sarcoma, Kaposi sarcoma, osteosarcoma (bone cancer), soft tissue sarcoma, uterine sarcoma); skin cancer; small intestine cancer; squamous cell carcinoma of the skin; testicular cancer; throat cancer (e.g. nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer); thymoma and thymic carcinoma; thyroid cancer; transitional cell cancer of the renal pelvis and ureter; urethral cancer; vaginal cancer; vascular tumors; vulvar cancer; and Wilms tumor and other childhood kidney tumors.
  • throat cancer e.g. nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer
  • thymoma and thymic carcinoma thyroid cancer
  • transitional cell cancer of the renal pelvis and ureter urethral cancer
  • autoimmune diseases include but are not limited to include but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-
  • AIDS Acquired Immunodeficiency Syndrome
  • alopecia areata ankylosing
  • the subject is receiving CAR T therapy to treat inflammatory disorders.
  • inflammatory disorders include but are not limited to, chronic and acute inflammatory disorders.
  • inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.
  • a subject is infused with cells disclosed herein (e.g., immune effector cells with CIR or CIR/CAR or mammalian cells with CIR or CIR/CAR disclosed herein).
  • cells e.g., immune effector cells or mammalian cells
  • cells e.g., immune effector cells or mammalian cells
  • cells e.g., immune effector cells or mammalian cells
  • are allogeneic i.e from a different animal of the same species.
  • cells e.g. immune effector or mammalian cells
  • are xenogeneic i.e. from an animal or a different species.
  • the administration of the cells described herein may be carried out in any convenient manner known to those of skill in the art.
  • the cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.
  • the cells described herein can also be administered using any number of matrices.
  • the present invention utilizes such matrices within the novel context of acting as an artificial lymphoid organ to support, maintain, or modulate the immune system, typically through modulation of T cells. Accordingly, the present invention can utilize those matrix compositions and formulations which have demonstrated utility in tissue engineering. Accordingly, the type of matrix that may be used in the compositions, devices and methods of the invention is virtually limitless and may include both biological and synthetic matrices. In one particular example, the compositions and devices set forth by U.S. Pat. Nos.
  • Matrices comprise features commonly associated with being biocompatible when administered to a mammalian host. Matrices may be formed from natural and/or synthetic materials. The matrices may be non-biodegradable in instances where it is desirable to leave permanent structures or removable structures in the body of an animal, such as an implant; or biodegradable. The matrices may take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, or nanoparticles.
  • matrices can be designed to allow for sustained release of seeded cells or produced active agent.
  • the matrix of the present invention is flexible and elastic, and may be described as a semisolid scaffold that is permeable to substances such as inorganic salts, aqueous fluids and dissolved gaseous agents including oxygen.
  • a matrix is used herein as an example of a biocompatible substance.
  • the current invention is not limited to matrices and thus, wherever the term matrix or matrices appears these terms should be read to include devices and other substances which allow for cellular retention or cellular traversal, are biocompatible, and are capable of allowing traversal of macromolecules either directly through the substance such that the substance itself is a semi- permeable membrane or used in conjunction with a particular semi-permeable substance.
  • a polypeptide includes a single polypeptide, and two or more polypeptides.
  • the culturing step as described herein can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
  • the culturing step as described further herein can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.
  • a skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of a disease, e.g., immuno-dysfunction).
  • a skilled medical professional can also change the identity and number (e.g., increase or decrease) of the mammalian cell or immune effector cell with CIR or CIR/CAR (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least the mammalian cell or immune effector cell with CIR or CIR/CAR (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).
  • compositions that contain at least one (e.g., one, two, three, or four) of the mammalian cell or immune effector cell with CIR or CIR/CAR described herein.
  • Two or more (e.g., two, three, or four) of any of the mammalian cell or immune effector cell with CIR or CIR/CAR described herein can be present in a pharmaceutical composition in any combination.
