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WO2013037714A1 - BIOMARQUEURS POUR LES TUMEURS DU SEIN DÉRIVÉS DE PEPTIDES ASSOCIÉS À Hsp70 - Google Patents

BIOMARQUEURS POUR LES TUMEURS DU SEIN DÉRIVÉS DE PEPTIDES ASSOCIÉS À Hsp70 Download PDF

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Publication number
WO2013037714A1
WO2013037714A1 PCT/EP2012/067565 EP2012067565W WO2013037714A1 WO 2013037714 A1 WO2013037714 A1 WO 2013037714A1 EP 2012067565 W EP2012067565 W EP 2012067565W WO 2013037714 A1 WO2013037714 A1 WO 2013037714A1
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hsp70
peptide
peptides
tumour
cancer
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Ursula Bond
Christina SIEBKE
Tharappel JAMES
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College of the Holy and Undivided Trinity of Queen Elizabeth near Dublin
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College of the Holy and Undivided Trinity of Queen Elizabeth near Dublin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G01N33/575—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

  • the present invention relates to methods of identifying tumour-specific peptide biomarkers, to peptides identified by these methods, and to methods of using these peptides in diagnostic assays and as targets in anti-tumour vaccine development.
  • the invention provides a panel of synthetic peptides derived from peptides associated with Heat Shock Protein 70 (HSP70) from cancer cell lines. Such peptides find use in the diagnosis and treatment of breast cancer.
  • HSP70 Heat Shock Protein 70
  • Cancer is the leading cause of death worldwide accounting for approximately 7 million deaths annually (WHO, world health statistics, 2008). Amongst women, breast cancer is the leading cause of cancer mortality, accounting for 16% of cancer deaths in adult women. Approximately one third of women diagnosed with breast cancer will succumb to the disease. While new forms of treatment have increased survival rates, there is an ongoing need for improvements in early cancer diagnosis and new treatment modalities.
  • Phage display technology has also been used to identify small peptides with tumour- specific binding (reviewed [1]).
  • a wide variety of peptides have been identified by in vitro or in vivo biopanning, which interact with protein or carbohydrate moieties overexpressed in tumour cells. While such approaches have identified a myriad of peptides, in the majority of cases (>80%), either poor discrimination between tumour and non-tumour cells or poor in vivo targeting limit their use as biomarkers.
  • the lack of discrimination between tumour and non-tumour cells most likely reflects the fact that while many tumour-associated biomarkers may be upregulated in tumour cells, few if any truly unique tumour specific biomarkers are present on the cell surface.
  • In vivo biopanning against xenografted tumours has also revealed a bias towards the identification of peptides specific to tumour vasculature rather than to the tumour itself.
  • Hsps Heat shock proteins
  • Hsps Heat shock proteins
  • Hsps are up-regulated in many tumour cells as a result of the induction of an adaptive stress response to adverse physiological and environmental conditions within the tumour microenvironment.
  • Hsps play key roles as molecular chaperones within the cell, assisting in protein folding and unfolding, protein transport and localisation and targeted proteasomal degradation of error-containing and/or misfolded proteins.
  • Hsps also interact with a number of client proteins either protecting them from proteasomal degradation or maintaining them in an inactive state.
  • a number of steroid receptors and key effectors of the apoptotic pathway belong to this latter category of proteins.
  • the interaction of Hsps with client proteins appears to assist in tumour growth and survival by inhibiting many anti-apoptotic pathways
  • Hsp70 is a highly conserved family of proteins that play essential cellular roles as protein chaperones during normal cellular growth and following exposure to environmental stresses. Through its substrate-binding domain (SBD), Hsp70 binds to a wide array of substrates, such as newly synthesised proteins, exposed regions of partially unfolded proteins or protein degradation products. Hsp70 also possesses ATPase activity and interacts with ATP through its nucleotide-binding domain. Hydrolysis of ATP to ADP increases the affinity of peptide substrates for the SBD.
  • SBD substrate-binding domain
  • Hsp70 While intracellular Hsp70 contributes to tumour growth and survival, counteractively, when present extracellularly, Hsp70 is capable of mounting potent adaptive and innate immune responses against the tumour cells from which it originated. Hsp70 and its associated peptides are taken up by antigen presenting cells (APCs) via specific receptors. The associated peptides are re-presented to T-cells by MHC class I molecules to elicit a CD8+ response against the tumour. It is solely the associated peptides that are responsible for this T-cell response as Hsps stripped of peptides are ineffective at stimulating adaptive immune responses.
  • APCs antigen presenting cells
  • Hsps can also directly activate T- lymphocytes through Toll-like 2 receptors on APCs and can activate the cytolytic and migratory activities of natural killer (NK) cells.
