Classifications

• • C—CHEMISTRY; METALLURGY
  • 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
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex

Definitions

• the invention relates to non-naturally selected (synthetic) polypeptide ligands which are selective for a given peptide-MHC complex or CDl -antigen complex, and which are derived from an ancestral TCR with specificity for a different antigen.
• synthetic TCR variable domains refers to any TCR variable domains which comprise germline TCR variable domain framework sequences in combination with mutations in one or more of the hypervariable regions of the variable domain.
• the essence of the invention described and claimed in PCT/GB2005/002078 is a method of changing the specificity of a given ancestral TCR for its ligand, by mutation of its variable domain, and selection of mutated variants which bind a different ligand.
• the ancestral TCR will be a heterodimeric TCR, usually an ⁇ TCR, and mutation may be in the variable domains of one or both chains.
• the mutated sequence(s) giving rise to the changed specificity may be determined, and those sequences may be incorporated in a desired TCR background using known methods of TCR expression and refolding.
• PCT/GB2005/002078 is the first demonstration that it is possible to alter the specificity of a given TCR, and it makes possible the creation of TCRs which bind any given antigen. This solves a long-standing problem in the art, since the identification of TCRs specific for a given antigen has previously relied on finding natural T-cells, presenting TCRs of the desired specificity. That has proved difficult, and often impossible, in the case of many desired antigens.
• the method of PCT/GB2005/002078 has enabled the creation of a new class of polypeptide ligands, specific for a given peptide-MHC complex or CDl -antigen complex, which are derived synthetically from an ancestral TCR of different specificity. That new class of polypeptides is the subject matter of this invention.
• the present invention provides a non-natural polypeptide lgand with specificity for a given peptide MHC complex or CDl -antigen complex, said polypeptide being derived from an ancestral TCR of different specificity by mutation of at least the variable domain thereof, provided that the given peptide-MHC complex is not (i)RLVDDFLLV-HLA-A*0201, (ii) ELTLGEFLKL-HLA-A*0201, (iii)LTLGEFLKL-HLA-A*0201 or (iv) VYGFVRACL-HLA-A*2401.
• polypeptides of the invention may be single chain polypeptides derived from one chain of an ancestral TCR, but currently it is preferred that they be heterodimeric TCRs in which one chain includes, or both chains include, a variable domain sequence or sequences derived from the ancestral TCR by mutation of the variable domain sequence or sequences of the corresponding chain or chains thereof.
• homodimeric polypeptides in which each chain includes a variable domain sequence or sequences derived from the ancestral TCR by mutation of the variable domain sequence or sequences of the corresponding chain thereof, are also included in the class of polypeptides of the invention.
• the new class of polypeptide ligands of the invention may be prepared by the methods disclosed in PCT/GB2005/002078.For convenience, those methods are briefly summarised below, and illustrated by the reproduced examples of PCT/GB2005/002078.
• members of a diverse library of nucleoproteins for example filamentous phage or ribosome particles, displaying on their surfaces polypeptides comprising diverse synthetic TCR variable domain sequences derived from a TCR which binds to a first peptide-MHC complex or CDl- antigen complex wherein diversity resides at least in the variable domains of the said polypeptides are contacted with a given peptide-MHC complex or CDl -antigen complex different from the first peptide-MHC complex or CDl -antigen complex, and binding is detected, for example by ELISA, between the library members and said given peptide-MHC complex or CDl -antigen complex.
• Library members detected as binding to the given peptide-MHC complex or CDl -antigen complex are isolated, and optionally multiplyied in an amplification process.
• EP-TCR error-prone PCR
• DNA shuffling techniques DNA shuffling techniques
• bacterial mutator strains such as XL-I -Red are convenient means of introducing mutations into the polypeptide sequences. It is particularly preferred if these mutations are introduced into a defined domain of the polypeptide, usually the variable domain of a TCR.
• mutations in the variable domain of a TCR particularly the complementarity-determining regions (CDRs) and/or framework regions are likely to be the most appropriate sites for the introduction of mutations leading to the production of a diverse library of nucleoproteins displaying the mutated polypeptides.
• EP-PCR is an example of a method by which such 'region-specific' mutations can be introduced into the ploypeptides.
• EP-PCR primers are used that are complementary to DNA sequences bordering the region to be mutated to amplify multiple copies of this region of the DNA that contain a controllable level of random mutations.
