Antibody Drug Conjugates in the Treatment of Urothelial Cancer: The Dawn of a New Era

1. Introduction

Urothelial cancer is the ninth most common cancer worldwide [1]. It is characterized by high recurrence and progression rates; thus, effective treatments are urgently needed. Its prognosis depends on the initial stage presentation. Most cases are diagnosed with non-muscle invasive disease (NMIBC), where local treatments are the most common form of therapy, usually a combination of intravesical instillations and maximum transurethral excision. For the rest of the patients with muscle-invasive disease, some form of systemic treatment will be administered in the course of their disease. Chemotherapy is the most widely used form of systemic treatment, and it can be administered as neoadjuvant or adjuvant treatment in the early stages, as a part of the bladder preservation approach, and in the advanced-metastatic setting. The agent associated with the most significant clinical benefit is cisplatin (usually as part of multidrug regimens). Agents such as carboplatin, taxanes, and vinflunine represent clinically meaningful alternatives for patients not fit or already exposed to cisplatin, especially in the later lines of therapy[2,3,4].

Immunotherapy -in the form of anti-PD-1/L1 monoclonal antibodies- has also been part of the therapeutic algorithm for urothelial cancer. Initially, it was used in the platinum refractory / cisplatin-ineligible setting with mixed results [5,6,7]. In the first-line setting, three of the four trials that tested the concurrent combination of immunotherapy with chemotherapy were negative. Until very recently, the current standard was the administration of 1st line chemotherapy with a platinum agent (cisplatin either or carboplatin), and for those patients who had not progressed, immunotherapy (agent: avelumab) is then administered as maintenance treatment (switched maintenance) following the scheme of Javelin Bladder 100 trial. Alternatively, for the cisplatin-eligible patients, there is also the choice of combining immunotherapy (agent: nivolumab) upfront with chemotherapy, following the scheme of the Checkmate 901 trial, the only positive trial of this combination so far [8,9]. Immunotherapy has also successfully increased the overall survival of patients with high-risk early disease when administered as an adjuvant treatment after cystectomy [10,11]. For the minority of patients harboring certain DNA alterations in their tumors, such as fibroblast growth factor receptor (FGFR) gene alterations, treatment with erdafitinib, a tyrosine kinase inhibitor, is also a valid option [12].

Despite these promising advances that significantly increased the therapeutic choices and offered a meaningful benefit to some patients, there are still unmet needs in the treatment of this highly lethal disease since its treatment in advanced stages has seen limited progress over the last few decades. We can expect a new class of drugs that can benefit patients irrespective of cisplatin eligibility, platinum resistance, or absence of PD-L1 positivity in the future, and this is precisely the case we will examine for antibody-drug conjugates.

2. Mechanisms of Action, Toxicity and Resistance

2.1. Mechanisms of Action

ADCs, or antibody-drug conjugates, are carriers that mimic the Trojan horse method in their complex mode of operation. Utilizing monoclonal antibodies, they can deliver high doses of cytotoxic payloads directly to tumor cells or the structural elements of the tumor microenvironment (TME). This allows them to exert their anti-tumor activity and cause cell death while having little to no effect on normal cells and selectively driving cancer cells to apoptosis. Three primary components define the ADCs architecture: the monoclonal antibody (mAb) that is rapidly released into the targeted tumor cells and ultra-selectively targets tumor antigens thus enchasing specificity; the cytotoxic payload engineered to break DNA or tubulin mechanism within the targeted cell, resulting in high efficacy, and finally the molecular binder that bridges the two, forming the antibody-drug conjugate. Immunoglobulin G is the hardware used the most for the antibody production of ADCs, taking advantage of their potential cross-reactivity, binding affinity, and immunogenicity. After exposure to ADCs, activation of immune responses is often observed, resulting in the generation of antibodies that ultimately attach to the ADC, undermining its role and effectiveness. Following this consideration, it is critical to point out the drug-to-antibody proportion as a factor that needs to be constantly assessed. Namely, a high ratio leads to the formation of aggregates and rapid destruction of the antibody-drug conjugate (ADC), while a low ratio leads to reduced effectiveness. Ultimately, the drug-antibody linkage should be adjusted to be stable enough while circulating in the bloodstream. A premature release of the cytotoxic payload in the serum can result in excessive toxicity and inevitably reduced effectiveness at the tumor site. Additionally, the linker must be capable of being cleaved after the ADC has been internalized into the target cell [13]. Upon target cells are at a proximal distance, the ADC (antibody-drug conjugate) identifies the antigens, attaches to the cell surface, and, through endocytosis, follows the internalization of the ADC-antigen complex into the cancer cell. The transitional path of the newly formed complex starts as an early endosome, followed by maturation into late endosomes, and ultimately fusing with lysosomes. As the procedure continues, either an enzymatic digestion in the lysosomes or a chemical process disengages the cytotoxic active payload from the monoclonal antibody (mAb), exposing targeted cells to detrimental effects by triggering cellular apoptosis or premature death [14]. In addition, the Fc region of the monoclonal antibody enhances immune-related cytotoxicity, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP) and complement-dependent cytotoxicity (CDC) [15]. Significant progress in Genetic engineering techniques, leading to improved antibody effectiveness in the Fc region [16]. The use of ADCs offers the opportunity to expose malignant cells to high doses of toxic anti-neoplastic agents. Even higher compared to what would be possible with a standard, non-antibody-directed infusion. At the same time, minimal toxicity is present to healthy cells surrounding the tumor microenvironment, given the lack of respective key antigens. An additional characteristic of ADC activity is the payload cell membrane permeability. An ability that could facilitate the apoptosis of bystander cells following the payload spillage from a targeted tumor cell. This is described as the Bystander effect. The capability, as mentioned above, may be affected by the drug (payload) to antibody ratio (DAR), which determines drug potency and serum clearance. The increased toxicity of bystander-killing payloads allows them to overcome tumor heterogeneity, enhancing the clinical potential of antibody-drug conjugates (ADC). Modern ADCs have been engineered to have a bystander-killing effect and a higher anti-neoplastic efficacy through linker optimization and conjugation [17]. This new drug category in comparison with standard chemotherapy, offers several advantages. This includes increased drug tolerability, significant cytotoxicity even at low drug concentrations, stability in the bloodstream and lysosomes, and reduced systemic toxicity—important characteristics that lead to broader therapeutic implementation [18]. Figure 1 summarizes the mode of ADC action

