RabMab 143-7 based Sandwich-ELISA

HPVE7 oncoproteins as new markers for detection of
cervical cancer and precancer
Dr. Pidder Jansen-Dürr
Innsbruck University
Innsbruck, Austria
[email protected]
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
HPV 1
HPV 6, 11
•
•
skin warts
genital warts
high-risk types
HPV 5, 8, 9, 12, 14,
15, 17, 19-25
skin
cancer
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
anti-16 E7
nuclei
merge
U
2
O
S
1
6
E
U
7
2
O
S
m
o
c
kFiedler 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 423 c
WT
C58G C91G C58/91
o
G
IF with rabbit polyclonal
antib.
• 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 epitope mapping: HPV-18 E7
protein
Western
blot
U
26 kd
17 kd
RabMab 143-7
unrelated rabbit antibodies
only secondary antibody
H
e
L
a
C
a
S
k
i
2
O
S
e
m
p
t
y
v
e
c
t
o
r
U
2
O
S
/
1
8
E
7
HPV E7
F
l
a
g 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
Detection
limit <RabMAb
1 pg 143-7
18 E7 protein
biotinylated
4.5
low-riskbiotinylated
E7 is not
detected
RabMAb 143-7
4.5
4.0
3.5
3.5
3.0
absorbance 450 nm
absorbance 450 nm
4.0
2.5
2.0
1.5
1.0
0.5
0.0
3.0
2.5
2.0
1.5
1.0
no
fg
fg
fg
10
0
25
0
pg
50
0
1
pg
pg
2.
5
5
pg
pg
pg
pg
10
25
50
pg
10
0
pg
25
0
50
0
1
ng
0.5
0.0
recombinant HPV-18 E7/well
18 E7
11 E7
250 pg/well
6 E7
no
protein
Cross-reactivity
with143-7
HPV-45 E7
biotinylated RabMAb
4,5
• 143-7-based sandwich ELISA
detects recombinant E7
proteins of HPV-18 and -45
absorbance 450 nm
4,0
3,5
3,0
• Detection limit < 1 pg
2,5
2,0
1,5
1,0
0,5
0,0
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
100 pg/well
protein
RabMab 143-7 based Sandwich-ELISA (ctd.)
• Decreasing numbers of HeLa cells (HPV-18 positive) added to U2OS cells (HPV-negative), total cell number 25.000
• Sandwich ELISA with cell lysates to detect HPV-18 E7 protein
HeLa cell titration
biotinylated RabMAb 143-7
Statistical evaluation (n = 5)
3.5
3.0
3.5
2.0
3.0
1.5
2.5
1.0
0.5
0.0
25,000 10,000 5,000
2,500
1,000
500
250
100
50
0
absorbance 450 nm
absorbance 450 nm
biotinylated RabMAb 143-7
2.5
2.0
1.5
1.0
0.5
HPV-18 positive HeLa cells/well
0.0
1.7
2.2
2.7
3.2
3.7
log10 HPV-18 positive HeLa cells/well
E7 signal from 500 HeLa cells detectable by sandwich
ELISA
4.2
4.7
Pilot clinical study with ELISA based on RabMab 1437
24 HPV- DNA negative +
14 HPV 18- DNA positive cervical
swabs
25
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 1437 (ctd.)
25
IIID
ar
bi
tr
ar
y
u
ni
ts
IVa
20
15
10
II
II
II /
IIID
V
5
IIID
IIID
IIID
5
6
IIID
IIID
IIID
IIID /
IVa
8
9
10
11
IVa
0
1
2
3
4
7
12
13
14
patient sample no.
Ehehalt et al., 2011
Pilot clinical study with ELISA based on RabMab 1437 (ctd.)
25
IIID
ar
bi
tr
ar
y
u
ni
ts
IVa
20
15
10
II
II
II /
IIID
V
5
IIID
IIID
IIID
5
6
IIID
IIID
IIID
IIID /
IVa
8
9
10
11
IVa
0
1
2
3
4
7
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
RabMab 31711 16 18
39
59
45
RabMab 381
18 33
39 45
59
RabMab 802 16 31
11
58
31 33 35 39 45 51 52 56 58 59
58
35
RabMab 84258 16 31 33 35
52 56 58 59
Cross-reactivity of RabMabs towards E7 proteins
Western blot (ctd.)
RabMab 2131
10
RabMab 5511 18
52
45
56
RabMab 583
16 31 33 35
16
RabMab 423 16
52
58
RabMab 191 11 18
51
56
RabMab 784
RabMab 146- 8
39
59
RabMab 128- 4
58
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
16 E7
4
18 E7
2
1.6
3
1.2
2
0.8
1
0
0.4
5pg
2.5pg
1pg
0.5pg
0
no E7
5pg
2.5pg
1pg
0.5pg
no E7
5pg
2.5pg
1pg
0.5pg
no
0
0.4
0.8
1.2
1.6
2
.4E7
5pgE7
2.5pg
1pg
0.5pg
no
0
1
2
3
4
45 E7
1.2
Detection limit: < 500 fg of hrE7 protein
0.8
0.4
0
5pg
2.5pg
1pg
0.5pg
no
0
1.6
0.4
0.8
1.2
.4E7
5pg
2.5pg
1pg
0.5pg
no E7
Sandwich ELISA based on RabMab cocktails
Cervical carcinoma cell lines
4
HeLa
CaSki
3
3
2
2
1
1
0
100000
50000
25000
10000
5000
1000
0
0
100000
50000
25000
10000
5000
1000
100000
50000
25000
10000
5000
1000
4
0
1
2
3
00000
100000
50000
25000
10000
5000
1000
0
1
2
3
4
00000
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
Detection limit: < 1000 tumor cells
100000
50000
25000
10000
5000
1000
0
0
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

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