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WO2025238364A1 - Récepteurs de lymphocytes t spécifiques du peptide wt1 restreint par hla-e et leurs utilisations - Google Patents

Récepteurs de lymphocytes t spécifiques du peptide wt1 restreint par hla-e et leurs utilisations

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Publication number
WO2025238364A1
WO2025238364A1 PCT/GB2025/051048 GB2025051048W WO2025238364A1 WO 2025238364 A1 WO2025238364 A1 WO 2025238364A1 GB 2025051048 W GB2025051048 W GB 2025051048W WO 2025238364 A1 WO2025238364 A1 WO 2025238364A1
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WIPO (PCT)
Prior art keywords
seq
tcr
cell
beta
alpha
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Pending
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PCT/GB2025/051048
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English (en)
Inventor
Andrew Mcmichael
Geraldine GILLESPIE
Simon BRACKENRIDGE
Hongbing Yang
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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Publication of WO2025238364A1 publication Critical patent/WO2025238364A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4242—Transcription factors, e.g. SOX or c-MYC
    • A61K40/4243—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the present invention relates to T cell receptors (TCRs) which are Wilm’s Tumour 1 (WT1) specific and HLA-E restricted.
  • TCRs T cell receptors
  • WT1 Tumour 1
  • HLA-E restricted HLA-E restricted
  • the non-classical human HLA class I molecule HLA-E regulates responses mediated by NK cells and a subset of CD8+ T cells by presenting a nonamer peptide, residues 3-11 of the classical HLA class la signal sequence (typically VMAPRTLVL, VL9), to the inhibitory receptor NKG2A-CD94 and its activating counterpart NKG2C-CD94 (Braud, V. et al, Eur J Immunol 1997. Braud, V.M., Nature 1998). Because the inhibitory receptor is dominant, NKG2A prevents NK cell mediated lysis of cells that co-express HLA class la and HLA-E molecules. Regulation of NK cell activity is likely the principal function of HLA-E.
  • Antigen specific HLA-E restricted CD8+ T cells are not commonly detected in infection or cancer, except in mycobacterial infection; the HLA-E restricted responses are relatively abundant to this microbe with more than 70 epitopes identified (Joosten S.A. et al., PLoS Pathog 6 2010). Furthermore, Mamu-E restricted T cell responses are prominent in Resus Macaques (RMs) immunized with rhesus cytomegalovirus (RhCMV) strain 68-1 vectored vaccines together with atypical MHC class II restricted CD8+T cell responses, while classical class la restricted CD8 T cell responses are absent (Hansen S G. et al, Science 2013 ).
  • RMs Resus Macaques
  • RhCMV rhesus cytomegalovirus
  • HLA-E restricted CD8+ T cell responses to HIV-1 peptides have been stimulated in vitro, using peripheral blood mononuclear cells (PBMCs) from HIV uninfected donors.
  • PBMCs peripheral blood mononuclear cells
  • HLA-E restricted peptide-specific CD8+ T cell clones are capable of recognising HIV infected cells and suppressing HIV replication in vitro (Yang, et al; Sci Immunol. 2021). Therefore, HLA-E restricted HIV-1 specific responses could also control and subsequently clear HIV-1 early after infection in humans. HLA-E restricted responses to cancer have also been poorly characterised.
  • WT1 expression is elevated on tumour cells including acute myeloid leukaemia, breast cancer, endometrial carcinoma, ovarian cancer, hepatocellular cancer, non-small cell lung cancer, colorectal cancer, soft tissue sarcoma, glioblastoma, astrocytoma, melanoma, and mesothelioma (Nakatsuka S., Mod Pathol. 2006).
  • tumour cells including acute myeloid leukaemia, breast cancer, endometrial carcinoma, ovarian cancer, hepatocellular cancer, non-small cell lung cancer, colorectal cancer, soft tissue sarcoma, glioblastoma, astrocytoma, melanoma, and mesothelioma (Nakatsuka S., Mod Pathol. 2006).
  • Over-expression of WT1 on many tumours makes it a good target for development of immunotherapy. No universal vaccine or treatment currently exists for such cancers.
  • TCR T-cell receptor
  • the TCR may be capable of binding to a peptide of amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in complex with HLA-E.
  • the TCR does not bind to a different peptide known to be bound and presented by HLA-E, such as VL9 (VMAPRTLVL).
  • the TCR may bind to a different peptide known to be bound and presented by HLA-E, such as VL9 (VMAPRTLVL), with a mean fluorescence intensity (MFI) of 10% or less, such as 5% or less, or 1% or less, than that of sequence RMFPNAPYL (SEQ ID NO: 1) in complex with HLA-E, when measured for example by flow cytometry.
  • MFI mean fluorescence intensity
  • the invention is based upon previous findings that classical HLA A, B and C class I molecules are down-regulated or absent in many tumour types, whereas the non- classical HLA-E is significantly up-regulated and can also be used as a biomarker of tumour invasiveness.
  • HLA-E overexpression has been shown to negatively interfere with innate immune surveillance, thereby protecting tumour cells from NK cell- mediated cytotoxicity.
  • the inventors identified a WT1 derived peptide RMFPNAPYL, which can bind HLA-E and which can be used to stimulate specific CD8 T cells.
  • HLA- E restricted responses to cancer have been poorly characterised.
  • Such an HLA-E restricted TCR overcomes the problem of genetic diversity experienced in classical MHCI restricted TCRs.
  • HLA-E is nonpolymorphic, there are only two common alleles in humans, which differ by only one amino acid which has no impact on peptide binding.
  • HLA-E restricted WT1 specific TCRs and T-cells comprising such TCRs can be used universally to treat or prevent cancers associated with WT1 overexpression.
