WO2023118820A1

WO2023118820A1 - Cancer specific t-cell receptors

Info

Publication number: WO2023118820A1
Application number: PCT/GB2022/053273
Authority: WO
Inventors: Andrew Sewell; Cristina RIUS; Gary DOLTON; Rachel WOOLLEY
Original assignee: Continuum Life Sciences Limited
Priority date: 2021-12-21
Filing date: 2022-12-16
Publication date: 2023-06-29

Abstract

The present invention provides novel T cell receptor (TCR) based molecules which are selective for IMP2. The TCR of the invention are of use for the diagnosis, treatment and prevention of IMP2 expressing cancerous diseases. Further provided are nucleic acids encoding the TCR of the invention, vectors comprising these nucleic acids, recombinant cells expressing the TCR and pharmaceutical compositions comprising the compounds of the invention.

Description

CANCER SPECIFIC T-CELL RECEPTORS

BACKGROUND TO THE INVENTION

A T-cell receptor (TCR) is composed of two different and separate protein chains, namely the TCR alpha and the TCR beta chain. The TCR alpha chain (TRA) comprises variable (V), joining (J) and constant (C) regions. The TCR beta chain (TRB) comprises variable (V), diversity (D), joining (J) and constant (C) regions. The rearranged V(D)J regions of both the TRA and the TRB chain contain hypervariable regions (CDR, complementarity determining regions) within a framework regions (or non-complementarity determining regions), among which the hypervariable CDR3 region determines the specific epitope recognition along with the CDR1 and CDR1 regions which are defined according to the TRAV or TRBV gene sequence. Each of the framework regions and CDR regions of the TRAV and TRBV chains are well defined and known in the art. At the C-terminal region both TRA chain and TRA chain contain a hydrophobic transmembrane domain and end in a short cytoplasmic tail. Typically, the TCR is a heterodimer of one alpha chain and one beta chain. This heterodimer can bind to MHC molecules presenting a peptide.

Within the context of the present invention, the TCR loci and genes are named using the International Immunogenetics (IMGT) TCR nomenclature (IMGT Database, www. IMGT.org; Giudicelli, V., et al., IMGT/LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T-cell receptor nucleotide sequences, Nucl. Acids Res., 34, D781-D784 (2006). PMID: 16381979; T-cell Receptor Factsbook, LeFranc and LeFranc, Academic Press ISBN 0-12- 441352-8).

T cell receptors (TCRs) are naturally expressed by CD4+ and CD8+ T-cells. TCRs are designed to recognize short peptide antigens that are displayed on the surface of antigen presenting cells in complex with Major Histocompatibility Complex (MHC) molecules (in humans, MHC molecules are also known as Human Leukocyte Antigens, or HLA) (Davis, et al., (1998), Annu Rev Immunol 16: 523-544.). CD8+ T-cells, which are also termed cytotoxic T-cells, specifically recognize peptides bound to MHC class I and are generally responsible for finding and mediating the destruction of diseased cells. CD8+ T-cells are able to destroy cancerous as well as virally infected cells; however, the affinity of TCRs expressed by cancer specific T- cells in the natural repertoire are typically low as a result of thymic selection, meaning that cancerous cells frequently escape detection and destruction. Novel immunotherapeutic approaches aimed at promoting cancer recognition by T-cells offer a highly promising strategy for the development of effective anticancer treatments.

Some TCRs specific to some cancer-associated peptide antigens have been described in the art. The efficiency and specificity of these TCRs mean that the practical applications of such TCRs are limited. There thus remains in the art a need for a TCR that can be used in a wider variety of cancer pathologies and with higher efficiency.

SUMMARY OF THE INVENTION

The above problem is solved by the provision of TCR that are specific to IMP2 protein, preferably to the IMP2 epitope NLSALGIFST (SEQ ID NO.l), which are residues 367-376 of IMP2. As shown here below, many and varied cancer pathologies are targeted, attacked and killed by T-cell containing TCR specific to IMP2.

The invention also provides for preferred variations of the TCR which are determined by the protein sequence in their TRA and/or TRB protein sequence.

The invention also provides for TCR constructs, nucleic acids, expression vectors containing said nucleic acids and cells that can be derived from or associated with the TCR according to the invention.

The TCR of the invention can be used in attacking, diagnosing and preventing neoplasia. The neoplasia can be malignant and thus cancerous.

NOTE ON SEQUENCE LISTINGS

A sequence listing associated with the instant disclosure has been submitted in ST26 format and is hereby incorporated by reference in its entirety where this is permitted. It should be noted that the SEQ ID NO. described in the present disclosure correspond to those in the sequence listing submitted.

FIGURES

The invention described here below makes reference to the following non-limiting Figures.

Figure 1 shows expression of IMP2 in a variety of cancer cells of different tissue origin.

Figure 2 demonstrates validation of the IMP2 NLSALGIFST epitope by IMP2 specific T-cells in which; Fig. 2A&B illustrate how HLA A*02 restricted IMP2 NLSALGIFST specific CD8 clone NLS.732 stains with IMP-2 tetramer and recognises exogenous NLSALGIFST peptide;

Fig. 2C shows the result of Lentiviral expression of HLA A*0201 and IMP2 genes in MOLT3 cells. IMP2 was expressed with co-marker rat (r)CD2;

Fig. 2D shows the result of overnight activation of CD8 clone NLS.732 with the MOLT3 cells: HLA A2 alone (-ve), HLA A2 + IMP2 and HLA A2 + collagen; and

Fig. 2E shows the result of overnight activation of CD8 clone NLS.732 with cancer cell lines +/- HLA A*02 expression (CRISPR-cas9 or transgene according to the key);

Figure 3 shows isolation of IMP2-specific T-cells recognise and kill cancer cells of different origin in which;

Fig. 3A shows the IMP2 line generated in Example 3 here below stained with irrelevant (SLYNTVATL from HIV pl7 gag) and NLSALGIFST tetramers;

Fig. 3B (right) shows flow based killing assay MIA PaCa-2 pancreatic cancer cells +/- HLA A*02 with CS001 IMP2 T-cell line after a 24h incubation at a T-cell to cancer cell ratio of 1:1; and

Fig. 3C shows real time killing by the IMP2 line of breast (MDA-MB-231) and pancreatic (PANC-1) cells lines +/- HLA A*02;

Figure 4 shows clonotypic dissection of the IMP2 specific TCRs in which;

Fig. 4A shows how the sorting of the T-cell line specific for IMP2 was sorted on a SONY MA900 with PE and APC conjugated IMP2 tetramers;

Fig. 4B shows pie charts for alpha and beta TCR chains with each segment representing a unique CDR3, with the total number of TCR chains shown in the centre of the pie chart; and

Fig. 4C and D show TCR alpha and beta CDR3s from the IMP2 tetramer sorted population;

Figure 5 shows NLSALGIFST- specific T-cell clones grown from IMP2-specific T-cell line in which;

Fig. 5A illustrates the reactivity of 35 T-cell clones grown from CS001 IMP2-specific T-cell line stained in Figure 4A to IMP2-derived NLSALGIFST peptide in overnight activation and supernatants used for MIP-lbeta ELISA with error bars depicting SEM of duplicates; and Fig. 5B shows results when clones were stained with HLA A*02 irrelevant (SLYNTVATL from HIV gag) and IMP2 tetramers;

Figure 6 shows the nucleotide sequences of the TCR alpha and beta chains from the IMP2 clones generated;

Figure 7 shows further nucleotide sequences of the TCR alpha and beta chains from the IMP2 clones generated; and

Figure 8 shows results of testing of the IMP-2 clones against cancer cells in which;

Fig. 8 A shows how clones representing four of the TCRs from the cloned T-cells tested against cancer cells +/- HLA A2; and

Fig. 8B shows flow based 4h TAPLO activation assay with two pancreatic cancer cell lines +/- HLA A2. MCF-7 and MDA-MB-231 CRISPR-Cas9 knockout of HLA A2. PC-3, AsPC-1 and MIA PaCa-2 expressing a HLA A2 transgene.

