EP4619425A1 - Protéines de fusion et leurs utilisations - Google Patents

Protéines de fusion et leurs utilisations

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Publication number
EP4619425A1
EP4619425A1 EP23889884.5A EP23889884A EP4619425A1 EP 4619425 A1 EP4619425 A1 EP 4619425A1 EP 23889884 A EP23889884 A EP 23889884A EP 4619425 A1 EP4619425 A1 EP 4619425A1
Authority
EP
European Patent Office
Prior art keywords
tgfbrii
cells
cell
car
seq
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
EP23889884.5A
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German (de)
English (en)
Inventor
Paul Joseph NEESON
Criselle Madona D'SOUZA
Jiang Zhu
Mengfei QIN
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.)
Peter MacCallum Cancer Institute
Original Assignee
Peter MacCallum Cancer Institute
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Priority claimed from AU2022903406A external-priority patent/AU2022903406A0/en
Application filed by Peter MacCallum Cancer Institute filed Critical Peter MacCallum Cancer Institute
Publication of EP4619425A1 publication Critical patent/EP4619425A1/fr
Pending legal-status Critical Current

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    • 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
    • A61K40/34—Antigenic peptides
    • 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
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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]
    • 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
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • 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
    • A61K2239/49—Breast
    • 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
    • A61K2239/58—Prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure relates generally to fusion proteins comprising a truncated transforming growth factor beta receptor 2 (TGFBRII) and one or more intracellular' co -stimulatory domains.
  • TGFBRII truncated transforming growth factor beta receptor 2
  • CAR-T cells Chimeric antigen receptor (CAR)-T cells have performed poorly to date in clinical trials in patients with advanced solid cancers. There are many potential reasons for this, including poor CAR-T cell persistence in the patient, heterogeneous target antigen expression, poor trafficking of CAR-T cells into the tumour and tumour microenvironment (TME)-induced immune suppression. This immune suppression is mediated by many soluble factors, including TGF-p.
  • TGF-P is highly expressed in advanced refractory tumours and has a profound effect on T cell effector function (cytotoxicity and cytokine secretion) and T cell proliferation.
  • TGF-P is expressed at higher levels across multiple cancer sub-types, where it inhibits effector T cell anti-tumour responses and is associated with T cell exhaustion.
  • increased levels of TGF-P can also increase FOXP3 expression levels skewing CD4 + T cell polarization to a suppressor phenotype (as mediated by regulatory T cells, Tregs).
  • TGF-P has immune-suppression effects across multiple immune cell types including myeloid cells and B cells.
  • TGF-P is also a key T cell homeostatic cytokine.
  • T cells undergo increased proliferation leading to enlarged lymphoid organs and immune related toxicity in mouse models (Oh et al., 2013, Journal of Immunology, 191(8): 3973- 3979).
  • TGF-P has also been a target for human CAR-T cells (Hartley and Abken, 2019, Clinical Translation Immunology, 8(6): el065), including by expressing a dominant negative TGF-PRII to abrogate TGF-P signalling and improve tumor control in vivo (Kloss et al., 2018, Molecular Therapy, 26(7): 1855-1866).
  • the present disclosure provides a fusion protein comprising: a. a truncated transforming growth factor beta receptor 2 (TGFBRII) comprising at least residues 1 to 187 of TGFBRII (SEQ ID NO: 1), wherein the truncated TGFBRII lacks a kinase domain; and b. one or more intracellular co-stimulatory domains.
  • TGFBRII transforming growth factor beta receptor 2
  • nucleic acid molecule encoding the fusion protein described herein.
  • a cell e.g., a population of cells
  • a cell comprising the nucleic acid molecule or vector described herein.
  • a pharmaceutical composition comprising the cell (e.g., a population of cells) described herein.
  • a method for the treatment of a subject having cancer comprising administering to the subject an effective amount of the cell (e.g., a population of cells) or the pharmaceutical composition described herein.
  • the cell e.g., a population of cells
  • the pharmaceutical composition described herein for use in a method for the treatment of cancer.
  • cell e.g., a population of cells
  • pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer.
  • a method of making a cell comprising introducing the nucleic acid molecule, or the vector described herein into a cell.
  • FIG. 1 shows that TGFBRII.41BB CAR-T cells exhibit superior cytotoxicity and cytokine production in the presence of TGF-p.
  • A A schematic representation of the TGFBRII.BB switch receptor gene construct for insertion in retrovirus, which includes LeY- CAR (CD28/CD3z) and the TGF-P receptor II extracellular domain fused with the intracellular domain of 4-1BB.
  • B A graphical representation of CAR (left panel) and TGFBRII.41BB (right panel) expression on CAR T cells assessed by flow cytometry using Flag-tag and Myc-tag, respectively.
  • C A graphical representation of luciferase activity (y- axis) of HEK293 cells transfected with TGFBRII.41BB and a NF-KB-driven luciferase reporter following treatment with TGF-P (ng/mE; x-axis). Data shown as mean ⁇ SEM of triplicate cultures.
  • D A series of graphical representations of percentage killing (%; y-axis) of DU-145 (LeY + prostate tumour cell line) after 16 hours co-culture with TGFBRII.41BB CAR-T cells or conventional CAR-T cells with or without TGF- P (x-axis), measured by 51 Cr release assays.
  • FIG. 2 shows that TGFBRII.41BB enhances proliferation of CAR-T cells in the presence of TGF-P through specific intracellular signalling.
  • A A graphical representation of proliferation of TGFBRII.41BB CAR-T cells and conventional CAR-T cells labelled with cell trace violet (CTV) at the 7 day time point when cultured in the presence of TGF-P and CAR stimulation by anti-idiotype antibody.
  • B A graphical representation of the percentage of divided of TGFBRII.41BB CAR-T cells and conventional CAR-T cells (%; y-axis) in baseline media (i.e., without TGF-P) or in the presence of 20 ng/mL TGF-P (x-axis).
  • (C) A graphical representation of the proliferation index of TGFBRII.41BB CAR-T cells and conventional CAR-T cells (%; y-axis), in baseline media (i.e., without TGF-P) or in the presence of 20 ng/mL TGF-P (x-axis).
  • (D) A graphical representation of histogram overlays of TGFBRII.41BB CAR-T cells and conventional CAR-T cells stained by MitoTracker Deep Red FM.
  • (E) A graphical representation of relative mitochondrial mass (%; y-axis) of TGFBRII.41BB CAR-T cells and conventional CAR-T cells calculated based on the MFI of MitoTracker after 7 day culture with same conditions as described above in (A).
  • (F) A graphical representation of expression levels of phosphorylated p38 MAPK (% MFI; y-axis) of TGFBRII.41BB CAR-T cells and conventional CAR-T cells, in baseline media (i.e., without TGF-P) or in the presence of 20 ng/mL TGF-P (x-axis).
  • (G) A graphical representation of the expression of phosphorylated SMAD2 (x-axis), in baseline media (i.e., without TGF-P) or in the presence of 20 ng/mL TGF-P (y-axis). Representative data from a triplicate assay. Two-tailed unpaired Mann- Whitney test, ns, p > 0.05; * p ⁇ 0.05; **p ⁇ 0.01.
  • Figure 3 shows the distinct transcriptional profile of TGFBRII.41BB CAR-T cells as compared to with conventional CAR-T cells in the presence of TGF-p.
  • A A heatmap depicting the top 100 differentially expressed genes (normalized expression) between activated conventional vs activated switch CAR-T cells when with TGF-p. Each row represents one gene.
