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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • A—HUMAN NECESSITIES
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  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
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  • C07K14/55—IL-2
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
  • A61K2039/5156—Animal cells expressing foreign proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

• the present invention relates to T cell receptors (TCRs) having the property of binding to SLLMWITQC-HLA-A*0201 and comprising at least one TCR ⁇ chain variable domain and/or at least one TCR ⁇ chain variable domain CHARACTERISED IN
• said TCR has a K D for the said SLLMWITQC-HLA-A*0201 complex of less than or equal to l ⁇ M and/or has an off-rate (k off ) for the SLLMWITQC-HLA-A*0201 complex of lxlO "3 S "! or slower.
• the SLLMWITQC peptide is derived from the NY-ESO-1 protein that is expressed by a range of tumours (Chen et al, (1997) PNAS USA 94 1914-1918).
• the Class I HLA molecules of these cancerous cells present peptides from this protein, including SLLMWITQC. Therefore, the SLLMWITQC-HLA-A2 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 peptide-HLA complex than native TCRs specific for that complex.
• TCRs having high affinity (K D ) of the interaction less than or equal to l ⁇ M, and/or a slower off-rate (k 0ff ) of lxl 0 "3 S "1 or slower, for the SLLMWITQC-HLA-A*0201 complex.
• K D affinity
• k 0ff off-rate
• the present invention provides a T-cell receptor (TCR) having the property of binding to SLLMW ⁇ TQC-HLA-A*0201 and comprising at least one TCR ⁇ chain variable domain and/or at least one TCR ⁇ chain variable domain CHARACTERISED IN
• said TCR has a K D for the said SLLMWITQC-HLA-A*0201 complex of less than or equal to l ⁇ M and/or has an off-rate (k off ) for the SLLMW ⁇ TQC-HLA-A*0201 complex of lxlO "3 S _1 or slower.
• the K D and/or (k 0f ) measurement can be made by any of the known methods.
• a preferred method is the Surface Plasmon Resonance (Biacore) method of Example 5.
• the interaction of a disulfide-linked soluble variant of the native 1G4 TCR (see SEQ ID NO: 9 for TCR ⁇ chain and SEQ ID NO: 10 for TCR ⁇ chain) and the SLLMWITQC-HLA-A*0201 complex has a K D of approximately lO ⁇ M, an off- rate (k 0ff ) of 1.28 x 10 "1 S '1 and a half-life of 0.17 minutes as measured by the Biacore- base method of Example 5.
• the native 1G4 TCR specific for the SLLMWITQC-HLA-A*0201 complex has the following Valpha chain and Vbeta chain gene usage: Alpha chain - TRAV21 Beta chain: - TRBV 6.5
• the native 1G4 TCR can be used as a template into which various mutations that impart high affinity and/or a slow off-rate for the interaction between TCRs of the invention and the SLLMWITQC-HLA-A*0201 complex can be introduced.
• the invention includes TCRs which are mutated relative to the native 1 G4 TCR ⁇ chain variable domain (see Figure la and SEQ ID No: 1) and/or ⁇ chain variable domain (see Figure lb and SEQ ID NO: 2) in at least one complementarity determining region (CDR) and/or variable domain framework region thereof. It is also contemplated that other hypervariable regions in the variable domains of the TCRs of the invention, such as the hypervariable 4 (HV4) regions, may be mutated so as to produce a high affinity mutant.
• HV4 hypervariable 4
• TCRs exist in heterodimeric ⁇ or ⁇ forms. However, recombinant TCRs consisting of a single TCR ⁇ or TCR ⁇ chain have previously been shown to bind to peptide MHC molecules.
• the TCR of the invention comprise both an ⁇ chain variable domain and an TCR ⁇ chain variable domain.
• the mutation(s) in the TCR ⁇ chain sequence and/or TCR ⁇ chain sequence may be one or more of substitution(s), deletion(s) or insertion(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.
• any ⁇ TCR that comprises similar Valpha and Vbeta gene usage and therefore amino acid sequence to that of the 1G4 TCR could make a convenient template TCR. It would then be possible to introduce into the DNA encoding one or both of the variable domains of the template ⁇ TCR the 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. •
• the TCRs of the invention include those in which one or more of the TCR alpha chain variable domain amino acids corresponding to those listed below are mutated relative to the amino acid occurring at these positions in the sequence provided for the native
• the TCR amino acid sequences herein are generally provided including an N-terminal methionine (Met or M) residue. As will be known to those skilled in the art this residue may be removed during the production of recombinant proteins. Furthermore, unless stated to the contrary, the soluble TCR and TCR variable domain sequences have been truncated at the N-terminus thereof. (Resulting in the lose of the N-terminal "K” and “NA” in the TCR alpha and beta chain sequences respectively.). As will be obvious to those skilled in the art these "missing" N-terminal TCR residues may be re-introduced into the TCRs of the present invention.
• variable domain is understood 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 ⁇ chains and either the TRBC1 or TRBC2 for TCR ⁇ chains.
• part of the diversity of the TCR repertoire is due to variations which occur in the amino acid encoded by the codon at the boundary between the variable domain, as defined herein, and the constant domain.
• the codon that is present at this boundary in the wild-type IG4 TCR sequence results in the presence of the Tyrosine (Y) residue at the C-terminal of the variable domain sequences herein.
• This Tyrosine replaces the N-terminal Asparagine (N) residue encoded by the TRAC gene shown in Figure 8A.
• Embodiments of the mvention include mutated TCRs which comprise mutation of one or more of alpha chain variable domain amino acids corresponding to: 20V, 51Q, 52S, 53S, 94P, 95T, 96S, 97G, 98G, 99S, 100Y, 1011 and 103T, for example the amino acids: 20A 51P/S/T or M 52P/F or G 53W/H or T 94H or A 95L/M/A/Q/Y/E/I/F/V/N/G/S/D or R 96L/T/Y/I/Q/V/E/X/A/W/R/G/H/D or K 97D/N/V/S/T or A 98P/H/S/T/W or A 99T/Y/D/H/Y/N/E/G/Q/K/A/I or R lOOF/M or D lOlP/T/ or M 103 A The numbering used above is the same as that
• Embodiments of the invention also include TCRs which comprise mutation of one or more of the TCR beta chain variable domain amino acids corresponding to those listed below, are relative to the amino acid occurring at these positions in the sequence provided for the native 1G4 TCR alpha chain variable domain of the native 1G4 TCR beta chain in Figure lb and SEQ ID No: 2.
