Nectin-4 Expression Research Articles

Abstract 6006: Modeling antibody drug conjugate sensitivity using urothelial carcinoma patient-derived models

Abstract Introduction: Enfortumab Vedotin (EV), an antibody-drug conjugate (ADC) targeting Nectin-4, has emerged as a new standard-of-care for patients with metastatic urothelial cancer (UC). Promising results have also been observed in bladder cancer (BLCA) patients with ADCs targeting HER2. To identify predictive biomarkers of ADC sensitivity, we characterized a large and expanding biobank of patient-derived organoid (PDO) and xenograft (PDX) models for Nectin-4 and HER2 expression, and Nectin-4 and HER2-targeted ADC sensitivity. Methods: We generated 19 PDOs and 34 PDXs that reflect the genomic and biologic heterogeneity of human UC. A subset of these models was further characterized using a multiplatform approach, including bulk and single-cell DNA and RNA sequencing and immunoblot for Nectin-4 and HER2 expression. We leveraged these models to study the biologic determinants of sensitivity to EV and the HER2-targeted ADC trastuzumab deruxtecan (T-DXd). PDX models were treated with EV (5 mg/kg IV on day 1, 3, 6) or T-DXd (10 mg/kg IV on day 1, 21, 42) or vehicle only as control. We also studied sensitivity to T-DXd and EV, as well as to their cytotoxic payloads (exatecan and vedotin) in vitro using paired PDO models. Results: We observed high degree of variability of HER2 and Nectin-4 expression across UC PDOs and PDXs. In PDO models, sensitivity to EV correlated strongly with Nectin-4 expression, whereas HER2 expression was not strongly correlated with sensitivity to T-DXd. We also noted a poor correlation between Nectin-4 expression in PDOs and PDXs derived from the same patient. A subset of models, for example, SMBO-104 (ERBB2 wildtype with low HER2 expression), exhibited significantly greater sensitivity to EV than to T-DXd. Conversely, T-DXd was significantly more active than EV in SMBO-170, a model with ERBB2 amplification (~40 copies) and HER2 3+ expression. Sensitivity to exatecan, an analogue of the T-DXd payload deruxtecan, as measured by inhibition of cell proliferation and induction of apoptosis, was more predictive of T-DXd sensitivity than HER2 expression level. Conclusion: In this study, we generated and biologically characterized UC patient-derived models with the goal of identifying predictive biomarkers of sensitivity to Nectin-4 and HER2-targeted ADCs. We observed significant heterogeneity of HER2 and Nectin-4 expression across models. Models were identified in which T-DXd was superior to EV, and vice versa. Sensitivity to the T-DXd cytotoxic payload was more predictive of PDX response than HER2 expression levels, with significant tumor regression observed with T-DXd in BLCA models with low HER2 expression. Our data suggest that clinical trials of HER2-targeted ADCs such as T-DXd should not be restricted to patients with HER2 overexpression and that novel biomarkers will be needed to identify those UC patients most likely to benefit from FDA-approved and investigational ADCs. Citation Format: Xinran Tang, Ziyu Chen, Jasmine Thomas, Karan Nagar, John Christin, Naryan Rustgi, Sizhi Gao, Elisa de Stanchina, Jonathan Coleman, Michael Shen, Hikmat Al-Ahmadie, Gopa Iyer, Kwanghee Kim, David Solit. Modeling antibody drug conjugate sensitivity using urothelial carcinoma patient-derived models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6006.

Abstract 4632: Lineage plasticity as a determinant of antibody-drug conjugate target expression in urothelial bladder cancer

Abstract Bladder cancers display a wide spectrum of morphologies that frequently co-exist within individual tumors. Several histologic variants, including plasmacytoid, neuroendocrine, and micropapillary subtypes, are associated with an increased bladder cancer recurrence risk and cancer-specific mortality. The molecular basis and therapeutic implications of this phenotypical plasticity remain poorly understood. Enfortumab vedotin (EV), an antibody-drug conjugate targeting Nectin-4, has also emerged as a new standard-of-care for metastatic bladder cancer patients, but the association between lineage plasticity as manifested by morphologic heterogeneity in bladder cancer, Nectin-4 expression and ADC response remains poorly defined. Previous DNA-based molecular profiling studies of histologic variants revealed that CDH1 mutations were the pathognomonic molecular alteration in plasmacytoid urothelial carcinoma, but no recurrent driver mutations have been found to be unique to the aberrant histology regions of other mixed histology subtypes. Thus, to explore the molecular basis for the lineage plasticity of bladder cancers with aberrant histology and to define the association between lineage plasticity manifested as morphologic heterogeneity and the expression of Nectin-4, HER2 and other potential ADC targets, we performed RNA-seq of pure urothelial carcinomas (n=118, not otherwise specified, UC-NOS) and bladder tumors with divergent differentiation or histologic subtypes (n=199). Our cohort included a subset of tumors in which RNA-seq was performed on microdissected UC-NOS and variant histology regions of the same tumor. Tumors with neuroendocrine, sarcomatoid and squamous histology had the lowest Nectin-4 expression, whereas histologic subtypes that retained a luminal transcriptional profile such as micropapillary, plasmacytoid and nested histology had higher levels of Nectin-4 expression. In mixed histology tumors, Nectin-4 and/or HER2 expression was often restricted to only one of the two histologic components. Gene Set Enrichment Analysis noted enrichment of distinct oncogenic pathway gene sets in different histologic variants. Preliminary investigation into the mechanism(s) of Nectin-4 regulation using bladder cancer cell lines and patient-derived organoids (PDOs) has identified targetable pathways which when inhibited revert lineage plasticity and induce Nectin-4 expression. In sum, lineage plasticity manifested as heterogenous histomorphologies was associated with heterogeneous Nectin-4 expression in some but not all histologic variants of bladder cancer. Ongoing studies using PDOs from bladder cancer patients with mixed histology will seek to identify molecular pathways whose inhibition leads to increased expression of Nectin-4 or other ADC targets such as HER2 and thus increased sensitivity of ADCs targeting these cell surface proteins. Citation Format: Jiaqian Luo, Sizhi P. Gao, Fengshen Kuo, Gamze Gokturk Ozcan, Merve Basar, Florestan Koll, Jacob E. Tallman, Syed M. Alam, Carissa E. Chu, Ziyu Chen, Eugene J. Pietzak, Gopakumar Iyer, Jonathan E. Rosenberg, David B. Solit, Hikmat Al-Ahmadie. Lineage plasticity as a determinant of antibody-drug conjugate target expression in urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4632.