• DNA sequences encoding mutated regions are inserted into the DNA sequences, which encode the non-mutagenised sections of the polypeptide, by ligation or overlapping PCR.
• the DNA encoding the polypeptide with mutated region can then be ligated onto DNA encoding a heterologous polypeptide in order to produce a fusion protein suitable for display.
• the expression vector utilised is either a phagemid or a phage genome vector in which the polypeptide DNA can be ligated to DNA encoding a surface protein, preferably the gill or gVIII surface protein.
• dimeric polypeptide such as a heterodimeric TCR
• only one of the polypeptide chains is ligated as aforesaid.
• the other chain is encoded in nucleic acid for co-expression with phagemid and helper phage nucleic acid, so that the expressed second chain finds and associates with the expressed phage with surface displayed first chain, hi the case of momnomeric polypeptides, as discussed in more detail above, properly positioned cysteines in the polypeptides, for example in the constant domains of a TCR, are helpful in causing the polypeptides, particularly the variable domains of a TCR, to adopt their functional positions, through the formation of a disulfide bond by those cysteines.
• the method also encompasses cases wherein the library of nucleoproteins comprises (a) a first set of nucleoproteins displaying on their surfaces polypeptides comprising diverse synthetic TCR ⁇ variable domain sequences, and/or (b) a second set of nucleoproteins displaying on their surfaces polypeptides comprising diverse synthetic TCR ⁇ variable domain sequences, and/or (c) a third set of nucleoproteins displaying on their surfaces polypeptides comprising diverse synthetic TCR ⁇ or ⁇ variable domain sequences, which polypeptides are associated in pairs with polypeptides also comprising diverse synthetic TCR ⁇ or ⁇ variable domain sequences.
• the nucleoproteins are filamentous phage particles
• the library includes or consists of members displaying polypeptides which are ⁇ dimeric TCRs ("dTCRs"), and optionally includes members displaying TCR ⁇ -chains and/or TCR ⁇ -chains and/or homodimeric ⁇ chains and/or homodimeric ⁇ chains.
• dTCRs ⁇ dimeric TCRs
• the nucleoproteins are filamentous phage particles
• the library includes or consists of members displaying TCR ⁇ -chains and/or TCR ⁇ -chains and/or homodimeric ⁇ chains and/or homodimeric ⁇ chains.
• the library comprises phage-displayed ⁇ dTCRs, the latter being dTCRs as disclosed in WO 03/020763, ie comprising
• 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
• first and second polypeptides being linked by a disulfide bond which has no equivalent in native ⁇ T cell receptors
• first or second polypeptides being linked by a peptide bond at its C- terminus to a surface exposed amino acid residue of the phage particle.
• first and second polypeptides are preferably linked by a disulfide bond between cysteine residues substituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 ofTRBCl*01 or TRBC2*01 or the non-human equivalent thereof.
• step (i) several members of the library can be contacted in parallel with the given pMHC or CDl -antigen and members which bind to the pMHC or CDl -antigen are identified, (ii) one or more members which bind to the given pMHC or CD 1 -antigen assessed in step (i) can be selected, and the variable domain sequences of the displayed TCRs is/are determined, (iii) soluble form TCRs incorporating the thus-determined variable domain sequences, can be created, (iv) the affinities and/or the off-rates for the given pMHC of these TCRs can be determined, and
• one or more TCRs having the desired affinity and/or off-rate determined in step (iv) can be selected.
• soluble TCR is understood to refer to any TCR that: (i) lacks the native transmembrane domain thereof and (ii) is not associated with a nuceloprotein and (iii) retains the ability to bind to a pMHC or CDl -antigen.
• the affinity and/or the off-rate measurements can be made by any appropriate method, hi a preferred embodiment the said affinities and/or the off-rates are determined by Surface Plasmon Resonance (SPR).
• SPR Surface Plasmon Resonance
• Example 3 herein provides details of an SPR-based (Biacore) method suitable for determining the affinities and off-rates for the interactions between soluble form TCRs and pMHCs or CDl -antigens.
• the displayed polypeptides may be derived from the given TCR(s) by mutation of at least one of its/their complementarity determining regions of the variable domains.
• the displayed polypeptides can be derived from the given TCR(s) by mutation of at least one of its/their CDR2 or CDR3 complimentarity determining regions of the variable domains.
• the TCR(s) which is/are the starting point for the creation of the library used will usually be naturally occurring.