  • the TCR may comprise an alpha chain variable domain and a beta chain variable domain comprising: a) a CDR3-alpha of AMSEAQEGGYQKVTF (SEQ ID NO: 3) or a CDR3- alpha with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 3, and a CDR3-beta of ASSPRPRAADTQYF (SEQ ID NO: 6) or a CDR3-beta with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 6; or b) a CDR3-alpha of VVNKPNDYKLSF (SEQ ID NO: 11) or a CDR3-alpha with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 11, and a CDR3-beta of ASSYP
  • the TCR may comprise an alpha chain variable domain comprising a CDR1 -alpha of NSAFQY (SEQ ID NO: 1), a CDR2-alpha of TYSSGN (SEQ ID NO: 2) and a CDR3- alpha of SEQ ID NO: 3; and/or a beta alpha chain variable domain comprising a CDR1- beta of MNHEY (SEQ ID NO: 4), a CDR2-beta of SMNVEV (SEQ ID NO: 5) and a CDR3-beta of SEQ ID NO: 6.
  • the TCR may comprise an alpha chain variable domain comprising a CDR1 -alpha of NSASQS (SEQ ID NO: 9), a CDR2-alpha of VYSSGN (SEQ ID NO: 10) and a CDR3- alpha of SEQ ID NO: 11; and/or a beta alpha chain variable domain comprising a CDR1- beta of MNHEY (SEQ ID NO: 12), a CDR2-beta of SVGEGT (SEQ ID NO: 13) and a CDR3-beta of SEQ ID NO: 14.
  • the TCR may comprise an alpha chain variable domain comprising a CDR1 -alpha of SSNFYA (SEQ ID NO: 17), a CDR2-alpha of MTLNGDE (SEQ ID NO: 18) and a CDR3 -alpha of SEQ ID NO: 19; and/or a beta alpha chain variable domain comprising a CDRl-beta of SNHLY (SEQ ID NO: 20), a CDR2-beta of FYNNEI (SEQ ID NO: 21) and a CDR3-beta of SEQ ID NO: 22.
  • the TCR may comprise an alpha chain variable domain comprising a CDR1 -alpha of DRGSQS (SEQ ID NO: 25), a CDR2-alpha of IYSNGD (SEQ ID NO: 26) and a CDR3- alpha of SEQ ID NO: 27; and/or a beta alpha chain variable domain comprising a CDRl- beta of SEHNR (SEQ ID NO: 28), a CDR2-beta of FQNEAQ (SEQ ID NO: 29) and a CDR3-beta of SEQ ID NO: 30.
  • the TCR may comprise an alpha chain variable domain comprising a CDR1 -alpha of NSAFQY (SEQ ID NO: 33), a CDR2-alpha of TYSSGN (SEQ ID NO: 34) and a CDR3- alpha of SEQ ID NO: 35; and/or a beta alpha chain variable domain comprising a CDR1- beta of SEHNR (SEQ ID NO: 36), a CDR2-beta of FQNEAQ (SEQ ID NO: 37) and a CDR3-beta of SEQ ID NO: 38.
  • the TCR may comprise an alpha chain variable domain comprising a CDR1 -alpha of DSAIYN (SEQ ID NO: 41), a CDR2-alpha of IQSSQRE (SEQ ID NO: 42) and a CDR3- alpha of SEQ ID NO: 43; and/or a beta alpha chain variable domain comprising a CDR1- beta of LGHDT (SEQ ID NO: 44), a CDR2-beta of YNNKEL (SEQ ID NO: 45) and a CDR3-beta of SEQ ID NO: 46.
  • the TCR may comprise an alpha chain variable domain comprising a variable segment encoded by the TRAV12-3 gene, a Joining segment encoded by the TRAJ13 gene, a CDRl-alpha of SEQ ID NO: 1, a CDR2-alpha of SEQ ID NO: 2 and a CDR3-alpha of SEQ ID NO: 3; and/or a beta chain variable region comprising a variable segment encoded by the TRBV27 gene, a Joining segment encoded by the TRBJ2-3 gene, a CDRl-beta of SEQ ID NO: 4, a CDR2-beta of SEQ ID NO: 5 and a CDR3-beta of SEQ ID NO: 6.
  • the TCR may also comprise an alpha chain TRAC constant domain sequence and a beta chain TRBC2 constant domain sequence.
  • the TCR may comprise an alpha chain variable domain comprising a variable segment encoded by the TRAV12-1 gene, a Joining segment encoded by the TRAJ20 gene, a CDRl-alpha of SEQ ID NO: 9, a CDR2-alpha of SEQ ID NO: 10 and a CDR3-alpha of SEQ ID NO: 11; and/or a beta chain variable region comprising a variable segment encoded by the TRBV6-2 gene, a Joining segment encoded by the TRBJ2-5 gene, a CDRl-beta of SEQ ID NO: 12, a CDR2-beta of SEQ ID NO: 13 and a CDR3-beta of SEQ ID NO: 14.
  • the TCR may also comprise an alpha chain TRAC constant domain sequence and a beta chain TRBC2 constant domain sequence.
  • the TCR may comprise an alpha chain variable domain comprising a variable segment encoded by the TRAV24 gene, a Joining segment encoded by the TRAJ28 gene, a CDRl-alpha of SEQ ID NO: 17, a CDR2-alpha of SEQ ID NO: 18 and a CDR3-alpha of SEQ ID NO: 19; and/or a beta chain variable region comprising a variable segment encoded by the TRBV2 gene, a Joining segment encoded by the TRBJ2-7 gene, a CDR1- beta of SEQ ID NO: 20, a CDR2-beta of SEQ ID NO: 21 and a CDR3-beta of SEQ ID NO: 22.
