T cell receptors specific for the NY-ESO-1 tumor antigen-HLA-A*02 complex

Abstract

The present invention relates to T cell receptors (TCRs) which bind the HLA-A*02 restricted peptide SLLMWITQC derived from the cancer antigen NY-ESO-1. Said TCRs may comprise mutations within the alpha and/or beta variable domains relative to a native NY-ESO-1 TCR. The TCRs of the invention are particularly suitable for use as novel immunotherapeutic reagents for the treatment of malignant disease.

Claims

1. A soluble T cell receptor (TCR) having the property of binding to SLLMWITQC (SEQ ID NO: 1) HLA-A*02 complex and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the alpha chain variable domain comprises an amino acid sequence that has at least 90% identity to SEQ ID NO: 12, and the beta chain variable domain comprises an amino acid sequence that has at least 90% identity to SEQ ID NO: 15, and the alpha chain variable domain sequence of amino acids residues 27-32 (CDR1), 50-57 (CDR2) and 91-107 (CDR3) and the beta chain variable domain sequence of amino acid residues 27-31 (CDR1), 49-55 (CDR2) and 93-106 (CDR3) are selected from one of the following: a) alpha chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 41, 54 and 43, respectively, and beta chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 59, 60 and 61, respectively; b) alpha chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 41, 54 and 43, respectively, and beta chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 59, 63 and 46, respectively; c) alpha chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 41, 54 and 57, respectively, and beta chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 59, 60 and 61, respectively; or d) alpha chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 41, 54 and 57, respectively, and beta chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 59, 63 and 46, respectively.

2. The TCR of claim 1, wherein the alpha chain variable domain and the beta chain variable domain are selected from one of the following: a) an alpha chain variable domain sequence provided in SEQ ID NO: 7 and a beta chain variable domain sequence provided in SEQ ID NO: 15; b) an alpha chain variable domain sequence provided in SEQ ID NO: 7 and a beta chain variable domain sequence provided in SEQ ID NO: 18; c) an alpha chain variable domain sequence provided in SEQ ID NO: 12 and a beta chain variable domain sequence provided in SEQ ID NO: 15; or d) an alpha chain variable domain sequence provided in SEQ ID NO: 12 and a beta chain variable domain sequence provided in SEQ ID NO: 18.

3. The TCR of claim 1, which is an alpha-beta heterodimer, wherein the TCR further comprises an alpha chain TRAC constant domain sequence and a beta chain TRBC1 or TRBC2 constant domain sequence, wherein, optionally, the alpha and beta chain constant domain sequences are modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2, or, wherein, optionally, the alpha and/or beta chain constant domain sequence(s) are modified by substitution of cysteine residues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the said cysteines forming a non-native disulfide bond between the alpha and beta constant domains of the TCR.

4. The TCR of claim 1, which is in single chain format of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, wherein Vα and Vβ are TCR α and β variable regions respectively, Cα and Cβ are TCR α and β constant regions respectively, and L is a linker sequence.

5. The TCR of claim 1, associated with a detectable label, a therapeutic agent or a pharmacokinetics modifying moiety.

6. A TCR anti-CD3 fusion comprising the TCR of claim 1, and an anti-CD3 antibody covalently linked to the C- or N-terminus of the alpha or beta chain of the TCR.

7. The TCR anti-CD3 fusion of claim 6, comprising an alpha chain variable domain selected from SEQ ID NO: 7 and 12 and comprising a beta chain variable domain selected from SEQ ID NO: 15 and 18, wherein, optionally the beta chain is linked to the anti-CD3 antibody sequence via a linker sequence, wherein, optionally, the linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 24), GGGSG (SEQ ID NO: 25), GGSGG (SEQ ID NO: 26), GSGGG (SEQ ID NO: 27), GSGGGP (SEQ ID NO: 28), GGEPS (SEQ ID NO: 29), GGEGGGP (SEQ ID NO: 30), and GGEGGGSEGGGS (SEQ ID NO: 31).

8. The TCR anti-CD3 fusion of claim 6, comprising an alpha chain variable domain consisting of an amino acid sequence of SEQ ID NO: 12 and a beta chain variable domain consisting of an amino acid sequence of SEQ ID NO: 15 and wherein the beta chain is linked to an anti-CD3 antibody via a linker sequence.

