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EP4615961A1 - Ciblage de l'irf2 pour une thérapie anticancéreuse - Google Patents

Ciblage de l'irf2 pour une thérapie anticancéreuse

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Publication number
EP4615961A1
EP4615961A1 EP23887239.4A EP23887239A EP4615961A1 EP 4615961 A1 EP4615961 A1 EP 4615961A1 EP 23887239 A EP23887239 A EP 23887239A EP 4615961 A1 EP4615961 A1 EP 4615961A1
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European Patent Office
Prior art keywords
cell
irf2
cells
modified
tumor
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German (de)
English (en)
Inventor
David Brooks
Sabelo LUKHELE
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University Health Network
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University Health Network
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
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    • 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
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2207/00—Modified animals
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
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    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes

Definitions

  • the invention relates to compositions for treating cancer and particularly to targeting IRF2 for cancer therapy, including T cell therapy.
  • Type I interferons IFN-I; IFNa/p
  • type II interferon IFNy
  • IFN-ls and IFN-II are also emerging as central regulators of both the chronic immune activation and the suppression that drive cancer progression (Boukhaled et al., 2021 ; Snell et al., 2017).
  • the efficacy of many types of anti-cancer therapies, including checkpoint blockades, are associated with increased IFN signaling (Boukhaled et al., 2021 ; Snell et al., 2017).
  • IFN-II increases PDL1 expression on the surface of tumor cells. PDL1 binds to PD1 on activated CD8 + T cells in the tumor microenvironment (TME), driving their apoptotic cell death (Dong et al., 2002).
  • IFN-ls signal through a dimeric IFNAR1/IFNAR2 receptor that activates the kinases Jak1 and Tyk2 to initiate STAT1 and STAT2 phosphorylation (among other pathways) to induce expression of hundreds of IFN-I stimulated genes (ISGs), including interferon regulatory factors (IRFs) (Lukhele et al., 2019).
  • IFN-ls signal through a dimeric IFNAR1/IFNAR2 receptor that activates the kinases Jak1 and Tyk2 to initiate STAT1 and STAT2 phosphorylation (among other pathways) to induce expression of hundreds of IFN-I stimulated genes (ISGs), including interferon regulatory factors (IRFs) (Lukhele et al., 2019).
  • IRFs interferon regulatory factors
  • IRF1 Central to these outcomes is the interplay between IRF1 and IRF2, known for their positive and negative regulation, respectively, of IFN-I and IFN-II signaling.
  • IRF1 is activated by IFN-II [as well as IFN-I and nuclear factor-kappa p (NF- K p)] to induce the pro-inflammatory and immune stimulatory functions critical to prevent tumor growth (Drew et al., 1995a; Harada et al., 1993).
  • IRF2 is constitutively expressed in many immune cells and is upregulated in response to either IFN-I or IFN- II (Harada et al., 1989; Taniguchi and Takaoka, 2001).
  • IRF2 antagonizes IRF1 by competing for binding to the same promoter elements of IFN-I and IFN-I l-inducible genes (Harada et al., 1989), and by inhibiting nuclear translocation of IRF1 (Wang et al., 2007).
  • IRF2 also interacts with NF-KP (Chae et al., 2008; Drew et al., 1995b), STAT1 (Rouyez et al., 2005), IRF8 (Bovolenta et al., 1994; Sharf et al., 1995), and IRF9 (Hida et al., 2000; Tanaka et al., 1993), factors that influence the ability of immune cells to control tumors.
  • NF-KP Choe et al., 2008; Drew et al., 1995b
  • STAT1 Rayez et al., 2005
  • IRF8 Bovolenta et al., 1994; Sharf et al., 1995
  • IRF9 Hida et al., 2000; Tanaka et al., 1993
  • IRF2 activates gene transcription in certain contexts (Vaughan et al., 1995; Vaughan et al., 1998; Yamamoto et al., 1994), for example, IRF2 cooperates with IRF1 to induce TLR3 in HeLa cells (Ren et al., 2015). Thus, IRF2 balances IFN stimulation by differentially inducing and antagonizing key transcriptional regulators.
  • IRF2 expression by tumor cells themselves generally correlates with development and progression of many human cancers, potentially through repressing cancer cell intrinsic IFN signaling (Chen et al., 2021a; Mei et al., 2017; Sakai et al., 2014; Wang et al., 2007; Yi et al., 2013).
  • IRF2 promotes tumor survival by inhibiting transcription of the IFNyR, thereby enhancing tumor-intrinsic resistance to IFN-II (Wang et al., 2008).
  • some tumor types downregulate IRF2 to evade immune targeting.
  • IRF2 directly represses PDL1 expression and activates components of the MHC-I pathway, both of which increase susceptibility to T cell mediated killing (Kriegsman et al., 2019; Yan et al., 2020).
  • IFNs IFN-I and II
  • IRF2 as a central regulator of CD8 + T cell exhaustion in cancer.
  • adoptive transfer of IRF2- deficient CD8 + T cells provided superior ability to control established tumors.
  • the CD8 + T cell exhaustion signature normally observed within the tumor was instead replaced with a program of functional cytotoxic T cells.
  • IRF2 is a CD8 + T cell-intrinsic nexus that translates signals from the inflammatory TME to adjust gene expression, attenuate cell activation and transcriptionally program T cell exhaustion to prevent tumor control.
  • a modified T cell engineered to have decreased IRF2 expression is provided.
  • the modified T cell described herein for use in the treatment of cancer.
  • the modified T cell described herein for use in adoptive cell therapy.
  • modified T cell described herein in the preparation of a medicament for the treatment of cancer.
  • a method of treating a subject with cancer comprising administering to the subject a therapeutically effective amount of the modified T cell described herein.
  • a method treating a subject with cancer comprising engineering the modified T cell described herein, in vivo in the subject.
  • a method treating a subject with cancer comprising administering to the subject a therapeutically effective amount of an inhibitor of IRF2 function or expression.
  • B Graph showing IRF2 expression (gMFI) in the spleens of naive (N) mice or from mice with MC38 tumors (T), as well as from tumor-infiltrating immune cells. Numbers next to the cell type indicate the fold change between IRF2 expression in the tumor compared to the spleens from those same mice. * p ⁇ 0.01 .
  • Data are representative of at least two independent experiments containing 5 or more mice per group in each experiment. A total of 5 human melanoma tumors were assessed for IRF2 expression., *p ⁇ 0.01 , ** p ⁇ 0.001 , *** p ⁇ 0.0001.
  • Tumor growth kinetics in WT or Irf - 1 - mice that received isotype control or anti-CD8 depleting antibody either (A) one day before (early CD8 + T cell depletion) or (B) 21 days after (late CD8 + T cell depletion) MC38 initiation.
  • Irf2-'- mice were used since WT mice had already reached endpoint by day 21. Shaded region indicates duration of antibody treatment.
  • C MC38 tumor growth in WT control (i.e., Irf2 +I+ , lightest), CD8-IRF2cWT (i.e., Irf 1 * CD8Cre + mice, black), CD8-IRF2cKO (IRF2-deficient only in CD8 + T cells; medium dark), or Irf2-'- (light) mice.
  • WT control i.e., Irf2 +I+ , lightest
  • CD8-IRF2cWT i.e., Irf 1 * CD8Cre + mice, black
  • CD8-IRF2cKO IRF2-deficient only in CD8 + T cells; medium dark
  • FIG. 1 IRF2-deficient CD8 + T cells resist exhaustion and maintain functionality in the TME.
  • FIG. 1 UMAP plots of CyTOF data showing PhenoGraph-defined clusters of WT and Irf2 ⁇ /_ CD8 + TILs on day 12 after MC38 initiation. The bar graph depicts the proportion of each cluster in WT and Irf2-'- mice.
  • the heatmap represents relative expression (normalized z-scores of the arcsinh transformed mean signal intensity; MSI) of the indicated protein in each cluster from panel A compared to the other clusters combined using Wilcoxon rank-sum test.
  • Data are representative of at least three independent experiments. In each experiment, tumors from 4-7 mice were pooled from WT or Irf2-'- mice to obtain sufficient numbers of CD8 + TILs for analysis. * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 , **** P ⁇ 0.0001.
  • IRF2 is highly expressed in activated and ISG-producing mouse and human CD8 + TILs .
  • CD8 + TILs were divided into IRF2 high (upper 30%) and low (lower 30%) levels of IRF2 expression.
  • CD8 + TILs were divided into IRF2 high and low fractions and then clustered as in panel A. Shown is one representative tumor. Bar graph depicts the proportion of each cluster in their respective groups.
  • mice MC38 data are representative of 3 independent experiments, each with at least 4 mice. * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.0001. Unpaired, two-tailed Student’s t-test used to analyze significance of cluster proportions between IRF2 high and IRF2 low groups.
  • FIG. 5 Transcriptional, epigenetic, and gene-binding profiling.
  • A WT and /rf2“ / “CD8 + TILs derived from scRNA-seq data and clustered using Seurat.
  • Bar graph depicts the proportion of each cluster within their respective group.
  • FIG. 6 IRF2 re-routes transcriptional networks and programming.
  • A Bar graph depicts z-scores of IPA-predicted upstream regulator molecules from the DEG dataset comparing c3. Irf2 ' to cO.WT CD8 + TILs. Upstream regulators predicted to be most enriched (Activated) in c3.lrf2-''- CD8 + T cells are shown at the top and those most activated in cO.WT at the bottom.