Abstract 2579: A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma

Abstract Introduction: Enfortumab vedotin (EV), an antibody-drug conjugate targeting nectin-4, offers hope for urothelial carcinoma patients (UC) but prolonged use leads to treatment-related toxicities often necessitating dose adjustments. How EV engages nectin-4 at the tumor and its relevance to efficacy is unknown. To understand the pharmacodynamics (target engagement) of EV at the tumor across FDA approved dosing levels, we developed a nectin-4 specific peptide-based radiotracer, [68Ga]AJ647, for positron emission tomography (PET) imaging. Methods: Six UC cell lines of luminal (HT-1376, UC-14, RT-112) and basal (UC-9, SCaBER, T24) morphological type were selected for in vitro and in vivo testing of [68Ga]AJ647 and nectin-4 target engagement by EV. Cell uptake and competitive binding assays were conducted to test [68Ga]AJ647 specificity for nectin-4 in the presence or absence of EV. Flow cytometry derived receptor density was used to correlate [68Ga]AJ647 uptake in cells with nectin-4 expression. Pharmacokinetics, biodistribution and specificity of [68Ga]AJ647 for nectin-4 expression was evaluated in six xenograft models by PET imaging and biodistribution studies. Target engagement by EV was quantified by measuring unoccupied nectin-4 levels by [68Ga]AJ647-PET in HT-1376 xenografts at different doses (15, 9, and 6 mg/kg) and time (1, 3, and 6 days). Results: [68Ga]AJ647 was generated in 20-30% radiochemical yield with >95% purity. In vitro assays showed distinctive uptake in cell lines (HT-1376 > UC-14 > RT-112; UC-9 > SCaBER > T24) that correlated with nectin-4 density (R2=0.85). PET and biodistribution studies showed tractable pharmacokinetics of [68Ga]AJ647 enabling high contrast images in 60 min and exhibiting nectin-4 dependent uptake in all the six xenografts. Uptake of [68Ga]AJ647 in UC cells reduced in a dose dependent manner in the presence of EV, indicating that [68Ga]AJ647 measures unoccupied nectin-4, and paving the way for EV target engagement evaluation. For target engagement evaluation, [68Ga]AJ647 accumulation in HT-1376 tumors in saline treated mice was assumed as 100% unoccupied nectin-4 levels. In mice treated with EV for 24 hrs (n=3-5), unoccupied nectin-4 levels were 21.1±11.6, 23.2±14.5, and 24.3±18.8 for 15, 9, and 6 mg/kg doses, respectively. Whereas on day 6, unoccupied nectin-4 levels showed significant differences at 29.5±15.8, 64.2±21.0, and 89.5±18.9 for 15, 9, and 6 mg/kg dose, respectively. These data indicate that FDA approved doses of EV show higher target engagement at 24 h but a dramatic reduction at lower doses over time. Conclusion: [68Ga]AJ647-PET quantifies variable nectin-4 expression in tumors. Furthermore, [68Ga]AJ647-PET quantifies EV therapy induced pharmacodynamics at the tumor. Such real-time ADC target engagement assessment could inform patient selection and dosing strategies to enhance efficacy and minimize toxicities. Citation Format: Akhilesh Mishra, Ajay K. Sharma, Kuldeep Gupta, Burles A. Johnson, David J. McConkey, Jeannie Hoffman-Censits, Sridhar Nimmagadda. A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2579.

Abstract 1872: A next generation treatment for Nectin-4 positive cancers - Preclinical characterization of LY4052031, an anti-Nectin-4 antibody, conjugated to a novel camptothecin payload

Abstract Nectin-4 is an immunoglobulin-like antigen overexpressed in a variety of cancers including bladder, breast, NSCLC, and gastric. Normal tissues including skin, salivary glands, bladder and esophagus express only low to medium levels of Nectin-4 making the protein an attractive target for cancer therapies. The approval of enfortumab vedotin-ejfv, an anti-Nectin-4 based antibody-drug conjugate (ADC) with a microtubule inhibitor payload, either as a single agent or in combination with pembrolizumab has been a significant advancement in the treatment of metastatic urothelial cancer. We have developed a novel anti-Nectin-4 ADC with a novel topoisomerase I (TOPO 1) inhibitor drug payload. Topoisomerase inhibitor payload-based ADCs offer potential advantages compared to microtubule payload-based ADCs including a different toxicity profile and the differentiated susceptibility to resistance mechanisms, such as P-gp mediated drug efflux. LY4052031 is a fully human monoclonal anti-Nectin-4 antibody conjugated to a novel TOPO 1 inhibitor by a cleavable peptide linker at a homogeneous drug antibody ratio (DAR) of 8:1. In vitro, both LY4052031 and its unconjugated parental antibody specifically bind to Nectin-4 expressing cell lines and are quickly and efficiently internalized. LY4052031 demonstrated potent cytotoxicity on tumor cells with high and low Nectin-4 expression. Additionally, bystander activity exerted by LY4052031 on antigen negative cells co-cultured with antigen positive cells was greater than that observed with the clinical benchmark enfortumab vedotin-ejfv. In vivo studies in xenografts expressing low to high levels of Nectin-4 showed significant tumor growth inhibition following treatment with LY4052031 as monotherapy. Overall, LY4052031 exhibits specificity, selectivity, potency, and effectiveness as a next generation therapy to treat Nectin-4 positive tumors. An IND submission is planned for 2024. Citation Format: Divya Sagar, Mohan Srinivasan, Kevin Lindquist, Qianxu Guo, Wailin Wong, Maria B. Lebron, Rose Marie Sattler, Jieyu Zhou, Whitney Helms, Jeffrey Boyles, Petra Verdino, Xuan Zhao, Yvonne Mak, Joshua Park, Rikke Holmgaard, Kyla Driscoll, Omar Duramad, Kristin Bedard. A next generation treatment for Nectin-4 positive cancers - Preclinical characterization of LY4052031, an anti-Nectin-4 antibody, conjugated to a novel camptothecin payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1872.