9. The TCR anti-CD3 fusion of claim 6, comprising an alpha chain variable domain consisting of an amino acid sequence of SEQ ID NO: 12 and a beta chain variable domain consisting of an amino acid sequence of SEQ ID NO: 15 and wherein the alpha chain is linked to an anti-CD3 antibody via a linker sequence.

10. A TCR anti-CD3 fusion according to claim 8, wherein the alpha chain variable domain consists of the amino acid sequence of SEQ ID NO: 12 and the beta chain variable domain consists of the amino acid sequence of SEQ ID NO: 15 and wherein the beta chain is linked to an anti-CD3 antibody via the linker sequence of SEQ ID NO: 24.

11. A pharmaceutical composition comprising the soluble TCR of claim 1, together with one or more pharmaceutically acceptable carriers or excipients.

12. The TCR of claim 1, wherein the TCR binds to the SLLMWITQC (SEQ ID NO: 1) HLA-A*02 complex with an affinity greater than 50 μM.

13. The TCR anti-CD3 fusion of claim 6, comprising an alpha chain consisting of SEQ ID NO: 37 and a beta chain consisting of SEQ ID NO: 38.

14. A TCR anti-CD3 fusion having the property of binding to SLLMWITQC (SEQ ID NO: 1) HLA-A*02 complex, wherein said TCR anti-CD3 fusion comprises an alpha chain amino acid sequence and a beta chain amino acid sequence pairing selected from the group consisting of: a) an alpha chain amino acid sequence at least 90% identical to SEQ ID NO: 32 and a beta chain amino acid sequence at least 90% identical to SEQ ID NO: 33; b) an alpha chain amino acid sequence at least 90% identical to SEQ ID NO: 35 and a beta chain amino acid sequence at least 90% identical to SEQ ID NO: 36; c) an alpha chain amino acid sequence at least 90% identical to SEQ ID NO: 37 and a beta chain amino acid sequence at least 90% identical to SEQ ID NO: 38; or d) an alpha chain amino acid sequence at least 90% identical to SEQ ID NO: 39 and a beta chain amino acid sequence at least 90% identical to SEQ ID NO: 40; and, wherein the alpha chain variable domain sequence of amino acids residues 27-32 (CDR1), 50-57 (CDR2) and 91-107 (CDR3) and the beta chain variable domain sequence of amino acid residues 27-31 (CDR1), 49-55 (CDR2) and 93-106 (CDR3) are selected from one of the following: (i) alpha chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 41, 54 and 43, respectively, and beta chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 59, 60 and 61, respectively; (ii) alpha chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 41, 54 and 43, respectively, and beta chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 59, 63 and 46, respectively; (iii) alpha chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 41, 54 and 57, respectively, and beta chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 59, 60 and 61, respectively; or (iv) alpha chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 41, 54 and 57, respectively, and beta chain variable domain CDR1, CDR2 and CDR3 sequences provided in SEQ ID NOs: 59, 63 and 46, respectively.

Description

(1) The invention is described below with reference to the following non-limiting figures and examples, in which:

(2) FIG. 1 provides amino acids sequences of the extracellular regions of native alpha and beta chain variable domains of the invention;

(3) FIG. 2 provides amino acid sequences of the soluble extracellular regions of native alpha and beta chains of the invention;

(4) FIG. 3 provides amino acid sequences of mutated TCR alpha chain variable regions of the invention;

(5) FIG. 4 provides amino acid sequences of mutated TCR beta chain variable regions of the invention;

(6) FIG. 5 provides amino acid sequences of ImmTAC molecules comprising TCR sequences of the invention;

(7) FIG. 6 provides amino acid sequences of ImmTAC molecules comprising TCR sequences of the invention;

(8) FIG. 7 provides amino acid sequences of ImmTAC molecules comprising TCR sequences of the invention;

(9) FIG. 8 provides amino acid sequences of ImmTAC molecules comprising TCR sequences of the invention;

(10) FIG. 9 shows potent and specific binding of ImmTAC molecules of the invention;

(11) FIG. 10 shows further potency testing for 2 ImmTAC molecules of the invention;

(12) FIG. 11 shows further specificity testing for 3 ImmTAC molecules of the invention; and

(13) FIG. 12 shows tumour cell killing by ImmTAC redirected T cells.