  • NK natural killer
  • Hsp70 Since Hsp70 is over-expressed in many tumours, harbours a large repertoire of antigenic as well as non-antigenic peptides and acts at the crossroads of many key intracellular processes, it represents a unique barcode of cellular activity in a cancer cell.
  • Hsp70-peptide complexes Hsp70-PCs
  • Our results show that Hsp70-PCs from different breast tumour cell lines interact with unique sets of peptides within the phage display library with little overlap.
  • a further object of the invention is to identify tumour specific biomarkers that can be used in the diagnosis of breast cancers and other forms of cancer.
  • a still further object of the invention is to provide a method of identifying anti-cancer therapeutics, utilising the novel peptides described herein, or which are identifiable by the method of the invention. It is a further object of the invention to develop methods and agents for targeted tumour destruction.
  • the identification of peptides with enhanced binding to tumour cells provides a vector for delivery of biological agents such as chemotherapeutic drugs, antibodies, radionucleotides, cell poisons, pro-drugs, immunostimulatory chemokines and other such biologies directly to tumour tissue for the specific purpose of destroying the tumour without systemic damage to the patient.
  • biological agents such as chemotherapeutic drugs, antibodies, radionucleotides, cell poisons, pro-drugs, immunostimulatory chemokines and other such biologies directly to tumour tissue for the specific purpose of destroying the tumour without systemic damage to the patient.
  • a further object of the invention is the identification of cellular proteins that are over- expressed in tumour cells which represent potential biomarkers for that specific tumour.
  • a peptide selected from the group consisiting of: ISTHGPL, TQRPDKS, STLNVLQ, TQKWPPH, HPLIHPD, APNETRS, TSIGTHS, LEGSKRS, STNISPM, THLPLRL, TQRPDKS, PRTSNPH, QDSKQNR, NRATLST, KVHNRFA, DPLNMYT, NIGKPHQ, TPTRLHY, DRVKSNK, NGNYSHS, NTKLHQS, HSGYLKS, NSPLNPS, HPQYKYL, DGPSRPM, NMVESRF, NYTLTST, and GHISRHL.
  • peptides ISTHGPL TQKWPPH, STLNVLQ, HPLIHPD and APNETRS are particularly preferred.
  • Abbreviations are as given by the lUPAC-IUB Joint Commission on
  • the peptide may be labelled.
  • the label may be a fluorescent label, biotin, horseradish peroxidase or the like.
  • the invention also provides an antibody raised against any of the peptides listed above.
  • the antibody may be a monoclonal or polyclonal antibody.
  • the antibody may also be labelled with a label as described above.
  • the invention further provides a diagnostic assay for determining susceptibility to or the existence of cancer in a patient.
  • the assay comprising a peptide selected from the group consisting of: ISTHGPL, TQRPDKS, STLNVLQ, TQKWPPH, HPLIHPD, APNETRS, TSIGTHS, LEGSKRS, STNISPM, THLPLRL, TQRPDKS, PRTSNPH, QDSKQNR, NRATLST, KVHNRFA, DPLNMYT, NIGKPHQ, TPTRLHY, DRVKSNK, NGNYSHS, NTKLHQS, HSGYLKS, NSPLNPS, HPQYKYL, DGPSRPM, NMVESRF, and NYTLTST, GHISRHL, alone or in combination with an antibody raised against such a peptide.
  • the diagnostic may be any conventional assay known to the skilled artisan.
  • Also provided is a method of determining if a patient is suffering from cancer comprising taking a sample from a patient and determining the level of binding to the sample, of a peptide selected from the group consisting of: ISTHGPL, TQRPDKS, STLNVLQ, TQKWPPH, HPLIHPD, APNETRS, TSIGTHS, LEGSKRS, STNISPM, THLPLRL, TQRPDKS, PRTSNPH, QDSKQNR, NRATLST, KVHNRFA, DPLNMYT, NIGKPHQ, TPTRLHY, DRVKSNK, NGNYSHS, NTKLHQS, HSGYLKS, NSPLNPS, HPQYKYL, DGPSRPM, NMVESRF, NYTLTST and GHISRHL, or an antibody raised against such a peptide, an elevated level thereof, compared to the level of binding to a control sample from an healthy individual, indicating the presence of a cancer.
  • the invention provides a vector(s) comprising a peptide as defined above.
  • the vector is suitable for delivery of biological agents such as chemotherapeutic drugs, antibodies, radionucleotides, cell poisons, pro-drugs, immunostimulatory chemokines and other such biologies directly to tumour tissue for the specific purpose of destroying the tumour without systemic damage to the patient.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a peptide as defined above conjugated to other chemical or biological agents.
  • the composition is suitable for the delivery of targeted therapy to tumour.
  • the composition may further comprise pharmaceutically acceptable carriers or excipients.
  • the invention provides a method of identifying tumour specific biomarker peptides comprising
  • step (c) subjecting the eluate from step (b) to an affinity purification step using anti-
  • HSP70 antibody and (d) subjecting the purified extract from step (c) to five or more rounds of biopanning.