Abstract 1891: Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window

Abstract Nectin-4 is a well validated tumor target, which is highly expressed in several solid cancers such as urothelial, head-and-neck, breast, lung, pancreatic, ovarian, and cervical cancer, while expression in normal tissues is limited. A first-generation antibody-drug conjugate targeting Nectin-4, called enfortumab vedotin-ejfv (EV), was granted accelerated FDA approval in 2019 for urothelial cancers, and is currently approved for frontline therapy in cisplatin-ineligible patients with locally advanced or metastatic urothelial carcinoma, providing groundbreaking improvement in patient survival. However, clinical use of EV has also revealed safety-driven limitations, which result in reduced and/or interrupted drug exposures impacting efficacy. To overcome these limitations in therapeutic window and to maximize on the potential of Nectin-4 as a tumor target beyond urothelial cancers, Adcentrx has developed ADRX-0706, a Nectin-4-targeting next-generation ADC with a drug-antibody ratio of 8. ADRX-0706 is comprised of a novel fully human IgG1k monoclonal antibody linked to an optimized tubulin inhibitor payload AP052 through a proprietary cleavable linker. ADRX-0706 has strong binding affinity and selectively induces cytotoxicity in Nectin-4-expressing tumor cells. Importantly, ADRX-0706 is designed to have improved bystander effect, which allows tumor regression even in cases of heterogeneous Nectin-4 expression. This translates to superior in vivo efficacy of ADRX-0706 over EV in multiple mouse xenograft models of other indications beyond bladder cancer. In a mouse clinical trial of patient-derived cervical cancers with a range of Nectin-4 expression levels, ADRX-0706 reaches a 73% ORR. Since peripheral neuropathy is a main platform toxicity for this payload class, ADRX-0706 is assessed in an in vitro co-culture model to evaluate its neurotoxic potential. While EV displays a 75% decrease in neurite outgrowth, ADRX-0706 strikingly has no impact at concentrations up to 1mg/ml, suggesting a lower risk of peripheral neuropathy in humans for ADRX-0706. In preclinical repeat dose toxicology study, ADRX-0706 reaches an HNSTD of 18mg/kg in NHPs well above therapeutic exposures in mouse pharmacology studies, indicating a tremendous increase in the therapeutic index compared to EV. In summary, the highly selective and potent anti-tumor activity in multiple tumor types and wide preclinical therapeutic index of ADRX-0706 support clinical evaluation of this next generation anti-Nectin-4 ADC. ADRX-0706 is currently in a phase 1a/b study (NCT06036121). Citation Format: Andrew M. Hau, Maria Shahmoradgoli, Dong Jun Lee, Wes Sisson, Anna Wang, Peter P. Challita, Erin Nye, Mario M. Kuo, Alexis Mahloch, Monica Leung, Oscar Betancourt, Alexander Chu-Kung, Maojun Guo, Hui Li, Sabine Rottmann, Pia M. Challita-Eid. Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1891.

Abstract 4009: Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy

Abstract Century Therapeutics is developing NECTIN4 targeted induced Pluripotent Stem Cell (iPSC) derived Chimeric Antigen Receptor (CAR) T cell therapy. NECTIN4 targeted antibody drug conjugate enfortumab vedotin, while generally well tolerated, causes severe skin adverse events in some patients, thought to be driven by on-target off-tumor toxicity against NECTIN4 displaying skin keratinocytes. We include a safety switch in our first clinical-stage iPSC-derived CAR-NK cell therapy, CNTY-101, which provides an antibody target for elimination of the engineered cells should that be necessary. An additional method for reducing the incidence and severity of this type of adverse event is through the incorporation of an inhibitory CAR, also known as a NOT-gate, directed against antigen expressed on normal skin keratinocytes, but the optimal surface protein to target with such an inhibitory CAR for a NECTIN4 targeted therapy is unknown. Here, we identify DSG1 (Desmoglein-1) as a top inhibitory CAR target for NECTIN4 targeted CAR-T cell therapy by conducting a multi-omics analysis of public single-cell RNAseq, bulk RNAseq, and protein microarray immunohistochemistry datasets. We find that DSG1 is constitutively and uniformly displayed on the surface of skin keratinocytes in the layers of epidermis where they display NECTIN4, and DSG1 gene expression in normal skin is uniformly high across individuals. Conversely, DSG1 mRNA and protein are rarely expressed in NECTIN4 expressing cancer indications including bladder, breast, non-small cell lung, ovarian, and pancreatic. Further, autoimmune antibodies specific to DSG1 have been described, suggesting that DSG1 is targetable by a CAR in situ. Finally, DSG1 is not expressed by our iPSC-derived CAR-T cells. While NECTIN4 expression is greatest in skin keratinocytes, epithelial cells in other tissues express NECTIN4 at lower levels. In this study, we compare the expression levels of NECTIN4 to targets associated with tissue and cell-type specific on-target off-tumor toxicity in primary CAR-T cell clinical trials. Our findings support the strategy of DSG1-directed inhibitory CAR incorporation into NECTIN4 specific CAR-T cell therapeutic candidates. Citation Format: Matthew S. Hall, Christopher M. Dower, Michael Miller, John Wheeler, Jill Carton, Ohad Manor, Hyam Levitsky. Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4009.