  • the pharmaceutical compositions may be formulated in any manner known in the art.
  • compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).
  • the compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbito)
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza® Corporation and Nova Pharmaceutical, Inc.).
  • biodegradable, biocompatible polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza® Corporation and Nova Pharmaceutical, Inc.
  • compositions containing the mammalian cell or immune effector cell with CIR or CIR/CAR described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, intratumoral, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).
  • parenteral e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, intratumoral, or intraperitoneal
  • dosage unit form i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage.
  • Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
  • a therapeutically effective amount of the one or more (e.g., one, two, three, or four) the mammalian cell or immune effector cell with CIR or CIR/CAR (e.g., any of the mammalian cell or immune effector cell with CIR or CIR/CAR described herein) will be an amount that treats the disease in a subject in a subject, or a subject identified as being at risk of developing the disease, decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human).
  • any of the mammalian cell or immune effector cell with CIR or CIR/CAR described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases).
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the disclosure also provides methods of manufacturing the mammalian cell or immune effector cell with CIR or CIR/CAR thereof for various uses as described herein.
  • scFv single-chain variable fragment
  • TIGIT T-cell immunoreceptor with Ig and ITIM domains
  • EGFRt truncated epidermal growth factor receptor
  • CD 19 FMC63 CAR containing either CD28 or 4- IBB costimulatory domain
  • Retrovirus particles were produced by transfecting 293GP packaging cells with the gene construct using LipofectamineTM 2000. Retrovirus containing supernatants were then collected and used for transducing human T cells. Prior to transduction with retrovirus, isolated CD3 + human T cells were activated for 2-3 days using CD3/CD28 DynabeadsTM at a 3:1 bead:cell ratio. Next, activated T cells were exposed to retronectin-bound retrovirus on two consecutive days. Following exposure to retrovirus DynabeadsTM were magnetically removed and transduced T cells were cultured and expanded in recombinant human IL-2. Transduction efficiency of T cells with CAR construct was assessed by staining with biotinylated CD19-Fc protein or biotinylated EGFR followed by secondary staining with fluorochrome-conjugated streptavidin.
  • CAR-T cells were co-cultured with CD19 + NALM-6 tumor cells at a 1:1 ratio for 18-22 hours. CAR-T cell immunophenotyping analysis was then performed. CAR-T cells were stained with antibodies against CD3, CD4, CD8, and EGFR. CAR-T cell activation state was measured by staining for CD69, CD25, CD38, PD-1, and CD107a. Dead cell exclusion was performed using Zombie AquaTM Fixable Viability dye. Data was acquired on a NovoCyte® 3005 flow cytometer (Agilent), and subsequent analysis was performed using FlowJoTM vlO.
  • Cytotoxicity was evaluated using a luciferase-based assay measuring bioluminescence.
  • NALM-6 tumor cells expressing GFP/firefly luciferase were co-incubated with non-transduced T cells, CD19.28z, CD19.28z.CIR, CD19.BBz, or CD19.BBz.CIR CAR-T cells for 18 hours at effector-to-target (E:T) ratios ranging from 10:1 to 1.25:1.
  • CAR-T cells were initially co-cultured with NALM-6 cells at a 1:1 ratio and then restimulated with fresh tumor cells very 2- 3 days.
  • CAR-T cells were co-cultured with NALM-6 tumor cells at a 1:1 ratio for 18-22 hours. Cocultures were incubated for 48 hours and supernatants were collected for cytokine and chemokine analyses by Luminex®. Single-cell secretome analysis for polyfunctionality.
  • CAR-T cells were co-cultured with NALM-6 tumor cells at a 2:1 ratio for 18-22 hours. Following incubation, CAR-T cells were collected and separated from tumor cells by negative selection using a Pan T cell Isolation Kit (Miltenyi). CAR-T cells were then subjected to Isolight® platform analysis (Bruker ⁇ ) using the Human Adaptive Immune Panel. CAR-T cells were loaded into an IsoCode Chip and incubated overnight.
  • CAR-T cells were co-cultured with NALM-6 tumor cells at a 2:1 ratio for 18-22 hours. Following incubation, CAR-T cells were collected and separated from tumor cells by positive selection using an anti-EGFR biotin antibody and anti-biotin microbeads (Miltenyi). CAR-T cells were then subjected to nCounter platform analysis (Nanostring®) using the CAR-T Characterization Panel. Gene expression data was normalized and analyzed using the ROSALIND online platform.
