Preclinical evaluation of the efficacy of anti-PVR antibody-drug conjugates combined with anti-PD-1 therapy for bladder cancer.

Loss of 15-Hydroxyprostaglandin Dehydrogenase Expression Contributes to Bladder Cancer Progression

Lentiviral interferon: A novel method for gene therapy in bladder cancer

BackgroundBladder cancer (BLCA) refers to the malignancy growth that spreads from the bladder linings to the bladder muscles. However, the impact of miR-22-3p and lncRNA TRPM2-AS on this tumor has generated divergent views in the literature. This research aimed to study the effects of lncRNA TRPM2-AS on BLCA and its interaction with miR-22-3p and GINS2 mRNA.MethodsqRT-PCR was employed to measure the expression of TRPM2-AS, miR-22-3p and GINS2 mRNA in bladder tissues and cells. The subcellular localization of TRPM2-AS in T24 and 5637 cell lines was identified using a cell fractionation system. Luciferase assay, RIP assay and RNA pull-down assay were later performed to validate the direct binding relationship between TRPM2-AS, miR-22-3p and GINS2 mRNA. Several experiments were conducted to determine the viability, proliferation, colony formation and apoptosis of the cell lines.ResultsFindings indicated that TRPM2-AS was significantly upregulated in BLCA tissues and cell lines. Apart from that, it was observed that TRPM2-AS knockdown significantly inhibited the viability, proliferation and colony formation of BCLA cells, but it promoted the apoptosis of the BCLA cells. A significant downstream target of TRPM2-AS, miR-22-3p was found to show a lower expression level in BLCA tissues and cell lines. However, the inhibition of miR-22-3p considerably enhanced BLCA cell phenotypes. As well as discovering that GINS2 mRNA was a downstream target of miR-22-3p and was significantly upregulated in BLCA, experimental results also indicated that the knockdown of GINS2 suppressed BLCA cell phenotypes.ConclusionThis research confirmed that TRPM2-AS could promote BCLA by binding to miR-22-3p to increase GINS2 expression. This novel interactome in BLCA cell lines might provide more insights into BLCA therapy.

AimBladder cancer (BLCA) is an urogenital system tumor with a high morbidity. We aimed to explore the function and potential mechanism of α-E-catenin (CTNNA1) in BLCA.MethodsThe CTNNA1 expression in BLCA tissues was detected using qRT-PCR and immunohistochemistry. QRT-PCR and Western blot were performed to measure the CTNNA1 expression in BLCA cell lines. CTNNA1 expression was up-regulated in T24 and UMUC-2 cells by CTNNA1 overexpression plasmid transfection. Cell proliferation, apoptosis, migration and invasion were respectively assessed by CCK-8 assay, flow cytometry, wound healing assay and transwell assay. The expression levels of epithelial–mesenchymal transition (EMT)-related factors were tested by qRT-PCR and Western blot. BLCA nude mice models were constructed to explore the effects of CTNNA1 on BLCA in vivo. Gene set enrichment analysis (GSEA) was proceeded to identify the CTNNA1-related pathways in BLCA.ResultsThe expressions of CTNNA1 were down-regulated in BLCA tissues and cell lines, and its low expression indicated poor prognosis of BLCA patients. CTNNA1 inhibited cell proliferation, migration, invasion and EMT and promoted cell apoptosis in BLCA cells. CTNNA1 enhanced E-cadherin expression and suppressed N-cadherin, snail, MMP2 and MMP9 expressions in BLCA cells, which suggested that CTNNA1 repressed EMT in BLCA cells. Moreover, CTNNA1 could inhibit tumor growth in vivo. CTNNA1 was positively associated with P53 and apoptosis pathways in BLCA cells.ConclusionCTNNA1 inhibited cell proliferation, migration, invasion and EMT and promoted cell apoptosis in BLCA via activating P53 and apoptosis pathways. CTNNA1 might be a novel target in BLCA therapy.