Figure 9 shows the results of 2 TCR according to the invention with the sequence listed in Table 4 binding to SEQ ID NO. 1 being detected with SPR

DESCRIPTION

In a first aspect, the present invention provides a T-cell receptor (TCR) having the property of binding to IMP2. Preferably, the TCR binds specifically to an IMP2 peptide, even more preferably which is in complex with HLA-A*02.

Binding specifically for a TCR means that it binds to an epitope with an affinity that allows the TCR to performs its desired function. This is generally defined by dissociation constants, as detailed below. Binding specifically also means that the TCR binds preferably to only one specific epitope and preferably shows no or substantially no cross-reactivity to another epitope and another protein.

IMP2 is also known as also known as insulin-like growth factor 2 (IGF2) messenger RNA- binding proteins (IGF2BP2). Within the context of the present invention, any sequence of IMP2 recognisable as such in the art is contemplated as within the scope of the present application. As is demonstrated in Fig.l and in Example 1, IMP-2 is strongly expressed in many cancer pathologies and thus the TCRs of the invention have a binding profile beneficial for therapeutic use. Cancer specific TCRs are rare in a natural repertoire and it therefore involves a large amount of skilful screening and analysis in order to identify suitable TCRs, as described in the examples section, below.

Preferably the TCR is specific to and binds to NLSALGIFST (SEQ ID NO.l). This sequence corresponds to amino acid residues 367-376 of IMP2, numbered according to Uniprot entry UniProtKB - Q9Y6M1 (IF2B2_HUMAN), isoform 1 (Q9Y6M1-2)), which is the canonical sequence.

The TCR according to the invention can be isolated and/or purified and may be soluble or membrane bound.

By soluble, it is meant that the transmembrane domains may be removed from the full length TCR polypeptide, such that it is not membrane bound. The TCR may be part of a fusion protein, such as a bispecific molecule, or joined to a tag for purification or identification or to a targeting moiety, referred to as a TCR construct herein. A TCR may be affinity matured to produce a TCR with higher than natural affinity. The TCR may be incorporated into an engineered T-cell for adoptive therapy.

Typically, the TCR recognizes the peptide fragment of the antigen when it is presented by a major histocompatibility complex (MHC) molecule. The human leukocyte antigen (HLA) system or complex is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. HLA-A*02 is one particular class I major histocompatibility complex (MHC) allele group at the HLA- A locus. HLA-A*0201 is a specific HLA-A*02 allele. Thus the TCR according to the invention preferably recognizes the HLA-A*02 bound form of the amino acid sequence of SEQ ID NO. 1, more preferably the HLA- A*0201 bound form of the amino acid sequence of SEQ ID NO. 1.

A TCR of the invention preferably has a KD for the IMP2 peptide antigen HLA complex as described above from 200pM to lOOnM about (i.e. +/- 10%). In an even more preferable version the upper limit of the KD range is lOOpM and/or the lower limit is IpM. However if the TCR is further affinity enhanced according to methods known in the art, the KD may be as low as single pM range if it is a soluble version while it preferably remains in the lower pM range when the TCR is present on the cell surface.

Methods to determine binding affinity (inversely proportional to the equilibrium constant KD) are known to those skilled in the art. Preferably, binding affinity is determined using Surface Plasmon Resonance (SPR), for example using a BIAcore instrument. To compare binding data between two samples measurements should be made using the same assay conditions (e.g. temperature), where possible.

The TCR according to the invention preferably comprises an amino acid sequence corresponding to that of a TRAV12-2 or TRAV12-3 alpha chain in its frame work and CDR1 and CDR2 region. The TCR according to the invention comprise an amino acid sequence having at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99% identity to a framework and/or CDR1 and/or CDR2 region of a TRAV 12-2 or a TRAV 12-3 chain sequence. A TCR of the invention may comprise a framework, CDR1 and CDR2 amino acid sequence of a TRAV29DV5 alpha chain sequence, in accordance with IMGT nomenclature, or a sequence having at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99% identity to a framework and CDR1 and CDR2 regions of a TRAV29DV5 sequence.

The TCR according to the invention preferably comprises a TRAV peptide sequence comprising a CDR3alpha sequence selected from any one of SEQ ID NO.s 2-21. The CDR3alpha sequence is preferably a sequence selected from the group of SEQ ID NO: 2, 3, 4, 9, 10 or 12.

The TCR of the invention preferably comprises a TRAJ sequence of any one of the TRAJ sequences listed in Figure 4C, according to IMGT nomenclature. More preferably, a TCR of the invention comprises an amino acid sequence of TRAJ31, TRAJ56, TRAJ27, TRAJ15, TRAJ13 or TRAJ43, according to IMGT nomenclature or a sequence having at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99% identity thereto.

The TCR according to the invention preferably comprises a sequence combination selected from those in Table 1 in its alpha variable domain, below, wherein the IMGT nomenclature is used to define the TRAV framework, CDR1 and CDR2 sequences and the TRAJ joining sequences, and the CDR3 alpha sequence of each TCR alpha chain variable domain is explicitly written out.

Table 1

The TCR according to the invention preferably comprises an amino acid sequence corresponding to that of a TRBV7-9, TRBV10-3, TRBV11-2, TRBV4-lor TRBV6-1 beta chain in its framework and CDR1 and CDR2 region. The TCR according to the invention comprise an amino acid sequence having at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99% identity to a framework and/or CDR1 and/or CDR2 region of a TRBV7-9, TRBV10-3, TRBV11-2, TRBV4-lor TRBV6-1 beta chain sequence.

The TCR according to the invention preferably comprises a TRBV peptide sequence comprising a CDR3beta sequence selected from any one of SEQ ID NO.s 22-57. The CDR3beta sequence is preferably a sequence selected from the group of SEQ ID NO: 22, 26, 31, 28 or 25.

A TCR beta chain variable domain also comprises a diversity region next to the joining region, encoded by TRBD genes. Preferably a TCR of the invention comprises a TRBD sequence comprising the sequence of TRBD1 or TRBD2, according to IMGT nomenclature or a sequence having at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99% identity thereto.