2.2. Toxicity

A fundamental part of the ADCs design is the ability to minimize the exposure of the cytotoxic compound to healthy tissues, resulting in a narrow band of rather manageable toxicities. Among the most frequent are vomiting, diarrhea, nausea, and fatigue. Apart from these, severe toxicities of G3 or G4 have been reported, such as Steven Johnson, peripheral neuropathy, and myelosuppression. There is an association with peripheral neuropathy for the ADCs that use cleavable linkers bound to tubulin inhibitors. Clinical manifestations may include tingling, numbness, or pain in the extremities. All cleavable linkers block tubulin polymerization and have a strong association with early payload release, resulting in a rather expected toxicity profile. Given that microtubules significantly participate in the axonal transport as an important partner to the maturation and growth of neurons. As a result of myelosuppression, anemia, neutropenia, and thrombocytopenia of any grade may occur. Neutropenia is predominant, mostly due to the off-target Fc receptor-mediated uptake of ADCs into immune cells. Other reported toxicities that could lead to dose adjustments mainly include hepatotoxicity (for payload MMAF, DM1, and calicheamicin), skin toxicity (for payload MMAE and PBD), and ocular toxicity (for payload MMAF and DM4). Upon administration, Sacituzumab govitecan and Trastuzumab deruxtecan may present gastrointestinal side effects. In addition, Trastuzumab deruxtecan has an established association with an increased risk of interstitial lung disease (ILD), in most cases, requires dose adjustment in order to even fatal outcomes. As these novel therapeutic options extend their implementations, it is of the utmost importance for clinicians to be able to identify potential life-threatening toxicities and deep pharmacogenetic analysis to identify SNPs or potential mutations in key genes. Aiming to improve the tolerability and efficacy of ADCs [18].