EXAMPLES

Example 1—Expression, Refolding and Purification of Soluble TCRs

(14) DNA sequences encoding the alpha and beta extracellular regions of soluble TCRs of the invention were cloned separately into pGMT7-based expression plasmids using standard methods (as described in Sambrook, et al. Molecular cloning. Vol. 2. (1989) New York: Cold spring harbor laboratory press). The expression plasmids were transformed separately into E. coli strain Rosetta (BL21 pLysS), and single ampicillin-resistant colonies were grown at 37° C. in TYP (+ampicillin 100 μg/ml) medium to an OD.sub.600 of ˜0.6-0.8 before inducing protein expression with 0.5 mM IPTG. Cells were harvested three hours post-induction by centrifugation. Cell pellets were lysed with BugBuster protein extraction reagent (Merck Millipore) according to the manufacturer's instructions. Inclusion body pellets were recovered by centrifugation. Pellets were washed twice in Triton buffer (50 mM Tris-HCl pH 8.1, 0.5% Triton-X100, 100 mM NaCl, 10 mM NaEDTA) and finally resuspended in detergent free buffer (50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM NaEDTA). Inclusion body protein yield was quantified by solubilising with 6 M guanidine-HCl and measuring OD.sub.280. Protein concentration was then calculated using the extinction coefficient. Inclusion body purity was measured by solubilising with 8M Urea and loading ˜2 μg onto 4-20% SDS-PAGE under reducing conditions. Purity was then estimated or calculated using densitometry software (Chemidoc, Biorad). Inclusion bodies were stored at +4° C. for short term storage and at −20° C. or −70° C. for longer term storage.

(15) For soluble TCR refolding, α and β chain-containing inclusion bodies were first mixed and diluted into 10 ml solubilisation/denaturation buffer (6 M Guanidine-hydrochloride, 50 mM Tris HCl pH 8.1, 100 mM NaCl, 10 mM EDTA, 20 mM DTT) followed by incubation for 30 min at 37° C. Refolding was then initiated by further dilution into 1 L of refold buffer (100 mM Tris pH 8.1, 400 mM L-Arginine HCL, 2 mM EDTA, 4 M Urea, 10 mM cysteamine hydrochloride and 2.5 mM cystamine dihydrochloride) and the solution mixed well. The refolded mixture was dialysed against 10 L H.sub.2O for 18-20 hours at 5° C.±3° C. After this time, the dialysis buffer was twice replaced with 10 mM Tris pH 8.1 (10 L) and dialysis continued for another 15 hours. The refold mixture was then filtered through 0.45 μm cellulose filters.

(16) Purification of soluble TCRs was initiated by applying the dialysed refold onto an anion exchange column (POROS® 50HQ) and eluting bound protein with a gradient of 0-500 mM NaCl in 20 mM Tris pH 8.1 over 50 column volumes using an AKTA® purifier (GE Healthcare). Peak TCR fractions were identified by SDS PAGE before being pooled and concentrated. The concentrated sample was then applied to a SUPERDEX® 75HR gel filtration column (GE Healthcare) pre-equilibrated in Dulbecco's PBS buffer. The peak TCR fractions were pooled and concentrated and the final yield of purified material calculated.

Example 2—Expression, Refolding and Purification of ImmTAC Molecules (Soluble TCR-Anti CD3 Fusion Proteins)

(17) ImmTAC preparation was carried out as described in Example 1, except that the TCR beta chain was fused via a linker to an anti-CD3 single chain antibody. In addition a cation exchange step was performed during purification following the anion exchange. In this case the peak fractions from anion exchange were diluted 20 fold in 20 mM IVIES (pH6.5), and applied to a cation exchange column (POROS® 50HS). Bound protein was eluted with a gradient of 0-500 mM NaCl in 20 mM IVIES. Peak ImmTAC fractions were pooled and adjusted to 50 mM Tris pH 8.1, before being concentrated and applied directly to the gel filtration matrix as described in Example 1.

