T cell receptors

Abstract

The present invention relates to T cell receptors (TCRs) that bind the HLA-A*02 restricted peptide SLLQHLIGL (SEQ ID NO: 1) derived from the germline cancer antigen PRAME. Said TCRs may comprise non-natural mutations within the alpha and/or beta variable domains relative to a native PRAME TCR. The TCRs of the invention are particularly suitable for use as novel immunotherapeutic reagents for the treatment of malignant disease.

Claims

1. A heterodimeric TCR-anti-CD3 antibody fusion molecule, wherein the alpha chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 25 and the beta chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 26; the alpha chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 27 and the beta chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 28; or the alpha chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 29 and the beta chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 30.

2. The heterodimeric TCR-anti-CD3 antibody fusion molecule of claim 1, wherein the alpha chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 25 and the beta chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 26.

3. The heterodimeric TCR-anti-CD3 antibody fusion molecule of claim 1, wherein the alpha chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 27 and the beta chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 28.

4. The heterodimeric TCR-anti-CD3 antibody fusion molecule of claim 1, wherein the alpha chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 29 and the beta chain of the fusion molecule has the amino acid sequence set forth in SEQ ID NO: 30.

5. A pharmaceutical composition comprising the heterodimeric TCR-anti-CD3 fusion molecule of claim 1, together with one or more pharmaceutically acceptable carriers or excipients.

6. A method of treating a human subject having cancer, comprising: administering to the subject a therapeutically effective dose of the pharmaceutical composition of claim 5.

7. The method of claim 6, wherein the cancer expresses the antigen PRAME.

8. The method of claim 7, wherein the cancer is selected from melanoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer, and head and neck cancer.

9. The method of claim 8, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

10. The method of claim 8, wherein the breast cancer is triple negative breast cancer.

11. The method of claim 6, wherein the pharmaceutical composition is administered by injection.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1—provides the amino acid sequence of the extracellular regions of the scaffold PRAME TCR alpha and beta chain.

(2) FIG. 2—provides the amino acid sequence of the extracellular regions of a soluble version of the scaffold PRAME TCR alpha and beta chain.

(3) FIG. 3—provides example amino acid sequences of mutated PRAME TCR alpha chain variable regions.

(4) FIG. 4—provides example amino acid sequences of mutated PRAME TCR beta chain variable regions.

(5) FIG. 5—provides amino acid sequences of ImmTAC molecules (TCR-anti-CD3 fusions) comprising certain mutated PRAME TCR variable domains as set out in FIGS. 3 and 4.

(6) FIG. 6—provides cellular data demonstrating potency and specificity of ImmTAC molecules of FIG. 5 comprising the mutated PRAME TCR variable domains as set out in FIGS. 3 and 4.

(7) FIG. 7 (panels a and b)—provide cellular data demonstrating specificity of ImmTAC molecules of FIG. 5, comprising the mutated PRAME TCR variable domains as set out in FIGS. 3 and 4.

(8) FIG. 8—provides cellular data demonstrating killing of PRAME positive melanoma cancer cells by ImmTAC molecules of FIG. 5, comprising the mutated PRAME TCR variable domains as set out in FIGS. 3 and 4.

(9) FIG. 9—provides cellular data demonstrating killing of PRAME positive lung cancer cells by ImmTAC molecules of FIG. 5, comprising the mutated PRAME TCR variable domains as set out in FIGS. 3 and 4.

(10) The invention is further described in the following non-limiting examples.

EXAMPLES

Example 1—Expression, Refolding and Purification of Soluble TCRs

(11) Method 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 harbour laboratory press). The expression plasmids were transformed separately into E. coli strain Rosetta (BL21 μLysS), or T7 Express, 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 00280. 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.

(12) 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, 800 or 1000 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.

(13) Purification of soluble TCRs was initiated by applying the dialysed refold onto a POROS® 50HQ anion exchange column and eluting bound protein with a gradient of 0-500 mM NaCl in 20 mM Tris pH 8.1 over 6 column volumes using an Akta® Pure (GE Healthcare). Peak TCR fractions were identified by SDS PAGE before being pooled and concentrated. The concentrated sample was then applied to a Superdex® 200 Increase 10/300 GL 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 Molecules)

(14) Method

(15) 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 MES (pH6.5), and applied to a POROS® 50HS cation exchange column. Bound protein was eluted with a gradient of 0-500 mM NaCl in 20 mM MES. 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

(16) 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.

