WO2023281256A1 - Cellule - Google Patents

Cellule Download PDF

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Publication number
WO2023281256A1
WO2023281256A1 PCT/GB2022/051731 GB2022051731W WO2023281256A1 WO 2023281256 A1 WO2023281256 A1 WO 2023281256A1 GB 2022051731 W GB2022051731 W GB 2022051731W WO 2023281256 A1 WO2023281256 A1 WO 2023281256A1
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cells
cell
tlr2
suitably
tlr4
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PCT/GB2022/051731
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English (en)
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Mala K MAINI
Laura J PALLETT
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Ucl Business Ltd
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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
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
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    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
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    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Definitions

  • the present invention relates to a means of modulating activity of a population of T cells for treating a disease.
  • the present invention relates to expanding CD14 + CD8 + T cells in vivo or inducing or engineering them in vitro for use in treating a disease.
  • the present invention further relates to engineered CD14 + CD8 + T cells, compositions comprising said T cells, methods of preparing said T cells and methods of treating disease using a CD14/TLR2/TLR4 ligand, stroma, CXCL12 or other inducer to expand or induce CD14 + CD8 + T cells or antibiotics to decrease them.
  • Organs that contact the environment form barriers between internal components of the body and external factors.
  • Such “barrier organs” are constantly exposed to non-self antigens, including pathogens, yet must maintain homeostatic immunological tolerance to avoid tissue damage from chronic immune activation.
  • Classical barrier organs are skin, gut, and lungs, but the liver can also be considered a type of barrier organ due to its blood supply from the gut via the portal vein.
  • barrier organs are capable of switching to be immunologically active, pathogens and tumours may exploit the tolerogenic state to evade immune responses.
  • the liver maintains a tolerogenic state despite constant exposure to microbes and microbial products transported from the gut via the portal vein.
  • diseases that may be fostered by the tolerogenic state of the liver include hepatitis B infection and hepatocellular carcinoma.
  • Tissue-resident immune cells are present in barrier organs, such as the liver, and may represent important factors in the balance between tolerance and immunological activity in those organs.
  • tissue-resident T cells expressing CD8 and CD14 have a dual character.
  • these CD14 + CD8 + T cells have immunoregulatory features (for example, homeostatic production of the immunosuppressive cytokine IL-10) that allow the cells to modulate the immune response of other immune cells.
  • the CD14 + CD8 + T cells have effector features, enabling the cells to mount a rapid antigen-specific pro-inflammatory response upon detection of antigen via engagement of their T cell receptor (TCR).
  • TCR T cell receptor
  • the CD14 + CD8 + T cells of the invention may mount a potent, rapid, local immune response to infection or tumours whilst maintaining a generally tolerogenic state. Such capabilities may be particularly useful in maintenance of tolerance whilst mounting pathogen/tumour defence in the context of barrier organs.
  • the invention relates to a means of modulating the activity of CD14 + CD8 + T cells for use in treatment of a disease.
  • the means may be a factor to which the CD14 + CD8 + T cells are responsive or may be CD14 + CD8 + T cells themselves.
  • the activity of the T cells that is modulated may relate to their immunomodulatory character and/or their effector character.
  • the invention provides a CD14/TLR2/TLR4 ligand or a CD14 + CD8 + T cell for use in treating a disease, wherein the CD14/TLR2/TLR4 ligand modulates the activity of a CD14 + CD8 + T cell.
  • the invention further provides an engineered T cell.
  • the engineered T cell may be an engineered CD14 + CD8 + T cell which comprises (i) a nucleic acid construct encoding TLR2 and/or TLR4; (ii) a nucleic acid construct encoding one or more tissue-retention molecules; and/or (iii) a nucleic acid construct encoding an exogenous TCR.
  • the engineered T cell may be an engineered CD8 + T cell which comprises a nucleic acid construct encoding (i) one or more of CD14, TLR2, and/or TLR4, and optionally (ii) a tissue-retention molecule and/or an exogenous TCR.
  • the engineered T cell may be an engineered CD14 + CD8 + T cell comprising a nucleic acid construct encoding an exogenous TCR.
  • the invention also provides a composition comprising an enriched population of CD14 + CD8 + T cells.
  • the invention also provides a pharmaceutical composition comprising a CD14 + CD8 + T cell.
  • the invention also provides a method of treating a disease which comprises administering a CD14/TLR2/TLR4 ligand or a CD14 + CD8 + T cell to a subject in need thereof.
  • the invention also provides a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of peripheral blood mononuclear cells (PBMCs) in vitro with a CD14/TLR2/TLR4 agonist.
  • the invention also provides a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of tissue-resident immune cells in vitro with a CD14/TLR2/TLR4 agonist.
  • the invention also provides a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of T cells in vitro with a CD14/TLR2/TLR4 agonist and/or a cytokine.
  • CD8 + T cells expressing CD14 are detectable in multiple tissue sites (and in various disease settings) in both humans and humanised mice, that are excluded from the blood ex vivo
  • MNP mononuclear phagocytes
  • TLR4 Toll-like receptor-4
  • MD-2 myeloid differentiation factor-2
  • TLR4:CD14 complex B Evaluation of the functionality of the TLR4:CD14 complex by culturing intrahepatic leukocytes in the presence or absence of 5 pg/mL fluorescently-labelled lipopolysaccharide (LPS) over time, prior to flow cytometric staining to determine cellular LPS-uptake.
  • LPS fluorescently-labelled lipopolysaccharide
  • Summary data depict the MFI of the bound
  • _PS AlexaFluor488 on intrahepatic CD14 CD8 + T-cells or CD14 + CD8 + T-cells (n 5) after in vitro culture for 30, 60 or 120 min with
  • CD14 + CD8 + T cells are enriched for markers associated with tissue residency and enhanced tethering
  • CD14 + CD8 + T cells exhibit markers associated with immune activation and immune regulation ex wYo
  • MFI mean fluorescence intensity
  • a Functional assessment of intrahepatic CD14-expressing T-cells by stimulating intrahepatic leukocytes with 1 pg/mL immobilised anti-CD3 and 5 pg/mL soluble anti-CD28 for 4 hr at 37 °C in the presence of 1 pg/mL brefeldin-A (to block cellular cytokine release).
  • H LA- A2- restricted epitopes for hepatitis B virus (HBV) and cytomegalovirus (CMV).
  • CD14 + CD8 + T cells can be expanded from a pre-existing population by alterations in the quantity of TLR ligand exposure in vivo and in vitro
  • FIG. 1 A Schematic depicting the human in vivo skin blister model.
  • Intradermal UV-killed E. coli UV- killed E. coli; strain: NCTC10418; source: Public Health England, UK
  • 1.5x10 7 UV-killed E. coli diluted in 100 pL of sterile saline was injected intradermally into a marked site on the ventral side of the right forearm of participants. From each participant the left forearm was used to raise a “naive” unexposed blister to analyse the exudate in the absence of UV-killed E. coli (baseline blister).
  • B Intrahepatic leukocytes were co-cultured with 0.3x10 6 UV-killed E. coli for various lengths of time and then assessed by flow cytometry.
  • Summary data depict the frequency of CD14 + CD8 + T-cells in the presence of the UV-killed E. coli compared to a media alone control.
  • Intrahepatic leukocytes were stained with a carboxyflorescein succinimidyl ester (CFSE)-dye and an APC-conjugated CD14 monoclonal antibody to identify pre-existing CD14-expressing T cells and co-cultured in in the presence of 0.3x10 6 UV-killed E. coli for 72 hr in vitro.
  • Representative flow cytometry plots depict the dilution of the CFSE dye as a measurement of proliferation of the CD14 + CD8 + T-cells during culture.
  • CD14 + CD8 + T cells can be generated in vitro from peripheral CD8 + T cells using TLR ligands, recombinant cytokines or interaction with hepatic stroma
  • TLR Toll-like receptor
  • the media and cytokines/chemokines (where applicable) were changed and re-supplemented at d 3.
  • Flow cytometric plots depict the expression of TLR2 and TLR4 on CD14 + CD8 + T cells compared to their CD8 + CD14 counterparts from a representative PBMC donor after hepatic stellate cell co-culture. Numbers on plots represent the MFI.
  • MNP mononuclear phagocytes
  • Monocytes (MNP; encompassing classical CD14 hi CD16 _ , non-classical CD14 + CD16 hi , and intermediate CD14 hi CD16 int monocytes) were isolated from peripheral blood using magnetic bead isolation or flow cytometric cell-sorting.
  • MNP classical CD14 hi CD16 _ , non-classical CD14 + CD16 hi , and intermediate CD14 hi CD16 int monocytes
  • G In vitro derivation of CD14-expressing CD8 + T cells: interaction with the hepatic stroma can be blocked by neutralisation of CXCR4.
  • ANOVA Kruskal- Wallis test
  • FIG. 8 Stromal cell derived CD14 + CD8 + T cells recapitulate enhanced TCR-induced effector function and show novel E. co///LPS induced functionality
  • Freshly isolated PBMC were co-cultured in the presence of 20 lU/mL recombinant human IL- 2 with primary human hepatic stellate cells for 4 d to induce CD14 expression. After induction of CD14 expression T cells were harvested and assessed by flow cytometry or Luminex array to determine their functionality: A in the presence or absence of 5 pg/mL fluorescently-labelled LPS at 37 °C for 120 min prior to flow cytometric staining to determine LPS-uptake.
  • Summary data depicts the MFI of
  • _PS AlexaFluor488 on FACS-sorted stromal-cell induced CD14 + CD8 + T- cells or CD14 CD8 + T-cells (n 5) after in vitro culture with labelled LPS, or media alone for 120 min.
  • B after stimulation with either media alone or 100 ng/ml LPS derived from E. coli for 4 hr in the presence of 1 pg/mL brefeldin-A (to block cellular cytokine release).
  • C after stimulation with either media alone or 0.3x10 6 UV-killed E.
  • Figure 9 Genetic engineering can be used to generate a population of CD14 + CD8 + T cells in vitro
  • a Activated T-cells were isolated by magnetic bead separation and were genetically engineered to express a TCR specific for the immunodominant HBc-derived epitope HBCI S -27 (C18; amino acid sequence: FLPSDFFPSV; SEQ ID NO. 18) or HBe-derived epitope HBei83- 91 (E183; amino acid sequence FLLTRILTI; SEQ ID NO 19).
  • Phoenix amphotropic packaging cells were transiently co-transfected using FuGENE with plasmids encoding the C18- or E183-specific TCR and an amphotrophic envelope.
  • Retroviral supernatants were collected for the transduction of donor T cells stimulated with 200 lU/ml IL-2 and 50 ng/ml immobilised anti-CD3. Activated lymphocytes were mixed with retroviral supernatants on retronectin coated plates. Cells were monitored for the expression of the endogenous recombinant TCR and after in vitro expansion in 100 lU/ml IL-2, 10 ng/ml IL-7 and 10 ng/ml IL-15, genetically modified T cells were sorted by flow cytometry.
  • Representative flow cytometry plots depict the expression of CD14 on gene-modified CD8 + T-cells after co-culture in the presence of 20 lU/mL recombinant human IL-2 with primary human hepatic stellate cells +/- the addition of autologous freshly isolated mononuclear phagocytes (MNP; monocytes; isolated using magnetic beads) for 4 d.
  • MNP mononuclear phagocytes
  • Isolated T cells were transfected by electroporation with the plasmid pDUO-hCD14-TLR4A (or GFP-empty control plasmid) designed to induce the co-expression of CD14 and TLR4 genes (InvivoGen). Electroporation was performed using Human T cell Nucleofactor Kit and the V- 024 program on Nucleofactor II device (Lonza). Flow cytometric plots depict the expression of CD14 and TLR4 respectively on pre-gated CD8 + T cells from a representative T cell donor 24 hr after transfection with the GFP-expressing control vector, or CD14/TLR4 construct by flow cytometry.