• nucleoproteins particularly filamentous phage and ribosome particles
• Methods for the preparation of libraries of single chain polypeptides displayed on nucleoproteins, particularly filamentous phage and ribosome particles are well known in the art, for example the art of antibody display libraries, and need no further discussion here.
• WO 2004/044004 describes the methods required to produce nucleoproteins displaying dTCRs.
• nucleic acids are required which encode (a) one chain of a desired polypeptide pair and (b) the other chain of the polypeptide pair fused to a nucleic acid sequence encoding a protein capable of forming part of the surface of a nucleoprotein particle.
• host cells maybe used transformed with an expression vector comprising nucleic acid encoding (a) one chain of the polypeptide pair and (b) the other chain of the dTCR polypeptide pair fused to a nucleic acid sequence encoding a protein capable of forming part of the surface of a nucleoprotein particle.
• the expression system comprises phagemid or phage genome vectors expressing nucleic acids (a) and (b).
• these phagemid or phage genome vectors is (are) those which encode bacteriophage gill or gVIII coat proteins.
• Transformed cells are incubated to allow the expression of the dTCR-displaying nucleoprotein particles.
• the nucleoprotein library members may be used in assays to identify polypeptide ligands variants with the desired affinity and/or off-rate characteristics. Any particles that possess the desired characteristics under investigation can then be isolated.
• the DNA encoding these TCRs can then be amplified by PCR and the sequence determined.
• WO 2004/044004 discloses several strategies for limiting the expression levels of an exogenous polypeptide from a given expression system in a host which may be suitable for the limiting the expression levels of a polypeptide, or one or both chains of a polypeptide pair.
• Another in-vitro display method to identify ancestral TCR-derived polypeptide ligands of the invention, comprising mutated variable domain sequences specific for a given target peptide-MHC complex or CDl antigen complex is ribosomal display.
• a DNA library is constructed that encodes a diverse array of mutated scTCRs or dTCR polypeptides using the techniques discussed above.
• the DNA library is then contacted with RNA polymerase in order to produce a complementary mRNA library.
• the mRNA sequences can then be ligated to a DNA sequence comprising a puromycin-binding site.
• mRNA encoding both chains of the TCR may be contacted with ribosom.es in- vitro under conditions allowing the translation of the TCR chains such that a ribosome displaying a dTCR is formed.
• ribosom.es in- vitro under conditions allowing the translation of the TCR chains such that a ribosome displaying a dTCR is formed.
• These scTCR- or dTCR-displaying ribosomes can then used for screening or in assays to identify TCR variants with specific enhanced characteristics. Any particles that possess the enhanced characteristics under investigation can then be isolated.
• the mRNA encoding these TCRs can then converted to the complementary DNA sequences using reverse transcriptase. This DNA can then be amplified by PCR and the sequence determined.
• Figure 1 details the DNA sequence of the pEX922-ILA vector
• Figure 2 provides a plasmid map of the pEX922-ILA vector
• Figure 3 a and 3b detail the DNA sequences of the soluble ILA TCR ⁇ and ⁇ chains as contained in the pEX922-ILA vector. Both of the DNA sequences contain a mutated codon encoding a cysteine residue required to form a non-native disulfide interchain bond in the expressed soluble TCR. Shading indicates the mutated cysteine codons.
• Figure 4a and 4b detail the amino acid sequences of the soluble ILA TCR ⁇ and ⁇ chains as encoded for by the DNA of figures 3 a and 3b respectively. Shading indicates the introduced cysteine residues.
• Figure 5 provides a schematic diagram representing the PCR products generated during the production of the ILA TCR-derived library.
• Figure 6 provides the Biacore response curve generated for the interaction of this soluble TCR Example 1- Mutagenesis of ILA-TCR CDRl, CDR2 and CDR3 regions
• the CDRl, 2 and 3 ⁇ and ⁇ variable regions of the ILA TCR were targeted for the introduction of mutations to investigate the possibility of generating high affinity mutants.
• Overlapping PCR was used to modify the sequence of ⁇ and ⁇ CDRl, 2 and 3 regions to introduce mutations.
• two PCR products were generated which flanked either side of the CDRl, 2 or 3 regions.
• the PCR products do not overlap and do not include the CDRl, 2 or 3 sequences to be mutated.
