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R E S E A R C H Open Access
Intrabodies targeting human papillomavirus
16 E6 and E7 oncoproteins for therapy of
established HPV-associated tumors
Francesca Paolini
1
, Carla Amici
2
, Mariantonia Carosi
3
, Claudia Bonomo
3
, Paola Di Bonito
4
, Aldo Venuti
1
and
Luisa Accardi
4*
Abstract
Background: The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on
the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular
homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the
infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular
transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We
previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7
proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy
against tumor progression of HPV16-positive cells.
Methods: Envisioning clinical application of the best characterized anti-HPV16 E6 and –HPV16 E7 scFvs, we verified
their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-
HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum
targeting sequence were delivered by injection followed by electroporation to three different preclinical models
using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc
cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second
model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model,
the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E
and caspase-3 staining of tumor section were performed.
Results: We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor
progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells
towards the apoptotic pathway.
(Continued on next page)
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* Correspondence: luisa.accardi@iss.it
4
Department of Infectious Diseases, Istituto Superiore di Sanità, 00161 Rome,
Italy
Full list of author information is available at the end of the article
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37
https://doi.org/10.1186/s13046-021-01841-w
(Continued from previous page)
Conclusion: Based on our study, two scFvs have been identified that could represent a safe and effective
treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to
be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed
for human treatment, to deliver scFvs to tumors was confirmed.
Keywords: HPV-associated cancer, Antitumor intracellular antibodies, Therapeutic single-chain antibody fragments,
HPV16 E6 and E7 oncoproteins, Apoptosis, Gene delivery by electroporation
Background
Papillomaviruses were the first class of viruses to be
associated with human cancer [1]. Out of over 200
Human Papillomaviruses (HPV) genotypes, only twelve
to fourteen, defined as high risk (HR) types, are etiologic-
ally involved in virtually all squamous cell carcinomas
(SCC) of the cervix, a high percentage of those in the
ano-genital area and an increasing fraction of the
head and neck cancers (HNSCC) [2,3].
The causal relation between HPV infection and cancer
allowed the development of prophylactic vaccines able
to prevent cancer but not intended to cure preexisting
infections [4]. Therefore, HR HPV genotypes can persist
unapparent after infection and cause the onset of lesions
and progression to cancer over years or decades, reliant
on co-factors [5].
The HPV oncogenicity is primarily dependent on the
continuous expression and activity of the E6 and E7 viral
proteins, which are Tumor-associated antigens (TAAs)
acting in concert to alter interrelated cellular processes
and promoting tumor development through the inter-
action with over 100 different cellular proteins [6]. E6 and
E7 are also recognized as tumor rejection antigens thus
representing valid targets for therapeutic vaccination.
Hence, two kinds of therapeutic approaches targeting the
E6 and E7 oncoproteins are usually implemented. The
first one is based on stimulation of the cell-mediated
immune response arming E6- and E7-specific CTLs able
of rejecting the HPV tumor [7]. The second one is based
on the direct blocking of their oncogenic activity through
specific monoclonal antibodies (mAbs) [8].
Therapeutic drugs based on mAbs are largely repre-
sented in the biotechnology industry, whereby the
European Medicines Agency and the US Food and Drug
Administration have approved ninety-eight antibody ther-
apies for the European or US market up to date and
sixteen are under review (Antibody Society. Approved
antibodies. Available at https://www.antibodysociety.org/
resources/approved-antibodies/)[9]. In this context, the
antibodies in single chain format (scFvs) are well repre-
sented due to characteristics which make them suitable to
multiple purposes, such as the capacity of effectively inhi-
biting different protein functions demonstrated by several
anticancer applications both in vitro and in vivo [10,11].
The little-sized scFv format allows ease of manipula-
tion as it comprises only the variable domains of the
heavy (VH) and light (VL) Immunoglobulin chains,
joined by a flexible linker. The scFv molecules can be
engineered according to the purpose, e. g. by grafting
the Complementarity Determining Regions (CDR) into
different plasmid scaffolds for expression in prokaryotic
cells for purification as proteins, or in eukaryotic cells,
by viral or non-viral vectors, even as intracellular
antibodies (intrabodies) targeting intracellular harmful
molecules [12,13].
It is notable that most therapeutic mAbs either in
single-chain or classical format approved so far or under
investigation for anti-cancer purposes, target proteins
localized on the surface of transformed or infected cells.
Instead, in the HPV system, the localization of oncopro-
teins only in the infected cells prompted us to develop
the potential therapeutic scFvs against the oncoproteins
as intrabodies.
We previously characterized several scFvs specific for
the E7 and E6 oncoproteins of HPV16 (16E7 and 16E6,
respectively) in terms of binding epitopes and biophys-
ical features. Two of them were expressed as intrabodies
in HPV16-positive tumor cells and showed antitumor
activity: the anti-16E7 scFv43M2SD with signal for
localization in endoplasmic reticulum (ER) and the anti-
16E6 scFvI7nuc with signal for nuclear localization
(NLS). Such scFvs were able to hamper cell proliferation
and favor apoptosis in vitro in cellular systems, and hin-
dered or delayed neoplastic growth in animal models, in
preventive setting [14–16].
In this study, we implement data supporting an effect-
ive use of scFv43M2 and scFvI7nuc in tumor therapy.
For this purpose, we investigated the antitumor effect of
scFvs delivered as intrabodies by electroporation (EP) to
HPV16-positive tumors implanted in mice. In addition
to being an effective in vitro gene transfer method, EP is
emerging as a method for delivery of chemotherapeutics
to human tissues [17]. Since a typical feature of HPV
cancer is the growth in well-defined areas, EP could rep-
resent a delivery system applicable to the treatment of
HPV lesions in humans.
