WO2023002204A1

WO2023002204A1 - T-cell receptor

Info

Publication number: WO2023002204A1
Application number: PCT/GB2022/051910
Authority: WO
Inventors: Peter Mason; Duncan HOWIE
Original assignee: Enara Bio Limited
Priority date: 2021-07-21
Filing date: 2022-07-21
Publication date: 2023-01-26

Abstract

The present disclosure relates to a T-cell receptor (TCR), a T-cell expressing the TCR; a polynucleotide and a vector encoding the TCR; a pharmaceutical composition comprising the TCR, T-cell, polynucleotide or vector; use of the TCR or T-cell, polynucleotide, vector or pharmaceutical composition to treat cancer; and a method of treating cancer using the TCR, T-cell, polynucleotide, vector or pharmaceutical composition.

Description

T-Cell receptor

Technical field

Background

The present invention relates to a T-cell effective for treating cancer, which recognizes cancer cells through the population-invariant major histocompatibility complex class I- related protein 1 (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 subset of patients. In contrast, the entire human population is thought to express MR1.

MR1 -restricted T-cells called mucosal-associated invariant T-cells (MAITs) are known to recognise intermediates of mycobacterial riboflavin biosynthesis. Recent studies have shown that there are also other types of MR1 -restricted T-cells that recognise different MR1 -bound ligands. Such T-cells have target specificity via MR1 but the TCR does not bind to MR1 per se or to MR1 loaded with known infectious disease agent ligands but recognises a cancer-specific ligand within the MR1 binding groove; MR1 presents a cancer-specific, or cancer-upregulated, ligand to the TCR.

The T-cell clone, MC.7.G5, disclosed in patent application PCT/GB2018/053045 published as WO2019081902, was discovered during a screen of T-cells from a healthy donor that was HLA mismatched for the adenocarcinoma alveolar basal epithelial cell line, A549 (ATCC^® reference CCL-185 for information). The experimental approach involved incubating T-cells with A549 cells then isolating and cloning T-cells that had proliferated in response to the A549 cells. Further investigations showed that the MC.7.G5 T-cell clone was able to recognise and kill cancers cells, including cancer cells from a number of organs and tissue types, thus showing the clone had potential for treating many types of cancer. Purified T-cells from the PBMCs of Stage IV melanoma patients which were lentivirally transduced with the heterologous MC.7.G5 TCR resulted in recognition and killing of autologous and non-autologous melanomas, but not healthy cells, the level of T-cell surface expression of the MC.7.G5 TCR, which comprises human constant alpha and beta regions, determined as frequency of the TCR positive cells quantified by antibody staining specific for the TCRV-beta subunit, has been demonstrated to be low in view of preferred therapeutic requirements.

An alpha beta TCR is a disulphide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (b) chains that associate with the invariant CD3 chain molecules to form a complete functioning TCR. T cells expressing this receptor are referred to as alpha beta T cells.

The alpha and beta chains are composed of extracellular domains comprising a Constant (C) region and a Variable (V) region. The Constant region is proximal to the cell membrane and extends to a transmembrane region and a short cytoplasmic tail, while the Variable region binds to the ligand (generally HLA presented).

The variable domain is formed of variable regions of both the TCR alpha-chain and beta- chain each of which has three hypervariable regions called complementarity determining regions (CDRs). There is also an additional area of variability on the beta-chain (HV4) that does not normally contact antigen and, therefore, is not considered a CDR. In general, the antigen-binding site is formed by the CDR loops of the TCR alpha-chain and beta-chain. CDR1a and CDR2a are encoded by the individual Va genes whereas CDR1p and CDR2p are encoded by the individual Vp genes. The CDR3 of the TCR alpha-chain is especially hypervariable due to the potential for nucleotide addition and removal around the joining of the V region and a Joining region. The TCR beta-chain CDR3 has even more capacity for variation as it can also include a diversity (D) gene. CDR3 is the main CDR responsible for recognizing HLA-bound antigenic peptides (the usual target for alpha-beta TCRs). However, in some cases CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the HLA-bound antigenic peptide, and CDR1 of the beta-chain may interact with the C-terminal part of the HLA-bound antigenic peptide.

In 2015 about 90.5 million people had cancer. About 14.1 million new cases occur a year (not including skin cancer other than melanoma). It causes about 8.8 million deaths (15.7%) of human deaths. It follows that there is a need to provide better and safer ways of treating or eradicating this disease. An immunotherapy that uses the body’s natural defence systems to kill aberrant tissue is acknowledged to be safer than chemical intervention but, to be effective, the immunotherapy must be cancer specific.

MC.7.G5 TCR was shown to advantageously provide a means of T-cell immunotherapy that is not HLA-restricted and could potentially be effectively administered to individuals regardless of HLA tissue type or suffering from many different types of cancer. An effective T-cell immunotherapy preferably demonstrates high levels of surface expression of the heterologous TCR in the transduced patient cells whilst maintaining safe levels of vector copy number and effective levels of specific potency to target cancer cells but not of healthy or non-target cells.

Brief summary of the invention

We have developed a TCR and encoding polynucleotide based on the recombinant engineering of the 7G5 TCR sequences to introduce changes to the TCR alpha and beta chain constant region sequences as well as to vary chain orientation within encoding polynucleotide sequences, as described herein. The T-cell expressed TCR possesses the resulting advantageous technical characteristics of improved surface expression in transduced T-cells with MR1 restricted cancer cell potency, specificity and demonstrates safety with respect to lack of healthy or normal cell recognition and safe levels of transduced viral copy number.

According to the present invention there is provided a tumour-specific T-cell receptor (TCR) comprising an alpha chain and beta chain, wherein the alpha chain comprises a variable region comprising a complementarity-determining region (CDR) (i.e. alpha chain CDR3, CDR3a) comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1; and a constant region (i.e. alpha chain constant region) which comprises or consists of a constant region of a murine TCR (i.e. a murine TCR constant region such as a murine alpha chain constant region) or variant thereof; and the beta chain comprises a variable region comprising a complementarity-determining region (CDR) (i.e. beta chain CDR3, CDR3p) comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2; and a constant region (i.e. beta chain constant region) which comprises or consists of a constant region of a murine TCR (i.e. a murine TCR constant region such as a murine beta chain constant region) or variant thereof. Optionally the tumour-specific T-cell receptor (TCR) binds a tumour antigen.

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.

"Naturally occurring" or “native”, which terms are interchangeable, when used with reference to a polypeptide or polynucleotide sequence means a sequence found in nature and not synthetically modified.

The term “artificial” or “engineered” when used with reference to a polypeptide or polynucleotide sequence means a sequence not found in nature which is, for example, a recombinantly produced sequence or synthetic modification of a natural sequence, or contains an unnatural polypeptide or polynucleotide sequence. The term “engineered” when used with reference to a cell means a cell not found in nature which is, for example, a recombinantly produced cell or a synthetic modification of a natural cell, for example, because it contains or expresses foreign elements and/or lacks natural elements.

The term “heterologous” 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 the the cell.

The term “domain” is generally used to refer to a part of the TCR formed of the corresponding region of the two chains. For example, the transmembrane regions of the a and b chains form the transmembrane domain.

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.

T-cell receptor (TCR)

A TCR of this invention comprises an a chain and a b chain. The extracellular region of each chain comprises three CDRs (CDR1, CDR2, CDR3) and four framework regions which are either side of the CDRs, and a constant region. A TCR has the extracellular domain as well as, at its C terminus, transmembrane and intracellular domains. An immature form of an entire TCR also comprises a leader peptide sequence at its N terminus which is removed after translation by cellular peptidases such as signal peptidase. Each chain has a connecting peptide region which links the transmembrane and intracellular regions to the extracellular domain at its C terminus.

According to the present invention there is provided a tumour-specific T-cell receptor (TCR) comprising an alpha chain and beta chain, wherein: the alpha chain comprises a variable region comprising a complementarity determining region (CDR) (i.e. alpha chain CDR3) comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1; and a constant region which comprises or consists of a constant region of a murine TCR (i.e. a murine TCR constant region) or variant thereof; and the beta chain comprises a variable region comprising a complementarity determining region (CDR) (i.e. beta chainCDR3) comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2; and a constant region which comprises or consists of a constant region of a murine TCR (i.e. a murine TCR constant region) or variant thereof. Optionally the tumour-specific T-cell receptor (TCR) binds a tumour antigen.

In a preferred embodiment of the invention, the alpha chain CDR3 comprises or consists of the sequence CAYRSAVNARLMF (SEQ ID NO: 1) or a CDR that shares at least 88% sequence identity therewith, such as 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In a preferred embodiment of the invention, the beta chain CDR3 comprises or consists of the sequence CASSEARGLAEFTDTQYF (SEQ ID No: 2) or a CDR that shares at least 88% sequence identity therewith, such as 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In a preferred embodiment of the invention, the alpha chain constant region comprises or consists of an alpha chain constant region of a murine TCR (i.e. a murine alpha chain constant region) or variant thereof or comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:11, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith). Hence the tumour specific TCR according to the invention preferably comprises an alpha chain comprising a constant region which comprises or consists of a constant region of a murine TCR (i.e. a murine TCR constant region) which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:11, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith).

In a preferred embodiment of the invention, the beta chain constant region comprises or consists of an beta chain constant region of a murine TCR (i.e. a murine beta chain constant region) or variant thereof or comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:17, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith). Hence the tumour specific TCR according to the invention preferably comprises a beta chain comprising a constant region which comprises or consists of a constant region (i.e. a murine TCR constant region)which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:17, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith).

Thus, an embodiment of the invention provides a tumour-specific T-cell receptor (TCR) or an alpha chain thereof comprising: a CDR (i.e. alpha chain CDR3), comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and an alpha chain constant region which comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region or alpha chain constant region of SEQ ID NO:11, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith). Optionally the tumour-specific T-cell receptor (TCR) or alpha chain thereof binds a tumour antigen. Hence the tumour specific TCR according to the invention preferably comprises an alpha chain comprising a variable region comprising a complementarity-determining region (CDR) (i.e. alpha chain CDR3) comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:11, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith).

An embodiment of the invention also provides a tumour-specific T-cell receptor (TCR) or a beta chain thereof comprising: a CDR comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and a beta chain constant region which comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region or beta chain constant region of SEQ ID NO:17, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith). Optionally the tumour-specific T-cell receptor (TCR) or beta chain thereof binds a tumour antigen. Hence the tumour specific TCR according to the invention preferably comprises a beta chain comprising a variable region comprising a complementarity-determining region (CDR) (i.e. beta chain CDR3) comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:17, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith).

A further embodiment of the invention provides a tumour specific T-cell receptor (TCR) according to the present invention wherein the TCR comprises: a CDR comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and an alpha chain constant region which comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region or alpha chain constant region of SEQ ID NO:11, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith); and a CDR comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and a beta chain constant region which comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region or beta chain constant region of SEQ ID NO:17, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith).

Hence the tumour specific TCR according to the invention preferably comprises an alpha chain comprising a variable region comprising a complementarity-determining region (CDR) (i.e. alpha chain CDR3) comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:11, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith), and a beta chain comprising a variable region comprising a complementarity-determining region (CDR) (i.e. beta chain CDR3) comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:17, (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith).

A further embodiment provides a tumour-specific T-cell receptor (TCR) according to the present invention wherein at least one amino acid is substituted in the alpha chain constant region and/or beta chain constant region, for example any one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 substitutions, for example with respect to the sequence of alpha chain constant region of SEQ ID NO:11 and/or the beta chain constant region of SEQ ID NO:17. Accordingly the at least one amino acid substituted in the alpha chain constant region may be a hydrophobic amino acid, preferably a valine or isoleucine. Preferably the alpha chain constant region comprises one or more of the substitutions selected from, S112L, M1141, G115V relative to SEQ ID NO:11 or comprises one or more of the substitutions selected from, L112, 1114, V115 relative to SEQ ID NO:11, preferably any of L112 and 1114 , L112 and V115, 1114 and V115, or L112, 1114 and V115. Preferably the alpha chain constant region comprises the sequence LVIV (SEQ ID NO:45), preferably at positions 112-115 relative to SEQ ID NO:11.

Accordingly, the beta chain constant region may comprise one or more of the substitutions selected from, Q135H, T161G, V164L, R170K, for example selected from any one of Q135H and T161G; Q135H and V164L; Q135H and R170K; T161G and V164L; T161G and R170K; V164L and R170K; Q135H, T161G and V164L; Q135H, T161G and R170K; Q135H, V164L and R170K; T161G, V164L and R170K; Q135H, T161G, V164L, R170K relative to SEQ ID N017.

According to the present invention the constant region of the TCR may comprise an alpha chain constant region comprising one or more of the substitutions selected from the substitutions S112L, M1141, G115V relative to SEQ ID NO:11 and a beta chain constant region comprising one or more of the substitutions selected from the substitutions, Q135H, T161G, V164L, R170K relative to SEQ ID NO:17. Preferably the TCR may comprise an alpha chain constant region comprising the substitutions S112L, M1141, G115V relative to SEQ ID NO:11 and a beta chain constant region comprising the substitutions, Q135H, T161G, V164L, R170K relative to SEQ ID NO:17.

A further embodiment of the invention provides a tumour specific T-cell receptor (TCR) according to the present invention wherein the TCR comprises an alpha chain constant region which comprises or consists of a constant region of SEQ ID NO:12, and/or a beta chain constant region which comprises or consists of a constant region of SEQ ID

NO:18.

In a further embodiment there is provided a tumour-specific T-cell receptor (TCR) according to the present invention comprising: a CDR comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1); and/or a CDR comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2).

In a further embodiment there is provided a tumour-specific T-cell receptor (TCR) according to the present invention comprising: a CDR comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1); and a CDR comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2).

Hence there is provided a tumour-specific T-cell receptor (TCR) according to the invention, comprising an alpha chain and beta chain, wherein the alpha chain comprises a variable region comprising a complementarity-determining region (CDR) (i.e. alpha chain CDR3) comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1) and/or a beta chain comprising a variable region comprising a complementarity-determining region (CDR) (i.e. beta chain CDR3) comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2).