A TCR of the invention preferably comprises a TRBJ sequence of any one of the TRBJ sequences listed in Figure 4D, according to IMGT nomenclature. More preferably, a TCR of the invention comprises an amino acid sequence of TRBJ1-1, TRBJ2-7 or TRBJ2-1 according to IMGT nomenclature, or a sequence having at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99% identity thereto. A TCR according to the invention preferably comprising a sequence combination selected from those in Table 2 in it beta chain variable domain, below, wherein the IMGT nomenclature is used to define the TRBV framework, CDR1 and CDR2 sequences, the TRBD diversity sequences, and the TRBJ joining sequences, and the CDR3beta sequence of each TCR beta chain is explicitly written out.

Table 2

The TCR of the invention preferably comprises one of the following combinations of alpha chain CDR3 and beta chain CDR3 sequences:

- a TRAV chain comprising a CDR3alpha sequence of SEQ ID NO: 2 and a TRBV chain comprising a CDR3beta sequence of SEQ ID NO: 22;

-a TRAV chain comprising a CDR3alpha sequence of SEQ ID NO: 3 and a TRBV chain comprising a CDR3beta sequence of SEQ ID NO: 22;

-a TRAV chain comprising a CDR3alpha sequence of SEQ ID NO: 4 and a TRBV chain comprising a CDR3beta sequence of SEQ ID NO: 26

-a TRAV chain comprising a CDR3alpha sequence of SEQ ID NO: 9 and a TRBV chain comprising a CDR3beta sequence of SEQ ID NO: 31

-a TRAV chain comprising a CDR3alpha sequence of SEQ ID NO: 10 and a TRBV chain comprising a CDR3beta sequence of SEQ ID NO: 28.

-a TRAV chain comprising a CDR3alpha sequence of SEQ ID NO: 12 and a TRBV chain comprising a CDR3beta sequence of SEQ ID NO: 25 Even more preferably, a TCR of the invention is selected from the group having the TCR alpha chain variable domain sequences and the TCR beta chain variable domain sequences in the combinations selected from the table below:

Table 3

The TCR of the invention may comprise TRAV and/or TRBV sequences that correspond to native TCR or may be engineered/non-natural TCR sequences. The native sequences may be referred to as wild-type or parental TCR domains or sequences. By native, it is meant TCRs that are isolated from the natural repertoire of a human subject after thymic selection; no mutations or alterations are made to the TCR, such as mutations to increase affinity or specificity.

Within the context of the present invention, the amino acid sequence of the TCR may comprise one or more phenotypically silent substitutions (or conservative amino acid substitutions). The concept of conservative amino acid substitutions is understood to be that codons encoding positively-charged residues (H, K, and R) are substituted with codons encoding positively- charged residues and codons encoding negatively- charged residues (D and E) are substituted with codons encoding negatively-charged residues and codons encoding neutral polar residues (C, G, N, Q, S, T, and Y) are substituted with codons encoding neutral polar residues and codons encoding neutral non-polar residues (A, F, I, L, M, P, V, and W) are substituted with codons encoding neutral non-polar residues. These variations can occur spontaneously (random mutagenesis occurring in nature) or can be introduced by directed mutagenesis. Those changes can be made without destroying the essential characteristics of these polypeptides. The ordinarily skilled artisan can readily and routinely screen variant amino acids and/or the nucleic acids encoding them to determine if these variations substantially reduce or destroy the ligand binding capacity by methods known in the art.

In another aspect of the invention, a TCR construct is provided. A TCR construct according to the invention can be made using the amino acid sequence of the TCR according to the invention and may be incorporated into or modified with a further molecule. The TCR construct according to the invention may comprise a detectable label, a therapeutic agent or pharmacokinetic modifying moiety. The TCR construct may include in some embodiments, a further binding moiety, as well as a TCR of the invention, such as to an additional peptide target, or to an immune effector molecule, such as an interleukin or a cytokine.

Non-limiting examples for detectable labels are radiolabels, fluorescent labels, nucleic acid probes, enzymes and contrast reagents. Therapeutic agents which may be associated with the TCR constructs include radioactive compounds, immunomodulators, enzymes or chemotherapeutic agents. The therapeutic agents could be enclosed by a liposome linked to the TCR construct so that the compound can be released slowly at the target site. This will avoid damaging during the transport in the body and ensure that the therapeutic agent, e.g. toxin, has maximum effect after binding of the TCR construct to the relevant antigen presenting cells. Other examples for therapeutic agents are peptide cytotoxins, i.e. proteins or peptides with the ability to kill mammalian cells, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase. Many more types of therapeutic agents are known to the person skilled in the art and can be adapted to make a TCR construct according to this invention.

The pharmacokinetic modifying moiety may be for example at least one polyethylene glycol repeating unit, at least one glycol group, at least one sialyl group or a combination thereof. The association of at least one polyethylene glycol repeating unit, at least one glycol group, at least one sialyl group may be caused in a number of ways known to those skilled in the art. The units are preferably covalently linked to the TCR. The pharmacokinetic modifying moiety may achieve beneficial changes to the pharamacokinetic profile of the therapeutic, for example improved plasma half-life, reduced or enhanced immunogenicity, and improved solubility.

Other useful functional moieties and modifications that are comprised within a TCR construct according to the invention include “suicide” or “safety switches” that can be used to shut off effector host cells carrying an inventive TCR in a patient’s body. An example is the inducible Caspase 9 (iCasp9) “safety switch” described by Gargett and Brown Front Pharmacol. 2014; 5: 235.

The TCR construct may involve truncating the alpha chain variable domain peptide sequence and/or the beta chain variable domain peptide sequence of the TCR of the invention, whilst still retaining the functional activity of the TCR of the TCR construct.

Another aspect of the invention is the provision of a nucleic acid encoding an alpha chain and/or beta chain of a TCR of the invention or according to a TCR construct of the invention. The nucleic acid may be cDNA, genomic DNA or the nucleic acid may be mRNA, The invention provides a nucleic acid encoding an alpha chain variable domain of a TCR of the invention. The invention also provides a nucleic acid encoding a beta variable chain domain of a TCR of the invention. A nucleic acid of the invention may encode the variable domain only, or the full alpha and/or beta chain of the TCR of the invention, which may or may not include the TRAC and/or TRBC domains. TCR or TCR construct according to the invention explained here above. The nucleic acid according to the invention encodes an alpha chain variable domain and/or a beta chain variable domain of a TCR of the invention. A nucleic acid of the invention, in some embodiments, also encodes a TCR constant domain of the alpha or beta chain, and in some embodiments, encodes an additional moiety in the case of a TCR construct of the invention being encoded. The nucleic acid may be cDNA, genomic DNA or the nucleic acid may be mRNA, The invention provides a nucleic acid encoding an alpha chain variable domain of a TCR of the invention. The invention also provides a nucleic acid encoding a beta variable chain domain of a TCR of the invention. A nucleic acid may encode both the alpha and beta chain variable domains of the invention. A nucleic acid of the invention may encode the variable domain only, or the full length alpha and/or beta chain of the TCR of the invention, which may or may not include the TRAC and/or TRBC domains. The nucleic acid can be a polymer of DNA or RNA, which can be single- stranded or double- stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, nonnatural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. The nucleic acid can be a polymer of DNA or RNA, which can be single-stranded or doublestranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, nonnatural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.