  • C shows IRF2 expression (gMFI) in dLNs of MC38 tumor-bearing WT mice following treatment beginning at day 9 with either isotype control, anti-IFNAR blocking, anti-IFNy blocking, or dual (anti-IFNy and IFNAR) blocking antibodies.
  • Data are representative of two independent experiments, each with at least 5 mice per treatment condition. *** p ⁇ 0.0001 , unpaired, two-tailed Student’s t-test.
  • Bar graph depicts the number of MC38-infiltrating CD8 + T cells in each cluster in their respective WT and Irf - 1 - groups. A total of 591 cells were analyzed in each group.
  • H Histogram showing BATF expression tumor-specific (P14) WT (black) and Irf - 1 - (light) CD8 + TILs.
  • the numbers in the histograms show the gMFI of BATF expression.
  • the graph summarizes gMFI of BATF expression of tumor-specific CD8 + TILs from five mice.
  • Data are representative of at least three independent experiments.
  • tumors from 4 - 7 mice were pooled from each group (WT or Irf2-'-) in order to obtain sufficient numbers of CD8 + TILs for analysis.
  • Bar graph depicts the number of MC38-infiltrating CD8 + T cells in each cluster in their respective IRF2 high and IRF2 low groups. A total of 3300 cells were analyzed in each group.
  • (D) Bar graph depicts the number of human melanoma-infiltrating CD8 + T cells in each cluster in their respective IRF2 high and IRF2 low groups. A total of 3000 cells were analyzed in each group.
  • E CyTOF plot showing gating of IRF2 high and IRF2 low fractions in human melanoma CD8 + TILs .
  • the flow plots show one representative tumor from the five melanoma samples.
  • F Bar plots show Spearman correlation (r) of IRF2 with the indicated protein in human melanoma CD8 + TILs.
  • Bar graph depicts the number of MC38-infiltrating CD8 + T cells in each cluster in their respective WT and Irf2 ' groups. A total of 679 cells were used in the scRNA-seq data analysis. Data representation is downsampled to 110 cells for each group.
  • Target/Background is the enrichment of motifs in the target peaks divided by the enrichment of those motifs in a random Homer simulated k-mer background; i.e., the number of target-peaks that have the indicated motif divided by the number of background-peaks that have the same motif.
  • CD8 T cells counts in naive and anti-CD3/CD28 stimulated conditions Absolute number of total T cells and CD8 T cells after 3 days incubation with anti- CD3/CD28 antibodies (i.e., activated cells) or medium alone (i.e., naive cells).
  • Human T cells were isolated and transfected with either nontargeting control ribonucleoprotein (RNP) gRNA/Cas9 complex or IRF2 RNP gRNA/Cas9 complex. The absolute number of CD8 T cells was calculated based on the total T cell manual counting followed by the percentage of CD8 T cells detected by flow cytometry.
  • RNP nontargeting control ribonucleoprotein
  • FIG. 13 CRISPR-mediated IRF2 deletion in primary human CD8 T cells -prior to culture.
  • Total T cells including CD4 T cells, CD4 Treg cells and CD8 T cells
  • T cells were isolated from primary human PBMC. Following isolation, the T cells were electroporated with IRF2 targeting (sglRF2) and control non-targeting (sgControl) sgRNAs and then cultured for 4 days.
  • the plots show IRF2 expression in the control and IRF2 targeted CD8 T cells 4 days after electroporation. Numbers in each plot show the percentage of IRF2 experessing CD8 T cells.
  • To achieve CRISPR deletion of IRF2 Primary human T cells were isolated using EasySep Human T cell isolation kit (STEMCELL, cat. no.
  • a crRNA targeting a non-essential gene was used for negative control (Alt-R® CRISPR-Cas9 Negative Control crRNA #1 ; IDT, cat. no. 1072544).
  • the RNP complex was mixed with the cell suspension and transferred into a 16-well nucleocuvette strip (Lonza, cat. no. V4XP-3032). Cells were transfected using program EH113 and buffer P3 on the 4D-Nucleofector system (4D- Nucleofector X unit, Lonza, cat. no. AAF-1003X).
  • FIG. 14 Activation of control and IRF2 deleted primary human CD8 T cells.
  • Total T cells including CD4 T cells, CD4 Treg cells and CD8 T cells
  • T cells were isolated from primary human PBMC. Following isolation, the T cells were electroporated with IRF2 targeting (sglRF2) and control non-targeting (sgControl) sgRNAs and then cultured for 4 days. IRF2 deletion was confirmed by flow cytometry.
  • the control and IRF2-deleted T cells were activated with anti-CD3 (10ug/ml), anti-CD28 (1 ug/ml) and IL-2 (20ng/ml) in T cell complete media. Cells that are kept naive have their media changed in place of activation.
  • Activated and naive cells are cultured for 3 days and are then assessed via flow cytometry.
  • the plots show Forward Scatter (FSC) and Side Scatter (SSC) of CD8 T cell in the indicated condition. This data show that without activation, the IRF2 deleted T cells remain in an unactivated/naive state similar to the control cells and that the IRF2-deleted T cells can be activated and maintained similar to control treated cells.
  • FSC Forward Scatter
  • SSC Side Scatter
  • FIG. 16 Assessment of IRF2 expression in CD8 T cells - Day 2 post-transfection. Optimization of Cas9/gRNA ribonucleoprotein (RNP) transfection for CRISPR/Cas9- Mediated IRF2 knockout (KO) in human T cells at different pulse codes (EH113: and CM137:). Half offset (left) or overlaid (right) histograms show IRF2 expression on human CD8 T cells transfected with nontargeting (nt) control RNP gRNA/Cas9 complex or IRF2 RNP gRNA/Cas9 complex. Flow cytometric analysis was performed on day 2 post-transfection.
  • RNP Cas9/gRNA ribonucleoprotein
  • FIG. 17 Assessment of IRF2 expression in CD4 T cells - Day 2 post-transfection. Optimization of Cas9/gRNA ribonucleoprotein (RNP) transfection for CRISPR/Cas9- Mediated IRF2 knockout (KO) in human T cells at different pulse codes (EH113: and CM137:). Half offset (left) or overlaid (right) histograms show IRF2 expression on human CD4 T cells transfected with nontargeting (nt) control RNP gRNA/Cas9 complex or IRF2 RNP gRNA/Cas9 complex. Flow cytometric analysis was performed at day 2 post-transfection.
  • RNP Cas9/gRNA ribonucleoprotein
  • FIG. 18 Assessment of IRF2 expression in CD8 T cells - Day 4 post-transfection. Optimization of Cas9/gRNA ribonucleoprotein (RNP) transfection for CRISPR/Cas9- Mediated IRF2 knockout (KO) in human T cells at different pulse codes (EH113: and CM137:). Half offset (left) or overlaid (right) histograms show IRF2 expression on human CD8hi T cells transfected with nontargeting (nt) control RNP gRNA/Cas9 complex or IRF2 RNP gRNA/Cas9 complex. Flow cytometric analysis was performed on day 4 post-transfection. Black histogram depicts no antibody staining control on the same flow cytometry channel as IRF2 (this is a way to measure inherent autofluorescence in the stain).
  • RNP Cas9/gRNA ribonucleoprotein
  • FIG. 19 Assessment of IRF2 expression in CD4 T cells - Day 4 post-transfection. Optimization of Cas9/gRNA ribonucleoprotein (RNP) transfection for CRISPR/Cas9- Mediated IRF2 knockout (KO) in human T cells at different pulse codes (EH113: and CM137:). Half offset (left) or overlaid (right) histograms show IRF2 expression on human CD4 T cells transfected with nontargeting (nt) control RNP gRNA/Cas9 complex or IRF2 RNP gRNA/Cas9 complex. Flow cytometric analysis was performed on day 4 post-transfection. Black histogram depicts no antibody staining control on the same flow cytometry channel as IRF2 (this is a way to measure inherent autofluorescence in the stain).
  • RNP Cas9/gRNA ribonucleoprotein
  • Figure 20 Assessment of IRF2 expression in CD8 T cells - Day 7 post-transfection. Optimization of Cas9/gRNA ribonucleoprotein (RNP) transfection for CRISPR/Cas9- Mediated IRF2 knockout (KO) in human T cells at different pulse codes (EH113: and CM137:). Half offset (left) or overlaid (right) histograms show IRF2 expression on human CD8 T cells transfected with nontargeting (nt) control RNP gRNA/Cas9 complex or IRF2 RNP gRNA/Cas9 complex. Flow cytometric analysis was performed on day 7 post-transfection.
  • RNP Cas9/gRNA ribonucleoprotein
  • Black histogram depicts no antibody staining control on the same flow cytometry channel as IRF2 (this is a way to measure inherent autofluorescence in the stain). Overall, this figure shows that the IRF2 deleted CD8 T cells can be maintained in culture for at least 7 days.
  • Figure 21 Assessment of IRF2 expression in CD4 T cells - Day 7 post-transfection. Optimization of Cas9/gRNA ribonucleoprotein (RNP) transfection for CRISPR/Cas9- Mediated IRF2 knockout (KO) in human T cells at different pulse codes (EH113: and CM137:). Half offset (left) or overlaid (right) histograms show IRF2 expression on human CD4 T cells transfected with nontargeting (nt) control RNP gRNA/Cas9 complex or IRF2 RNP gRNA/Cas9 complex. Flow cytometric analysis was performed on day 7 post-transfection.
  • RNP Cas9/gRNA ribonucleoprotein
  • Black histogram depicts no antibody staining control on the same flow cytometry channel as IRF2 (this is a way to measure inherent autofluorescence in the stain). Overall, this figure shows that the IRF2 deleted CD4 T cells can be maintained in culture for at least 7 days.