  • the cell extract is a cell extract from a cell line derived from the cancer tissue or may be derived from tumour tissue.
  • a protein cell extract is applied to the ADP-agarose column in step (b).
  • the bound protein may be eluted with ADP.
  • the anti-Hsp70 antibody may be a human anti-Hsp70 mouse monoclonal antibody.
  • the ADP- agarose eluate may be further purified by anti-Hsp70 antibody affinity chromatography.
  • chromatography column may be incubated with a PhD- C7C phage library.
  • a portion of the total amplified phage stock from round one can be incubated with recombinant human Hsp70 to exclude phage binding to the Hsp70 moiety of the Hsp70- PCS.
  • biopanning Preferably five rounds of biopanning are conducted but as many as ten or more rounds may be performed. Thus there may be six or seven or eight or nine or ten rounds of bio-panning. It is not conventional to conduct more than four rounds of bio-panning, and by conducting more rounds, the present inventors have been able to identify peptides that are not otherwise identifiable.
  • a subtractive step of incubating a portion of the amplified phage stock with recombinant Hsp70 is included after each of the first five rounds of biopanning.
  • the eluted phage is amplified in E. coli.
  • the phage may be eluted from each round of biopanning using ATP.
  • the tumour cell lines are selected from MDA-M B-231, MCF7 and the non- tumour breast cell line MCF12A or from solid tumour tissue extract from a patient.
  • Biopanning is an affinity selection technique which selects for peptides that bind to a given target. Biopanning involves 4 major steps for peptide selection. The first step is to prepare phage display libraries, which involves inserting gene segments into a region of the bacteriophage genome, so as to display random peptides on the surface of the M 13 viron as part of the minor coat protein PIN . The next step is a capturing step, involving conjugating the phage library to the desired target. This procedure is termed panning. It utilizes binding interactions so that only specific peptides presented by bacteriophage are bound to the target.
  • a washing step follows the capturing step to wash away the unbound phages from a solid surface. Only the bound phages with strong affinity remain.
  • the final step involves an elution step where the bound phages are eluted through a change in pH and/or specific elution with a competing peptide or other competing molecule.
  • the resulting peptides produced by the bacteriophage are specific, and the phages can be used to infect bacteria once again to produce phage libraries.
  • the four steps can be repeated many times resulting in strong affinity binding peptides to the target.
  • the recombinant phage is thus expressing a random oligopeptide library to identify "recogniser” peptides of known and unknown molecules in tumour cells.
  • the target binding phages or their peptides can then be screened to identify " recogniser” peptides that differentially bind to human breast tumour, or other types of tumours as opposed to normal tissue.
  • the method of the present invention is based on the M13 phage-based random synthetic peptide display system, used in conjunction with tumour-derived peptides sequestered as a dedicated pool of antigens by cellular stress-chaperones in the tumour cell, or known, or unknown tumour antigens.
  • tumour-derived peptides sequestered as a dedicated pool of antigens by cellular stress-chaperones in the tumour cell, or known, or unknown tumour antigens.
  • Hsps are known to chaperone processed peptides from protein products of both tumour and normal cells. Hsps can present some of these peptides to dendritic cells and can illicit adaptive cytotoxic T-lymphocyte responses against antigenic peptides. Moreover Hsp- peptide complexes " Hsp-PCs" have anti-tumour properties that are dependent on the associated peptides. The anti-tumour activity is manifested through the activation of immune responses against the tumours.
  • Hsp70- PC preparations from tumour cells are natural reservoirs of tumour-associated antigens. Additionally, Hsp70, acts at the cross-roads of many intracellular pathways in its role as a major chaperone protein. Through its peptide binding domain, Hsp70 interacts with a large repertoire of proteins either assisting in folding of nascent polypeptides, unfolding of aberrantly folded protein, in protein transport and localisation and finally, through its interaction with the proteasome, in protein degradation [2]. Dysregulation of cellular protein homeostasis is associated with tumour development [3] and the pool of peptides associated with Hsp70 represents a unique barcode of protein metabolism in tumour cells.
  • biopanning was carried out using the Hsp-PCs from a breast tumour cell line.
  • a series of potential Hsp-PC recognisers were identified. The inventors have shown that the recognisers bind specifically to cellular proteins that are known to be up-regulated in tumour cells, specifically GRP78, the eukaryotic
  • translation initiation factor 3 complex el F-3
  • EF-alphal a member of the elongation factor family EF-alphal.
  • the inventors further show that the identified recogniser peptides bind to the tumour cells from which they were derived.
  • FIG. 1 Hsp70-PC purification.
  • A Lane M, Molecular weight markers, Lane 1, recombinant Human Hsp70 ( ⁇ ), Lanes 2-4, ADP-agarose column eluate ( ⁇ g) from MCF-7, MDA-M B-231 and MCF-12A cells respectively.
