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T CELL RECEPTOR LIKE ANTIBODIES

20200071406 ยท 2020-03-05

    Inventors

    Cpc classification

    International classification

    Abstract

    Antibodies and fragments thereof which bind to peptide-MHC complexes are described. In particular, an antibody binding to a peptide-MHC complex comprising a peptide of p53 and a MHC Class I molecule comprising -chain encoded by an HLA-A*24 allele is claimed. Also disclosed are compositions comprising such antibodies and fragments, and uses and methods of treating, preventing and diagnosing cancers using the same.

    Claims

    1. An antibody or antigen binding fragment, optionally isolated, which is capable of binding to a peptide-MHC complex comprising a peptide of p53 and an MHC class I molecule.

    2. The antibody or antigen binding fragment according to claim 1, wherein the MHC class I molecule comprises an MHC class I -chain encoded by an HLA-A*24 allele.

    3. The antibody or antigen binding fragment according to claim 1 or claim 2, wherein the peptide of p53 comprises, or consists of, the amino acid sequence of SEQ ID NO:75, or a variant having thereof having one or two or three amino acid substitutions in the amino acid sequence.

    4. The antibody or antigen binding fragment according to any one of claims 1 to 3, comprising the amino acid sequences i) to vi): TABLE-US-00006 i)LC-CDR1: (SEQIDNO:46) X.sub.1GSX.sub.2SNIGX.sub.3X.sub.4YX.sub.5X.sub.6X.sub.7; (SEQIDNO:29) TGTSSDVGGYNYVS; or (SEQIDNO:21) RASQSIGTDLA; ii)LC-CDR2: (SEQIDNO:47) GNX.sub.8NRPS; (SEQIDNO:22) DASNRAT; or (SEQIDNO:30) DVSSRPS iii)LC-CDR3: (SEQIDNO:48) QSYDSX.sub.9LSX.sub.10X.sub.11WV; (SEQIDNO:23) QQRSNWPPT; or (SEQIDNO:31) SSYTVFSTLV; iv)HC-CDR1: (SEQIDNO:49) SGGYYWX.sub.12; or (SEQIDNO:50) X.sub.13YYX.sub.14H; v)HC-CDR2: (SEQIDNO:51) YIYYSGX.sub.15TYYNPSLKS; or (SEQIDNO:52) WX.sub.16X.sub.17PX.sub.18SX.sub.19X.sub.20TX.sub.2YAQKFQG; vi)HC-CDR3: (SEQIDNO:53) ENFGX.sub.22X.sub.23DX.sub.24; (SEQIDNO:39) EGADGIYYFDY; or (SEQIDNO:45) DTYGHDY; or a variant thereof in which one or two or three amino acids in one or more of the sequences i) to vi) are replaced with another amino acid; wherein X.sub.1=T or A, X.sub.2=S or Y, X.sub.3=A or D, X.sub.4=G or D, X.sub.5=D or E, X.sub.6=V or T, X.sub.7=H or N, X.sub.8=N or T, X.sub.9=N or S, X.sub.10=Absent or D, X.sub.11=A or T, X.sub.12=S or A, X.sub.13=G or D, X.sub.14=M or I, X.sub.15=S or T, X.sub.16=I or M, X.sub.17=N or S, X.sub.18=N or D, X.sub.19=A or G, X.sub.20=G or A, X.sub.21=N or Y, X.sub.22=A or S, X.sub.23=F or Y, and X.sub.24=H or Y.

    5. The antibody or antigen binding fragment according to claim 4, wherein LC-CDR1 is one of TGSSSNIGADYETH (SEQ ID NO:17), AGSYSNIGDDYETH (SEQ ID NO:20), TGSSSNIGAGYDVH (SEQ ID NO:24), TGSSSNIGAGYDVN (SEQ ID NO:27), TGTSSDVGGYNYVS (SEQ ID NO:29) or RASQSIGTDLA (SEQ ID NO:21).

    6. The antibody or antigen binding fragment according to claim 4 or claim 5, wherein LC-CDR2 is one of GNTNRPS (SEQ ID NO:18), GNNNRPS (SEQ ID NO:25), DASNRAT (SEQ ID NO:22) or DVSSRPS (SEQ ID NO:30).