  • the TCR may also comprise an alpha chain TRAC constant domain sequence and a beta chain TRBC2 constant domain sequence.
  • the TCR may comprise an alpha chain variable domain comprising a variable segment encoded by the TRAV12-2 gene, a Joining segment encoded by the TRAJ26 gene, a CDRl-alpha of SEQ ID NO: 25, a CDR2-alpha of SEQ ID NO: 26 and a CDR3-alpha of SEQ ID NO: 27; and/or a beta chain variable region comprising a variable segment encoded by the TRBV7-9 gene, a Joining segment encoded by the TRBJ2-7 gene, a CDRl-beta of SEQ ID NO: 28, a CDR2-beta of SEQ ID NO: 29 and a CDR3-beta of SEQ ID NO: 30.
  • the TCR may also comprise an alpha chain TRAC constant domain sequence and a beta chain TRBC2 constant domain sequence.
  • the TCR may comprise an alpha chain variable domain comprising a variable segment encoded by the TRAV12-3 gene, a Joining segment encoded by the TRAJ33 gene, a CDRl-alpha of SEQ ID NO: 33, a CDR2-alpha of SEQ ID NO: 34 and a CDR3-alpha of SEQ ID NO: 35; and/or a beta chain variable region comprising a variable segment encoded by the TRBV7-9 gene, a Joining segment encoded by the TRBJ2-7 gene, a CDRl-beta of SEQ ID NO: 36, a CDR2-beta of SEQ ID NO: 37 and a CDR3-beta of SEQ ID NO: 38.
  • the TCR may also comprise an alpha chain TRAC constant domain sequence and a beta chain TRBC2 constant domain sequence.
  • the TCR may comprise an alpha chain variable domain comprising a variable segment encoded by the TRAV21 gene, a Joining segment encoded by the TRAJ15 gene, a CDRl-alpha of SEQ ID NO: 41, a CDR2-alpha of SEQ ID NO: 42 and a CDR3-alpha of SEQ ID NO: 43; and/or a beta chain variable region comprising a variable segment encoded by the TRBV3-1 gene, a Joining segment encoded by the TRBJ2-1 gene, a CDRl-beta of SEQ ID NO: 44, a CDR2-beta of SEQ ID NO: 45 and a CDR3-beta of SEQ ID NO: 46.
  • the TCR may also comprise an alpha chain TRAC constant domain sequence and a beta chain TRBC2 constant domain sequence.
  • the TCR may comprise an alpha chain variable domain of a sequence of QKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSG NKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSEAQEGGYQKVTFGIGTK LQVIP (SEQ ID NO: 7) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 7, and a beta chain variable domain of a sequence of
  • the TCR may comprise an alpha chain variable domain of a sequence of RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSG NEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNKPNDYKLSFGAGTTVTVR A (SEQ ID NO: 15) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 15, and a beta chain variable domain of a sequence of
  • NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE GTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYPGRQTQYFGPG TRLLVL (SEQ ID NO: 16) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 16.
  • the TCR may comprise an alpha chain variable domain of a sequence of ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGD EKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCATGGSYQLTFGKGTKLSVIP (SEQ ID NO: 23) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 23, and a beta chain variable domain of a sequence of
  • EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISE KSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSDYALTYEQYFGPGTRLT VT (SEQ ID NO: 24) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 24.
  • the TCR may comprise an alpha chain variable domain of a sequence of QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGD KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKPHGQNFVFGPGTRLSVLP (SEQ ID NO: 31) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 31, and a beta chain variable domain of a sequence of
  • DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQ LEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLDSSYEQYFGPGTRL TVT (SEQ ID NO: 32) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 32.
  • the TCR may comprise an alpha chain variable domain of a sequence of QKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSG NKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSGRMDSNYQLIWGAGTK LIIKP (SEQ ID NO: 39) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 39, and a beta chain variable domain of a sequence of
  • SEQ ID NO: 40 DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQ LEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLDSSYEQYFGPGTRL TVT (SEQ ID NO: 40) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 40.
  • the TCR may comprise an alpha chain variable domain of a sequence of KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQRE QTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRLAGTALIFGKGTTLSVSS (SEQ ID NO: 47) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 47, and a beta chain variable domain of a sequence of
  • SEQ ID NO: 48 DTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMYWYKQDSKKFLKIMFSYNNKE LIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASTITGTGYEQFFGPGTRL TVL (SEQ ID NO: 48) or a sequence with at least 90% identity, such as 90%, 95%, 96%, 97%, 98%, 99% of 100% identity to SEQ ID NO: 48.
  • the TCR may comprise an alpha chain TRAC constant domain sequence and/or a beta chain TRBC1 or TRBC2 constant domain sequence.
  • the constant domain may be from another species, such as mouse TRAC and or TRBC, or from a human.
  • a mouse constant domain may allow for monitoring of the survival of T cells comprising such a TCR when injected in vivo, for example.
  • the hybrid TCRs will also only pair together, which avoids hybrid TCR alpha or beta chain molecules pairing with the host cell TCRs alpha or beta chain molecules.
  • the TCR may be a recombinant TCR.
  • the TCR may be a soluble TCR.
  • the TCR may be isolated.
  • nucleic acid encoding one or more, TCR disclosed herein, such as one, two, three, four, five, six or more TCRs disclosed herein.
  • the nucleic acid may be a recombinant nucleic acid.
  • the nucleic acid may be isolated.
  • the recombinant nucleic acid may be a DNA or RNA molecule.
  • the DNA may be a cDNA.
  • the nucleic acid may be non-naturally occurring and/or purified and/or engineered.
  • a vector comprising a nucleic acid disclosed herein.
  • the vector may be a TCR expression vector.
  • the vector may be a plasmid.