(17) BIAcore Method

(18) 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.

(19) 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 between 50-100 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.

(20) Octet Method

(21) 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+cone), where “response” is the equilibrium binding in nm at each TCR concentration (cone) and Rmax is the maximum binding response at pHLA saturation.

(22) 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 the Native TCR

(23) A soluble native 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 the PRAME peptide SLLQHLIGL (SEQ ID NO: 1) and immobilised onto a BIAcore sensor chip.

(24) Results

(25) Binding was determined at various concentrations and the K.sub.D value for the interaction was determined to be 141 μM. Cross reactivity (specificity) was assessed against a panel of 14 irrelevant peptide HLA-A*02 complexes using the equilibrium BIAcore method of Example 3. The 14 irrelevant pHLAs were divided into three groups and loaded onto one of three flow cells, to give approximately 1000 RU of each pHLA per flow cell. 30 μL of soluble wild type TCR was injected at concentrations of 130 and 488 μM over all flow cells at a rate of 20 μL/min. No significant binding was detected at either concentration indicting that the native TCR is specific for the SLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1).

(26) These data indicate that this native TCR has characteristics that are suitable for use as a starting sequence for engineering high affinity therapeutic TCRs.

Example 5—Binding Characterisation of Certain Mutated TCRs of the Invention

(27) The mutated TCR alpha and beta variable domain amino acid sequences, provided in FIGS. 3 and 4 respectively (SEQ ID NOs: 6-24), were used to prepare ImmTAC molecules. Note that inclusion of a glycine residue at the start of the alpha chain (−1 position relative to the numbering of SEQ ID NO: 2) was found to improve cleavage efficiency of the N terminal methionine during production in E. coli. Inefficient cleavage may be detrimental for a therapeutic since it may result in a heterogeneous protein product and or the presence of the initiation methionine may be immunogenic in humans. Full amino acid sequences of ImmTAC molecules comprising the following alpha and beta chains are provided in FIG. 5 a28b50—ImmTAC1 a79674—ImmTAC2 a79b46—ImmTAC3

(28) The molecules were prepared as described in Example 2 and binding to SLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1) was determined according to Example 3.

(29) Results

(30) The data presented in the table below show that ImmTAC molecules comprising the indicated TCR variable domain sequences recognised SLLQHLIGL-HLA-A*02 complex (“SLLQHLIGL” disclosed as SEQ ID NO: 1) with a particularly suitable affinity and/or half-life.

(31) TABLE-US-00012 α chain β chain k.sub.D t.sub.1/2 a28 (SEQ ID NO: 6) b50 (SEQ ID NO: 9) 391 pM 1.8 h a28 (SEQ ID NO: 6) b60 (SEQ ID NO: 19) 261 pM 2.8 h a28 (SEQ ID NO: 6) b74 (SEQ ID NO: 17) 182 pM 3.7 h a28 (SEQ ID NO: 6) b75 (SEQ ID NO: 20) 214 pM 5.1 h a28 (SEQ ID NO: 6) b57 (SEQ ID NO: 10) 83 pM 8.3 h a28 (SEQ ID NO: 6) b58 (SEQ ID NO: 21) 79 pM 8.9 h a79 (SEQ ID NO: 7) b46 (SEQ ID NO: 11) 31.8 pM 29.2 h a109 (SEQ ID NO: 8) b46 (SEQ ID NO: 11) 170 pM 7.31 h a79 (SEQ ID NO: 7) b63 (SEQ ID NO: 22) 79 pM 10.8 h a79 (SEQ ID NO: 7) b64 (SEQ ID NO: 12) 138 pM 6.38 h a79 (SEQ ID NO: 7) b66 (SEQ ID NO: 23) 89 pM 9.16 h a79 (SEQ ID NO: 7) b67 (SEQ ID NO: 13) 47 pM 12.69 h a79 (SEQ ID NO: 7) b69 (SEQ ID NO: 14) 52 pM 20.41 h a79 (SEQ ID NO: 7) b71 (SEQ ID NO: 15) 87 pM 14.89 h a79 (SEQ ID NO: 7) b58 (SEQ ID NO: 21) 23.1 pM 28.7 h a79 (SEQ ID NO: 7) b73 (SEQ ID NO: 16) 132 pM 4.6 h a79 (SEQ ID NO: 7) b74 (SEQ ID NO: 17) 53.3 pM 12.5 h a79 (SEQ ID NO: 7) b75 (SEQ ID NO: 20) 57.7 pM 16.9 h a79 (SEQ ID NO: 7) b76 (SEQ ID NO: 24) 11.8 pM 58.3 h a79 (SEQ ID NO: 7) b77 (SEQ ID NO: 18) 77.9 pM 8.6 h