    7. The antibody or antigen binding fragment according to any one of claims 4 to 6, wherein LC-CDR3 is one of QSYDSNLSAWV (SEQ ID NO:19), QSYDSNLSDTWV (SEQ ID NO:26), QSYDSSLSAWV (SEQ ID NO:28), QQRSNWPPT (SEQ ID NO:23) or SSYTVFSTLV (SEQ ID NO:31).

    8. The antibody or antigen binding fragment according to any one of claims 4 to 7, wherein HC-CDR1 is one of SGGYYWS (SEQ ID NO:32), SGGYYWA (SEQ ID NO:35), SGGYYWS (SEQ ID NO:40), GYYMH (SEQ ID NO:37), or DYYIH (SEQ ID NO:43).

    9. The antibody or antigen binding fragment according to any one of claims 4 to 8, wherein HC-CDR2 is one of YIYYSGSTYYNPSLKS (SEQ ID NO:33), YIYYSGTTYYNPSLKS (SEQ ID NO:41), WINPNSAGTNYAQKFQG (SEQ ID NO:38) or WMSPDSGATYYAQKFQG (SEQ ID NO:44).

    10. The antibody or antigen binding fragment according to any one of claims 4 to 9, wherein HC-CDR3 is one of ENFGAFDH (SEQ ID NO:34), ENFGSYDY (SEQ ID NO:36), EGADGIYYFDY (SEQ ID NO:39), or DTYGHDY (SEQ ID NO:45).

    11. The antibody or antigen binding fragment according to any one of claims 1 to 10, having at least one light chain variable region incorporating the following CDRs: TABLE-US-00007 LC-CDR1: (SEQIDNO:17) TGSSSNIGADYETH LC-CDR2: (SEQIDNO:18) GNTNRPS LC-CDR3: (SEQIDNO:19) QSYDSNLSAWV; LC-CDR1: (SEQIDNO:20) AGSYSNIGDDYETH LC-CDR2: (SEQIDNO:18) GNTNRPS LC-CDR3: (SEQIDNO:19) QSYDSNLSAWV; or LC-CDR1: (SEQIDNO:21) RASQSIGTDLA LC-CDR2: (SEQIDNO:22) DASNRAT LC-CDR3: (SEQIDNO:23) QQRSNWPPT; or LC-CDR1: (SEQIDNO:24) TGSSSNIGAGYDVH LC-CDR2: (SEQIDNO:25) GNNNRPS LC-CDR3: (SEQIDNO:26) QSYDSNLSDTWV; or LC-CDR1: (SEQIDNO:27) TGSSSNIGAGYDVN LC-CDR2: (SEQIDNO:25) GNNNRPS LC-CDR3: (SEQIDNO:28) QSYDSSLSAWV; or LC-CDR1: (SEQIDNO:29) TGTSSDVGGYNYVS LC-CDR2: (SEQIDNO:30) DVSSRPS LC-CDR3: (SEQIDNO:31) SSYTVFSTLV.

    12. The antibody or antigen binding fragment according to any one of claims 1 to 11, having at least one heavy chain variable region incorporating the following CDRs: TABLE-US-00008 HC-CDR1: (SEQIDNO:32) SGGYYWS HC-CDR2: (SEQIDNO:33) YIYYSGSTYYNPSLKS HC-CDR3: (SEQIDNO:34) ENFGAFDH; or HC-CDR1: (SEQIDNO:35) SGGYYWA HC-CDR2: (SEQIDNO:33) YIYYSGSTYYNPSLKS HC-CDR3: (SEQIDNO:34) ENFGAFDH; or HC-CDR1: (SEQIDNO:32) SGGYYWS HC-CDR2: (SEQIDNO:33) YIYYSGSTYYNPSLKS HC-CDR3: (SEQIDNO:36) ENFGSYDY; or HC-CDR1: (SEQIDNO:35) SGGYYWA HC-CDR2: (SEQIDNO:33) YIYYSGSTYYNPSLKS HC-CDR3: (SEQIDNO:36) ENFGSYDY; or HC-CDR1: (SEQIDNO:37) GYYMH HC-CDR2: (SEQIDNO:38) WINPNSAGTNYAQKFQG HC-CDR3: (SEQIDNO:39) EGADGIYYFDY; or HC-CDR1: (SEQIDNO:40) SGGYYWS HC-CDR2: (SEQIDNO:41) YIYYSGTTYYNPSLKS HC-CDR3: (SEQIDNO:42) ENFGAFDY; or HC-CDR1: (SEQIDNO:43) DYYIH HC-CDR2: (SEQIDNO:44) WMSPDSGATYYAQKFQG HC-CDR3: (SEQIDNO:45) DTYGHDY.

