Proof of concept study with an HER

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OncoImmunology
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Proof of concept study with an HER-2 mimotope
anticancer vaccine deduced from a novel AAVmimotope
library platform
Josef Singer, Krisztina Manzano-Szalai, Judit Fazekas, Kathrin Thell, Anna
Bentley-Lukschal, Caroline Stremnitzer, Franziska Roth-Walter, Margit
Weghofer, Mirko Ritter, Kerstin Pino Tossi, Markus Hörer, Uwe Michaelis &
Erika Jensen-Jarolim
To cite this article: Josef Singer, Krisztina Manzano-Szalai, Judit Fazekas, Kathrin Thell, Anna
Bentley-Lukschal, Caroline Stremnitzer, Franziska Roth-Walter, Margit Weghofer, Mirko Ritter,
Kerstin Pino Tossi, Markus Hörer, Uwe Michaelis & Erika Jensen-Jarolim (2016) Proof of concept
study with an HER-2 mimotope anticancer vaccine deduced from a novel AAV-mimotope library
platform, OncoImmunology, 5:7, e1171446, DOI: 10.1080/2162402X.2016.1171446
To link to this article: https://doi.org/10.1080/2162402X.2016.1171446
© 2016 The Author(s). Published with
license by Taylor & Francis Group, LLC©
Josef Singer, Krisztina Manzano-Szalai,
Judit Fazekas, Kathrin Thell, Anna Bentley-
Lukschal, Caroline Stremnitzer, Franziska
Roth-Walter, Margit Weghofer, Mirko Ritter,
Kerstin Pino Tossi, Markus Hörer, Uwe
Michaelis, and Erika Jensen-Jarolim.
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Apr 2016.
Published online: 30 Jun 2016.
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ORIGINAL RESEARCH
Proof of concept study with an HER-2 mimotope anticancer vaccine deduced from
a novel AAV-mimotope library platform
Josef Singera,b,*, Krisztina Manzano-Szalaib,c,*, Judit Fazekasa,c, Kathrin Thella,b, Anna Bentley-Lukschala,
Caroline Stremnitzera, Franziska Roth-Walterc, Margit Weghoferd, Mirko Ritterd, Kerstin Pino Tossid, Markus H€orerd,
Uwe Michaelisd,e, and Erika Jensen-Jarolima,b,c
aCenter of Pathophysiology, Infectiology and Immunology, Institute of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna,
Austria; bBiomedical International RCD GmbH, Vienna, Austria; cComparative Medicine, Messerli Research Institute of the University of Veterinary
Medicine Vienna, Medical University Vienna, and University Vienna, Vienna, Austria; dMediGene AG, Martinsried, Germany; eImevaX GmbH Munich,
Germany
ARTICLE HISTORY
Received 27 January 2016
Revised 18 March 2016
Accepted 21 March 2016
ABSTRACT
Background: Anticancer vaccines could represent a valuable complementary strategy to established
therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target
for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously
generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled
to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to
avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and
optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotopedisplay
system, which can be directly used for vaccination. Methods: An AAV capsid display library was
constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2
capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to
immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype
composition and functional properties of these were tested. Finally, prophylactically immunized mice
were challenged with human HER-2 transfected mouse D2F2/E2 cells. Results: HER-2 mimotope AAVvaccines
induced antibodies specific to human HER-2. Two clones were selected for immunization of mice,
which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors
significantly, as compared to controls. Conclusion: In this study, a novel mimotope AAV-based platform
was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The
example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish
active immunotherapy for breast-cancer patients.
KEYWORDS
AAV; adeno-associated virus;
cancer vaccine; HER-2;
mimotope
Introduction
Ever since the first promising vaccination experiments against
cancer by the New York surgeon William B. Coley (1862–
1936), who injected inoperable sarcoma patients with bacteria
and thereby accomplished a cure rate of around 10%,1 cancer
research has been aiming to develop active immunotherapies
against cancer. The “Coley’s toxin” vaccine was evidently
unspecific and clinical effects were likely due to the response of
macrophages with the release of pro-inflammatory cytokines
such as IL-12.2 Nevertheless, such adjuvant therapies may certainly
help to overcome tumor-induced immunosuppression
and may initiate an active immune reaction against the malignant
cells.3
Current targeted anticancer immunotherapies are directed
against tumor-associated antigens, which are highly overexpressed
on malignant cells, but scarcely expressed in normal tissue.4