  • B Western blot of gel from panel A incubated with anti-Hsp70 antibody
  • C Anti-Hsp70 affinity purified MDA-M B- 231-derived Hsp70-PC. The ADP agarose eluate was applied to an anti Hsp70-A/G agarose column. The bound proteins were eluted by boiling in SDS sample buffer.
  • Lane M Molecular weight marker
  • Lane 2 ADP-agarose column eluate ( ⁇ g)
  • Lane 3 Anti- Hsp70/Protein A/G eluate (half of total sample).
  • the two additional bands present in lane 3 correspond to the reduced heavy and light chains of the IgG anti-Hsp70 antibody.
  • Figure 3 1ST peptide binding proteins.
  • the peptide 1ST was covalently linked to amino- coated paramagnetic beads and incubated with total cell lysate from MDA-M B-231 cells. Following washing of the column, the bound beads were eluted by boiling in SDS protein sample buffer. Lane M, Molecular weight marker, Lane 1, ADP-agarose eluate fraction (lC ⁇ g), Lane 2, Total eluate from IST-peptide linked paramagnetic beads, Lane 3, Total eluate from paramagnetic beads only.
  • FIG. 4 Immunofluorescent staining with 1ST peptide.
  • MDA-MB-231 and MCF12A cells were fixed with paraformaldehyde and incubated with lOC ⁇ g/mL of biotinylated 1ST peptide or a control peptide (LSTSSTV). The cells were washed and incubated with ExtrAvidin-Cy3. Cells were counterstained wit 4',6-diamidino-2-phenylindole (DAPI) at a concentration of ⁇ / ⁇ . Slides were examined using either a Nikon Eclipse E400 microscope or an Olympus confocal microscope. Magnification 200x.
  • Top Panel Tissues incubated with anti-Hsp70 antibody.
  • Bottom Panel Tissues incubated with biotinylated 1ST peptide and then anti-biotin antibody.
  • Antibody binding was detected using the Bond Polymer Refine Detection Kit (Leica Microsystems) and visualized using DAB (3' 3'-diaminobenzidine tertahydrochloride). Panels A and B are the same case. Magnification (A, xlOO, B, x400, C, x 200).
  • Seed cultures of metastatic breast adenocarcinoma cell lines MDA-MB-231 and MCF-7 were propagated in RPMI-1640 supplemented with 2mM Glutamine, 10% fetal calf serum as previously described [4].
  • the immortilised non-tumourigenic breast epithelial cells, MCF12A was grown in RPMI-1640 supplemented with 2mM Glutamine, 10% fetal calf serum, 20ng/mL human epidermal growth factor, O.Olmg/mL human insulin and 500ng/mL hydrocortisone.
  • An antibiotic/antimycotic solution (Sigma Chemical Co., Poole U.K) was added during growth.
  • Hsp70-Peptide Complex Purification and Characterisation Approximately 10 8 MDA- MB-231, MCF-7 and MCF-12A cells were lysed using CelLytic M reagent (Sigma Chemical Co., Poole, UK) using the manufacturer's instructions. The protein supernatants were then adjusted to give a final buffer concentration of 20mM Tris— acetate pH7.5, 20mM NaCI, 3mM MgCI 2 , 5mM DTT and 0.5mM phenyl methyl sulphonyl fluoride (PMSF). Approximately 12 mg of protein cell extract was applied to an ADP-agarose column (Sigma Chemical Co., Poole, UK) as previously described [5].
  • the bound proteins were eluted with 3mM ADP and then applied to an Amicon Ultra- 15 centrifugal filter unit spin column (Millipore) with a cut off of lOkDa and centrifuged at 4000 rpm at 4°C.
  • Protein samples were separated on 12% sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE) and stained with Coomassie Brilliant Blue or transferred to nitrocellulose membranes as previously described [6].
  • the membranes were incubated with human anti-HSP70 mouse monoclonal antibody (Sigma Chemical Co., Poole, UK) at a dilution of 1:5000 and then with Horse-Radish peroxidase conjugated goat anti-mouse-lgG antibody (Sigma Chemical Co., Poole UK) at a dilution of 1:20,000.
  • Antibody binding was detected using a luminol-based chemiluminescent peroxidase substrate (Sigma Chemical Co., Poole UK).
  • the ADP-agarose-eluate was further purified by anti-Hsp70 antibody affinity chromatography. Approximately 10 ⁇ g of protein eluate from the ADP-agarose-column was incubated with a 10-fold excess of monoclonal anti-HSP70 antibody (Sigma Chemical Co., Poole, UK) in Tris-Buffered Saline (TBS). Protein A/G agarose (Ultralink; Pierce Thermosciientific Co., Rockford Illinois) was added to the HSP70-PC-antibody complex and the mixture was incubated at 4°C overnight. The sample was spun gently to pellet the Protein A/G agarose and the supernatant was retained as the unbound protein fraction.