2.3. Resistance

Inevitably, drug resistance occurs under treatment pressure. Malignant cells have a well-known ability to develop resistance mechanisms to avoid drug control. The exact mechanisms contributing to acquired ADC resistance are mostly unknown, but it is hypothesized that they may be related to individual parts of ADCs. The mechanisms related to this ability could be categorized into antigen down-regulation end or high-regulation, the drug efflux pumps, lysosomal function abnormality, and irregularity of signaling pathways controlling the cell cycle. Antigen-related resistance has a profound impact on the efficacy of ADCs. In this context, an association was spotted between treatment with brentuximab vedotin and antigen levels affecting the ADCs efficacy, whose multiple treatment cycles correlated with CD30 downregulation and, consequently, increased tumor cell resistance to MMAE. This example demonstrates how the presence of appropriate antigen ligands could potentially challenge the hypothesis of modulating the sensitivity of ADCs. Current research emphasizes that ligands, like neuregulin, which facilitates the heterodimerization of HER2 with HER3 and HER4, may reduce the efficacy of TDM-1. According to the following resistance-induced theory, ABC transporters are a family of transmembrane proteins that facilitate drug efflux across cell membranes. This is significant for ADCs, too, because these ABC transporters are substrates for many of the cytotoxic agents used in ADCs. Their role is to pump out various functional components from the tumor cellular cytoplasm, including therapeutic pharmaceutical payloads. That evacuation procedure increases resistance to potentially curative treatment. Given this mechanism, it is observed that cyclic dosing of TDM-1 induces an increase in ABCC1 transporter levels, resulting in acquired drug resistance. In addition to the above, impaired lysosomal function notably reduces the ADCs' therapeutic capability. This escape mechanism is involved in the acidification of lysosomal PH, especially those with non-cleavable and lysosomal protease-sensitive cleavable linkers. When linkers are cleaved by lysosomes, an acid hydrolase begins to take place, releasing a cytotoxic payload into the cytoplasm of targeted tumor cells. Malignant cells often acquire the ability to emit this process upon constant therapeutic exposure, altering the pH of the lysosomal compartment. This clever technique aims to slow down the proteases' catabolic procedure. Nevertheless, perturbations in signaling pathways involved in cell cycle regulation and alterations in apoptotic regulation may also modulate tumor cell sensitivity to ADC. In addition to the mechanisms mentioned above for ADCs, endocytic uptake is crucial; it can occur through various pathways such as clathrin-caveolin-independent endocytosis, clathrin-mediated (CME), and caveolin-mediated. Caveolae-mediated endocytosis responds to trastuzumab etamine (TDM-1) as a recently described resistance mechanism. It is of great importance to understand drug resistance mechanisms and implement effective strategies to overcome them. Regarding antigen down-regulation or deletion, novel ultra-specific combinations with other targeted cytotoxic molecules could be an appropriate response. Bi-payloads and immune-stimulating payloads may contribute to increased ADC effectiveness. New bispecific antibodies bearing characteristics of increasing tumor cell affinity, enhanced internalization efficiency, and notable resistance management could be future options. For cases exhibiting endocytosis deficiency, developing ADCs that do not internalize but instead directly release their payload into the tumor cell for absorption could also be a promising approach [18].

3. ADCs and Urothelial Cancer

3.1. Nectin 4

Nectins are calcium-independent immunoglobulin-like transmembrane molecules belonging to a family of four members that mediate cell-cell adhesion functions. Nectin 4 is highly expressed in embryonal and placentas tissues. Nectin 4 is also overexpressed in various human cancer types such as gastric, breast, ovarian, lung, pancreatic, and bladder. Nectin 4 is a significant contributor to cell growth and highly malignant tumor phenotype since its expression is implicated in cell migration and invasion. Various studies in cases of urothelial cancer indicate a level of expression around 80% for bladder cancer and 65,7% for upper urinary tract cancer, with strong expression recognized as an adverse prognostic factor [20,21,22].

3.1.1. Enforumab Vedotin

Enfortumab Vedotin (EV) is an ADC targeting Nectin 4; it is the most widely used ADC in urothelial cancer and the only one- so far- with regulatory approval in both the EU and the USA. EV contains a fully human IgG1 monoclonal antibody (AGS-22M6), which binds with high affinity against Nectin 4 and is conjugated with a cleavable linker to its payload monomethyl auristatin E (MMAE), a microtubule-disrupting toxin. The resulting DAR is approximately 4. Targeted delivery of MMAE results in cell-cycle arrest and apoptosis [22,23,24].

The clinical development of EV began in 2011 with its phase I trial (NCT01409135). This trial, which enrolled 42 patients with metastatic urothelial cancer (mUC), provided evidence about EV's safety and antitumor activity, paving the way for its further clinical use. Subsequent phase I dose escalation and expansion study EV101 (NCT02091999), established the dosing scheme of EV. EV has a half-life of approximately two days and is administered on days 1,8 and 15 of every 28-day cycle at a dose of 1.25mg/m2 [24,25].

EV-201 was a two-cohort, single-arm phase II trial and is considered the pivotal trial of EV in mUC after platinum and anti-PD1/PDL1 therapy. Cohort I enrolled 128 patients previously treated with platinum and anti-PD-1/L1 therapy. Patients were heavily pretreated, with a median of three systemic therapies (range: 1-6). The objective response rate was 44% (95% CI,35.1% to 53.2%), the median time to response was 1.84 months, the median duration of response was 7.6 months. 12% of the patients achieved complete response, and for them, the duration of response ranged from 3.6 to 11.3 months. At the time of analysis, 44% of all responders had ongoing responses. Responses were observed irrespective the site of metastasis, the presence of poor prognostic characteristics and regardless the of patient's response to prior anti-PD-1/L1 therapy. Cohort II enrolled 91 cisplatin-ineligible patients who were previously treated with only anti-PD-1/L1 therapy. The objective response rate was 52% (95% CI 41–62), median duration of response (DOR) was 10.9 months. The median progression-free survival (PFS) was 5.8 months for both cohorts, and the median overall survival (OS) was 11.7 months for cohort I and 14.7 months for cohort II [26,27].