Example 3—Binding Characterisation

(18) Binding analysis of purified soluble TCRs and ImmTAC molecules to the relevant peptide-HLA complex was carried out by surface plasmon resonance, using a BIAcore 3000 or BIAcore T200 instrument, or by biolayer interferometry, using a ForteBio Octet instrument). Biotinylated class I HLA-A*02 molecules were refolded with the peptide of interest and purified using methods known to those in the art (O'Callaghan et al. (1999). Anal Biochem 266(1): 9-15; Garboczi, et al. (1992). Proc Natl Acad Sci USA 89(8): 3429-3433). All measurements were performed at 25° C. in Dulbecco's PBS buffer, supplemented with 0.005% P20.

(19) BIAcore

(20) Biotinylated peptide-HLA monomers were immobilized on to streptavidin-coupled CM-5 sensor chips. Equilibrium binding constants were determined using serial dilutions of soluble TCR/ImmTAC injected at a constant flow rate of 30 μl min.sup.−1 over a flow cell coated with ˜200 response units (RU) of peptide-HLA-A*02 complex. Equilibrium responses were normalised for each TCR concentration by subtracting the bulk buffer response on a control flow cell containing an irrelevant peptide-HLA. The K.sub.D value was obtained by non-linear curve fitting using Prism software and the Langmuir binding isotherm, bound=C*Max/(C+KD), where “bound” is the equilibrium binding in RU at injected TCR concentration C and Max is the maximum binding.

(21) For high affinity interactions, binding parameters were determined by single cycle kinetics analysis. Five different concentrations of soluble TCR/ImmTAC were injected over a flow cell coated with ˜100-200 RU of peptide-HLA complex using a flow rate of 50-60 μl min.sup.−1. Typically, 60-120 μl of soluble TCR/ImmTAC was injected at a top concentration of 100-200 nM, with successive 2 fold dilutions used for the other four injections. The lowest concentration was injected first. To measure the dissociation phase buffer was then injected until ≥10% dissociation occurred, typically after 1-3 hours. Kinetic parameters were calculated using BIAevaluation® software. The dissociation phase was fitted to a single exponential decay equation enabling calculation of half-life. The equilibrium constant K.sub.D was calculated from k.sub.off/k.sub.on.

(22) Octet

(23) Biotinylated peptide-HLA monomers were captured to 1 nm on to (SA) streptavidin biosensors (Pall ForteBio) pre-immobilised with streptavidin. The sensors were blocked with free biotin (2 μM) for 2 minutes. Equilibrium binding constants were determined by immersing the loaded biosensors into soluble TCR/ImmTAC serially diluted in a 96-well or 384-well sample plate. Plate shaking was set to 1000 rpm. For low affinity interactions (μM range) a short association (˜2 minutes) and a short dissociation time (˜2 minutes) was used. Binding curves were processed by double reference subtraction of reference biosensors loaded with irrelevant pHLA using Octet Data Analysis Software (Pall ForteBio). Responses (nm) at equilibrium were used to estimate the K.sub.D value from steady state plots fitted to the equation Response=Rmax*conc/(KD+conc), where “response” is the equilibrium binding in nm at each TCR concentration (conc) and Rmax is the maximum binding response at pHLA saturation.

(24) For high affinity interactions (nM-pM range), kinetic parameters were determined from binding curves at ≥3 TCR/ImmTAC concentrations typically 10 nM, 5 nM and 2.5 nM. The association time was 30 minutes and the dissociation time 1-2 hours. Binding curves were processed by double reference subtraction of reference biosensors loaded with irrelevant pHLA and blocked with biotin. Kinetic parameters k.sub.on and k.sub.off were calculated by global fitting directly to the binding curves using Octet Data Analysis Software (Pall ForteBio). K.sub.D was calculated from k.sub.off/k.sub.on and the dissociation half-life was calculated from t.sub.1/2=0.693/k.sub.off.