• amino acids referred to which may be mutated are:18M, 50G, 51 A, 52G, 531, 55D, 56Q, 70T, 94Y, 95V and 97N, for example: 18V 50S or A 51V or I 52Q 53T or M 55R 56R 701 94N or F 95L 97G or D
• the numbering used above is the same as that shown in Figure lb and SEQ FD No: 2
• TCRs comprising one of the mutated alpha chain variable domain amino acid sequences shown in Fig 6 (SEQ ID Nos: 11 to 83). Phenotypically silent variants of such TCRs also form part of this invention.
• TCRs comprising one of the mutated beta chain variable domain amino acid sequences shown in Figures 7 or 13. (SEQ ID Nos: 84 to 99 or 117 to 121). Phenotypically silent variants of such TCRs also form part of this invention.
• TCRs exist in heterodimeric ⁇ or ⁇ forms. However, recombinant TCRs consisting of ⁇ or ⁇ homodimers have previously been shown to bind to peptide MHC molecules. Therefore, one embodiment of the invention is provided by TCR ⁇ or TCR ⁇ homodimers.
• TCRs of the invention comprising the alpha chain variable domain amino acid sequence and the beta chain variable domain amino acid sequence combinations listed below, phenotypically silent variants of such TCRs also form part of this invention:
• TCR of the invention comprising: the alpha chain variable domain shown in the SEQ ID NO: 49 and the beta chain variable domain shown in the SEQ ID NO: 94, or phenotypically silent variants thereof.
• TCRs of the invention comprising the variable domain combinations detailed above further comprise the alpha chain constant region amino acid sequence shown in Figure 8a (SEQ LD NO: 100) and one of the beta chain amino acid constant region sequences shown in Figures 8b and 8c (SEQ ID NOs: 101 and 102) or phenotypically silent variants thereof.
• the term "phenotypically silent variants" is understood to refer to those TCRs which have a K D for the said SLLMWITQC-HLA-A*0201 complex of less than or equal to l ⁇ M and/or have an off-rate (k 0ff ) of lxlO "3 S "1 or slower.
• k 0ff off-rate
• Such trivial variants are included in the scope of this invention. Those TCRs in which one or more conservative substitutions have been made also form part of this invention.
• the TCRs of the invention are in the form of either single chain TCRs (scTCRs) or dimeric TCRs (dTCRs) as described in WO 04/033685 and WO 03/020763.
• a suitable scTCR form comprises a first segment constituted by an amino acid sequence corresponding to a TCR ⁇ chain variable domain, a second segment constituted by an amino acid sequence corresponding to a TCR ⁇ chain variable domain sequence fused to the N terminus of an amino acid sequence corresponding to a TCR ⁇ chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
• the first segment may be constituted by an amino acid sequence corresponding to a TCR ⁇ chain variable domain
• the second segment may be constituted by an amino acid sequence corresponding to a TCR ⁇ chain variable domain sequence fused to the N terminus of an amino acid sequence corresponding to a TCR ⁇ chain constant domain extracellular sequence
• the above scTCRs may further comprise a disulfide bond between the first and second chains, said disulfide bond being one which has no equivalent in native ⁇ T cell receptors, and wherein the length of the linker sequence and the position of the disulfide bond being such that the variable domain sequences of the first and second segments are mutually orientated substantially as in native ⁇ T cell receptors.
• the first segment may be constituted by an amino acid sequence corresponding to a TCR ⁇ chain variable domain sequence fused to the N terminus of an amino acid sequence corresponding to a TCR ⁇ chain constant domain extracellular sequence
• the second segment may be constituted by an amino acid sequence corresponding to a TCR ⁇ chain variable domain fused to the N terminus of an amino acid sequence corresponding to TCR ⁇ chain constant domain extracellular sequence
• a disulfide bond may be provided between the first and second chains, said disulfide bond being one which has no equivalent in native ⁇ T cell receptors.
• the linker sequence may link the C terminus of the first segment to the N terminus of the second segment, and may have the formula -PGGG- (SGGGG) n -P- wherein n is 5 or 6 and P is proline, G is glycine and S is serine.
• a suitable dTCR form of the TCRs of the present invention comprises a first polypeptide wherein a sequence corresponding to a TCR ⁇ chain variable domain sequence is fused to the N terminus of a sequence corresponding to a TCR ⁇ chain constant domain extracellular sequence, and a second polypeptide wherein a sequence corresponding to a TCR ⁇ chain variable domain sequence fused to the N terminus a sequence corresponding to a TCR ⁇ chain constant domain extracellular sequence, the first and second polypeptides being linked by a disulfide bond which has no equivalent in native ⁇ T cell receptors.
• the first polypeptide may comprise a TCR ⁇ chain variable domain sequence is fused to the N terminus of a sequence corresponding to a TCR ⁇ chain constant domain extracellular sequence, and a second polypeptide wherein a sequence corresponding to a TCR ⁇ chain variable domain sequence is fused to the N terminus a sequence corresponding to a TCR ⁇ chain constant domain extracellular sequence, the first and second polypeptides being linked by a disulfide bond between cysteine residues substituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 of TRBC1*01 or TRBC2*01 or the non-human equivalent thereof.
• the dTCR or scTCR form of the TCRs of the invention may have amino acid sequences corresponding to human ⁇ TCR extracellular constant and variable domain sequences, and a disulfide bond may link amino acid residues of the said constant domain sequences, which disulfide bond has no equivalent in native TCRs.
• the disulfide bond is between cysteine residues corresponding to amino acid residues whose ⁇ carbon atoms are less than 0.6 nm apart in native TCRs, for example between cysteine residues substituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 of TRBC1*01 or TRBC2*01 or the non-human equivalent thereof.
• Other sites where cysteines can be introduced to form the disulfide bond are the following residues in exon 1 of TRAC*01 for the TCR ⁇ chain and TRBC1*01 or TRBC2*01 for the TCR ⁇ chain:
• the dTCR or scTCR form of the TCRs of the invention may include a disulfide bond between residues corresponding to those linked by a disulfide bond in native TCRs.
• the dTCR or scTCR form of the TCRs of the invention preferably does not contain a sequence corresponding to transmembrane or cytoplasmic sequences of native TCRs.
• a TCR of the invention is associated with at least one polyalkylene glycol chain(s). This association may be cause in a number of ways known to those skilled in the art.