  • Figure 10 HBV/HCC TCR-re-directed T cells are super-responders following CD14 acquisition
  • the invention provides a CD14/TLR2/TLR4 ligand or a CD14 + CD8 + T cell for use in treating a disease, wherein the CD14/TLR2/TLR4 ligand modulates activity of a CD14 + CD8 + T cell.
  • treating a disease means to ameliorate the effects of a disease in a subject suffering from said disease.
  • the invention may cure the disease completely, eradicating the effects of the disease. Alternatively, the effects of the disease may be reduced but not completely eradicated.
  • the effects of the disease may include symptoms of the disease adverse to the well-being of the subject suffering from the disease.
  • the effects of the disease may additionally or alternatively include the physiological or cellular effects of the disease, regardless of whether or not they are accompanied by symptoms in the subject (for example, presence of virus in the cells of the subject).
  • the disease is a disease of a tissue or organ. In some embodiments of the invention the disease is a disease of a barrier organ.
  • barrier organ refers to an organ of the body that contacts the environment, thereby forming a barrier between internal components of the body and external factors. Such “barrier organs” maintain homeostatic immunological tolerance despite constant exposure to non-self antigens.
  • the aetiology of the disease involves exploitation of the tolerogenic state of the barrier organ.
  • Classical barrier organs are skin, gut and lungs but the liver can be regarded as a semi barrier organ due to its contact with gut products carried to the liver in the portal vein.
  • the disease may be an infection or an infection-related disorder, such as an infection or an infection-related disorder of a tissue or organ, such as a barrier organ.
  • infection-related disorder refers to secondary diseases caused by a primary infection, but which are different from the disease caused directly by the primary infection.
  • HBV Hepatitis B virus
  • HCC hepatocellular carcinoma
  • the disease may be a viral infection or a viral infection-related disorder, such as a viral infection or a viral infection- related disorder of a tissue or organ - such as a barrier organ.
  • the barrier organ is selected from liver, skin or lung.
  • the barrier organ is liver.
  • the barrier organ is skin.
  • the barrier organ is lung.
  • the organ is spleen.
  • the barrier organ is liver and the disease is selected from Hepatitis B virus (HBV) infection, Hepatitis C virus (HCV) infection, cytomegalovirus (CMV) infection, malaria, liver cancer such as hepatocellular carcinoma (HCC) or a secondary metastatic tumour, non-viral hepatitis such as non-alcoholic steatohepatitis (NASH), autoimmune liver disease, primary biliary sclerosis, liver transplant rejection and bacterial peritonitis.
  • HBV Hepatitis B virus
  • HCV Hepatitis C virus
  • CMV cytomegalovirus
  • malaria liver cancer
  • liver cancer such as hepatocellular carcinoma (HCC) or a secondary metastatic tumour
  • non-viral hepatitis such as non-alcoholic steatohepatitis (NASH)
  • autoimmune liver disease primary biliary sclerosis
  • liver transplant rejection and bacterial peritonitis.
  • the disease is liver cancer.
  • the disease is HCC.
  • the disease is HBV infection.
  • the disease is HCV infection.
  • the disease is CMV infection.
  • the disease is malaria.
  • the disease is skin cancer.
  • the disease is a secondary metastatic tumour in the liver.
  • the disease is non-viral hepatitis.
  • the disease is NASH.
  • the disease is alcoholic liver disease.
  • the disease is autoimmune liver disease.
  • the disease is primary biliary cirrhosis.
  • the disease is liver transplant rejection.
  • the disease is eczema.
  • the disease is bacterial peritonitis.
  • CD14 + CD8 + T cell refers to a T cell expressing the proteins cluster of differentiation 14 (CD14) and cluster of differentiation 8 (CD8). Whether cells express cell surface proteins, such as CD14 and CD8, can be determined by techniques known in the art, such as flow cytometry. Antibodies for detecting cell surface molecules such as CD14 and CD8 are known in the art and commercially available. An example of a monoclonal CD14 antibody is that available from BD Bioscience (clone M5E2, catalogue no. 561391). An example of a monoclonal CD8 antibody is that available from Biolegend (clone RPA-T8, catalogue no. 301046).
  • CD8 is a transmembrane glycoprotein formed from a pair of polypeptides, CD8-a and O ⁇ d-b, which acts as a co-receptor with the TCR in antigen recognition.
  • the extracellular domain of CD8 interacts with a MHC molecule class I on an antigen-presenting cell, thereby binding the TCR closely to the antigen-presenting cell.
  • CD8 is also involved in T cell signalling through the interaction of its cytoplasmic domain with intracellular components.
  • CD8 is a marker for cytotoxic T cells.
  • CD8-0 SEQ ID NO. 1
  • the CD8-a polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 1 or a functional fragment thereof.
  • the CD8-a polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 1 or a functional fragment thereof.
  • the CD8-a polypeptide comprises SEQ ID NO. 1 or a functional fragment thereof.
  • the CD8-a polypeptide may be a variant of SEQ ID NO. 1 , for example a natural variant.
  • the CD8-a polypeptide is an isoform of SEQ ID NO. 1.
  • O ⁇ d-b polypeptide An illustrative sequence of a O ⁇ d-b polypeptide is provided by UniProtKB database entry P10966-1 , presented below as SEQ ID NO. 2.
  • the O ⁇ d-b polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 2 or a functional fragment thereof.
  • the O ⁇ d-b polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 2 or a functional fragment thereof.
  • the O ⁇ d-b polypeptide comprises SEQ ID NO. 2 or a functional fragment thereof.
  • the O ⁇ d-b polypeptide may be a variant of SEQ ID NO. 2, for example a natural variant.
  • the O ⁇ d-b polypeptide is an isoform of SEQ ID NO. 2.
  • CD14 is a plasma membrane-localised pattern recognition receptor. Pattern recognition receptors recognise conserved molecular structures known as pathogen- or damage- associated molecular patterns (PAMPs or DAMPs) found in pathogens. In response to binding of DAMPs, pattern recognition receptors signal to induce an anti-pathogenic response from the cell. CD14 acts as a co-receptor with Toll-like receptor 4 (TLR4) in the detection of bacterial lipopolysaccharide (LPS). CD14 can also act as a co-receptor for other DAMPs in combination with alternative TLRs such as TLR2.
  • TLR4 Toll-like receptor 4
  • LPS bacterial lipopolysaccharide
  • CD14 polypeptide An illustrative sequence of a CD14 polypeptide is provided by UniProtKB database entry P08571-1 , presented below as SEQ ID NO. 3.
  • CD14 (SEQ ID NO. 3)
  • the CD14 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 3 or a functional fragment thereof.
  • the CD14 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 3 or a functional fragment thereof.
  • the CD14 polypeptide comprises SEQ ID NO. 3 or a functional fragment thereof.
  • the CD14 polypeptide may be a variant of SEQ ID NO. 3, for example a natural variant.
  • the CD14 polypeptide is an isoform of SEQ ID NO. 3.
  • the CD14 + CD8 + T cell expresses TLR2 and/or TLR4 and/or MD2.
  • TLR2 and TLR4 are members of the toll-like receptor (TLR) family.
  • TLRs are T-cell surface proteins that recognise pathogenic molecules. Recognition of such molecules triggers signalling pathways leading to a pro-inflammatory immune response.
  • TLR4 functions with CD14 to recognise bacterial lipopolysaccharide (LPS).
  • LPS bacterial lipopolysaccharide
  • TLR2 functions with CD14 to recognise other DAMPs.
  • TLR2 a TLR2 polypeptide is provided by UniProtKB database entry 060603-1, presented below as SEQ ID NO. 4.
  • TLR2 (SEQ ID NO. 4)
  • the TLR2 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 4 or a functional fragment thereof.
  • the TLR2 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 4 or a functional fragment thereof.
  • the TLR2 polypeptide comprises SEQ ID NO. 4 or a functional fragment thereof.
  • the TLR2 polypeptide may be a variant of SEQ ID NO. 4, for example a natural variant.
  • the TLR2 polypeptide is an isoform of SEQ ID NO. 4.
  • TLR2 antibody An example of a monoclonal TLR2 antibody is that available from BD Bioscience (clone 11 G7, catalogue no. 742770).
  • TLR4 an illustrative sequence of a TLR4 polypeptide is provided by UniProtKB database entry 000206-1, presented below as SEQ ID NO. 5.
  • TLR4 (SEQ ID NO. 5)
  • the TLR4 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 5 or a functional fragment thereof.
  • the TLR4 polypeptide comprises SEQ ID NO. 5 or a functional fragment thereof.
  • the TLR4 polypeptide may be a variant of SEQ ID NO. 5, for example a natural variant.
  • the TLR4 polypeptide is an isoform of SEQ ID NO. 5.
  • An example of a monoclonal TLR4 antibody is that available from BD Bioscience (clone HTA125, catalogue no. 743392).
  • CD14 + CD8 + T cell expresses MD-2.
  • An illustrative sequence of a MD-2 polypeptide is provided by UniProtKB database entry Q9Y6Y9, presented below as SEQ ID NO. 14.
  • the MD-2 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 14 or a functional fragment thereof.
  • the MD-2 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 14 or a functional fragment thereof.
  • the MD-2 polypeptide comprises SEQ ID NO. 14 or a functional fragment thereof.
  • the MD-2 polypeptide may be a variant of SEQ ID NO. 14, for example a natural variant.
  • the MD-2 polypeptide is an isoform of SEQ ID NO. 14.
  • TLR2 antibody An example of a monoclonal TLR2 antibody is that available from ThermoFisher Scientific (clone 9F1 B1, catalogue no. MA5-15765).
  • the CD14 + CD8 + T cell further expresses one or more of Foxp3, CTLA-4, IL-2 and/or IL-10.
  • CD14 + CD8 + T cell further expresses Foxp3.
  • Foxp3 is the abbreviated name of the forkhead box P3 protein.
  • FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of the regulatory pathway in the development and function of regulatory T cells.
  • An illustrative sequence of a Foxp3 polypeptide is provided by UniProtKB database entry Q9BZS1-1, presented below as SEQ ID NO. 6.
  • the Foxp3 polypeptide comprises an amino acid sequence which is at
  • the Foxp3 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 6 or a functional fragment thereof.
  • the Foxp3 polypeptide comprises SEQ ID NO. 6 or a functional fragment thereof.
  • the Foxp3 polypeptide may be a variant of SEQ ID NO. 6, for example a natural variant.
  • the Foxp3 polypeptide is an isoform of SEQ ID NO. 6.
  • CTLA-4 cytotoxic T lymphocyte-associated protein 4
  • SEQ ID NO. 7 An illustrative sequence of a CTLA-4 polypeptide is provided by UniProtKB database entry P16410-1 , presented below as SEQ ID NO. 7.
  • CTLA-4 (SEQ ID NO. 7)
  • the CTLA-4 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 7 or a functional fragment thereof.
  • the CTLA-4 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 7 or a functional fragment thereof.
  • the CTLA-4 polypeptide comprises SEQ ID NO. 7 or a functional fragment thereof.
  • the CTLA-4 polypeptide may be a variant of SEQ ID NO. 7, for example a natural variant.
  • the CTLA-4 polypeptide is an isoform of SEQ ID NO. 7.
  • the CD14 + CD8 + T cell further expresses IL-2.
  • the CD14 + CD8 + T cell further expresses IL-10.