The nucleic acids according to the invention are preferably recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication. The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art or commercially available (e.g. from Genscript, Thermo Fisher and similar companies). The nucleic acid can comprise any nucleotide sequence which encodes any of the recombinant TCRs, polypeptides, or proteins, or functional portions or functional variants thereof.

Within the context of the present invention, the nucleic acid according to the invention also comprises a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein. The nucleotide sequence which hybridizes under stringent conditions preferably hybridizes under high stringency conditions, which means the nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions which would distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches from a random sequence that happened to have a few small regions (e.g., 3-10 bases) that matched the nucleotide sequence. Such small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions would include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70° C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any of the TCRs described herein. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

The nucleic acid according to the invention may be modified or altered without altering its recognized overall function. Useful modifications in the overall nucleic acid sequence may be codon optimization. Alterations may be made which lead to conservative substitutions within the expressed amino acid sequence. These variations can be made in complementarity determining and non-complementarity determining (framework) regions of the amino acid sequence of the TCR chain that do not affect function. Usually, additions and deletions should not be performed in the CDR3 region.

The nucleic acid encoding the alpha chain of a TCR of the invention may comprise a nucleic acid sequence of SEQ ID NO: 59, 61, 62, 64, 66 or 68, The nucleic acid encoding the beta chain of a TCR of the invention may comprise a nucleic acid sequence of SEQ ID NO: 60, 63, 65, 67 or 69.

Another aspect of the invention is the provision of a vector comprising a nucleic acid of the invention.

The vector is preferably a plasmid, shuttle vector, phagemide, cosmid, expression vector, retroviral vector, adenoviral vector or particle and/or vector to be used in gene therapy.

An expression vector is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable host cell where synthesis of the encoded polypeptide can take place. An expression vector may be a nucleic acid that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e.g. a nucleic acid of the invention). The expression vector may comprise DNA or RNA and/or comprise liposomes. Said vector may be a plasmid, shuttle vector, phagemide, cosmid, expression vector, retroviral vector, lentiviral vector, adenoviral vector or particle and/or vector to be used in gene therapy. Said vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. Said vector may also include one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. Said vector preferably is an expression vector that includes a nucleic acid according to the present invention operably linked to sequences allowing for the expression of said nucleic acid. Said vector is preferably a retroviral, more specifically a gamma-retroviral or lentiviral vector.

Another aspect of the invention is the provision of a cell harbouring a vector of the invention, preferably a TCR expression The cell according to the invention may harbour a vector according to the invention as described above.

The cell according to the invention may be a mammalian cell, such as an immune cell, such as a peripheral blood lymphocyte, such as a T-cell. Preferably the cell is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The cell may be a natural killer cell or a T-cell. Preferably, the cell is a T-cell. The T-cell may be a CD4+ or a CD8+ T- cell, more preferably a CD8+ T-cell. Said T-cell according to the invention may also be a stem cell like memory T-cell (TCSM).

The cell according to the invention may be isolated or non-naturally occurring. The cell according to the invention preferably comprises the nucleic acid according to the invention or the expression vector according to the invention. The exogenous nucleic acid according to the invention or the expression vector according to the invention may be transfected (or transduced) i.e. the process by which an exogenous nucleic acid sequence is introduced in a host cell, e.g. in a eukaryotic host cell. Examples of such methods include electroporation, microinjection, gene gun delivery, lipofection, superfection and the mentioned infection by retroviruses or other suitable viruses for transduction or transfection.

Another aspect of the invention refers to pharmaceutical composition comprising the TCR, TCR construct, nucleic acid, vector and/or cell according to the invention. The TCR, TCR construct, nucleic acid, vector and/or cell according to the invention are treated as the active compound in the pharmaceutical composition.

The pharmaceutical composition comprises said active compound in doses mixed with an acceptable carrier or carrier material, which is a non-toxic material, which does not interfere with effectiveness of the biological activity of the active component. Such a composition can (in addition to the active component and the carrier) include filling material, salts, buffer, stabilizers, solubilizers and other materials, which are known state of the art.

The pharmaceutical composition may contain additional components which enhance the activity of the active component or which supplement the treatment. Such additional components and/or factors can be part of the pharmaceutical composition to achieve synergistic effects or to minimize adverse or unwanted effects. Techniques for the formulation or preparation and application/medication of active components of the present invention are published in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. An appropriate application is a parenteral application, for example intramuscular, subcutaneous, intramedular injections as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. The intravenous injection is the preferred treatment of a patient. The pharmaceutical composition is preferably an infusion or an injection. Said injectable composition is, for example, a fluid comprising at least one active ingredient, e.g. an expanded T-cell population (for example autologous or allogenic to the patient to be treated) expressing a TCR. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like. Such injectable compositions that are useful for use with the fusion proteins of this disclosure are conventional; appropriate formulations are well known to those of ordinary skill in the art.

Another aspect of the invention provides the TCR according to the invention, the TCR construct according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention and/or the pharmaceutical composition according to the invention for use in medicine, preferably for use in the treatment, prophylaxis and/or diagnosis of neoplasia.

Another aspect of the invention provides for the use of a TCR according to the invention, the TCR construct according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention and/or the pharmaceutical composition according to the invention in the manufacture of a medicament for treating, preventing and/or diagnosing of neoplasia.

Another aspect of the invention provides a method of treating, preventing and/or diagnosing of neoplasia, comprising administering to a patient in need thereof a TCR according to the invention, a TCR construct according to the invention, a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention and/or a pharmaceutical composition according to the invention. A method of treating may also include the administration of an additional anti-neoplastic agent, separately, in combination or sequentially. Another aspect of the invention provides a method of treating, preventing and/or diagnosing of neoplasia , comprising administering to a patient in need thereof a TCR according to the invention, a TCR construct according to the invention, a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention and/or a pharmaceutical composition according to the invention. A method of treating may also include the administration of an additional anti-neoplastic agent, separately, in combination or sequentially.

Another aspect of the invention provides an injectable formulation for administering to a human subject comprising a TCR according to the invention, a TCR construct according to the invention, a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention and/or a pharmaceutical composition according to the invention.

Neoplasia is an abnormal mass of tissue that forms when cells grow and divide more than they should or do not die when they should. Malignant or potentially malignant neoplasms are generally referred to as cancer and benign neoplasms are not referred to as cancer. The cancer is preferably for liver, breast, bone, cervical, kidney, prostate, lung, brain, ovarian or a blood cancer. The cancer is even more preferably pancreas, breast and/or prostate cancer.