Candidate targets comprise growth factor receptors5 that are
important during embryonic development6 and silenced in adults,
but overexpressed again in some cancers.7 The human epidermal
growth factor receptor-2 (HER-2) is a ligand-less receptor tyrosine
kinase typically amplified in breast, gastric and esophageal cancer.8
Via homo- or hetero-dimerization with related molecules, HER-2
mediates proliferative and anti-apoptotic signals, ultimately leading
to unfavorable courses of the disease.9 Therefore, HER-2 belongs to
the most prominent targets for specific anticancer therapies,
proven many times by the clinically used anti-HER-2 monoclonal
antibodies trastuzumab (Herceptin®, Roche) or pertuzumab
(Perjeta®, Roche).10-11 As the serum half-life of these monoclonal
antibodies is around 2–4 weeks, depending on the dosage12-13
(t1/2D 16,4 d for trastuzumab14 and 18 d for pertuzumab15), repetitive
applications in tri-weekly intervals are necessary.16 Even after
CONTACT Erika Jensen-Jarolim [email protected]
Supplementary data of this article can be accessed on the publisher’s website.
*These authors contributed equally to this work.
Published with license by Taylor & Francis Group, LLC © Josef Singer, Krisztina Manzano-Szalai, Judit Fazekas, Kathrin Thell, Anna Bentley-Lukschal, Caroline Stremnitzer, Franziska Roth-Walter,
Margit Weghofer, Mirko Ritter, Kerstin Pino Tossi, Markus H€orer, Uwe Michaelis, and Erika Jensen-Jarolim.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
ONCOIMMUNOLOGY
2016, VOL. 5, NO. 7, e1171446 (11 pages)
http://dx.doi.org/10.1080/2162402X.2016.1171446
being chimerized or humanized, these antibodies are recognized as
foreign proteins by the immune system and severe side effects may
occur during treatments due to hypersensitivity reactions.17-20
Hence, these therapies have to be applied under strict medical
supervision and often under cortisol or anti-histamine premedication,
21 making them laborious for patients as well as oncologists.
Last but not the least, they are expensive for the health-care system
and may encourage development of class-based treatment. Consequently,
turning passive immunotherapies with monoclonal antibodies
into active vaccines triggering the patient’s own immune
system to produce antibodies of the same specificity, would be
highly desirable and could overcome many of the aforementioned
obstacles.22
However, HER-2, due to its pivotal role in ductal morphogenesis
of the human mammary gland23 and its involvement in
repair mechanisms of myocardial cells,24-25 is a self-antigen.
Therefore, immunization strategies against HER-2 have to vanquish
the patient’s self-tolerance. Small B-cell epitope mimicking
peptides, so called “mimotopes,” could be an interesting
option to break tolerance, as they do not share consensus
sequence with the mimicked epitopes, but possess the same
structure due to similar amino acid charges.26 Thus, immunization
with mimotopes induces antibodies that recognize the natural
antigen only via molecular mimicry. In previous studies,
our group selected mimotopes from phage display libraries and
demonstrated their high specificity and immunogenicity in
mice.26-32 As it is not possible to use filamentous phages for
vaccinating patients, the mimotope peptides had to be produced
synthetically and to reconstitute their immunogenicity,
chemical coupling to antigenic carriers such as keyhole limpet
hemocyanin28 or tetanus toxoid33 was necessary. During this
process substantial loss of the mimicry potential of the epitope
was observed.26 Therefore, suitable vehicles are called for,
which guarantee safe and stable display while maintaining
immunogenicity. We propose in this study AAV as novel mimotope
display systems, as they could be proven to induce strong
humoral34 and cellular35 immune responses.36
AAV are small (25 nm), non-enveloped single-stranded
DNA viruses37-38 which belong to the genus Dependovirus—
they need a helper virus to facilitate their replication. Appropriate
helper viruses for AAV are adenoviruses or herpes simplex
virus.38 As AAV are non-pathogenic, they were long-neglected
in medical research,38 a fact that suddenly reversed when Hermonat
and Muzyczka used an AAV-based vector to express
foreign genes in mammalian tissue culture cells in 1984.39
Within the last 30 y, AAV were established as vectors for gene
therapy and their experimental use ranges from the cardiovascular
system40 over neurodegenerative disorders41 to bone
defects, cartilage lesions or rheumatoid arthritis42 as well as
infectious diseases.36 In parallel, AAV or their self-assembled
capsid proteins alone as virus-like particles (AAVLP) were
employed as a display system for vaccines,36 because the virus