• These two PCR products are then included in a further PCR reaction in the presence of mutagenic oligos which contain regions of homology with both tlie CDRl, 2 or 3 flanking PCR products.
• the presence of the mutagenic oligos in the PCR reaction in conjunction with outside flanking primers allows the stitching of the two PCR fragments to incorporate either CDRl, 2 or 3 mutations.
• PCR 40.5 ⁇ l water, 5 ⁇ l 10x Pfu buffer, l ⁇ l YOL13 primer lOpM/ ⁇ l, l ⁇ l (272) primer lOpM/ ⁇ l, l ⁇ l 1OmM dNTPs, IOng pEX922-ILA and 0.5 ⁇ l of Pfu polymerase.
• the PCR was cycled as follows 30 cycles of 95 degrees 15 sec, 55 degrees 15 sec and 72 degrees for 2 min. PCR product was run on a 1.6% TBE agarose gel, a band of the correct size excised and purified using the Qiagen gel extraction kit.
• PCR7 As above substituting the primers 278 and 238.
• PCR12 As above substituting the primers 292 and 238.
• the pEX922-ILA vector contains DNA encoding the ILA TCR ⁇ and ⁇ chains in a soluble form containing mutated codons encoding non-native cysteine residues.
• Figure 1 details the DNA sequence of the pEX922-ILA vector
• Figure 2 provides a plasmid map of this vector
• Figure 3 a and 3b detail the DNA sequences of the soluble ILA TCR ⁇ and ⁇ chains as contained in the pEX922-ILA vector.
• Figure 4a and 4b detail the amino acid sequences of the ILA TCR ⁇ and ⁇ chains as encoded for by the DNA of figures 3a and 3b respectively.
• V ⁇ CDR3 mutagenesis For the V ⁇ CDR3 mutagenesis the following PCR products were generated using the pEX922-ILA vector as template and the primers described below. The same PCR conditions were used as detailed above,
• PCRl 5 As above substituting the primers 299 and 250.
• PCRl 8 As above substituting the primers 299 and 259.
• PCR22 As above substituting the primers 252 and 22.
• PCR26 As above substituting the primers 265 and 22.
• PCR products described above are subsequently used in the overlap PCRs described below to generate full length CDRl, 2 or 3 mutated V alpha or V beta chains which can then be cut and cloned into the phage display vector pEX922.
• These PCR reactions were carried out as follows. (See Figure 5 for a schematic diagram representing the PCR products generated during the production of the ILA TCR-derived library)
• PCR A 39.5 ⁇ l water, 5 ⁇ l 1Ox Pfu buffer, l ⁇ l 299 primer lOpM/ ⁇ l, l ⁇ l 22 primer lOpM/ ⁇ l, l ⁇ l 1OmM dNTPs, IOng PCR14, IOng PCR21 (see map above) IOng and 0.33 pM of oligo 248 and 0.5 ⁇ l of Pfu polymerase.
• the PCR was cycled as follows 30 cycles of 95 degrees 15 sec, 55 degrees 15 sec and 72 degrees for 2 min. PCR product was run on a 1.6% TBE agarose gel, a band of the correct size excised and purified using the Qiagen gel extraction kit
• PCR F As above (PCR A) but substitute mutagenic oligo with 263 0.33 pM and the PCR templates with IOng PCRl 9, IOng PCR26 (see map above) IOng.
• PCR G As above (PCR A) but substitute mutagenic oligo with 264 0.33 pM and the PCR templates with IOng PCRl 9, IOng PCR26 (see map above) IOng.
• PCR A but substitute mutagenic oligo with 264 0.33 pM and the PCR templates with IOng PCRl 9, IOng PCR26 (see map above) IOng.
• V ⁇ CDR2 mutagenesis the following overlap PCRs were carried out.
• PCR H As above (PCR A) but substitute mutagenic oligo with 267 0.33 pM and the PCR templates with IOng PCR20, IOng PCR27 (see map above) IOng.
• PCR product was run on a 1.6% TBE agarose gel, a band of the correct size excised and purified using the Qiagen gel extraction kit PCR K: As above (PCR J) but substitute mutagenic oligo with 274 0.33 pM.
• PCR O As above (PCR J) but substitute mutagenic oligo with 279 0.33 pM and the PCR templates with IOng PCRl, IOng PCR8 (see map above) IOng.
• PCR S As above (PCR A) but substitute mutagenic oligo with 291 0.33 pM and the PCR templates with IOng PCR5, IOng PCR12 (see map above) IOng.
