BINDING MOLECULES SPECIFIC FOR HBV ENVELOPE PROTEIN

Abstract

The present invention relates to specific binding molecules that bind the HLA-A*02 restricted peptide GLSPTVWLSV (SEQ ID NO: 1) derived from HBV envelope protein. The specific binding molecules may comprise alpha and/or beta TCR variable domains and may comprise non-natural mutations within the alpha and/or beta variable domains relative to a native TCR. The specific binding molecules of the invention are particularly suitable for use as novel immunotherapeutic reagents for the treatment of infectious or malignant disease.

Claims

1. A specific binding molecule having the property of binding to GLSPTVWLSV (SEQ ID NO: 1) HLA-A*02 complex and/or GLSPTVWLSA (SEQ ID No: 17) HLA-A*02 complex and comprising a TCR alpha chain variable domain and/or a TCR beta chain variable domain each of which comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 where FR is a framework region and CDR is a complementarity determining region, wherein (a) the alpha chain CDRs have the following sequences: TABLE-US-00014 (SEQ ID NO: 18) CDR1 - DRGSQS (SEQ ID NO: 19) CDR2 - IYSNGD (SEQ ID NO: 20) CDR3 - CAVRNYNTDKLIF optionally with one or more mutations therein, and/or (b) the beta chain CDRs have the following sequences: TABLE-US-00015 (SEQ ID NO: 21) CDR1 - MNHEY (SEQ ID NO: 22) CDR2 - SVGAGI (SEQ ID NO: 23) CDR3 - CASSYATGGTGELFF optionally with one or more mutations therein.

2. The specific binding molecule of claim 1, wherein the alpha chain variable domain framework regions comprise the following sequences: FR1—amino acids 1-26 of SEQ ID NO: 2 FR2—amino acids 33-49 of SEQ ID NO: 2 FR3—amino acids 56-88 of SEQ ID NO: 2 FR4—amino acids 102-111 of SEQ ID NO: 2 or respective sequences having at least 90% identity to said sequences, and/or the beta chain variable domain framework regions comprise the following sequences: FR1—amino acids 1-26 of SEQ ID NO: 3 FR2—amino acids 32-48 of SEQ ID NO: 3 FR3—amino acids 55-90 of SEQ ID NO: 3 FR4—amino acids 106-114 of SEQ ID NO: 3 or respective sequences having at least 90% identity to said sequences.

3. The specific binding molecule of any one of claim 1 or 2, wherein one or more of the mutations in the alpha chain CDRs is selected from (with reference to the numbering of SEQ ID NO: 2): TABLE-US-00016 Wild type Mutation N53 D V91 A N95 K K98 L

4. The specific binding molecule of claim 3, wherein the alpha chain CDRs have one of the following groups of mutations (with reference to the numbering of SEQ ID NO: 2): TABLE-US-00017 1 V91A N95K K98L 2 N53D V91A N95K K98L

5. The specific binding molecule of claim 2 or claim 3, wherein the alpha chain has CDR sequences selected from: TABLE-US-00018 CDR1 CDR2 CDR3 DRGSQS IYSNGD (SEQ ID CAVRNYNTDKLIF (SEQ ID No: 18) No: 19) (SEQ ID No: 20) IYSDGD (SEQ ID CAARNYKTDLLIF No: 24) (SEQ ID No: 25)

6. The specific binding molecule of any preceding claim, wherein one or more of the mutations in the beta chain CDRs is selected from (with reference to the numbering of SEQ ID NO: 3): TABLE-US-00019 Wild type Mutation M27 L N28 S E30 G V50 L E102 V or D or L

7. The specific binding molecule of claim 6, wherein the beta chain CDRs have one of the following groups of mutations (with reference to the numbering of SEQ ID NO: 3): TABLE-US-00020 1 M27L E30G E102V 2 N28S E30G E102D 3 E102L 4 V50L E102D 5 M27L N28S E30G E102D