  • NSG mice NOD-scid IL2Ry nu11 mice were purchased from The Jackson Laboratory. Mice were housed according to institutional guidelines. Animal experimentation protocols were performed with approval of the University of Virginia Institutional Animal Care and Use Committee. Six- to eight-week-old NSG mice were i.v. injected with 0.5xl0 6 NALM-6 (GFP + /Luciferase + ) tumor cells. 2-3 days later, mice were i.v. injected with 10xl0 6 CD19 CAR or CD19 CAR.CIR (CAR/CIR) T cells. Tumor growth and progression was monitored using weekly bioluminescent imaging. Bioluminescent tumor data was obtained on a LagoX (Spectral Instruments Imaging) instrument following intraperitoneal injection of D-luciferin (Revvity). Data and Statistical Analysis.
  • EXAMPLE 1 Model of chimeric inhibitory receptor (CIR).
  • Severe cytokine release syndrome correlates with rapid CAR-T cell expansion accompanied by a robust and multi-dimensional proinflammatory state.
  • CIR chimeric inhibitory receptor
  • CAR chimeric inhibitory receptor
  • CIR activity will attenuate the supraphysiologic immune response responsible for sCRS yet allow continued anti-tumor activity ( Figure 2 and 3).
  • the CIR should: (a) be specifically activated by increased levels of a proinflammatory cytokine; (b) dampen overall CAR-T cell activation to blunt inflammatory cytokine production and reduce myeloid cell activation; yet (c) retain CAR-T cell anti-tumor functions.
  • EXAMPLE 2 Normal expansion and proliferation of CD19.28z.CIR CAR-T cells coexpressing CIR domain during in vitro manufacture
  • CIR containing an inhibitory receptor signaling domain is expected to lead to overall attenuation in the activation level of CAR-T cells co-expressing the CIR when CAR-T cells are undergoing stimulation by target expressing tumor cells.
  • CD19.28z or CD19.28z.CIR CAR- T cells were exposed to CD19 + NALM-6 leukemia cells in vitro at a 1:1 effector cell to target cell ratio for 24 hours.
  • CAR-T cells were then stained and labeled with fluorescent antibodies against T cell surface markers to identify CD4 and CD8 subsets as well as multiple known markers of activation including CD69, CD25, CD38, PD-1, and CD107a. Labeled cells were then analyzed on a flow cytometer. Representative flow cytometry plots for CD69 and CD38 on CD4 + and CD8 + CAR-T cells are shown for non-transduced (NT) T cells, CD19.28z CAR-T cells, and CD19.28z.CIR CAR-T cells either stimulated with NALM-6 cells or left unstimulated ( Figure 5A).
  • NT non-transduced
  • CD19.28z CAR-T cells CD19.28z.CIR CAR-T cells either stimulated with NALM-6 cells or left unstimulated
  • the overall levels of activation for CAR-T cells were then evaluated by calculating the percentage of CD4 + and CD8 + CAR-T cells expressing a specified number of activation markers, ranging from 0 to 5 markers, with 5 markers indicating the most highly activated cells.
  • CIR-expressing CAR-T cells exhibited significantly reduced percentages of cells expressing 4 and 5 activation markers and significantly increased percentages of cells expressing only 1 and 2 activation markers (Figure 5B).
  • EXAMPLE 4 In vitro cytotoxicity of CD19.28z CAR-T cells expressing CIR receptor against CD19 + leukemia cell line NALM-6.
  • EXAMPLE 5 CD19.28z.CIR CAR-T cells exhibit attenuated cytotoxicity kinetics which is dependent upon CIR-mediated binding to IFN-gamma.
  • CD 19 CAR-T cells expressing a CIR domain show equal tumor cell cytotoxicity during short-term exposure to tumor cells.
  • prolonged exposure to tumor cells through repetitive tumor cell challenges would lead to the increasing and/or continual presence of interferon-gamma (IFN-y).
  • IFN-y interferon-gamma
  • CD 19 CAR-T cell anti-tumor activity could be beneficial in that CD 19 CAR-T cell antitumor activity is attenuated but not abrogated, resulting in a more tempered CD 19 CAR-T cell response with reduced cytokine release and toxicity.
  • repeated and prolonged signaling through the CIR could result in a substantial loss of anti-tumor activity and control of tumor cell growth.
  • CD19.28z or CD19.28z.CIR CAR-T cells were cultured with NALM-6 tumor cells for three consecutive challenges and the presence of tumor cells was measured at various timepoints over the course of 96 hours. As expected, both CAR-T cell groups exerted equal cytotoxicity after the first challenge.
  • CD19.28z.CIR CAR-T cells exhibited a delay in tumor cell killing relative to standard CD19.28z CARs, however, CD19.28z.CIR CAR-T cells eventually reached the maximum level of cytotoxicity (Figure 7A).
  • CD19.28z.CIR CAR-T cells Upon the third tumor cell challenge, CD19.28z.CIR CAR-T cells once again showed attenuated cytotoxicity, however the levels of cytotoxicity were equivalent at the end of the time course. Thus, the presence of a CIR domain results in attenuated cytotoxicity kinetics of CD19.28z CAR-T cells.
  • CD19.28z.CIR (scFv-less) CAR-T cells which lack to ability to recognize IFN-y through the CIR domain, did not exhibit an attenuation of cytotoxicity kinetics and were equivalent to standard CD19.28z CAR-T cells ( Figure 7B). This result demonstrates that IFN-y binding by the CIR is required for attenuated cytotoxicity of CD19.28z CAR-T cells co-expressing a CIR domain.