  • IRF2 deletion increases ISG upregulation in response to IFN
  • T cells were electroporated with IRF2 deleting sgRNAs or control sgRNAs and cultured for 4 days (time for IRF2 protein to be downregulated). The T cells were then stimulated with media (unstim), IFN
  • Expression of ISG 15 measured by flow cytometry comparing IFNp-stimulated, IFNv-stimulated and unstimulated T cells within Irf2-deleted and non-deleted CD4 (left) and CD8 (right) T cells. Numbers indicate geometric mean fluorescence intensity for each histogram.
  • FIG. 23 Total T cells were negatively selected from viably frozen human PBMC. Different volumes of control (WT) or IRF2-targeted (KO) Cas9 RNP mix were then tested for IRF2 deletion in CD8 and CD4 T cells. IRF2 expression was analyzed by flow cytometry two and four days after CRISPR-Cas9 RNP treatment. Electroporation for IRF2 RNP versus control RNP by electroporation was performed in the presence of an Enhancer. Following treatment, the cells were cultured in complete medium + human IL-7. The number in each histogram indicates the percentage of IRF2- expressing cells.
  • the data show that IRF2 can be deleted in 91% of CD8 T cells and 84% of CD4 T cells by four days after CRISPR-Cas9 deletion.
  • the 2.5ul Cas9-RNP concentration is used in subsequent experiments, although both concentrations tested effectively delete IRF2 from human, primary CD8 and CD4 T cells.
  • FIG. 24 Total T cells were negatively selected from viably frozen human PBMC.
  • a carrier DNA (enhancer) was added to the control (WT) or IRF2-targeted (KO) CRISPR- Cas9 reaction, and the IRF2 deletion efficiency (%) in human CD8 and CD4 T cell was analyzed by flow cytometry 4 days later. Following treatment, the cells were cultured in complete medium + human IL-7. The number in each histogram indicates the percentage of IRF2-expressing cells. The data show that the reaction works quite effectively without the enhancer, but that the deletion efficiency is increased using the enhancer.
  • FIG 25 Total T cells were negatively selected from viably frozen human PBMC.
  • the T cells were then electroporated with control (WT) or IRF2-targeted (KO) Cas9 RNP mix either a single time (as in figures 1 and 2) or two times. After 2 days, half of the cells were electroporated again for the control or the IRF2-targeted RNP. After 4 days (6 days in total), all cells were assessed for IRF2 expression by flow cytometry. Following treatment, the cells were cultured in complete medium + human IL-7. The number in each histogram indicates the percentage of IRF2-expressing cells. No enhancer was used in these experiments. The 2-times treatment worked somewhat better, but also substantially decreased viability of the cells. Therefore, the single electroporation approach is used in subsequent protocols.
  • Jurkat T cells are an immortal human T lymphocyte line.
  • Jurkat T cells were thawed, expanded and electroporated with the control (WT) or IRF2-targeted (KO) Cas9-RNP in the presence of an Enhancer. Cells were seeded in complete medium + human IL-7. After 4 days, single cells were FACSorted and subsequently expanded. One of these control (top) and 5 of these IRF2-targeted clones were again assessed for IRF2 expression by flow cytometry. The second IRF2-deleted clone will be used for further experiments. Use of these Jurkat T cell clones provides controls for IRF2 expression. These will be used to as controls to confirm IRF2 expression in the human T cells.
  • FIG. 27 Total T cells were isolated from viably frozen PBMCs by negative selection. Electroporation with control (WT) or IRF2-targeted Cas9-RNP versus control RNP was performed in the presence of an Enhancer. Cells were seeded in complete medium + human IL-7 and after 4 days, IRF2 expression was measured by flow cytometry. In this experiment, the IRF2-targeted Cas9-RNP deleted IRF2 expression in 90% of CD8 T cells and 83% of CD4 T cells. On day 7 after the initial CRISPR-Cas9 treatment, the cells were split and treated with media alone or stimulated with 1000 Units IFN[3.
  • ISG15 and PD-L1 two IFN-stimulated proteins
  • flow cytometry The data show that IRF2-deletion led to increased expression of ISG15 and to a lesser extent PD-L1 following IFN[3 treatment.
  • the increased PDL1 expression is consistent with the data showing that mice with IRF2 deleted CD8 T cells exhibited enhanced responses interferons.
  • ISG15 and PD-L1 two IFN-stimulated proteins
  • flow cytometry The data show that IRF2-deletion led to slightly increased expression of ISG15 and PD-L1 following IFNy treatment.
  • the increased PDL1 expression is consistent with the data showing that mice with IRF2 deleted CD8 T cells exhibited enhanced responses to interferons.
  • FIG. 29 Total T cells were isolated from viably frozen PBMCs by negative selection. Electroporation with control (WT) or IRF2-targeted Cas9-RNP versus control RNP was performed in the presence of an Enhancer. Cells were seeded in complete medium + human IL-7 and after 4 days, IRF2 expression was measured by flow cytometry. In this experiment, the IRF2-targeted Cas9-RNP deleted IRF2 expression in 90% of CD8 T cells and 83% of CD4 T cells. On day 7 after the initial CRISPR-Cas9 treatment, the cells were split and treated with media alone or stimulated with 1000 Units IFN[3 + 1000ng IFNy.
  • ISG15 and PD-L1 two IFN- stimulated proteins
  • flow cytometry The data show that IRF2- deletion led to increased expression of ISG15 and to a lesser extent PD-L1 following IFN[3 treatment.
  • the increased PDL1 expression is consistent with the data showing that mice with IRF2 deleted CD8 T cells exhibited enhanced responses to interferons.
  • FIG. 30 Total T cells were isolated from viably frozen PBMCs by negative selection. Electroporation with control (WT) or IRF2-targeted Cas9-RNP versus control RNP was performed in the presence of an Enhancer. Cells were seeded in complete medium + human IL-7. After 4 days, CRISPR-mediated IRF2 deletion was confirmed by flow cytometry (CD8 T cells: 90% deleted for IRF2). Half of the cells were kept unstimulated, while the other half was activated for 3 days with anti-CD3 (2ug/mL), anti-CD28 (1 ug/mL) and IL-2 (20ng/mL). (A) Numbers in each plot indicate the percent of live CD8 T cells. (B) Histograms show the FSC of unstimulated and anti-CD3 + CD28 stimulated CD8 T cells. Increase in FSC indicates CD8 T cell activation and blasting.
  • FIG. 31 CRISPR-mediated IRF2 deletion in mouse tumor-specific (P14) CD8 T cells.
  • P14 T cells are murine T cells that transgenically express rearranged TCR genes recognizing the LCMV-GP33-41 peptide presented in the context of murine H2Db.
  • P14 T cells were isolated by negative selection from the spleen of naive P14 mice. The P14 T cells were then electroporated with control (WT) or IRF-2-targeted (KO) Cas9- RNP and cultured in complete medium + mouse IL-7. After 4 days, all cells were assessed for IRF2 expression by flow cytometry. The number in each histogram indicates the percent of P14 T cells that express IRF2.
  • mice receiving control or IRF2-targeted P14 T cells were stimulated with anti-CD3 + anti-CD28 antibodies for 24 hours. Then 350,000 of the control or IRF2-targeted P14 cells were adoptively transferred into mice with established MC38-GP tumors (day 8 after tumor inoculation).
  • MC38 is an adenocarcinoma tumor cell line that we engineered to express the LCMV-GP1-100 peptide sequence.
  • the tumor size in mice receiving control or IRF2-targeted P14 T cells was measured on day 8 (just prior to adoptive P14 T cell transfer) and on day 23 after tumor implantation. Error bars represent mean ⁇ SEM. One-way ANOVA for multiple comparisons.
  • IFNs Interferon Regulatory Factor 2
  • IRF2 Interferon Regulatory Factor 2
  • CD8 + T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor, instead enabling sustained effector functions that promoted long-term tumor control, and increased responsiveness of immune-checkpoint and adoptive cell therapies.
  • the long-term tumor control by IRF2-deficient CD8 + T cells required continuous integration of both IFN-I and IFN-II signals.
  • IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a new target to enhance cancer control.
  • a modified T cell engineered to have decreased IRF2 expression In an aspect therefore, there is provided a modified T cell engineered to have decreased IRF2 expression.
  • T cells may be modified using methods of genetic engineering (also referred to as genetic modification or genetic manipulation) that modify and manipulate a cell’s genes using techniques known in the art.
  • genetic engineering also referred to as genetic modification or genetic manipulation
  • the modified T cell has been engineered using virus-mediated knockdown. In some embodiments, the modified T cell has been engineered using CRISPR/Cas9.
  • the T-cell is a regulatory T cell.
  • the T-cell is an effector T cell.
  • the T-cell is CD4+.
  • the T-cell is CD8+.
  • the T-cell is CD4- and/or CD8-.
  • the T-cell is a CAR T cell.
  • the IRF2 gene has been knocked-down in the modified T cell.
  • the IRF2 gene, or portions thereof, have been deleted in the modified T cell.
  • the T cell is a human T cell.
  • the modified T cell has improved anti-tumor activity compared to the corresponding wild-type T-cell.
  • the modified T cell has improved resistance to T cell exhaustion compared to the corresponding wild-type T-cell.
  • the modified T cell described herein for use in the treatment of cancer.
  • the modified T cell described herein for use in adoptive cell therapy.
  • modified T cell described herein in the preparation of a medicament for the treatment of cancer.