Example 4—Binding Characterisation of a Soluble Non-Mutated TCR of the Invention

(25) A soluble wild-type TCR was prepared according to the methods described in Example 1 and binding to pHLA analysed according to Example 3. The amino acid sequences of the alpha and beta chains corresponded to those shown in FIG. 2. Soluble biotinylated HLA-A*02 was prepared with either the wild-type (SLLMWITQC (SEQ ID NO: 1)) or heteroclitic NY-ESO-1 peptide (SLLMWITQV SEQ ID NO: 34), and immobilised onto a BIAcore sensor chip. KD values were determined to be 5.2 μM and 4.3 μM respectively. No significant binding was detected against 15 irrelevant peptide HLA-A*02 complexes.

(26) These data indicate that the TCR binds to the target with a suitable affinity and specificity. These TCR chains therefore provide a useful scaffold for the identification of further TCRs of the invention.

Example 5—Binding Characterisation Soluble High Affinity TCRs and ImmTAC Molecules of the Invention

(27) Soluble mutated TCRs and ImmTAC molecules were prepared as described in Examples 1 and 2, and binding characteristics determined according to Example 3. The TCR alpha and or beta chains contained mutations in at least one CDR region relative to the CDR sequences shown in FIG. 2 (SEQ ID NOs:41 to 46). The amino acid sequences of certain mutated TCR alpha and beta chain variable regions of the invention are provided in FIGS. 4 and 5 respectively. The table below provides binding characteristics for soluble TCRs and/or ImmTAC molecules comprising the indicated alpha and beta variable regions. Binding was measured using the heteroclitic NY-ESO-1 peptide (SLLMWITQV (SEQ ID NO: 34)).

(28) TABLE-US-00027 Alpha Beta Format Method chain SEQ chain SEQ (soluble/ (Biacore/ Binding parameters ID NO: ID NO: ImmTAC) Octet) KD T.sub.1/2 (a12) 6 (b12) 14 ImmTAC Biacore 202 pM 19.2 h (a12) 6 (b5) 13 soluble Biacore 1.5 nM 163.6 min (a12) 6 (b52) 15 ImmTAC Biacore 244 pM 14.2 h 2 (b5) 13 soluble Octet nd 1.3 min 2 (b12) 14 soluble Biacore 3.8 nM 72.33 min 2 (b12) 14 ImmTAC Biacore 1.6 nM 1.92 h (a24I) 8 (b52) 15 ImmTAC Biacore 65 pM 33.54 h (a24I) 8 (b65I) 19 ImmTAC Octet 324 pM 8.71 h (a24I) 8 (b12) 14 ImmTAC Biacore 125 pM 17.9 h (a24I) 8 (b65) 18 ImmTAC Octet 325 pM 6.8 h (a24I) 8 (b56) 16 ImmTAC Octet 253 pM 6.65 h (a24I) 8 (b56I) 17 ImmTAC Octet 249 pM 9.05 h (a24I) 8 (b67) 20 ImmTAC Octet 252 pM 9.49 h (a24I) 8 (b67I) 21 ImmTAC Octet 256 pM 10.08 h (a24I) 8 (b68) 22 ImmTAC Octet 273 pM 7.38 h (a24I) 8 (b68I) 23 ImmTAC Octet 303 pM 6.59 h (a24I) 8 (b5) 13 soluble Biacore 2.7 nM 54.8 min (a24) 7 (b52) 15 ImmTAC.sup.1 Biacore 18.6 pM 78 h (a24) 7 (b65) 18 ImmTAC.sup.2 Biacore 76 pM 24.2 h (a28) 9 (b12) 14 ImmTAC Biacore 120 pM 22.8 h (a28) 9 (b5) 13 soluble Biacore 1.7 nM 155.8 min (a28) 9 (b52) 15 ImmTAC Octet 361 pM 7.13 h (a78I) 10 (b52) 15 ImmTAC Octet 210 pM 7.6 h (a78I) 10 (b67) 20 ImmTAC Octet 173 pM 6.9 h (a78I) 10 (b68) 22 ImmTAC Octet 334 pM 4.7 h (a78I) 10 (b12) 14 ImmTAC Octet 272 pM 7.3 h (a82I) 11 (b65) 18 ImmTAC Biacore 135 pM 17.7 h (a82I) 11 (b52) 15 ImmTAC Biacore 30.8 pM 72 h (a82I) 11 (b67) 20 ImmTAC Octet 226 pM 8.5 h (a82I) 11 (b68) 22 ImmTAC Octet 476 pM 4.1 h (a82I) 11 (b12) 14 ImmTAC Octet 547 pM 5.3 h (a82) 12 (b52) 15 ImmTAC.sup.3 Biacore 40 pM 64.2 h (a82) 12 (b65) 18 ImmTAC.sup.4 Biacore 166 pM 18.7 h (a86) 51 (b71) 52 ImmTAC Biacore 39.8 15.7 h nd = not determined .sup.1Corresponds to ImmTAC1 from Example 6, full alpha and beta chain sequences are provided in FIG. 5 .sup.2Corresponds to ImmTAC2 from Example 6, full alpha and beta chain sequences are provided in FIG. 6 .sup.3Corresponds to ImmTAC3 from Example 6, full alpha and beta chain sequences are provided in FIG. 7 .sup.4Corresponds to ImmTAC4 from Example 6, full alpha and beta chain sequences are provided in FIG. 8