  • B A graphical representation of log-fold change and FDR (Benjamini-Hochberg adjusted p-value ⁇ 0.05). Genes with negative log-fold-change are more highly expressed in conventional CAR-T cell with TGF-p. Differentially expressed genes associated with effector functions and chemotaxis are shown in (C) and (D) as heatmaps.
  • E A graphical representations of a gene ontology (GO) analysis of differentially expressed genes with Log2FC ⁇ 1 and BH-adjusted p-value ⁇ 0.05. GO term on the y-axis plotted by the gene ratio matched in each pathway on x-axis. Each dot represents one pathway. The size of the dot represents the number of counts in matched GO pathway and is coloured by the BH-adjusted p-value.
  • FIG. 4 shows that TGFBRII.41BB receptor signalling induced a unique gene signature and reduced the TGF-P-induced immunosuppressive signal in TGFBRII.41BB CAR-T cells.
  • A A graphical representation of the non-overlapped differentially expressed genes of activated conventional CAR-T cells (with TGF-P versus no TGF-P) and activated TGFBRII.41BB CAR-T cells (with TGF-P versus no TGF-P).
  • B A heatmap showing the gene expression associated with T cell activating signal from uniquely upregulated genes differentially expressed in TGFBRII.41BB CAR-T cells.
  • (C) A graphical representation of log-fold change and FDR (Benjamini-Hochberg adjusted p-value ⁇ 0.05). Genes with positive log-fold-change are more highly expressed in TGFBRII.41BB CAR-T cell with TGF-P, with several differential expressed genes from (B) highlighted in the plot. NS, not significant.
  • (D) A graphical representation of the non-overlapped differentially expressed genes of activated conventional CAR-T cells (with TGF-P versus no TGF-P) and activated TGFBRII.41BB CAR-T cells (with TGF-P versus no TGF-P) from the genes shown in (C).
  • (C) A graphical representation of survival (%; y-axis) and time (day; x-axis) for mice treated with TGFBRII.41BB CAR-T cells or conventional CAR-T cells. Significance for Kaplan-Meier survival analysis was determined by log-rank Mantel-Cox test.
  • (D) A graphical representation of the percentage of human CAR-T cells in the peripheral blood (CD3+ cells/mL blood) at 17 days post-treatment with TGFBRII.41BB CAR-T cells or conventional CAR-T cells (x-axis).
  • (E) A graphical representation of the level of TGF-P 1 (pg/mL tumor; y-axis) at 17 days post-treatment with TGFBRII.41BB CAR-T cells or conventional CAR-T cells (x-axis) evaluated by AlphaLISA assay. Data shown are the mean ⁇ SEM of 3 mice per group.
  • FIG. 6 shows endogenous TGF-P type I and type II receptor expression in CAR-T cells.
  • CD33 is a myeloid marker absent from T cells (barely detectable) and was chosen as negative control.
  • Con conventional CAR-T cells
  • Switch TGFBRII.41BB CAR-T cells
  • B271, 295 and 296 are healthy donor numbers.
  • FIG. 7 shows that TGF-P altered the gene expression profile of conventional CAR-T cells.
  • A A schematic representation of the group comparison between TGF-P treated and untreated conventional CAR-T cells.
  • the heatmap was constructed by Pearson correlation clustering of genes and Euclidean clustering of samples.
  • (D) A graphical representation of gene expression of TGF- P treated and untreated conventional CAR-T cells shown as log2-fold change (x-axis) and - logio(p-value) (y-axis). Genes with negative log2-fold-change are more highly expressed in conventional CAR-T cell without TGF- . Several differential expressed genes from (C) are highlighted in the plot.
  • E A graphical representation of gene expression of TGF- treated and untreated conventional CAR-T cells shown as log2-fold change (x-axis) and -logio(p- value) (y-axis). Genes with negative log2-fold-change are more highly expressed in conventional CAR-T cell without TGF-p. Genes associated with T cell proliferation, Thl differentiation and cell survival are highlighted. NS, not significant.
  • FIG. 8 shows that TGF-P induced different responses in conventional CAR-T cells and TGFBRII.41BB CAR-T cells.
  • A A schematic representation of the group comparison between activated TGFBRII.41BB CAR-T cells and activated conventional CAR-T cells.
  • the heatmap was constructed by Pearson correlation clustering of genes and Euclidean clustering of samples.
  • FIG. 9 shows that TGF-P induced enhanced activation and T effector gene expression in TGFBRII.41BB CAR-T cells but not in conventional CAR-T cells.
  • B A graphical representation of gene expression of TGF-P treated TGFBRII.41BB CAR- T cells and conventional CAR-T cells shown as log2-fold change (x-axis) and -logio(p- value) (y-axis).
  • FIG. 10 shows that TGFBRII.II receptor signalling induced a unique gene signature in TGFBRII.41BB CAR-T cells.
  • A A graphical representation of the nonoverlapped differentially expressed genes of activated conventional CAR-T cells (with TGF- P versus no TGF- ) and activated TGFBRII.41BB CAR-T cells (with TGF- versus no TGF- P).
  • (C) A graphical representation of gene expression of TGF-P treated TGFBRII.41BB CAR-T cells and untreated TGFBRII.41BB CAR-T cells shown as log2-fold change (x-axis) and -logio(p-value) (y-axis). Genes with positive log2-fold-change are more highly expressed in TGFBRII.41BB CAR-T cells with TGF-p. Several differentially expressed genes from (B) are highlighted in the plot. NS, not significant.
  • FIG. 11 shows that TGFBRII.41BB receptor reduced the TGF-P-induced immunosuppressive signal in TGFBRII.41BB CAR-T cells.
  • A A graphical representation of the non-overlapped differentially expressed genes of activated conventional CAR-T cells (with TGF-P versus no TGF-P) and activated TGFBRII.41BB CAR-T cells (with TGF-P versus no TGF-P).
  • the heatmap was constructed by Pearson correlation clustering of genes and Euclidean clustering of samples.
  • C A graphical representation of gene expression of TGF-P treated conventional CAR-T cells and untreated conventional CAR-T cells shown as log2-fold change (x-axis) and -logio(p-value) (y-axis). Genes with positive log2-fold-change are more highly expressed in conventional CAR-T cells with TGF-p. Several differentially expressed genes from (B) are highlighted in the plot. NS, not significant.
  • Figure 12 shows the structure of the fusion proteins relative to wild-type
  • TGFBRII A schematic representation of wild-type TGFBRII.
  • B A schematic representation of the fusion proteins described herein, which comprise truncated TGFBRII (i.e., comprising residues 1-199, 1-220 and 1-243 of SEQ ID NO: 1).
  • C A schematic representation of the fusion proteins described herein, which comprise intracellular costimulatory domains 4- IBB, 0X40, ICOS or CD40.
  • Figure 13 shows the in vitro function of CAR-T cells engineered with fusion protein receptors comprising different intracellular domains (ICD).
  • ICD intracellular domains
  • A A graphical representation of percentage killing (%lysis, y-axis) of CAR-T cells (with different ICD, x- axis) against the LeY + prostate cancer cell line DU-145 after 16 hours co-culture, measured by 51 Cr release assays. Data shown as the mean ⁇ SEM of triplicate cultures.
  • B A graphical representation of TNF-a-producing (%, y-axis) CAR-T cells (with different ICD, x-axis) when stimulated with LeY anti-idiotype antibody and TGF-P, measured by AlphaLISA assay. Data shown as mean ⁇ SEM of triplicate cultures and is representative of one donor.
  • FIG. 14 shows that fusion proteins can form homodimers.