  • a method of treating a subject with cancer comprising administering to the subject a therapeutically effective amount of the modified T cell described herein.
  • a method treating a subject with cancer comprising engineering the modified T cell described herein, in vivo in the subject.
  • the method further comprises treating the subject with a checkpoint inhibitor.
  • the method further comprises treating the subject with anti-PDL1 and/or anti-PD1 blockade therapy.
  • a method treating a subject with cancer comprising administering to the subject a therapeutically effective amount of an inhibitor of IRF2 function or expression.
  • inhibitor refers to molecules having the ability to specifically inhibit, wholly or partially, the expression or function of IRF2.
  • inhibitors can include, without limitation, small molecules, antibodies, and nucleic acids (e.g. antisense oligonucleotides, siRNA, shRNA, miRNA...etc.).
  • therapeutically effective amount refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.
  • pharmaceutically acceptable carrier means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.
  • auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.
  • RNA-seq Single-cell RNA-seq, ATAC-Seq and CUT&Tag data have been deposited at GEO. Accession numbers are listed Table 1 : Key Resource Table.
  • mice C57BL/6 mice were purchased from The Jackson Laboratory or the breeding colony at the Princess Margaret Cancer Center, University Health Network (PMCC, UHN).
  • Irf ⁇ - mice (Matsuyama et al., 1993) were kindly provided by Dr. Tak Mak at the PMCC (UHN). Briefly, the Irf?-'- were generated by replacing exon 3 of the Irf2 gene (which encodes amino acids 30 - 63 and is part of the DNA-binding domain of the protein) with a neomycin resistance cassette.
  • mice The Irf2 fll/fl mice (described below) were crossed with CD8a-Cre (E8iii-Cre) transgenic mice (C57BL/6-Tg(CD8a-cre)1 ltan/J, Stock No. 008766, The Jackson Laboratory) to delete IRF2 from peripheral CD8a-expressing T cells.
  • LCMV-GP33-specific CD8 + TCR transgenic (P14) mice have been described previously (Brooks et al., 2006). Mice were housed under specific pathogen-free conditions at the PMCC (UHN). Mouse handling conformed to the experimental protocols approved by the OCI Animal Care Committee at the PMCC (UHN). Experiments were performed using sex and age matched male and/or female mice. Mice used were between 7 and 10 weeks old at the initiation of each experiment.
  • a targeting construct was designed to conditionally delete the 3 rd exon of the mouse interferon regulatory factor 2 gene.
  • primers based on mouse mlrf2 genomic sequence (GenBank Accession No. NC_000074) to use in PCR from mouse C57BL/6 genomic DNA (Jackson Laboratory, Bar Harbor, Maine).
  • PCR primers 5'- GCA CTT AGC GAT CGC AGC TGC TCC TTG GAC CAA TGA CCT T -3'(Jrf2 AsiSI sense) and 5'- AAG TTA AAT CGA TAG AAG ACT CCT GGC GCA TGC TCA GTC -3' (Jrf2 Clal antisense) were used to amplify a 4497 bp 5’ homology-arm fragment (corresponding to mlrf2 intron 2 sequence) from 200 ng of C57BI/6J genomic DNA using the PfuUltra II fusion HS DNA polymerase (Agilent Technologies, Santa Clara CA).
  • a 4555 bp 3’ homology-arm fragment (encompassing exon 4, exon 5, and part of intron 5 of mlrf2) was amplified from C57BI/6J Genomic DNA using the PCR primers 5'- TGG ACC AGT TTA AAC ATA TTG GAA GCT CGT CTC TGC -3' (Jrf2 Pmel sense) and 5'- ATT TAT GCG GCC GCT CAC TTC CTG GAT GAA CAT GGC -3' (Jrf2 Notl antisense).
  • PfuUltra II fusion HS DNA polymerase was also used to amplify a 1146 bp fragment from C57BI/6J genomic DNA spanning the targeted mlrf2 exon 3 using the primers 5' TTC TGG TCT TAA TTA ACT TTA GCA GGA CTA GGA TTA CAG 3' (mlrf2 Ex3 Pad sense) and 5' AAT ATG ATT AAT TAA AAG GTC CAC ATC TAA AGA TAT CTC C 3' (mlrf2 Ex3 Pad antisense).
  • the resulting PCR products were gel-purified using the Nucleospin® Gel and PCR Clean-up system (Machery-Nagel Gmbh & Co., Germany), and TA-overhangs were added via a 20-minute incubation with Taq DNA polymerase (New England BioLabs, Ipswich, MA) at 68°C in the presence of 1 mM dNTPs and 1X Buffer.
  • Nucleospin® Gel and PCR Clean-up system Machery-Nagel Gmbh & Co., Germany
  • TA-overhangs were added via a 20-minute incubation with Taq DNA polymerase (New England BioLabs, Ipswich, MA) at 68°C in the presence of 1 mM dNTPs and 1X Buffer.
  • the fragments were then TA-cloned into pCR2.1-TOPO (Invitrogen, San Diego, CA) and subcloned into a modified pBluescript II KS (Stratagene, La Jolla, CA) vector containing a PGK- neomycin cassette flanked by both LOXP and FRT sequences.
  • pCR2.1-TOPO Invitrogen, San Diego, CA
  • KS Stratagene, La Jolla, CA
  • a diphtheria toxin (OTA) gene was inserted 3’ of the long arm to negatively select against non- homologous targeting. Insert sequence was validated using fluorescent dideoxynucleotide sequencing and automated detection (ABI/Perkin Elmer, Forest City, CA).
  • Targeted disruption of the murine Irf2 gene in ES cells The mlrf2 conditional targeting vector (25 pg) was linearized with Notl restriction endonuclease at the 3’ end of the 3’homology-arm and electroporated into C57BI6/N ES cells (NIH Knockout Mouse Project (KOMP) repository; University of California Davis) using a Bio-Rad Gene Pulser, 0.34 kV, and 0.25 mF. ES cell culture was carried out as previously described (Hakem et al., 1996).
  • homologous recombinants were identified by 5’ and 3’ flanking PCR and confirmed by sequencing following published protocols (Hakem et al., 1996). Homologous recombination at the 5’ homology-arm was confirmed by TerraTM PCR Direct (Takara Bio, Mountain View, CA, USA) amplification of a 4657 bp fragment using the primers I rf2 5PCR Sense: 5’ GCC AGG CCA TTT GTT TAG GAA TGC AGG AG -3’, in the flanking sequence of mlrf2 intron 2, and the vector-specific primer PCRA antisense: 5'- CGA CGG TCA ACG AGC AGT CCA GCG TAT CC -3'.
  • mice were produced by microinjection of independent mlrf2 + /- ES cell clones into E3.5 C57BL/6J blastocysts and transferred to pseudopregnant foster mothers. Chimeric males were mated with C57BI/6J females (Jackson Laboratory). Germ line transmission of the mutant allele was confirmed by PCR analysis of tail DNA from mice with an agouti coat color.
  • the PGK-Neo cassette was removed from mouse Irf2 +t mice by crossing with Flp-deleter mice (Jackson Laboratory stock #009086) (Farley et al., 2000) and PCR genotyping and sequence validation of recombination at the FRT sites.
  • MC38 tumor cells derived from C57BL/6 murine colon adenocarcinoma were a generous gift from Dr. Daniel de Carvalho at the PMCC (UHN) and were cultured in McCoy 5A medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin, 1% streptomycin and 1% L-glutamine.
  • FBS heat-inactivated fetal bovine serum
  • penicillin 1%
  • streptomycin 1% L-glutamine
  • 1x10 5 B16-F10 tumor cells were injected s.c. into one hind leg of each mouse.
  • the MMTV-PyMT cell line was a generous gift from Dr. Christopher Page at the PMCC (UHN) and was cultured in McCoy 5A medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin, 1% streptomycin and 1% L- glutamine.
  • FBS heat-inactivated fetal bovine serum
  • penicillin 1%
  • streptomycin 1% L- glutamine
  • LCMV-GP33-41 -specific CD8 + P14 T cells were isolated from the spleens and lymph nodes of C57BL/6 and Irf2 ' transgenic mice by negative selection (Stem Cell Technologies). For pre-tumor transfer, 2x10 5 naive P14 T cells were transferred i.v. in the retro-orbital sinus. For therapeutic transfer experiments, P14 T cells were activated for 24 hours using plate-bound anti-CD3 (clone 2C11 , 10pg/mL) and soluble anti- CD28 (1 g/mL) antibodies. Cells were then counted and 2x10 5 P14 cells were transferred i.v. in the retro-orbital sinus nine days following tumor implantation.
  • tumors were cut into small fragments and digested in RPMI media containing collagenase I (100U/mL, ThermoFisher), DNasel (10ug/mL, Sigma) and 2% FBS in a gentleMACS dissociator (Cat#130-093-235 Miltenyi Biotec) using the gentleMACS program 37c_m_TDK_1.
  • Cells were filtered through a 70pm pre-separation filters (Cat#130-095-823 Miltenyi Biotec) and subjected to red blood cell lysis.
  • CD45 + tumor infiltrating lymphocytes were isolated from the digested single-cell suspensions using Mouse CD45 (TIL) Microbeads (Cat#110-021- 618 Miltenyi Biotec), autoMACS columns (Cat#130-021 -101 Miltenyi Biotec) and an autoMACS Pro Separator (Miltenyi Biotec) following the manufacturer’s instructions. Purified CD45 + single-cell suspensions from tumors from at least 5 mice per condition were pooled before staining.
