Background: Optimizing sequential use of Antibody Drug Conjugates (ADCs) is an area of unmet need and of rising clinical importance. With the recent approvals of sacituzumab govitecan (SG) for HR+/HER2- and triple negative metastatic breast cancer (MBC) as well as trastuzumab deruxtecan (T-DXd) for HER2-low MBC, many patients are now candidates for multiple ADCs. However, given potential cross-resistance based on antibody target vs payload (Coates et al, Cancer Discov. 2021), optimal sequencing remains uncertain. We evaluated the safety and efficacy of ADC after ADC for patients with HER2 negative MBC. Methods: We included all patients at an academic institution treated with more than one ADC for MBC. Each line of ADC beyond the first was evaluated for presence of the same “antibody target” or “payload” compared to prior ADC. Clinicopathological information was gathered by chart review. We defined “cross-resistance” as progressive disease (PD) at time of first restaging on second ADC. Progression-free survival (PFS) was evaluated as time from start of treatment to disease progression or death from any cause. All PFS estimation was done using the KM method. All pairwise comparisons across ADC were done using a Wilcoxon Rank Sum test to allow for divergences from normality in progression times. Significance was declared as a type I error less than 0.05. Results: A total of 193 patients with MBC were treated with ADCs between August 2014-February 2023. Among these,32 patients were identified as having received more than one ADC (HR+/HER2- = 13, TNBC = 19, HER2 low = 22). Median age at time of second ADC was 57.1 years (range 31.3-88.6) and patients had received a median of 4 lines (range 2-12) of prior treatment before initiation of second ADC. The median PFS on the first ADC used (ADC1) was significantly longer at 7.55 months (95% CI 3.22-10.25) compared to a median of 2.53 months on the second ADC (ADC2) (95% CI 1.38-4.14) (p=0.006). PFS for ADC2 with antibody target change was 3.25 months (95% CI 1.38 months, n/a) compared to 2.30 months with no target change (95% CI 1.38 months, n/a) (p=0.16). At time of first imaging, cross-resistance was present in 17 cases (53.1%), absent in 12 (37.5%), and not evaluable in 3 cases. When the second ADC contained the same antibody target as the first, cross-resistance was present in 9/13 cases (69.2%), compared to 8/16 cases (50.0%) when the second ADC targeted a different tumor antigen. Similarly, differences were noted based on payload switch vs not. Conclusions: This study highlights a subset that had cross-resistance to ADC after ADC, while others had durable responses on latter lines of therapy, particularly if a different antibody target was utilized. Further research is needed to validate these findings and discern mechanisms of clinical resistance to guide optimal sequencing of ADC-based treatment options.