  • IL-2 and IL-10 are cytokines, which bind to receptors on immune cells and thereby stimulate downstream signalling that modulates activity of the immune cells.
  • IL-2 induces Foxp3 and IL-10 expression and is critical for maintenance of tolerance through regulatory T cells.
  • IL-10 has a variety of effects on immunoregulation and inflammation, including suppression of expression of cytokines and MHC class II molecules.
  • IL-2 (SEQ ID NO. 8) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVE FLNRWITFCQSIISTLT
  • the IL-2 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 8 or a functional fragment thereof.
  • the IL-2 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 8 or a functional fragment thereof.
  • the IL-2 polypeptide comprises SEQ ID NO. 8 or a functional fragment thereof.
  • the IL-2 polypeptide may be a variant of SEQ ID NO. 8, for example a natural variant.
  • the IL-2 polypeptide is an isoform of SEQ ID NO. 8.
  • the IL-10 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 9 or a functional fragment thereof.
  • the IL-10 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 9 or a functional fragment thereof.
  • the IL-10 polypeptide comprises SEQ ID NO. 9 or a functional fragment thereof.
  • the IL-10 polypeptide may be a variant of SEQ ID NO. 9, for example a natural variant.
  • the IL-10 polypeptide is an isoform of SEQ ID NO. 9.
  • the CD14 + CD8 + T cell further expresses one or more tissue-retention molecules.
  • tissue-retention molecule refers to a molecule that causes the immune cell expressing it to localise to, and be retained in a particular tissue in vivo.
  • a tissue-retention molecule may be referred to as a “tissue-retention signal” or an “organ homing molecule”.
  • a tissue-retention molecule may target the cell to a barrier organ.
  • the tissue-retention molecule may target the cell to liver.
  • the tissue-retention molecule may target the cell to skin.
  • the tissue-retention molecule may target the cell to lung.
  • the tissue-retention molecule may target the cell to spleen.
  • a tissue-retention molecule may localise an immune cell to a particular tissue by causing the immune cell to be retained in the tissue.
  • the tissue-retention molecule may be a cell surface receptor, such as a cell adhesion molecule.
  • a cell adhesion molecule binds characteristic molecules on target tissues, thereby adhering the immune cell expressing the adhesion molecule to that specific tissue.
  • a cell adhesion molecule may to characteristic molecules in the extracellular matrix of a specific tissue.
  • tissue-retention molecule may localise an immune cell to a particular tissue by facilitating recruitment of the immune cell to the organ.
  • the tissue-retention molecule may be a chemokine receptor to which chemokines (chemotactic cytokines) bind, signalling to attract the cell to a particular tissue.
  • the one or more tissue-retention molecules are selected from CD69, CD103, CXCR3, CXCR6, CXCR4, CD49a, CD49b, CD11a and ICAM.
  • the one or more tissue-retention molecules are selected from CD69, CD103, CD49a, CD49b, CXCR3, CXCR4 and CXCR6.
  • the one or more tissue-retention molecules are selected from CD69, CD103, CXCR3 and CXCR6.
  • CD14 + CD8 + T cell expresses CD69.
  • CD69 is a transmembrane protein associated with both regulatory T cells and memory T cells.
  • CD69 is a negative regulator of sphingosine 1 phosphate receptor-mediated T cell egress.
  • CD69 expression contributes to retention of the CD14 + CD8 + T cells in liver.
  • An illustrative sequence of a CD69 polypeptide is provided by UniProtKB database entry Q07108-1 , presented below as SEQ ID NO. 10.
  • CD69 (SEQ ID NO. 10)
  • the CD69 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 10 or a functional fragment thereof.
  • the CD69 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 10 or a functional fragment thereof.
  • the CD69 polypeptide comprises SEQ ID NO. 10 or a functional fragment thereof.
  • the CD69 polypeptide may be a variant of SEQ ID NO. 10, for example a natural variant.
  • the CD69 polypeptide is an isoform of SEQ ID NO. 10.
  • CD14 + CD8 + T cell expresses CD103.
  • CD103 is a transmembrane protein, specifically an integrin a sub-unit (integrin aE). Integrins are cell adhesion molecules. CD103 expression contributes to retention of T cells in tissues by allowing the T cell to bind epithelial cells expressing e-cadherin, the ligand for aEb7 integrin. Human liver epithelia (hepatocytes) express e-cadherin. Suitably, CD103 expression contributes to retention of the CD14 + CD8 + T cells in liver. T RM cells in human lung express CD103. Suitably, CD103 expression contributes to retention of the CD14 + CD8 + T cells in lung.
  • An illustrative sequence of a CD103 polypeptide is provided by UniProtKB database entry P38570-1, presented below as SEQ ID NO. 11.
  • CD103 (SEQ ID NO. 11)
  • the CD103 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 11 or a functional fragment thereof.
  • the CD103 polypeptide comprises SEQ ID NO. 11 or a functional fragment thereof.
  • the CD103 polypeptide may be a variant of SEQ ID NO. 11, for example a natural variant.
  • the CD103 polypeptide is an isoform of SEQ ID NO. 11.
  • CD14 + CD8 + T cell expresses CD49a.
  • CD49a is an integrin alpha subunit. It makes up half of the a1b1 integrin duplex.
  • CD49a expression contributes to retention of the CD14 + CD8 + T cells in liver.
  • T RM cells in human lung express CD49a.
  • CD49a expression contributes to retention of the CD14 + CD8 + T cells in lung.
  • CD49a binds to collagen-IV in the extracellular matrix.
  • An illustrative sequence of a CD49a polypeptide is provided by UniProtKB database entry P56199, presented below as SEQ ID NO. 15.
  • CD49a (SEQ ID NO. 15)
  • the CD49a polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 15 or a functional fragment thereof.
  • the CD49a polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 15 or a functional fragment thereof.
  • the CD49a polypeptide comprises SEQ ID NO. 15 or a functional fragment thereof.
  • the CD49a polypeptide may be a variant of SEQ ID NO. 15, for example a natural variant.
  • the CD49a polypeptide is an isoform of SEQ ID NO. 15.
  • CD14 + CD8 + T cell expresses CD49b.
  • CD49b is an integrin alpha subunit. It makes up half of the a2b1 integrin duplex.
  • CD49b expression contributes to retention of the CD14 + CD8 + T cells in liver.
  • An illustrative sequence of a CD49b polypeptide is provided by UniProtKB database entry P17301, presented below as SEQ ID NO. 17.
  • CD49b (SEQ ID NO. 17)
  • the CD49b polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 17 or a functional fragment thereof.
  • the CD49b polypeptide comprises SEQ ID NO. 17 or a functional fragment thereof.
  • the CD49b polypeptide may be a variant of SEQ ID NO. 17, for example a natural variant.
  • the CD49b polypeptide is an isoform of SEQ ID NO. 17.
  • CXCR3 is a chemokine receptor.
  • CXCR3 ligands include CXCL9, CXCL10 and CXCL11. Human hepatocytes and hepatic endothelium have been shown to express CXCR3 ligands.
  • CXCR3 expression contributes to retention of the CD14 + CD8 + T cells in liver.
  • CXCR3 is critical for directing T RM cells to virally infected mouse skin.
  • CXCR3 expression contributes to retention of the CD14 + CD8 + T cells in skin.
  • An illustrative sequence of a CXCR3 polypeptide is provided by UniProtKB database entry P49682-1, presented below as SEQ ID NO. 12.
  • the CXCR3 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 12 or a functional fragment thereof.
  • the CXCR3 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 12 or a functional fragment thereof.
  • the CXCR3 polypeptide comprises SEQ ID NO. 12 or a functional fragment thereof.
  • the CXCR3 polypeptide may be a variant of SEQ ID NO. 12, for example a natural variant.
  • the CXCR3 polypeptide is an isoform of SEQ ID NO. 12.
  • CXCR6 is a chemokine receptor.
  • CXCR6 ligands include CXCL16.
  • Human liver cells express CXCL16.
  • CXCR6 expression contributes to retention of the CD14 + CD8 + T cells in liver.
  • An illustrative sequence of a CXCR6 polypeptide is provided by UniProtKB database entry 000574-1, presented below as SEQ ID NO. 13.
  • the CXCR6 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 13 or a functional fragment thereof.
  • the CXCR6 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 13 or a functional fragment thereof.
  • the CXCR6 polypeptide comprises SEQ ID NO. 13 or a functional fragment thereof.
  • the CXCR6 polypeptide may be a variant of SEQ ID NO. 13, for example a natural variant.
  • the CXCR6 polypeptide is an isoform of SEQ ID NO. 13.
  • CXCR4 is a chemokine receptor.
  • CXCR4 is specific for stromal-derived-factor- 1 (SDF-1/CXCL12).
  • SDF-1/CXCL12 stromal-derived-factor- 1
  • CXCR4 expression contributes to retention of the CD14 + CD8 + T cells in liver.
  • An illustrative sequence of a CXCR4 polypeptide is provided by UniProtKB database entry P61073-1 , presented below as SEQ ID NO. 16.
  • the CXCR4 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 16 or a functional fragment thereof.
  • the CXCR4 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 16 or a functional fragment thereof.
  • the CXCR4 polypeptide comprises SEQ ID NO. 16 or a functional fragment thereof.
  • the CXCR4 polypeptide may be a variant of SEQ ID NO. 16, for example a natural variant.
  • the CXCR4 polypeptide is an isoform of SEQ ID NO. 16.
  • the CD14 + CD8 + T cell has been isolated and enriched to form a CD14 + CD8 + T cell composition.
  • isolated and enriched means to separate out CD14 + CD8 + T cells from a heterogeneous mixture of multiple types of cells, thereby increasing the proportion of the cells in the separated population that are CD14 + CD8 + T cells.
  • the separated population of CD14 + CD8 + T cells comprises at least 70 % CD14 + CD8 + T cells, such as 75 %, 85 %, 90 % or 95 % CD14 + CD8 + T cells.
  • T echniques for isolating specific cell types from a heterogeneous population of cells are known in the art. Examples include use of immuno-magnetic beads and fluorescence-activated cell sorting (FACS).
  • isolating the population of Tregs comprises using immuno-magnetic beads.
  • immuno-magnetic beads Various companies (e.g. Miltenyi Biotec, Stem Cell Technologies, ThermoFisher Scientific) offer kits comprising immuno-magnetic beads for isolation of specific types of T cells. These isolation kits make use of antibodies widely available in the art to T cell surface proteins such as CD8, CD25, CD49b and others.
  • CD8 + cells may be first negatively selected by incubating the cell population with biotin-conjugated antibodies to markers of non-CD14 + CD8 + T cells and removing these cells using anti-biotin magnetic beads.
  • CD8 + cells may be positively selected by incubation with anti-CD8-labelled beads.
  • FACS is a form of flow cytometry which is well-known in the art. During FACS, cells are suspended in fluid and streamed through a detection system that analyses various characteristics. Cells can be sorted according to their characteristics using this method. In particular, in FACS, molecules are marked using fluorescent antibodies and cells sorted according to their degree of fluorescence, which indicates level of expression of the particular molecule (see Adan et al. Flow cytometry: basic principles and applications Crit. Rev. Biotechnol. 2017 Mar;37(2):163-176, incorporated herein by reference).
  • the CD14 + CD8 + T cell may have been isolated from the subject to be treated.
  • Isolating cells from the subject may involve taking a sample from the body of the subject, such as a blood sample or a tissue sample from an organ.
  • a sample of liver tissue can be taken using liver biopsy techniques known in the art.
  • Such a sample is typically a heterogeneous population of cells.