  • A A graphical representation of the fusion proteins tested, where wild-type TGFBRII receptor was fused with eGFP (R2 WT-eGFP ) and the TGF-P binding motif with varying intracellular non-kinase regions (SIS: residues 1 to 199 of TGFBRII, SEQ ID NO: 1; MIS: residues 1 to 220 of TGFBRII, SEQ ID NO: 1; and FIS: residues 1 to 243 of TGFBRII, SEQ ID NO: 1) fused with either eGFP or mCherry.
  • FIG. 1 A graphical representation of a FACS-based FRET assay showing both homodimerization of two SIS receptors (circles and solid line) and heterodimerisation of a SIS receptor and a wild-type TGFBRII receptor (crosses and dotted line).
  • C A graphical representation of a FACS-based FRET assay showing both homodimerization of two MIS receptors (circles and solid line) and heterodimerisation of a MIS receptor and a wild-type TGFBRII receptor (crosses and dotted line).
  • Figure 15 shows that TGFBRII.41BB CAR-T cells significantly enhanced tumor control in vivo in a breast cancer model.
  • B A graphical representation of survival (%; y-axis) and time (day; x-axis) for mice treated with TGFBRII.41BB CAR-T cells or conventional CAR-T cells. Significance for Kaplan-Meier survival analysis was determined by log-rank Mantel-Cox test.
  • SEQ ID sequence identifier number
  • SEQ ID NO: 1 shows the amino acid sequence of full length human transforming growth factor beta receptor 2 (TGFBRII).
  • SEQ ID NO: 2 shows the amino acid sequence of the TGFBRII extracellular domains, including signal peptide.
  • SEQ ID NO: 3 shows the DNA sequence encoding the TGFBRII extracellular domains of SEQ ID NO: 2.
  • SEQ ID NO: 4 shows the amino acid sequence of the TGFBRII transmembrane domain.
  • SEQ ID NO: 5 shows the DNA sequence encoding the TGFBRII transmembrane domain of SEQ ID NO: 4.
  • SEQ ID NO: 6 shows the amino acid sequence of the TGFBRII intracellular non-kinase region.
  • SEQ ID NO: 7 shows the DNA sequence encoding the TGFBRII intracellular non-kinase region of SEQ ID NO: 6.
  • SEQ ID NO: 8 shows the amino acid sequence of a truncated human TGFBRII comprising residues 1 to 199 of SEQ ID NO: 1.
  • SEQ ID NO: 9 shows the amino acid sequence of a truncated human TGFBRII comprising residues 1 to 220 of SEQ ID NO: 1.
  • SEQ ID NO: 10 shows the amino acid sequence of a truncated human TGFBRII comprising residues 1 to 243 of SEQ ID NO: 1.
  • SEQ ID NO: 11 shows the amino acid sequence of fusion protein comprising the truncated human TGFBRII of SEQ ID NO: 8 and the 4-1BB intracellular co-stimulatory domain (i.e., TGFBRII199.41BB).
  • SEQ ID NO: 12 shows the amino acid sequence of fusion protein comprising the truncated human TGFBRII of SEQ ID NO: 9 and the 4-1BB intracellular co-stimulatory domain (i.e., TGFBRII220.41BB).
  • SEQ ID NO: 13 shows the amino acid sequence of fusion protein comprising the truncated human TGFBRII of SEQ ID NO: 10 and the 4-1BB intracellular co-stimulatory domain (i.e., TGFBRII243.41BB).
  • SEQ ID NO: 14 shows the amino acid sequence of fusion protein comprising the truncated human TGFBRII of SEQ ID NO: 8 and the 0X40 intracellular co-stimulatory domain (i.e., TGFBRII199.OX40).
  • SEQ ID NO: 15 shows the amino acid sequence of fusion protein comprising the truncated human TGFBRII of SEQ ID NO: 8 and the ICOS intracellular co-stimulatory domain (i.e., TGFBRII199.ICOS).
  • SEQ ID NO: 16 shows the amino acid sequence of fusion protein comprising the truncated human TGFBRII of SEQ ID NO: 8 and the CD40 intracellular co-stimulatory domain (i.e., TGFBRII199.CD40).
  • SEQ ID NO: 17 shows the DNA sequence encoding the fusion protein of SEQ ID NO: 11.
  • SEQ ID NO: 18 shows the DNA sequence encoding the fusion protein of SEQ ID NO: 12.
  • SEQ ID NO: 19 shows the DNA sequence encoding the fusion protein of SEQ ID NO: 13.
  • SEQ ID NO: 20 shows the DNA sequence encoding the fusion protein of SEQ ID NO: 14.
  • SEQ ID NO: 21 shows the DNA sequence encoding the fusion protein of SEQ ID NO: 15.
  • SEQ ID NO: 22 shows the DNA sequence encoding the fusion protein of SEQ ID NO: 16.
  • SEQ ID NO: 23 shows the amino acid sequence of the 4- IBB intracellular costimulatory domain.
  • SEQ ID NO: 24 shows the DNA sequence encoding the 4- IBB intracellular costimulatory domain.
  • SEQ ID NO: 25 shows the amino acid sequence of the 0X40 intracellular costimulatory domain.
  • SEQ ID NO: 26 shows the DNA sequence encoding the 0X40 intracellular costimulatory domain.
  • SEQ ID NO: 27 shows the amino acid sequence of the ICOS intracellular costimulatory domain.
  • SEQ ID NO: 28 shows the DNA sequence encoding the ICOS intracellular costimulatory domain.
  • SEQ ID NO: 29 shows the amino acid sequence of the CD40 intracellular costimulatory domain.
  • SEQ ID NO: 30 shows the DNA sequence encoding the CD40 intracellular costimulatory domain.
  • SEQ ID NO: 31 shows the DNA sequence of the pSAMEN vector comprising nucleic acid sequences encoding TGFBRII199.41BB.
  • SEQ ID NO: 32 shows the DNA sequence of a pSAMEN vector comprising nucleic acid sequences encoding LeY-CD34BEND10-TGFBRII199.41BB.
  • SEQ ID NO: 33 shows the DNA sequence of a pSAMEN vector comprising nucleic acid sequences encoding LeY-CD34BEND10-TGFBRII220.41BB.
  • SEQ ID NO: 34 shows the DNA sequence of a pSAMEN vector comprising nucleic acid sequences encoding LeY-CD34BEND10-TGFBRII243.41BB.
  • SEQ ID NO: 35 shows the DNA sequence of a pSAMEN vector comprising nucleic acid sequences encoding LeY-CD34BEND10-TGFBRII199.0X40.
  • SEQ ID NO: 36 shows the DNA sequence of a pSAMEN vector comprising nucleic acid sequences encoding LeY-CD34BEND10-TGFBRII.ICOS.
  • SEQ ID NO: 37 shows the DNA sequence of a pSAMEN vector comprising nucleic acid sequences encoding LeY-CD34BEND10-TGFBRII199.CD40.
  • the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the single letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single letter codes.
  • sequence database identifiers e.g., GenBank ID, EMBL-Bank ID, DNA Data Bank of Japan (DDBJ) ID, etc.
  • DDBJ DNA Data Bank of Japan
  • fusion proteins comprising a truncated TGFRBII and one or more intracellular co-stimulatory domains are capable of improving the function of cell therapies (e.g., CAR T cell therapies) by converting immunosuppressive TGF-P signalling to an activation signal, which increases cytotoxic function, cytokine secretion and proliferation of immune effector cells (e.g., T cells, NK cells, macrophages).
  • cell therapies e.g., CAR T cell therapies
  • an activation signal which increases cytotoxic function, cytokine secretion and proliferation of immune effector cells (e.g., T cells, NK cells, macrophages).
  • a fusion protein comprising: a. a truncated transforming growth factor beta receptor 2 (TGFBRII) comprising at least residues 1 to 187 of TGFBRII (SEQ ID NO: 1), wherein the truncated TGFBRII lacks a kinase domain; and b. one or more intracellular co-stimulatory domains.
