Immunological Analysis of Lymph Node Tissue After Intralymphatic Immunotherapy: A Prospective Case Control Study
Phase: EARLY_PHASE1
Conditions: Hypersensitivity, Hay Fever, Rhinitis, Allergic, Seasonal, Rhinoconjunctivitis
Sponsor: University of Zurich

 
Interventional study with investigational medicinal 
product (IMP) 
 
Clinical Study Protocol 
Immunological analysis of lymph node tissue after intralymphatic 
immunotherapy: A prospective case control study 
 
Study Type: 
Interventional study with investigational medicinal product (IMP) 
for intralymphatic allergen immunotherapy. 
Study Categorisation: 
Clinical trial, Risk category B.  
Study Registration: 
Registry on SNCTP via BASEC (Nr:…) 
Study Identifier: 
ILIT-FNA-2023 
Principle Investigator 
Professor Peter Schmid-Grendelmeier, MD, 
Allergy Unit, Department of Dermatology,  
University Hospital Zurich 
The Circle 59,  
8058 Zurich-Flughafen, Switzerland  
Mail : peter.schmid@usz.ch 
Tel. +41 44 255 30 78 
Sponsor-: 
Professor Pål Johansen, PhD,  
Department of Dermatology 
University Hospital Zurich 
Rämistrasse 100 
8091 Zurich, Switzerland 
Mail: pal.johansen@usz.ch 
Tel. +41 44 255 8616 
Investigational Product:  
         Grass pollen extract (Polvac ) produced by Allergy Therapeutics  
 
 
Protocol Version and Date: 
         Version 2, 06/06/2023 
 
CONFIDENTIAL 
The information contained in this document is confidential and the property of the sponsor. The 
information may not - in full or in part - be transmitted, reproduced, published, or disclosed to others 
than the applicable Competent Ethics Committee(s) and Regulatory Authority(ies) without prior written 
authorisation from the sponsor except to the extent necessary to obtain informed consent from those 
who will participate in the study. 
 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 2 of 39 
Signature Page 
 
Study number 
BASEC Number 2023-00821 
Study Title 
Immunological analysis of lymph node tissue after intralymphatic 
immunotherapy: A prospective case control study 
 
The Principle-Investigator, the sponsor and the trial statistician have approved the protocol version 2 
(dated.2023), and confirm hereby to conduct the study according to the protocol, current version of the 
World Medical Association Declaration of Helsinki, the ICH-GCP guidelines and the local legally 
applicable requirements. 
 
The Principle Investigator and the sponsor generally oversees the whole project and at all sections of 
the project (hospital and laboratories). Especially, but not exclusively, it is assures that  
• 
approval is obtained from ethical review board and from Swissmedic,  
• 
that all involved investigator or project partners are informed and educated,  
• 
that the project is financially secured,  
• 
that the investigational product is provided,  
• 
that responsible and qualified medical investigators are available,  
• 
that biological samples obtained from patients are process by qualified personnel as planned,  
• 
that all documentation is obtained, filed and stored as planned,  
• 
that contracts are made when required (e.g. monitoring) and  
• 
that reporting is made to authorities. 
 
We have read and understood this trial protocol and agree to conduct the trial as set out in this study 
protocol, the current version of the World Medical Association Declaration of Helsinki, ICH-GCP 
guidelines and the local legally applicable requirements. 
 
Principle Investigator 
Professor Peter Schmid-Grendelmeier, MD 
Department of Dermatology, University Hospital Zurich 
 
 
 
 
Place/Date 
 
Signature 
 
 
 
Sponsor  
Professor Pål Johansen, PhD 
Department of Dermatology, University Hospital Zurich  
 
 
 
 
 
Place/Date 
 
Signature 
 
 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 3 of 39 
Table of Contents 
 
STUDY SYNOPSIS ................................................................................................................................. 6 
ABBREVIATIONS ................................................................................................................................... 8 
STUDY SCHEDULE ................................................................................................................................ 9 
1. 
STUDY ADMINISTRATIVE STRUCTURE .................................................................................... 10 
1.1 
Sponsor ....................................................................................................................................... 10 
1.2 
Medical Investigators................................................................................................................... 10 
1.3 
Statistician ("Biostatistician") ....................................................................................................... 11 
1.4 
Laboratory ................................................................................................................................... 11 
1.5 
IT Management ........................................................................................................................... 12 
1.6 
Data Safety Monitoring Committee ............................................................................................. 12 
1.7 
Partners at the USZ Allergy Unit ................................................................................................. 12 
1.8 
Further Partners .......................................................................................................................... 12 
2. 
ETHICAL AND REGULATORY ASPECTS .................................................................................. 13 
2.1 
Study registration ........................................................................................................................ 13 
2.2 
Categorisation of study................................................................................................................ 13 
2.3 
Independent Ethics Committee (IEC).......................................................................................... 13 
2.4 
Competent Authorities (CA) ........................................................................................................ 13 
2.5 
Ethical Conduct of the Study ....................................................................................................... 13 
2.6 
Declaration of interest ................................................................................................................. 13 
2.7 
Patient Information and Informed Consent ................................................................................. 13 
2.8 
Participant privacy and confidentiality ......................................................................................... 14 
2.9 
Early termination of the study ...................................................................................................... 14 
2.10 Protocol amendments ................................................................................................................. 14 
3. 
BACKGROUND AND RATIONALE .............................................................................................. 15 
3.1 
Background and Rationale .......................................................................................................... 15 
3.2 
Investigational Product (treatment) and Indication ...................................................................... 16 
3.3 
Preclinical Evidence .................................................................................................................... 16 
3.4 
Clinical Evidence to Date ............................................................................................................ 16 
3.5 
Rationale for the dosage, route, regimen .................................................................................... 17 
3.6 
Explanation for choice of comparator (or placebo) ..................................................................... 17 
3.7 
Risks / Benefits ............................................................................................................................ 17 
3.8 
Justification of choice of study population ................................................................................... 18 
4. 
STUDY OBJECTIVES ................................................................................................................... 18 
4.1 
Overall Objective ......................................................................................................................... 18 
4.2 
Primary Objective ........................................................................................................................ 19 
4.3 
Secondary Objectives ................................................................................................................. 19 
4.4 
Safety Objectives ........................................................................................................................ 19 
5. 
STUDY OUTCOMES ..................................................................................................................... 19 
5.1 
Primary Outcome ........................................................................................................................ 19 
5.2 
Secondary Outcomes .................................................................................................................. 19 
5.3 
Other Outcomes of Interest ......................................................................................................... 19 
5.4 
Safety Outcomes ......................................................................................................................... 19 
6. 
STUDY DESIGN ............................................................................................................................ 19 
6.1 
General study design and justification of design ......................................................................... 19 

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6.2 
Methods of minimising bias ......................................................................................................... 20 
6.2.1 Randomisation .................................................................................................................... 20 
6.2.2 Blinding procedures ............................................................................................................ 20 
6.2.3 Other methods of minimising bias ....................................................................................... 20 
6.3 
Unblinding Procedures (Code break) .......................................................................................... 20 
7. 
STUDY POPULATION .................................................................................................................. 21 
7.1 
Eligibility criteria ........................................................................................................................... 21 
7.2 
Recruitment and screening ......................................................................................................... 21 
7.3 
Assignment to study groups ........................................................................................................ 22 
7.4 
Criteria for withdrawal / discontinuation of participants ............................................................... 22 
8. 
STUDY INTERVENTION ............................................................................................................... 22 
8.1 
Identity of Investigational Products ............................................................................................. 22 
8.1.1 Experimental Intervention ................................................................................................... 22 
8.1.2 Control Intervention (standard/routine/comparator treatment) ........................................... 23 
8.1.3 Packaging, Labelling and Supply (re-supply) ..................................................................... 23 
8.1.4 Storage Conditions.............................................................................................................. 23 
8.2 
Administration of experimental and control interventions ........................................................... 23 
8.2.1 Experimental Intervention ................................................................................................... 23 
8.2.2 Control Intervention ............................................................................................................. 25 
8.3 
Dose modifications ...................................................................................................................... 25 
8.4 
Compliance with study intervention ............................................................................................. 25 
8.5 
Data Collection and Follow-up for withdrawn participants .......................................................... 25 
8.6 
Trial specific preventive measures .............................................................................................. 25 
8.7 
Concomitant Interventions (treatments) ...................................................................................... 26 
8.8 
Study Drug Accountability ........................................................................................................... 26 
8.9 
Return or Destruction of Study Drug ........................................................................................... 26 
9. 
STUDY ASSESSMENTS ............................................................................................................... 26 
9.1 
Study flow chart ........................................................................................................................... 26 
9.2 
Assessments of outcomes .......................................................................................................... 26 
9.2.1 Assessment of primary outcome ......................................................................................... 26 
9.2.2 Assessment of secondary outcomes .................................................................................. 28 
9.2.3 Assessment of other outcomes of interest .......................................................................... 28 
9.2.4 Assessment of safety outcomes ......................................................................................... 28 
9.2.5 Assessments in participants who prematurely stop the study ............................................ 28 
9.3 
Procedures at each visit .............................................................................................................. 28 
9.3.1 Visit 1: Screening visit (1 hour) ........................................................................................... 28 
9.3.2 Visit 2 (120 minutes) ........................................................................................................... 29 
9.3.3 Visit 3 (30 minutes) ............................................................................................................. 29 
9.3.4 Visit 4 (10 minutes) ............................................................................................................. 29 
9.3.5 Visit 5 (15-20 minutes) ........................................................................................................ 29 
10. SAFETY ......................................................................................................................................... 29 
10.1 Drug studies ................................................................................................................................ 29 
10.1.1 Definition and assessment of (serious) AEs and other safety related events .................... 29 
10.1.2 Reporting of SAE and other safety related events .............................................................. 31 
10.1.3 Follow up of AEs and SAEs ................................................................................................ 31 
10.2 Assessment, notification and reporting on the use of radiation sources ..................................... 31 

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11. STATISTICAL METHODS ............................................................................................................. 32 
11.1 Hypothesis ................................................................................................................................... 32 
11.2 Determination of Sample Size ..................................................................................................... 32 
11.3 Statistical criteria of termination of trial ....................................................................................... 32 
11.4 Planned Analyses ........................................................................................................................ 32 
11.5 Handling of missing data and drop-outs...................................................................................... 32 
12. QUALITY ASSURANCE AND CONTROL .................................................................................... 32 
12.1 Data handling and record keeping / archiving ............................................................................. 32 
12.1.1 Case Report Forms ............................................................................................................. 32 
12.1.2 Specification of source documents ..................................................................................... 33 
12.1.3 Record keeping / archiving ................................................................................................. 33 
12.2 Data management ....................................................................................................................... 33 
12.3 Monitoring .................................................................................................................................... 33 
12.4 Audits and Inspections ................................................................................................................ 33 
12.5 Confidentiality, Data Protection ................................................................................................... 33 
12.6 Storage of biological material and related health data ................................................................ 33 
13. PUBLICATION AND DISSEMINATION POLICY.......................................................................... 34 
14. FUNDING AND SUPPORT ............................................................................................................ 34 
14.1 Funding ....................................................................................................................................... 34 
14.2 Other Support .............................................................................................................................. 34 
14.2.1 Cooperation with other institutes ......................................................................................... 34 
15. INSURANCE .................................................................................................................................. 34 
16. REFERENCES ............................................................................................................................... 35 
17. APPENDICES ................................................................................................................................ 38 
 