  • CD14 + CD8 + T cells may be isolated from the heterogeneous population using the techniques known in the art and described herein.
  • the CD14 + CD8 + T cell has been isolated and enriched from a tissue or organ, optionally wherein the CD14 + CD8 + T cell has been isolated and enriched from the tissue or organ of the subject to be treated.
  • the CD14 + CD8 + T cell has been isolated and enriched from the same organ as the infection or infection-related disease. In some embodiments of the invention the CD14 + CD8 + T cell has been isolated and enriched from a barrier organ. Optionally, the CD14 + CD8 + T cell may have been isolated and enriched from a barrier organ of the subject to be treated.
  • the T cell has been engineered to express CD14, TLR2 and/or TLR4.
  • the expression “engineered to express” means that the T cell has been manipulated by human intervention such that it expresses a particular molecule. Techniques are known in the art for manipulating cells to express particular molecules. Such techniques include introducing into a cell an exogenous polynucleotide encoding the molecule of interest. Accordingly, the engineered T cell may comprise an exogenous polynucleotide encoding CD14, TLR2 and/or TLR4. The engineered T cell may comprise an exogenous polynucleotide encoding CD14. The engineered T cell may comprise an exogenous polynucleotide encoding TLR2. The engineered T cell may comprise an exogenous polynucleotide encoding TLR2.
  • exogenous polynucleotide is a polynucleotide that originates outside the T cell.
  • the exogenous polynucleotide may be introduced into the T cell as part of an expression vector. Accordingly, the exogenous polynucleotide may be contiguous with expression vector elements, such as a promoter, which facilitate expression of the protein encoded by the exogenous polynucleotide.
  • Techniques for introducing exogenous polynucleotides into cells are known in the art, and include transfection and transduction methods. Transfection is the process of introducing nucleic acids into a cell by non-viral methods.
  • Transduction is the process of introducing an exogenous polynucleotide into a cell via a viral vector.
  • Viral delivery systems include but are not limited to adenovirus vector, an adeno-associated viral (AAV) vector, a herpes viral vector, retroviral vector, lentiviral vector, baculoviral vector.
  • CD14 comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 3 ora functional fragment thereof.
  • the CD14 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 3 or a functional fragment thereof.
  • the CD14 polypeptide comprises SEQ ID NO. 3 or a functional fragment thereof.
  • the CD14 polypeptide may be a variant of SEQ ID NO. 3, for example a natural variant.
  • the CD14 polypeptide is an isoform of SEQ ID NO. 3.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 3 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 3 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 3 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • TLR2 comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 4 ora functional fragment thereof.
  • the TLR2 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 4 or a functional fragment thereof.
  • the TLR2 polypeptide comprises SEQ ID NO. 4 or a functional fragment thereof.
  • the TLR2 polypeptide may be a variant of SEQ ID NO. 4, for example a natural variant.
  • the TLR2 polypeptide is an isoform of SEQ ID NO. 4.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 4 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 4 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 4 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • TLR4 comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 5 ora functional fragment thereof.
  • the TLR4 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 5 or a functional fragment thereof.
  • the TLR4 polypeptide comprises SEQ ID NO. 5 or a functional fragment thereof.
  • the TLR4 polypeptide may be a variant of SEQ ID NO. 5, for example a natural variant.
  • the TLR4 polypeptide is an isoform of SEQ ID NO. 5.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 5 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 5 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 5 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • the T cell has been engineered to express one or more tissue-retention markers.
  • the one or more tissue-retention markers are selected from CD49a, CD49b, CD69, CD103, CXCR3, CXCR4 and CXCR6.
  • the T cell has been engineered to express CD49a.
  • the CD49a polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 15 or a functional fragment thereof.
  • the CD49a polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 15 or a functional fragment thereof.
  • the CD49a polypeptide comprises SEQ ID NO. 15 or a functional fragment thereof.
  • the CD49a polypeptide may be a variant of SEQ ID NO. 15, for example a natural variant.
  • the CD49a polypeptide is an isoform of SEQ ID NO. 15.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 15 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 15 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 15 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • the T cell has been engineered to express CD49b.
  • the CD49b polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 17 or a functional fragment thereof.
  • the CD49b polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 17 or a functional fragment thereof.
  • the CD49b polypeptide comprises SEQ ID NO. 17 or a functional fragment thereof.
  • the CD49b polypeptide may be a variant of SEQ ID NO. 17, for example a natural variant.
  • the CD49b polypeptide is an isoform of SEQ ID NO. 17.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 17 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 17 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 17 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • the T cell has been engineered to express CD69.
  • the CD69 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 10 or a functional fragment thereof.
  • the CD69 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 10 or a functional fragment thereof.
  • the CD69 polypeptide comprises SEQ ID NO. 10 or a functional fragment thereof.
  • the CD69 polypeptide may be a variant of SEQ ID NO. 10, for example a natural variant.
  • the CD69 polypeptide is an isoform of SEQ ID NO. 10.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 10 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 10 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 10 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • the T cell has been engineered to express CD103.
  • the CD103 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 11 or a functional fragment thereof.
  • the CD103 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 11 or a functional fragment thereof.
  • the CD103 polypeptide comprises SEQ ID NO. 11 or a functional fragment thereof.
  • the CD103 polypeptide may be a variant of SEQ ID NO. 11 , for example a natural variant.
  • the CD103 polypeptide is an isoform of SEQ ID NO. 11.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 11 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 11 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 11 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • the T cell has been engineered to express CXCR3.
  • the CXCR3 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 12 or a functional fragment thereof.
  • the CXCR3 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 12 or a functional fragment thereof.
  • the CXCR3 polypeptide comprises SEQ ID NO. 12 or a functional fragment thereof.
  • the CXCR3 polypeptide may be a variant of SEQ ID NO. 12, for example a natural variant.
  • the CXCR3 polypeptide is an isoform of SEQ ID NO. 12.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 12 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 12 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 12 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • the T cell has been engineered to express CXCR6.
  • the CXCR6 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 13 or a functional fragment thereof.
  • the CXCR6 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 13 or a functional fragment thereof.
  • the CXCR6 polypeptide comprises SEQ ID NO. 13 or a functional fragment thereof.
  • the CXCR6 polypeptide may be a variant of SEQ ID NO. 13, for example a natural variant.
  • the CXCR6 polypeptide is an isoform of SEQ ID NO. 13.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 13 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 13 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 13 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • the T cell has been engineered to express CXCR4.
  • the CXCR4 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO. 16 or a functional fragment thereof.
  • the CXCR4 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 16 or a functional fragment thereof.
  • the CXCR4 polypeptide comprises SEQ ID NO. 16 or a functional fragment thereof.
  • the CXCR4 polypeptide may be a variant of SEQ ID NO. 16, for example a natural variant.
  • the CXCR4 polypeptide is an isoform of SEQ ID NO. 16.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 80% identical to SEQ ID NO. 16 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO. 16 or a functional fragment thereof.
  • the engineered T cell comprises a nucleic acid construct encoding an amino acid sequence SEQ ID NO. 16 or a functional fragment thereof.
  • the sequence of the nucleic acid construct may vary substantially without altering the sequence of the encoded polypeptide owing to the redundancy of the genetic code.
  • the invention also provides an engineered CD14 + CD8 + T cell which comprises (i) a nucleic acid construct encoding TLR2 and/or TLR4; and/or (ii) a nucleic acid construct encoding one or more tissue-retention molecules.
  • the engineered T cell may comprise a nucleic acid construct encoding one or more of the polypeptides described herein.
  • the engineered T cell may comprise a nucleic acid construct encoding one or more of TLR2, TLR4, CD69, CD103, CXCR3, and/or CXCR6.
  • the T cell may be engineered to express an exogenous TOR directed against, for example, a cancer-associated antigen or epitope or HBV antigen or epitope.
  • the T cell may be a CD8 + T cell which is engineered to express one or more of the polypeptides described herein.
  • the engineered T cell may comprise a nucleic acid construct encoding one or more of CD14, TLR2, TLR4, CD69, CD103, CXCR3, and/or CXCR6.
  • the invention also provides an engineered CD14 + CD8 + T cell comprising a nucleic acid construct encoding an exogenous TOR.
  • engineered CD14 + CD8 + T cell expresses an exogenous TOR.
  • the exogenous TOR is specific for the HBc-derived epitope HBcis-27or the HBe-derived epitope HBei83-9i.
  • the exogenous TOR is specific for the HBe-derived epitope HBei83-9i.
  • An engineered CD14 + CD8 + T cell expressing an exogenous TOR specific for HBei83-9i may be referred to herein as an env183 + CD14 + CD8 + T cell.
  • Expression of an exogenous TOR specific for the HBc-derived epitope HBC IS -27 or the HBe-derived epitope HBei83-9i in a T cell is known in the art, and described in for example Gehring et al. 2011, Journal of Hepatology vol. 55, 103-110.
  • a CD14/TLR2/TLR4 ligand is an extracellular molecule that associates with at least one of a CD14 molecule, a TLR2 molecule and/or a TLR4 molecule on the surface of a T cell.
  • CD14, TLR2 and TLR4 are T cell surface proteins that recognise pathogenic molecules. Such recognition triggers signalling pathways leading to an immune response.
  • CD14 functions with TLR4 to bind LPS and with TLR2 to bind other DAMPs.
  • the CD14/TLR2/TLR4 ligand may bind TLR2 in a dimer with other TLR proteins, such as a TLR2/TLR1 heterodimer or a TLR2/TLR6 heterodimer.
  • CD14 may be associated with TLR2, such as in a TLR2/TLR1 heterodimer or a TLR2/TLR6 heterodimer.
  • the CD14/TLR2/TLR4 ligand may bind a TLR4 homodimer.
  • CD14 may be associated with TLR4, such as a TLR4 homodimer.
  • the CD14/TLR2/TLR4 ligand may also be referred to as a CD14 ligand, a TLR2 ligand, a TLR4 ligand or a TLR2/TLR4 ligand.
  • the term “CD14/TLR2/TLR4 ligand” encompasses CD14 ligands, TLR2 ligands, TLR4 ligands and TLR2/TLR4 ligands.
  • the CD14/TLR2/TLR4 ligand may be from a pathogen.
  • the CD14/TLR2/TLR4 ligand may be a DAMP.
  • the CD14/TLR2/TLR4 ligand may be a synthetic molecule.
  • the CD14/TLR2/TLR4 ligand may be an antibody or an antibody fragment, a peptide or a small molecule.
  • the CD14/TLR2/TLR4 ligand is a CD14/TLR2/TLR4 agonist and enhances the activity of a CD14 + CD8 + T cell.
  • the term “agonist” refers to a molecule that binds to a receptor and stimulates a downstream signalling pathway.
  • a CD14/TLR2/TLR4 agonist is a CD14/TLR2/TLR4 ligand that, when it binds CD14, TLR2 and/or TLR4, stimulates a signalling pathway downstream of CD14, TLR2 and/or TLR4.
  • the CD14/TLR2/TLR4 agonist may also be referred to as a CD14 agonist, a TLR2 agonist, a TLR4 agonist and/or a TLR2/TLR4 agonist.
  • the term “CD14/TLR2/TLR4 agonist” encompasses CD14 agonists, TLR2 agonists, TLR4 agonists and TLR2/TLR4 agonists.
  • the CD14/TLR2/TLR4 agonist is selected from pharmaceutically acceptable UV-killed E.
  • LPS lipopolysaccharide
  • MPLA monophosphoryl lipid A
  • LTA lipoteichoic acid
  • diacyl lipoproteins such as Pam2CSK4 and/or triacyl lipoproteins such as Pam3CSK4.