As it concerned safety, EV-201 trial revealed a distinct pattern of toxicity for EV, with peripheral neuropathy, rash, hyperglycemia, and infusion-related reactions prespecified as composite terms. Peripheral neuropathy occurred in 50% of patients, almost all (94%) of which were grade 2 or less, with peripheral sensory more common than motor neuropathy. Treatment-related rash occurred in 48% of patients, most of which were low grade, with onset in the 1^st cycle. Only 2 patients discontinued treatment because of rash (one case was reported as Stevens-Johnson syndrome). Hyperglycemia occurred in 11% of patients, with only one grade IV case discontinuing treatment [26,27].

After the promising results of those earlier phase trials, the randomized phase 3 EV-301 trial evaluated EV as compared with chemotherapy, in mUC patients who had previously received treatment with a platinum compound and an anti-PD-1/L1 agent. The trial enrolled 608 patients in a 1:1 ratio to receive EV or chemotherapy (docetaxel, paclitaxel or vinflunine). The primary endpoint was OS, and after 11.1 months of median follow-up, treatment with EV resulted in 30% lower risk of death (hazard ratio, 0.70; 95% confidence interval [CI], 0.56 to 0.89; p= 0.001), indicating significantly longer OS with EV. Treatment with EV resulted in significantly longer PFS with 38% lower risk of progression or death (95% CI, 0.51 to 0.75; p<0.001). Median OS and PFS were 12.88 and 5.55 months respectively for EV vs 8.97 and 3.71 for the chemotherapy group. Among patients who responded to treatment, the EV group confirmed more than double the responses from the chemotherapy group (40.6% vs 17.9%). There were no new or unexpected toxicity events, and the already described toxicity of EV was regarded as mild to moderate and overall manageable [28]. Based on the OS data of this confirmatory trial, the U.S. FDA granted EV regular approval on July 9, 2021. In May 2022 EV was authorized by the EMA for use across E.U. [29,30].

There is accumulated evidence that the major drugs with clinical activity in mUC ADCs and anti-PD-1/L1 can act synergistically. The biological rationale behind this hypothesis is quite strong. ADCs have the potential to induce immunogenic cell death, and thus the release of damage-associated molecular patterns (DAMPs). DAMPs are easily recognized by elements of innate and adaptive immunity and via cross-presentation by cytotoxic T cells. These T cells are also a prime target for immune-modulating agents such as anti-PD-1/L1, increasing their antigen (tumor)-specific cytotoxicity. Thus, by combining EV with an anti-PD-1 like pembrolizumab the antitumor activity is enhanced versus either agent alone, since their respective immune properties act complementary. Based on these theories many clinical trials explored the possibility of combining these agents.

EV-103 (NCT03288545) was the first trial to assess the combination of EV and pembrolizumab in previously untreated advanced urothelial cancer (aUC) patients. EV-103 is a three-cohort (dose escalation, cohorts A and K), phase Ib/II study evaluating the safety, tolerability, and antitumor activity of first-line EV in combination with pembrolizumab. Cohort A enrolled 45 cisplatin-ineligible aUC/mUC patients. Confirmed ORR after a median of 9 cycles was 73.3% with a CR rate of 15.6%. The median DOR and OS were 25.6 and 26.1, respectively. No new safety signals were reported, and the safety profile was manageable. Since this combination appears to offer higher antitumor activity than that of carboplatin-based chemotherapy it received Breakthrough Therapy designation by the U.S FDA in February 2020. Cohort K randomized 149 patients to receive EV plus pembrolizumab or EV monotherapy in a 1:1 ratio. The combination arm exhibited a confirmed ORR of 64.5%, with rapid (median time to response was two months) and durable responses, with 65.4% of responders maintaining a response at 12 months [31,32].

EV-302 is a phase III, randomized trial comparing the efficacy of EV plus pembrolizumab combination with that of platinum-based chemotherapy in the first-line setting. The trial enrolled 886 patients in a 1:1 ratio to receive EV and pembrolizumab or platinum-based chemotherapy, cisplatin, or carboplatin -depending on their status as cisplatin eligible- with gemcitabine. Treatment was to be continued until occurrence of disease progression, unacceptable toxicity or completion of the maximum cycles, (six for chemotherapy, 35 for pembrolizumab); maintenance therapy was permitted according to the local standard. The primary endpoints were PFS and OS [33].