The CDRs described hereinabove represent the CDR3s (i.e. alpha chain CDR 3 / CDR3a or beta chain CDR3 / CDR3p) of the TCR which are the main CDRs responsible for recognizing processed antigen or ligand. The other CDRs (CDR1a (alpha chain CDR1), CDR2a (alpha chain CDR2), CDR1p (beta chain CDR1) and CDR2p (beta chain CDR2)) are encoded by the germline. Therefore, the invention further concerns a TCR also including one or more of these other CDRs i.e. CDR1 a, CDR2a, CDR1 b or CDR2p.

Accordingly, in a preferred embodiment the TCR according to the present invention comprises one or more, including any combination of, CDRs comprising or consisting of the following CDRs:

TSESDYY (CDR1a) SEQ ID NO:3; or a variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto,

ATEN (CDR2a) SEQ ID NO:4; ora variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto,

MGHDK (CDR1p) SEQ ID NO:5; or a variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto, and

SYGVNS (CDR2p) SEQ ID NO:6. or a variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto.

Hence there is provided a tumour-specific T-cell receptor (TCR) according to the invention, comprising an alpha chain and beta chain, wherein the alpha chain comprises a variable region comprising a CDR1 comprising or consisting of TSESDYY (CDR1a) SEQ ID NO:3 or variant thereof; and/or a CDR2 comprising or consisting of ATEN (CDR2a) SEQ ID NO:4 or variant thereof and / or a beta chain comprising a variable region comprising a CDR1 comprising or consisting of MGHDK (CDR1 b) SEQ ID NO:5 or variant thereof; and/or a CDR2 comprising or consisting of SYGVNS ^ϋE2b) SEQ ID NO:6 or variant thereof; wherein said variant CDR has at least one substitution, addition or deletion, for example two, with respect to the respective CDR sequence.

Reference herein to a tumour-specific TCR is to a TCR that specifically recognises a tumour cell or a tumour-cell ligand / antigen, in the context of MR1, and is activated by same but is not activated by a non-tumour cell or a non-tumour-cell ligand /antigen, in the context of MR1. The tumour specific T-cell receptor (TCR) of the invention preferably specifically binds to tumour, preferably in the context of MR1 , for example MR1 of SEQ ID NO:44. Preferably the tumour specific T-cell receptor (TCR) of the invention specifically binds a tumour ligand / antigen or tumour-specific ligand / antigen, preferably in the context of MR1 , for example where the ligand / antigen is bound and/or presented by MR1 , preferably on the tumour or tumour cells. Such ligand or antigen may be a a biomolecule expressed by the tumour or fragment thereof, for example a peptide / polypeptide or nucleotide/polynucleotide or metabolite of tumour cell metabolism.

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, for example a non-tumour cell or a non- tumour-cell ligand /antigen, for example in the context of MR1. The term “being specifically bound” is to be interpreted in a similar sense.

As used herein, the term “specifically recognises” when used in relation to a TCR in the context of a tumour cell or a tumour-cell ligand / antigen means that the TCR may specifically bind to and/or be specifically reactive to said tumour cell or a tumour-cell ligand / antigen, for example in the context of MR1. As used herein, the term “specifically reactive” when used in relation to the binding of A to B means that A is activated when bound to B, to an extent that greatly exceeds that of the binding of A to other molecules not intended to be targeted by A. For example, the TCR or T-cell comprising the TCR, is activated when bound to the tumour cell or a tumour-cell ligand / antigen. The determination as to whether the TCR specifically recognises, or if it specifically binds to and/or is specifically reactive to a tumour cell or a tumour-cell ligand / antigen, for example in the context of MR1 , may be discovered by determining if upon interaction with the tumour cell or a tumour-cell ligand / antigen, for example in the context of MR1, the TCR, which is an immune cell presented receptor, induces the activation of the immune cell which presents the receptor (or in the case of a soluble receptor TCR induces an immune reaction) to a higher level than the activation measured in the absence of tumour cell or a tumour-cell ligand / antigen, for example in the context of MR1 or for example a non-tumour cell or a non-tumour-cell ligand /antigen, for example in the context of MR1. For example, such immune cell or T cell activation can be evaluated with any of the following measurements: cytokine release (IFN gamma orTNF alpha), chemokine release, immune cell proliferation, immune cell expression of activation markers, immune cell target cell killing, induction of transcription factors or of reporter genes of the immune cell.

In a preferred embodiment of the invention the TCR is an ab TCR having an a chain and a b chain and the CDR of the a chain (i.e. alpha chain CDR3) comprises or consists of the CDR: CAYRSAVNARLMF (SEQ ID NO: 1) or a CDR that shares at least 88% identity therewith, such as 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%; and the CDR of the b chain (i.e beta chain CDR3) comprises or consists of the CDR: CASSEARGLAEFTDTQYF (SEQ ID No: 2) or a CDR that shares at least 88% identity therewith, such as 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Accordingly, the TCR according to the present invention may comprise one or both of the aforementioned CDRs and in a preferred embodiment comprises both of the CDRs.

In an embodiment, the tumour-specific T-cell receptor (TCR) according to the present invention comprises a chain, preferably alpha chain, with CDRs comprising or consisting of:

CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith); TSESDYY (SEQ ID NO:3) or variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto; and ATEN (SEQ ID NO:4) or variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto; optionally wherein the alpha chain constant region comprises or consists of a constant region of SEQ ID NO:12. In an embodiment, the tumour-specific T-cell receptor (TCR) according to the present invention comprises a chain, preferably beta chain, with CDRs comprising or consisting of:

CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith); MGHDK (SEQ ID NO:5) or variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto; and SYGVNS (SEQ ID NO:6) or variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto, optionally wherein the beta chain constant region comprises or consists of a constant region of SEQ ID NO:18.

CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith); TSESDYY (SEQ ID NO:3) or variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto; and ATEN (SEQ ID NO:4) or variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto; and comprises a chain, preferably a beta chain, with CDRs comprising or consisting of:

CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith); MGHDK (SEQ ID NO:5) or variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto; and SYGVNS (SEQ ID NO:6) or variant CDR having at least one substitution, addition or deletion, for example two, with respect thereto, optionally wherein the alpha chain constant region comprises or consists of a constant region of SEQ ID NO: 12, and/or the beta chain constant region comprises or consists of a constant region of SEQ ID NO:18.

As noted above, the TCR of the present invention is unconventional in that it is not HLA- restricted, rather it binds to a tumour-specific ligand / antigen in the context of MR1, an alternative MHC-related molecule. Hitherto, it was thought that MR1 -restricted ab T-cells were exclusively mucosal-associated invariant T cells (MAIT cells), however, we demonstrate herein that a further type of MR1 -restricted T-cells exists that does not express the semi-invariant MAIT TCR a chains, moreover, advantageously, these T-cells and their TCRs are tumour specific (i.e. respond to tumour cells but not non-tumour cells) but, surprisingly, are able to identify many tumour types of different origin or tissue type and so have pan-cancer therapy potential as are not HLA-restricted.

In an embodiment of the present invention the tumour-specific T-cell receptor (TCR) is preferably not expressed by or associated with a mucosal-associated invariant T cell (MAIT cell).

The specific mechanism that allows killing of many types of cancer cells is still to be elucidated. Nevertheless, it may be hypothesized (and without being limited by theory) that the T-cell of the invention is able to bind to surface-displayed MR1 molecules liganded with a by-product (or by-products) of an aberrant tumour-specific metabolic pathway(s) (for example, an altered pathway arising from epigenetic changes associated with neoplastic transformation), with the type of aberrant metabolism being a common feature amongst different types of cancer.

The sequence of the a chain variable region is as follows:

AQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQ NATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRSAVNARLMFGDGTQLVVKP (SEQ ID NO:7), (CDRs shown in underlined bold type). The variable region of the a chain results from recombination of genes TRAV38.2/DV8 and TRAJ31. In SEQ ID NO: 7, residues 1 -26 are framework region 1 , residues 27-33 are CDR1 (shown in underlined bold type), residues 34-58 are framework region 2, residues 59-62 are CDR2 (shown in underlined bold type), residues 63-91 are framework region 3, residues 92-104 are CDR3 (shown in underlined bold type), residues 105-114 are framework region 4

The sequence of the b chain variable region is as follows:

EADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKG DLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEARGLAEFTDTQYFGPGTRLTVL (SEQ ID NO:8), (CDRs shown in underlined bold type). The variable region of the b chain results from recombination of genes TRBV25.1 and TRBJ2.3.

In SEQ ID NO: 8, residues 1 -26 are framework region 1 , residues 27-31 are CDR1 (shown in underlined bold type), residues 32-48 are framework region 2, residues 49-54 are CDR2 (shown in underlined bold type), residues 55-90 are framework region 3, residues 91-108 are CDR3 (shown in underlined bold type), residues 109-117 are framework region 4.

In an embodiment, the tumour-specific T-cell receptor (TCR) according to the present invention comprises an alpha chain variable region comprising or consisting of SEQ ID NO:7 or a variant alpha chain variable region which has at least 88% sequence identity (such as 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.) to the alpha chain variable region of SEQ ID NO: 7, preferably 100% identity to SEQ ID NO:7.

In an embodiment, the tumour-specific T-cell receptor (TCR) according to the present invention comprises a beta chain variable region comprising or consisting of SEQ ID NO:8 or a variant alpha chain variable region which has at least 88% sequence identity (such as 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.) to the alpha chain variable region of SEQ ID NO: 8, preferably 100% identity to SEQ ID NO:8.

In a preferred embodiment there is provided a TCR according to the present invention which comprises: (a) an alpha chain comprising a variable region comprising a CDR3 sequence of SEQ ID NO: 1 or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), a CDR1 sequence of SEQ ID NO:3 and CDR2 sequence of SEQ ID NO: 4 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:11 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith), and a beta chain comprising a variable region comprising a CDR3 sequence of SEQ ID NO: 2 or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), a CDR1 sequence of SEQ ID NO: 5 and CDR2 sequence of SEQ ID NO: 6 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:17 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith); or

(b) an alpha chain comprising a variable region comprising or consisting of SEQ ID NO:7 or a variant variable region which has at least 88% sequence identity to the alpha chain variable region of SEQ ID NO: 7 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to constant region of SEQ ID NO:11; and a beta chain comprising a variable region comprising or consisting of SEQ ID NO:8 or a variable region which has at least 88% sequence identity to the beta chain variable region of SEQ ID NO: 8 and constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:17; or

(c) an alpha chain comprising a variable region comprising or consisting of SEQ ID NO:7 or a variant variable region which has at least 88% sequence identity to the alpha chain variable region of SEQ ID NO: 7 and a constant region which comprises or consists of a constant region of SEQ ID NO:12; and a beta chain comprising a variable region comprising or consisting of SEQ ID NO:8 or a variable region which has at least 88% sequence identity to the beta chain variable region of SEQ ID NO: 8 and constant region which comprises or consists of a constant region of SEQ ID NO:18;or

(d) an alpha chain comprising or consisting of SEQ ID NO:13 minus the N- terminal leader sequence (i.e. residues 1-20 of SEQ ID NO:13) or a variant alpha chain which has at least 88% sequence identity to the alpha chain of SEQ ID NO: 13 minus the N-terminal leader sequence and a beta chain comprising or consisting of SEQ ID NO: 19 minus the N-terminal leader sequence (i.e. residues 1-19 of SEQ ID NO:19) or a variant beta chain which has at least 88% sequence identity to the beta chain of SEQ ID NO: 19 minus the N-terminal leader sequence, or

(e) an alpha chain comprising or consisting of SEQ ID NO:14 minus the N- terminal leader sequence (i.e. residues 1-20 of SEQ ID NO:14) and a beta chain comprising or consisting of SEQ ID NO: 20 minus the N-terminal leader sequence (i.e. residues 1-19 of SEQ ID NO:20).

According to the above description where a variant sequence is described as having at least 88% sequence identity to a comparator sequence SEQ ID NO:X, this means having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith. Likewise where a variant sequence is described as having at least 80% sequence identity to a comparator sequence SEQ ID NO:Y, this means having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith.

In an embodiment there is provided a TCR according to the present invention wherein the TCR is MR1 -restricted and/or that specifically recognises a tumour cell or a tumour cell ligand / antigen, in the context of MR1. In yet another preferred embodiment of the invention the TCR is part of a chimeric receptor preferably having the functionality described herein.

TCR polypeptide sequences of the invention 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.

Sequence comparisons

For the purposes of comparing two closely-related polypeptide sequences, the “% sequence identity" between a first sequence and a second sequence may be calculated. Polypeptide sequences are the to be the same as or identical to other polypeptide sequences, if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C- terminus for polypeptides. The terms “identical” or percentage “identity”, in the context of two or more polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues that are the same (i.e. at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity e.g. 100% sequence identity over a specified region), when compared and aligned for maximum correspondence over a comparison window. Suitably, the comparison is performed over a window corresponding to the entire length of the reference sequence.

For sequence comparison, one sequence acts as the reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percentage sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, refers to a segment in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Nat’l. Acad. Sci. USA 85:2444, by computerised implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel etal., eds. 1995 supplement)).

An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et ai, 1977, Nuc. Acids Res. 25:3389-3402 and Altschul et ai, 1990, J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et ai, supra). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

A “difference” between sequences refers to an insertion, deletion or substitution of a single residue in a position of the second sequence, compared to the first sequence. Two sequences can contain one, two or more such differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If the identical sequences are 17 amino acid residues long, two substitutions in the second sequence results in a sequence identity of 88.2%.

Alternatively, for the purposes of comparing a first, reference sequence to a second, comparison sequence, the number of additions, substitutions and/or deletions made to the first sequence to produce the second sequence may be ascertained. An addition is the addition of one residue into the first sequence (including addition at either terminus of the first sequence). A substitution is the substitution of one residue in the first sequence with one different residue. A deletion is the deletion of one residue from the first sequence (including deletion at either terminus of the first sequence).

Sequence variants

The term “amino acid” refers to any one of the naturally occurring amino acids, as well as amino acid analogues and amino acid mimetics that function in a manner which is similar to the naturally occurring amino acids. Naturally occurring amino acids are those 20 L- amino acids encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. The term “amino acid analogue” refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e. , an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group but has a modified R group or a modified peptide backbone as compared with a natural amino acid. Examples include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium and norleucine. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Suitably an amino acid is a naturally occurring amino acid or an amino acid analogue, especially a naturally occurring amino acid and in particular one of those 20 L-amino acids encoded by the genetic code.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

In an embodiment, the amino acid sequence of the TCR or tumour-specific binding fragment thereof is artificial. Without limitation, and as discussed further herein, the TCR or tumour-specific binding fragment thereof may comprise at least one mutation to remove a cysteine residue by replacement with another residue and/or to introduce a cysteine residue by replacement of another residue with cysteine.