The cancer to be treated may be a solid or liquid tumour, which preferably expresses IMP2.

Benign neoplasms may grow large but do not spread into, or invade, nearby tissues or other parts of the body. As such, the aforementioned use for the treatment of a neoplasia may be considered as the elimination of a benign neoplasia. The benign neoplasia may be uterine fibroids, osteophytes and melanocytic nevi (skin moles), warts and papilloma. Said treatment may be considered medically necessary or simply for cosmetic purposes.

The invention also provides a method of producing a TCR of the invention, comprising maintaining a cell according to the invention under optimal conditions for expression of the TCR of the invention, and isolating the TCR chain(s).

EXAMPLES

In the following sections, the following methods were used to obtain the results mentioned in the Example section below Peripheral blood mononuclear cells

PBMCs were isolated from whole blood by standard density gradient centrifugation using Histopaque-1077 (Merck Group) and used immediately for tetramer enrichment of IMP2 specific T-cells. 4-digit HLA typing of the sample was performed by Nottingham University; the sample is heterozygous at the HLA-A allele, expressing HLA A*02:01 and A*01:01. Antibody staining (clone BB7.2, BioRad, Hercules, CA, US) confirmed expression of HLA A*02. All human tissue was obtained and handled in accordance with Cardiff University’s guidelines to comply with the UK Human Tissue Act 2004.

Cancer cell culture

AsPC-1 (pancreatic adenocarcinoma), PC-3 (prostate adenocarcinoma) and MDA-MB-231 (breast adenocarcinoma) were cultured in R10 (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U ml-1 Penicillin, 100 pg ml^-1 Streptomycin and 2 mM L-glutamine (Merck Group, Darmstadt, Germany) at 37°C/5% CO2. MIA PaCa-2 and PANC-1 (pancreatic carcinoma) were cultured in D10-F12 (as for R10, but with DMEM-F12 (Merck Group)). Adherent cancer cell lines were passaged when 70-90% confluent by detachment using Dulbecco's phosphate-buffered saline (D-PBS) (Merck Group) with 2 mM EDTA, followed by 10-50% of the cells being seeded back for culture. Cell lines were routinely checked for mycoplasma using the Myco Alert™ kit (Lonza, Basel, Switzerland).

T-cell culture and cloning

T cell clones and lines were cultured in T-cell media (RPMI 1640 media supplemented with 10% FBS, 100 U ml^-1 penicillin, 100 pg ml^-1 streptomycin, 2 mM L-glutamine, lOmM HEPES buffer, IX MEM non-essential amino acids, ImM sodium pyruvate (ThermoFisher Scientific), 25 ng/ml IL- 15 (Miltenyi Biotec, Bergisch Gladback, Germany) and either 20 or 200 lU/ml IL-2 (Aldesleukin, brand name Proleukin; Prometheus, San Diego, CA)), depending on the stage of culture at 37°C/5% CO2. T-cells were stimulated for expansion every 2-5 weeks with irradiated (3,000-3,100 cGy) allogeneic Peripheral Blood Mononuclear Cells (PBMCs) from three donors (from the Welsh Blood Service) and 1.2 pg/mL of phytohemagglutinin (PH A; Thermo Scientific). To clone T-cells, lines were plated in 96U wells at 0.5, 1 and 3 cells per well in 100 pL of T-cell media (as above with 20 lU/mL IL-2) and stimulated for expansion as above. After 7 days 100 pL of T-cell media was added to each well. On day 14, 50% of the media was removed and 5 xlO⁴ irradiated PBMCs from 3 donors, with a final concentration of 1 p/mL of phytohemagglutinin. On day 21, half of the media from each well was changed and from day 28 T-cell clones screened for reactivity towards cancer cells. T-cells were cultured for a minimum of 2 weeks post-restimulation before being used for experiments.

HLA A*02 knock-out of cancer cells

Oligonucleotides (IDT, Coralville, IA, USA) encoding guide RNA 5’- CCAGAGCCCCCGAGAGTAGC-3’ to target the HLA A*02:01 gene were cloned in to pLentiCRISPR v2 (Addgene 52961; gift from Feng Zhang). Lentiviral particles were generated using envelope plasmid pMD2.G (Addgene 12259; gift from Didier Trono) and packaging plasmid psPAX2 (Addgene 12260; gift from Didier Trono). PC-3, PANC-1 and MDA-MB- 231 are HLA A*02:01⁺ and knockout cell lines were created followed by cloning to ensure pure populations for the assays.

HLA A*0201 transgene expression in cancer cells

Codon-optimised HLA A*0201 genes were synthesised (Genewiz, Essex, UK) and cloned into a 3rd generation lentiviral plasmid pELNS (Kind gift from James Riley): Xbal-Kozak-HLA A*02-Stop-Xhol. For transfection, the envelope plasmid (pMD2.G) and packaging plasmids (pMDLg/pRRE and pRSV-REV) were used. PC-3, MIA PaCa-2 and AsPC-1 are HLA A*02:01^neg and transgene expression of HLA A*02 was maintained >90% by flow cytometry sorting or by using fluorochrome conjugated anti-HLA A*02 Ab and anti-fluorochrome magnetic microbead according to the manufacturer’s instructions (Miltenyi Biotec).

Lentiviral production and transduction

Lentiviral particles were generated using HEK293Ts and calcium chloride precipitation and unconcentrated lentiviral supernatant used immediately or stored at -80°C. HEK293Ts were cultured in D10 (as for R10, with DMEM as the based media from ThermoFisher Scientific). Cell lines were seeded (50-100 xlO⁴ cells/well respectively) in a 24 well plate a day before transduction. Prior to transduction, medium was aspirated, and 2 ml of lentiviral supernatant was added with 8 pg/ml +/- polybrene (Santa Cruz Biotechnology, Dallas, TX, US) (polybrene is toxic to some cell lines hence performing transduction +/-) then subjected to spinfection (by centrifugation at 500 g for 2 hours) followed by incubation at 37°C/5% CO2 overnight. The lentivirus-containing medium was then replaced with fresh medium the next morning. After 7 days, cells were tested for transgene expression or knockout via antibody staining and flow cytometry analysis.

Killing assays Flow cytometry based killing assays were previously described (Crowther, M.D. et al. Genome- wide CRISPR-Cas9 screening reveals ubiquitous T-cell cancer targeting via the monomorphic MHC class I-related protein MR1. Nat Immunol 21, 178-185 (2020). Briefly, cancer cells were incubated +/- T-cells in 96U well plates in 0.2 mL of T-cell media for 24h. Prior to harvest 2 xlO⁴ CFSE labelled C1R suspension cells (cultured in R10) were added to each well followed by staining with VIVID, and CD3 and CD8 Abs to exclude T-cells and dead cells, leaving viable cancer and CFSE labelled cells to calculate the percentage of killing using the equation below. +

/ / cancer cell events with T-cells CFSE CS 1R events

% killing = 100 - ) x 100 cancer cell events without T-cells CFSE CS1R events

IncuCyte® S3 Live-Cell Analysis System with cancer cells labelled with 0.75 pM CytoLight Rapid Red and IX caspase 3/7 green according to the manufacturer’s instructions (Sartorius, Gottingen, Germany). Flat bottomed 96U well tissue culture plates were sourced from Corning (product number 3595) as recommended by Sartorius. Four images per well were taken every 6 min for 48 h. For analysis T-cells were excluded based on their size and an overlap of green and red signal (dying cancer cells) displayed versus time.