 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
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STUDY SYNOPSIS   
Principle 
investigator 
Professor Peter Schmid Grendelmeier, MD 
Sponsor 
Professor Pål Johansen, PhD 
Study Title: 
Immunological 
analysis 
of 
lymph 
node 
tissue 
after 
intralymphatic 
immunotherapy: A prospective case control study 
Study ID: 
ILIT-FNA-2023 
Version/Date: 
Version 2, 30.05.2023   
Trial registration: 
Registry on SNCTP via BASEC (Nr: 2023 - 00821) 
Study category 
and Rationale 
Interventional study with investigational medicinal product (IMP) for 
intralymphatic allergen immunotherapy. Clinical trial, Risk category B. 
Clinical Phase: 
Not applicable 
Background and 
Rationale: 
IgE-mediated allergy can be treated by subcutaneous allergen-specific 
immunotherapy (SCIT) (1-3). However, SCIT is not used by the majority of 
allergic individuals, mainly due to the long duration of the therapy and the risk 
of allergic reactions associated with allergen administration (4-6). 
Intralymphatic immunotherapy (ILIT), the administration of the allergens 
directly into a lymph node, could reduce treatment time, number of injections 
and adverse events. ILIT could raise patient compliance and improve allergy 
management. Clinical trials with only three monthly intralymphatic injections 
have shown comparable efficacy and safety to that of three years of SCIT (7).  
While efficacy of ILIT is becoming increasingly recognized, the mechanism by 
which ILIT acts is not known. The advantages of ILIT are typically explained 
by a more potent and efficient delivery of allergen to the lymphatic system. 
However since a different tissue is targeted than when using SCIT or SLIT, it 
may be assumed that other cells are directly involved or providing bystander 
effects. Moreover, while the existing ILIT trials have investigated long term 
clinical effects as well long term antibody and T-cell responses, early 
immunological events have not been investigated so far. Therefore, the current 
study aims to analyse and characterize local and systemic immunological 
changes upon a single ILIT injection in allergy patients receiving AIT as 
standard of care. 
For this, we will collect lymph node tissue and blood within one month of a 
single ILIT injection. One group of patients will receive a SCIT injection as a 
reference to ILIT. Cells and serum will be analysed by standard diagnostic 
methods as well as modern immunological methods. The study may highlight 
the immune properties of ILIT and potentially further propagate ILIT as a valid 
alternative to SCIT. 
Objective(s): 
The study seeks primarily to determine hallmarks in immunological responses 
(analysis of inflammatory cell and proteins) in lymph nodes and in peripheral 
blood after allergen ILIT and SCIT. 
Outcome(s): 
1) Fine needle aspiration (FNA) material of lymph node tissue at baseline 
and 2 or 6 or 24 hours after allergen ILIT or SCIT will be evaluated for 
changes in cell populations and cell functions. 
2) Immunological changes (analysis of cell populations and functions ex vivo 
and in vitro) in peripheral blood at baseline, after 2 or 6 or 24 hours, after 7 
days and after 28 days of allergen ILIT or SCIT. 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 7 of 39 
Study design: 
This is a prospective, comparative, clinical study. We will recruit 30 grass-
pollen allergic patients undergoing pre-seasonal short-term SCIT scheme with 
Polvac™ Grass+Rye at the USZ Allergy Unit. 4 weeks after last SCIT injection 
and after reaching maintenance SCIT dose (2000 U), the patients will receive 
an additional allergen injection as part of the study. Patients will be randomized 
into the 3 different groups with 10 participants per group. Patients will receive 
a single ultrasound-guided injection with Polvac™ Grass+Rye ILIT (0.1 ml, 400 
U) or SCIT (0.5 ml, 2000 U). 
FNA of lymph node tissue will be conducted at baseline and at 2, 6 or 24 hours 
after allergen injection. Venous blood will be sampled at baseline, after 2, 6 or 
24 hours, after 7 days, and after 28 days of allergen injection.  
Inclusion / 
Exclusion 
criteria: 
Included are patients between 18 and 55 years of age with grass-pollen 
allergy and undergoing pre-seasonal short-term-scheme Polvac Grass+Rye 
SCIT at the USZ Allergy Unit for treatment of allergic rhinoconjunctivitis.  
Exclusion criteria are significant allergy to house dust mite or pet animal 
allergens, uncontrolled asthma, upper airway disease, severe pulmonary 
disease, recent allergic reactions, immunosuppression, cardiovascular 
disease, malignant disease, conspicuous lymph node size or morphology, 
use of ACE-blockers and any other diseases or conditions rendering the 
treatment of anaphylactic reactions difficult.  
Measurements 
and procedures: 
A single injection of grass pollen allergen extract (Polvac™ Grass+Rye) will be 
administered by ILIT or SCIT. FNA of material from lymph node tissue will be 
aspirated 6 hours after allergen injection. Opposite inguinal lymph node tissue 
will be aspirated by FNA as a baseline comparison. Venous blood will be 
sampled at baseline, after 2, 6 or 24 hours, after 7 days, and after 28 days of 
allergen injection. For safety reasons, as to exclude respiratory cases, peak 
flow measurement will be performed before treatment and 40 minutes after 
treatment. 
Study Product / 
Intervention:  
A single dose of grass pollen allergen extract (Polvac™ Grass+Rye from 
Bencard AG, Switzerland) will be injected via ultrasound-guided ILIT or SCIT 
route. Different doses will be injected according to the 2 study arms: (i) ILIT 0.1 
ml, 400 U or SCIT 0.5 ml, 2000 U.  
Control 
Intervention: 
No control intervention is planned. 
Number of 
Participants with 
Rationale: 
For this pilot, unblinded study with three treatment groups, no sample size 
calculation is made. We assume that 10 patients per treatment group will 
enable us to describe immunological features of the three treatments. 
Study Duration: 
01.09.2023 to 31.12.2024 
Study Schedule: 
Plan: October 2023 First-Participant-In; March 2024 Last-Participant-Out.  
Investigators: 
Emma Widmer, Department of Dermatology, University of Zurich, 
Raemistrasse 100, 8091 Zurich, Switzerland 
Lara Šošić, MD, Department of Dermatology, University of Zurich, 
Raemistrasse 100, 8091 Zurich, Switzerland 
Claudia Lang, MD, USZ Allergy Unit, The Circle 59, 8058 Zurich-Flughafen, 
Switzerland 
Study Centre(s): 
Department of Dermatology, University Hospital Zurich, Zurich 
Statistical 
Considerations: 
All data are analysed and reported with descriptive statistics.  
GCP Statement: 
This study will be conducted in compliance with the protocol, the current 
version of the Declaration of Helsinki, ICH-GCP, as well as all national legal 
and regulatory requirements.  
 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 8 of 39 
ABBREVIATIONS 
AE 
Adverse Event  
ASR 
Annual Safety Report 
ARC 
Allergic Rhinoconjunctivitis 
BASEC 
Business Administration System for Ethical Committees, 
(https://submissions.swissethics.ch/en/) 
CA 
Competent Authority (e.g. Swissmedic) 
CRF 
Case Report Form  
ClinO 
Ordinance on Clinical Trials in Human Research (in German: KlinV, in French: 
OClin, in Italian: OSRUm) 
eCRF 
Electronic Case Report Form  
CTCAE 
Common terminology criteria for adverse events 
DSUR 
Development safety update report 
FNA 
Fine needle aspiration 
GCP 
Good clinical practice 
IB 
Investigator’s Brochure 
Ho 
Null hypothesis 
HRA 
Federal Act on Research involving Human Beings (in German: HFG, in French: 
LRH, in Italian: LRUm) 
IEC 
Independent Ethics Committee 
IMP 
Investigational Medicinal Product 
IIT 
Investigator-initiated Trial 
ILIT 
Intralymphatic immunotherapy 
ITT 
Intention to treat 
PI 
Principal Investigator  
SCIT 
Subcutaneous Immunotherapy 
SDV 
Source Data Verification  
SmPc 
Summary of Product Characteristics 
SOP 
Standard Operating Procedure 
SPC 
Summary of product characteristics 
SUSAR 
Suspected Unexpected Serious Adverse Reaction  
TMF 
Trial Master File  
USZ 
University Hospital Zurich 
 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 9 of 39 
STUDY SCHEDULE  
Table 1. Illustrating the study schedule with all interventions divided into the five visits. Patients will 
receive 6 SCIT injections as standard of care (not part of the study). 4-6 weeks later, the patients will be 
included in the clinical trials for assessment of immunological characteristics after a single ILIT or SCIT 
with subsequent fine needle aspiration (FNA), adverse event (AE) monitoring, and venous blood 
collection. 
 
Visit / time point 
1 
Screening 
2  
AIT 
3 
FNA 
4  
Follow-up 
5  
Follow up 
< 6 weeks 
of last SCIT 
Day 0 
Day 0-1 
Day 7 
Day 28 
Required personnel 
Nurse/MD 
MD 
MD 
Nurse/MD 
Nurse/MD 
Informed consent 
x 
 
 
 
 
Randomization 
x 
 
 
 
 
Medical history 
x 
 
 
 
 
Physical exam 
x 
 
 
 
 
Incl./excl. criteria 
x 
 
 
 
 
Blood samples 
 
x 
x 
x 
x 
Pregnancy test 
 
x 
 
 
 
AEs/Concomitant medication 
 
x 
 
 
x 
Peakflow measurement 
 
x* 
 
 
 
Injection: Study drug administration 
 
x 
 
 
 
FNA (2x) 
 
 
x** 
 
 
AE monitoring (1h observation at 
allergy unit after injection) 
 
x 
 
 
 
Dispensing rescue medication 
 
x 
 
 
 
* Peakflow measurement will be performed immediately before injection and 40 minutes after injection 
** The FNA is performed 2, 6 or 24 hours after the Polvac ILIT or SCIT, and the injected (ILIT) or draining (SCIT) 
lymph node is used for FNA. For baseline reference, a contralateral lymph node is also aspirated. 
 
 
 
 
 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 10 of 39 
1. STUDY ADMINISTRATIVE STRUCTURE  
1.1 Sponsor  
Professor Pål Johansen, PhD  
Department of Dermatology  
University Hospital Zurich  
Rämistrasse 100, 8091 Zurich, Switzerland  
Mail: pal.johansen@usz.ch 
Tel. +41 44 255 86 16 
 
1.2 Medical Investigators 
Principle medical investigator 
Peter Schmid-Grendelmeier, MD (Head of Allergy Unit)  
Allergy Unit, Department of Dermatology, USZ Flughafen  
The Circle 59, 8058 Zurich-Flughafen, Switzerland  
Mail : peter.schmid@usz.ch 
Tel. +41 44 255 30 78  
 
Responsible medical investigator:  
Claudia Lang, MD (Oberärztin)  
Allergy Unit, Department of Dermatology, USZ Flughafen,  
The Circle 59, 8058 Zurich-Flughafen, Switzerland  
Mail: claudia.lang@usz.ch 
Tel: +41 43 254 14 85  
 
Medical investigator: 
Marie-Charlotte Brüggen, MD, PhD 
Allergy Unit, Department of Dermatology, USZ Flughafen  
The Circle 59, 8058 Zurich-Flughafen, Switzerland  
Mail: Marie-Charlotte.Brueggen@usz.ch 
Tel. Phone: +41 81 417 33 72 
 
Medical investigator: 
Emma Widmer 
Department of Dermatology, University Hospital Zurich  
Rämistrasse 100, 8091 Zurich, Switzerland  
Mail: emma.widmer@usz.ch 
Tel. +41 76 779 27 65 
 
Medical investigator:  
Lara Šošić, MD  
Department of Dermatology, University Hospital Zurich  
Rämistrasse 100, 8091 Zurich, Switzerland  
Mail: lara.sosic@usz.ch 
Tel. +41 44 255 99 52 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 11 of 39 
 
Study nurse:  
Stephanie Bösch  
Allergy Unit, Department of Dermatology, USZ Flughafen  
The Circle 59, 8058 Zurich-Flughafen, Switzerland  
Mail: stephanie.boesch@usz.ch 
 
1.3 Statistician ("Biostatistician")  
Nicole Graf, PhD 
Graf Biostatics 
Amelenweg 5, 8400 Winterthur 
Mail: graf@biostatistics.ch 
 
1.4 Laboratory 
Reihane Ziadlou, PhD 
Swiss Institute of Allergy and Asthma Resarch 
Hochgebirgsklinik Davos 
Herman-Burchard-Str. 9, 7265 Davos-Wolfgang, Switzerland 
Mail: reihane.ziadlou@usz.ch 
Tel: +41 81 410 08 44 
 
Agathe Duda  
Department of Dermatology, USZ Flughafen  
Raemistrasse 100, 8091 Zurich, Switzerland  
Email: agathe.duda@usz.ch 
Tel: +41 44 255 396 22 01 
 
Muriel Rentschler  
Department of Dermatology, University Hospital Zürich  
Raemistrasse 100, 8091 Zurich, Switzerland  
Email: Muriel.rentschler@usz.ch 
Tel: +41 43 253 401 22 07 
 
Daniel Hug 
Department of Dermatology, University Hospital Zürich  
Raemistrasse 100, 8091 Zurich, Switzerland  
Email: daniel.hug@usz.ch 
Tel: +41 43 254 14 85 
 
Marta Paolucci, PhD 
Department of Dermatology, University Hospital Zurich  
Rämistrasse 100, 8091 Zurich, Switzerland  
Mail: marta.paolucci@usz.ch  
Tel. +41 77 909 40 65 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 12 of 39 
1.5 IT Management 
Not applicable. 
1.6 Data Safety Monitoring Committee  
Not applicable. 
 
