WO2022248881A1 - Novel protein-ligand complex - Google Patents
Novel protein-ligand complex Download PDFInfo
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- WO2022248881A1 WO2022248881A1 PCT/GB2022/051361 GB2022051361W WO2022248881A1 WO 2022248881 A1 WO2022248881 A1 WO 2022248881A1 GB 2022051361 W GB2022051361 W GB 2022051361W WO 2022248881 A1 WO2022248881 A1 WO 2022248881A1
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- cells
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- oligoadenylate
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- ligand
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 229940045136 urea Drugs 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 229950004393 visilizumab Drugs 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
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- C12Y207/07—Nucleotidyltransferases (2.7.7)
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- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
Definitions
- Novel protein-ligand complex Field of the invention This invention relates inter alia to an isolated MR1-ligand complex, its use in raising and discovering antibodies, T-cell receptors and T-cells (and other killer cells), and use of these and related therapy modes (such as BiTEs and CAR-T-cells) in the treatment or prevention of cancer. Products for the treatment or prevention of autoimmune diseases are also provided.
- Background WO2019/081902 (Sewell et al) and Crowther et al (2020) describe a new class of T- cell (called “K43A sensitive MR1-T-cells”) effective for treating cancer, which recognize cancer cells through population-invariant major histocompatibility complex class related protein MR1.
- Ligands of MAIT cells are described in WO2015/149130 (Corbett et al) and include, for example, the substance 5-OP-RU.
- K43A sensitive MR1-T-cells such as clone MC.7.G5 (disclosed in WO2019/081902), have target specificity via MR1, but the T-cell receptors (TCRs) of such T-cells do not bind to MR1 per se or to MR1 loaded with known bacterial ligands, rather, they apparently recognise a cancer-specific ligand within the MR1 binding groove; MR1 presents a cancer-specific, or cancer-upregulated, ligand to the TCR.
- a feature of clone MC.7.G5 as described in the Sewell/Crowther disclosures is the apparent importance of the K43 residue for TCR binding since MC.7.G5 did not significantly recognise target cells expressing the MR1 protein containing the K43A mutation (but not the wild type MR1).
- Different MR1-T-cells which do tolerate the K43A mutation have been described (Vacchini et al (2020)).
- the K43A-sensitive MR1-T-cells were found to target a wide range of cancer cell lines implying that the cancer-specific, or cancer-upregulated, ligand is common to a wide range of different types of cancers.
- the MR1 protein structure (PDB id 4PJ9) is shown as a light grey ribbon and/or surface, with 4 water molecules identified crucial for successful docking shown as light grey dotted spheres.
- FIG 13 – (A) Docking results of 2',5'-oligoadenylate tetramer triphosphate, (B) ‘Side’ view of MR1-ligand binding, (C) ‘Top’ view of MR1-ligand binding, (D) ‘Top’ view of MR1-ligand binding with 5-OP-RU superimposed.
- Figure 14 - Docking results of 5-OP-RU (PubChem 3D: CID 86289574). In this figure the 5-OP-RU structure is provided in stick representation in black.
- the polypeptides and polynucleotides used in the present invention are isolated.
- polypeptide or polynucleotide is one that is removed from its original environment.
- a naturally occurring polypeptide or polynucleotide is isolated if it is separated from some or all of the coexisting materials in the natural system.
- a polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of its natural environment.
- a polypeptide is isolated if it is, for example, a recombinant polypeptide.
- Naturally occurring or “native” or “natural”, which terms are interchangeable, when used with reference to a polypeptide or polynucleotide sequence means a sequence found in nature and not synthetically or recombinantly produced or modified or introduced, for example exogenously or heterologously, into a system or native system for example as found in an existing biological system.
- artificial when used with reference to a polypeptide or polynucleotide sequence means a sequence not found in nature i.e.
- engineered when used with reference to a polypeptide (such as a TCR) or cell means a polypeptide or cell not found in nature which is, for example, a synthetic polypeptide or protein or polynucleotide, a synthetic modification of a natural polypeptide or cell, for example, because it contains or expresses foreign residues or elements and/or lacks natural residues or elements.
- heterologous or “exogenous” when used with reference to the relationship of one polynucleotide or polypeptide to another polynucleotide or polypeptide indicates that the two or more sequences are not found in the same relationship to each other in nature.
- T-cell effector function domain means a domain associated with the effector function of the T-cell as opposed to a domain associated with the target binding function of the T-cell and includes, for example, a CD3zeta intracellular signalling domain or a co-stimulatory domain.
- Other types of immune cell receptor e.g.
- NK receptors include Ly49, NCR (natural cytotoxicity receptors), CD16.
- intracellular domain or region is used interchangeably with the term “cytoplasmic” domain or region and in the literature this is sometimes referred to as the “cytosolic” domain or region.
- polynucleotide means a polymeric macromolecule made from nucleotide monomers particularly deoxyribonucleotide or ribonucleotide monomers in natural form or in the form of analogues comprising one or more unnatural backbone residues, linkages or bases.
- a polynucleotide which is a DNA comprises units composed of deoxyribose, phosphate and bases selected from guanine, adenine, cytosine and thymidine.
- a polynucleotide which is an RNA comprises units composed of ribose, phosphate and bases selected from guanine, adenine, cytosine and uracil.
- MR1 protein means the protein MR1 (UniProt accession no.
- ligand means a binding molecule.
- a ligand for MR1 is a molecule capable of binding to MR1.
- a ligand for MR1 may be associated, or be capable of associating with, with a binding pocket on the MR1 molecule.
- T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity and also involved in the adaptive immune system.
- the T cells can include, but are not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g. , TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and gamma-delta T cells.
- helper T cells include, but are not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g. , TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated in
- the term “specifically binding” in relation to the binding of A to B means that A binds to B, for example at or within a respective specific binding site, domain or pocket, with an affinity typically associated with the binding of ligands to receptors or typically associated with molecules of the immune system, such as antibodies and T-cell receptors, optionally of the binding affinity level of micromolar or nanomolar affinity, such that the affinity of binding of A to B greatly exceeds that of the binding of A to other molecules not intended to be targeted by A.
- the term “being specifically bound” is to be interpreted in a similar sense.
- antibody protein means an antibody, an antibody fragment, an antibody conjugated to an active moiety, a fusion protein comprising one or more antibody fragments, or a derivative of any of the aforementioned.
- Antibody fragments are most suitably antigen binding fragments of antibodies.
- derivatives include conjugated derivatives e.g. an antibody or antibody fragment conjugated to another moiety.
- moieties include chemically inert polymers such as PEG.
- Antibodies may include monoclonal antibodies and polyclonal antibodies, preferably monoclonal antibodies. The monoclonal antibodies can be, for example, mammalian (e.g.
- antibody fragments including Fab, Fab2, scFv fragments and the like, scFv-Fc, single domain antibody, diabody, dsFv, Fab', minibody, diabody, single-chain antibody molecules, especially such fragments which bind antigens.
- VHH variable portion
- the term “4-chain monoclonal antibody” means a monoclonal antibody of conventional type having two heavy chains and two light chains, said heavy and light chains being paired to form at their extremities two variable regions which each constitute different antigen-binding sites.
- the 2’,5’-oligoadenylate molecules of use according to the invention are molecules containing n adenosine nucleosides linked 2’,5’ via a phosphodiester linkage wherein the number n is typically in the range 2-6, e.g.2-5, e.g.2, 3 or 4.
- the 5’ end of a 2’,5’-oligoadenylate molecule may typically be provided with one or more (e.g. one to three) phosphate groups e.g.
- Derivatives of 2’,5’-oligoadenylate molecules are also of use according to the invention.
- Example derivatives are those where the 2’,5’-oligoadenylate molecule has been conjugated to a small carbonyl-containing molecule, preferably wherein the small carbonyl-containing molecule is glyoxal or methylglyoxal.
- the conjugation referred to is typically via reaction of the amine sidechain of at least one adenine of the 2’,5’-oligoadenylate molecule with the carbonyl of the carbonyl- containing molecule e.g. the terminal carbonyl of glyoxal or methylglyoxal to form a Schiff base.
- the immune cell engaging portion suitably comprises an antibody protein which is capable of binding and/or specifically binding to any one or more of CD3, CD5, CD16, CD16a, CD16b, NKp30, NKp46, NKG2D and DNAX Accessory Molecule-1 (DNAM-1) on immune cells for example on immune cells, for example T-cells, T-lymphocytes or NK cells, preferably CD3 on immune cells.
- Anti-CD3 monoclonal antibodies are known in the art e.g. muromonab-CD3, otelixizumab, teplizumab and visilizumab.
- the antibody protein which is capable of specifically binding to CD3 on immune cells may for example be an scFv e.g.
- cancers which may be treated according to the invention include solid tumours and blood cancers, for example cancers selected from lung, melanoma (e.g. skin melanoma), bone, breast, blood (e.g. leukemia), prostate, kidney, bladder, cervical, ovarian and colorectal cancers, and in particular selected from lung, melanoma (e.g. skin melanoma), bone and breast cancers.
- Cancer may be primary cancer or metastatic cancer.
- Genetic modification of cell lines would also be used to demonstrate 2’,5’- oligoadenylate molecules as MR1-binding ligands.
- Overexpression of OAS would be performed (preferably with an inducible, e.g., tet-responsive promoter) and influence of OAS expression on increased surface MR1 would be assessed, with or without exogenous supply of 2’,5’-oligoadenylate molecule (pulsing) or OAS agonists.
- genetic manipulation of MR1 to introduce wild type MR1 to be overexpressed in MR1-negative cells and assessed for surface presentation following 2’,5’-oligoadenylate molecule pulsing or OAS agonism would be performed.
- T cells that recognize the MR1-2’,5’-oligoadenylate molecule complex can be identified by multiple methods using blood-derived T cells from normal subjects and cancer patients. For example, na ⁇ ve of patient T cells can be incubated with MR1- 2’,5’-oligoadenylate molecule complexes and then specifically activated T cells can be identified by using flow-cytometry based methods such as detection of activation- induced TNF ⁇ (TAPI-0-based assay).
- TAPI-0-based assay detection of activation- induced TNF ⁇
- antibodies can be generated through the use of phage- or yeast-display technology (Sheehan & Marasco, 2015), which are based on large libraries of antibody-like reactive molecules that can be screened with the MR1-2’,5’-oligoadenylate molecule complex to discover antibody complementary determining paratopes that react with MR1.
- phage- or yeast-display technology Sheehan & Marasco, 2015
- individual clones of phage or yeast found in large libraries displaying diverse scFv or Fab fusions can be selected for their ability to bind the MR1-2’,5’- oligoadenylate molecule complexes, and the selected antibody fragments can be reconstructed into functional antibodies for further use.
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Abstract
There is provided inter alia an isolated MR1 -ligand complex comprising (a) an MR1 protein and (b) a ligand molecule selected from a 2',5'-oligoadenylate molecule and a derivatised 2',5'-oligoadenylate molecule as ligand for the MR1 protein which is capable of being specifically bound by a T-cell receptor.
Description
Novel protein-ligand complex Field of the invention This invention relates inter alia to an isolated MR1-ligand complex, its use in raising and discovering antibodies, T-cell receptors and T-cells (and other killer cells), and use of these and related therapy modes (such as BiTEs and CAR-T-cells) in the treatment or prevention of cancer. Products for the treatment or prevention of autoimmune diseases are also provided. Background WO2019/081902 (Sewell et al) and Crowther et al (2020) describe a new class of T- cell (called “K43A sensitive MR1-T-cells”) effective for treating cancer, which recognize cancer cells through population-invariant major histocompatibility complex class related protein MR1. The identification of this new type of T-cell stemmed from experiments searching for T-cells recognising cancer cells without the requirement for a specific Human Leukocyte Antigen (HLA). The HLA locus is highly variable with over 17,000 different alleles having been described today. As such, any therapeutic approach that works via an HLA, can only be effective in a minority of patients. In contrast, the entire human population expresses MR1 which means that MR1 targeted TCRs should be effective across most of the population. The main type of MR1-restricted T-cells that are known are called mucosal-associated invariant T-cells (MAITs). MAITs are known to recognise intermediates of mycobacterial riboflavin biosynthesis. Ligands of MAIT cells are described in WO2015/149130 (Corbett et al) and include, for example, the substance 5-OP-RU. K43A sensitive MR1-T-cells, such as clone MC.7.G5 (disclosed in WO2019/081902), have target specificity via MR1, but the T-cell receptors (TCRs) of such T-cells do not bind to MR1 per se or to MR1 loaded with known bacterial ligands, rather, they apparently recognise a cancer-specific ligand within the MR1 binding groove; MR1 presents a cancer-specific, or cancer-upregulated, ligand to the TCR. A feature of clone MC.7.G5 as described in the Sewell/Crowther disclosures is the apparent importance of the K43 residue for TCR binding since MC.7.G5 did not significantly recognise target cells expressing the MR1 protein containing the K43A mutation (but not the wild type MR1). Different MR1-T-cells which do tolerate the K43A mutation have been described (Vacchini et al (2020)).