Abstract 6899: Enfortumab vedotin (EV): Efficacy comparison with trastuzumab deruxtecan (T-DXd) and sacituzumab govitecan (SG) and its potential for combination benefit in bladder cancer XPDX models

Abstract EV is a Nectin-4 targeting antibody-drug conjugate (ADC) with an MMAE payload, recently approved for bladder cancer treatment. We established and characterized 18 bladder XPDX models representing primary and metastatic disease from naïve or clinically treated patients, some with actionable mutations. To better understand the potential additive benefit of EV in bladder cancer, we evaluated the ADC alone and in combination with agents targeting ERBB2, PIK3CA or FGFR3. In addition, we tested EV alone in ST5420B, a model established from a patient who progressed on EV after five cycles, as well as ST975B, a model which harbors a novel NECTIN4 fusion. 18 bladder XPDX models were established in immune-deficient mice, including 5 from newly diagnosed patients (2 from primary site), and 13 from recurrent disease (2 from primary site). Nectin-4, Trop2 protein and ERBB2 receptor expression was tested, and models profiled using WES and RNAseq. For in vivo studies, all models were evaluated against cisplatin, T-DXd, SG, and EV. Models with actionable mutations were tested with alpelisib, erdafitinib, and neratinib alone and in combination with EV. Endpoints in all studies included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C<0%) versus Day 0 tumor volume was also reported. Most models stained positive for Nectin-4 and Trop-2. FGFR3 variants were found in five models and ERBB2 mutations in two while several models reported PIK3CA variants; deletions in RB1 and CDKN2A were common. In vivo studies reported differential responses to cisplatin with the most sensitive models established from treatment-naïve patients. EV and SG tested alone reported activity in several models; EV added benefit in some combination treatments. We have characterized a panel of bladder XPDX models and benchmarked them against T-DXd, EV and SG alone and EV in combination with targeted therapies. This data is a valuable tool in further developing EV and identifying novel therapies for bladder cancer. Citation Format: Johnnie Flores, Heaven Sessions, Alyssa Simonson, Natalia Banos, Tahmineh Rouzbahani, Armando Diaz III, Jim Lund, Emiliano Calvo, Victor Moreno, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Scott Ulmer, Luis Rodriguez, Michael Wick. Enfortumab vedotin (EV): Efficacy comparison with trastuzumab deruxtecan (T-DXd) and sacituzumab govitecan (SG) and its potential for combination benefit in bladder cancer XPDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6899.

Abstract 6785: KIYA-PREDICT™ XPDX: An ex vivo 3D spheroid platform for modeling in vivo responses to multiple classes of oncology therapeutics

Abstract Patient-derived xenografts (PDX) are a powerful and commonly used tool in preclinical drug development as they represent an in vivo platform for screening drug response in human tumors that recapitulate many molecular and histological features of the original patient tumor. However, these models are costly and time-intensive, necessitating the development of ex vivo platforms for assessing drug response to PDX-derived tumors to de-risk in vivo studies by aiding in model selection and reducing the number of animals and costs associated with in vivo experimentation. The KIYA-PREDICT™ platform is a clinically validated, ex vivo 3D cell culture platform with demonstrated ability to predict patient response to chemotherapy in high grade glioma and ovarian cancer. Here, we apply this platform to profiling XPDX from XenoSTART’s comprehensive library of well-characterized models as a tool for assessing drug response and mechanism of action ex vivo while also aiding in model selection for in vivo studies. Over 100 XPDX models have been profiled ex vivo for response to several classes of cancer therapeutics, including chemotherapy, targeted therapy, and antibody-drug conjugates (ADCs), demonstrating robust concordance with in vivo drug response. CDK4/6 inhibitors palbociclib and ribociclib evaluated ex vivo in breast XPDX display strong correlation to in vivo response and recapitulate palbociclib resistance. Similarly, G12C inhibitors sotorasib (AMG-510) and adagrasib (MRTX849) screened against a panel of non-small cell lung cancer XPDX ex vivo match in vivo response and drug resistance. Finally, ex vivo and in vivo responses to FDA approved ADC enfortumab vedotin were evaluated in a panel of bladder cancer PDX, with ex vivo and in vivo responses showing good concordance and expected modulation based on Nectin-4 expression levels. The combination of the KIYA-PREDICT™ platform with XenoSTART’s XPDX catalogue is a flexible and powerful tool that can be utilized for streamlining preclinical drug development for a wide range of therapeutic classes. Citation Format: Aaron L. Carlson, Natalie W. Dance, Kimberly J. Burgess, Danielle Y. Nadeau, Melissa Millard, Alyssa Simonson, Armando Diaz, Michael Wick, Teresa M. DesRochers. KIYA-PREDICT™ XPDX: An ex vivo 3D spheroid platform for modeling in vivo responses to multiple classes of oncology therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6785.

Re: Assessment of PD-L1, TROP2, and NECTIN-4 Expression in Penile Squamous Cell Carcinoma

Re: Assessment of PD-L1, TROP2, and NECTIN-4 Expression in Penile Squamous Cell Carcinoma

Downregulation of endogenous nectin1 in human keratinocytes by herpes simplex virus 1 glycoprotein D excludes superinfection but does not affect NK cell function.

Many viruses downregulate their cognate receptors, facilitating virus replication and pathogenesis via processes that are not yet fully understood. In the case of herpes simplex virus 1 (HSV1), the receptor binding protein glycoprotein D (gD) has been implicated in downregulation of its receptor nectin1, but current understanding of the process is limited. Some studies suggest that gD on the incoming virion is sufficient to achieve nectin1 downregulation, but the virus-encoded E3 ubiquitin ligase ICP0 has also been implicated. Here we have used the physiologically relevant nTERT human keratinocyte cell type - which we have previously shown to express readily detectable levels of endogenous nectin1 - to conduct a detailed investigation of nectin1 expression during HSV1 infection. In these cells, nectin1, but not nectin2 or the transferrin receptor, disappeared from the cell surface in a process that required virus protein synthesis rather than incoming virus, but did not involve virus-induced host shutoff. Furthermore, gD was not only required but was sufficient for nectin1 depletion, indicating that no other virus proteins are essential. NK cells were shown to be activated in the presence of keratinocytes, a process that was greatly inhibited in cells infected with wild-type virus. However, degranulation of NK cells was also inhibited in ΔgD-infected cells, indicating that blocking of NK cell activation was independent of gD downregulation of nectin1. By contrast, a superinfection time-course revealed that the ability of HSV1 infection to block subsequent infection of a GFP-expressing HSV1 was dependent on gD and occurred in line with the timing of nectin1 downregulation. Thus, the role of gD-dependent nectin1 impairment during HSV infection is important for virus infection, but not immune evasion, which is achieved by other mechanisms.

