EV is an ADC that is comprised of a human monoclonal antibody against nectin-4 conjugated to monomethyl auristatin E (MMAE), an inhibitor of microtubule formation, via a protease-cleavable linker (16, 17). Nectin-4 is a transmembrane protein that is highly expressed in multiple solid tumors, namely urothelial, gastric, and breast carcinomas, and is a marker of poor prognosis (18–20). When EV binds nectin-4, it becomes internalized and releases MMAE which disrupts microtubule formation and leads to cell-cycle arrest in tumor cells. Clinically, EV has shown promising efficacy in treatment of mUC. EV-201 was a single-arm, phase II clinical study of EV monotherapy that demonstrated a 52% overall response rate (ORR) in patients with mUC who had received previous CPI treatment without chemotherapy, highlighting the efficacy of EV in patients with limited treatment options (21, 22). The phase III trial EV-301 showed that EV monotherapy improved medial overall survival (mOS) (12.9 vs. 9.0 months; hazard ratio [HR] 0.70) and median progression-free survival (mPFS) (5.6 vs. 3.7 months; HR 0.62) compared to standard chemotherapy post-platinum and post-CPI over a median follow-up of 24 months. The ORR was 40.6% with EV compared to 17.9% with chemotherapy. Skin toxicity, hyperglycemia, and peripheral neuropathy are adverse events of particular interest when using EV.

EV leapt from the third line to the first line setting after EV-302 showed an improvement in OS in patients treated with EV plus pembrolizumab (EV-P) regardless of cisplatin eligibility (10). A new benchmark for median survival was set at 31.5 months vs 16.1 months with platinum doublet chemotherapy (hazard ratio 0.47; 95% CI 0.38 to 0.58, P<0.001). A total of 67.7% of patients responded to EV-P compared to 44.4% of patients who received chemotherapy; this includes a complete response (CR) rate of 29.1%. The improvement in survival was seen in both cisplatin-eligible and ineligible patients, a finding that has since made cisplatin eligibility criteria, largely in the first-line setting, a relic of the past (23). We know from an updated analysis presented at ASCO 2025 that of patients who achieved a CR, 74.3% of them maintained it 24 months later, indicating the durability of this response and opening the door to a potential cure in mUC (24).

Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase inhibitor involved in cell proliferation and differentiation that is overexpressed in many solid tumors including urothelial carcinoma (25, 26). Trastuzumab deruxtecan (T-DXd) is a HER2-directed ADC composed of an anti-HER2 antibody, topoisomerase I inhibitor payload, and a tetrapeptide-based cleavable linker (27, 28). In 2023, DESTINY-PanTumor02, a phase II trial, demonstrated that T-DXd had durable antitumor activity across many HER2 positive tumor types, including bladder carcinoma (n=41). The response rate was 56.3% in patients with UC with HER2 IHC 3+ and 35.0% in those with IHC 2+ (11). The tissue-agnostic approval of this agent in cancers with HER2 IHC 3+ has led to routine testing for this biomarker in the later line setting of mUC. Recent data from a phase Ib, non-randomized study showed that combining T-DXd with nivolumab, a PD-1 inhibitor, had synergistic anti-tumor activity in HER2-positive mUC (29). Pneumonitis remains an adverse event of interest with use of this drug.

Disitamab vedotin (DV) is also a HER2-targetng ADC with a MMAE payload, like EV. Initially, DV monotherapy demonstrated potent antitumor activity in patients with HER2-positive locally advanced or metastatic UC who had progressed on at least one line of chemotherapy (30). In combined analysis of two-phase II clinical trials, DV monotherapy achieved an ORR of 50.5% of patients with HER2-positive, locally advanced or metastatic UC who had progressed on prior therapies (31–33). The median PFS was 5.9 months, and the median OS was 14.2 months. Beyond monotherapy, DV has been evaluated in combination with CPI to enhance antitumor activity (34–36). When combined with toripalimab in an HER2 unselected population within a phase Ib/2 trial, the ORR was 76% in treatment-naïve patients, 83.3% in those who were HER2 IHC 3+/2+, and even 33.3% in those who were completely negative for HER2 expression on IHC (32). An ongoing phase 3 study of DV plus pembrolizumab versus chemotherapy in patients with HER2-expressing mUC may further elucidate its role in the treatment of mUC (37).