The results, obtained with three different HPV tumor
models, confirmed the ability of anti-16E6 and –16E7
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 2 of 11
scFvs to induce a marked inhibition of tumor growth.
Importantly, the anti-tumor treatment was associated
with the presence of large apoptotic areas in tumors,
substantiating the hypothesis that the scFv-induced per-
turbation of the E6 or E7 activity can trigger cell death
pathways in HPV16+ tumors as already reported in vitro
in HPV16+ cells [16].
Methods
ScFv constructs and cell lines
Selection of the anti-16E7 scFv43 from the ETH-2
library and scFvI7 from the SPLINT library was
previously described [14,16]. ScFv43 was subjected to
site-directed mutagenesis to improve stability and the
new antibody used thereafter was named scFv43M2 [18].
By subcloning, the scFvs were provided with signals for
intracellular localization: scFv43M2 was provided with
SEKDEL for retention in ER, and scFvI7 with NLS for
nuclear localization. The resulting scFvs were named
scFv43M2SD and scFvI7nuc. Two recombinant plasmids
expressing irrelevant anti-β-galactosidase scFvs, respectively
provided with signals for nuclear targeting (scFvR4nuc) or
secretory leader sequence (R4sec), provided by A. Cattaneo
[19] were used as controls.
The murine TC-1 cell line, derived from primary lung
epithelial cells co-transformed with the HPV16 E6-E7
and activated c-H-Ras oncogenes [20], and C3 cell line,
derived from embryonic mouse cells transformed with
full HPV16 genome and activated Ras oncogene [21],
were grown in RPMI 1640 with 10 mmol/L of HEPES, 1
mmol/L of sodium pyruvate supplemented with 2 mmol/
L of nonessential amino acids and 10% FCS. Both cell
lines are passages of the original clones and were
routinely checked for the presence of HPV sequence and
resistance to the G418 antibiotic selection (0.4 μg/ml).
They are able to establish subcutaneous tumors in
C57BL/6 syngeneic mice, providing models of human
HPV16-associated neoplasms. TC-1-LUC cells were ob-
tained by infection with a lentivirus containing the firefly
luciferase gene that was generated according to standard
procedures [22]. Cells were cultivated in the presence of
10 μg/ml Blasticidin (Merck, Italy) and those with stable
LUC expression selected by luciferase assay screening.
Therapeutic setting: intra-tumor scFv delivery in mice by
electroporation
Six-eight week-old female C57BL/6 mice were obtained
from Charles River Laboratories Italia, divided into four
groups and maintained under specific pathogen-free
conditions at the Experimental Animal Department of
the Regina Elena National Cancer Institute (Rome, Italy).
All experimental procedures were approved by the
Institutional Animal Care of the Regina Elena National
Cancer Institute and by the Government Committee of
National Ministry of Health (85/2016-PR) and were car-
ried out in accordance with EU Directive 2010/63/EU for
animal experimentation. In consideration of the ethical
suggestions to minimize the number of animals, 4 mice
per treatment were used. Three different in vivo experi-
ments were performed, using 5 × 10
4
TC-1 Luc, 2 × 10
5
TC-1 or 5 × 10
5
C3 cells, respectively. Tumor cells were
injected subcutaneously (s. c.) into the right inner flank of
mice. One week after cancer cell injection, tumors were
measurable and the first treatment was administered
intra-tumor. Briefly, mice were anesthetized and 50 μgof
scFvI7nuc or scFv43M2SD -expressing plasmids, diluted
in sterile 0.9% saline solution, were injected centrally into
the tumor using a 1ml syringe with a 30-gauge needle.
Recombinant plasmids expressing the irrelevant anti-β-
galactosidase scFv provided with NLS (R4nuc) or
secretory sequence (R4sec) were used as negative controls,
respectively. Immediately after DNA injection, tumors
were subjected to electroporation using a BTX ECM 830
square wave generator (Harvard Apparatus) to deliver one
unidirectional pulse (100 V/cm, 50 ms) with a BTX twee-
zertrode array. This intra-tumor delivery setting was
repeated once a week for three times, for a total of four
treatments. According to the experimental plan, tumor
growth was monitored once or twice a week by digital
caliper measurements, or weekly by in vivo imaging. Mice
were euthanized after one week from the last treatment
for ethical reasons, to avoid animal suffering. In the
bioluminescent approach, tumor burden was quantified
by measuring the luciferase activity. Briefly, mice were
anesthetized and intraperitoneal injection of 150 mg/kg of
D-luciferin (Caliper, PerkinElmer, Italy) was performed.
Ten minutes later, light emission was acquired for 5 min.
Signal was detected using the IVIS Lumina II CCD camera
system and analyzed by the Living Image 15 2.20 software
package (Caliper Life Sciences, Milan, Italy). Photon emis-
sion was measured in specific regions of interest (ROI)
and expressed as photon/second/cm
2
/steradian (p/s/cm
2
/
sr). Higher signal intensity represents higher tumor mass.
Tumor volumes were calculated by caliper measurement
according to the formula V = L x W
2
× 0.52, where V is
tumor volume, L is tumor length, W is tumor width.
Histochemical and Immunohistochemical analysis of TC-1
tumors
The presence of necrotic areas (Fig. 4) in tumor sections
was evaluated by routine Hematoxylin/Eosin (H&E)
staining by two independent and blinded pathologists.
For immunohistochemical staining, five μm-thick sec-
tions of paraffine-embedded mouse tumors were treated
according to the procedure developed by Bonnet et al.