Antibody-drug conjugates (ADCs) have emerged as a transformative therapeutic modality in oncology, offering unprecedented precision in targeting tumor cells while sparing healthy tissues. In bladder cancer, a malignancy with high recurrence rates and limited treatment options, ADCs have demonstrated remarkable efficacy by targeting specific tumor-associated antigens such as NECTIN-4 and Human Epidermal Growth Factor Receptor 2 (HER2). This review provides a comprehensive evaluation of the current landscape of ADC-based therapies for bladder cancer, focusing on their mechanisms of action, clinical efficacy, and safety profiles. We systematically analyze 232 clinical trials from 2004 to 2025, revealing a significant upward trend in ADC research, particularly following the Food and Drug Administration's (FDA) accelerated approval of Enfortumab vedotin in 2019. Our findings highlight the predominance of HER2, NECTIN4, and PD-1 as the most extensively studied molecular targets, with a growing interest in combining ADCs with immune checkpoint inhibitors. Geographically, the United States and China lead in ADC clinical trials, reflecting robust research investment and infrastructure. espite the promising advancements, challenges such as toxicity management, patient stratification, and trial design remain critical. This review underscores the importance of continued innovation in ADC technology and personalized approaches to overcome these limitations, ultimately paving the way for more effective and safer treatment options for bladder cancer patients. The future of ADC therapy in bladder cancer is bright, with immense potential to revolutionize the standard of care and improve patient outcomes globally.

Objective To study the expression and significance of PIM-1 in bladder transitional cell carcinoma.Methods Expression and localization of PIM-1 in human normal and malignant bladder specimens were examined by Immunohistochemistry and the PIM-1 staining score was also compared with several clinicopathologic parameters; then PIM-1 expression was also validated in five bladder cancer cell lines by western blotting and immunohistochemistry assays.Subsequent knockdown of PIM-1 was achieved by lentivirus encoding small interfering RNA,and the effect of PIM-1 on bladder cell survival was further assessed by colony formation assays,finally the anti-apoptosis protein bcl-2 and p-BAD were examined by WB.Results When compared with normal epithelium,PIM-1 was overexpressed in bladder cancer epithelium (2/21 to 38/45) (Pvalue ＜ 0.01 ),and the expression level was even higher in invasive bladder cancer than Non-invasive bladder cancer specimens ( 19/20 to 19/25 ) (P value ＜0.01 ).PIM-1 was also detected in all the bladder cancer cell lines examined.Moreover,the knockdown of PIM-1 significantly inhibited bladder cancer cell growth which associated with the reduction of bcl-2 and p-BAD.Conclusion PIM-1 is up-regulated in bladder transitional cell carcinoma specimens and associated with the tumor stage,more importantly PIM-1 is much higher in invasive bladder transitional cell carcinoma than no-invasive bladder transitional cell carcinoma.Further more,PIM-1 is highly expressed in bladder cancer cell lines and downregulated PIM-1 can inhibit the growth of bladder cancer cells and is involve in the increase of apoptosis,therefore PIM-1 may play a role in bladder cancer initiation and progression which may be a potential candidate as target for novel therapy in bladder cancer. Key words: Bladder carcinoma; Kirase; PIM-1

