HPVE7 oncoproteins as new markers for detection of cervical cancer and precancer

HPVE7 oncoproteins as new markers for detection of cervical cancer and precancer Dr. Pidder Jansen-Dürr Innsbruck University Innsbruck, Austria p.jansen-duerr@uibk.ac.at

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, 19-25 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

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

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

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?

hre7 protein: key in cervical carcinogenesis E7 E6 Prediction: E7 protein levels should be high in tumor cells

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

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/2005/026731 Can we use this information for cervical cancer screening?

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)

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 11 16 18 31 33 35 39 45 51 52 56 58 59 Recombinant E7 proteins (5 mg each) on a silver-stained gel

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

Unmatched sensitivity of RabMab 42-3 for IF detection of HPV-16 E7 Rabbit polyclonal antibodies (WO/2005/026731) RabMab42-3 (WO/2011/101122)

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

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

Performance of RabMAb 143-7

RabMab 143-7 recognizes an epitope in the HPV-18 E7 N-terminus Pepscan analysis RabMab 143-7 RabMAb 143-7 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

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

RabMab 143-7 based Sandwich-ELISA absorbance 450 nm 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1 ng Detection limit < 1 pg 18 E7 protein biotinylated RabMAb 143-7 500 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 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 biotinylated RabMAb 143-7 18 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 143-7 16 E7 18 E7 31 E7 33 E7 35 E7 39 E7 45 E7 51 E7 52 E7 56 E7 58 E7 59 E7 no 143-7-based sandwich ELISA detects recombinant E7 proteins of HPV-18 and -45 Detection limit < 1 pg 100 pg/well protein

E7 signal from 500 HeLa cells detectable by sandwich ELISA RabMab 143-7 based Sandwich-ELISA (ctd.) Decreasing numbers of HeLa cells (HPV-18 positive) added to U2- OS cells (HPV-negative), total cell number 25.000 Sandwich ELISA with cell lysates to detect HPV-18 E7 protein HeLa cell titration 3.5 biotinylated RabMAb 143-7 Statistical evaluation (n = 5) absorbance 450 nm 3.0 2.5 2.0 1.5 1.0 0.5 0.0 25,000 10,000 5,000 2,500 1,000 500 250 100 50 0 HPV-18 positive HeLa cells/well absorbance 450 nm 3.5 3.0 2.5 2.0 1.5 1.0 0.5 biotinylated RabMAb 143-7 0.0 1.7 2.2 2.7 3.2 3.7 4.2 4.7 log 10 HPV-18 positive HeLa cells/well

Pilot clinical study with ELISA based on RabMab 143-7 25 24 HPV- DNA negative + 14 HPV 18- DNA positive cervical swabs ar bi tr ar y u ni ts 20 15 10 5 0 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 patient sample no. Ehehalt et al., 2011

Pilot clinical study with ELISA based on RabMab 143-7 (ctd.) 25 IIID IVa ar bi tr ar y u ni 20 15 10 ts II II II / IIID V 5 IIID IIID IIID IIID IIID IIID IIID / IVa IVa 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 patient sample no. Ehehalt et al., 2011

Pilot clinical study with ELISA based on RabMab 143-7 (ctd.) 25 IIID IVa ar bi tr ar y u ni 20 15 10 ts II II II / IIID V 5 IIID IIID IIID IIID IIID IIID IIID / IVa IVa 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 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

RabMAbs against other hre7 types

Cross-reactivity of RabMabs towards E7 proteins Western blot Purified E7 proteins used in Western blot 11 16 18 31 33 35 39 45 51 52 56 58 59 RabMab 31- RabMab 38-711 16 18 39 45 1 18 33 39 45 59 59 RabMab 80- RabMab 84-11 2 16 31 35 2 16 31 33 35 52 56 58 59 58 58 58

Cross-reactivity of RabMabs towards E7 proteins Western blot (ctd.) RabMab 21-10 31 52 RabMab 55-11 18 45 56 RabMab 58-3 16 31 33 35 52 58 16 RabMab 19-1 11 18 51 56 RabMab 42-3 16 RabMab 78-4 58 RabMab 146-8 39 59 RabMab 128-4 51 56

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

Sandwich ELISA based on RabMab cocktails Recombinant E7 proteins 4 3 2 1 16 E7 2 18 E7 1.6 1.2 0.8 0.4 0 2.5pg 1pg 0.5pg no 012340 E7 5pg 2.5pg 1pg 0.5pg no E7 0 2.5pg 1pg 0.5pg no 20 0.4 0.8 1.2 1.6 E7 5pg 2.5pg 1pg 0.5pg no E7 1.2 0.8 45 E7 Detection limit: < 500 fg of hre7 protein 0.4 0 2.5pg 1pg 0.5pg no 1.6 0.4 0.8 1.2 E7 5pg 2.5pg 1pg 0.5pg no E7

4 3 Sandwich ELISA based on RabMab cocktails Cervical carcinoma cell lines HeLa 3 CaSki 2 2 1 1 0 100000 50000 25000 10000 5000 1000 0 0 100000 50000 25000 10000 5000 1000 0 0100000 50000 25000 012340 0100000 50000 25000 012340 1.98 1.96 1.94 1.92 1.9 1.88 1.86 1.84 1.82 1.8 1.78 1.76 1.74 1.72 1.7 1.68 1.66 1.64 1.62 1.6 1.58 1.56 1.54 1.52 1.5 1.48 1.46 1.44 1.42 1.4 1.38 1.36 1.34 1.32 1.3 1.28 1.26 1.24 1.22 1.2 1.18 1.16 1.14 1.12 1.1 1.08 1.06 1.04 1.02 1 0.98 0.96 0.94 0.92 0.9 0.88 0.86 0.84 0.82 0.8 0.78 0.76 0.74 0.72 0.7 0.68 0.66 0.64 0.62 0.6 0.58 0.56 0.54 0.52 0.5 0.48 0.46 0.44 0.42 0.4 0.38 0.36 0.34 0.32 0.3 0.28 0.26 0.24 0.22 0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 U2-OS 100000 50000 25000 10000 5000 1000 0 Detection limit: < 1000 tumor cells

Towards a pan-hre7 ELISA detection kit All-in-one E7 detection ELISA

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

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: 2012-2015) Land Tirol