Sacituzumab govitecan (SG) is an ADC that is composed of an anti-trophoblast cell surface protein (Trop-2) IgG1 monoclonal antibody linked to an SN-38 payload. SN-38 is a metabolite of irinotecan, a topoisomerase I inhibitor (38). The phase II trial TROPHY-U-01 studied the efficacy of SG in patients with mUC who were heavily pretreated with both chemotherapy and immunotherapy. In the single-arm study, SG demonstrated an ORR of 28% with mPFS of 5.4 months and OS of 10.9 months. The study showed that SG had sustained clinical benefit independent of PD-L1 expression or prior response to checkpoint inhibition, leading to the accelerated FDA approval of SG for patients with mUC (39). More recently, TROPiCS-04 was a phase III randomized trial that compared SG to TPC (paclitaxel, docetaxel, or vinflunine) in the same patient population (40). Unfortunately, SG did not result in a statistically significant improvement in OS or PFS when compared to standard chemotherapy. However, the ORR was higher at 23% for SG compared to single-agent chemotherapy (14%). There was an enrichment in neutropenic deaths occurring within the SG-containing arm, most occurring within the first month of treatment, which may have at least in some part contributed to the negative results of this phase 1 study. Only 21% of patients received granulocyte colony stimulating factor (G-CSF) as primary prophylaxis was not mandated. This negative trial led to voluntary withdrawal of the accelerated approval status for SG by the manufacturer. Questions remain about the future of SG in mUC.

Datopotamab deruxtecan (Dato-DXd) is another ADC that targets Trop-2 with a topoisomerase I inhibitor payload. Trop-2 is overexpressed in several epithelial tumors, including UC, and is a marker of aggressive tumor behavior (41, 42). TROPION-PanTumor01 is an ongoing, phase 1, multicohort study investigating Dato-DXd in multiple tumor types and the early data from mUC was presented in 2025. A total of 40 patients were treated with the drug in the study, resulting in an ORR of 25%, disease control rate (DCR) of 77.5%, and a 6-month duration of response rate of 76.2%. This study was conducted in a heavily pre-treated population with 50% of patients having received at least 3 prior lines of therapy in the metastatic setting (43). Pneumonitis and stomatitis are the most concerning adverse events with this agent (44).

Sacituzumab tirumotecan (Sac-TMT) is another Trop-2 targeting ADC linked to a topoisomerase I payload. A phase I trial of this agent was conducted in a patient population previously treated with prior platinum doublet chemotherapy and immunotherapy (45). In the patients who received sac-TMT in the second line (N = 11), the ORR was 45.5%; it was 26.3% in those which received it in the third line and beyond (N = 38). The primary adverse events that occurred were anemia (38.8%) and decreased neutrophil count (28.6%), however there were no treatment-related deaths to date.

ADC and FGFR inhibitors target specific antigens on the surface of tumor cells. Therefore, a common proposed mechanism of resistance is tumor cell alteration of its surface antigen expression level. Loganzo et al. studied the mechanisms by which breast cancer cell lines develop resistance to ADCs through persistent drug exposure and found that downregulation of HER2 antigen or increased ABCC1 protein expression were the driving mechanisms (57). This preclinical study suggested that tumor heterogeneity in HER2 expression may correlate to ADC efficacy and thereby to clinical outcomes. These results were corroborated in the KRISTINE and ZEPHIR clinical trials which found that tumors with increased heterogeneity in HER2 expression prior to treatment with ADCs had worse clinical outcomes when compared to patients with tumors of low heterogeneity (58, 59).

To date, several studies have found that NECTIN-4 amplification predicts EV response in mUC. Klümper et al. demonstrated that NECTIN-4 protein expression, independent of gene amplification, is downregulated during metastatic progression of mUC and that its expression correlates with EV response (60). This study suggests that NECTIN-4 amplification can be used as a genomic biomarker for patients with mUC undergoing EV therapy to predict clinical response. Similarly, alteration of target antigen is a common resistance mechanism against FGFR inhibitors. Gatekeeper mutations in FGFR3, such asV555M, following administration of FGFR inhibitors in non-small cell lung cancer can interfere with drug binding, leading to eventual erdafitinib resistance (61, 62).

After binding to its surface antigen, ADCs are internalized into tumor cells via receptor-mediated endocytosis. ADC efficacy is reliant on target-mediated endocytosis to deliver its cytotoxic payload at sufficient concentrations to cause tumor cytotoxicity (63). There are several endocytosis pathways that mediate ADC uptake: clathrin-mediated endocytosis (CME), clathrin-independent endocytosis, and caveolae-mediated endocytosis (64, 65). CME is a receptor-mediated endocytosis process and is the most thoroughly studied in ADC transport. Briefly, the ADC-antigen complex gets internalized through clathrin-coated pits which invaginate and form vesicles with endosomes. The ADC is then trafficked through the endosomal-lysosomal pathway in which the cytotoxic payload is released. Sung et al. developed in vitro ADC-resistant breast cancer cells lines that internalize ADCs into caveolin-1 (CAV1)-coated vesicles which alters their trafficking to lysosomes (66). ADC colocalization into CAV1 vesicles reduced therapy response and was a negative biomarker for patient response. These results underscore the importance of understanding endocytosis pathways as alterations can impact therapeutic outcomes.

