High Affinity Melan-A T cell receptors
The present inveaitiDn relates to T cell receptors (TCRs) having His property' of faindiiis to AAGIGrLTV-HLA-A*0201 and comprismg at least one TCR a chain variable domain and/or at least one TCR p chain vaziable domain CHARACTERISED IN THAT said TCR has a KD for the said AAGIGILTV-HLA-A*0201 con^lex of less than or equaJ to 3 fiM and/or an ofi-rate (kos) of 1 x 10'^ S'l or SIOWCT
Backsroimd to the Invention
The AAGIGELTV peptide is derived from the Melan-A (Mart-1) protein that is expressed by the majority of fresh melanoma samples and approximately 60% of Melanomacelllines, as well as nomial melanocytes. {(CoxiUee? a/., (1994)/ Exp. Med. 180: (1) 1-4) and Kawakami et at., (1994) PNAS USA 91. 3515) The Class I HLA molecules of these cancerous cells present peptides from tiiis protein, including
AAGIGILTV (SEQ ID NO: 43) (Melan-Azvos). The AAGIGILTV-HLA-A*0201
complex appears to be an immuno-dominant tai^et for Melanoma-specific T cells. ((Kawakami etal., (1994) FNAS USA 91: 3515) and (Rjvoltim etal, (1995) J. Immunol 154: 2257) Tlierefore, this peptide-HLA complex provides a cancer marker that TCRs can target, for example for the purpose of delivering cytotoxic or immuno-
stimulatory agents to the cancer cells. However, for that purpose it would be desirable
if the TCR had a higher affinity and/or a slower off-rate for the p^rtide-HLA complex flian native TCRs specific for iimt complex.
Brief Deacrjptiop of the Invention
This invention makes available for -fee first time TCRs havii^ high affinity (KD) of lie interactioii. less than or equal to 3|iM and/or ao off-rate (kosd of I x 10"^ S'l or slower for the AAGIGILTV-HLA-A*020i complex. Sudi TCRs are usefiil, eifiier alone or associated with a terapieutic agent for targeting cancer cells presenting that complex.

Detailed Description of the Invention
The present invention provides a T-cell receptor (TCR) having the property of binding to AAGIGILTV-HLA-A*02Ol and comprising at least one TCR a chain variable domain and/or at least one TCR p chain variable domain CHARACTERISED IN TEIAT said TCR has a KD for the said AAGIGILTV-HLA-A*0201 con^lex of less than or equal to 3fiM and/or an off-rate (TiofEt of 1 x 10'^ S'l or slovrar.
In a farther embodiment the present invention said TC3ls have a KD for tiae AAGIGILTV-HLA-A*0201 complex of less than or equal to IMM.
The Kd msasuraiDent can be made by any of the known mefeods. A preferred meHiod is the Surface Plasmon Resonance (Biacore) method of Example 4.
For comparison, the interaction of a disulfide-linked soluble variant of the native MEL TCR (see SEQ ID NO: 9 for TCR a chain and SEQ ID NO: 10 for TCR p chmn) and the AAGIGILT\'-HLA-A*0201 complex has a KQ of approximately 4jiM as measured by the Biacore-base method of Example 4.
The native MEL TCR specific for the AAGIGILTV-HLA-A*0201 complKt has the following Valpha chain and Vbeta chain gene usage (using the terminology of the T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, see below):
Alpha chain- TRAV 12-2 Beta chain:- TRBV30
The native MEL TCR can be used as a tsaplsAe into which various mutations tiiat impart hi^ afBnity and/or a slow off-rate for fee interaction betweeai TCRs of the inventian and Ihe AAGIGILTV-HLA-A*0201 complex can be introduced. Thus the iaventimi includes TCRs wiich are mutated relative to the native MEL TCR a chain variable region (see Figure laandSEQ IDNo: 1) and/or fl chain variable region (see

Figure 1 h and SBQ ID NO: 2) in est least one compiementarity detenmning region (CDR) and/or variable region framework region thereof It is also contemplated that other hypCTvariable regions in the variable regions of the TCRs of Hie in'vnntion, such as the hypervariable 4 (HV4) regions, may be mutated so as to produce a higb affinity mutant
Native TCR^ racist in heterodimeric op or yb forms. However, recombinant TCRs consisting of a single TCR a or TCR p chain have previously been shown to bind to peptide MHC molecules.
In one embodiment the TCR of the invention comprise both an a chain variable domain and an TCR p chin variable domain
As will be obvious to those skilled in the art the midation(s) in the TCR a chain sequeence and/or TCR P chain sequence may be one or more of substitution(s), deietion(s) or insenion(s). These mutations can be carried out using any appropriate method including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation independent cloning (LIC) procedures. These methods are detailed in many of the standard molecular biology texts. For further details regarding polymerase chain reaction (PCR) mutagenesis and restriction enzyme-based cloning see (Sambiook & Russell, (2001) Molecular Cloning — A Laboratory Manual (5"* Ed.) CSHL Press) Rnther information on LIC procedures can be found in (Rashtchian, (1995) Citrr Opin Biotechml 6 (1): 30-6). Phage display provides one means fay which liraries of TCR variants can be generated. Mefiiods suitable for the phage display and subsequent screening of libraries of TCR variants eadi containing a non-native disulfide interdiain bond are detailKi in (Li eta!., (2005) Nature Biotech 23 (3): 349-354) and WO 2004/04404.
It should be noted that any ctp TCR that conqnises similar Valpha and Vbeta gene usage and Hierefore amino acid sequmce to that of the MEL TCR could make a convenient template TCR It would then be possible to introduce into the DNA

encoding one or both of the variable regions of the texaplate ap TCR fee changes required to produce the mutated high affinity TCRs of the invention. As will be obvious to those skilled in the art, the necessary mutations could be introduced by a number of methods, for example site-directed mutagenesis.
Unless stated to the contrary, the TCR amino acid sequences herein are generally provided including an N-temiinal methionine (Met or M) residue. As will be known to tiiose skilled in the art iMs residue may be removed during the production of recombinant proteins. As will also be obvious to Ihose skilled in the art, it may be possible to truncate the sequences provided at the C-terminus and/or N-terminus thereof by i, 2, 3, 4, 5 or more residues, without substantially affecting tiie pMHC binding characteristics of the TCR, all such trivial variants are enconmpassed by the present invention.
As used herein the term ''variable region" is underatood to encompass all amino acids of a given TCR which are not included within the constant domain as encoded by the TRAC gene for TCR a chains and either the TRBCl or TRBC2 for TCR p chains. (T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-S)
As used herein the term "variable domain" is understood to encompass all amino acids of a given TCR wiiich are included within the amino add sequence encoded by a TRAV gene for TCR a chains and a TRBV gene for TCR p diains. (T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-B)
Embodiments of the invention include mutated TCRs which coinprise mutatioQ of one or more of alpha chain variable region amino acids corresponding to: 28D,29R, 30G, 31S, 49M, 511. 53S. 54N, 72Y, 94V, 95A, 96G, 97K, 98S, and 99T, for example the mutations corresponding to:


TTie numbering used above is the same as that shown m Figure la and SEQ ID No: J
Bmbodimcnts of the inveaition indude mutated TCRs which comprise mutation of one or more of beta chain variable region amino acids corresponding to: 45L, 51S, 52V, 53G, 541, 761, lOOG, lOlT, 102G, 103E, 104L and 105F, using the numbering shown in SEQ ID NO: 2 is/are mutated, for example the mutations corresponding to:



The numbeaing used above is the same asthat shown in Figure lb and SEQ ID No: 2
Further preferred embodiments of the inventiojn are jffovided by TCRs con^jrisiiig one of the mutated alpha chain variable region amino acid sequences shown in Figure 6 (SEQ ID Nos: 11 to 24) or Figure 20 (SEQ ID NOs: 47 to 53). Phenatypicany sOent variants of such TCRs also form part of tiiis invention.
Further preferred embodiments of the invention are provided by TCRs compring one of the mutated beta chain variable region amino acid sequences shown in Figure 21 (SEQ ID Nos: 54 to 67). Pheaotypically silent variants of such TCRs also form part of
this invention.
Native TCRs exist in iieterodimKic ap or yS forms. However, recombinant TCRs consisting of ota or pp homodimers have previously been shown to bind to pqitide MHC molecules. Therefore, one embodimeol of the invention is provided by TCR aa or TCR pp homodimers.
Further preferred embodiments are provided by TCRs of the invention comprising thfe alpha chain variable region amino acid sequence and the beta chain variable region amino acid sequence combinations listed below, phenotypically silent variants of such TCB^ also ferm part of this invention;

Preferred embodiments provide TCRs of the invention cominising:
the alpha chain variable region shown in ffae SEQ ID NO: 11 and the beta chain variable region shown in the SEQ ID NO: 2,
Aealphachaic variable region shown in die SEQ ID NO: 47 and the beta chain
variable region shown in the SEQ ID NO: 2.
the alpha chain variable region shownio fee SEQ ID NO: 4K and the beta chain variable region shown in the SEQ ID NO: 2.
the alpha chain variable region ^lown in tiw SEQ ID NO: 53 and the beta chain variable region shown in the SEQ ID NO: 2.
the alpha chain variable region shown in the SEQ ID NO: 11 and fee beta chain variable region shown in fee SEQ ID NO: 54.
the alpha chain variable region sho^ism. in fee SEQ ED NO: 11 and the beta chain variable region shown in the SEQ ID NO: 55.
the alpha chain variable region shown in fee SEQ ID NO: 11 and the beta chain variable region shown m fee SEQ ID NO: 56.

the alpha chain variable region shown in liie SEQ ID NO: ] 1 and the beta chain
variable re^on shown in the SEQ E) NO: 57.
the alpha chain variable region shown in the SEQ ID NO: 31 and the beta chain variable region shown m the SEQ E) NO: 58.
the alpha chain variable region shown in the SEQ ID NO: 11 and the beta cdiain variable region shown in the SEQ ID NO: 62.
the alpha chain variable region shown in the SEQ ID NO: 11 and the beta chain variable region shown in the SEQ ID NO: 65.
the alpha ohmn variable region shown in the SEQ ID NO: 11 and the beta frhgin variable region shown in the SEQ ID NO: 66,
Or phsnotypicaBy silent variants of any of the above TCRs.
In another prefsrred embodiment TCRs of the invention comprisiDg the variable region combinadons detailed above further comprise H^ alpha chain constant region aniino add sequiMice shown in Figure 7a (SEQ ID NO: 25) and tme of the beta chain amino acid constant re^on sequences shown in Figures 7b and 7c (SEQ ID NOs; 26 and 27) or phenotypicaUy silent variants thereof.
As used herein the tenn "phenotypicaUy silent variants" is understood to refer to Ihose TCRs which have a Kp for the said AAGIGILTV-HLA-A»0201 complex of less than or equal to SjiM. For example, as is known to those skilled in the art, it may be possible to produce TCRs ibst incorporate minor changes in the constant and/or variable regions fliereof compared to those detailed above without altraing the affinity andyor off-rate for the interaction with the AAGIGILTV-HLA-A*0201 complex. Such trivial variants are included in ■Qie scope of this invention. Those TCRs in vAach one or more conservative substitutians have been made also fram pari of tins invention