According to the present invention the tumour-specific T-cell receptor (TCR) may comprise or consist of an amino acid sequence which is artificial, for example the amino acid sequence of the TCR may be artificial or recombinant.

In an embodiment the tumour-specific T-cell receptor (TCR) according to the present invention comprises an alpha chain variable region and/or beta chain variable region wherein at the least one amino acid is substituted, added or deleted relative to the alpha chain variable region of SEQ ID NO:7 and/or the beta chain variable region of SEQ ID NO: 8 respectively. Preferably the at least one amino acid is (are) located in a framework region or a CDR, alternatively wherein the at least one amino acid is not located in any CDR. Thus, particularly, any and all additions, substitutions and deletions of amino acids are in a framework region. In an embodiment the tumour-specific T-cell receptor (TCR) according to the present invention comprises an alpha chain constant region and/or beta chain constant region wherein at least one amino acid is substituted, added or deleted relative to the alpha chain constant region of SEQ ID NO:11 and/or the beta chain constant region of SEQ ID NO: 17 respectively.

Variations in sequence can be in the form of additions, substitutions and deletions, especially substitutions. For example, additions can be at the N and/or C termini of sequences and deletions can be at the N and/or C termini of sequences. There may, for example, be 1 or 2 (particularly 1) additions, substitutions or deletions in the sequence of SEQ ID NO: 1. There may, for example, be 1 or 2 (particularly 1) additions, substitutions and deletions in the sequence of SEQ ID NO: 2. There may, for example, be up to 24 e.g. up to 20 e.g. up to 15 e.g. up to 10, 9, 8, 7 or 6 e.g. up to 5 e.g. 4, 3, 2 or 1) additions, substitutions and deletions in the sequences of SEQ ID NO:

7 and/or SEQ ID NO: 8 and/or SEQ ID NO:11 and/or SEQ ID NO: 17.

Substitutions are suitably conservative substitutions. The following eight groups each contain amino acids that are typically conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins 1984).

Suitably, sequence variations do not significantly adversely affect the ability of the TCR or fragment thereof to bind to its target epitope on the tumour or to bind to the tumour, tumour ligand / antigen, for example its binding affinity of the variant is 75% or more e.g. 80% or more e.g. 85% or more e.g. 90% or more e.g. 95% or more e.g. 98% or more e.g. 99% or more of that of the TCR that in soluble form has a and b chain variable regions of SEQ ID NOs: 7 and 8 respectively. Suitably, sequence variations, for example in the constant region do not significantly adversely affect the ability of the TCR to be expressed on a cell surface or demonstrate antitumour activity, for example as measured by expression levels of TCR when transduced into T-cells or as measured by cytokine production of said transduced T-cells in response to tumour cells. For example, the expression levels of TCR or antitumour activity of the variant is preferably 75% or more e.g. 80% or more e.g. 85% or more e.g. 90% or more e.g. 95% or more e.g. 98% or more e.g. 99% or more of that of the TCR having a and b chain constant regions of SEQ ID NOs: 12 and 18 respectively.

The following variants formed of sequence mutations are contemplated in particular:

Particularly useful mutations are mutations made to one or both chains (i.e. one or both of the a and b chains) of a TCR of the invention to reduce the likelihood that the chain or chains will pair with a chain or chains of the TCR naturally produced by a T-cell. For example, mutations may be made in the constant region of the extracellular region to promote pairing of the recombinant chains and reduce pairing with an endogenous TCR. One such example contemplated for the TCR according to the present invention is the introduction into the constant region of the a and b chains of one or more new cysteine (C) residues such that for example the modified TCR may form a disulphide bridge not otherwise present in the TCR. According to the present invention a suitable variant sequence of SEQ ID NO: 11 (murine alpha chain constant region) or SEQ ID NO: 12 (murine alpha chain constant region variant), residue 48 (Threonine) may be mutated to C, T48C. According to the present invention a suitable variant sequence of SEQ ID NO: 17 (murine constant beta chain) or SEQ ID NO: 18 (murine constant beta chain variant), residue 57 (Serine) is mutated to C, S57C. The mutations T48C in SEQ ID NO: 11 or SEQ ID NO: 12 and S57C in SEQ ID NO: 17 or SEQ ID NO: 18 can lead to the formulation of a new disulphide bridge in the heterodimer TCR which can increase the stability of the TCR.

In an embodiment, the tumour-specific T-cell receptor (TCR) according to the present invention comprises an alpha chain wherein the alpha chain constant region can comprise one or more of the substitutions selected from, S112L, M114I, G115V relative to SEQ ID NO:11, preferably two, further preferably all three substitutions and/or comprises a beta chain constant region which can comprise one or more of the substitutions selected from, Q135H, T161G, V164L, R170K relative to SEQ ID NO:17, preferably two or three further preferably all four substitutions. Preferably the alpha chain constant region of the TCR according to the invention comprises the sequence LVIVLRILLLKVAGFNLLMTL (SEQ ID NO:31), in an embodiment, the alpha chain constant region can comprise the sequence LVIVLRILLLKVAGFNLLMTL (SEQ ID NO:31) from positions 112 to 137 relative to SEQ ID NO:11, e.g. in SEQ ID NO:11.

Additionally, as contemplated in the present invention, the tumour-specific T-cell receptor (TCR) according to the present invention can comprise sequences intended for expression in a bacterial host and may be provided with an initial M (methionine) residue.

Preferably the tumour-specific T-cell receptor (TCR) according to the present invention is MR1 -restricted. Preferably the tumour-specific T-cell receptor (TCR) according to the present invention is not expressed by or associated with a mucosal-associated invariant T cell (MAIT cell).

Preferably the tumour specific TCR according to the invention is translated as a preproprotein or immature protein, i.e. a full-length polypeptide product of mRNA that must be processed to generate the mature protein. Preferably the preproprotein comprises or consists of a sequence comprising,

(i) an alpha chain comprising; an alpha chain signal sequence comprising or consisting of a sequence which has at least 88% sequence identity to the sequence of SEQ ID NO: 42 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), a variable region comprising a CDR3 sequence of SEQ ID NO: 1 or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), a CDR1 sequence of SEQ ID NO:3 and CDR2 sequence of SEQ ID NO: 4 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to constant region of SEQ ID NO:11 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), and

(ii) a beta chain comprising; a beta chain signal sequence comprising or consisting of a sequence which has at least 88% sequence identity to the sequence of SEQ ID NO: 43 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith); a variable region comprising a CDR3 sequence of SEQ ID NO: 2 or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), a CDR1 sequence of SEQ ID NO: 5 and CDR2 sequence of SEQ ID NO: 6 and constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:17 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith); optionally wherein any one or more of SEQ ID Nos: 3, 4, 5 and 6 may comprise 1 or 2 (particularly 1) additions, substitutions and deletions in the said sequence, and further comprising

(iii) a self cleaving protein sequence, optionally a 2A peptide sequence, for example selected from any one of a P2A, T2A, E2A or F2A sequence, for example any one of SEQ ID Nos: 27, 36, 37, 38; optionally comprising a linker sequence on the N-terminal of the self cleaving protein sequence / P2A sequence, for example SEQ ID NO: 39, and further optionally (iv) a Furin cleavage site sequence, for example any of SEQ ID Nos: 40 or 41.

Preferably the preproprotein comprises a sequence comprising,

(i) an alpha chain comprising; an alpha chain signal sequence comprising or consisting of a sequence which has at least 88% sequence identity to the sequence of SEQ ID NO: 42 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith), a variable region comprising or consisting of SEQ ID NO:7 or a variant variable region which has at least 88% sequence identity to the alpha chain variable region of SEQ ID NO: 7 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith) and constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:11 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith), and

(ii) a beta chain comprising; a beta chain signal sequence comprising or consisting of a sequence which has at least 88% sequence identity to the sequence of SEQ ID NO: 43 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith); a beta chain comprising a variable region comprising or consisting of SEQ ID NO:8 or a variable region which has at least 88% sequence identity to the beta chain variable region of SEQ ID NO: 8 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), and constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:17 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,

94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith); and further comprising

Further preferably the preproprotein comprises a sequence comprising, (i) an alpha chain comprising or consisting of a sequence which has at least 88% sequence identity to the sequence of SEQ ID NO: 14 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith), and (ii) a beta chain comprising or consisting of a sequence which has at least 88% sequence identity to the sequence of SEQ ID NO: 20 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith e.g. 100% sequence identity therewith), and (iii) a self cleaving protein sequence, optionally a 2A peptide sequence, for example selected from any one of a P2A, T2A, E2A or F2A sequence, for example any one of SEQ ID Nos: 27, 36, 37, 38; optionally comprising a linker sequence on the N-terminal of the self cleaving protein sequence / P2A sequence, for example SEQ ID NO: 39, and further optionally (iv) a Furin cleavage site sequence, for example any of SEQ ID Nos: 40 or 41.

Preferably the preproprotein comprises the alpha chain sequence situated N-terminal to the beta chain sequence, further preferably the preproprotein comprises the beta chain sequence situated N-terminal to the alpha chain sequence. Preferably the alpha chain sequence and beta chain sequence are separated from each other by the self cleaving protein sequence, optionally including the linker sequence and further optionally the furin cleavage site sequence. Optionally the preproprotein comprises a purification tag for example any of a MYC tag with amino acid sequence EQKLISEEDL (SEQ ID NO: 33), a FLAG tag with sequence DYKDDDDK (SEQ ID NO: 34) or an HA tag with sequence YPYDVPDYA (SEQ ID NO: 35).

Polynucleotides and vectors

According to a further aspect there is provided a polynucleotide or nucleic acid sequence encoding the TCR of the present invention. Such a polynucleotide or nucleic acid sequence encoding the TCR of the present invention may encode both the alpha and beta chains of a TCR of the present invention. There is also provided a polynucleotide or nucleic acid sequence encoding the alpha chain (e.g. only the alpha chain) of a TCR of the present invention. There is also provided a polynucleotide or nucleic acid sequence encoding the beta chain (e.g. only the beta chain) of a TCR of the present invention.

According to the present aspect the polynucleotide or nucleic acid sequence encoding the TCR according to the present invention comprises a nucleic acid sequence encoding the TCR in a single open reading frame or two distinct open reading frames encoding the alpha chain and beta chain respectively, the order of the sequences encoding the chains may be alpha beta or beta alpha, preferably beta alpha.

According to the present aspect, a polynucleotide or nucleotide sequence encoding the a chain of the TCR according to the invention may comprise the sequence comprising or consisting of SEQ ID NO:21. According to the present aspect, a polynucleotide or nucleotide sequence encoding the b chain of the TCR according to the invention has the sequence comprising or consisting of SEQ ID NO:22. The sequences of SEQ ID NO: 21 and SEQ ID NO:22. do not include a stop codon. A suitable stop codon e.g. TAA, TAG, TGA can typically be included at the C terminus in a construct including this sequence unless a run through translation of the sequence as part of a fusion or construct is required. According to the present aspect, a polynucleotide encoding the a chain of the TCR according to the invention may comprise a sequence which is a JIN transmembrane sequence for example SEQ ID NO:30 or which encodes a JIN transmembrane sequence for example SEQ ID NO:31.

According to the present aspect the polynucleotide may comprise a nucleic acid sequence encoding the TCR according to the invention and a heterologous promoter, for example a constitutive promoter sequence or a cytomegalovirus (CMV) promoter sequence or elongation factor 1a (EF1 alpha) promoter sequence, or other transcription control element operably linked thereto. Preferably the promoter is an EF-1 alpha promoter for example the EF-1 alpha promoter sequence can have the sequence comprising or consisting of SEQ ID NO:23. Preferably the promoter, for example an EF-1 alpha promoter, is operably linked to the nucleic acid sequence or sequences encoding the TCR of the present invention, e.g. encoding the alpha chain and/or beta chain of the invention.

According to the present aspect the polynucleotide may comprise a nucleic acid sequence comprising or encoding any one or more of (i) a 2A peptide sequence or self cleaving protein sequence, for example selected from a sequence encoding any one of a P2A, T2A, E2A or F2A sequence, for example encoding any one of SEQ ID Nos: 27, 36, 37, 38, (ii) a furin cleavable linker sequence orfurin cleavage site sequence, for example a sequence comprising or consisting of SEQ ID NO:25 or encoding either of SEQ ID Nos: 40 or 41 (iii) a WPRE mutation sequence optionally mut6WPRE, for example a sequence comprising or consisting of SEQ ID NO:29 (iv) a Kozak consensus sequence, for example a sequence comprising or consisting of either SEQ ID NO: 24 or SEQ ID NO: 32. Optionally the polynucleotide may comprise a nucleic acid sequence comprising or encoding all of (i) to (iv). Optionally the polynucleotide may further comprise a GCAC sequence or motif (e.g. cis- acting element GCAC).

According to the present aspect the self cleaving protein sequence or 2A peptide sequence is selected from a T2A, E2A, F2A, or P2A sequence, for example encoding any one of SEQ ID Nos: 27, 36, 37, 38, optionally including a GSG (Gly-Ser-Gly) linker sequence on the N-terminal of the 2A peptide sequence. Preferably the 2A peptide sequence is a P2A sequence optionally including a GSG (Gly-Ser-Gly) linker sequence on the N-terminal of the 2A peptide sequence, for example a sequence comprising or consisting of SEQ ID NO: 26 or encoding either SEQ ID NO: 27 or SEQ ID NO: 28.

According to the present aspect the polynucleotide or nucleic acid sequence encoding the TCR comprises a nucleotide sequence encoding a TCR alpha chain and a TCR beta chain wherein the alpha chain nucleotide sequence is 5’ to the beta chain nucleotide sequence (alpha-beta), or alternatively wherein the beta chain nucleotide sequence is 5’ to the alpha chain nucleotide sequence (beta-alpha), preferably beta- alpha orientation.