  • nontransduced T cells, CD19.28z CAR-T cells, and CD19.28z.CIR CAR-T cells were first exposed in vitro to recombinant human IFN-y (0.05ng/ml-50ng/ml) for 24 hours. T cells were then cultured with target tumor cells to evaluate cytotoxicity. After 6 hours, cytotoxicity of CD19.28z.CIR CAR-T cells was substantially blunted relative to standard CD19.28z CAR-T cells in an IFN-y dose-dependent manner (Figure 7C, left).
  • EXAMPLE 6 CIR signaling in CD19.28z CAR-T cells results in attenuated production of cytokines and chemokines.
  • CD19.28z and CD19.28z.CIR CAR-T cells were stimulated in vitro by NALM-6 tumor cells for 24 hours, cell-free culture supernatants were collected, and subsequently analyzed by Luminex® to measure the levels of specific cytokines and chemokines.
  • CD19.28z.CIR CAR-T cells tended to secrete lower levels of several proinflammatory cytokines including IFN-y, IL-2, TNF-CX, IL-17A and IL-4 relative to standard CD19.28z CAR-T cells, while the secretion of anti- inflammatory cytokines like IL- 13 and IL- 10 were unchanged ( Figure 8A).
  • a more comprehensive evaluation of additional cytokines and chemokines showed a strong trend towards a decrease in the secretion of effector, stimulatory, and inflammatory mediators by CD19.28z.CIR CAR-T cells relative to standard CD19.28z CARs ( Figure 8B).
  • EXAMPLE 7 Overall polyfunctionality of CD19.28z CAR-T cells co-expressing CIR is not compromised by CIR signaling.
  • Polyfunctional CAR-T cells are CAR-T cells that secrete two or more cytokines or chemokines. Polyfunctional CAR-T cells are associated with favorable clinical outcomes and successful CAR-T cell expansion following patient infusion. Poly functionality, specifically the polyfunctionality strength index (PSI), in CAR-T cells is a measure of the percent of polyfunctional cells and the mean level of proteins secreted. Thus, CAR-T cells with a higher PSI are associated with improved clinical outcomes.
  • PSI polyfunctionality strength index
  • Nanostring® nCounter gene expression profiling demonstrates significant regulation of CAR-T cell pathways by the presence of CIR signaling.
  • the overall attenuation of the activation state of CD19.28z CAR-T cells expressing a CIR domain was confirmed as genes in multiple activation pathways were less enriched relative to CD19.28z CARs, including JAK/STAT, MAPK and PI3K Signaling, NFkB, and Costimulatory Molecules pathways following exposure to tumor cells (Figure 11).
  • the NFAT signaling pathway was markedly less enriched in CD19.28z.CIR CAR-T cells relative to CD19.28z CAR-T cells.
  • Additional pathways that were reduced included NOTCH, TGF-beta, and Wnt Signaling.
  • CD19.28z CAR-T cells led to significant metabolic shifts across many gene pathways involved in cellular metabolism. Overall, CD19.28z.CIR CAR-T cells exhibited reductions in these metabolic pathways, potentially a result of the attenuated activation as changes in T cell metabolism are induced naturally following activation. This altered metabolic phenotype may confer enhanced metabolic fitness to CAR-T cells. Lastly, the exhaustion phenotype of CD19.28z.CIR CAR-T cells was unchanged relative to CD19.28z CAR-T cells, however, the presence of the CIR domain reduced the expression of genes related to apoptosis.
  • EXAMPLE 10 CIR-regulated CD19.28z CAR-T cells exhibit similar anti-tumor activity and survival in vivo relative to CD19.28z controls.
  • CAR-T cell anti-tumor efficacy and disease control is typically demonstrated using a standard in vivo mouse model where NOD.Cg-Prkdc ⁇ 1 112rg tmlw j 1 /SzJ (NSG) mice are injected with NALM-6 leukemia tumor cells followed by administration of CD19 CAR-T cells.
  • NSG NOD.Cg-Prkdc ⁇ 1 112rg tmlw j 1 /SzJ mice
  • NALM-6 (GFP + /Luciferase + ) tumor cells were first injected into mice and two days later mice received either non-transduced T cells, CD19.28z CAR-T cells, or CD19.28z.CIR CAR-T cells via intravenous injection (Figure 12A). Weekly live-animal bioluminescent imaging was conducted to monitor tumor burden over the course of the experiment. Tumor burden and disease rapidly increased in mice that were administered control non-transduced T cells and required humane euthanasia by day 13 post-treatment (Figure 12B-D).
  • EXAMPLE 11 CIR-mediated attenuation of CD19 CAR-T cell responses is observed in vivo in tumor-bearing mice.
  • EXAMPLE 13 In vitro cytotoxicity of CD19.4-lBBz CAR T cells expressing CIR receptor against leukemia cell line NALM-6 demonstrates similar leukemia control.
  • EXAMPLE 14 Diminished pro-inflammatory cytokine/chemokine secretion by bystander autologous macrophages and DCs during in vitro CRS-assay.
  • EXAMPLE 15 Self-regulating CD19.BBz CAR-T cells expressing anti-IFN-y/TIGIT CIR exhibit similar anti-tumor activity and survival but improved persistence in vivo