serotype 2 (AAV2) tolerates the insertion of small peptides into
its VP3 capsid protein.43
With regards to oncology, promising results for AAVLPbased
vaccines were obtained especially against the tumorigenic
virus Human Papilloma Virus (HPV). Nieto et al. could demonstrate
that upon prophylactic vaccination with AAVLP displaying
the L2 epitope of HPV, high titers of antibodies were
induced in mice and rabbits, resulting in neutralization of infections
with several HPV types in a pseudovirion infection assay.44
Especially intriguing, however, would be a combination of
the high-density display AAV system with a library allowing
the selection of mimotopes from a repertoire of peptides with
an antibody of interest. We addressed here whether in this way
AAV mimotope vaccines could be created that at the same
time would be useful as immunogen, using HER-2 as a model
target.
Results
Selection of HER-2 mimotopes from an AAV library using
trastuzumab
AAV-mimotope clones (schematic representation in Fig. 1A,
amino acid sequences depicted in Table 1) were selected based
on their ability to bind to the monoclonal anti-HER-2 antibody
trastuzumab. Extensive description of the selection algorithm
and the results can be found in the Supplementary section.
Immunization with HER-2 mimotope AAV clones
AAV-mimotope clones were next selected based on their ability
to induce HER-2 specific antibodies. After four rounds of
immunization without any observed local or systemic side
effects, HER-2 specific antibodies were detectable in sera of
immunized mice, with significantly higher levels compared to
na€ıve mice in 5 out of 7 tested mimotope groups. When compared
to wtAAV immunized animals, mice from 3 mimotope
groups, DMD4 (p < 0 .05), DMD6 (p < 0 .001) and DDD19
(p < 0 .001), displayed significantly higher levels of HER-2 specific
IgG antibodies (Fig. 1B). To prove the specificity of the
induced antibodies, immunohistochemical stainings were performed
with HER2-overexpressing and non-expressing tumor
cells. As depicted in Fig. 1C, staining with IgG from sera of
immunized mice showed a membrane specific pattern in HER-
2 transfected D2F2/E2 cells only, whereas the parental cell line
D2F2, negative for human HER-2, remained unstained. Specificity
was tested using purified IgG antibodies in ELISA against
rHER-2, but also against two other known tumor-associated
antigens, EGFR and CEA, or against BSA for control purposes.
The HER-2 mimotope clones DMD4 and DMD6 induced specific
anti-HER-2 antibodies (Fig. 2A), which reacted significantly
higher compared to antibodies purified from naive mice
(p < 0 .001), or antibodies purified from the DMD1 or DMD2
groups (p < 0 .001 for both clones). Only background reactivity
against control proteins sEGFR, sCEA or BSA was measured
for all treatment groups (Fig. 2A).
Epitope specificity is particularly essential for cancer immunotherapy,
because antibodies against HER-2 can act either
tumor-promoting or -inhibiting, even when directed against
the same molecule.26,45 Thus, the second line of screening was
done by means of a tetrazolium-based cell proliferation assay
to exclude mimotopes that induce antibodies either with insufficient
tumoricidic effects or favoring tumor growth. Here,
purified antibodies from sera of immunized mice were
employed for incubation of HER-2 overexpressing BT474 cells.
After 72 h, cell viability was measured (Fig. 2B). Clones DMD1,
e1171446-2 J. SINGER ET AL.
DMD4 and DMD6 mediated growth inhibition; DMD2,
DDD19 and DMM44 had only minor effects on tumor growth,
but also antibodies purified from wtAAV immunized or naive
mice showed tumor growth inhibition to some extent. Also
antibodies induced by rHER-2, which are not restricted to the
trastuzumab epitope and thus a mix of tumor-promoting and
Figure 1. HER-2 mimotope AAV model and immunogenicity evaluation. (A) Model of the AAV vector displaying HER-2 specific mimotopes fused to its capsid protein VP3.
Mimotopes are inserted between amino acid positions 587 and 588, which resemble a peak in the capsid, for optimal display. (B) AAV vectors displaying HER-2 mimotopes
are able to induce HER-2 specific antibodies. Screening of sera from mice, immunized with AAV-displaying HER-2 mimotopes and Al(OH)3 as adjuvant, against the
extracellular domain of human HER-2. Depicted are means (n D 2/mouse serum) & SD; values of MIS4 were statistically analyzed (One-way ANOVA, Tukey’s post-test).
(C) Antibodies induced by AAV clone DMD4 recognize HER-2 on the surface of cancer cells.Immunofluorescence staining of D2F2/E2 cells, overexpressing human HER-2
with sera of mice immunized with AAV clone DMD4 (PIS D Pre-Immune Serum, MIS 3 D MIS after three immunizations). After immunization with the HER-2 specific mimotope,