  • Antibodies directly conjugated to metal tags were purchased from Fluidigm. Purified unconjugated antibodies were labeled with metal tags at the SickKids-UHN Flow and Mass Cytometry Facility using the MaxPar Antibody Labeling Kit from Fluidigm. CyTOF staining was performed as previously described(Snell et al., 2021). Briefly, mouse single cell suspensions (up to 5x10 6 cells) were pulsed with 12.5 mM Cisplatin (BioVision) in PBS for 1 min at room temperature (RT) prior to quenching with CyTOF staining media (Mg + /Ca + HBSS containing 2% FBS (Multicell), 10mM HEPES (Corning), and FBS underlay.
  • CyTOF staining media Mg + /Ca + HBSS containing 2% FBS (Multicell), 10mM HEPES (Corning), and FBS underlay.
  • Heatmaps were generated from arcsinh-transformed median of spectral indices (MSI) values, plotted in R using the viridis color package and the ggplots package.
  • the CyTOF data was clustered and analyzed using the Phenograph(Levine et al., 2015) algorithm together with the R implementation of UMAP. An equal number of cells was used for clustering, determined based on the lowest common denominator between WT and Irf - 1 - tumor infiltrating CD8 + T cells.
  • Flow cytometry and intracellular cytokine stimulation were generated from arcsinh-transformed median of spectral indices (MSI) values, plotted in R using the viridis color package and the ggplots package.
  • the CyTOF data was clustered and analyzed using the Phenograph(Levine et al., 2015) algorithm together with the R implementation of UMAP. An equal number of cells was used for clustering, determined based on the lowest common denomin
  • Single-cell suspensions of cells from the indicated tissue or tumor were stained with a zombie aqua viability stain (Biolegend) at 4°C and then surface stained using antibodies against targeted molecules CD45 (30-F11), CD45.2 (104), CD45.1 (A20), CD8a (53-6.7), CD4 (GK1.5), Tcr(3 (H57-597), PD1 (29F.1A12), Lag3 (C9B7W), CD39 (24DMS1), PDL1 (10F.9G2), B220 (RA3-6B2), CD62L (MEL-14), CD25 (PC61), CD86 (GL-1), CD11C (3.9), CD11 b (M1/70), CD44 (IM7), NK1.1 (PK136), Ly6C (HK1.4), Ly6G (1A8).
  • CD45 (30-F11), CD45.2 (104), CD45.1 (A20), CD8a (53-6.7), CD4 (GK1.5), Tcr(3 (H57-597), PD1 (29F.1
  • Single-cell suspensions of tumor cells were counted and restimulated with 2 mg/ml of MHC class l-restricted LCMV peptide GP33-40 in standard complete T cell medium (RPMI supplemented with 10% FBS, 1% penicillin and streptomycin, HEPES, 1% Sodium pyruvate, 1% non-essential amino acids, 1% L-glutamine and 2- mercaptoethanol) containing 50 U/mL recombinant murine IL-2 and 1 mg/mL brefeldin A (Sigma), at 37°C.
  • standard complete T cell medium RPMI supplemented with 10% FBS, 1% penicillin and streptomycin, HEPES, 1% Sodium pyruvate, 1% non-essential amino acids, 1% L-glutamine and 2- mercaptoethanol
  • IFN[3 stimulation of mouse cells Single-cell suspensions of splenocytes from naive mice were counted and incubated with 100U/ml_ IFN[3 for 8 hours in T cell RPMI medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin and 1% L-glutamine. Cells were then surface stained for CD8 (53-6.7), CD4 (GK1.5), B220 (RA3-6B2) and then intracellularly stained for FoxP3 (MF-14) and IRF2 [EPR4644(2), Abeam],
  • CD8 + and CD4 + T cells were depleted by administering 250pg of anti-CD8a (2.43) antibody or anti-CD4(GK1 .5) one day prior to tumor injection.
  • 250pg of anti-CD8a (2.43) antibody or isotype control (LTF-2) was injected from 3 weeks after MC38 tumor injection, for a total of three anti-CD8a treatments (every 2 days).
  • 500pg of anti-PDL1 (10F.9G2) or isotype control (LTF-2) antibody treatment was initiated i.p.
  • mice were treated every 2 days with 500pg of anti-IFNAR (MAR1-5A3) of isotype control (mouse lgG1) for the first 2 treatments and 250pg for subsequent treatments.
  • 500pg of anti-IFNy (XMG1.2) was administered every 2 days. Isotype control antibodies were injected in a similar fashion.
  • Single-cell suspensions of tumor cells were counted and stained with a zombie aqua viability dye (Biolegend) and then surface stained with anti-CD45.2 (A20) antibody. Stained cells were washed and resuspended in RPMI medium supplemented with 10% FBS, 1% penicillin and streptomycin and 1% L-glutamine. At least 8 mice were pooled per group and CD45 + tumor-infiltrating cellsimmune cells were FACSorted on a Moflo Astrios (Beckman Coulter). Sorted cells were labelled with sample multi-plexing antibodies and AbSeq Ab-Oligos (antibody-oligonucleotides) using the BD Mouse Immune Single-Cell Multiplexing Kit (Cat.
  • Seurat analyses Data was further analysed using the R Seurat (v 4.0.4) package (Hao et al., 2021). To filter out low quality cells and poorly expressed genes, we applied the following quality control steps: First, cells with ⁇ 500 read counts, with >25% mitochondrial reads, and expressing ⁇ 700 and >7000 genes in Tumor WT were removed. Second, cells with ⁇ 500 read counts, with >20% mitochondrial reads, and expressing ⁇ 700 and >6000 genes in Tumor IRF2 Z - were excluded. Third, genes expressed in ⁇ 10 cells in each condition were also excluded. Finally, doublets were removed using the Chord R package (v1.0.0).
  • RNA assay Gene expression (RNA assay) data was then normalized using the logNormalize method and the antibody-derived tags (ADTs) assay data using the CLR method. RNA assay was scaled and adjusted by regressing out cell cycle genes and mitochondrial proportion. PCA was performed on most variable genes.
  • WNN weighted nearest neighbor
  • PCs principal components
  • CD8 + T cells from both WT and Irf2 / were identified using both RNA and ADT and reclustered (0.4 resolution). Markers of each cluster were identified for RNA and ADT assays using the FindAIIMarkers functions with default parameters.
  • RNA gene expression was also analyzed using SeqGeq software (v.1.6 or 1.7, BD Biosciences).
  • GSEA Gene set enrichment analysis
  • Irf2 ' vs cO.WT were identified by the FindMarkers function, as mentioned previously, without filtering any genes, and were ranked by sign(log2FC) * -Iog10(pval), where log2FC is the Iog2 fold change of average of gene expression in IRF2 /_ and average in WT and pval represents the P-value calculated using Wilcoxon rank-sum test.
  • Enrichment Map To identify biological processes enriched in cO.WT and c3.lrf2 ' cells, we performed a GSEA analysis for RNA assay data and Gene Ontology Biological Processes gene sets using the GSEA application with default parameters. An Enrichment Map of the GSEA results was generated using the Enrichment Map Visualization tool from the GSEA application. Gene sets included in the Enrichment Map have a P-value ⁇ 0.005 and an FDR ⁇ 0.05 and a cutoff of 0.5 for the overlap coefficient used as a similarity metric between the different gene sets. Enrichment Map was illustrated in Cytoscape (Shannon et al., 2003).
  • Upstream regulator analysis Predicted upstream regulator analysis of differentially expressed genes of c3.lrf2-''- vs cO.WT was performed using the Ingenuity Pathway Analysis software (Qiagen) according to the developer’s instructions.
  • SCENIC analyses' We assessed regulon activity in cO.WT and c3.1 rf 2 ⁇ - CD8 + T cells by performing an analysis of the gene regulatory network using the pyscenic implementation of SCENIC (Aibar et al., 2017) consisting of three steps: 1) identifying regulons based of TF-targets co-expression using GRN, 2) pruning regulons to keep direct targets of TFs using RcisTarget, and finally 3) assessing regulon activity score (RAS) for each regulon on cellular level using AUCell. Due to the stochastic nature of GRN, SCENIC might generate slightly different results on each run.
  • regulons having an FDR ⁇ 0.05, an absolute value of Iog2 fold change (c3./rf2- / - 1 cO.WT) > 0.25 and regulons active in at least 10% of the cells of either of the two compared clusters.
  • Single-cell suspensions of WT, Irf ⁇ -, CD8-IRF2cWT and CD8-IRF2cKO MC38- infiltrating CD8 + T cells were isolated using autoMACS (Miltenyi Biotec), washed and treated with DNase I, and nuclei isolated according to the 10x Genomics nuclei isolation for single cell ATAC sequencing protocol.
  • AT AC libraries were prepared and sequenced (to a target depth of 25,000 reads per cell) on the Novaseq6000 at the Princess Margaret Genomics Center, following the 10x Genomics sequencing workflow and protocol.
  • Reads alignment and quantification base calls were generated using Illumina RTA v3.4.4 as bcl files which were then converted into fastq files using bcl2fastq v2.20 with default parameters. Reads in fastq files were aligned using cellranger-atac count (v 2.0.0) to the reference mm10 genome (i.e., refdata-cellranger-arc-mm10-2020-A- 2.0.0). The chemistry parameter of cellranger-atac count function was set to ARC-v1 , since only scATAC-Seq data was analysed from the multiome data.