  • the CD14/TLR2/TLR4 agonist is LPS.
  • LPS is a component of the outer membrane of Gram-negative bacteria that can induce an immune response in humans.
  • LPS consists of a “core” oligosaccharide linked to O antigen and Lipid A.
  • the core oligosaccharide consists of about 9 sugar residues whilst the O antigen is a longer polysaccharide.
  • Lipid A consists of sugar residues linked to fatty acid chains.
  • LPS is recognised by TLR4 in combination with CD14, MD-2 and LPS-binding protein (LBP) on the surface of T cells and thereby stimulates an immune response.
  • LPS may be referred to as a TLR4 ligand and/or a TLR4 agonist.
  • UV-killed E. coli may be used as a source of LPS.
  • LPS is commercially available, for example LPS-EK (LPS from E. coli strain K12) from Invivogen.
  • the CD14/TLR2/TLR4 agonist is MPLA.
  • MPLA is a form of the Lipid A component of LPS. Particular forms of Lipid A can stimulate an immune response, and the ability of LPS to do so depends on the Lipid A within its structure.
  • MPLA is recognised by TLR4 on the surface of T cells.
  • MPLA may be referred to as a TLR4 ligand and/or a TLR4 agonist.
  • MPLA is commercially available, for example MPLA-SM (MPLA from Salmonella minnesota strain R595) from Invivogen. MPLA is in widespread use as a vaccine adjuvant, making MPLA particularly suitable for use in the present invention.
  • the CD14/TLR2/TLR4 agonist is Pam2CSK4.
  • Pam3CSK4 is a synthetic diacylated lipopeptide (LP).
  • Pam2SCK4 is recognised by TLR2 on the surface of T cells.
  • Pam2CSK4 may be referred to as a TLR2 ligand and/or a TLR2 agonist.
  • Pam2CSK4 is commercially available, for example from Invivogen.
  • the CD14/TLR2/TLR4 agonist is Pam3CSK4.
  • Pam3CSK4 is a synthetic triacylated lipopeptide (LP) that mimics the acylated amino terminus of bacterial LPS.
  • LP triacylated lipopeptide
  • Pam3SCK4 is recognised by TLR2 on the surface of T cells, in particular by the TLR2/TLR1 heterodimer.
  • Pam3CSK4 may be referred to as a TLR2 ligand and/or a TLR2 agonist.
  • Pam3CSK4 is commercially available, for example from Invivogen.
  • the CD14/TLR2/TLR4 agonist is LTA.
  • LTA is a surface-associated molecule from Gram-positive bacteria. LTA is recognised by TLR2 on the surface of T cells. Thus LTA may be referred to as a TLR2 ligand and/or a TLR2 agonist.
  • LTA is commercially available, for example LTA-BS (LTA from Bacillus subtilis) from Invivogen.
  • the CD14/TLR2/TLR4 agonist is UV-killed E.coli. UV-killed E. coli are recognised by TLR2 and TLR4 on the surface of T cells. Thus UV-killed E.coli may be referred to as a TLR2/TLR4 ligand and/or a TLR2/TLR4 agonist.
  • LPS, MPLA and UV-killed E.coli are TLR4 ligands and TLR4 agonists.
  • LTA, Pam2CSK4, Pam3CSK4 and UV-killed E.coli are TLR2 ligands and TLR2 agonists.
  • the present invention provides a TLR2 ligand or a TLR4 ligand for use in treating a disease, wherein the TLR2 ligand or TLR4 ligand modulates activity of a CD14 + CD8 + T cell.
  • the present invention provides a TLR2 agonist or a TLR4 agonist for use in treating a disease, wherein the TLR2 agonist or TLR4 agonist modulates activity of a CD14 + CD8 + T cell.
  • the CD14/TLR2/TLR4 agonist may be targeted to a specific tissue of interest (e.g. liver) in order to engage the cell type of interest locally.
  • a specific tissue of interest e.g. liver
  • the CD14/TLR2/TLR4 agonist is administered in combination with a CD3 agonist.
  • CD3 is a protein complex expressed at the surface of T cells consisting of four polypeptides (CD3y, CD36 and two CD3s). CD3 functions as a co-receptor with the TCR, associating with the TCR and a z-chain polypeptide to generate a T cell activation signal. Accordingly, CD3 is required for T cell activation.
  • a CD3 agonist is a molecule that binds to CD3 and stimulates a signalling pathway downstream of CD3, such as a pathway associated with T cell activation.
  • the CD3 agonist is an antibody.
  • An example of an antibody that binds to CD3 is OKT3.
  • modulates activity means to increase or to decrease.
  • modulates means increasing the activity of T cells.
  • the expressions “enhances” and “increases” are synonymous.
  • modulates activity means decreasing the activity of T cells.
  • the term “activity” refers to a function of a T cell. Examples of such functions include proliferation, cytokine secretion and cytotoxic activity. Accordingly, “modulating the activity of a T cell” may mean to cause that T cell to proliferate, or to inhibit proliferation of that T cell. Alternatively or additionally, “modulating the activity of a T cell” may mean to inhibit proliferation of that T cell or to inhibit secretion of cytokines by that T cell.
  • the activity of CD14 + CD8 + T cells is increased by increasing the number of CD14 + CD8 + T cells, for example by inducing proliferation of CD14 + CD8 + T cells.
  • the number of CD14 + CD8 + T cells may be increased by inducing T cells that do not express CD14 to express CD14.
  • T cells that do not express CD14 may be induced to express CD14 by treating the T cells with a CD14/TLR2/TLR4 ligand, such as LPS.
  • the number of CD14 + CD8 + T cells may be increased by inducing T cells that express CD14 to proliferate.
  • the number of CD14 + CD8 + T cells may be increased by supplying or administering CD14 + CD8 + T cells to a subject.
  • an initial population of CD14 + CD8 + T cells is supplemented by the addition of further CD14 + CD8 + T cells.
  • the number and/or activity of CD14 + CD8 + T cells is decreased by administering antibiotics to the subject.
  • the CD14/TLR2/TLR4 agonist enhances proliferation of the CD14 + CD8 + T cell, and/or enhances immunomodulatory activity of the CD14 + CD8 + T cell and/or enhances an effector response of the CD14 + CD8 + T cell.
  • the CD14/TLR2/TLR4 agonist enhances proliferation of a CD14 + CD8 + T cell.
  • To enhance proliferation means to increase the number of CD14 + CD8 + T cells, such as by stimulating the T cells to divide. Proliferation of T cells may be measured by techniques known in the art, such as measuring Ki67 expression, CFSE staining or cell counting.
  • the number of CD14 + CD8 + T cells may have increased 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more.
  • the CD14 + CD8 + T cells treated with the CD14/TLR2/TLR4 agonist according to the invention may proliferate two or more times more rapidly than CD14 + CD8 + T cells not treated with the CD14/TLR2/TLR4 agonist, such as three times more rapidly, such as five times more rapidly, such as ten times more rapidly.
  • CD14 + CD8 + T cells not treated with the CD14/TLR2/TLR4 agonist may not proliferate at all, whereas CD14 + CD8 + T cells treated with the CD14/TLR2/TLR4 agonist do proliferate.
  • the CD14/TLR2/TLR4 agonist enhances the immunomodulatory activity of a CD14 + CD8 + T cell.
  • Immunomodulatory activity refers to the ability to influence the activity of other cells of the immune system.
  • CD14 + CD8 + T cells with enhanced immunomodulatory activity have an increased ability to influence the activity of other immune cells.
  • Immunomodulatory activity is facilitated by secretion of factors that signal to other immune cells, such as cytokines which bind to receptors on the surface of immune cells and alter cellular activity.
  • cytokines which bind to receptors on the surface of immune cells and alter cellular activity.
  • enhanced immunomodulatory activity may mean increased secretion of factors such as cytokines.
  • the enhanced immunomodulatory activity may be in reference to a CD14 + CD8 + T cell population.
  • the enhanced immunomodulatory activity may refer to the immunomodulatory activity of a population of cells enriched for CD14 + CD8 + T cells as described herein, for example by isolating or engineering CD14 + CD8 + T cells or treating a starting population of CD14 + CD8 + T cells with a CD14/TLR2/TLR4 agonist, compared to a population of cells which has not been enriched for CD14 + CD8 + T cells.
  • the immunomodulatory activity of CD14 + CD8 + T cells may be immunostimulatory or immunosuppressive.
  • Immunostimulatory activity means that the CD14 + CD8 + T cells increase the activity of certain other immune cells. Immunostimulation may result in enhancement of an antigen-specific proinflammatory response by the immune system.
  • Immunosuppressive activity means that the CD14 + CD8 + T cells decrease the activity of certain other immune cells. Immunosuppression may result in suppression, reduction or prevention of an antigen-specific proinflammatory response by the immune system.
  • Both immunostimulation and immunosuppression may be mediated by either increased or decreased secretion of factors such as cytokines.
  • immunostimulatory activity of CD14 + CD8 + T cells may be increased by increased secretion of an immunostimulatory factor or decreased secretion of an immunosuppressive factor.
  • immunosuppressive activity of CD14 + CD8 + T cells may be increased by increased secretion of an immunosuppressive factor or decreased secretion of an immunostimulatory factor.
  • T regs are a type of T cell that has immunomodulatory activity. Accordingly, treatment with a CD14/TLR2/TLR4 agonist may alter the characteristics of the CD14 + CD8 + T cells to be more like the characteristics of a Treg. Treatment with a CD14/TLR2/TLR4 agonist may enhance the Treg characteristics of the CD14 + CD8 + T cell. For example, treatment with a CD14/TLR2/TLR4 agonist may result in the CD14 + CD8 + T cells secreting the same factors as Tregs.
  • the CD14 + CD8 + T cell has an enhanced effector response compared to a CD14 CD8 + T cell.
  • the “effector response” may also be referred to as an antigen-specific proinflammatory or cytotoxic response.
  • the CD14 + CD8 + T cell has an enhanced antigen-specific proinflammatory response compared to a CD14 CD8 + T cell.
  • the “effector response” or “antigen-specific proinflammatory response” refers to a number of physiological and cellular effects facilitated by the immune system in response to a stimulus such as a pathogen or an autoantigen. Examples of such effects include increased proliferation of T cells, secretion of cytokines and expression of molecules that facilitate cytotoxicity. Assays are known in the art for measuring such indicators of the strength of an effector response.
  • An enhancement of the effector response means an increase in any of these effects. For example, a relative increase in cytokine secretion would be indicative of an enhanced effector response.
  • the CD14 + CD8 + T cell has enhanced IL-10 production compared to a CD14 CD8 + T cell.
  • IL-10 production refers to synthesis and secretion of the IL-10 cytokine. IL-10 production may be enhanced by increasing the number of CD14 + CD8 + T cells producing IL-10. The number of CD14 + CD8 + T cells producing IL-10 may be increased by inducing IL-10 production in CD14 + CD8 + T cells otherwise not producing IL-10. Alternatively or additionally, the number of CD14 + CD8 + T cells producing IL-10 may be increased by causing CD14 + CD8 + T cells already producing IL-10 to proliferate.
  • the number CD14 + CD8 + T cells producing IL-10 increases by at least about 10%, such as about 20%, such as about 30%, such as about 40%, such as about 50%, such as about 60%, such as about 70%, such as about 80%, such as about 90%, such as about 100%.
  • the number CD14 + CD8 + T cells producing IL-10 increases by at least 2-fold, such as 3-fold, such as 4-fold, such as 5-fold, such as 10-fold.