  • Hsp70-PCs Hsp70-peptide complexes
  • Proteins separated by SDS-PAGE were stained with Coomassie Brillant Blue. The stained bands were digested in situ using the Trypsin Profile IGD Kit (Sigma Chemical Co., Poole, UK), as per manufacturer's instructions. Reduction and alkylation of the separated proteins was performed in-gel using the ProteoPrep Reduction and Alkylation Kit (Sigma Chemical Co., Poole, UK). The trypsin-digested peptides were concentrated and re-suspended in 0.1% (v/v) trifluoroacetic acid. Mass spectrophotometry analysis was carried out using an Applied Biosystems 4800 Plus MALDI-TOF/TOF. Proteins were identified from the Swiss-Prot database using the search tool Mascot. A protein score of >90 and a confidence interval of ⁇ 99% were used as the criteria for identifying significant hits.
  • one quarter of the total amplified phage stock from round 1 was first incubated with recombinant human Hsp70 (100 ⁇ g/ml) in TBS for 30 min at room temperature to exclude phage binding to the Hsp70 moiety of the Hsp70-PCs.
  • This subtractive step was repeated after each of the first five rounds of bio-panning.
  • Other subtractive steps were carried out to remove non-specific plastic-binding or blocking buffer-binding phage.
  • the unbound phage fraction was then incubated for lhr at room temperature with approximately 2 ⁇ g of the ADP-agarose eluate, bound to anti-Hsp70-Protein A/G Ultralink matrix.
  • the unbound phages were removed and the agarose was washed with 100 column volumes of TBS-T.
  • the remaining bound phage were eluted as described above.
  • the eluted phage were amplified in E. coli.
  • the third round of bio-panning was carried out in the same manner as round 1.
  • a further two rounds of bio-panning were carried out under conditions identical to round 2 except that (a) the blocking solution was alternated between BSA and casein and (b) the fifth round eluate phage was not amplified.
  • Hsp70-PC Recogniser Peptides and their Interacting Proteins Individual phage clones were picked, amplified and sequenced after rounds 4, 5, 6 and 10 in order to monitor phage enrichment during the experiment. Phage single strand DNA (ssDNA) was extracted with sodium iodide as per phage library manufacturer's instructions. PCR was performed to verify the presence of inserts in the phage DNA using the primers 5'TGGTTGTTGTCATTGTCGGCG3' and 5' G CCCTCATAGTTAG CGTAACG 3' . The insert region was sequenced (GATC Biotech AG, Germany) using the primer 5-CCCTCATAGTTAGCGTAACG-3. Generally between 12 and 24 phage clones were sequenced from each round.
  • Short peptides (deciphered from the insert DNA in phage present in the final round eluate) were synthesized by Peptide 2.0 Inc. (Chantilly, VA, U.S.A) or GL Biochem Ltd (Beijing, PRC).
  • the synthetic peptides contained the flanking cysteine residues and two glycine residues at the C- terminus end and a biotin moiety at the N-terminal end.
  • non-biotinylated synthetic peptides were covalently linked via the carboxyl group to amino-coated paramagnetic beads (Kisker Biotech GmbH & Co., Steinfurt, Germany) using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC; Sigma Chemical Co., Poole UK) at lOmg/ml in 0.1M phosphate buffer, pH 7.4. After 3hr, the un-reacted material was inactivated and removed by washing in TBS, and peptide-linked magnetic beads were recovered using magnetic separation and re-suspended in PBS at a concentration of lmg/ml. As a negative control an aliquot of beads were processed similarly in the absence of any synthetic peptides and re-suspended at the same density in PBS.
  • peptide-linked or control beads were blocked in 1% (w/v) casein in TBS for 2hrs. Following washes with TBS, the beads were incubated for lhr with 2mg of soluble protein extract from MDA-MB-231 breast tumour cells. The beads and any peptide-bound proteins were separated from unbound proteins through magnetisation. The bead-bound fraction was washed in TBS, then in TBS containing 0.5M NaCI, and finally in TBS alone. Beads were re-suspended in Laemmli SDS-sample buffer [7] and boiled prior to analysis by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue and protein bands were trypsinised in situ and mass spectrometry analysis was performed as described above.
  • tissue sections were obtained under ethical guidelines.
  • the tissue sections underwent antigen retrieval using Bond Epitope Retrieval Solution 2 (Leica Microsystems) prior to incubation with the biotinylated peptides (200 ⁇ g/ml) or anti Hsp70 antibody (1:500 dilution).
  • Bound peptide was detected using an anti-biotin antibody (Leica Microsystems).