Another common mechanism of ADC resistance is elimination of the drug by the overexpression of ATP-binding cassette (ABC) transporters, which actively efflux the cytotoxic payloads out of the cancer cells. ABCB1 (P-glycoprotein) and ABCC1 (multidrug resistance-associated protein 1 [MDR1]) are the most well-characterized ABC transporters, which rely on ATP hydrolysis to translocate ADC payloads across cell members and thereby reduce its intracellular concentration. Corbett et al. showed that upregulation of ABCB1 and ABCC1 transporters was associated with resistance to ADC-containing pyrrolobenzodiazepine (PBD) and that inhibiting these transporters could restore drug sensitivity in previously resistant cancer cells (67). Similarly, T-DM1–resistant cells had an increased expression of ABC transporters despite preserved HER2 overexpression, and pharmacologic inhibition of these transporters reinstated T-DM1 responsiveness (68).

Upregulation of drug-efflux pumps correlates to enhanced toxic payload resistance. Studies have found that upregulation of drug efflux transporters such as P-glycoprotein is associated with resistance to MMAE, the payload used in EV. Chang et al. created EV-resistant bladder cancer cell lines in vitro through upregulation of P-glycoprotein and TGF-β genes which led to decreased sensitivity to MMAE (69). They found that resistance to EV was largely attributable to resistance to the payload MMAE rather than downregulation of surface antigen Nectin-4.

Activation of alternative signaling pathways is another mechanism of resistance to ADCs and FGFR inhibitors. One prominent mechanism involves the activation of PI3K/AKT/mTOR pathway which promotes tumor cell survival and proliferation. This pathway has been most prominently studied with trastuzumab which showed that PIK3CA mutations resulted in decreased sensitivity to the ADC, and that adding a PI3K inhibitor to trastuzumab had enhanced anti-tumor activity in HER2-positive metastatic breast cancer (70, 71). In mUC, upregulation of the PI3K/AKT/mTOR pathway correlates with increased EV resistance and enhanced tumor cell survival (72). Another critical bypass signaling pathway involves the TGF-β signaling pathway, which can induce the epithelial-mesenchymal transition (EMT) and enhanced tumor metastasis. In urothelial cancer, TGF-β signaling has been implicated in resistance to EV.

Similarly, resistance to FGFR3 inhibitors in urothelial carcinoma can occur through activation of downstream signaling pathways. Hosni et al. demonstrated that adipocyte precursor-derived neuregulin 1 (NRG1) promotes resistance to FGFR inhibition by activating the epidermal growth factor receptor 3 (ERBB3; also known as HER3) signaling pathway, which can bypass the inhibited FGFR3 pathway to sustain tumor cell proliferation (73). In another study, Weickhardt et al. showed that increased expression of phosphorylated ERBB3 is a key resistance mechanism in FGFR3-dependent bladder cancer and that dual targeting of FGFR3 and ERBB3 delayed the reactivation of pERBB3 and enhanced FGFR inhibitor efficacy (74). Better understanding of these signaling pathways is crucial in developing therapies that counteract resistance mechanisms and improve clinical outcomes.

The TME consists of network of stromal cells, immune cells, extracellular matrix, and soluble factors that interact with tumor cells. The TME plays an important role in tumor progression, metastasis, and immune escape which engenders resistance to ADCs. In addition to direct cytotoxicity, ADCs can induce immunogenic cell death (ICD), which is the release of damage-associated molecular proteins (DAMPs) such as calreticulin, ATP, and HMGB1 from dying tumor cells (75, 76). These DAMPs then activate dendritic cells (DCs) which in turn activate T cells to generate a robust antitumor response. Tumor cells can create an immunosuppressive TME phenotype through the recruitment of regulatory T cells (Tregs), MDSCs, and secretion of cytokines such as TGF-B. These changes decrease the effectiveness of immune effector cells, thereby inhibiting ICD and reducing the efficacy of ADCs. Recent studies have found that ADCs conjugated with payloads known to be strong modulators of the immune microenvironment such as pyrrolobenzodiazepine or tubulysin more effectively induce ICD, thereby synergizing with CPI (77, 78).

