HPVE7 oncoproteins as new markers for detection of cervical cancer and precancer
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1 HPVE7 oncoproteins as new markers for detection of cervical cancer and precancer Dr. Pidder Jansen-Dürr Innsbruck University Innsbruck, Austria
2 Human Papillomaviruses (HPVs) and cervical lesions HPVs infect basal proliferating epithelial cells of either the skin or mucosa > 100 different genotypes have been described today low-risk types high-risk types HPV 1 skin warts HPV 5, 8, 9, 12, 14, 15, 17, skin cancer HPV 6, 11 genital warts HPV 16, 18, 31, 33, 35, 45, 58 cervical cancer cervical cancer is one of the most common cancers in women worldwide Link between hrhpv infection and cervical cancer firmly established by Harald zur Hausen & colleagues Nobel Prize in Medicine or Physiology 2008
3 Progression from a productive high-risk HPV infection to malignancy M Thomas et al., Oncogene, 2008 The E6 and E7 oncoproteins are required for tumorigenesis
4 Current procedures in cervical cancer screening Detection of abnormal cells Papanicolao staining of cells in cervical smears Liquid based cytology high rate of false- positive and false- negative results Detection of viral nucleic acids high- risk HPV DNA high- risk HPV E6/E7 mrna no discrimination between transient infections and the onset of cervical cancer
5 New tools for cervical cancer screening are urgently needed p16ink4a as surrogate marker for hr HPV infection Cervical swabs of healthy women contain p16ink4a-positive cells p16/ki67 double staining? E7 proteins of high-risk HPV types: new markers for cervical cancer?
6 hre7 protein: key in cervical carcinogenesis E7 E6 Prediction: E7 protein levels should be high in tumor cells
7 Antibodies to HPV-16 E7 - immunofluorescence U 2 - O S 1 6 EU 72 - O S m o c k anti-16 E7 nuclei merge Fiedler et al., 2004; FASEB J. & WO/2005/026731
8 HPV-16 E7 is highly expressed in cervical cancer a-hpv-16 E7 a-hpv-16 E7/competed Fiedler et al., 2004; FASEB J. & WO/2005/ Can we use this information for cervical cancer screening?
9 Viral high-risk E7 proteins: new markers for cervical cancer? E7 protein is necessary to directly inactivate cellular tumor suppressors HPV-16, HPV-18 and HPV-45 E7 oncoproteins are expressed continously in CxCa (Ressler et al., 2007; Clin. Cancer Res.) and cervical adenocarcinoma ( Dreier et al., 2011; Virology) Aim of the study: to detect E7 proteins of hrhpv types in cervical smears Rabbit monoclonal antibodies (RabMabs) against E7 proteins as key diagnostic tools immunofluorescence detection of E7 proteins in liquid-based cytology (example: RabMab 42-3) sandwich ELISA to quantitate E7 protein levels in conventional pap smears (example: RabMab 143-7)
10 Recombinant E7 oncoproteins E7 proteins of the most prevalent hrhpv types were expressed in E.coli and purified HPV-11 E7 (low-risk virus) was used as a control Recombinant E7 proteins (5 mg each) on a silver-stained gel
11 Rabbit monoclonal antibodies (RabMAbs) for detection of hrhpv E7 proteins E7 proteins of the 12 most prevalent hrhpv types (coverage > 99%) were used to immunize rabbits Rabbit monoclonal antibodies (RabMabs) produced > 20 RabMabs were obtained that cover the full spectrum of hrhpv E7 proteins
12 Unmatched sensitivity of RabMab 42-3 for IF detection of HPV-16 E7 Rabbit polyclonal antibodies (WO/2005/026731) RabMab42-3 (WO/2011/101122)
13 RabMab 42-3 recognizes a conformational epitope in the HPV-16 E7 zinc finger PepScan analysis Cysteine residues 58/61 and 91/94 stabilize a Zn finger structure in the C-terminal domain of HPV-16 E7
14 RabMab 42-3 recognizes a conformational epitope in the HPV-16 E7 zinc finger (ctd.) IF analysis with RabMab 42-3 c o WT C58G C91G IF with rabbit polyclonal antib. C58/91 G RabMab 42-3 recognizes an epitope in the HPV-16 E7 C-terminus Destabilization of the Zn finger by single Cys mutations abolishes IF signal RabMab 42-3 stains endogenous E7 protein in Caski cells (Dreier et al. 2011) LBC analysis underway
15 Performance of RabMAb 143-7