membrane specific staining can be seen. Pre-Immune sera and serum of mice immunized with wtAAV alone do not show any specific staining on D2F2/E2 cells.
ONCOIMMUNOLOGY e1171446-3
-inhibiting ones, were not able to mediate significant growth
inhibition. Upon statistical evaluation, only antibodies induced
by clones DMD1 and DMD6 were able to reach significance
when compared to untreated cells (p < 0 .01 for DMD1 and
p < 0 .05 for DMD6).
Finally, antibodies induced by clone DMD15 were tumorpromoting
(Fig. S1). This effect, however, was drastic as BT474
cells showed 4-fold faster proliferation compared to untreated
cells (p <0 .01) and underlines the importance of tumor cell
proliferation inhibition assays as part of the screening procedure
of novel anticancer vaccines.
Analyzing the subclass immune responses after
vaccination of BALB/c mice
Clones DMD4 and DMD6 had performed best overall throughout
the screening steps and were thus chosen for monitoring
the IgA, IgG1, IgG2a, IgG2b and IgE responses during a further
immunization experiment in BALB/c mice.
Both DMD4 and DMD6 induced negligible levels of HER-2
specific IgA (Fig. 3A), whereas DMD4, less DMD6, induced
IgG1 against HER-2 (p < 0 .05 for DMD4 compared to the
naive group, Fig. 3B). Neither DMD4 nor DMD6 induced large
extents of HER-2 specific IgG2a and IgG2b (Figs. 3C–D). However,
IgG2a levels in DMD4 immunized mice were significantly
elevated after four rounds of immunization compared to the
animals of the naive group (p < 0 .001; Fig. 3C, right panel).
Interestingly, this effect was not observed, when the DMD4
group was compared to the group immunized with wtAAV, as
this group also showed elevated levels of IgG2a antibodies.
As for IgE, again DMD4 induced significant levels of HER-2
specific IgE, as compared to naive (p < 0 .001) and wtAAV
immunized animals (p < 0 .01), but vaccination with DMD6
did not lead to a significant IgE anti HER-2 immune response
(Fig. 3E).
The positive control group, being vaccinated four times with
the extracellular domain of HER-2, showed significant antibody
levels of HER-2 specific IgG1, IgG2a, IgG2b and IgE isotypes,
but not IgA (Figs. 3A–E).
To determine the effect of different adjuvant agents on the
elicited immune response, a cohort of BALB/c mice was immunized
with the HER-2 mimotope AAV clone DMD2, alongside
several different adjuvants, being either in clinical use or in
clinical trials. DMD2 plus aluminum-hydroxide (Alum),
monophosphoryl-Lipid A (MPL), or a combination of both,
induced higher levels of IgG specific for HER-2 as compared to
ODN-1826 or Alum plus ODN-1826 (Fig. S2A). This IgG
response seems to be constituted predominantly by IgG1
antibodies (Fig. S2B). As for all other investigated isotypes
(IgG2a, IgG2b and IgM), no differences were observable
(Figs. S2C–E).
The memory effects of the mimotope-AAV vaccine were
also investigated: the immune response in immunized mice
remained stable until day 137 and was not dependent on the
type of adjuvant used (Fig. S3).
Tumor graft trial
Immunized mice were grafted with 2 £ 106 D2F2/E2 cells,
overexpressing human HER-2. In all mice, tumors could be
detected after 5 d. In control groups (naive mice and mice
immunized with wtAAV), tumors grew exponentially. In clear
contrast, the tumors in mice vaccinated with either rHER-2, or
HER-2 mimotope AAV clones DMD4 or DMD6 grew significantly
slower (p < 0 .01 for DMD4 compared to naive mice;
p < 0 .05 compared to wtAAV treated animals; p < 0 .05 for
DMD6 compared to naive as well as wtAAV immunized mice;
Fig. 4A). The mean tumor volumes at day 12 of the trial and
standard errors of the mean were as follows: DMD4 D 139,
27 mm3 § 40, 45; DMD6 D 156, 13 mm3 § 54, 99; wtAAV D
289, 61 mm3 § 67, 91; rHER-2 D 124, 11 mm3 § 51, 11 and
naive mice D 309, 09 mm3 § 128, 59, respectively.
When the first mouse reached a tumor volume larger than
300 mm3, the experiment was terminated and all mice were
euthanized. Fig. 4B shows representative pictures of explanted
tumors and Fig. 4C displays a graph of the tumor weights in
grams, where mice of the rHER-2 and DMD6 immunized
groups presented the lowest tumor weights.
Thus, vaccination with both tested vaccines, DMD4 and
DMD6, distinctly showed a protective effect in BALB/c mice,
comparable to vaccination with human HER-2.
Discussion
Although HER-2 is highly successfully targeted by passive
immunotherapies in clinical oncology, no active anticancer vaccine
against HER-2 is in clinical use yet. However, an active
anti-HER-2 vaccine could be complementary to passive immunotherapy
and especially relevant in a prophylactic setting in
high-risk families, in early stage and in minimal residual disease,
