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Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors


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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. 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.
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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
, Carla Amici
, Mariantonia Carosi
, Claudia Bonomo
, Paola Di Bonito
, Aldo Venuti
Luisa Accardi
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:
Department of Infectious Diseases, Istituto Superiore di Sanità, 00161 Rome,
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
(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
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
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 [1416].
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].
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
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
TC-1 Luc, 2 × 10
TC-1 or 5 × 10
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
/steradian (p/s/cm
sr). Higher signal intensity represents higher tumor mass.
Tumor volumes were calculated by caliper measurement
according to the formula V = L x W
× 0.52, where V is
tumor volume, L is tumor length, W is tumor width.
Histochemical and Immunohistochemical analysis of TC-1
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 Students 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.
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
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
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
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
/steradian (p/s/cm
/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
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
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
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
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,
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 [1416] .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 34 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 Students 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.
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.
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 ().
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
We thank Dr. Isabella Sperduti for the support in the statistical analysis.
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.; writingoriginal draft preparation, L.A.;
writingreview 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.
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
HPV Unit, UOSD Tumor Immunology and Immunotherapy, IRCCS Regina
Elena National Cancer Institute, 00144 Rome, Italy.
Department of Biology,
University of Rome Tor Vergata, 00133 Rome, Italy.
Anatomy Pathology Unit,
Department of Research, Diagnosis and Innovative Technologies, IRCC
S-Regina Elena National Cancer Institute, 00144 Rome, Italy.
Department of
Infectious Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy.
Received: 18 September 2020 Accepted: 13 January 2021
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... We then investigated the I7nuc antitumour activity in mouse models for HPV tumours based on the injection of HPV16-positive tumour cells in C57BL6 mice. The scFv capability to either prevent cancer development from scFv-expressing tumour cells, or to hinder cancer progression by delivery to already established tumours, was evaluated [71,72] ( Figure 4). We observed a clear impairment of tumour growth in all mice injected with TC-1 tumour cells expressing I7nuc by retroviral transduction before inoculation into mice, with 60% of them completely protected from tumour onset for the 4 months of observation time [71]. ...
... The result was confirmed using two different HPV16-positive tumour cells, namely C3 and TC-1 cells, and also by employing higher amounts of TC-1 cells to mimic tumours that are more aggressive. Through histology and immunohistochemistry, we showed that the antitumour activity is based on the induction of tumour cell death by apoptosis [72]. ...
... In addition, the scFv43M2SD intracellular expression was able to inhibit significantly and specifically the proliferation of different HPV16-positive cell lines [85]. The scFv43M2 was then tested in vivo in mouse HPV tumour models, demonstrating the ability to counteract tumour progression both when administered to tumour cells before their injection into mice and when administered to already implanted tumours [72,85] (Figure 4). ...
Full-text available
In recent decades, recombinant antibodies against specific antigens have shown great promise for the therapy of infectious diseases and cancer. Human papillomaviruses (HPVs) are involved in the development of around 5% of all human cancers and HPV16 is the high-risk genotype with the highest prevalence worldwide, playing a dominant role in all HPV-associated cancers. Here, we describe the main biological activities of the HPV16 E6, E7, and E5 oncoproteins, which are involved in the subversion of important regulatory pathways directly associated with all known hallmarks of cancer. We then review the state of art of the recombinant antibodies targeted to HPV oncoproteins developed so far in different formats, and outline their mechanisms of action. We describe the advantages of a possible antibody-based therapy against the HPV-associated lesions and discuss the critical issue of delivery to tumour cells, which must be addressed in order to achieve the desired translation of the antibodies from the laboratory to the clinic.
... Recently, Paolini et al. reported the delivery of plasmids encoding an antibody in single-chain format (scFv) against the HPV-16 E6 and E7 proteins in three different murine preclinical models [157]. They demonstrated the efficient antitumor response induced by scFv delivered as intrabodies by electroporation, with the induction of a delayed tumor progression and large apoptotic areas in tumors. ...
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Recently, the interest in using nucleic acids for therapeutic applications has been increasing. DNA molecules can be manipulated to express a gene of interest for gene therapy applications or vaccine development. Plasmid DNA can be developed to treat different diseases, such as infections and cancer. In most cancers, the immune system is limited or suppressed, allowing cancer cells to grow. DNA vaccination has demonstrated its capacity to stimulate the immune system to fight against cancer cells. Furthermore, plasmids for cancer gene therapy can direct the expression of proteins with different functions, such as enzymes, toxins, and cytotoxic or proapoptotic proteins, to directly kill cancer cells. The progress and promising results reported in animal models in recent years have led to interesting clinical results. These DNA strategies are expected to be approved for cancer treatment in the near future. This review discusses the main strategies, challenges, and future perspectives of using plasmid DNA for cancer treatment.