ABC transporter expression to predict therapeutic effect of enfortumab vedotin in urothelial cancer.

693 Background: Enfortumab vedotin (EV) is an antibody-drug conjugate consisting of anti-Nectin-4 antibody and is approved for metastatic urothelial cancer. Predictors of therapeutic benefit of EV have not been established since nectin-4 expression varies widely with the course of treatment. ATP Binding Cassette (ABC) transporters which export the administered anti-cancer drugs out of the cells have been postulated as one of the mechanisms of therapeutic resistance to EV. In this study, we evaluated the expression of Nectin-4 and ABC transporters in primary biopsy tissues, primary radical resection tissues, and metastatic sites from urothelial cancer patients and evaluated their association with prognosis after EV therapy. Methods: We studied 16 patients who received enfortumab vedotin for metastatic urothelial cancer. Biopsy of primary tissue, radical surgery of primary tumor, and resection of metastatic sites was performed in 16, 7, and 4 patients, respectively. In these specimens, the expression of nectin-4 and ABC transporters was evaluated by immunostaining. Among ABC transporters, we evaluated MDR1 (ABCB1), MRP1 (ABCC1), and BCRP (ABCG2) since they have been reported to be associated with anticancer drug resistance. Staining intensity on the cell membrane was classified into 0 to 3 according to the evaluation criteria of HER2. We investigated the relationship between the expression of nectin-4 and ABC transporters and survival after EV therapy. Results: Nectin-4 was positive in all patients (score 3 in 12 patients, score 1 in 4 patients) in primary biopsy. All patients tested negative for MDR1. MRP1 was positive in 11 (score 2 in 2 patients and score 1 in 9 patients), and BCRP was positive in 14 (score 2 in 10 patients, score 1 in 4 patients). Nectin-4 expression decreased in radical surgery tissues and metastatic sites. On the other hand, ABC transporter expression remained unchanged or rather increased in radical surgery tissues and metastatic sites (Table). Patients with both MRP1 and BCRP expression in the primary biopsy specimen (n=8) had significantly worse PFS and OS after EV therapy while nectin-4 expression in the primary biopsy specimen was not associated with survival. Conclusions: Patients with ABC transporter expression had a poor prognosis after EV therapy, although nectin-4 expression in primary biopsies was not associated with prognosis. Nectin-4 expression tended to decrease, while the ABC transporter remained unchanged or enhanced as the disease progressed. [Table: see text]

TROP-2, NECTIN-4 and predictive biomarkers in sarcomatoid and rhabdoid bladder urothelial carcinoma.

The surface protein TROP-2/TACSTD2 and the cell adhesion protein NECTIN-4/NECTIN4 are responsible for the efficacy of anticancer therapies based on antibody-drug conjugates (ADC) targeting intracellular microtubules. In contrast with common histologic subtypes of bladder urothelial carcinoma (BUC), little is known of TROP-2 and NECTIN-4 expression in sarcomatoid and rhabdoid BUC. In this study, we aimed to analyze TROP-2 and NECTIN-4 expression and additional predictive biomarkers by immunohistochemistry and fluorescence in situ hybridization (FISH) on 35 undifferentiated BUC (28 sarcomatoid and 7 rhabdoid). Wide genomic investigation was also performed on 411 BUC cases of the PanCancer Atlas, focusing on genes related to the microtubule pathways. Seven of 35 (20%) undifferentiated BUC showed expression of TROP-2. NECTIN-4 was expressed in 10 cases (29%). Seven cases (20%) co-expressed TROP-2 and NECTIN-4. HER-2 FISH was amplified in 5 cases (14%) while HER-2 immunoexpression was observed in 14 cases (40%). PD-L1 scored positive for combined proportion score (CPS) in 66% of cases and for tumor proportion score (TPS) in 51% of cases. Pan-NTRK1-2/3 was elevated in 9 cases (26%) and FGFR-2/3 was broken in 7 of 35 cases (20%). Of 28 sarcomatoid BUC, 9 (32%) were negative for all (TROP-2, NECTIN-4, PD-L1, HER-2, FGFR and pan-NTRK) biomarkers and 3 (11%) expressed all five biomarkers. Among cases with rhabdoid dedifferentiation, 1 of 7 (14%) showed activation of all biomarkers, whereas 2 of 7 (28%) showed none. The mRNA analysis identified microtubule-related genes and pathways suitable for combined ADC treatments in BUC. Sarcomatoid and rhabdoid BUC do harbor positive expression of the ADC targets TROP-2 or NECTIN-4 in a relatively modest subset of cases, whereas the majority do not. Different combinations of other positive biomarkers may help the choice of medical therapies. Overall, these findings have important clinical implications for targeted therapy for BUC.

Comparison of tissue based FGFR mutation detection by Therascreen FGFR with UroTyper FGFR and ADC test and relevance for potential co-targeting with TROP2 and NECTIN4: Preview of Bladder BRIDGister.