8. The specific binding molecule of claim 6 or 7, wherein the beta chain has CDR sequences selected from: TABLE-US-00021 CDR1 CDR2 CDR3 LNHGY (SEQ ID SVGAGI (SEQ ID No: CASSYATGGTGVLFF No: 26) 22) (SEQ ID No: 30) MSHGY (SEQ ID SLGAGI (SEQ ID No: CASSYATGGTGDLFF No: 27) 29) (SEQ ID No: 31) MNHEY (SEQ ID CASSYATGGTGLLFF No: 21) (SEQ ID No: 32) LSHGY (SEQ ID CASSYATGGTGELFF No: 28) (SEQ ID No: 23)

9. The specific binding molecule of any preceding claim, which has one of the following combinations of alpha chain and beta chain CDRs: TABLE-US-00022 Alpha chain Beta chain CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 DRGSQS IYSNGD CAARNYKTDLLIF LNHGY SVGAGI CASSYATGGTGVLFF DRGSQS IYSNGD CAARNYKTDLLIF MSHGY SVGAGI CASSYATGGTGDLFF DRGSQS IYSNGD CAARNYKTDLLIF MNHEY SVGAGI CASSYATGGTGLLFF DRGSQS IYSNGD CAARNYKTDLLIF MNHEY SLGAGI CASSYATGGTGDLFF DRGSQS IYSNGD CAARNYKTDLLIF LSHGY SVGAGI CASSYATGGTGDLFF DRGSQS IYSDGD CAARNYKTDLLIF MSHGY SVGAGI CASSYATGGTGDLFF DRGSQS IYSDGD CAARNYKTDLLIF LSHGY SVGAGI CASSYATGGTGDLFF

10. A specific binding molecule as claimed in any preceding claim, wherein the alpha chain variable domain comprises any one of the amino acid sequences of SEQ ID NOs: 4-6 and the beta chain variable domain comprises any one of the amino acid sequences of SEQ ID NOs: 7-11.

11. A specific binding molecule claimed in any preceding claim wherein the alpha chain variable domain and the beta chain variable domain are selected from the amino acid sequences of: TABLE-US-00023 Alpha chain variable domain Beta chain variable domain SEQ ID No: 4 SEQ ID No: 7 SEQ ID No: 4 SEQ ID No: 8 SEQ ID No: 4 SEQ ID No: 9 SEQ ID No: 4 SEQ ID No: 10 SEQ ID No: 4 SEQ ID No: 11 SEQ ID No: 5 SEQ ID No: 8 SEQ ID No: 5 SEQ ID No: 11 SEQ ID No: 6 SEQ ID No: 8

12. A specific binding molecule as claimed in any preceding claim, which is an alpha-beta heterodimer, having an alpha chain TRAC constant domain sequence and a beta chain TRBC1 or TRBC2 constant domain sequence.

13. A specific binding molecule as claimed in claim 12, wherein the alpha and beta chain constant domain sequences are modified by truncation or substitution to delete a native disulphide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

14. A specific binding molecule as claimed in claim 12 or claim 13, wherein 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 disulphide bond between the alpha and beta constant domains of the TCR.

15. A specific binding molecule as claimed in any preceding claim, 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.

16. A specific binding molecule as claimed in any preceding claim associated with a detectable label, a therapeutic agent or a PK modifying moiety.

17. A specific binding molecule as claimed in claim 16, wherein an anti-CD3 antibody is covalently linked to the C- or N-terminus of the alpha or beta chain of the TCR, optionally via a linker sequence.

18. A specific binding molecule as claimed in claim 17, wherein the linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 33), GGGSG (SEQ ID NO: 34), GGSGG (SEQ ID NO: 35), GSGGG (SEQ ID NO: 36), GSGGGP (SEQ ID NO: 37), GGEPS (SEQ ID NO: 38), GGEGGGP (SEQ ID NO: 39), GGEGGGSEGGGS (SEQ ID NO: 40) and GGGSGGGG (SEQ ID NO:41).

19. A specific binding molecule-anti-CD3 fusion molecule wherein the alpha chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 4-6 and the beta chain variable domain comprises an amino acid sequence selected from SEQ ID NO: 7-11, and wherein the anti-CD3 antibody is covalently linked to the N-terminus or C-terminus of the TCR beta chain via a linker sequence selected from SEQ ID NOs: 33-41.