... A scFv intrabody targeting VEGFR2/KDR was selected from an immune phage display library of mice [63]. Recently, intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for the treatment of established HPV-associated tumors were generated from a synthetic human antibody phage display library [64]. One scFv against 16 E7 was targeted to the ER and interferes with the binding of E7 to retinoblastoma tumor suppressor (pRb). ...
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Tumor cells are characterized by overexpressed tumor-associated antigens or mutated neoantigens, which are expressed on the cell surface or intracellularly. One strategy of cancer immunotherapy is to target cell-surface-expressed tumor-associated antigens (TAAs) with therapeutic antibodies. For targeting TAAs or neoantigens, adoptive T-cell therapies with activated autologous T cells from cancer patients transduced with novel recombinant TCRs or chimeric antigen receptors have been successfully applied. Many TAAs and most neoantigens are expressed in the cytoplasm or nucleus of tumor cells. As alternative to adoptive T-cell therapy, the mRNA of intracellular tumor antigens can be depleted by RNAi, the corresponding genes or proteins deleted by CRISPR-Cas or inactivated by kinase inhibitors or by intrabodies, respectively. Intrabodies are suitable to knockdown TAAs and neoantigens without off-target effects. RNA sequencing and proteome analysis of single tumor cells combined with computational methods is bringing forward the identification of new neoantigens for the selection of anti-cancer intrabodies, which can be easily performed using phage display antibody repertoires. For specifically delivering intrabodies into tumor cells, the usage of new capsid-modified adeno-associated viruses and lipid nanoparticles coupled with specific ligands to cell surface receptors can be used and might bring cancer intrabodies into the clinic.
... It provides a direction for the study of new forms of viral gene expression that play a role in the pathogenesis of tumor viruses. Paolini et al. (64) described the expression profile of HPV-16 E7 regulated circRNA in cervical cancer cells for the first time, which directly proved that viral oncogenes may affect the abundance of multiple circRNA and promote the growth of HPV positive cancer cells. These studies have increased the understanding of circRNA and virus-related tumor mechanisms. ...
Circular RNA (circRNA) is a large class of covalently closed circular RNA. As a member of competitive endogenous RNA (ceRNA), it participates in the regulation of circRNA-miRNA-mRNA network and plays an important role in the regulation of physiology and pathology. CircRNA is produced by the reverse splicing of exon, intron or both, forming exon or intron circRNA. Studies have shown that circRNA is a ubiquitous molecule, which exceeds the linear mRNA distributed in human cells. Because of its covalent closed-loop structure, circRNA is resistant to RNase R, which is more stable than linear mRNA; circRNA is highly conserved in different species. It was found that circRNA competitively adsorbs miRNA, as a miRNA sponge, to involve in the expression regulation of a variety of genes and plays an important role in tumor development, invasion, metastasis and other processes. These molecules offer new potential opportunities for therapeutic intervention and serve as biomarkers for diagnosis. In this paper, the origin, characteristics and functions of circRNA and its role in tumor development, invasion and metastasis, diagnosis and prognosis are reviewed.
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Human Papillomavirus 16-associated cancer, affecting primarily the uterine cervix but, increasingly, other body districts, including the head–neck area, will long be a public health problem, despite there being a vaccine. Since the virus oncogenic activity is fully ascribed to the viral E6 and E7 oncoproteins, one of the therapeutic approaches for HPV16 cancer is based on specific antibodies in single-chain format targeting the E6/E7 activity. We analyzed the Complementarity Determining Regions, repositories of antigen-binding activity, of four anti-HPV16 E6 and -HPV16 E7 scFvs, to highlight possible conformity to biophysical properties, recognized to be advantageous for therapeutic use. By epitope mapping, using E7 mutants with amino acid deletions or variations, we investigated differences among the anti-16E7 scFvs in terms of antigen-binding capacity. We also performed computational analyses to determine whether length, total net charge, surface hydrophobicity, polarity and charge distribution conformed well to those of the antibodies that had already reached clinical use, through the application of developability guidelines derived from recent literature on clinical-stage antibodies, and the Therapeutic Antibodies Profiler software. Overall, our findings show that the scFvs investigated may represent valid candidates to be developed as therapeutic molecules for clinical use, and highlight characteristics that could be improved by molecular engineering.
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To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson’s disease (α-synuclein), Alzheimer’s disease (amyloid-β) or Huntington’s disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.
Full-text available
Purpose: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation. Methods: The Nefmut gene was fused in frame at its 3'-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay. Results: The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv. Conclusion: Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.