EV-302 is a positive trial in both its co-primary endpoints. Patients treated with EV and pembrolizumab had 55% lower risk of disease progression or death (HR, 0.45; 95% CI, 0.38 to 0.54; p<0.001) and 53% lower risk of death (HR, 0.47; 95% CI, 0.38 to 0.58; p<0.001) compared with the patients treated with chemotherapy. The median duration of PFS was 12.5 months for the EV-pembrolizumab group vs 6.3 months for the chemotherapy group, and the median was OS 31.5 months and 16.1 months, respectively. PFS and OS was consistent in all prespecified patient groups regardless of cisplatin eligibility status or PDL-1 positivity. At the 12-month time point, 78.2% of patients in the EV-pembrolizumab group were alive vs 61.4% in the chemotherapy group. ORR was higher in the EV-pembrolizumab group with 67.7% vs 44.4% in the chemotherapy group; 29.1% of patients achieved complete response vs 12.5% in the chemotherapy group. Among responders, 67.3% and 59.6% were still in remission in the EV-pembrolizumab group at 12 and 18 months, respectively, vs 35.2% and 19.3% for the chemotherapy group. The median duration of response in the EV-pembrolizumab group was not reached at the time of the data cutoff point (August 8, 2023). At the time of data cutoff, almost one-third of patients in the EV-pembrolizumab arm were still on treatment, vs none in the chemotherapy group. The most common subsequent therapy was platinum-based chemotherapy for the former and anti-PD-1/L1 for the latter, including 143 patients who received maintenance therapy (135 of them avelumab). Regarding safety, the toxicity profile was consistent with what was already known from previous trials for each agent. Fewer grade 3 or higher adverse events were reported for the patients in the EV-pembrolizumab group (55.9%) than those in the chemotherapy group (69.5%). Despite that fact, more patients discontinued treatment in the EV-pembrolizumab group (29.5%) than in the chemotherapy group (21.4%) as a result of treatment-related adverse events with the most common peripheral sensory neuropathy and anemia, respectively. Treatment-related deaths occurred in 4 patients in each group (<1%) [33,34].

3.2. TROP-2

TROP-2 is a transmembrane glycoprotein that is overexpressed among different types of epithelial cancers, such as urothelial carcinoma, while maintaining low expression in normal tissues [35]. Its presence correlates with poor prognosis and aggressive tumor behavior [19,36,37].

3.2.1. Sagituzumab Govitecan

Sacituzumab Govitecan(SG) utilizes α variation of humanized mAb against TROP-2, namely hRS7 IgG1κ conjugated with the active metabolite of irinotecan SN-38. Its role is to inhibit nuclear topoisomerase I (Topo-I), a milestone for DNA stability [38]. Sacituzumab Govitecan's engineered design is notable for its fast breakdown once it reaches the TROP-2-expressing cells and enchased linker stability in the bloodstream. The result is improved specificity and effectiveness [37,39]. The stable topoisomerase inhibitor uses a breakable linker to connect itself to a monoclonal antibody for therapy to be employed directly into the malignant cells without an off-target effect [40]. This unique design has increased importance because the cleavable nature of the linker almost guarantees that the cytotoxic payload is delivered within the tumor microenvironment [40]. Consequently, the desired outcome of minimal systemic exposure and toxicity is attained. Following the rationale mentioned above, the clinical effectiveness of SG was studied in locally advanced or mUC disease settings, including patients refractory to platinum-based chemotherapy and a checkpoint inhibitor (CPI) treatment. IMMU-132-01, a Phase I/II basket study, resulted in a median PFS of 7.3m and OS 18.9m; the median DOR was 12.6 months. Complete response (CR) for 2 subjects and 12 partial responses (PR), ORR 31%. Promising outcomes for SG, resulting in clinical efficacy for patients with mUC, including local relapsed or visceral disease, after receiving a standard chemotherapy regimen and anti-PD-1 or PD-L1 inhibitor therapies [41].