or even to protect the next generation.46 Anticancer-vaccines
that lead to apoptosis of cancer cells may besides the
antibody-mediated effects47 also activate the T-cellular branch
via antigen crosspresentation and recruition of CD8C48,49
depending on the induced isotype.50
Moreover, we propose that anticancer vaccines, especially in
combination with immune checkpoint-inhibitors may be
another breakthrough in oncology. Overcoming the strong
immunosuppressive tumor environment may also be supported
by using a similar, but not identical antigen for immunization,
such as a mimotope.
Mimotopes are small epitope-mimicking peptides that via
molecular mimicry are able to induce epitope-specific antibodies.
Hence, mimotopes for clinically approved antibodies could
be effective tools to induce the desired epitope specificity in
an active vaccination approach.26-32,51 However, the small
Table 1. Amino acid sequences of tested mimotopes.
Clone ID Amino acid sequence
DMD1 RLVPVGLERGTVDWV
DMD2 TRWQKGLALGSGDMA
DMD4 QVSHWVSGLAEGSFG
DMD6 LSHTSGRVEGSVSLL
DMD15 LDSTSLAGGPYEAIE
DDD19 HVVMNWMREEFVEEF
DMM44 SWASGMAVGSVSFEE
e1171446-4 J. SINGER ET AL.
Figure 2. Specificity and functionality testing of antibodies purified from sera of immunized mice. (A) AAV-mimotope induced antibodies recognize HER-2, but not tumorassociated
antigens EGFR, CEA or control protein BSA. Antibodies (c D 1 mg/mL) purified from sera of na€ıve mice and mice immunized with candidate particles were
screened for their reactivity toward known tumor antigens HER-2, EGFR, CEA as well as the control protein BSA in ELISA. Antibodies induced by mimotopes DMD4
and DMD6 show significant higher IgG reactivity toward rHER-2 than naive mice (p < 0.001) and mice immunized with candidates DMD1 (p < 0 .001) and DMD2
(p < 0 .001). Toward all other antigens only background reactivity is visible. Bars represent mean values & SD (n D 2); One-way ANOVA, Tukey’s post-test. (B) Purified antibodies
of immunized mice to different degrees inhibit growth of human HER-2 overexpressing mammary carcinoma cells. Tetrazolium-based proliferation assays with
HER-2 overexpressing BT474 cells are depicted upon incubation for 72 h with purified antibodies (c D 1 mg/mL) of sera from mice immunized with candidate particles.
Bars displaying mean values & SD, analyzed by means of Kruskal–Wallis test plus Dunns post-test.
ONCOIMMUNOLOGY e1171446-5
mimotope peptides need to be fixed to an immunogenic carrier
to elicit an immune response.28,33
Up to now this has been a methodological problem in mimotope
vaccine production when translating phage-displayed
mimotopes to a vaccine, involving peptide synthesis and chemical
coupling to an immunogenic carrier. Our study presents an
AAV library that enables selection of mimotope AAV clones,
which can be used without further modifications as a high-density
display system for vaccination (Fig. 1A). Selections of the
AAV library with trastuzumab (Herceptin®) rendered HER-2
mimotopes, which triggered antibody formation against the
same epitope as trastuzumab. Seven mimotopes were used for
Figure 3. Subclass analysis of HER-2 specific antibodies induced by selected mimotope AAV clones DMD4 and DMD6 by ELISA. Left Panel: a-e: IgA, IgG1, IgG2a, IgG2b or
IgE antibody determination before (PIS) and after each immunization (MIS 1-4). Right panel: Antibody levels after four immunization rounds (MIS 4); Box and whiskers
plot, displaying minimum and maximum values. Each serum was measured in duplicate, eight mice per group § SD; Kruskal–Wallis test, Dunns post-test.
e1171446-6 J. SINGER ET AL.
Figure 4. Readout of tumor graft trial. (A) Mice immunized with either rHER-2, DMD4 or DMD6 particles display significantly slower and smaller tumor growth compared
to naive mice and mice immunized with wtAAV only. Tumor growth curve of grafted HER-2 overexpressing D2F2/E2 cells; at day 12, DMD4 immunized mice had significantly
smaller tumors compared to those of the naive (p < 0.01) and of the wtAAV group (p < 0.05). DMD6 treated mice also developed significantly smaller tumors
(p < 0.05 compared to naive group, p < 0.05 compared to wtAAV). Mice immunized with rHER-2 also developed significantly smaller tumors compared to both wtAAV
treated (p < 0.01) or naive mice (p < 0.01). Tumor size at day 12 was analyzed by One-way ANOVA alongside Tukey’s post-test; displayed are mean tumor volume values
§ standard error of the mean (SEM). (B) Representative macroscopic pictures of explanted tumors. (C) Tumors of mice immunized with mimotope DMD6 or rHER-2 had
significantly lower weight compared to tumors of animals receiving wtAAV. Diagram displaying weight of explanted tumors in grams; Box and whiskers plot, whiskers