The K43A-sensitive MR1-T-cells were found to target a wide range of cancer cell lines implying that the cancer-specific, or cancer-upregulated, ligand is common to a wide range of different types of cancers. There is great interest in identifying the ligand for the TCR of MR1-T-cells including K43A-sensitive MR1-T-cells. Knowledge of the identity of this ligand would enable further TCRs and antibodies to be raised, as well as other therapeutic modalities, all of which could be useful e.g. in the treatment or prevention of cancer. The work of the present inventors has identified such a ligand. Summary of the invention The inventors have identified that 2’-5’-oligoadenylate molecules are ligands for MR1 and the MR1-2’-5’-oligoadenylate molecule complex is recognised by K43A sensitive MR1-T-cells and K43A sensitive MR1-T-cells bind to the complex in structural molecular models. Structural modelling studies described in the Examples section shows that 2’,5’-oligoadenylate molecules bind in the ligand binding pocket formed in the groove between the α1 and α2 domains of the heavy chain of MR1. Without being limited by theory it is believed that cancers either constitutionally, through genomic instability (e.g. DNA damage response, radiotherapy, chemotherapy, immunotherapy or otherwise), release double-stranded DNA (“dsDNA”) or double stranded RNA ((“dsRNA”) and this ds nucleic acid aberrantly accumulates in the cytosol of tumor cells. A variety of different classes of molecular pattern-recognition receptors (PRRs), including RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), NOD-like receptors (NLRs), and the cyclic GMP-AMP (cGAMP) synthase (cGAS) and the 2’-5’-oligoadenylate synthetase (OAS) family of nucleotidyltransferases. Enzymes such as OAS bind RNA in the cytosol and generate linear 2’-5’-linked oligoadenylate messenger molecules which, in turn, activate RNase L or Stimulator of Interferon Genes (STING) immune signaling, respectively resulting in the downstream production of type 1 interferons and other cytokines. STING, Stimulation of Interferon Genes (STING), is an ER-resident protein encoded by TMEM173. As a canonical oligoadenylate sensor, on exposure to its ligand, STING multimerises (e.g. dimerises) and is translocated to different cellular compartments, whereupon it activates downstream pathways (e.g., TANK- binding kinase 1 (TBK1) which further phosphorylates interferon regulatory
transcription factor 3 (IRF3); NF-κB is also liberated). STING is an important mediator of the innate immune response to pathogens and in cancer. Oligoadenylate molecules are widely distributed intra- and extracellularly, to neighbouring cells via gap junctions or extracellularly through vesicles or through a variety of transporters (e.g. LRRC8 and SLC19A1). MR1 has the capacity to load a variety of potential ligands, mainly but not limited to metabolites, either by binding to de novo synthesized MR1 or through binding to previously synthesized forms which continuously recycle to the cell surface, presenting ligands. MR1 has broad low-level expression across multiple cancer and tissue types. These 2’,5’-oligoadenylate molecules can access MR1 rich compartments and in a steric conformation that has a stabilising affinity through binding in the MR1 ligand-binding pocket; stabilise the MR1 structure leading it to traffic to the plasma membrane and present the antigen; thus, 2’,5’-oligoadenylate molecules are ligands for MR1 and are expected to be recognised by K43A sensitive MR1-T-cells such as MC.7.G5. Thus, according to the invention, there is provided an isolated MR1-ligand complex comprising (a) an MR1 protein and (b) a ligand molecule selected from a 2’-5’- oligoadenylate molecule and a derivatised 2’,5’-oligoadenylate molecule as ligand for the MR1 protein which is capable of being specifically bound by a T-cell receptor. Brief description of the figures Figure 1 - 2',5'-Oligoadenylate structural formula from the KEGG database (https://www.genome.jp/dbget-bin/www_bget?C20935). Figure 2 – 2D structure of the 2’,5’-oligoadenylate dimer monophosphate Figure 3 – 2D structure of the 2’,5’-oligoadenylate dimer triphosphate Figure 4 – 2D structure of the 2’,5’-oligoadenylate trimer monophosphate Figure 5 – 2D structure of the 2’,5’-oligoadenylate trimer triphosphate Figure 6 – 2D structure of the 2’,5’-oligoadenylate tetramer monophosphate Figure 7 – 2D structure of the 2’,5’-oligoadenylate tetramer triphosphate
For the following docking results and structural representations, the MR1 protein structure (PDB id 4PJ9) is shown as a light grey ribbon and/or surface, with 4 water molecules identified crucial for successful docking shown as light grey dotted spheres. Proposed 2',5'-oligoadenylate ligands for MR1 are provided in stick representation in black; superimposed 5-OP-RU ligand structures are provided in stick representation in grey. Figure 8 – (A) Docking results of 2',5'-oligoadenylate dimer monophosphate, (B) ‘Side’ view of MR1-ligand binding, (C) ‘Top’ view of MR1-ligand binding, (D) ‘Top’ view of MR1-ligand binding with 5-OP-RU superimposed. Figure 9 – (A) Docking results of 2',5'-oligoadenylate dimer triphosphate, (B) ‘Side’ view of MR1-ligand binding, (C) ‘Top’ view of MR1-ligand binding, (D) ‘Top’ view of MR1-ligand binding with 5-OP-RU superimposed. Figure 10 – (A) Docking results of 2',5'-oligoadenylate trimer monophosphate, (B) ‘Side’ view of MR1-ligand binding, (C) ‘Top’ view of MR1-ligand binding, (D) ‘Top’ view of MR1-ligand binding with 5-OP-RU superimposed. Figure 11 – (A) Docking results of 2',5'-oligoadenylate trimer triphosphate, (B) ‘Side’ view of MR1-ligand binding, (C) ‘Top’ view of MR1-ligand binding, (D) ‘Top’ view of MR1-ligand binding with 5-OP-RU superimposed. Figure 12 – (A) Docking results of 2',5'-oligoadenylate tetramer monophosphate, (B) ‘Side’ view of MR1-ligand binding, (C) ‘Top’ view of MR1-ligand binding, (D) ‘Top’ view of MR1-ligand binding with 5-OP-RU superimposed. Figure 13 – (A) Docking results of 2',5'-oligoadenylate tetramer triphosphate, (B) ‘Side’ view of MR1-ligand binding, (C) ‘Top’ view of MR1-ligand binding, (D) ‘Top’ view of MR1-ligand binding with 5-OP-RU superimposed. Figure 14 - Docking results of 5-OP-RU (PubChem 3D: CID 86289574). In this figure the 5-OP-RU structure is provided in stick representation in black.
Detailed description of the invention Definitions Suitably, the polypeptides and polynucleotides used in the present invention are isolated. An “isolated” polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally occurring polypeptide or polynucleotide is isolated if it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of its natural environment. A polypeptide is isolated if it is, for example, a recombinant polypeptide. "Naturally occurring" or “native” or “natural”, which terms are interchangeable, when used with reference to a polypeptide or polynucleotide sequence means a sequence found in nature and not synthetically or recombinantly produced or modified or introduced, for example exogenously or heterologously, into a system or native system for example as found in an existing biological system. The term “artificial” when used with reference to a polypeptide or polynucleotide sequence means a sequence not found in nature i.e. not natural, which is, for example, a synthetic polypeptide or protein or polynucleotide, a synthetic modification of a natural sequence, or contains an unnatural polypeptide or polynucleotide sequence, or for example comprises modified subunits such as modified or derivative forms of amino acids or nucleotides, such as modified bases, modified elements or modified linkages between such elements, amino acids or nucleotides. The term “engineered” when used with reference to a polypeptide (such as a TCR) or cell means a polypeptide or cell not found in nature which is, for example, a synthetic polypeptide or protein or polynucleotide, a synthetic modification of a natural polypeptide or cell, for example, because it contains or expresses foreign residues or elements and/or lacks natural residues or elements. The term “heterologous” or “exogenous” when used with reference to the relationship of one polynucleotide or polypeptide to another polynucleotide or polypeptide indicates that the two or more sequences are not found in the same relationship to each other in nature.
The term “heterologous” when used with reference to the relationship of one polynucleotide or polypeptide sequence to a cell means a sequence which is not isolated from, derived from, expressed by, associated with or based upon a naturally occurring polynucleotide or polypeptide sequence found in, or endogenous to, the said cell. As used herein, the term “chimeric” means, in the context of a polypeptide or polynucleotide, an artificial polypeptide or polynucleotide that is engineered to contain elements (e.g. sequences of amino acids or nucleotides) of more than one origin. The term “domain”, when used with reference to a TCR or T cell receptor protein as used herein, is generally used to refer to a part of the TCR or T cell receptor protein formed of the corresponding region of the two chains comprising said domain. For example, the transmembrane regions of the α and β chains of αβTCRs form the transmembrane domain. The term “T-cell effector function domain” means a domain associated with the effector function of the T-cell as opposed to a domain associated with the target binding function of the T-cell and includes, for example, a CD3zeta intracellular signalling domain or a co-stimulatory domain. Other types of immune cell receptor (e.g. for B cells, NK cells or NKT cells), for example antibody proteins have analogous immune cell effector function domains, NK receptors include Ly49, NCR (natural cytotoxicity receptors), CD16. The term “intracellular” domain or region is used interchangeably with the term “cytoplasmic” domain or region and in the literature this is sometimes referred to as the “cytosolic” domain or region. As used herein, the term “polynucleotide” means a polymeric macromolecule made from nucleotide monomers particularly deoxyribonucleotide or ribonucleotide monomers in natural form or in the form of analogues comprising one or more unnatural backbone residues, linkages or bases. Typically, a polynucleotide which is a DNA comprises units composed of deoxyribose, phosphate and bases selected from guanine, adenine, cytosine and thymidine. Typically, a polynucleotide which is an RNA comprises units composed of ribose, phosphate and bases selected from guanine, adenine, cytosine and uracil.
As used herein, the term “MR1 protein” means the protein MR1 (UniProt accession no. Q95460) from human or other mammal (such as mouse, rat or bovine), particularly from human, as well as derivatives and fragments thereof which are capable of binding ligand as heavy chain when non-covalently associated with a β2- microglobulin protein as light chain and thus comprising, in particular, the α1, α2 and α3 domains of the heavy chain. Other variants of non- β2-microglobulin MR1s are also described and may present oligoadenylate molecules (Lion et al (2013). The polypeptide sequence of human MR1 is provided herein as SEQ ID NO: 1:
As used herein, the term “β2 microglobulin protein” means the protein β2 microglobulin (UniProt accession no. P61769) from human or other mammal (such as mouse, rat or bovine), particularly from human, as well as derivatives and fragments thereof which are capable of binding ligand, as light chain when non- covalently associated with an MR1 protein as heavy chain. The polypeptide sequence of human β2 microglobulin protein is provided herein as SEQ ID NO: 2:
As used herein, the term “ligand” means a binding molecule. A ligand for MR1 is a molecule capable of binding to MR1. In the context of the present invention, a ligand for MR1 may be associated, or be capable of associating with, with a binding pocket on the MR1 molecule. Such a binding pocket will typically have a size and shape and contain appropriate residues for the binding of the ligand. The ligand may be a 2’,5’-oligoadenylate molecule or derivatised 2’,5’-oligoadenylate molecule as herein described. In the context of the present invention the term “binding” will typically mean non-covalent binding i.e. means binding by means of ionic, hydrophobic or Van-de-Waals interactions however binding covalently e.g. via Schiff base linkages is not excluded, for example with residue K43 of MR1.