[23]. In brief, deparaffinized and rehydrated sections on
polylysine-coated glass slides were subjected to epitope
retrieval in sodium citrate buffer, pH 6, for 30 min at
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 3 of 11
97 °C. After inhibition of endogenous peroxidase, the
sections were incubated in humidity chamber overnight
at 4 °C with rabbit anti-human/mouse active caspase-3
antigen affinity-purified polyclonal antibody (AF835;
B&D Systems, Italy) diluted 1: 2000 in antibody diluent
solution. Primary antibody detection and stain were
performed in an automated apparatus (Leica BOND-III)
with secondary biotinylated universal antibody at a
dilution of 1:1000.
After the last washing, sections were H&E counter-
stained according to standard procedures. By this 3,3′-
Diaminobenzidine (DAB)-H&E-staining (brown and
blue-pink, respectively) procedure, apoptotic cells appear
brown whereas necrotic cells have pink stained (eosin)
nuclei and cytoplasm.
Statistical analysis
Two-tailed Student’s t-test using the GraphPad Prism 8
software was used for correlation data. Two-way repeated
measures ANOVA was applied for multiple measure-
ments using SPSS Statistics software version 21. A
p< 0.05 was considered statistically significant.
Results
Inhibition of tumor growth by anti-E6 and-E7 scFvs
delivered as intrabodies
In this study, to design a treatment mimicking the therapy
of already established HPV lesions in humans, we tested
the anti-tumor potential of two previously characterized
anti-16E6 and -16E7 scFvs in three different therapeutic
settings of HPV-associated experimental tumors.
Firstly, the anti-16E6 and –16E7 scFvs ability to ham-
per development of TC-1 tumors was evaluated. C57/
BL6 mice were inoculated s. c. in the right flank with
0.5 × 10
5
TC-1-LUC tumor cells. After one week, when
tumors were palpable, recombinant plasmids expressing
scFvI7nuc or scFv43M2SD were injected intra-tumor,
immediately followed by EP at the injection site. The
procedure was repeated for 3 times at 1-week intervals,
for a total of 4 treatments. The irrelevant anti-β-
galactosidase scFvR4nuc and scFvR4sec were used as
negative controls. Tumor growth was monitored weekly
by measurement of luciferase activity.
All mice treated with the irrelevant vectors showed
progressive tumor growth. Conversely, tumor develop-
ment was greatly slowed down in mice treated with
scFv43M2SD or scFvI7nuc. Three weeks after tumor
challenge, the difference between the luminescent signal
of mice treated with the specific scFvs and the respective
controls became significant. Luminescent signal quantifi-
cation of pooled data from animals developing tumors
are summarized in Fig. 1. A clear delay in tumor pro-
gression was evidenced upon intra-tumor treatment with
anti-16E6 and -16E7 scFvs. Mice were followed for 4
weeks from tumor challenge, when the experiment was
interrupted for ethical reasons to avoid animal suffering.
Images of the mice treated weekly are shown in supple-
mentary data (Fig. 1s).
Secondly, in order to evaluate antitumor scFv activity in
a more aggressive tumor condition, an additional experi-
ment was performed by inoculating 2 × 10
5
TC-1 cells s.c.
in the mice right flank. One week after tumor inoculum,
Fig. 1 Antitumor therapeutic effect of the anti-16E6 scFvI7nuc and anti-16E7 scFv43M2SD on TC-1-Luc tumors. The graph shows the luminescent
signal quantification of pooled data from tumors developed by injection of 5 × 10
4
TC-1-LUC cells in C57/BL6 mice. The scFvI7nuc (I7nuc) and
scFv43M2SD (M2SD), and the irrelevant anti-β-galactosidase scFvR4nuc (R4nuc) and scFvR4sec (R4sec) as negative controls, were delivered to
tumors of randomized groups of mice, four times at one-week interval. Tumor growth was monitored with the IVIS® Lumina imaging system.
Photon emission was measured in specific regions of interest (ROI) and expressed as photon/second/cm
2
/steradian (p/s/cm
2
/sr). The difference
between the mean values of photon emission of therapeutic scFvs versus their controls was statistically significant (p= 0.0195 for I7 nuc, p= 0.049
for 43M2SD) as calculated at T5. T is the time point in weeks after tumor cell challenge
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 4 of 11
mice were injected intratumor with scFv43M2SD or
scFvI7nuc, immediately followed by EP at the injection
site. The treatment was repeated 3 times at one-week
intervals, in parallel with monitoring of tumor size by
caliper, which was continued for one week after the last
treatment. Tumor volumes of single mice are shown in
Fig. 2. Significant delay of tumor development was
obtained in all mice treated with both scFvs targeting the
oncoproteins but not in the control mice receiving irrele-
vant scFvs. In Fig. 2, the averages of tumor weight at the
time of mice sacrifice are also shown. The difference be-
tween relevant scFvs and their controls confirmed efficacy
and specificity of the intrabody treatment even in a more
aggressive tumor setting.
Thirdly, the effect of scFvI7nuc was tested in C57/BL6
mice inoculated with 5 × 10
5
C3 cells, which are tumor
cells harbouring the full HPV16 genome, to extend
applicability of the scFv therapy to a different HPV16-
positive solid tumor. Similar to previous experiments,
intratumor scFvs injection and electroporation in mice
were started one week after tumor challenge but treat-
ment was repeated twice at 1-week intervals. Tumor size
was measured by caliper at different time intervals. As
shown in Fig. 3, effective tumor growth inhibition was
obtained by delivery of scFvs even in this HPV tumor
model, strengthening the feasibility of such methodology
for the treatment of HPV tumors. Two-way repeated
measures ANOVA analysis showed high significance
with p= 0.017 for treatment and p= 0.0001 for linear
trend.