Introduction: Current ADC development focus on a small number of tumor-associated antigens (TAA). In recognizing heterogeneity of cancers and acquired resistance evolved in response to therapies, we have strategized our ADC development by focusing on discovering and develop novel TAAs. Methods: Integrative mining of large-scale omics databases was carried out to discover TAA. In-house antibody discovery and engineering platforms were employed for antibody discovery. In vitro assays were used to screen antibodies of high affinity, high specificity, efficient internalization and developability. A MMAE based ADC was evaluated in vitro for stability, cytotoxicity and bystander effect and in vivo in CDX models for anti-tumor efficacy. PK/TK and GLP toxicology studies were carried out in monkeys. Results: CDCP1 was identified to be significantly up-regulated in large proportions of cancer cells across many solid tumors, which was comparable to or higher expression than targets of the marketed ADC drugs. It is associated with cancer progression, metastasis and contributed to resistance to cancer therapies. We discovered and humanized a CDCP1-binding antibody (HY0001) which shows high affinity and specificity, and displays high efficiency of internalization and yet no agonist activity. We developed an ADC preclinical candidate (HY0001a) by conjugating HY0001 with vcMMAE at a DAR=4. HY0001a was rapidly internalized upon binding to CDCP1+ cancer cells and colocalized with lysosomes in 1∼2 hours after endocytosis. In vitro cytotoxicity assays with CDCP1+ cancer cell lines, HY0001a showed high cytotoxicity activities (IC50 = 0.05∼10 nM) in a target- and dose-dependent manner with strong bystander effect. HY0001a demonstrated strong anti-tumor efficacy in a target- and dose-dependent (1-5 mg/kg) manner when tested across multiple CDX models, including prostate, breast, pancreatic, colon, ovarian and bladder cancers. Like MMAE, HY0001a, but not HY0001, could arrest proliferative cancer cells in their G2M phase and significantly increase the number of apoptotic cells. HY0001a significant induced the apoptotic pathway in the tumor tissue of MDA-MB-231 CDX model. HY0001a is stable in biomatrix and possesses desired PK profiles (T1/2= 2-4 days in the CDX models and 2-3 days in cynomolgus monkeys). HY0001a showed toxicity profiles, similar to the marketed MMAE-based ADC drugs. We have completed development of the CMC processes and production of lots to enable the IND submission and early clinical trials. Conclusions: We identified CDCP1 as an excellent ADC TAA with great potential against multiple cancers. We discovered an antibody (HY0001) of high affinity, cancer cell-specific and efficiently internalization, and completed development of a preclinical candidate MMAE based ADC (HY0001a), which showed a great rationale for further clinical investigation to provide a novel cancer therapy. Citation Format: Wenjun Yan, Yang Zhang, Mengyun Zhang, Bo An, Yujing Zhang, Qing Xiong, Guifeng Chen, Ao Fu, Dan Sun, Xiaoqiang Cai, Huaqin Chen, Mingxia Geng, Haibo Wang, Chengwei Yan, Yike Yuan, Tao Wei. HY0001a: A novel antibody-drug conjugate (ADC) targeting CUB domain containing protein 1 (CDCP1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4256.

The cell surface antigen Lymphocyte antigen 75 (LY75, CD205, DEC-205) is over-expressed in several tumor histotypes. It is a type I C-type lectin receptor (CLR), normally expressed on various APC subsets, characterized by a cytoplasmic domain containing protein motifs crucial for endocytosis and internalization upon ligation. These features make the antigen ideal to be exploited as a target for a novel ADC. MEN1309 is a humanized IgG1 antibody directed against the cell surface antigen Ly75, conjugated through a cleavable linker to a potent maytansinoid microtubule disruptor, DM4. In this study, we evaluated the in vitro and in vivo (xenografts and PDX) efficacy of MEN1309 in different tumor histotypes. A PK/PD relationship was also investigated in tumor-bearing mice. IHC demonstrated high prevalence of Ly75 in human pancreatic, triple negative breast, and bladder cancers, as well as in diffuse large B-cell lymphoma. In vitro experiments showed that cytotoxic activity of MEN1309 was in nM/sub nM range against several lymphoma, pancreatic, bladder and triple-negative breast cancer (TNBC) cell lines. Moreover, MEN1309 exhibited high cell-killing ability against cells having either strong as well as low to moderate antigen expression. In vivo, MEN1309 at 2.5-5 mg/kg (schedule varying from single dose, q7dx3, or q21dx3) showed an impressive antitumor activity, resulting in complete and long lasting responses in most of the xenograft models representing lymphoma, TNBC, bladder and pancreatic cancers, expressing the antigen at high but also at low levels. No treatment related toxicity in terms of change of body weight and death events were detected. Moreover, the administration of (i) isotype control-DM4, (ii) the non-conjugate antibody IgG1 and (iii) the free toxin DM4 (at a dosage corresponding to the equimolar concentration linked at 10 mg/kg ADC) showed little to no therapeutic efficacy on tumor growth. In TNBC patient-derived xenograft (PDX) model (coming from a heavily pre-treated patient and expressing high level of the antigen Ly75), MEN1309 (5 mg/kg q21dx3) showed a complete tumor regression. Finally, in the pancreatic adenocarcinoma xenograft model HPAFII, the pharmacokinetics profile in serum of MEN1309 at 5 mg/kg was characterized and it was qualitatively correlated, using immunofluorescence, with the occurrence of phosphorylation of Serine 10 of H3 Histone in cancer cells, as a pharmacodynamic (PD) marker of DM4 activity on microtubules. Initial ADC exposure was noteworthy and was followed by a relatively fast decline. In parallel with the decay of the serum ADC concentrations there was a progressive increase in the number of positive cells showing the PD marker for mitotic arrest. Overall, our data suggest that MEN1309 is a selective and potent novel antitumoral ADC and it deserves to enter into aPhase I study for a variety of Ly75 positive tumor histotypes. Citation Format: Mario Bigioni, Giuseppe Merlino, Cristina Bernadó Morales, Rossana Bugianesi, Attilio Crea, Rosanna Manno, Joaquin Arribas, Rachel Dusek, Nickolas Attanasio, Keith Wilson, Christian Rohlff, Monica Binaschi. MEN1309, a novel antibody drug conjugate (ADC) targeting Ly75 antigen, induces complete responses in several xenografts of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2630. doi:10.1158/1538-7445.AM2017-2630

