Delta-like ligand 3–targeted radioimmunotherapy for neuroendocrine prostate cancer

133 Objectives: Transdifferentiation of prostate adenocarcinoma to Neuroendocrine Prostate Cancer (NEPC) has emerged as one of the leading causes of resistance to androgen deprivation therapy (ADT) [1]. The patients have aggressive disease with visceral metastasis and have short survival time (7-10 months) [2]. Genomic and proteomic characterization of biopsy samples of NEPC lesions indicates loss of androgen receptor (AR) signaling [3]. Therefore, current PET imaging agents such as 18F-FDHT and 68Ga-PSMA11 that rely on functional AR cannot be used. Our goal is to develop a PET based molecular imaging agent that can uniquely identify NEPC lesions. Taking advantage of highly specific expression of Delta-like ligand 3 (DLL3) in NEPC lesions, we have developed zirconium-89 labeled immunoPET agent that can specifically identify NEPC lesions. Methods: We have used well characterized NCI-H660 cell line as representative NEPC model and compared it with AR dependent LNCaP and AR independent PC3 and DU145 cell lines. qPCR was used to measure relative levels of AR-regulated gene transcripts (AR, PSMA, PSA) and NEPC marker DLL3 and normalized to β-actin in the cell lines. Relative protein levels of AR regulated genes and DLL3 were measured by western blot analysis of cellular extracts using β-actin as our control. ImmunoPET agent - 89Zr-SC16 - was developed through the conjugation of the DFO chelator to SC16 (DLL3 specific) mAb and radiolabeled with zirconium-89. Saturation binding assay was performed on the cell line NCI-H660 to determine Bmax and Kd values. For in vivo PET imaging and biodistribution studies, NCI-H660 (DLL3/+) or DU145 (DLL3/-) xenografts were established in 6-8 week old male athymic nude mice. The mice were administered with 89Zr-SC16 and imaged at 24, 48, 72, 96, and 120 h post injection on a PET scanner and at chosen time points mice were euthanized and organs collected for biodistribution studies. Results: Saturation binding assay reveals that Kd =0.35 nM and Bmax = 863 fm/106 cells for 89Zr-SC16. In vitro studies indicated that NCI-H660 cell line was positive for DLL3 and negative for AR, PSA, and PSMA both at transcriptional and translational level. As expected all other cells except PC3 were negative for DLL3. Invivo PET imaging with 89Zr-SC16 showed clear delineation of NCI-H660 (DLL3/+) tumor xenografts. Biodistribution studies showed tumor uptake of 18.4 ± 3.8 %ID/g in the NCI-H660 tumors compared to 5.5 ± 0.5 %ID/g in the DU145 tumors, demonstrating the selective accumulation of the radiotracer in the DLL3-expressing tumors. Conclusions: Our findings demonstrate that only NEPC cells selectively express DLL3 and using DLL3 targeting PET agent 89Zr-SC16 we can non-invasively and uniquely identify NEPC lesions in vivo. Acknowledgement: Partial funding for these studies was provided by 2019 Geoffrey Beene Cancer Research Center Grant.

5029 Background: TheNotch ligand Delta like ligand 3 (DLL3) is aberrantly expressed on the cell surface of small cell lung cancer (SCLC), and the DLL3-antibody drug conjugate, Rova-T, has shown promise for patients with SCLC (Rudin et al, Lancet Onc 2017). NEPC is a late stage subtype of castration resistant prostate cancer with limited therapeutic options. Based on clinical and molecular similarities with SCLC, we investigated expression of DLL3 and the use of Rova-T in NEPC. Methods: We evaluated mRNA and/or protein expression of DLL3 in a cohort of 395 patients (535 samples) ranging from benign prostate (BEN), localized prostate adenocarcinoma (PCA), castration resistant adenocarcinoma (CRPC), and NEPC and correlated with pathologic and genomic features. Prostate cancer cell lines and patient-derived organoids were treated with Rova-T (SC16LD6.5) in vitro and in vivo. Results: DLL3 was expressed at the mRNA and/or protein level in 0/143 BEN (0%), 4/266 PCA (1%), 8/76 CRPC (10%), 33/50 NEPC (66%). DLL3 IHC was of higher intensity in NEPC and co-localized with classical NE marker expression (SYP, CGA). DLL3 was amongst the most differentially expressed genes by RNA-seq in NEPC versus CRPC (p = < 0.0001, fold change = 71), correlated with ASCL1 expression (r = 0.88) and RB1 genomic loss (83%), and inversely with AR expression. Although treatment with the Notch inhibitor DAPT suppressed Notch target gene expression in NEPC, DAPT did not have significant effect on cellular proliferation. siRNA knockdown of DLL3 or DAPT did not alter AR signaling or NE markers. Rova-T (SC16LD6.5) was active in DLL3-positive NEPC cell lines with an IC50 of 580pM compared to the control IgG1LD6.5 (IC50 = 6.3nM), whereas CRPC lines were insensitive. Conclusions: DLL3 is a cell surface protein aberrantly expressed in the majority of NEPC and a subset of CRPC, and is not expressed in primary prostate cancer or benign tissues. The DLL3 antibody-drug conjugate Rova-T demonstrates preferential preclinical activity in NEPC compared to prostate adenocarcinoma. These data support further investigation of Rova-T as a potential therapeutic agent for NEPC. A phase I trial with dedicated NEPC arm is currently accruing patients (NCT02709889).