20. A specific binding molecule-anti-CD3 fusion molecule as claimed in claim 19, comprising an alpha chain amino acid sequence selected from SEQ ID NOs: 12, 14, and 15, or an alpha chain amino acid sequence that has at least 90% identity, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity, to the amino acid sequences as set forth in SEQ ID NOs: 12, 14 and 16, and a beta chain amino acid sequence selected from SEQ ID NOs: 13 and 16, or a beta chain amino acid sequence that has at least 90% identity, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity, to the amino acid sequences as set forth in SEQ ID No: 13 and 16.

21. A specific binding molecule-anti CD3 fusion molecule as claimed in claim 20, comprising (a) an alpha chain amino acid sequence corresponding to SEQ ID NO: 12 and beta chain amino acid sequence corresponding to SEQ ID NO: 13; (b) an alpha chain amino acid sequence corresponding to SEQ ID NO: 14 and beta chain amino acid sequence corresponding to SEQ ID NO: 13; or (c) an alpha chain amino acid sequence corresponding to SEQ ID NO: 15 and beta chain amino acid sequence corresponding to SEQ ID NO: 13. (d) an alpha chain amino acid sequence corresponding to SEQ ID NO: 14 and beta chain amino acid sequence corresponding to SEQ ID NO: 16.

22. A nucleic acid encoding a TCR alpha chain and/or a TCR beta chain as claimed in any one of the preceding claims.

23. An expression vector comprising the nucleic acid of claim 22.

24. A cell harbouring (a) an expression vector as claimed in claim 23 encoding TCR alpha and beta variable chains as claimed in any one of claims 1 to 21, in a single open reading frame, or two distinct open reading frames; or (b) a first expression vector which comprises nucleic acid encoding the alpha variable chain of a TCR as claimed in any one of claims 1 to 21, and a second expression vector which comprises nucleic acid encoding the beta variable chain of a TCR as claimed in any one of claims 1 to 21.

25. A non-naturally occurring and/or purified and/or engineered cell, especially a T-cell, presenting a specific binding molecule as claimed in any one of claims 1 to 18.

26. A pharmaceutical composition comprising a specific binding molecule as claimed in any one of claims 1-18, or a specific binding molecule-anti CD3 fusion molecule as claimed in any one of claims 19-21, or a cell as claimed in claim 24 or 25, together with one or more pharmaceutically acceptable carriers or excipients.

27. The specific binding molecule of any one of claims 1 to 18, or specific binding molecule-anti-CD3 fusion molecule of any one of claims 19-21, or nucleic acid of claim 22, pharmaceutical composition of claim 26 or cell of claim 24 or 25, for use in medicine, preferably in a human subject.

28. The specific binding molecule of any one of claims 1 to 18, or specific binding moleculr-anti-CD3 fusion molecule of any one of claims 19-21, or nucleic acid of claim 22, pharmaceutical composition of claim 26 or cell of claim 24 or 25, for use in a method of treating chronic HBV infection or a cancer or tumour resulting from chronic HBV infection, preferably in a human subject.

29. A method of treating a human subject having chronic HBV infection or a cancer or tumour resulting from chronic HBV infection comprising administering to said subject in need thereof a pharmaceutically effective dose of a pharmaceutical composition according to claim 26.

30. A method of producing a specific binding molecule according to any one of claims 1 to 18, or a specific binding molecule-anti-CD3 fusion molecule according to any one of claims 19-21, comprising a) maintaining a cell according to claim 24 or 25 under optimal conditions for expression of the specific binding molecule chains and b) isolating the specific binding molecule chains.

Description

DESCRIPTION OF THE DRAWINGS

[0137] FIG. 1—provides the amino acid sequence of the extracellular regions of a soluble version of the scaffold TCR alpha and beta chain.

[0138] FIG. 2—provides example amino acid sequences of mutated TCR alpha chain variable regions.

[0139] FIG. 3—provides example amino acid sequences of mutated TCR beta chain variable regions.

[0140] FIG. 4—provides amino acid sequences of TCR-antiCD3 fusions comprising certain mutated TCR variable domains as set out in FIGS. 2 and 3.

