Patent ID: 12195526

EXAMPLES

The inventors describe in the following Examples the generation and characterisation of monoclonal antibodies against DBD region point mutants of P53. The antibodies are shown to be highly specific for the individual p53 hot-spot mutations, and the utility of the antibodies in a variety of biochemical and histological assays is demonstrated.

Example 1: Immunogen Design and Production

Attempts to generate antibodies against specific p53 mutants using a large array of protocols have not been entirely successful, due likely to lack of efficient expression of the mutant epitopes, resulting lack of specificity for the mutant p53 polypeptides.

The inventors therefore utilized the TrxA protein with a protruding body, in which was placed three copies of the mutant p53 mutation (i.e. R175H, R248Q or R273H), with variable lengths of the amino acid sequence flanking the mutation. The mutant p53 polypeptide amino acid sequences were inserted into the active site of TrxA with flanking flexible Gly-Ser-Gly-Ser-Gly (SEQ ID NO:236) linkers separating the antigen sequence and the TrxA sequence. Shorter Gly-Ser-Gly linkers were also inserted between each mutant p53 sequence.

TrxA is a widely-used fusion partner inEscherichia coliexpression systems for enhancing protein expression levels, solubility and thermal stability, in which the active site (Cys33-Gly34-Pro35-Cys36) (SEQ ID NO:91) protrudes from the protein body into solution (LaVallie et al, 2000 Methods Enzymol 326:322-340; Young et al., 2012 Biotechnol. J. 7:620-634). The presence of a restriction (RsrII) site on the DNA sequence coding for this active site provides an insertion point for internal peptide fusions which can be presented on the surface of TrxA, and has been successfully exploited for production of antibodies by insertion of the antigen within the solvent-accessible loop on the TrxA scaffold (Barrell et al., 2004 Protein Expr. Purif. 33:153-159).

TrxA scaffold harbouring the mutant p53 (R175H, R273H and R248Q) tri-peptide sequences cloned into the pJexpress404 vector, were obtained from DNA2.0 (Menlo Park, CA, USA). The coding sequences were designed with a C-terminus His6-tag to facilitate protein purification by immobilized metal affinity chromatography (IMAC) and custom optimized toE. colipreferred codons. Authenticity of the synthetic coding fragments was verified by DNA sequencing.

The amino acid sequences for the immunogens used to raise the antibodies are shown inFIG.1A to1C.

Homology modeling predicted that inserting the mutant p53 antigen sequences into the active site of the TrxA scaffold offers a viable presentation strategy to increase the immunogenicity of the peptide sequence, with the mutant p53 antigen sequence extending away from the TrxA protein, and with the mutated residues exposed in the solvent accessible loop. The predicted 3D structure of the TrxR175H, TrxR248Q and TrxR273H immunogens by Swiss Model are shown (FIG.2A to2C), revealing the exposed, solvent accessible mutant p53 antigenic regions.

E. colihost strain BL21 (DE3) trxB (Novagen, Merck Millipore, Darmstadt, Germany) was used for the expression of the recombinant Trxp53mutant constructs. Mini-scale expression studies to determine solubility of the protein were performed as described in Liew et al. 2014 Biochimie 89, 21-29. For large scale purification, recovery soluble and insoluble peptides were recovered by native and denaturing IMAC with integrated on-column refolding into phosphate buffer respectively, performed as described in Liew et al. 2014. Protein quantitation was performed using the BCA assay (Pierce, Rockford, IL, USA) and purity was assessed by SDS-PAGE analysis.

Expression of the TrxR175H immunogen was induced inE. coli, and was expected to be of ˜18.8 kDa for TrxR175H, which was expressed in both the soluble and insoluble forms (FIG.3A), with higher partitioning into the insoluble fraction. Analysis by mass spectrometry of purified TrxR175H revealed an actual molecular mass of ˜18,700 Da, consistent with loss of the first methionine residue (calculated mass ˜18,707 Da) (FIG.4A). Previous reports have shown that TrxA fusions containing a Ser residue in the second position of its amino acid sequence allowed for efficient cleavage of the first methionine (131 Da), presumably by endogenous methionine aminopeptidase inE. coli(Liew et al., 2007). Similarly, TrxR273H was expressed primarily in inclusion bodies in the insoluble fraction, at the expected size of ˜18,200 Da (FIG.3C). The TrxR248Q was found to partition to the insoluble protein fraction, and migrated at an unexpectedly lower molecular weight position of 15 kDa on SDS-PAGE (FIG.3B), and mass spectrometric analyses revealed a mass of 17,352 Da (FIG.4B). The coding sequence of the construct was verified by DNA sequencing and the expressed protein could be purified by immobilized metal affinity chromatography (IMAC), which indicated that the His6 tag located at the carboxyl terminus of TrxR248W was intact and that the full length protein was translated, and suggested that the lower molecular mass may be a result of unexpected proteolytic cleavage byE. coliproteases, as has been demonstrated (Carrio et al., 1999 Biochim. Biophys. Acta 1434, 170-176; Corchero et al., 1997 Biochem. Biophys. Res. Commun. 237, 325-330), downstream of Ala20 at the N-terminus and corresponds to a 166-residue protein moiety and its sodium form (17,324/17,347 Da). In any case, the unexpected proteolysis byE. coliproteases highlights a distinct advantage of nesting the antigen sequence within TrxA instead of the terminal regions of the fusion protein.

Example 2: Initial Screening to Identify p53 Monoclonal Antibodies Against Specific p53 Mutants

Three groups of five, 8 week old Balb/c female mice (Biological Resource Center, Singapore) were inoculated with the Trxp53mutant peptides. The first immunisation was performed intraperitoneally with Sigma Adjuvant System (Sigma), followed by five intraperitoneal and subcutaneous injections at 3 week intervals. One week after the fourth immunization, blood was taken from each mouse via cheek bleed using a lancet (MEDIpoint International Inc.). Blood samples were centrifuged for 10 min at 1600 rpm and serum was aspirated and stored at 4° C., for subsequent enzyme-linked immunosorbent assay (ELISA) analyses against the full length R175H, R273H and R248Q mutant p53 proteins.