  • the vector may be a viral vector, such as a retroviral vector, lentiviral vector or adenoviral vector.
  • the vector may comprise nucleic acid disclosed herein, encoding in a single open reading frame, or two distinct open reading frames, the alpha chain and the beta chain respectively.
  • a cell or population of cells comprising and/or encoding one or more TCR, nucleic acid or vector disclosed herein.
  • the cell or population of cells may harbour a first expression vector which comprises nucleic acid encoding the alpha chain of a TCR disclosed herein, and a second expression vector comprising nucleic acid encoding the beta chain of a TCR disclosed herein.
  • the cell may be a T-cell.
  • the T-cell may be a CD4+ T cell.
  • the T-cell may be a CD8+ T cell.
  • the T-cell may be a Mucosal associated invariant T cell (MAIT cell),
  • the cell may be a natural killer cell, myeloid or monocyte cell.
  • the cell or population of cells may include a non-naturally occurring and/or purified and/or or engineered cell, especially a T-cell such as a CD8+ T-cell, expressing/presenting a TCR disclosed herein.
  • T cells with nucleic acid such as DNA, cDNA or RNA
  • T-cells expressing the TCRs disclosed herein will be suitable for use in adoptive therapy-based treatment of or prevention of cancer.
  • suitable methods by which adoptive therapy can be carried out see for example Rosenberg et al, (2008) Nat Rev Cancer 8(4): 299-308).
  • the cells or population of cells may be isolated and/or recombinant and/or non-naturally occurring and/or engineered.
  • composition comprising one or more TCR, nucleic acid, vector, cell or population of cells of the invention.
  • TCR TCR
  • nucleic acid vector
  • cell or population of cells or pharmaceutical composition of the invention for use in medicine.
  • TCR TCR
  • nucleic acid vector, cell or population of cells, or pharmaceutical composition of the invention for use in treating or preventing a disease or disorder associated with upregulated WT1 expression.
  • a TCR, nucleic acid, vector, cell or population of cells, or pharmaceutical composition of the invention for use in treating or preventing cancer in a subject.
  • the cancer may be any cancer which is associated with WT1 expression or over expression.
  • a method of treating or preventing cancer in a subject comprising administering to the subject one or more TCR, nucleic acid, vector, cell or population of cells, or composition of the invention.
  • TCR TCR
  • nucleic acid vector
  • cell or population of cells or pharmaceutical composition of the invention in the manufacture of a medicament for treating or preventing cancer in a subject.
  • the cancer may be acute myeloid leukaemia, breast cancer, endometrial carcinoma, ovarian cancer, hepatocellular cancer, non-small cell lung cancer, colorectal cancer, soft tissue sarcoma, glioblastoma, astrocytoma, melanoma, or mesothelioma.
  • the one or more further therapeutic agent may be an agent known for the treatment and/or prevention of cancer.
  • the one or more further therapeutic agent may be a cytotoxic agent.
  • the one or more further reagent could be an immune checkpoint inhibitor.
  • the one or more further therapeutic agent may be an immunomodulatory agent, such as IL-2, IFN-gamma, an anti-CD3 antibody.
  • any TCR of the invention may be a bispecific TCR.
  • the bispecific TCR may bind, in addition to a WT1 peptide, such as RMFPNAPYL, a second or further target.
  • the TCR may further comprise an anti-CD3 targeting moiety.
  • the TCR may further comprise an anti-CD2 targeting moiety.
  • the TCR may further comprise an anti-CTLA4 targeting moiety.
  • the TCR may further comprise an anti-CD28 targeting moiety.
  • the TCR may further comprise an anti-ICOS targeting moiety.
  • the TCR may further comprise an anti-TNF receptor superfamily member targeting moiety.
  • TCRs may be used, as soluble targeting agents for the purpose of delivering cytotoxic or immune effector agents to neoplastic cells (Lissin, et al., (2013). "High-Affinity Monoclonal T-cell receptor (mTCR) Fusions. Fusion Protein Technologies for Biophamaceuticals: Applications and Challenges". S. R. Schmidt, Wiley; Boulter, et al, (2003), Protein Eng 16(9): 707-71 1; Liddy, et al. ,(2012), Nat Med 8 : 980-987), or alternatively they may be used to engineer T cells for adoptive therapy (June, et al., (2014), Cancer Immunol Immunother 63(9): 969-975).
  • the invention is in part based on the finding that human CD8+ T cells can be generated to respond to a WT1 peptide presented by HLA-E. Importantly, the inventors demonstrate for the first time that HLA-E restricted WT1 specific T cells are capable of inducing the death of WT1 expressing cells.
  • Binding of the homologous signal peptide and recognition by the NKG2-CD94 receptors on NK cells is conserved for the closely related non-classical MHC molecules, H-2Qa- 1 in mice, and Mamu-E in rhesus macaques, furthermore, HLA-E and Mamu-E are specifically targeted by human and rhesus cytomegaloviruses (CMVs), thus, regulation of NK cell activity appears to be the principal function of the MHC-E family of molecules across species.
  • CMVs human and rhesus cytomegaloviruses
  • HLA-E restricted CD8+ T cells are driven by HLA-E restricted CD8+ T cells; only a few are currently known. These include HLA-E restricted T cells specific for mycobacterial peptide antigens are found in most adult humans and more than 70 epitopes have been identified; The HLA-E restricted response is a major component of the CD8+ T cell response to this microbe.
  • Mamu-E restricted T cell responses were identified in rhesus macaques vaccinated with rhesus cytomegalovirus strain 68-1, which was recombinant for all SIV genes except Vif (RhCMV 68-1-SIV) (S. G.