    13. An antibody or antigen binding fragment, optionally isolated, which is capable of binding to a peptide-MHC complex comprising a peptide of p53 and an MHC class I molecule, comprising a light chain and a heavy chain variable region sequence, wherein: the light chain comprises a LC-CDR1, LC-CDR2, LC-CDR3, having at least 85% overall sequence identity to LC-CDR1: one of X.sub.1GSX.sub.2SNIGX.sub.3X.sub.4YX.sub.5X.sub.6X.sub.7 (SEQ ID NO:46), TGTSSDVGGYNYVS (SEQ ID NO:29) or RASQSIGTDLA (SEQ ID NO:21); LC-CDR2: one of GNX.sub.8NRPS (SEQ ID NO:47), DASNRAT (SEQ ID NO:22) or DVSSRPS (SEQ ID NO:30); LC-CDR3: one of QSYDSX.sub.9LSX.sub.10X.sub.11WV (SEQ ID NO:48), QQRSNWPPT (SEQ ID NO:23) or SSYTVFSTLV (SEQ ID NO:31); and the heavy chain comprises a HC-CDR1, HC-CDR2, HC-CDR3, having at least 85% overall sequence identity to HC-CDR1: one of SGGYYWX.sub.12 (SEQ ID NO:49) or X.sub.13YYX.sub.14H (SEQ ID NO:50); HC-CDR2: one of YIYYSGX.sub.15TYYNPSLKS (SEQ ID NO:51) or WX.sub.16X.sub.17PX.sub.18SX.sub.19X.sub.20TX.sub.2YAQKFQG (SEQ ID NO:52); HC-CDR2: one of ENFGX.sub.22X.sub.23DX.sub.24 (SEQ ID NO:53), EGADGIYYFDY (SEQ ID NO:39) or DTYGHDY (SEQ ID NO:45); wherein X.sub.1=T or A, X.sub.2=S or Y, X.sub.3=A or D, X.sub.4=G or D, X.sub.5=D or E, X.sub.6=V or T, X.sub.7=H or N, X.sub.8=N or T, X.sub.9=N or S, X.sub.10=Absent or D, X.sub.11=A or T, X.sub.12=S or A, X.sub.13=G or D, X.sub.14=M or I, X.sub.15=S or T, X.sub.16=I or M, X.sub.17=N or S, X.sub.18=N or D, X.sub.19=A or G, X.sub.20=G or A, X.sub.21=N or Y, X.sub.22=A or S, X.sub.23=F or Y, and X.sub.24=H or Y.

    14. An antibody or antigen binding fragment, optionally isolated, which is capable of binding to a peptide-MHC complex comprising a peptide of p53 and an MHC class I molecule, comprising a light chain and a heavy chain variable region sequence, wherein: the light chain sequence has at least 85% sequence identity to the light chain sequence of one of SEQ ID NOs:1 to 7, and; the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence of one of SEQ ID NOs:8 to 16.

    15. The antibody or antigen binding fragment according to any one of claims 1 to 14, which displays antibody-dependent cell-mediated cytotoxicity (ADCC) to cells comprising or expressing peptide-MHC complex comprising a peptide of p53 and an MHC class I molecule.

    16. The antibody or antigen binding fragment according to any one of claims 1 to 15, which is internalised by cells comprising or expressing peptide-MHC complex comprising a peptide of p53 and an MHC class I molecule.

    17. The antibody or antigen binding fragment according to any one of claims 1 to 16, which is a fully human antibody or a fully human antibody fragment.

    18. The antibody or antigen binding fragment according to any one of claims 1 to 17, conjugated to a drug moiety or a detectable moiety.