  • Peaks annotation and inference of IRF2 motifs The TFBStools (v 1 .32.0) R package in combination with position weight matrices (PWM) taken from the JASPAR2020 (v 0.99.10) database were used to infer occurrence of IRF2 motifs within each peak. Promoters were predicted using the GenomicFeatures (v 1.46.1) R package as 3000 bases upstream and 3000 bases downstream the transcription start site. Peaks that occur at least once in at least 10 cells were considered accessible.
  • Peaks were called using MACS2 v2.2.7.1 (Zhang et al., 2008) with the following parameters: ‘-q 0.01 -nomodel --shift -100 -extsize 200'.
  • Genome-wide IRF2 transcription binding site accessibility was predicted at a p-cutoff of 0.0005 using the TFBStools v1.28.0 R package (Tan and Lenhard, 2016) in coordination with position frequency matrices obtained via the JASPAR2020 v0.99.10 database (Fornes et al., 2020). Bedgraph files from each sample and a bed file of all predicted IRF2 binding sites were visualized using IGV v2.10.3. Additionally, pre-processed normalized counts for the ATAC data were obtained from GSE89308.
  • Peak annotations were cross validated using the ChlPSeeker v1.26.2 R package (Yu et al., 2015). Differential accessibility was inferred using DESeq2 v1.30.1 (Love et al., 2014) using the apeglm v1.12.0 R package (Zhu et al., 2019) for the effect size shrinkage estimations. Finally, all gene-set enrichment analysis and over-representation analyses were done using the clusterprofiler v3.18.1 R package (Yu et al., 2012).
  • Naive CD8 + T cells were isolated from the spleens and lymph nodes of C57BL/6 by negative selection (Stem Cell Technologies). Isolated CD8 + T cells were activated for 3 days using 5pg/mL plate-bound anti-CD3 (16-0031-86 Invitrogen) and 2pg/mL soluble anti-CD28 (16-0281-86 Invitrogen) antibodies in 10% RPMI complete media containing 100U/ml rhlL2 (200-02 PeproTech). Nuclei were isolated and CUT&Tag performed using the protocol from Epicypher (EpiCypher® CUTANATM Direct-to-PCR CUT&Tag Protocol v1.6 Revised: 11.04.2021).
  • Samples were aligned to the mm 10 genome using bowtie v2.4.1 with the following parameters: ‘--local -very-sensitive -no-mixed -no-discordant -phred33 -I 10 -X 700’ and preprocessed using samtools v1.10.
  • a bedgraph file was generated by scaling the reads by a scaling factor defined as 1 ,000,000/number_of_aligned_reads. Regions overlapping the ENCODE mm10 blacklist regions, as defined by Amemiya et al. (Amemiya et al., 2019) were then removed.
  • Peaks were called using SEACR v1.3 (Meers et al., 2019) with a signal_threshold of 1 and run in 'stringent' and 'norm' mode. IRF2 motifs were then mapped to peaks using the catalogue of predicted IRF2 motifs in the mm10 genome described above. By calculating the proportion of peaks containing an IRF2 motif in the IRF2 sample, we established a minimum quantile-based cut-off of 0.81 for the totalsignal which corresponded to the largest increase in IRF2-motif containing peaks.
  • the DNA sequences corresponding to the IRF2 peaks were derived using the bedtools v2.27.1 getfasta tool (Quinlan and Hall, 2010), which was then used as an input for the HOMER v4.8 findMotifsGenome.pl (Duttke et al., 2019) tool with a size parameter of 200. Analysis of transcription factor motifs enriched in IRF2 target peaks was performed using HOMER and ranked based on their Target/Background scores.
  • CRISPR RNAs Pre-designed CRISPR RNAs (crRNAs) targeting IRF2 were annealed to transactivating crRNAs (tracrRNAs) to form a guide RNA (gRNA).
  • the gRNAs were further complexed with Streptococcus pyogenes Cas9 protein (InvitrogenTM TrueCutTM Cas9 Protein v2. Cat.no. A36499) to form the CRISPR-Cas9-gRNA-ribonucleoprotein (RNP) complex at a molar ratio of 1 :2.5 i.e., 30 pmol Cas9 protein to 75 pmol of gRNA).
  • RNP CRISPR-Cas9-gRNA-ribonucleoprotein
  • a combination of different crRNAs targeting IRF2 were used (see table below).
  • a crRNA targeting a non- essential gene (IDT, Ref. 442435748) was annealed with Cas9 at the same molar ratio.
  • 10 6 to 10 7 freshly isolated (primary human or mouse) T cells were resuspended in 20 pl buffer P3 (P3 Primary Cell 4D-Nucleofector X Kit S, Lonza, cat. no. V4XP-3032).
  • the cell suspension, RNP complex mix and where indicated 4uM of an electroporation enhancer non-homologous ssDNA oligonucleotide, IDT, cat. 1075916) were combined, transferred into a 16-well nucleocuvette strip (Lonza, cat. no.
  • transfection control or IRF2-deleted T cells were activated for 3 days with anti-CD3 (2ug/mL), anti-CD28 (1 ug/mL) and IL-2 (20ng/mL).
  • transfection control or IRF2-deleted T cells were treated with media alone or stimulated with 1000 Units IFN(3 and/or 1000ng IFNy.
  • P14 T cells are murine T cells that transgenically express rearranged TCR genes recognizing the LCMV-GP33-41 peptide presented in the context of murine H2D b .
  • P14 T cells were isolated by negative selection from the spleen of naive P14 mice. The P14 T cells were then electroporated with control (WT) or IRF-2-targeted (KO) Cas9-RNP and cultured in complete medium + mouse IL-7. After 4 days, all cells were assessed for IRF2 expression by flow cytometry to determine percent IRF2 deletion.
  • the control or IRF2-targeted P14 T cells were stimulated with anti-CD3 + anti-CD28 antibodies for 24 hours. Then 200,000 to 350,000 of the control or IRF2-targeted P14 cells were adoptively transferred into mice with established MC38-GP tumors (day 8 after tumor inoculation).
  • MC38 is an adenocarcinoma tumor cell line that we engineered to express the LCMV-GP-MOQ peptide sequence.
  • IRF2 is expressed across immune subsets and its deficiency enables tumor control
  • IRF2 immune cell expression within the TME
  • WT IRF2-sufficient wild-type mice
  • MC38 colorectal adenocarcinoma cells and isolated total tumor-infiltrating cells 14 days after tumor initiation.
  • Cells were then analyzed by mass cytometry (CyTOF) with a panel identifying all major and most minor immune cell populations (data not shown).
  • IRF2 was widely expressed across immune cells from mouse MC38 tumors (Fig 1A).
  • TILs T tumor-infiltrating lymphocytes
  • Fig 1 B T tumor-infiltrating lymphocytes
  • human melanoma tumor-infiltrating immune cells possessed a broad IRF2 expression pattern (Fig 1C), indicating conserved immune-wide IRF2 expression within the mouse and human TME.
  • Irf2 ⁇ l - mice had prolonged survival and enhanced control of minimally immunogenic B16-F10 melanoma and an orthotopic polyoma middle T antigen (PyMT) breast tumor (Fig 1 E), indicating that the absence of IRF2 enables control of diverse tumor types.
  • CD4 + T cell subsets in Irf - 1 - mice based on their increased expression of IRF2 within the tumor (Fig 1 B).
  • CD8 + T cell depletion prior to MC38 initiation abolished the tumor control observed in isotype antibody treated Irf - 1 - mice (Fig 2A), underscoring a key role of CD8 + T cells in enabling tumor control in the lrf2- , ⁇ mice.
  • CD8 + T cell depletion in Irf - 1 - mice 3 weeks after MC38 tumor injection rapidly led to rebound of tumor growth (Fig 2B), indicating that CD8 + T cells actively and continually maintained the long-term tumor control in Irf2 ' mice.
  • CD8 + TILs The number of CD8 + TILs was similar between WT and 1 ⁇ 2-'- CD8 + T cells (Fig 8C), indicating comparable expansion/maintenance of the CD8 + TILs.
  • Irf2 floxed mice we created Irf2 floxed mice and crossed them with CD8-E8iii-Cre mice, to generate mice that only lack IRF2 expression in the CD8 + T cells (termed CD8-IRF2cKO mice; Fig 8D).
  • the lack of IRF2 expression in CD8-IRF2cKO mice did not affect the expansion or survival of CD8 + TILs as their numbers were similar to WT (Fig 8E).
  • the CD8- IRF2cKO mice efficiently controlled MC38 tumors in a manner like 1 ⁇ 2-'- mice (Fig 2C), indicating that /rf2-deletion in CD8 + T cells specifically enabled the long-term tumor control.
  • MC38-GP tumor cells To further test the direct role of IRF2 in CD8 + T cells toward tumor control, we engineered MC38 cells to stably express the MHC-I Derestricted lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) 33 -4i epitope (referred to as MC38-GP tumor cells).
  • LCMV lymphocytic choriomeningitis virus
  • GP tumor-GP tumor cells MHC-I Derestricted lymphocytic choriomeningitis virus
  • GP tumor-GP tumor cells MHC-I Derestricted lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) 33 -4i epitope.
  • LCMV lymphocytic choriomeningitis virus
  • GP tumor-GP tumor cells we adoptively transferred naive WT or Irf2 ' LCM -GP 3 3-4i-specific (i.e., tumor
  • mice that received the Irf ⁇ '-PM T cells exhibited enhanced tumor control (Fig 2D). Both the WT and Irf ⁇ -P T cells maintained similar tumor infiltration prior to divergence in tumor sizes (Fig 8F). Further, whereas all mice receiving WT P14 T cells reached endpoint by day 21 , the mice that received Irf2-'- P14 T cells were all alive at day 21 , and 60% were still alive at 25 days (Fig 8G), indicating that a small fraction of Irf2 ' tumor-specific CD8 + T cells can effectively inhibit tumor growth.