One significant mechanism involves upregulation of immune checkpoint molecules, such as programmed death-ligand 1 (PD-L1), which inhibits the cytotoxic activity of immune cells. This upregulation of PD-L1 can be a response to the inflammatory response induced by ADCs and creates an immunosuppressive TME, leading to an adaptive resistance mechanism. These changes decrease the effectiveness of immune effector cells, thereby reducing the efficacy of ADCs. As discussed above, combining ADCs or FGFR inhibitors with CPIs can potentially overcome this resistance mechanism.

Ouyang et al. explored the impact of FGFR3 alterations in bladder cancer TME and demonstrated that mutant FGFR3 indirectly induces an immunosuppressive TME by increasing serine synthesis. This activates the PI3K/Akt pathway and suppresses macrophage immunostimulatory functions, shifting them toward an immune-inert phenotype (79). Targeting PI3K in FGFR3 tumors reversed the macrophage phenotype and demonstrated synergistic antitumor activity when combined with erdafitinib. Overcoming these immunosuppressive modulations of TME through direct inhibitors is a potential strategy to enhance the effectiveness of ADC and FGFR3-inhibitors in mUC.

Overcoming tumor resistance to ADCs and FGFR3 inhibitors to increase clinical response rate requires a multifaceted approach. One promising strategy is the use of combination therapies. Combining ADCs with ICI both leverages the cytotoxic effects of ADCs while also activating the host’s immune system to target UC tumor cells. EV was initially approved as a single agent in the third line setting of mUC after prior platinum and prior CPI therapy. EV-302 study, a phase III randomized clinical trial, showed that when combined with pembrolizumab, EV had an overall response rate (ORR) of 67.7% compared to 45.2% in EV monotherapy arm in patients with untreated la/m UC (10, 80). These results highlight the synergistic and not just additive mechanism of action between EV and pembrolizumab and led to a change in the standard of care management of mUC (10, 81). However, despite these promising results, it remains uncertain whether the observed benefits represent true synergy or reflect additive effects from two separate agents. Additionally, overlapping toxicities such as rash, peripheral neuropathy, and immune-related adverse events require careful management and may limit broader applicability in frailer populations.

SC is another ADC which has been studied in combination with CPI. Results of the JAVELIN Bladder Medley trial were presented at ASCO 2025, a phase 2 study investigating maintenance avelumab plus SG versus avelumab alone. The study showed improved PFS but there was no significant difference in OS at the time of this interim analysis and there was toxicity incurrent in the investigational arm consistent with the known profile of SG (82). These findings underscore that not all ADC–ICI combinations yield clear survival advantages, and further data are needed to determine whether improved progression data translate to durable, clinical benefit. There is an enrichment of Trop-2 expression in variant histology and ongoing trials in rare bladder histology are of interest. The SMART trial is investigating SG with or without atezolizumab in locally advanced unresectable or metastatic rare genitourinary cancers including small cell, squamous cell, and adenocarcinoma of the bladder (NCT06161532) (83). The phase 1 DAD trial showed that combination EV and SG is possible. Of the 23 enrolled patients, the response rate was 70% with a manageable safety profile once prophylactic G-CSF was added (84). The DAD-IO trial builds on this concept and is a phase 2 trial of combination EV, SG and pembrolizumab in the first-line setting (85).

Similarly, overcoming resistance to FGFR inhibitors can also be achieved through combination therapies targeting complementary pathways. The BISCAY trial combined durvalumab, a PD-L1 inhibitor, with FGFR inhibitors in mUC but did not show enhanced efficacy over durvalumab monotherapy (86). In contrast, the NORSE study evaluated the combination of erdafitinib with cetrelimab, a PD-1 inhibitor, in FGFR-altered mUC, which had a ORR of 54.5% and a 12-month OS rate of 68%, compared to an ORR of 44.2% and a 12-month OS rate of 56% for erdafitinib monotherapy (87). This study could be interpreted as suggesting that combining FGFR inhibition with ICIs may enhance therapeutic efficacy by reprogramming the TME to support antitumor immunity. Although, it is not clear whether the efficacy is simply additive because of the two classes of agents. Moreover, long-term toxicity profiles of these combinations remain incompletely characterized, particularly given overlapping risks such as hyperphosphatemia, ocular events, and immune-related toxicities. Lastly, targeting bypass signaling pathways that contribute to resistance is another effective strategy. The phosphoinositide 3-kinase (PI3K) pathway has been identified as a key determinant of resistance to FGFR inhibitors (88). Inhibition of PI3K with agents such as BKM120 has been shown to act synergistically with FGFR inhibitors, enhancing their efficacy in urothelial carcinoma cell lines harboring FGFR mutations (88–90). However, these findings are largely preclinical, and translating synergistic activity into clinical benefit will require further evaluation of combinatorial dosing and scheduling in future trials.