In one hroad aspect, the TCRs of the inveniion are in the farm of either singie chain TCRs (scTCRs) oir dimsric TCRs (dTCRs) as describsd in WO 04/033685 aafl WO
03/020763,
A suitable scTCR form comprises a first segment constituted by an anuno acid sequence coiresponding to a TCR a chain variable region, a second segment constituted by an amino acid sequence coiresponding to a TCR p chain variable region sequence fiised to the N terminus of an amino acid sequaice corresponding to a TCR p chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of ^ second segment
Alternatively the first segment may be constituted by an amino acid sequence corresponding to a TCR P chain variable region; the secotui segment may be constituted by an amino acid sequence corresponding to a TCR a chain vari^le region sequence fused to the N terminus of an amino acid sequence correspondinE to a -
TCR a chain constant domain extracellular sequeoce
The above scTCRs may further comprise a disulfide bond between the first and second chains, said disulfide bond b^g one which has no equivalent ui native tzpT cell recepiors, and ■wherein the length of the linker sequence and 4e position of the disulfide bond being such that the variable domain sequences of the first and second segments are mutually orientated sufastantiaUy as in native ap T cell receptors.
More specifically the first segment may be constituted by an amino acid sequence corresponding to a TCR a chain variable region sequence fiised to the N terminus of an amino acid sequence corresponding to a TCR a chain constant domain extracellular sequence, the second segment may be constituted by an amino acid sequence corresponding to a TCR p chain variable region fused to the N terminus of an amino acid sequence corresponding to TCR p chain constant domain extracellular sequence,

and a disulfide bond mEy be provided between ib& first and sectmd cJiaans, said disulfide bond being one which has no equivalait in native aP T cdl receptors.
In the above scTCR forms, the linker segufince may link the C terminos of the first segment to liie N tenniniw of the secoaid segmentj and may have fee fiMmula -PGGG-(SGGGG)r-P- whaeic n is 5 or 6 and P is proline, G is ^ycane and S is serine.

A suitable dTCR form of flie TCRs of the present invention comprises a first polypqitide herein a sequence corresponding to a TCR. a chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR a chain constant domain extracellular sequence, and a second polypepti^ wherein a sequence corresponding to a TCR P chain variable region sequence fused lo the N terminus a sequence corresponding to a TCR p chain constant domain extracelhilar seqiience, the first and second polypeptides being linked bj' a disulfide bond -which has no equivalent in native aP T cell receptors.
The first polypeptide may comprise a TCR a chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR a chain constant domain extracellular sequence, and a second polypeptide wherein a sequence correspondii^ to a TCR p chain variable region sequence is fused to the N terminus a sequence corresponding to a TCR P chain constant domain extracellular sequence, &e first and second polypeptides being linked by a disulfide bond between cysteine residues substituted for Tfar 4& of exon 1 of TRAC*01 and Ser 57 of exon 1 ofTRBC!*Ql or TRBC2*01 or the non-human equivalent liiereof. CTRAC" etc. nomenclature herein as per T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8)

Tlie dTCai or scTC3l form of the TCRs of the invention may have amino add sequences corresponding to human a.^ TCR extraoelMar constant and variable region sequences, and a disulfide bond may link amino acid residues of tiie said constant domain sequences, whidi disulfide bond has no equivalent in native TCRs. Ths disulfide bond is between cysteine residues corresponding to amino acid residues whose p carbon atoms are less &an 0.6 nm apart in native TCRs, for example between cysteine residues substituted for Tlir 48 of exon I ofTRAC*01 andSer57of exon I ofTRBCi'Ol orTRBC2*0I or the non-human equivalent thereof Other sites w^iere cysteines can be introduced to form the disulfide bond are the foIlowii:g residues in exon 1 of TRAC*01 for the TCR a chain and TRBCl *01 or TRBC2*01 for the TCR p chain;

Specific embodiments of the invention provided a TCR of the invention which is a dTCR comprising
a first polypeptide wherein a sequence corresponding to a TCR a chain variable region sequence is fused to the N tcnninus of a sequence corresponding to a TCR a chain constant domain extraceEular sequence, and
a second polypeptide wtierein a sequence corresponding to a TCR p chain variable region sequence fused to the N tramimis a sequence corresponding to a TCR p chain constant domain extracellular seqiience,

the &st and second polypeptides bemg linked by a disulfide bond which has no equivalent in nati-\^ op T ceU between cj'steine residues substituted for "nir 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 of TRBC1*01 or T:RBC2*01 or the non-human equivalent thereof,
In addition to the non-native disulfide bond refen^ to above, the dTCR or scTCR form of the TCRs of the invention may include a disulfide bond between residues corresponding to tiiose linked by a disulfide bond in native TCRs.
The dTCR or scTCR form of the TCRs of the invention fnKfeiably does not contain a sequence corresponding to transmembrane or cj^oplasmic sequences of native TCRs.
Currently preferred embodiments of the invention provide soluble TCRs comprising:
the alpha chain amino acid sequence of SEQ ID NO: 29 and beta chain amino acid sequence SEQ ID NO: 10.
me alpha chain amino acid sequence of SEQ ID NO; 68 and beta chain amino acid sequence SEQ ID NO: lO.'
the alpha chain amino acid sequence of SEQ ID NO; 69 and beta chain amino acid sequence SEQ ID NO: 10.
the alpha cham amino acid sequence of SEQ ID NO: 70 and beta chain amino acid sequence SEQ ID NO: 10.
the alpha chain amino acid sequence of SEQ ID NO: 29 and beta chain amino acid sequence SEQ ID NO: 71.
the alpha chain amino acid sequence of SEQ ID NO; 29 and beta, chain amino acid sequence SEQ ID NO: 72.

the alpim chain amino acid sequence of SEQ ED NO: 29 and "beta chain amino add ssgumce SEQ ID NO: 73.
the alpha chain amino add sequence of SEQ ID NO: 29 and beta chain amino acid sequence SEQ ID NO: 74.
the alpha chain amino acid sequence of SEQ ID NO: 29 and beta chain amino add sequence SEQ ID NO: 75.
the alpha chain amino add sequence of SEQ ID NO: 29 and beta diain amino add sequence SEQ ID NO: 76.
the alpha chain amino add sequence of SEQ ID NO: 29 and beta diain amino add sequence SEQ ID NO: 77.
the alpha chain amino acid sequence of SEQ ID NO: 29 and beta chain amino acid sequence SEQ ID NO; 78.
Also provided is a nudeic acid or nudeic acids encoding TCRs of the invention. Such a nudeic acid or nudeic adds may be provided in a form which has been adapted for expression in a prokaryote or eukaryote host cdl. Suitable host cells include, but are not limited to, bacterial, yeast, mammalian or insect cells. For example, the host cell may be a human T cell or a himian haematopoietic stem cell.
Such adapted nudeic add or nucldc adds is/are mutated to reflect liie codon preference of the host cell in to -wiack it is introduced. Ilie mutations introduced are silent mutations v/bidh do not affect the amino acid sequence of llie polypeptide or polypE^Jtides Hiereby encoded. GeneArt (Regensburg, Germany) offer a suitable nudeic add c^rtimisation service (GeneOptimizer'™). WO 2004/059556, owned by GesneAit, provides fiirther details of tiie optimisation process.

FurflieEr cuireaitly preferred embodiiaeints of the inventitm are provided by nudeic adds consisting of one of liie full-iength TCR a chain DNA sequences of SEQ ID Nos 33,35 or 37 (Figures 12a, 13a, or 14a, respectively) and the TCR p chain DKA sequence of SEQ ID No 39 (Shown in Figure I5a). Anucleic acid cornplementary to any of the foregoing, or a corresponding RNA sequence also forms part of this invention. Furthermore, as will be obvious to those sldllsd in tlie art sudi nucleic acid or nucleic acids encoding TCRs of ti^ invention may also comprise noo-coding (intron) sequences.
The fuU-length wild-type and high affinity MEL TCR chain DNA sequences of SEQ ID Nos: 31,33,35,37 and 39 encode the amino acid sequences of SEQ ID Nos: 32, 34,36, 38, and 40 respectively. (Figures lib, 12b, I3b, 14b and 15b respectively)
TTae amino acids sequences of SEQ ID Nos; 33, 35 and 37 comprise the irigh afBnity MEL TCR alpha chain variable regions of SEQ ID Nos: 11,15 and 23 respectively.
As will be obvious to those skilled in the art such full-length TCR chain DNA sequences encode for the following sequences:
A leader sequence and the extracellular, transmembrane, and cyKipiasmic TCR
sequences.
The fiill-lengfli DNA sequences provided herein also include restriction enzyme recognition sequences to fecilitate Ugation into the vector of choice,
PEOylated TCR Monomers
In one particular embodiment a TCR of the invention is associated wifli at least one poJ)falkyIene glycol chain(s). This association may be cause in a number of ways known to those skilled in the art In a pre&ired embodiment the polyalkylene chain(5) is/are covalently linked to the TCR. In a furflwr embodiment the polyelhylene glycol

chains of the present aspect of ihs nrvention comprise at least two polyetiiyleaie
repealing units.
Multivalent TCR Complexes
One aspect of the invention provides a multivaleiit TCR complex comprising at least two TCRs of the inventiorL In one wnbodiment of this aspect, at least two TCR molecules are linked via linker moieties to form muhivalrait complexes. Preferably the complexes are water soluble, so the linker moiety should be selected accordingly. Furthermore, it is preferable that the linker moiety should be capable of attadmient to defined positions on the TCR molecules, so that the structural diversity of the complexes formed is minimised. One embodiment of the present aspect is provided by a TCR complex of the invention vAierein the polymra: diain or perptidic linker sequence extends between amino acid residues of each TCR Tftiich are not located in a variable region sequence of the TCR.
Since the complexes of the invention may be for use in medicine, the linker moieties should be chosen wi& due regard to their pharmaceutical suiTability, for example their immunogenioity.
Examples of linker moieties vAiich fulfil the above demrable criteria are known in the art, for exan^le the art of linking antibody fragments.
There are two classes of linker that are preferred far use in liie production of multivalent TCR molecules of ti^ present invention. A TCR complex of the invention in which the TCRs are linlad by a polyalkylene glycol chain provides one embodiment of the present aspect
The first are hydrophilic polymers sudi as polyalkylaie glycols. The most commonly used of this class are based on polyethylene glycol or PEG, the structure of "which is shown below.


