Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein belonging to the folate receptor family, is highly expressed in metastatic castration-resistant prostate cancer (mCRPC), and increases following androgen deprivation therapy (ADT). PSMA is a validated diagnostic and therapeutic target for radioligands and antibody-drug conjugates (ADCs). TD001 is a novel ADC composed of a deimmunized anti-PSMA IgG1 monoclonal antibody (HuJ591) conjugated to a highly stable protease-cleavable proprietary linker and the potent Topo I inhibitor exatecan payload. The LD038 linker-payload leverages exatecan’s ability to stabilize Topo I-DNA complexes and induce DNA damage and cell death. We tested TD001 in 2D and 3D cell cultures and in vivo in human CRPC xenograft models with different levels of PSMA expression. We evaluated cell internalization, payload release, and drug activity by flow cytometry, immunohistochemistry, and immunofluorescence microscopy. Free payload levels were measured 24 h post treatment in plasma and tumors by LC-MS/MS. TD001 showed efficient binding and rapid internalization within 1-3 h followed by lysosomal localization and payload release in PSMA-expressing cells. Due to the quick and effective payload release, TD001 exhibited selective and potent activity with the induction of Topo I-mediated DNA damage (gamma-H2AX) and apoptosis (cleaved caspase 3). TD001 had no effect on PSMA-negative cells. This was supported by 3D cell culture models, which demonstrated rapid diffusion into the extracellular matrix, and rapid internalization of TD001 into PSMA-expressing tumor cells. TD001 was evaluated in vivo in three CRPC xenograft models with different PSMA expression patterns: the high/homogenous (∼104 ligands/cell) PSMA-expressing LNCaP-abl cell line-derived xenograft (CDX) castrated mouse model, the intermediate/heterogeneous (∼102-104 ligands/cell) PSMA-expressing 22Rv1 CDX castrated mouse model, and the AR-positive, high/homogenous (∼105 ligands/cell)-PSMA-expressing patient-derived xenograft (PDX) C5 mouse model. The LD038 linker-payload in TD001 resulted in high free payload release within tumor tissue, triggering DNA damage, cell death, and growth arrest. Notably, tumor uptake and a very high tumor/plasma ratio (70-100 fold) of exatecan were similar 24 h after IV administration in all three in vivo models despite substantial differences in PSMA expression and heterogeneity. These findings highlight the ability of TD001 to deliver effectively and selectively the payload exatecan to PSMA-positive prostate tumors, resulting in potent antitumor activity in different CRPC models, including those with intermediate and heterogenous PSMA expression. Daniela Impellizzieri, Elisa Storelli, Atik Balla, Simone Mosole, Cristina Dongilli, Federico Jauk, Roberta Frapolli, Lavinia Morosi, Maurizio D'Incalci, Jemila Houacine, Morris Rosenberg, Bérangère Deleglise, Esteban Cvitkovic, Carlo V. Catapano. Improved tumor penetration and cytotoxic payload release with TD001, a novel PSMA-targeting ADC with optimized linker-payload composition, in PSMA-expressing CRPC CDX castrated mouse models [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 314.