• the polyalkylene chain(s) is/are covalently linked to the TCR.
• the polyethylene glycol chains of the present aspect of the invention comprise at least two polyethylene repeating units.
• One aspect of the invention provides a multivalent TCR complex comprising at least two TCRs of the invention.
• at least two TCR molecules are linked via linker moieties to form multivalent complexes.
• the complexes are water soluble, so the linker moiety should be selected accordingly.
• the linker moiety should be capable of attachment 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 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 the TCR.
• the linker moieties should be chosen with due regard to their pharmaceutical suitability, for example their immunogenicity.
• linker moieties which fulfil the above desirable criteria are known in the art, for example the art of linking antibody fragments. There are two classes of linker that are preferred for use in the production of multivalent TCR molecules of the present invention.
• a TCR complex of the invention in which the TCRs are linked by a polyalkylene glycol chain provides one embodiment of the present aspect.
• hydrophilic polymers such as polyalkylene glycols.
• the most commonly used of this class are based on polyethylene glycol or PEG, the structure of which is shown below.
• n is greater than two.
• suitable, optionally substituted, polyalkylene glycols include polypropylene glycol, and copolymers of ethylene glycol and propylene glycol.
• Such polymers may be used to treat or conjugate therapeutic agents, particularly polypeptide or protein therapeutics, to achieve beneficial changes to the PK profile of the therapeutic, for example reduced renal clearance, improved plasma half-life, reduced immunogenicity, and improved solubility.
• therapeutic agents particularly polypeptide or protein therapeutics
• Such improvements in the PK profile of the PEG-therapeutic conjugate are believe to result fi-om the PEG molecule or molecules forming a 'shell' around the therapeutic which sterically hinders the reaction with the immune system and reduces proteolytic degradation.
• the size of the hydrophilic polymer used my in particular be selected on the basis of the intended therapeutic use of the TCR complex.
• 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, pentaerythritol, sorbitol and lysine.
• the polymer will have a chemically reactive group or groups in its structure, for example at one or both termini, and/or on branches from the backbone, to enable the polymer to link to target sites in the TCR.
• This chemically reactive group or groups may be attached directly to the hydrophilic polymer, or there may be a spacer group/moiety between the hydrophilic polymer and the reactive chemistry as shown below: Reactive chemistry-Hydrophilic polymer-Reactive chemistry
• a TCR complex of the invention in which 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 the invention in which the polyalkylene glycol chain comprises at least two polyethylene glycol repeating units provides a further embodiment of the present aspect.
• hydrophilic polymers linked, directly or via a spacer, to reactive chemistries that may be of use in the present mvention. These suppliers include Nektar Therapeutics (CA, USA), NOF Corporation (Japan), Sunbio (South Korea) and Enzon Pharmaceuticals (NJ, USA).
• hydrophilic polymers linked, directly or via a spacer, to reactive chemistries that may be of use in the present invention include, but are not limited to, the following:
• a wide variety of coupling chemistries can be used to couple polymer molecules to protein and peptide therapeutics.
• the choice of the most appropriate coupling chemistry is largely dependant on the desired coupling site.
• the following coupling chemistries have been used attached to one or more of the termini of PEG molecules (Source: Nektar Molecular Engineering Catalogue 2003): N-maleimide Vinyl sulfone Benzotriazole carbonate Succinimidyl proprionate Succinimidyl butanoate Thio-ester Acetaldehydes Acrylates Biotin Primary amines
• non-PEG based polymers also provide suitable linkers for multimerising the TCRs of the present invention.
• linkers for multimerising the TCRs of the present invention.
• moieties containing maleimide termini linked by 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 function to produce simple linkers or multimerisation domains onto which TCR molecules can be attached.
• the biotin / streptavidin system has previously been used to produce TCR tetramers (see WO/99/60119) for in-vitro binding studies.
• stepavidin is a microbially-derived polypeptide and as such not ideally suited to use in a therapeutic.
• a TCR complex of the invention in which the TCRs are linked by a peptidic linker derived from a human multimerisation domain provides a further embodiment of the present aspect.
• a multivalent TCR complex of the invention comprising at least two TCRs provides a final embodiment of this aspect, wherein at least one of said TCRs is associated with a therapeutic agent.
• a TCR (or multivalent complex thereof) of the present invention may alternatively or additionally comprise a reactive cysteine at the C-terminal or N- terminal of the alpha or beta chains thereof.
• the TCR of the invention may be associated with a therapeutic agent or detectable moiety.
• said therapeutic agent or detectable moiety may be covalently linked to the TCR.
• said therapeutic agent or detectable moiety is covalently linked to the C-terminus of one or both TCR chains.
• the scTCR or one or both 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 SLLMWITQC-HLA-A*0201 complex which method comprises contacting the TCR ligand with a TCR (or a multimeric high affinity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand.
• fluorescent streptavidin can be used to provide a detectable label.
• a fluorescently-labelled TCR tetramer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying theSLLMWITQC-HLA-A*0201 complex for which these high affinity TCRs are specific.
• TCR-specific antibodies in particular monoclonal antibodies.
• anti-TCR antibodies such as ⁇ Fl and ⁇ Fl, which recognise the constant domains of the ⁇ and ⁇ chains, respectively.
• a TCR (or multivalent complex thereof) of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a toxic moiety for use in cell killing, 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 compared to a non-multimeric wild-type or T cell receptor heterodimer of the invention.
• the multivalent TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent TCR complexes having such uses.
• the invention also provides a method for delivering a therapeutic agent to a target cell, which method comprises contacting potential target cells with a TCR or multivalent TCR complex in accordance with the 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 theSLLMWITQC-HLA- A* 0201 complex and having the therapeutic agent associated therewith.
• the soluble TCR or multivalent TCR complex of the present invention can be used to deliver therapeutic agents to the location of cells presenting a particular antigen. This would be useful in many situations and, in particular, against tumours.
• a therapeutic agent could be delivered such that it would exercise its effect locally but not only on the cell it binds to.
• one particular strategy envisages anti-tumour molecules linked to TCRs or multivalent TCR complexes according to the invention specific for tumour antigens.
• therapeutic agents could be employed for this use, for instance radioactive compounds, enzymes (perform for example) or chemotherapeutic agents (cis-platin for example).
• radioactive compounds for instance
• enzymes perform for example
• chemotherapeutic agents cis-platin for example.