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Nearly all cases of cervical cancer are initiated by persistent infection with high-risk strains of human papillomavirus (hr-HPV). When hr-HPV integrates into the host genome, the constitutive expression of oncogenic HPV proteins E6 and E7 function to disrupt p53 and retinoblastoma regulation of cell cycle, respectively, to favor malignant transformation. HPV E6 and E7 are oncogenes found in over 99% of cervical cancer, they are also expressed in pre-neoplastic stages making these viral oncoproteins attractive therapeutic targets. Monoclonal antibodies (mAbs) represent a novel potential approach against the actions of hr-HPV E6 and E7 oncoproteins. In this report, we describe the utilization of anti-HPV E6 and HPV E7 mAbs in an experimental murine model of human cervical cancer tumors. We used differential dosing strategies of mAbs C1P5 (anti-HPV 16 E6) and TVG701Y (anti-HPV E7) administered via intraperitoneal or intratumoral injections. We compared mAbs to the action of chemotherapeutic agent Cisplatin and demonstrated the capacity of mAbs to significantly inhibit tumor growth. Furthermore, we investigated the contribution of the immune system and found increased complement deposition in both C1P5 and TVG701Y treated tumors compared to irrelevant mAb therapy. Taken together, the results suggest that anti-HPV E6 and E7 mAbs exert inhibition of tumor growth in a viral-specific manner and stimulate an immune response that could be exploited for an additional treatment options for patients.
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Background Endoplasmic reticulum stress has a profound effect on cancer cell proliferation and survival, and also has the capacity to activate cells of the adaptive immune system. Multimodal treatment methods that utilize and combine conventional cancer therapies with antigen-specific immunotherapies have emerged as promising approaches for the treatment and control of cancer. However, it is not well known whether endoplasmic reticulum stress-inducing agents can influence the efficacy of tumor antigen-targeting vaccines. Methods In the past, we developed a therapeutic human papillomavirus (HPV) DNA vaccine that encodes for calreticulin (CRT) linked to the HPV16 E7 antigen (CRT/E7). In this study, we utilize the CRT/E7 and further encode for an endoplasmic reticulum (ER) stress-inducing agent, 3-bromopyruvate (3-BrPA), in a preclinical model, by harnessing its potential to enhance HPV16 E7-specific CD8+ T cell immune responses as well as antitumor effects against E7-expressing tumors (TC-1 cells). E7-specific CD8+ T cells were added to evaluate the cytotoxicity of luciferase-expressing TC-1 tumor cells treated with 3-BrPA in vitro, as measured with an IVIS Luminescence Imaging System. We also determined the levels of ER stress markers in 3-BrPA-treated TC-1 cells. TC-1 tumor-bearing mice were treated with either 3-BrPA (10 mg/kg, intraperitoneal injection) and/or CRT/E7 DNA vaccine (30 μg/mouse). Results Treatment of E7-expressing TC-1 tumor cells with 3-BrPA induced significantly higher in vitro cytotoxicity and resulted in upregulation of endoplasmic reticulum stress markers (CHOP and GRP78). More importantly, combination treatment of 3-BrPA and the CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC-1 tumor-bearing mice. Conclusions Our data indicate that 3-BrPA can enhance therapeutic HPV vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation and provide novel strategies for the treatment of HPV-associated diseases.
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HPVs (human papillomaviruses) infect epithelial cells and their replication cycle is intimately linked to epithelial differentiation. There are over 200 different HPV genotypes identified to date and each displays a strict tissue specificity for infection. HPV infection can result in a range of benign lesions, for example verrucas on the feet, common warts on the hands, or genital warts. HPV infects dividing basal epithelial cells where its dsDNA episomal genome enters the nuclei. Upon basal cell division, an infected daughter cell begins the process of keratinocyte differentiation that triggers a tightly orchestrated pattern of viral gene expression to accomplish a productive infection. A subset of mucosal-infective HPVs, the so-called 'high risk' (HR) HPVs, cause cervical disease, categorized as low or high grade. Most individuals will experience transient HR-HPV infection during their lifetime but these infections will not progress to clinically significant cervical disease or cancer because the immune system eventually recognizes and clears the virus. Cancer progression is due to persistent infection with an HR-HPV. HR-HPV infection is the cause of > 99.7% cervical cancers in women, and a subset of oropharyngeal cancers, predominantly in men. HPV16 (HR-HPV genotype 16) is the most prevalent worldwide and the major cause of HPV-associated cancers. At the molecular level, cancer progression is due to increased expression of the viral oncoproteins E6 and E7, which activate the cell cycle, inhibit apoptosis, and allow accumulation of DNA damage. This review aims to describe the productive life cycle of HPV and discuss the roles of the viral proteins in HPV replication. Routes to viral persistence and cancer progression are also discussed.