    19. The antibody or antigen binding fragment according to any one of claims 1 to 18, further comprising an antibody or antigen binding fragment specific for a target other than a peptide-MHC complex.

    20. The antibody or antigen binding fragment according to claim 19, wherein the target other than a peptide-MHC complex is an immune cell surface molecule.

    21. A chimeric antigen receptor (CAR) comprising an antigen binding fragment according to any one of claims 1 to 20.

    22. An in vitro complex, optionally isolated, comprising an antibody, antigen binding fragment or CAR according to any one of claims 1 to 21 bound to a peptide-MHC complex comprising a peptide of p53 and an MHC class I molecule.

    23. A composition comprising the antibody, antigen binding fragment or CAR according to any one of claims 1 to 21 and at least one pharmaceutically-acceptable carrier.

    24. An isolated nucleic acid encoding the antibody, antigen binding fragment or CAR according to any one of claims 1 to 21.

    25. A vector comprising the nucleic acid of claim 24.

    26. A cell comprising the nucleic acid according to claim 24 or the vector according to claim 25.

    27. A method for making an antibody, antigen binding fragment or CAR according to any one of claims 1 to 21, comprising culturing the cell of claim 26 under conditions suitable for the expression of the antibody or antigen binding fragment or CAR.

    28. An antibody, antigen binding fragment, CAR, composition, nucleic acid, vector or cell according to any one of claims 1 to 21, or 23 to 26 for use in therapy, or in a method of medical treatment.

    29. An antibody, antigen binding fragment, CAR, composition, nucleic acid, vector or cell according to any one of claims 1 to 21, or 23 to 26 for use in the treatment or prevention of a cancer.

    30. Use of an antibody, antigen binding fragment, CAR, composition, nucleic acid, vector or cell according to any one of claims 1 to 21, or 23 to 26 in the manufacture of a medicament for treating or preventing a cancer.

    31. A method of treating or preventing a cancer, comprising administering to a subject a therapeutically or prophylactically effective amount of the antibody, antigen binding fragment, CAR, composition, nucleic acid, vector or cell according to any one of claims 1 to 21, or 23 to 26.

    32. A method of treating or preventing a cancer in a subject, comprising: (a) isolating at least one cell from a subject; (b) modifying the at least one cell to express or comprise the antibody, antigen binding fragment, CAR, nucleic acid or vector according to any one of claims 1 to 21, or 24 to 26 and; (c) administering the modified at least one cell to a subject.

    33. A method of treating or preventing a cancer in a subject, comprising: (a) isolating at least one cell from a subject; (b) introducing into the at least one cell the nucleic acid according to claim 24 or the vector according to claim 25, thereby modifying the at least one cell and; (c) administering the modified at least one cell to a subject.

    34. A kit of parts comprising a predetermined quantity of the antibody, antigen binding fragment, CAR, composition, nucleic acid, vector or cell according to any one of claims 1 to 21, or 23 to 26.

    35. A method of diagnosing a disease or a condition in a subject, the method comprising contacting a sample containing, or suspected to contain, peptide-MHC complex with an antibody or antigen binding fragment according to any one of claims 1 to 21 and detecting the formation of a complex of antibody, or antigen binding fragment, and the peptide-MHC complex.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0247] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

    [0248] FIG. 1. Light chain variable domain sequences for anti-p53-A24 antibody clones. CDRs are underlined and shown separately.

    [0249] FIG. 2. Heavy chain variable domain sequences for anti-p53-A24 antibody clones. CDRs are underlined and shown separately.

    [0250] FIG. 3. Table showing light chain CDR sequences for anti-p53-A24 antibody clones.

    [0251] FIG. 4. Table showing heavy chain CDR sequences for anti-p53-A24 antibody clones.

    [0252] FIG. 5. Tables showing light chain CDR sequences for anti-p53-A24 antibody clones and consensus sequences, for (A) LC-CDR1, (B) LC-CDR2 and (C) LC-CDR3.

    [0253] FIG. 6. Tables showing heavy chain CDR sequences for anti-p53-A24 antibody clones and consensus sequences, for (A) HC-CDR1, (B) HC-CDR2 and (C) HC-CDR3.