As used herein, the term “MR1-ligand complex” means a complex formed by the binding of a ligand for MR1 to MR1, specifically a binding in the binding pocket for said ligand. Binding of ligand to or within the binding groove or pocket of MR1 may constitute specific binding for example such that the ligand is presented for recognition by a further binding partner, e.g. by an immune cell receptor. In this form, the MR1-ligand complex presents the ligand as an antigen that is capable of being recognised by immune cell receptors, such as T cells, T cell receptor proteins or T cell TCRs. As used herein, the term “immune cell receptor” means a receptor that is capable of being expressed on the surface of an immune cell and that has a binding function i.e. the function of binding to a target antigen epitope on a target cell and an effector function i.e. the function of eliciting a functional behaviour of the immune cell, such as cell killing or recruitment of other cells, such as other cells of the immune system in response to the binding event. When an immune cell receptor binds to a target antigen epitope, for example an MR1 presented ligand, on a target cell (e.g. a cancerous cell) and the effector function results, this may be referred to herein as “recognition” i.e. the immune cell receptor recognises the target antigen epitope on the target cell. The immune cell may be a T-cell in which case the immune cell receptor is a T-cell receptor (TCR) or may be a B-cell in which case the immune cell receptor is an antibody protein. As used herein, the term “T-cell receptor protein” means a protein or complex of proteins which is a T-cell receptor formed of two chains, typically an α chain and a β chain in the case of αβ T-cells or a γ chain and a δ chain in the case of γδ T-cells, or a fragment of said receptor, capable of recognising a target antigen epitope. The chains of a T-cell receptor protein typically comprise a variable region and a constant region. The variable region of a chain which, when the two chains are paired binds to the target antigen epitope, typically contains 3 CDRs and 4 framework regions. The constant region typically comprises an extracellular region, a connecting peptide region, a transmembrane region and an intracellular region. Depending on the context, the receptor protein may be useful in soluble form i.e. in the form of a fragment of a T-cell receptor in which the constant region comprises an extracellular region but lacks a transmembrane region and an intracellular region. The soluble form of a T-cell receptor protein lacks an effector function. Single chain
formats where the two chains or fragments thereof are linked to form a single polypeptide and which are capable of recognising a target antigen epitope are also embraced by the term “T-cell receptor protein”. As used herein, the term “immune cell” includes T-cell, NK-cell and NKT-cell. For example, according to the present invention the immune cells can be cells of the lymphoid lineage, comprising B, T or natural killer (NK) cells. The immune cells may be cells of the lymphoid lineage including T cells, Natural Killer T (NKT) cells, γδ T- cells and precursors thereof including embryonic stem cells, and pluripotent stem cells (e.g, those from which lymphoid cells may be differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity and also involved in the adaptive immune system. According to the present invention the T cells can include, but are not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g. , TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and gamma-delta T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T- lymphocytes capable of inducing the death of infected somatic or tumour cells. A subject’s own T cells may be genetically modified to target specific antigens through the introduction of a heterologous TCR. Preferably, the modified immune cell is a T cell optionally a CD4+T cell or a CD8+T cell. Accordingly, the immune cells may be T-cells, optionally CD4+ T cells or CD8+ T cells, or the immune cells may be a population of modified T-cells, optionally CD4+ T cells; or CD8+ T cells, or a mixed population of CD4+ T cells and CD8+ T cells. As used herein, the term “T-cell” includes CD4+ T-cells and CD8+ T-cells and especially includes cytotoxic T-cells and γδ T-cells. As used herein, the term “NK-cell” includes umbilical cord blood NK cells, iPSC derived NK cells, PMBC derived NK cells and NK cells from NK92, YTS and NKL cell lines. As used herein, the term “NKT-cell” includes cells that have properties of both T cells and NK cells. These cells are traditionally thought to recognise the non-
polymorphic CD1d molecule presenting self and foreign lipids and glycolipids. They constitute only approximately 1% of all peripheral blood T cells. As used herein, the term “immune cell engaging protein” means an artificial bispecific molecule being capable of binding (e.g. specifically binding) (i) an antigen and (ii) a molecule expressed on the surface of an immune cell. Typically, an immune cell engaging protein binds an extracellular part of an immune cell receptor protein expressed on the surface of an immune cell, for example to any one or more of CD3, CD5, CD16, CD16a, CD16b, NKp30, NKp46, NKG2D and DNAX Accessory Molecule-1 (DNAM-1) on immune cells for example on T-cells, T-lymphocytes or NK cells. As used herein, the term “chimeric immune cell receptor protein” means an artificial molecule comprising the effector domains of an immune cell receptor and having the binding region of an antibody protein. Typically, a chimeric immune cell receptor protein comprises the effector domains of an immune cell receptor (including the transmembrane and intracellular domains of an immune cell receptor) fused to an scFv, said scFv being capable of specifically binding an antigen on a target cell. As used herein, the term “MR1-T-cell” means a T-cell expressing on its surface a T- cell receptor which binds to and/or recognises an MR1-ligand complex. The term “K43A sensitive MR1-T-cell” means an MR1-T-cell which, when its T-cell receptor comprises the K43A mutation, is incapable of (or substantially impaired in) binding to and/or recognising the MR1-ligand complex normally bound and/or recognised by an MR1-T-cell. As used herein, the term “specifically binding” in relation to the binding of A to B means that A binds to B, for example at or within a respective specific binding site, domain or pocket, with an affinity typically associated with the binding of ligands to receptors or typically associated with molecules of the immune system, such as antibodies and T-cell receptors, optionally of the binding affinity level of micromolar or nanomolar affinity, such that the affinity of binding of A to B greatly exceeds that of the binding of A to other molecules not intended to be targeted by A. The term “being specifically bound” is to be interpreted in a similar sense. As used herein, the term “antibody protein” means an antibody, an antibody fragment, an antibody conjugated to an active moiety, a fusion protein comprising one or more
antibody fragments, or a derivative of any of the aforementioned. Antibody fragments are most suitably antigen binding fragments of antibodies. Examples of derivatives include conjugated derivatives e.g. an antibody or antibody fragment conjugated to another moiety. Such moieties include chemically inert polymers such as PEG. Antibodies may include monoclonal antibodies and polyclonal antibodies, preferably monoclonal antibodies. The monoclonal antibodies can be, for example, mammalian (e.g. murine) or avian, chimeric or reverse chimeric, for example, human/mouse or human/primate chimeras, humanized antibodies or fully human antibodies. Antibodies may be produced in a non-human species (e.g., rodent) genetically modified to have elements of a human immune system. Suitable antibodies include an immunoglobulin, such as IgG, including IgG1, IgG2, IgG3 or IgG4, IgM, IgA, such as IgA1 or IgA2, IgD, IgE or IgY. Suitable antibodies also include single chain antibodies. Also included are antibody fragments including Fab, Fab2, scFv fragments and the like, scFv-Fc, single domain antibody, diabody, dsFv, Fab', minibody, diabody, single-chain antibody molecules, especially such fragments which bind antigens. Also embraced are single domain antibodies and heavy chain only antibodies derived from Camelids (e.g. llamas) and sharks and fragments thereof such as the variable portion (VHH). As used herein, the term “4-chain monoclonal antibody” means a monoclonal antibody of conventional type having two heavy chains and two light chains, said heavy and light chains being paired to form at their extremities two variable regions which each constitute different antigen-binding sites. As used herein, the term “scFv” means a single chain variable fragment that may be engineered to substitute for one of the antigen-binding domains of a 4-chain monoclonal antibody in which the variable region of the heavy chain (VH) is connected by a linker to the variable region of the light chain (VL). As used herein, the term “cancerous” means, in relation to a cell, a cell of malignant character typically associated with the behaviour of uncontrolled or dysregulated proliferation. 2’,5’-Oligoadenylate molecules and their derivatives OAS enzymes transfer an adenosine monophosphate (AMP) unit from the AMP donor substrate (adenosine triphosphate, ATP) to the 2’-hydroxyl group of an AMP acceptor substrate (ATP or a preformed 2’-5’A oligomer). This produces a 2’-5’A
dimer (pppA(2’-5’)A) or an elongated 2’-5’A oligomer (pppA((2’-5’)A)n), as well as one molecule of pyrophosphate (PPi). Adenosine monophosphate (AMP), also known as 5'-adenylic acid, is a nucleotide. AMP consists of a phosphate group, the sugar ribose, and the nucleobase adenine; it is an ester of phosphoric acid and the nucleoside adenosine. The 2’,5’-oligoadenylate molecules of use according to the invention are molecules containing n adenosine nucleosides linked 2’,5’ via a phosphodiester linkage wherein the number n is typically in the range 2-6, e.g.2-5, e.g.2, 3 or 4. The 5’ end of a 2’,5’-oligoadenylate molecule may typically be provided with one or more (e.g. one to three) phosphate groups e.g. one phosphate group or three phosphate groups. Derivatives of 2’,5’-oligoadenylate molecules are also of use according to the invention. Example derivatives are those where the 2’,5’-oligoadenylate molecule has been conjugated to a small carbonyl-containing molecule, preferably wherein the small carbonyl-containing molecule is glyoxal or methylglyoxal. The conjugation referred to is typically via reaction of the amine sidechain of at least one adenine of the 2’,5’-oligoadenylate molecule with the carbonyl of the carbonyl- containing molecule e.g. the terminal carbonyl of glyoxal or methylglyoxal to form a Schiff base. MR1-ligand complex As noted above, the invention provides an isolated MR1-ligand complex comprising (a) an MR1 protein and (b) a ligand molecule selected from a 2’,5’-oligoadenylate molecule and a derivatised 2’,5’-oligoadenylate molecule as ligand for the MR1 protein which is capable of being specifically bound by a T-cell receptor (herein after the “isolated complex of the invention”). Suitable MR1 in the complex comprises the heavy chain of MR1 and thus, in particular, comprises the α1, α2 and α3 domains of MR1, optionally wherein the MR1 has an amino acid sequence which has sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO:1. Suitably the heavy chain of MR1 in the complex is non-covalently associated with a β2 microglobulin protein as light chain. Alternatively, an artificial single-chain
construct may be created in which the heavy chain of an MR1 protein in the complex is covalently linked to a β2 microglobulin protein for example via a linker peptide, optionally wherein the β2 microglobulin protein has an amino acid sequence which has sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO:2. According to the present invention the 2’,5’-oligoadenylate molecule or derivatised 2’,5’-oligoadenylate molecule is, for example, any of those shown in any of Figures 2 to 7 or set out in clauses 4 to 7 herein or as set out below with reference to anti- cancer applications. For use in anti-cancer applications or for selection of a binding and/or recognising immune cell, T cell, immune cell engaging protein, or T cell receptor protein /TCR as described below (i.e. targeting said complex with an immune cell receptor protein or immune cell presenting such receptor protein, an immune cell engaging protein, or an antibody) suitably the 2’,5’-oligoadenylate molecule is any one of the following species: a) 2’,5’-oligoadenylate dimer monophosphate, b) 2’,5’-oligoadenylate dimer triphosphate, c) 2’,5’-oligoadenylate trimer monophosphate, d) 2’,5’-oligoadenylate trimer triphosphate, e) 2’,5’-oligoadenylate tetramer monophosphate, f) 2’,5’- oligoadenylate tetramer triphosphate; or any one of these molecules (a) to (f) that has been conjugated to a small carbonyl-containing molecule, preferably the small carbonyl-containing molecule is either glyoxal or methylglyoxal. Antibody proteins The invention also provides an antibody protein which specifically binds to the isolated complex of the invention (herein after an “antibody protein of the invention”). In one embodiment the antibody protein is a 4-chain monoclonal antibody. In another embodiment the antibody protein is an scFv. In another embodiment the antibody protein is a construct which comprises two or more scFvs, for example, two or more scFvs linked in series. Antibody proteins of the invention may be raised by conventional methods, e.g. phage/yeast display or involving immunisation an experimental animal (such as rabbit, mouse, rat, guinea pig, hamster, llama etc.) with an MR1-ligand complex as immunogen, optionally together with an immunostimulant (e.g. specol), and
obtaining antibodies or antibody producing cells from said animal (e.g. from PBMCs or from spleen). Monoclonal antibodies may be obtained by fusing said antibody producing cells with immortal cells to generate corresponding antibody producing hybridomas therefrom. Antibody proteins can be selected and cloned using conventional phage or yeast display technology and can be modified (e.g. to humanise or to introduce stability conferring mutations) and produced by conventional molecular biology and genetic engineering technology. Antibodies which are partially or completely human can also be produced in experimental animals which have genes of the human immune system. Immune cell engaging proteins The invention also provides an immune cell engaging protein which is capable of targeting a cell expressing a cell presenting on its surface an MR1-ligand complex comprising (a) a cell targeting portion comprising an antibody protein of the invention and (b) an immune cell engaging portion. The immune cell may as herein described and for example may be a T-cell, an NK-cell or an NKT cell and in particular is a T-cell. Thus, an embodiment, the immune cell engaging protein is a bispecific T-cell engaging protein (BiTE). The immune cell engaging portion suitably comprises an antibody protein which is capable of binding and/or specifically binding to any one or more of CD3, CD5, CD16, CD16a, CD16b, NKp30, NKp46, NKG2D and DNAX Accessory Molecule-1 (DNAM-1) on immune cells for example on immune cells, for example T-cells, T-lymphocytes or NK cells, preferably CD3 on immune cells. Anti-CD3 monoclonal antibodies are known in the art e.g. muromonab-CD3, otelixizumab, teplizumab and visilizumab. The antibody protein which is capable of specifically binding to CD3 on immune cells may for example be an scFv e.g. a scFv derived from one of the aforementioned anti-CD3 monoclonal antibodies. The antibody portion of the cell targeting portion comprising an antibody protein of the invention may for example be an scFv.. Preferably the immune cell engaging protein is a bispecific T-cell engaging protein The bispecific T-cell engaging protein may be of a formula: X-Ll '-Y or a formula: Y-Ll '-X, wherein: X comprises an antibody or antibody fragment recognising the immune cell (immune cell engaging portion); LI ' comprises the one or more linkers; and Y comprises a second antibody or antibody fragment recognising a targeted MR1-ligand complex, particularly the MR1 ligand complex according to the invention, particularly as
expressed and/or presented on the surface of a cell, for example a cancer, cancerous or tumour cell. X and/or Y may comprise a human, human engineered, humanized, chimeric antibody or fragment. X and/or Y may comprise a human engineered antibody or antibody fragment. X may comprise a fully human antibody or fully human antibody fragment. X and/or Y may comprise any one of; one or more Fv, one or more Fc, one or more Fab, one or more (Fab')2, one or more single chain Fv (scFv), one or more diabodies, or more triabodies, one or more tetrabodies, one or more bifunctional hybrid antibodies, one or more CDR1, one or more CDR2, one or more CDR3, one or more combinations of CDR's, one or more variable regions. X and/or Y may comprise one or more framework regions. X and/or Y may comprise one or more constant regions. X and/or Y may comprise one or more heavy chains. X and/or Y may comprise one or more light chains. X and/or Y may comprise one or more and variable regions. X and/or Y may comprise one or more alternative scaffold non-antibody molecules. X and/or Y may comprise a combination of any of Fv, Fc, Fab, (Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, alternative scaffold non-antibody molecules. Preferably X and/ or Y may comprise a Fab fragment or single chain Fv (scFv), preferably single chain Fv (scFv). Y may comprise at least a portion of an antibody, preferably scFv or Fab fragment. Y may comprise at least a portion of an antibody or antibody fragment that binds to an antigen on a lymphocyte or to an antigen on a B- cell or B-cell progenitor or to an antigen on a cancerous, cancer or tumour cell, particularly wherein said cell or cells present the MR1-ligand complex according to the present invention. X may comprise at least a portion of an antibody or antibody fragment that binds to an antigen selected from any of CD3, CD5, CD16, CD16a, CD16b, NKp30, NKp46, NKG2D and DNAX Accessory Molecule-1 (DNAM-1) on immune cells for example on T-cells, T-lymphocytes or NK cells, preferably CD3 on immune cells. Chimeric immune cell receptor protein The invention also provides a chimeric immune cell receptor protein which comprises (a) a cell targeting portion comprising an antibody protein according to the invention and (b) a portion comprising immune cell effector function domains (hereinafter a “chimeric T-cell receptor protein of the invention”).