• PCR U As above (PCR J) but substitute mutagenic oligo with 2970.33 pM and the PCR templates with IOng PCR28, IOngPCR29 (see map above) IOng.
• ILA CDRl, 2 and 3 alpha mutagenic phage display library pooled ⁇ -chain PCR fragments (J,K,L,M,N,O,P,Q,R,S,T and U) were digested with Nco I and Notl and re-purified using a Qiagen kit and the recipient vector was prepared by digesting pEX922 with Nco I and Notl followed by gel purification using a Qiagen kit.
• ILA CDRl, 2 and 3 beta mutagenic phage display library pooled ⁇ -chain PCR fragments (A,B ; C,D,E,F,G,H and I) were digested with Nco /and Notl and re-purified using a Qiagen kit and the recipient vector was prepared by digesting pEX922 with Nco I and Notl followed by gel purification using a Qiagen kit.
• V alpha and V beta libraries were ligated and transformed separately.
• the cells were re-suspended immediately with 960 ⁇ l of SOC medium at 37 0 C and plated on a 244mm x244mm tissue culture plate containing YTE (15g Bacto-Agar, 8g NaCl, 1Og Tryptone, 5g Yeast Extract in 1 litre) supplemented with lOO ⁇ g/ml ampicillin and 2% glucose. The plate was incubated at 3O 0 C over night. The cells were then scraped from the plates with 5 ml of DYT (16g Trytone, 1Og Yeast extract and 5gNaCl in 1 litre, autoclaved at 125 0 C for 15 minutes) supplemented with 15% glycerol.
• DYT 16g Trytone, 1Og Yeast extract and 5gNaCl in 1 litre, autoclaved at 125 0 C for 15 minutes
• DYTag containing 100 ⁇ g/ml of ampicillin and 2% glucose
• the culture was grown until OD(600nm) reached 0.5.
• 100 ml of the culture was infected with helper phage (M13 K07 (Invitrogen), or HYPER PHAGE (Progen Biotechnik, GmbH 69123 Heidelberg), and incubated at 37°C water bath for 30 minutes.
• the medium was replaced with 100 ml of DYTak (DYT containing 100 ⁇ g/ml ampicillin and 25 ⁇ g/ml of kanamycin).
• the culture was then incubated with shaking at 300 rpm and 25 0 C for 20 to 36 hours.
• Example 2 Isolation of TCRs that bind to (Telomerase)RL VDDFLL V -HLA-A *0201, (SurvivinJELTLGEFLKL -HLA-AW201, or (Survivin) LTLGEFLKL -HLA- A*0201 complex from a TCR phage display library derived from the ILA TCR
• A*0201 complex from the ILA Telomerase phage display library described above was carried out as follows. The initial panning was carried utilising the selection of phage particles (prepared as described above) displaying mutant TCRs derived from the ILA TCR.
• Streptavidin-coated paramagnetic beads were pre-washed according to manufacturer's protocols. Phage particles, displaying mutated ILA TCR at a concentration of 10 to 10 cfu in 3% powdered milk- PBS 5 were pre-mixed with either biotinylated (Telomerase)RLVDDFLLV -HLA-A*0201, (Survivin)ELTLGEFLKL -HLA-A*0201 , or (Survivin)LTLGEFLKL -HLA-
• the mixture of ILA TCR-displaying phage particles and either (Telomerase)RLVDDFLLV -HLA-A*0201, (Survivin)ELTLGEFLKL -HLA-A*0201, or (Survivin)LTLGEFLKL -HLA-A* 0201 complex was incubated for one hour at room temperature with gentle rotation, and the TCR-displaying phage particles bound to (Telomerase)RLVDDFLLV -HLA-A*0201, (Survivin)ELTLGEFLKL -HLA- A*0201, or (Survivin)LTLGEFLKL -HLA-A*0201complex.
• Phage biotynlated HLA complexes were rescued for 5 minutes with 100 ml of streptavidin-coated (Roche) magnetic beads which had been blocked with de-biotynalyted 3% Milk powder PBS, After capture of the phage particles, the beads were washed a total of six times (three times in PBS-0.1 % tween20 and three times in PBS) using a Dynal magnetic particle concentrator.