• the toxin could be inside a liposome linked to streptavidin so that the compound is released slowly. This will prevent damaging effects during the transport in the body and ensure that the toxin has maximum effect after binding of the TCR to the relevant antigen presenting cells.
• cytotoxic agents i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents.
• agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolmide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; • peptide cytotoxins, i.e. proteins or fragments thereof with the ability to kill mammalian cells.
• radio-nuclides i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of ⁇ or ⁇ particles, or ⁇ rays, including but not limited to, iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; chelating agents may be used to facilitate the association of these radio-nuclides to the high affinity TCRs, or multimers thereof; « prodrugs, including but not limited to, antibody directed enzyme pro-drugs; • immuno-stimulants, i.e.
• moieties which stimulate immune response Including but not limited to, cytokines such as IL-2 and IFN, Superantigens and mutants thereof, TCR-HLA fusions and chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, etc, antibodies or fragments thereof, complement activators, xenogeneic protein domains, allogeneic protein domains, viral/bacterial protein domains, viral/bacterial peptides and anti-T cell determinant antibodies (e.g. anti-CD3 or anti-CD28).
• cytokines such as IL-2 and IFN
• TCR-HLA fusions and chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, etc
• antibodies or fragments thereof complement activators, xenogeneic protein domains, allogeneic protein domains, viral/bacterial protein domains, viral/bacterial peptides and anti-T cell determinant antibodies (e.g. anti-CD3 or anti-CD28).
• Antibody fragments and variants/analogues which are suitable for use in the compositions and methods described herein include, but are not limited to, the following.
• Antibody Fragments As is known to those skilled in the art, it is possible to produce fragments of a given antibody which retain substantially the same binding characteristics as those of the parent antibody. The following provides details of such fragments:
• Minibodies - These constructs consist of antibodies with a truncated Fc portion. As such they retain the complete binding domains of the antibody from which are derived.
• Fab fragments comprise a single immunoglobulin light chain covalently- linked to part of an immunoglobulin heavy chain. As such, Fab fragments comprise a single antigen combining site. Fab fragments are defined by the portion of an IgG that can be liberated by treatment with papain. Such fragments are commonly produced via recombinant DNA techniques. (Reeves et al, (2000) Lecture Notes on Immunology (4th Edition) Published by Blackwell Science)
• F(ab') fragments comprise both antigen combining sites and the hinge region from a single antibody.
• F(ab') 2 fragments are defined by the portion of an IgG that can be liberated by treatment with pepsin. Such fragments are commonly produced via recombinant DNA techniques. (Reeves et al, (2000) Lecture Notes on Immunology (4tth Edition) Published by Blackwell Science)
• Fv fragments - comprise an immunoglobulin variable heavy domain linked to an immunoglobulin variable light domain.
• Fv designs have been produced. These include dsFvs, in which the association between the two domains is enhanced by an introduced disulfide bond.
• scFVs can be formed 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 immunoglobulin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced.
• NanobodiesTM have also been multimerised to form diabodies and triabodies (Maynard et al., (2000) Annu RevBiomedEng 2 339-376) NanobodiesTM - These constructs, marketed by Ablynx (Belgium), comprise synthetic single immunoglobulin variable heavy domain derived from a camelid (e.g. camel or llama) antibody.
• Domantis (Belgium) comprise an affinity matured single immunoglobulin variable heavy domain or immunoglobulin variable light domain.
• the defining functional characteristic of antibodies in the context of the present invention is their ability to bind specifically to a target ligand. As is known to those skilled 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 the compositions and methods of the present invention include, but are not limited to, the following.
• Protein scaffold-based binding polypeptides - This family of binding constructs comprise mutated analogues of proteins which contain native binding loops.
• Affibodies marketed by Affibody (Sweden), which are based on a three-helix motif derived from one of the IgG binding domains of Staphylococcus aureus Protein A.
• Evibodies marketed by EvoGenix (Australia) which are based on the extracellular domains of CTLA-4into which domains similar to antibody binding loops are grafted.
• Cytokine Traps marketed by Regeneron Pharmaceuticals (US), graft cytokine receptor domains into antibody scaffolds.
• Fibronectin Fibronectin, Green fluorescent protein (GFP) and ankyrin repeats.
• GFP Green fluorescent protein
• ankyrin repeats As is known to those skilled in the art antibodies or fragments, variants or analogues thereof can be produced which bind to various parts of a given protein ligand.
• anti-CD3 antibodies can be raised to any of the polypeptide chains from which this complex is formed (i.e. ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ CD3 chains)
• Antibodies which bind to the ⁇ CD3 chain are the preferred anti-CD3 antibodies for use in the compositions and methods of the present invention.
• Soluble TCRs or multivalent TCR complexes of the invention may be linked to an enzyme capable of converting a prodrug to a drug. This allows the prodrug to be converted to the drug only at the site where it is required (i.e. targeted by the sTCR).
• the localisation in the vicinity of tumours or metastasis would enhance the effect of toxins or immunostimulants.
• the vaccine antigen could be localised in the vicinity of antigen presenting cells, thus enhancing the efficacy of the antigen.
• the method can also be applied for imaging purposes.
• an isolated cell presenting a TCR of the invention.
• said cell may be a T cell.
• 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, together with a pharmaceutically acceptable carrier;
• the invention also provides a method of treatment of cancer comprising administering to a subject suffering 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 of cells presenting at least one TCR of the invention.
• 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, in the preparation of a composition for the treatment of cancer.
• Therapeutic or imaging TCRs in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier.
• This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.
• the pharmaceutical composition may be adapted for administration by any appropriate route, for example parenteral, transdermal or via inhalation, preferably a parenteral (including subcutaneous, intramuscular, or, most preferably intravenous) route.
• a parenteral route for example parenteral, transdermal or via inhalation, preferably a parenteral (including subcutaneous, intramuscular, or, most preferably intravenous) route.
• Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
• Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. Additional Aspects
• a scTCR or dTCR (which preferably is constituted by constant and variable sequences corresponding to human sequences) of the present invention maybe provided in substantially pure form, or as a purified or isolated preparation. For example, it may be provided in a form which is substantially free of other proteins.
• the invention also provides a method of producing a high affinity TCR having the property of binding to SLLMWITQC-HLA-A*0201.
