Urothelial carcinoma (UC) of the bladder is the fourth most frequent cancer of male adults in the United States, with approximately 81,200 cases expected in 2022 (1). Outcomes are driven by the stage at which the disease is diagnosed, with a 5-year overall survival (OS) rate greater than 90% for patients diagnosed with non-muscle invasive bladder cancer (NMIBC), 50-82% for muscle invasive bladder cancer (MIBC) and 5% for metastatic disease (2). Currently approved management strategies for metastatic disease include chemotherapy such as GC (gemcitabine and cisplatin or carboplatin) or MVAC (methotrexate, vinblastine, doxorubicin, and cisplatin), immunotherapy (pembrolizumab, atezolizumab, or avelumab), or targeted therapy (erdafitinib for patients with certain FGFR3 or FGFR2 mutations). These therapies are limited by the toxicities associated with active chemotherapy regimens, the low response rates seen with immunotherapy, and the limited number of patients harboring the relevant biomarker. Fortunately, several novel therapies have been introduced in the management of advanced and metastatic disease that have the potential to improve these outcomes. Specifically, the introduction of antibody-drug conjugates (ADCs) such as enfortumab vedotin (EV) and sacituzumab govitecan (SG) have expanded treatment options for patients with advanced UC. Predictive biomarkers that could help to forecast outcomes following the use of specific therapies have not been incorporated into clinical practice. As treatment options are added to the clinician’s armamentarium, biomarkers become valuable in prioritizing active therapies for individual patients. For example, both EV and SG are approved as third line therapies for metastatic UC. Biomarker-driven data may aid in therapy selection between these two agents. Data from preclinical models and secondary analyses of clinical trials using ADCs suggest that several biomarkers exist that may improve therapy selection. Thus, in this review we will cover currently available information supporting the potential use of biomarkers in the management of advanced UC.

ADCs were first synthesized in the 1960s when small molecule chemotherapies were linked to immune gamma globulins (3). Since then, nine ADCs have been approved by the FDA for use in cancers including UC, lymphoma, acute myelogenous leukemia, and breast cancer (4, 5). ADCs are composed of a monoclonal antibody that binds to specific target antigens on the surface of tumor cells linked to anticancer agents such as chemotherapy drugs (6). By delivering these anticancer agents directly to the target cells, ADCs increase therapeutic efficacy while decreasing potential systemic toxicities (7). Many of the antibodies used in the synthesis of ADCs are composed of immunoglobulin G and, instead of optimizing their ability to elicit an immune response, these antibodies are selected based on binding affinity, potential cross-reactivity, and immunogenicity (7, 8). Activation of immune responses following exposure to ADCs can lead to the formation of additional antibodies that bind to the ADC and compromise its efficacy (9, 10). Thus, due to its long half-life, stability in the serum and wide distribution in the intra and extravascular compartments, immunoglobulin G is an ideal candidate in the construction of ADCs (8). The ratio between the drug and the antibody must also be addressed, as a high ratio results in aggregation and clearance of the ADC while a low ratio results in low efficacy following drug delivery (11). Finally, the linker between the drug and the antibody must be optimized to allow stability in the circulation. Early release in the serum can lead to undue toxicity and decreased effect at the tumor (12, 13). However, in most cases the linker must also be able to be cleaved once the ADC is internalized into the target cell (14). Despite these requirements, the introduction of ADCs into clinical practice has been increasing significantly over the past decade. Table 1 summarizes ADCs that have been studied in UC and their mechanism of action. Table 2 summarizes ongoing studies investigating the role of ADCs in UC. Unfortunately, many of these ADCs have been studied independent of biomarker testing to predict treatment response, toxicities and potential resistance mechanisms.

EV is a fully human monoclonal antibody conjugated by a protease-cleavable linker to monomethyl auristatin E (MMAE). The delivery of MMAE to the tumor cell results in inhibition of microtubule formation and cell death through interruption of cell division (15, 16). The antibody component of EV binds to Nectin-4 on the surface of the cell, an immunoglobulin-like transmembrane protein that participates in the formation of the adherens junction (17–20). Nectin-4 is enriched in placental and embryonic tissues as well as squamous epithelial tissues such as the skin where it plays a role in cellular adhesion and immune evasion (21, 22). Nectin-4 is also overexpressed in several cancers including UC as well as breast, ovarian, lung and gastric carcinomas (14, 17, 23–25). In upper tract UC, upregulation of Nectin-4 is associated with poor outcomes, including a shortened progression free survival (26). By targeting Nectin-4, EV delivers MMAE directly to the tumor cells. Although response to EV has not been proven to correlate with levels of Nectin-4, in preclinical models resistance to EV can be induced through downregulation of Nectin-4 suggesting a relationship between Nectin-4 levels and EV sensitivity (19).