Example 6—Potency and Specificity Characterisation of Certain Mutated TCRs of the Invention

(32) ImmTAC molecules comprising the same TCR variable domain sequences as set out in Example 5 were assessed for their ability to mediate potent and specific redirection of CD3.sup.+ cells against PRAME positive cancer cells. Interferon-γ (IFN-γ) release was used as a read out for T cell activation. Full amino acid sequences of ImmTAC molecules comprising the following alpha and beta chains are provided in FIG. 5 a28b50—ImmTAC1 a79674—ImmTAC2 a79b46—ImmTAC3

(33) Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturers instructions. Briefly, 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. Peripheral blood mononuclear cells (PBMC), isolated from fresh donor blood, were used as effector cells and plated at 50,000 cells per well in a volume of 50 μl (the exact number of cells used for each experiment is donor dependent and may be adjusted to produce a response within a suitable range for the assay). ImmTAC molecules were titrated to give final concentrations of 10 nM, 1 nM, 0.1 nM, 0.01 nM and 0.001 nM, 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 instructions. 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, 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% Tween-20, 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. No more than 15 mins 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 a CTL analyser with Immunospot software (Cellular Technology Limited).

(35) In this example, the following cancer cells lines were used as target cells:

(36) TABLE-US-00013 Mel624 (melanoma) PRAME + ve HLA-A*02 + ve Granta519 (hemo-lymphocytic) PRAME − ve HLA-A*02 + ve SW620 (colon carcinoma) PRAME − ve HLA-A*02 + ve HT144 (melanoma) PRAME + ve HLA-A*02 − ve
Results

(37) Each of the ImmTAC molecules, comprising the alpha and beta variable domains indicated in the table below, demonstrated potent activation of redirected T cells in the presence of antigen positive Mel624 cells. In each case, EC50 values were calculated from the data and are shown in the table below. In addition, each ImmTAC molecule demonstrated minimal or no recognition of two antigen negative, HLA-A*02 positive cells, at a concentration of up to 1 nM. The ImmTAC molecules also demonstrated no recognition of PRAME positive cells that are HLA-A*02 negative (data not shown). FIG. 6 shows representative data from four of the ImmTAC molecules listed in the table below.

(38) TABLE-US-00014 α chain β chain EC.sub.50 (SEQ ID NO) (SEQ ID NO) (mel624) a28 (SEQ ID NO: 6) b50 (SEQ ID NO: 9) 34.8 pM a28 (SEQ ID NO: 6) b60 (SEQ ID NO: 19) 31.7 pM a28 (SEQ ID NO: 6) b74 (SEQ ID NO: 17) 24.3 pM a28 (SEQ ID NO: 6) b75 (SEQ ID NO: 20) 13.9 pM a28 (SEQ ID NO: 6) b57 (SEQ ID NO: 10) 13.4 pM a28 (SEQ ID NO: 6) b58 (SEQ ID NO: 21) 12 pM a79 (SEQ ID NO: 7) b46 (SEQ ID NO: 11) 18.6 pM a109 (SEQ ID NO: 8) b46 (SEQ ID NO: 11) 60.1 pM a79 (SEQ ID NO: 7) b63 (SEQ ID NO: 22) 22.9 pM a79 (SEQ ID NO: 7) b64 (SEQ ID NO: 12) 27.5 pM a79 (SEQ ID NO: 7) b66 (SEQ ID NO: 23) 16.7 pM a79 (SEQ ID NO: 7) b67 (SEQ ID NO: 13) 26.3 pM a79 (SEQ ID NO: 7) b69 (SEQ ID NO: 14) 39.8 pM a79 (SEQ ID NO: 7) b71 (SEQ ID NO: 15) 31.8 pM a79 (SEQ ID NO: 7) b58 (SEQ ID NO: 21) 10.6 pM a79 (SEQ ID NO: 7) b73 (SEQ ID NO: 16) 23.1 pM a79 (SEQ ID NO: 7) b74 (SEQ ID NO: 17) 9.55 pM a79 (SEQ ID NO: 7) b75 (SEQ ID NO: 20) 23.6 pM a79 (SEQ ID NO: 7) b76 (SEQ ID NO: 24) 17.2 pM a79 (SEQ ID NO: 7) b77 (SEQ ID NO: 18) 13.8 pM