Landscapes

  • Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente divulgation concerne de nouveaux récepteurs inhibiteurs chimériques (CIR), pour administrer une signalisation inhibitrice ciblée, et des méthodes de fabrication et d'utilisation de ceux-ci. Les lymphocytes T CAR exprimant le CIR conçus pour inhiber l'IFN gamma médiateur inflammatoire ont présenté une réduction significative des niveaux du médiateur inflammatoire et de l'activation immunitaire en aval tout en n'affectant pas négativement la destruction des lymphocytes T CAR/CIR des cellules tumorales in vivo.
PCT/US2024/053822 2023-11-01 2024-10-31 Nouveaux lymphocytes t car autorégulés et leurs utilisations Pending WO2025096730A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363595225P 2023-11-01 2023-11-01
US63/595,225 2023-11-01

Publications (1)

Publication Number Publication Date
WO2025096730A1 true WO2025096730A1 (fr) 2025-05-08

Family

ID=93566392

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/053822 Pending WO2025096730A1 (fr) 2023-11-01 2024-10-31 Nouveaux lymphocytes t car autorégulés et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2025096730A1 (fr)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5199942A (en) 1991-06-07 1993-04-06 Immunex Corporation Method for improving autologous transplantation
US5626561A (en) 1995-06-07 1997-05-06 Gore Hybrid Technologies, Inc. Implantable containment apparatus for a therapeutical device and method for loading and reloading the device therein
US5843069A (en) 1995-06-07 1998-12-01 Gore Hybrid Technologies, Inc. Implantable containment apparatus for a therapeutical device and method for loading and reloading the device therein
US5902745A (en) 1995-09-22 1999-05-11 Gore Hybrid Technologies, Inc. Cell encapsulation device
US5980889A (en) 1993-08-10 1999-11-09 Gore Hybrid Technologies, Inc. Cell encapsulating device containing a cell displacing core for maintaining cell viability
WO2013022739A1 (fr) 2011-08-05 2013-02-14 Leonard Joshua N Architecture de capteurs extracellulaires modulaires pour biocapteurs à base de cellules
WO2016138034A1 (fr) 2015-02-24 2016-09-01 The Regents Of The University Of California Commutateurs transcriptionnels déclenchés par une liaison et procédés d'utilisation associés
WO2021035093A1 (fr) * 2019-08-20 2021-02-25 Senti Biosciences, Inc. Récepteur inhibiteur chimérique
WO2021061791A1 (fr) 2019-09-24 2021-04-01 The Regents Of The University Of California Récepteurs notch avec un effecteur de transcription contenant un doigt de zinc
WO2021168317A1 (fr) * 2020-02-20 2021-08-26 Senti Biosciences, Inc. Architectures de récepteurs chimériques inhibiteurs