Two independent and blinded pathologists analysed
tumor histology after scFv treatment. H&E staining of
tumor sections showed wide areas of necrosis in the
scFv-treated tumors (Fig. 4a, panels 43M2SD, I7nuc)
that were almost absent in those treated with unspecific
intrabodies (Fig. 4a, panels R4sec, R4nuc).
Apoptosis and necrosis are the two major cell death
pathways, and can be distinguished based on
Fig. 2 Antitumor therapeutic effect of the anti-16E6 scFvI7nuc and anti-16E7 scFv43M2SD on higher inoculum of TC-1 cells. C57/BL6 mice were
injected subcutaneously with 2 × 10
5
TC-1 cells and treated intratumor with plasmids expressing the anti-16E6 scFvI7nuc and anti-16E7 scFv43M2SD or
irrelevant scFvs (CTR scFvs). Each line represents a different mouse within the same treatment group. Two types of control mice treated with R4nuc
and R4sec were combined into a single group consisting of 4 total mice to keep the number of mice to a minimum. Treatment was performed four
times at one-week intervals, in parallel with tumor size monitoring by caliper measurement. T is the time point in weeks after tumor cell challenge.
Tumor growth is expressed as the tumor volume in mm
3
at the indicated time points. Significant pvalues with respect to the corresponding controls
are reported on the graphs. In the histogram, the mean weight ±SD of the tumors treated with therapeutic scFvs, and their controls, excised after
mice sacrifice, is reported. Differences are significant with p= 0.004 for scFvI7nuc and p= 0.006 for scFv43M2SD
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 5 of 11
morphological and molecular criteria [24]. To verify
which of these two processes takes place following treat-
ment with intrabodies in vivo, an immunohistochemical
staining protocol was applied to discriminate between
apoptotic and necroptotic cell death through a single
staining procedure on tissue sections of TC-1 tumors
treated with scFvI7nuc, scFv43M2SD or scFvR4nuc and
scFvR4sec as described in Materials and Methods.
As shown in Fig. 4b, only apoptotic death was revealed
in the tumors treated with the anti-E7, or anti-E6 intra-
bodies (panels 43M2SD and I7nuc), while rare apoptotic
areas were detected in tumors treated with unspecific
intrabodies (panels R4sec and R4nuc). Both pathologists
scored the same percentage of apoptotic areas, corre-
sponding to 60% for scFv43M2SD and 30% for scFvI7-
nuc. A percentage of 5 and 15% was detected in tumors
treated with the R4sec and R4nuc irrelevant intrabodies,
respectively.
Discussion
HR HPVs, together with environmental and genetic
cofactors, can cause cancer in different body districts.
The E6 and E7 oncoproteins of HR HPVs play a key
role in cellular transformation and maintenance of
the transformed status. This circumstance and the
unique localization of E6/E7 within tumors can guide
therapeutic approaches that are safe and precise be-
cause they target such oncoproteins.
The approach we used relies on specific scFvs character-
ized in previous studies [14–16] .Confocal microscopy
results suggested that a key mechanism underlying the an-
titumor activity of both the anti-16E6 scFvI7nuc targeting
cell nucleus and the anti-16E7 scFv43M2SD targeting ER
intrabodies is the delocalization of oncoproteins. Further-
more, through immunological assays and Surface Plasmon
Resonance (SPR), we showed that the activity of the
anti-16E7 scFv43M2SD depends to some extent on
interference with the E7/pRb binding [18], whereas the
anti-16E6 scFvI7nuc activity seems to be at least partly
related to the p53 rescue, with consequent increase of
cell death due to necrosis and apoptosis [16]. Interest-
ingly, a p53 rescue suggestive of cross-reaction was
observed after transfection with scFvI7nuc of Me180
cell lines, which harbor the HR HPV68 and express an
E6 protein with homology of 61.43% to the 16E6, and
not after transfection of HeLa cells, harboring the
HPV18 and expressing an E6 protein with homology of
57.97% to the 16E6 [25,26]. This observation suggests the
advisability of testing possibly available scFvs, against
more than one related HPV genotype, to investigate
potential broader activity spectra.
Recently, we also compared our scFvs to Clinical-Stage
Therapeutic antibodies (CSTs) by computational analysis,
Fig. 3 Antitumor therapeutic effect of the anti-16E6 scFvI7nuc on C3 tumors. The scatter plot shows the volume of C3 tumors raised in C57/BL6
mice treated with scFvI7nuc (I7nuc) or the unspecific scFvR4nuc (R4nuc). Each point represents the mean volume ± SD of 4 different mice,
measured by caliper every 3–4 days. T is the time point in weeks after tumor cell challenge. At the end point (T4), differences are statistically
significant (p= 0.0005) by two-tailed Student’s t-test. Two-way repeated measures ANOVA analysis showed p= 0.017 for treatment and p= 0.0001
for linear trend. Data were expressed as means ± standard deviations (SD). Of note, the tumor volume of R4nuc mice had an unexpected
increase in the last 3 days prior to the measurement at T4. Despite mice were in good health, tumor burden exceeded the size allowed by our
internal ethics and all mice were immediately sacrificed
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 6 of 11
Fig. 4 Treatment with anti-16E6 and -16E7 scFvs induces cell death by apoptosis in tumor mass. H&E staining (panels a) and active caspase-3
(cleaved caspase) staining (panels b) of HPV tumor sections from mice treated with scFvI7nuc (I7nuc), scFv43M2SD (43M2SD) and the irrelevant
scFvR4nuc (R4nuc) and scFvR4sec (R4sec) are presented. Tumors from one representative mouse for each treatment group are shown. Referring
to the numbering shown in Fig. s1, mouse # 3501 for I7nuc, #3504 for R4nuc, #3520 for M2SD and #3514 for R4sec were used. With H&E staining,
wide areas of necrosis are visible in tumors treated with both therapeutic scFvs, while rare signs of necrosis are detected in tumors treated with
the control scFvs (a). With active caspase-3 staining, brown-stained areas meaningful of apoptotic cell death are clearly visible in tumors treated
with both therapeutic scFvs while rare apoptotic areas are detected in tumors treated with the control scFvs (b)
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 7 of 11
and observed that they have properties well-conforming
antibodies which have already reached Phase I clinical
trial [27].