The mouse with the highest serum antibody titer was selected as the spleen donor for fusion. The selected mice (one for each p53 mutation) received a final boost by intravenous injection of the Trxp53mutant peptide without adjuvant. Mouse myeloma SP2/0 cell line was used as the fusion partner. One week before fusion, cells were cultured in RPMI (Gibco) and 10% FBS until they attained >70% confluency in the logarithmic phase. The spleen cells of the immune mice were removed under sterile conditions. Generation, selection and cloning of hybridoma cells were performed using the ClonaCell-HY Hybridoma Cloning kit (STEMCELL Technologies) according to the manufacturer's protocol.

Hybridoma clones secreting anti-mutant p53 mAbs were selected by ELISA with 96-well plates coated with recombinant full length p53 protein harboring the R175H, R273H and R248Q mutations respectively. Thioredoxin peptide was used as negative control. Supernatant collected from individual hybridoma wells were tested on ELISA plates. 10% fecal bovine serum (FCS) was used for blocking and antibody dilution. PBS with 0.05% Tween 20 was used for washes. After washing, IgGs were detected using 1:5000 goat anti-mouse IgG conjugated to HRP (Biorad) in PBST with 10% FCS. Plates were developed with 1×TMB ELISA substrate solution (Sigma). Absorbance was measured at 650 nm with EnVision Plate Reader (Perkin Elmer).

Data from at least three independent mice are presented for each mutation. Initial ELISA screening using the mutant or wild-type p53 protein or the mutant p53 peptide fragment revealed that for each of the hybridomas producing antibody recognising mutant p53, the antibodies were specific for the respective mutations and did not cross-react with the wildtype p53 protein (FIG.5A-5C). The antibodies were more sensitive to the whole mutant p53 protein as compared to the peptide fragments.

Further analyses using all peptides and mutant proteins to further confirm specificity also showed that the three hybridoma clones producing monoclonal antibody against the R175H mutant p53 were highly specific (clones 7B9, 10C8 and 4H5), and did not cross react with the R248Q or R273H mutant proteins and peptides (FIG.6A). Similar results were obtained with the clones against the R248Q (clones 4H2 and 3G11) and R273H (clone 13E4) (FIGS.6B and6C).

To determine the epitopes targeted by the individual hybridoma clones, peptide phage display analysis was performed using the antibodies against the three p53 mutants.

An M13 phage library (New England Biolabs) encoding random 12-mer peptides at the NH2 terminus of pIII coat protein (2.7×109 sequences) was used. 50 nm purified antibody was coated on 96 well maxisorp plates (Nunc). The wells were incubated with blocking buffer (PBS, 0.5% Tween20, 2% BSA) for 1 h at room temperature, washed with washing buffer (PBS, 1% Tween20, 2% BSA), and incubated in washing buffer at room temperature with 4×1010phages. Bound phages were eluted with 0.2 M glycine (pH 2.2) and neutralized with 1 M Tris (pH 9.1). The eluted phages were amplified according to the manufacturer's instructions.

The selection process was repeated for three cycles. Phage plaques from the final round were selected, amplified as described by the manufacturer and sequenced. The peptides displayed on the selected phages were deduced from analysis of results from DNA sequencing. Epitopes targeted by individual antibodies were obtained by determination of consensus sequences from alignment of peptide sequences using Clustal Omega multiple sequence alignment tool.

For the hybridomas against the R175H mutant p53, the consensus sequence was “HCPHH”, in which the first Histidine was the mutation that replaces the Arginine residue in the wildtype p53 (FIG.7A). Almost all clones from all hybridomas against R175H mutant p53 captured this sequence (FIG.8A). The consensus sequences for the R248Q antibody clones were “SV . . . HY” (positions 215-216 and 233-234;FIG.8A) for clone 3G11, and “RP” (positions 249-250;FIG.7B) for clone 4H2. The consensus sequence for anti-R273H clone 13E4 was “VH” (positions 272-273;FIG.7C).

To determine the crucial amino acids of the epitope, two sets of individually alanine substituted 13 amino acid peptides corresponding from Met169 to Arg181 (MTEVVRHCPHHER) of R175H mutant p53 protein, and Arg267 to Gly 279 (RNSFEVHVCACP) of R273H mutant p53 protein were chemically synthesised and obtained from Bio Basic Inc. Peptides were conjugated with an N-terminal Biotin and individually incubated on a 96 well Streptavidin coated ELISA plate (Pierce, Thermo Scientific) at 10 ug/ml for 1 hour. After three rounds of washing, the plates were incubated with anti-R175H 10C8, 4H5 and 7B9 and anti-R273H 13E4 respectively at 1 ug/ml for overnight at 4° C. The plates were incubated for an hour at 37° C. with secondary anti-mouse IgG-HRP after washing. After incubation, plates were washed three times prior to application of soluble HRP substrate for 5 minutes and absorbance at 650 nm was determined with Envision plate reader (Perkin Elmer).

The results for the alanine scans for the R175H antibody clones 4H5, 7B9 and 10C8 are shown inFIGS.9A-9C, and the results for the R273H antibody clone 13E4 are shown inFIG.9D. Mutations within p53 peptide sequences were identified as critical to the mAb epitope if they did not support reactivity of the test mAb but did support reactivity of other antibodies.

BALB/c mice were given a single 0.25_mL intraperitoneal (IP) injection of Incomplete Freund's Adjuvant (Sigma Chemical Co.). Fourteen days later, mice were injected with a single IP injection of 4×105in a volume of 0.5 mL of the hybridoma cells, after which they were examined daily for development of ascites fluid as determined by abdominal distention. Seven to ten days after the injection of hybridoma cells, mice were anesthetized and the ascites fluid was collected aseptically from anesthetized mice by abdominal paracentesis with an 18-22 gauge needle by gravity flow into sterile centrifuge tubes. Digital pressure was gently applied to the abdomen and the position of the mouse was altered as needed to facilitate removal of the ascites fluid. Ascites was pooled for each individual cage of mice. The isotype of the antibody clones was determined from hybridoma supernatant using a mouse mAb isotyping kit (Roche) according to the manufacturer's instructions. The ascitic fluids were diluted at a ratio of 1:10 with PBS and IgGs were purified via Protein G column chromatography (GE Healthcare). Antibody was eluted from the column through 5 ml of elution buffer containing 0.2M Tris-Glycine pH2.7. The eluted fractions were dialyzed against 0.05 mM PBS, pH 7.4. Confirmation of the purified antibodies was performed by SDS-PAGE under reducing conditions.

