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Reach α-synuclein-dependent neurodegeneration: clinical development of therapeutic AFFITOPE vaccines for Parkinson’s disease and multisystem atrophy

Final Report Summary - SYMPATH (Reach α-synuclein-dependent neurodegeneration: clinical development of therapeutic AFFITOPE vaccines for Parkinson’s disease and multisystem atrophy)

Executive Summary:
The SYMPATH project was initiated to address two synucleinopathies for which no causal therapy exists, namely Parkinson’s disease (PD) and Multiple System Atrophy (MSA). Ultimately, both lead to patient disability and death, which along with the number of affected individuals (PD) and the age of disease onset (MSA, PD), define their high medical need status. Neuropathological hallmark lesions of these synucleinopathies are the so-called Lewy bodies in PD and glial cytoplasmic inclusions in MSA respectively, which primarily contain α-synuclein (aSyn). Multiple lines of evidence strongly suggest that the aggregated (toxic) form of aSyn plays a central role in the pathogenesis of synucleinopathies. It is therefore assumed that lowering the aggregated aSyn has the potential to modify the underlying pathophysiology of both PD and MSA.
The SYMPATH consortium successfully initiated an unprecedented clinical approach, termed TANDEM strategy to address the main aim of advancing the clinical development of therapeutic vaccine candidates (AFFITOPE®s PD01A and PD03A) targeting aSyn-driven neurodegenerative diseases, namely PD and MSA. This used the synergy resulting from applying two vaccine candidates in two complementary indications linked through their pathology. TANDEM PD/MSA capitalised on (i) excellent clinical research centres and their associated national/European networks, (ii) platform methods assessing aggregated aSyn as a candidate biomarker and (iii) preliminary clinical experience with PD01A, the first aSyn targeting vaccine ever tested in humans. Its core was formed by two phase I studies testing PD01A in MSA and PD03A in PD/MSA. Importantly, trial design (duration, endpoints, vaccine dose and schedule) was well positioned to ensure collection of initial biomarker data in parallel to the clinical results of the PD- and MSA trajectories.
Using this approach SYMPATH was able to 1) demonstrate the chemical stability of both vaccine candidates which is a prerequisite for clinical studies 2) demonstrate the safety and tolerability of both vaccine candidates in PD and MSA patients 3) establish and validate a broad range of assays to evaluate and monitor the immunogenicity and specificity of the AFFITOPE® technology 4) demonstrate a dose-dependent immune response against the PD03A vaccine and also the aSyn epitope in PD patients 5) demonstrate immunogenicity of both vaccine candidates in MSA patients 6) successfully adapt the sFIDA technology (surface-based fluorescence intensity distribution analysis) for quantitative analysis of aggregated α-Syn in human biological samples 7) found a spin-off company for the diagnostic biomarker assay 8) advance the exploitation strategy for both AFFITOPE® vaccines and the biomarker programme 9) create with FAHR-MIT a model for similar events in future.
Professional project management and dissemination significantly contributed to the successful collaboration of the SYMPATH partners, with 6 peer-reviewed publications and additional clinical trial results publications expected. Public awareness of SYMPATH was addressed with a website, press releases and a video. SYMPATH achieved its aim of addressing the general public and researchers through two significant events, namely the FAHR-MIT bike tour, which brought together patients, clinicians and policy makers, and the SYMPATH symposium, which presented SYMPATH results in the context of world-leading experts.
In summary, SYMPATH was highly successful and met essentially all objectives on schedule. SYMPATH brought together unique expertise and infrastructure with leading experts in clinical PD and MSA research as well as biomarker assay development to achieve world-class collaborative research. The socio-economic impact of this project cannot be overestimated. Independently of the success of the vaccine candidates in the clinical studies, medical practitioners could already benefit from the effective diagnostic methods developed in the short-mid-term.
Project Context and Objectives:
The SYMPATH project was initiated to address two synucleinopathies for which no causal therapy exists, namely Parkinson’s disease (PD) and Multiple System Atrophy (MSA). Ultimately, both lead to patient disability and death, which along with the number of affected individuals (PD) and the age of disease onset (MSA, PD), define their high medical need status.
PD is, behind Alzheimer’s disease, the second most common neurodegenerative disease of the elderly (approx. 1.2 Mio European patients). Until recently, PD was considered a motor disease characterised by akinesia/bradykinesia, rigidity and rest tremor; but its non-motor symptoms are now well established. Notably, some of the non-motor symptoms (loss of the sense of smell, constipation) start well before classical motor symptoms are established. PD is now seen as a systemic disease of the nervous system; however, this new interpretation is not yet reflected in the way PD is diagnosed. At present PD diagnosis is still based largely on the motor symptoms. Unfortunately, the early non-motor symptoms are not specific enough to establish a PD diagnosis. This would for example require biological markers. Despite various efforts, there are as yet no established biomarkers that would support clinicians in making their diagnosis or which would guide rational drug development. Candidate biomarkers include CSF markers, such as levels of aSyn species and imaging markers. However, to date there has only been limited assessment. In 2010, the Michael J. Fox Foundation launched the landmark study ‘Parkinson’s Progression Markers Initiative (PPMI), a comprehensive programme to define the natural disease course based on clinical and biological data. The study aims to integrate clinical and biomarker data into a new diagnostic algorithm allowing the earlier detection of disease with higher specificity.