Wherein n is greater than two. However, others are based cm aQia suitable, optionally substitnted, polyalkylene glycols include polypropyleaie glycol, and copolymers of ethylene glycol and propylene glycol.
Sudi polymers may be used to treat or conjugate thwi^eutic agents, particularly polypeiptide or proteiii ther^Mfutics, to achieve beneficial changes to tiie PK profile of the tberapeutic, for example reduced renal clearance, improved plasma half-life, reduced immunogenicity, and improved solubility. Such in^irovements in the PK profile of the PEG-then^wutic conjugate are believt to result fixmi the PEG molecule or molecules fomiing a 'shell' around the therapeutic wiiich sterically hinders the reaction with the immune system and reduces proteolytic degiadaticm. (Casey et al, (2000) Tumor Targetting 4 23 5-244) The size of the hydrophilic polymer used my in particular be selected on the basis of tiie intended therapeutic use of the TCR complex. Thus for sample, where the product is intended to leave the circulatiDn and penetrate tissue, for example for use in the treatmrait of a tumour, it may be advantageous to use iow molecular wei^ polymers in the order of 5 KDa. There are numerous review papers and books that detail the use of PEG and similar molecules in pharmaceutical fonnulations. For sxample, see Harris (1992) Polyethylene Glycol Chsmistty -Biotechnical and Biomedical Applications, Plenum, New York, NY. or Harris & Zaiipskj' (1997) Chemistry' and Biological Applications of PolyetiiylMie Glycol ACS Books, Washington, D.C.
The polymer used can have a linear or branched conformation. Branched PEG molecules, or derivatives thereof can be induced by the addition of branching moieties including glycerol and glycerol oligomers, peaitaerytfaritol, sorbitol and lysine.
I Usually, the polymer will have a chemically reactive groi^ or groups in its structure, for example at one or both termini, and/or on branches from the baddxme, to enable

the poiymsr to link to target sites in the TCR. This ohemiGally reactive erotg> or groups may fas attached directly to the hydrophilic polymer, or there may be a spacer grotip/moiet;' between liie hydrophilic poIyniH- and Hie reactive chranistry as shown below:
Reactive chemistry-Hydrophilic polymer-Reactive chemistry
Reactive chemistry-Spacer-Hydrophilic polymer-Spacer-Reactive chemistry
"Rie spacer used in the f onnation of constructs of the type outlined above may be any organic moiety that is a non-reactive, chemically stable, chain, Siu;h ^lacers include, by are not limited to the following:
-(CH:),,- wherein n = 2 to 5
-(CH:)3NHCOCCH2)2
A TCR complex of the invention in wiiich a divalent alkylene spacer radical is located
between the polyalkylene glycol chain and its point of attachment to
a TCR of the complex provides a further embodiment of the present aspect.
A TCR complex of tiie invention in which liw polyall^iene glycol chain comprises at least two polyethylene glycol repeating units provides a iurthCT ranbodiment of the
present aspect
There are a number of commercial supphers of hydrophilic polymers linked, directly or via a spacer, to reactive chemistries that may be of use in the present inventioru Tliese sijppEers include Nektar TherapeutiGS (CA, USA), NOF Corporation (Japsai), Sunbio (South Korea) and Enzon Pharmaceuticals (NJ, USA).
Commercially available hydrophilic polymers linked, directiy OT via a spacer, to reactive diemistries that may be of use in the present invention include, but are not limited to, the following:


A wide variety of coupling chffinistries can be used to couple polymer molecules to protein and peptide tiier^wutics. The choice of the most apfffopiiate coupling chemistry is largely depmdant cm the desired coupling site. For example, the

following coupling chemistries have beai used attached to one or mors of the temuni of PEG molscules (Source: Nsktar Molecular ^gmeering Catalogue 2003):

As stated above non-PEG based polymers also provide suitable linksis for
multjmerising the TCRs of the present invention. For example, moiedes containing maleimide termini linked bj' aliphatic chains such as BMH and BMOE (Pierce,
products Nos. 22330 and 22323) can be used.
Peptidic linkers are the other class of TCR linkers. These linkers are comprised of chains of amino acids, and fimction to produce simple Unkers or multimerisation domains onto which TCR molecules can be attached. Tlie biotin / streptavidin sj-stem has previously been used to prodiice TCR tetramers (see WO/99/60119) for in-vitro binding studies. However, strepavidin is a microbially-derived polypeptide and as such not ideally suited to use in a ther^eutic.
A TCR complex of the invention in which the TCRs are linked by a peptidic linker derived &om a human multimOTsatioD dcnnain provides a fiirdur embodiment of the present aspect
Tliere are a number of human proteins that contain a multimerisation domain that could be used in the production of multivalent TCR con^jlexes. For example the

tetramerisation dotnain of p53 which has been utilised to produce tetramers of scFv antibodj' fragments which exhibited increased ssram psrsistaice and significantly reduced off-rate compared to the monomtdc scFv SagmenL (Willuda et al. (2001) J, Biol Chem. 276(17): 14385-14392) Haemoglobin also has a tetramerisalion domain thai could potentially be used for this kind of application,
A multivalent TCR complex of tiie invention comprising at least two TCRs provides a final embodiment of this aspect, wherein at least one of said TCRs is associnted with a tiier^eutic agent
In one aspect a TCR (or multivalent complex thereof) of the present inveaition may altenialiveiy or additionally comprise a reactive cysteine at fbe C-tenninal or N-temiioal of the alpha or beta chains thereof.
Diagnostic and therapeutic Use
In one aspect the TCR of the invention may be associated with a therapsunc agent or detectable moiety. For example, said therapeutic agrait or detectable moietj'- may be covalently linked to the TCR-
In one embodimenl of the invention said ther^ieutic agem or (tetectable moiety is covalently linked to the C-terminus of one or both TCR chains.
In one aspect the scTCR or one or bofli of the dTCR chains of TCRs of the present invention may be labelled with an detectable moiety, for example a label that is suitable for diagnostic purposes. Such labelled TCRs are useful in a method for detecting a AAGIGILTV-HLA-A*0201 complex Triiich method comprises contacting the TCR ligand with a TCR (or a mulfimeric h^ affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand. In tetrameric TCR complexes fomied for exany)le, using biotinylated heterodimeiB, Sucarscent streptavidin can be used to iH-ovide a detectable label. Such a fluoresceaitly-IabeUed

TCR tetramsx is suitable for use in FACS analysis, for example to detect antigeiJ presenting cells oairying theAAGIGILTV-HLA-A*0201 con^jlex fcH" windi these hi^ affinitj' TCRs are specific.
Another manner ia istibh the soluble TCRs of the present invention may be detected is by the use of TCR-specific antibodies, in particulai monoclonal antibodies. There are many commercially available anti-TCR antibodies, such as dFl and pH, wbich recognise tiie constant (kanains of the a and p chains, respectively.
In a further aspect s TCR (or multivalent compleK thereof) of liie present invention may alternatively or additionally be associated with (e.g. co'v^entiy or otherwise linked to) a thenqjcutic agent ■w^ch may be, for example, a toxic moiety for use in cell Idlliagi or an immune effector molecule such as an interleukin or a cytokine. A multivalent TCR complex of the invention may have enhanced binding capability for a TCR ligand conqaared to a non-multimeric wild-type or T cell receptor beterodimer of die invention. Thus, the muMvaient TCR complexes according to tiie invention are particularly useful for tracking or targeting cells presenting particular antigens in viiro or in vivo, and are also useful as intamediates for ■flie production of further multivalent TCR conyjlexes having such uses. These TCRs or multivalent TCR con^ilexes may therefore be provided in a phannaceutically acceptable formulation for use in vivo.
The invaition also provides a medjod for delivering a therapeutic agrait to a target cell, wWch metbod comprises contacting potential target cells witii a TCR or multivalent TCR complex in accordance witii 1he invention under conditions to allow attachment of the TCR or multivalent TCR complex to the target cell, said TCR or multivalent TCR complex being specific for the AAGIGILTV-HLA-A*0201 complex and havmg Hie therapeutic ^ent associated tiierewith.
In particular, the soluble TCR or multivalent TCR complex of the prraent invention can be used to deliver therapeutic agraits to the location of cells presentii^ a particular antigen. This wotild be use^ in many situations and, in particular, against tumours.

A therapsutic agemt could be delivered sucb tiiat it would exercise its effect locally but not only on the cell it binds to. Tius, one particular strategy envisages anti-tumour molecujes linVed to TCRs or multivalent TCR complexes according to lie iaventioii specific for tamour antigens.
Many therapeutic agents could be employed for this use, for instance radioactive compounds., enzymes (perforin for example) or chemother^wutic agents fcis-plstiii for example). To ensure that toxic effects are exercised in the desired locatioE the toxin could be inside a liposome linked to streptavidin so that the compound is released slowly. This will prevent dfP"agTP£ effects during lie transport in the body and ensure that the toxin has rnaxiirmni effect after binding of the TCR to l±ie relevant antigen presentmg cells.
Other suitable thra^ieutic agmts include:
• smaU molecule cjTiotoxic agaits, i.e. compounds with the abilitj' to kill TP!^TTJTns^^^fln ceils having a molecular weight of less Ibac 700 daitons. Surdi compoimds could also contain toxic meials capable of having a cytotoxic effsct Furthermore, it is to be understood thai these small molecule cytotoxic agents also include pro-dnigs, i.e. compounds that decay or are converted undsT physiological conditions to release cytotoxic agents. Examples of such agents incluite cis-platin, maytansine derivatives, rachelmycin, calicheamidn, docstaxel, stoposids, gemcitaime, ifosfemide, irinotecai:, melphalan, mitoxantcone, sorfimer sodiumpiiotofiiD H, temozolmide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e. piuteins or feagmeote thereof with the ability to Idll mammalian cells. Includii^ but not limited to, ricin, diphfceria toxin, pseudomonas bacterial exotoxin A, DNAase ard RNAase; radio-nuciides, i.e. tmstabie isotopes of elements which decay wi& ttie concurrant emission of one or more of a oi p particles, or y lays. including but not limited to, iodine 131,ifasniam 186, indium 111, yttrium 90, bismuHi 210 and 213, actinium 225 and astatine 213; chelating agerrts may be used to

facilitate liie association of these radio-nuclides to tiae high affinity TCRs, or miiltimers thereof,
• prodrugs, including but not linaited to, antibody directed emyme pro-drugs;
■ innmuno-stinmlants, i.e. moieties which stimulate immune response. Including but not limited to, cytokines such as IL-2 and IFN, si^>srantigeiis and mutants tiiereof, pHLA complexes and chemokines such as IL-8, platelet fectoT 4, melanoma growth stimulatory protein, etc, antibodies or fragments thereof, complement activators, xenogeneic protein domains, allogeneic protein domains, viral/bacteiial protein domains, viral/bacterial peptides and anti-T cell determinant antibodies {e.g. anti-CD3 or anti-CD28).
Functional antibody fra^nents and variants
Antibody fragments and variants/analogues '^lich are suitable for use in tiie
compositions ami methods described h^ein include, but are not limited to, ^ following.
Antibody Fragments
As is knowTi to those skilled in the art, it is possible to produce fragments of a given antibody which retain sirfjstantiaUy the same bindir^ characteristics as those of the parent antibody. The following provides details of such fragments:
Ivfinibodies — These constructs consist of antibodies with, a truncated Fc portion. As such they retain tiie complete binding domains of the antibody from which are derived.
Fab fragments - These ccni^se a smgle immunoglobulin light chain covalently-linked to part of an immunoglobulin heavy t^iain. As such. Fab fragments comimse a single antigen combinir^ site. Fab fragments are defined by the portion of an IgG that can be liberated by treatment wilii pqjain. Sudi fragments are cotmnonly produced via