Example 3: Evaluation of Specificity of mAbs Against Specific p53 Hot-Spot Mutants by Biochemical Approaches

The first attempt to determine specificity of the p53 hot-spot mutant-specific antibodies were made by evaluating their effectiveness in immunoblot assays. p53 null H1299-cells stably expressing or transiently transfected with the six common hot-spot p53 mutations (R175H, R245S, R248Q and R248W, R249S, R273H and R282W) were used for the initial analysis.

50 μg of cell lysates were loaded into each well of a 4-12% Bis-Tris SDS polyacrylamide precast gels (Invitrogen). The protein marker used was Precision Plus Protein™ Standards Dual Colour (Bio-Rad, Hercules, CA). SDS-PAGE gels were ran at constant voltage of 60 volts (V) until the protein bands exceeded the stacking gel, after which the gel was continuously ran at 100V until the dye front reaches the bottom. For immunoblotting, protein transfer was carried out on the iBlot™ Drying Blotting system (Invitrogen) for 10 minutes at 20-25V onto nitrocellulose membranes. The membrane was washed three times for 10 minutes each with PBST (phosphate buffered saline (PBS) containing 0.05% Tween20 (Bio-Rad, Hercules, CA), and non-specific binding was blocked using 4% non-fat milk in PBST buffer for 1 hour with gentle agitation. Subsequently, the membrane was washed three times for 10 minutes each with PBST. Excess PBST after the washing step was removed before hybridoma supernatant was added. The primary antibody was incubated under gentle agitation at 4° C. overnight. The membrane was washed three times for 10 minutes each with PBST to remove unbound primary antibodies. 1:5000 goat anti-mouse IgG conjugated to HRP (Biorad) in PBST with 10% FCS was used for detection. The secondary antibody was incubated under gentle agitation for 1 hr at room temperature. Unbound secondary antibodies were washed off in the above mentioned manner before visualization using Clarity western blot ECL substrate (Biorad). Densitometric analysis was performed using Odessey Fc (Licor).

All three mutant-specific antibodies were able to detect their corresponding mutant p53 proteins expressed in H1299 cells, without detecting the other mutants or the wildtype p53 that were abundantly expressed, as determined using the pan-p53 antibody DO1 (FIG.11).

Of particular significance was the finding that the anti-R248Q antibody was unable to detect the closely related R248W mutant, which comprises a different mutation at the same residue, highlighting the very high specificity of the antibodies.

Immunoprecipitation analyses were also performed using the p53 mutant-specific antibodies, followed by detection with the anti-p53 rabbit antibody CM1, which again revealed that the antibodies were specific in bringing down only the respective mutant proteins (FIG.12). Reverse immunoprecipitation with CM1 antibody followed by immunoblotting with the mutant-specific antibodies also gave similar results, with extreme specificity (FIG.12).

The inventors next determined the ability of these mAbs to recognize endogenous p53 in a large number of human tumor cell lines that express the wild-type protein or the various hot-spot mutants (FIG.13). Direct immunoblotting with the mutant specific mAbs was able to detect only the respective mutant p53 proteins in the tumor cell lines, though all cell lines expressed large amounts the various mutant p53, or the wild-type p53 that was induced by UV-irradiation and detected by the DO1 antibody (FIG.14), confirming the specificity of the antibodies.

Immunoprecipitation of the endogenous proteins indicated the same trend for the R175H-specific antibody (FIG.15, left panel). Both the R248Q and the R273H antibodies were also able to specifically detect only their respective mutant proteins when used for immunoblot detection after the primary immunoprecipitation with the pan-p53 CM1 antibody (FIG.15, middle and right panels). However, when used directly for immunoprecipitation followed by immunodetection with CM1, these two antibodies detected some other p53 mutants as well, likely due to non-specific binding under the conditions tested. Nonetheless, the high level of specificity in direct immunoblotting and upon immunoprecipitation with a pan-p53 antibody highlights the specificity of these antibodies against their respective mutant p53 antigens.

Example 4: Specificity of mAbs Against Specific p53 Hot-Spot Mutants in Immunofluorescence Analyses

To test the specificity of the mutant-specific antibodies by immunofluorescence staining, the inventors again utilized the H1299-cells overexpressing the wild-type p53 or the various p53 mutants, or tumor cells lines that express endogenous mutant p53 proteins.

Fixed, transfected cells on 96 well plates were subjected to permeabilization with 0.4% Triton X-100 for 20 minutes. After rinsing with PBS, cells were blocked with 5% BSA in PBSTritonX (PBSTX) for 20 min, followed by incubation in hybridoma supernatant at 4° C. overnight. IgGs were detected using 1:1000 goat Alexaflor 488 Donkey anti-mouse IgG conjugated (Life technologies) in PBSTX with 1% BSA. Then, cells were counterstained with DAPI, and viewed with Incell Analyzer (GE Healthcare).

A distinct nuclear staining pattern was observed with the mutation-specific antibodies, which detected only their respective mutant proteins when overexpressed (FIG.16), or in the endogenous state in tumor cell lines (FIGS.17A and17B), though all cells expressed the various p53 forms in abundance, as determined by staining with either the DO1 or the CM1 antibodies.

As observed with analysis by immunoblot and immunoprecipitation, the antibody against the R248Q mutant was extremely specific, and was unable to detect the related R248W mutant protein (FIG.17B).

Example 5: Analyses of Human Tumor Samples Using mAbs Against the Specific p53 Hot-Spot Mutants by Immunohistochemistry

To determine the effectiveness of the p53 mutant-specific antibodies in paraffin-embedded tissues, the inventors analyzed a large number of human tumor samples by immunohistochemical (IHC) analysis. Firstly, a series of tumor samples with known p53 mutations, confirmed by DNA sequencing (FIG.18A), were examined using the mutant-specific antibodies.