1.7 Partners at the USZ Allergy Unit  
Chief Nurse of the Allergy unit:  
Elvira Schmid-Schwotzer  
Allergy Unit, Department of Dermatology, USZ Flughafen  
The Circle 59, 8058 Zurich-Flughafen, Switzerland  
Mail : elvira.schmid@usz.ch 
Tel. +41 44 255 24 41 
 
1.8 Further Partners 
Clinical Director  
Thomas M. Kündig, MD, Professor 
Department of Dermatology, University Hospital Zurich  
Rämistrasse 100, 8091 Zurich, Switzerland  
Mail: thomas.kuendig@usz.ch 
Tel. +41 78 609 35 36 
 
Scientific advisor 
Hans Jürgen Hoffmann, PhD, Professor  
Department of Clinical Medicine  
Aarhus University 
Juul Jensens Boulevard 99, 8200 Aarhus N, Denmark 
Mail hjh@clin.au.dk 
Tel. +45 2818 8147 
 
Investigational Medicinal Product  
Allergy Therapeutics 
Dominion Way, Worthing, West Sussex, BN14 8SA, United Kingdom 
Tel. +44 1903 844 700 
 
Investigational Medicinal Product  
Bencard AG 
Tumigerstrasse 71, 8606 Greifensee, Switzerland 
Email: info@bencard.ch 
Tel: +41 44 942 00 13 
 
 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 13 of 39 
2. ETHICAL AND REGULATORY ASPECTS  
The clinical study will only begin once approval from CEC and Swissmedic has been received. Any 
additional requirements imposed by the authorities will be implemented. 
2.1 Study registration  
The trial will be registered on SNCTP via BASEC. 
2.2 Categorisation of study  
Interventional study with investigational medicinal product (IMP) for intralymphatic allergen 
immunotherapy. Clinical trial, Risk category B.  
Allocation to Risk Category B because of off-label use of the allergen, i.e., intralymphatic administration 
and dosage change of investigational medicinal product (IMP). 
2.3 Independent Ethics Committee (IEC)  
Approval will be requested from the cantonal ethical committee (Kantonale Ethikkomission Zürich) via 
BASEC and by the CA Swissmedic. 
The Sponsor will report any premature termination or interruption of the trial to the IEC and Swissmedic 
within 15 days. The regular end of the study is reported by the Sponsor to the IEC as well to Swissmedic 
within 90 days, the final study report will be submitted within one year after study end. The Sponsor will 
submit an annual safety report to the IEC.  
Substantial amendments are only implemented after approval of the IEC and CA respectively. 
Under emergency circumstances, deviations from the protocol to protect the rights, safety and well-
being of human subjects may proceed without prior approval of the Sponsor and the IEC/CA. Such 
deviations shall be documented and reported to the Sponsor and the IEC/CA as soon as possible. 
All non-substantial amendments are communicated to the CA as soon as possible if applicable and to 
the IEC within the ASR.  
2.4 Competent Authorities (CA)  
Approval will be requested from the CA Swissmedic according to study category B before the start of 
the clinical trial.  
2.5 Ethical Conduct of the Study  
The study will be carried out in accordance to the protocol and with principles enunciated in the current 
version of the Declaration of Helsinki, the guidelines of Good Clinical Practice (GCP) issued by ICH, 
and Swiss Law and Swiss regulatory authority’s requirements. The IEC and regulatory authorities will 
receive Annual Safety (ASR) and interim Reports and be informed about study stop/end in agreement 
with local requirements.  
2.6 Declaration of interest  
The Department of Dermatology of the University Hospital of Zurich (Director Professor Dr. med. 
Thomas Kündig) will support the study with infrastructure and overhead. 
Prof. Thomas Kündig has served as medical advisor for Allergy Therapeutics (Worthing, UK), which is 
providing the investigational drug (Polvac™ Grass+Rye) to be used in this study. The Sponsor of this 
study, Pål Johansen, has received research funding from Allergy Therapeutic for pre-clinical research 
with products or developments from Allergy Therapeutics. However, the efficacy of the study drug 
(Polvac™ Grass+Rye) itself in comparison to other study drugs plays no role in this study for which 
reason the relationship to Allergy Therapeutics should have no influence on the overall analysis of the 
study.  
Other researchers participating in this study have no conflicts of interest. No bias in the study is expected 
from conflicts of interest. 
2.7 Patient Information and Informed Consent 
The medical investigators explains to each study participant the nature of the study, its purpose, the 
procedures involved, the expected duration, the potential risks and benefits and any discomfort it may 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 14 of 39 
entail. Each subject is informed that the participation in the study is voluntary and that he/she may 
withdraw from the study at any time and that withdrawal of consent will not affect his/her subsequent 
medical assistance and treatment. The subjects are informed that he/she can ask any question, and 
consult with family members, friends, their treating physicians, or other experts before deciding about 
their participation in the study. Enough time is given to the subjects before signing the informed consent 
form.  
The subjects are informed that authorised individuals other than their treating physician may examine 
his/her medical records. 
All subjects are given a subject information sheet and a consent form describing the study and providing 
sufficient information for the subjects to make an informed decision about their participation in the study.  
The formal consent of a subject, using the approved consent form, is obtained before the subject is 
submitted to any investigation procedure.   
The subject should read, understand, and voluntarily agree before signing and dating the informed 
consent form, and is given a copy of the signed document. The consent form is signed and dated by the 
subject and the Principle-Investigator (or his designee). The signed consent form it is retained as part 
of the investigation records. 
The patients will be compensated for potential travel cost only. 
2.8 Participant privacy and confidentiality  
The investigators affirm and uphold the principle of the participant's right to privacy and that they shall 
comply with applicable privacy laws. Especially, anonymity of the participants shall be guaranteed when 
presenting the data at scientific meetings or publishing them in scientific journals.  
Individual subject medical information obtained as a result of this study is considered confidential and 
disclosure to third parties is prohibited. 
For data verification purposes, authorised representatives of the Sponsor, a competent authority (e.g. 
Swissmedic), or the competent ethics committee (Kantonale Ethikkomission Zürich) may require direct 
access to parts of the medical records relevant to the study, including participants’ medical history. 
The identity of the participants in the study will be coded with a participant number. The handling of data, 
including data quality control, will comply with regulatory guidelines (e.g. International Conference on 
Harmonisation (ICH) and Good Clinical Practice (GCP). The project will comply with the General Data 
Protection Regulation (GDPR) and The Data Protection Act.  
2.9 Early termination of the study  
The Principle-Investigator or the Sponsor may terminate the study prematurely according to certain 
circumstances, for example: 
• 
ethical concerns, 
• 
insufficient participant recruitment, 
• 
when the safety of the participants is doubtful or at risk, respectively, 
• 
alterations in accepted clinical practice that make the continuation of a clinical trial unwise,  
• 
early evidence of benefit or harm of the experimental intervention  
The Sponsor will report any premature termination of the trial to the IEC.  
2.10 Protocol amendments 
Substantial amendments are only implemented after approval of the IEC and CA respectively. 
Under emergency circumstances, deviations from the protocol to protect the rights, safety and well-
being of human subjects may proceed without prior approval of the sponsor and the IEC/CA. Such 
deviations will be documented and reported to the sponsor and the IEC/CA as soon as possible. 
All non-substantial amendments are communicated to the CA as soon as possible if applicable and to 
the IEC within the ASR.  
 