When used alone, pembrolizumab and SG have shown effectiveness in controlling solid tumors and have acceptable safety characteristics that do not overlap significantly. These treatments have been tested in patients with platinum-relapsed/refractory mUC. The hypothesis that a combination of these two agents would be secure and efficacious in this context was tested in cohorts of the TROPHY-U-01 trial. This is a phase II trial (NCT03547973) aimed to determine if the combination of sacituzumab govitecan (SG) with pembrolizumab could increase the (ORR and DOR, leading to improved clinical benefit rates (CBRs). The trial enrolled patients who had not previously been treated with checkpoint inhibitors and had progressive disease mUC after receiving platinum-based chemotherapy in or within 12 months after the neoadjuvant stage. The initial report with a median follow-up of 9.1 months resulted in an ORR of 27% (31 of 113; 95% CI, 19.5 to 36.6); 77% decrease in measurable disease, mDOR of 7.2 months (95% CI, 4.7 to 8.6 months), mPFS of 5.4 months (95% CI, 3.5 to 7.2 months) and OS 10.9 months (95% CI, 9.0 to 13.8 months). With longer follow-up, it was pointed out that ORR remained high at 28%. Subject to confirmation of its therapeutic benefits from Cohort 1 of the TROPHY-U-01 trial, the fast-tracked designation led to accelerated approval by the FDA for the use of SG in treating mUC that has already been treated with platinum-based chemotherapy and a checkpoint inhibitor (CPI) treatment [42].

Cohort 3 of TROPHY-U-01 study included 41 patients, 78% with visceral metastases. A median follow-up of 14.8 months resulted in ORR of 41% (95% confidence interval 26.3 to 57.9) and CR of 20%, CBR was 46% (95% CI 30.7 to 62.6), Median DOR was 11.1 months (95% CI 4.8 to not estimable). Median PFS was 5.3 months (95% CI 3.4 to 10.2), and median OS reached 12.7 months (range, 10.7-NE). To fully explore SG's potential, cohort 3 additionally explored SG in combination with a range of other treatments, including cisplatin, avelumab maintenance in first-line cisplatin-ineligible mUC, and zimberelimab an anti PD-1 monoclonal antibody [43]. TROPiCS-04, a phase III confirmatory study of SG monotherapy in mUC in refractory disease after platinum-based chemotherapy and ICI. At the 2024 ASCO annual meeting, the published results did not meet the initial hypothesis, concluding a negative study. Although the investigators observed trends in minimal numerical improvement of OS and secondary endpoints of PFS and ORR were also shown [44]. SURE-01 (NCT05226117) designed to evaluate sacituzumab govitecan (SG) as neoadjuvant therapy before radical cystectomy. Given higher toxicity rate than expected the authors applied a protocol amendment consisting of a dose reduction and G-CSF prophylaxis. As a result, the preliminary data is encouraging. Demonstrated hypothesis-generating complete responses with monotherapy sacituzumab govitecan-hziy (Trodelvy) as neoadjuvant treatment following surgery in patients with muscle-invasive bladder cancer (MIBC)[15]. Future research should focus on investigating the pharmacokinetics and dynamics of the linker technology. This could lead to the creation of improved versions that are more effective and less toxic [37].

3.2.2. Datopotamab Deruxtecan

Datopotamab Deruxtecan (Dato-DXd, DS-1062a) is a novel humanized anti-TROP2 IgG1 monoclonal antibody ADC, representing a significant advancement in the therapeutic landscape of urothelial carcinoma through its targeted approach. It is designed to selectively deliver a Topo I inhibitor intracellularly, namely Dato-DXd via a tetra peptide-based cleavable linker. Applying the DXd-ADC technology cluster with the optimized DAR of 4 is anticipated to expand the potential therapeutic capability [45]. This theory was tested in the ongoing phase 1 TROPION-PanTumor01 study. Preliminary results as published in 2024 ASCO Genitourinary Cancers Symposium suggested an encouraging antitumor activity as salvage therapy for unrespectable advanced or metastatic UC after first line. Confirmed ORR of 27.8% (95% CI 9.7–53.5), DCR of 77.8% (95% CI 52.4–93.6) [46].

3.3. HER2

HER2, renowned for its involvement in the development of breast cancer, is also associated with urothelial carcinoma, where its expression can range widely, from 23.42% HER2 1+, 54.95% HER2 2+ to 4.50% HER2 expression 3+. This disparity in expression levels has a significant impact on both prognosis and response to treatment [47]. According to the recently released study at the 2024 ASCO GU conference, there is an inverse correlation between the expression of HER2 IHC and the PD-L1 CPS score [48].