Mouse tumour sections from HT29 xenograft mouse model with p53 R273H genotype and tumour sections generated from p53R172H mutant cell lines were processed into paraffin blocks by the Advanced Molecular Pathology Laboratory (AMPL), Institute of Molecular and Cell Biology. Wax sections of 5 μm were then embedded onto glass slides (Leica Biosystems) and dried for an hour on a 50° C. hot plate. Sections were deparaffinized in xylene (ChemTech Trading) and rehydrated through descending percentages of ethanol (ChemTech Trading) into water. Tissue sections were heated with Target Retrieval Solution, pH9 (Dako) for antigen exposure, then rinsed in PBS. Endogenous peroxidase was blocked with 2% (v/v) hydrogen peroxide (Merck) in PBS for 30 min, rinsed with PBS. Sections were blocked with 10% (v/v) goat serum (Dako) in PBS for 1 h then incubated with biotinylated primary antibodies clones at 4° C. overnight. Sections were washed with water then rinsed in PBS before detection with streptavidin-HRP antibody (BioLegend). Antigen-antibody interaction was then visualized using 3,3-diaminobenzidine as a substrate, and the sections were lightly counterstained with hematoxylin before dehydrating and mounting in Cytoseal 60 synthetic resin (Richard-Allan Scientific™, Fisher Scientific). Slides were imaged under bright field using the Axiolmager (Zeiss) light microscope and analyzed with AxioVision Rel 4.8 software (Carl Zeiss AG).

As was the case in the immunofluorescence analyses, the three mutant-specific antibodies stained the samples with the respective mutations in p53, but not the sample with either wild-type p53 or with other mutations (FIG.18B), demonstrating specificity in the IHC setting.

The inventors further evaluated several tumor microarrays from colon, breast (triple negative), lung, prostate and renal tumors by staining with these antibodies. Representative results from staining with the pan-p53 antibody suitable for IHC staining (DO7) and the R175H-specific antibodies are shown (FIG.19). Three groups of samples emerged: one that was stained both by DO7 and the R175H mAb; one that was only positive for DO7; and the last that was negative for both antibodies (FIG.21). The highest level of DO7 staining was for in the triple negative breast, lung and colon cancer groups, confirming previous data that these cancers have a higher mutation rate for p53 (Olivier et al., 2004 IARC Sci Publ 157:247-270), and are therefore positive for staining by anti-p53 antibodies. Staining by the R175H-mAb mirrored that of DO7 in the first group, suggesting that both the antibodies were recognizing the same cells in this group. A few samples were sequenced to determine the p53 mutational status, and found that all samples that were stained by both antibodies carried a R175H mutation. By contrast, samples that were stained by DO7 only had mutations in other residues of p53 but not on R175, and the samples that were negative for both antibodies had no mutations in p53.

Human triple negative breast cancer samples B41, B89, B98, B27 and B52 were stained with anti-p53 antibodies D07, 11D1, or the anti-R175H antibody for analysis by immunohistochemistry. Tumor samples B41, B89 and B98 comprising the R175H mutation (CAG—underlined) were positive for staining by all three antibodies (FIG.20A). Tumor sample B27 not comprising the R175H mutation (CGC—underlined) was only stained by the anti-p53 antibodies DO7 and 11D1 (and not by the anti-R175H antibody), and sample B52 was not stained by any of the anti-p53 antibodies (FIG.20B).

These data collectively indicate the specificity of these mutant-specific antibodies in paraffin-embedded clinical samples.

Example 6: Comparison of Effectiveness of the Human p53 Mutant-Specific Antibodies with Equivalent Mouse Mutants

Finally, the inventors investigated if the antibodies are able to detect the corresponding mouse mutations. The human sequences corresponding to the three mutations studied here are highly homologous in mouse p53 (FIG.22), and the sequences around the R175 residue are similar to the equivalent mouse R172 residue (Olive et al, 2004 Cell 119, 847-860). Hence, the inventors utilized mouse embryonic fibroblasts (MEFs) from the R172H mice, which contain an equivalent mutation to the R175H in humans (Lang et al., 2004 Cell 119, 861-872), or MEFs with an unrelated mutation R246S, which is equivalent to the human R4249S (Lee et al., 2012 Cancer Cell 22, 751-764).

Direct immunoblotting indicated that the anti-R175H antibody was able to detect only the mutant p53 from the R172H MEFs, but not from the R246S or the wild-type MEFs, although these latter cells expressed significant amounts of p53 as determined by the pan-p53 antibody, CM5 (FIG.23A). Immunoprecipitation with the R175H-specific antibody followed by immunoblotting also demonstrated that the antibody was indeed specific and can detect the mouse R172H mutant protein (FIG.23B). The inventors also tested the ability of the R175H-specific antibody by immunofluorescence (FIG.23C) and IHC (FIG.23D) analyses, which confirmed its specificity, highlighting the utility of these antibodies in different species.

Immunostaining for IHC analyses was performed on formalin fixed paraffin-embedded (FFPE) 5 μm sections. Antigen retrieval was performed using with Dako Tris/EDTA target retrieval solution, pH9. Blocking was performed with DAKO 10% goat serum. Secondary antibody was Dako Envision™+/HRP. Develop with DAKO liquid DAB+. Images were captured with a Zeiss Axiolmager upright microscope using 40× objective lens.

Example 7: Monoclonal Anti-Mutant p53 Antibodies

7.1 R175H Mutant p53 Antibody Clones 4H5, 7B9 and 10C8

Anti-R175H p53 mouse monoclonal antibodies were raised by immunising mice with immunogen comprising three copies of the R175H p53 mutation inserted in the active site sequence of TrxA, as described in Example 1 (seeFIG.1A).

Hybridoma clones producing anti-R175H p53 mouse monoclonal antibodies were obtained. The amino acid sequences of the light and heavy chain variable domain sequences were determined and are shown inFIGS.24and25. The DNA sequences encoding the light and heavy chain variable domain sequences for the antibodies are shown inFIG.27.

The CDRs were predicted using VBASE2 (Retter et al. Nucleic Acids Research (2005) 33 (Database issue): D671-674, incorporated by reference hereinabove).

7.2 R248Q Mutant p53 Antibody Clones 3G11 and 4H2

Anti-R248Q p53 mouse monoclonal antibodies were raised by immunising mice with immunogen comprising three copies of the R248Q p53 mutation inserted in the active site sequence of TrxA, as described in Example 1 (seeFIG.1B).