displaying minimum and maximum values (eight mice/group). Analysis by means of Kruskal–Wallis test and Dunns post-test.
ONCOIMMUNOLOGY e1171446-7
immunizing mice and optimal candidates classified with
respect to (i) antibody induction capacity (Fig. 1B), (ii) specificity
of the induced antibodies (Fig. 2A) and (iii) growth inhibitory
potential when applied to HER-2 overexpressing cancer
cells (Fig. 2B). In spite of the fact that we used trastuzumab for
mimotope selections, clone DMD15 induced antibodies which
acted tumor-promoting (Fig. S1), underlining the importance
of our screening algorithm. Based on their good performance,
two candidate molecules, DMD4 and DMD6, were chosen for
prophylactic vaccination in mice prior to subcutaneous tumor
grafting with HER-2 overexpressing syngeneic D2F2/E2 cells.
Both immunized groups showed significantly slower tumor
growth compared to control groups (Fig. 4A).
In order to elucidate the type of the induced humoral immune
response, sera of mice were taken after each immunization round
and screened in ELISA for HER-2 specific antibodies. As can be
seen in Fig. 3, the immune response is mainly mediated via IgG1
antibodies and to some extent via IgE, whereas IgA and IgG2b levels
were not significantly elevated. This might be explained by the
fact, that BALB/c mice show a genetic bias toward Th2 immunity.
52-53 Independent of the employed adjuvant, ranging from the
typical Th2 adjuvant Al(OH3) (Alum)54 to Th1 adjuvants such as
MPL 55 or ODN,56 the immune response was stable and all adjuvants
triggered mostly IgG1 antibodies (Fig. S2). However, groups
receiving AAV alongside Alum or MPL (or in combination) had
higher IgG levels of HER-2 specific antibodies. Based on these
results, Alum, the adjuvant most used in clinical practice, was
selected for all further immunizations.
Taken together, these results indicate that from our AAV
library platform potent HER-2 mimotope AAV vaccines can be
generated and directly used for vaccination. These HER-2 vaccines
did not cause any local or systemic side effects in the
immunized mice. Our observations are in line with the clinical
experience from the use of AAV for gene therapy (e.g., in inherited
retinal disorders and hemophilia B), where those vectors
displayed favorable safety profiles (reviewed in 57,58). However,
vaccination approaches using viral vectors are often considered
dangerous due to their content of viral DNA. In this regard,
AAV exhibit significant advantages, as they are non-pathogenic
and inherently replication defective,59 as they need concomitant
helper viruses to cause an infection.38 Therefore, usage of
adeno-associated virus-like particles (AAVLP), consisting only
of the virus capsid as display system instead of the whole virus,
will enhance safety. In a previous study, AAVLP had no anaphylactogenic
potency even when employed in an allergy
mouse model where high levels of antibodies including IgE and
IgG1 were induced.60
Thus, the concept of AAVLP-based vaccines is of high interest
in the field of cancer vaccines, with a great potential for prophylactic
vaccination of high-risk patients, or those with
minimal residual disease.
Materials and methods
Cell lines
The human BT474 cell line is a human ductal mammary carcinoma
cell line, first described by E. Lasfargues and W.G. Coutinho
in 1978,61 distributed by ATCC (American Type Culture
Collection; Cat-No: BT474 or HTB-20TM, respectively), and
was a kind gift of Prof Thomas Grunt from the Institute of
Cancer Research of the Medical University of Vienna. BT474
cells were authenticated by short tandem repeat profiling
(PowerPlex® 16 Loci Service, LGC Standards).
Mouse mammary carcinoma cells D2F2/E2 are derived from
the D2F2 cell line62 by stable transfection of human HER-263
and were kindly provided by Prof Wei-Zen Wei (Karmanos
Cancer Institute, Wayne State University School of Medicine,
Detroit, Michigan, USA).
AAV particle production and candidate selection
Methods for production of the AAV library and selection of
trastuzumab specific AAV clones are described in detail within
the supplementary information. In short, an AAV capsid
library was produced by transfection of HEK293T cells with a
plasmid library encoding for AAV and containing a random
peptide insertion of 15 amino acids with a diversity of 109
between amino acid position 587 and 588 of the capsid protein
VP3 (see schematic Fig. 1A). Resulting AAV capsids with different
peptide insertions were selected for this study based on
their reactivity toward trastuzumab (for detailed information
see Supplemental Materials and Results).
Animals
For immunization and tumor graft experiments, 6–8 weeks old
female BALB/c mice were obtained from Charles river and kept