According to the present aspect the polynucleotide or nucleic acid sequence encoding the TCR comprises a nucleotide sequence encoding a TCR alpha chain and a TCR beta chain in which the TCR alpha chain and TCR beta chain sequences are separated from each other by a 2A peptide sequence and/or a furin cleavable linker sequence or furin cleavage site, for example SEQ ID NO:25, preferably wherein the 2A peptide sequence is a P2A sequence optionally comprising a GSG (Gly-Ser-Gly) linker sequence on the N-terminal of the 2A peptide sequence, for example a sequence comprising or consisting of SEQ ID NO: 26 or encoding either SEQ ID NO: 27 or SEQ ID NO: 28 According to the present aspect, the invention provides a polynucleotide or nucleic acid sequence encoding the TCR according to the present invention which comprises a nucleic acid sequence encoding the a chain of the TCR according to the invention comprising or consisting of SEQ ID NO:21 and a nucleic acid sequence encoding the b chain of the TCR according to the invention comprising or consisting of SEQ ID NO:22 and further comprises a EF-1 alpha promoter sequence comprising or consisting of SEQ ID NO:23 preferably operably linked thereto, a furin cleavable linker sequence or furin cleavage site sequence comprising or consisting of SEQ ID NO:25, a P2A peptide sequence comprising or consisting of SEQ ID NO: 26 or encoding either SEQ ID NO:

27 or SEQ ID NO: 28, wherein the TCR alpha chain and TCR beta chain sequences are separated from each other by the P2A peptide sequence and/or furin cleavable linker sequence or furin cleavage site, and further optionally a WPRE mutation sequence comprising or consisting of SEQ ID NO:29; optionally wherein the alpha chain nucleotide sequence is 5’ to the beta chain nucleotide sequence (alpha-beta), or alternatively wherein the beta chain nucleotide sequence is 5’ to the alpha chain nucleotide sequence (beta-alpha), preferably beta-alpha orientation. Optionally the polynucleotide may further comprise a Kozak consensus sequence, for example a sequence comprising or consisting of either SEQ ID NO: 24 or SEQ ID NO: 32.

The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to a polymeric macromolecule made from nucleotide monomers particularly deoxyribonucleotide or ribonucleotide monomers. The term encompasses polynucleotides containing known nucleotide analogues or modified backbone residues or linkages, which are naturally occurring and non-naturally occurring, which have similar properties as the reference polynucleotide, and which are intended to be metabolized in a manner similar to the reference nucleotides or are intended to have extended half-life in the system. Examples of such analogues include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Suitably the term “polynucleotide” refers to naturally occurring polymers of deoxyribonucleotide or ribonucleotide monomers. Suitably the polynucleotides of the invention are recombinant. “Recombinant” means that the polynucleotide is the product of at least one of cloning, restriction or ligation steps, or other procedures that result in a polynucleotide that is distinct from a polynucleotide found in nature (e.g., in the case of cDNA). In an embodiment the polynucleotide of the invention is an artificial polynucleotide sequence (e.g., a cDNA sequence or polynucleotide sequence with non-naturally occurring codon usage or produced recombinantly). In one embodiment, the polynucleotides of the invention are DNA. Alternatively, the polynucleotides of the invention are RNA.

DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) refer to polynucleotides having a backbone of sugar moieties which are deoxyribosyl and ribosyl moieties respectively. The sugar moieties may be linked to bases which are the 4 natural bases (adenine (A), guanine (G), cytosine (C) and thymine (T) in DNA and adenine (A), guanine (G), cytosine (C) and uracil (U) in RNA). As used herein, a “corresponding RNA” is an RNA having the same sequence as a reference DNA but for the substitution of thymine (T) in the DNA with uracil (U) in the RNA. The sugar moieties may also be linked to unnatural bases such as inosine, xanthosine, 7-methylguanosine, dihydrouridine and 5-methylcytidine. Natural phosphodiester linkages between sugar (deoxyribosyl/ribosyl) moieties may optionally be replaced with phosphorothioates linkages. Suitably polynucleotides of the invention consist of the natural bases attached to a deoxyribosyl or ribosyl sugar backbone with phosphodiester linkages between the sugar moieties.

In an embodiment the polynucleotide of the invention is a DNA, including single- or double-stranded DNA and straight-chain or circular DNA (i.e. plasmid DNA).

Due to the degeneracy of the genetic code, a large number of different, but functionally identical polynucleotides can encode any given polypeptide. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such polynucleotide variations lead to “silent” (sometimes referred to as “degenerate” or “synonymous”) variants, which are one species of conservatively modified variations. Every polynucleotide sequence disclosed herein which encodes a polypeptide also enables every possible silent variation of the polynucleotide. One of skill will recognise that each codon in a polynucleotide (except AUG, which is ordinarily the only codon for methionine, and UGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a polynucleotide that encodes a polypeptide is implicit in each described sequence and is provided as an aspect of the invention.

Degenerate codon substitutions may also be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ai, 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8:91- 98).

Codons of the polynucleotide sequences of the invention may be altered in order that sequence variants of the TCR are expressed as discussed above. In an embodiment, up to 5 codons are altered e.g. one, two or three e.g. one or two e.g. one codons are altered such that a different amino acid is encoded where the codon alteration occurs. Codon alterations may involve the alteration of one, two or three bases in the polynucleotide according to the amino acid alteration to be achieved. Suitably, codons encoding residues of the CDRs are not altered.

In an embodiment, the polynucleotides of the invention are codon optimised for expression in a human host cell, particularly, an immunoresponsive cell such as for example a T-cell.

According to a yet further aspect of the invention there is provided a vector encoding the TCR of the invention and/or comprising the polynucleotide or nucleic acid sequence according to the above aspect of the invention which encodes the TCR of the invention. Specifically, there is provided a vector for delivery of the polynucleotide to cells, particularly immunoresponsive cells, comprising a polynucleotide encoding the TCR of the invention.

As noted above, since a complete TCR comprises an a chain and a b chain, two vectors each comprising a polynucleotide encoding a chain of the TCR may be provided or a vector comprising a polynucleotide encoding both chains of the TCR may be provided. Yet further, the two chains of the TCR may be linked by a cleavable peptide linker (e.g. a linker that cleaves in a T-cell) and the vector may comprise a polynucleotide which encodes both chains of the TCR and the linker, for example a self cleaving protein sequence, optionally a 2A peptide sequence, for example selected from any one of a P2A, T2A, E2A or F2A sequence, for example any one of SEQ ID Nos: 27, 28, 36, 37, 38; optionally comprising a linker sequence on the N-terminal of the self cleaving protein sequence / 2A peptide sequence, for example SEQ ID NO: 39, and further optionally (iv) a Furin cleavage site sequence, for example any of SEQ ID Nos: 40 or 41.

The or each vector should suitably comprise such elements as are necessary for permitting transcription of a translationally active RNA molecule in the host 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.

The vector may be, for example, a viral vector such as a lentiviral vector. Other examples of viral vectors include vectors derived from g-retrovirus, 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.

A suitable polynucleotide of the invention, and which may be comprised in the vector of the invention, may encode a TCR, for example a tumour specific TCR comprising an alpha chain and beta chain according to the invention, which comprises any of:

(a) an alpha chain comprising a variable region comprising a CDR3 sequence of SEQ ID NO: 1 or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), a CDR1 sequence of SEQ ID NO:3 and CDR2 sequence of SEQ ID NO: 4 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:11 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), and a beta chain comprising a variable region comprising a CDR3 sequence of SEQ ID NO: 2 or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2 (e.g. at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith), a CDR1 sequence of SEQ ID NO: 5 and CDR2 sequence of SEQ ID NO: 6 and constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:17 (e.g. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith); or

(c) an alpha chain comprising a variable region comprising or consisting of SEQ ID NO:7 or a variant variable region which has at least 88% sequence identity to the alpha chain variable region of SEQ ID NO: 7 and a constant region which comprises or consists of a constant region of SEQ ID NO:12; and a beta chain comprising a variable region comprising or consisting of SEQ ID NO:8 or a variable region which has at least 88% sequence identity to the beta chain variable region of SEQ ID NO: 8 and constant region which comprises or consists of a constant region of SEQ ID NO:18; or

(d) an alpha chain comprising or consisting of SEQ ID NO:13 minus the N- terminal leader sequence (residues 1-20) or a variant alpha chain which has at least 88% sequence identity to the alpha chain of SEQ ID NO: 13 minus the N-terminal leader sequence and a beta chain comprising or consisting of SEQ ID NO: 19 minus the N-terminal leader sequence (residues 1-19) or a variant beta chain which has at least 88% sequence identity to the beta chain of SEQ ID NO: 19 minus the N-terminal leader sequence, or (e) an alpha chain comprising or consisting of SEQ ID NO:14 minus the N- terminal leader sequence (residues 1-20) and a beta chain comprising or consisting of SEQ ID NO: 20 minus the N-terminal leader sequence (residues 1-19).

According to the above description where a variant sequence is described as having at least 88% sequence identity to a comparator sequence SEQ ID NO:X, this means having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith. Likewise where a variant sequence is described as having at least 88% sequence identity to a comparator sequence SEQ ID NO:Y, this means having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

The polynucleotide may suitably encode an immature TCR which comprises an N- terminal leader sequence (residues 1-20 of SEQ ID NO: 13 or 14 and residues 1-19 of SEQ ID NO: 19 or 20) which N-terminal leader sequence is removed by cellular peptidases (such as signal peptidase) to produce the mature form.

A suitable vector of the invention comprises the aforementioned polynucleotide and is, for example, a TCR expression vector, for example a viral vector as disclosed above such as a lentiviral vector or such as described in Figure 2. Other examples of viral vectors include vectors derived from g-retrovirus, 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 immune cells, such as T-cells with TCR genes. Accordingly, a polynucleotide or vector of the invention, which encodes a tumour specific TCR comprising an alpha chain and beta chain according to the invention, may comprise any one or more of the following features or elements;

(a) a 3' long terminal repeat (LTR), for example a self-inactivating 3' long terminal repeat (LTR) from HIV-1,

(b) one or more stop codons, preferably 2 or 3,

(c) a 5' long terminal repeat (LTR), for example a 5' long terminal repeat (LTR) from HIV-1, preferably truncated,

(d) a nucleotide sequence encoding a TCR alpha chain according to the present invention and as herein above defined, for example a sequence comprising or consisting of SEQ ID NO:21 ,

(e) at least one ATCA motif

(f) at least one initiation codon, for example ATG>TTG motif

(g) a nucleotide sequence encoding a TCR beta chain according to the present invention and as herein above defined, for example a sequence comprising or consisting of SEQ ID NO:22,

(h) an enhancer sequence, for example a human cytomegalovirus immediate early enhancer sequence,

(i) a promoter sequence, for example a human cytomegalovirus (CMV) immediate early promoter,

(j) a polypurine tract and central terminationjs_Episequence, for example a cPPT/CTS central polypurine tract and central termination sequence of HIV-1,

(k) a start codon and associated intron, for example an EF-1 -alpha intron A - intron upstream of the start codon of human EF-1 -alpha, (L) a promoter sequence, for example a EF-1 -alpha promoter, e.g. a strong constitutive promoter for human elongation factor EF-1 -alpha, for example a sequence comprising or consisting of SEQ ID NO:23,

(m) a Furin cleavage site sequence, for example a sequence comprising or consisting of

SEQ ID NO:25,

(n) a GCAC motif,

(o) a GCTGA>ATCAT motif,

(p) a viral packaging signal, for example a packaging signal of human immunodeficiency virus type 1 HIV-1 Psi,

(q) a nucleotide sequence encoding a Jin transmembrane domain TMD, for example a sequence comprising or consisting of SEQ ID NO:30, optionally as part of the nucleotide sequence encoding a TCR alpha chain according to the present invention,

(r) a KOZAK sequence, for example a sequence comprising or consisting of SEQ ID

NO:24 or 32,

(s) a nucleotide sequence encoding a the self cleaving protein sequence / 2A peptide sequence, for example P2A ( 2A peptide from porcine teschovirus-1 polyprotein), for example a sequence comprising or consisting of SEQ ID NO:26,

(t) an origin of replication, for example a high-copy-number ColE1/pMB1/pBR322/pUC origin of replication and / or an SV40 origin of replication,

(u) a Rev response element (RRE), for example a RRE of HIV-1 ,

(v) a terminator sequence, for example a rrnG terminator, e.g. a transcription terminator from the E. coli ribosomal RNA rrnG operon,

(x) a WPRE mutant 6 motif, for example a sequence comprising or consisting of SEQ ID NO:29; preferably the polynucleotide or vector comprises each or all of the foregoing features (a) to (x) and optionally may further comprise a selectable marker, for example an antibiotic resistance marker, for example a kanamycin resistance marker. Production of TCR receptors

TCRs of the invention 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, Science, 223:1299- 1301, Sakamar and Khorana, 1988, Nucl. Acids Res., 14:6361-6372, Wells et al., 1985, Gene, 34:315-323 and Grundstrom et al., 1985, Nucl. Acids Res., 13:3305-3316) 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, Annu. Rev. Biochem. 67:99-134). 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 or fungal), 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. Other example purification tags include a MYC tag with amino acid sequence EQKLISEEDL (SEQ ID NO: 33), a FLAG tag with sequence DYKDDDDK (SEQ ID NO: 34) or an HA tag with sequence YPYDVPDYA (SEQ ID NO: 35)

The polypeptides of the invention may be produced ex vivo in immunoresponsive cells such as T-cells as discussed below.

T-cells and T-cell clones

According to the present invention there is provided a cell or immunoresponsive cell expressing or presenting a tumour-specific T-cell receptor (TCR) or a heterologous tumour-specific T-cell receptor (TCR) according to the invention. The invention further provides a cell or immunoresponsive cell harbouring the polynucleotide of the invention or the vector of the invention for example which comprises the polynucleotide of the invention.

Accordingly there is provided a cell or immunoresponsive cell harbouring the polynucleotide or the vector of the present invention wherein the cell or immunoresponsive cell expresses or presents a tumour-specific T-cell receptor (TCR) or a heterologous tumour-specific T-cell receptor (TCR) according to the invention.

The invention further provides a cell or immunoresponsive cell, wherein the cell or immunoresponsive cell is transduced with a (heterologous) polynucleotide or vector according to the invention and preferably expresses or presents a tumour-specific T-cell receptor (TCR) or a heterologous tumour-specific T-cell receptor (TCR) according to the invention. The immunoresponsive cells may be a cell of the lymphoid lineage including T cells, Natural Killer T (NKT) cells, and precursors thereof including embryonic stem cells, and pluripotent stem cells from which lymphoid cells may be differentiated. Preferably the immunoresponsive cells are T cells which can for example 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 effector memory T cells such as TEM cells and TEMRA cells, Regulatory T cells or suppressor T cells, Natural killer T cells, Mucosal associated invariant T cells, TILs (tumour infiltrating lymphocytes) and gamma-delta T cells. Preferably, the immunoresponsive cell is a T cell optionally a CD4⁺T cell or a CD8⁺T cell. Accordingly, the immunoresponsive cell may be T-cells, optionally CD4+ T cells or CD8+ T cells, or the immunoresponsive cell 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.