[0141] FIG. 5—provides comparative binding data for soluble WT TCR against alanine substituted peptides.

[0142] FIG. 6—provides cellular data demonstrating potency and specificity of TCR-antiCD3 fusion molecules comprising the mutated TCR variable domains as set out in FIGS. 2 and 3.

[0143] FIG. 7—provides cellular data demonstrating killing of antigen position cells by TCR-antiCD3 fusion molecules comprising the mutated TCR variable domains as set out in FIGS. 2 and 3.

[0144] FIG. 8—provide cellular data further demonstrating specificity of TCR-antiCD3 fusion molecules comprising the mutated TCR variable domains as set out in FIGS. 2 and 3.

[0145] FIG. 9—provides cellular data demonstrating that TCR-antiCD3 fusion molecules comprising the mutated TCR variable domains as set out in FIGS. 2 and 3 mediate a reduction in percentage of infected cells.

[0146] The invention is further described in the following non-limiting examples.

EXAMPLES

Example 1

Expression, Refolding and Purification of WT TCR in Soluble Format

[0147] Method

[0148] DNA sequences encoding the alpha and beta extracellular regions of a soluble TCR (corresponding to the amino acid sequences in FIG. 1) 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 pLysS). For expression, cells were grown in auto-induction media supplemented with 1% glycerol (+ampicillin 100 μg/ml and 34 μg/ml chloramphenicol) at 230 rpm at 37C for 2 hours before reducing the temperature to 30C overnight. Cells were subsequently harvested by centrifugation. Cell pellets were lysed with BugBuster protein extraction reagent (Merck

[0149] 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 OD2so. Protein concentration was then calculated using the extinction coefficient. Inclusion body purity was measured by solubilising with 8 M 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.

[0150] For soluble TCR refolding, α and β chain-containing inclusion bodies were first mixed and diluted into 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 refold buffer (100 mM Tris pH 8.1, 800 or 400 mM L-Arginine HCL, 2 mM EDTA, 4 M Urea, 6.5 mM cysteamine hydrochloride and 1.9 mM cystamine dihydrochloride) and the solution mixed well. The refolded mixture was dialysed against 10 L H.sub.2O per L of refold for 18-20 hours at 5° C.±3° C. After this time, the dialysis buffer was twice replaced with10 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.

[0151] Purification of soluble TCRs was initiated by applying the dialysed refold onto a POROSO 50HQ anion exchange column and eluting bound protein with a gradient of 0-500mM 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 Soluble TCR-antiCD3 Fusion Molecules

[0152] Method

[0153] Preparation of soluble TCR-antiCD3 fusion molecules 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, the concentration of the redox reagents in the refolding step were 1 mM cystamine dihydrochloride, 10 mM cysteamine hydrochloride). Finally, a cation exchange step was added following the anion exchange step. In this case, the peak fractions from anion exchange were diluted 20-fold in 40 mM MES, and applied to a POROS® 50HS cation exchange column. Bound protein was eluted with a gradient of 0-500 mM NaCl in 40 mM MES. Peak fractions were pooled and adjusted to 200mM Tris pH 8.1, before being concentrated and applied directly to the gel filtration matrix as described in Example 1.

Example 3

Binding Characterisation

[0154] Binding analysis of purified soluble TCRs and fusion molecules to peptide-HLA complex was carried out by surface plasmon resonance, using either a BIAcore 8K, BIAcore 3000 or BIAcore T200 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.

[0155] BIAcore Method

[0156] Biotinylated peptide-HLA monomers were immobilized on to streptavidin-coupled CM-5 of Biotin CAPture sensor chips. Equilibrium binding constants were determined using serial dilutions of soluble TCR or fusion molecules injected at a constant flow rate of 10-30 μl min.sup.−1 over a flow cell coated with ˜500 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 no 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.

[0157] For high affinity interactions, binding parameters were determined by single cycle kinetics analysis. Five different concentrations of soluble TCR or fusion protein were injected over a flow cell coated with ˜50-200 RU of peptide-HLA complex using a flow rate of 50-60 μl min .sup.1. Typically, 60-200 pl of soluble TCR or fusion molecule was injected at a top concentration of between 2-100 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 injected until 10% dissociation occurred, typically after 1 3 hours. Kinetic parameters were calculated using the manufacturer's software. The dissociation phase was fitted to a single exponential decay equation enabling calculation of half-life. The equilibrium constant KID was calculated from k.sub.off/k.sub.on.