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3. BACKGROUND AND RATIONALE  
3.1 Background and Rationale  
Allergic rhinitis is an important public health concern; it affects more than 20% of Western populations 
(8). According to the definition proposed by the ARIA (Allergic Rhinitis and its Impact on Asthma) 
Initiative, allergic rhinitis is an IgE-mediated inflammatory reaction of the nasal epithelium with the 
corresponding symptoms, which is precipitated by exposure to an allergen (9). IgE mediated allergy can 
be treated by subcutaneous (SCIT) or sublingual (SLIT) allergen-specific immunotherapy (1-3). While 
SLIT comprises daily tablets or drops for three years, SCIT is performed by weekly-to-monthly injections 
over the same time period, resulting in 18 to approx. 50 injections, depending on the chosen scheme. 
However, AIT, and especially SCIT is not used by the majority of allergic individuals with allergic rhinitis, 
mainly due to the long duration of the therapy, each injection associated with a doctor visit and the risk 
of severe systemic allergic reactions associated with the allergen administration (4-6). 
Intralymphatic immunotherapy (ILIT), the administration of the allergens directly to an inguinal lymph 
node, is an investigational method developed to shorten treatment duration and improve safety while 
maintaining efficacy. 
Clinical trials with only three intralymphatic injections that are given with 1-2 month intervals showed the 
same effect as 3 years of subcutaneous or sublingual immunotherapy (7). Indeed, the treatment effect 
as measured by clinical symptoms, allergen provocation tests and symptom-medication scores have 
shown comparable efficacy and safety to that of three years of SCIT (10-12) 
The effects were typically explained by the more efficient delivery of allergen to the lymphatic system 
with ILIT as compared to other routes (13, 14). Other routes (SLIT and SCIT) depend on the drainage 
and uptake of injected allergen into lymph node for antigen presentation, resulting in only a minor fraction 
of the administered allergen arriving in the lymph node. Moreover, because fewer and lower doses are 
used in ILIT than in SCIT, less AIT-associated side effects may be expected. Hence, ILIT could raise 
patient compliance and improve management of allergies in many patients afraid or reluctant to receive 
conventional AIT. 
While efficacy of ILIT is becoming increasingly recognized, the mechanism by which ILIT acts is not 
known and has not been investigated. Since a different tissue is targeted than when using SCIT or SLIT, 
it may be assumed that other cells are directly involved or providing bystander effects. For instance, 
antigen-presenting cells in the subcutaneous (SCIT) or mucosal (SLIT) tissues are surely different from 
those residing in the lymph nodes. Since the presentation of proteins in vaccines and immunotherapy 
products differs in quality and effect size between different antigen-presenting cells (e.g. macrophages, 
dendritic cells, monocytes, B cells and neutrophils), antigen presentation after ILIT, SLIT and SCIT 
mostly likely differs. Moreover, inflammatory cells such as neutrophils have versatile bystander functions 
and contribute to adaptive immunity by various way. The migration of neutrophils from the periphery (as 
in SCIT) to lymphoid organs has been linked to upregulation of the chemokine receptor CCR7, but it is 
unknown to what extent such inflammatory cells get involved when allergen is injected directly to lymph 
nodes (ILIT). Therefore, the current study aims to analyse and characterise local and systemic 
immunological changes upon a single ILIT injection in grass-pollen allergic patients receiving 
immunotherapy. We will characterize the immunological potential induced by ILIT and compare this to 
the immunological events observed upon SCIT. By these means, we expect to reveal some of the 
mechanisms by which ILIT exerts its effects and thereby address unmet needs in AIT. The present small 
study with limited patient numbers has a focus on the major ILIT-induced changes in leukocyte numbers, 
activation, migration and function. The results should inform the strategy for bigger follow-up studies to 
optimize ILIT timing and dosing. 
The following question will be addressed:  
• 
Is immunity by SCIT and ILIT generated by the same mechanisms and only weaker in SCIT 
due to less allergen reaching the lymph nodes or does ILIT result in more efficient activation of 
specialized lymph node cells, different from what is seen after SCIT? 
• 
Which innate immune cells are involved in ILIT and SCIT? 
• 
Why does SCIT require higher doses and longer treatment times as compared to ILIT?  
• 
How do innate and adaptive immune responses correlate? 
 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
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3.2 Investigational Product (treatment) and Indication   
Polvac Grass+Rye, a grass- allergen extract with microcrystalline tyrosine (MCT) adjuvant for 
treatment of allergic rhinitis will be used. The active ingredient is a standardized grass pollen allergen 
extract from 13 species of grass combined with the excipient MCT adjuvant as from Allergy Therapeutics 
Ltd (cf. Appendix 001 for further details). The allergen extract has been treated with glutaraldehyde as 
to reduce allergic side effects, i.e., to increase the safety. Bencard AG (Greifensee, Switzerland) is 
providing the product in Switzerland. Polvac, is a marketed product that has been approved by the 
Swiss medical product agency Swissmedic (#46489 and #65479) and adheres to GMP. The product 
consists of needle-like crystalline structures of an average size of 20 micrometers (15). Polvac is 
approved for SCIT, which includes 6 pre-seasonal injections with 1-2 week intervals for 3-4 years, 
adding up to a total of 18 to 24 injections for the treatment to be completed (16-18). In the current study, 
for SCIT, Polvac will be used as described by the provider (Appendix 001). For ILIT, the samples will 
be processed as described by the provider, but administered at one fifth of the dose used for SCIT.  
3.3 Preclinical Evidence   
ILIT has been successfully performed in various mouse models using purified ovalbumin, bee venom 
allergen extract or purified phospholipase A1 (Api m 1), recombinant Fel d 1 cat allergen, and birch and 
grass pollen allergen extracts (19-25). One ILIT study in mice revealed that approximately 100-fold 
higher allergen doses were found in lymph nodes after ILIT as compared with a subcutaneous injection 
of the same allergen dose (20). Accordingly, similar therapeutic effects after ILIT with Api m 1 could be 
reached with only 1% of dose needed after SCIT with the same allergen (20). In another mouse ILIT 
study, only 0.1% of the SCIT dose was required to obtain comparable results (20, 26). 
3.4 Clinical Evidence to Date  
ILIT has been tested in clinical phase I and II studies in patients with sensitization to grass and tree 
allergens (7, 27-39). In addition, ILIT has been tested for treatment of allergic rhinitis in patients 
sensitised to house dust mite, cats and dogs (ref). Generally, three ILIT injections are performed with a 
time interval of 4-12 weeks between the injections (33, 40), as to allow for the development of antigen-
specific immune responses, similar to the immunological mechanism of childhood vaccines. Shorter 
intervals showed no clinical improvement despite indications of immunological tolerance (31, 32, 40). 
Three recent systematic reviews and meta-analysis of 10-17 clinical trials about ILIT for allergic 
rhinoconjunctivitis showed a benefit toward symptom alleviation and reduced medication use, as well 
as comparable safety and efficacy to both SCIT and SLIT (10-12). One clinical investigation 
demonstrated that ILIT with only 1/1000th of the SCIT dose produced comparable immunological or 
clinical effects (7), and the follow-up of the same study patients showed long-term allergen tolerance of 
ILIT in 29 patients that received grass pollen ILIT 19 years earlier (41). Most recently, a five year follow-
up DBPC study in patients that received birch and grass pollen ILIT was published (42). The authors 
reported long-lasting effect of ILIT as measured by the registration of combined symptoms and 
medications score (cSMS), a nasal provocation test, and assessment of immunological parameters such 
as IgG4 and IgE. One study concluded that high-dose ILIT (up to 10’000 SQ-U, corresponding to the 
maintenance of SCIT) was safe in patients that recently underwent SCIT, suggesting high-dose ILIT in 
patients as add-on treatment for patients receiving SCIT could be an option to further improve symptom 
control (28). 
To date, there has been one published clinical ILIT trial where Polvac™ ILIT was applied in 19 hay fever 
patients (43). One double-blind, placebo controlled clinical trial with 60 patients receiving Polvac™ 
Grass+Rye ILIT or placebo ILIT is ongoing in our clinic in Zurich (BASEC Nr. 2021-02301), with no 
severe adverse events occurring after a total of 180 injections, 90 thereof in the Polvac™ group. Local 
adverse events (AEs) were transient and included erythema and swelling at the site of injection (Fig. 1). 
Typically, more AEs were observed or perceived after second or third ILIT than after the first ILIT. , 
Since the study is still blinded, no segregation of AEs in placebo and active treatment is not yet possible. 
This trial, which goal it is to test the treatment efficacy of Polvac™ ILIT, is performed with the same PI 
(Pål Johansen) and with one of the study doctors (Lara Sosic) as the planned new project described in 
this study protocol and ethics application. 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 17 of 39 
 
Figure 1. Registration of adverse events within an ongoing placebo-controlled study of Polvac ILIT in patients with 
allergic rhinitis due to sensitisation to grass pollen allergens (BASEC Nr. 2021-02301). Safety parameters were 
analysed by clinician 40 minutes post ILIT or by patients 1, 2 and 3 days post ILIT. The pie charts illustrate the 
percentage and numbers of events graded as none (blue), mild (red) and dark red (moderate). In the current project 
and application, a comparable injection will be performed in a comparable patient cohort. In the current study, 
however, only one injection will be made, while in the illustrated study, three injections were made (Lara Sosic, 
unpublished data). 
3.5 Rationale for the dosage, route, regimen  
We will include grass pollen allergy patients with pre-seasonal short-term and pre-seasonal injection 
scheme with Polvac SCIT at the USZ Allergy Unit (Head: Prof. Peter Schmid-Grendelmeier). After the 
patients have received the full cycle of SCIT (six injections) as a part of standard care, they will be 
randomized into one of the two study groups for one additional injection, which is to be given 4-6 weeks 
after the last SCIT cycle injection. Depending on the group allocation, patients will receive allergen by 
SCIT (or by ILIT. Figure 2 illustrates schematically the study design and Figure 3 shows the flow chart 
for the study participants. 
Polvac™ Doses: 
• 
SCIT (n=15): 0.5 ml Polvac (max maintenance dose, 2000 U). 
• 
ILIT (n=15): 0.1 ml Polvac (400 U). 
The SCIT dose is chosen as this is the recommended and standardized treatment dose for patients 
receiving SCIT with Polvac for treatment of grass pollen allergy. The ILIT dose is 1/5th of the 
maintenance SCIT dose. This has been safely administered in four patients treated off-label with 
Polvac at the USZ allergy in 2023 (Lara Sosic and Peter Schmid, personal communication). These 
four patients received three injections in a dose escalating manner and with four-week intervals. The 
doses were 1/15th, 1/10th and finally 1/5th of the maintenance SCIT dose. An ILIT dose of 1/15th of the 
maintenance SCIT dose was safely administered three times in 30 patients in the aforementioned 
ongoing study with Polvac ILIT (BASEC 2021-02301). 
3.6 Explanation for choice of comparator (or placebo)  
No comparator or placebo is used in the current project.  
3.7 Risks / Benefits  
Benefits 
• 
The study should improve our understanding of the immune responses stimulation after SCIT 
and ILIT, and could lead to novel developments in AIT, and especially, ILIT.  
• 
All study participants will get one additional AIT injection (SCIT or ILIT), which may contribute 
to and even further consolidate the success of their therapy. 
• 
Results from previous studies suggest that an ILIT injection on top of a full SCIT cycle may 
further improve the therapeutic efficacy.  