Accordingly, the present invention provides an immunoresponsive cell, or engineered immunoresponsive cell, preferably a T-cell which expresses or presents a TCR, or tumour specific TCR, of the invention. According to an aspect of the invention there is provided an immunoresponsive cell or engineered immunoresponsive cell, preferably a T-cell which expresses or presents a TCR of the invention, in particular which expresses or presents a TCR which comprises:

Suitably the cell is a T-cell which is a CD8+ T-cell.

In an embodiment the immunoresponsive cell or T-cell is engineered. For example, the invention provides an immunoresponsive cell or T cell wherein the cell or cells are transduced with a heterologous polynucleotide encoding the TCR of the invention or by a vector of the invention e.g. the immunoresponsive cell or T cell is transduced with a polynucleotide or vector according to the present invention, for example which encodes or expresses the tumour specific TCR of the invention. Preferably said tumour specific TCR is a heterologous tumour specific TCR, i.e. heterologous to the immunoresponsive cell or T-cell which is transduced.

In an embodiment, the cell, immunoresponsive cell or T-cell comprises a vector comprising a polynucleotide encoding the TCR of the invention and or such that, the cell, immunoresponsive cell or T-cell expresses or presents the TCR of the invention.

According to an aspect of the invention there is provided an ex vivo process comprising (i) obtaining immunoresponsive cells orT-cells from a patient, (ii) transforming or transducing the immunoresponsive cells orT-cells with a polynucleotide or heterologous polynucleotide of the invention or by a vector of the invention so that they express a tumour-specific T-cell receptor (TCR) according to the invention; and (iii) reintroducing the transformed or transduced immunoresponsive cells or T-cells into the patient. The terms transform and transduce are used and understood as equivalent herein.

There is also provided a process comprising reintroducing transduced immunoresponsive cells or T-cells into a patient wherein the transduced immunoresponsive cells or T-cells are obtained from the patient and transduced ex vivo with a polynucleotide or heterologous polynucleotide of the invention or by a vector of the invention so that they express a tumour-specific T-cell receptor (TCR) according to the invention. The patient in question suitably is a cancer patient particularly a human cancer patient, e.g. a patient or subject suffering from or having cancer.

The immunoresponsive cells or T-cells may optionally be expanded before or (more preferably) after step (ii) above. The immunoresponsive cells or T-cells may be expanded by multiple methods, e.g. by treatment with IL-2 and/or antibodies against CD3 and CD28.

In a further embodiment which is a variant of the aforementioned processes, the transduced immunoresponsive cells or T-cells which are administered to the patients were not originally from the same patient (i.e. they are allogeneic cells), e.g. may be obtained from a different subject or donor, optionally a healthy subject or donor and/or may be immunoresponsive cells or T-cells derived from stem cells obtained from a different subject or donor, optionally a healthy subject or donor.

The immunoresponsive cells or T-cells that are introduced into the patient after transformation may be polyclonal or monoclonal. In the latter case, a particular transduced clone is selected for expansion before administering the cells to the patient.

There is also provided a method of treatment of cancer comprising administering to a patient in need thereof transduced immunoresponsive cells or T-cells wherein the transduced cells are immunoresponsive cells or T-cells which have been obtained from the patient and transduced ex vivo with a polynucleotide or heterologous polynucleotide of the invention or by a vector of the invention so that they express a tumour-specific T- cell receptor (TCR) according to the invention. There are also provided transduced immunoresponsive cells orT-cells for use in the treatment of cancer wherein the transduced cells are immunoresponsive cells orT-cells which have been obtained from the patient and transduced ex vivo with a polynucleotide or heterologous polynucleotide of the invention or by a vector of the invention so that they express a tumour-specific T-cell receptor (TCR) according to the invention.

There is also provided use of the aforementioned transduced immunoresponsive cells or T-cells in the manufacture of a medicament for the treatment of cancer.

Preferably the cancer is selected from any one of colorectal, lung cancer (including non small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), kidney, prostate, bladder, cervical, melanoma (skin), bone, breast (including metastatic breast cancer), ovarian, blood cancer, myeloma, acute lymphoblastic leukaemia (ALL), melanoma, glioblastoma, or cholangiocarcinoma. Preferably the cancer is selected from any one of colorectal, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), melanoma (skin), breast (including metastatic breast cancer), ovarian, acute lymphoblastic leukaemia (ALL), glioblastoma, or cholangiocarcinoma. A further example cancer is brain cancer e.g. astrocytoma.

As noted above, since a complete TCR comprises an a chain and a b chain, the T-cells may (i) be transduced with two polynucleotides each encoding a chain of the TCR or with two vectors each comprising a polynucleotide encoding a chain of the TCR or (ii) may be transduced with a polynucleotide (e.g. a transposon) encoding both chains of the TCR or with a vector comprising a polynucleotide encoding both chains of the TCR. Yet further, the two chains of the TCR may be linked by a cleavable peptide linker (e.g. a linker that cleaves in a T-cell) and so the immunoresponsive cells or T-cells may be transduced with a polynucleotide which encodes both chains of the TCR and the linker or with a vector comprising a polynucleotide which encodes both chains of the TCR and the linker such that the two chains of the TCR are produced in the immunoresponsive cells orT-cells.

In an embodiment, expression of the endogenous TCR in the immunoresponsive cell or T-cell is inhibited or prevented. Various methods can be employed to silence the genes encoding the a and b chains of the endogenous TCR. For example, the genes encoding the endogenous TCR may be silenced at the RNA level by an siRNA approach. Thus, an inhibitory or interfering RNA, such as a short hairpin RNA or other double stranded RNA comprising a region complementary to the gene to be silenced (e.g. comprising a sequence of 18-25 preferably 20-25 based paired nucleotides having complementarity to a region in the gene to be silenced) may be administered to the immunoresponsive cells or T-cells. The same vector to that of the TCR or a different one may be used. Alternatively, the genes encoding the endogenous TCR may be deleted or disrupted e.g. using a CRISPR approach. Thus, the elements of CRISPR/Cas9 including the Cas9 enzyme and an appropriate guide RNA selected for disruption or deletion of the genes in question may be administered to the T-cells, for example electroporation of a complex containing the Cas9 enzyme and the guide RNA. Alternatively, the Cas9 enzyme and the guide RNA components could be transduced with a vector.

Compositions

The invention provides a pharmaceutical composition comprising the TCR, polynucleotide, vector, cell or immunoresponsive cell of the invention and a pharmaceutically acceptable carrier. Suitably the pharmaceutical composition is formulated under sterile conditions and is suitable for parenteral administration. For parenteral administration, the carrier preferably comprises water and may contain buffers for pH control, stabilising agents e.g., surfactants and amino acids and tonicity modifying agents e.g., salts and sugars.

Cancer treatment

In a preferred embodiment the TCR, polynucleotide, vector, cell or immunoresponsive cell of the invention or pharmaceutical composition according to the invention is used to treat cancer, e.g. in a patient in need thereof, e.g. a cancer patient particularly a human cancer patient, e.g. a patient, individual or subject suffering from or having cancer, particularly any cancer selected from: colorectal, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), kidney, prostate, bladder, cervical, melanoma (skin), bone, breast (including metastatic breast cancer), ovarian, blood cancer, myeloma, acute lymphoblastic leukaemia (ALL), melanoma, glioblastoma, cholangiocarcinoma, liver, thyroid, endometrial, esophageal or head and neck cancer. A further example cancer to be treated is brain cancer e.g. astrocytoma.

According to a yet further aspect of the invention there is provided the use of the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention to treat cancer.

According to a yet further aspect of the invention there is provided a method of treating cancer comprising administering the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention to an individual or subject to be treated. For example in an individual or subject (patient) in need thereof, e.g. suffering from or having cancer.

More generally, there is provided a method of treating cancer in a subject comprising administering a therapeutically effective amount of the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention to the subject. For example e.g. in an individual or subject (patient) in need thereof, e.g. suffering from or having cancer.

Except where the context indicates otherwise, reference herein to “tumour” includes a reference to “cancer”.

The cancer may include solid tumours and/or blood cancers, but in particular colorectal, lung (including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), kidney, prostate, bladder, cervical, melanoma (e.g. skin melanoma), bone, breast (including metastatic breast cancer), ovarian or blood cancer (e.g. leukemia), myeloma, acute lymphoblastic leukaemia (ALL), glioblastoma, cholangiocarcinoma, liver, thyroid, endometrial, esophageal or head and neck. A further example cancer is brain cancer e.g. astrocytoma.

According to a yet further aspect of the invention there is provided the use of the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention in the manufacture of a medicament to treat cancer. More generally, there is provided use of a TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention in the manufacture of a medicament to treat cancer.

There is also provided the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention for use in the treatment of cancer.

Equally well, it is also envisaged that all embodiments of the present invention that may be used to treat cancer may potentially be used to prevent cancer. Thus, corresponding methods and uses and substances for use to prevent cancer are provided as an aspect of the invention.

Combinations

According to a yet further aspect of the invention there is provided a combination therapeutic for the treatment of cancer comprising: a) the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention in combination with b) a further cancer therapeutic agent.

More generally, there is provided a pharmaceutical composition comprising: a) the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention; and b) an anti-tumour agent.

Alternatively, the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention may be administered separately, simultaneously or sequentially with an anti-tumour agent.

According to a yet further aspect of the invention there is provided a method of treating cancer comprising administering the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention separately, simultaneously or sequentially with an anti-tumour agent to an individual or subject to be treated. For example in an individual or subject (patient) in need thereof, e.g. suffering from or having cancer.

More generally, there is provided a method of treating cancer in a subject comprising administering a therapeutically effective amount of the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention separately, simultaneously or sequentially with an anti-tumour agent to the subject. For example e.g. in an individual or subject (patient) in need thereof, e.g. suffering from or having cancer.

According to a yet further aspect of the invention there is provided the use of the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention in the manufacture of a medicament to treat cancer wherein said medicament is for administration separately, simultaneously or sequentially with an anti-tumour agent.

There is also provided the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention for use separately, simultaneously or sequentially with an anti-tumour agent in the treatment of cancer.

According to a yet further aspect of the invention there is provided the use of the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention separately, simultaneously or sequentially with an anti-tumour agent to treat cancer.

Further cancer therapeutic agents/anti-tumour agents that may be included in a combination therapy include immune check point inhibitors e.g. selected from PD-1 inhibitors, such as pembrolizumab, (Keytruda) and nivolumab (Opdivo), PD-L1 inhibitors, such as atezolizumab (Tecentriq), avelumab (Bavencio) and durvalumab (Imfinzi) and CTLA-4 inhibitors such as ipilimumab (Yervoy), other immune stimulants such as interferons (e.g. interferon a, b or y), steroids e.g. prednisolone and alkylating agents such as platinum-based anti-neoplastic agents e.g. cisplatin, carboplatin and oxaliplatin. In a preferred method of the invention the TCR, polynucleotide, vector, cell or immunoresponsive cell or pharmaceutical composition according to the invention is administered in combination with an anti-tumour agent such as, but not limited to, a bispecific.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprised or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Sequences and Figure Legends

CAYRSAVNARLMF (SEQ ID NO: 1) alpha chain CDR3

CASSEARGLAEFTDTQYF (SEQ ID NO: 2); beta chain CDR3 TSESDYY (SEQ ID NO:3); CDR1 alpha.

ATEN (SEQ ID NO:4); CDR2 alpha.

MGHDK (SEQ ID NO:5); CDR1 beta.

SYGVNS (SEQ ID NO:6); CDR2 beta. AQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQ

NATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRSAVNARLMFGDGTQLVVKP

(SEQ ID NO:7) Alpha Chain variable region, CDRs (bold and underlined).

EADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKG DLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEARGLAEFTDTQYFGPGTRLTVL (SEQ ID NO:8) Beta Chain variable region, CDRs (bold and underlined).

NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSV IGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:9) Human Alpha Chain constant region sequence

NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSV IGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:10) Human Alpha Chain constant region variant sequence

NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKS NGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLR ILLLKVAGFNLLMTLRLWSS (SEQ ID NO:11) Murine Alpha Chain constant region sequence

NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKS NGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRIL LLKVAGFNLLMTLRLWSS (SEQ ID NO:12) Murine Alpha Chain constant region variant sequence (LVIV amino acids 112-115)

MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWY

KQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYR

SAVNARLMFGDGTQLWKP NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTME SG T FITDKTVLDMKA MDSKSNGA I A WSNQT SFTCQDIFKETNA TYPSSD VPCDA TLT EK SFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:13) TCR Alpha Chain (variable and constant regions) comprising the murine alpha chain constant region sequence (italics), CDRs (bold and underlined), signal sequence (underlined)

MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWY KQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYR SAVNARLMFGDGTQLVYKP NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTME SG TFIT DKTVLDMKA MDSKSN GA I A WSNQ TSFTCQDIFKETNA TYPSSD VPCDA TL TEK SFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID N0:14) TCR Alpha Chain (variable and constant regions) comprising the murine alpha chain constant region variant sequence (italics), CDRs (bold and underlined), signal sequence (underlined)

EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVST

DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRA

KPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVK

RKDSRG

SEQ ID NO:15) Human Beta Chain constant region sequence

EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCT DPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRA KPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVK RKDSRG (SEQ ID NO:16) Human Beta Chain constant region variant sequence

EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVST DPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVT QNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLWMAMVKRKNS SEQ ID NO:17) Murine Beta Chain constant region sequence EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVST DPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVT QNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO:18) Murine Beta Chain constant region variant sequence

MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDP GMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEAR G LAEFJDJQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKA TL VCLARGFFPD HVELSWWVNGKEVHSG VSTDPQA YKESNYSYCLSSRLRVSA TFWHNPRNHFRCQ V QFHGLSEEDKWPEGSPKPVTQNISAEA WGRADCGITSASYQQG VLSA TIL YEILLGKA T L YA VL VSTL WMAMVKRKNS

(SEQ ID N0:19) TCR Beta Chain, CDRs (bold and underlined) including murine beta chain constant region sequence (italics), signal sequence (underlined).

MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDP GMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEAR G LAEFJDJQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKA TL VCLARGFFPD HVELSWWVNGKEVHSG VSTDPQA YKESNYSYCLSSRLRVSA TFWHNPRNHFRCQ V QFHGLSEEDKWPEGSPKPVTQNISAEA WGRADCGITSASYHQG VLSA TIL YEILLGKA T L YA VL VSGL VLMAMVKKKNS

(SEQ ID NO:20) TCR Beta Chain, CDRs (bold and underlined) including murine beta chain constant region variant sequence (italics), signal sequence (underlined)

ATGGCTTGTCCTGGATTTCTGTGGGCCCTCGTGATCAGCACCTGTCTGGAATTCAG

CATGGCCCAGACCGTGACACAGTCCCAGCCTGAGATGTCTGTGCAAGAGGCCGA

GACAGTGACCCTGAGCTGCACCTACGATACCAGCGAGAGCGACTACTACCTGTTC

TGGTACAAGCAGCCTCCTAGCCGGCAGATGATCCTGGTTATCAGACAAGAGGCCT

AT AAG C AG C AG AAC G C C AC C G AG AAC AG ATT C AG C GT G AACTT C C AG AAG G C C G C

CAAGAGCTTCAGCCTGAAGATCAGCGATAGCCAGCTGGGCGACGCCGCCATGTAC

TTTTGCGCCTATAGAAGCGCCGTGAACGCCCGGCTGATGTTTGGAGATGGCACAC

AGCTGGTGGTCAAGCCCAACATTCAGAACCCTGAGCCTGCCGTGTACCAGCTGAA GGACCCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGATTTCGACAGCCAG AT C AAC GT G C C C AAG AC C AT G G AAAG C G G C AC CTT CAT C AC C G AC AAG AC AGT G C T G GAC AT G AAG G C CAT G G AC AG C AAG AG C AAC G G C G C C ATT GCCTGGTC C AAC C A GACCAGCTTCACGTGCCAGGACATCTTCAAAGAGACAAACGCCACCTATCCTAGC AGCGACGTGCCCTGTGATGCCACACTGACCGAGAAGTCCTTCGAGACAGACATGA AC CT GAATTT C C AG AAC CTG CT C GT GAT C GTG CT G AG AAT C CTG CT GCTG AAG GT G GCCGGCTTCAACCTGCTGATGACACTGAGACTGTGGTCCTCC (SEQ ID N0:21)

TCR Alpha Chain, including murine alpha chain constant region variant sequence.

ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGA

TGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAGAA

AAT C AC C CT GG AAT G C AGC C AG AC C AT G G G C C AC GAC AAG AT GTACTG GTATC AG

CAGGACCCCGGCATGGAACTGCACCTGATCCACTACAGCTACGGCGTGAACAGCA

CCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGC

ACTTCCCACTGACACTGGAAAGCGCTAGACCCAGCCACACCAGCCAGTACCTGTG

TGCCTCTTCTGAGGCCAGAGGCCTGGCCGAGTTTACCGACACACAGTACTTTGGC

CCTGGCACCAGACTGACCGTGCTGGAAGATCTGAGAAACGTGACCCCTCCTAAGG

TGTCCCTGTTCGAGCCTAGCAAGGCCGAGATCGCCAACAAGCAGAAAGCCACACT

CGTGTGTCTGGCCAGGGGCTTCTTTCCCGATCACGTGGAACTGTCTTGGTGGGTC

AACGGCAAAGAGGTGCACAGCGGCGTCAGCACAGATCCCCAGGCCTACAAAGAG

AGCAACTACTCCTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGC

ACAACCCCAGAAACCACTTCAGATGCCAGGTCCAGTTTCACGGCCTGAGCGAAGA

GGACAAGTGGCCTGAGGGCTCTCCCAAGCCTGTGACACAGAATATCTCTGCCGAA

GCCTGGGGCAGAGCCGATTGTGGAATTACCAGCGCCAGTTACCACCAGGGCGTG

CTGTCTGCCACAATCCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGTACGCCG

TGCTGGTGTCTGGCCTGGTCCTGATGGCCATGGTCAAGAAGAAGAACAGC (SEQ

ID NO:22) TCR Beta Chain, including murine beta chain constant region variant sequence

GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAG

TTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTA

AACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAG AACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCC

GCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGG

GTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTT

GATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAA

GGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGC

CGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTC

TAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTC

TTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGG

CGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGC

GAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTC

TGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGG

CCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTG

CAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA

C C C AC AC AAAG G AAAAG G G C CTTT C C GTC CT C AG CCGTCGCTT CAT GT G ACT CCA

CGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTAC

GT C GTCTTT AG GTT G G G G G GAG G G GTTTT ATG C GAT G G AGTTT C C C C AC ACT GAG

TG G GTG GAG ACT G AAGTT AG G C C AG CTTG G C ACTT GAT GT AATT CTC CTTG G AATT

TGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG

TTTTTTTCTT C C ATTT CAGGTGTCGT G A (SEQ ID NO:23) EF1 -alpha promoter

ATCAGCCGCCACC (SEQ ID NO:24) KOZAK including ATCA sequence

CGGGCCAAGAGA (SEQ ID NO:25) Furin cleavage site sequence

TCTGGATCTGGCGCCACCAACTTCTCCCTCCTCAAGCAGGCCGGCGACGTCGAGG AGAACCCCGGCCCC (SEQ ID NO:26) P2A sequence

ATNFSLLKQAGDVEENPGP (SEQ ID NO:27) P2A sequence

GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:28) P2A with N-terminal GSG

AAT C AAC CT CTG GATT AC AAAATTT GT G AAAG ATT G ACT G GT ATT CTTAACTATGTT GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCT TCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATG

AGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGA

CGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACT

TTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCC

GCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGG

GGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGC

GGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCC

GCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGAC

GAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO:29) WPRE mut 6

CTCGTGATCGTGCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGA TGACACTG (SEQ ID NO:30) JIN Transmembrane Sequence

LVIVLRILLLKVAGFNLLMTL (SEQ ID N0:31) JIN Transmembrane Sequence

GCCGCCACC (SEQ ID NO:32) KOZAK lacking ATCA sequence

EQKLISEEDL (SEQ ID NO: 33), MYC tag

DYKDDDDK (SEQ ID NO: 34), FLAG tag

YPYDVPDYA (SEQ ID NO: 35), HA tag.

EGRGSLLTCGDVEENPGP (SEQ ID NO:36) T2A, self cleaving protein sequence. QCTNYALLKLAGDVESNPGP (SEQ ID NO:37) E2A, self cleaving protein sequence. VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:38) F2A, self cleaving protein sequence. GSG (SEQ ID NO:39) Linker RAKR (SEQ ID NO:40) Furin cleavage site

RX(R/K)R (SEQ ID NO:41) Furin cleavage site, where X is any naturally occurring amino acid & only a single residue and where R/K indicates that residue R or K are interchangeable at the represented position..

MACPGFLWALVISTCLEFSM (SEQ ID NO:42) signal sequence TCR alpha chain MTIRLLCYMGFYFLGAGLM (SEQ ID NO:43) signal sequence TCR beta chain

RTHSLRYFRL GVSDPIHGVP EFISVGYVDS HPITTYDSVT RQKEPRAPWM AENLAPDHWE RYTQLLRGWQ QMFKVELKRL QRHYNHSGSH TYQRMIGCEL LEDGSTTGFL QYAYDGQDFL IFNKDTLSWL AVDNVAHTIK QAWEANQHEL LYQKNWLEEE CIAWLKRFLE YGKDTLQRTE PPLVRVNRKE TFPGVTALFC KAHGFYPPEI YMTWMKNGEE IVQEIDYGDI LPSGDGTYQA WASIELDPQS SNLYSCHVEH CGVHMVLQVP QESETIPLVM KAVSGSIVLV IVLAGVGVLV WRRRPREQNG AIYLPTPDR [SEQ ID NO: 44], polypeptide sequence of mature wildtype human MR1 polypeptide.

LVIV (SEQ ID NO:45) Alpha Chain constant region variant sequence

Figure Legends

Figure 1 : The lentiviral construct (pELNS) insert for expression of the non-optimised / human 7G5 TCR construct with human constant regions, V alpha SEQ ID NO:7, V beta SEQ ID NO:8, C alpha SEQ ID NO:9, C beta SEQ ID NO:15.

Figure 2: BA Mur Jin 7G5 TCR cassette in pSF-lenti plasmid with optimised murinised constant regions, V alpha SEQ ID NO:7, V beta SEQ ID NO:8, C alpha SEQ ID NO:12,

C beta SEQ ID NO:18.

Figure 3: 7G5 TCR surface expression on primary T cells for construct variants

Figure 4: IFNy secretion by transduced CD3+ cells in response to antigen positive cells

Figure 5. Auto-reactivity data for optimised 7G5 TCR cassette provides no evidence for altered specificity of significant TCR mis-pairing - [A] Donor 34 IFN gamma endpoint, [B] Donor 35 IFN gamma endpoint, [C] Donor 35 TNF alpha endpoint.

Figure 6. Summary of cassette results against selection criteria tested across three T- cell donors for cassette transduction, donors 33, 34, 35.

Figure 7: shows data from an IFNy ELISA evaluating effective activation of a 7G5 TCR BA-Mur-Jin TCR-T when co-cultured for 48 hours with a variety of cancer cells including the lung cancer cell line A549, B-cell precursor Acute lymphoblastic leukaemia cell (ALL-EB1), a low passage, primary-like lung cancer cell line “Lung-EB1” and the breast cancer cell line “Breast-EB1”. This contrasts with the lack of T-cell activation against A549 cells where MR1 polypeptide has been knocked out by CRISPR (A549.MR1.KO).

Figure 8 Mouse tumour disease model and treatment with optimised construct 7G5 BA- Mur-Jin transduced T cells [A] Two T-cell donor data for 7G5-BA-Mur-Jin 0.5E7 / 1 E7 dose (NTD = non=transduced) measuring tumour volume vs days post implant [B] Two T-cell donor data for 7G5-BA-Mur-Jin 0.5E7 / 1E7 dose (NTD = non=transduced) measuring mean bodyweight vs days post implant. D41 , D43 indicates donor 41 and 43 respectively.

Other features of the present invention will become apparent from the following examples. Unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose

EXAMPLES

In the following section the term 7G5 indicates a TCR having the variable region alpha and beta sequences of SEQ ID NO: 7 and 8 respectively. The different TCR constructs produced in this section vary in the nature of the alpha and beta chain constant region sequences which in the case of the non-optimised 7G5 construct are of human origin (for example SEQ ID NO: 9 or 10 and 15 or 16) or in the optimised 7G5 are murinised (for example SEQ ID NO: 11 or 12 and 17 or 18) and may contain a JIN sequence (SEQ ID NO: 31) in the C alpha region, the orientation of the alpha and beta chains in the encoding constructs is also varied.

Example 1 - Non-optimised 7G5 TCR construct, T-cell transduction

Previous studies disclosed in patent application PCT/GB2018/053045 published as WO201 9081902 described a TCR targeting MR1 expressing cancer cells, identified in T-cell clone MC.7.G5, and comprised of a TRAV38.2/DV8 TRAJ31 a-chain paired with a TRBV25.1 TRBJ2.3 b-chain. Purified T-cells from the PBMCs of Stage IV melanoma patients were lentivirally transduced with the 7G5 TCR, which resulted in recognition and killing of autologous and non-autologous melanomas, but not healthy cells. The killing was specific to MR1 as the 7G5 TCR transduced cells did not lyse MR1 knockout melanomas.

For the purposes of T-cell transduction the 7G5 TCR was synthesised with full length alpha and beta TCR chains (comprising variable alpha region SEQ ID NO:7 + constant human alpha region SEQ ID NO:9, variable beta region SEQ ID NO:8 + constant human beta region SEQ ID NO: 15) separated by a self-cleaving 2A sequence. The TCR was cloned into the third generation pELNS lentiviral vector (Figure 1) which contains rCD2 separated from the TCR by a second 2A self-cleavage sequence. Lentiviral particles were generated by calcium chloride transfection of HEK293T cells and concentrated by ultra-centrifugation. Post therapy PBMCs were obtained from TIL patients MM909.1 1 and MM909.24 and CD8 and CD4 T cells purified by magnetic enrichment (Miltenyi Biotec). T cells were subsequently activated by overnight incubation with CD3/CD28 beads (Dynabeads; Life Technologies) at a 3: 1 bead-to-T-cell ratio. T-cells were then transduced with the 7G5 construct TCR in the presence of 5 pg/mL polybrene (Santa Cruz Biotechnology). T cells that had taken up the virus were magnetically enriched with anti-rCD2 antibody and anti-PE magnetic beads, according to manufacturer's instructions (Miltenyi Biotec). 14 days post transduction, T cells were expanded by standard methodology. Transduced T cells were shown to be functionally active as described above however the level of T-cell surface expression of the non optimised 7G5 TCR, determined as frequency of the TCR positive cells quantified by flow cytometric v-beta staining using antibody specific for the TCRV-beta subunit, was demonstrated to be relatively low.

Example 2 - Optimised 7G5 TCR constructs

7G5 TCR cassette variants were cloned into a pSF-lenti transfer plasmid backbone (Figure 2), which was modified to include an EF-1 alpha promoter. The TCR cassette was designed to contain the TCR chains from the MR1 -specific 7G5 clone in beta-alpha order with a P2A and furin cleavable linker, a JIN transmembrane mutation (e.g. comprising the LVIV sequence (SEQ ID NO:45), see SEQ ID NO:31) to enhance hydrophobicity and constant domains murinised to improve TCR pairing and surface expression. The same basic vector system was used for variant constructs with different chain orientations or lacking the murinisation or Jin sequence.

Example 3 - TCR surface expression on primary T cells for optimised 7G5 construct variants

CD3+ T-cells were magnetically isolated from healthy donor PBMCs and activated and expanded in the presence of CD3/CD28 Dynabeads. One day later, cells were transduced with comparable virus particle number based on p24 measurement for the different variants as indicated: AB Mur Jin, BA Mur, BA Jin, and BA Mur Jin, where the first two letters indicate the orientation of the TCR alpha and beta chains in the plasmid, and Mur stands for murinisation of the constant domains and Jin indicating a respective transmembrane mutation in the alpha constant region. TCR expression was assessed 9 days later by staining with a Vp-specific antibody and Flow-Cytometry analysis (Figure 3).