  • Antibody binding was detected using the Bond Polymer Refine Detection Kit (Leica Microsystems) and visualized using DAB (3' 3'- diaminobenzidine tertahydrochloride) as the chromogen.
  • Tissues were counterstained with hematoxylin.
  • Hsp70 and its associated peptides can be isolated by ADP-agarose column chromatography [5].
  • Whole cell lysates were prepared from the breast tumour cell lines MDA-MB-231, MCF7 and the non-tumour breast cell line MCF12A and applied to an ADP-agarose affinity column. The bound proteins were eluted as previously described [5]. As shown in Fig. 1A, four prominent bands of molecular weights 70, 41, 17 and 15 kDa. were retained on the column.
  • the band migrating at 70 kDa. which in fact represents a number of closely migrating proteins, was confirmed as the Hsp70 family of proteins by Western blot using an anti- Hsp70 antibody (IB).
  • IB anti- Hsp70 antibody
  • the bands were excised from the gel and digested in situ with trypsin.
  • the peptide products of trypsin digestion were identified by MALDI-TOF peptide mass fingerprinting and the proteins were identified from the Swiss-Prot database using the search tool Mascot (Table 1).
  • the closely migrating bands at 70 kDa. all represented members of the Hsp70 family including the stress-induced Hsp70, the constitutively expressed Hsp71 and the mitochondrion-associated Hsp70, GRP-75.
  • the co-eluting band at 41 kDa. was identified as actin while the 17 and 15 kDa. bands corresponded to Nucleotide Diphosphate Kinase A and B respectively. The latter three proteins are known to interact with ADP, thus explaining their retention on the ADP-agarose column.
  • Table 1 MALDI-TOF peptide mass fingerprinting of ADP-agarose binding proteins.
  • Hsp70-PCs To purify Hsp70-PCs from the co-eluting proteins, the ADP-agarose bound fraction was incubated with anti-Hsp70 monoclonal antibody and the complex was then retained on a Protein A/G- agarose column. The column was washed to remove unbound proteins and both the bound and unbound samples were separated by SDS-polyacrylamide electrophoresis and stained Coomassie Brilliant Blue (Fig. IC). Hsp70 was retained on the anti-Hsp70 column (IC, lane 3) and could be separated from the other ADP-binding proteins, which were evident in the unbound fraction (data not shown).
  • peptides and polypeptides associated with Hsp70 represent a unique barcode of cellular activity in a cancer cell
  • the bio-panning protocol incorporated both of the purification steps outlined above. Firstly, the ADP-agarose column eluate from MDA-MB-231 and MCF7 cells, isolated as described above, was separately bound to microtitre plates and incubated with the PHD-C7C phage display library. This library displays random-7-mer peptides, flanked by cysteine residues that are expressed as part of the minor coat protein pill of the M13 phage virion.
  • the spontaneous formation of a disulphide bond between the cysteine residues results in the display of a constrained cyclic peptide.
  • the bound phage were eluted and amplified.
  • Amplified phages from the first round of bio-panning were subsequently incubated with immunoaffinity-purified Hsp70-PCs. This two-step bio- panning was repeated once more.
  • a further six rounds of bio-panning was carried out with immunoaffinity-purified Hsp70-PCs until a clear enrichment in the sequenced phage DNA was evident.
  • Phages interacting with the Hsp70 moiety of the Hsp70- peptide complexes were removed using a subtractive bio-panning step with recombinant Hsp70. This subtractive step was repeated after each of the first five rounds of bio-panning. Individual phage were isolated and sequenced after rounds four, five, six and ten to evaluate sequence enrichment. The peptide sequences identified after rounds four, five, six and ten of bio-panning from each of the breast tumour cell lines are shown in Table 2.
  • a single peptide ISTHGPL dominated the final phage pool from the MDA-MB-231 cell line.
  • Four unique peptides were identified in the final phage pool from the MCF-7 bio- panning.
  • the peptide sequence STLNVLQ. (STL) was present in 50% of the phages (Table 2).
  • the other isolated peptides in the final MCF-7 bio-panning round were TQ.KWPPH (TQK), APNETRS (APN) and HPLIHPD (HPL).
  • TQK TQK
  • APN APNETRS
  • HPLIHPD HPLIHPD
  • the peptides are non-polar and are positively charged or neutral (Fig. 2). Since the peptides displayed on the enriched phage pool bind to the peptides/polypeptides associated with Hsp70, they were termed Hsp70-PC "Recogniser Peptides".
  • a number of the newly identified peptides bore sequence homology to two 12-mer peptides, independently isolated from bio-panning against the ADP- agarose eluted fraction from MDA-MB-231 cells [4] using a different phage display library (PH.D-12; New England Biolabs, MA., USA).
  • the common motifs include LNV, WPPH and TQ.RPXK, the latter of which was present in a peptide identified in round 5 of bio-panning with Hsp70-PCs from MDA-MB-231 cells (Table 2).