The cell targeting portion is extracellular when the receptor protein is expressed at the surface of an immune cell and the portion comprising immune cell effector function domains is intracellular when the receptor protein is expressed at the surface of an immune cell. Typically, the chimeric immune cell receptor protein (e.g. chimeric antigen receptor (CAR) proteins) will comprise a transmembrane portion e.g. derived from CD8, CD16 or CD28. The transmembrane portion is transmembrane when the receptor protein is expressed at the surface of an immune cell. Chimeric NK receptor proteins may alternatively comprise a transmembrane portion e.g. derived from DAP12, 2B4, NKp44, NKp46 or NKG2D. The immune cell may, for example be a T-cell, an NK-cell or an NKT cell and in particular is a T-cell. Suitably the portion comprising an immune cell effector function domain comprises a CD3zeta intracellular signalling domain. Suitably the portion comprising an immune cell effector function domain also comprises one or more co-stimulatory domains. Co-stimulatory domains may for example be or be derived from the intracellular portions of proteins selected from CD28, OX40, 4-1BB, DAP12, DAP10 and 2B4, especially selected from the intracellular portions of CD28, OX40 and 4-1BB, For chimeric antigen receptor proteins designed for use in T cells, said co- stimulatory domains may for example be or be derived from the intracellular portions of proteins selected from CD28, OX40 or 4-1BB (such as a combination of the intracellular portions of CD28 and 4-1BB or CD28 and OX40). For chimeric antigen receptor proteins designed for use in NK cells, said co-stimulatory domains are for example selected or derived from the intracellular portions of CD28, 4-1BB, DAP12, DAP10 and 2B4. For chimeric antigen receptor proteins designed for use in NKT cells, said co-stimulatory domains may for example be or be derived from the intracellular portions of proteins selected from CD28 and 4-1BB (such as a combination of CD28 and 4-1BB). Chimeric immune cell receptor proteins may also be expressed by immune cells in conjunction with cytokines e.g. IL-2, IL-5 or IL-12 or other costimulatory ligands. Further details of the domain structure and generation of chimeric immune cell receptor proteins may be gleaned from Xie et al (2020).
T-cell receptor proteins The invention also provides an isolated T-cell receptor protein which is capable of specifically binding to the isolated complex of the invention which T-cell receptor protein is not a T-cell receptor protein described in WO2019/081902 (University College Cardiff Consultants Ltd) such as MC.7.G5, specifically an antibody protein having an alpha and a beta chain having the sequences recited in Figure 3 thereof (hereinafter a “T-cell receptor protein of the invention”). The T-cell receptor protein is also not a T-cell receptor protein described in WO2018/162563 (Universität Basel) since the T-cell receptor proteins described in this patent application are believed not to be T-cell receptors of K43A sensitive MR1-T-cells. The differences include that the Basel MR1-T-cells are: (1) not cancer- specific; (2) they activate in response to monocyte-derived DC (see B in Figure 8 of Lepore et al (2017)) and (3) do not require Lysine 43 for target recognition (see Figure 4B). These MR1-T-cells are common and supposedly occur at a frequency between 1:2500 and 1:5000 whereas K43A sensitive MR1-T-cells are rarer. T-cell receptor proteins of the invention may be raised and selected by priming and stimulating primary T-cells, particularly CD8+ T-cells, with antigen presenting cells (APCs) loaded with the MR1 ligand, suitably a 2’-5’-oligoadenylate molecule or a derivatised 2’-5’-oligoadenylate molecule as herein described, or naturally expressing the MR1-ligand complex, and selecting T-cells that respond specifically to the APCs. APCs include dendritic cells, B-cells, and immortalized cell lines. Such T-cell cloning methods have been described. Alternatively, multimerization of the soluble, refolded, MR1-ligand complex could be used to directly label T-cells with such T-cell receptor proteins, for example using tetramers or dextramers. Additionally, soluble T-cell receptor protein libraries could be generated, using methods such as phage display, and the soluble, refolded MR1-ligand monomers could be used to pan these libraries to select and enrich for specific T-cell receptor proteins. T-cell receptor proteins of the invention may also be raised and selected by identification of T cell clones from human donors that display activity against (including killing of) cancer cells that are of different HLA types, and then confirming
that the reactivity of such cells is dependent on MR1 expression, but not the HLA types expressed by the tumour cells (see Crowther et al. (2020)). Polynucleotides The invention provides isolated polynucleotide which encode proteins of the invention described herein (hereinafter “a polynucleotide of the invention”). For example, the invention provides an isolated polynucleotide which encodes a chimeric immune cell receptor of the invention. The invention also provides an isolated polynucleotide which encodes a T-cell receptor protein according to the invention. Vectors The invention provides a vector comprising a polynucleotide of the invention (hereinafter a “vector of the invention”). Suitably the vector is a viral vector, such as a lentiviral vector or a retroviral vector (e.g. γ-retrovirus). Other examples of viral vectors include vectors derived from adenovirus, adeno-associated virus (AAV), alphavirus, herpes virus, arenavirus, measles virus, poxvirus or rhabdovirus. DNA molecules, for example transposons, may also be suitable vectors to transduce T- cells with TCR genes. Vectors should suitably comprise such elements as are necessary for permitting transcription of a translationally active RNA molecule in the host cell, e.g. T-cell, such as a promoter and/or other transcription control elements such as an internal ribosome entry site (IRES) or a termination signal. A “translationally active RNA molecule” is an RNA molecule capable of being translated into a protein by the host cell’s translation apparatus. Engineered immune cells The invention also provides an engineered immune cell which expresses on its surface a chimeric immune cell receptor protein of the invention or T-cell receptor protein of the invention. Suitably the engineered immune cell is an engineered T- cell and the chimeric immune cell receptor protein is a chimeric antigen receptor protein. The invention also provides an engineered immune cell which expresses on its surface a T-cell receptor protein of the invention. Thus, suitably the engineered cell is a CAR-T-cell or TCR T-cell.
The engineered immune cell may, for example be an engineered T-cell, NK-cell or NKT cell and in particular is an engineered T-cell, preferably an engineered T-cell engineered to express on its surface a heterologous TCR or chimeric immune cell receptor protein. For example, the invention provides an engineered immune cell (such as an engineered T-cell) which is transduced with a vector of the invention. This vector may be, for example a lentiviral vector encoding the T-cell receptor protein or derivative thereof or chimeric immune cell receptor protein or derivative thereof, as a transgene. Non-viral delivery vectors may also be used for example, transposons. Enriched T-cells may be activated with specific reagents such as antibodies to CD3 and CD28 or other known activation markers and transduced with the vector encoding the T-cell receptor protein or derivative thereof or chimeric immune cell receptor protein or derivative thereof, using established methods. Treatment or prevention of cancer The invention provides a method of treatment or prevention of cancer which comprises administering to a patient in need thereof an immune cell engaging protein of the invention. Suitably the immune cell engaging protein is a T-cell engaging protein. The invention also provides a method of treatment or prevention of cancer which comprises administering to a patient in need thereof an engineered immune cell of the invention. Suitably the engineered immune cell is an engineered autologous immune cell. Suitably the immune cell is a T-cell such as an engineered autologous T-cell. The invention also provides an immune cell engaging protein of the invention for use in the treatment or prevention of cancer. It also provides use of an immune cell engaging protein of the invention in the manufacture of a medicament for the treatment or prevention of cancer. The invention also provides an engineered immune cell of the invention for use in the treatment or prevention of cancer. It also provides use of an engineered immune cell of the invention in the manufacture of a medicament for the treatment or prevention of cancer. Thus, in a further aspect of the present invention, there is provided an ex vivo process comprising (i) obtaining immune cells from a patient, (ii) optionally
expanding the immune cells (iii) introducing a heterologous polynucleotide of the invention or a vector of the invention into the immune cells to produce modified immune cells which express an T-cell receptor protein or a chimeric immune cell receptor protein of the invention; and (iii) reintroducing said modified immune cells into the patient. In yet a further aspect of the present invention, there is provided a method of treatment or prevention of cancer comprising administering to a patient in need thereof transduced immune cells wherein the immune cells are immune cells that have been obtained from said patient and a heterologous polynucleotide of the invention or a vector of the invention has been introduced into the immune cells, such that the immune cells express a T-cell receptor (TCR) protein or a chimeric immune cell receptor protein of the invention. Suitably the immune cells are T-cells. Suitably the chimeric immune cell receptor protein is a chimeric T-cell receptor protein. The invention also provides said transduced immune cells for use in the treatment or prevention of cancer. It also provides use of said transduced immune cells in the manufacture of a medicament for the treatment or prevention of cancer. According to this aspect of the invention, immune cells such as T-cells are first obtained from the patient or can be generated allogeneically. One suitable method is to collect PBMCs by apheresis. Alternatively, PBMCs may be isolated by density centrifugation. T-cells may thereafter be enriched from the PBMC fraction using positive or negative selection. An example of positive selection would be anti-CD3 or anti-CD4 or anti-CD8 microbeads. Negative selection could be achieved using antibody coated microbeads that specifically bind non-T-cell immune cells present in the PBMC fraction. Other methods of enrichment besides microbeads could be used, for example fluorescent activated sorting. T-cells may be expanded by ex vivo by culturing with media containing standard stimulatory cytokines such as IL-2, IL-7, IL-15, IL-21 and mixtures thereof. The invention also provides a method of treatment or prevention of cancer which comprises administering to a patient in need thereof a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule. The invention also provides a method of treatment or prevention of cancer which comprises administering to a patient in need thereof an agent which mimics the 2’,5’-oligoadenylate molecule or
the derivatised 2’,5’-oligoadenylate molecule or stimulates the production of a 2’,5’- oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo. The invention also provides a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’- oligoadenylate molecule for use in the treatment or prevention of cancer. It also provides use of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in the manufacture of a medicament for the treatment or prevention of cancer. The invention also provides an agent which mimics the 2’,5’-oligoadenylate molecule or the derivatised 2’,5’-oligoadenylate molecule or stimulates the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo for use in the treatment or prevention of cancer. It also provides use of an agent which mimics the 2’,5’-oligoadenylate molecule or the derivatised 2’,5’-oligoadenylate molecule or stimulates the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo in the manufacture of a medicament for the treatment or prevention of cancer. An agent which mimics the 2’,5’-oligoadenylate molecule or the derivatised 2’,5’-oligoadenylate molecule may include for example any of phosphorothioate/phosphodiester 2′,5′-oligoadenylate derivatives, 2',5'-phosphorothioate trimer and tetramer 2′,5′-oligoadenylate analogues or 5’-O-monophosphates thereof, xyloadenosine-substituted 2′, 5′- oligoadenylates or derivatives disclosed in WO1993017692A. Agents which stimulate the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’- oligoadenylate molecule may include synthetic double-stranded or single stranded RNA, e.g. poly(I), poly(A) or poly(U) or·poly(C), such as single-stranded or double- stranded RNA aptamers. According to the present invention the 2’,5’-oligoadenylate molecule or the derivatised 2’,5’-oligoadenylate molecule to be administered or produced in vivo can be, for example, any one of the following a) 2’,5’-oligoadenylate dimer monophosphate, b) 2’,5’-oligoadenylate dimer triphosphate, c) 2’,5’-oligoadenylate trimer monophosphate, d) 2’,5’-oligoadenylate trimer triphosphate, e) 2’,5’- oligoadenylate tetramer monophosphate, f) 2’,5’-oligoadenylate tetramer triphosphate; or any one of the foregoing 2’,5’-oligoadenylate molecules or derivatised 2’,5’-oligoadenylate molecules that has been conjugated to a small carbonyl-containing molecule, preferably wherein the small carbonyl containing molecule is either glyoxal or methylglyoxal.
