Background: SG is an antibody-drug conjugate (ADC) with accelerated FDA approval for aUC refractory to platinum-based chemotherapy and immune checkpoint inhibitor (ICI). SG activity following treatment with EV, the most commonly used ADC in the post-platinum/ICI setting, and biomarkers of response to SG are not well described. We hypothesized that SG would be active after EV in aUC. Methods: Patients (pts) treated with SG monotherapy who had next-generation sequencing (NGS), were identified in the retrospective UNITE study. Observed response rate (ORR) was determined for evaluable pts with scans after ≥1 SG cycle. All pts were assessed for baseline clinical characteristics, as well as molecular biomarkers including tumor mutational burden, somatic gene alterations (alt) in ≥ 10% of pts and presence of ≥1 DNA damage response (DDR) mutations. ORRs were compared with Chi-squared test. Kaplan Meier method and cox proportional hazard (cph) models were used to assess progression-free survival (PFS) and overall survival (OS) from SG start. Pt characteristics and molecular biomarkers were evaluated in univariate analyses (UVA), followed by evaluation of biomarkers in separate multivariate cph analyses (MVA), while accounting for clinical features. Results: Among 90 pts treated with SG at 9 US sites, 78 had NGS data. Median age at SG start was 68, 65 (72%) men, 73 (81%) Caucasian, 56 (62%) with pure urothelial histology, 60 (67%) primary bladder tumor and 67 (74%) ECOG PS 0/1. Most (n=84, 93%) received SG after EV, and 33 (37%) had ≥4 prior therapies for aUC. ORR with SG was 23% (15/66); 24% in pts with prior CR/PR/SD on EV and 14% after primary PD on EV. Median follow-up from SG start was 8.7 mos, PFS and OS were 3.5 mos and 7.1 mos. In pts with NGS, ORRs to SG were higher in MTAP altered pts (50% vs 19%; p = 0.05). In UVA, low neutrophil to lymphocyte ratio (NLR), high Hgb, no prior smoking history, < 3 prior therapies, as well as BRCA2 and DDR alts were associated with longer OS (p<0.05 for all). In MVA, pts with alt in either TP53 or MDM2 had longer OS. Conclusions: SG has notable activity after EV in heavily pretreated pts with aUC, with ORR consistent with SG phase 2 trial data in post-platinum/ICI only pts. We identified several clinical and genomic biomarkers, including TP53/MDM2 alt, that are potentially associated with improved outcomes with SG treatment.