    [0254] FIG. 7. Nucleotide sequences encoding VL regions for the anti-p53-A24 antibody clones.

    [0255] FIG. 8. Nucleotide sequences encoding VH regions for the anti-p53-A24 antibody clones.

    [0256] FIG. 9. Graphs showing the results of ELISA analysis of binding of anti-p53-A24 antibody clones to (A) p53125-134-A24 monomers, and (B) negative control antigen.

    [0257] FIG. 10. Graphs showing binding of (A) P1C1, (B) P1 H4 and (C) P2B4 to HLA*A24-expressing HT29 cells, unpulsed (1), pulsed with irrelevant peptides hTERT324-332, hTERT.sub.461-469, WT1.sub.235-243, WT1.sub.417-425 or p53.sub.204-212 (2 to 6), or pulsed with p53.sub.125-134 (7).

    [0258] FIG. 11. Graphs showing binding of P1C1 to (A) wild-type MDA-MB-231 cells (left panels) or HLA*A24-transduced MDA-MB-231 cells (right panels), either pulsed with p53 peptide (top panels) or unpulsed (bottom panels), and (B) HLA*A24+ SoaS2 cells, before or after pulsing with various hTERT, WT1 or p53 peptides. MFI histograms are shown.

    [0259] FIG. 12. Table showing binding affinity of antibody P1C1-derived antibody clones to p53-A24.

    [0260] FIG. 13. Graphs showing binding of P1C1-derived clones, the germline version (P1C1_gl) and 2 affinity-matured clones (P1C1_dm and P1C1_tm) to unpulsed HT29 cells. MFI histograms are shown; the narrow, sharp peak to the left represents the negative control (secondary antibody only) and the broader peak to the right represents the test clone.

    [0261] FIG. 14. Bar charts showing specific killing of HLA*A24 positive cells in an ADCC assay in the presence of P1C1_gl or P1C1_tm. Mean cytotoxicity in triplicate SD is shown.

    [0262] FIG. 15. Graphs and bar chart showing internalisation of the antibody complexed to p53-A24 by HT29 pulsed cells. (A) MFI curves of HT29 cells incubated with pH-sensitive dye-labelled P1C1_tm (left panels) or a labelled isotype control antibody (right panels) on ice (top panels) or at 37 C. (bottom panels). (B) MFI values of cells for cells incubated with pH-sensitive dye-labelled P1C1_tm or a labelled isotype control antibody at different time points.

    [0263] FIG. 16. Bar chart showing increased cytotoxic effect of PNU and PBD on HT29 cells in the absence (PNU/PBD 2dary only) or presence (PNU/PBD) of P1C1_tm antibody. Cells were incubated with P1C1_tm and PNU or PBD cytotoxic drugs conjugated to anti-Fc antibodies. Three concentrations of antibodies were tested, for each of them the ratio P1C1_tm/anti-Fc antibody was 1:1. Shown is the proportion of surviving cells after 72 hours of incubation compared to the number of cells in wells left untreated.

    [0264] FIG. 17. Photographs showing in vivo specificity of P1C1_tm antibody. The antibody was used to track human tumour cell lines injected in NSG mice. (A) HT29 cells expressing p53 and HLA*A24 were seeded in the right flank of the animals, and HLA*A24-/p53+control cells were seeded in the left flank. (B) HT29 cells expressing p53 and HLA*A24 were seeded in the right flank of the animals, and HLA*A24+/p53-control cells were seeded in the left flank. Representative images from in vivo imaging of established tumours at the indicated number of hours after injection of fluorescently-labelled P1C1_tm antibody are shown.

    [0265] FIG. 18. Graph showing % cytolysis of HT29 tumour cells by p53 CAR T cells, control T cells, or no T cells. % cytolysis was measured over 40 hours using an impedance-based T cell-mediated cytotoxicity assay (xCELLigence).

    [0266] FIG. 19. Anti-p53-A24/CD3 bispecific antibodies were evaluated for their ability to induce tumour cell killing via human primary T cells. (A) Two different formats of bispecific antibody were tested: BsAb1 and BsAb2. (B) The target-specific cytotoxicity of BsAb1 and BsAb2 was tested against HLA-A24+/p53 mutant+ cell line HT29 (BsAb1: right hand curve, BsAb2: left hand curve) and HLA-A24+/p53null cell line SaOS2.