679 Background: In view of the efficacy of FGFR targeting in early and advanced bladder cancer, as has been shown for erdafitinib in the THOR and NORSE trial, molecular testing of FGFR mutations and fusions will soon become clinical routine worldwide. Therascreen FGFR testing has been approved as companion diagnostic to select patients for erdafitinib. However, technical problems and shortages as well as a limited number of detectable mutations indicate the need of FGFR test systems that can be performed easily & fast worldwide. The objective was to evaluate concordance of UroTyper FGFR with Therascreen FGFR mutation testing and unravel molecular characteristics of FGFR mutated tumors to assess potential co-targeting options for erdafitinib to justify subsequent adopted clinical trials with the Bladder BRIDGister framework. Methods: FFPE tumor tissues from 100 TURB samples were prospectively collected as part of the Bladder BRIDGister. RNA from FFPE tissues were extracted by commercial kits to assess Therascreen FGFR & UroTyper FGFR MODAPLEX (BIOTYPE GmbH, Dresden) in conjunction with relative gene expression of subtyping markers (i.a.. PPARG), ADC targets (TROP2, NECTIN4) and CPI targets by RT-qPCR systems. Hierarchical clustering, Spearman correlation tests were done by JMP 9.0.0 (SAS software). Results: After exclusion of FGFR fusion samples, a total of 94 samples was available for comparative mutation analysis. Both PCR based FGFR tests concordantly identified S249C (n=15), R248C (n=2) and G370C (n=1) mutations, while the results for Y373C were in part discrepant with Therascreen FGFR detecting 9 mutations, while UroTyper FGFR did detect 6 mutations. Further examination of the 3 discrepant Y373C cases by SNaPshot and Uromonitor FGFR test revealed that the Therascreen FGFR test resulted in 3 false positive results. Moreover, one sample was invalid from Therascreen FGFR test. Overall, the concordance was very good and reached a kappa value of 0.92. Interestingly, FGFR3 mutations by UroTyper FGFR test were positively associated not only with FGFR3 mRNA overexpression (r=0.6699, p<0.0001), but also with TROP2 and NECTIN4 (r=0.5026 and r=0.4406, p<0.0001) indicating, that combinations of FGFR targeting with ADC targeting of TROP2 or NECTIN4 could have strong synergistic effects. Hierarchical clustering revealed a homogenous FGFR3 mutated group of tumors simultaneously co-expressing the target genes TROP2, NECTIN4 and PPARG. Conclusions: PCR-based FGFR assessment by Therascreen and UroTyper is highly concordant and enables fast, local FGFR assessment within few hours. FGFR3 mutations are associated with increased TROP2 & NECTIN4 expression indicating potential synergistic options which warrants further exploration as part of molecularly stratified clinical trials.

Activity of antibody-drug conjugates (ADCs) with radiation in preclinical bladder cancer models.

664 Background: Antibody-drug conjugates (ADCs) are a novel class of therapeutics that combine a tumor cell targeting antibody with a cytotoxic payload. Two ADCs are currently approved in the US for treatment of advanced bladder cancer: enfortumab vedotin (EV) and sacituzumab govitecan (SG). Radiation therapy (RT) plays a central role in trimodality therapy (TMT), a curative treatment approach for muscle-invasive bladder cancer (MIBC). However, the biological activity of ADCs combined with RT in preclinical MIBC models has not been reported. Methods: We use a molecularly annotated panel of human MIBC preclinical models to test the impact of RT on expression of ADC targets and define the combined activity of EV and SG with RT. We also test the in vivo activity of combining ADCs with image-guided, fractionated RT in bladder cancer flank xenograft mouse models. Results: Radiation has variable impact on expression levels of nectin-4 and trop-2, the targets of EV and SG, respectively, as determined by immunoblot, immunofluorescence microscopy, and immunohistochemistry (IHC). Whereas modest dose-dependent increases in nectin-4 and/or trop-2 levels were observed in some models, no significant changes were observed in other models. Importantly, RT did not lead to a significant decrease in nectin-4 or trop-2 expression in any of the models. EV and SG showed additive or synergistic cell killing when combined with RT across preclinical models in vitro. Combining EV or SG with fractionated RT in vivo was well-tolerated, showed improved tumor control, and prolonged survival in bladder cancer flank xenograft mouse models compared to either ADC or RT alone. Conclusions: ADCs demonstrate combination activity with RT across a panel of molecularly diverse bladder cancer preclinical models. These studies provide preclinical data supporting clinical trials to investigate the safety and efficacy of combining ADCs with RT as a bladder-preserving therapy for MIBC.

Sequencing of erdafitinib (erda) and enfortumab vedotin (EV) in patients (pts) with fibroblast growth factor receptor (FGFR2/3) altered (alt) advanced urothelial cancer (aUC): Analysis of UNITE database.

616 Background: The optimal therapy sequencing in aUC with FGFR2/3 alt is unclear. FGFR inhibition may increase Nectin-4 expression. We hypothesized that outcomes with EV would be more favorable when given after E compared to before E in pts with aUC harboring FGFR2/3 genomic alt. Methods: Pts with FGFR2/3 alts who received EV only (EVo), E then EV (E->EV), or EV then E (EV->E) were included. Overall survival (OS) measured from E or EV start (based on agent given first) and progression-free survival (PFS) measured from EV start were assessed with KM method and group comparison by log rank test with Cox proportional hazards regression model. Variables with p ≤0.2 were included in multivariate analysis (MVA). Wilcoxon rank sum and Fisher’s exact test were used to compare continuous and categorical variables, respectively. Observed response rate (ORR) was compared between groups using χ2 test. Results: Among 633 pts from 16 US sites, 487 had NGS data, and 94 (19%) had FGFR2/3 alterations with 24 receiving E->EV, 15 EV->E, and 55 EVo. Median age: 72 yrs; 87% Caucasian, 65% primary bladder tumor, 73% pure urothelial histology, 76% BMI <30, 83% ECOG PS 0-1, 60% visceral metastases (mets) with 23% liver mets, 93% prior IO, and 63% prior Plt. Pts treated with E->EV vs EV->E had similar baseline characteristics, but at EV start ECOG PS 0-1 was 93% in EV->E vs 67% in E->EV (p=0.04). ORR to EV for E->EV pts was 32% (CI- 12%-51%) vs 67% (CI 54%-91%) for EV->E pts (p=0.04). ORR for EVo was 49%. Median OS was 21 months (mo) E->EV, 19 mo EV->E, and 12 mo EVo; median PFS was 5, 5, 6 mo, respectively. Univariate analysis (UVA) and MVA results are shown in Table. In MVA, longer OS was seen in pts who received both EV and E vs EVo. Conclusions: We showed longersurvival in pts with FGFR2/3 alt aUC who received both EV and E regardless of sequence, which may reflect guarantee-time bias. Additional limitations include retrospective nature, modest sample size, no central scan review, lack of data for pts who received E only, selection and confounding biases. Larger prospective studies are needed to determine optimal sequencing and putative biomarkers. [Table: see text]

Outcomes with enfortumab vedotin in patients with advanced urothelial carcinoma and histology variants: Analysis of the UNITE study.