Viability and surface antibody staining

5 xlO⁴ cells were typically stained per 5 mL fluorescence activated cell sorting (FACS) tube. Cells were washed with 3 mL of PBS then stained for 5 minutes at room temperature in the dark with 2 pL of 1 :40 diluted LIVE/DEAD®Fixable Violet Dead Stain Kit (VIVID; Thermo Fisher Scientific). Followed by staining with human FcR Blocking Reagent (Miltenyi Biotec) for 10 minutes at room temperature before the stain with fluorochrome-conjugated antibody of interest for 20 minutes on ice and in the dark: anti-CD8-APC, -APC Vio770™ or -FITC (clone BW135/80, Miltenyi Biotech), anti-CD3 -peridinin chlorophyll (clone BW264/56, Miltenyi Biotech) or anti-HLA A*02 (BB7.2, BioRad). The cells were then washed with 3 mL of PBS and (if needed) fixed in 4% paraformaldehyde (PFA) for 20 minutes on ice. Cells were then washed and resuspended in PBS and subsequently acquired on a NovoSampler Pro (Agilent, Santa Clara, CA, US) or BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, US) and analysed using FlowJo software (Tree Star, Inc.; Ashland, OR). Compensation was performed by single staining with fluorochrome-conjugated antibody of interest on anti-mouse Ig Compensation Particles (BD Biosciences) or by using cells with appropriate tetramer or antibody. Tetramer staining

Manufacture of biotinylated monomeric pMHC-molecules and multimerization to from tetramer with fluorescently labelled streptavidin was done as previously described in Rius, C. et al. Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T-cell Clonotypes and Underestimate Antigen-Reactive T-cell Populations. J Immunol 200, 2263- 2279 (2018). Premium grade PE and APC conjugated streptavidin were sourced from Thermo Fisher Scientific. T-cell clones or lines (0.5-1 xlO⁵) or PBMCs (3-4 xlO⁶) were pre-treated with the PKI (50 nM) Dasatinib (Axon Medchem, VA, USA), for 5-60 min (typically 30 min) at 37°C in 100 pL of PBS with 2% FBS (FACS buffer). Tetramers were spun in a microfuge to remove aggregates (full speed for 1 minute), and then 0.5 pg (with respect to pMHC component) added directly to each sample without washing or pre-chilling, followed by incubation for 30 min on ice and in the dark. Cells were washed with 3 mL of PBS then stained with VIVID and antibodies as above. Tetramer staining was enhanced by addition to the antibody staining step of 0.5 pg (10 pg/mL) of mouse anti-PE unconjugated Ab (clone PE001, BioLegend, London, UK) or anti-APC unconjugated Ab (clone APC003, BioLegend) to match the fluorescence of the tetramer being used (ref. Dolton, G. et al. Optimised peptide-MHC multimer protocols for detection and isolation of autoimmune T-cells. Frontiers in immunology doi: 10.3389/fimmu.2018.01378 (2018)). Samples for analysis were fixed if needed, as above.

Tetramer Enrichment from whole PBMCs

PBMCs were treated and incubated with PKI and tetramers as above. Cells were washed in 3mL of chilled MACS buffer (D-PBS, 0.5% bovine serum albumin (both Merck Group), and 2 mM EDTA, pH 7.2-7.5). Anti-PE magnetic microbeads were used according to the manufacturer’s instructions, whereby 80 pL of MACS buffer and 20 pL of beads were used per 1 xlO⁷ cells, with no scaling down for lower cell numbers. Positive cells were collected by centrifugation (400g for 5 min) and incubated overnight in a single well of a 96 U well plate, in T-cell media. Cells were expanded in the same well with PHA and allogenic PBMCs, as described above.

Enzyme-linked immunosorbent assay (ELISA)

T cells were rested in R5 (R10 but with 5% FBS) for 24 hours before the assay. Typically, 3 x 10⁴ T-cells and 6 x 10⁴ target cells (1:2 ratio) were co-cultured per well of a 96U well plate in 100 pL R5 and incubated overnight. Supernatants were then collected the following morning and the concentration of macrophage inflammatory protein 1-P (MIP1-P) was quantified using the respective detection kit (R&D Systems, Bio-Techne Minneapolis, MN, USA), according to the manufacturer’s instructions.

TNF Processing Inhibitor-0 (TAPI-0) Assay

T-cells were rested in R5 (R10 but with 5% FBS) for 24 hours before the assay. Typically, 3xl0⁴ T-cells and 6xl0⁴ target cells (1:2 ratio), along with TAP-I 0 mixture (consisting of 30pM TAP-I 0 (Santa Cruz Biotechnology), IpL anti-CD107a PE (clone H4A3, BD Biosciences) and 1.2pL anti -TNF PE-Vio770 (clone cA2, Miltenyi Biotec)) were co-cultured per well 96U plate in lOOpL R5. T-cells alone condition was used as a negative control, whereas positive control comprised of T-cells activated with 2pL Dynabeads Human T- Activator CD3/CD28 (Thermo Fisher Scientific). The assay plate was then centrifuged at 400g for 5 minutes and placed in the incubator for 4-6 hours at 37°C/5% CO2. After incubation, cells were washed with 3 mL PBS and stained with VIVID for 5 minutes at room temperature. Followed by staining with human FcR Blocking Reagent for 10 minutes at room temperature and then with the appropriate mixture of T-cell surface antibodies (CD3 and CD8) for 20 minutes on ice. Cells were washed with 3 mL PBS and resuspended before acquisition on NovoSampler Pro (Agilent). Alternatively, cells were fixed using 50pL of 4% paraformaldehyde solution for 20 minutes on ice, washed with 3mL PBS before samples were acquired. Data were then analysed using FlowJo Software.

Flow cytometry-based cell sorting for culture and sequencing

Antibody and tetramer labelled cells were sorted based on purity on a SONY MA900 using a lOOpM sorting chip (Sony Biotechnology, San Jose, CA, US) and captured in appropriate culture media. Sorted cells were then washed and resuspended in the respective culture media with 25pg/mL of Amphotericin B (Thermo Fisher Scientific) and lOpg/mL of Ciprofloxacin (Ciproxin, Bayer, Leverkusen, Germany). T-cells for sequencing were lysed as described below.