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 18 of 39 
• 
The study participants receiving an ILIT injection would get to know a new route of AIT. In the 
future, they may use this as basis for discussion with their treating allergist regarding a 
possible change in the treatment regimen, as an alternative to SCIT. The benefits being 
reduced treatment times and lower allergen doses with potentially less AIT-associated side 
effects.   
Risks 
The SmPc for Polvac is the reference sheet for evaluation of AEs (see Appendix 002). The following 
known SCIT-associated are mentioned  
• 
AE: itch or swelling and rubor at the injection site that resolves without treatment by next day 
(27%, 125 events in 462 injections of 154 patients)  
• 
AE: allergic symptoms - urticaria, tightness of chest, drop in FEV1 >40% (2%, 11 of 462 
injections) treated with an antihistamine or a short acting beta-2 agonist. Every clinic offering 
AIT is equipped to deal with this AE, and patients are required to stay in the clinic for 60 
minutes for observation to deal with these incidents.  
• 
Unexpected AE: some few patients reported fatigue within 6 hours  
• 
Severe AE: systemic allergic symptoms that required hospitalization: drop in blood pressure, 
generalized urticaria or deterioration by 40% of peak flow or FEV1 (0.2%, 1 of 462 injections)  
• 
Generation of autoimmune antibodies may be a potential long-term effect of AIT. This has not 
been reported in any of the AIT trials with Polvac.  
• 
One study reported one patient with hypothyroidism with auto-antibodies among 72 patients 
treated by ILIT (patients were not treated with Polvac but with a comparable alum-based 
product).  
• 
In all AIT procedures (ILIT, SCIT or SLIT), there is a risk of anaphylactic reactions that 
typically happens within minutes of the injections. This is a SAE that will required 
hospitalization.  
• 
The fine-needle aspiration (FNA) of the previously injected lymph node is a minimally invasive 
procedure and comparable to the ILIT procedure, hence, the potential AEs of ILIT injection 
and FNA are similar: infection and bleeding (hematoma). These problems are very rare, and 
antiseptic and ultrasound-guided procedures further minimizes the risk. 
All SAE will be reported to the sponsor within 24 hours. SAEs will be followed up until one month after 
the end of the treatment period.  
To minimize the risk of any allergic AEs, the patients will receive an emergency set (“Allergie Notfallset”) 
for self-medication in case of side effects after the patient has left the study site. The emergency set 
consists of two tablets of Xyzal® 5 mg and two tablets of Spiricort® 50 mg (Appendix 003). Patients will 
be advised on when and how to use the emergency set. Furthermore after ILIT treatment the patients 
have to stay one hour at the allergy unit for observation.  
3.8 Justification of choice of study population  
We plan to include 30 participants with allergic rhinitis due to sensitization to grass pollen allergens. We 
chose to include patients already undergoing AIT and not sensitized-only patients as to reduce risk of 
allergic side effects and to enable the injection of a higher allergen dose. As compared to a low dose, a 
high allergen dose is expected to trigger stronger immune responses thereby facilitating assessment of 
such responses, the primary objective of the study. 
4. STUDY OBJECTIVES  
4.1 Overall Objective 
The overall purpose of this study is to widely characterize immunological changes induced by ILIT and 
to evaluate these changes in comparison to SCIT by descriptive statistics. In brief, the study will assess 
quantity and quality of ILIT- and SCIT-induced immunological changes in lymph nodes and in blood. 
The two following theories will be investigated addressing the question: Why does SCIT require higher 
doses and longer than ILIT?  
• 
Quantitative theory: Immunity by SCIT and ILIT is generated by the same cell types and 
mechanisms, but only a fraction of the SCIT allergen arrives in the regional lymph nodes, hence 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 19 of 39 
weaker effect of SCIT. 
• 
Qualitative theory: ILIT results in a more efficient activation of specialized lymph node cells, 
different from the vaccination effect activated after SCIT. 
The current study is a pilot study including small patient numbers as a basis for bigger follow up studies 
with a focus on the major ILIT induced immunological changes. The study seeks primarily to determine 
hallmarks in immunological response (analysis of cell populations and cell culture expression patterns) 
in lymph nodes and peripheral blood after ILIT versus SCIT. 
4.2 Primary Objective 
The primary objective is to compare inflammatory responses in blood and lymph nodes from patients 
receiving grass pollen allergen ILIT or SCIT. No single primary endpoint is defined, as numerous 
readouts will be performed as part of a wide screening of inflammatory responses.  
4.3 Secondary Objectives 
As a single secondary endpoint is not defined in this pilot study, primary and secondary endpoints melt 
together in the overall objective of analysing a wide panel of inflammatory responses to a single ILIT or 
SCIT.  
4.4 Safety Objectives 
Although the risk is low when AIT is performed appropriately, allergic reactions do occasionally occur. 
Part of the inflammatory characterization of ILIT and SCIT will therefore be to assess also local and 
systemic reaction associated with ILIT and SCIT treatment.  
5. STUDY OUTCOMES  
5.1 Primary Outcome 
The FNA material of lymph node tissue at baseline and 2, 6 or 24 hours after allergen injection (via ILIT 
or SCIT) will be evaluated for changes in cellular composition and functions. 
Immunological changes in peripheral blood at baseline, after 2, 6 or 24 hours, after 7 days and after 28 
days of allergen ILIT or SCIT will be analysed by measuring cellular composition and functions in whole 
blood and in culture blood cells as well as inflammatory proteins and antibodies in serum. 
5.2 Secondary Outcomes 
To analyse the effect of dose on the measured immunological and inflammatory responses after a single 
ILIT injection (400 U).  
To analyse differences between ILIT and SCIT-induced inflammatory and immunological responses. 
5.3 Other Outcomes of Interest 
None. 
5.4 Safety Outcomes 
To analyse local and systemic reactions after ILIT and SCIT (assessed by clinician during on site 
monitoring 60 min after the intervention and by study participant at home for the next two days). 
6. STUDY DESIGN  
6.1 General study design and justification of design  
This is a prospective, comparative, clinical open label pilot study. From patients with grass pollen allergy 
who undergo a pre-seasonal SCIT scheme with Polvac™ Grass+RyeSCIT at the USZ Allergy Unit, 30 
patients will be recruited for the study that is illustrated in Figure 2.  
Four to six weeks after end of SCIT treatment (at maximal maintenance dose), the patients will be 
randomized to one out of two treatment groups and receive one additional allergen injection. Fifteen 
patients will be allocated to receive another subcutaneous injection with 0.5 ml (2000 U) Polvac™ 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 20 of 39 
Grass+Rye. The other 15 patients will be allocated to receive an intralymphatic injection of 0.1 ml 
Polvac™ Grass+Rye with 400 U allergen. Both SCIT and ILIT injections are performed by ultrasound-
guidance as to assure injection into the lymph node (ILIT) or 1 cm next to the lymph node (SCIT).  
The FNA of lymph node tissue will be conducted at 2, 6 or 24 hours after allergen injection. At the same 
time, FNA of a contralateral lymph nodes will be performed as baseline. Venous blood will be sampled 
at all visits: at baseline as well as after 2, 6 or 24 hours, after 7 days and after 28 days of the allergen 
ILIT or SCIT. The FNA is performed by ultrasound-guidance. 
 
Figure 2. Study design illustrated schematically the open-label clinical pilot study for assessment of immunological 
and inflammatory responses to a single ILIT or SCIT in patients receiving grass pollen immunotherapy with Polvac 
Grass+Rye. 
 
6.2 Methods of minimising bias  
6.2.1 
Randomisation  
All patients who give consent for participation and who fulfil the inclusion criteria will be randomized to 
either SCIT or ILIT treatment group with a 1:1 allocation. The randomisation list will be computer-
generated by a statistician using a permuted block design (groups of six). Before trial initiation, 
randomisation envelopes will be given to the study physician who will then be responsible of envelope 
storage and appropriate use. Allocation concealment will be ensured as the randomisation envelopes 
will not be opened before the patient has been recruited into the trial, which takes place after screening 
has been completed. 
6.2.2 
Blinding procedures  
Not applicable. The study is not blinded. 
6.2.3 
Other methods of minimising bias  
Patients will be randomized into two different groups. We will avoid age bias by choosing only patients 
from 18 to 55 years. Neutral recruiting will take place at USZ Allergy Unit.  
6.3 Unblinding Procedures (Code break)  
Not applicable. The study is not blinded. 

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Page 21 of 39 
7. STUDY POPULATION  
7.1 Eligibility criteria  
Participants fulfilling all of the following inclusion criteria are eligible for the study: 
• 
Patients who have seasonal grass-pollen-induced rhinoconjunctivitis as confirmed by patient 
history and type-1-sensitization to grass-pollen in skin and/or serum 
• 
Patients that undergo pre-seasonal short-term scheme with Polvac™ SCIT at the USZ Allergy 
Unit in autumn and winter 2023 for treatment of allergic rhinoconjunctivitis. 
• 
Informed Consent as documented by signature. 
• 
Patients are between 18 and 55 years of age when they sign the informed consent. 
The presence of any one of the following exclusion criteria will disqualify the patient from participating 
the study: 
 
Allergy related exclusion criteria:  
• 
Known or suspected allergy to additives to the study product 
• 
Known intolerance or allergy to phenol  
• 
Planned depot steroid injection for treatment of allergic rhinoconjunctivitis  
• 
Uncontrolled asthma or severe asthma with post bronchodilator FEV1<70%, decided by the 
investigator  
• 
Pulmonary disease with post bronchodilator FEV1 < 70 % of predicted  
• 
Pulmonary disease, perennial or seasonal, with daily use of more than 800 microgram inhaled 
budesonide/day (or equivalent) 
• 
Treatment with omalizumab or other biologics for allergy, AD, urticaria or asthma.  
• 
Allergic reaction within the last 4 days or anaphylaxis within last month before planned ILIT or 
SCIT injection.  
Exclusion criteria based on serious comorbidities as judged by the recruiting physician:  
• 
Autoimmune or collagen disease  
• 
Disease or conditions rendering the treatment of anaphylactic reactions difficult (symptomatic 
coronary heart diseases, severe arterial hypertension, and treatment with beta-blockers)  
• 
Concomitant infection with fever or other signs/symptoms of an acute or chronic infection at 
treatment visit  
• 
Vaccination of any kind two weeks before or after the ILIT or SCIT injections.  
• 
Known cardiovascular disease, i.e., not even NYHA class I.  
• 
Use of ACE-blockers.  
• 
Recent or on-going hepatic or renal disease  
• 
Any malignant disease  
• 
Conspicuous lymph node size or morphology 
• 
Immuno- or chemotherapy during the last 15 years. 
• 
Increased bleeding tendency  
• 
Any other than study medication with an effect of interfering with the immune response  
• 
Chronic obstructive or restrictive lung disease  
• 
Skin diseases with barrier defect in the inguinal areas  
• 
Alcohol or drug abuse  
• 
Women who are pregnant and breast feeding 
7.2 Recruitment and screening  
We will recruit 30 patients directly at the USZ Allergy Unit. All 30 participants must have a type-1-
sensitisation to grass pollen allergens and be undergoing pre-seasonal short-term SCIT scheme with 
Polvac™ at the USZ Allergy Unit in winter 2023/2024.  
Patients will be invited to a screening visit 1 to 60 days before the intervention, where an extensive 
interview will be conducted and the informed consent will be signed by the patient and the medical 
investigator. The informed consent will be uploaded to the clinical information system (KISIM) and filed 

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Page 22 of 39 
into the trial master file (TMF). At the screening visit there will be a medical interview assessing all 
inclusion and exclusion criteria and a short clinical exam (auscultation of the lungs and the heart, blood 
pressure measurement). 
7.3 Assignment to study groups  
The 30 patients will be randomized to the two different equally-sized treatment groups (ILIT 400 U and 
SCIT 2000 U), with each 15 patients per group.  
7.4 Criteria for withdrawal / discontinuation of participants  
If a patient is seen to be ineligible for study intervention because of change in inclusion or exclusion 
criteria, the patient may be excluded from the trial at the discretion of the physician. The patient can at 
any time chose to withdraw the informed consent and thereby be released from the study. Replacement 
of a study participant is possible if the timeframe of 4-6 weeks between last dose of the short-term 
scheme and start of the additional allergen injection in the framework of the study can be complied. 
Replacement of a participant that has withdrawn consent is possible within the time frame of the study. 
8. STUDY INTERVENTION  
8.1 Identity of Investigational Products 
Four weeks after patients have received the full cycle of SCIT with Polvac™ Grass+Rye as a part of 
standard care treatment, they will be randomized to receive one additional treatment.  
All study participants will receive again the same allergen extract (Polvac™ Grass+Rye). Depending on 
the group allocation, they will receive the allergen by the subcutaneous (SCIT) or by intralymphatic (ILIT) 
injections. 
The following Polvac doses are used: 
• 
SCIT (n=15): 0.5 ml Polvac (2000 U). 
• 
ILIT (n=15): 0.1 ml Polvac (400 U). 
8.1.1 
Experimental Intervention 
The treatment medication (Polvac Grass+Rye) will be prepared aseptically as described (Appendix 
001). The medication that is stored with the pharma team GCP lab in a temperature-logged fridge (2-
8 °C) is placed at room temperature 2 hours prior to use. The medical investigator will order the drug 
from the pharma team to be provided on the day of treatment or on the day before treatment if treatment 
will take place early next morning (before 09:00 a.m.). Table 2 summarizes the characteristics of the 
Polvac Grass+Rye preparation used in standard subcutaneous (SCIT) administration and the 
experimental intralymphatic (ILIT) administration. 
Table 2. Characterisation of Polvac Grass+Rye preparation used in standard subcutaneous (SCIT) administration 
and the experimental intralymphatic (ILIT) administration. 
 