Hybridoma clones producing anti-R248Q p53 mouse monoclonal antibodies were obtained. The amino acid sequences of the light and heavy chain variable domain sequences were determined and are shown inFIGS.28and29. The DNA sequences encoding the light and heavy chain variable domain sequences for the antibodies are shown inFIG.31. The CDRs were predicted using VBASE2 as above.

7.3 R273H Mutant p53 Antibody Clone 13E4

Anti-R273H p53 mouse monoclonal antibodies were raised by immunising mice with immunogen comprising three copies of the R273H p53 mutation inserted in the active site sequence of TrxA, as described in Example 1 (seeFIG.1C).

A hybridoma clone producing anti-R273H p53 mouse monoclonal antibodies was obtained. The amino acid sequences of the light and heavy chain variable domain sequences were determined and are shown inFIGS.32and33. The DNA sequences encoding the light and heavy chain variable domain sequences for antibody clone 13E4 are shown inFIG.34.

The CDRs were predicted using VBASE2 as above.

Example 8: Characterisation of Monoclonal Anti-Mutant p53 Antibodies

8.1 R175H Mutant p53 Antibody Clones 4H5, 7B9 and 10C8

Western blot analysis was performed on cell extracts obtained from R172H mouse thymic lymphoma cell line cells, T47D cells, WiDr cells and DKO cells using cell culture supernatant of hybridoma clones 4H5, 7B9 and 10C8. The results are shown inFIG.35. Antibodies from each clone were specific for R175H mutant p53.

Immunofluorescence analyses were also performed using R172H mouse thymic lymphoma cell line cells, TKO cells, C6 cells, H1299 cells, T47D cells, and H1299 cells transfected with a construct expressing R175H mutant p53, using cell culture supernatant of hybridoma clones 4H5, 7B9 and 10C8. The results are shown inFIG.36A to36C. Antibodies from each clone were specific for mouse R172H mutant p53.

The epitope recognised by the antibodies 4H5, 7B9 and 10C8 is shown in the context of human R175H p53 inFIG.10.

The antibody clones were further investigated for their ability to recognise R175H p53 by immunohistochemical (IHC) analysis of mouse intestinal tissue sections obtained from p53 knockout mice or irradiated, R172H p53 positive mice, using cell culture supernatant of hybridoma clones 4H5, 7B9 and 10C8. The results are shown inFIG.37. The antibodies were able to detect R172H mutant p53 in mouse intestinal tissue sections by IHC.

Antibody clone 4H5 was further analysed for ability to visualise and monitor R175H-positive cancer in vivo. Mouse tumour cells derived from P53R172H/R172Hmice were transfected with luciferase gene and used to establish a tumour model. Mice were injected IV with 100 μg of XenoLight CF750-labelled anti-R175H mutant p53 antibody clone 4H5, and imaged by IVIS analysis at 6 h, 24, 72 h and 7 days post-injection. The results of the experiment are shown inFIG.38. The antibody was demonstrated to be suitable for visualisation and monitoring tumour in vivo.

8.2 R248Q Mutant p53 Antibody Clones 3G11 and 4H2

Western blot analysis was performed on cell extracts obtained from TKO cells, and HCC70 cells (which possess the R248Q mutation) using cell culture supernatant of hybridoma clones 3G11 and 4H2. The results are shown inFIG.39. The antibodies were specific for R248Q mutant p53.

Immunofluorescence analyses were also performed using TKO cells, HCC70 cells and OVCAR3 cells (which possess the R248Q mutation), using cell culture supernatant of hybridoma clones 3G11 and 4H2. The results are shown inFIGS.40A and40B. Antibodies from each clone were specific for R248Q mutant p53.

The epitopes recognised by antibody clones 3G11 and 4H2 were analysed by peptide phage display analysis (FIGS.8B and7B). The epitopes recognised by the antibodies are shown in the context of human R248Q p53 inFIG.10.

8.3 R273H Mutant p53 Antibody Clone 13E4

Western blot analysis was performed on cell extracts obtained from T47D cells and MB468 cells (which possess the R273H mutation) using cell culture supernatant of hybridoma clone 13E4. The results are shown inFIG.41. Antibody from clone 13E4 was specific for R273H mutant p53.

Immunofluorescence analysis was also performed using T47D cells, and WiDr cells expressing R273H mutant p53, using cell culture supernatant of hybridoma clone 13E4. The results are shown inFIG.42. Antibody from clone 13E4 was specific for R273H mutant p53.

The epitope recognised by 13E4 antibody is shown in the context of human R273H p53 inFIG.10.

Antibody clone 13E4 was further analysed for ability to visualise and monitor R273H-positive cancer in vivo. Cells of the p53-R273H-mutant HT29 tumour cell line were transfected with luciferase gene and used to establish a tumour model. Mice were injected IV with 100 μg of XenoLight CF750-labelled anti-R273H mutant p53 antibody clone 13E4, and imaged by IVIS analysis at 72 hours post-injection. The results of the experiment are shown inFIG.43. The antibody was demonstrated to be suitable for visualisation and monitoring tumour in vivo.

Example 9: Chimeric Mouse Fv-Human IgG1 Fc Anti-Mutant p53 Antibodies

Mouse Fv-Human IgG1 Fc chimeric versions of the anti-mutant p53 antibodies were prepared.

Variable regions of the heavy and light chains were cloned from parental 4H5, 7B9, 10C8, 3G11, 4H2 and 13E4 mouse monoclonal antibody clones into pTT5 vectors each containing the human IgG1 constant region.

Mouse-human chimeric heavy- and light-chain plasmids were co-transfected into HEK293-6e cells at 1 g total plasmid per million cells, using 2 μL of 293-fectin transfection reagent per μg of plasmid. Culture supernatant containing secreted chimeric antibodies was harvested and purified using protein G agarose beads, 4 to 6 days post-transfection.

Chimeric antibody was eluted off beads using 0.1 M glycine-HCl (pH 2.7) neutralised with 1 M Tris (pH 9.0) and dialysed into PBS.

The chimeric antibodies were determined to be able to recognise their respective mutant p53 by ELISA (test concentration 1 ng/μL).