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HPV is the cause of almost all cervical cancer and is responsible for a substantial fraction of other anogenital cancer and oropharyngeal cancer. Understanding the HPV-attributable cancer burden can boost programs of HPV vaccination and HPV-based cervical screening. Attributable fractions (AF) and the relative contributions of different HPV types were derived from published studies reporting on the prevalence of transforming HPV infection in cancer tissue. Maps of age-standardized incidence rates of HPV-attributable cancers by country from GLOBOCAN 2012 data are shown separately for the cervix, other anogenital tract, and head and neck cancers. The relative contribution of HPV16/18 and HPV6/11/16/18/31/33/45/52/58 was also estimated. 4.5% of all cancers worldwide (630,000 new cancer cases per year) are attributable to HPV: 8.6% in women and 0.8% in men. AF in women ranges from <3% in Australia/New Zealand and the USA to >20% in India and sub-Saharan Africa. Cervix accounts for 83% of HPV-attributable cancer, two-thirds of which occur in less developed countries. Other HPV-attributable anogenital cancer includes 8,500 vulva; 12,000 vagina; 35,000 anus (half occurring in men); and 13,000 penis. In the head and neck, HPV-attributable cancers represent 38,000 cases of which 21,000 are oropharyngeal cancers occurring in more developed countries. The relative contributions of HPV16/18 and HPV6/11/16/18/31/33/45/52/58 are 73% and 90%, respectively. Universal access to vaccination is the key to avoiding most cases of HPV-attributable cancer. The preponderant burden of HPV16/18 and the possibility of cross-protection emphasize the importance of the introduction of cheaper vaccines in less developed countries. This article is protected by copyright. All rights reserved.
Conventional antibodies used for cancer therapies can only target the extracellular epitopes of tumor-associated antigens (TAAs); however, they struggle to enter cancer cells to interact with intracellular TAAs. Intrabodies are the engineered single-chain antibodies that can be transferred to the living cells or expressed within the cells and target the intracellular TAAs, owing to their nanosizes, enhanced motility and specific and potent binding affinities for the TAAs. Intrabody-based technology is a supplement to the current gene silencing technologies that can regulate a variety of biochemical processes and cellular functions and could be applicable for clinical treatment of tumors. Here, we review the development and current status of the intrabodies for the targeted treatment of cancers.
Directing an antigen to the endoplasmic reticulum (ER) improves the antigen-specific immune response, revealing a potentially useful strategy in cancer immunotherapy using tumor-associated antigens (TAAs). This can be achieved by fusing the antigen to an ER chaperone protein, such as calreticulin (CRT). We previously reported the antitumor response by fusing the CRT signal peptide (SP) and its ER retention sequence (KDEL) to full-length human papillomavirus type 16 (HPV-16) E6 and E7 antigens, obtaining a potent antitumoral effect. In this article, we compare the antitumor response due to the use of each signal (SP and/or KDEL) fused to HPV16 E6 and E7 antigens in a DNA vaccination model. Using both SP and KDEL signals promotes higher interferon (IFN)-γ production and a faster antitumor response than using only the SP, resulting in better tumor growth restraint and higher survival, indicating that the KDEL addition to an ER-directed antigen helps by shortening the time to response. Meanwhile, antigens without signals or only the KDEL signal showed no induction of antigen-specific IFN-γ or antitumor response. Our results indicate that directing the E6E7m antigen to the ER by the SP signal is sufficient to promote an efficient antitumor response. Importantly, this effect is stronger and faster when the antigen also has an ER retention sequence, such as the KDEL signal.
HPV16 persistent infection is a well-known condition that precedes human cancer development. High risk HPV E5 proteins cooperate with E6/E7 oncogenes to promote hyper-proliferation of infected cells leading to possible cancer progression. Thus, presence of E5 viral transcripts could be a key marker of active infection and, in turn, a target of immunotherapy. Purpose of the study is to detect E5 transcripts in clinical samples and to explore the activity of novel anti-HPV16 E5 DNA vaccines. HPV transcripts were detected by PCR with specific primers encompassing the splice-donor sites of E5 transcript. For E5-based immunotherapies, two E5-based versions of DNA vaccines carrying whole E5 gene or a synthetic multiepitope gene were improved by fusion to sequence of PVX coat protein. These vaccines were challenged with a new luminescent animal model based on C3-Luc cell line. E5 transcripts were detected in clinical samples of women with HPV positive low-grade SIL, demonstrating the validity of our test. In C3 pre-clinical mouse model, vaccine candidates were able to induce a strong cellular immunity as indicated by ELISPOT assays. In addition, E5-CP vaccines elicited strong anti-tumor effects as showed by decreased tumor growth monitored by animal imaging. The tumor growth inhibition was comparable to those obtained with anti-E7 DNA vaccines. In conclusion, detection of E5 transcripts in clinical samples indicates that E5 is a possible target of immunotherapy. Data from pre-clinical model demonstrate that E5 genetic immunization is feasible, efficacious and could be utilized in clinical trials.