• the TCR (i) comprises at least one TCR ⁇ chain variable domain and/or at least one
• TCR ⁇ chain variable domain (ii) has a K D for the said SLLMWITQC-HLA- A*0201 complex of less than or equal to l ⁇ M and/or an off-rate (k 0ff ) for the SLLMWITQC-HLA-A*0201 complex of lxlO "3 S "1 or slower, wherein the method comprises:
• Figure la and lb details the alpha chain variable domain amino acid and beta chain variable domain amino acid sequences of the native 1G4 TCR respectively.
• Figures 2a and 2b show respectively the DNA sequence of soluble versions of the native 1G4 TCR ⁇ and ⁇ chains.
• Figures 3 a and 3b show respectively the 1G4 TCR ⁇ and ⁇ chain extracellular amino acid sequences produced from the DNA sequences of Figures 2a and 2b.
• Figures 4a and 4b show respectively the DNA sequence of soluble versions of the 1G4 TCR ⁇ and ⁇ chains mutated to include additional cysteine residues to form a non- native disulphide bond.
• the mutated codon is indicated by shading.
• Figures 5 a and 5b show respectively the 1G4 TCR ⁇ and ⁇ chain extracellular amino acid sequences produced from the DNA sequences of Figures 4a and 4b.
• the introduced cysteine is indicated by shading.
• Figure 6 details the alpha chain variable domain amino acid sequences of the high affinity 1G4 TCR variants.
• Figure 7 details the beta chain variable domain amino acid sequences of the high affinity 1G4 TCR variants.
• Figure 8a details the amino acid sequence of a soluble form of TRAC.
• Figure 8b details the amino acid sequence of a soluble form of TRBC1.
• Figure 8c details the amino acid sequence of a soluble form of TRBC2.
• Figure 9 details the DNA sequence of the pEX954 plasmid.
• Figure 10 details the DNA sequence of the pEX821 plasmid.
• Figure 11 details the DNA sequence of the pEX202 plasmid.
• Figure 12 details the DNA sequence of the pEX205 plasmid.
• FIG. 13 details further beta chain variable domain amino acid sequences of the high affinity 1 G4 TCR variants.
• Figure 14a details the alpha chain amino acid sequences of a preferred soluble high affinity 1G4 TCR variant.
• Figure 14b details the beta chain amino acid sequences of a preferred (c58c61) soluble high affinity 1G4 TCR variant utilising the TRBCl constant domain.
• Figure 14c details the beta chain amino acid sequences of a preferred (c58c61) soluble high affinity 1G4 TCR variant utilising the TRBC2 constant domain.
• Figure 14d details the beta chain amino acid sequences of a preferred (c58c61) soluble high affinity 1G4 TCR using the TRBC2 encoded constant region fused via a peptide linker to wild-type human IL-2.
• Figure 15a shows FACs staining of T2 cell pulsed with a range of NY-ESO-analogue SLLMWITQV peptide concentrations using the high affinity c58c61 1G4 TCR-IL-2 fusion proteins.
• Figure 15b shows FACs staining of T2 cell pulsed with a range of NY-ESO-derived
• Figure 16 shows FACs staining of SK-MEL-37, ScaBER, J82, HcT119 and Colo 205 cancer cells transfected with an SLLMWITQC peptide producing ubiquitin minigene
• Figure 17 shows ELISPOT data demonstrating the ability of soluble high affinity c58c61 1G4 TCR to inhibit CTL activation against the MEL-624 cancer cell.
• Figure 18 shows ELISPOT data demonstrating the ability of soluble high affinity c58c61 1G4 TCR to inhibit CTL activation against the SK-MEL-37 cancer cell.
• Figure 19 shows inhibition of T cell activation against peptide pulsed T2 cells by the soluble c58c61 high affinity 1G4 TCR as measured by LFN ⁇ production.
• Figure 20 shows lack of inhibition of T cell activation against peptide pulsed T2 cells by the soluble wild-type 1G4 TCR as measured by TFN ⁇ production.
• Figure 21 shows tumor growth inhibition caused by soluble c58c61 high affinity 1G4 TCR-IL-2 immunoconjugates.
• Figure 22 shows the number of SLLMWITQC-HLA-A*0201 antigens on the surface of Mel 526, Mel 624 and SK-Mel-37 cancer cells as determined by fluorescent microscopy.
• the visualisation of cell-bound biotinylated soluble c58c61 high affinity 1G4 TCRs was facilitated by conjugation with streptavidin-R phycoerythrin (PE).
• Example 1 Production of a soluble disulfide-linked TCR comprising the native 1G4 TCR variable domain
• RNA isolation Total RNA was isolated from 10000 clonal T cells by re-suspension in lOO ⁇ l tri- reagent (Sigma) and processing of the lysate according to the manufacturer's instructions. After the final precipitation the RNA was re-dissolved in 12.5 ⁇ l RNAse free water.
• RNA was mixed and incubated for 1 hour at 37°C. cDNA was then stored at -80°C.
• the above cDNA was used as template.
• a panel of forward primers covering all possible alpha and beta variable chains was used to screen for, and amplify by PCR, alpha and beta chains genes.
• Primer sequences used for TCR chain gene amplification were designed from the NCBI website (http://www.ncbi.nlm.nih.gov/Entrez/) using accession numbers obtained from the T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8.
• Alpha -chain forward primers were designed to contain a Clal restriction site and the universal alpha chain reverse primer a Sail restriction site.
• Beta-chain forward primers were designed to contain aAsel restriction site and universal beta reverse primer an Agel restriction site.
• Recipient vectors for the TCR gene fragments were based on a pGMT7 parent plasmid, which contains the T7 promoter for high level expression in E.coli strain BL21-DE3(pLysS) (Pan et ⁇ l, Biotechniques (2000) 29 (6): 1234-8)
• Alpha chain purified PCR products were digested with Clal and Salll and ligated into pEX954 (see figure 9) cut with Clal and Xhol.
• Beta chain purified PCR products were digested with Asel and Agel and ligated into pEX821 (See Figure 10) cut with Ndel/Agel.
• the cut PCR product and cut vector were ligated using a rapid DNA ligation kit
• Ligated plasmids were transformed into competent E.coli strain XLl-blue cells and plated out on LB/agar plates containing lOOmg/ml ampicillin. Following incubation overnight at 37°C, single colonies were picked and grown in 10 ml LB containing lOOmg/ml ampicillin overnight at 37°C with shaking. Cloned plasmids were purified using a Miniprep kit (Qiagen) and the insert was sequenced using an automated DNA sequencer (Lark Technologies).