Current PD treatments are based on the dopamine deficit, which is associated with the classical motor symptoms. Current dopamine replacement strategies include levodopa (the precursor of dopamine), dopamine receptor agonists, monoamine oxidase B (MAOB)- and catechol O-methyltransferase inhibitors. Even 50 years after its introduction, levodopa remains the cornerstone of PD therapy. All currently approved PD drugs are of symptomatic benefit only. Moreover, their long-term use is associated with side effects and complications. Thus, there are currently several areas of medical need. Firstly, we lack biomarkers that would allow a reliable and early diagnosis. Secondly, there is still no disease-modifying agent. Thirdly, measures to combat dyskinesias (movement disorders) induced by current treatments are limited. Finally, there are only a few therapeutic options for non-motor symptoms.
MSA is a rare neurodegenerative disease that rapidly progresses, leading on average to death within 10 years after disease onset. Diagnosis is established based on consensus criteria defining three categories: definite, probable and possible MSA, which while lacking sensitivity, are characterized by high specificity. The clinical course of MSA is also well established, with MSA-P (MSA subtype characterised by Parkinsonian features) typically progresses faster than PD. As is the case in PD, there is no compelling biomarker programme with established markers informing on diagnosis, prognosis or response to therapy. MSA therapy is scarce. Only Parkinsonism and dysautonomia (malfunction of the autonomic nervous system), but not cerebellar ataxia (damaged cerebellum) are amenable to symptomatically acting drugs. Of note, levodopa, which is used for Parkinsonism, is only of transient benefit in a third of MSA patients leaving the vast majority of them unresponsive in the long term. In conclusion, current management of MSA patients is based on empirical evidence and as with PD, a disease-modifying agent is urgently needed.
Neuropathological hallmark lesions of these synucleinopathies are the so-called Lewy bodies in PD and glial cytoplasmic inclusions in MSA respectively, which primarily contain α-synuclein (aSyn). aSyn is a presynaptic protein, that is normally soluble, widely expressed in central neurons and an essential component of synaptic transmission. Multiple lines of evidence strongly suggest that the aggregated (toxic) form of aSyn plays a central role in the pathogenesis of synucleinopathies. It is therefore assumed that lowering the aggregated aSyn has the potential to modify the underlying pathophysiology of both PD and MSA.
Active vaccination using the whole aSyn molecule has been reported and demonstrated to modify the disease course in an animal model. However, the approach could not be translated to the human setting due to the high risk of autoimmunity.
Therefore, AFFITOPE® vaccines were developed to overcome both humoral and cellular autoimmunity. This is achieved by two integral features 1) The AFFITOPEs® limited length precludes T cell activation and, thus, cellular autoimmunity 2) AFFITOPEs® physically differ from the structures they target, which allows control of the fine specificity of the ensuing Abs and, therefore, humoral autoimmunity. The technology with its safety components, has been successfully validated in Alzheimer’s disease, with no reported case of autoimmunity in >350 treated patients.
Applying the AFFITOME® technology to aSyn yielded several AFFITOPES®. AFFITOPEs® PD01A and PD03A are synthetically produced aSyn mimicking peptides of eight amino acids in length, including a cysteine, which is chemically conjugated to keyhole limpet hemocyanine (KLH) and formulated with Alhydrogel (Alum). Peptides were found by screening a peptide library with an antibody (Ab) specific for human aSyn. Further selection criteria included recognition of both monomeric- and aggregated aSyn but no cross-reaction with β-synuclein (bSyn), a close homologue to aSyn co-expressed at central synapses. The two adjuvant components (KLH and Alum) of PD01A and PD03A have a long history of safe human use.
In pre-clinical studies PD01A and PD03A were able to induce specific immune responses against full-length aSyn in several animal species but not to bSyn, proposed to exert neuroprotective functions. In both aSyn-based mouse models, vaccination with PD01 was found to be capable of altering the dynamics of aSyn oligomerization/aggregation and to modify the course of the disease. Pre-clinical evaluation of candidates PD01A and PD03A demonstrated them to reduce cerebral aSyn levels, prevent aSyn-driven neuropathology and improve functional deficits in several transgenic models. PD01A is already being tested in a phase I study in PD patients. It is the first PD immunotherapeutic ever tested in human beings. Preliminary results demonstrated a favourable safety profile. Currently, there are only a couple of other aSyn-specific Abs in mid-to-end stage preclinical development.
The OVERALL AIM of SYMPATH was therefore to advance the clinical development of the therapeutic vaccine candidates (AFFITOPE®s PD01A and PD03A) targeting aSyn-driven neurodegenerative diseases, namely PD and MSA.