Mouse-Fv-Human IgG1 Fc chimeric versions of anti-R175H p53 antibody clones 4H5, 7B9 and 10C8 were also investigated for ability to recognise R175H p53 by IHC analysis of mouse intestinal tissue sections obtained from irradiated, R172H p53 positive mice. The results are shown inFIG.44. Chimeric versions of the antibodies were able to detect R172H mutant p53 in mouse intestinal tissue sections by IHC.

Example 10: Analysis of the Ability of Anti-p53 Mutant Antibody to Treat Cancer In Vivo

The inventors investigated the ability of the monoclonal anti-mutant p53 antibodies to treat cancer in vivo.

Briefly, mice were injected with 5×106SKBR3 cells (human breast cancer cells) carrying a luciferase gene. SKBR3 cells carry the R175H mutation in p53 (see e.g.FIG.13). The luciferase gene allowed monitoring of tumor growth in vivo, by detection of luciferase activity. Four days after mice have been injected with the SKBR-3 cells, mice were treated either with 100 μl of anti-R175H antibody, or 100 μl of IgG antibody control. Injections were repeated every four days (as indicated by the arrows inFIG.45). Tumor growth was monitored throughout the experiment by measuring luciferase luminescence. The results are shown inFIG.45, which demonstrates an anti-cancer effect in mice treated with the anti-R175H antibody, as evidenced by reduced levels of luciferase activity as compared to the control treatment group.

Example 11: Analysis of the Ability of the Immunogens to be Used to Generate an Immune Response to Mutant p53 Polypeptides

The inventors also investigated the ability of the immunogens described in Example 1 to be used as vaccines, for stimulating an immune response against p53 mutant polypeptides.

Groups of mice were immunised with the immunogens, and the polyclonal antibody response was analysed by ELISA to determine whether the immunogens can be used as vaccines to trigger antibody responses.

The results are shown inFIGS.46A-46E. Immunization with the different mutant p53 immunogens is shown to induce the production of antibodies which are highly specific for peptide corresponding to the respective p53 mutant (FIGS.46A,46C and46E), and which display minimal interaction with wildtype human p53 (FIGS.46B,46D and46F). These results demonstrate that the mutant p53 immunogens are capable of triggering an antibody response, and that this immunity is specific to the respective p53 mutant.

The inventors next investigated whether the antibodies generated in response to immunisation with the immunogens were capable of recognising mutant p53 polypeptides. Sera was obtained from mice injected with the R175H, R248Q or R273H immunogens described in Example 1, and analysed for ability to recognise mutant p53 polypeptides by immunofluorescence analysis. The results are shown inFIGS.47A-47C, which demonstrate that the antibodies generated in mice in response to immunisation with immunogen were capable of recognising the corresponding mutant p53 polypeptide.

Example 12: Conclusion

The inventors have successfully generated p53 mutant-specific antibodies against three commonly-occurring p53 hot-spot mutations in the DBD region—the R175H, R248Q and R273H (Vikhanskaya et al., Nucleic Acids Res (2007) 35:2093-2104). The antibodies are characterised, and their utility in a variety of biochemical and histological assays is demonstrated, as is their usefulness to treat cancer in vivo.

The inventors have for the first time been able to generate antibodies capable of specifically binding to single point mutants of p53, which do not cross-react with wildtype p53, raised using immunogen in which antigen expression is enhanced by the provision of multiple copies of the region containing the mutation, displayed by protrusion from the protein body into solution, using TrxA as a fusion partner. This approach consistently led to the generation of mAb clones against several p53 mutants, with high specificity and selectivity.

The present experimental examples demonstrate generation of antibodies to three of the most common hot-spot mutations found in p53, namely R175H, R248Q and R273H. The mAbs generated against these mutants were specific in their ability to discern between the intended antigens and other mutations or the wildtype p53 protein, in a variety of techniques, ranging from immunoblotting, immunoprecipitation, immunofluorescence and immunohistochemistry. The inventors have moreover demonstrated the ability to inhibit growth of tumor cells comprising the corresponding p53 mutation in a human xenograft cancer mouse model in vivo.

Furthermore, the antibodies were able to detect the corresponding mutations in other species such as mouse, as demonstrated with the R175H mutation, and thus, should be applicable to the other mouse mutants, given the sequence conservation across species (see e.g.FIG.22), thereby proving to be valuables tools for fundamental research.

The mutant-specific antibodies are useful tools to dissect out the individual and combined roles of both the wild-type and mutant p53 proteins in the same cell, potentially even at the single cell level, and during the clonal evolution of the cancer cell.

The utility of the mutant-specific mAbs for IHC analysis of human tumor samples highlights that these mAbs are very useful tools in pathological analyses in determining p53 status, which could be easily implemented and is significantly cost effective compared to DNA-sequencing technologies.

The TrxA presentation system utilised in the present examples will be useful for generating mAbs against other mutations in p53 which can be of clinical utility, and also for generating mAbs against other mutations found in tumor suppressors and oncogenes. Monoclonal antibodies which are able to discriminate between proteins differing by only a single amino-acid could be clinically useful as diagnostic and therapeutic agents.

Moreover, the inventors have demonstrated the usefulness of the immunogens for vaccination strategy, demonstrating the ability to induce an antibody response capable recognising mutants of p53.

In a therapeutic context, such mutant-specific antibodies are likely to be very safe as they will not have any side-effects in normal cells of patients that do not carry the mutation. It is expected that such antibodies would be superior to the currently available general antibodies against proteins that are either overexpressed or deregulated in disease.

Example 13: Effect of Mutant-Specific Anti-p53 Antibody on Tumour Growth In Vivo

The inventors next investigated the effect of administration of a monoclonal p53 mutant-specific antibody on growth of cancer cells in in vivo.

Briefly, SCID mice were injected subcutaneously at their flanks with 5×106luciferase labelled SKBR cells (which carry the R175H mutation in p53-see e.g.FIG.13)). From day 4, mice were injected intravenously every 4 days with 100 μl of monoclonal antibody specific for the R175H mutant p53 (a175) or isotype control antibody (IgG). Tumour growth was monitored by measuring luciferase activity.

The results of the experiment are shown inFIG.48. A strong anti-cancer effect was observed in mice treated with the monoclonal antibody specific for the R175H mutant p53.

