Target engagement and immunogenicity of an active immunotherapeutic targeting pathological α-synuclein: a phase 1 placebo-controlled trial

Parkinson’s disease (PD) lacks approved disease-modifying therapies. Pathological, aggregation-prone forms of α-synuclein (αSyn) are central to PD pathology, and immunotherapies—both passive monoclonal antibodies and active vaccines—have shown promise in preclinical models. However, two phase 2 trials of anti-αSyn monoclonal antibodies failed to demonstrate clinical efficacy, underscoring the need for optimized trial design and robust biomarkers of target engagement. UB-312 is an active immunotherapeutic vaccine utilizing a synthetic T-helper peptide carrier platform designed to elicit a B cell humoral response against a C-terminal αSyn epitope while avoiding T cell-mediated cytotoxicity. Preclinical work showed selective binding of UB-312-induced antibodies to pathological oligomeric and fibrillar αSyn and neuroprotective effects, and Part A of a phase 1 program in healthy volunteers established safety and immunogenicity with detectable serum and CSF antibodies. Part B (reported here) evaluated safety, tolerability, and immunogenicity of two UB-312 prime-boost regimens in PD and explored target engagement using the αSyn seed amplification assay (αSyn-SAA) along with clinical scales (MDS-UPDRS, MoCA).

Prior studies demonstrate that both passive and active αSyn immunotherapies can reduce αSyn pathology and functional deficits in animal models. Despite this, phase 2 trials of prasinezumab and cinpanemab in early PD did not meet primary efficacy endpoints. Lessons from Alzheimer’s disease emphasize the importance of biomarkers for target engagement (e.g., amyloid PET). For PD, αSyn-SAA has emerged as a sensitive and specific assay to detect pathological αSyn seeds in CSF, support diagnosis, and discriminate among synucleinopathies. The UB-312 platform derives from synthetic T-helper peptides linked to target epitopes, aiming to overcome immune tolerance while minimizing T cell cytotoxicity. Part A in healthy volunteers showed UB-312 was safe up to a 300/300/300 µg prime-boost regimen, induced dose- and time-dependent antibodies detectable in serum and CSF (average CSF/serum ratio ~0.2%), and did not alter total αSyn levels, consistent with preferential binding to pathological forms rather than monomeric αSyn.

Design: Phase 1, single-center, randomized, double-blind, placebo-controlled trial (Part B) at the Centre for Human Drug Research (CHDR), The Netherlands. Conducted per the Declaration of Helsinki and ICH-GCP; ethics approval by BEBO, Assen, The Netherlands. ClinicalTrials.gov: NCT04075318. Participants: Adults 40–85 years with idiopathic PD (Hoehn & Yahr ≤ III), BMI 18–32 kg/m², stable on PD and concomitant medications, and able to undergo procedures. Screening included MRI to exclude structural abnormalities and DaT scan when needed to confirm dopaminergic deficit. Key exclusions: significant medical/neurological/psychiatric comorbidities compromising safety, HIV/HCV/HBV infection, autoimmune disease, anergy, allergy, prior anti-αSyn antibody/vaccine trials, atypical parkinsonism, freezing/falls/orthostatic hypotension at screening, DaT scan inconsistent with PD, other neurological disease. Randomization and blinding: Two cohorts (each n=10) randomized 7:3 (UB-312:placebo) to UB-312 300/100/100 µg or 300/300/300 µg prime-boost regimens versus placebo. Randomization by independent statistician (SAS 9.4). Syringes prepared by an independent unblinded pharmacist; participants and site staff were blinded. Interventions and visits: Intramuscular deltoid injections at weeks 1, 5, and 13. Follow-up at weeks 2, 6, 9, 14, 21 (end of treatment), 29, 37, and 45 (end of study). Safety assessments at each visit: TEAEs, concomitant meds, vitals; physical/neurological exams (weeks 1, 5, 13, 21, 29, 37, 45); triplicate ECGs (weeks 1, 5, 13 pre- and 6 h post-dose; week 21); safety labs (weeks 1, 5, 13, 17, 21); pregnancy tests (weeks 1, 5, 13 pre-dose); urine drug/alcohol tests at week 1 pre-dose; safety phone call day after each administration. Participants maintained 7-day diaries for solicited local/systemic reactions after each dose. CSF and biomarker sampling: CSF collected at weeks 1 (baseline), 21, 45 per CHDR SOPs; samples processed within 2 h, aliquoted within 60 min, stored at −80 °C; samples with RBCs discarded. Assays included CSF and serum anti-αSyn antibodies against the C-terminal epitope (αSyn97–135) and full-length αSyn; antibodies against vaccine components (CpG1, T-helper peptide); free and total αSyn in blood and CSF; cytokines (blood weeks 1, 5, 13, 21); T cell ELISpot (weeks 1, 17); HLA (week 1). Clinical scales: MoCA and MDS-UPDRS parts II and III (all ON state) at weeks 1, 21, 45; H&Y at screening and repeated during study. Exploratory biomarker assays: αSyn seed amplification assay (αSyn-SAA) per Shahnawaz et al. with modifications for kinetic evaluation per Concha-Marambio et al.; baseline serial dilutions (threefold up to 1:729) to determine optimal dilution; longitudinal kinetics at a single 1:5 dilution (triplicates). SAA conditions included recombinant αSyn, PIPES buffer, NaCl, sarkosyl, and silicon nitride beads with cyclic agitation and thioflavin T fluorescence readouts. Dot blots assessed binding of postimmunization IgG fractions/affinity-purified antibodies to aggregated and monomeric αSyn. CSF pS129-αSyn measured by immunomagnetic reduction (MagQu kit) in duplicate. Outcomes: Primary—safety and tolerability (TEAEs, labs, exams, ECGs) and immunogenicity (anti-αSyn antibodies in blood and CSF). Exploratory—changes in MDS-UPDRS-II/III and MoCA; target engagement via αSyn-SAA kinetic parameters (e.g., maximum fluorescence Fmax). Seroconversion defined as development of quantifiable antibodies post-vaccination in those below LLOQ at baseline. Populations and statistics: Safety population = all randomized and exposed (identical to mITT). Per-protocol (PP) = those receiving all planned vaccinations without major protocol deviations (n=20 through week 13; n=19 thereafter). Descriptive analyses for safety and immunogenicity; trial not powered for between-regimen comparisons. For SAA and clinical scales, two-way ANOVA with mixed-effects model for time and treatment; within-group analyses adjusted per Benjamini, Krieger, Yekutieli; unpaired t-tests for between-group differences at time points. Data reported as means ± s.e.m. unless noted. Exploratory post hoc analyses compared individuals with versus without detectable CSF anti-αSyn titers at week 21.