The agent which stimulates the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule according to the invention in vivo can be, for example, an agonist of an oligoadenylate synthase enzyme (OAS) such as OAS1/OASL, OAS2 or OAS3. Example agonists of OAS include a small RNA molecule, for example any of VAI RNA from adenovirus, REX / TAR RNA of HIV1 virus, poly(I)n, poly(A)n or poly(U)n. The patient is suitably a human patient. In the context of a method of treatment of cancer, a patient is a subject suffering from cancer. In the context of a method of prevention of cancer, a patient is a subject in whom cancer is to be prevented. Treatment or prevention of other diseases The invention also provides a method of treatment or prevention of an autoimmune disease which comprises administering to a patient in need thereof an agent which inhibits the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’- oligoadenylate molecule in vivo. The invention also provides an agent which inhibits the production of a 2’,5’- oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo for use in the treatment or prevention of an autoimmune disease. It also provides use of an agent which inhibits the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo in the manufacture of a medicament for the treatment or prevention of an autoimmune disease. The agent which inhibits the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo can be, for example, an inhibitor of oligoadenylate synthase (OAS) activity, for example such as OAS1/OASL, OAS2 or OAS3 activity. Inhibitors include molecules that exert a competitive inhibition in the ATP binding sites of 2′-5′ oligoadenylate synthetase proteins. Example inhibitors of OAS activity include 3-[([1,2,4]triazolo[4,3-a]guinoxalin-4-yl)amino]benzamide compounds such as for example any of ZINC06786354, ZINC 06900324, ZINC 09561289 or (4-phenylpiperazin-l-yl)-[3-(tetrazolo[1,5-a]guinoxalin-4- ylamino)phenyl]methanone compounds such as for example any of ZINC09561285, ZINC09561278 or ZINC09561296 as disclosed in Karen J. Gonzalez, Computational Biology and Chemistry, 2020, Vol 85, issue C, In silico identification of potential inhibitors against human 2’-5’- oligoadenylate synthetase (OAS) proteins. Divalent
metal ions such as copper, iron and zinc ions also have been shown to strongly inhibit the enzymatic activity of 2′-5′ oligoadenylate synthetase and are suitable inhibitors. The patient is suitably a human patient. In the context of a method of treatment of an autoimmune disease, a patient is a subject suffering from an autoimmune disease. In the context of a method of prevention of an autoimmune disease, a patient is a subject in whom autoimmune disease is to be prevented. Methods of raising T-cells The invention provides an in vitro method of obtaining T-cells capable of specifically binding to cells which present an MR1-ligand complex on their surface, preferably the complex or MR1-ligand complex is an MR1-ligand complex according to the invention, which comprises selecting T-cells obtained from a donor, particularly a human donor, e.g. by apheresis, which recognize an MR1-ligand complex e.g. by testing if said T-cells recognise (i) an MR1-ligand complex presented on a cell (such as a dendritic cell or other suitable antigen presenting cell) exogenously loaded with the ligand or the MR1-ligand complex or (ii) said complex in monomer or multimeric form, preferably the complex or MR1-ligand complex is an MR1-ligand complex according to the invention,. Suitably the complex includes β2-microglobulin. Such recognition can be determined by conventional methods such as measuring T cell activation (for example via interferon gamma release assays) or T cell mediated killing (for example via measuring target cell death using flow cytometry assays with known markers of cell death such as 7-aminoactinomycin D) in response to the MR1-ligand complex. Said multimeric forms include tetramers, dextramers and streptavidin based multimers. T-cells could be directly isolated using labelled multimers, for example fluorescently labelled multimers that would stain the MR1- ligand-specific TCR on the surface of the T-cell clone, or T-cells capable of specifically binding to cells which present an MR1-ligand complex on their surface, preferably the complex or MR1-ligand complex is an MR1-ligand complex according to the invention, and thus allow subsequent cell sorting based on fluorescence to allow for expansion of the T-cell. T-cells so selected can be amplified by well-known methods. The invention also provides a T-cell obtained by the aforesaid method.
The present invention further provides a method for identification of an immune cell, for example as defined herein, for example a T-cell, B-cell, NK cell, NKT cell, capable of recognizing or binding to or reactive to an MR1-ligand complex according to the invention in an immune cell sample or preparation, comprising providing a sample or preparation which comprises immune cells, or an immune cell containing sample or preparation, contacting immune cells of said immune cell containing sample or preparation with said MR1-ligand complex and isolating an immune cell that is capable of recognizing or binding to or reactive to said MR1-ligand complex, optionally wherein the immune cell is reactive as judged by proliferation response, or production or expression of an immune cell receptor, for example antibody protein or TCR or NK cell receptor such as natural cytotoxity receptor,or chimeric immune cell receptor protein, or cytokine production for example by production of interferon- gamma or interleukin (IL)-10 or granzyme B or TNFα, for example as assayed by tetramer staining, intracellular staining or ELISPOT (enzyme-linked immunospot assay), optionally wherein the MR1-ligand complex is the MR1-ligand complex according to the invention, preferably the identified immune cell may be further isolated. Also provided is an immune cell identified and/or isolated according to the method of the invention, preferably the immune cell is a T-cell or a B-cell, alternatively an NK cell or NKT cell. Accordingly, the method of the invention may further provide a method for obtaining or isolating an immune cell receptor, for example an antibody protein or TCR or NK cell receptor such as natural cytotoxity receptor, or chimeric immune cell receptor protein, capable of recognizing or binding or specifically binding to an MR1-ligand complex of the invention and/or cells which present an MR1-ligand complex according to the invention, which comprises identifying or isolating an immune cell according to the forgoing method and obtaining or isolating an immune cell receptor capable of binding or specifically binding to a MR1-ligand complex therefrom and/or nucleotide sequence encoding said immune cell receptor. According to this method of the invention there is further provided an isolated immune cell receptor or nucleotide sequence encoding said immune cell receptor, optionally wherein the immune cell receptor is a TCR (T cell receptor) or antibody protein, preferably a TCR. Accordingly the method of the invention further provides a recombinant immune cell comprising and/or expressing the nucleotide sequence encoding said immune cell receptor and/or producing or expressing said immune cell receptor.
Methods of raising antibodies The invention provides an in vitro method of raising antibodies capable of specifically binding to cells which present an MR1-ligand complex on their surface which comprises immunising an experimental animal (such as rabbit, mouse, rat, guinea pig, hamster, llama etc.) with an MR1-ligand complex as immunogen, optionally together with an immunostimulant (e.g. specol), and obtaining antibodies or antibody producing cells from said animal (e.g. from PBMCs or from spleen). See further details in the “Antibody proteins” section above. The invention also provides an antibody obtained by the aforesaid method. Agents and uses The invention also provides a method of treatment or prevention of an autoimmune disease which comprises administering to a patient in need thereof an agent which is not a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’- oligoadenylate molecule and which binds MR1 and prevents the formation of a MR1-ligand complex on the surface of cells and hence prevents the binding of T- cells to said cells. In one embodiment, the agent competes with a natural 2’,5’- oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule for binding to MR1 in the same binding site. The invention also provides said agent which is not a natural a 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule and which binds MR1 and prevents the formation of a MR1-ligand complex on the surface of cells and hence prevents the binding of T-cells to said cells for use in the treatment or prevention of an autoimmune disease. It also provides use of said agent which is not a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’- oligoadenylate molecule and which binds MR1 and prevents the formation of a MR1-ligand complex on the surface of cells and hence prevents the binding of T- cells to said cells in the manufacture of a medicament for the treatment or prevention of an autoimmune disease. The invention also provides a method of treatment or prevention of cancer which comprises administering to a patient in need thereof an agent which is not a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule and which binds MR1 to form a MR1-agent complex on the surface of cells and which
complex is capable of specifically binding a T-cell. In one embodiment, the agent competes with a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’- oligoadenylate molecule for binding to MR1 in the same binding site. The invention also provides said agent which is not a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule and which binds MR1 to form a MR1-agent complex on the surface of cells and which complex is capable of specifically binding a T-cell for use in the treatment or prevention of cancer. It also provides use of said agent which is not a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule and which binds MR1 to form a MR1-agent complex on the surface of cells and which complex is capable of specifically binding a T-cell in the manufacture of a medicament for the treatment or prevention of cancer. An agent which is not a natural 2’,5’- oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule the 2’,5’-oligoadenylate molecule or the derivatised 2’,5’-oligoadenylate molecule may include for example any of phosphorothioate/phosphodiester 2′,5′-oligoadenylate derivatives, 2',5'-phosphorothioate trimer and tetramer 2′,5′-oligoadenylate analogues or 5’-O-monophosphates thereof, xyloadenosine-substituted 2′, 5′- oligoadenylates or derivatives disclosed in WO1993017692A. Cancer Exemplary cancers which may be treated according to the invention include solid tumours and blood cancers, for example cancers selected from lung, melanoma (e.g. skin melanoma), bone, breast, blood (e.g. leukemia), prostate, kidney, bladder, cervical, ovarian and colorectal cancers, and in particular selected from lung, melanoma (e.g. skin melanoma), bone and breast cancers. Cancer may be primary cancer or metastatic cancer. Autoimmune diseases Exemplary autoimmune diseases which may be treated according to the invention include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, multiple sclerosis, psoriasis, Crohn’s disease, ulcerative colitis, uveitis, cryopyrin-associated periodic syndromes, Muckle-Wells syndrome, juvenile idiopathic arthritis, chronic obstructive pulmonary disease and Aicardi-Goutieres syndrome.
Combination therapy In the treatment or prevention of diseases, different products described herein may be used in combination and one or more products described herein may be used in combination with other treatments (or preventions) for the given disease. For example, in the treatment or prevention of cancer one or more of the following substances can be combined: (a) an agent which stimulates the production of a 2’,5’- oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo; (b) a T-cell engaging protein of the invention and (c) and engineered T-cell of the invention, particularly (a) plus (b) or (a) plus (c). Any of the products for treatment or prevention of cancer described herein may be used in combination with another anti-cancer drug e.g. selected from alkylating agents (e.g. nitrogen mustard analogues, nitrosoureas, alkyl sulfonates, platinum containing compounds, ethylemines, and imidazotetrazines), cytotoxic antibiotics (e.g. anthracyclines, actinomycins), plant alkaloids and other natural products (e.g. campthotecin derivatives, epipodophyllotoxins, taxanes, and vinca alkaloids), antimetabolites (e.g. cytidine analogues, folic acid analogues, purine analogues, pyrimidine analogues, urea derivatives) and drugs for targeted therapy (e.g. kinase inhibitors, monoclonal antibodies and other immunotherapies) or radiotherapy. Immunotherapies include check point inhibitors such as anti-PD1, anti-PD-L1 and anti-CTLA-4 antibodies such as ipilumamab, nivolumab, pembrolizumab and atezolizumab. Diagnosis The invention also provides a method of determining whether a cell is cancerous which comprises determining whether said cell expresses on its surface an MR1- ligand complex comprising (a) MR1 and (b) a ligand molecule selected from a 2’,5’- oligoadenylate molecule and a derivatised 2’,5’-oligoadenylate molecule as ligand for MR1. The step of determining whether said cell expresses on its surface an MR1-ligand complex suitably includes the step of detecting the binding of an antibody protein to said MR1-ligand complex. The antibody protein may, in particular, be an antibody protein of the invention linked to a detectable label such as a fluorescent label.