Example 14: Preparation of Mouse-Human Chimeric Anti-p53 Mutant Antibodies

The inventors prepared chimeric mouse-human versions of the anti-R175H antibody clones 4H5, 7B9 and 10C8, the anti-R273H antibody clone 13E4 and the anti-R248Q antibody clones 3G11 and 4H2.

DNA encoding the variable heavy and light chains of the parental mouse monoclonal antibodies were cloned from parental mouse monoclonal antibodies into separate pTT5 vectors each containing the human constant region. Mouse-human chimeric heavy- and light-chain plasmids were co-transfected into HEK293-6e cells at 1 μg total plasmid per 1×106cells, using 2 μl of 293-fectin transfection reagent per microgram of plasmid.

4-6 days after transfection, the cell culture supernatant containing the secreted mouse-human chimeric antibodies was harvested, and the antibodies were purified using protein G agarose beads. The chimeric mouse-human antibodies were eluted off beads using 0.1 M glycine-HCl (pH 2.7) neutralised with 1 M Tris (pH 9.0) and dialysed into PBS. The mouse-human chimeric antibodies comprise mice Fv and human Fc.

Mouse-human chimeric anti-p53 mutant R175H antibody clone VL sequences are shown inFIGS.49and51, and the VH sequences are shown inFIGS.50and52.

Mouse-human chimeric anti-p53 mutant R273H antibody clone 13E4 VL sequence is shown inFIGS.53and55, and the VH sequence is shown inFIGS.54and56.

Mouse-human chimeric anti-p53 mutant R248Q antibody clone VL sequences are shown inFIGS.57and59, and the VH sequences are shown inFIGS.58and60.

Example 15: Characterisation of Mouse-Human Chimeric Anti-p53 Mutant Antibodies

Mouse-Human Chimeric Anti-p53 R175H Antibodies

The mouse human chimeric anti-p53 R175H antibodies were analysed by ELISA for binding to R175H mutant p53.

The results are shown inFIG.61. The chimeric anti-p53 R175H antibodies (MH 4H5, MH 7B9, MH 10C8) antibodies produced positive signals between 0.1 and 1 ng/μl concentrations, bound specifically only to human p53 R175H full length protein and not human p53 R273H full length, human p53 wild-type full length, or mouse p53 wild-type full length protein. Commercial anti-p53 antibody (1C12) was included as a positive control antibody.

Binding was also analysed by western blot, and the results are shown inFIG.62. Detection of denatured endogenous p53 in human cell lines via western blot (antibodies were used at a concentration of 1 ng/μl) confirmed the specificity of the chimeric anti-p53 R175H antibodies (MH 4H5, MH 7B9, MH 10C8). The antibodies only detected p53 from the SKBR3 cell line which harbours the R175H mutation in p53, and not from the p53-null H1299 cell line, the wildtype 53 cell lines MCF7 and A549, nor the R273H cell lines A431 and SW480.

The antibodies were further analysed for their ability to bind specifically to R175H mutant p53 by immunohistochemical analysis of binding to different cancer cell lines fixed in 4% paraformaldehyde and embedded in paraffin. The mouse human chimeric anti-p175H antibodies were shown only to stain SKBR3 cells. Representative images from the analysis using MH 7B9 are shown inFIG.63.

Mouse-Human Chimeric Anti-p53 R273H Antibody

The mouse human chimeric anti-p53 R273H antibody 13E4 was analysed by ELISA for binding to R273H mutant p53.

The results are shown inFIG.64. The chimeric anti-p53 R175H antibody MH 13E4 bound to human p53 R273H full length protein much more than it bound to thioredoxin, human p53 R175H full length and human p53 wild-type full length protein.

Binding was also analysed by western blot, and the results are shown inFIG.65. Detection of denatured endogenous p53 in human cell lines via western blot (antibodies were used at a concentration of 1 ng/μl) confirmed the specificity of MH 13E4, which only detected p53 from the A431 and SW480 cell lines which harbour the R273H mutation in p53, and not from the p53-null H1299 cell line, the wildtype 53 cell lines MCF7 and A549, nor the R175H cell line SKBR3.

The antibody was further analysed for ability to bind specifically to R273H mutant p53 by immunohistochemical analysis of binding to different cancer cell lines fixed in 4% paraformaldehyde and embedded in paraffin. MH 13E4 was found only to stain cells harbouring the R273H mutation (i.e. A431 cells)—seeFIG.66.

Mouse-Human Chimeric Anti-p53 R248Q Antibodies

The mouse human chimeric anti-p53 R248Q antibodies were analysed by ELISA for binding to R248Q mutant p53. The antibodies were used in the experiments at at final concentration of 1 ng/μl.

The results are shown inFIG.67. The chimeric anti-p53 R248Q antibodies (MH 3G11 and MH 4H2) antibodies were found to bind to full-length R248Q p53, and not to full-length R175H or R273H p53, or full-length wildtype human or mouse p53. Commercial anti-p53 antibody (1C12) was included as a positive control antibody.

Example 16: Evaluation of Anti-p53 Mutant Antibodies as Diagnostic Antibodies for Tumor Imaging In Vivo

The inventors next investigated whether anti-p53 mutant antibodies were useful for tumor imaging in vivo.

Briefly, 100 μg of fluorescently-labeled R273H specific mAb MH 13E4 (or fluorescently labelled IgG control), was injected intravenously into mice bearing HT29 tumors (which harbour the R273H mutation in p53). Mice were imaged using the IVIS Sprectrum in vivo imaging system for trafficking mAbs 72 hours after antibody injection.

The results are shown inFIG.68. MH 13E4 specifically detected R273H mutant p53-positive HT29 xenograft tumours in nude mice.

In a separate experiment, 100 μg of fluorescently-labeled R175H specific mAb MH 4H5 or MH 7B9, was injected i.v. into mice bearing R175H mutant p53-positive clone32 tumors. Mice were imaged using the IVIS Sprectrum in vivo imaging system for trafficking mAbs at 6 h and 24 h, and on Days 2, 3 and 7 following antibody injection. The clone 32 cells express luciferase, and so the location of the tumor cells could be analysed by detection of luciferase activity.