  • TGFBRII transforming growth factor beta receptor 2
  • protein protein
  • peptide and “polypeptide” are used interchangeably herein to refer to a polymer of amino acid residues linked together by peptide (amide) bonds.
  • amide peptide bonds
  • the terms refer to a protein, peptide, or polypeptide of any size, structure or function.
  • fusion protein refers to a recombinant protein comprising two or more polypeptides, which are not normally associated in nature, but the respective amino and carboxy termini may be joined together by direct or indirect binding to form one contiguous polypeptide.
  • fusion protein is used interchangeably with "switch receptor” when referring to the fusion protein as described herein.
  • transforming growth factor beta receptor 2 or "TGFBRII” means the receptor for transforming growth factor beta (TGF-P).
  • TGF-P is an immunosuppressive soluble factor that is highly expressed in advanced tumors (Mariathasan et al., 2018, Nature, 554(7693): 544-548) and inhibits effector T cell anti-tumor responses (e.g., cytotoxicity and cytokine secretion) and T cell proliferation.
  • the complete human TGFBRII sequence is shown in SEQ ID NO: 1.
  • truncated TGFBRII refers to a TGFBRII comprising at least residues 1 to 187 of TGFBRII (SEQ ID NO: 1), wherein the truncated TGFBRII lacks a kinase domain (i.e., residues 244-567 of SEQ ID NO: 1).
  • the truncated TGFBRII may comprise at least residues 1 to 187 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 188 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 189 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 190 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 191 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 192 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 193 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 194 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 195 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 196 of TGFBRII (SEQ ID NO: 1), at least residues 1 to 197 of TGFBRII (SEQ ID NO: 1)
  • the truncated TGFBRII comprises at least residues 1 to 199 of TGFBRII (SEQ ID NO: 1).
  • the truncated TGFBRII comprises at least residues 1 to 220 of TGFRBII (SEQ ID NO: 1).
  • the truncated TGFBRII comprises at least residues 1 to 243 of TGFRBII (SEQ ID NO: 1).
  • the truncated TGFBRII comprises, consists or consists essentially of any one the amino acid sequences of SEQ ID NOs: 8-10, or an amino acid sequence having at least 90% sequence identity thereto. Accordingly, the sequence may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 8-10.
  • the fusion protein comprises one or more intracellular costimulatory domains. In an embodiment, the fusion protein comprise one intracellular costimulatory domain. In another embodiment, the fusion protein comprises two intracellular co-stimulatory domains.
  • Examples of intracellular co-stimulatory domains include an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signalling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49
  • the intracellular co-stimulatory domain is a functional signalling domain of a protein selected from the group consisting of 4- IBB, 0X40, ICOS and CD40.
  • the intracellular co-stimulatory domain comprises, consists or consists essentially of any one the amino acid sequences of SEQ ID NOs: 23, 25, 27 and 29, or an amino acid sequence having at least 90% sequence identity thereto. Accordingly, the sequence may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 23, 25, 27 and 29.
  • the fusion protein comprises, consists, or consists essentially of any one of the amino acid sequences of SEQ ID NOs: 11-16, or an amino acid sequence having at least 90% sequence identity thereto. Accordingly, the sequence may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 11-16.
  • nucleic acid molecule encoding the fusion protein described herein.
  • nucleic acid or “nucleic acid molecule” mean a single- or double-stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues or natural nucleotides, or mixtures thereof, and can include molecules comprising coding and non-coding sequences of a gene, sense and antisense sequences and complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.
  • nucleotide refers to the nucleotides adenosine, guanosine, cytidine, thymidine and uridine, each of which comprise a nucleotide base attached to a ribose ring.
  • adenine / adenosine uracil / uridine
  • guanine / guanosine cytosine / cytidine
  • thymidine / thymine C
  • non-naturally occurring may be interchangeably used herein to refer to nucleotides or nucleic acid molecules that are distinguished from their naturally occurring counterparts.
  • the nucleic acid molecule of the present disclosure may be recombinant, synthetic, or comprise mixtures of naturally and non-naturally occurring nucleotides.
  • Non-naturally occurring nucleotides or nucleotide analogs may be modified at the ribose, phosphate and/or base moiety.
  • the terms “encode”, “encoding” and the like refer to the capacity of a nucleic acid molecule to provide for another nucleic acid or a polypeptide.
  • a nucleic acid molecule is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide.
  • Such a nucleic acid molecule may include a coding sequence or both a coding sequence and a non-coding sequence.
  • the terms "encode,” "encoding” and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.
  • a processed RNA product e.g., mRNA
  • the fusion protein is encoded by a codon optimized nucleic acid sequence for expression in particular cells, e.g., eukaryotic cells.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Various species exhibit particular bias for certain codons of a particular amino acid.
  • Codon bias i.e., differences in codon usage between organisms
  • Codon bias often correlates with the efficiency of translation of mRNA, which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, e.g., the "Codon Usage Database" available at www.kazusa.ogp/codon/.
  • the nucleic acid molecule comprises one or more non-natural occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, boranophosphate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2' and 4' carbons of the ribose ring, or bridged nucleic acids (BNA).
  • LNA locked nucleic acid
  • modified nucleotides include 2'-0-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2'-fluoro analogs.
  • modified bases include, but are not limited to, 2-aminopurine, 5 -bromo-uridine, pseudouridine ( ), N 1 -methylpseudouridine (me lx F), S- methoxyuridine(SmoU), inosine, 7-methylguanosine.
  • the nucleic acid molecule is a recombinant nucleic acid molecule.
  • nucleic acid molecules of the present disclosure may be produced using any method in the art, including synthetically or by recombinant techniques such as expression of polynucleotide constructs encoding the components.
  • the nucleic acid molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 17-22, or a nucleotide sequence having at least 90% sequence identity thereto. Accordingly, the sequence may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the nucleotide sequences of SEQ ID NOs: 17-22.
  • nucleotide sequence identity is known to persons skilled in the art, illustrative examples of which include computer programs that employ algorithms such as protein BLAST (Altschul et al., 1997, Nucleic Acids Research, 25: 3389- 3402).
  • nucleic acid molecule described herein.
  • the vectors can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into a host cell genome.
  • Vectors may be replication competent or replication-deficient.
  • Exemplary vectors include, but are not limited to, plasmids, cosmids, and viral vectors, such as adeno-associated virus (AAV) vectors, lentiviral, retroviral, adenoviral, herpesviral, parvoviral and hepatitis viral vectors.
  • AAV adeno-associated virus
  • the choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • the vector is suitable for use in biotechnology.
  • Vectors suitable for use in biotechnology would be known to persons skilled in the art, illustrative examples of which include viral vectors derived from adenovirus, adeno- associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, self-amplifying single-strand RNA (ssRNA) viruses such as alphavirus (e.g., Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis, Ml), and flavivirus (e.g., Kunjin virus, West Nile virus, Dengue virus), rhabdovirus (e.g., rabies, vesicular stomatitis virus), measles virus, Newcastle Disease virus (NDV) and poxivirus as described by, for example, Lundstrom (2019, Diseases, 6: 42).
  • alphavirus e.g., Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis, Ml
  • flavivirus e.
  • the vector is a plasmid or a viral vector.
  • non-viral delivery vehicles of the genome editing systems as described herein, and components thereof.
  • Suitable non-viral delivery vehicles will be known to persons skilled in the art, illustrative examples of which include using lipids, lipid-like materials or polymeric materials, as described by , e.g., Rui et al. (2019, Trends in Biotechnology, 37(3): 281-293), and nanoparticles/nanocarriers, as described by, e.g., Nguyen et al. (2020, Nature Biotechnology, 38: 44-49).