  • mice with established PyMT breast tumors with anti-PDL1 blocking antibody were treated with established PyMT breast tumors with anti-PDL1 blocking antibody. Although PyMT breast tumors are controlled better in Irf2 ' compared to WT mice, the tumors still progress, allowing the opportunity for therapeutic intervention to control established tumors.
  • Anti-PDL1 treatment was initiated at day 15, at a time after T cell priming and when tumors in WT and Irf2 ' mice had reached ⁇ 50mm 3 but were still comparable in size.
  • PDL1 blockade in WT mice induced a 2-fold reduction in PyMT tumor growth, whereas a 10-fold reduction was observed in mice (Fig 2E, 8H).
  • the enhanced efficacy of PDL1 blockade was also observed in the CD8-IRF2cKO mice (Fig 8I), indicating that IRF2-deficiency within CD8 + T cells enhances the efficacy of anti-PDL1 immunotherapy.
  • IRF2-deficient CD8 + T cells resist exhaustion and maintain functionality in the TME
  • the Irf?-'- CD8 + T cells exhibited increased expression of CD80, SLAMF1 , Blimpl , Ki67 and CD25 [a protein that associates with an enhanced effector phenotype (Kalia et al., 2010)] compared to their WT counterparts (Fig 3B, 9B).
  • TCF1 + PD1 + CD8 + T cells that have been shown to be capable of self-renewing as well as generating terminally differentiated cytotoxic T cells (Im et al., 2016; Miller et al., 2019; Siddiqui et al., 2019; Utzschneider et al., 2016; Wu et al., 2016) were also comparable between the WT and Irf - 1 - CD8 + T cells (Fig 9D), indicating that IRF2 deficiency does not deplete the TCF1 + stem-like population (Miller et al., 2019; Siddiqui et al., 2019).
  • IR-int population co-expressed Ki67 with BATF and Blimpl (Fig 3H, Fig 9G, 9H), proteins that are associated with sustained effector function (Chen et al., 2021 b; Shin et al., 2009; Xin et al., 2015).
  • the BATF interacting partner, IRF4 that cooperatively limits T cell exhaustion to favor robust effector functions in the tumor (Seo et al., 2021) and in chronic viral infections (Grusdat et al., 2014; Xin et al., 2015), was highly expressed in the Irf2-'- IR-int population (Fig 9I).
  • the Irf2-’- CD8 + T cells expressed increased levels of multiple activation proteins (Fig 3G), indicating that even within the phenotypically similar populations, the IRF2-deficient CD8 + T cells exhibited increased activation and proliferation, and decreased exhaustion profiles.
  • CD8 + T cells expressed the cytolytic protein granzyme B (GzmB) specifically within the IR-int population and did so at higher single cell expression levels (Fig 3J).
  • GzmB cytolytic protein granzyme B
  • WT and Irf - 1 - TILs were stimulated ex vivo with tumor-specific GP33-41 peptide on day 12 after MC38-GP tumor initiation (when tumor sizes were similar).
  • a larger proportion of Irf - 1 - CD8 + TILs produced IFNy and TNFa, with the increase specifically within the IR-int cells (Fig 3K).
  • Irf2-'- CD8 + T cells coexpressed TNFa and IFNy, unlike their WT CD8 + T cell counterparts (Fig 9J).
  • Irf2-'- CD8 + T cells exhibit lower expression levels of IRs, increased expression of cytotoxic molecules, and elevated polyfunctional cytokine production within the TME.
  • IRF2 is preferentially expressed in activated and ISG-producing CD8 + T cells from mouse and human tumors
  • IRF2 expression was analyzed in CD8 + TILs.
  • CD8 + T cells were clustered based on the upper and lower third of IRF2 expression, distinct enrichment patterns emerged (Fig 4A, 10A, 10B).
  • the IRF2-low cells were enriched in TCF1 + clusters (c5, c7, c8) that also expressed low amounts of most activation-induced proteins (Fig 4A-C, 10B), consistent with a less activated state.
  • c4, c6 and almost exclusively c1 were enriched in the IRF2-high fraction and expressed the highest levels of CD44, CD39, CD69, PD1 , Ki67, BATF, GzmB, Helios, and Tbet (Fig 4A-C, 10B).
  • the IRF2-high subsets also expressed increased levels of the ISG Protein Kinase R (PKR), suggesting that IRF2 expression is linked to the strength of IFN-I signaling (Fig 4C).
  • PSR Protein Kinase R
  • the cluster with the highest amount of IRF2 (c4) is also the most activated/terminally differentiated, and is almost entirely absent from the IRF2-low fraction (Fig 4D, 4E, 10E). Indeed, in total melanoma-infiltrating CD8 + T cells, expression of these activation proteins positively correlated with IRF2 expression (Fig 10F). Overall, in both mouse and human CD8 + TILs, IRF2-high expressing cells were enriched for co-expression of all activation-induced proteins measured (Fig 4G + , with IRF2 highest expressed in the most activated subsets.
  • Cluster 0 (accounting for half of the WT cells) was largely absent in the Irf2 ' cells, while c3 accounted for half of the Irf2 ' CD8 + TILs and was almost absent in WT CD8 + TILs (Fig 5A, 11 A).
  • the abundance of c1 , c2, and c4 were largely comparable between WT and Irf - 1 - cells (Fig 5A).
  • the TCF1 + c2 was largely comprised of naive CD8 + T cells [Tcf7 + , SelT, Lef1 + , Cx3crT, PdcdT, EntpdT, Havcr , cd44'°, itgaf (encoding Cd11a), and Icos] and potentially some Tcf7 + , Satb1 + regenerative, stem-like cells, and was equally present in both the WT and /rf? 7- CD8 + TILs (Fig 5A, 5B, and data not shown), suggesting that Irf2 ⁇ does not skew the differentiation of the TCF1 + populations.
  • Cluster 4 are y ⁇ 5 T cells that were present at the same frequencies in WT and /rf2- / - CD8 + TILs (Fig 5A, 5B).
  • cO cO.WT
  • Tox a genes associated with immune dysfunction. These genes included Tox, Nr4a2 [which functions with Tox to drive exhaustion (Chen et al., 2019; Seo et al., 2019)], Irf8 (Mognol et al., 2017), Nt5e [encoding CD73 (Briceno et al., 2021)], Klrel [which negatively regulates cytotoxicity (Westgaard et al., 2003)], Crbn [which is associated with decreased CD8 + T cell activation and effector function (Hesterberg et al., 2020)], and Il2rb [which has been shown to drive terminal exhaustion in chronic viral infections (Beltra et al., 2016)] (Fig 5B, 5C, and data not shown).
  • the predominating ZrfZ 7- CD8 + TIL c3 (c3.lrf2 ⁇ 1 -) exhibited increased expression of cytotoxic genes (Gzma, Gzmb, Gzmk, Stx11, Srgri), inflammatory cytokines and receptors [Cc/3, H2ra, Il12rb2, Tnfrsf4 (encoding 0X40)], NFkb-signaling factors (Trafl, Nfkbid, Nfkbiz, Nfkbia), factors that sustain effector functionality [Batf (Chen et al., 2021b; Grusdat et al., 2014; Xin et al., 2015)], Ttc39c [(encoding Bach2) (Yao et al., 2021)] and numerous ISGs (e.g., Bst2, Ifitl, lfitm1/2, lsg15, Slfnl, lrf7, CD274, G
  • the WT CD8 + TILs also expressed some of the cytotoxic and immune-stimulatory genes, such as Gzmb, Prf1 and Ifng, however they did so at reduced levels compared to the /rf2- z - cells (both in proportions and at a single-cell expression) and with a large fraction of the WT cells co-expressing Tox (Fig 5E). Since the RNA expression is from the cells directly ex vivo (i.e., no in vitro stimulation), the increased levels of Ifng, Prf1 and Gzmb RNAs represent increased production of these anti-tumor factors by the Irf2 CD8 + TILs.
  • the RNA expression is from the cells directly ex vivo (i.e., no in vitro stimulation)
  • the increased levels of Ifng, Prf1 and Gzmb RNAs represent increased production of these anti-tumor factors by the Irf2 CD8 + TILs.
  • CD8 + TILs were enriched in the signature of genes upregulated in Tox-deficient CD8 + T cells (Khan et al., 2019) (Fig 5F + ).
  • IRF2 epigenetically regulates CD8 + TILs we performed ATAC-seq on CD8 + TILs from WT vs /rfZ 7- mice or from CD8- IRF2cWT and CD8-IRF2cKO mice.
  • CUT&Tag Cleavage Under Targets & Tagmentation
  • IRF2 interacted with many genes including those associated with immune dysfunction (Tox, Nr4a3, Lag3, Ctla4), immune-stimulatory and pro- inflammatory functions (Statl, Traf2, Nfkbid, Nfkbie, Ifnab, Tbx21, Prdml, Cd3e, Il12rb1), protein translation (Eif2ak4, Eifla, Eif3e, Eif3h, Eif4e3), as well as numerous ISGs (Isg15, lsg20, Ifitl, Bst2, Usp18, CD274, Gbps2-10) (Fig 5J, and data not shown).
  • IRF2 binding genes were also enriched in motifs for other transcription factors (TFs).
  • HOMER motif analysis indicated that the IRF2 target genes were enriched for TF motifs involved in immune activation (IRF1 , ISRE, BATF, AP-1 , PRDM1 , IRF4) as well as repression of effector responses including Fli1 (Chen et al., 2021c) (Fig 11 C, and data not shown).