Polvac Grass+Rye  
SCIT 
Polvac Grass+Rye  
ILIT  
 
Route of injection 
subcutaneous 
intralymphatic 
 
Initial U/ml 
4000 U/ml 
4000 U/ml 
 
Dilution 
none 
none 
 
Dose volume / injection 
0.5 ml 
0.1 ml 
 
Phl p 5 / injection 
900 ng 
180 ng 
 
U / injection 
2000 
400 
 
 
To prepare Polvac™ for SCIT (2000 U), the 1 ml medication vial (4000 U/ml) is shaken well for 10 
seconds. The head of the vial is disinfected and 0.5 ml of the medication is drawn into a 1 ml syringe 
with 23G hypodermic needle assuring no air in the syringe or needle. The medication is used no later 
than 4 hours after the preparation. Replica of the SCIT dose or of the ILIT high dose (see below) are 
made if more than one injection is to be performed. The remaining medication is discarded. The time 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 23 of 39 
point for preparation is documented in the CRF. 
To prepare Polvac™ for ILIT (400 U), the 1 ml medication vial (4000 U/ml) is shaken well for 10 seconds. 
The head of the vial is disinfected and 0.1 ml of the medication is drawn into a 1 ml syringe with 19G 
hypodermic needle. The medication is used no later than 4 hours after the preparation. Before injection, 
the needle is changed to a 23G hypodermic needle, assuring no air in the syringe or needle. Replica of 
the dose are made if more than one ILIT is to be performed, and the remaining medication is discarded. 
The time point for preparation is documented in the drug accountability log (Appendix 004). 
Commercially available Polvac™ will be purchased from Bencard AG (Greifensee, Switzerland) and 
kept in a temperature-logged fridge (2-8 °C).  
After preparation of the syringes, these will be labelled with the study name (ILIT-FNA-2023), patient 
number, and the expiry time point (e.g., “ILIT-FNA-2023, #21, Exp 21.12.23, 10:30h”).  
8.1.2 
Control Intervention (standard/routine/comparator treatment)  
No control intervention is used in this study.  
8.1.3 
Packaging, Labelling and Supply (re-supply)  
All medication is stored in a temperature-logged fridge with the pharma team GCP lab at the Department 
of Dermatology.  
The medical investigator will request needed medication from the pharma team at the Department of 
Dermatology.  
The treatment medication will be prepared aseptically under laminar flow between 30 minutes and 4 
hours before use. After preparation, the medication is discarded or it is stored in a temperature-logged 
fridge with the pharma team at the Department of Dermatology.  
The IMP Polvac has market authorization (MA) and has been released for the market. However, as 
the IMP Polvac will be administrated in different doses for the study, a simplified Investigator Medicinal 
Product Dossier (sIMPD) is provided (Appendix 003). The drug substance remains unchanged. 
8.1.4 
Storage Conditions  
Polvac will be kept in a temperature-logged fridge (2-8 °C) in the Department of Dermatology. The 
medication is collected and brought to room temperature at least 30 minutes and not longer than 4 hours 
before administration. 
The time points for removal of medication from fridge and the administration time are documented in a 
drug accountability log (one for all patients). 
8.2 Administration of experimental and control interventions  
8.2.1 
Experimental Intervention  
8.2.1.1 
Allergen Injection 
The immunotherapy is performed by an experienced physician at the USZ Circle Allergy Unit 
(Department of Dermatology). Both SCIT and ILIT injections are performed using 1 ml syringes with 
23G needles and for both SCIT and ILIT, inguinal lymph nodes are identified using ultrasound guidance. 
For SCIT, a volume of 0.5 ml undiluted Polvac is injected subcutaneously ca. 1 cm from a lymph node 
(Fig. 3). For ILIT a volume of 0.1 ml undiluted Polvac is injected directly into a lymph node. The lymph 
nodes are tagged by marking the site of injection, ultrasound position and video recording of injection 
with the ultrasound machine. 

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Figure 3: By ILIT administration, the study drug is injected into an inguinal lymph node under ultrasound guidance 
(top image), which allows targeting of the needle to the lymph node (lower left). In the current study, the study 
drug, Polvac Grass+Rye, is injected into an inguinal lymph node (ILIT) or it is injected subcutaneously ca. 1 
cm from an inguinal lymph node (SCIT) is illustrated in the scheme (lower right). Two, six or 24 hours after 
injection, a fine-needle aspiration (FNA) of the treated (LIT) or involved (SCIT) LN is performed. As baseline 
control, the contralateral LN is also aspirated (green dot).  
 
8.2.1.2 
Fine needle aspiration (FNA) 
The FNA is minimally invasive and an alternative to more invasive methods such as incisional or 
excisional biopsy for sampling of lymph node tissue (Fig. 3). Just recently, we used FNA in a similar 
way on sentinel lymph nodes in skin cancer patients (44). 
An experienced investigator will perform the FNA. The skin above the lymph node, will be disinfected 
and a sterile needle (23-25g), attached to the 5 ml syringe, fixed in a FNA gun, will be inserted into the 
lymph node cortex under colour Doppler ultrasound guidance while avoiding important tissues and 
organs, such as large blood vessels and nerves. The syringe plunger is drawn to set the seal at a 2 ml 
scale in order to maintain negative pressure in the syringe. After rotating and aspirating the syringe 5 
times, the needle is rotated to aspirate in a fan shape way at different trajectories in the lymph node. 
The needle is withdrawn when ca. 0.1 ml tissue is present in the syringe. The puncture site is covered 
with sterile gauze and pressure is applied with appropriate force for 10 min. Patients are observed for 
30 min of observation and released when no adverse reaction.  
The FNA sample will be rinsed with dimethyl sulfoxide (DMSO)-containing live cell freezing medium 
(10% DMSO and 90% fetal bovine serum (FBS)), put into a cryotube and frozen in -80°C. 

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8.2.1.3 
Blood collection and preservation of cells and blood serum 
First venous blood collection is performed at baseline before the immunotherapy. Second blood 
collection is performed 2, 6 or 24 hours post injection. Third and fourth blood collection are performed 7 
and 28 days after immunotherapy. Each time, 25 ml venous blood is collected for preparation of PBMCs 
and 5 ml blood is collected for preparation of serum. Both samples are processed by routine in-house 
methods within 2 hours for preparation of serum and cells. The produced serum is aliquoted in 1 ml 
samples and frozen at – 20 °C for later analysis. Whole blood is centrifuged on Ficoll and the PBMCs 
isolated and washed in PBS before re-suspended in DMSO-containing live cell freezing medium. 1 ml 
aliquots of the cells are slow-frozen in cryotubes and finally stored at -80 °C before further analysis.  
8.2.2 
Control Intervention 
No control intervention is used in this pilot study.  
8.3 Dose modifications  
We already have experiences with dosing Polvac from an ongoing study using Polvac at our 
department (BASEC Number: 2021-02301) and with off-label use of up-dosing Polvac. The ILIT doses 
used in the trial described in the current protocol is based on the experience from the past trial (133 U) 
and from off-label treatment of four patients in February to April 2023 (133 U, 200 U and 400 U). 
We inject the study dose after patients finished a full cycle of pre-seasonal short-term SCIT scheme with 
Polvac™ (the patients have therefore reached and tolerated the maximal maintenance dose of 2000 U). 
The maximal dose in the current study will not exceed the maintenance dose for SCIT (2000 U) and will 
be much lower for ILIT (400 U ) and should therefore be well tolerated by the study participants.  
8.4 Compliance with study intervention  
Monitoring of subject compliance will not be done. We only include patients who have finished pre-
seasonal SCIT with six injections at the USZ Allergy Unit. Patients who opt for SCIT are usually highly 
motivated because their burden of allergy-suffering is high. The effort for the patient associated with our 
study is relatively modest. The five personal visits include 1) screening visit (which can be combined 
with a SCIT injection already planned), 2) allergen injection, 3) FNA and blood samplings 2-24 hours 
post injection, 4) follow-up after 7 days, and 5) follow-up after 28 days. We expect that the patients will 
be motivated for the study, because they potentially will benefit from this one additional injection and 
might also benefit from future ILIT applications (such as off-label interventions).  
8.5 Data Collection and Follow-up for withdrawn participants  
In accordance with the Declaration of Helsinki, the investigator must explain to the patient that they have 
the right to withdraw from the study at any time, and that this will in no way prejudice their future 
treatment. We are required by law to report on adverse events and other side effects; this law takes 
precedence over the GDPR as it concerns public safety. Even if a patient withdraws from the project, 
we will have to keep safety data after treatment. However, all other clinical data that do not relate to 
safety of the applied treatment, will be deleted at a written request of a patient. A subject that 
discontinues will always be asked about the reason(s) for discontinuation and the presence of any 
adverse events. The timing and reason for any kind of withdrawal will be recorded in the patient medical 
file (KISIM) and deposited in the TMF. The sponsor should inform Swissmedic and IEC within 24 hours 
after decision of a treated patient to discontinue the study and also the consequences for the study 
persons as well as for the continued investigations will be declared.  
Intervention (SCIT or ILIT) within the study is not necessary for the routine therapy for the patients, 
therefore, the study intervention don’t need to be repeated in case of a withdrawal. 
8.6 Trial specific preventive measures 
All the preventive measures described below will be recorded in the CRF. 
Prior to the allergen injection, a pregnancy testing (if applicable) will be performed.   
The patients will receive an emergency set (“Allergie Notfallset”) to take home in case of systemic side 
effects (Appendix 003). The emergency set consists of two tablets of Xyzal® 5 mg (Levocetirizine 
dihydrochloride) and two tablets of Spiricort® 50 mg (Prednisolone). Patients will be given an instruction 
about when and how to use the emergency set. Fenistil gel 1% will be given for local allergic reactions 
restricted to the site of injection. 
After ILIT treatment the patients have to stay one hour at the allergy unit for observation. 

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During the study (from allergen injection to blood samplings after 28 d) patients cannot receive another 
allergen injection. To avoid allergen exposure from the environment, the study allergen is seasonal, and 
therefore not in the environment during the whole study period.  
8.7 Concomitant Interventions (treatments)  
Besides the allergen administration (via ILIT or SCIT), the FNA (2x) and blood collection (4x) a 
pregnancy test, 2x peak flow measurement and a physical examination will be performed. 
8.8 Study Drug Accountability  
Commercially available Polvac™ will be purchased from Bencard AG (Switzerland). The study drug will 
be kept in a temperature-logged fridge (2-8 °C) with the pharma team at the Department of Dermatology. 
The pharma team will keep a log for received and used study medication. 
The medical investigator will order drug from the pharma team, which will log study drug provision with 
date and time point (DD.MM.YYYY, HH:MM h). The time point for preparation of study drug is 
documented in the log with the pharma team and in the CRF (ILIT 400U and for SCIT 2000 U; directly 
syringed by the medical investigator).  
8.9 Return or Destruction of Study Drug 
At the end of the study, rest doses of Polvac are kept in the research lab in Wagistrasse 18 for use in 
non-clinical research. 
9. STUDY ASSESSMENTS  
9.1 Study flow chart 
The study flow chart is illustrated in Figure 4. 
9.2 Assessments of outcomes  
9.2.1 
Assessment of primary outcome  
FNA material of lymph node tissue at baseline and 6 hours after allergen injection (via ILIT or SCIT) will 
be evaluated for changes in lymph node cell populations and cell culture expression patterns. 
Immunological changes (analysis of cell populations and cell cultures) in peripheral blood at baseline, 
after 2, 6 and 24 hours and at 7 and 28 days after allergen injection (via ILIT or SCIT). 
9.2.1.1 
Analysis of allergen-specific antibodies in serum by ImmunoCAP 
The concentrations of IgE, IgG and IgG4 against grass-pollen allergens as well as tryptase are 
determined in serum using the ImmunoCAP system (ThermoFisher Scientific, Phadia, Waltham, US-
MA) according to the manufacturer’s instructions. This is a routine diagnostic method in our department.  
9.2.1.2 
Analysis of innate cell reactions in serum by antibody arrays 
PBMCs cryotubes are thawed and immediately washed in pre-warmed culture medium supplemented 
with calf serum, glutamate and antimicrobial agents before restimulated with allergen in vitro. Firstly, the 
cells are cultured for up to 5 days in the presence of grass pollen allergens and the supernatants are 
analyzed for cytokines, e.g. IL-2, IL-4, IL-5, IFN-gamma, IL-10, and TGF-beta. The cytokines are 
measured by ELISA using a TECAN plate reader. Secondly, and based on the ELISA results, the cells 
are cultured with allergens and transferred to multiscreen HTS membrane plates for detection of 
cytokine-secreting cells by ELISPOT using a AID EliSpot/FluoroSpot Reader.  
9.2.1.3 
Analysis of T-cell activation by flow cytometry 
PBMCs are also analyzed by flow cytometry after allergen-restimulation in vitro. Briefly, the cells are 
typically cultured 18-20h in the presence of grass pollen allergens, the last 4-6 hours of the incubation, 
the compound brefeldin A is added to prevent the golgii apparatus to release newly produced proteins. 
Then, the cell as stained for with fluorescent antibodies to detect lymphocytes, lymphocyte activation 
surface proteins. The cell are then fixed, permeabilised and stained to detect intracellular transcription 
factors such as Foxp3, STAT-1 and STAT-3 and STAT-6 as well as intracellular cytokines such as IL-4, 
IL-10 and IFN-gamma. The exact cytokines may depend on the result from the above-mentioned ELISA 

ILIT-FNA-2023, Ver2, 06/06/2023 
 
Page 27 of 39 
studies. The cells are acquired on a BD LSR-Fortessa Cell analyzer and the data analyzed using FlowJo 
LCC software.  
 