Example 4

Binding Characterisation of the Soluble WT TCR

[0158] The soluble WT TCR, having the amino acid sequences shown in FIG. 1, was prepared according to the methods described in Example 1. The yield of purified protein was 9.1 mg/L. Binding parameters were calculated based on equilibrium binding constants according to Example 3. pHLA complexes were prepared comprising either the cognate peptide, a known viral variant of the peptide, or irrelevant peptides.

[0159] Results

[0160] The soluble WT TCR bound to the cognate peptide GLSPTVWLSV-HLA-A*02 complex with a K.sub.D of 1.44 μM+/−0.076 μM (Bmax=303; R.sup.2=0.997). The same TCR bound to the variant peptide GLSPTVWLSA-HLA-A*02 with a KID of 1.23 μM+/−0.07 μM (Bmax=152; R.sup.2 =0.996). These data indicate that the soluble VVT TCR can be used to target both the natural and variant peptide.

[0161] Specificity of the soluble WT TCR was assessed against a panel of 24 irrelevant peptide HLA-A*02 complexes that are naturally presented. The irrelevant pHLAs were divided into three groups and loaded onto one of three flow cells. Soluble wild type TCR was injected at concentrations of 68.3 and 6.8 μM over all flow cells. No significant binding was detected at either concentration indicting that the soluble WT TCR is specific for theHLA-A*02 complex.

[0162] Additional specificity assessment was carried out using a panel of peptides in which each residue of the GLSPTVWLSV peptide was sequentially replaced with alanine. Relative binding to each of the alanine substituted peptides was determined and the resulting data are shown in FIG. 5. Alanine substitutions in the central part of the peptide result in loss of TCR binding. A low tolerance for substitutions in the central part of the peptide indicate that the TCR has a high specificity and therefore presents a low risk for cross-reactivity with alternative peptides.

Example 5

Binding Characterisation of Mutated Soluble TCRs Fused to Anti-CD3

[0163] The mutated TCR alpha and beta variable domain amino acid sequences provided in FIGS. 2 and 3 respectively (SEQ ID NOs: 4-11) were used to prepare TCR-antiCD3 fusion molecules. Preparation was carried out according to Example 2. FIG. 4 provides full amino acid sequences of four of these TCR-antiCD3 fusion molecules indicated below. The yield of each of these four fusion molecules is shown in brackets [0164] a19b03 (6.02 mg/L) [0165] a13b03 (4.13 mg/L) [0166] a01 b03 (3.78) mg/L) [0167] a13b09 (3.46 mg/L)

[0168] Binding to peptide-HLA-A*02 complex was determined according to Example 3.

[0169] Results

[0170] The data presented in the table below show that fusion molecules comprising the indicated TCR variable domain sequences recognised the GLSPTVWLSV-HLA-A*02 complex with a supra-physiological binding affinity and half-life.

TABLE-US-00012 Alpha chain Beta chain K.sub.D (pM) t.sub.1/2 (hr) a01 (SEQ ID NO: 4) b02 (SEQ ID NO: 7) 21 5.76 a01 (SEQ ID NO: 4) b03 (SEQ ID NO: 8) 16 11.28 a01 (SEQ ID NO: 4) b04 (SEQ ID NO: 9) 19 10.78 a01 (SEQ ID NO: 4) b05 (SEQ ID NO: 10) 18 9.63 a01 (SEQ ID NO: 4) b09 (SEQ ID NO: 11) 61.6 4.5 a13 (SEQ ID NO: 5) b03 (SEQ ID NO: 8) 25.9 14.33 a13 (SEQ ID NO: 5) b09 (SEQ ID NO: 11) 67.1 6.1 a19 (SEQ ID NO: 6) b03 (SEQ ID NO: 8) 21 14.20

Example 6

Potency and Specificity Characterisation of Mutated Soluble TCRs Fused to Anti-CD3

[0171] T Cell Activation

[0172] Fusion molecules comprising the TCR variable domain sequences as set out in FIGS. 2 and 3 were assessed for their ability to mediate potent and specific activation of CD3+ T cells against cells presenting the GLSPTVWLSV-HLA-A*02 complex. Interferon-γ (IFN-γ) release was used as a read out for T cell activation.