EV was granted accelerated FDA approval in December of 2019 for the treatment of patients with advanced UC that progressed following prior treatment with chemotherapy and immunotherapy. This approval was based on the results of a phase II single arm trial evaluating EV as third line therapy. The overall response rate in this study was 44% (95% confidence interval (CI) 35.1 – 53.2) with a median response duration of 7.6 months (95% CI 6.3 – not reached) (15). EV was fully approved following the completion of the phase III EV-301 study comparing EV to physician’s choice chemotherapy (docetaxel, paclitaxel or vinflunine). Compared to chemotherapy, EV significantly prolonged meaningful outcomes with similar toxicity rates. More specifically, the median progression free survival with EV was 5.55 months versus 3.71 months (HR 0.62; 95% CI 0.51 to 0.75, p<0.01) and the median overall survival was 12.88 months versus 8.97 months (HR 0.7; 95% CI 0.56 – 0.89, p=0.001) (27).

SG is a humanized antibody against Trop-2 that is linked with a hydrolysable linker to the small molecule chemotherapy agent SN-38 (32, 33). An inhibitor of topoisomerase I (topo-I), SN-38 acts to destabilize the interaction between this protein and the DNA resulting in lethal DNA double strand breaks during replication that cannot be repaired (34). Trop-2 is a glycoprotein found on the cellular membrane of trophoblastic cells during development as well as stratified squamous epithelial cells in the skin, esophagus, tonsillar crypts and uterine cervix where it plays a role in calcium signalling, proliferation and several other regulatory signalling pathways (35–38). Trop-2 is also overexpressed in epithelial cancers including up to 83% of UC where it is associated with aggressive disease and poor prognosis (36, 37, 39). SG is currently approved for use in triple negative breast cancer (TNBC) and UC, however continued approval for use in bladder cancer patients will depend on the outcomes observed in the phase III TROPiCS-04 trial (NCT04527991).

Previously referred to as ASG15-ME, sirtratumab vedotin (SV) is a humanized IgG2 antibody against SLITRK6 linked by a protease cleavable linker to MMAE. SLITRK6 is a transmembrane protein that is overexpressed in UC, breast and lung cancer as well as glioblastoma multiforme (47, 48). In a phase I trial of 51 patients with metastatic UC, 93% stained positively for SLITRK6 (48). However, evaluation of whether expression of this therapeutic target would serve adequately as a predictive biomarker of treatment response remains lacking.

Several HER-2 targeted therapies are under investigation in the treatment of UC. Although initial trials with lapatinib and trastuzumab did not show efficacy, more recent trials with ADCs show promise (49–51). Trastuzumab emtansine (T-DM1) is composed of an anti-HER2 antibody joined with a non-cleavable linker to emtansine, a microtubule inhibitor. Preclinical models suggest efficacy in UC cells that overexpress HER2. Although a phase II basket trial showed no response in UC patients, several studies are ongoing (NCT02999672, NCT02675829) (52). A higher response rate of 61% in UC patients was seen in a phase II trial of disitamab vedotin (RC48-ADC), an ADC composed of a humanized anti-HER2 antibody conjugated to MMAE (53, 54). A phase II trial studying MRG002, another ADC composed of a humanized anti-HER2 antibody conjugated to MMAE, has identified a safe treatment dose and has observed an overall response rate of 65% (NCT04839510) (55). Other ADCS that target HER-2 currently under investigation in UC include trastuzumab duocarmazine and trastuzumab deruxtecan. The majority of these trials require HER-2 expression levels that are 3+ by IHC or positive by FISH. However, recent studies of trastuzumab deruxtecan in breast cancer suggest these definitions may be broadened, as responses to this drug were seen in patients with HER2-low tumors (defined as 1+ by ICH or 2+ with negative FISH testing) (56). Studies suggest HER-2 is overexpressed in at most 20% of UCs, but evaluation for HER2-low status has not been investigated (4, 57). Based on these findings, validation of HER-2 levels as a predictive biomarker to aid in therapy selection will be critical.

In 2021 the American Society of Clinical Oncology released a Report on Progress Against Cancer that underscored the importance of including both tissue and blood-based biomarkers in clinical trials. Clinically useful biomarkers should aid physicians and other health care professionals in predicting response to treatment, potential toxicities and resistance mechanisms to therapy (58). Treatment with ADCs are particularly well suited to the integration of biomarkers, as their efficacy depends on the presence of both the antibody target and sensitivity to the delivered anticancer agent. However, trials that have completed and are currently recruiting patients have by and large forgone this potentially fruitful avenue of investigation. Although initial studies suggested high expression of the ADC target, further retrospective and cohort trials identified heterogeneity in expression of proteins such as Nectin-4 and Trop-2 that appear to be critical for ADC efficacy. These raise questions regarding whether expression levels in the primary tumor can be used to evaluate patients with metastatic disease, as well as whether patients with alternative tissue histologies should undergo further testing prior to therapy initiation. Prior therapeutic exposures may also alter biomarker expression, suggesting repeat biopsies may be necessary. Biomarkers remain an untapped resource in the clinical management of patients with advanced UC, and as Jindal et al. showed in their retrospective study of UC we likely have only uncovered the tip of the iceberg in identifying potentially beneficial biomarkers (31).

SF and DV contributed equally to the conceptualization, writing, and editing of this article. All authors contributed to the article and approved the submitted version.