Figure 4. Patient flow chart illustrating study visits with timing, interventions, and assessments for patients treated 
by ILIT or SCIT for grass pollen-mediated allergic rhinoconjunctivitis.  

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9.2.1.4 
Multiparameter cellular analysis by mass cytometry (CyTOF) 
PBMCs as well as lymph node cells are also analyzed by mass spectrum cytometry (45, 46) Again, 
cryotubes are thawed and cell immediately washed with complete medium. The cells are incubated with 
a viability dye washed extensively, and stained for a wide panel of extracellular antigens that will enable 
us to detect the phenotype of all possible leucocytes. After fixation and permeabilisation, the cells are 
patient-wise barcoded using the commercial Barcoding Kit (Fluidigm) and pooled before staining for 
intracellular antigens. Cell DNA is then stained using Cell-ID™ Intercalator-Ir (Fluidigm) overnight at 
4°C, frozen at -80°C using a cryopreservation box, thawed, supplemented with Four Element Calibration 
Beads (Fluidigm) and acquired on a Helios™ mass cytometer. Files are concatenated, normalized, and 
debarcoded using the CyTOF software v6.7.1014. The data are analyzed by descriptive statistics as 
well as multivariant test methods with dimension reduction, e.g. PCA, t-SNA and UMAP (47). 
9.2.2 
Assessment of secondary outcomes 
Differences in immunological response after injection of ILIT 400 U and SCIT 2000 U via two routes to 
demonstrate a possible route dependency. 
9.2.3 
Assessment of other outcomes of interest 
None. 
9.2.4 
Assessment of safety outcomes 
9.2.4.1 
Adverse events  
For reporting of adverse event information see 10.1.2.  
To minimise the risk for AE the following precautions are carried out: 
• 
The patients will receive an emergency set (“Allergie Notfallset”) to take home in case of 
allergic side effects. The emergency set consists of two tablets of Xyzal® 5 mg and two 
tablets of Spiricort® 50 mg. Patients will be advised on when and how to use the emergency 
set. 
• 
The patients will receive Fenistil gel 1% to use in case of local allergic side effects 
• 
After the allergen injection the patients have to stay one hour at the allergy unit for observation 
of systemic allergic reactions as well as allergic skin reaction oat the site of injection.  
• 
The patients will be advised to call the medical investigators upon any delayed AEs or 
otherwise medical emergency if systemic allergic reactions (e.g. asthma). 
9.2.4.2 
Laboratory parameters 
Before the allergen injection a pregnancy testing (if applicable) is performed. Pregnant participants are 
excluded from the clinical study. 
9.2.4.3 
Vital signs 
A physical examination is done in a semi-structured way; several items are checked on a list in the CRF, 
and details can be entered if required.  
The following vital signs will be assessed (e.g. heartbeat, lung auscultation and blood pressure). 
9.2.5 
Assessments in participants who prematurely stop the study 
Most allergic events are resolved within a week, and we do not expect to have to follow patients 
for longer. If an AE is registered in a patients CRF this will be addressed by attending staff at the next 
visit. Patients will be able to contact staff by e-mail during the trial. If a patient should experience allergic 
symptoms severe enough to warrant hospitalization this will be recorded in the CRF.  
Our injection in the framework of the study is not necessary for the routine therapy for the patients, 
therefore they don’t need to be repeated in case of a withdrawal. 
9.3 Procedures at each visit 
9.3.1 
Visit 1: Screening visit (20 minutes) 
Completed until 02.02.2024 (-29 to 1 days before Visit 2) 
• 
Interview with medical investigator 
o 
Medical history 
o 
Check of Inclusion/Exclusion criteria 

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o 
Physical examination 
• 
Reading and signing of informed consent by participant and medical investigator 
9.3.2 
Visit 2 (120 minutes) 
Completed until 03.02.2024 (at least 1 day after screening visit) 
• 
Pregnancy testing (if applicable) 
• 
Peak Flow before and 40 min after injection 
• 
Venous blood collection before injection (5 ml serum and 25 ml EDTA) 
• 
Injection of Polvac Grass+Rye (SCIT 2000U or ILIT 400 U) 
• 
Immediately after injection 
o 
1 h observation at the allergy unit of the University Hospital Zurich 
o 
Dispense of rescue medications 
• 
2, 6 or 24 hours after allergen injection the following parameters are obtained: 
o 
FNA of ILIT-treated inguinal lymph node or lymph node adjacent to SCIT injection 
o 
FNA of non-involved inguinal lymph node on the opposite side, which had not been 
treated (baseline measurement) 
o 
Venous blood collection (5 ml serum and 25 ml EDTA) 
9.3.3 
Visit 3 (30 minutes) 
Completed until 04.02.2024 (2, 6 or 24 hours after SCIT/ILIT)) 
• 
Venous blood collection before injection (5 ml serum and 25 ml EDTA) 
• 
FNA of ILIT-treated inguinal lymph node or lymph node adjacent to SCIT injection 
• 
FNA of non-involved inguinal lymph node on the opposite side, which had not been treated 
(baseline measurement) 
9.3.4 
Visit 4 (10 minutes) 
Completed until 24.03.2024 (7 days after injection) 
• 
Venous blood collection (5 ml serum and 25 ml EDTA). 
9.3.5 
Visit 5 (20 minutes) 
Completed until 31.03.2024 (28 days after injection) 
• 
Venous blood collection (5 ml serum and 25 ml EDTA). 
• 
Study termination 
10. SAFETY  
10.1 Drug studies 
During the entire duration of the study, all adverse events (AE) and all serious adverse events (SAEs) 
are collected, fully investigated, and documented in source documents and case report forms (CRF).  
10.1.1 Definition and assessment of (serious) AEs and other safety related events 
An AE is any untoward medical occurrence in a patient, or a clinical investigation participant 
administered a pharmaceutical product and which does not necessarily have a causal relationship with 
the study procedure. An AE can therefore be any unfavourable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal 
(investigational) product, whether or not related to the medicinal (investigational) product. [ICH E6 1.2] 
A SAE is classified as any untoward medical occurrence that: 
• 
results in death, 
• 
is life-threatening, 
• 
requires in-patient hospitalization or prolongation of existing hospitalisation, 
• 
results in persistent or significant disability/incapacity, or 
• 
is a congenital anomaly/birth defect. 
In addition, important medical events that may not be immediately life-threatening or result in death, or 
require hospitalisation, but may jeopardise the patient or may require intervention to prevent one of the 
other outcomes listed above should also usually be considered serious. [ICH E2A] 

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Examples of such events are intensive treatment in an emergency room or at home for allergic 
bronchospasm, blood dyscrasias or convulsions that do not result in hospitalisation, or development of 
drug dependency or drug abuse.  
SAEs should be followed until resolution or stabilisation. Participants with ongoing SAEs at study 
termination (including safety visit) will be further followed up until recovery or until stabilisation of the 
disease after termination.  
10.1.1.1 Assessment of Causality 
Investigators and Sponsor make a causality assessment of the event to the study drug, based on the 
criteria listed in the ICH E2A guidelines: 
Relationship 
Description 
Unrelated 
This category is applicable to those AEs which, after careful medical consideration 
at the time of evaluation, are judged to be clearly and incontrovertibly due to 
extraneous causes (disease, environment, etc) and do not meet the criteria for 
study medication relationship listed under remote, possible, or probable. 
Unlikely 
In general, this category is applicable to those AEs which, after careful medical 
consideration at the time they are evaluated, are judged to be remotely related to 
the test study medication. An AE may be considered remote if, or when, for 
example: (i) it does not follow a reasonable temporal sequence from 
administration; (ii) it could readily have been produced by the patient’s clinical 
state, environmental or toxic factors, or other modes of therapy administered to 
the patient; (iii) it does not follow a known response pattern to the suspected study 
medication. 
Plausible 
(Must have the first 2 criteria): This category is applicable to those AEs which, 
after careful medical consideration at the time they are evaluated, the connection 
with the test study medication administration appears unlikely but cannot be ruled 
out with certainty. An AE may be considered plausible if, or when: (i) it follows a 
reasonable temporal sequence from administration; (ii) it could readily have been 
produced by the patient’s clinical state, environmental or toxic factors, or other 
modes of therapy administered to the patient; (iii) it follows a known response 
pattern to the suspected study medication. 
Probable 
(Must have the first 3 criteria): This category is applicable to those AEs, which, 
after careful medical consideration at the time they are evaluated, the connection 
with the test study medication administration appears to, with a high degree of 
certainty, be related to the test study medication. An AE may be considered 
probable if: (i) it follows a reasonable temporal sequence from administration; (ii) 
it could not be reasonably explained by the known characteristics of the patient’s 
clinical state, environmental or toxic factors, or other modes of therapy 
administered to the patient; (iii) it disappears or decreases on cessation or 
reduction in dose. There are important exceptions when an AE does not disappear 
upon discontinuation of the study medication, yet study medication-relatedness 
exists, e.g. bone marrow depression, fixed study medication eruptions, and 
tardive dyskinesias; (iv) it follows a known response pattern to the suspected 
study medication; (v) it reappears upon rechallenge. 
Not assessable 
When not possible to assign the event to any of the criteria mentioned above. 
10.1.1.2 Unexpected Adverse Drug Reaction 
An “unexpected” adverse drug reaction is an adverse reaction, the nature or severity of which is not 
consistent with the applicable product information (e.g. Investigator’s Brochure for drugs that are not yet 
approved and Product Information for approved drugs, respectively). [ICH E2A] 
10.1.1.3 Suspected Unexpected Serious Adverse Reactions (SUSARs) 
The Principle-Investigator evaluates any SAE that has been reported regarding seriousness, causality, 
and expectedness. If the event is related to the investigational product and is both serious and 
unexpected, it is classified as a SUSAR.  
10.1.1.4 Assessment of Severity 
For all AEs, serious as well as non-serious, the investigator must make an assessment of severity: 