Patients with metastatic castration-resistant prostate cancer (mCRPC) who progress on androgen deprivation therapy (ADT) have limited therapeutic options. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein highly expressed in mCRPC and is a validated target for precision medicine approaches. TD001 is a novel antibody-drug conjugate (ADC) composed of a deimmunized anti-PSMA IgG1 monoclonal antibody (HuJ591) conjugated to a highly stable protease-cleavable topoisomerase I inhibitor (exatecan) proprietary linker-payload (LD038) with a drug-to-antibody ratio (DAR) of 8. TD001 was evaluated in vivo in human cell line-derived xenograft (CDX) models with high PSMA expression (LNCaP-abl; ∼104 ligands/cell) or intermediate/heterogeneous PSMA expression (22Rv1; ∼102-104 ligands/cell) in castrated NRG mice, and in the high PSMA-expressing (∼105 ligands/cell) patient-derived xenograft (PDX) model C5 in non-castrated NRG mice. Of note, PSMA expression increased upon castration in both CDX models, mimicking the clinical condition of the target patient population. After single IV administration across a wide range of doses (up to 20 mg/kg), TD001 achieved dose-dependent, potent tumor growth inhibition (TGI) not only in the high-PSMA LNCaP-abl CDX and C5 PDX models but also in the intermediate-PSMA 22Rv1 CDX model. Rapid responses and high TGI (>90%) after a single administration at optimal doses were seen across all models. Upon tumor regrowth, PSMA expression was unchanged between treated and vehicle control groups, indicating no impact on target expression. Recurrent tumors remained similarly responsive to rechallenge with TD001. Of note, fractionation of a single TD001 dose into 2 or 3 doses administered every 10 days resulted in persistent tumor regression (TGI ≥90% for 60-70 days) and extended survival compared to the equivalent higher single dose. TD001 was well tolerated with no body weight loss or other signs of toxicity, nor mortality at all doses after single and repeated dosing regimens. Alternative dosing schedules, dose response, and tumor/plasma ratio result in a 2-3 fold increase in therapeutic index (TGI ≥90% for up to 60 days). TD001 appears as a best-in-class anti-PSMA ADC with its novel linker-payload (LD038) and exhibits potent antitumor activity in preclinical models with a broad range of PSMA expression, representing a highly promising treatment for PSMA-expressing CRPC patients. The efficacy of TD001 in preclinical in vivo castrated CDX and PDX models supports further investigation in clinical settings. Daniela Impellizzieri, Elisa Storelli, Atik Balla, Simone Mosole, Cristina Dongilli, Federico Jauk, Roberta Frapolli, Lavinia Morosi, Maurizio D'Incalci, Jemila Houacine, Morris Rosenberg, Mohamed Bekradda, Esteban Cvitkovic, Carlo V. Catapano. Long-term tumor growth inhibition and extended survival with TD001, a novel optimized PSMA-targeting ADC, in PSMA-expressing CRPC CDX castrated mouse models [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 311.

Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein belonging to the folate receptor family, is highly expressed in metastatic castrate-resistant prostate cancer (CRPC), and increases following androgen deprivation therapy. TD001 is a novel antibody-drug conjugate (ADC) composed of a deimmunized anti-PSMA IgG1 monoclonal antibody (HuJ591), which binds to the extracellular domain of PSMA, conjugated to a proprietary cleavable topoisomerase I inhibitor exatecan linker-payload (LD038), with a drug-to-antibody ratio (DAR) of 8. Flow cytometry and immunofluorescence microscopy show TD001 is internalized and cleaved rapidly, colocalizing with lysosomes in PSMA-expressing tumor cells. TD001 (10-20 mg/kg IV) was evaluated in a human CRPC cell line-derived xenograft (CDX) model, LNCaP-abl with high PSMA expression, in castrated NSG mice, with a range of tumor sizes (from 150 to 700 mm3) at the time of treatment. TD001 gave dose-dependent and sustained potent activity (tumor growth inhibition [TGI] >80%) with single dosing. Sustained inhibition of regrowth for 20 days was observed after TD001 10 mg/kg even in large tumors up to 400 mm3. Strong antitumoral activity of a single dose of TD001 10 mg/kg was also observed even in tumors >400 mm3 at the time of treatment. Tumor regrowth was faster in larger tumors (mean volume ∼300-400 mm3) compared to smaller tumors (mean volume ∼200 mm3). PSMA expression was unchanged in tumor regrowth between treated and vehicle control in the CDX castrated mouse model after treatment with TD001 up to 20 mg/kg, indicating no impact on target expression. Fractionation of a single TD001 dose into 2 or 3 doses administered every 10 days resulted in a 2-3 fold higher therapeutic index (TGI >90%) compared to the equivalent higher single isodose. No mortality or weight loss was reported. Pharmacokinetics/pharmacodynamic correlations of TD001 are described separately. TD001 appears as a best-in-class anti-PSMA Topo I inhibitor ADC (exatecan payload), and exhibited potent and sustained antitumor activity, including in large tumors, and represents a promising new treatment for PSMA-expressing CRPC patients. Roberta Frapolli, Ezia Bello, Marina Meroni, Daniela Impellizzieri, Elisa Storelli, Carlo V. Catapano, Lavinia Morosi, Maurizio D'Incalci, Jemila Houacine, Esteban Cvitkovic. Dose-response and tumor size at time of treatment with TD001, a novel ADC against PSMA, drive potent tumor growth inhibition in a CRPC CDX castrated mouse model [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 315.