  • cells are engineered to express the fusion protein and a chimeric antigen receptor (CAR) or T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T-cell receptor
  • the nucleic acid molecules encoding the fusion protein and the CAR or TCR may be comprised in separate vectors or within the same vector, e.g., the vectors shown as SEQ ID NOs: 32-37.
  • each nucleic acid molecule When multiple nucleic acid molecules are combined within the same vector, the expression of each nucleic acid molecule may be controlled by the same promoter or different promoters according to the optimal stoichiometry of the different components of the fusion protein, CAR or TCR disclosed herein.
  • the polynucleotide encoding the fusion protein will be operably linked to a first promoter and the polynucleotide encoding a CAR operably linked to a second promoter.
  • promoter refers to an array of nucleic acid control sequences that direct the transcription of the polynucleotide.
  • Suitable promoters would be known to persons skilled in the art, illustrative examples of which include retroviral LTR elements, constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, or P-actin, inducible promoters, such as those containing Tet-operator elements, and/or tissue specific promoters.
  • nucleic acid molecules are not comprised within the same vector, the nucleic acid molecules may be comprised in separate vectors, or one nucleic acid molecule (e.g., encoding the fusion protein) may be comprised in a vector and a second nucleic acid molecule (e.g., encoding a CAR or TCR) is provided to the cell using non-viral delivery vehicles.
  • one nucleic acid molecule e.g., encoding the fusion protein
  • a second nucleic acid molecule e.g., encoding a CAR or TCR
  • the nucleic acid molecules may comprise other additional regulatory elements or sequences. Suitable regulatory sequences would be known to persons skilled in the art, illustrative examples of which include leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, and enhancer or activator sequences. It is also contemplated herein that the polypeptides comprises elements and sequences associated with protein localization and interactions.
  • the present disclosure provides a cell (e.g., a population of cells) comprising the nucleic acid molecule or vectors described herein.
  • Cells according to the present disclosure include any cell into which the nucleic acid molecules or vectors described herein may be introduced and expressed. It is not intended that use of the nucleic acid molecule or vectors disclosed herein be limited by cell type. Accordingly, the cells of the present disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archaeal cells, eubacterial cells and the like.
  • cell refers to an individual cell, cell line, cell culture or population of cells that comprise the nucleic acid molecule or vectors described herein, or that is capable of expressing the fusion protein described herein.
  • population of cells may refer to homogenous cell populations comprising cells that each comprise the nucleic acid molecule or vectors described herein, or that is capable of expressing the fusion protein described herein, or heterogeneous cell populations that may comprise progeny of a single parental cell. Due to natural, accidental or deliberate mutation, the progeny cells may not necessarily be identical in morphology or in genome to the original parental cell, but may be capable of expressing the fusion protein described herein.
  • the cells contemplated herein may be derived from any species, particularly a vertebrate, and even more particularly a mammal.
  • Suitable vertebrates that fall within the scope of the disclosure include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes), rodents (e.g., mice rats, guine
  • the cell is an immune effector cell.
  • Suitable immune effector cells would be known to persons skilled in the art, illustrative examples of which include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and bone marrow-derived phagocytes.
  • the immune effector cell is an autologous immune effector cell.
  • T cells isolated from the subject may be contacted with the genome editing systems described herein and cultured ex vivo for a time and under conditions suitable for the integration of the heterologous nucleotide sequence, before being reinfused back into the subject in accordance with the method of treatment described herein.
  • the immune effector cell is an allogenic immune effector cell.
  • allogenic refers to any material derived from a different animal of the same species as the subject to whom the material is administered.
  • the cell is a T cell.
  • Suitable T cells would be known to persons skilled in the art, illustrative examples of which include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, activated T lymphocytes or tumour infiltrating lymphocytes (TILs).
  • Illustrative populations of T cells suitable for use in particular embodiments include but are not limited to helper T cells (HTL; CD4 + T cell), a cytotoxic T cell (CTL; CD8 + T cell), CD4 + CD8 + T cell, CD4 CD8" T cell, or any other subset of T cells.
  • T cells suitable for use in particular embodiments include but are not limited to T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR and if desired, can be further isolated by positive or negative selection techniques.
  • the cell is a NK cell.
  • Suitable NK cells would be known to persons skilled in the art, illustrative examples of which include NK tolerant , NK cyt0t0Xlc , NK regulatory cells and memory-like NK cells.
  • Illustrative populations of NK cells suitable for use in particular embodiments include but are not limited to NK cells expressing one or more of the following markers: CD27, CD1 lb, CD56, CD 16, and if desired, can be further isolated by positive or negative selection techniques.
  • isolated refers to a cell, which is substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment, e.g., whole blood.
  • Methods for the isolation of immune effector cells from whole blood would be known to persons skilled in the art, illustrative examples of which include the isolation of T cells from whole blood using the Ficoll-Paque method.
  • the cell e.g., a population of cells
  • the cell may be provided with the nucleic acid molecules or vectors described herein using any suitable method known in the art. Such methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like. The skilled person would readily understand and adapt any such method taking consideration of whether the nucleic acid molecules are provided as polynucleotides or vectors.
  • the term "recombinant cell” as used herein refers to a cell which comprises the vectors described herein.
  • the term “recombinant cell” includes the specific cell and the progeny of the cell.
  • the cell has been engineered to express a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T-cell receptor
  • chimeric antigen receptor or “CAR” as used herein mean a recombinant polypeptide comprising at least an antigen-binding domain that is linked, via hinge and transmembrane domains, to an intracellular signalling domain.
  • the antigen-binding domain is a functional portion of the CAR that is responsible for transmitting information within the cell to regulate cellular activity via defined signaling pathways.
  • the antigen-binding domain may comprise an antibody or antibody fragment thereof.
  • antibody is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multi-specific antibodies (e.g., bispecific antibodies), and single variable domain antibodies so long as they exhibit the desired biological activity.
  • antibody includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof e.g., IgM). Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, Cm, CH2 and Cm-
  • Each light chain comprises a light chain variable region (which may be abbreviated as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CLI).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of an antibody may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
  • antibody is an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • the heavy-chain constant regions that correspond to the different classes of immunoglobulins are called a, 5, 8, y, and p, respectively.
  • the subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.
  • an “antigen-binding fragment” may be provided by means of arrangement of one or more CDRs on non-antibody protein scaffolds.
  • “Protein scaffold” as used herein includes but is not limited to an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human or primate origin, or which may be an artificial chimera of human and primate constant regions.
  • the protein scaffold may be an Ig scaffold, for example an IgG, or IgA scaffold.
  • the IgG scaffold may comprise some or all the domains of an antibody (i.e., CHI, CH2, CH3, VH, VL).
  • the antigen binding protein may comprise an IgG scaffold selected from IgGl, IgG2, IgG3, IgG4 or IgG4PE.
  • the scaffold may be IgGl.
  • the scaffold may consist of, or comprise, the Fc region of an antibody, or is a part thereof.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigenbinding fragment,” as used herein.
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • an antigenbinding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CHI; (ii) VH-CH2; (iii) VH-CHS; (iv) VH-CHI-CH2; (V) VH-CHI-CH2-CH3, (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2, (X) VL-CH3; (xi) VL-CHI-CH2; (xii) VL-CHI-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment of an antibody may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • antigen-binding fragments may be monospecific or multi-specific e.g., bispecific).
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multi-specific antigen-binding molecule format may be adapted for use in the context of an antigen-binding fragment of an antibody using routine techniques available in the art.
  • the antigen-binding domain comprises an antibody fragment.