3.3.1. Trastuzumab Deruxtecan

Fam-trastuzumab deruxtecan-nxki (T-DXd) builds upon the concept of targeted HER2 therapy but with enhanced efficacy and a broader application spectrum. Concists of an anti-HER2 ADC that combines an antibody, a tetrapeptide-based cleavable linker and a payload of topoisomerase I inhibitor (DXd). T-DXd harbors a twin antitumor effect. In the first place by inhibiting the double-stranded DNA breakage and through topoisomerase cancer cell apoptosis. Second, it acts on proximal cancer cells with low HER2 expression through the "bystander effect" [49]. Amplification of HER2 In UC range from 6% to 17%, while incidence ranges from 6% to 80% [50]. In the past various trials tested anti-HER2 therapies for treatment of UC. Nevertheless, they have failed. Targeting HER2 with ADCs may represent a more promising strategy [51]. Given the ability of ADCs to employ cytotoxic therapy to tumor cells with low HER2 levels (IHC 1+, 2+ with FISH test negative), HER2 positive report on UC will be subject to greater scrutiny [52]. DESTINY-PanTumor01 a phase II trial tested patients expressing HER2 activating mutations in a context of metastatic disease from different primary sites. Patients with urothelial malignancy achieved objective response (ORR) of 29% [53]. Following DESTINY-PanTumor02 an open-label phase II basket trial (NCT04482309) with 267 patients expressing receptors pocitivity HER2 3+ or 2+, originated from different primary sites, who received monotherapy of Fam-trastuzumab deruxtecan-nxki [54]. Patients with bladder malignancy marked an overall response rate (ORR) of 39% (95% CI 24.2–55.5%). Of note is the fact that patients with IHC HER2 3+ disease reported ORR of 56% vs 35% for IHC HER2 2+ disease. Based on these results On April 5, 2024, T-DXd is the first HER2-targeted ADC to be granted the Food and Drug Administration accelerated approval for adult patients with unresectable or metastatic HER2-positive (IHC 3+) solid tumors including UC who have received prior systemic treatment and have no satisfactory alternative treatment options [55]. Currently T-DXd is a treatment option for patients with mUC in the US. This option highlights the need to define HER2 protein expression in this malignancy and ensure routine testing.

3.3.2. Disitamab Vedotin

Disitamab vedotin (DV) also known as RC48, contains a monoclonal antibody that targets HER2 with higher affinity than trastuzumab, its valine- citrulline linker is cleavable, ensuring on-target cytotoxicity upon endocytosis by the tumor cell, and its payload is MMAE. The drug has intracellular cell killing as well as bystander effect on adjacent cells. Its DAR is 4 to 1 [56,57].

Phase I trial of DV enrolled 4 patients with HER2+ mUC; in this trial, two patients reported with partial response and two with stable disease, proving the drug's antitumor activity. The clinical development of DV continued with two open-label phase II trials, both conducted in China, RC48-C005 (ClinicalTrials.gov identifier: NCT03507166) and RC48-C009 (ClinicalTrials.gov identifier: NCT03809013) studies. In these very similar studies, 107 patients with HER2-positive (IHC 2+/3+) laUC/mUC were enrolled and had previously progressed on at least one line of systemic chemotherapy. DV was administered intravenously every 2 weeks at a dose of 2mg/kg. The primary end-point was ORR. 65% of patients had received at least two lines of previous systematic treatment, and 25% had received an anti-PD-1/L1 treatment. ORR was 50%, with 32% of patients achieving stable disease as their best response, and 18% experienced disease progression. The ORR was numerically higher for patients with higher HER2 expression. The median DoR was 7.3 months, median PFS was 5.9 months and median OS was 14.2 months. In terms of safety, the most reported TRAE resulting in treatment discontinuation was peripheral sensory neuropathy (11.2%), no grade 4 or 5 TRAEs were observed. Peripheral sensory neuropathy and leukopenia were the most common grade 3 TRAEs reported. Based on these results, DV was approved in China in January 2022 for platinum-refractory patients with mUC [57,58,59,60]. DV has also gained Breakthrough therapy designation by the US FDA since September 2020.

DV has also been examined in other -smaller- phase II trials. In the NCT04073602 trial, DV was administered in 19 patients with tumors lacking HER2 expression. ORR was 26%, indicating a certain degree of response even in the absence of HER2 expression. In addition to monotherapy, DV-based combinations also have been explored. The combination with toripalimab, an anti-PD-1 antibody, resulted in a response rate of 73%, in the NCT04264936 phase II trial, by the cutoff date of 18 November 2022. This trial had enrolled 41patiens, 61% of them without prior exposure to any treatment. Median PFS was 9.2 months, and 2-year OS rate is 63.2%. A phase III study is ongoing to compare this combination with the standard of care [57,61,62]. Currently, there are also two larger trials testing the combination of DV with pembrolizumab in non-Chinese patients. RC48G001 (NCT04879329) is a phase II trial, with three cohorts. Patients in cohorts A and B with tumors expressing HER2+/ HER2 low, respectively, are previously treated with platinum-containing therapy and will receive DV monotherapy, while patients in cohort C are treatment-naïve and will receive either monotherapy or the combination of DV with pembrolizumab. DV-001 (NCT05911295), is a phase III randomized trial, where previously untreated patients with HER2-expressing laUC/mUC will receive in a 1:1 ratio DV and pembrolizumab or platinum-containing chemotherapy (maintenance with avelumab is permitted). The primary endpoints are PFS and OS [63,64]. Outside the context of prospective clinical trials, DV combinations with anti-PD-1/L1 has also been tested in patients with muscle-invasive and la UC, with promising results [65].