    EXAMPLES

    [0267] In the following Examples, the inventors describe isolation and characterisation of antibodies capable of binding to p53 peptide:MHC class I complex.

    Example 1

    Isolation of anti-p53:MHC Class I Complex Antibodies

    [0268] The p.sub.125-134 peptide was attached to soluble HLA*A2402 (p53-A24) to form a soluble peptide MHC complex (pMHC). Antibodies capable of binding to this pMHC complex were then isolated from a human antibody phage display library via in vitro selection. Out of 190 clones screened, the 36 clones showing the highest binding to the pMHC by ELISA assay were isolated, and 4 amongst them were cloned into IgG format for further characterisation: P1C1, P1H4, P1A8 and P1B11.

    [0269] The amino acid sequences for the light chain and heavy chain variable regions are shown in FIGS. 1 and 2, respectively.

    Example 2

    Avidity and Specificity for p53-A24

    [0270] Antibody clones P1C1, P1 H4, P1A8 and P1 B11 were analysed by ELISA assay for binding to p53-A24 monomers, and for binding to irrelevant antigen, to determine avidity and specificity.

    [0271] With the exception of P1A8, all clones were shown to bind to p53-A24 with high affinity (FIG. 9A). The antibodies also displayed zero or limited non-specific binding (FIG. 9B).

    Example 3

    Specific Binding to p53 on HLA*A24-Expressing Cells

    [0272] The ability to recognise and bind p53-A24 pMHC expressed at the cell surface was measured using HT29 cells. These cells constitutively express HLA*A24.

    [0273] Briefly, HT29 cells were pulsed with the p53.sub.125-134 peptide, peptide selected from a panel of irrelevant peptides, or were unpulsed for 1 hour at room temperature. Cells were then incubated with P1C1, P1 H4 or P2B4, and binding was measured by flow cytometry using a secondary labelled antibody.

    [0274] The results are shown in FIGS. 10A to 10C. P1C1, P1H4 or P2B4 antibodies only bound to cells that had been pulsed with the p53.sub.125-134 peptide, demonstrating their specificity for the p53.sub.125-134 antigen (FIGS. 10A to 10C). Moreover, the fact that the antibodies did not bind to unpulsed HT29 cells suggests they only bind to HLA*A24 presenting p53 antigen, and does not bind to HLA*A24 not presenting p53.

    Example 4

    Specificity for HLA*A24 MHC Class I Molecule and for p53-A24

    [0275] To confirm the specificity for the HLA*A24 haplotype, binding was assessed on cells expressing a different HLA*A type: MDA-MB-231 cells, which constitutively express HLA*A02 and p53. MDA-MB-231 cells were also transduced to express HLA*A24.

    [0276] Binding of P1C1 was then assessed on transduced and nontransduced MDA-MB-231 cells, which were either pulsed with the p53.sub.125-134 peptide or unpulsed. Binding was measured by flow cytometry using a secondary labelled antibody.

    [0277] The results are shown in FIG. 11A. P1C1 bound only to cells expressing HLA*A24 (i.e. transduced MDA-MB-231), and no binding was observed on nontransduced MDA-MB-231 cells expressing HLA*A02, confirming the specificity for p53.sub.125-134 peptide presented by HLA*A24.

    [0278] Similar experiments were conducted using SaoS2 cells which are p53-negative. These cells constitutively express HLA*A24. The cells were pulsed with various peptides of p53, WT1, hTERT or unpulsed. Analysis of binding of P1C1 antibody to the cells by bound exclusively cells that were pulsed with p53.sub.125-134, confirming the specificity of the antibody for this antigen (FIG. 11 B).

    [0279] Taken together, these results demonstrate the specificity of the antibody for p53.sub.125-134 presented by HLA*A24.

    Example 5

    Affinity Matured Antibodies

    [0280] The P1C1 sequence was reverted to a germline framework to give clone P1C1_gl, which subsequently underwent affinity maturation. 2 affinity matured clones were retained for the heavy chain (2E3 and 1 E11), and 1 affinity matured clone was retained for the light chain (1G7).