based on authorization of the Animal Ethics Committee of the
Medical University according to the Austrian, European Union
and FELASA guidelines for animal care and protection (GZ:
BWMF-66.009/0003-II/3b/2011).
Immunization trials
Mice were immunized in immunization experiments according to
the scheme in Fig. S4A; respective groups received subcutaneously
10 mg of either AAV displaying HER-2 mimotopes or wild type
AAV (wtAAV) adsorbed to 50 mL AlOH3 (Imject!Alum Adjuvant,
Pierce Protein Biology Products, Thermo Scientific; Cat-No:
77161) C 50 mL PBS. Mice were immunized four times in twoweek
intervals. Serum samples were taken prior to the first immunization
(PISDpre immune serum) and 10 d after each immunization
round (MIS1-4D mouse immune serum 1-4).
For evaluation of the effects of different adjuvants, only one
candidate clone, DMD2, was used; the time points of immunization
and serum sampling were scheduled as depicted in
Fig. S4B. In this trial, the long-term effects of the AAV based
mimotope vaccine were also evaluated. Hence, multiple blood
samples were taken after the last round of immunization
(MIS3-7) and the immune response was monitored until day
137 (Fig. S4B).
Tumor graft experiments
2 £ 106 D2F2/E2 cells were grafted subcutaneously (Day 0) in
immunizedmice (see scheme in Fig. S4C). Mice were observed regularly
for tumor growth as well as tumor-related side effects such as
e1171446-8 J. SINGER ET AL.
ceasing to ingest food or weight loss, which did not occur in a single
mouse during the whole study. Tumor size was monitored by caliper
measurement and tumor volume was calculated by means of
the following formula: V (mm3) D d2 (mm2) £ D (mm)/2, where
d stands for the smallest and D for the largest diameter of the
tumor. Endpoint of the tumor graft trial was defined as when the
tumor of the first mouse reached 300mm3. Then, allmicewere sacrificed
and tumors were taken for further analysis.
Data handling and statistics
ELISAs measuring antibody responses (Fig. 1B) and specificity of
induced antibodies (Fig. 2A) were done in duplicates and data of
MIS4 were analyzed by means of one-way ANOVA with Tukey’s
post-test. EZ4U® cell viability assays (Fig. 2B) were calculated
using Kruskal–Wallis test due to the non-Gaussian distribution
of the values in connection with Dunns post-test. Cell viability
evaluation in Fig. S3 was analyzed using the Mann–Whitney test.
Subclass analysis of induced anti-HER-2 antibodies (Fig. 3) in
MIS4 was analyzed again by Kruskal–Wallis test in connection
with Dunns post-test. In the tumor graft trial, size of mouse
tumors was measured by caliper (Fig. 4A) and values of day 12
were analyzed by means of one-way ANOVA with Tukey’s posttest.
Tumor weight (Fig. 4B) was analyzed using Kruskal–Wallis
test in connection with Dunns post-test. For all experiments significance
was accepted at p< 0.05 (), p< 0.01 () and p< 0.001
(). All statistic calculations were performed using GraphPad
Prism4 Software (GraphPad).
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors would like to express their gratitude to all members of the
Jensen-Jarolim lab for critical and fruitful discussions, as well as to Anna
Willensdorfer and Erika Bajna for excellent technical support. Moreover,
we thank Amelie Wein for proofreading our manuscript.
Funding
This work was supported by MediGene AG (Martinsried, Germany) and
Biomed Int. R C D GmbH (Vienna, Austria), and in part by the Austrian
Science Fund (FWF) grants P23398-B11 and W1205-B09. Erika Jensen-
Jarolim is shareholder of Biomed Int. RCD GmbH. The study was funded
by grants from Medigene AG to Erika Jensen-Jarolim.
Notes
A patent has been filed by Medigene and Biomedical International RCD
GmbH, Vienna, Austria: “Anti-HER-2 vaccine based upon AAV derived
multimeric structures,” WO2013/037961. Markus H€orer, Mirko Ritter and
Kerstin Pino Tossi are inventors of the AAV related patent application
WO 2008/145400 “Mutated structural protein of a parvovirus” and related
patent applications. Kerstin Pino Tossi is an employee of Medigene AG.
This does not alter our adherence to all the journal’s policies on sharing
data and materials.
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We have highlighted some of the most popular subjects we handle above. Those are just a tip of the iceberg. We deal in all academic disciplines since our writers are as diverse. They have been drawn from across all disciplines, and orders are assigned to those writers believed to be the best in the field. In a nutshell, there is no task we cannot handle; all you need to do is place your order with us. As long as your instructions are clear, just trust we shall deliver irrespective of the discipline.