The overall SYMPATH aim encompassed the following objectives:
• Initiate safety and efficacy (exploratory) testing of PD01A and PD03A within a Phase I study in MSA (Work Package 2, WP2)
• Initiate safety and efficacy (exploratory) testing of PD03A within a Phase I study in PD (WP3)
• Collect biomaterial and relevant data to generate a biobank (WP4)
• Develop assays for the assessment of target engagement based on biomaterial from Phase I studies (WP4)
• Develop an exploitation strategy to advance clinical development of a therapeutic aSyn vaccine beyond safety and initial efficacy testing (WP5)
To this end, SYMPATH implemented a collaborative project with funding by the European Commission’s Seventh Framework Programme (Grant Agreement no. 602999), organised into four scientific components or work packages (WP1, WP2, WP3 and WP4), one exploitation component (WP5), one dissemination component (WP6) and one management component (WP7).
The SYMPATH consortium successfully initiated an unprecedented clinical approach, termed TANDEM strategy. This used the synergy resulting from applying two vaccine candidates in two complementary indications linked through their pathology. TANDEM PD/MSA capitalised on (i) excellent clinical research centres and their associated national/European networks, (ii) platform methods assessing misfolded aSyn as a candidate biomarker and (iii) preliminary clinical experience with PD01A, the first aSyn targeting vaccine ever tested in humans. Its core was formed by two phase I studies testing PD01A in MSA and PD03A in PD/MSA. Importantly, trial design (duration, endpoints, vaccine dose and schedule) was well positioned to ensure collection of initial biomarker data in parallel to the clinical results of the PD- and MSA trajectories.
SYMPATH focused on PD01A and PD03A, two AFFITOPE® vaccine candidates developed by the innovative AFFITOME® technology of AFFiRiS AG. Both are peptide-protein conjugate vaccines and first-in-class candidates. They were designed to elicit antibodies neutralising misfolded aSyn, but sparing compensatory β-synuclein, thus adding to their safety.
For Objective 1 (Initiate safety and efficacy (exploratory) testing of PD01A and PD03A within a Phase I study in MSA), SYMPATH implemented a Phase I clinical study in MSA at two study centres, CHUB and CHUT to evaluate the safety of PD01A and PD03A and perform exploratory efficacy testing.
For Objective 2 (Initiate safety and efficacy (exploratory) testing of PD03A within a Phase I study in PD), SYMPATH implemented a Phase I clinical study in PD at two study centres, MUI and Prosenex to evaluate the safety of PD03A and perform exploratory efficacy testing.
For Objective 3 (Collect biomaterial and relevant data to generate a biobank), SYMPATH collected biological samples (CSF and serum) from patients as part of the SYMPATH clinical trials, with samples not exposed to the IMP targeted for biobanking.
For Objective 4 (Develop assays for the assessment of target engagement based on biomaterial from Phase I studies), SYMPATH developed and optimised a range of assays required for the evaluation of the immune response and specificity of the AFFITOPE®-induced antibodies.
For Objective 5 (Develop an exploitation strategy to advance clinical development of a therapeutic aSyn vaccine beyond safety and initial efficacy testing), SYMPATH developed an exploitation plan for vaccine and biomarker development to define the next steps for product development.
In order to address these objectives SYMPATH brought together eight partners from three European countries. Our multidisciplinary consortium combined the unique expertise and infrastructure of groups with leading experts in PD, MSA, aSyn biology as well as biomarker assay development. This combination excellently positioned the consortium to achieve its main aim of advancing the clinical development of therapeutic vaccine candidates (AFFITOPE®s PD01A and PD03A) targeting aSyn-driven neurodegenerative diseases, namely PD and MSA.
Project Results:
Work Package 1, ‘Production of GMP PD01A material and set-up of a GMP production process for PD03A’.
WP1 aimed to ensure the availability of vaccine batches of both PD01A and PD03A, manufactured under Good Manufacturing Practice (GMP) conditions for the intended clinical studies.
The objectives of WP1 were the following:
• Generation of a GMP-grade Drug Product (DP) batch of the conjugate vaccine PD01A according to already established protocols
• Manufacturing of GMP-grade PD03 peptide
• Definition of a GMP production process for PD03A Drug Substance (DS) including appropriate analytics
• Generation of a GMP-grade Drug Product (DP) batch of the conjugate vaccine PD03A
• Demonstration of stability (in vitro and in vivo methods) and immunological potency
At the beginning of the SYMPATH project, AFFiRiS AG successfully established the GMP production processes of the vaccine candidates PD01A and PD03A at the Contract Manufacturing Organisation (CMO) for clinical application and was able to deliver the vaccine batches for use in patients at the clinical trial sites.
The release of the clinical grade vaccine batches for PD01A and PD03A involved the following steps:
1. Production of DS. GMP-grade peptide and DS (peptide–KLH conjugate) manufacturing.
2. DP Formulation and Filling. DP contains a conjugate (DS) and Alhydrogel as an adjuvant.
3. Labelling, Packaging and Distribution to the study sites.
4. Final Release of DP. It consists of two steps: a technical release by the CMO and a release by a qualified person at AFFiRiS AG.