(29) These data demonstrate TCR alpha and beta chain sequences of the invention produce soluble TCRs and ImmTAC molecules with binding characteristics suitable for the development of immunotherapeutic reagents.

Example 6—Potent and Specific T Cell Redirection by ImmTAC Molecules of the Invention

(30) ImmTAC molecules containing alpha and beta variable chain sequences of the invention were tested for their ability to mediate potent and specific redirection of CD3+ T cells by ELISPOT assay, using interferon-γ (IFN-γ) secretion as a read out for T cell activation.

(31) The sequences of the alpha and beta chains of the four ImmTAC molecules tested are provided in FIGS. 5-8.

(32) Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences). Target cells were prepared at a density of 1×10.sup.6/ml in assay medium (RPMI 1640 containing 10% heat inactivated FBS and 1% penicillin-streptomycin-L-glutamine) and plated at 50,000 cells per well in a volume of 50 μl. The following target cell lines were used in this example: NCI-H1755 (NY-ESO-1.sup.+ve; HLA-A*02.sup.+ve) human lung cancer cell line (supplied by ATCC, cat. no: CRL-5892) HAo5 (NY-ESO-1.sup.−ve; HLA-A*02.sup.+ve) human cardiac cells (supplied by Promocell, cat. no: C-12271)

(33) Peripheral blood mononuclear cells (PBMC), isolated from fresh donor blood, were used as effector cells and plated at a concentration of 40,000 cells per well in a volume of 50 μl. Varying concentrations of ImmTAC were used, spanning the anticipated clinically relevant range, and added to the well in a volume of 50 μl.

(34) Plates were prepared according to the manufacturer's instruction. Target cells, effector cells and ImmTAC molecules were added to the relevant wells and made up to a final volume of 200 μl with assay medium. All reactions were performed in triplicate. Control wells were also prepared with the omission of either ImmTAC, effector cells, or target cells. The plates were then incubated overnight (37° C./5% CO.sub.2). The next day the plates were washed three times with wash buffer (1×PBS sachet, containing 0.05% P20, made up in deionised water). Primary detection antibody was then added to each well in a volume of 50 μl. Plates were incubated at room temperature for 2 hours prior to being washed again three times. Secondary detection was performed by adding 50 μl of diluted streptavidin-HRP to each well and incubating at room temperature for 1 hour and the washing step repeated. Plates were then washed twice with 200 μl PBS (pH 7.4). No more than 15 min prior to use, one drop (20 μl) of AEC chromogen was added to each 1 ml of AEC substrate and mixed and 50 μl added to each well. Spot development was monitored regularly and plates were washed in tap water to terminate the development reaction. The plates were then allowed to dry at room temperature for at least 2 hours prior to counting the spots using an CTL analyser with Immunospot software (Cellular Technology Limited).

(35) The data presented in FIG. 9 show that ImmTAC molecules containing alpha and beta variable chain sequences of the invention are able to mediate potent T cell redirection against HLA-A*02.sup.+ve cancer cells expressing target antigen. No activity was observed against HLA-A*02.sup.+ve antigen negative cells. These data indicate that ImmTAC molecules are target cell specific within the clinically relevant concentration range (≤1 nM).