recombinant DNA techniques. (Reeves et al., (2000) Lecture Notes an Immunology (4th Edition) Published byBlaito^ Sciaice)
F(ab'): Segments — liese comprise both aniigen combining sites and tie hinge re^on from a single antibody. F(ab'):&aginEiits are degned by Ifae portion of an IgG that can be liberated by treatment -with pepsin. Such &agiiientE are commonly produced via recombinant DNA techniques. (Reeves et al, (2000) Lecture Notes on Immunology (4th Edition) Published bj' Blackwell Science)
Fv fragments - These comprise an immunoglobulin variable heavy domain linked to an immunoglobulin variable light domain. A mmiber of Fv designs have been pmduced. These include dsFvs, in which the association between the two domains is enhanced by an introduced disulfide bond. Altemslively, scFvs can be fomied using a peptide linker to bind the two domains together as a single polypeptide. Fvs constructs containing a variable domain of a heavy or light immxmoglobuiin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced. FV have also been rouItinKirised to form diabodies and triabodies (Maynard et al., (2000) Anm Rev BiomedEng 2 339-376)
Nanobodies'Tw - These construcE, mflrbeted by Abljux (Belgium), cornprise synthetic single immunoglobulin variable heavy domain derived from a camelid (e.g. camel or llama) antibody.
Domain Antibodies - These constructs, marketed by Domantis (Belgium), cott^mse an affinity matured single immunoglobulin variable heavj' domain or immunoglobulin variable light domain.
Antibody vmiants and analogues
The defining functional characteristic of antibodies in ttie context of the present invention is their ability to bind specifically to a target ligand. As is known to those

skilied in the art it is possible to engineer such binding characteristics into a range of other proteins. Examples of antibody variants and analogues suitable for use in -flie compositions and meibods of the present inveotiDn include but are not limited K>, the
fDllowing.
Protein scaffold-based binding polypeptSdes - This femity of binding constructs comprise mutated analogues of proteins which contain native binding loops. Examples include Affibodies, mariceted by AfSbody (Sweden), ^^ch are based on a three-helix motif derived from one of the IgG binding domains of Stc^hylococcus aureus Protein A. Another example is provided by Evibodies, marketed by EvoGenix (Australia) w^iich are based on the extracellular domains of CTLA-4iiito vriiich domains similar to antibody binding loops are grafted. A final exanq>Ie, Cytokine Tr^s marketed by Regeneron Phaimaceuticals (US), graft cytokine receptor domains into antibody scaffolds. (Nygrenera/., (2O00) Current Opinion in Structural biology 1:463-469) provides a review of the uses of scaffolds for engineering novel binding sites in proteins. This review mentions the following protems as sources of scaffolds: CPl zinc finger, Tendami^iat, Z domain (a protein A analogue), PSTl, Coiled coils, lACI-Dl and cytochrome 0552- Othsr protein scaffold studies have reported the use of Fibronectin, Green fluorescent protein (GFP) and ank^Tin r^eats.
As is known to those skilled in the art antibodies or fragments, variants or analogues thereof can be produced v/indh bind to various parts of a given protean ligand. For example, anti-CD3 antibodies can be raised to any of the polypeptide diains from which this complex is formed (i.e.Y, S, E, C. a°t3 "H CD3 chains) Antibodies wbich bind to the £ CD3 chain are the preferred anli-CD3 anCibodieE for use in the composilions and methods of liie present inventioiL
Soluble TCRs or multivalrait TCR conqilexes of the inrandon may be linked to an enzyme capable of converting a imxhug to a drug. This allows the prodrug to be converted to the drug only at the site "wtere it is required O-e. targeted by the sTCR).

It is expescted that the high afSnity AAGIGILTV (SEQ K) NO; 43)-HLA-A*0201 specific TCRs disclosed hsrein may be used in methods for lie diagnosis and treatment of cancer.
For caEicer treatmsnt, the localisation in the vicinity of tumours or metastasis wo'Lild enhance the effect of toxins or immunostimulants. For vaccine delivery, fee vacdne antigsn could be localised in the vicinits' of antigen presenting cells, liius enhancing the efficacy of the antigen. The method can also be ^jplied for imaging pfurposes.
One emfaotSniKit is jHovided by an. isolated cell presaitii^ a. TCR of Ibe invention. For K^ample, said cell may he a human T cell or a human haematopoietic stem cell Furtl^ embodiments of the invention are provided by a pharmaceutical composition con^nising:
a TCR or a multivalent TCR complex of the invention (optionally associated with a ther^ieutic agent), or a plurality of cells presenting at least one TCR of the invention, or a nucleic acid or nucleic acids encoding a TCR of the invention together with a pharmaceutically acceptable carrier,
Tie invention also provides a mefliod of ti'eatinent of cancer comprising administering to a sul:^ect suSering such cancer disease an effective amount of a TCR or a multivalent TCR complex of the invention (optionally associated with a therapeutic agent), or a plurality ofcells presenting at least one TCR of the invention, or a nucleic acid or nucleic adds encoding a TCR of ^le invention. In a related embodiment the invention provides for the use of a TCR or a multivalent TCR complex of the invention (optionally associated with a therapeutic agent), or a plurality of cells presenting at least one TCR of the invention, or a nucleic acid or nucleic acids encoding a TCR of the invMition in the preparation of a con^osition for the treatment of cancer.

As will be obvious to those skilled in the ait, ths cancers that are amenable to treatment by compositions comprising the TCRs of the invention will be Meian-A^ cancers.
Therapeutic or im^ng TCRs in acccmlance wili the invention will usually be siropiied as part of a sterile, phamiaceutical composition "whici will nanHaBy include a phannaoeuncally acceptable carrier. This pharmaceutical con^Qsition ipa;^ be in any suitable form, (depeodingiqwn the desiredrnetbodofadmimsteringittoapatient). It maj' be provided in unit dosage form, will geaierally be provided in a sealed container and m^ be jMcvided as part of a kit Suidi a kit would normally (although not necessarily) include instructions for use. It may include a plurali^ of said mat dosage ftnms.
The pharmaceutical composition Tosy be adapted &tr administration by amy qjpropriate route, for exarr^ile parenteral, transderaial or via inbalation, preferably a parenteral (including subcutaneous, intramuscular, or, most preferably intravenous) route. Sudi
con^)osirions may be prepared by any method known in ihs art of phannacj', for example by mixing the active ii^redient with the canier(s) or exdpient(s) under sterile conditions.
Dosages of the substances of the present invention can vary betwe^i wide limits, depending i^xm the disease or disorder to be treated, the age and ccmdition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be usei
Additional Aspects
A scTCR or dTCR (i^ttdi preferably is constituted by constant and variable sequences corresponding to hiunan sequences) of tiie present invention may be provided in substantially pure form, or as a purified or isolated preparatioiL For eKanq)le, it may be provided in a form i^ch is substantially &ee of oftierpratems.
The invention also provides a method of identifying a hi^ affinity TCR. having the property of binding to AAGIG]LTV-HLA-A*0201 CHARACTEiaSED IN THAT tiie

TCR (i) comprises at least one TCR a chain variable domain and/or at least one TCR p chain variable domain and (ii) has a K^ for 4e said AA.GIGILTV-HIA-A*0201 complex of less than μM said meftiod compsing:
(a) the production of a diverse library of TCRs comprisiiig tiie a and p chain variable domains of tiae MEL TCR wherein one or botti of tiie a and p chain variable domains comprise a mntation(s);
(b) contacting said diverse library of TCRs with AAGIGILTV-HLA-A*0201 under conditions suitable to allow &e binding of ttie TCRs to AAGIGILTV-HLA.-A*0201 ;and
(c) measuring the IQs of the interaction.
Preferred features of each aspect of the invention are as ftff each of tiie olher aspects mutatis mutandis. The prior art documents mentioned herein are incoiporstKi to the
fullest extent permitted by law.
Examples
The invention is furfiier described in the following examples, wiiich do not limit the scope of &e invention in aiiy way.
ReferMice is made in the following to the accompanying drawing in istoch:
Figure la and lb provide tiie alpha chain variable region amino acid and beta chain variable region amino acid sequences of the native MEL TCR respectivdy.
Figures 2a and 2b provide respectively the DNA sequence of soluble versions of the native MEL TCR a and p chains.
Figures 3a and 3b provide respectively Hie MEL TCR a and p chain extracellular amino acid sequences produced firan tiie DNA sequences of Figures 2a and 2b.

Figures 4E and 4b j^ovide respectivsly the DNA sequsnce of soluble versJons of Ihe MEL TCR ex and p chains mutated to include additional cyssteine residues to form a non-native disulphide bond. The mutated codon in each chain is indicated by shading and the introduced restriction enzyme recognition sites are uaderlined.
Figures 5a and 5b show respectively tiie MEL TCR a and p chain extracellular amino acid sequences produced from the DNA sequences of Figures 4a and 4b. The introduced cysteine in each chain is indicated by shading.
Figure 6 provides the alpha chain variable region amino acid sequences of high affinity MEL TCR variants. The mutated residues are underlined
Figure 7a provides the amino acid sequence of a truncated form of TRAC.
Figure 7b provides the amino acid sequence of a truncated fonn of TRBC1.
Figure 7c provides the amino acid sequenceof a truncated form of TRBC2.
Figure 8a provides tiK plasmid tasp of liie pEX202 plasmii
Figure 8b provides the DNA sequence of the pEX202 plasmid.
Figure 9a. details &s alpha diain amino acid sequences of a preferred soluble high affinity MEL TCR variant
Figure 9b details the beta chain amino acid sequences of Hie wild-type soluble MEL TCR using the TRBC2 encoded constant region fused via a peptide linker to wild-type human E.-2. The linker and IL-2 sequences are in italics.