  • the invention provides an opportunity to produce HLA-E restricted WT1 specific TCRs and T-cells expressing such TCRs from third party blood donors on an industrial scale, providing an “off the shelf’ opportunity to negate the effects of T-cell exhaustion in the host.
  • Mutagenesis can be carried out using any appropriate method including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation independent cloning (LIC) procedures. These methods are detailed in many of the standard molecular biology texts. For further details regarding polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd Ed.) CSHL Press. Further information on ligation independent cloning (LIC) procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6.
  • PCR polymerase chain reaction
  • LIC ligation independent cloning
  • the TCR may be an alpha-beta heterodimer or may be in single chain format.
  • Single chain formats include aP TCR polypeptides of the Va-L-V , Vp-L-Va, Va-Ca-L-Vp, Va-L-Vp-Cp or Va-Ca-L-Vp-Cp or Vp-Cp-L-Va-Ca types, wherein Va and VP are TCR a and P variable regions respectively, C and P are TCR a and P constant regions respectively, and L is a linker sequence.
  • the TCR may be in soluble form (i.e. having no transmembrane or cytoplasmic domains).
  • TCRs for use in adoptive therapy may contain a disulphide bond corresponding to that found in nature between the respective alpha and beta constant domains, additionally or alternatively a non-native disulphide bond may be present.
  • a disulphide bond corresponding to that found in nature between the respective alpha and beta constant domains, additionally or alternatively a non-native disulphide bond may be present.
  • a TCR disclosed herein may also be part of a multivalent complex of the TCR.
  • Such multivalent TCR complexes may be particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo and are also useful as intermediates for the production of further multivalent TCR complexes having such uses.
  • Identity as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs.
  • Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).
  • a program such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are suitable.
  • the percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions.
  • the "best alignment” is an alignment of two sequences which results in the highest percent identity.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.
  • An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad . Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5877.
  • the NBUAST and XBUAST programs of Altschul, et al. (1990) J. Mol. Biol. 2 15:403-41 0 have incorporated such an algorithm.
  • Gapped BUAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Gapped BLAST
  • PSI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).
  • the ALIGN program version 2.0 which is part of the CGC sequence alignment software package has incorporated such an algorithm.
  • Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8.
  • ktup is a control option that sets the sensitivity and speed of the search.
  • TCR specificity can be measured in vitro, for example, in cellular assays known to the skilled person, such as by measuring the binding of multimer (eg tetramer) HLA-E- peptide complexes.
  • the TCRs may be in soluble form and/or may be expressed on the surface of T cells, either naturally as with the T cell clones or expressed on third party T cells after transfer of DNA encoding the TCRs (eg by retrovirus or lentivirus transduction).
  • Recognition by T cells bearing the TCRs disclosed herein, may be determined by measuring the level of T cell activation after exposure to HLA- E plus WT1 peptide on cells or other antigen. .
  • Minimal recognition of antigen negative target cells is defined as a level of T cell activation of less than 20%, preferably less than 10%, preferably less than 5%, and more preferably less than 1%, of the level produced in the presence of antigen positive target cells, when measured under the same conditions and at a therapeutically relevant TCR concentration.
  • functional assays be performed to provide information on TCR specificity and activity.
  • in vitro assays may be used, which are usually short term (such as 6 hours) and which may show a low (2-10%) , moderate ( 11-50%) or high (51-100%) fraction of cloned T cells or TCR transduced T cells responding for example via CD69 expression or Interferon-gamma production) depending on affinity of peptide binding to HLA-E and affinity or avidity of TCR binding to the peptide-HLA-E complex .
  • These assays may show only low level responses or binding, but significantly above background levels. Longer term functional assays may be utilised, such as by assaying for killing of AML cells by TCR bearing T cells in vitro.
  • a ‘good’ clone will show about 20-100% cell lysis over 24-48 hours at a killer to target cell ratio of about 1 : 1 or 2: 1. Or for Testing virus-specific T cells may provide 50-100% inhibition in an assay spread over 24-96 hours. These functional assays show that the target of interest is being recognised biologically by the TCR. The skilled person will appreciate that these are just a few, non-limiting examples of assays to determine specificity and activity of TCR clones.
  • a therapeutically relevant concentration may be defined as a TCR concentration of 10 ⁇ 9 M or below, and/or a concentration of up to 100 times, preferably up to 1000 fold, greater than the corresponding EC50 value.
  • Antigen positive cells may be obtained by peptide-pulsing using a suitable peptide concentration to obtain a low level of antigen presentation (for example, 10’ 9 M peptide, as described in Bossi et al. (2013) Oncoimmunol. 1;2 (1 l):e26840) or, they may naturally present said peptide.
  • antigen negative cells are human cells.
  • a soluble TCR would be a bispecific or multi-specific TCR, comprising one antigen binding portion binding the WT1 peptide as defined herein, and comprising a further antigen binding portion binding a cell-specific antigen, such as CD3.
  • a cell-specific antigen such as CD3.
  • a TCR disclosed herein (which may be associated with a detectable label or therapeutic agent or expressed on a transfected T cell) or a cell or population of cells disclosed herein may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients.
  • an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers.
  • the compositions of the invention will preferably be administered intravenously, for example comprising cells bearing one or more TCR of the invention, or soluble bispecific TCRs as defined herein.
  • Suitable formulations for intravenous administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • compositions may be provided in unit dosage form, a sealed container, or as part of a kit, which may include instructions for use and/or a plurality of unit dosage forms. Suitable dose range for a TCR disclosed herein may ultimately determined by a physician.
  • agents may be formulated together in the same formulation or may be formulated into separate pharmaceutical compositions.
  • the separate compositions may be administered concurrently, sequentially, or separately.