(36) Further assessment of potency was carried out for ImmTAC molecules 3 and 4 to determine EC50 values. ELISpot assays were performed as described above with ImmTAC concentrations ranging from 10.sup.−13 M to 10.sup.−8 M. Data were analysed using Prism 5.0 software (GraphPad) to calculate EC50 values. Values were determined as 95 μM and 121 μM for ImmTAC molecules 3 and 4 respectively (FIG. 10). These data confirm the ability of these ImmTAC molecules to mediate a potent redirected T cell response.

Example 7—Further Specificity Testing of ImmTAC Molecules of the Invention

(37) Further specificity testing of ImmTAC molecules was carried out against a panel of normal cells. ImmTAC molecules 2-4 (FIGS. 6-8) are used in this example. Interferon-γ (IFN-γ) secretion was used as a read out for T cell activation.

(38) Assays were performed using an IFN-γ DuoSet ELISA kit (R&D Systems, Cat No: DY285) and carried out as instructed by the manufacturer. Briefly, IFN-γ was diluted to 10,000 μg/ml and 2 fold dilutions made to produce a standard curve. Target cells were counted and plated at 10,000 cells per well in 10 ul in assay media. ImmTAC molecules were diluted to give final concentrations of 2 nM and 1 nM in 10 ul per well. A control sample without ImmTAC was also prepared. Effector PBMCs were thawed and plated at 10,000 cells/well in 10 ul. The plates were incubated for 48 h before being developed and read.

(39) In this example, NCI-H1755 cells were used as an antigen positive control and HTC-116 cells were used as an antigen negative control. The cell panel included cardiomyocytes (CM12, CM5 and CM10), aortic endothelial cells (HAo5), airway epithelial cells (HCAEC2 and HCAEC5) skeletal muscle myoblasts (HSkMM3) and HPF9 cells. All cell lines were HLA-A*02.sup.+ve. Assays were performed in triplicate.

(40) The data presented in FIG. 11 demonstrate minimal IFN-γ production in the presence of cell lines derived from normal tissues, relative to antigen positive cancer cell lines, within the therapeutically relevant ImmTAC concentration range. These data indicate that ImmTAC molecules of the invention have a high level of specificity and are therefore particularly suitable for therapeutic use.

Example 8—Potent Killing of Tumour Cells by ImmTAC Redirected T Cells

(41) The ability of ImmTAC molecules of the invention to mediate potent redirected T cell killing of antigen positive tumour cells was investigated using the IncuCyte platform (Essen BioScience). This assay allows real time detection by microscopy of the release of Caspase-3/7, a marker for apoptosis.

(42) Assays were performed using the CellPlayer 96-well Caspase-3/7 apoptosis assay kit (Essen BioScience, Cat. No. 4440) and carried out according the manufacturers protocol. Briefly, target cells (NCI-H1755—NYESO.sup.+ve HLA A*02.sup.+ve or HCT-116—NYESO.sup.−ve HLA A*02.sup.+ve) were plated at 5000 cells per well and incubated overnight to allow them to adhere. ImmTAC solutions were prepared at concentrations between 2.16 nM and 8.8 μM. 25 ul of each concentration was added to the relevant well. PBMCs were used as effector cells and plated at 50,000 per well in 50 ul. A control sample without ImmTAC was also prepared. NucView assay reagent was made up at 30 uM and 25 ul added to every well (giving 5 uM final conc). The plate was placed in the IncuCyte instrument and images taken every 2 hours (1 image per well) over 3 days. The number of apoptotic cells in each image was determined and recorded as no. objects per mm.sup.2. Assays were performed in triplicate.

(43) The data presented in FIG. 12 show real-time killing of tumour cells by ImmTAC redirected T cells. Results are presented for ImmTAC3 and ImmTAC4. Both ImmTAC molecules shows T cell redirected killing of antigen positive tumour cells at concentrations as low as 26 μM. ImmTAC3 shows T cell redirected killing below 10 μM. Low level killing of antigen negative cells is only observed at the highest concentration (2.16 nM).

(44) These data confirm that ImmTAC3 and immTAC4 mediate potent redirected T cell killing of antigen positive tumour cells within the therapeutically relevant concentration range.