Delivering proteins to the cytosol is challenging and
the same is true also in the case of scFvs. In vivo gene
therapy using DNA delivery is a well-established proced-
ure, with clinical trials in progress and some already
available drugs such as the ZOLGENSMA® (onasemno-
gene abeparvovec-xioi), recently approved by FDA for
muscular spinal atrophy. In this study, intrabodies were
expressed directly in the target tumor cells by electro-
porating tumors with scFv-expressing recombinant
plasmids. However, direct delivery of scFvs as proteins is
also feasible [28].
The main goal of this study was to validate the antitu-
mor activity of the previously characterized scFvI7nuc
and scFv43M2SD in animal preclinical models that
mimic a condition as close as possible to that occurring
in humans, where a tumor lesion is already established
at the time of diagnosis. The ability of counteracting
tumor growth by intratumor expression of the scFvs was
investigated in different mouse models.
In the first HPV tumor model used, the TC-1-LUC
cells, which allow monitoring of tumor growth in
tumor-bearing mice through luminescence imaging,
were employed [29]. By luciferase measurements, we
could visualize a decreased luciferase activity related to
both the scFvI7nuc and scFv43M2SD expression, not ob-
served in cells expressing the scFvR4nuc and scFvR4sec
used as negative controls.
A critical situation of more advanced tumor progression
was then simulated by using a fourfold higher number of
normal TC-1 cells to implant HPV tumor in syngeneic
C57/BL6 mice. Tumor growth inhibition was obtained even
under these more aggressive experimental conditions.
To ensure that the scFv therapeutic efficacy did not
depend on the animal model, the effect of scFvI7nuc
was evaluated in a mouse model based on C3 cells,
which express slightly higher levels of E6 and E7 with
regard to TC-1 cells [30]. The results were comparable
to those obtained with TC1 cells. It is to note that all
the treatments have been started on well-established
tumors while many studies reporting more effective im-
munotherapies on similar models have been conducted
at an early stage of tumor development [31]. Interest-
ingly, the level of tumor inhibition in the TC-1 model
was similar to that obtained by inducing cell apoptosis
with different treatments [32].
The incomplete inhibition of tumor growth stresses
the importance of dosage and timing for efficacy of the
scFv treatment, and does not exclude that increased
doses of scFvs or a different treatment schedule might
result in complete tumor inhibition. Of note, the scFv
delivery system based on the electroporation of
recombinant plasmids has high safety features that make
it suitable for use in humans as well. However, different
delivery systems may even increase efficacy of the treat-
ment with scFvs. In this perspective, we are exploring an
exosomes-based delivery system which has the capacity
of auto-implementing [33].
Histological observation of tumors treated with thera-
peutic scFvs highlighted the presence of large areas of
necrosis that could be due to scFv-induced apoptosis
and were almost absent in tumors treated with the ir-
relevant scFvs. Staining of tumor sections for active
caspase-3, which is a major player in the apoptotic
process, revealed a high percentage of caspase-3 positive
areas in tumors treated with therapeutic scFvs compared
to controls. This finding indicates a direct involvement
of the scFvs in the apoptotic process thus suggesting a
possible biological reason for their therapeutic effect.
The apoptotic process induced in mice tumors by scFv
treatment is in agreement with data previously obtained
in vitro in HPV16-positive cell lines, where we demonstrated
the scFvI7nuc involvement in hampering E6-dependent p53
degradation and rescuing pro-apoptotic activity of the tumor
suppressor [16]. The results presented here conclusively
demonstrate that both treatments with anti-16E6 or -16E7
intrabodies induce strong apoptosis in tumors.
As far as concerns the scFv43M2SD action, we believe
that it may have effects in more than one direction. We
have already demonstrated that the scFv43M2SD binding
to E7 interferes with the pRb binding and degradation, thus
increasing the pRb intracellular pool and reestablishing
anti-proliferative activity of the tumor suppressor [15,18].
However, non-nuclear activity of pRb was reported in the
induction of mitochondrial apoptosis via direct interaction
of pRB with Bax [34]. Accordingly, a fraction of endogen-
ous pRB is constitutively associated with mitochondria.
Hence, it could be hypothesized that the decreased pRb
degradation in 43M2SD-expressing cells may increase
mitochondrial pRb levels thereby inducing the intrinsic
pathway of apoptosis.
Nevertheless, it cannot be excluded that scFv43M2SD,
in virtue of the SEKDEL signal, may exert anticancer ac-
tivity also through a mechanism involving the ER stress.
Indeed, over-expression of proteins binding to the SEKD
EL receptors on ER was shown to induce the release of
important mediators of ER homeostasis and the ER
stress response [35,36]. Both the ER stress response and
the activation of the mithocondrial pathway of apoptosis
may contribute to the tumor growth inhibition after the
scFv43M2SD delivery to tumor cells.
In Fig. 5, a schematic representation of the hypothet-
ical and demonstrated effects of intrabodies targeting
the oncoproteins, is shown.