(39) These data demonstrate that ImmTAC molecules comprising mutated TCR variable domain sequences of the invention can mediate potent and specific T cell redirection against PRAME positive, HLA-A*02 positive, cancer cells, in a concentration range suitable for therapeutic use.

Example 7—Further Specificity Characterisation of Certain Mutated TCRs of the Invention

(40) To further demonstrate the specificity of ImmTAC molecules comprising the mutated TCR sequences, further testing was carried out using the same ELISPOT methodology as described in Example 6, with a panel of normal cells derived from healthy human tissues as target cells. Normal tissues included cardiovascular, renal, skeletal muscle, pulmonary, vasculature, hepatic and brain. In each case antigen positive Mel624 cancer cells were used as a positive control.

(41) The data presented in this example includes ImmTAC molecules comprising the following TCR alpha and beta chains a28b50 a79674 a79b46 a79677

(42) The full amino acid sequences of ImmTAC molecules comprising a28b50, a79674 and a79b46 are provided in FIG. 5 (ImmTAC 1-3 respectively)

(43) Results

(44) The data presented in FIG. 7 (panel a) demonstrate that ImmTAC molecules comprising mutated alpha and beta chain a28b50 and a79b46 show minimal reactivity against a panel of 8 normal cells relative to antigen positive cancer cells at a concentration up to 1 nM. Likewise, the data in FIG. 7 (panel b) demonstrate that ImmTAC molecules comprising a28b57 and a79b46 show minimal reactivity against a panel of 4 normal cells relative to antigen positive cancer cells at a concentration up to 1 nM.

Example 8—Cancer Cell Killing Mediated by Certain Mutated TCRs of the Invention

(45) The ability of ImmTAC molecules comprising the mutated TCR sequences 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.

(46) Method

(47) 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 (Mel624 (PRAME+ve HLA-A*02+ve) or NCI-H1755) were plated at 10,000 cells per well and incubated overnight to allow them to adhere. ImmTAC molecules were prepared at various concentrations and 25 μl of each was added to the relevant well such that final concentrations were between 1 μM and 100 μM. Effector cells were used at an effector target cell ratio of 10:1 (100,000 cells per well). A control sample without ImmTAC was also prepared along with samples containing either effector cells alone, or target cells alone. NucView assay reagent was made up at 30 μM and 25 μl added to every well and the final volume brought to 150 μl (giving 5 μM final cone). 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 apoptotic cells per mm.sup.2. Assays were performed in triplicate.

(48) The data presented in this example includes ImmTAC molecules comprising the following TCR alpha and beta chains a28b50 a79674 a79b46

(49) The full amino acid sequences of ImmTAC molecules comprising a28b50, a79674 and a79b46 are provided in FIG. 5 (ImmTAC 1, 2 and 3 respectively).

(50) Results

(51) The data presented in FIGS. 8 and 9 shows real-time killing of antigen positive cancer cells (Melanoma cell lines Mel624 in FIG. 8 and Lung cancer cell line NCI-H1755 in FIG. 9) in the presence of ImmTAC molecules comprising the mutated TCR sequences, at a concentration of 100 pM or lower. No killing was observed in the absence of ImmTAC molecules.