652 Background: Enfortumab vedotin (EV) is approved for patients (pts) with advanced urothelial carcinoma (aUC) but outcomes in pts with histology variants (HV) have not been well described. We hypothesized that presence of HV would impact EV treatment outcomes. Methods: UNITE is a multi-site retrospective study of pts with aUC treated with targeted agents, including 650 pts treated with EV regimens. We compared outcomes to EV monotherapy in pts with pure UC (pUC) relative to pts with any HV and with specific HVs. In pts with scans after > 1 cycle of EV, observed response rate (ORR) was assessed using χ2 test and logistic regression. Median progression-free survival (PFS) and overall survival (OS) from EV start were measured using the Kaplan Meier method and Cox proportional hazard test. Results: A total of 566 pts (366 with pUC; 200 with HV) from 17 US sites were treated with EV monotherapy. Median age was 70 years, 71% men, 87% Caucasian, 74% with ECOG PS 0/1 and 28% with liver mets. Median follow up was 21 mos from EV start, ORR was 49%, mPFS and mOS were 5.8 mos (95% CI: 5.29-6.67) and 12.2 mos (95% CI: 11.5 – 13.8), respectively. Pts with pUC had more visceral mets (69% vs 60%, p = 0.04) and liver mets (32% vs 21%, p =0.003) than pts with HV. No significant difference in outcomes were seen between pUC and pts with HV component (1-100%). Pts with predominant HV (50-99%; N=35), had numerically lower ORR relative to pUC pts (36% vs 52% OR 0.31, p <0.09), but no difference in mPFS or mOS. Pts with pure HV (pHV) (100%: N=14) had inferior outcomes relative to pts with pUC (ORR 0% vs 52%, p<0.001; mOS HR: 2.96 (95% CI: 1.60 – 5.46), p <0.001; mPFS HR: 1.9 (95% CI: 1.06 – 3.39), p=0.03). ORRs for specific HV are listed in the table. No significant difference in treatment outcomes were seen in pts with pUC vs any specific HV, except for NE HV which had inferior ORR (0% vs 52%; p=0.003). Conclusions: Responses to EV are noted in some aUC pts with HV, except for NE (any component) and pHV. Certain HV notable for poor outcomes, such as sarcomatoid and plasmacytoid, demonstrated responses to EV. These hypothesis-generating findings require further validation in studies with adequate sample size for specific HV, and in particular pHV. Evaluation of biomarkers, including Nectin4 expression, in HV is paramount. [Table: see text]

Enfortumab vedotin (EV) as monotherapy in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC): Results of stage I of a phase 2 trial.

152 Background: EV is an antibody-drug conjugate consisting of a fully human antibody directed against the extracellular domain of Nectin-4, which is conjugated to a microtubule-disrupting agent, monomethyl auristatin E (MMAE) by a protease-cleavable linker. In animal models of prostate cancer, metastatic lesions showed high membranous Nectin-4 expression (PMID: 30767361). We aimed to investigate EV in pts with mCRPC. Methods: This IRB-approved, investigator-initiated, single-arm, single-center, phase 2 study administered EV at 1.25 mg/kg IV on days 1, 8, and 15 of 28 days cycle. Key eligibility criteria: ≥18 years age, mCRPC with histologically/cytologically confirmed adenocarcinoma without small cell histology, performance status ≤ 1, adequate organ function, prior treatment (Rx) with ≥3 cycles of docetaxel and at least one androgen-receptor pathway inhibitor, received/refused all therapies shown to improve overall survival except cabazitaxel which is exclusionary. Primary objective: To assess antitumor activity of EV as determined by one of the following: 1) objective response by RECIST 1.1 in those with measurable disease, 2) confirmed conversion of circulating tumor cell count (CTC) to <5/7.5 mL blood, 3) PSA decline of ≥ 50%, and 4) stable disease for ≥ 6 months per PCWG3 modified RECIST 1.1. Simon’s two-stage design was used. The null hypothesis (H0) that true response rate (RR) is 25% is tested against a one-sided alternative. In stage I if ≤3 responses were observed of 11 pts, the study will be stopped. Otherwise, 23 additional pts will be accrued (total 34). H0 will be rejected if ≥13 responses are observed in 34 pts (type I error rate 0.046, power 0.854 when true RR is 50%). Results: Herein, we present results of 11 pts treated on stage I of study. All pts were non-Hispanic white with median age of 72 years (range 43-78). Median number of prior systemic therapies in addition to castration: 5 (range 3-8). Median follow-up of the cohort was 9 months (range 1-13). 4/11 had RECIST measurable disease and 7/11 had CTC ≥5 at baseline. Overall 7/11 (64%) pts had protocol-defined response (5/11 PSA 50% responses, 3/4 objective response, 4/7 confirmed CTC response and 2/11 ≥ 6 months on Rx). Of the 9 pts who discontinued Rx, 8 discontinued due to disease progression (1 withdrew consent). Median number of completed cycles were 6 (range: 2-12) and median radiographic progression-free survival was 5.5 months (95% CI 5.3-not reached). Most common grade 3/4 treatment-related adverse events (TRAEs) were anemia, AST increase, dry skin, fatigue, hyperglycemia, rash, leucopenia (1 pt each) and nausea and decrease in neutrophil (2 pts each). 8 pts had dose interruptions and dose reductions. No Grade 5 TRAEs. Conclusions: EV showed promising efficacy and an acceptable safety profile in pts with heavily treated refractory mCRPC. Further enrollment in stage 2 of the trial is ongoing. Clinical trial information: NCT04754191 .

A phase II open-label study of enfortumab vedotin in patients with previously treated locally advanced, recurrent, or metastatic pancreatic adenocarcinoma (EPIC).