- Enrollment and disposition: 41 screened; 20 enrolled and dosed; 19 received all three vaccinations (one in 300/100/100 µg cohort missed third due to SAE); all completed follow-up. mITT = 20; PP = 20 through week 13 and 19 thereafter. Baseline characteristics were comparable across groups. - Safety and tolerability: TEAEs occurred in 14/14 UB-312 recipients and 5/6 placebo recipients. Most TEAEs were mild or moderate; common events included headache, local pain after lumbar puncture, and fatigue. Three SAEs occurred in two patients; one SAE (deep venous thrombosis of the left leg 50 days after the second 300/100/100 µg dose) was considered possibly related; all SAEs resolved without sequelae. No safety signals in ECGs, vital signs, or clinical laboratory assessments. No severe local injection-site reactions; no postvaccination brain MRIs were performed. - Immunogenicity (primary): Robust, time-dependent serum anti-αSyn (C-terminal epitope αSyn97–135) responses observed, peaking at week 29 and remaining above baseline at week 45. Mean serum titers (log-dilution factor, logDF) increased from baseline by 1.398 (300/100/100 µg) and 1.354 (300/300/300 µg) and peaked at week 29 at 2.520 and 2.133 logDF, respectively. Seroconversion occurred in 12/13 participants who received all three doses (5/6 in 300/100/100 µg; 7/7 in 300/300/300 µg). CSF epitope-specific antibodies were detectable in 5/13 participants at week 21 (4/6 in 300/100/100 µg; 1/7 in 300/300/300 µg); CSF titers were 0 at baseline and reached 0.182 and 0.032 logDF at week 21 for the two regimens, respectively; detectable at week 45 in only two participants. UB-312 did not induce antibodies against full-length αSyn compared with placebo. - Exploratory clinical outcomes: MoCA and MDS-UPDRS-II/III scores were generally stable with no significant between-group differences over time. - αSyn-SAA target engagement: At baseline, 19/20 CSF samples were αSyn-SAA positive; median maximal dilution factor 32.40 (range 197.1). Longitudinally, maximum fluorescence (Fmax) showed a significant change from baseline overall (F=6.622 (1.541–22.35), P=0.009): placebo showed a nonsignificant +2.8% increase, UB-312 300/100/100 µg showed a significant −19.8% decrease (P<0.05), and UB-312 300/300/300 µg showed a nonsignificant −15.2% decrease at week 45. At week 45, Fmax was significantly lower in the 300/100/100 µg group versus placebo (P<0.05). One participant in 300/300/300 µg converted from αSyn-SAA positive at baseline/end of treatment to negative at end of study. - Post hoc analyses by CSF antibody status: Individuals with detectable CSF antibodies (n=5) exhibited a more pronounced and statistically significant reduction in Fmax versus those without (n=13) (time effect F=12.77 (1.73–26.82), P=0.0002; treatment × time F=6.755 (2–31), P=0.0037). CSF pS129-αSyn levels differed significantly at end of study between patients with versus without detectable CSF antibodies (Bonferroni-adjusted P=0.0154). MDS-UPDRS-II change from baseline showed a significant improvement in those with detectable CSF antibodies versus others (F=12.94 (1.569–24.32), P=0.0004; interaction P=0.016), while MDS-UPDRS-III showed no significant difference. - Mechanistic support: Postimmunization IgG fractions from Part A healthy volunteers bound aggregated αSyn (MSA-, PD-, and recombinant-derived) with low binding to monomeric αSyn and delayed aggregation kinetics in SAA, including when spiked into PD CSF.