Although the E6 and E7 action has been extensively
studied, it should not be excluded that in the future new
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 8 of 11
functions performed in other cellular compartments will
be discovered. In this regard, the implementation of new
constructs to provide these or other scFvs with different
signals for intracellular localization may represent a use-
ful opportunity. Different antigen-specific scFvs can bind
to different epitopes of the same protein thus potentially
interfering with different protein functions. This may
have therapeutic relevance since the pro-tumor action is
a mosaic of activities that could be prevented as a whole
or even modulated separately. Furthermore, since the
two oncoproteins exert a concerted action, the concomi-
tant inhibition of E6 and E7 may be more efficacious
than the inhibition of only one of them; for this reason,
it could be useful to study the effect of anti-16E6 and
anti-16E7 scFvs in combination.
Conclusion
This study shows in preclinical models the efficacy of
two intrabodies against the E6 and E7 oncoproteins in
counteracting the growth of HPV16 tumors. This repre-
sents a step forward for the treatment of HPV tumors,
but further studies will be needed to optimize delivery,
doses, timing and number of administrations for their
translation to the clinic. The localized expression of the
transgene increases the safety of the treatment, which
can be repeated as many times as necessary since the
plasmid vectors are free from undesired pathogenicity
and immunogenicity. Furthermore, the effectiveness of
electroporation, a methodology allowed for human treat-
ment, to deliver scFvs to tumors was confirmed.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s13046-021-01841-w.
Additional file 1: Fig. s1. Antitumor effect of scFvs delivered to TC-1-luc
HPV tumors. Imaging of single mice challenged with TC-1-luc tumors and
treated with scFvI7nuc, scFv43M2SD or scFvR4nuc and R4sec as controls.
Fig. 5 Hypothetical mechanisms of induction of apoptosis by the anti-16E6 and anti-16E7 intrabodies. The binding of scFvI7nuc (I7nuc) to E6 can
inhibit the cytoplasmic degradation of p53. The restored levels of nuclear p53 may activate the transcription of pro-apoptotic genes including
Puma, Noxa, Bak and Bax and the subsequent loss of mitochondrial membrane potential, with downstream activation of the executor caspase 3,
finally leading to cell death. The binding of scFv43M2SD (43M2SD) to E7 can inhibit its translocation to nucleus and the subsequent inactivation
of the Retinoblastoma tumor suppressor (pRB) thus restoring the control of E2F transcription factors by pRb. At the same time, the binding of
scFv43M2SD can inhibit the association of E7 with the cullin 2 complex (CUL2) and the recruitment of pRB for ubiquitination. Increased levels of
pRB, regardless of its role as a transcriptional regulator, can directly activate the BAX apoptosis regulator at mitochondrial level and promote cell
death [34]. The binding of scFv43M2SD to KDEL receptors of ER may also induce ER stress-related molecules which can cause apoptosis by
triggering the activation of caspases
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 9 of 11
Treatments were delivered four times at one-week intervals (from T1 to T4).
Mice are indicated by numbers on the side. T is the time in weeks after
tumor cell challenge. Luminescence was quantified as described in Methods
at indicated time points before the sacrifice of mice for ethical reasons (†).
Abbreviations
HPV: Human Papillomavirus; HR: High Risk; SCC: Squamous Cell Carcinoma;
HNSCC: Head and Neck Squamous Cell Carcinoma; TAA: Tumor Associated
Antigen; CTL: Cytotoxic T-Lymphocyte; mAbs: Monoclonal Antibodies;
VH: Variable Heavy region; VL: Variable Light region; CDR: Complementarity
Determining Region; ER: Endoplasmic Reticulum; NLS: Nuclear Localization
Signal; s. c.: Subcutaneously; EP: Electroporation; H&E: Hematoxylin/Eosin;
DAB: 3,3′-diaminobenzidine; SPR: Surface Plasmon Resonance; CSTs: Clinical
Stage Therapeutics
Acknowledgements
We thank Dr. Isabella Sperduti for the support in the statistical analysis.
Authors’contributions
Conceptualization, L.A. and P.D.B.; methodology, L.A., P.D.B. and F.P.;
validation, L.A., C.A. and A. V.; investigation, L.A., F.P., M.A.C. and C.B.;
resources, L.A., P.D.B. and A. V.; writing—original draft preparation, L.A.;
writing—review and editing, L.A., C.A., A.V., F.P. and P.D.B; visualization, L. A.
and C.A.; supervision, L.A. and A.V.; project administration, L.A.; funding
acquisition, L.A., A.V. and P.D.B. All authors read the article and gave consent
to its publication. The author(s) read and approved the final manuscript.
Funding
This research was funded by intramural funds of the Istituto Superiore di
Sanità and IRCCS Regina Elena National Cancer Institute.
Availability of data and materials
The data that support the findings of this study are available from the
corresponding author, upon reasonable request.
Ethics approval and consent to participate
All experimental procedures were approved by the Institutional Animal Care
of the Regina Elena National Cancer Institute and by the Government
Committee of National Ministry of Health (85/2016-PR) and were carried out
in accordance with EU Directive 2010/63/EU for animal experimentation.
Consent for publication
Not applicable.
Competing interests
The authors declare no conflict of interest.
Author details
1
HPV Unit, UOSD Tumor Immunology and Immunotherapy, IRCCS Regina
Elena National Cancer Institute, 00144 Rome, Italy.
2
Department of Biology,
University of Rome Tor Vergata, 00133 Rome, Italy.
3
Anatomy Pathology Unit,
Department of Research, Diagnosis and Innovative Technologies, IRCC
S-Regina Elena National Cancer Institute, 00144 Rome, Italy.
4
Department of
Infectious Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy.