Figure 10 providss the Biacore response carve generated for the interaction of a wild-type soluble disulfide-linked MEL TCE and HtA-.'^A.GIGILTV-HLA-A*0201
Figures Ua and 1 lb provide the full-higth wild-tj'pe MEL JCR alpha r-hr^'m DNA seqience mutated in order to produce enhanced expression in human cells and the amino acid sequence thereby enclosed respectively.
Figures 12a and 12b provide the fuU-lenglii high affinity cl MEL TCR a^ha chain DNA sequence mutated in order to produce enhanced ejq>ression in human cells and the amino acid sequence thereby encoded respectively.
Figures 13a and ISbprovidethefull-lengdihighafEnrty eld MEL TCR alpha chain DNA sequence mutated in order to produce oihanced ej^iression in human cells and the amino acid sequence thereby encoded respectively.
Figures 14a and 14b provide the full length high affinity c9 MEL TCR a^ha diain sequence mutated in order to produce enhanced expression in hmnan cells and the amino acid sequoice thereby encoded respectively.
Figures 15a ami 15b provide the full-length c9 MEL TCR alpha chain sequence mutated in order to produce enhanced exprcssion in human cells and UtR amino acid sequence thereby encoded respectively.
Figure 16 Provides an ELISPOT assay demonstrating the ability of a soluble disulfide-linked version of the high afSnity cl WT Mel TCR to inhibit the activation of a Mel-specific CTL Clone.
Figures 17a and 17b provide the fidl-length wild-type MEL TCR a^ha cdiain ORF encoding and wild-type MEL TCR beta chain ORF encoding DNA sequences
respectively,

Figure 18 provides the full-length cl MEL TCR alpha chain ORF encoding DNA sequence con^Kising wild-tj-pe DKA codons except for those encjoding the nautated amino acids.
Figure 19a pro\'ides FACS data on the level of TCR expression adiicved by transfsction of Jurkat ceUs wilh non-codon-optimsed DKA encoding a cl alpha / WT beta MEL TCR.
Figure 19b provides FACS data on the level of TCR expression achieved fay transfection of Juikat cells with codon-f^rtimsed DNA encoding a cl alpha / WT beta
MEL TCR
Figure 20 provides the amino acid sequences of the variable regions of additional hi^ afEinity MEL TCR alpha chains. The mutated residues are underlined
Figure 21 provides the amino acid sequraices of the variable regions of high afBniTy MEL TCR beta chains. The mutated residues are UKierhned
Figure 22 provides the amino acid sequences of soluble high afEinity MEL TCR alpha chains comprising with a non-native cysteine residue. The non^iative oysteine residue is hi^iUghted and tne mutated residues are underlined.
Figure 23 provides tiie amino acid sequsices of soluble high affinity MEL TCR beta chains comprising with a non-native cysteine residue. The non-native cj-steine residue is highlighted and the mutated residues are undefined.

Example I - Production of a soluble disulfide-lmked TCR comprising the native MEL variable domains
Figures 4a and 4b provide the DNA sequences of soluble disuifide-Iinked a^ha and beta chains from the wad-type MEL TCR which is specific for the AAGIGILTV-HLA-A*0201 complex. These DNA sequsuces can be synthesised de-novo by a number of contract research companies, for example GeneArt (Regensbarg, Germany). Restriction enzyme recognition sites are also added to these DNA sequKices in order to facilitate ligation of these DNA sequences into pGMT7-based expression plasmids, which contain the T7 promoter for high level e^iression in. E.coli strain BL21-DE3(pLysS) (Pan et al. Biotechniques (2000) 29 (6): 1234-8)
The DNA sequences encoding each TCR chain cut with Ndel and Hindlll are ligated into separate pEX202 pGMT7-based vectors, -Which are also cut with Ndel and HindUI. (See Figure 8a for the plasmid map of pEX202, and Figure Sb for the DNA sequence of this vector (SEQ E) NO: 28))
Restriction enzyme recognition sites as introduced into DNA encoding the soluble wild-type MEL TCR chains:
Ndel- CATATG HindlH' AAGCTT
Ligation
Ligated plasmids are transformed into con^etent E.coli strain XLl -blue cells and plated out on LB/agar plates containing 1 OOmg/ml ampidllin. Following incubation ovwnight at 37^, smgle colonies are picked and grown in 10 ml LB contaiTiing lOOn^ml ampicillin overmght at 37°C with sbaldiig. Cloned plasmids are purified using a Isfiniprep kit (Qiagen) and the insert is sequenced using an automated DNA sequencer (Laric Technologies).

Figures 5a and 5b show respectively the soluble disulfide-liuked wild-typs MEL. TCR a and p chaiE extracellular amino acid sequsocss produced from the DNA sequences of Figures 4a and 4b. The restrictioii enzyme recognition sequences in these DNA sequences are underlined.
Example 2- Production of high affinity variants of the soluble disulfide linked MEL TCR
The soluble disulfide-Iinked native MEL TCR produced as described in Example 1 can be used a template from which to produce the TCRs of the invention \\4iich have an increased afBnitj' for the AAGIGILTV (SEQ ID NO: 43) -HLA-A*0201 complex.
Phage display is one means by which iibrariK of HTV Gag TCR variants can be generated in order to identify high affinity mutants. For example, the TCR phage
display and screraiing methods described in (Li et cd., (2005) Nature Biotech 23 (3): 349-3 54) can be adapted and applied to HI\" Gag TCRs.
The amino sequences of the mutated TCR alpha variable regions which, when combined with the wild-type MEL beta variable region, demonstrate hi^ affinity for the AAGIGILTV-HLA-A*0201 complex are listed in Figure 6. (SEQ ID Nos: 11-24) As is known to those skilled in the art thie necessary codon changes required to produce these mutated chains can be introduced into the DNA encoding these chains by site-directed mtitagenesis. (QaickChangeTM Site-Directed Mutagenesis Kit from Stratagene)
Briefly, this is achieved by using primers that incorporate liie desired codon change(s) and the pEX202 plasmids containing the relevant MEL TCR diain DNA as a tonplate for the mutagoiesis:
Mtitagenesis is carried out using the following conditions: 50ng plasmid tsanplate, Iμl of lOmM dNTP, 5 [il of lOx Pfu DNA polymerase buffer as supplied by the

manufacturer, 25 pmol of fwd primer, 25 pmol of rev primer, liil pfo DNA polymerase ID total volume 50 [il. After an initial denaturation step of 2 Tn^T^)= at 95C, flie reaction is subjected to 25 cycles of denaturation (95C, 10 sees), annealing (55C 10 sees), and elongation (72C, 8 mins). The resulting product is dig^ted with Dpnl restriction enzympme to remove the tauplate plasmid and transformed into E. coli sSrain XLl-blue. MucagMiesis was verified by sequencing.
Example 3 - Expression, refolding and purification of soluble TCR
The pEX202 eiqiressicm plasmids containing the MEL TCR a-chains and MEL TCR ^-chains as prepared in Examples 1 or 2 are transformed separately into E.coli strain BL21pLysS, and single ampicillin-resistant colonies are grown at ST'C in TYP (ampiciilic 100|j,g/ml) medium to ODeoo of 0.4 before inducing protein expression with O.SmM IPTG. Cells are harvested three hours post-induction by centrifiigation for 30 minutes at 4000rpm in a Beckman J-6B. Cell pellets are re-suspended in a buffer containing 50mM Tris-HCL 25% (w/v) sucrose, ImMNaEDTA, O.lVo (w/v) NaAzide, lOmM DTT, pH S.O. After an ovemi^i freeze-thaw step, re-suspended cells are sonicated in I minute bursts for a total of around 10 minutes in a Milsonix XL2020sonicatoT using a standard 12mm diameter probe. Inclusion body pellets are recovered by centrifiigation for 30 minutes at 13000rpm in a Beckman J2-21 centrifuge. Three detergent washes are then carried out to remove eel! debris and membrane components. Each lime the inclusion body pellet is homogaiised in a Triton buffer (SOmM Tris-HCI, 0.5% Triton-XlOO, 200mM NaCI, lOmM NaEDTA, 0.1% (w/v) NaAzide, 2mMDTT,pH 8.0) before being pelleted by centrifiigatioD for 15 minutes at ISOOOrpm in a Beckman J2-21. Detergent and salt is 1hen removed by a similar wash m the foUowmg buffer: 50mM Tris-HCI, ImM NaEDTA, 0.1% (yffv) NaAzide, 2mM DTT, pH 8.0. Finally, the inclusion bodies were divided into 30 mg aiiquots and frozen at -TO^C. Inclusion body protein yield is quantjtated by solubilismg with 6M guanidine-HQ and measurement with a Bradford dye-bindmg assay (PerBio).

Approjdmatsly 30mg of TCR p chain and 60mg of TCR a chain solubilissd indusion bodies are thawed from frozen stocks, samples were then mixed and the mixture diluted into 15ml of a guanidine soludon (6 M Guanidine-hydrochloiide, IQmM Sodium Acetate, l0mM EDTA), to rasure complete cdiain de-naturation. The guanidine solution containing fully reduced and denatured TCR chains is then injected into 1 litre of the following refolding buffer: lOOmM Tris pH 8.5, 400inM L-Arginine, 2mM EDTA, 5inM reduced Glutathione, 0.5mM oxidised Glutathione, 5M urea, 0.2mM PMSF. The redox couple (2-mCTC^)toethylaniine and cystamine (to final concentrations of 6.6mM and 3.7mM, respectively) are added approximately 5 minutes before addition of the draiatured TCR chains. The solution is left for 5 hrs ± 15minutes. The refolded TCR is dialysed in Spectr^jor 1 membrane (Spectrum; Product No. 132670) against 10 L 10 mM Tris pH 8.1 at 5°C ± 3°C for 18-20 hours. After this time, the dialysis fauHer is changed to fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at S^C ± 3°C for anotha- 20-22 hours.
sTCR is separated from degradation products and impurities by loading the dialysed refold onto a POROS 50HQ anion exchange column and sluling bound protein wiih a gradient of 0-500mM NaCI over 50 column volumes using an Akta purifier (Pharmacia). Peak fractions are stored at 4''C and amdysed by Coomassae-staiiffid SDS-PAGE before being pooled and conceotratsd. Finally, the sTCR is purified and charBcterised using a Superdex 200HR gel filtration column pre-equiKfarated in HBS-EP buffer (10 mM HEPES pH 7.4,150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40). The peak eluting at a relative molecular weight of approximately 50 kD& is pooled and concentrated prior to characterisatioD by BlAcore surfece plasmon resonance analysis.
Exarrple 4 - Biacore surface plasmon resonance characterisation ofsTCR binding to
specific pMHC