3.4. EPCAM

3.4.1. Oportuzumab Monatox (Vicineum)

EpCAM (epithelial cell adhesion molecule) is a transmembrane glycoprotein that is overexpressed in various human tumors, including urothelial carcinomas. Targeting the extracellular domain of EpCAM is clinically relevant, and clinical trials in urothelial cancer have been underway since 2010. The drug used is oportuzumab monatox (OM), known as vicineum, a fusion protein of a humanized single-chain antibody against EpCAM linked to Pseudomonas exotoxin. OM binds to the surface of EpCAM-positive cancer cells and exotoxin releases apoptosis. OM is used in BCG-refractory non-muscle invasive bladder cancer (NMIBC) patients as intravesical instillation, thus reducing systemic toxicity [56,66,67].

Phase I study of OM enrolled 64 patients with BCG-refractory or intolerant patients with grade 2 or 3, stage Ta/T1 transitional cell carcinomas or in-situ carcinomas. Patients were administered 6 weekly instillations, achieving CR at a rate of 39% at the 12-week endpoint. No patients were removed from the study due to toxicity. The subsequent open-label phase II study enrolled 46 patients and reported similar outcomes, with 44% of the patients achieving CR. The phase III registrational trial of OM, the VISTA study, enrolled 133 patients divided in three cohorts, two depending on CIS recurrence from BCG treatment and the third recruited patients with papillary recurrence < 6 months from BCD treatment. Intrvesical instillation of 30mg OM was administered two times per week for 6 weeks followed by weekly for 6 weeks and then every 2 weeks for up to 2 years. The primary end-point was CR rate and duration of response in Cohort 1 (CIS recurrence < 6 months from BCG treatment). At 3 months,the primary end point of CR was 39% in cohort 1. Treatment was well tolerated with only 4% experiencing treatment-related grade ≥ 3 toxicity, most commonly urinary tract infections. In July 2022, the US FDA rejected the approval of Vicineum, leading to the pausing of its further development by the manufacturer Sesen Bio [66,68,69,70].

Another phase I trial combines OM with durvalumab (an anti-PD-L1 checkpoint inhibitor) in high-grade NMIBC after BCG failure (NCT03258593). The trial enrolled 15 patients, and its final results were recently presented at the 2024 American Urological Association meeting. The combination was well tolerated and resulted in a 47% CR rate at the 12-week time point, with three patients showing no evidence of disease for more than 12 months [71].

3.5. Tissue Factor

3.5.1. Tisotumab Vedotin

Tissue factor (TF) is highly expressed on the surface of many solid tumor cells, including ovarian, prostate, bladder, esophageal, endometrial, and lung tumors. Tisotuman vedotin (TV) comprises a human monoclonal antibody specific for TF conjugated to the microtubule-disrupting agent MMAE via a protease-cleavable linker.

TV has been examined in the phase I-II InnovaTV 201 trial, a multicenter dose-escalation and dose-expansion trial to assess its safety administration in patients. The trial enrolled in both phases a total of 174 patients with metastatic solid tumors, of whom 17 had bladder cancer. Of the 15 bladder cancer patients eligible for response analysis, 4 (26,7%) demonstrated objective response. Overall, the trial has shown that TV is safe to administer and has an encouraging antitumor activity in heavily pretreated patients with tumors known to express TF. Its safety profile is consistent with other MMAE-containing ADCs, with epistaxis and conjunctivitis appearing more frequently [72]. Over the next years, the development of TV focused more on patients with cervical cancer. In April 2024, the FDA granted full approval to TV for the treatment of patients with recurrent or metastatic cervical cancer who have disease progression on or after treatment with chemotherapy.

A list of approved indications of ADCs in urothelial cancer can be found in Table 1.

5. Conclusions

The advent of ADCs has opened new avenues for the treatment of this aggressive cancer type, providing hope for more personalized and effective therapies. ADCs represent a promising approach in the arsenal against cancer, particularly urothelial carcinoma. Still, their successful integration into clinical practice requires a nuanced understanding of their mechanism, including the critical bystander effect, and a strategic approach to overcoming the inherent challenges associated with this innovative therapy.