  • IRF2 independently and/or through transcription factor complexes
  • IRF2 interacts with genes enriched in a variety of pathways including IFN signaling, TNFO/NF-K
  • Regulators for other pro-inflammatory and immune-activating pathways were also predicted to be activated in the 1 ⁇ - cells, including CD3 (TCR), cytokines involved in sustained T cell function (IL2, IL21 , IL12), NF-K
  • upstream regulators for networks controlling cellular proliferation and metabolism were inhibited in the Irf2-'- cells (Fig 6A, and data not shown).
  • IFN signaling was increased in mouse CD8 + T cells above its constitutive expression levels (Fig 7A), and many (but not all) IFN-I and IFN-II signaling-associated molecules were increased at the protein level in /rf2- z - CD8 + T cells (Fig 7B), demonstrating that in vivo in the TME, IRF2 restricts the level of IFN-I and IFN-II signaling by CD8 + T cells.
  • IFNs To next determine whether IFNs continue to increase IRF2 expression in the context of cancer, we used anti-IFNAR and anti-IFNy antibodies to respectively block IFN-I and IFN-II signaling in WT mice with established MC38-tumors. Blocking either IFN-I or IFN-II signaling alone, and particularly in combination, substantially reduced IRF2 levels in CD8 + T cells (Fig 7C). Thus, IFNs induce and actively sustain the heightened IRF2 expression by CD8 + T cells, which subsequently feeds-back to limit IFN signaling.
  • IRF2 is a CD8 + T cell-intrinsic feedback inhibitor that translates IFN signals from the TME to transcriptionally program T cell exhaustion and, consequently, prevents long-term tumor control.
  • IRF2 IRF2-deleted CD8 T cells can be efficiently activated (Figs 12-15) which is necessary for transduction of the CAR T constructs. Additionally, referring to Figs 16-21 , we show knockdown on IRF2 expression in human CD4 and CD8 T cells (at Days 2, 4, and 7 post-transfection).
  • FIG 22 shows expression of ISG 15 measured by flow cytometry comparing IFN
  • FIGS 27-29 demonstrate that IRF2-deletion lead to increased expression of the interferon-stimulated proteins ISG 15 and to a lesser extent PD-L1 following IFN
  • the increased PDL1 expression is consistent with the data showing that murine IRF2-deleted CD8 T cells exhibited enhanced responses to interferons.
  • the data in Figure 30 demonstrates that primary human T cells deleted of IRF2 can be activated, induced to blast, and have viability similar to their electroporation control CRISRP-Cas9 T cell counterparts following anti-CD3 + anti- CD28 stimulation (mimicking TCR and CD28 mediated stimulation).
  • Figure 31 demonstrates that compared to control tumor-specific CD8 T cells, adoptive transfer of CRISPR-Cas9 IRF2-deleted murine tumor-specific CD8 T cells delay growth of established MC38 adenocarcinoma tumors and prolong survival of tumor-bearing mice.
  • IFNs molecularly switch from pro-inflammatory to suppressive outcomes has long been a subject of interest.
  • IRF2 as a keystone factor that translated IFN signals within the TME to temper inflammation. This feedback inhibition then suppressed the CD8 + T cell response and allowed tumor escape from immune control. In essence, IRF2 re-routed IFN induced transcriptional programming from pro- to antiinflammatory signaling. In the absence of IRF2, these exhaustion promoting functions of IFNs were bypassed and CD8 + T cells instead retained high levels of anti-tumor activity and were able to effectively control tumor growth within the otherwise suppressive TME.
  • IFN-I signaling would continue to promote robust CD8 + T cell effector function is counter-intuitive based on the established role of prolonged IFN signaling to instead switch to drive immune suppression and CD8 + T cell exhaustion in multiple chronic disease contexts.
  • the fact that this entire shift in the IFN programming focuses down to a single molecule was also unexpected, given the diversity of the ISGs driven by IFN signaling.
  • the similarity between IRF2 expression and distribution in mouse and human CD8 + TILs implies a conserved role of IRF2.
  • IRF2 is a foundational feedback mechanism that quells IFN-induced inflammatory reactions, and as a result, is a central regulator of T cell exhaustion in cancer.
  • IRF2 insulin receptor 2
  • Blimpl a transcription factor that directly mediate and counter T cell exhaustion
  • BATF a transcription factor that directly mediate and counter T cell exhaustion
  • IRF4a NFAT
  • Nr4a NFAT
  • Tox a transcription factor that directly mediate and counter T cell exhaustion
  • cytotoxic genes include Blimpl , BATF, IRF4, NFAT, Nr4a and Tox.
  • proinflammatory cytokines as well as factors that sustain effector CD8 + T cells functions were enhanced following deletion of IRF2.
  • the CD8 + T cells lose the Tox gene expression signature and resist exhaustion but resist the overstimulation-induced cell death in the TME.
  • IRF2 regulates a multi-directional response that instills exhaustion, while also suppressing the expression of multiple transcriptional nodes that sustain effector function in times of chronic antigen stimulation.
  • Irf2-'- CD8 + T cells strong candidates for adoptive CAR T cell therapies at cell numbers well below those required for WT CD8 + T cells.
  • modulating IRF2 represents a new target for multiple therapeutic modalities aimed to enhance tumor-specific T cell functions and control tumor growth.
  • IRF2 is a key factor that negatively regulates IFN signaling, immune activation, and CD8 + T cell function, ultimately impeding the ability to control multiple mouse tumors.
  • IRF2 expression is similar in mouse and human melanoma TILs.
  • Irf - 1 - CD8 + T cells for adoptive CAR T cell therapies are anticipated to be effective against human cancers.
  • IRF2 may synergize with immune- checkpoint therapies to fight less immunogenic tumors.
  • Cancer control is a complex process, involving a variety of IRF2-expressing cell types that are also capable of IFN signaling. It is likely that IRF2 in these other cells also contribute to the anti-tumor immune response and cancer control.
  • IRF-2 regulates NF-kappaB activity by modulating the subcellular localization of NF-kappaB. Biochemical and biophysical research communications 370, 519-524.
  • NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530-534.
  • BATF regulates progenitor to cytolytic effector CD8(+) T cell transition during chronic viral infection. Nat Immunol 22, 996-1007.
  • Tumor-associated B7- H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8, 793-800.
  • NF kappa B and interferon regulatory factor 1 physically interact and synergistically induce major histocompatibility class I gene expression. J Interferon Cytokine Res 15, 1037-1045.
  • Interferon regulatory factor-2 physically interacts with NF- kappa B in vitro and inhibits NF-kappa B induction of major histocompatibility class I and beta 2-microglobulin gene expression in transfected human neuroblastoma cells. Journal of neuroimmunology 63, 157-162.
  • JASPAR 2020 update of the open-access database of transcription factor binding profiles. Nucleic Acids Res 48, D87-D92.
  • IRF2 interferon regulatory factor 2
  • IRF4 and BATF are critical for CD8(+) T-cell function following infection with LCMV.
  • Cell Death Differ 21 1050-1060.
  • the tumor suppressor gene Brcal is required for embryonic cellular proliferation in the mouse. Cell 85, 1009-1023.
  • the NF-kappaB regulator Bcl-3 restricts terminal differentiation and promotes memory cell formation of CD8+ T cells during viral infection.
  • MSigDB The Molecular Signatures Database
  • MSigDB Molecular signatures database 3.0. Bioinformatics 27, 1739-1740. Lou, X., Sun, S., Chen, W., Zhou, Y., Huang, Y., Liu, X., Shan, Y., and Wang, C. (2011). Negative feedback regulation of NF-kappaB action by CITED2 in the nucleus. J Immunol 186, 539-548.
  • IFN regulatory factor-2 cooperates with STAT1 to regulate transporter associated with antigen processing- 1 promoter activity. Journal of immunology 174, 3948-3958.
  • TOX is a critical regulator of tumour-specific T cell differentiation. Nature 571 , 270-274. 60.
  • IFN regulatory factor (IRF)-1 and IRF-2 involvement of IFN regulatory factor (IRF)-1 and IRF-2 in the formation and progression of human esophageal cancers. Cancer research 67, 2535-2543.
  • clusterprofiler 4.0 A universal enrichment tool for interpreting omics data. Innovation (Camb) 2, 100141.
  • IRF-2 The oncogenic transcription factor IRF-2 possesses a transcriptional repression and a latent activation domain. Oncogene 9, 1423-1428. 85. Yan, Y., Zheng, L, Du, Q., Yan, B., and Geller, D.A. (2020). Interferon regulatory factor 1 (IRF-1) and IRF-2 regulate PD-L1 expression in hepatocellular carcinoma (HOC) cells. Cancer Immunol Immunother 69, 1891- 1903.
  • Interferon regulatory factor (I RF)-1 and IRF- 2 are associated with prognosis and tumor invasion in HCC. Annals of surgical oncology 20, 267-276.
  • clusterProfiler an R package for comparing biological themes among gene clusters. OMICS 16, 284-287.
  • ChlPseeker an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31 , 2382-2383.

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Abstract

La présente invention concerne des lymphocytes T modifiés pour présenter une diminution de l'expression de l'IRF2, ainsi que leur procédé de fabrication et d'utilisation dans le cadre d'une thérapie anticancéreuse.
EP23887239.4A 2022-11-09 2023-11-08 Ciblage de l'irf2 pour une thérapie anticancéreuse Pending EP4615961A1 (fr)

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