In vitro evaluation of the immune-skewing potential of different candidate building blocks for nanoparticle-based allergen immunotherapy for treatment of allergy*

AIT is the only causal treatment for allergy that targets the immunological
basis of the disease. All other treatments are symptomatic treatments, either
typical pharma treatments such as antihistamines or corticosteroids or
biologicals such as anti-IgE. Upon cessation of administration of such symptom
medication, symptoms return rapidly. AIT is the only treatment with documented
sustained reduction of symptoms after stopping the treatment. However, to
achieve this state of prolonged tolerance, a very long treatment protocol of 3
to 5 years is required. For subcutaneous AIT this means that patients have to
visit an outpatient clinic for their monthly injection for years. This burden
is one of the reasons that the majority of patients resort to symptom
medication. In addition, current AIT frequently gives allergic side-effects
because allergen is administered essentially in its native (symptom-triggering)
conformation. To increase acceptance of AIT it is clear that significant
reduction of the burden of treatment duration and frequency and of side-effects
would be a major step forward.
Immunologically, successful AIT transforms an allergen-specific
IgE/Th2-dominated inflammatory immune response into an allergen-specific
anti-inflammatory response dominated by regulatory T-cells, regulatory B-cells
and IgG4 antibodies. During current AIT protocols, allergen extracts, usually
adsorbed to aluminum hydroxide, are subcutaneously administered. To induce the
desired persistent anti-inflammatory response dominated by IgG4, this treatment
is given for 3 to 5 years. Besides the duration of the treatment, chronic
exposure to aluminum hydroxide is increasingly
considered as undesirable. In recent years it has become clear that AIT is
likely to be more effective at younger age when the immune system may still be
more receptive to immune modulation. In addition, it has been shown that AIT in
young patients with allergic rhinitis prevents the development of allergic
asthma. Although there is not really convincing evidence that chronic exposure
to aluminum hydroxide is detrimental, there is a demand for alternatives, in
particular when AIT will increasingly be used at younger age.

In summary, there is a need to develop alternatives for current generation AIT
products that 1) require less injections to achieve an effective reduction of
symptoms and 2) are not dependent on addition of aluminum hydroxide. There are
essentially four potential building blocks for the design of an improved AIT
vaccine:

a. The allergen, either as a complete extract or as purified major allergens.
The allergen can be modified to a) decrease allergenicity (hypo-allergenicity:
less side-effects) and b) target it more effectively to (receptors on) antigen
presenting cells.
b. Adjuvants, to more effectively induce allergen-specific anti-inflammatory
immune responses, both with respect to kinetics (quicker) and persistence
(memory that can be triggered ideally by natural exposure or by occasional
booster injections) than is currently achieved with aluminum hydroxide.
c. A vehicle for formulation and administration of allergen, to replace
aluminum hydroxide as a depot for allergen that helps shielding off IgE-binding
sites
d. Antibody approaches to more effectively target the allergen to the
appropriate antigen-presenting cells in the skin such as dendritic cells (DCs)
and Langerhans cells (LCs).

In the frame of three projects at Amsterdam UMC, we are exploring possibilities
to improve AIT for both respiratory and food allergy. The three projects
largely overlap with respect to timelines. We have combined these three
projects in a single protocol because experiments to be carried out with moDCs
generated from voluntary blood donations will be used in identical experiments.
In fact, candidate vaccine components from the three different projects can and
will be compared in single experiments to establish which (combination of)
approaches is most promising to improve AIT. The three projects are:

1) Dendritic cell targeting for resetting immune balance (DC4BALANCE).
This is a 4 years* project with a start date on January 1, 2019, ending
December 31, 2022. Due to Covid-19 restrictions in 2020/21, the end date may
move up to one year to December 31 2023. Pilot experiments have been carried
out with blood donations from subjects with unknown demographic and clinical
background (buffy coats from Sanquin / volunteers through BACON). Since it is
known that DCs from allergic and from non-allergic donors possess different
immune-skewing properties, it is difficult to draw firm conclusions from the
experiments. There is a need to perform these experiments with blood from
donors with known clinical background.