Production of antibody proteins, immune cell engaging proteins, chimeric immune cell receptor proteins, T-cell receptor proteins and other polypeptides of the invention Antibody proteins, immune cell engaging proteins, chimeric immune cell receptor proteins, T-cell receptor proteins and other polypeptides of the invention described herein can be obtained and manipulated using the techniques disclosed for example in Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual 4th Edition Cold Spring Harbour Laboratory Press. In particular, artificial gene synthesis may be used to produce polynucleotides (Nambiar et al. (1984), Sakamar and Khorana, (1988), Wells et al. (1985) and Grundstrom et al. (1985)) followed by expression in a suitable organism to produce polypeptides. A gene encoding a polypeptide of the invention can be synthetically produced by, for example, solid-phase DNA synthesis. Entire genes may be synthesized de novo, without the need for precursor template DNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. Products can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity (Verma and Eckstein (1998)). These relatively short segments are readily assembled by using a variety of gene amplification methods (Methods Mol Biol., 2012; 834:93-109) into longer DNA molecules, suitable for use in innumerable recombinant DNA-based expression systems. In the context of this invention one skilled in the art would understand that the polynucleotide sequences encoding the TCRs and fragments thereof described in this invention could be readily used in a variety of protein production systems, including, for example, viral vectors. For the purposes of production of polypeptides of the invention in a microbiological host (e.g., bacterial such as E coli or fungal such as yeast), polynucleotides of the invention will comprise suitable regulatory and control sequences (including promoters, termination signals etc) and sequences to promote polypeptide secretion suitable for protein production in the host. Similarly, polypeptides of the invention could be produced by transducing cultures of eukaryotic cells (e.g., Chinese hamster ovary cells or drosophila S2 cells) with polynucleotides of the invention which have been combined with suitable regulatory and control sequences (including promoters,
termination signals etc) and sequences to promote polypeptide secretion suitable for protein production in these cells. Improved isolation of the polypeptides of the invention produced by recombinant means may optionally be facilitated through the addition of a purification tag at one end of the polypeptide. An example purification tag is a stretch of histidine residues (e.g.6-10 His residues), commonly known as a His-tag. Further aspects of the invention are defined by the following clauses: 1. An isolated MR1-ligand complex comprising (a) an MR1 protein and (b) a ligand molecule selected from a 2’,5’-oligoadenylate molecule and a derivatised 2’,5’-oligoadenylate molecule as ligand for the MR1 protein which is capable of being bound and/or specifically bound by a T-cell receptor or antibody protein, optionally wherein the T-cell receptor is an MR1 specific T- cell receptor. 2. The isolated complex according to clause 1 comprising the heavy chain of MR1, optionally wherein the MR1 has an amino acid sequence which has sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to SEQ ID NO:1. 3. The isolated complex according to clause 2 wherein the heavy chain is non- covalently associated with a β2 microglobulin protein as light chain or is covalently linked to a β2 microglobulin protein in an artificial single-chain construct, optionally wherein the β2 microglobulin protein has an amino acid sequence which has sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% SEQ ID NO:2. 4. The isolated complex according to any one of clauses 1 to 3 wherein the ligand molecule is selected from any one of: a. 2’,5’-oligoadenylate dimer monophosphate, b. 2’,5’-oligoadenylate dimer triphosphate, c. 2’,5’-oligoadenylate trimer monophosphate, d. 2’,5’-oligoadenylate trimer triphosphate, e. 2’,5’-oligoadenylate tetramer monophosphate, and f. 2’,5’-oligoadenylate tetramer triphosphate.
5. The isolated complex according to any one of clauses 1 to 3 wherein the ligand molecule is a derivatised 2’,5’-oligoadenylate molecule. 6. The isolated complex according to clause 4 wherein the derivatised 2’,5’- oligoadenylate molecule is a 2’,5’-oligoadenylate molecule derivatised with a small carbonyl-containing molecule. 7. The isolated complex according to clause 6 wherein the carbonyl-containing molecule is glyoxal or methylglyoxal. 8. An antibody protein which binds or specifically binds to the isolated complex according to any one of clauses 1 to 7. 9. The antibody protein according to clause 8 which is a 4-chain monoclonal antibody or fragment thereof, optionally any one of Fab (fragment antigen binding), scFv (single chain fragment variable), scFv-Fc, single domain antibody, diabody, dsFv, Fab', (Fab’)2, minibody, diabody, single-chain antibody molecule. 10. The antibody protein according to clause 8 or 9 which is an scFv. 11. An immune cell engaging protein which is capable of targeting a cell presenting on its surface an MR1-ligand complex comprising (a) a cell targeting portion comprising an antibody protein according to any one of clauses 8 to 10 and (b) an immune cell engaging portion. 12. The immune-cell engaging protein according to clause 11 wherein the immune cell is selected from any one of a T-cell, natural killer (NK) cell, Natural Killer T (NKT) cell, Tumour Infiltrating Lymphocyte (TIL), optionally wherein the immune cell may be a CD4+ T cell or CD8+ T cell, or may be a population of T-cells, optionally CD4+ T cells; or CD8+ T cells, or a mixed population of CD4+ T cells and CD8+ T cells, preferably the immune cell is a T-cell. 13. The immune cell engaging protein according to clause 11 or 12 wherein the immune cell engaging portion that is a T-cell engaging portion comprises an antibody protein which is capable of specifically binding to CD3 on T-cells. 14. The immune cell engaging protein according to clause 13 wherein the antibody protein which is capable of binding or specifically binding to CD3 on T-cells is an scFv. 15. A chimeric immune cell receptor protein which comprises (a) a cell targeting portion comprising an antibody protein according to any one of clauses 8 to 10 and (b) a portion comprising immune cell effector function domains.
16. A chimeric immune cell receptor protein according to clause 15 wherein the immune cell is a T-cell. 17. The chimeric immune cell receptor protein according to clause 16 wherein the portion comprising T-cell effector function domains comprises a CD3zeta intracellular signalling domain. 18. The chimeric immune cell receptor protein according to clause 16 or clause 17 wherein the immune cell is a T-cell and the portion comprising T-cell effector function domains comprises one or more co-stimulatory domains. 19. An isolated T-cell receptor protein which is capable of specifically binding to the isolated complex according to any one of clauses 1 to 7, optionally which T-cell receptor protein is not a T-cell receptor protein described in WO2019/081902. 20. An engineered immune cell which expresses on its surface an antibody protein of any of clauses 8 to 10, a chimeric immune cell receptor protein according to any one of clauses 15 to 18 or a T-cell receptor protein according to clause 19. 21. An engineered immune cell according to clause 20 wherein the immune cell is selected from any one of a T-cell, natural killer (NK) cell, Natural Killer T (NKT) cell, Tumour Infiltrating Lymphocyte (TIL), or the immune cell may be optionally a CD4+ T cell or CD8+ T cell, or may be a population of T-cells, optionally CD4+ T cells; or CD8+ T cells, or a mixed population of CD4+ T cells and CD8+ T cells, preferably the immune cell is a T-cell. 22. An isolated polynucleotide which encodes an antibody protein of any of clauses 8 to 10, a chimeric immune cell receptor protein according to any one of clauses 15 to 18 or a T-cell receptor protein according to clause 19. 23. A vector comprising a polynucleotide according to clause 22. 24. The vector according to clause 23 which is a viral vector, for example a lentiviral vector or a retroviral vector (e.g.γ-retrovirus) or a vector derived from adenovirus, adeno-associated virus (AAV), alphavirus, herpes virus, arenavirus, measles virus, poxvirus or rhabdovirus, preferably the vector is a lentiviral vector. 25. An engineered immune cell which comprises or is transduced with a vector according to clause 23 or clause 24 or which comprises or is transduced with a polynucleotide according to clause 22.
26. A method of treatment or prevention of cancer which comprises administering to a patient in need thereof an antibody protein of any of clauses 8 to 10 or an immune cell engaging protein according to any one of clauses 11 to 14. 27. A method of treatment or prevention of cancer which comprises administering to a patient in need thereof an engineered immune cell according to any one of clauses 20, 21 or 25. 28. A method according to clause 27 wherein the engineered immune cell is an engineered autologous immune cell, optionally wherein the immune cell is selected from any one of a T-cell, natural killer (NK) cell, Natural Killer T (NKT) cell, Tumour Infiltrating Lymphocyte (TIL), optionally wherein the immune cell may be a CD4+ T cell or CD8+ T cell, or may be a population of T-cells, optionally CD4+ T cells; or CD8+ T cells, or a mixed population of CD4+ T cells and CD8+ T cells, preferably the immune cell is a T-cell. 29. A method of obtaining T-cells capable of binding or specifically binding to cells which present an MR1-ligand complex on their surface which comprises selecting T-cells obtained from a donor which recognize an MR1-ligand complex e.g. by testing if said T-cells recognise (i) an MR1-ligand complex presented on a cell exogenously loaded with the MR1-ligand complex or (ii) said complex in monomer or multimeric form, optionally wherein the complex is a complex according to any one of clauses 1 to 7. 30. A method of obtaining T-cells capable of binding or specifically binding to cells which present an MR1-ligand complex which comprises priming and stimulating T-cells, particularly primary T-cells, particularly CD8+ T-cells, with antigen presenting cells (APCs) loaded with ligand and selecting T-cells that specifically bind to the APCs, optionally wherein the MR1-ligand complex is the MR1-ligand complex according to any one of clauses 1-7. 31. A method for identification of a T-cell reactive to an MR1-ligand complex in a T-cell containing preparation which comprises providing a T-cell containing preparation, contacting T-cells of said T-cell containing preparation with said MR1-ligand complex and isolating a T-cell that is reactive to said MR1-ligand complex, optionally wherein the T-cell is reactive as judged by proliferation response or cytokine production for example by production of interferon- gamma or interleukin (IL)-10 or granzyme B or TNFα, for example as assayed by tetramer staining, intracellular staining or ELISPOT (enzyme-linked
immunospot assay), optionally wherein the MR1-ligand complex is the MR1- ligand complex according to any one of clauses 1-7. 32. A T-cell obtained by the method any one of clauses 29 to 31. 33. A method of obtaining a T-cell receptor protein capable of binding or specifically binding to cells which present an MR1-ligand complex which comprises obtaining T-cells according to the method of clause 29 or clause 30 or isolating T-cells according to clause 31 and obtaining the T-cell receptor protein from said T-cells, optionally wherein the MR1-ligand complex is the MR1-ligand complex according to any one of clauses 1-7. 34. An isolated T-cell receptor protein obtained by the method of clause 33, optionally wherein the T-cell receptor protein is a TCR (T cell receptor). 35. A method of raising antibodies capable of binding or specifically binding to cells which present an MR1-ligand complex on their surface which comprises immunising an experimental animal with an MR1-ligand complex according to any one of clauses 1 to 7 as immunogen, optionally together with an immunostimulant, and obtaining antibodies or antibody producing cells from said animal. 36. An antibody obtained by the method of clause 35. 37. A method of treatment or prevention of an autoimmune disease which comprises administering to a patient in need thereof an agent which is not a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’- oligoadenylate molecule and which binds MR1 and prevents the formation of a MR1-ligand complex on the surface of cells and hence prevents the binding of T-cells to said cells. 38. The method according to clause 37 wherein the agent competes with a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule for binding to MR1 in the same binding site. 39. A method of treatment or prevention of cancer which comprises administering to a patient in need thereof an agent which is not a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule and which binds MR1 to form a MR1-agent complex on the surface of cells and which complex is capable of binding or specifically binding a T-cell, a T-cell receptor (TCR) or antibody protein.
40. The method according to clause 39 wherein the agent competes with a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule for binding to MR1 in the same binding site. 41. A method of treatment or prevention of cancer which comprises administering to a patient in need thereof a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule, for example a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule. 42. A method of treatment or prevention of cancer which comprises administering to a patient in need thereof an agent which stimulates the production of a 2’,5’- oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo. 43. A method of treatment or prevention of an autoimmune disease which comprises administering to a patient in need thereof an agent which inhibits the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’- oligoadenylate molecule in vivo. 44. The method according to any one of clauses 37 to 43, or any one of clauses 38 and 40-43, wherein the 2’,5’-oligoadenylate molecule is a 2’,5’- oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule according to any one of clauses 4 to 7. 45. A method of determining whether a cell is cancerous which comprises determining whether said cell expresses on its surface an MR1-ligand complex comprising (a) MR1 and (b) a ligand molecule selected from a 2’,5’- oligoadenylate molecule and a derivatised 2’,5’-oligoadenylate molecule as ligand for MR1 optionally wherein the ligand is a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule according to any one of clauses 4 to 7. 46. A method according to clause 45 wherein the step of determining whether said cell expresses on its surface an MR1-ligand complex includes the step of detecting the binding of an antibody protein to said MR1-ligand complex. 47. A method of clause 46 wherein the antibody protein is an antibody protein according to any one of clauses 8 to 10 linked to a detectable label.