A surface plasmon resonance biosensor (Biacore 3000'™ ) was used to analyse the binding of a soluble MEL TCRs to the cognate psptide-MHC ligaod. This was facilitated by producing single pMHC complexes (described below) -which were immobilised to a streptavidin-coated binding surface in a srani-oriented feshion, allowing efScient iesang of the binding of a soluble T-csU receptor to up to four different pMHC (immobilised on separate flow cells) simultaneously. Manual injection of HLA complex allows the precise level of hnmobiiised class I molecules to be manipulated easily.
Biotinylated class I HLA-A*0201 molecules were refolded in vitro from bacterially-
expressed inclusion bodies containing the constituent subunit proteins and synthetic petide, followed by purification and in vitro enzymatic biotinylation (O'Calla^ian et al (1999) ATUII. Biochem. 266: 9-15). HLA-A*0201-heavy chain was e:qiressed wilh a C-terminal biotiaylation tag which replaces the transmembrane and cytoplaanic
domains of the protein in an appropriate construct, hiciusion body expression levels of-75 mg/Iitre bacterial culture were obtained. The MHC light-chain or p2-microgiobuiin was also expressed as inclusion bodies in. E. coli from an Eroproprime construct, at a level of ~500 mg/litre bacterial culture.
E. coli cells were lysed and inclusion bodies are purified to approximately 80% purity. Protein from inclusion bodies was denatured in 6 M guanidine-HCl, 50 mM Tris pH 8.1,100 mMNaCl, 10 mM DTT, 10 mMSDTA, and was refolded at a conceBtradon
of 30 mg/litre heavy chain, 30 mg/litre p2m into 0.4 M L-ArgJnine-HCl, 100 toM Tris pH 8.1, 3.7 mM cystamine, 6.6mM p-cysteamine, 4 mg/ml of fee AAGIGILTV peptide required to be loaded by the HLA-A*02C1 molecule, by addition of a single pulse of denatured protem mto refold buffer at < 5°C. Refolding vras allowed to reach completion at 4''C for at least 1 hour.
Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strengtti of the solution sufficiently. The protem solution was fcen filtrared through a 1 .μm cellulose acetate filter and loaded

onto a POROS 50HQ anion sxcfaangs column (8 ml bed voltime). Protein was eluted with a linear 0-500 mM NaCI gradieait HLA-A*0201-psprids complex eluted at approximatpily 250 mM NaCl, and peak fractions were coBected, a cooktail of protease inhibitors (Calbiochem) was added and liie fractions were chilled on ice,
Biotinylatioii tagged pMHC molecules were buffer exchanged into 10 mM Tris pH 8.1,5 mM NaCl using a Pharmacia fast desalting column equilibrated in the same biiffer. Immediately upon elution, the protein-containing fractions wwre chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added; I mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgC12, and 5 ug/ml BirA enzyme (purified accordingtoO'Callaghan er al. (1999)^na/. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature ovemigiit
The biotinylated pHLA-A*0201 molecules were purified using gel filtration chromatographj'. A Pharmacia Superdex 75 HR 10/30 cohmm was pre-equilibrated with filtered PBS and 1 ml of the biotinylaation reaction mixture was loaded and the column wss developed with PBS at Q.5 ml/min. BiotinybtsdpHLA-A*Q201 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was detemuned using a Coomassie-binding assay (PeiBio) and aliquots of biotinylated pHLA-A*0201 molecules were stored frozen at -20°C. Streptavidin was immobilised by standard amine compling methods.
Such nnmobilised complexes are enable of binding both T-celi receptors and fee corecerptor CDSaa, both of with may be injected in the soluble phase. Specific binding of TCR is obtained even at low concentrations (at least 40)ig/nil), inq)lying the TCR is relatively stable. The pMHC binding properties of sTCR are observed to be qualitatively and quantitatively similar if sTCR is used eithK in the soluble or immobilised phase, "niis is an important control for partial activity of soluble species and also suggests that biotin^ated pMHC complexes are biologically as active as non-biotinylated complexes.

The interactioiis betwsaj soluble MEL TCRs containing a novel inter-tiiain bond and its cognate pMHC or an irrelevant pMHC combination, the production of-wAich is described above, were analysed on a Biacore 3000'™ surfece plasmon resonancs (SPR) bioseaisor. SPR measures dianges in re&active index exjressed in response units (RU) near a sensor surfece within a grnail flow cell, a principle that can be used to ttetect receptor ligand intsracrions and to analyse their afSnity and kinetic parameters. The probe flow cells were prepared by immobilising the individiial HLA-peptide complexes in separate flow cells via binding between the biotin cross linked onto |32m and streptavidin -vMcb. have been diemicaily cross linked to the activated surface of the flow cells. The assay was then performed by passing sTCR over &e surfeoes of the different flow cells at a constant flow rate, measuring the SPR response in doing so.
To measure Equilibrium bijiding constant
Serial dilutions of the wild-tj-pe or mutated MEL sTCR were prepared and injected at constant flow rate of 5 jil min-1 over two different flow cells; one coated with ~I000 RU of specific AAGIGILTV-HLA-A*Q201 complex, the second coaied with-1000 RU of non-specific HLA-A2 -peptide complex. Response was normalised for e^di concentration using the measurement from liie control cell. Normalised data response was plotted versus concentration of TCR san^jle and fitted to a faypexbola in order to calculate the equilibrium binding constant, KD- (Price & Dwek, Principies and Problems in Physical Chemistry for Biochemists {2"^ Edition) 1979, Clarendon Press, Oxford).
To measure Kinetic Parameters
For high affinity TCRs KD -W^ determined by experimentally measuring the dissociafion rate constant, kd, and the association rate constant, ka. The equilibrium constant KD was calculated as kd/ka.
TCR was injected over two diSerent cells one coated with -300 RU of specific HLA-A2-AAGIGILTV complex, tiie second coated wilii -300 RU of non-specific HLA-A2

-peptide complex. Flow rale was ast at 5Q μl/min Typically 250 \il of TCR at ~3 μM concentration was injected. Buffer was then, flowed over until tiK response had returned to baseline, Kinetic parameters were calcidaled using Biaevalnation software. The dissociation phase was also fitted to a single exponential decay equation enabling calculation of half-life.
Results
The interaction between a soluble disulfide-linked wild-type MEL TCR (consisting of the a and p TCR chains detailed in SEQ ID NOs 9 and 10 respectively) and 4e AAGIGrLTV-HLA-A*0201 complex was analysed using the above methods and demonstrated a KD of 4 jiM. (See Figure 12 for Biacore response curve)
TCRs containing the variable region use sperafied in the followiag table have a Ku of less than or equal to 3 \iM. Based on experiHice with high afSnity TCRs other than the present MEL TCRs (see for exan^le Li el a!.. Nature Biotech 2005 23 (3): 349-354) it is expected that some or all of the TCRs specified in Ihe foUowir^ table wiU have koff of 1 x 10'"' S'l or slower, and indeed that has been shown to be the case by the preparation of soluble TCRs comprising these variable domains. (See Table 1 below)

Table 1 - Biacore data for the interaction of high affinity soluble disuifide-Iinked MEL TCRs comprising defined variable regions and the cognate AAGIGILTV-HLA-
5 A*0201 peptide-MHC.
Example 5 - Production of a soluble high qffimty MEL TCR -WT human IL-2 fusion protein
10 The methods substantially as described in Stamples 1 to 3 can be used to produce a soluble high affinity MEL TCR -WT human IL-2 fusion protein. Briefly, the DNA encoding the desired linker and WT human IL-Z are adtted into the 3' end of liie DNA sequence of the soluble disulfide-linked wild-type MEL TCR beta chain immediately prior to the TAA ("Stop") codon. Figure 9b provides the amino add sequence of a
15 fusion protein comprising a disulfide-linked wild-type MEL TCR beta chain fused to

WT human TL-2 via linker ssqusnce.CSEQ ID NOi 30) The linker and IL-2 portion of this fusion protein are intiicated in italics. Ths DNA encoding thiis oonstnict can then be ligated into pEX202. tlie soluble high affinity MEL TCR- IL-2 fusion protein can then be expressed by combimng this beta chain fusion protein with a sohoble high affinity disulfide-linked MEL TCR alpha chain containing any of the variable regions detailed in Figure 6 (SEQ ID MOs: 11 - 24-) using the methods substantially as described in Example 3. For example. Figure 9a (SEQ ID NO: 29) proiades the amino acid sequence of a soluble high afEinitj' disulfide-linked MEL TCR alpha chain containing the variable region detailed in SEQ ID NO: 11.
Example 6 —EUSPOT assay for assessing in-vitro inhibition ofcyto-taxic Tcell (CTL) activation by soluble high affinity Mel ClcWTMel TCRs
The following method provides a means of assessii^ the abihty of Mel cl cWT hi^ afanity Mel TCRs to inhibit the acrivadon AAGIGILTV-HLA-A*0201 reactive T cell clones.
The soluble Mel cl cWT high affinity Mel TCR utilised m this experiment contained the Mel TCR alpha chain variable domain and WT Mel TCR beta chain variable regions of (SEQ ID NO; 11) and (SEQ ID NO: 2) respectively. The full amnio acid sequences of the TCR alpha and beta chains of this soluble disulfide-linked TCR are
provided by Figure 9a (SEQ ID NO:29) and Figure 5b (SEQ ID NO:10) respectively.
Reagents:
Assay media: 10% PCS (heat-inactivated, Gibco, cso^ lOlOS-165), 88% RPMI1640 (Gibco, cal# 42401-018), 1% giutamine (Gibco, cat* 25030-024) and 1% penicillin/streptomycin (Gibco, oaSM 15070-063).
Wash bufer: 0.01 M PBS/0.05% Tween 20 (1 satdiet of Phosrihate buffered salme with Tween 20, pH7.4 from Sigma, Cat M P-3563 dissolved in I litre distilled water

gives final composition 0.01 M PBS, 0.138 MNaCl, 0.0027 M KCL 0.O5 % Tween 20).
PBS (Gibco, cal#10010-015).
Diaclons EliSpot kit (IDS) EliSpot kit contains all other reagents required i.e. capttire and detection antibodies, sldimned milk powder, BSA, streptavidin-alkaline phosphatase, BCIP/NBT solution (Human IFK-7 PVDF Eli-spot 20 x 96 wells with plates (IDS cat# DC-856.051.020, DC-856.000.000.
The following method is based on the maaufacturers instructions supplied with each kit but contains some alterations.
Method
100 )J1 capture antibody was diluted in 10 ml sterile PBS perplate. 100 |iJ diluted capture antibody was aiiquoted iuio each well and left ovemight at 4C, or for 2 hr at room temperature. The plates were then washed three times with 450 μl wash buffer, Ultrawash 96-weIl plate washer, (Thenno Life Sciences) to remove excess capture antibody. l00μlof 2% skimmed milk was then added to each well. (One vial of skimmed milk powder as supplied with the ELISPOT kit was dissolved in 50 ml sterile PBS). The plates were the: incubated at room tempsrature for two hours before washing washed a further three times with 450|il wash buffer, Ultrawaah 96--welI plate washer, (Thermo Life Sciences)
Mel 624 and Mel 526 target cancer cells were detached &om thrar tissue culture fiadcs using trypsin, washed once by centrifiigation (280 x g for 10 mintites) in assay media and re-suspended at lxl06 /ml in the same media. 50ul of this suspension was then added to the assay plate to give a total target cell number of 50,000 cells/well.
ELAGlGILTV-pulaed T2 tai^et cells were also used as a Control. This analogue peptide was used as it has a higher afBnity for HLA-A*0201 than fee WT peptide.