• Figures 4a and 4b show respectively the DNA sequence of soluble versions of the 1G4 TCR ⁇ and ⁇ chains mutated to include additional cysteine residues to form a non- native disulphide bond.
• Figures 5a and 5b show respectively the NY-ESO TCR ⁇ and ⁇ chain extracellular amino acid sequences produced from the DNA sequences of Figures 4a and 4b
• the soluble disulfide-linked native 1G4 TCR produced as described in Example 1 can be used a template from which to produce the TCRs of the invention which have an increased affinity for the SLLMWITQC (SEQ ID NO: 125) -HLA-A*0201 complex.
• Mutagenesis was carried out using the following conditions : 50ng plasmid template, l l of lOmM dNTP, 5 ⁇ l of lOx Pfu DNA polymerase buffer as supplied by the manufacturer, 25 pmol of fwd primer, 25 pmol of rev primer, l ⁇ l pfu DNA polymerase in total volume 50 ⁇ l. After an initial denaturation step of 2 mins at 95C, the reaction was subjected to 25 cycles of denaturation (95 C, 10 sees), annealing (55C 10 sees), and elongation (72C, 8 mins). The resulting product was digested with Dpnl restriction enzyme to remove the template plasmid and transformed into E. coli strain XLl-blue. Mutagenesis was verified by sequencing.
• Alpha chain - c-jun leucine zipper The construct was made by PCR stitching.
• the plasmid coding for the high affinity TCR alpha chains and containing the code for the introduced inter-chain di-sulfide bridge was used as template.
• PCR with the following two primer pairs generated the desired variable domain.
• the plasmid pEX202 (see Figure 11), coding for a wild type affinity TCR alpha chain fused to human c-jun leucine zipper domain and not containing the code for the introduced inter-chain di-sulfide bridge, was used as template. PCR with the following primer pair generated the desired constant domain.
• the two products were mixed and diluted 1000 fold and 1 ⁇ l was used as template in a 50 ⁇ l PCR with 5'-TRAV21 fwd and 3 '-alpha rev primers.
• the resulting PCR product was digested using restriction enzymes Asel and Xmal and ligated into pEX202 cut with Ndel and Xmal .
• PCRs were carried out using the following conditions : 50 pg plasmid template, 1 ⁇ l of
• TRBV6-5 fwd tctctcattaatgaatgctggtgtcactcagacccc
• the plasmid pEX205 (see Figure 12), coding for a wild type affinity TCR beta chain fused to the human c-fos leucine zipper domain and not containing the code for the introduced inter-chain di-sulfide bridge, was used as template. PCR with the following two primers generated the desired constant domain gene fragment.
• TRBC rev aagcttcccggggtctgctctaccccaggc
• the two products were mixed and diluted 1000 fold and 1 ⁇ l was used as template in a 50 ⁇ l PCR with TRBV6-5 fwd and TRBC rev primers. PCRs were carried out as described above.
• the resulting PCR product was digested using restriction enzymes Asel and Xmal and ligated into pEX205 cut with Ndel and Xmal.
• Example 4 Expression, refolding and purification of soluble TCR
• the expression plasmids containing the mutated ⁇ -chain and ⁇ -chain respectively as prepared in Examples 1, 2 or 3 were transformed separately into E.coli strain BL21pLysS, and single ampicillin-resistant colonies were grown at 37°C in TYP (ampicillin lOO ⁇ g/ml) medium to OD 60 o of 0.4 before inducing protein expression with 0.5mM IPTG. Cells were harvested three hours post-induction by centrifugation for 30 minutes at 4000 ⁇ m in a Beckman J-6B.
• Cell pellets were re-suspended in a buffer containing 50mM Tris-HCl, 25% (w/v) sucrose, ImM NaEDTA, 0.1% (w/v) NaAzide, lOmM DTT, pH 8.0. After an overnight freeze-thaw step, re-suspended cells were sonicated in 1 minute bursts for a total of around 10 minutes in a Milsonix XL2020 sonicator using a standard 12mm diameter probe. Inclusion body pellets were recovered by centrifugation for 30 minutes at 13000 ⁇ m in a Beckman J2-21 centrifuge. Three detergent washes were then carried out to remove cell debris and membrane components.
• Triton buffer 50mM Tris-HCl, 0.5% Triton-XlOO, 200mM NaCl, lOmM NaEDTA, 0.1 % (w/v) NaAzide, 2mM DTT, pH 8.0
• TCR ⁇ chain and 60mg of TCR ⁇ chain solubilised inclusion bodies were thawed from frozen stocks, samples were then mixed and the mixture diluted into 15ml of a guanidine solution (6 M Guanidine-hydrochloride, lOmM
• guanidine solution containing fully reduced and denatured TCR chains was then injected into 1 litre of the following refolding buffer: lOOmM Tris pH 8.5, 400mM L- Arginine, 2mM EDTA, 5mM reduced Glutathione, 0.5mM oxidised Glutathione, 5M urea, 0.2mM PMSF.
• the redox couple (2-mercaptoethylamine and cystamine (to final concentrations of 6.6mM and 3.7mM, respectively) were added approximately 5 minutes before addition of the denatured TCR chains. The solution was left for 5 hrs ⁇ 15minutes.
• the refolded TCR was dialysed in Spectrapor 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 buffer was changed to fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at 5°C ⁇ 3°C for another 20-22 hours.
• sTCR was separated from degradation products and impurities by loading the dialysed refold onto a POROS 50HQ anion exchange column and eluting bound protein with a gradient of 0-500mM NaCl over 50 column volumes using an Akta purifier (Pharmacia). Peak fractions were stored at 4°C and analysed by Coomassie-stained
• the sTCR was purified and characterised using a Superdex 200HR gel filtration column pre-equilibrated 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 kDa was pooled and concentrated prior to characterisation by BIAcore surface plasmon resonance analysis.
• a surface plasmon resonance biosensor (Biacore 3000TM ) was used to analyse the binding of a sTCR to its peptide-MHC ligand. This was facilitated by producing single pMHC complexes (described below) which were immobilised to a streptavidin- coated binding surface in a semi-oriented fashion, allowing efficient testing of the binding of a soluble T-cell receptor to up to four different pMHC (immobilised on separate flow cells) simultaneously. Manual injection of HLA complex allows the precise level of immobilised class I molecules to be manipulated easily.