Are your writers competent enough to handle my paper?

Our essay writers are graduates with bachelor's, masters, Ph.D., and doctorate degrees in various subjects. The minimum requirement to be an essay writer with our essay writing service is to have a college degree. All our academic writers have a minimum of two years of academic writing. We have a stringent recruitment process to ensure that we get only the most competent essay writers in the industry. We also ensure that the writers are handsomely compensated for their value. The majority of our writers are native English speakers. As such, the fluency of language and grammar is impeccable.

What if I don’t like the paper?

There is a very low likelihood that you won’t like the paper.

Reasons being:

• When assigning your order, we match the paper’s discipline with the writer’s field/specialization. Since all our writers are graduates, we match the paper’s subject with the field the writer studied. For instance, if it’s a nursing paper, only a nursing graduate and writer will handle it. Furthermore, all our writers have academic writing experience and top-notch research skills.
• We have a quality assurance that reviews the paper before it gets to you. As such, we ensure that you get a paper that meets the required standard and will most definitely make the grade.

In the event that you don’t like your paper:

• The writer will revise the paper up to your pleasing. You have unlimited revisions. You simply need to highlight what specifically you don’t like about the paper, and the writer will make the amendments. The paper will be revised until you are satisfied. Revisions are free of charge
• We will have a different writer write the paper from scratch.
• Last resort, if the above does not work, we will refund your money.

Will the professor find out I didn’t write the paper myself?

Not at all. All papers are written from scratch. There is no way your tutor or instructor will realize that you did not write the paper yourself. In fact, we recommend using our assignment help services for consistent results.

What if the paper is plagiarized?

We check all papers for plagiarism before we submit them. We use powerful plagiarism checking software such as SafeAssign, LopesWrite, and Turnitin. We also upload the plagiarism report so that you can review it. We understand that plagiarism is academic suicide. We would not take the risk of submitting plagiarized work and jeopardize your academic journey. Furthermore, we do not sell or use prewritten papers, and each paper is written from scratch.

When will I get my paper?

You determine when you get the paper by setting the deadline when placing the order. All papers are delivered within the deadline. We are well aware that we operate in a time-sensitive industry. As such, we have laid out strategies to ensure that the client receives the paper on time and they never miss the deadline. We understand that papers that are submitted late have some points deducted. We do not want you to miss any points due to late submission. We work on beating deadlines by huge margins in order to ensure that you have ample time to review the paper before you submit it.

Will anyone find out that I used your services?

We have a privacy and confidentiality policy that guides our work. We NEVER share any customer information with third parties. Noone will ever know that you used our assignment help services. It’s only between you and us. We are bound by our policies to protect the customer’s identity and information. All your information, such as your names, phone number, email, order information, and so on, are protected. We have robust security systems that ensure that your data is protected. Hacking our systems is close to impossible, and it has never happened.

How our Assignment Help Service Works

1. Place an order

You fill all the paper instructions in the order form. Make sure you include all the helpful materials so that our academic writers can deliver the perfect paper. It will also help to eliminate unnecessary revisions.

2. Pay for the order

Proceed to pay for the paper so that it can be assigned to one of our expert academic writers. The paper subject is matched with the writer’s area of specialization.

3. Track the progress

You communicate with the writer and know about the progress of the paper. The client can ask the writer for drafts of the paper. The client can upload extra material and include additional instructions from the lecturer. Receive a paper.

4. Download the paper

The paper is sent to your email and uploaded to your personal account. You also get a plagiarism report attached to your paper.

GET A PERFECT SCORE!!!

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