TPS719 Background: Nectin-4 is a type I transmembrane protein member of the nectin family involved in the formation and maintenance of adherens junctions. In contrast to its limited expression in normal tissues, aberrant overexpression of nectin-4 has been identified in various solid tumors, including in pancreatic adenocarcinoma. About 71% of human PDAC specimens stain positive and 13% stain strongly positive for nectin-4. Mechanistically, nectin-4 has been associated with various pro-oncogenic cellular processes, including the promotion of tumor cell proliferation and metastasis via WNT- βcatenin pathway activation. It interacts with ERBB2/HER2 and activates Rac1 (small G protein) to ultimately upregulate PI3K-AKT pathway signaling, leading to cell survival and proliferation. Enfortumab vedotin (EV) is an antibody-drug conjugate that delivers the microtubule-disrupting agent monomethyl auristatin E (MMAE) to cells expressing Nectin-4. EV is FDA approved as a single agent for the treatment of adult patients with locally advanced or metastatic urothelial cancer who have previously received a programmed death receptor-1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitor and platinum-containing chemotherapy based on the EV-301 Trial which demonstrated overall survival benefit. Taken together, the above data and the efficacy of EV seen in bladder cancer warrants the evaluation of EV in advanced pancreatic adenocarcinoma. Methods: Design: Open-label, phase II single-arm trial of EV (n=28) in previously treated locally advanced, recurrent, or metastatic pancreatic adenocarcinoma. Treatment schedule: EV 1.25 mg/kg administered IV on D1, D8, D15 every 28 days until disease progression or unacceptable toxicity. Imaging assessment will be done every 8 weeks. A biopsy prior to therapy and a biopsy on treatment (between days 15-21 of cycle 1) is required. Eligibility: Patients with previously treated, locally advanced, recurrent, or metastatic pancreatic adenocarcinoma. Inclusion criteria also include ECOG PS 0-1 and at least 1 line of prior therapy. Objectives: The primary objective is to determine anti-tumor activity by overall response rate using RECIST 1.1. Secondary objectives include safety, duration of response, disease control rate, progression-free survival and overall survival. Exploratory objectives include Nectin-4 expression (H-score) in tumor tissue and the relationship between the mutational profile of the tumor and response. Statistical Plan: A Simon’s two-stage Minimax design will be used. In the first stage, 18 pts will be accrued. If there is less than 1 response in these 18 pts, the study will be stopped for futility. Otherwise, 10 additional pts will be accrued for a total of 28 pts. The null hypothesis will be rejected if 3 or more responses are observed in 28 pts. Enrollment is currently ongoing. Clinical trial information: NCT05915351 .

Nectin-4 expression in a subset of cutaneous adnexal carcinomas: A potential target for therapy with enfortumab vedotin.

Enfortumab vedotin (EV) is an antibody-drug conjugate directed against Nectin-4 that is used to treat urothelial carcinoma. Nectin-4 is inherently expressed in the skin and adnexal structures. Since therapeutic options for cutaneous adnexal carcinomas are limited, we sought to evaluate Nectin-4 expression in adnexal carcinomas and benign adnexal neoplasms to identify tumors that are potentially targetable with EV. Eight sebaceous carcinomas (seven periocular and one lymph node metastasis), eight digital papillary adenocarcinomas, seven squamoid eccrine ductal carcinomas, eight poromas, eight trichilemmomas, and seven sebaceous adenomas were subjected to immunohistochemical staining for anti-Nectin-4 antibody. H-scores for Nectin-4 expression were calculated. Benign adnexal neoplasms had a significantly lower mean (±SD) Nectin-4 H-score (142.6 ± 39.1) than did the adnexal carcinomas (198 ± 90.8; p = 0.006). Nectin-4 was expressed in 91% (21/23) of adnexal carcinomas. Sebaceous carcinomas frequently exhibited high expression of Nectin-4 (88% [7/8]), with a mean (±SD) H-score (258.1 ± 58.4) significantly higher than those for digital papillary adenocarcinomas (197.5 ± 52.5; p = 0.035) and squamoid eccrine ductal carcinomas (131.4 ± 114.1; p = 0.031). Sebaceous carcinomas also had significantly higher H-scores than did sebaceous adenomas (186.4 ± 25.0; p = 0.013). Increased Nectin-4 expression in a subset of cutaneous adnexal carcinomas, particularly sebaceous carcinomas, reveals that EV is a potential therapeutic option for these tumors.

Associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 with molecular subtypes, PD-L1 expression, and FGFR3 mutational status in two advanced urothelial bladder cancer cohorts.

Treatment options for advanced urothelial carcinoma (aUC) rapidly evolved: besides immunomodulative therapeutic options and inhibitors targeting Fibroblast growth factor receptor (FGFR) alterations, two new antibody-drug conjugates (ADC), sacituzumab govitecan (SG) and enfortumab vedotin (EV), have been approved. However, little is known about the associations of specific aUC properties and the surface target expression of TROP2 and NECTIN-4. Our aim was to characterize associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 protein and gene expression with morphomolecular and clinicopathological characteristics of aUC in two large independent cohorts. The TCGA BLCA (n = 405) and the CCC-EMN (n = 247) cohorts were retrospectively analysed. TROP2/TACSTD2 and NECTIN-4/NECTIN-4 are highly expressed at the protein and transcript level in aUC, and their expression status did not correlate with patient survival in both cohorts. NECTIN-4/NECTIN-4 expression was higher in luminal tumours and reduced in squamous aUCs. NECTIN-4 was negative in 10.6% of samples, and 18.4% of samples had low expression (H-score <15). The TROP2 negativity rate amounted to 6.5%. TACSTD2 and NECTIN-4 expression was reduced in neuroendocrine-like and/or protein-based double-negative tumours. TROP2- and NECTIN-4-negative tumours included one sarcomatoid and four neuroendocrine aUC. FGFR3 alterations and PD-L1 expression on tumour and immune cells did not associate with TROP2 or NECTIN-4 expression. TACSTD2/TROP2 and NECTIN-4/NECTIN-4 are widely expressed in aUC, independent of FGFR3 alterations or PD-L1 expression, thus representing a suitable target for ADC treatment in the majority of aUC. The expression loss was associated with aggressive morphomolecular aUC subtypes, i.e. neuroendocrine(-like) and sarcomatoid aUC.