[0173] Method

[0174] Assays were performed using a human IFN-γ ELISPOT kit (BD Biosciences) according to the manufacturer's 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). Fusion molecules were titrated to give final concentrations of 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM and 0.0001 nM, (spanning the anticipated clinically relevant range), and added to the well in a volume of 50 μl.

[0175] Plates were prepared according to the manufacturer's instructions. Target cells, effector cells and fusion 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 fusion molecules. 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).

[0176] In this example, the following cells lines were used as target cells: [0177] PLC/PRF/5 (antigen positive)

[0178] PLC/PRF/5 is human hepatocellular carcinoma cell line with integrated HBV genome. The GLSPTVWLSV peptide is naturally presented by these cells (as determined by Mass Spectrometry). [0179] HepG2 (antigen negative)

[0180] HepG2 is human cell line derived from a liver hepatocellular carcinoma

[0181] Both of the cells lines were transduced with genes encoding for HLA-A*02 132M complex

[0182] Results

[0183] Each of the fusion molecules tested demonstrated potent activation of T cells in the presence of antigen positive cells. Ec50 values were calculated from the data and are shown in the table below. The fusion molecules demonstrated no recognition of antigen negative HLA-A*02 positive cells. FIG. 6 shows representative data from four of the fusion molecules listed in the table below(note that value shown were obtained using different effector donors (i.e. not one donor that is common to all).

TABLE-US-00013 Alpha chain Beta chain Ec50 (pM) a01 (SEQ ID NO: 4) b02 (SEQ ID NO: 7) 5.72 a01 (SEQ ID NO: 4) b03 (SEQ ID NO: 8) 15.4 a01 (SEQ ID NO: 4) b04 (SEQ ID NO: 9) 10.9 a01 (SEQ ID NO: 4) b05 (SEQ ID NO: 10) 11 a01 (SEQ ID NO: 4) b09 (SEQ ID NO: 11) 14.1 a13 (SEQ ID NO: 5) b03 (SEQ ID NO: 8) 13.7 a13 (SEQ ID NO: 5) b09 (SEQ ID NO: 11) 8.3 a19 (SEQ ID NO: 6) b03 (SEQ ID NO: 8) 1.6

[0184] These data demonstrate that fusions molecules comprising mutated TCR variable domain sequences of the invention can mediate potent (Ec50 in low μM range) and specific T cell activation against antigen positive cells.

[0185] Target Cell Killing

[0186] The ability of fusion molecules comprising the mutated TCR sequences to mediate potent T cell mediated 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.

[0187] Method

[0188] Assays were performed using the CellPlayer 96-well Caspase-3/7 apoptosis assay kit (Essen BioScience, Cat. No. 4440) and carried out according the manufacturer's protocol. Briefly, PLC/PRF/5 cells were stained with CellTracker DeepRed before plating to allow for 2-colour analysis and subsequently plated at 10,000 cells per well and incubated overnight to allow them to adhere. Fusion molecules were prepared at various concentrations and 25 μl of each was added to the relevant well such that final concentrations were between 100 fM and 10 nM. Effector cells were used at an effector target cell ratio of 10:1 (100,000 cells per well). A control sample without fusion 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 1.25 μM final conc). The plate was placed in the IncuCyte instrument and images taken every 3 hours (1 image per well) over 5 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.

[0189] Results

[0190] The data presented in FIG. 7 show real-time killing of antigen positive cells in the presence of fusion molecules comprising the mutated TCR variable chain sequences indicated on each graph (for clarity only three concentrations are shown). In each case, target cell killing was observed at concentrations of 100 μM or lower. No killing was observed in the absence of fusions molecules.