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5199942A (en) 1991-06-07 1993-04-06 Immunex Corporation Method for improving autologous transplantation
US5980889A (en) 1993-08-10 1999-11-09 Gore Hybrid Technologies, Inc. Cell encapsulating device containing a cell displacing core for maintaining cell viability
US5626561A (en) 1995-06-07 1997-05-06 Gore Hybrid Technologies, Inc. Implantable containment apparatus for a therapeutical device and method for loading and reloading the device therein
US5787900A (en) 1995-06-07 1998-08-04 Gore Hybrid Technologies, Inc. Method for loading and reloading a therapeutical device in a vascularized implantable containment apparatus
US5843069A (en) 1995-06-07 1998-12-01 Gore Hybrid Technologies, Inc. Implantable containment apparatus for a therapeutical device and method for loading and reloading the device therein
US5913998A (en) 1995-06-07 1999-06-22 Gore Hybrid Technologies, Inc. Method of making an implantable containment apparatus for a therapeutical device
US5902745A (en) 1995-09-22 1999-05-11 Gore Hybrid Technologies, Inc. Cell encapsulation device
WO2013022739A1 (fr) 2011-08-05 2013-02-14 Leonard Joshua N Architecture de capteurs extracellulaires modulaires pour biocapteurs à base de cellules
WO2016138034A1 (fr) 2015-02-24 2016-09-01 The Regents Of The University Of California Commutateurs transcriptionnels déclenchés par une liaison et procédés d'utilisation associés
WO2021035093A1 (fr) * 2019-08-20 2021-02-25 Senti Biosciences, Inc. Récepteur inhibiteur chimérique
WO2021061791A1 (fr) 2019-09-24 2021-04-01 The Regents Of The University Of California Récepteurs notch avec un effecteur de transcription contenant un doigt de zinc
WO2021168317A1 (fr) * 2020-02-20 2021-08-26 Senti Biosciences, Inc. Architectures de récepteurs chimériques inhibiteurs

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL.: "National Center for Biotechnology Information", 1990, UNITED STATES NATIONAL INSTITUTES OF HEALTH
AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY
AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1992
BRASH ET AL., MOL. CELL BIOL., vol. 7, 1987, pages 2031 - 34
CELICHOWSKI PIOTR ET AL: "Tuning CARs: recent advances in modulating chimeric antigen receptor (CAR) T cell activity for improved safety, efficacy, and flexibility", JOURNAL OF TRANSLATIONAL MEDICINE, vol. 21, no. 1, 15 March 2023 (2023-03-15), pages 197, XP093042135, Retrieved from the Internet <URL:https://translational-medicine.biomedcentral.com/counter/pdf/10.1186/s12967-023-04041-6.pdf> [retrieved on 20250205], DOI: 10.1186/s12967-023-04041-6 *
COBB DUSTIN A ET AL: "Expression of a Novel Chimeric Inhibitory Receptor Attenuates CD19 CAR T Cells: A Promising Strategy for Toxicity Mitigation", 11 February 2025 (2025-02-11), XP093249251, Retrieved from the Internet <URL:https://www.postersessiononline.eu/173580348_eu/congresos/ASH2023/aula/-SUN_3436_ASH2023.pdf> [retrieved on 20250211] *
COBB DUSTIN A ET AL: "Expression of a Novel Chimeric Inhibitory Receptor Attenuates CD19 CAR T Cells: A Promising Strategy for Toxicity Mitigation", BLOOD, vol. 142, no. Supplement 1, 2 November 2023 (2023-11-02), AMSTERDAM, NL, pages 3436 - 3436, XP093249245, ISSN: 0006-4971, DOI: 10.1182/blood-2023-186464 *
COBB DUSTIN ET AL: "Expression of a Novel Chimeric Inhibitory Receptor Attenuates CD19 CAR T Cells: A Promising Strategy for Toxicity Mitigation", TRANSPLANTATION AND CELLULAR THERAPY, ELSEVIER, AMSTERDAM, NL, vol. 30, no. 2, 7 February 2024 (2024-02-07), XP087465130, ISSN: 2666-6367, [retrieved on 20240207], DOI: 10.1016/J.JTCT.2023.12.146 *
GREENWUTS: "Protecting Groups in Organic Synthesis", vol. 7, 1991, JOHN WILEY AND SONS
JOHNSTON, NATURE, vol. 346, 1990, pages 776 - 77
MILLER, NATURE, vol. 357, 2000, pages 455 - 60
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443
PEARSONLIPMAN, PROC. NAT'L. ACAD. SCI. USA, vol. 85, 1988, pages 2444
REMINGTON: "The Science and Practice of Pharmacy", 2006, LIPINCOTT WILLIAMS AND WILKINS, pages: 958 - 89
ROSENBERG ET AL., NEW ENG. J. OF MED., vol. 319, no. 1676, 1988
SMITHWATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482