One strategy to overcome resistance is reprogramming the immune-suppressive features within the TME. For instance, MDSCs, tumor-associated macrophages (TAMs), and regulatory T cells (Tregs) facilitate an immunosuppressive TME which limits drug efficacy. Targeting these immune-suppressive cells with immune-modulating therapies such as CSF1R inhibitors can deplete TAMs. Resistance to FGFR inhibitors is mediated by the TME through promotion of TAMs which can secrete factors that activate the FGFR pathway, leading to tumor cell survival. Another approach involves remodeling the extracellular matrix (ECM) which can act as a physical barrier to drug delivery. Enzymatic degradation of the ECM by hyaluronidase can enhance the penetration of DCs within the tumor (91, 92). Normalization of tumor vasculature through anti-angiogenic agents such as vascular endothelial growth factor (VEGF) inhibitors or EGFR tyrosine kinase inhibitors (TKIs), such as osimertinib, may be useful.

Moreover, the TME can influence the expression of drug efflux pumps and enzymes that deactivate the cytotoxic payload. Resistance mechanisms that increase drug excretion by overexpressing drug efflux pumps can be overcome by implementing direct inhibitors of these pumps. Tariquidar, an ATPase inhibitor of P-glycoprotein drug efflux pump, has been used in combination with chemotherapy to increase drug exposure in resistance cancers, including renal cell carcinoma (93, 94). While no studies yet exist combining ADCs with drug efflux pump inhibitors, Cabaud et al. demonstrated that resistance to an anti-nectin 4 ADC could be reversed by tariquidar in preclinical breast cancer model (95).

One of the most direct strategies to circumvent resistance to ADCs and FGFR inhibitors is structural modification of these drugs to create next-generation compounds capable of overcoming established resistance mechanisms. There are three components of ADCs that can be altered to prevent resistance: antigen targeting, improved linker technology, and payload delivery. Novel bispecific ADCs that target two tumor cell antigens can reduce the likelihood of resistance due to antigen loss in tumor cells with low antigen expression. Dual targeting of HER2 by biparatopic ADCs has been shown to enhance toxin delivery to breast cancer cells with significant intratumor heterogeneity of HER2 expression (96). Preliminary results from a phase I study on a HER2 bispecific ADC, zanidatamad zovodotin, showed an ORR of 28% with disease control rate of 72% across multiple cancer types (97). Additionally, new bispecific ADCs that selectively bind two different antigens, such as Trop2 and Nectin4, are being studied in UC to enhance its selectivity and minimize off target effects (98). In addition, novel technologies like the bicycle drug conjugate (BDC) may be helpful to this end. These molecules are tiny with a short plasma half-life, holding the potential for increased tumor exposure and decreased normal tissue activity. Zelenectide pevedotin is a BDC highly selective for Nectin-4 and linked to MMAE. This agent is currently being studied within the DURAVELO-2 phase 2/3 study of the agent as monotherapy as well as in combination with pembrolizumab versus platinum doublet chemotherapy (99).

Secondly, linker structure can be modified to further optimize ADCs. Recent advancements have led to the development of cleavable linkers that are sensitive to specific TME conditions that ensure the payload is released within tumor cells. Incorporation of hydrophilic XTEN polypeptides in linker design have resulted in ADCs with extended half-lives (100). Lastly, payload delivery can be modified. Li et al. studied how the concentration of payload release affects ADC efficacy and found that the higher rates of payload delivery correlated to enhanced killing of neighboring tumor cells that did not express the target antigen, also known as the bystander effect (101). Creation of dual-drug ADCs, ADCs that deliver two distinct cytotoxic payloads, has shown synergistic antitumor activity in UC mouse models (102–104). Ongoing research aims to optimize the potential of dual-drug ADCs to overcome resistance where single-drug ADCs have failed in refractory tumor types.

Development of next generation of FGFR inhibitors is currently ongoing. Resistance to these agents often arise from secondary mutations in the FGFR kinase domain such as gatekeeper mutations like V555M in FGFR3 that reduce drug binding. Futibatinib, an irreversible FGFR1–4 inhibitor, forms covalent bonds with the kinase and maintains efficacy even in patients with solid tumors that have acquired resistance to prior FGFR inhibitors (105). Similar to ADCs, dual-targeted inhibitors are being engineered that can bypass resistance of activating signaling pathways such as EGFR, P13K/AKT, and MAPK pathways. Dual targeting of FGFR3 and ERBB3 showed to overcome resistance of FGFR3-fusion driven bladder cancer (74).