[0191] Safety Screening Against High Risk Normal Tissue

[0192] To further demonstrate the specificity of fusion molecules comprising the mutated TCR sequences, further testing was carried out using the same ELISPOT methodology as described above, using a panel of normal cells derived from healthy human tissues as targets.

[0193] In a first experiment, a TCR-antiCD3 fusion comprising a19b03 mutated TCR variable domains was tested at three different concentrations (2 nm, 1 nM and 0.1 nM). Two lots of cells from each normal tissue were used as targets, and effector T cells were obtained from 3 different donors. Control measurements were made using a sample without fusion molecule and a sample in which normal cells were replaced with PLC/PRF/5 (antigen positive) cells at a single concentration of fusion molecule (1 nM).

[0194] In a second experiment, four different TCR-antiCD3 fusions were used comprising the following mutated chains (a01b03, a01b02, a01b04, a01b05). The same three concentrations of fusion molecule were used (2nm, 1 nM and 0.1 nM). A single lot of cells from each normal tissue were used as targets and effector T cells were obtained from a single donor. Control measurements were made using a sample without fusion molecule and a sample in which normal cells were pulsed with 10 μM peptide (antigen positive) and a fusion molecule included at a single concentration of 0.1 nM.

[0195] Results

[0196] In both experiments, T cell activation against normal cells was observed at a similar level to background (i.e. taken as sample without fusion molecule).

[0197] FIG. 8a shows data from the first experiment for one lot of normal cells from two different tissues and for three different donors (labelled Donorl-3). FIG. 8b shows data from the second experiment for two different tissues. The dotted line in each graph indicates the background level.

[0198] These data indicate that fusion molecules comprising the TCR variable domains shown in FIGS. 2 and 3 show no material cross reactivity against a panel of cells derived from normal tissues.

[0199] TCR-antiCD3 fusions described have properties that make them particularly suitable for therapeutic use.

Example 7

Potent T Cell Activation Against HBV Infected Cells by Soluble TCRs Fused to Anti-CD3

[0200] To demonstrate that that the TCR-antiCD3 fusions of the invention can redirect T cell activity towards HBV infected cells, a HBV infection model was established.

[0201] Briefly, the HLA-A*02:01 positive hepatocellular carcinoma (HCC) cell line, HepG2, was transfected with the receptor NTCP. Cells were subsequently infected with 200 MOI of HBV, genotype D (1×108 genome copies of lot 03072018, ImQuest BioSciences). Infected cells were then co-cultured with pan T cells obtained from donor blood, in the presence or absence of TCR-antiCD3 fusion. The percentage of infected cells remaining was quantified using PrimeFlow (Invitrogen) to detect Hepatitis B surface antigen (HBsAg) RNA.

[0202] TCR-antiCD3 fusions comprising a19b03 and a01b03 were used in this example. In addition, a TCR-antiCD3 specific for an alternative, non-HBV peptide, was used as a negative control

[0203] Method

[0204] On day 7 of infection supernatant was removed from wells containing infected HepG2-NTCP cells before pan T cells, with or without TCR-antiCD3 fusion, were added for co-culture. Effector T cells were added at a ratio of 10:1 according to the initial number of HepG2-NTCP plated for infection, and TCR-antiCD3 fusion added at a final concentration of either 1 nM or 0.1 nM. Cells were cultured for 5 days. At the end of co-culture, infected cells were removed from culture plates by trypsin, and surface stained for CD45 and a fixable viability dye (eFluor 780). Following this, cells were fixed and permeabilised for staining of Hepatitis B surface antigen (HBsAg) RNA using the PrimeFlow probeset VF1-6000704. Staining of the housekeeping gene RPL13A was used a positive control for the staining procedure (Probeset VA4-13187). Stained cells were run on the MACSQuant X flow cytometer and analysed by FlowJov10 to quantify the percentage of HBsAg expressing cells.

[0205] Results

[0206] FIG. 9 show that both a19b03 and a01 b03 TCR-antiCD3 fusions lead to an approximately 60% reduction in the % of infected cells at 1 nM fusion. The effect is titratable with 0.1 nM giving a 34% reduction.

[0207] These data indicate that fusion molecules comprising the TCR variable domains shown in FIGS. 2 and 3 can effectively clear infected cells.