T-cell receptor sequencing

Up to 1,000,000 T-cells were washed in PBS and resuspended in RLT lysis buffer (Quiagen) supplemented with 40 mM Dithiothreitol (DTT). Total mRNA from T-cell clones and lines was extracted using the RNEasy Micro Plus Kit (Qiagen, Heidelberg, Germany) according to manufacturer’s instructions. Full-length cDNA was generated using 5’ SMARTer™ (Switching Mechanism At 5’ end of RNA Transcript) RACE (Rapid Amplification of cDNA Ends) kit (Takara Bio, Kusatsu, Shiga, Japan). TCR cDNA was then amplified using a two- step Nested PCR designed to capture the whole variable (V) region of TCR a- or P- chains using forward primers binding to 5’ SMARTer Oligo, and reverse chain- specific primers targeting the 3’ constant domain of the TCR. On the second amplification step, “internal” primers were designed to anneal upstream of the sequence amplified by the first set of primers. Internal primers were also designed to include universal adaptors for multiplex sequencing. Resulting amplified PCR products were purified by gel extraction (Monarch®, New England BioLabs, Ipswich, MA, USA) and DNA concentration measured using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). Libraries were run on an Illumina MiSeq instrument using the MiSeq v2 reagent kit (Illumina, San Diego, CA, USA) and TCR gene usage determined using MiXCR software (v3.0.3).

Example 1 - IMP2 Cancer cell culture

As shown in Figure 1, Expression of IMP2 in a variety of cancer cells of different tissue origin was tested against insulin. RNA sequence data taken from the TRON Cell Line Portal (http ://celllines .tronmainz.de) . INS(Insulin) values were included as a baseline. Based on the results, breast, pancreatic and prostate cancer cells lines were selected for the study and engineered with CRISPR-Cas9(MDA-MB-231andPANC-l) or transgene (PC-3, MIAPaCa- 2andAsPCl) to provide isogenic cancer cell lines with and without HLAA*02:01 to study IMP2 specific T-cells. The M0LT3 line (IMP2 low expression) was selected for epitope validation (Figure 2).

Example 2 - Role of IMP2 derived epitope NLSALGIFST in cancer pathologies

Previous studies on tumour-infiltrating lymphocyte (TIL) used to treat two patients and ex vivo staining of peripheral blood mononuclear cells (PBMC) showed that they contained large numbers of IMP2-specific T-cells. Over 1 in 50 CD8 T-cells bound to an HLA A*02:01- NLSALGIFST tetramer. ALWGPDPAAA from preproinsulin was used as an HLA A*02 irrelevant tetramer. The successful treatment of end-stage solid cancer patients with TIL containing large numbers of activated IMP2-specific T-cells and the long-term persistence of these cells in vivo without any obvious pathology instils confidence that targeting IMP2 in vivo will be safe.

A T-cell clone (NLS.732) was generated from a donor and reactive to NLSALGIFST. This is shown by the fact that it stains and is reactive with IMP2 tetramer and responds to a titration of exogenous NLSALGIFST peptide (Figs. 2 A and B). The NLSALGIFST epitope was validated by expression of IMP2 and HLA A*02:01 genes in the M0LT3 cell line (Figure 2C) (one of very few cancer cell lines that does not naturally express IMP2 (ref. Example 1). Collagen was used as an irrelevant protein, also expressed with the rCD2 co-marker.

IMP2 T-cell clones react towards the HLA A*02⁺ MOLT3 cell line expressing IMP2, confirming that the epitope is naturally processed and presented (Figure 2D).

Supernatants were used in Figs. 2D and 2E for MIP-10 EEISA with error bars depicting SEM of duplicates. The IMP2 -reactive T-cell clone NLS.732 recognises cancer cells of different tissue origins, including cervical, prostate and kidney cancer cell lines providing they express HLA A*02 (Figure 2E), showing that the IMP2 derived epitope NLSALGIFST is a suitable target for a wide variety of cancer pathologies.

Example 3 - T-cells with proven cancer killing capabilities are IMP2-specific T-cells

T-cells from the PBMCs of a long-term cancer survivor were enriched with phycoerythrin (PE)-conjugated HLA A*02-NLSALGIFST tetramer and anti-PE magnetic microbeads, followed by expansion with allogeneic PBMCs and PHA, creating an 80% IMP2 tetramer positive T-cell line. This is shown in Figure 3 A. T-cell clones generated from this line respond to exogenous IMP2 NLSALGIFST peptide.

Flow cytometry-based activation assays (according to Betts, M.R. et al. Sensitive and viable identification of antigen- specific CD8+ T-cells by a flow cytometric assay for degranulation. J Immunol Methods 281, 65-78 (2003) and Haney, D. et al. Isolation of viable antigen- specific CD8+ T-cells based on membrane -bound tumor necrosis factor (TNF)-alpha expression. J Immunol Methods 369, 33-41 (2011)) with the IMP2 T-cell line demonstrated HLA A*02 dependent recognition of prostate (PC-3) and pancreatic (MIA PaCa-2 and ASPC-1) cancer cells. This is shown in Fig. 3B.

Cytotoxicity of the IMP2 line towards breast (MDA-MB-231) and pancreatic (PANC-1, AsPC- 1 and MIA PaCa-2) cancer cell lines was demonstrated with flow cytometry based (Figure 3B) or real-time (Figure 3C) killing assays. Each of the cell lines is HLA A*02^neg and engineered to express HLA A*0201 as a transgene and lines maintained at >90% HLA A*02⁺ based on Ab staining.

Example 4 - HLA A*02-NLSALGIFST T-cell is dominated by TRAV12-2 and TRAV12-3 TCRs

The IMP2-specific T-cell line from Example 3 was re-sorted by HLA A*02-NLSALGIFST tetramer staining in two dimensions (PE and allophycocyanin; APC as shown in Figure 4A. TCRs were analyzed by Next Generation Sequencing. Bulk sorting and T-cell receptor (TCR) sequencing of the gated populations as shown in Figure 4A revealed 20 TCR alpha chains and 37 TCR beta chains as shown in Figure 4B. TCR variable (V) (arc on the right) and joining (J) (arc on the left) gene rearrangements are displayed, with the dominant clonotypes annotated. TCRs from clones grown from the IMP2 line are indicated by arrows that are indicated according to the key.

The IMP2 line was dominated by two TCR alpha chains: TRAV12-3 TRAJ31 CDR3a- CAIDNARLMF (59.77%) and TRAV29DV5 TRAJ56 CDR3a-CAAEGPGANSKLTF (26.65%), and one TCR beta chain TRBV7-9 TRBJ1-1 CDR3p-CASSRGPMGTEAFF (46.94%) (Figure 4C).

The other TCRs exhibited a TCR alpha bias towards TRAV12-2 (16/20 of the TCRs, Figure 4C); IMP2 NLSALGIFST peptide-reactive T-cell clones grown from the IMP2 line had their TCRs sequenced, which are indicated on the table according to the key.

clones of a naturally sourced IMP2-specificT-cellline

The T-cell line from Example 3 and 4 was cloned by limiting dilution and produced 35 clones that grew well, with 15 of them reacting convincingly to exogenous IMP2 peptide (Figure 5A). T-cells alone (-ve control) and PHA (+ve control) conditions are also displayed.