Similar Documents

Publication Publication Date Title
JP7460675B2 (ja) Ｐｄ－１－ｃｄ２８融合タンパク質および医療におけるその使用
JP7439002B2 (ja) ヒト化抗ｃｄ１９キメラ抗原受容体を使用するがんの処置
TWI728308B (zh) 一種結合ｂｃｍａ的嵌合抗原受體（ｃａｒ）及其應用
JP7109789B2 (ja) 融合タンパク質を使用したｔｃｒの再プログラム化のための組成物及び方法
US20210128617A1 (en) SYNTHETIC CARS TO TREAT IL13R-alpha-2 POSITIVE HUMAN AND CANINE TUMORS
CA3107675A1 (fr) Amelioration de l&#39;efficacite et de la securite de therapies cellulaires adoptives
EP3805270B1 (fr) Cellule car-t anti-cd19 améliorée
CA3111384A1 (fr) Compositions de cellules allogeniques et methodes d&#39;utilisation
EP3228702A1 (fr) Cellule effectrice immune transgénique à double cible gpc3 et asgpr1 et son utilisation
JP2022548778A (ja) 線維芽細胞活性化タンパク質（ｆａｐ）を標的とすることによる腫瘍組織の破壊の方法
WO2017219934A1 (fr) Lymphocyte t cytotoxique capable d&#39;exprimer un anticorps avec efficacité et stabilité, et ses utilisations
EP4022035A1 (fr) Méthodes et compositions pour la modification et l&#39;administration de lymphocytes
JP2023553419A (ja) 遺伝子操作細胞およびその使用
JP2024514933A (ja) Ｍｈｃクラスｉ及びｉｉ発現の下方調節のための人工マイクロｒｎａ埋込ｓｈｒｎａを発現するｃａｒ ｎｋｔ
TW202342734A (zh) 具有抗-cd19/抗-cd22嵌合抗原受體之基因工程細胞及其用途
WO2019182996A1 (fr) Protéines de fusion anticorps-interféron pour améliorer les thérapies par transfert cellulaire adoptif de lymphocytes t pour le traitement du cancer
JP2022526187A (ja) 造血細胞の細胞傷害性を促進するための組成物及び方法
CN113549157B (zh) 双靶向嵌合抗原受体及其应用
US20250108069A1 (en) Chimeric adaptor polypeptides
US20200155600A1 (en) Methods and compositions for the treatment of cancer
WO2025096730A1 (fr) Nouveaux lymphocytes t car autorégulés et leurs utilisations
CN118055944A (zh) 调节Bcl-2增强嵌合抗原受体癌症免疫疗法功效
EP4599843A1 (fr) Combinaison de car et cd200r modifié
TW202444909A (zh) 用於腫瘤治療之car修飾劑的生成
CN119894923A (zh) 包含tmigd2共刺激结构域的嵌合抗原受体及其相关使用方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24809099

Country of ref document: EP

Kind code of ref document: A1