  • the term “therapeutically effective amount” refers to the total amount of the agent or each active component of the pharmaceutical composition or method that is sufficient to provide patient benefit, i.e., prevention or amelioration of the condition to be treated, a reduction in symptoms, an increase in rate of healing, or a detectable change in the levels of a substance in the treated or surrounding tissue.
  • patient benefit i.e., prevention or amelioration of the condition to be treated, a reduction in symptoms, an increase in rate of healing, or a detectable change in the levels of a substance in the treated or surrounding tissue.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in concurrently, sequentially, or separately.
  • the agents or compositions may be delivered at intervals ranging from about 24 hours to about 2 days, to about 1 week, to about 2 weeks, to about 3 weeks to about 1 month to about 2 months, to about 3 months, to about 4 months, to about 5 months, to about 6 months, to about 12 months, or more.
  • the scheduling of such dosage regimens can be optimized by the practitioner.
  • the agents or compositions may be administered using a treatment regimen comprising one or more doses, wherein the treatment regimen is administered over 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, 14 days, 30 days, 1 month, 2 months, 3 months, 6 months, 12 months or more.
  • patient or “subject,” as used interchangeably herein, refers to any mammal, preferably a human.
  • any aspect of the invention may be in vivo, ex vivo or in vitro.
  • the skilled person will appreciate that preferred features of any one embodiment and/or aspect of the invention may be applied to all other embodiments and/or aspects of the invention.
  • Figure 1 - shows priming and cloning of HLA-E restricted WTl-specific CD8+ T cells from an HLA-A0201 negative heathy donor.
  • PBMCs were stimulated with autologous activated dendritic cells and the WT1 peptide for 9 days.
  • HLA-E restricted WT1 specific CD8+ T cells were identified using HLA-E-WT1 tetramer conjugated to fluorochrome-PE or-APC.
  • B WT1 tetramer-PE+/APC+ cells were sorted for single cell cloning, and positive clones were identified using WT1 tetramer.
  • TCR T cell receptor
  • Figure 2 - shows the full TCR alpha chain V- J region and beta chain V-D-J amino acid sequences of the T cell receptors of the invention.
  • the leader sequences are shown in italics, the CDR1 regions in turquoise (first highlighted), the CDR2 regions in pink (second highlighted), and the CDR3 regions in yellow (third highlighted).
  • Figure 3 - shows functional analysis of HLA-E restricted WT1 -specific CD8+ T cell clones in response to naturally presented WT1 epitope of AML cell lines.
  • IFNy, TNF-a, CD107a/b, IL-4, IL-13, IL-17, and CTLA-4 expression by 14 WT1 clones upon stimulation with AML cell lines P31FUJ, N0M01 and M0LM13 were assessed using flow cytometry-based readouts with a Staphylococcal endotoxin B (SEB) positive control; responsive clones could be detected using multiple functional readouts (A and B).
  • SEB Staphylococcal endotoxin B
  • FIG. 4 - shows elimination of AML cells by WT1 clones.
  • AML cells were cultured with WTl-specific CD8+ T cell clones or an irrelevant control clone (HLA-E restricted HIV GAG RL9 specific clone) at an E: T ratio of 1 : 1.
  • the number of AML cells after 48 hours of coculture was quantified and normalised (N) using count bright beads by Flow Cytometry. The percentage reduction of AML cells in the presence of the WT1 clones was calculated according to the formula:
  • FIG. 5 Shows antigenic stimulation of CD8+ T cells from an HLA-A0201 negative heathy donor which were transduced with the C14, C18 and C20 T cell receptors.
  • the CD8+ transductants were stained with anti-mouse TCR VB antibody, anti-CD8 and Live/Dead Fixable Aqua to confirm the expression the WT1 TCRs on CD8+ T cells.
  • CD8+ transductants were stimulated with AML cell line P31FUJ for 9 hours with or without additional WT1 peptide at a CD 8 T cell: AML ratio of 1 : 1 for 6 hours. PMA-ionomycin was used as a maximum stimulation control. Bars indicate mean of frequencies of TNF-a+ or CD107a/b+ WT1 CD8+ transductants. Error bars indicate SD. Quite weak but significant responses were seen in this short term activation assay.
  • Figure 6 shows the lytic function of HLA-E-WT1 specific TCR transduced CD8 T cells in a cell lysis assay.
  • A Three WT1 TCRs, cl4, cl8 and c30B were transduced into primary CD8+ T cells. CD8+ transductants were stained with HLA-E-VL9 (VMAPRTVLL) tetramer initially, washed with PBS, and then stained with anti -mouse TCR N (i antibody. No cross reactivity with the VL9 peptide was seen.
  • VMAPRTVLL HLA-E-VL9
  • B The xCELLigence assay which measures adherence of the target cells to a poly-l-lysing coated electronic chip over 0-50 hours.
  • Lysed cells fall off the chip which can be measured.
  • the assay enables functional measurements over 48 hours, which may favour low affinity T cell receptor-target interactions. No peptide was added for these assays.
  • the figure shows killing of AML cell lines by WT1 TCR transductants by the xCELLigence Real Time Cell Analysis (RTCA).
  • E:T effector cell to target cell
  • C % WTl-specific TCR mediated cytolysis of AML cells by CD8+ T transductants of the 3 WT1 TCRs. The calculation of % cytolysis was described in Materials and Methods.
  • the xCELLigence assay which measures adherence of the target cells to a poly-l-lysing coated electronic chip over 0-50 hours. Lysed cells fall off the chip which can be measured. The assay enables functional measurements over 48 hours, which may favour low affinity T cell receptor-target interactions. No peptide was added for these assays.
  • the figure shows killing of AML cell lines by WT1 TCR transductants by the xCELLigence Real Time Cell Analysis (RTCA).