16 RabMab recognizes an epitope in the HPV-18 E7 N-terminus Pepscan analysis RabMab RabMAb epitope mapping: HPV-18 E7 protein unrelated rabbit antibodies only secondary antibody 26 kd - 17 kd - Western blot H e L a C a S k i U - 2 O S e m p t y v e c t o r U - 2 O S / 1 8 E 7 - F l a g HPV E7 Actin
17 Specific detection of endogenous HPV-18 E7 by immunofluorescence and IHC Immunofluorescence HPV E7 DNA Immunohistochemistr y Merge HeLa SCC AC NSE NGE CaSki U-2OS
18 RabMab based Sandwich-ELISA absorbance 450 nm ng Detection limit < 1 pg 18 E7 protein biotinylated RabMAb pg 250 pg 100 pg 50 pg 25 pg 10 pg 5 pg 2.5 pg 1 pg 500 fg 250 fg 100 fg recombinant HPV-18 E7/well no low-risk E7 is not detected absorbance 450 nm biotinylated RabMAb E7 11 E7 6 E7 no 250 pg/well protein absorbance 450 nm 4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0 0,5 0,0 Cross-reactivity with HPV-45 E7 biotinylated RabMAb E7 18 E7 31 E7 33 E7 35 E7 39 E7 45 E7 51 E7 52 E7 56 E7 58 E7 59 E7 no based sandwich ELISA detects recombinant E7 proteins of HPV-18 and -45 Detection limit < 1 pg 100 pg/well protein
19 E7 signal from 500 HeLa cells detectable by sandwich ELISA RabMab based Sandwich-ELISA (ctd.) Decreasing numbers of HeLa cells (HPV-18 positive) added to U2- OS cells (HPV-negative), total cell number Sandwich ELISA with cell lysates to detect HPV-18 E7 protein HeLa cell titration 3.5 biotinylated RabMAb Statistical evaluation (n = 5) absorbance 450 nm ,000 10,000 5,000 2,500 1, HPV-18 positive HeLa cells/well absorbance 450 nm biotinylated RabMAb log 10 HPV-18 positive HeLa cells/well
20 Pilot clinical study with ELISA based on RabMab HPV- DNA negative + 14 HPV 18- DNA positive cervical swabs ar bi tr ar y u ni ts patient sample no. Ehehalt et al., 2011
21 Pilot clinical study with ELISA based on RabMab (ctd.) 25 IIID IVa ar bi tr ar y u ni ts II II II / IIID V 5 IIID IIID IIID IIID IIID IIID IIID / IVa IVa patient sample no. Ehehalt et al., 2011
22 Pilot clinical study with ELISA based on RabMab (ctd.) 25 IIID IVa ar bi tr ar y u ni ts II II II / IIID V 5 IIID IIID IIID IIID IIID IIID IIID / IVa IVa patient sample no. Problematic samples revisited: Cytological diagnosis (PapII) incorrect for patients #1 & #2; multiple hrhpv infections occurred in patients # 5,6,9,10,11
23 RabMAbs against other hre7 types
24 Cross-reactivity of RabMabs towards E7 proteins Western blot Purified E7 proteins used in Western blot RabMab 31- RabMab RabMab 80- RabMab
25 Cross-reactivity of RabMabs towards E7 proteins Western blot (ctd.) RabMab RabMab RabMab RabMab RabMab RabMab RabMab RabMab
26 Towards a pan-hre7 ELISA detection kit RabMabs recognizing other E7 proteins of hrhpv types Combination of different RabMabs in ELISA plates Use recombinant E7 proteins to test specificity and sensitivity
27 Sandwich ELISA based on RabMab cocktails Recombinant E7 proteins E E pg 1pg 0.5pg no E7 5pg 2.5pg 1pg 0.5pg no E pg 1pg 0.5pg no E7 5pg 2.5pg 1pg 0.5pg no E E7 Detection limit: < 500 fg of hre7 protein pg 1pg 0.5pg no E7 5pg 2.5pg 1pg 0.5pg no E7
28 4 3 Sandwich ELISA based on RabMab cocktails Cervical carcinoma cell lines HeLa 3 CaSki U2-OS Detection limit: < 1000 tumor cells
29 Towards a pan-hre7 ELISA detection kit All-in-one E7 detection ELISA
30 Conclusions E7 proteins of high-risk HPV types as new markers for cervical cancer Rabbit monoclonal antibodies to hrhpv E7 proteins as key diagnostic tools sandwich ELISA for E7 detection in cervical smears Proof of the principle OK, assay optimization underway Clinical validation underway
31 Acknowledgements IBA, Dept MZB Haymo Pircher Daniela Ehehalt Ruth Greussing Michael Neuhaus Hans-Peter Viertler Tyrolean Cancer Research Institute Barbara Lener Kerstin Dreier Evi Huetter Christina Metzger Andreas Kaiser Collaborations Andreas Widschwendter (UFK Innsbruck) Andreas Kaufmann (Charite Berlin) Theo Agorastos (Univ. Thessaloniki, Greece) Isabel Koch (Mikrogen GmbH, Germany) Catherine Muller (Biosynex SA, France) Funding Austrian Science Funds (FWF) European Union (IP INCA; PIPAVIR: ) Land Tirol
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