  • IL-10 production may be enhanced by increasing the amount of IL-10 synthesised and secreted by individual CD14 + CD8 + T cells, thereby increasing the amount of IL-10 produced by the population of CD14 + CD8 + T cells as a whole.
  • the amount of IL-10 produced by a given CD14 + CD8 + T cell increases by at least about 10%, such as about 20%, such as about 30%, such as about 40%, such as about 50%, such as about 60%, such as about 70%, such as about 80%, such as about 90%, such as about 100%.
  • the amount of IL-10 produced by a given CD14 + CD8 + T cell increases by at least 2-fold, such as 3-fold, such as 4-fold, such as 5-fold, such as 10-fold.
  • the CD14/TLR2/TLR4 agonist enhances IFNy, TNFa, MIR1b and/or IL-2 production by the CD14 + CD8 + T cell.
  • IFNy, TNFa, MIR1b (also known as CCL4) and IL-2 are all cytokines, which bind to receptors on immune cells, stimulating downstream signalling that modulates activity of the immune cells.
  • the CD14/TLR2/TLR4 agonist enhances the capability of the CD14 + CD8 + T cell to generate chemokines which chemoattract innate and adaptive effector cells.
  • the CD14/TLR2/TLR4 agonist enhances IL-8 and/or CXCL1 production by the CD14 + CD8 + T cell.
  • the CD14/TLR2/TLR4 agonist enhances CD107a expression by the CD14 + CD8 + T cell.
  • the CD14/TLR2/TLR4 agonist enhances antigen-specific IFNy (interferon gamma) production.
  • the CD14/TLR2/TLR4 agonist enhances antigen-specific TNFa (tumour necrosis factor alpha) production.
  • the CD14/TLR2/TLR4 agonist enhances antigen-specific MIR1b (macrophage inflammatory protein 1 beta) production.
  • the CD14/TLR2/TLR4 agonist enhances antigen-specific IL-2 (interleukin 2) production.
  • the CD14/TLR2/TLR4 agonist enhances antigen-specific IL-8 (interleukin 8) production.
  • the CD14/TLR2/TLR4 agonist enhances antigen-specific CXCL1 (also known as GRO alpha) production.
  • the CD14/TLR2/TLR4 agonist enhances CD107a expression.
  • the CD14/TLR2/TLR4 agonist enhances IFNy, TNFa, MIP1 b, IL-2, IL-8 and/or CXCL1 production by the CD14 + CD8 + T cell by any or all means selected from: increasing the amount of IFNy, TNFa, MIP1 b, IL-2, IL-8 and/or CXCL1 produced by individual cells, inducing cells to begin producing IFNy, TNFa, MIP1 b, IL-2, IL-8 and/or CXCL1 and inducing proliferation of cells already producing IFNy, TNFa, MIP1 b, IL-2, IL-8 and/or CXCL1.
  • the present invention provides a composition comprising an enriched population of CD14 + CD8 + T cells.
  • Techniques for enriching specific cell types from a heterogeneous population of cells are known in the art. Examples include use of immuno-magnetic beads and fluorescence activated cell sorting (FACS).
  • An “enriched population of CD14 + CD8 + T cells” refers to a population of cells comprising a high proportion of CD14 + CD8 + T cells.
  • the enriched population of CD14 + CD8 + T cells comprises at least 70 % CD14 + CD8 + T cells, such as 75 %, 85 %, 90 % or 95 % CD14 + CD8 + T cells.
  • the enriched population of T cells may have been sourced from an initial population that was a heterogeneous mixture of multiple types of cells.
  • CD14 + CD8 + T cells may be isolated from this initial population to produce a cell population with an increased proportion of CD14 + CD8 + T cells.
  • the present invention also provides a pharmaceutical composition comprising a CD14 + CD8 + T cell.
  • Such a pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • a pharmaceutically acceptable carrier diluent, excipient or adjuvant.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s) and other carrier agents.
  • compositions typically should be sterile and stable under the conditions of manufacture and storage.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent.
  • a pharmaceutical composition of the present invention may include pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers which are nontoxic to the subjects at the dosages and concentrations employed.
  • such a composition can further comprise a pharmaceutically acceptable carrier or excipient for use in the treatment of disease that that is compatible with a given method and/or site of administration, for instance for parenteral (e.g. sub-cutaneous, intradermal, or intravenous injection) or intrathecal administration.
  • a pharmaceutically acceptable carrier or excipient for use in the treatment of disease that is compatible with a given method and/or site of administration, for instance for parenteral (e.g. sub-cutaneous, intradermal, or intravenous injection) or intrathecal administration.
  • Administration of the pharmaceutical composition can be accomplished using any of a variety of routes that make the CD14/TLR2/TLR4 ligand or CD14 + CD8 + T cell comprised in the pharmaceutical composition bioavailable to the subject.
  • the composition can be administered by oral and parenteral routes, intraperitoneally, intravenously, subcutaneously, transcutaneously, intramuscularly, via local delivery for example by catheter or stent.
  • a CD14/TLR2/TLR4 ligand may be administered intramuscularly, intradermally, intravenously, intranasally or orally.
  • composition may be produced using current good manufacturing practices (cGMP).
  • composition may comprise one or more further pharmaceutically active compounds.
  • the composition may comprise an organic solvent, such as but not limited to, methyl acetate, dimethyl sulfoxide (DMSO), N.Ndimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide, including mixtures or combinations thereof.
  • organic solvent such as but not limited to, methyl acetate, dimethyl sulfoxide (DMSO), N.Ndimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide, including mixtures or combinations thereof.
  • the pharmaceutical composition is endotoxin free.
  • the CD14 + CD8 + T cell further expresses TLR2 and/or TLR4.
  • the CD14 + CD8 + T cell further expresses one or more of Foxp3, CTLA-4, IL-2 and/or IL-10.
  • the CD14 + CD8 + T cell further expresses Foxp3.
  • the CD14 + CD8 + T cell further expresses CTLA-4.
  • the CD14 + CD8 + T cell further expresses IL-2.
  • the CD14 + CD8 + T cell further expresses IL-10.
  • the CD14 + CD8 + T cell further expresses one or more tissue-retention markers.
  • the one or more tissue-retention markers are selected from CD69, CD103, CXCR3 and CXCR6.
  • the CD14 + CD8 + T cell further expresses CD69.
  • the CD14 + CD8 + T cell further expresses CD103.
  • the CD14 + CD8 + T cell further expresses CXCR3.
  • the CD14 + CD8 + T cell further expresses CXCR6.
  • the present invention provides a method of treating a disease which comprises administering a CD14/TLR2/TLR4 ligand or a CD14 + CD8 + T cell to a subject in need thereof.
  • PBMCs peripheral blood mononuclear cells
  • tissue-associated lymphocytes from a subject
  • a PBMC is any blood cell with a round nucleus found within the circulating pool of blood, rather than sequestered in the bone marrow, liver, spleen or lymphatic system.
  • PBMCs consist of monocytes and lymphocytes (T cells, B cells and NK cells).
  • T cells, B cells and NK cells monocytes and lymphocytes
  • Techniques for isolation of PBMCs from whole blood are known in the art.
  • PBMCs can be separated from a blood sample by addition of a density gradient medium, such as Ficoll (GE Healthcare), followed by centrifugation.
  • the different types of cells in the blood separate out into different layers, including a layer containing the PBMCs.
  • the lymphocytes may be tissue-resident lymphocytes.
  • a tissue-resident lymphocyte sample can be obtained by taking a sample from the tissue of interest.
  • a sample of liver- resident lymphocytes may be obtained by performing a liver biopsy or a surgical liver sample.
  • Techniques are known in the art for culturing and expanding cells obtained from a subject such that the cells can be manipulated or treated to render the cells useful for treatment of the subject.
  • the cytokine is a cytokine that induces CD14 expression.
  • the cytokine recapitulates the functional features of CD14 + CD8 + T cells in vitro.
  • the cytokine is IL-33, IFNy, TNFa, CXCL12 and/or an II_-15/TQRb combination, preferably CXCL12.
  • the cytokine is a combination of any of IL-33, IFNy, TNFa, CXCL12, IL-15 and TGF$.
  • step (ii) comprises treating the cells with a CD14/TLR2/TLR4 agonist.
  • step (ii) comprises treating the cells with a CD14 agonist.
  • step (ii) comprises treating the cells with a TLR4 agonist.
  • step (ii) comprises treating the cells with a TLR2 agonist.
  • step (ii) comprises treating the cells with a cytokine.
  • step (ii) comprises treating the cells with IL-33.
  • step (ii) comprises treating the cells with IFNy.
  • step (ii) comprises treating the cells with TNFa.
  • step (ii) comprises treating the cells with CXCL12.
  • step (ii) comprises treating the cells with I ⁇ -15/TQRb combination.
  • step (ii) comprises co culturing the cells with hepatic stroma and mononuclear phagocytes (MNPs).
  • MNPs mononuclear phagocytes
  • the method according to the invention further comprises the step of isolating CD14 + CD8 + T cells from the cells obtained from step (ii) prior to administration to the subject.
  • the method of treating disease according to the invention further comprises the step of treating the cells with a TCR agonist prior to administering the cells to the subject.
  • the method according to the invention further comprises the step of expanding the CD14 + CD8 + T cells obtained by the isolation step prior to administration to the subject.
  • step (iii) administering the cells obtained from step (ii) to the subject.
  • the method according to the invention further comprises the step of expanding the cells obtained from step (ii) prior to administration to the subject.
  • the method according to the invention further comprises the step of engineering the CD14 + CD8 + T cells obtained from step (ii) to express TLR2 and/or TLR4 prior to administration to the subject.
  • the present invention provides a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of peripheral blood mononuclear cells (PBMCs) in vitro with a CD14//TLR2/TLR4 agonist and/or a cytokine.
  • PBMCs peripheral blood mononuclear cells
  • the invention also provides a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of T cells in vitro with a CD14/TLR2/TLR4 agonist and/or a cytokine.
  • the CD14/TLR2/TLR4 agonist is selected from UV-killed E. coli, LPS, MPLA, LTA, Pam2CSK4 and/or Pam3CSK4 as described herein.
  • the cytokine is a cytokine that induces CD14 expression.
  • the cytokine is IL-33, IFNy, TNFa, CXCL12 and/or an II_-15/TQRb combination.
  • the cytokine is a combination of any of IL-33, IFNy, TNFa, CXCL12, IL-15 and TGF .
  • the method comprises treating a population of PBMCs in vitro with a CD14/TLR2/TLR4 agonist.
  • the method comprises treating a population of PBMCs in vitro with a CD14 agonist.
  • the method comprises treating a population of PBMCs in vitro with a TLR4 agonist.
  • the method comprises treating a population of PBMCs in vitro with a TLR2 agonist.
  • the method comprises treating a population of PBMCs in vitro with a cytokine.
  • the method comprises treating a population of PBMCs in vitro with IL-33.
  • the method comprises treating a population of PBMCs in vitro with IFNy.
  • the method comprises treating a population of PBMCs in vitro with TNFa.
  • the method comprises treating a population of PBMCs in vitro with CXCL12.
  • the method comprises treating a population of PBMCs in vitro with an II_-15/TQRb combination.
  • the method comprises co-culturing the PBMCs with hepatic stroma and MNPs.
  • the present invention also provides a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of tissue-resident immune cells in vitro with a CD14/TLR2/TLR4 agonist and/or a cytokine.
  • the cytokine is a cytokine that induces CD14 expression.
  • the cytokine is IL-33, IFNy, TNFa, CXCL12 and/or an I ⁇ -15/TQRb combination.
  • the cytokine is a combination of any of IL-33, IFNy, TNFa, CXCL12, IL-15 and TGF .