Conclusions: Transduction experiments achieved improved levels of TCR expression. T-cells transduced with the BA_Mur_Jin cassette display the highest TCR levels on their membrane of between 40 and 50%. TCR expression of BA_Mur and AB_Mur_Jin is comparable. T-cells transduced with BA_Jin constructs express lowest TCR levels on their membrane comparable to the non-optimised 7G5. It was also found that viral copy number (VCN) did not exceed VCN values of 2 at the highest transduction concentration of lentiviral vector (LVV) indicating, this is well within recognised safe therapeutic values.

Example 4 - IFNy secretion by transduced CD3+ cells in response to antigen positive cells

T cells were isolated from healthy donor, transduced with TCR constructs BA Mur Jin or BA Mur, and expanded as previously described. 7G5 TCR+ and non-transduced T cells were then co-cultured with MR1- expressing or MR1 knock out A549 cells. IFNy- secretion was measured by ELISPOT. T cells only and in presence of anti-CD3/CD28 stimulation were included as negative and positive controls (Figure 4). Conclusions: Clear and specific IFNy response against target cells was detected which is most apparent for the BA Mur Jin construct. Additionally, there was no detectible cytokine secretion above background (untransduced T-cells) detected when TCR-T cell were co-cultured with autologous PBMCs indicating TCR safety.

Example 5: Demonstrating lack of optimised TCR construct autoreactivity

To test for off-target autoreactivity potentially induced by TCR mis-pairing, an autoreactivity assay was performed. 50,000 PBMC from the same donor used to produce TCR-T were incubated with TCR-T at a 1:1 and 2:1 effectontarget ratio followed by IFNg and TNFa ELISpot. In this experiment the number of VB25+ cells measured on day 10 after transduction was taken as a reference. This was used to calculate the ratio between the cassette with the higher VB25 positivity (ba Mur Jin) and the one with the lower VB25 positivity (ba Mur) in each donor and the corresponding number of untransduced cells to add to the ba Mur Jin sample in order to equalize the % of VB25+ cells in the BA Mur sample. No IFNy or TNFa was observed above background (PBMC alone or untransduced UT) for any transduced T cells tested [Figure 5]

Conclusions:

The autoreactivity assay demonstrated with 2 donors shows no evidence for cytokine release above background when transduced cells co-cultured with autologous PBMC. The data supports broad specificity and lack of significant mis-pairing with the TCR cassette designs and particularly for the ba Mur Jin construct, this indicates safety as a therapeutic construct.

Example 6: Viral Copy Number Determination

Lentiviral vector (LVV) for each construct BA_Jin, BA_Mur and BA_Mur_Jin was titrated across 3 donor T-cell samples at 7.5E6 CD8+ T-cells and viral copy number (VCN) was determined as well as surface TCR expression level.

Viral Copy Number (VCN) was determined using a qPCR/dPCR method. This method amplifies, in the same reaction, both a virus structural gene or the transgene of interest and a single copy gene of the human genome (2 copies per diploid genome). Following amplification of both genes in the same reaction the copy number of each gene in the starting material can be determined. A TaqMan probe based dPCR method was used to determine VCN by amplifying the viral packaging signal sequence (Psi) and a region of the single copy human gene Ribonuclease P protein subunit p30 (RPP30) in a multiplex reaction. The probe against Psi was conjugated with FAM for detection in the green channel, whereas the probe for RPP30 was conjugated with HEX for detection in the yellow channel in a multiplex assay. The dPCR reaction was setup using 0.8 mM forward primer, 0.8 mM reverse primer, 0.4 pM probe and 100 ng total DNA input per well in a 8.5K partition 96 well nanoplate using the QIAcuity dPCR platform from Qiagen. At the end of the thermal cycling phase, plates were imaged using the green and yellow channels, and the number of partitions that contain the target genes (Psi and/or RPP30) were designated as positive partitions (p) and partitions that do not have the target genes were designated as negative partitions. The number of positive and negative partitions were counted and used in Poisson statistics to estimate the concentration of Psi and/or RPP30 copies per positive partition, at 95% confidence interval, using the following equation:

Target copies per partition = -ln(1-p)ⁿ

The copy number of each of the target genes in the starting material (copies/mI) was then calculated from the total number of positive partition and the volume of each partition (pre defined volume).

By taking the 2 RPP30 copies per diploid human genome into consideration, the VCN/cell was calculated from the ratio between the copy number of Psi gene/mI and the copy number of RPP30/pl

Psi copies/mΐ

VCN / cell = 2x RPP30 copies/mΐ

VCN was normalized with % transduction efficiency, determined using flow cytometry, to establish VCN/ transduced cell. VCN

VCN / transduced cell = - : - — - — — xlOO transduction efficiency (%)

Conclusions: All TCR cassettes had VCN values of <2 at highest transduction concentration of LVVs. BA-Jin, BA_Mur and BA_Mur_Jin, VCN values were comparable at highest LVVs concentrations, however BA_Mur_Jin remained the optimal cassette for the 7G5 TCR based on the VCN data, BA_Mur_Jin yielded a comparable higher surface TCR expression and slightly higher VCN whilst still at the safe level of VCN value of <2 [Figure 6]

Example 7 - IFNY secretion by transduced Donor T-cells cells in response to a range of cancer cell lines

The reactivity of the optimised MR1 -restricted TCR-T cell products transduced with the optimised 7G5 TCR BA-Mur-Jin construct, was tested against a range of cancer cell lines including non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), Breast cancer, Melanoma, Ovarian cancer, Colorecta cancer, Acute lymphoblastic leukaemia (ALL), Cholangiocarcinoma and Glioblastoma [Figure 7]

Standard functional assays assessing T-cell activation and T-cell-mediated killing were utilised to characterise the reactivity of the optimised cancer-targeting MR1 -restricted TCR-T cell product expressing the 7G5 TCR BA-Mur-Jin construct. In such assays, the MR1 TCR-T was co-cultured with cancer cell lines for up to 72 hr. Specific activation of the 7G5 TCR BA-Mur-Jin TCR-T cells was assessed by production of IFNy by ELISA. Specific activation of TCR-T cell products as evaluated by IFNy release is known to be highly correlated with other functional readouts such as T-cell mediated in vitro killing of cancer cell lines, as well as in vivo efficacy.

T cells were isolated from two healthy donors, Donor 1 and Donor 2, the cell populations were divided for transduction or for use as comparator non-transduced controls.

The isolated T-cells were transduced with the optimised 7G5 TCR BA-Mur-Jin construct, activated and expanded in the presence of CD3/CD28 Dynabeads (Human T- activator, 3:1 bead:cell ratio). 7G5 TCR BA-Mur-Jin transduced + and non-transduced T cells were then co-cultured for 48 hours with MR1- expressing or MR1 knock out A549 cells which were included as controls. IFNY-secretion was measured by ELISPOT, T cells only were included as negative controls. Levels of recorded IFNY-secretion above the T-cell only control level are indicative of specific response to the cancer cell tested in Figure 7 (above the dotted line).

Conclusions: Clear and specific 7G5 TCR BA-Mur-Jin TCR-T cell IFNy response against target cells was detected which is most apparent for the, ALL-EB1 , Breast-EB1 , Lung-EB1 and A549-Lung cancer cells. Additionally, there was no detectible cytokine secretion above background (untransduced T-cells) detected when TCR-T cell were co cultured with autologous PBMCs indicating TCR safety for therapeutic treatment. The data from both donors supports the utility of in the treatment of 7G5 TCR BA-Mur-Jin TCR-T in the treatment of cancer, including non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), Breast cancer, Melanoma, Ovarian cancer, Colorecta cancer, Acute lymphoblastic leukaemia (ALL), Cholangiocarcinoma and Glioblastoma, [Figure 7] Levels of recorded IFNY-secretion above the T-cell only control level were also recorded indicative of TCR specific response to a brain cancer astrocytoma cell line tested (data not shown).

Example 8 In-Vivo efficacy of 7G5 BA-Mur-Jin in a solid tumour model

Malignant melanoma cell line A375 (ATCC CRL-1619IG-2) was engineered to overexpress MR1 and beta-2-microglobulin, (A374-MR1 cells). T cells were obtained from two donors Donor 41 and Donor 43, donor T cell populations were divided into non-transduced populations and transduced populations which were transduced with the optimised 7G5-BA-Mur-Jin cassette construct (BA-Mur-Jin).

56 NSG {NOD. Cg-Prkdcscidll2rgtm1 WjllSzJ) mice aged 6 to 8 weeks were subcutaneously (s.c.) implanted with 5x10⁶ A375-MR1 cells on Day 0. On Day 1 mice were randomised into 7 groups of 8 animals. Mice in Group 1 received vehicle (saline) and mice in Groups 2 and 3 received 2.3x10⁷ non-transduced T cells from Donor 41 and Donor 43, respectively, via intravenous injection. Mice in Groups 4 and 6 received 1.0x10⁷ (2.3x10⁷ total T cells) or 0.5x10⁷ (1.15 x 10⁷ total T cells) Donor 41 7G5-BA- Mur-Jin-positive T cells, respectively, and mice in Groups 5 and 7 received 1.0x10⁷ (2.3x10⁷ total T cells) or 0.5x10⁷ (1.15 x 10⁷ total T cells) Donor 437G5-BA-Mur-Jin- positive T cells, respectively, via intravenous injection. Mice were weighed and tumour volume was measured using callipers three times weekly. Mice were removed from the study when the tumour volume was >1000mm3, the tumour measured more than 15mm in any direction, was classed as ulcerated, if there was only one mouse left in the group or when the study ended on Day 33.

Results: No adverse effects or weight loss were observed after T cell injection on Day 0, regardless of type of T cells injected. All mice in all groups gained weight over the duration of the study [Figure 8B] Mice treated with Donor 41 or 437G5-BA-Mur-Jin T cells at either of the concentrations used, showed a significantly improved overall survival compared to Donor 41 and Donor 43 non-transduced T cells, respectively. Six mice from Group 4 (D41 7G5-BA-Mur-Jin 1.0E7), four mice from Group 6 (D41 7G5-BA- Mur-Jin 0.5 E7) and all mice in Groups 5 and 7 (D437G5-BA-Mur-Jin 1.0E7 and D43 7G5-BA-Mur-Jin 0.5E7, respectively), were remaining when the study was terminated on Day 33.

Treatment with Donor 41 or 437G5-BA-Mur-Jin transduced T cells at either of the concentrations used, resulted in reduced A375-MR1 tumour growth [Figure 8A] From Day 9 until the study ended on Day 33, mice treated with the low (0.5x10⁷) or high (1.0x10⁷) dose of Donor 437G5-BA-Mur-Jin transduced T cells (Groups 7 and 5, respectively), showed a statistically significant reduction in tumour growth compared to Donor 43 non-transduced (NTD) treated mice. Furthermore, the tumour volume for mice treated with the low or high dose of Donor 41 7G5-BA-Mur-Jin transduced T cells was significantly reduced from Day 14 until Day 21 (low dose) or Day 26 (high dose) compared to mice receiving non-transduced T cells from Donor 41. In contrast, all untreated control animals (Group 1: A375-MR1) and mice treated with non-transduced T cells from donor 41 (Group 2: D41 NTD) and donor 43 (Group 3: D43 NTD) had been removed from the study by Day 21. At this point, all mice in Groups 4-7 remained in the study and 5 tumours in Group 4 (D41 7G5-BA-Mur-Jin 1.0E7), 7 tumours in Group 5 (D437G5-BA-Mur-Jin 0.5E7), 1 tumour in Group 6 (D41 7G5-BA-Mur-Jin 1.0E7) and 7 tumours in Group 7 (D437G5-BA-Mur-Jin 0.5E7) were no longer visible. On Day 33, when the study ended, there were 6 mice left in Group 4, out of which 1 did not have a visible tumour. There were 8 mice left each in Groups 5 and 7, out of which 4 from group 5 and 1 in Group 7 did not have measurable tumours. In Group 6, there were 4 mice remaining, out of which 1 did not have a measurable tumour.

Conclusion: Mice tolerated the intravenous injection of 7G5-BA-Mur-Jin T cells well with no adverse effects detected on the day of injection. Injection of Donor 41 or 43 7G5-BA-Mur-Jin transduced T cells resulted in significantly reduced A375-MR1 tumour growth [Figure 8A] Furthermore, the overall survival was significantly increased for mice treated with 7G5-BA-Mur-Jin T cells compared to mice receiving vehicle or non- transduced T cells from either donor. Mice treated with 7G5-BA-Mur-Jin T cells from Donor 43 showed the lowest level of tumour burden on Day 21 where 14 out of 16 mice had no visible tumours. Furthermore, at the end of the study, 50% of animals in the group treated with the highest dose of 7G5-BA-Mur-Jin T cells (1.0x10⁷), still did not present a visible tumour. Additionally there was no significant measurable deficit to mean bodyweight over the period of the treatment for mice dosed with the optimised 7G5 BA-Mur-Jin construct transduced T cells indicating safety and lack of off target effects on the animals [Figure 8B] The results support the safety and effectiveness of 7G5 BA-Mur-Jin T cells in treating and preventing cancer or tumour including solid tumour where the cancer or cancer cells present MR1.

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

1. A tumour-specific T-cell receptor (TCR) comprising an alpha chain and beta chain, wherein: the alpha chain comprises a variable region comprising a complementarity determining region (CDR) comprising or consisting of CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1; and a constant region which comprises or consists of a murine TCR constant region or variant thereof; and the beta chain comprises a variable region comprising a complementarity determining region (CDR) comprising or consisting of CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2; and a constant region which comprises or consists of a murine TCR constant region or variant thereof.

2. The tumour-specific T-cell receptor (TCR) according to claim 1 wherein the alpha chain constant region comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:11, and the beta chain constant region comprises or consists of a constant region which has at least 80% sequence identity to the murine TCR constant region of SEQ ID NO:17.

3. The tumour-specific T-cell receptor (TCR) according to either claim 1 or 2 wherein at least one amino acid is substituted in the alpha chain constant region and/or beta chain constant region.

4. The tumour-specific T-cell receptor (TCR) according to claim 3 wherein the at least one amino acid substituted in the alpha chain constant region is a hydrophobic amino acid, preferably a valine or isoleucine.