• HLA-A*0201 molecules were refolded in vitro from bacterially- expressed inclusion bodies containing the constituent subunit proteins and synthetic peptide, followed by purification and in vitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal Biochem. 266: 9-15).
• HLA-A*0201-heavy chain was expressed with a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains of the protein in an appropriate construct.
• Inclusion body expression levels of -75 mg/litre bacterial culture were obtained.
• the MHC light-chain or ⁇ 2- micro globulin was also expressed as inclusion bodies in E.coli from an appropriate 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
• 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 strength of the solution sufficiently.
• the protein solution was then filtered through a 1.5 ⁇ m cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCl gradient. HLA-A* 0201 -peptide complex eluted at approximately 250 mM NaCl, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
• Biotinylation tagged pMHC molecules were buffer exchanged into 10 mM Tris pH
• BirA enzyme purified according to O'Callaghan et al. (1999) Anal. Biochem. 266: 9-
• Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pHLA-A*0201 molecules were stored frozen at -20°C. Streptavidin was immobilised by standard amine coupling methods.
• Such immobilised complexes are capable of binding both T-cell receptors and the coreceptor CD8 ⁇ , both of which may be injected in the soluble phase. Specific binding of TCR is obtained even at low concentrations (at least 40 ⁇ g/ml), implying the TCR is relatively stable.
• the pMHC binding properties of sTCR are observed to be qualitatively and quantitatively similar if sTCR is used either in the soluble or immobilised phase. This is an important control for partial activity of soluble species and also suggests that biotinylated pMHC complexes are biologically as active as non- biotinylated complexes.
• SPR measures changes in refractive index expressed in response units (RU) near a sensor surface within a small flow cell, a principle that can be used to detect receptor ligand interactions and to analyse their affinity and kinetic parameters.
• the probe flow cells were prepared by immobilising the individual HLA- peptide complexes in separate flow cells via binding between the biotin cross linked onto ⁇ 2m and streptavidin which have been chemically cross linked to the activated surface of the flow cells. The assay was then performed by passing sTCR over the surfaces of the different flow cells at a constant flow rate, measuring the SPR response in doing so.
• Serial dilutions of WT 1G4 sTCR were prepared and injected at constant flow rate of 5 ⁇ l min-1 over two different flow cells; one coated with -1000 RU of specific SLLMWITQC-HLA-A*0201 complex, the second coated with -1000 RU of nonspecific HLA-A2 -peptide complex.
• Response was normalised for each concentration using the measurement from the control cell. Normalised data response was plotted versus concentration of TCR sample and fitted to a hyperbola in order to calculate the equilibrium binding constant, K D . (Price & Dwek, Principles and Problems in Physical Chemistry for Biochemists (2 nd Edition) 1979, Clarendon Press, Oxford).
• Kp was determined by experimentally measuring the dissociation rate constant, kd, and the association rate constant, ka.
• the equilibrium constant K D was calculated as kd/ka.
• TCR was injected over two different cells one coated with -300 RU of specific HLA-
• A2-nyeso peptide complex the second coated with -300 RU of non-specific HLA-A2 -peptide complex.
• Flow rate was set at 50 ⁇ l/min. Typically 250 ⁇ l of TCR at -3 ⁇ M concentration was injected. Buffer was then flowed over until the response had returned to baseline.
• Kinetic parameters were calculated using Biaevaluation software. The dissociation phase was also fitted to a single exponential decay equation enabling calculation of half-life.
• the interaction between a soluble disulfide-linked native 1G4 TCR (consisting of the and ⁇ TCR chains detailed in SEQ ID NOs 9 and 10 respectively) and the SLLMWTTQC-HLA-A*0201 complex was analysed using the above methods and demonstrated a K D of 15 ⁇ M and a k of of 1.28 x 10 "1 S "1 .
• the TCRs specified in the following table have a K D of less than or equal to 1 ⁇ M and/or a k off of 1 x 10 "3 S " l or slower.
• Example 6 In-vitro cell staining using a high affinity c58c61 NY-ESO TCR- IL-2 fusion protein.
• T2 lymphoblastoid cells were pulsed with the NY-ESO-derived SLLMWITQC, NY- ESO-analogue SLLMWITQV peptide, or an irrelevant peptide at a range of concentrations (10 "5 - 10 "10 M) for 180 minutes at 37'C.
• the NY-ESO-analogue SLLMWITQV peptide (V-variant peptide) was used as this peptide is known to have a higher affinity for the binding cleft of the HLA-A*0201 complex than the native NY- ESO-derived SLLMWITQC peptide.
• TCR-IL-2 After pulsing, cells were washed in serum-free RPMI and 5 x 10 5 cells were incubated with high affinity c58c61 NY-ESO TCR- IL-2 fusion protein for lOmin at room temperature, followed by secondary anti-IL-2 mAb conjugated with PE (Serotec) for 15min at room temperature. After washing, bound TCR-IL-2 was quantified by flow cytometry using a FACS Vantage SE (Becton Dickinson). Controls, also using peptide-pulsed T2 cells were included where TCR-IL- 2 was omitted.
• Figure 14a details the amino acid sequence of the alpha chain of the c58c61 NY-ESO TCR. (SEQ ID NO: 122).
• Figure 14c (SEQ ID NO: 124) details the amino acid sequence of the beta chain of the c58c61 NY-ESO TCR using the TRBC2 encoded constant region.
• Figure 14d (SEQ ID NO: 125) details the amino acid sequence of the beta chain of the c58c61 NY-ESO TCR using the TRBC2 encoded constant region fused via a peptide linker to wild- type human IL-2.
• the alpha and beta chain variable domain mutations contained within the soluble c58c61 1G4 TCR - IL-2 fusion protein correspond to those detailed in SEQ ID NO: 49 and SEQ JD NO: 94 respectively.
• SEQ FD NOs: 121 -125 have been provided in a form which includes the N-terminal methionine (M) and the "K” and "NA” residues omitted in the majority of the other TCR alpha chain and beta chain amino acid sequences.
• SK-MEL-37, ScaBER, J82, HcT119 and Colo 205 cancer cells transfected with a NY-ESO-derived SLLMWITQC peptide expressing ubiquitin minigene construct were used.
• the cancer cells were transfected using substantially the methods described in (Rimoldi et al, (2000) J. Immunol. 165 7253-7261). Cells were labelled as described above.