Received: 18 September 2020 Accepted: 13 January 2021
References
1. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical
application. Nat Rev Cancer. 2002;2:342.
2. de Martel C, Plummer M, Vignat J, Franceschi S. Worldwide burden of
cancer attributable to HPV by site, country and HPV type. Int J Cancer. 2017;
141(4):664–70.
3. Castellsagué X, Alemany L, Quer M, Halec G, Quirós B, Tous S, et al. HPV
Involvement in Head and Neck Cancers: Comprehensive Assessment of
Biomarkers in 3680 Patients. J Natl Cancer Inst. 2016;108(6):djv403 [cited
2020 Aug 14] Available from: https://pubmed.ncbi.nlm.nih.gov/26823521/.
4. Human Papillomavirus Vaccines [Int. Drugs and Lactation Database
(LactMed). National Library of Medicine (US); 2006 [cited 2020 Aug 14].
Available from: http://www.ncbi.nlm.nih.gov/pubmed/30000765.
5. Graham SV. The human papillomavirus replication cycle, and its links to
cancer progression: A comprehensive review. Clin Sci Portland Press Ltd.
2017;131:2201–21 [cited 2020 Aug 14] Available from: https://pubmed.ncbi.
nlm.nih.gov/28798073/.
6. Cohen PA, Jhingran A, Oaknin A, Denny L. Cervical cancer. Lancet Lancet
Publishing Group. 2019;393:169–82 [cited 2020 Aug 14] Available from:
https://pubmed.ncbi.nlm.nih.gov/30638582/.
7. Yan J, Reichenbach DK, Corbitt N, Hokey DA, Ramanathan MP, McKinney KA,
et al. Induction of antitumor immunity in vivo following delivery of a novel
HPV-16 DNA vaccine encoding an E6/E7 fusion antigen. Vaccine. 2009;27(3):
431–40 [cited 2020 Aug 14] Available from: https://pubmed.ncbi.nlm.nih.
gov/19022315/.
8. Jiang Z, Albanese J, Kesterson J, Warrick J, Karabakhtsian R, Dadachova E,
et al. Monoclonal Antibodies Against Human Papillomavirus E6 and E7
Oncoproteins Inhibit Tumor Growth in Experimental Cervical Cancer. Transl
Oncol. 2019;12(10):1289–95 [cited 2020 Jun 27] Available from: https://
pubmed.ncbi.nlm.nih.gov/31325765/.
9. Antibody therapeutics approved or in regulatory review in the EU or
US - The Antibody Society . [cited 2020 Aug 14]. Available from:
https://www.antibodysociety.org/resources/approved-antibodies/.
Accessed 14 Sept 2020.
10. Accardi L, Di Bonito P. Antibodies in single-chain format against tumour-
associated antigens: present and future applications. Curr Med Chem. 2010;
17(17):1730–55.
11. Frenzel A, Schirrmann T, Hust M. Phage display-derived human antibodies
in clinical development and therapy. mAbs. Taylor and Francis Inc. 2016;8:
1177–94 [cited 2020 Sep 15] Available from: /pmc/articles/PMC5058633/
?report=abstract.
12. Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single
domains. Nat Biotechnol. 2005;23:1126–36 [cited 2020 Sep 15] Available
from: https://pubmed.ncbi.nlm.nih.gov/16151406/.
13. Lin Y, Chen Z, Hu C, Chen ZS, Zhang L. Recent progress in antitumor
functions of the intracellular antibodies. Drug Discovery Today. Elsevier Ltd.
2020;25 [cited 2020 Aug 14]. Available from: https://pubmed.ncbi.nlm.nih.
gov/32112969/.
14. Accardi L, Donà MG, Di Bonito PGC. Intracellular anti-E7 human antibodies
in single-chain format inhibit proliferation of HPV16-positive cervical
carcinoma cells. Int J Cancer. 2005;116(4):564–70.
15. Accardi L, Paolini F, Mandarino A, Percario Z, Di Bonito P, Di Carlo V, et al. In
vivo antitumor effect of an intracellular single-chain antibody fragment
against the E7 oncoprotein of human papillomavirus 16. Int J Cancer. 2014;
134(11):2742.
16. Amici C, Visintin M, Verachi F, Paolini F, Percario Z, Di Bonito P, et al. A
novel intracellular antibody against the E6 oncoprotein impairs growth of
human papillomavirus 16-positive tumor cells in mouse models.
Oncotarget. 2016;7(13):15539.
17. Young JL, Dean DA. Electroporation-Mediated Gene Delivery. Adv Genet.
2015;89:49–88 [cited 2020 Sep 10] Available from: https://pubmed.ncbi.nlm.
nih.gov/25620008/.
18. Accardi L, Donà MG, Mileo AM, Paggi MG, Federico A, Torreri P, et al.
Retinoblastoma-independent antiproliferative activity of novel intracellular
antibodies against the E7 oncoprotein in HPV 16-positive cells. BMC Cancer.
2011;11(1):17 Available from: http://www.biomedcentral.com/1471-2407/11/17.
19. Lener M, Horn IR, Cardinale A, Messina S, Nielsen UB, Rybak SM, et al.
Diverting a protein from its cellular location by intracellular antibodies: The
case of p21Ras. Eur J Biochem. 2000;267(4):1196–205 [cited 2020 Aug 20]
Available from: https://pubmed.ncbi.nlm.nih.gov/10672031/.
20. Lin KY, Guarnieri FG, Staveley-O’Carroll KFLH, August JT, Pardoll DMWT. Treatment
of established tumors with a novel vaccine that enhances major
histocompatibility class II presentation of tumor antigen. Cancer Res. 1996;56:21–6.