OTX015 has no significant effect on non-malignant human fibroblasts. (a) Representative pictures of human fibroblasts treated for 72 h with 500 nM OTX015 or DMSO control. Scale bar=100 µm. (b) Growth of fibroblasts treated with 500 nM OTX015 (black) or DMSO control (grey) monitored over 96 h using the xCELLigence system. The slope (1/h) of both curves was calculated, and did not show a significant difference (right) using the Student's t test.

Supplemental Table S1: Cell lines and growth medium.

Supplementary Figure Legends 1-2 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

Supplemental Figures S6. Supplemental Figures S6: OTX015 effects on the production of IL-4 and IL-10 in DLBCL cell lines.

OTX015 reduces cell proliferation and induces apoptosis in MYCN amplified human xenografts. (a) Representative pictures of IMR5 xenograft tumor sections stained with hematoxylin/eosin, and immunohistochemical staining for apoptotic cells (cleaved caspase 3) and proliferating cells (Mib-1/Ki 67) are shown. Scale bar=250 µm. The relative fraction of positively stained cells for cleaved caspase 3 (b) and Mib-1 (c) were calculated from three representative images from each xenograft tumor and are shown as box plots. Statistical difference between groups was assessed by Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Differentially expressed genes in IMR5 cells after OTX015 and JQ1 treatment respectively

Supplementary Figure 2 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

Supplemental Figures S1-2. Supplemental Figures S1: effects of OTX015 on cell cycle and cell growth in DLBCL cell lines. Supplemental Figures S2: effects of OTX015 on apoptosis in DLBCL cell lines.

Westermann et al. as well as Valentijn et al. MYCN target gene list and expression changes after OTX015 treatment as well as BRD4 binding

GSEA of cancer gene sets after treatment with OTX015 and/or JQ1(GSEA, Broad institute)

OTX015 effect on cell viability positively correlates with MYCN status. (a) IC50 of the IMR 5, Chp 134, Chp 212, SK N-BE(2), IMR-32, SK-N-BE, NB69, SK N AS and GI-M-EN neuroblastoma cell lines measured using MTT assays. (b) Maximum reduction of viability in IMR 5, Chp 134, Chp 212, SK N-BE(2), IMR-32, SK-N-BE, NB69, SK N AS and GI-M-EN cells treated for 72 h with 6 µM OTX015 and measured using MTT assays. (c) Relative MYCN mRNA (top) and protein (bottom) expression in all analyzed neuroblastoma cell lines. (d/e) The IC50 values of the cell lines shown in (a) were plotted against MYCN mRNA expression (d) or MYCN protein expression (e), which is shown in (c). Both graphs show weak anti-correlative tendencies in both cell lines (MYCN mRNA p=0.22, MYCN protein p=0.16).

GSEA of gene sets describing general cellular processes after treatment with OTX015 and/or JQ1(GSEA, Broad institute)

Supplementary Figure 1 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

List of all genes from hg19 with changes in BRD4 binding using BRD4- ChIP-seq in IMR5 cells with changes after treatment with OTX015

BET inhibition preferentially disrupts expression of genes associated with super-enhancers. (a) ChIP-seq profiles for H3K27Ac binding at representative super-enhancer-associated gene loci, including MYCN, NCOR2, BCOR and GLI2, in MYCN-amplified IMR-5 cells. The x-axis shows genomic position and the y-axis the histone mark signal in units of reads per million per base pair (rpm/bp). The gene model is depicted below and scale bars above the binding profiles. (b) The Fisher exact test shows enrichment of super-enhancer associated genes among the downregulated genes (logFC < 0 and q < 0.05) by OTX015 or JQ1 treatment. (c) Cumulative distribution plots of the top 200 super-enhancer-associated genes and their fold-change measured using genes which are differentially regulated in IMR-5 cells by OTX015 treatment, JQ1 treatment or the overlap between these datasets (left to right). Plots show a shift towards negative logFC for super-enhancer associated genes (blue curves) compared to genes not associated with super-enhancers (black curves). (d) Box plots of log2-fold changes in the top 200 genes associated with super-enhancers (SE) and regular enhancers (non-SE) in genes differentially regulated (compared to DMSO-treated controls) in IMR-5 cells treated with OTX015, JQ1 or the overlap from both datasets (left to right). Statistical significance was determined using a two-sided Mann-Whitney test.

Supplemental Figures S7. Supplemental Figures S7. Effects of JQ1 and OTX015 on BRD4 binding to MYD88 promoter region in DLBCL cell lines.

All H3K27Ac peaks called in non-promoter regions in IMR5