These psptide-pulsed cells were washed once by oentrifugadon (280 x g for 10 minutes) in assay media and re-suspended at IxlOVml in the same media. 50ul of tiiis suspension was Ihen added to the assay piale to give a total target cell number of 50,000 celis/weU.
A T cell clone (KA/C5) (efiector cells), raised by autologous stimulatioD with -the ELAGIGILTV peptide, was harvested by centrifogaticm (280 x g for 10 min) and re-suspHidedat 1x105 Delis/ml in assay media to give 5000 cells/well-when 50fil was added to the assay plate.
The soluble Mel cl cWT high affinity Mel TCR high afSnity Mel TCRs were diluted in assay media at a 3x concramation to give a Ix final when 50ul was added to tiie plate in a final volume of 150μ1. The concentraticm range of high afEinrty Mel TCRs tested was luM- InM.
Wells containing the following were then prepared, (the final reaction volimie in each well was lOOμl):
Test samples (added in order)
50 μl Mel 624 or Mei 526 target cells
50μl of the desired concentration of soluble high affinity Mel cl cWT TCRs.
SOul KA/C5 T cell clone effector cells.
Negative Controls
50 μl target cells
SOul of HM: highest concentratioD of soluble high affinity Me! cl cWT TCRs. 50 μl assay media
And
50μl effector cells
50Μ1 of the highest concentration soluble high affinity Mel cl cWT TCRs 50μ1 assay media


The plates were then incubated ovemight at 37°C/5% CO2- "Hie plates were feem washed six times with wash buffer before tapping crat excess bufEisr. 550 ^l distilled water was then added to each vial of detection antibody siqjphed with the ELISPOT id: to prepare a diluted soluticm. 100 pJ of the diluted detection antibody solution was then further diluted in 10 ml PBS^l % BSA per piate and 100 ^d of tlie diluted detection antibody solutiOD was aliquoted into each welL The plates were thea incubated at room temperature for 90 minutes.
After this time the plates wra:e washed three times with wash buffer (three times witii 450 Lii wash buSer, Ultrawash 96-well plate washer (Thermo Life Sciences) and tapped dry. 10 lil streptavidin-Alkaline phosphatase was then diluted with 10 ml with PBS/1% BSA per plate and 100 μl of Ihe diluted streptavidin was added to each well and incubated at room tnnpcrature for 1 hr. The plates were then wa^ed again three times with 450 pi wash buffer and tapped dry.
100 μl of the BCIP/NBT supplied solution was added to each well and the plates are covered in foil and left to develop for 5 - 15 miu. The plates were checked regularly during this praiod for spot forrmation in order to decide when to tenninate the reaction.

The plates were then washed thoroughly in tap water and shaken before being taken apart and left to dry on the bench.
Once dry the plates were read using an ELISPOT reader (Autoimmun Diagnotist2ka, Germany).
The number of spots that appeared in each well is proportional to the number of T cells activated. Therefore, any decrease in the number of spots in the wells containing the high affinity Mel TCR mdicates inhibition of KA/C5 CTL Clone activation.
Results
As shown in Figure 16 the soluble cl cWT high afBnity Md TCRs were effective at mhibiting KA/C5 CTL clone activatioiL These data indicate that 100% inhibition of CTL activation was achieved using 1μM soltible cl cWT high afRnity Mel TCRs.
Example 7 - Comparison of TCR expression levels on Jurkat cells transfected with codon-optimised and non-codor optimised DNA encoding a high affinit}- (cla WT^) MEL TCR
4x10^ Jurkat cells grown in RMPI containing 10% heat-inactivated fetal calf serum medium cells were washed in serum-free medium and transfected with either
a) 5\ig of endotoxin-fi^e plasmid pChieo containing floe non-codon optimised sequence encoding MELa cl mutant full length TCR chain plus 5μg of endotoxin-free plasmid pCI containing the non-codon optimised sequence encoding MELp wt full length TCR chain (TheORFBofliese sequences are puvidedm Figure 18 (SEQID NO: 46) and Figure 17b (SEQ ID NO: 45) respectively); or
b) 5 |ig of endotoxin-free plasmid pCIneo contahring an ORF codon-optimised MELa cl mutant Ml length TCR chain plus Sμg of endotoxin-free plasmid pCI containing an ORF codon-oplimised MELp wt full length TCR chain (The ORFs of these sequences

are provided in Figure 12a (SEQ ID NO: 33) and Figure 15a (SEQ ID NO: 39) respectively),
Transfection was achieved by electroporation using 0.4cm cuvettes using conditions of 0.27 kV and 975 μF in a BioRad Genepulser apparatus.
Cells were placed in 6ml of RPMI containing 20% heat-inactivated fstai calf Serum at 37 C for 72 hours.
Cells were stained in a volume of lOOtil PBS using liil (0.54jig) of PE-iabelled streptavidin p/HLA-A2 tetramer (peptide was either heteroclytic MEL perptide ELAGIGILTV or a negative control NY-ESO peptide SLLMWITQC). After 20 minutes at room temperature the cells were washed once in Sml RPMI and re-suspended in SO0p,i RPMI and analysed on a FC500 Beckman Coulter instrument
Results
FACs seining data shown in Figure !9a and Figure 19b are the level of cognate pMHC tetramer staining obtained for Jurkat cells transfected with the non-codon optimised and codon optiniised DNA encoding 2ie cl alpha / WT beta MEL TCR. These data demonstrate that a high level of TCR surface ra^aression was achieved using liie codon optimised DNA compared to that achieved using the correspondii^ non-codon optimised DNA.

1. A T-cell receptor (TCR) having the property of binding to ^A.GIGILTV-HLA-A*020] and comprising at least one TCR a chain variable domain and/or at least one TCK p drnin variable domain CHARACTERISED IN THAT said TCR has a Kb for the said AAGIGILTV-HLA-A*0201 complex of less than or equal to 3 uM andyor an off-rate (kgid of 1 x 10'^ S"l or slower.
2. A T-ceil receptor (TCR) as claimed in claim I CHARACTHUSED IN THAT said TCR has a KD for the said AAGIGILTV-HLA-A* 0201 complex of less than or equal to 1 pM.
3. A TCR. as dahned in claim 1 or claim 2 comprising both an a chain variable domain and EUI TCR p chain variable domain.
4. A TCR as claimed in claim 1 or claim 2 which is an cm or pp homodimei.
5. A T-celi receptor (TCR) as claimed in any of the preceding claims wiiCTein the said KD and/or koff is/are as measured by Surfece Plasmon Resonance.
6. A TCR as claimed in any of the preceding daims whidi is mutated relative to the native MEL TCR Q chain variable region (SEQ ID No: I) and/or p chain variable region (SEQ ID NO; 2) in at least one con^)lemaitarity determining region hereof.
7. A TCR as claimed in any of the p-eceding claims vs^ch is mutated relative to iht native MEL TCR a chain variable region (SEQ ID No: 1) and/ot p chain variable region (SEQ ID NO: 2) in at least one variable region fiamewoik region thereof
8. A TCR as claimed in any of Ihe preceding claims wfaerran one or more of alpha chain variable region amino acids 28D, 29R, 30G, 31S, 49M, 511, 53S, 54N, 72Y,

94V, 95A, 96G, 97K, 9SS, and 99T using the numbsriiig shown in SEQ ID NO: 1 is/are mutated.
9. A TCR as claimed in any of liie preceding claims whraein one or mcec of beta ahain variable region amino adds 45L, 51S, 52V, 53G, 541 761, lOOG, lOIT, 1020, 103E, 104L and 105F, using the nmnbsring shown in SEQ ID NO: 2 is,'are mutated.

12. A TCR as claimed in any of tiaims 1 to 7 comprising one of the alpha cb^
variable region amino add sequaices of SEQ ID Nos: 13 to 24 or 47 to 53.
13. A TCR as claimed in any of claims 1 to 7 or 12 oomprisii^ one of the beta
chain variable region amino acid sequences of SEQ ID Nos: 54 to 67.
14. A TCR as daimed in claim 3 comprising the alpha and beta chain variable
region pairings shown in the foilowir^ table:

15. ATCRas claimed in claim 3 comprising the alpha chain variable region
shown in the SEQIDNO: 11 and Ihe beta chain variable region shown in the SEQ ID
NO: 2.
16. A TCR as claimed in claim 3 comprisii^ the alpha chain variable region
shown in the SEQ ID NO 47: and the beta drain variable region shown in the SEQ ID
NO: 2.
17. A TCR as claimed in claim 3 comprising the alpha chain variable region
shown in the SEQ JD NO: 48 and the beta chain variable region shown in the SEQ ID
NO: 2.
18. ATCRas claimed in claim 3 coniprising the alpha chain variable region
shown in liie SEQ ID NO: 53 and Ihe beta f^Tniii variable region shown in the SEQ ID NO: 2.
19. ATCRas claimed in claim 3 compriang the alpha chain variable region
shown in the SEQ ID NO: U and the beta chain variable region shown in the SEQ ID
NO: 54.

20. A TCR as claimed in claim 3 comprising iiie alpha diain vaxiablt region shown in the SEQ ID NO: 11 and the beta chain variable region shown in the SEQ E) NO: 55.
21. A TCR as claimed in claim 3 comprising the alpha chain variable region shown in the SEQ ID NO: 11 and the beta chain variable region shown in tiie SEQ ID NO: 56.
22. A TCR as claimed in claim 3 comprising the a^iha chain variable regitai shown in the SEQ ID NO: 11 and die beta chain variable region shown in tiie SEQ ID NO; 57.
23. A TCR as claimed in claim 3 comprising tiie alpha chain variable region shown in the SEQ ID NO: II and the beta chain variable region shown in the SEQ ID NO: 58.
24. A TCR as claimed in claim 3 comprising the alpha chain variable region shown in the SEQ ID NO: 11 and "die beta chain variable regiori. shown m the SEQ ID
NO: 61.
25. A TCR as claimed in claim 3 com^msing the alpha chain variable region
shown in the SEQ ID NO: 11 and the beta chain variable region shown in the SEQ ID
NO: 65.
26. A TCR as claimed in claim 3 comprising the alpha chain variable region shown in the SEQ ID NO: 11 and die beta chain variable region shown in the SEQ ID NO: 66.
27. A TCR as claimed in any preceding claim further comprising the alpha chain constant region atninn acid sequesnce shown in SEQ ID NO: 25, and/or one of the beta chain amino acid constant region sequences shown in SEQ ID NOs: 26 and 27.