  • the antigen-binding domain may comprise a scFv consisting of a VL and VH sequence of a monoclonal antibody (mAb) specific for a tumor cell surface molecule (i.e., tumor antigen).
  • mAb monoclonal antibody
  • the CAR comprises an antigen binding domain that binds specifically to an antigen selected from the group consisting of CD7, CD 19, CD20, CD22, CD30, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD133, CD138, CD171, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSCA, PSMA, Glycolipid F77, Her2, EGFR, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, Claudin 6, Claudin 18.2, Lewis Y (LeY), GRP-78, EphA2, CEA, CEACAM5, ROR1, FAP and combinations thereof.
  • an antigen selected from the group consisting of CD7, CD 19, CD20, CD22, CD30, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD133, CD138, CD171, BCMA (CD269), GPC2, GPC3, FGFR4,
  • T-cell receptor or "TCR” as used herein mean a recombinant or naturally-occurring heterodimeric polypeptide comprising an alpha polypeptide chain (i.e., alpha chain, a chain) and a beta polypeptide chain (i. e. , beta chain, chain), which is capable of binding to a peptide antigen bound to MHC.
  • alpha polypeptide chain i.e., alpha chain, a chain
  • beta polypeptide chain i. e. , beta chain, chain
  • the TCR binds specifically to an antigen selected from the group consisting of 707- AP, AFP, ART-4, BAGE, Bcr-abl, CAMEL, CAP-1, CASP-8, CDC27m, CDK4/m, CEA, CT, Cyp-B, DAM, EGFRvlll, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, GplOO, HAGE, HER-2/neu, HLA-A, HPV, HSP70-2M, HST-2, hTERT, hTRT, iCE, KIAA0205, LAGE (L antigen), LDLR/FUT, MAGE, MART-l/Melan-A, MCI R, Myosin/m, MUC1, MUM-1, MUM -2, MUM -3, NA88-A, NY-ESO-1, P15, pl90 minor, Pml/RARa, PRAME, PSA,
  • an antigen selected from
  • the present disclosure provides a method of making a cell (e.g. , a population of cells), comprising introducing the nucleic acid molecule or vector described herein into a cell.
  • a cell e.g. , a population of cells
  • the nucleic acid molecule or vector may be introduced into the cell using any method known in the art, illustrative examples of which include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like.
  • the method further comprises introducing a nucleic acid molecule or vector encoding a CAR or TCR into the cell.
  • composition comprising the cell, or population of cells described herein.
  • composition refers to a composition that is in a form that allows the biological activity of the active ingredient (i.e., a cell expressing the fusion protein described herein) to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered.
  • the pharmaceutical composition comprises a population of the cells in sufficient number to administer a dosage of 10 4 to 10 9 cells/kg body weight per dose. Accordingly, the pharmaceutical composition may comprise a population of the cells in sufficient number to administer a dosage of 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 cells/kg body weight per dose.
  • the pharmaceutical composition comprises a population of the cells in sufficient number to administer a dosage of 10 5 to 10 6 cells/kg body weight per dose, including all integer values within those ranges.
  • periodic re-administration of the pharmaceutical composition may be required to achieve a desirable therapeutic effect.
  • the exact amounts and rates of administration of the pharmaceutical composition will depend on a number of factors, examples of which are described elsewhere herein, such as the subject’s age, body weight, general health, sex and dietary requirements, as well as any drugs or agents used in combination or coincidental with the administration of the composition. Where multiple divided doses are required, these may be administered hourly, daily, weekly, monthly or at other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. Alternatively, a continuous infusion strategy can be employed.
  • the pharmaceutical composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.
  • compositions disclosed herein may be prepared according to conventional methods well known in the pharmaceutical industries, such as those described in Remington’s Pharmaceutical Handbook (Mack Publishing Co., NY, USA), comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.
  • pharmaceutically acceptable carrier means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain anti-oxidants, buffers and solutes to render the composition isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, anti-fungal and anti-bacterial agents, isotonic and absorption agents, and the like.
  • the pharmaceutical composition further comprises one or more immune adjuvants.
  • immune adjuvant refers to a compound or substance that is capable of enhancing a subject’s immune response to the immunogen including, for example, the subject's antibody response to the immunogen.
  • An immune adjuvant may therefore assist to enhance the immune response to an engineered T cell in a subject, compared to the administration of the engineered T cell or in the absence of the immune adjuvant.
  • Suitable immune adjuvants will be familiar to persons skilled in the art, illustrative examples of which include an inhibitor of the PDL-1 : PD-1 axis, a TLR3 agonist, a 4-1BB agonist, a TLR7 agonist, an inhibitor of TIM-3, and an inhibitor of CTLA-4.
  • the pharmaceutical composition may be coadministered with one or more other agents suitable for the treatment or amelioration of symptoms associated with cancer, such as a solid tumor, illustrative examples of which include surgery, chemotherapy (e.g., anastrozole, bicalutamide, bleomycin sulfate, busulfan, busulfan injection, capecitabine, N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, dactinomycin, daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide
  • chemotherapy e.g., anastrozo
  • Such combinations may be administered simultaneous with the pharmaceutical composition or concurrently with the pharmaceutical composition.
  • the cell e.g., a population of cells
  • pharmaceutical compositions described herein may be adapted for the treatment of diseases and disorders that are characterized by immunosuppression mediated by TGF-p.
  • T cells co-expressing the fusion protein and a CAR z.e., TGFBRII.41BB CAR-T cells
  • TGF-P levels were increased relative to conventional CAR T cells.
  • the cell e.g., a population of cells
  • the pharmaceutical compositions described herein may be useful in the treatment of cancer.
  • the present disclosure provides a method for the treatment of a subject having cancer, the method comprising administering a therapeutically effective amount of the cell (e.g., a population of cells) or the pharmaceutical composition described herein.
  • the present disclosure provides the use of the cell (e.g., a population of cells) or the pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer.
  • the present disclosure provides the cell (e.g., a population of cells) or the pharmaceutical composition described herein for use in the treatment of cancer.
  • the therapeutic regimen for the treatment of cancer can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the type, size, stage and receptor status of the tumor in addition to the age, weight and general health of the subject. Another determinative factor may be the risk of developing recurrent disease. For instance, for a subject identified as being at high risk or higher risk or developing recurrent disease, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is deemed at a low or lower risk of developing recurrent disease. Similarly, for a subject identified as having a more advanced stage of cancer, for example, stage III or IV disease, a more aggressive therapeutic regimen may be prescribed as compared to a subject that has a less advanced stage of cancer.
  • cancer means any condition associated with aberrant cell proliferation. Such conditions will be known to persons skilled in the art.
  • the cancer is a primary cancer (e.g., a tumor).
  • the cancer is a metastatic cancer.
  • cancers examples include breast cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, sarcoma and the like.
  • cancer and “tumor” may be used interchangeably herein, e.g., encompassing both solid and diffuse or circulating tumors.
  • the cancer is a TGF-P-expressing cancer.
  • TGF-P-expressing cancers would be known to persons skilled in the art, illustrative examples of which include breast cancer, melanoma, carcinoid, cervical cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, endometrial cancer, renal cancer, glioma, skin cancer, head and neck cancer, stomach cancer, liver cancer, testis cancer, lung cancer, thyroid cancer, lymphoma and urothelial cancer.
  • the TGF-P-expressing cancer is prostate cancer.
  • the TGF-P-expressing cancer is breast cancer.
  • subject refers to any mammal, including livestock and other farm animals (such as cattle, goats, sheep, horses, pigs and chickens), performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals and humans.