Use of Fluorescence In Situ Hybridization to Predict Response to Bacillus Calmette-Guérin Therapy for Bladder Cancer: Results of a Prospective Trial

MAINTENANCE BACILLUS CALMETTE-GUERIN IMMUNOTHERAPY FOR RECURRENT TA, T1 AND CARCINOMA IN SITU TRANSITIONAL CELL CARCINOMA OF THE BLADDER: A RANDOMIZED SOUTHWEST ONCOLOGY GROUP STUDY

Background: Nectin-4 is a tumor-associated antigen, which was observed in multiple cancer types, including bladder, breast, lung, pancreatic and ovarian cancer. Enfortumab Vedotin (EV) is the first approved nectin-4 antibody-drug conjugate (ADC) with MMAE as its payload for the treatment of urothelial carcinoma (UC). However, beyond UC, EV hasn’t been approved for other indications. In addition, severe skin adverse reactions were reported in EV-treated patients. There are several new nectin-4 ADCs in development but haven’t entered confirmative clinical studies so far. To overcome the limitations of efficacy and safety, we developed a novel mechanism nectin-4 ADC GLR1059, with a payload that was more effective for potential indications with nectin-4 expression and had lower toxicity, including skin adverse reactions. Methods: For in vitro studies, binding affinity and specificity, internalization, cancer cell killing, bystander effect and serum stability were performed. For in vivo studies, efficacy studies were conducted on cell line-derived xenografts (CDX) of multiple cancer types, e.g. breast cancer (including triple-negative breast cancer), and urothelial carcinoma. Pharmacokinetic (PK) properties were studied in BALB/c mice and tumor-bearing nude mice, and safety studies were conducted in humanized nectin-4 mice. Results: GLR1059 is a nectin-4 ADC composed of a novel humanized IgG1 monoclonal antibody, a cleavable linker and a microtubule targeting agent (not auristatin derivatives). Moreover, to generate DAR4 ADCs with higher homogeneity, the payload was site-specifically conjugated to the antibody via a disulfide bond bridge. In surface plasmon resonance (SPR) assays, GLR1059 had strong and specific binding to human nectin-4, and it did not bind to nectin-1, nectin-2, or nectin-3. Furthermore, GLR1059 exhibited efficient internalization and potent activity in nectin-4 expressing cell lines and effective bystander activity. In a panel of CDX models, GLR1059 demonstrated compelling anti-tumor activity across bladder cancer and breast cancer (including TNBC), with tumor growth inhibition rates (TGI) significantly higher than EV (approximately 2-4 times). In the tumor-bearing mice PK study, the Cmax of free payload in tumor tissue was approximately 100-fold higher than that in the blood (131±14ng/g vs 1.04±0.27ng/mL). GLR1059 demonstrated longer survival and less weight loss in the single- and repeated-dose toxicity studies in humanized nectin-4 mice. Notably, GLR1059 significantly reduced the incidence and severity of skin toxicity compared with EV (1/6 vs 6/6). Conclusion: Preclinical data show that GLR1059 has the potential to achieve better efficacy and safety, and the different drug mechanisms provide new options to overcome drug resistance of current nectin-4 ADCs. Citation Format: Jiao Jiao, Yingjie Sun, Jiaxing Wang, Liming Che, Menghan Cui, Wenjie Shao, Hairui Yuan, Yan Huang, Nannan Zhai, Yuling Tang, Xiaomei Zheng, Siyao Li. GLR1059, next-generation nectin-4-targeted ADC with a novel mechanism-of-action payload, demonstrated significantly potent anti-tumor efficacy and reduced toxicity in preclinical evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1567.