The results are shown inFIG.69. MH 4H5 and MH 7B9 antibodies detected R175H mutant p53 and were retained in the tumours for up to 7 days. Specificity of the antibodies for the tumour cells is demonstrated by detection of luciferase at the same position as the antibodies.

In a further experiment, it was investigated whether anti-p53 mutant R175H antibodies could detect spontaneously arising R175H p53 tumours. 100 μg of fluorescently-labeled R175H specific mAb MH 4H5 was injected i.v. into mice having the murine R172H mutation in murine p53. Mutant p53R172Hmice are highly susceptible to the spontaneous development of tumours harbouring the R172H mutation in murine p53. Mice were imaged using the IVIS Sprectrum in vivo imaging system for trafficking mAbs on Day2 and Day3 following antibody injections.

The results are shown inFIG.70. MH 4H5 was able to detect spontaneously occurring mutant murine p53 R172H tumours.

Example 17: Evaluation of Therapeutic Utility of Anti-p53 Mutant Antibodies to Treat Cancer In Vivo

The inventors next investigated whether the anti-p53 mutant antibodies were useful as a treatment for cancer in vivo.

Anti-p53 Mutant R175H Antibodies

In a first experiment, the therapeutic effect of administration of the monoclonal antibody 13E4 was analysed in a HT 29-luciferase xenograft tumor model. Briefly, nude Balb/c mice (n=3) were subcutaneously inoculated with 5×106HT29-luc cells on Day 0, and followed by i.v. injection of 15 mg/kg of control IgG or 13E4 mAb on Days 3, 7, 11, 14, 18 and 21. Mice were analysed at Day 28. Tumour growth was determined by measuring average photon intensity.

A schematic representation of the treatment schedule is shown inFIG.71A. The results of the experiment are shown inFIG.71B. Mice treated with 13E4 mAb showed an inhibition of 81.62% tumour size as compared to mice treated with IgG control antibody.

In a second experiment, the therapeutic effect of administration of the monoclonal antibody 13E4 was analysed in a HT 29-luciferase xenograft tumor model. Briefly, nude Balb/c mice (n=3) were subcutaneously inoculated with 5×106HT29-luc cells on Day 0 on each flank, and followed by i.v. injection of 10 mg/kg of control IgG or 13E4 mAb on Days 4, 7, 11, 14, 18, 21, 25, 28, 32 and 35. Mice were analysed on Days 7, 14, 21, 28 and 35. The tumour volume was measured every week following inoculation by luminescence imaging of luciferase expressing HT29 tumour cells. At the end of the experiments tumours were excised from mice and the mass of the tumours was recorded.

A schematic representation of the treatment schedule is shown inFIG.72A, and the results of the experiment are shown inFIGS.72B,72C and72D. Mice treated with 13E4 mAb showed an inhibition of 87.3% tumour size as compared to mice treated with IgG control antibody, and the tumor mass was approximately 2.5 time less than the weight of tumours obtained from mice treated with control IgG.FIG.72Dshows inhibition of tumor growth by 13E4 over time.

In a further experiment, the anti-p53 mutant R175H antibody 4H5 was analysed for its ability to inhibit growth of spontaneously-occurring murine p53 mutant R172H-positive cancer. Briefly, a mouse tumor cell line, clone32, was generated from p53R172H mutant mouse. 3×106clone32 cells were injected into syngeneic B6 mice on Day 0, and 100 μg of 4H5, 7B9 (mAb specific to R175H), 13E4 (specific to R273H) and 11D10 (reactive to both human and mouse p53) were i.v. injected into mice on Days 3, 6, 9, 12, 15, 18, 21 and 24. Mice were sacrifice on Day 25 for analysis and tumor measurement.

A schematic representation of the treatment schedule is shown inFIG.73A, and the results of the experiment are shown inFIG.73B. The average of tumor weight in 4H5 (R175H mAb) treated mice was 75% less than the weight of tumors in the control IgG treated group. 7B9 (R175H mAb), 13E4 (R273H mAb), 11D10 (p53 mAb) did not show significant effects on the weight on the syngeneic mouse tumor.

Example 18: Evaluation of Immunogens Used to Raise p53 Mutant Antibodies as Vaccine Candidates

The inventors next investigated whether the immunogens used to raise the p53 mutant-specific antibodies of the present invention were capable of vaccinating subjects against the development of p53 mutant cancers.

Wildtype BALB/C or B6 mice and mutant p53R172H mice were injected with TrxR175H protein (see Example 1) on Days 0, 21, 42, 63 and 84. Serum was collected 7 days after each injection and analysed by ELISA, cell staining and western blot. Antigens used for ELISA analysis were thioredoxin protein (Trx), Trx-R175H protein or the full-length R175H mutant p53 protein. On day 87, p53 R175H-positive tumor cells were injected into the mice.

A schematic representation of the experimental procedures is shown inFIG.74A. The results of the ELISA analysis are shown inFIG.74B, and show that serum obtained at the indicated time points (after the first and second immunizations) reacted with TrxR175H protein and the full-length R175H mutant p53 protein.

FIGS.74C and74Dshow the results of staining of SKBR3 cells (which harbour the R175H mutation) and TKO cells (p53 null) with serum obtained from the first and second bleeds from 10 different mice (M #1-M #10). Antibody in the serum of TrxR175H immunized mice showed positive staining on p53R175H expressing SKBR3 cells but not the p53 knock out TKO cells.

FIG.74Eshows the results of western blot analysis of reactivity of anti-p53R175H antibodies in the serum of mice immunised with TrxR175H against cell lysates from SKBR3 cells. Serum was used at a 1:1000 dilution. Lanes A-E contain serum from five different mice, and lane F is a positive control containing antibody DO1. The results show that after the second injection all of the mice analysed contained antibody specific for full-length R175H mutant p53 protein.

FIG.74Fshows the results of the analysis of the levels of cells of different immune cell subsets following immunisation with Trx, TrxR175H or PBS. 3 days after the 5th injection mice were sacrificed and splenocytes were analysed by flow cytometry. The percentage of T cells was found to be increased by immunisation with TrxR175H or Trx as compared to PBS in p53R172H/R172Hmutant mice.

Taken together, the ELISA, cell staining, western blot and flow cytometry data demonstrate that TrxR175H can effectively elicit both T cell and B cell responses to mutant R175H p53.