Some deviation from the original plan occurred in the course of the project. Due to delays in the clinical studies several additional batches of the vaccines were required to be produced over the course of the project to ensure continuous supply of the clinical sites with GMP-grade clinical trial material. This ensured that it was possible for the clinical studies to successfully complete the treatment phase and proceed to the study analysis.
Adequate in-process controls were set-up and used during the manufacturing process. Quality control measures were implemented by the CMO and defined together with AFFiRiS AG.
In order to be able to analyse the clinical vaccine batches, several assays (in vitro as well as in vivo) had to be set up. In vitro analytical methods were performed at the CMO- and AFFiRiS AG. The analytical methods were similar in many respects to those prepared for PD01A, therefore there was a limited amount of PD03A-specific set-up necessary for the production of the PD03A study material. Stability analyses took place at regular intervals over a 36-month period and were completed for the first batches of PD01A and PD03A. In vivo analysis was performed at AFFiRiS AG, respectively.
The results of completed stability programs clearly demonstrated that both vaccines comply with predefined stability settings at assessed time points. For the additional vaccine batches produced the assessment is ongoing and will continue beyond the project’s lifetime.

Work package 2, ‘Set-up of a phase I study assessing safety and efficacy (exploratory) of PD01A/PD03A in MSA’
Since no disease modifying treatment for MSA is currently available and MSA is a deteriorating fatal disease the unmet medical need is undoubtedly high.
WP2 aimed to investigate the safety as well as the initial immunological-, biological- and clinical activity of repeated immunisations with PD01A and PD03A in early MSA patients.
The objectives of WP2 were the following:
• Set-up of clinical trial including submission of study documents to authorities
• Conduction of the clinical trial including close out, analysis and report of results
In the first months of the project, all the essential study documents for the regulatory authorities and Ethics committee were written and submitted, some of which directly referred to the GMP produced batch of PD01A and PD03A generated in WP 1. For certain tasks, such as insurance, statistical analysis, data management and medical writing, qualified subcontractors were identified and engaged/bound by contracts. The two French study sites Boudreaux (CHUB) and Toulouse (CHUT) are centres of expertise for MSA: Both are members of the European MSA study group, which is a consortium of scientific investigators from academic research centres in Europe and Israel who are committed to clinical trial activity and other research studies aimed at improving the treatment of MSA. Furthermore, CHUB leads a registry of MSA patients.
Based on their expertise and network both centres were able to recruit 30 MSA patients, as planned, for screening. Since all patients fulfilled the predefined in- and exclusion criteria, all were included in the study. After randomisation (with a ratio of 2:2:1) they were allocated into three groups, which were treated with PD01A (12 patients), PD03A (12 patients) or with a placebo (6 patients). Patients were blinded to the group assignment and received 5 subcutaneous applications of 75ug vaccine each time following a 4-weekly interval with the one exception being the last injection, which occurred after 24 weeks. 24 patients (corresponding to 80% of those recruited) completed the study by attending in total 9 visits and 5 phone interviews over a 12-month time period. Patients were assessed by clinical rating scales covering the motor and autonomic nervous system, which is most affected by MSA, as well as cognitive function, depression, overall global impression and quality of life. These assessments took place at three-time points: inclusion, month 6 and month 12 after the first immunisation. For safety assessment purposes and for the assessment of exploratory outcomes of the clinical trial they also received a brain MRI (at study inclusion, month 6 and month 12) and cerebrospinal fluid (CSF) was collected (at study inclusion and at month 9).
The primary endpoint for the clinical trial was that safety would be successfully met, and to this end both vaccines were overall well tolerated. A secondary objective followed by the study was also to investigate the clinical and immunological effects.
The entire study was conducted according to GCP standards with the study quality ensured by regular monitoring visits at both sites. Furthermore, an audit at CHUT, dedicated specifically to the trial master file (TMF) was also conducted.
Considering the fact that clinical trials addressing MSA are rare, this study represents an important contribution to the clinical research of this orphan disease. Moreover, it is the first study applying the concept of active immunisation against aSyn in MSA patients.

Work Package 3, ‘Set-up of a phase I study assessing safety and efficacy (exploratory) of PD03A in PD’.
PD is the second most frequent neurodegenerative disease and its prevalence/patient numbers are increasing due to increased live expectancy. PD imposes a high and increasing burden on society and health care systems. Currently there is no treatment available that can interrupt the early pathological processes of PD. aSyn aggregation and cell to cell transmission is assumed to be one of those early processes and therefore holds great therapeutic potential. Currently, there are two therapeutic monoclonal antibodies targeting aSyn in PD in clinical trials.
WP3 aimed to assess the safety as well as the initial immunological-, biological- and clinical activity of repeated immunisations with PD03A in early PD patients.