5. The tumour-specific T-cell receptor (TCR) according to either of claims 3 or 4 wherein the alpha chain constant region comprises one or more of the substitutions selected from, S112L, M114I, G115V relative to SEQ ID NO:11.

6. The tumour-specific T-cell receptor (TCR) according to claim 5 wherein the alpha chain constant region comprises one or more of the substitutions selected from, L112, 1114, V115 relative to SEQ ID NO:11.

7. The tumour-specific T-cell receptor (TCR) according to either of claims 3 to 6 wherein the alpha chain constant region comprises the sequence LVIV (SEQ ID NO:45) or comprises the sequence of SEQ ID NO:31.

8. The tumour-specific T-cell receptor (TCR) according to any previous claim wherein the beta chain constant region comprises one or more of the substitutions selected from, Q135H, T161G, V164L, R170K relative to SEQ ID NO:17.

9. The tumour-specific T-cell receptor (TCR) according to any previous claim wherein the alpha chain constant region comprises or consists of a constant region of SEQ ID NO: 12, and/or the beta chain constant region comprises or consists of a constant region of SEQ ID NO:18.

10. The tumour-specific T-cell receptor (TCR) according to any previous claim wherein the TCR comprises one or more of the following CDRs comprising or consisting of:

TSESDYY (SEQ ID NO:3); or variant CDR having at least one substitution, addition or deletion, with respect thereto,

ATEN (SEQ ID NO:4); or variant CDR having at least one substitution, addition or deletion, with respect thereto,

MGHDK (SEQ ID NO:5); or variant CDR having at least one substitution, addition or deletion, with respect thereto, and

SYGVNS (SEQ ID NO:6) or variant CDR having at least one substitution, addition or deletion, with respect thereto.

11. The tumour-specific T-cell receptor (TCR) according to claim 10 wherein the TCR or tumour-specific binding fragment comprises an alpha chain with CDRs comprising or consisting of:

CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1; TSESDYY (SEQ ID NO:3); and ATEN (SEQ ID N0:4);

12. The tumour-specific T-cell receptor (TCR) according to claim 10 or claim 11 wherein the TCR comprises a beta chain with CDRs comprising or consisting of:

CASSEARGLAEFTDTQYF (SEQ ID NO: 2) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2;

MGHDK (SEQ ID NO:5); and SYGVNS (SEQ ID NO:6).

13. The tumour-specific T-cell receptor (TCR) according to any previous claim wherein the TCR comprises 3 alpha chain CDRs comprising or consisting of:

CAYRSAVNARLMF (SEQ ID NO: 1) or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1);

TSESDYY (SEQ ID NO:3); and ATEN (SEQ ID NO:4); and comprises 3 beta chain CDRs comprising or consisting of:

MGHDK (SEQ ID NO:5); and SYGVNS (SEQ ID NO:6).

14. The tumour-specific T-cell receptor (TCR) according to any one of claims 1 to 13 wherein the TCR comprises an alpha chain variable region comprising or consisting of:

AQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEA YKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRSAVNARLMFGD GTQLVVKP (SEQ ID NO:7); or a variant alpha chain variable region which has at least 88% sequence identity to the alpha chain variable region of SEQ ID NO:

7.

15. The tumour-specific T-cell receptor (TCR) according to claim 14 wherein said TCR comprises an alpha chain variable region comprising or consisting of SEQ ID NO:7.

16. The tumour-specific T-cell receptor (TCR) according to any one of claims 1 to 15 wherein said TCR comprises a beta chain variable region comprising or consisting of:

EADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVN STEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEARGLAEFTDTQY FGPGTRLTVL (SEQ ID NO:8); or a variant beta chain variable region which has at least 88% sequence identity to the beta chain variable region of SEQ ID NO:

8

17. The tumour-specific T-cell receptor (TCR) according to claim 16 wherein said TCR comprises a beta chain variable region comprising or consisting of SEQ ID NO:8.

18. The tumour-specific T-cell receptor (TCR) according to any one of claims 1 to 17, wherein the amino acid sequence of the TCR is artificial.

20. The tumour-specific T-cell receptor (TCR) according to claim 18, wherein the alpha chain variable region and/or the beta chain variable region has at least one amino acid which is substituted, added or deleted relative to the alpha chain variable region of SEQ ID NO:7 and/or the beta chain variable region of SEQ ID NO: 8 respectively.

20. The tumour-specific T-cell receptor (TCR) according to claim 20, wherein the at least one amino acid is (are) located in a framework region or a CDR.

21. The tumour-specific T cell receptor (TCR) according to claim 20, wherein the at least one amino acid is not located in a or any CDR.

22. The tumour-specific T-cell receptor (TCR) according any one of claims 18 to 21 , wherein in the alpha chain constant region and/or the beta chain constant region has at least one amino acid is substituted, added or deleted relative to the alpha chain constant region of SEQ ID NO:11 and/or the beta chain constant region of SEQ ID NO: 17 respectively.

23. The tumour-specific T-cell receptor (TCR) according to any previous claim comprising:

(a) an alpha chain comprising a variable region comprising a CDR3 sequence of SEQ ID NO: 1 or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 1 , a CDR1 sequence of SEQ ID NO:3 and a CDR2 sequence of SEQ ID NO: 4 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:11, and a beta chain comprising a variable region comprising a CDR3 sequence of SEQ ID NO: 2 or a variant CDR which has at least 88% sequence identity to the CDR of SEQ ID NO: 2, a CDR1 sequence of SEQ ID NO: 5 and a CDR2 sequence of SEQ ID NO: 6 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:17; or

(b) an alpha chain comprising a variable region comprising or consisting of SEQ ID NO:7 or a variant variable region which has at least 88% sequence identity to the alpha chain variable region of SEQ ID NO: 7 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to constant region of SEQ ID NO:11; and a beta chain comprising a variable region comprising or consisting of SEQ ID NO:8 or a variable region which has at least 88% sequence identity to the beta chain variable region of SEQ ID NO: 8 and a constant region which comprises or consists of a constant region which has at least 80% sequence identity to the constant region of SEQ ID NO:17; or

(c) an alpha chain comprising a variable region comprising or consisting of SEQ ID NO:7 or a variant variable region which has at least 88% sequence identity to the alpha chain variable region of SEQ ID NO: 7 and a constant region which comprises or consists of a constant region of SEQ ID NO:12; and a beta chain comprising a variable region comprising or consisting of SEQ ID NO:8 or a variable region which has at least 88% sequence identity to the beta chain variable region of SEQ ID NO: 8 and a constant region which comprises or consists of a constant region of SEQ ID NO:18;or

(d) an alpha chain comprising or consisting of SEQ ID NO:13 minus the N- terminal leader sequence (residues 1-20 with reference to SEQ ID NO:13) or a variant alpha chain which has at least 88% sequence identity to the alpha chain of SEQ ID NO: 13 minus the N-terminal leader sequence and a beta chain comprising or consisting of SEQ ID NO: 19 minus the N-terminal leader sequence (residues 1-19 with reference to SEQ ID NO:19) or a variant beta chain which has at least 88% sequence identity to the beta chain of SEQ ID NO: 19 minus the N-terminal leader sequence, or

(e) an alpha chain comprising or consisting of SEQ ID NO:14 minus the N- terminal leader sequence (residues 1-20 with reference to SEQ ID NO:14) and a beta chain comprising or consisting of SEQ ID NO: 20 minus the N- terminal leader sequence (residues 1-19 with reference to SEQ ID NO:20).

24. The tumour-specific T-cell receptor (TCR) according to any one of claims 1 to 23 wherein the TCR is MR1 -restricted.

25. The tumour-specific T-cell receptor (TCR) according to any one of claims 1 to 24 wherein the TCR is not expressed by or associated with a mucosal-associated invariant T cell (MAIT cell).

26. A polynucleotide encoding the TCR according to any one of claims 1 to 25.

27. A polynucleotide encoding the TCR according to claim 26 comprising a nucleic acid sequence encoding the TCR in a single open reading frame or two distinct open reading frames encoding the alpha chain and beta chain respectively.

28. The polynucleotide according to claim 26 or 27 wherein the polynucleotide comprises a nucleic acid sequence encoding the TCR and a heterologous promoter, optionally an EF-1 alpha promoter sequence, or other transcription control element operably linked thereto.

29. The polynucleotide according to any one of claims 26 to 28 wherein the polynucleotide comprises a nucleic acid sequence comprising or encoding any one or more of (i) a cleavable linker sequence, (ii) a furin cleavable linker sequence (iii) a WPRE mutation sequence optionally mut6WPRE (iv) Kozak consensus sequence.

30. The polynucleotide according to claim 29 wherein the cleavable linker sequence is a 2A peptide sequence optionally selected from a T2A, E2A, F2A or P2A sequence further optionally including a GSG (Gly-Ser-Gly) linker sequence on the N-terminal of the 2A peptide sequence.

31. The polynucleotide according to any one of claims 26 to 30 wherein the nucleic acid sequence encoding the TCR comprises a nucleotide sequence encoding a TCR alpha chain and a TCR beta chain wherein the alpha chain nucleotide sequence is 5’ to the beta chain nucleotide sequence (alpha-beta), alternatively wherein the beta chain nucleotide sequence is 5’ to the alpha chain nucleotide sequence (beta-alpha).

32. The polynucleotide according to any one of claims 26 to 31 wherein the nucleic acid sequence encoding the TCR comprises a nucleotide sequence encoding a TCR alpha chain and a TCR beta chain in which the TCR alpha chain and TCR beta chain sequences are separated from each other by a 2A peptide sequence and/or a furin cleavable linker sequence, optionally wherein the 2A peptide sequence is a P2A sequence, optionally including a GSG (Gly-Ser-Gly) linker sequence on the N-terminal of the 2A peptide sequence.

33. A vector comprising a polynucleotide according to any one of claims 26 to 32 optionally wherein the vector is a TCR expression vector.

34. The vector according to claim 33 wherein the vector is a viral vector optionally a lentiviral vector.

35. A cell or immunoresponsive cell expressing or presenting a heterologous tumour- specific T-cell receptor (TCR) according to any one of claims 1 to 25.

36. A cell or immunoresponsive cell harbouring the polynucleotide of any one of claims 26 to 32 or the vector of claim 33 or 34.

37. A cell or immunoresponsive cell harbouring the polynucleotide of any one of claims 26 to 32 or the vector of claim 33 or 34 wherein the cell or immunoresponsive cell expresses or presents a heterologous tumour-specific T-cell receptor (TCR) according to any one of claims 1 to 25.

38. The cell or immunoresponsive cell according to any one of claims 35 to 37, wherein the cell or immunoresponsive cell is transduced with a polynucleotide or heterologous polynucleotide according to any one of claims 26 to 32 or by a vector according to either one of claims 33 or 34.

39. An ex vivo process comprising (i) obtaining immunoresponsive cells from a patient, (ii) optionally expanding the immunoresponsive cells (iii) transducing the immunoresponsive cells with a polynucleotide or heterologous polynucleotide according to any one of claims 26 to 32 or by a vector according to either one of claims 33 or 34 so that they express a tumour-specific T-cell receptor (TCR) according to any one of claims 1 to 25; and (iii) reintroducing said transduced immunoresponsive cells into the patient.

40. A method of treatment of cancer comprising administering to a patient in need thereof transduced immunoresponsive cells wherein the transduced immunoresponsive cells are immunoresponsive cells which have been obtained from the patient and transduced ex vivo with a heterologous polynucleotide according to any one of claims 26 to 32 or by a vector according to either one of claims 33 or 34 so that they express a tumour-specific T-cell receptor (TCR) according to any one of claims 1 to 25.

41. A pharmaceutical composition comprising the TCR according to any one of claims 1 to 25, the polynucleotide according to any one of claims 26 to 32, a vector according to either one of claims 33 or 34, the cell or immunoresponsive cell according to any one of claims 35 to 38 and a pharmaceutically acceptable carrier.

42. A pharmaceutical composition according to claim 41 , wherein the pharmaceutical composition is formulated under sterile conditions and is suitable for parenteral administration.

43. A pharmaceutical composition comprising a) the TCR according to any one of claims 1 to 25, the polynucleotide according to any one of claims 26 to 32, a vector according to either one of claims 33 or 34, the cell or immunoresponsive cell according to any one of claims 35 to 38, or pharmaceutical composition according to either one of claims 41 or 42, and b) an anti-tumour agent.

44. A method of treating cancer in a subject comprising administering a therapeutically effective amount of the TCR according to any one of claims 1 to 25, the polynucleotide according to any one of claims 26 to 32, a vector according to either one of claims 33 or 34, the cell or immunoresponsive cell according to any one of claims 35 to 38, or pharmaceutical composition according to any one of claims 41 to 43, to the subject.

45. A method of treating cancer in a subject comprising administering a therapeutically effective amount of the TCR according to any one of claims 1 to 25, the polynucleotide according to any one of claims 26 to 32, a vector according to either one of claims 33 or 34, the cell or immunoresponsive cell according to any one of claims 35 to 38, or pharmaceutical composition according to any one of claims 41 to 43, separately, simultaneously or sequentially with an anti-tumour agent to the subject.

46. The method according to any one of claims 40, 44 or 45 wherein said cancer is selected from the group consisting of any one of colorectal, lung cancer (including non small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), kidney, prostate, bladder, cervical, melanoma (skin), bone, breast (including metastatic breast cancer), ovarian, blood cancer, myeloma, acute lymphoblastic leukaemia (ALL), melanoma, glioblastoma, cholangiocarcinoma or brain cancer e.g. astrocytoma.

47. The cell or immunoresponsive cell according to any one of claims 35 to 38, the ex vivo process of claim 39, the pharmaceutical composition of any of claims 41 to 43 or the method according to any of claims 40 or 44 to 46 wherein the immunoresponsive cell is selected from B cells, TILs (tumour infiltrating lymphocytes), natural killer (NK) cells and T cells, such as CD4⁺ T cells or CD8⁺ T cells.

48. The cell or immunoresponsive cell according to any one of claims 35 to 38, the ex vivo process of claim 39, , the pharmaceutical composition of any of claims 41 to 46 or the method according to any of claims 40 or 44 to 46 wherein the immunoresponsive cell is a population of immunoresponsive cells optionally a population of CD4+ T cells; or CD8+ T cells, or a mixed population of CD4+ T cells and CD8+ T cells.