• the culture was infected with 100 ⁇ l of 2TYAG containing 5 x 10 9 pfu helper phages, and incubated at 37 0 C for 60 minutes. 5 ⁇ l of the infected culture was added to 200 ⁇ l of 2TY AK ("TYAG + 100 ⁇ g/ml Ampicillin and 50 ⁇ g/ml Kanomycin) The plates were incubated at 25°C for 20 to 36 hours with shaking at 300 rpm. The cells were precipitated by centrifugation at 3000g for 10 minutes at 4 0 C. Supernatants were used to screen for high affinity TCR mutants by phage ELISA.
• Phage clones which bound to either (Telomerase)RL VDDFLLV -HLA-A* 0201, (Survivin)ELTLGEFLKL -HLA-A*0201, or (Survivin)LTLGEFLKL -HLA- A* 0201 complex were found during the ELISA screening as determined by their strong ELISA signals (O.D. 600 0.3-1) relative to control wells (O.D. 600 0.05).
• the wild- type ILA TCR from which the library was derived was not capable of binding to any of these pMHC complexes to an extent that was detectable by the above ELISA assay. This therefore ' demonstrates that TCRs with specificities differing from those of the parental ILA TCR used to construct the mutated phage display library had been isolated from said library.
• Such an approach should also allow one to isolate TCRs from a phage display library derived from a TCR with a particular pMHC specificity that are capable of binding to peptides presented by a different MHC.
• the general approach involves phagemid DNA encoding the identified TCR being isolated from the relevant E.coli cells using a Mini-Prep kit (Quiagen, UK). PCR amplification can be carried out using the phagemid DNA as template and a set of primers designed to amplify the soluble TCR ⁇ and ⁇ chain DNA sequences encoded by the phagemid. The full range of primers required can be deduced by reference to the TCR ⁇ and TCR ⁇ Vand C sequences.
• the PCR product is then digested with appropriate restriction enzymes and cloned into an E. coli expression vector with corresponding insertion sites.
• the amplified TCR ⁇ and ⁇ chain DNA sequences (which include, as described above, codons encoding the cysteines required to form the introduced constant domain interchain disulfide bond) are then used to produce a soluble TCR as described in WO 03/020763. Briefly, the two chains are expressed as inclusion bodies in separate E.coli cultures. The inclusion bodies are then isolated, de-natured and re-folded together in vitro.
• a surface plasmon resonance biosensor (BIAcore 3000TM ) was used to analyse the binding of an 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.
• Biotinylated class I pMHC 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).
• MHC-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-microglobulin 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 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA, and was refolded at a concentration of 30 mg/litre heavy chain, 30 mg/litre ⁇ 2m into 0.4 M L-Arginine-HCl, 100 mM Tris pH 8.1, 3.7 mM cystamine, mM cysteamine, 4 mg/ml of the peptide required to be loaded by the MHC, by addition of a single pulse of denatured protein into refold buffer at ⁇ 5 0 C. Refolding was allowed to reach completion at 4 0 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 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-A2 -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 8.1, 5 mM NaCl using a Pharmacia fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgC12, and 5 ⁇ g/ml BirA enzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem. 266: 9- 15). The mixture was then allowed to incubate at room temperature overnight.
• Biotinylated pMHC molecules were purified using gel filtration chromatography. A Pharmacia Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min. Biotinylated pMHC 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 determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pMHC molecules were stored frozen at -2O 0 C. Strep tavidin was immobilised by standard amine coupling methods.
• PerBio Coomassie-binding assay
• 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.
• K D 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 ILAKFLHWL-HLA-A* 0201 complex, the second coated with -300 RU of nonspecific 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.
• Example 4 -Isolation of an HLA-A24- VYGFVRACL binding TCR from an A6 TCR- derived phage display library.
• a second phage displayed TCR library was created using the procedures of Examples 1 and 2 of the inventor's co-pending application WO 2004/044004.
• the A6 TCR from which this library was derived is specific for HLA- A2- LLFGYPVYV.
• This library was panned against HLA- A24 loaded with the Telomerase-derived VYGFVRACL peptide busing the phage ELISA method described above.
• the DNA encoding the displayed TCR was then isolated from the binding phage particles, and used to produce a soluble dTCR was described above.
• the soluble TCR containing an introduced disulfide interchain bond was shown to bind to HLA- A24- VYGFVRACL with an affinity (Kd) of 1.6 ⁇ M.
• Figure 6 provides the Biacore response curve generated for the interaction of this soluble TCR.

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