The objectives of WP3 were the following:
• Set-up of the clinical trial including submission of study documents to authorities
• Conduction of the clinical trial including close out, interpretation of results and reporting
This study was initiated in November 2014 (the date the first informed consent was signed) after all study documents required by the competent authorities were submitted and accepted. In parallel, providers of insurance, statistical analysis, data management and medical writing with relevant experience and expertise were identified and contracted.
38 patients with early PD were screened at the two study sites in Austria (MUI and Prosenex). Apart from 2 patients all 36 qualified to participate in the trial (with a mean age of 58 years). After randomisation into three equal groups (1:1:1), they were treated with either low dose (15ug) PD03A, high dose (75ug) PD03A or a placebo containing only the adjuvant Alhydrogel (12 patients each group). Patients were blinded to their group identity and were allowed to continue using their standard PD medication throughout the study.
4 subcutaneous applications were given at 4 weeks intervals followed by a fifth booster vaccine after another 24 weeks.
35 patients (corresponding to 97% of the patients recruited) completed the study by attending a total of 11 visits with detailed physical and neurological examination and 5 phone interviews one week after each injection. In addition, DAT-SPECT imaging to measure changes in dopamine receptors and volumetric measurements of the relevant brain regions by MRI were assessed. Including the follow up period the entire study lasted 12 months.
The safety and tolerability endpoint was successfully met: PD03A was well tolerated at both doses. There were no study drug related serious adverse events (SAE), nor any suspected unexpected serious adverse reactions (SUSAR). Most adverse events were limited to the injection site and were of a mild nature.
A secondary objective of the study was the investigation of the clinical and immunological effects. To this end various clinical scores measuring motor as well as non-motor symptoms of PD (including depression, cognition problems, and overall quality of life), were used. In addition, the progression of PD was measured on the modified Hoehn & Yahr scale. At each visit a serum sample was taken for immunogenicity testing. A robust humoral immune response directed against the vaccine conjugates was induced in both groups.
To ensure study quality, both sites were regularly monitored. Furthermore, an audit at each site and an audit dedicated specifically to the trial master file (TMF) also took place.
Given that this was the first study of PD03A in man, it represents an important contribution to the future of PD therapy.

Work Package 4, ‘Set-up of a biomarker programme in the sense of target engagement/companion marker’.
The diagnosis of aSyn-related disorders is largely based on clinical symptoms. However, clear and definitive confirmation of the disease still requires a pathological examination at the autopsy. Since PD shares common neuropathological, cognitive and clinical profiles with several others neurodegenerative disorders, misdiagnosis occurs quite frequently. Thus, the development of diagnostic biomarker-based tool for the early and precise diagnose is of high medical need.
Within WP4 SYMPATH aimed to develop a solid biomarker platform.
The objectives for WP4 were the following:
• Generation of a repository of biological samples collected before, during and after vaccination of MSA-/PD patients allowing correlation with clinical course
• Analysis of the vaccination-induced immune response based on test systems developed to characterise and measure levels of specific serum antibodies
• Development of assays to assess target engagement and evaluation of collected samples
• Correlation of biological test results with clinical parameters to test their potential as diagnostic, prognostic and/or theragnostic markers
All these elements are prerequisite for the successful establishment of a diagnostic tool, not only specifically for the AFFITOPE® approach, but also beyond that with focus on the diagnosis of synucleinopathies.
The collection of biological material (CSF and Serum samples) and imaging data from the clinical trials was successfully completed and will be managed by AFFiRiS AG as a sample repository for future purposes.
During the course of the project, AFFiRiS AG established several ELISA (enzyme-linked immunosorbent assay) assays, which enable the precise characteristic and measurement of the antibodies induced upon the AFFITOPE®-treatment. Such measurements conferred the ability to estimate and monitor the immune response through-out the study and evaluate the specificity of the induced antibodies to bind the aSyn target epitope. In both studies (AFF009 and AFF0011) serum samples for use in testing the immunogenicity of vaccine were taken at each visit. In AFF009 the immune response against the peptide moiety of PD01A and PD03A, the related epitope on aSyn and the carrier protein was successfully detected and quantified. In AFF011 the immune response against the peptide moiety of PD03A, its related epitope on aSyn and the carrier protein were also successfully detected and quantified.
In addition, AFFiRiS AG invested further work in the establishment of a method that allows the precise characterisation of the antibodies’ in vitro target engagement to monomeric and aggregated (toxic) forms of aSyn. However, in the course of the assays set-up, AFFiRiS AG faced certain limitation in the measurements due to unspecific aSyn reactivity detected prior to the AFFITOPE® treatment. Consequently, AFFiRiS AG worked intensively on an additional set up, which enabled the discrimination between the unspecific aSyn reactivity present prior to exposure and the specific AFFITOPE®-induced aSyn reactivity occurring after treatment. The successful establishment of the assay provided the possibility of isolating the AFFITOPE®-induced antibodies, and significantly, characterising their binding capacity to different aSyn species. AFFiRiS AG proceeded with further assay development and also established a method for the characterisation of the preferential binding of the AFFITOPE®-induced antibodies to either aggregated aSyn or monomer. These evaluations detect crucial characteristic features that highlight the specificity of the approach, in particular towards the aggregated form of aSyn.