    [0281] A double-mutant comprising the substitutions present in both 2E3 and 1E11 was constructed, designated clone P1C1_dm. A triple-mutant comprising the substitutions present in 2E3, 1 E1 1 and 1G7 was generated, designated clone P1C1_tm.

    [0282] The affinity of different P1C1-derived clones for p53-A24 complex was measured via Surface Plasmon Resonance analysis. The results are shown in FIG. 12, and show that P1C1_tm has 10-fold higher affinity for p53-A24 as compared to P1C1_gl.

    [0283] The ability of P1C1_dm, P1C1_tm and P1C1_gl to bind to unpulsed HT29 cells was analysed by flow cytometry. P1C1_dm and P1C1_tm clones showed a higher binding to unpulsed HT29 cells than the original germline clone (FIG. 13).

    Example 6

    In Vitro Activity: Induction of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

    [0284] To assess the ability of the antibodies to induce ADCC, HLA*A24 positive HT29 were mixed with PBMCs at a ratio of 1:10, and incubated overnight in the presence or absence of P1C1_gl or P1C1_tm, and cell killing was measured. Cells were either unpulsed, or pulsed with the p53 peptide.

    [0285] The specificity of induction of ADCC by the antibodies was also assessed using HLA*A24-negative MDA-MB-231 cells, nontransduced or transduced with HLA*A24.

    [0286] The results are shown in FIG. 14. Dose-dependent killing was observed to be more efficient in p53 peptide pulsed HLA*A24-positive cells as compared to unpulsed cells. No significant ADCC was observed in HLA*A24-negative MDA-MB-231 cells. P1C1_tm exhibits more potent ADCC as compared to P1C1_gl.

    Example 7

    Internalisation of P1C1_tm by HT29 Cells

    [0287] P1C1_tm antibody was labelled with pH-sensitive pHrodo-Red dye and incubated with p53 peptide-pulsed HT29 cells at 37 C. or on ice for various periods of time, before internalisation was analysed by flow cytometry. Internalised antibodies produce significant fluorescence as compared to surface bound antibodies due to the acidic environment in the endosomes.

    [0288] The results are shown in FIGS. 15A and 15B. Upon binding to p53-A24 pMHC, the antibody is internalised with the complex when the complex is recycled, as evidenced by fluorescence shift in HT29 cells pulsed with p53 peptide incubated at 37 C. in the presence of P1C1_tm antibody (FIG. 15A, bottom left panel). This shift was not observed with a non-specific control antibody (FIG. 15A, bottom right panel), or in cells incubated on ice and hence not recycling the p53-A24 pMHC (FIG. 15A, top left panel).

    Example 8

    Drug Delivery to Tumour Cells

    [0289] Internalisation of the antibody with the p53-A24 complex may be used as a tool for drug delivery to specifically target tumour cells. This was tested using drug-conjugated secondary antibodies binding to P1C1_tm. Briefly, HT29 cells were incubated with P1C1_tm and anti-human Fc specific secondary antibodies conjugated with cytotoxic drugs PNU159682 (PNU) or pyrrolobenzodiazepine (PBD). After 72 hours, cell viability was analysed by MTT assay. Some cells were incubated only with the drug-conjugated antibodies, in the absence of P1C1_tm, as controls.

    [0290] The results of the experiments are shown in FIG. 16. P1C1_tm was found to dramatically improve the cytotoxic effect of PNU and PBD. The increased effect is likely due to the internalisation of the drugs as part of the immune complex of P1C1_tm/drug-conjugated anti-Fc antibody bound to the p53-A24 complex.

    [0291] These data suggest that P1C1_tm could be used in the treatment of cancer for targeted drug delivery of drugs to tumours, and to increase the efficacy of drugs by forcing their internalisation into the targeted cells.

    Example 9

    In Vivo Imaging of HT29 in NSG Mice

    [0292] One of the applications of the TCR like antibodies will be to help diagnose cancer by identifying tumour cells which cross-present intracellular molecules via MHC class I, in the present case p53.