• Figure 15a shows FACs staining of T2 cell pulsed with a range of NY-ESO-analogue SLLMWITQV peptide concentrations using the high affinity c58c61 1G4 TCR-IL-2 fusion proteins.
• Figure 15a shows FACs staining of T2 cell pulsed with a range of NY-ESO-analogue SLLMWITQV peptide concentrations using the high affinity c58c61 1G4 TCR-IL-2 fusion proteins.
• Figure 15b shows FACs staining of T2 cell pulsed with a range of NY-ESO-derived
• Figure 16 shows FACs staining of SK-MEL-37, ScaBER, J82, HcT119 and Colo 205 cancer cells transfected with an SLLMWITQC peptide producing ubiquitin minigene
• the following assay was carried out to demonstrate that the soluble high affinity c58c61 NY-ESO TCR was capable of inhibiting activation of an SLLMWITQC-HLA- A*0201 specific CTL clone (1G4). TFN- ⁇ production was used as the read-out for CTL activation.
• R10 Assay media 10% FCS (heat-inactivated, Gibco, cat# 10108-165), 88% RPMI 1640 (Gibco, cat# 42401-018), 1% glutamine (Gibco, cat# 25030-024) and 1% penicillin/streptomycin (Gibco, cat# 15070-063).
• Peptide (obtained from various sources) initially dissolved in DMSO (Sigma, cat# D2650) at 4mg/ml and frozen. Wash buffer: 0.01M PBS/0.05% Tween 20 (1 sachet of Phosphate buffered saline with Tween 20, pH7.4 from Sigma, Cat. # P-3563 dissolved in llitre distilled water gives final composition 0.01M PBS, 0.138M NaCl, 0.0027M KC1, 0.05% Tween 20). PBS (Gibco, cat#10010-015).
• the EliSpot kit contains all other reagents required i.e. capture and detection antibodies, skimmed milk powder, BSA, streptavidin-alkaline phosphatase, BCIP/NBT solution (Human IFN-g 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 instructions supplied with each kit but contains some alterations.
• MEL-624 and SK-MEL-37 melanoma cell lines were treated with trypsin for 5 minutes at 37 °C. The cells are then washed and re-suspended in R10 media.
• TFN- ⁇ production was used as the read-out for CTL activation.
• RIO Assay media 10% FCS (heat-inactivated, Gibco, cat# 10108-165), 88% RPMI 1640 (Gibco, cat# 42401-018), 1% glutamine (Gibco, cat# 25030-024) and 1% penicillin/streptomycin (Gibco, cat# 15070-063).
• Peptide (obtained from various sources) initially dissolved in DMSO (Sigma, cat#
• Wash buffer 0.01M PBS/0.05% Tween 20 (1 sachet of Phosphate buffered saline with Tween 20, pH7.4 from Sigma, Cat. # P-3563 dissolved in 1 litre distilled water gives final composition 0.01M PBS, 0.138M NaCl, 0.0027M KC1, 0.05% Tween 20).
• the ELISA kit contains all other reagents except BSA (Sigma), required i.e. capture and detection antibodies, skimmed milk powder, streptavidin-HRP, TMB solution (Human IFN-g Eli-pair 20 x 96 wells with plates. The following method is based substantially on the instructions supplied with each kit.
• ELISA plates were prepared according to the manufacturers instructions. (Diaclone kit, Immunodiagnostic systems, UK T2 cell line target cells were washed and re-suspended in RIO media with or without varying concentrations (lOOnM - lOpM) of SLLMWITQC peptide, then incubated for l hour at 37°C, 5%CO 2 .
• the plates were then incubated for 48 hours at 37°C, 5% CO 2 .
• the ELISA was then processed according to manufacturer's instructions.
• Example 11 In-vivo tumour targeting using a high affinity c58c61 IG4 TCR-IL-2 fusion protein
• mice Fifty female nude mice (HARLAN, France) were used in this trial. All animals were injected subcutaneously with the human melanoma tumour- forming cell line (SK-MEL-37) which had been stably transfected with a NY-ESO peptide/ubiquitin minigene construct in ensure enhanced expression of the appropriate class I-peptide target at the cell surface. Tumors were allowed to grow in the animals for 5 days to allow tumour development prior to commencement of treatment.
• SK-MEL-37 human melanoma tumour- forming cell line
• the rats then received the following i.v. bolus dosage of c58c61 high affinity NY-ESO TCR IL-2 fusion protein:
• Doses ranged between 0.02 and 1.0 mg/kg high affinity 1G4 TCR/IL-2 fusion proteins in PBS, administered at 5, 6, 7, 8, 11, 13, 17, 20, 24, 28, and 30 day post-tumor engraftment.
• a control treatment group was included where PBS alone was substituted for the TCR/IL-2 immunoconjugate.
• the TCR/IL-2 immunoconjugate exhibited a clear dose-dependent anti-tumor effect as shown by the tumour growth curves depicted in Figure 21.
• Example 12 Quantification of cell surface TCR ligands by fluorescence microscopy using high affinity c58c61 1G4 TCR
• HLA-A2-tax peptide-specific high affinity TCR in PBS/Mg containing 0.5% BSA albumin) for 30 min at 4°C.
• TCR solution was removed, and cells were washed three times with 500 ⁇ l of PBS/Mg.
• Cells were incubated in 200 ⁇ l of streptavidin-PE solution (5 ⁇ g ml "1 streptavidin-PE in PBS/Mg containing 0.5% BSA) at room temperature in the dark for 20 min. Streptavidin-PE solution was removed and cells were washed five times with 500 ⁇ l of PBS/Mg. Wash media was removed, and cells kept in 400 ⁇ l of imaging media before imaging by fluorescence microscopy.
• Fluorescence microscopy Fluorescent microscopy was carried out using an Axiovert 200M (Zeiss) microscope with a 63x Oil objective (Zeiss).
• a Lambda LS light source containing a 300W Xenon Arc lamp (Sutter) was used for illumination, and light intensity was reduced to optimal levels by placing a 0.3 and a 0.6 neutral density filter into the light path.
• Excitation and emission spectra were separated using a TRITC/Dil filter set (Chroma).
• Cells were imaged in three dimensions by z-stack acquisition (21 planes, 1 ⁇ m apart). Image acquisition and analysis was performed using Metamo ⁇ h software (Universal Imaging) as described (Irvine et al, Nature (419), p845-9, and Purbhoo et al, Nature Immunology (5), p524-30.).

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