In parallel, FZ Jülich worked on the establishment of the sFIDA (surface-based fluorescence intensity distribution analysis) assay for measuring aSyn aggregates (oligomers) in biological compartments to demonstrate the in vivo target engagement. FZ Jülich successful generated aSyn oligomers for calibration and also evaluated several aSyn-specific antibodies, which serve as a capture and detection tool for the sFIDA technology. Several evaluations confirmed the final combination of antibodies that were selected for the assay. Based on the precise selection and specific signals, the sFIDA assay enables the measurement of aSyn oligomers in biological compartments such as serum and CSF. The method development was completed with all accompanying quality controls and the sFIDA assay was applied to the measurement of aggregated aSyn levels in the collected CSF and serum samples from patients from both SYMPATH clinical trials.
As a result of the successful establishment of all planned and required assays, the analysis of biological samples from both clinical trials for long-term immunogenicity (titers against the peptide moiety used for immunisation), cross-reactivity (aSyn target epitope), in vitro (ELISA) and in vivo target engagement (sFIDA) were successfully undertaken. Measurements were performed after the completion of the clinical trials on the biological samples that were at collected at selected time points throughout both clinical trials.
In conclusion, the data from biological samples was included in the final clinical study reports (CSRs) and confirmed the AFFITOPE® approach in PD and MSA patients as being immunogenic and aSyn (target) specific.
Due to delays occurring in the start of both clinical studies and a subsequent delay in the availability of the final CSRs, the correlation of biological test results (ELISAs and sFIDA) with clinical parameters and other types of correlations were not feasible within the project timelines. Further activities (post-hoc analyses related to antibody characterisation) will be undertaken to enhance our understanding of the biomarker data and to establish aggregated (or misfolded) aSyn as a theragnostic/prognostic marker. This will be crucial for further clinical studies with PD-AFFITOPE® vaccines, offering the possibility of using the assays established within SYMPATH (ELISAs and sFIDA) further, even with potential in other non-AFF clinical studies as measuring, prognostic and diagnostic tools (sFIDA).

Work Package 5, ‘Exploitation’.
WP5 was aimed at the exploitation of the results coming out of the SYMPATH project.
The objectives for WP5 were the following:
• Developing an exploitation plan to ensure effective translation of the generated know-how into a medical product (PD01A and PD03A). This includes looking for partnership with a larger pharmaceutical company to further develop the PD-AFFITOPE® drug candidate towards the next clinical phase and the regulatory authorisation.
• Establishing an IP/exploitation strategy plan for biomarker/companion diagnostic development by the consortial partners involved. This includes exploration of patentability of the project’s results, especially biomarker (misfolded aSyn) investigated in WP4.
Concerning the PD01A and PD03A PD-AFFITOPE®s, AFFiRiS AG initiated and completed several preparatory steps, which should ensure successful partnering and the securing of external financing for the aSyn programme. During the course of the project, AFFiRiS AG has initiated and maintained contacts with major pharmaceutical companies from the neurodegeneration field in order to explore potential partnerships. Furthermore, AFFiRiS AG attended several international partnering events to present the aSyn product portfolio. In the final year of the SYMPATH project AFFiRiS AG actualised and finalised the documents needed for the due diligence process with potential partners. Moreover, AFFiRiS AG, with support from consortial partners, further progressed the planning of the next steps for clinical product development. The availability of SYMPATH clinical study results contributed substantially to these planning activities and the overall completeness of the aSyn programme portfolio. The major next step is to reach a decision on a lead compound (PD01 or PD03), as it is too cost-intensive to advance both vaccine candidates towards the next clinical phase. A thorough comparison of the results coming from the SYMPATH clinical studies in addition to those from the non-SYMPATH clinical study AFF008 (PD01A in PD), is a prerequisite for this decision.
It is important to mention, that AFFiRiS AG protected and maintained its Intellectual Property (IP) regarding the AFFITOPE® approach for targeting aSyn in synucleinopathies prior to the SYMPATH program (Background IP). In these IP measures the sequences of PD01 and PD03, which are also used in the biomarker program, were included and protected.
All of these activities increase the chances of obtaining successful partnering for the aSyn programme, which would thus assure the further clinical development of the PD-AFFITOPE® lead candidate.
Concerning the biomarker development, several experimental assays were established by AFFiRiS AG and sFIDA assay was finalised by FZ Jülich. They were successfully applied in the two clinical studies AFF009 and AFF011 within the SYMPATH program as evaluation tools for the exploratory efficacy of the trials in terms of immune response and target engagement. Therefore, their potential use beyond the SYMPATH project as diagnostic and monitoring tools will be extensively followed up and further investigated.