Examples Example 1 - Docking studies demonstrating oligoadenylate molecules as ligands for MR1 To determine whether oligoadenylate molecules are cancer-specific MR1 ligands, a docking experiment was performed using the macromolecular structure of MR1 and utilising CCDC GOLD docking software with Hermes 2020.2 visualizer (Jones et al (1997)). The MR1 structure for docking was extracted from RCSB PDB Protein Data Bank (PDB ID 4pj9 – the structure of human MR1-5-OP-RU in complex with human MAIT TRAJ20 TCR, Sidonia et al (2014)). This structure is shown for example in ribbon form in Figure 8. For the oligoadenylate molecular docking analysis, the following steps were taken. Firstly, the structures of the MAIT TCR and known MR1 ligand 5-OP-RU were removed from the overall macromolecular structure file, leaving only the MR1 protein structure. All missing hydrogen atoms were then added to the MR1 protein structure to ensure an explicit representation of the protein. The inventors observed in this MR1 structure, as well as other high-resolution structures of MR1, that four water molecules are consistently bound to the same regions of the MR1 protein. In addition, in the PDB ID 4pj9 MR1 structure, two more water molecules were found presented within 3Å of the site where the 5-OP-RU ligand is known to be bound. All these water molecules were assumed to contribute to the stabilization of the MR1 binding pocket as well as to the increase in the stability of the protein-ligand complex. To validate this assumption, the effect of these water molecules was confirmed during the GOLD docking parameter setting test runs, where the known MR1 ligand 5-OP-RU was docked into the MR1 protein structure. The location and orientation of the docked 5-OP-RU ligand was compared to the experimentally derived location and orientation from the original crystal structure.4 water molecules (identifiers HOH441, HOH443, HOH445 and HOH458) were identified as crucial for the success of the docking of the known 5-OP-RU ligand and therefore were included in the docking run for the oligoadenylate molecule structures (using the toggle function in GOLD). The likely binding pocket for the oligoadenylate molecules to MR1 was estimated using the location and orientation of 5-OP-RU ligand in the
original crystal structure file, with the pocket encompassing residues of the MR1 protein within 6Å of 5-OP-RU. The Configuration Template in CCDC GOLD was left in default settings. CHEMPLP Docking Scoring Function under default parameters was used to assess the goodness of the ligand fit in the MR1 pocket. Suitable three-dimensional (3D) structures of 2’,5’-oligoadenylate molecules for docking experiments were not available in the Protein Data Bank and thus the inventors generated appropriate 3D structures to enable these studies. Firstly, the PubChem database was searched for two-dimensional (2D) structures of 2’,5’- oligoadenylate molecules. This search identified a 2D structure for 2’,5’- oligoadenylate trimer monophosphate (PubChem CID 107918) but no structures were identified for other 2’,5’-oligoadenylates of interest. The inventors utilized PubChem Sketcher V.2.4 ((https://pubchem.ncbi.nlm.nih.gov//edit3/index.html).) to generate additional 2’,5’-oligoadenylate molecule structures, as well as all their triphosphate forms. These structures were guided by the generic 2’,5’-oligoadenylate molecule structural formula available in KEGG database (https://www.genome.jp/dbget-bin/www_bget?C20935; the structural formula is shown in Figure 1). The 2D structures of various 2’,5’-oligoadenylate molecules are shown in Figures 2-7. Secondly, 3D structures for the 2’,5’-oligoadenylate molecule structures were generated in Frog2 (Miteva MA, Guyon F, Tufféry P. Frog2: Efficient 3D conformation ensemble generator for small compounds. Nucleic Acids Res.2010 Jul;38), server using the generated PubChem Sketcher 2D structure representations (also known as ‘SMILES’ which are PubChem Sketcher structures). The following setting was used to generate all ligand conformations on sdf format. The algorithm was set to perform energy minimization step in AMMOS and produce 50 conformers per ligand. The absolute Van der Waals energy score to accept a conformer as correct was set to Emax = 100. The maximum number of monte-Carlo steps to sample a compound conformation was set to 100. The energy window to accept conformations with the lowest energy conformation generated as a reference was set to 50. This process yielded a set of 3D structures of various 2’,5’-oligoadenylate molecules suitable for docking into the MR1 protein structure. The quality of the docking of the various 2’5’-oligoadenylate molecule structures to the MR1 protein structure was assessed via the CHEMPLP Fitness score defined by the CCDC GOLD software, as well as visual inspection of the fit of the molecules into
the known MR1 pocket. The CHEMPLP Fitness score for the known MR1 ligand 5- OP-RU was re-generated and used as a reference (Figure 14, the 3D reference conformer for 5-OP-RU was downloaded from National Center for Biotechnology Information (NCBI) PubChem database). The following scores were observed for the different docking tests: Table 1: CHEMPLP Fitness Scores from docking studies.
All investigated ligands received higher docked fitness scores compared to the MR1 confirmed ligand 5-OP-RU. This suggest that 2’,5’-oligoadenylate molecules are potential MR1 ligands. As evidenced in Figures 8-13, the di-, tri- and tetramer 2’,5’- oligoadenylate structures are highly flexible chains able to fill a similar space in the MR1 binding pocket as the experimentally observed 5-OP-RU (Figure 14). As noted above, there is a class of T-cells, referred to as K43A sensitive MR1-T- cells, which have activity against a range of cancer cell lines. The susceptibility of certain cancer cell lines to these T cells is significantly, but not entirely, diminished if the lysine-43 residue of the MR1 protein is mutated to alanine. It is also known that a covalent Schiff base bond is formed between lysine-43 and a carbonyl-group present on the known MR1- ligand 5-OP-RU, with the carbonyl group of 5-OP-RU arising due to a reaction between methylglyoxal and 5-amino-6-D-ribitylaminouracil (5-A-RU) (Corbett et al, Nature.2014 May 15;509(7500):361-5). The inventors explored the structures of MR1 docked with the 2’,5’-oligoadenylate molecule ligands with a view to rationalising a link between 2’,5’-oligoadenylate molecule structures as cancer-specific ligands of MR1 and the observed importance of lysine-43 of MR1 in
modulating K43A sensitive MR1-T-cell activity. Whilst the docking experiments do not identify significant interactions between lysine-43 and the 2’,5’-oligoadenylate molecule structures, the inventors found that lysine-43 is well orientated in the binding pocket with space for a carbonyl group-containing small molecule adduct such as methylglyoxal to be conjugated to the various 2’,5’-oligoadenylate molecule structures (Figures 8-13) typically via reaction of the amine sidechain of at least one adenine of the 2’,5’-oligoadenylate molecule with the carbonyl of the carbonyl- containing molecule. These observations provide a plausible explanation for the observed importance of lysine-43 for the anti-cancer activity of K43A sensitive MR1- T-cells. Overall, these macromolecular docking studies provide compelling evidence that 2,5- oligoadenylate molecules and their derivatives could be ligands for MR1. Example 2 - Demonstrating that 2’,5’-oligoadenylate molecules are ligands of MR1 Known MR1 ligands (e.g. acetyl-6-FP) have been shown to stabilise intracellular pools of ligand-free (‘empty’) MR1 when co-incubated with MR1-expressing cells at varying concentrations over time. This stabilisation then leads to trafficking of the MR1-ligand complex to the cell surface which can be detected via antibody detection methods such as flow cytometry, using antibodies to MR1. Ac-6-FP (and other synthetic molecules) were important in discovering bacterially produced ligands that mediate the anti-bacterial activity of MR1-restricted mucosal invariant T cells (MAITs). Importantly, although Ac-6-FP can upregulate cell-surface expression of MR1, the Ac-6-FP-liganded MR1 is not a target for MAITs (Kjer-Nielsen et al., Nature, 2012). Rather, biologically relevant ligands for MAIT cells appear to be the product of condensation reactions of riboflavin precursors with small molecules (Corbett et al. (2014); Awad et al. (2020)). To demonstrate a role of 2’,5’-oligoadenylate molecules in liganding MR1, MR1- expressing cells can be pulsed with titrating amounts of 2’,5’-oligoadenylate molecules as disclosed hereinover a time course (1 – 48 hr) and surface expression of MR1 can be assessed by flow cytometry. Cancer cell lines known to express MR1 and beta-2-microglobulin with detectable MR1 at the surface would be tested first. Known MR1 ligands (e.g. Ac-6-FP) would be run in parallel as a control. Cells that
are thought to have little-to-no surface expression such as normal primary cells would then also be tested. An increase in MR1 at the surface observed at any point or concentration of 2’,5’-oligoadenylate molecule would indicate the 2’,5’- oligoadenylate molecule was a potential ligand for MR1. To further validate the association of the mammalian 2’,5’-oligoadenylate molecule pathway with MR1-ligand cell surface presentation, key components of the 2’,5’- oligoadenylate pathway can be modulated. OAS is responsible for generating 2’,5’- oligoadenylate molecules from AMP and ATP in the presence of cytosolic ds nucleic acid, therefore agonism of OAS could lead to an increase in MR1- 2’,5’- oligoadenylate molecule. Cancer cell lines would be treated with an OAS agonist such as disclosed herein and MR1 surface levels assessed using methods such as flow cytometry. An increase in surface MR1 in treated cells would be consistent with an increase in 2’,5’-oligoadenylate molecule production via OAS where a 2’,5’- oligoadenylate molecule is a ligand of MR1. Genetic modification of cell lines would also be used to demonstrate 2’,5’- oligoadenylate molecules as MR1-binding ligands. Overexpression of OAS would be performed (preferably with an inducible, e.g., tet-responsive promoter) and influence of OAS expression on increased surface MR1 would be assessed, with or without exogenous supply of 2’,5’-oligoadenylate molecule (pulsing) or OAS agonists. Further, genetic manipulation of MR1 to introduce wild type MR1 to be overexpressed in MR1-negative cells and assessed for surface presentation following 2’,5’-oligoadenylate molecule pulsing or OAS agonism would be performed. In addition, mutant forms of MR1, such as the well-described K43A mutation, could also be introduced into MR1-negative cell lines to demonstrate the importance of certain amino acids known to be involved in ligand binding in the MR1 binding groove are important in liganding of 2’,5’-oligoadenylate molecule. The MR1-β2-microglobulin complex can be generated in a soluble form and refolded with MR1 ligands.2’,5’-oligoadenylate molecules would be provided in excess with refolding of MR1- β2-microglobulin soluble protein, and the interaction could be stabilised through UV irradiation, which should cross-link the nucleotide to the protein. This refolded 2’,5’-oligoadenylate molecule -MR1- β2-microglobulin soluble complex would be assessed by mass spectrometry and/or grown as crystals and
subject to X-ray crystallography to confirm that 2’,5’-oligoadenylate molecules are bona fide ligands of MR1. Example 3 - Demonstrating the function of MR1-bound 2’,5’-oligoadenylate molecules as targets for cancer-reactive T cells To demonstrate 2’,5’-oligoadenylate molecules can bind MR1 and are ligands for MR1-T-cells reactive to cancer cells, functional assays with known cancer-reactive MR1-T-cells would be used with optimal concentrations and time-points as determined in the previous set of experiments. Cancer-reactive MR1-T-cell clones (or primary T cells transduced to express the TCR from such clones) would be co- cultured with cancer cell lines that had been pulsed with 2’,5’-oligoadenylate molecule as well as untreated target cells. Following co-culture for 24 – 48 hr, killing of the target cell lines would be assessed (Flow cytometry and/or xCELLigence methods), and activation of the MR1-T-cells would be assessed by evaluation of T cell activation markers by using assays such as production of IFNγ (e.g., by ELISpot) and/or TNFα (TAPI-0 flow cytometry-based assay). Increased target cell death and/or MR1-T-cell activation in the presence of targets incubated with 2’,5’- oligoadenylate molecules over untreated targets would demonstrate that the MR1- 2’,5’-oligoadenylate molecule complex is an antigen for MR1-T-cells. Similar to the approach described above (Example 2) for modulating OAS activity, the expectation of these studies is that inhibition of OAS should lead to a loss in MR1- 2’,5’-oligoadenylate molecule complex, and therefore reduce interactions with anti-cancer MR1-T cells. Target cancer cell lines would be treated with an OAS inhibitor, such as RU.521, and a dose-dependent inhibition of cytotoxicity and/or T cell activation would be observed when treated cells are co-incubated with anti- cancer MR1-T-cells in functional assays. Likewise, treating cancer cell lines with an OAS agonist would increase the activity of anti-cancer MR1-T-cells when incubated with the treated cells, as measured by target cell cytotoxicity and/or T cell activation markers. Genetic manipulation of target cell lines would also be used to demonstrate the hypothesis (see Example 2). Briefly, modulation, genetic knock-down, or knock-out of OAS (2’,5’-oligoadenylate synthetase) in cell lines would be performed which would be expected to lead to a reduction in target cell death and/or T cell activation