  • the method comprises treating a population of tissue-resident immune cells in vitro with a CD14/TLR2/TLR4 agonist.
  • the method comprises treating a population of tissue-resident immune cells in vitro with a CD14 agonist.
  • the method comprises treating a population of tissue-resident immune cells in vitro with a TLR4 agonist.
  • the method comprises treating a population of tissue-resident immune cells in vitro with a TLR2 agonist.
  • the method comprises treating a population of tissue-resident immune cells in vitro with a cytokine.
  • the method comprises treating a population of tissue-resident immune cells in wfrowith IL-33.
  • the method comprises treating a population of tissue- resident immune cells in vitro with IFNy.
  • the method comprises treating a population of tissue-resident immune cells in vitro with TNFa.
  • the method comprises treating a population of tissue-resident immune cells in vitro with CXCL12.
  • the method comprises treating a population of tissue-resident immune cells in vitro with an I ⁇ -15/TQRb combination.
  • the method comprises co-culturing the tissue-resident immune cells with hepatic stroma. In some embodiments of the invention the method comprises the step of isolating (a) CD8 + T cells or (b) CD14 + CD8 + T cells to provide an enriched cell sample and treating the enriched cell sample in vitro with the agent.
  • the method comprises treating a sample enriched in CD14 + CD8 + T cells in vitro with a CD14/TLR2/TLR4 agonist.
  • the method comprises treating a sample enriched in CD14 + CD8 + T cells in vitro with a CD14 agonist.
  • the method comprises treating a sample enriched in CD14 + CD8 + T cells in vitro with a TLR4 agonist.
  • the method comprises treating a sample enriched in CD14 + CD8 + T cells in vitro with a TLR2 agonist.
  • the method comprises treating a sample enriched in CD14 + CD8 + T cells in vitro with IL-33.
  • the method comprises treating a sample enriched in CD14 + CD8 + T cells in vitro with IFNy.
  • the method comprises treating a sample enriched in CD14 + CD8 + T cells in vitro with TNFa.
  • the method comprises treating a sample enriched in CD14 + CD8 + T cells in vitro with CXCL12.
  • the method of preparing a composition of CD14 + CD8 + T cells according to the invention further comprises the step of treating the cells with a TCR agonist.
  • the method according to the invention further comprises the step of isolating CD14 + CD8 + T cells following treatment with the CD14/TLR2/TLR4 agonist and/or cytokine.
  • the method of preparing a composition of CD14 + CD8 + T cells according to the invention comprises genetically engineering the cells to express an exogenous TCR.
  • the exogenous TCR is specific for the HBc-derived epitope HBcis- 27 or the HBe-derived epitope HBei83-9i.
  • the method comprises the following steps:
  • the cells may be genetically engineered using any genetic engineering technique known in the art, such as those described herein.
  • PBMCs or tissue-resident immune cells are treated in vitro with a cytokine to produce CD14 + CD8 + T cells, as described herein.
  • Cytokines may also be referred to as chemokines.
  • chemokines are used interchangeably in the context of the present invention.
  • the cytokine used in the methods of the invention is a cytokine that induces expression of CD14.
  • the cytokine used in the methods of the invention is IL-33 (interleukin 33).
  • IL-33 is expressed by mainly stromal cells and its expression is upregulated following proinflammatory stimulation.
  • IL-33 is a ligand for the receptors ST2 (IL-1 RL1) and IL-1 receptor accessory protein, and drives production of T helper 2-associated cytokines.
  • Recombinant IL- 33 is commercially available, for example from Abeam.
  • the cytokine used in the methods of the invention is IFNy (interferon y).
  • IFNy interferon y
  • IFNY is secreted by activated T cells and natural killer cells, and has pleiotropic immunologic effects.
  • IFNY is a ligand for the IFNY receptor. Recombinant IFNY is commercially available, for example from Sigma-Aldrich.
  • the cytokine used in the methods of the invention is TNFa (tumour necrosis factor a).
  • TNFa is an inflammatory cytokine produced by T cells and macrophages during acute inflammation.
  • TNFa is a ligand for the receptors TNFR-1 and TNFR-2.
  • Recombinant TNFa is commercially available, for example from Sigma-Aldrich.
  • the cytokine used in the methods of the invention is CXCL12 (CXC motif chemokine 12).
  • CXCL12 is also known as stromal cell-derived factor 1 (SDF1). Stromal cells secrete CXCL12, which influences chemotaxis of leukocytes.
  • CXCL12 is a ligand for the receptors CXCR4 and CXCR7. Recombinant CXCL12 is commercially available, for example from ThermoFisher.
  • the cytokine used in the methods of the invention is an I ⁇ -15/TQRb combination.
  • IL-15 interleukin 15
  • IL-2 is similar to IL-2 and is a ligand for the IL-15 receptor, which shares some sub-units with the IL-2 receptor.
  • IL-15 is expressed by multiple cell types and has pleiotropic immunologic effects, including regulating activation and expression of T cells and NK cells.
  • TQRb transforming growth factor b
  • Recombinant IL-15 and TQRb are commercially available, for example from R&D systems.
  • PBMCs or tissue-resident immune cells are treated in vitro with an agent to produce CD14 + CD8 + T cells, as described herein.
  • such methods comprise co-culturing the cells (PBMCs or tissue- resident immune cells) with hepatic stroma and mononuclear phagocytes (MNPs).
  • PBMCs or tissue- resident immune cells hepatic stroma and mononuclear phagocytes
  • the treatment of the cells with a CD14/TLR2/TLR4 agonist and/or a cytokine may be effected by co-culturing the cells with hepatic stroma and MNPs.
  • hepatic stroma refers to primary human hepatic stellate cells. These are liver pericytes (mesenchymal cells) that transdifferentiate upon activation in vivo into myofibroblasts, responsible for producing the extracellular matrix driving liver fibrosis and cirrhosis.
  • Hepatic stroma may be prepared by methods known in the art.
  • primary human hepatic stellate cells pHSC
  • pHSC primary human hepatic stellate cells
  • the pHSCs transdifferentiate into activated myofibroblasts expressing the hallmark alpha smooth muscle actin (aSMA).
  • Hepatic stellate cells may be isolated from liver tissue samples using density centrifugation with an Optiprep (Sigma Aldrich; Merck) density gradient using a protocol such as described in Singh et al. Sci Rep. 2017 17; 7(1): 5514 “TRAIL regulatory receptors constrain human hepatic stellate cell apoptosis” and in Pallett et al. Nat Med. 201521(6): 591-600 “Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells”.
  • Pre-isolated hepatic stellate cells can be expanded and frozen down for later use. Once thawed, hepatic stellate cells can be cultured and expanded in vitro in stellate cell media (ScienCell Research Laboratories) for co-culture experiments.
  • MNPs may be isolated from peripheral blood using magnetic bead isolation using commercially available kits (for example, Pan Monocyte Isolation Kit, human 130-096-537 from Miltenyi Biotec). Alternatively, MNPs may be isolated by flow cytometric cell sorting based on expression of HLA-DR and CD14 and a lack of lineage defining markers for lymphocytes (T cell, B cells and NK cells CD3/CD19/CD56 respectively). MNPs can be cultured in RPMI media, which is readily available commercially.
  • the term “MNPs” encompasses classical CD14 hi CD16 _ monocytes, non-classical CD14 + CD16 hi monocytes and intermediate CD14 hi CD16 int monocytes.
  • the hepatic stroma comprising primary human hepatic stellate cells isolated from healthy liver margins and expanded in vitro, supports expansion of CD14 + CD8 + T cells through secretion of the cytokine CXCL12 and possibly other factors that influence gene expression, such as extracellular matrix components that bind receptors such as CD49a/b on CD14 + CD8 + T cells. Co-culturing with hepatic stroma and MNPs thereby recapitulates the extracellular milieu of the liver to produce CD14 + CD8 + T cells.
  • additional agents are added to the co-culture with the hepatic stroma.
  • the method comprises co-culturing the cells with hepatic stroma, MNPs and IL-2.
  • PBMCs or tissue-resident immune cells are treated in vitro with a TCR agonist, as described herein.
  • a TCR agonist is a molecule which binds to a TCR on the T cell surface and stimulates a signalling pathway downstream of the TCR, such as a pathway associated with T cell activation.
  • Treatment of CD14 + CD8 + T cells with a TCR agonist in vitro pre-activates the T cells to enhance their effector response and improve treatment of disease.
  • the cells are treated with the TCR agonist simultaneously with the CD14/TLR2/TLR4 agonist and/or the cytokine. In some embodiments, the cells are treated with the TCR agonist after treatment with the CD14/TLR2/TLR4 agonist and/or the cytokine.
  • the TCR agonist is a CD3 agonist.
  • the CD3 agonist is an antibody.
  • Anti-CD3 antibodies are available commercially (such as OKT3, ThermoFisher cat. # 16-0037-85).
  • the TCR agonist is a CD28 agonist.
  • the CD28 agonist is an antibody.
  • Anti-CD28 antibodies are available commercially (such as ThermoFisher cat. # 16- 0289-85).
  • the TCR agonist is selected from an MHC peptide, an anti-CD3 antibody and an anti-CD28 antibody.
  • the TCR agonist is an MHC peptide.
  • An MHC peptide is a peptide that binds to the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • the MHC peptide is the HBc-derived epitope HBCI 8 -27, which has the amino acid sequence FLPSDFFPSV (SEQ ID NO. 18).
  • HBci8-2 7 is also referred to herein as “C18 peptide”.
  • HBc Hepatitis B core
  • the MHC peptide is the HBe-derived epitope HBei 8 3-i9i, which has the amino acid sequence FLLTRILTI (SEQ ID NO. 19).
  • HBe is hepatitis B e-antigen.
  • HBei 8 3-i9i is also referred to herein as ⁇ 183 peptide”.
  • HBCI 8 -27 and HBei 83 -i9i may be used, for example, to pre-activate HBV-specific T cells engineered with an exogenous TCR specific for HBCI 8 -27 and HBei 8 3-i9i respectively.
  • ABD alcoholic liver disease
  • NASH non-alcoholic steatohepatitis
  • Sal. gland salivary gland.
  • CD14 + CD8 + T cells are present in multiple tissues, including barrier organs such as the skin and liver, but are excluded from the blood.
  • Example 2 - CD14 + CD8 + T cells co-express components of the LPS co-receptor and are responsive to TLR ligands ex vivo
  • CD14 + CD8 + T cells express significantly more TLR4, TLR2 and MD-2 than CD14 + CD8 + T cells.
  • TLR4:CD14 complex Functionality of the TLR4:CD14 complex was evaluated by culturing intrahepatic leukocytes with fluorescently labelled LPS, prior to flow cytometric staining to determine LPS-uptake (Figure 2B). The data show that over time CD14 + CD8 + T cells take up increasing amounts of LPS, whilst CD14 CD8 + T cells do not.
  • Example 3 - CD14 + CD8 + T cells are enriched for markers associated with tissue residency and enhanced tethering
  • markers associated with tissue residency and enhanced tethering by intrahepatic CD14 CD8 + T cells and CD14 + CD8 + T cells was assessed using flow cytometry (Figure 3).
  • CD14 + CD8 + T cells have significantly higher expression of each of these markers than CD14 CD8 + T cells.
  • markers associated with immune activation and immune regulation by intrahepatic CD14 CD8 + T cells and CD14 + CD8 + T cells was assessed using flow cytometry (Figure 4).
  • CD14 + CD8 + T cells have significantly higher expression of each of these markers than CD14 CD8 + T cells.