    [0293] To assess the usefulness of P1C1_tm as a diagnostic antibody, HT29 cells (positive for both A24 and p53), and nontransduced MDA-MB-231 cells (expressing p53 but negative for A24) or SaoS2 cells (expressing A24 but not p53) were implanted into flanks of NSG mice, as shown schematically in FIGS. 17A and 17B and explained in the Figure legend.

    [0294] Tumours were allowed to establish and grow up to 100-200 mm.sup.3 before 50 g of AF680-labelled P1C1_tm antibody was administered intravenously. Tumour labelling was captured 48 and 120 hours later by in vivo fluorescent imaging.

    [0295] As shown in FIGS. 17A and 17B, the antibody allowed the detection of the HLA*A24+ HT29 cells but did not track the HLA*A02 expressing tumour (FIG. 9A), nor the HLA*A24+ tumour cells not expressing p53 (FIG. 9B)

    Example 10

    T cell-Mediated Cytotoxicity Against HT29 Tumour Cells

    [0296] T cells expressing a chimeric antigen receptor (CAR) comprising a TCR like antigen binding fragment were assessed for their ability to kill HT29 tumour cells.

    [0297] T cells from healthy donors were activated by addition of TransAct (CD3/CD28 agonists) and maintained at a density of 1-210.sup.6 cells/ml in TexMACS medium supplemented with 50 ng/mL IL2 for a duration of 72 hr. Upon stimulation, cells were electroporated with or without lentiviral-based p53 CAR plasmid (2 g per million cells).

    [0298] Due to a considerable amount of cell death caused by plasmid DNA electroporation, apoptotic cells (Annexin V+) were depleted from cell culture by immunomagnetic negative selection at 48 hr post-electroporation.

    [0299] Cells were rested in culture for additional 24 hr before being assessed in a T cell-mediated cytotoxicity assay (xCELLigence). In this assay, cell index values of tumour cells are measured. Cell index is determined by the impedance of current across the transistor plate caused by tumour cell adherence.

    [0300] Using the xCELLigence impedance-based system, continuous tumour cell killing was evaluated over 40 hours. HT29 tumour cells were plated in a 96-well, resistor-bottomed plate at 15,000 cells per well in complete growth media. After 18-24 hours, 3,750 effector T cells (1:4 seeding ratio) were added, at which point cell index values correlating to HT29 adherence were normalized. Impedance-based measurements of the normalized cell index were recorded every 10 minutes and converted into % cytolysis. Data present the mean (standard deviation) of triplicates.

    [0301] The results are shown in FIG. 18. The p53 CAR T cells were able to kill significantly more of the HLA*A24 positive HT29 tumour cells compared to control T cells.

    Example 11

    Anti-p53-A24/CD3 Bispecific Antibody Cytotoxicity In Vitro Against HT29 Tumour Cells

    [0302] Two different formats of anti-p53-A24/CD3 bispecific antibody (BsAb) cocultured with human primary T cells and tumour cells and assessed for their ability to induce tumour cell killing. The two BsAb formats (1 and 2) are shown in FIG. 19A.

    [0303] Tumour cells (target cells) were pre-labelled with Oregon Green and cultured in 96-well half-area flat-bottom plate overnight for cell adhesion. The following day, primary T cells isolated from human PBMCs were added with Effector to Target ratio 10 to 1 (E:T=10:1). BsAbs, with 10-fold titration for 8 points, were also prepared and added to each well to reach to the indicated final concentration. The plate was then incubated in 37C, 5% CO2, for 3 days. For FACS readout, tumour cells were gently detached with Accutase and stained with Propidium iodide (PI) to label the dead cells. The BsAb-induced cytotoxicity was measured with FACS readout after 3-day coculture using MACSQuant Analyzer. % cytotoxicity equals to the cell count of dead cells divided by the cell count of target cells.

    [0304] The results are shown in FIG. 19B. In HLA-A24+/p53 mutant+ cell line HT29, target-specific cytotoxicity is observed with both bispecific constructs. BsAb2 (EC50=0.48nM; left hand curve) shows higher potency than BsAb1 (EC50=3.05nM; right hand curve). On the other hand, no non-specific cytotoxicity is observed in both constructs in the cell killing of HLA-A24+/p53null cell line SaOS2, showing the specificity of the bispecific constructs for targeting the p53-A24 complex.