The project is funded by Health Holland under the TKI-PPP and focuses on
several diseases, one being house dust mite allergy. The candidate vaccine
building blocks to study immune modulatory characteristics in this project are:


• Purified house dust mite allergens Der p 1 and Der p 2 and house dust mite
extract
• Vitamin D3, retinoic acid and a TGFb mimetic peptide as candidate
anti-inflammatory adjuvants
• Nanoparticles: liposomes of various composition and PLGA nanoparticles as
alternative for aluminum hydroxide. By loading the liposomes/nanoparticles with
allergen, it is shielded off from contact with mast cells when injected,
decreasing the risk of side-effects.
• Antibodies against receptors on DCs, for more effective targeting to the
right antigen-presenting cells.

2) Sialylation van allergens - mode of action for improved immunotherapy using
novel immune tolerizing pathways (SIALLERGEN). This is a 4 years* project
starting November 1, 2019, ending by December 31, 2023. Due to Covid-19
restrictions in 2020/21, the end date may move up to June 30, 2024. Based on
previous findings this project is continued, funded by Eurostars, starting
January 2025, ending December 2027.

The project is also funded by Health Holland under TKI-PPP and focuses on house
dust mite allergy and peanut allergy. The central hypothesis of the project is
that sialylated antigens/allergens effectively induce a regulatory
anti-inflammatory immune response. This effect is thought to be mediated by
Siglec receptors on DCs. The candidate vaccine building blocks to study immune
modulatory characteristics in this project therefore are:

• For house dust mite: purified Der p 1 and Der p 2, and sialylated versions
thereof
• For peanut: purified Ara h 1, Ara h 2, Ara h 3 en Ara h 6 and sialylated
versions thereof
• Sialylated liposomes loaded with house dust mite allergens Der p 1 and/or Der
p 2 or with peanut allergens Ara h 1 and/or Ara h 2. The advantage of this
approach may again be that the allergen is shielded off from contact with mast
cells when injected, decreasing the risk of side-effects.

3) Evaluation of plant-based bioparticles surface-expressing (ANGANY).This
project is a 3-year project starting in October 2019 and ending by September
2022. Based on previous findings an additional plant-based bioparticle project
is funded by Amsterdam UMC under TKI-PPP starting in August 2024 and ending in
July 2026.

The project is funded by a Canadian biotech company from Quebec City, ANGANY
Inc. The concept is a plant-based nanoparticle approach. The plant bioparticles
are exposing allergens on the surface and are rich in glucosylceramide which is
thought to induce anti-inflammatory responses. Surprisingly, in pilot
experiments the multivalent surface expression showed decreased capacity to
induce basophil activation, possibly explained by sterically unfavorable
presentation to achieve IgE cross-linking. In mice, the bioparticles induced a
more vigorous IgG response than aluminum hydroxide adsorbed allergens. The
candidate vaccine building blocks to study immune modulatory characteri

Allergen immunotherapy (AIT) is currently performed with aluminum
hydroxide-adsorbed allergen extracts. This approach is effective, but requires
a long burdensome treatment protocol with a relatively high frequency of
allergic side-effects. There is a need to increase safety and to reduce the
duration and frequency of administration during treatment protocols.
Modification of allergens, application of novel adjuvants, nanoparticles and
cell-targeting strategies are amongst the possible strategies to reach improved
safety and efficacy. Pre-clinical evaluation of such innovations requires blood
samples from allergic patients and appropriate controls. The aim is to enroll
volunteers for donation of blood samples for the isolation of PBMCs and
collection of serum. This will allow to make steps towards:

1. Safer AIT: shielding off allergen from interaction with mast cells by
application of nanoparticles, high-density presentation on nanoparticles,
sialylation of allergens altering their IgE-binding surface.
2. More effective induction of an anti-inflammatory regulatory response
(Tregs/Bregs/IgG4): addition of adjuvants, modification by sialylation,
presentation in context of glycosylceramide containing plant bioparticles.
3. Replacement of aluminum hydroxide: liposomes, PLGA, plant bioparticles.