  • CD14 + CD8 + T cells were clonal expanded in response to viral antigens by stimulating T cells with MHC/peptide multimers loaded with well-described HLA-A2-restricted epitopes for hepatitis B virus (HBV) and cytomegalovirus (CMV).
  • HBV-specific and CMV- specific CD14 + CD8 + T cells expanded more than global (HBV- and CMV-negative) T cells
  • Example 6 - CD14 + CD8 + T cells can be expanded from a pre-existing population by alterations in the quantity of TLR ligand exposure in vivo and in vitro
  • a human in vivo skin blister model was used to model in vivo gram-negative bacteria exposure.
  • UV-killed E. coli were injected intradermally into a marked site on the ventral side of the right forearm of participants. From each participant the left forearm was used to raise a “naive” unexposed blister to analyse the exudate (baseline blister). After 24 hr a suction blister was used to remove the inflammatory exudate from the LPS-exposed injection site from the right forearm and the frequency of CD14 + CD8 + T cells in blood or skin blister aspirate measured (Figure 6A). The proportion of CD14 + CD8 + T cells increased in response to UV- killed E. coli specifically in blister fluid.
  • Example 7 - CD14 + CD8 + T cells can be generated in vitro from peripheral CD8 + T cells using TLR ligands, recombinant cytokines or interaction with hepatic stroma
  • Freshly isolated PBMCs were co-cultured for 4 d with recombinant human IL-2 and with various other molecules, and the frequency of CD14 + CD8 + T cells assessed by flow cytometry (Figure 7).
  • MNP mononuclear phagocytes
  • Example 8 Stromal cell derived CD14 + CD8 + T cells recapitulate enhanced TCR- induced effector function and show novel E. co///LPS induced functionality
  • stromal cell derived CD14 + CD8 + T cells could recapitulate the enhanced TCR-induced effector function
  • freshly isolated PBMC were co-cultured in the presence of recombinant human IL-2 with primary human hepatic stellate cells for 4 d to induce CD14 expression.
  • After induction of CD14-expression T cells were harvested and assessed by flow cytometry or Luminex array ( Figure 8) to determine their functionality: A in the presence or absence of fluorescently-labelled LPS prior to flow cytometric staining to determine LPS-uptake, B after stimulation with either media alone or LPS derived from E.
  • brefeldin-A to block cellular cytokine release
  • C after stimulation with either media alone or UV-killed E. coli for 24 hr in the presence of brefeldin-A (to block cellular cytokine release) prior to assessment of cytokine expression
  • D after stimulation with either media alone or anti-CD3 and anti-CD28 for 4 hr in the presence of brefeldin-A (to block cellular cytokine release) prior to assessment of cytokine expression.
  • Example 9 Genetically engineered T cells for adoptive immunotherapy (TCR- redirected T cells) can undergo myeloid reprogramming in vitro to express CD14
  • T cells were isolated by magnetic bead separation and were genetically engineered to express a TCR specific for the immunodominant HBc-derived epitope HBCI 8 -27 (C18; amino acid sequence: FLPSDFFPSV; SEQ ID NO. 18) or HBe-derived epitope HBei83-9i (E183; amino acid sequence FLLTRILTI; SEQ ID NO. 19).
  • packaging cells were transiently co transfected with plasmids encoding the C18- or E183-specific TCR and an amphotrophic envelope. Retroviral supernatants were collected for the transduction of donor T cells stimulated with IL-2 and anti-CD3.
  • Activated lymphocytes were mixed with retroviral supernatants on retronectin-coated plates.
  • Cells were monitored for the expression of the endogenous recombinant TCR and after in vitro expansion in IL-2, IL-7 and IL-15, genetically modified T cells were sorted by flow cytometry.
  • Representative flow cytometry plots depict the expression of CD14 on gene-modified CD8 + T-cells after co-culture in the presence of recombinant human IL-2 with primary human hepatic stellate cells +/- the addition of autologous freshly isolated mononuclear phagocytes (MNP; monocytes) in either direct contact for 4 d.
  • MNP mononuclear phagocytes
  • C18 T cells identified by flow cytometry by staining for the murine TCRp from the construct
  • C18 T cells co-cultured ⁇ the addition of autologous MNP (as above in Figure 9A) were stimulated with cognate C18 peptide (FLPSDFFPSV; SEQ ID NO. 18) for 16hrwith brefeldin-A.
  • CD14-expressing CD8 + T cells were derived in vitro using lentiviral constructs.
  • Peripheral blood T cells were isolated from freshly isolated PBMC by magnetic bead separation.
  • Isolated T cells were transfected by electroporation with the plasmid pDUO-hCD14-TLR4A (or GFP- empty control plasmid) designed to induce the co-expression of CD14 and TLR4 genes.
  • Flow cytometric plots depict the expression of CD14 and TLR4 respectively on pre gated CD8 + T cells from a representative T cell donor 24 hr after transfection with the GFP- expressing control vector, or CD14/TLR4 construct by flow cytometry.
  • Example 10 In vitro induction of CD14 + on TCR-redirected T cells optimises their anti-tumour/anti-viral function
  • liver cancer such as hepatocellular carcinoma (HCC) or a secondary metastatic tumour
  • HCC hepatocellular carcinoma
  • HCV Hepatitis B virus
  • HCV Hepatitis C virus
  • CMV cytomegalovirus
  • malaria non- viral hepatitis such as non-alcoholic steatohepatitis (NASH), alcoholic liver disease
  • CD14/TLR2/TLR4 ligand or CD14 + CD8 + T cell for use according to any preceding paragraph wherein the CD14 + CD8 + T cell expresses TLR2 and/or TLR4.
  • a CD14/TLR2/TLR4 ligand or CD14 + CD8 + T cell for use according to any preceding paragraph wherein the CD14 + CD8 + T cell expresses one or more of Foxp3, CTLA-4, IL-2 and/or IL-10.
  • a CD14/TLR2/TLR4 ligand or CD14 + CD8 + T cell for use according to any preceding paragraph wherein the CD14 + CD8 + T cell expresses one or more tissue-retention molecules.
  • CD14/TLR2/TLR4 agonist enhances proliferation of a CD14 + CD8 + T cell or CD14 + CD8 + T cell population, and/or enhances immunomodulatory activity of the CD14 + CD8 + T cell or CD14 + CD8 + T cell population.
  • CD14/TLR2/TLR4 agonist enhances I L-10 production by the CD14 + CD8 + T cell orCD14 + CD8 + T cell population.
  • LPS lipopolysaccharide
  • MPLA monophosphoryl lipid A
  • LTA lipoteichoic acid
  • a CD14 + CD8 + T cell for use according to any of paragraphs 1 to 10 wherein the
  • CD14 + CD8 + T cell has been isolated and enriched to form a CD14 + CD8 + T cell composition, optionally wherein the CD14 + CD8 + T cell has been isolated from the subject to be treated.
  • An engineered CD14 + CD8 + T cell which comprises (i) a nucleic acid construct encoding a TLR2 and/or TLR4; (ii) a nucleic acid construct encoding one or more tissue- retention molecules; and/or (iii) a nucleic acid construct encoding an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • An engineered CD8 + T cell which comprises a nucleic acid construct encoding one or more of (i) CD14, TLR2, and/or TLR4, and optionally (ii) a tissue-retention molecule and/or an exogenous TCR.
  • An engineered CD14 + CD8 + T cell comprising a nucleic acid construct encoding an exogenous TCR.
  • composition comprising an enriched population of CD14 + CD8 + T cells.
  • a pharmaceutical composition comprising a CD14 + CD8 + T cell.
  • a composition or pharmaceutical composition according to paragraph 34 wherein the one or more tissue-retention molecules is selected from CD69, CD103, CXCR3 and CXCR6.
  • a method of treating a disease which comprises administering a CD14/TLR2/TLR4 ligand or a CD14 + CD8 + T cell to a subject in need thereof.
  • step (iii) administering the cells obtained from step (ii) to the subject.
  • a method according to paragraph 37 which further comprises the step of treating the cells with a TCR agonist prior to administering the cells to the subject.
  • TCR agonist is selected from an MHC peptide, an anti-CD3 antibody and an anti-CD28 antibody.
  • cytokine is IL-33, IFNy, TNFa, CXCL12 and/or an IL-15/TGFp combination.
  • step (ii) comprises co culturing the cells with hepatic stroma and mononuclear phagocytes (MNPs).
  • a method according to any of paragraphs 37 to 41 which further comprises the step of isolating CD14 + CD8 + T cells from the cells obtained from step (ii) prior to administration to the subject.
  • a method according to paragraph 42 which further comprises the step of expanding the CD14 + CD8 + T cells obtained by the isolation step prior to administration to the subject.
  • step (iii) administering the cells obtained from step (ii) to the subject.
  • a method according to paragraph 44 which further comprises the step of expanding the cells obtained from step (ii) prior to administration to the subject.
  • a method according to any of paragraphs 43 to 45 which further comprises the step of engineering the CD14 + CD8 + T cells obtained from step (ii) to express TLR2 and/or TLR4 prior to administration to the subject.
  • a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of peripheral blood mononuclear cells (PBMCs) in vitro with a CD14/TLR2/TLR4 agonist and/or a cytokine.
  • PBMCs peripheral blood mononuclear cells
  • a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of tissue-resident immune cells in vitro with a CD14/TLR2/TLR4 agonist and/or a cytokine.
  • a method for preparing a composition of CD14 + CD8 + T cells which comprises treating a population of T cells in vitro with a CD14/TLR2/TLR4 agonist and/or a cytokine. 50. A method according to any of paragraphs 47 to 49 which further comprises treating the population of cells with a TCR agonist.
  • TCR agonist is selected from an MHC peptide, an anti-CD3 antibody and an anti-CD28 antibody.
  • cytokine is IL-33, IFNy, TNFa, CXCL12 and/or an II_-15/TQBb combination, preferably CXCL12.
  • a method according to any of paragraphs 47 to 52 which comprises co-culturing the cells with hepatic stroma and MNPs.
  • a method according to any of paragraphs 47 to 53 which comprises the step of isolating (a) CD8 + T cells or (b) CD14 + CD8 + T cells to provide an enriched cell sample and treating the enriched cell sample in vitro with the CD14/TLR2/TLR4 agonist and/or cytokine.
  • a method according to paragraph 54 which comprises genetically engineering the cells to express an exogenous TCR.
  • a method according to any of paragraphs 47 to 57 which further comprises the step of isolating CD14 + CD8 + T cells following treatment with the CD14/TLR2/TLR4 agonist and/or cytokine.

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Abstract

La présente invention concerne un moyen de modulation de l'activité d'une population de lymphocytes T pour le traitement d'une maladie. En particulier, la présente invention concerne un ligand CD14/TLR2/TLR4 ou un lymphocyte T CD14+CD8+ pour son utilisation dans le traitement d'une maladie. La présente invention concerne en outre des lymphocytes T CD14+CD8+, des compositions comprenant ces lymphocytes T, des procédés de préparation de ces lymphocytes T et des procédés de traitement de maladies utilisant un ligand CD14/TLR2/TLR4 ou un lymphocyte T CD14+CD8+.
PCT/GB2022/051731 2021-07-07 2022-07-06 Cellule WO2023281256A1 (fr)

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WO2009136874A1 (fr) * 2008-05-09 2009-11-12 Agency For Science, Technology And Research Récepteur des cellules t (tcr) exogène réactif à un épitope du hbv et ses utilisations
WO2019099479A1 (fr) * 2017-11-14 2019-05-23 Memorial Sloan-Kettering Cancer Center Cellules immunoréactives sécrétant il-33 et leurs utilisations

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