  • the pools of peptides displayed on virions enriched at earlier stages of the bio-panning process were also identified.
  • Table 2 a series of unique peptides were identified following rounds 4, 5 and 6 of biopanning.
  • Certain peptide motifs present in peptides in the final round of biopanning were evident in peptides identified in the earlier rounds, for example the motifs THxPL and ISxH present in the peptide ISTHGPL was also found in the fourth round peptide THLPLRL and the sixth round peptide GHISRHL respectively.
  • the motif SxLN present in STL.NVLQ. was present in the earlier round peptide NSPLNPS.
  • the peptide HPLIHPD present in the 10 th round eluate from the MCF-7 bio-panning was also evident in earlier round.
  • the 1ST peptide flanked by cysteine residues and a di-Glycine linker at the C-terminus, was chemically synthesised with or without a biotin tag. The presence of the cysteine residues enables the peptide to retain its cyclic nature as displayed on the pil l virion protein.
  • the 1ST peptide interacts with GRP78 and the Translation factors, elF-3 and EF1A.
  • the 1ST peptide linked covalently to paramagnetic beads, was incubated with total cellular lysates from MDA-MB-231 cells. The unbound proteins were removed by extensive washing. The bound proteins were eluted by solubilisation of the 'pull down' complex in SDS-sample buffer [7]. Five major co-migrating protein bands (bands 4-8) and a number of minor bands were evident in the samples eluted from cell lysates incubated with paramagnetic beads with or without the 1ST peptide (Fig. 3, lanes 2 and 3).
  • the peptide mass fingerprinting of the proteins uniquely pulled down with the 1ST peptide identified three subunits of the eukaryotic translation initiation factor 3 (elF3A, B and L; bands 1, 2 and 4) and a member of the elongation factor EF1A family, EF1A-3 (band 7). Additionally, GRP78, a member of the Hsp70 family was also pulled down by the 1ST beads (band 3).
  • the proteins common to both the IST-beads and the control beads (Fig. 3, lanes 2 and 3, bands 5, 6 and 8) represented cytoskeleton-associated proteins such as Tubulin, Actin and Vimentin.
  • the ability of the 1ST peptide to bind to breast tumour cells was investigated by firstly examining the binding of 1ST to paraformaldehyde-fixed cell lines in vitro.
  • the cell lines M DA-M B-231 (tumourigenic) and MCF12A (non-tumourigenic) were incubated with biotinylated 1ST peptide and binding was detected using Cy3-conjugated ExtrAvidin.
  • I ncubation with the 1ST peptide produced a distinctive cellular staining pattern, with staining restricted to the cytoplasm (Fig. 4). I n many cells, a crescent shaped perinuclear halo staining pattern was evident (Fig. 4 A and B).
  • Fig. 5A The staining patterns obtained with both anti-Hsp70 antibody and the 1ST peptide are illustrated in Fig. 5.
  • the Hsp70 antibody showed positive staining in tumour epithelial cells (Fig. 5A). Staining with the 1ST peptide was weaker but the same areas of the tumour which were anti-Hsp70 positive also stained with the 1ST peptide (Fig. 5B, panels 1-3).
  • Fig. 5B panels 1-3
  • breast tumour tissue showing positive staining for Hsp70 were also positive for 1ST staining (data not shown).
  • Hsp70 acts at the cross-roads of many intracellular pathways in its role as a major chaperone protein. Through its peptide binding domain, Hsp70 interacts with a large repertoire of proteins either assisting in folding of nascent polypeptides, unfolding of aberrantly folded protein, in protein transport and localisation and finally, through its interaction with the proteasome, in protein degradation. Dysregulation of cellular protein homeostasis is associated with tumour development and the pool of peptides and polypeptides associated with Hsp70 represents a unique barcode of protein metabolism in tumour cells. Previous studies have demonstrated that this peptide pool contains many antigenic peptides.
  • Hsp70 directly presents these peptides to antigen presenting cells where they are re-presented by MHC class 1 molecules to elicit T-cell responses against the tumour.
  • MHC class 1 molecules MHC class 1 molecules to elicit T-cell responses against the tumour.
  • Stocki et al, [10] used mass spectrometry fingerprinting to identify peptides associated with Hsp70 from the leukemia cell line, K562.
  • One of the identified peptides, which contains a HLA-A*-3 antigenic epitope was derived from dermcidin, a protein that purportedly promotes tumour cell survival.
  • the pool of peptides associated with HSP70 in tumour cells may act as a novel untapped source of tumour biomarkers and used phage display bio-panning to identify small peptides that bind to this peptide pool.
  • ADP-agarose chromatography for purifying Hsp70-PCs, we identified three additional proteins, namely, actin and nucleoside diphosphate kinase A and B, co- eluted along with the Hsp70 family of proteins.