Bladder and Upper Tract Urothelial Cancer

Bladder cancer (BLCA) is a prevalent urological malignancy that exhibits a high degree of tumor heterogeneity and morbidity. Tumor angiogenesis, a vital hallmark of cancer, greatly influences the tumor microenvironment (TME). The emergence of anti-angiogenic drugs has provided a new turning point in cancer treatment. An integrated machine learning system was constructed to build the angiogenesis-related gene signatures (ARGS). ARGS was used to assess TME status in BLCA. Pharmacophore construction was employed to construct pharmacophore features of highly cytotoxic drug payload combinations for antibody-drug conjugates (ADCs). In addition,we developed a natural compound using artificial intelligence-driven drug design technology.This compound exhibits anti-angiogenic effects in BLCA and serves as a highly cytotoxic drug payload for ADCs. Multi-dimensional machine learning was used to screen biomarkers for evaluating the post-treatment effects of drug therapy in BLCA. The ARGS consists of 12 angiogenesis-related genes associated with prognostic risk in BLCA. The ARGS divides BLCA patients into high-risk and low-risk groups. Significant TME remodeling was identified in the high-risk BLCA cohort and demonstrated a strong association with tumor angiogenesis. Expression levels of key immune checkpoint markers significantly differed between BLCA risk groups. Saikosaponin D (SSD) shows promising potential as a novel ADC drug for anti-angiogenic treatment in BLCA. Multi-dimensional machine learning results indicate that MYH11 is the most likely biomarker for evaluating the post-treatment effects of SSD therapy. SSD may potentially treat tumors by regulating angiogenesis in BLCA. The detection of MYH11 can be used to assess the therapeutic effectiveness of SSD in BLCA.

Rab1A protein has been identified to be highly expressed in a number of malignant tumor tissues and to participate in the regulation of tumor development, but no data concerning bladder cancer have been described at present. The present study measured the expression of Rab1A in bladder cancer tissues and cell lines, and analyzed its clinical significance for patients with bladder cancer. A total of 153 pairs of bladder cancer tumor tissues and adjacent cancer healthy tissues were included in the present study. Western blot analysis and immunohistochemistry were used to measure the expression of Rab1A protein in normal bladder and bladder cancer cell line, and bladder cancer and normal adjacent tissues. SPSS 20.0 software was used for statistical analysis and mapping of survival curves in patients with bladder cancer. The expression levels of Rab1A protein in normal bladder cells and tissues was significantly decreased compared with that in bladder cancer cells and tissues, and it was significantly associated with tumor size, histological grade, tumor-node-metastasis (TNM) stage, lymph node metastasis and remote metastasis in 153 patients with bladder cancer. Cox regression analysis demonstrated that the expression of Rab1A protein in bladder cancer tissues was an independent risk factor for prognosis (overall risk=0.549; 95% confidence interval=0.139-0.916). The 5-year survival rate of patients with bladder cancer with high expression levels of Rab1A protein was 48.613%, which was significantly decreased compared with the rate of patients with low expression 75.31% (P<0.05). The expression of Rab1A in bladder cancer tissues and cell lines was upregulated, and its expression increased with increasing TNM stages. It was also associated with the metastasis of tumor cells and negatively affected the survival time of patients with bladder cancer.

N-Acetylcysteine Augments The Cellular Redox Changes and Cytotoxic Activity of Internalized Mycobacterium Bovis in Human Bladder Cancer Cells