Background: Immunotherapy is an ineffective approach in molecularly unselected mCRPC, and improving patient selection for targeted treatment in this setting is an unmet medical need. We evaluated the antitumour activity of pembrolizumab in a subset of molecularly selected mCRPC with putative phenotypes associated with immunotherapy sensitivity. Methods: PERSEUS1 (NCT03506997) is a multicentre, open-label, single arm, phase II trial in patients suffering from mCRPC progressive on at least one approved treatment for mCRPC, and who had at least one of the following molecular profiles: mismatch repair deficiency (MMRd); bi-allelic CDK12 loss; high microsatellite instability with either MMRd, a high CD3 count, or high mutational load (HIMUT); HIMUT with high CD3 or a deleterious mutation in a DNA repair gene; or DNA repair defects with high CD3. Patients were treated with pembrolizumab 200mg intravenously every 3 weeks until progression, unacceptable toxicity, or 2 years. The primary endpoint was composite response rate by 24-weeks based on either: iRECIST, circulating tumour cell (CTC) count conversion from ≥5 to <5/7.5ml, or PSA decline ≥50%. The trial used a two-stage Simon Minimax design; H_a=40%, H₀=20%. We report stage 1, with target n=24 participants and >5 responses required to warrant progression to stage 2. Progression-free survival (PFS), overall survival (OS) and safety using CTCAE v4.0 were secondary endpoints. Results: From Nov-2018 to May-2023, 25 participants were enrolled, with 24 reaching response-evaluability. Median follow-up was 28.4 months. Median age of evaluable patients was 69 years, and patients had received 1-4 previous lines of treatment. Participants received a median of 6 cycles of pembrolizumab (range 2–13). Seven out of 24 patients (29.2%) had a composite response by 24 weeks (95% CI 12.6%-51.1%). Responses according to iRECIST, CTC and PSA criteria were 3/24 (12.5%), 3/24 (12.5%) and 4/24 (16.7%) respectively. Median PFS was 4.2 months (95% CI 3.0–8.3). Median OS was 14.5 months (95% CI 9.9–18.4). Of the 3 radiological responses, 2 patients were MMRd, including one with complete metabolic response (iRECIST partial response (PR); 49 months, ongoing) and another with PR (8.3 months PFS). Eight patients (33.3%) experienced grade 3-4 toxicity. This led to discontinuation in two patients: one (patient with complete metabolic response) with immunotherapy-induced colitis, and another with immune-related myocarditis and pneumonitis. Conclusions: Pembrolizumab showed prolonged antitumour activity against a subset of molecularly selected mCRPC. Although the analysis met the pre-specified threshold of composite response criteria, due to slow accrual and modest clinical relevance in most responders, it was decided not to continue to stage 2 of the trial. Clinical trial information: NCT03506997.