  • RTCA Real Time Cell Analysis
  • A Representative cell index (ID) curve of target AML cell line N0M01 cocultured with TCR transductants of WT1 clones cl8, c20 and c41 or an irrelevant TCR at various effector cell to target cell (E:T) ratios.
  • PBMCs Human peripheral blood mononucleated cells
  • the acute myeloid leukaemia cell line P31LUJ, N0M01, U937 and M0LM13 http s : //de pm ap . org/po rtal/
  • ATCC https://www.atcc.org/cell.
  • Monoclonal antibodies against human HLA-E (clone 3D 12) conjugated to APC and antihuman CD3/APC-Cy7, CD4/PerCP-Cy5.5, CD8/BV421 are from BioLegend. Streptavidin/PE and /APC were purchased from BD Biosciences.
  • the WT1 peptide, residuesl26-135, RMLPNAPYL was predicted to bind to HLA-E using an algorithm described in Walters et al (Walters, L.C. et al, Nat Commun 2018; Walters L.C. et al., Eur J Immunol, 2020) and by NET-MHC.
  • Synthetic 9 amino acid WT1 (RMLPNAPYL) peptide was generated by Fmoc (9- fluorenylmethoxy carbonyl) chemistry to a purity of 85% (Genscript, Hong Kong).
  • HLA-E*01 03-WT1 tetramers were generated via conjugation to streptavidin-bound APC (Biolegend, San Diego) or PE (Biolegend, San Diego) at a Molar ratio of 4: 1. HLA-E tetramers staining of CD8 + T cells
  • PBMCs post WT1 priming or CD8+ T cell clones were stained with tetramers at 0.5ug per IxlO 6 cells for 45 minutes at room temperature (RT) in the dark.
  • RT room temperature
  • cells were subsequently stained with Live/Dead Fixable Aqua and flow antibodies to surface markers for 20 min at RT in the dark.
  • Cells were then washed and fixed with 2% paraformaldehyde, and then acquired using a LSR Fortessa (BD Biosciences). The data were analyzed using FlowJo software vl0.4 (Tree Star).
  • PBMCs peripheral blood mononuclear cells
  • AIM-V medium Invitrogen
  • DC dendritic cell
  • GM-CSF GM-CSF
  • IL-4 500U/ml, Miltenyi Biotech Ltd
  • TNF-a (lOOOU/ml, R&D Systems), IL- ip (lOng/ml, R&D Systems) and prostaglandin E2 (PGE2 IpM, Merck) were added together with RL9HIV peptide (20pM, GenScript), IL-7 (5ng/ml, R&D Systems) and IL-15 (5ng/ml, R&D Systems).
  • IL-2 was added at a concentration of 500IU/ml.
  • HLA-E RL9 tetramer staining was evaluated on day 9.
  • PBMCs post-WTl priming were stained with HLA-E tetramers initially. Cells were subsequently washed with PBS and stained with Live/Dead Fixable Aqua, anti-CD3- APC-Cy7, anti-CD4-PerCP-Cy5.5, anti-CD8-BV421, anti-CD94-FITC and anti-CD56- BV510 for 30 mins at RT in the dark.
  • CD3 + CD4"CD56"CD94"CD8 Tetramer + T cells were live sorted using FACSAria III (BD Biosciences).
  • Sorted cells were seeded into 384-well plates at 0.4 cells per well with irradiated (45 Gy) allogeneic feeder cells (3 healthy donors, 2xl0 6 cells/mL) stimulated with PHA (Ipg/mL) and IL-2 (500 U/mL) cultured in complete media (CM) containing RPMI 1640, 10% AB human sera (UK National Blood Service), 1% penicillin/streptomycin, 1% glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 0.1% beta-mercaptoethanol. 12 days later, T cell clones were further expanded with feeder cells and PHA/IL-2.
  • CM complete media
  • T cell clones were confirmed as clones by TCR sequencing showing a single TCR B chain with one or two TCR a chains. Determination of T cell clone specificity
  • HLA-E-WT1 tetramer Each clone was shown to bind the HLA-E-WT1 tetramer. They were also tested with HLA-E bound to the dominant self peptide VL9, derived from the signal sequence 3-11 of HLA A,B,C class I proteins. Clones 14, 18 and 20 were negative for HLA-E-VL9 staining. Clones 41 and 30 (before separation into 30A and 3 OB) showed additional HLA-E VL9 staining although they and all other clones were negative for expression of the HLA-E VL9 specific NKG2-CD94 receptor. This result demonstrates that their TCRs bound both WT1 and VL9 peptides bound to HLA-E.
  • RNA of CD8 clones was extracted using a RNeasy Plus Mini Kit (Qiagen).
  • TCR libraries using around lOOng RNA were prepared using a SMARTer Human TCR a/b Profiling Kit (Takara Bio) according to the manufacturer instructions based on SMART and 5 ’RACE techniques. Subsequently, full length TCR alpha and beta chains were sequenced using a Miseq Reagent Kit v3 (600-cycle) on an Miseq sequencer (Illumina).
  • Raw BCL files were converted to FASTQ format using bcl2fastq (v2.20.0.422).
  • TCR sequences were then reconstructed using MiXCR (v3.0.13), using the mixer analyze amplicon command, and only productive TCRs were included.
  • MiXCR output files were parsed into R (v4.0.1) using tcR (v2.3.2).
  • TCRs were filtered based on clone counts to retain only laip or 2a ip paired TCRs for each clone. Clonality was confirmed by the uniqueness of TCR sequences, where each clone showed only one TCR P chain.