The study aims at recruiting healthy subjects (n=20) and subjects with allergic
rhinitis (n=40), or peanut allergy (n=20) that are prepared to give 50-200 ml
of blood on multiple occasions (up to maximally 20 times in 4 years) for
pre-clinical evaluation of novel approaches for AIT. Between subsequent blood
donations a period of at least 2 months is required. Patients with allergic
(caused by house dust mite, cat or [grass] pollen sensitization) and
non-allergic rhinitis and patients with peanut allergy will be approached
either retrospectively or prospectively. Retrospective recruitment will take
place via the ENT department at the AMC. The database kept at the ENT
department, containing a subgroup of patients that have consented to be
approached for future allergy-associated research projects, will be used. To
comply with GDPR legislation, CTcue will be used to select patients that have
consented without jeopardizing the privacy of those that have not provided such
consent. For prospective recruitment, allergic subjects will be recruited via
the allergy outpatient clinic of the ENT department of Amsterdam UMC or the
Internal Medicine department of the Amsterdam UMC and Huid Medisch Centrum
Paasheuvelweg, where they will be asked whether they are interested to
participate in the study when they come for their normal consultation visits.
Likewise, prospective recruitment of allergic subjects also occurs via
advertisements (e.g. on notification boards) in the AMC, Amsterdam UMC intranet
and social media of the student association of the medical faculty at the AMC
(MFAS). Healthy subjects not having chronic inflammatory diseases will be
approached via www.link2trials.com, MFAS social media, the Amsterdam UMC
intranet and via advertisements on notification boards of outpatient clinic
departments not primarily seeing patients with inflammatory diseases (e.g.
orthopedic surgery). Advertisements for allergic and healthy subjects will
contain contact details from the laboratory of Experimental Immunology.
Prospectively recruited subjects do not need to go through the ENT department,
which will save them time and effort. All subject that enter the study via the
ENT department will receive a skin prick test (SPT). While subject that enter
the study via the Department of Experimental Immunology will be characterized
via the measurement of allergen-specific IgE serum levels. Subjects that enter
via the Internal Medicine-Allergy outpatient clinic at Amsterdam UMC or Huid
Medisch Centrum Paasheuvelweg already have a doctor-diagnosed allergy based on
history, confirmed with skin prick test and/or IgE levels and/or a provocation
test. The choice of methods depends on the material and expertise available at
the clinical and laboratory departments, making inclusion into the study highly
efficient for both the subjects and the clinicians/researchers. However, these
different methods provide us with the same information regarding the allergic
background of the patients. Subjects that enter the study via the Department of
Experimental Immunology could still be invited for a SPT to confirm previous
IgE serum level results. For mass-spectometry experiments, 500 ml of blood is
required. Subjects will be recruited via similar routes as mentioned above.
After a 500 ml blood donation, a subsequent donation can only take place after
a period of at least 4 months. During the study, subjects may at each moment
withdraw from participation, without giving a reason for their withdrawal. Upon
withdrawal of subjects, new candidate subjects may be approached for
replacement. Subjects will be provided with a patient information sheet and
will be asked to sign informed consent. Subjects will be given 50 euro
reimbursement for the blood donation and reimbursement of their travel costs.

Patients will contribute to increasing the knowledge in the field of innovation
of AIT for the treatment of allergic diseases, In the future this may provide
new safer and more effective treatments that benefit the allergic patient.
Disadvantage of volunteering is minimal. Blood drawing can cause a low level of
pain and may occasionally results in a hematoma. Skin tests are safe and
adverse events are uncommon. Vasovagal reactions (pallor, sweating, faintness)
may appear in up to 4 out of 10,000 patients. Systemic allergic reactions (such
as e.g. an asthma episode) are even less common and may be seen in 1.5-2.2 out
of 10,000 patients. No fatalities have been reported. As this test is performed
under the observation of the study personnel, adequate treatment of those
reactions can be effectively initiated.