21. Feltkamp MC, Smits HL, Vierboom MP, Minnaar RPD, Jongh BM, Drijfhout JW,
ter Schegget J, Melief CJ, Kast WM. Vaccination with cytotoxic T lymphocyte
epitopecontaining peptide protects against a tumor induced by human
papillomavirus type 16-transformed cells. Eur J Immunol. 1993;23:2242–9.
22. Paolini F, Curzio G, Cordeiro MN, Massa S, Mariani L, Pimpinelli F, de Freitas
AC, Franconi R,Venuti A. HPV 16 E5 oncoprotein is expressed in early stage
carcinogenesis and can be a target of immunotherapy. Hum Vaccin
Immunother. 2017;13(2):291–7.
Paolini et al. Journal of Experimental & Clinical Cancer Research (2021) 40:37 Page 10 of 11
23. Bonnet MC. Immunohistological Tools to Discriminate Apoptotic and
Necrotic Cell Death in the Skin. In: Methods in molecular biology (Clifton,
NJ). Methods Mol Biol. 2013. p. 135–42. [cited 2020 Aug 14] Available from:
https://pubmed.ncbi.nlm.nih.gov/23733574/.
24. Edinger AL, Thompson CB. Death by design: Apoptosis, necrosis and
autophagy. Curr Opin Cell Biol. 2004;16:663–9 [cited 2020 Aug 14] Available
from: https://pubmed.ncbi.nlm.nih.gov/15530778/.
25. Howie HL, Katzenellenbogen RA, Galloway DA. Papillomavirus E6 proteins.
Virology. 2009;384:324–34.
26. PaVE: Papilloma virus genome database. [cited 2020 Aug 17]. Available
from: https://pave.niaid.nih.gov/#search/pv_specific_blast.
27. Amici C, Donà MG, Chirullo B, Di Bonito P, Accardi L. Epitope Mapping and
Computational Analysis of Anti-HPV16 E6 and E7 Antibodies in Single-Chain
Format for Clinical Development as Antitumor Drugs. Cancers (Basel). 2020;
12(7):1803 [cited 2020 Aug 14] Available from: https://pubmed.ncbi.nlm.nih.
gov/32640530/.
28. Zhang C, Ötjengerdes RM, Roewe J, Mejias R, Marschall ALJ. Applying
Antibodies Inside Cells: Principles and Recent Advances in Neurobiology,
Virology and Oncology. BioDrugs Adis. 2020;34:435–62 [cited 2020 Aug 20]
Available from: https://pubmed.ncbi.nlm.nih.gov/32301049/.
29. Hung CF, Calizo R, Tsai YC, He L, Wu TC. A DNA vaccine encoding a single-
chain trimer of HLA-A2 linked to human mesothelin peptide generates anti-
tumor effects against human mesothelin-expressing tumors. Vaccine. 2007;
25(1):127–35.
30. Paolini F, Massa S, Manni I, Franconi R, Venuti A. Immunotherapy in new
pre-clinical models of HPV-associated oral cancers. In: Human Vaccines and
Immunotherapeutics. Landes Bioscience. 2013. p. 534–43. [cited 2020 Aug
14] Available from: https://pubmed.ncbi.nlm.nih.gov/23296123/.
31. Venuti A, Curzio G, Mariani L, Paolini F. Immunotherapy of HPV-associated
cancer: DNA/plant-derived vaccines and new orthotopic mouse models.
Cancer Immunology, Immunotherapy. Springer Science and Business Media
Deutschland GmbH. 2015;64:1329–38 [cited 2020 Sep 7] Available from:
/pmc/articles/PMC4554738/?report=abstract.
32. Kim JE, Lee JI, Jin DH, Lee WJ, Bin PG, Kim S, et al. Sequential treatment of
HPV E6 and E7-expressing TC-1 cells with bortezomib and celecoxib
promotes apoptosis through p-p38 MAPK-mediated downregulation of
cyclin D1 and CDK2. Oncol Rep. 2014;31(5):2429–37 [cited 2020 Sep 7]
Available from: https://pubmed.ncbi.nlm.nih.gov/24627094/.
33. Ferrantelli F, Arenaccio C, Manfredi F, Olivetta E, Chiozzini C, Leone P, et al.
The intracellular delivery of anti-HPV16 E7 scFvs through engineered
extracellular vesicles inhibits the proliferation of HPV-infected cells. Int J
Nanomedicine. 2019;14:8755–68 [cited 2020 Aug 14] Available from: /pmc/
articles/PMC6844212/?report=abstract.
34. Hilgendorf KI, Leshchiner ES, Nedelcu S, Maynard MA, Calo E, Ianari A, et al.
The retinoblastoma protein induces apoptosis directly at the mitochondria.
Genes Dev. 2013;27(9):1003–15 [cited 2020 Aug 14] Available from: /pmc/
articles/PMC3656319/?report=abstract.
35. Pérez-Trujillo JJ, Robles-Rodríguez OA, Garza-Morales R, García-García A,
Rodríguez-Rocha H, Villanueva-Olivo A, et al. Antitumor Response by
Endoplasmic Reticulum-Targeting DNA Vaccine Is Improved by Adding a
KDEL Retention Signal. Nucleic Acid Ther. 2018;28(4):252–61 [cited 2020 Aug
14] Available from: https://pubmed.ncbi.nlm.nih.gov/29733248/.
36. Lee SY, Oh JY, Kang TH, Shin HS, Cheng MA, Farmer E, et al. Endoplasmic
reticulum stress enhances the antigen-specific T cell immune responses and
therapeutic antitumor effects generated by therapeutic HPV vaccines. J
Biomed Sci. 2019;26(1):1–8.
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