Twelve of the clones grew sufficiently to be stained with HLA A*02 IMP2 tetramer, confirming their specificity (Figure 5B). The mean fluorescence intensity (MFI) of staining is displayed. The same key indicators represents clones expressing the same TCR (see TCR sequencing in Figure 4).

TCR sequencing of 14 of the 15 clones (clone 26 died) revealed 13 ‘true’ clones (clone 6 was not clonal) and 5 unique TCRs (Figures 4-7).

Seven of the clones (3, 7, 8, 19, 22, 28 and 33) co-express the two dominant TRAV12-3 and TRAV29DV5 TCR alpha chains seen in the IMP2 line, and the dominant TRBV7-9 TCR beta chain (details above).

Clones 1 and 31 express TRAV12-2 TRAJ43 CDR3a-CAGNNNDMRF and TRBV10-3 TRBJ2-7 CDR3p-CAIGTGGTYEQYF. Clone 4 expresses TRAV12-2 TRAJ15 CDR3a- CAVNQAGTALIF and TRBV6-1 TRBJ2-1 CDR3p-CASREPGLGVNEQFF. The amino acid sequence of the CDR3a of clone 4 appeared twice in the sequencing results for the IMP2 line (frequencies of 0.94% and 0.06%), but with different nucleotide sequences, which could be attributed to either an independent TCR rearrangement or sequencing error. Clones 16 and 23 express TRAV12-2 TRAJ27 CDR3a-CAVDAGKSTF and TRBV11-2 TRBJ2-7 CDR3p-CASSVPGASYEQYF. Finally, clone 27 expresses TRAV12-2 TRAJ43 CDR3a-CAVTPRYQKVTF and TRBV10-3 TRBJ2-7 CDR3p-CASSQDGAGAYEQYF.

The nucleotide sequences of the TCRs from the clones are shown in Figures 6 and 7.

Example 6 - Preliminary testing of the IMP2 clones against cancer cells

Four of the cloned TCRs according to Example 5 were used in preliminary activation assays with various cancer cells +/- HLA A*02 expression.

Reactivity towards breast, prostate and pancreatic cancer cell lines is most marked for clone 3 (IMP2 line dominant TCR with 2 alpha chains, found in 7 sister clones) and clone 23 (second IMP2 line dominant alpha chain expressed by clones 16 and 23) as shown in Figure 8. Overnight activation and supernatants used for MIP-ip ELISA with error bars depicting SEM of duplicates.

Example 7 - Demonstration of TCR binding affinity

Surface Plasmon Resonance (SPR) experiments were carried out on 2 preferred embodiments of the invention to demonstrate binding to IMP2.

Codon optimization and removal of TM regions of SEQ ID NOs 60, 61, 64 and 65 was carried out for expression in CHO cells. This produced TCR having the following relevant sections as detailed above in Table 3 and shown here below in Table 4:

Table 4

The resultant TCR were biotinylated in vivo by co-transfection with biotin ligase enzyme DNA. Soluble TCRs (EXTCR1 and EXTRC2) were harvested from conditioned media and dialysed into SPR buffer before analysis.

CM5 chips were used to immobilize streptavidin via NH2. Biotinylated EXTCR1 and EXTRC2 were loaded onto streptavidin coated chip (-200 RU each). pHLA presenting SEQ ID NO. 1 lowed over TCR loaded chips (10 concentrations in two-fold serial dilution). Chip coated with streptavidin (no TCR) used as reference and subtracted from all data sets.

The results of the SPR analysis for EXTRC1 and EXTRC2 are shown in Figure 9. Western blotting against biotin using Strep tavidin-HRP was also carried out to confirm that both the alpha and beta chain were being expressed for both TCRs.

Using a 1-to-l model from the data in Figure 9, one can see that EXTRC1 and EXTRC2 have a KD of 71.1 pM and 16.3 pM respectively as reported in Table 5 below.

Table 5

Claims

1. A TCR that binds specifically to IMP2.

2. The TCR according to claim 1, wherein the TCR binds specifically to NLSALGIFST (SEQ ID NO. 1) or a fragment thereof.

3. The TCR according to claims 1 or 2, wherein the TCR alpha chain variable domain comprises the sequence of a TRAV 12-2 or a TRAV 12-3 or a TRAV29DV5 chain or a sequence having at least 90% identity thereto.

4. The TCR according to any of claims 1-3, wherein the TCR alpha chain variable domain comprises a CDR3 sequence selected from the group consisting of SEQ ID NO. 2-21.

5. The TCR according to any of claims 1-4, wherein the TCR beta chain variable domain comprises a CDR3 sequence selected from the group consisting of SEQ ID NO. 22-58.

6. The TCR of any one of claims 1 to 5, wherein the alpha chain variable domain comprises a combination of sequences selected from the group consisting of:

7. The TCR of any one of claims 1 to 6, wherein the beta chain variable domain comprises a combination of sequences selected from the group consisting of:

8. A TCR construct comprising a TCR according to any one of claims 1-7.

9. A nucleic acid encoding the TCR alpha chain variable domain and/or TCR beta chain variable domain of the TCR of any one of claims 1-7 or the TCR construct of claim 8.

10. The nucleic acid according to claim 9, wherein the nucleic acid is selected from the group consisting of SEQ ID NO. 59-69.

11. A vector comprising the nucleic acid of claim 9 or 10.

12. The vector of claim 11, wherein the vector is an expression vector.

13. A cell harbouring the TCR according to any of claims 1-7, the TCR construct according to claim 8, the nucleic acid according to any of claims 9 or 10 or the vector of claims 11 or 12.

14. The cell according to claim 13, wherein the cell is a leukocyte.

15. A pharmaceutical composition comprising the TCR according to any of claims 1-7, the TCR construct according to claim 8, the nucleic acid according to any of claims 9 or 10 or the vector of claims 11 or 12 or the cell according to claim 13 or 14 and a pharmaceutically acceptable carrier.

16. The TCR according to any of claims 1-7, the TCR construct according to claim 8, the nucleic acid according to any of claims 9 or 10 or the vector of claims 11 or 12, the cell according to claim 13 or 14 or the pharmaceutical composition according to claim 15 for use as a medicament.

17. The TCR according to any of claims 1-7, the TCR construct according to claim 8, the nucleic acid according to any of claims 9 or 10 or the vector of claims 11 or 12, the cell according to claim 13 or 14 or the pharmaceutical composition according to claim 15 for use in diagnosing, prophylaxis and/or the treatment of neoplasia.

18. The TCR according to any of claims 1-7, the TCR construct according to claim 8, the nucleic acid according to any of claims 9 or 10 or the vector of claims 11 or 12, the cell according to claim 13 or 14 or the pharmaceutical composition according to claim 15 for use according to claim 18, wherein the neoplasia is malignant.

19. The TCR according to any of claims 1-7, the TCR construct according to claim 8, the nucleic acid according to any of claims 9 or 10 or the vector of claims 11 or 12, the cell according to claim 13 or 14 or the pharmaceutical composition according to claim 15 for use according to claim 18, wherein the malignant neoplasia is breast, pancreatic and/or prostate cancer.