FZ Jülich has further protected its sFIDA technology by filing the patent application WO2016146093A1. The invention relates to a method for producing a standard for detecting protein aggregates from a protein misfolding disease, such as Alzheimer’s or Parkinson’s disease. Furthermore, FZ Jülich substantially advanced the commercial potential of its proprietary sFIDA technology by creating a spin-off company ‘attyloid’ to be founded by Q1/2018. The board of directors of FZ Jülich and the entrepreneurs of the spin-off are currently negotiating to stipulate the terms of an exclusive license in order to transfer foreground and background IP from this project to the new company.
The company will be founded by first quarter of 2018 and will offer its proprietary sFIDA technology for oligomer-based diagnostics of neurodegenerative orders. sFIDA is fully automated, amenable to high-throughput and features a wide dynamic range covering five orders of magnitude. The technology can be applied to direct measurement of target engagement in clinical trials and, once a causal therapy is available, for routing diagnostics for Alzheimer’s and Parkinson’s disease. Moreover, sFIDA was adapted for monitoring aggregation of biopharmaceutical products and counting single viral particles. ‘Attyloid’ will immediately start offering the sFIDA technology as a service to clients from pharma, biotech and academia. At a later stage of business development, diagnostic kits and devices will be developed and distributed to establish the sFIDA platform technology for routine applications in clinical settings.
The SYMPATH consortium will continue with its exploitation strategies beyond the project’s lifetime in accordance with the established exploitation plans.
Potential Impact:
Well-established clinical trial management and conduct structures within WP2 and WP3 have ensured successful completion of the clinical studies within the project’s lifetime. It has defined the logical next development step for one of the PD-AFFITOPE® vaccine candidates for synucleinopathies. Diagnostic assays, successfully established by AFFiRiS AG, will accompany the AFFITOPE® technology development in the future. This is a crucial element in ensuring maximum efficiency in the progression of the successful vaccine candidate through further development and commercialisation to being effective immunotherapeutic to treat synucleinopathies.
PD and MSA are diseases of the elderly, affecting approximately 1,6% of individuals aged 65 or more and are as such one of the major threats to healthy aging. An estimated $14.4 billion a year in the United states (as of 2010) is lost due to PD and the lack of a curative treatment puts enormous psychological and practical strain on patients and their families. Only the perspective of a curative treatment has the potential to reduce this burden. A therapeutic vaccine has the potential to revert disease progression thus promoting healthy aging for a considerable number of patients. This would substantially improve the quality of life for those patients and their families.
As the mean onset of PD is around 65 years of age (55 years for MSA), indirect costs incurred through lost productivity currently account for only about 10% of the burden of PD and MSA. However, with the prospect that the retirement age in the developed world might be raised, there is a high potential that this burden could increase considerably. An effective treatment would allow direct and indirect costs associated with disease management to be minimised. It is therefore very likely that the chosen approach would prove to be competitive with any other curative treatment and could therefore provide a significant impact in terms of cost savings and contribute to the sustainability of global health care systems.
Lack of earlier diagnosis and the inability to differentiate from similar diseases present serious problems in the care of PD patients. In the biomarker programme, FZ Jülich is set to commercialise its proprietary sFIDA technology by creating the spin-off company “attyloid”. Thus, this assay can in future be applied to the direct measurement of target engagement in further clinical trials and, once a causal therapy is available, for routing diagnostics for Alzheimer’s and Parkinson’s disease. The availability of a suitable biomarker will enable not only the monitoring of vaccination effects on clinical parameters, but also the assessment of vaccination effects on biological markers, possibly reflecting pathology.
The spin-off activities aim to generate economic and social value and contribute to improving the competitiveness of national and EU industry. In the light of demographic changes, a dramatic increase in age-related disorders such as Alzheimer’s and Parkinson’s disease is projected, associated with a high demand for a causal therapy and accurate diagnostics. Thus, the socio-economic impact of this project cannot be overestimated. Independently of the success of the vaccine candidates in the clinical studies, medical practitioners could already benefit from this effective diagnostic method in the short-mid-term.
The expected impacts of the project results were strengthened by dissemination activities, which have strong interactions with exploitation, as they set in place or investigate key mechanisms for the successful deployment and adoption of the key project aims and results coming out of the SYMPATH project.

Main dissemination activities and exploitation of results
Professional project management and dissemination of results significantly contributed to the successful collaboration of the SYMPATH beneficiaries. The Coordinator encouraged all participants to actively disseminate their project aims and results to colleagues, stakeholders and the wider public.
At the beginning of the project, a logo and a corporate identity for the project was created in order to identify the project. It is used together with the Grant Agreement number HEALTH.2013.1.3-3 602999 and the FP7 logo (until use of the FP7 logo was officially discontinued) on all printed and electronic material for the public and for any other official contact.