  • CD8 clones were rested in CM before coculture with AML cells. The coculture was incubated at the CD8: AML ratio of 1 : 1 for 9 hours. For maximal functionality testing, CD8 clones were also independently treated with PMA/Ionomycin. Brefeldin A (5p.g/ml) and GolgiStop (5p.g/ml) were added after 1 hour during incubation. Anti-CD107a-BV421 and anti-CD107b-BV421 were supplemented at beginning of coculture.
  • Cells were stained with Live/Dead Fixable Aqua and flow antibodies surface markers (anti-human CD3 and anti-human CD8) in PBS, and then fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences). Intracellular staining (ICS) was performed with fluorochrome-conjugated antibodies against TNFa, IFN-y, MIP-ip, IL-4, IL-13, CTLA-4 and CD137 in Permwash solution. Cells were acquired on a LSRFortessa (BD Biosciences) and analyzed using FlowJo vl0.4 (Tree Star).
  • ICS Intracellular staining
  • P31FUJ cells were gated on live /CD3-/CD8- cells.
  • the number of P31FUJ cells were normalised by the number CountBright beads acquired from the same well.
  • % Reduction of P31FUJ cells was calculated to data obtained with control CD8+ T cell clones using the following formula: (1 -normalised number of AML cells co-cultured with WT1 clone/ normalised number of AML cells co-cultured with control clone) x 100.
  • TCR alpha and beta VDJ regions were amplified and assembled into a pHR-SIN backbone with the murine TCR alpha and beta constant regions using the HiFi DNA Assembly cloning kit (NEB).
  • Lentiviruses were generated by transfecting the TCR-containing plasmid together with packaging plasmids pMDG- VSVG and pCMV-dR8.91 into HEK 293T cells by TurboFectin (Origene).
  • CD8+ T cells were isolated from cone PBMC and activated with 1 : 1 of CD3/CD28 Dynabeads (Thermo Fisher) for 2 days, then transduced with lentiviruses.
  • Mouse TCRp+CD8+cells were sorted (BD Fusion) and expanded for a further 17 days before subsequent functional analysis of TCR transductants.
  • a real-time cellular impedance monitoring technology xCELLigence® was adapted to measure the potency of WT1 TCR transductants mediated cytotoxicity.
  • the xCeLLigence® platform utilizes gold microelectrodes embedded in the bottom of microtitre wells to monitor the status of adherent cells. When seeded alone, target adherent cells proliferation rate is recorded as increase in the impedance-related Cell Index (CI) over time. When non-adherent immune cells are added to adherent target cells, their cytolytic activity causes the adherent target cells to round up and detach, consequently reducing CI value. Because the AML cells are not adherent, the tethering reagent anti-CD71 is used to immobilise the AML cells on the well bottoms of the E- plate following the manufacturer’s instructions.
  • AML cell culturing media 50 .l of AML cell culturing media was added to each well of the tethering reagent anti-CD71 pre-treated E-Plate (Agilent) to obtain background impendance measurement displaced as Cell Index.
  • AML cells were seeded at a density of 100,000 cells /well in a volume of 50pl media.
  • the E-plate was left in the cell culture hood for 30 minutes to allow the AML cells to adhere on the electrode surface before transferred to the xCelligence RTCA instrument (Agilent) inside a cell culture incubator at 37° C with 5% CO2. Data recording was initiated immediately at 10 minutes intervals for the entire duration of the experiment.
  • Example 1 identification of functional HLA-E restricted WTI -specific CD8+ T cell clones able to induce killing of WTl-expressing cancer cells.
  • the inventors identified HLA-E restricted WTI -specific CD8+ T cell clones, by coculturing PBMCs of a healthy donor with autologous dendritic cells and WTI peptide.
  • WTI specific CD8+ T cells were sorted for single cell cloning ( Figure 1), and individual clones sequenced ( Figure 2).
  • Three clones, 14, 18 and 20 were shown to be specific for HLA-E bound to the WTI peptide and did not recognise HLA-E bound to the VL9 self HLA ABC signal that is the predominant peptide bound to HLA-E in normal healthy cells.
  • T-cell clones Functionality and responsiveness of identified T-cell clones was confirmed on AML cells naturally presenting WTI (P31FUJ, N0M01 and M0LM13), using IFNy, TNF-a, CD107a/b, IL-4, IL-13, IL-17, and CTLA-4 expression as a readout ( Figure 4).
  • AML cells were cultured with the WTl-specific CD8+ T cell clones or an irrelevant clone (HLA-E restricted HIV GAG RL9 clone). AML cells were demonstratively killed in the presence of the with multiple WTl-specific CD8+ T cell clones (Figure 5).
  • TCRs were then successfully cloned and transduced into primary CD8+ T cells from an HLA-A0201 negative heathy donor. TCR clones were shown to not cross-react with the self-peptide VL9, which would provide clinical benefits of reduced or no off target effects. CD8+ transductants were stimulated by AML cell lines. T-cells transduced with TCRs from clones 14, 18 and 30B killed the AML cells in the absence of added WT1 peptide, demonstrating their efficacy and that in the AML cells the peptide was supplied by endogenous antigen processing (Figure 6).
  • HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391 :795-799.
  • HLA-E-restricted, Gag-specific CD8(+) T cells can suppress HIV-1 infection, offering vaccine opportunities. Sci Immunol.

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Abstract

L'invention concerne des TCR restreints par WT1 et des TCR restreints par HLA-E, ainsi que des acides nucléiques codant pour le TCR et des vecteurs comprenant un tel acide nucléique. L'invention concerne également des utilisations d'un tel TCR dans le traitement ou la prévention du cancer.
PCT/GB2025/051048 2024-05-16 2025-05-15 Récepteurs de lymphocytes t spécifiques du peptide wt1 restreint par hla-e et leurs utilisations Pending WO2025238364A1 (fr)

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