of anti-cancer MR1-T-cells due to a loss of 2’,5’-oligoadenylate molecule as a ligand for MR1. Conversely, overexpression of OAS (assuming dsDNA substrate is not limiting) in cancer cell lines would lead to an increase in target cell death and/or T cell activation with anti-cancer MR1-T-cells. Soluble 2’,5’-oligoadenylate molecule -MR1- β2-microglobulin complexes generated would be made into multimeric structures (e.g. tetramer, dextramer, etc). These multimers would be used to stain anti-cancer MR1-T-cells to demonstrate TCR- recognition of 2’,5’-oligoadenylate molecules in complex with MR1. In addition, these multimers (or monomers) would be titrated into co-culture assays of MR1+ cancer cell lines and anti-cancer MR1-T-cells to demonstrate a dose-dependent blocking of functional activity (IFNγ and/or TNFα release) or cytotoxicity. Example 4 – Preparation and characterization of a MR1-2’,5’-oligoadenylate molecule ligand complex MR1 is unable to assemble with β2 microglobulin in the absence of a ligand, however, the subunits obtained from inclusion bodies of E.coli expressing the individual MR1 heavy chain and β2 microglobulin can be mixed together and refolded with ligands as described by Corbett et al (2014). Briefly, the inclusion bodies containing these 2 proteins are mixed with ligand in a refolding solution (for example: 0.1 M Tris, pH 8.5, 2 mM EDTA, 0.4 M arginine, 0.5 mM oxidized glutathione and 5 mM reduced glutathione) in the presence of various 2’,5’- oligoadenylate molecules. The refolded MR1-2’,5’-oligoadenylate molecule antigen complexes can then be purified by sequential application of DEAE anion exchange, gel filtration, and MonoQ anion exchange chromatography (Corbett et al. (2014)). Refolding can also be accomplished in the presence of the riboflavin intermediate 5- A-RU and methylglyoxal or glyoxal to produce the MR1 liganded to the MAIT-ligand, 5-OE-RU, described by Corbett et al. (2014) to confirm the conditions used for folding and subsequent purification. Confirmation that the 2’,5’-oligoadenylate molecule -liganded MR1 molecules contain the co-incubated 2’,5’-oligoadenylate molecule can be accomplished by using mass spectroscopy under conditions that resolve the 2’,5’-oligoadenylate molecules incubated with the exclusion products, or the 5-OE-RU (Corbett et al. (2014)) in the case of 5-OE-RU-liganded MR1 complex. Side-by-side evaluation of purified 2’,5’-
oligoadenylate molecules can be used to confirm the identity of the spectra assigned to the liganded 2’,5’-oligoadenylate molecules/5-OE-RU molecule. Purified 2’,5’-oligoadenylate molecule -liganded MR1 complexes can be tested for their ability to activate cancer-reactive MR1-T-cell clones (or primary T cells transduced to express the TCR from such clones), by methods that include TNFα (TAPI-0 flow cytometry-based assay). Alternatively, intracellular cytokine staining can be used to demonstrate that the cancer-reactive MR1 T cells are activated by treatment with the MR1-2’,5’-oligoadenylate molecule complexes. Lack of activation of MAIT cells (or primary cells transduced to express the TCR of MAIT cells) can also be evaluated. Both types of T cells (cancer-reactive and MAIT) can be tested on 5-OE-RU-liganded MR1, 2’,5’-oligoadenylate molecule -liganded MR1, and the non- activating MR1-Ac-6-FP complexes, to confirm the specificity of MR1 ligand activation. Example 5 - Identification of T-cells that recognise MR1-2’,5’-oligoadenylate molecule ligand complexes T cells that recognize the MR1-2’,5’-oligoadenylate molecule complex can be identified by multiple methods using blood-derived T cells from normal subjects and cancer patients. For example, naïve of patient T cells can be incubated with MR1- 2’,5’-oligoadenylate molecule complexes and then specifically activated T cells can be identified by using flow-cytometry based methods such as detection of activation- induced TNFα (TAPI-0-based assay). Alternatively, intracellular cytokine staining can be used to identify if T cells incubated with the MR1-2’,5’-oligoadenylate molecule complex are activated. Appropriate controls for these studies can include the non- activating MR1-Ac-6FP complex which is incapable of activating T cells specific for MR1 ligands (Kjers-Nielsen et al., Nature, 2012). Studies conducted side-by-side with 5-OE-RU-liganded MR1 can also be conducted. In these studies, co-staining of activated cells with antibodies specific for TRAV1-2 could be used to demonstrate that MAIT cells are only activated with the 5-OE-RU-liganded MR1, and that the cells reactive to the MR1-2’,5’-oligoadenylate molecule complex are not MAIT-like.
Example 6 - Preparation of antibodies against MR1-2’,5’-oligoadenylate molecule ligand complex. Antibodies that specifically recognize the MR1-2’,5’-oligoadenylate molecule ligand complex could be generated by immunisation of an experimental animal with an MR1-2’,5’-oligoadenylate molecule ligand complex. Alternatively, antibodies can be generated through the use of phage- or yeast-display technology (Sheehan & Marasco, 2015), which are based on large libraries of antibody-like reactive molecules that can be screened with the MR1-2’,5’-oligoadenylate molecule complex to discover antibody complementary determining paratopes that react with MR1. Briefly, individual clones of phage or yeast found in large libraries displaying diverse scFv or Fab fusions can be selected for their ability to bind the MR1-2’,5’- oligoadenylate molecule complexes, and the selected antibody fragments can be reconstructed into functional antibodies for further use. Counter screening of the positive phage/yeast clones with MR1 (K43A) mutant refolded without ligand and MR1 folded with various MAIT ligands can be used to eliminate MR1-2’,5’- oligoadenylate molecule complex-reactive phage/yeast clones that are not specific for the MR1-2’,5’-oligoadenylate molecule complex. References Awad et al (2020) Nature Immunology 21:400-411 Crowther et al (2020) Nature Immunology, 21, 178-185 Grundstrom et al. (1985) Nucl. Acids Res., 13, 3305-3316) Jones et al (1997) J. Mol. Biol., 267, 727-748 Lion et al (2013) Eur. Journ Immunology 43(5) 1363-1373 Lepore et al (2017) eLife 6, e24476 Nambiar et al. (1984) Science, 223, 1299-1301 Ren, et al. (2015) Cell Reports, 11 (1) 1 – 12 Sakamar and Khorana (1988) Nucl. Acids Res., 14, 6361-6372 Vacchini et al (2020) Frontiers in Immunology, 11, 1-8 Verma and Eckstein (1998) Annu. Rev. Biochem., 67, 99-134
Wells et al. (1985) Gene, 34, 315-323 Xie et al (2020) EBioMedicine, 59, 102975 Corbett et al. (2014) Nature 509, 361-365 Kato, K. et al (2018) Nat Commun 9, 4424 Kjers-Nielsen et al. (2012) Nature 491, 717-723 Lama et al (2019) Nature Communications 10, 2261 Sun et al. (2013) Science 339, 786-791 Sidonia B.G. et al (2014) J Exp Med 211 (8): 1585–1600 Sheehan & Marasco (2015) Microbiol Spectrum 3(1):AID-0028-2014. doi:10.1128 /microbiolspec.AID-0028-2014. Zhang X et al (2013) Molecular Cell 51(2):226-235 Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps. All patents, patent applications and references mentioned throughout the specification of the present invention are herein incorporated in their entirety by reference. The invention embraces all combinations of preferred and more preferred groups and suitable and more suitable groups and embodiments of groups recited above.
Claims
Claims 1. An isolated MR1-ligand complex comprising (a) an MR1 protein and (b) a ligand molecule selected from a 2’,5’-oligoadenylate molecule and a derivatised 2’,5’-oligoadenylate molecule as ligand for the MR1 protein which is capable of being bound by a T-cell receptor.
2. The isolated complex according to claim 1 comprising the heavy chain of MR1.
3. The isolated complex according to claim 2 wherein the heavy chain is non- covalently associated with a β2 microglobulin protein as light chain or is covalently linked to a β2 microglobulin protein in an artificial single-chain construct.
4. The isolated complex according to any one of claims 1 to 3 wherein the ligand molecule is selected from any one of: a. 2’,5’-oligoadenylate dimer monophosphate, b. 2’,5’-oligoadenylate dimer triphosphate, c. 2’,5’-oligoadenylate trimer monophosphate, d. 2’,5’-oligoadenylate trimer triphosphate, e. 2’,5’-oligoadenylate tetramer monophosphate, and f. 2’,5’-oligoadenylate tetramer triphosphate. 5. The isolated complex according to any one of claims 1 to 3 wherein the ligand molecule is a derivatised 2’,
5’-oligoadenylate molecule.
6. An antibody protein which binds to the isolated complex according to any one of claims 1 to 5.
7. An immune cell engaging protein which is capable of targeting a cell presenting on its surface an MR1-ligand complex comprising (a) a cell targeting portion comprising an antibody protein according to claim 6 and (b) an immune cell engaging portion.
8. A chimeric immune cell receptor protein which comprises (a) a cell targeting portion comprising an antibody protein according to claim 6 and (b) a portion comprising immune cell effector function domains.
9. An isolated T-cell receptor protein which is capable of specifically binding to the isolated complex according to any one of claims 1 to 5 optionally which T- cell receptor protein is not a T-cell receptor protein described in WO2019/081902.
10. An engineered immune cell which expresses on its surface a chimeric immune cell receptor protein according to any claim 8 or a T-cell receptor protein according to claim 9.
11. An isolated polynucleotide which encodes a chimeric immune cell receptor protein according to claim 8 or a T-cell receptor protein according to claim 9.
12. A vector comprising a polynucleotide according to claim 11.
13. An engineered immune cell which comprises or is transduced with a vector according to claim 12 or which comprises or is transduced with a polynucleotide of claim 11.
14. An antibody protein according to claim 6, an immune cell engaging protein according to claim 7 or an engineered immune cell according to claim 10 or 13 for use in the prevention or treatment of cancer.
15. A method of treatment or prevention of cancer which comprises administering to a patient in need thereof an antibody protein of claim 6, an immune cell engaging protein according to claim 7 or an engineered immune cell according to claim 10 or 13.
16. A method of obtaining T-cells capable of specifically binding to cells which present an MR1-ligand complex on their surface which comprises selecting T- cells obtained from a donor which recognize an MR1-ligand complex e.g. by testing if said T-cells recognise (i) an MR1-ligand complex presented on a cell exogenously loaded with the MR1-ligand complex or (ii) said complex in monomer or multimeric form.
17. A method of obtaining T-cells capable of binding to cells which present an MR1-ligand complex which comprises priming and stimulating T-cells, particularly primary T-cells, particularly CD8+ T-cells, with antigen presenting cells (APCs) loaded with ligand and selecting T-cells that specifically bind to the APCs, wherein the MR1-ligand complex is the MR1-ligand complex according to any one of claims 1-5.
18. A method for identification of a T-cell reactive to an MR1-ligand complex in a T-cell containing preparation which comprises providing a T-cell containing preparation, contacting T-cells of said T-cell containing preparation with said MR1-ligand complex and isolating a T-cell that is reactive to said MR1-ligand complex, wherein the MR1-ligand complex is the MR1-ligand complex according to any one of claims 1-5.
19. A T-cell obtained by the method of any one of claims 16 to 18.
20. A method of obtaining a T-cell receptor protein capable of specifically binding to cells which present an MR1-ligand complex which comprises obtaining T- cells according to the method of claim 16 or claim 17 or isolating T-cells according to claim 18 and obtaining the T-cell receptor protein from said T- cells.
21. An isolated TCR obtained by the method of claim 20.
22. A method of raising antibodies capable of specifically binding to cells which present an MR1-ligand complex on their surface which comprises immunising an experimental animal with an MR1-ligand complex according to any one of claims 1 to 5 as immunogen, optionally together with an immunostimulant, and obtaining antibodies or antibody producing cells from said animal.
23. An antibody obtained by the method of claim 22.
24. A method of treatment or prevention of cancer which comprises administering to a patient in need thereof a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule or an agent which stimulates the production of a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule in vivo.
25. A method of treatment or prevention of cancer which comprises administering to a patient in need thereof an agent which is not a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule and which binds MR1 to form a MR1-agent complex on the surface of cells and which complex is capable of specifically binding a T-cell or T-cell receptor, optionally wherein the agent competes with a natural 2’,5’-oligoadenylate molecule or a natural derivatised 2’,5’-oligoadenylate molecule for binding to MR1 in the same binding site.
26. A method of determining whether a cell is cancerous which comprises determining whether said cell expresses on its surface an MR1-ligand complex comprising (a) MR1 and (b) a ligand molecule selected from a 2’,5’- oligoadenylate molecule and a derivatised 2’,5’-oligoadenylate molecule as ligand for MR1 optionally wherein the ligand is a 2’,5’-oligoadenylate molecule or a derivatised 2’,5’-oligoadenylate molecule according to any one of claims 1 to 5.
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