2S. A TCR as claimed in any preceding claim -^iiicti is a dimKic T cell receptor (dTCR) or a single chain T cell recqrtor (scTCR).
29. A TCR as claimed in of claims 5 to 28 vrfiidi is an scTCR con^nisiiig
a first segment constituted by an amino acid sequence corresponding to a TCR a chain variable region
a second segment constituted by an pminn acid sequence coirespoiuiing to a TCR (3 chain variable region sequence fused to the N tenninus of an amino acid sequence corresponding to a TCR p chain constant domain Extracellular sequence, and
a linker sequence linking the C taminus of the fcst segment to the N tenninus
of the second segment.
30. A TCR as claimed in any of claims 5 to 28 which is an scTCR comprisii^
a nrst segment consiituled by an amino acid sequence corresponding to a TCR p diain variable region
a second segment constituted bj' an amino acid sequraice corresponding to a TCR a chain variable region sequeuce fiised to tiK N terminus of an amino acid sequence corres^jonding tc a TCR a chain constant domain extracellular sequence, and
a linker sequence linking the C terminus of tbe first segment to ttie N termiims of the second segment.
31. A TCR as claimed in olaiir 29 or 30 further comprising a disulfide bond
between bond between the first and second diains, said disulfide bond being one

which has no equivalent in native apT cell receptors, and ■wherein tiie length of the linksr ssqusnce and the position of the disulfide bond being such that Ihe variable domain seqiKnces of Ihe first and second segments are mutual];' orientated substantially as in native aP T cell leocptors.
32. A scTCR as claimed in any of claims 29 to 31 ■wierein in the binding part the linkra: sequmce links the C terminus of the fiist segment to the N terminus of the second segment
33. A scTCR as claimed in any of claims 29 to 32 wherein in the binding part the linker sequence has the formula -P0GG-(SGGGG)5-P- (SEQ ID NO: 41) or -PGGG-(SGGGG)6-P- (SEQ ID NO: 42) wherein P is proline, G is glycine and S is serine.
34. A TCR as claimed in any of claims 1 to 3 or 5 to 28 which is a dXCR comprising
a first polypeptide wherein a sequence corresponding to a TCR a chain variable region sequence is fused to tiie N terminus of a sequence corresponding to a TCR a chain constant domain extracellular sequence, and
a second polypeptide wherein a sequence corresponding to a TCR p chain variable
region sequence fused to the N terminus a sequence corresponding to a TCR p chain constant domain extracellular sequence,
the first and second polypeptides being linked by a disulfide bond which has no equivalent in native ap T cell receptors.
35. A TCR as claimed in claim 34 wherein the disulfide bond links amino acid
residues of the said constant domain sequences, wfaioh disulfide bond has no
equivalent in native TCRs.

36. A TCR as claimed in claim 35 -wherein the said disulfide bond is bstwssn cystsiQe residues corresponding to amino acid residues PAIOSS p carbon aftoms are less than 0.6 nm apart in nativs TCRs.
37 A TCR as claimed in claim 35 wheereia the said disulfide bond is between C5'steine residues substituted for Thr 48 of sxon 1 ofrRAC*01 and SsrSTof exon 1 ofTRBCl*01 orTRBC2*01 or the oon-human equivalent thereof.
38. A TCR as clidmed in any of claims 1 to 3 or 5 to 28 \'rfiich is a dTCR
comprising
a first polypeptide wherein a sequence corresponding to a TCR a chain variable region sequence is fused to the N terminus of a sequence correspcpnding to a TCR a chain constant domain e:ctracellular sequence, and
a second potypepride wherein a sequence corresponding to a TGR p chain variable region sequence fused to the N trarninus a sequence corresponding to a TCR p chain constant domain extracellular sequaice,
the first and second polypeptides beii^ linked by a disulfide bond -wbidh has no equivalent in native ap T cell between cysteine residues substituted for Thr 48 of exon 1 DfTRAC*01 and Ser57 of exm 1 ofTRBCl*01 orTE3C2*01 or the non-human equivalent thereof.
39. ATCRasclaimedinanyofclaimsMtoSSwhereinthedTCRoTScTCR binding part includes a disulfide bond between residues corresponding to those linked by a disulfide bond in native TCRs.
40. A TCR as claimed m any of claims 14 to 39 isterein the dTCR or scTCR binding part does not contain a sequence corresponding to transmembrane or cytoplasmic sequeaices of native TCRs.

41. A soluble TCR comprising the alpha cliam ammo acid sequences of SEQ ID NO: 29 and beta chain amino acid sequsaice of SBQ ID NO: 10
42. A soluble TCR comprising the alpha chian amino acid sequence of SEQ ID NO: 68 and beta chain amino acid sequence SEQ ID NO: 10.
43. A soluble TCR comprising the alpha chain amino acid sequence of SEQ ID NO: 69 and beta chain amino acid sequence SEQ ID NO: 10.
44. A soluble TCR comprising Ihe a^ha chain amino acid sequence of SEQ ID NO: 70 and beta chain amino acid sequence SEQ ID NO: 10.
45. A soluble TCR comprising the alpha chain amino acid sequence of SEQ ID
NO: 29 and beta chain amino acid sequence SEQ ID NO: 71.
46. A soluble TCR compri^ng the alpha chain amino acid sequcaice of SEQ ED NO: 29 and beta chain amino acid sequence SEQ ID NO: 72.
47. A soluble TCR comprising ihe alpha chain amino acid sequence of SEQ ID NO: 29 and beta diam amino acid sequence SEQ ID NO: 73.
48. A soluble TCR conqaising die alpha chain amino acid sequence of SEQ ID NO; 29 and beta chain amino acid sequence SEQ ID NO: 74.
49. A soluble TCR con^sing the alpha chain amino acid sequence of SEQ ID NO: 29 and beta clain amino acid sequence SEQ ID NO: 75.
50. A soluble TCR comprising the alpha cdiain amino acid sequence of SEQ ID NO: 29 and beta chain amino acid sequence SEQ ID NO: 76.

51. A soluble TCR cxamprising the alpha chain amino acid ssqences of SEQ ID NO: 29 and beta chain amino acid sequence SEQ ID NO: 77.
52. A soluble TCR comjffising the alpha diain artuno acid sequence of SEQ ID NO: 29 and beta cbam amino acid sequence SEQ ID NO: 7S.
53. A TCR as claimed in any preceding claim wherein the TCR is associated with at least one poiyalkylene glycol diain(s).
54. A TCR as claimed in claim 53 wherein the poiyalkylene glycol chain(s) is/are covalently linked to the TCR.
55. A TCR as claimed in claim 53 or claim 54 wherein the poiyalkylene glycol chain(s) coinprise(s) at least two polyethylene glycol repeating units.
56. A TCR as claimed in any preceding claim further comprising a reamve cysteine at the C temuna] or N-terminal of the alpha or beta chains thereof.
57 A TCR as claimed m any preceding claim associated with a thCTapeutic agent or detectable moiety.
58. A TCR as claimed in claim 57 wherein the TCR is covalently linked to a thempeutic agent or detectable moiety.
59. A TCR as claimed in claim 57 -wherein the ther^eutic agent or detectable moiety is covalKitly linked to ^ C terminus of one or both TCR chains.
60 A TCR as claimed in any of claims 57 to 59 associated with a feerapeutic agent which is an immune effisctor molecule.

61. A TCR as claimed in claim 60 where in the inunune effector molsculs is a cytoldne.
62. A TCR as claimed in claim 60 wherein the immune effector molecule is IL-2, or a functional variant or fragment thereof.
63. ATCRasclaimedinany ofclaimsS? to 59 wherein the therapeutic agent is a cytotoxic agent.
64. ATCRasclaimedinanyof claims 57 to 49 wherein tttt therapeutic agent is a
radionuclide.
65. A multivalent TCR complex comprising at least two TCRs as claimed in any of the preceding claims.
66. A multivalent TCR complex comprising at least two TCRs as claimed ia any of the preceding claims linked by a non-peptidic polymer chain or a peptidic linka-sequence.
67. A TCR complex as claimed in claim 65 wherein the polymer chain or peptidic linker sequence extends between amino acid residues of each TCR which are not located in a variable region sequence of fee TCR.
68. A TCR complex as claimed in either of claims 66 or 67 in which the TCRs are linked by a polyalkylene glycol chain or a peptidic linker derived from a human multimerisation domain.
69. A TCR complex as claimed in claim 68 wherein a divalent alkylene spacer radical is located between the polyalkylene glycol chain and its point of attachment to a TCR of the complex.

70. A TCR complex as claimed in claim 68 or claim 69 wherein the polyalkylsns
glycol chain comprises at least two polyethylenes glycol repeating units.
71. A multivalent TCR complex comprising at least two TCSls as claimed in any
■ of claims 1 to 56-wherein (:) at least one of said TCRs is associated with, a therapeutic
agent as claimed in any of claims 57 to 64.
72. An isolated cell presenting a TCR as defined in any of claims 1 to 52.
73. An isolated cell as claimed in claim 72 which is a himian T cell or a hionan haematopoietic stem cell.
74. A nucleic acid or nucleic acids encoding a TCR as claimed in any of the preceding claims.
75. A nucleic acid or nucleic acids as claimed in claim 74, adapted for expression in a bacterial, yeast, mammalian or lEisect cell.
76. A nucleic acid or nucleic acids as claimed in claim 74 adapted for expression in a human T cell or a human haematopoietic stem cell.
77. Nucleic acid as claimed in claim 76 consisting of one of the full-lengfii TCR a chain DNA sequences of SEQ ID Nos 33, 35 or 37 and the TCR p chain DNA sequence of SEQ ID No 39 or a nucleic acid complKnentary thereto or a correspondii^ RNA sequraice.
78. A pharmaceutical composition comprising a TCR or a multivaleirt TCR complex as claimed in any of claims 1 to 71, or a plurality of cells as claimed in claims 72 or 73,OTanucleicacidornucleicacidsasdaimedinany ofdaims74to 77 together with a phannaceudcally acceptable cairiex.

79 A method of treatment of cancer comprising admiiiistermg to asutgect
suffering such caacsr an effective amouat of a TCR or a multivaleiiit TCR complex as
claimsdinany of claims 1 to 71, or a plurality of csll as claimed in claim E72 or 73,
or a nucleic acid or nucleic-acids as claimed in any of claims 74 to 77.
80 The use of a TCR or a multivalent TCR complex as claimed in any of claims 1
to 71, or a plurality of cells as claimed in claims 72 or 73, or a nucleic acdd or nucleic
acids as claimed in any of claims 74 to 77 in the pweraration of a con^osition for the
treatment of cancer.
81. A method of identifying a high afEinity TCR having the property of binding to AAGIGILTV-HLA-A*0201 CHARACTERISED IN THAT the TCR (i) comprises at least one TCR ci chain variable domain and/or at least one TCR p chain variable domain and (ii) has a K^ for tiie said AAGIGILrV-HLA-A*020I conqjJex of less than j^iM said method comprising:
(a) the production of a diverse iibrarj' of TCRs comprising the a and p
chain variable domains of the MEL TCR wiiereni one or both of the a
and ^ chain variable domains comprise a mutatioii(s);
(b) contacting said diverse library of TCRs with AAGIGILTV-HLA-
A*0201 under conditions suitableto allow tiw binding of tiie TCRs to
AAGIGILTV-HLA-A*0201:and
(c) measuring the KD of liie interaction.