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• 
Mild: awareness of sign or symptom, but easily tolerated and cause no interference with 
daily activities. 
• 
Moderate: discomfort enough to cause interference with daily activities. 
• 
Severe: inability to perform normal daily activities. 
10.1.2 Reporting of SAE and other safety related events  
10.1.2.1 Reporting of SAEs 
The study doctor or the principal investigator will report all SAEs immediately and within a maximum of 
24 hours to the Sponsor of the study (via notification by phone/email) The Sponsor will re-evaluate the 
SAE and return the form to the site. 
The study doctor or the PI will report SAEs resulting in death to the Ethics Committee via BASEC 
within 7 days. The sponsor-representative will inform all SAEs to Swissmedic within 7 days of the 
event.  
10.1.2.2 Reporting of SUSARs 
A SUSAR will be reported by the principal investigator to the Ethics Committee via BASEC and to 
Swissmedic by the Sponsor within 7 days, if the event is fatal, or within 15 days (all other events). 
10.1.2.3 Reporting of immediate safety and protective measures 
All suspected new risks and relevant new aspects of known adverse reactions that require immediate 
safety-related measures, are reported by the study doctor to the Sponsor within 24 hours.  
The principal investigator will notify the Ethics Committee and the sponsor will notify the Swissmedic 
within 7 days if immediate safety and protective measures had to be taken during the trial conduct. The 
circumstances necessitating the safety-protective will also be reported.  
10.1.2.4 Reporting and handling of pregnancies 
Before the allergen injection a pregnancy testing (if applicable) is performed. Pregnant participants are 
excluded from the clinical study. All interventions except one blood sampling (28 days) are done on the 
day of pregnancy testing. 
Any pregnancy within 28 days after allergen injection will be reported to the Sponsor within 24 hours. 
The course and outcome of the pregnancy should be followed up carefully, and any abnormal outcome 
regarding the mother or the child should be documented and reported. 
10.1.2.5 Periodic reporting of safety 
The ASR / Development safety update report (DSUR) is written in compliance to ICH Harmonised 
Guideline E2F for commercial and non-commercial sponsor.  
An ASR / DSUR is submitted once a year to the IEC via Investigator and to Swissmedic via Sponsor. 
The start date for the ASR/DSUR is the date the sponsor’s first authorisation to conduct a clinical trial. 
The ASR / DSUR is submitted to the IEC and to Swissmedic throughout the duration of the clinical trial, 
and the last ASR / DSUR submission will cover the Last Patient Last Visit.  
The sponsor will submit an annual report of SUSAR and SAE to Swissmedic as well as to the 
Pharmacovigilance department of the medicine supplier Allergy Therapeutics.  
10.1.3 Follow up of AEs and SAEs 
Most allergic events are resolved within a week, and we do not expect to have to follow patients 
for longer. If an adverse event is registered in a patients CRF this will be addressed by attending staff 
at the next visit. Patients will be able to contact staff by e-mail during the trial. If a patient should 
experience allergic symptoms severe enough to warrant hospitalization this will be recorded in the CRF.  
10.2 Assessment, notification and reporting on the use of radiation sources 
Not applicable. 

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11. STATISTICAL METHODS  
11.1 Hypothesis 
This study is a pilot study which goal is to describe the effect of ILIT on the immune cell composition 
and immune cell function in lymph nodes and in blood. We will compare this with the effect SCIT on the 
same, but not to confirm a hypothesis of a certain difference.  
We will use a wide screening with several methods to describe the immune responses. 
11.2 Determination of Sample Size  
Not applicable for a pilot study.  
11.3 Statistical criteria of termination of trial  
There is no statistical criteria for stopping the study.  
11.4 Planned Analyses  
The investigators and the appointed statistician will be responsible for the statistical analysis of the 
collected data. The statistical software used will be Graph Pad Prism®, SPSS®, or open-source 
software “R”.  
We will apply descriptive statistics to describe frequencies, proportions and numbers for the various 
analysis to be performed. The data will be described with an average (mean or median) and with 
variances (confidential intervals or standard deviations or standard errors) depending on the type of 
data. 
Data will typically be illustrated with scatter plots or with box plots with 90 and 10% whiskers. For 
multiparametric analysis, the results from different tests will transformed/normalized (e.g. z-score 
normalization) to generate heat maps, and for the hierarchical clustering of treatments and phenotypes, 
we will apply The R Project with Euclidean metrics.  
For comparative testing of the different treatment groups (SCIT versus ILIT), ANOVA is applied for 
continuous parametric data that fulfil the criteria for ANOVA. Kruskal-Wallis analysis are applied for non-
parametric ranked data.  
For changes over time within patients, dependent (paired) t-test may be used. No sex/gender difference 
analysis is planned. 
11.5 Handling of missing data and drop-outs  
There will be no imputation of missing data. We will not use false data. There will not be unused data. 
We will strive to publish all data. 
12. QUALITY ASSURANCE AND CONTROL  
The Sponsor is responsible for implementing and maintaining quality assurance and quality control 
systems with written SOPs and Working Instructions. The Sponsor is responsible for proper training of 
all involved study personnel. 
12.1 Data handling and record keeping / archiving  
12.1.1 Case Report Forms  
Study data is recorded in Case Report Forms on paper (pCRF). Data entry will be done by the 
investigators who are responsible for the correctness of the data. If a person is enrolled to participate in 
the study, the personal record will be encoded, and the person given a randomization number. For each 
enrolled study participant a pCRF is maintained. All patient information and test results are recorded in 
the CRF. Only the participant number and the year of birth will be entered in the pCRF. The encryption 
process used for the pCRF are in accordance with Swiss guidelines for encryption. Hence, only the 
investigators and authorized persons have access to data.  
The pCRFs are kept current to reflect subject status at each phase during the course of study. The 
investigators at the site are responsible for pCRF entries and have to sign their entries with initials and 
date of the entry according the specifications of the pCRF. 

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The informed consent will be obtained in wet ink during the screening visit and then uploaded to the 
clinical information system (KISIM) and filed into the TMF for recordkeeping. 
Data transfer in excel tables for statistical evaluation is checked by double data entry. A printout of the 
excel sheet is added to the study documents and every data change in the electronic data should be 
assigned a new printout.  
The randomization list will be stored electronically as a series of files labelled with the participant number 
that contains information on the randomization, with password-protected, logged access and an audit 
trail that will be stored electronically for 15 years. As this is an open-label trial, any physician can access 
the randomization list, and request details of the allocation of an individual patient. 
12.1.2 Specification of source documents  
All data will be registered in a series of pCRFs including patient questionnaires and laboratory analysis 
that will be considered as source data. The source documents include the electronic medical records 
on site, as well as the study-specific documents and data collected during the visits, which are recorded 
directly via questionnaires or by the study-specific tests. The relevant source documents from the 
electronic medical records are printed out, signed by the investigator and collected in a separate folder. 
This folder is stored in a lockable cabinet to which only the study team has access. 
Data from the pCRF are filed in to the TMF for recordkeeping and additionally in the clinical information 
system (KISIM). 
12.1.3 Record keeping / archiving  
All study documents (essential documents and site documents) are archived in the Department of 
Dermatology of the University Hospital Zurich for a minimum of 10 years after study termination or 
premature termination of the clinical trial.  
12.2 Data management  
The project site will retain all essential documents according to national legal and regulatory 
requirements, including source data and CRF. Source data are all information in original records and 
certified copies of original records of clinical findings, observations, or other activities necessary for the 
reconstruction and evaluation of this project. All essential documents are stored until 10 years after the 
termination of the project, during which time the project leader guarantees access and availability of the 
data at any time. 
12.3 Monitoring  
The trial monitoring in Zurich is done by the Clinical Trials Center, USZ. 
12.4 Audits and Inspections  
The investigators and sponsor allow direct access to source documents for monitoring, audit and 
inspection by GCP units, health authorities, ethics committees, data authorities and other medical 
autothrities. All involved parties must keep the participant data strictly confidential. 
12.5 Confidentiality, Data Protection  
Health-related personal data captured during this project from participants are strictly confidential and 
disclosure to third parties is prohibited. Coding will safeguard participants' confidentiality.  
The electronic case report forms (CRFs) will be entered into an excel-based database. Confidentiality 
will be ensured by entering only coded data into the analysis set. The coded data will be password-
protected and accessible to the sponsor, the medical investigators and the project researchers. The 
statistician will obtain copies of the Excel database after the data entry has been completed. Coded 
research data are not considered part of the essential documents and will be stored on a central data 
server at the USZ with restricted access: \\fs-group\der_forschungen\DER_FL_Immunotherapie  
The code for data generated at USZ is kept with the sponsor. 
12.6 Storage of biological material and related health data  
Data from this trial will be stored for 10 years in electronic form after completion of the trial. The collected 
blood and FNA samples are to be used and analysed exclusively in the current study. No material or 
data transfer to other or future projects are foreseen. The collected blood samples and FNA in this study 

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is not identified by participant name but by a unique participant number and is appropriately stored in a 
restricted area only accessible to the authorized personnel.  
13. PUBLICATION AND DISSEMINATION POLICY  
A clinical study report will be prepared covering clinical aspects and summarizing all findings of the 
clinical study. The content has to be treated as strictly confidential. The information gathered in this 
study is considered confidential and is the property of the sponsor. Within 12 months of completion of 
the trial, trial results will be entered into the international clinical trials registry (SNCTP and 
clinicaltrials.gov). 
We aim to publish the results of this study in a peer reviewed journal, irrespective of the outcome.  
There is a publication delay of up to 3 months while partners or interested parties may evaluate the 
commercial potential of results from this clinical trial. Authorship will follow Vancouver rules.  Partners 
in the project will be authors on the publication; contributors will be mentioned under acknowledgements 
(there is a group of researchers that is credited without being authors. that is where they may be).  
We confirm if gender effects are observed, they will be published in the final study report. If none are 
observed, this will be published as well. 
14. FUNDING AND SUPPORT  
14.1 Funding  
The Department of Dermatology of the University Hospital of Zurich (Professor Thomas Kündig) will 
support the study with CHF ca. CHF 30’000 if required through University funds. The sponsor will 
guarantee for ca. 50’000 through overhead from various project-unrelated contracted research with 
biotech.  
The Truus und Gerrit van Riemsdijk Stifung, Vaduz will support the study with 20’000 CHF. 
We submitted a project-grant application to EMDO Stiftung (CHF 50’000), and we are planning an 
application to Theodor und Ida Herzog Egli Stiftung for the same amount (submission deadline 
30.4.2023).  
14.2 Other Support  
14.2.1 Cooperation with other institutes 
All clinical and experimental infrastructures required to conduct the study are available in Department 
of Dermatology, USZ/UZH and at the associated lab at SIAF, Davos.  
We will collaborate with Prof. Dr. med. Peter Schmid-Grendelmeier and his team the Allergy Unit USZ 
CIRCLE for screening, recruitment, treatment and monitoring of patients included in the study. Such a 
collaboration is already existing as part of already running randomized double-blind placebo controlled 
trial on the efficacy of grass pollen ILIT. Hence, all personnel and infrastructure required in the current 
project, is already established and available. 
We will also collaborate closely with the UZH-Institute SIAF, Davos, for the analysis of blood and lymph 
node cells by mass cytometry (CyTOF). Since four years, SIAF as being building of expertise on CyTOF 
through Dr. Reihane Ziadlou and Prof Dr. med. Charlotte Brüggen.  
Finally, we are and will consult Prof. Hans Jürgen Hoffmann from the Department of Respiratory 
Diseases and Allergy at the University of Aarhus, Denmark. Prof. Hoffmann is worldwide perhaps 
recognized as the most experienced researcher on ILIT. We have discussed the trial design for the 
current study with Prof. Hoffmann. 
15. INSURANCE  
Insurance will be provided by the Sponsor /  
Patients will be covered by an insurance (Pauschalvertrag) for clinical trials, issued by Zurich 
Versicherungs-Gesellschaft AG. The policy number is 14.970.888.  

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17. APPENDICES 
001. Product description Polvac™ 
 
 
002. SmPC PolvacTM  
 
003. Product description of rescue medication  
1. Product description “Allergie-Notfallset” 
2. Product description “Fenistil Gel 1%”  
 
004. Drug Accountability Log 
 
005. CRF and questionnaire for participants 
 
006. Patient questionnaires in German (days 0-3 post ILIT) 
 
 
 

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