Background: B7-H3/CD276, a member of the B7 superfamily, is highly expressed in prostate cancer (PCa) and is associated with rapid biochemical recurrence and early metastases. B7-H3 is the only checkpoint candidate to have a presumptive androgen receptor binding site, suggesting interaction with the androgen axis. Enoblituzumab (MacroGenics) is an investigational humanized Fc-optimized B7-H3–targeting antibody that induces antibody dependent cellular cytotoxicity (ADCC). Methods: In this phase 2 single-arm biomarker-rich neoadjuvant trial, men with operable intermediate- and high-risk localized prostate cancer (Grade Groups 3-5) were enrolled to evaluate the safety, anti-tumor efficacy, and immunogenicity of enoblituzumab when given prior to prostatectomy. Patients received enoblituzumab (15 mg/kg IV weekly x 6) prior to surgery. Prostate glands were harvested 2 weeks after the last dose, and were examined for pathologic and immunologic endpoints. The co-primary outcomes were safety and PSA0 at 1 year post-op. Pre-planned secondary outcomes were PSA and Gleason grade group change from biopsy to prostatectomy. Results: 32 men were enrolled. Grade 3/4 adverse events occurred in 12% of patients. One patient developed a grade-3 infusion reaction, and one had immune myocarditis that improved with steroids. Pre-prostatectomy PSA declines of >10% were observed in 31% of patients (95% CI: 16-50%). PSA0 at 1 year post-op was seen in 66% of men (95% CI: 47-81%). Median time to PSA recurrence was not reached, with a median follow-up of 30 months. Gleason group upgrade, no change, and downgrade was observed in 13%, 37%, and 50% of patients. Gleason grade group changes were significantly associated with enoblituzumab treatment compared to 1:1 matched historical controls (p=0.023). Tumor microenvironment profiling by NanoString GeoMx spatial proteomics and PanCancer IO 360 mRNA expression analysis revealed post-treatment upregulation of CD8+ T cells, PD-1/PD-L1 expression, and immune activation (granzyme B, IFN signaling, myeloid inflammation). There was a significant association between CD8+ T-cell increases and Gleason grade group declines. First-in-human antigen spread profiling revealed no safety concerns. TCR sequencing showed focused peripheral expansion of tumor associated T-cell clones that correlated with PSA0 at 1 year. Whole exome and RNAseq data, and clinical correlations, will be presented. Conclusions: In this neoadjuvant trial, inhibition of B7-H3 with enoblituzumab demonstrated favorable safety and encouraging activity in localized PCa patients. Data suggest robust intratumoral induction (adaptive upregulation) of immune checkpoints, T-cell activation, and myeloid inflammation. Enoblituzumab-induced peripheral expansion of tumor associated T-cell clones may be associated with tumor control. Clinical trial information: NCT02923180.