  • livestock and other farm animals such as cattle, goats, sheep, horses, pigs and chickens
  • performance animals such as racehorses
  • companion animals such as cats and dogs
  • laboratory test animals such as cats and dogs.
  • the subject is a human.
  • the subject is an adult. In another embodiment, the subject is a child.
  • the term “effective amount” typically refers to an amount of the cell, or population of cells, or pharmaceutical composition described herein that is sufficient to affect one or more beneficial or desired therapeutic outcomes (e.g., reduction in tumor size).
  • beneficial or desired therapeutic outcomes may be measured using clinical techniques known in the art, illustrative examples of which include the measurement of imaging biomarkers, tumor size (e.g., as measured by anatomical imaging modalities, such as CT or MRI), quantification of the presence of inflammatory mediators (e.g., Interleukin- 1, TNF, TGF-P, etc.).
  • An “effective amount” can be provided in one or more administrations. The exact amount required may vary depending on factors such as the nature and severity of the cancer to be treated, and the age and general health of the subject.
  • treat means relieving, reducing, alleviating, ameliorating or otherwise inhibiting the severity and/or progression of cancer, or a symptom thereof, in a subject. It is to be understood that the terms “treat”, “treating”, “treatment” and the like, as used herein, do not imply that a subject is treated until clinical symptoms of cancer have been eliminated or are no longer evident (e.g., elimination of solid tumor mass and associated metastatic lesions, if any). Said treatment may also reduce the severity of cancer by preventing progression or alleviating the symptoms associated with cancer.
  • prevent means inhibit, hinder, retard, reduce or otherwise delay the development of cancer and/or progression of cancer, or a symptom thereof, in a subject.
  • the term “prevent” and variations thereof does not necessarily imply the complete prevention of the specified event. Rather, the prevention may be to an extent, and/or for a time, sufficient to produce the desired effect. Prevention may be inhibition, retardation, reduction or otherwise hindrance of the event, activity or function. Such preventative effects may be in magnitude and/or be temporal in nature.
  • the anti-LeY scFv-CD3 ⁇ -CD28 CAR (LeY CAR) construct was used in this study as described by Williford et al. (2019, Scientific Advances, 5(12): p.eaayl357).
  • 3 receptor 2 extracellular and transmembrane domains and the 4- IBB intracellular domain fusion protein (e.g., SEQ ID NOs: 17-19) was synthesized by Genscript (Piscataway, NJ, USA).
  • Genscript Procataway, NJ, USA.
  • the DNA fragment was then cloned into the pSAMEN retroviral vector containing the LeY CAR (e.g., SEQ ID NOs: 32-34).
  • HEK293 cells were transduced with retrovirus to express the TGFBRII.41BB fusion protein.
  • Transduced cells were sorted and subsequently transfected with pGL4.73[hRluc/SV40] (E6911, Promega, Madison, WI, USA) and pGL4.32[luc2P/NF-KB- RE/Hygro] (E8491, Promega) by Lipof ectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.
  • Transfected cells were then treated with either plain media or media containing 100 ng/ml TGH-
  • Retroviral packaging cell line PG13-LeY-CAR and PG13-LeY-TGFBRII.41BB CAR was prepared and used in this study as described by Williford et al. (2019, supra).
  • Packaging cells were cultured with RPMI 1640 (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS to produce retrovirus-containing media for transduction.
  • Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor huffy coats (Australian Red Cross Blood Service) by density gradient centrifugation (Ficoll- Paque, GE Healthcare Life Science). Healthy donor packs were accessed from the ARCBS via the Peter Mac HREC approval number 01-14.
  • Chromium release assay was performed as described by Davenport et al. (2015, Cancer Immunology Research, 3(5): 483-494). Briefly, target cells (LeY-i- OVCAR-3 and HER2+ MB-468) were labeled with 100 pCi Chromium-51 (51Cr, PerkinElmer) for one hour at 37°C and subsequently co-cultured with conventional or TGFBRII.41BB CAR-T cells or empty vector T cells in triplicate wells at an effector:target ratio of 2:1 with or without 10 ng/ml human TGH-
  • the conventional and TGFBRII.41BB CAR-T cells were incubated with or without 100 ng/ml human TGF-
  • the pre-warmed fixation buffer (BioLegend, #420801) was added to the cell culture at the time points indicated. After 15 minutes incubation at 37°C, the cells were washed with FACS buffer and re-suspended in pre-chilled True-Phos Perm Buffer (BioLegend, #425401) and kept at -20°C for 2 hours. After a wash, the cells were stained with the antibodies for the phosphorylation markers according to the manufacturer’s instructions.
  • the antibodies used in the FACS staining were CD3-BUV395 (BD, #564001), Flag-tag-PerCP/Cy5.5 (BioLegend, #637325), Flag-tag- PE (BioLegend, #637310), Myc- tag-PE(Cell Signaling Technology, #2233S), pERKl/2-PE/Cy7 (BioLegend, #369515), pNFkB-PE (Cell Signaling Technology, #5733S), pSMAD2-AF488(Cell Signaling Technology, #56532S), pMAPKp83-PE (BioLegend, #690203).
  • Raw sequence reads were quality-controlled using FastQC module (version 0.11.8) and undergone trimming using Cutadapt (version 3.4) in the Galaxy platform.
  • the single-end reads were aligned to the human reference genome (hgl9) using HISAT2 (version 2.2.1, Galaxy) and quantified using HTSeq.
  • Gene normalization was performed by edge R package (version 3.35.0) with library size adjustment by Trimmed Mean of M-values method (Robinson & Oshiack, 2010, Genome Biology, 11(3): R25).
  • Differential expression analysis (DEG) was performed using Limma-Voom workflow (limma version 3.46.0). Genes with consistent extreme-low count (less than 2 CPM) across 85% samples were discarded to reduce the noise in the downstream analysis.
  • NSG mice NSG mice aged between 6-8 weeks were injected s.c. on the right flank with 1 x 10 6 DU-145 cells in 100 pl of PBS on Day 14.
  • the mice were irradiated by 0.5 Gy.
  • Mice received three doses of 1 x 10 7 conventional or TGFBRII.BB CAR-T cells i.v. on Days 1, 2, and 3 with cytokine support (IL-2, 50 000 Ul/dose). Tumor size was measured by using calipers and presented as the product of perpendicular diameters.
  • mice were bled for blood sampling to investigate the persistence of CD3 + human T cells in the peripheral blood.
  • Tumors were also collected from a group of three mice per treatment to assess the TGF-
  • FFPE Formalin fixed paraffin embedded
  • the TGFBRII.BB fusion protein enhances CAR-T cell cytotoxicity in the presence of TGF-p
  • the TGFBRII.41BB fusion protein was designed by fusing a truncated TGFBRII comprising the extracellular and transmembrane domain of TGFBRII with the intracellular domain of 4-1BB. A Myc-tag was added to the N-terminal of the TGFBRII.41BB fusion protein. By using a T2A self-cleaving peptide, the TGFBRII.41BB fusion protein was engineered to co-express a CAR and TGFBRII.41BB fusion protein in T cells ( Figure 1 A).
  • T cells were shown to constitutively co-express the CAR and TGFBRII.41BB fusion protein as indicated by Flag-tag and Myc-Tag ( Figure IB).
  • a NF-KB driven luciferase reporter was introduced to TGFBRII expressing HEK293 cells. Thereafter, signaling derived from the TGFBRII.41BB fusion protein was assessed by treating the reporter HEK293 cells with external TGF ⁇ p.
  • luciferase activity significantly increased when the cells were treated with TGF-P, indicating that the TGFBRII.41BB fusion protein was activated by TGF-P and subsequently induced NF-KB signaling (Figure 1C).