The study met its primary prespecified endpoints, demonstrating that UB-312 was generally safe and well tolerated in patients with PD and induced time-dependent anti-αSyn antibody responses detectable in serum and, in a subset, in CSF. The presence of CSF antibodies indicates that vaccine-elicited antibodies can cross the blood–brain barrier. Exploratory biomarker analyses showed reductions in αSyn-SAA Fmax, particularly with the 300/100/100 µg regimen and in individuals with detectable CSF antibody titers, supporting in vivo target engagement of pathological αSyn seeds. These biomarker changes are consistent with the vaccine’s intended selectivity for aggregated forms of αSyn and align with preclinical and healthy volunteer data. Observed antibody titers were lower in patients with PD than in healthy volunteers from Part A, potentially reflecting target-mediated redistribution/clearance to pathological tissues where aggregated αSyn is present, or disease-related immune alterations (e.g., reduced B cell numbers). Dosing strategies for active immunization are complex; a high prime with lower boosts (300/100/100 µg) appeared at least as immunogenic, if not more so, than repeated high boosts (300/300/300 µg), suggesting regimen optimization could further enhance responses and CSF exposure. While clinical scales did not differ overall between treatment groups in this small phase 1 study, post hoc analyses indicated that patients with detectable CSF antibodies exhibited both stronger biomarker effects and improvement in MDS-UPDRS-II, providing hypothesis-generating signals linking central antibody exposure, target engagement, and potential functional benefit. αSyn-SAA is not yet validated as a quantitative measure, and fluorescence units are arbitrary; nevertheless, these data suggest a promising biomarker framework for assessing target engagement in PD immunotherapy trials.

This first-in-patient, randomized, placebo-controlled phase 1 trial of the active αSyn-targeting immunotherapeutic UB-312 achieved its primary goals of demonstrating safety, tolerability, and immunogenicity in PD. Vaccine-elicited antibodies were detectable in serum and in CSF for a subset of participants, and exploratory αSyn-SAA analyses indicated reduced seeding activity consistent with target engagement, particularly among those with detectable CSF antibodies. Together with supportive preclinical and Part A healthy volunteer data, these findings justify continued clinical development of UB-312 as a potential disease-modifying therapy for PD. Future studies should optimize dosing regimens and boosting schedules to increase CSF antibody exposure, extend treatment duration, further validate and deploy αSyn-SAA and complementary biomarkers of target engagement, and evaluate clinical efficacy across broader synucleinopathy populations.

- Small sample size (n=20; 13 completed full UB-312 vaccination), typical for a first-in-patient phase 1 but limiting power for efficacy comparisons and subgroup analyses. - Single-center design and a relatively homogeneous cohort; all participants were White, which may limit generalizability. - Patients were not preselected for αSyn-SAA positivity or genetic predispositions, which could influence responsiveness to αSyn-targeted therapies. - CSF lipoprotein levels were not measured to explicitly rule out interference with αSyn aggregation; a 1:5 dilution was used to mitigate potential inhibition. - CSF anti-αSyn antibodies were detectable by titer assay in some patients, but concentrations were below the lower limit of quantification by concentration-based assays, reflecting assay sensitivity differences and overall low CSF exposure; higher exposures may be needed for consistent effects. - The study was not powered for clinical efficacy; clinical scales showed no between-group differences overall. - No postvaccination brain MRI assessments were conducted; safety imaging was available only if clinically indicated. - Short-to-moderate follow-up (45 weeks) and limited dosing regimens; longer treatment and further dose optimization are warranted.