  • the co-eluting proteins are known ADP-binding proteins.
  • ADP-agarose chromatography is not sufficient to generate pure heat shock protein-peptide complexes.
  • Hsp70-PCs were therefore further purified from this fraction by anti-Hsp70 antibody affinity chromatography.
  • the affinity-purified Hsp70-PCs from two different breast epithelial tumour cell lines were used as targets for phage display bio-panning and distinct pools of peptides interacting with Hsp70-PCs were identified for each cell line. Rigorous steps were taken to rule out the selection of phages binding to the Hsp70 protein itself.
  • the identification of unique 'recogniser' peptides for Hsp70-PCs from each cell line indicates that (a) the pool of peptides associated with Hsp70 differs in each cell line and (b) that the isolated peptides are not recognising the Hsp70 backbone of the Hsp70-PC complexes.
  • a yeast two-hybrid based approach was used to identify small peptide inhibitor molecules of Hsp70 from HeLa cells [11]. None of the peptides identified in that study were identical or similar to the peptides reported in this study, indicating that the 'recogniser' peptides are more likely derived from the associated peptides and not to the Hsp70 protein itself.
  • the isolated peptides were non polar and positively charged or neutral. Most interesting was the finding that some of the newly isolated peptides bore homology to a set of peptides previously identified from the bio-panning of a 12- mer linear peptide phage display library using an ADP-agarose purified HSP70-PC fraction from MDA-MB-231 as the target [4], Bond et al unpublished data).
  • the discovery of common amino acid motifs amongst peptides isolated with two different libraries, each containing approximately lxlO 11 individual peptide sequences, provides veracity to the use of phage display bio-panning as an approach to identifying biologies that interact specifically with Hsp70-PCs.
  • the 1ST peptide was characterized in more detail as it was the single peptide present in the final round of bio-panning against MDA-MB-231 cells.
  • Two classes of proteins were identified as IST-interacting proteins. Firstly, components of the translational machinery, in particular, subunits of the initiation factor elF3 and a member of the elongation factor 1A were identified. Over-expression of elF3 subunits has been demonstrated in a number of cancer tissues [12] and may contribute to tumourigenesis through the up-regulation of global protein synthesis or alternatively by affecting the translation of key mRNAs encoding proteins required for cell cycle regulation.
  • elF3A is a key regulator of a subset of mRNA including p27 (kipl), tyrosinated a-tubulin and ribonucleotide reductase M2 subunit, all of which play central roles in the regulation of the cell cycle [13].
  • the over-expression of individual subunits of elF3 appears to be sufficient to result in the malignant transformation of the immortal NIH3T3 cell line [14].
  • up- regulation of this protein complex plays a pivotal role in tumourigenesis.
  • the second translation machinery component identified was EFla3, a member of the elongation factor la family. This protein is 99% identical to EFlal and 96% identical to EFla2. Both EFlal and EFla2 have been identified as oncoproteins and are over-expressed in breast and ovarian tumour cells [15, 16].
  • the third 1ST interacting protein identified in this study is GRP78, a member of the HSP70 family of proteins. Like elF3 and the EFla family, GRP78 is also over-expressed in a number of cancers and the presence of autoantibodies to GRP78 in cancer patient sera is associated with higher pathological tumour grades, poor prognosis and drug resistance in breast cancers [17].
  • Hsp70-associated peptides represent a reservoir of peptides unique to each cell, which through their association with over- expressed Hsp70, can serve as useful source of cancer biomarkers.
  • the prototype peptide 1ST interacts with a number of key cellular proteins that are over-expressed in cancer cells and binds to the tumour tissue from which it was derived.
  • the methodology employed here can be used in cancer research to identify novel short peptides for diagnostic and therapeutic uses.
  • shock protein 70-peptide complexes by ADP-affinity chromatography shock protein 70-peptide complexes by ADP-affinity chromatography.

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Abstract

La présente invention concerne des procédés d'identification de biomarqueurs peptidiques spécifiques d'une tumeur, les peptides identifiés par ces procédés, et des procédés d'utilisation de ces peptides dans des dosages diagnostiques et en tant que cibles dans la mise au point d'un vaccin antitumoral. La présente invention concerne en outre un ensemble de peptides synthétiques dérivés de peptides associés à la protéine de choc thermique 70 (HSP70) de lignées de cellules cancéreuses. Ces peptides sont utiles dans le diagnostic et le traitement du cancer du sein.
PCT/EP2012/067565 2011-09-12 2012-09-07 BIOMARQUEURS POUR LES TUMEURS DU SEIN DÉRIVÉS DE PEPTIDES ASSOCIÉS À Hsp70 Ceased WO2013037714A1 (fr)

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CN113631572A (zh) * 2019-03-06 2021-11-09 Ghp科学有限公司 用于治疗用途的grp78和/或hsp70抑制剂

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