  • the TGFBRII.41BB fusion protein was assembled with a EeY-CAR, which targets the Eewis Y antigen on tumor cells.
  • EeY-CAR-T cells lysed EeY + DU-145 prostate cancer cells.
  • the % lysis of DU-145 cells was significantly decreased.
  • TGF-P induced a significant increase in % lysis of DU145 cells, further indicating that the TGFBRII.41BB fusion protein was activated by TGF-
  • TGF-[3 the cytokine production by conventional and TGFBRII.41BB LeY- CAR-T cells also differed. After 16-hours co-culture in the presence or absence of TGF-
  • the TGFBRII.41BB fusion protein improves CAR-T cell proliferation and altered mitochondrial biogenesis while retaining endogenous TGF- signaling
  • TGF-P induces a different response in TGFBRII.41BB CAR-T cells
  • RNA-seq was performed using TGF- treated conventional and TGFBRII.41BB CAR-T cells.
  • the expression (i.e., transcription level) of endogenous TGF-P receptors was assessed by using the normalized RNA-seq data ( Figure 6).
  • Figure 6 the normalized RNA-seq data
  • Figure 7 the conventional CAR-T cell response to TGF- P at the transcriptional level, where TGF-P significantly repressed the expression of genes associated with T cell activation, migration, and cytotoxicity
  • TGFBRII.41BB CAR-T cells also showed a distinct profile of T cell chemotaxis, including CCR1, CCR5, CCL5, IL16, ITGB7 and ITGA1 ( Figure 3D).
  • the chemokine receptor gene, CXCR3 known to drive the migration and trafficking of cytotoxic T cells to solid tumors, also displayed significant upregulation in switch CAR-T cells.
  • conventional CAR-T cells were characterized by high expression of CXCL13, CCL20 and CXCR4 ( Figure 3D).
  • GO analysis was also performed to further investigate the molecular pathways involved in the TGF-P induced transformation of TGFBRII.41BB CAR-T cells.
  • TGFBRII.41BB CAR-T cells were characterized by gene sets associated with the T cell activation and cytolysis (Figure 3E). Pathways associated with T cell migration and trafficking (e.g., chemotaxis, chemokine- and integrin-mediated signaling pathway) were also upregulated by TGF-P in TGFBRII.41BB CAR-T cells versus the conventional counterparts.
  • TGFBRII.41BB fusion protein converts inhibitory signals to unique activation signals
  • TGFBRII.41BB CAR-T cells All the 58 upregulated genes in conventional CAR-T cells were also found in TGFBRII.41BB CAR-T cells, indicating the TGFBRII.41BB fusion protein did not completely compromise the endogenous TGF-P signaling, consistent with the in vitro data presented above (Figure 2E).
  • TGFBRII.41BB CAR- T cells increased expression of genes associated with T cell activation (TNFSF4, CAMK4 W&NFATC2), chemotaxis (EPH A ! and ITGB7) and immune synapse formation (PAK1 and MYH9) ( Figures 4B and 4C).
  • TGFBRII.41BB CAR-T cells were also characterized by increased SLC3A2 and SLC7A5, which encode the amino-acid transporters required for proliferation and effector differentiation in T cells (see, e.g. , Sinclair et al., 2013, Nature Immunology, 14(5): 500-508).
  • TGFBRII.41BB CAR-T cells have enhanced anti-tumor activity in vivo in a prostate cancer model
  • Fusion proteins comprising different intracellular domains enhance CAR-T cell cytotoxicity in the presence of TGF-
  • fusion proteins were produced comprising a range of different intracellular domains, specifically TGFBRII.OX40 (SEQ ID NO: 14), TGFBRII.ICOS (SEQ ID NO: 15) and TGFBRII.CD40 (SEQ ID NO: 16).
  • CAR-T cells were engineered to express each of these fusion proteins, using the method described in Example 1.
  • TGFBRII.ICOS To assess the cytotoxicity of these CAR-T cells, TGFBRII.ICOS, TGFBRII.CD40, TGFBRII.OX40 and TGFBRII.41BB CAR-T cells were co-cultured with the prostate cancer cell line, DU-145, in the presence of exogenous TGF-p.
  • CAR-T cells expressing these fusion proteins significantly increased percentage lysis of tumor cells, indicating that the fusion proteins were activated by TGF-P and subsequently enhanced CAR-T cell-mediated killing of the tumor cells (Figure 13 A).
  • the cytokine production by conventional and fusion protein-expressing CAR-T cells also differed, where fusion protein-expressing CAR-T cells significantly increased production of TNF-a ( Figure 13B).
  • Fusion proteins with different length of intracellular non-kinase region were tagged with either eGFP or mCherry and co-transfected in pairs into HEK293 cells to assess their homodimerization. Heterodimerization was examined by co-transfection of R2 WT eGFP (wildtype TGFBRII tagged with eGFP) along with fusion proteins tagged mCherry.
  • TGFBRII.41BB CAR-T cells have enhanced anti-tumor activity in vivo in a breast cancer model
  • MDA-MB468 breast cancer model established MDA-MB468 tumors develop resistance to CAR-T cell therapy due to high level of TGF-[3 in vivo.
  • NSG mice were engrafted with MDA-MB-468 tumor cells and treated with three doses of CAR-T cells, supported with human IL-2.
  • TGFBRII.41BB CAR-T cells showed significantly improved tumor control up to day 30 post-infusion and better survival relative to treatment with conventional CAR-T cells, which showed no significant difference compared with the control treated mice ( Figure 15).
  • TGF-[3] has a profound direct effect on T cell effector function, proliferation and homeostasis, and also on CD4 + T cell and myeloid cell functional polarization to a suppressor phenotype. In addition, TGF-
  • TGFBRII.41BB fusion protein As described herein, this issue has been addressed by engineering a novel fusion protein comprising a truncated TGFRBII and one or more intracellular co-stimulatory domains.
  • the TGFBRII.41BB fusion protein described herein transduced an activation signal via the 4-1BB intracellular co-stimulatory domain. This led to increased proliferation, cytotoxicity and cytokine secretion in vitro and also improved control over established prostate and breast tumors in vivo.
  • the TGFBRII.41BB fusion protein induced a unique transcriptional profile in the LeY-CAR-T cells, which was distinct from that generated by the endogenous TGF-[3RII.
  • the TGFBRII.41BB CAR-T cells described herein express both the TGFBRII.41BB fusion protein and the endogenous TGF- P receptor.
  • the data presented herein demonstrate that the TGFBRII.41BB fusion protein is dominant, and actively signals in the context of a target antigen expressing tumor cell, i.e., the tumor microenvironment.
  • the signaling from the TGFBRII.41BB fusion protein did not interfere with that of the endogenous TGF-P receptor, which should be sufficient to avoid the serious toxicity that has previously been associated with approaches for inhibiting the immunosuppressive function of TGF-p.
  • TGFBRII.ICOS transduced an activation signal via their respective intracellular domains. This led to increased proliferation, cytotoxicity and cytokine secretion in vitro.
  • signaling from the TGFBRII.ICOS, TGFBRII.CD40 or TGFBRII.OX40 fusion proteins did not interfere with that of the endogenous TGF-P receptor, which should be sufficient to avoid the serious toxicity that has previously been associated with approaches for inhibiting the immunosuppressive function of TGF-p.

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Abstract

La présente invention concerne de manière générale des protéines de fusion comprenant un récepteur 2 du facteur de croissance transformant bêta tronqué (TGFBRII) et un ou plusieurs domaines co-stimulateurs intracellulaires.
EP23889884.5A 2022-11-14 2023-11-14 Protéines de fusion et leurs utilisations Pending EP4619425A1 (fr)

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