On the web, public awareness of SYMPATH was addressed by the project website www.sympath-project.eu. The website content was regularly updated and includes key background information about the project and its beneficiaries. All content was available in three languages: English, German and French. Additionally, project fact sheets are available in 3 languages for external stakeholders allowing information to be obtained at a glance. The media center section provided visitors with the latest publications, press releases and dissemination activities of SYMPATH, as well as the SYMPATH video clip, which was released to coincide with the SYMPATH symposium to generate greater exposure for the project and increased impact. The website formed the information hub for the SYMPATH public event: SYMPATH FAHR-MIT, a bike tour with Parkinson’s patients across Austria. A separate SYMPATH website was created for the publicity of, and registration for, the SYMPATH scientific symposium.
A major step for the dissemination of the project and as a lasting tribute to the achievements and impact created by the SYMPATH project, was achieved through the creation and launch of the SYMPATH ‘gateway’ website. The concept for the new ‘gateway’ website was to provide a project overview with a focus on: partners, results, collaboration and impact. This would require neither update nor maintenance in the future and would represent a digital final results summary, with the considerable advantage of continued visibility and the possibility to easily update if required. The focus of the ‘gateway’ website is on large images and graphic elements interspersed with clearly structured texts about partners, strategy, outcomes and dissemination activities. The text was kept short to provide a quick summary overview to the whole project. As with the previous SYMPATH website, the new ‘gateway’ website also provided full compatibility for viewing on mobile devices, including mobile phones and tablets.
The original project website was updated throughout the reporting period, including a major update during the 3rd official reporting to keep pace with the latest technology trends. This relaunch included the implementation of a new theme with increased speed for an optimised user experience. The original website is now embedded in the new ‘gateway’ website, and thus still accessible if desired.
The Consortium also took measures to provide SYMPATH exposure to the public through conventional PR media work and new media, which complemented the public access website, particularly for those that do not have internet access.
The scientific community has a vested interest in keeping up with SYMPATH progress and results to build on the body of knowledge that currently exists. This was achieved in part through conventional scientific dissemination means, i.e. peer reviewed publications and presentations at international conferences. 6 peer-reviewed publications were published during the project funding period as part of the project and partners have presented SYMPATH work world-wide 63 times at international scientific conferences. As the major outcomes of the SYMPATH clinical trials only became available towards the end of the project it is expected that a number of additional publications will be published in the near future.
The SYMPATH project had two major dissemination highlights, namely the public event ‘SYMPATH FAHR-MIT’ and the scientific event ‘SYMPATH Symposium on Targeting Synucleinopathies’.
‘SYMPATH-FAHR-MIT’ took place over seven consecutive days (Saturday 30th May 2015 to Friday 6th June 2015) and comprises a bike tour across Austria with Parkinson patients, aimed to engage the public with the EU-funded SYMPATH project and Parkinson’ disease in general. Daily events comprised of a cycling leg followed by a reception event for the cyclists including speeches by local dignitaries and an interactive exhibition. The event was accompanied by extensive media and press work to raise awareness, which successfully generated a significant amount of media coverage, including a number of radio interviews and slots on television. SYMPATH FAHR-MIT was featured on: 64 web sites, 18 paper-based publications, 7 radio slots and 3 TV slots were recorded. Post tour events included an invitation by the president of the Vienna City Council to a reception at the Vienna City Hall and a presentation event for a photographic reportage book prepared as a commemoration of the event.
The SYMPATH Symposium on Targeting Synucleinopathies, which took place on the 28th March 2017, was conceived to bring together world-leading experts to discuss recent progress in developing new potential biomarkers and therapeutic concepts, including vaccines, for Parkinson’s disease (PD) and multiple system atrophy (MSA). The speakers represented all areas of the SYMPATH project supplemented by additional experts in the field. The symposium was successful in attracting a high-calibre of speaker, which was reflected in attracting 115 attendees, of which 25 were international, including 4 from the USA. In addition to the scientific presentations the symposium also hosted a poster session and an interactive exhibition, which stimulated additional interest in many researchers of the Vienna Biocenter who interacted with the exhibition material.

Future perspectives
Efficient collaboration of partners will be maintained beyond the end of the project to foster advancement of the AFFITOPE® vaccines to further product development.
SYMPATH will publish the settings and results of the clinical trials thus to make sure the concept will be shared with the scientific community.
In summary, successful implementation of SYMPATH results will lead to the establishment of a novel and unprecedented treatment approach. Therapeutic immunisation using AFFITOPE®-based vaccines deliver a novel, safe and cost-effective treatment modality exhorting disease modification in PD and MSA. Availability of such a drug on the market would constitute not only a scientific breakthrough, but also fundamentally change the management and burden of synucleinopathies.
List of Websites:
Website:
http://www. sympath-project.eu/

Contact details:

Project Coordinator
AFFiRiS AG
Karl-Farkas-Gasse 22, 1030 Vienna, Austria
E-mail: office@affiris.com
Phone: +43-1-7981575-300
Fax: +43-1-7981575-311

biolution - SYMPATH office
Helmut-Qualtinger-Gasse 2/2
1030 Vienna, Austria
E-mail: office@sympath-project.eu
Phone: +43-1-786 95 95
Fax: +43-1-786 95 95 20