Background: Neuroendocrine Prostate Cancer (NEPC) is an aggressive variant of prostate cancer that can arise de novo or emerge during prostate cancer progression as a mechanism of therapy resistance. The development of effective therapies for patients with NEPC is a clinical unmet need. Delta-Like Ligand 3 (DLL3) is a negative regulator of Notch signaling and aberrantly expressed on the cell surface in the majority of NEPC and other small cell carcinomas. HPN328 is a DLL3-targeted Tri-specific T cell Activating Construct (TriTAC) that binds to DLL3 on tumor cells, CD3 on T cells, and human serum albumin. Here, we evaluated the specificity and anti-tumor activity of HPN328 in NEPC preclinical models. Methods: Immunohistochemistry (IHC) was used to evaluate DLL3 expression in prostate cancer preclinical models. We co-cultured DLL3-positive and DLL3-negative tumor cells in vitro with either human PBMCs or T cells and used CellTiter-Glo and live-cell imaging to examine tumor cell viability after the exposure to HPN328. We conducted flow cytometry to examine T cell activation. Patient-derived organoid xenografts were used to determine the anti-tumor activity of HPN328 in vivo and we applied digital spatial profiling (Nanostring) to examine effects on immune cell infiltration. Results: HPN328 suppressed cell growth and induced cell death in DLL3-positive prostate cancer organoid cell line models at a range of 0.7nM - 10nM with the presence of human T cells. Flow cytometry analysis illustrated that T cell activation markers, such as CD25, were upregulated in a dose- and time-dependent manner. In vivo studies demonstrated robust anti-tumor activity of HPN328 in causing tumor regression and stable disease after stopping the treatment in DLL3-positive models, but not in DLL3-negative models. Sustained anti-tumor effects were observed without adverse events in mice. Conclusions: The DLL3-targeted TriTAC HPN328 demonstrates robust and specific anti-tumor activity in DLL3-positive NEPC preclinical models in vitro and in vivo. T cell engagers targeting DLL3 represent a promising therapeutic strategy for NEPC. Acknowledgements: This research is funded by a Helen Trailblazers Award from the Helen Gurley Brown Foundation/DFCI Presidential Initiative (H.B.). Citation Format: Sheng-Yu Ku, Yasutaka Yamada, Patrick Ng, Laura Sun, Himisha Beltran. DLL3-targeted T cell engager therapy (HPN328) for neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2896.

Imaging with [89Zr]Zr-DFO-SC16.56 anti-DLL3 antibody in patients with high-grade neuroendocrine tumours of the lung and prostate: a phase 1/2, first-in-human trial

Treatment-induced neuroendocrine prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer. Using the 89Zr-labeled delta-like ligand 3 (DLL3) targeting antibody SC16 (89Zr-desferrioxamine [DFO]-SC16), we have developed a PET agent to noninvasively identify the presence of DLL3-positive NEPC lesions. Methods: Quantitative polymerase chain reaction and immunohistochemistry were used to compare relative levels of androgen receptor (AR)-regulated markers and the NEPC marker DLL3 in a panel of prostate cancer cell lines. PET imaging with 89Zr-DFO-SC16, 68Ga-PSMA-11, and 68Ga-DOTATATE was performed on H660 NEPC-xenografted male nude mice. 89Zr-DFO-SC16 uptake was corroborated by biodistribution studies. Results: In vitro studies demonstrated that H660 NEPC cells are positive for DLL3 and negative for AR, prostate-specific antigen, and prostate-specific membrane antigen (PSMA) at both the transcriptional and the translational levels. PET imaging and biodistribution studies confirmed that 89Zr-DFO-SC16 uptake is restricted to H660 xenografts, with background uptake in non-NEPC lesions (both AR-dependent and AR-independent). Conversely, H660 xenografts cannot be detected with imaging agents targeting PSMA (68Ga-PSMA-11) or somatostatin receptor subtype 2 (68Ga-DOTATATE). Conclusion: These studies demonstrated that H660 NEPC cells selectively express DLL3 on their cell surface and can be noninvasively identified with 89Zr-DFO-SC16.

e15057 Background: Delta-like ligand 3 (DLL3) is expressed on the tumor cell surface of some neuroendocrine prostate cancer (NEPC). We assessed the safety and feasibility of the DLL3-targeted imaging tracer [ 89 Zr]Zr-DFO-SC16.56, which is composed of the anti-DLL3 antibody SC16.56 conjugated to p-SCN-Bn-deferoxamine (DFO) and zirconium-89 in patients with NEPC. Methods: We conducted an open-label, first-in-human study of immunoPET/CT imaging with [ 89 Zr]Zr-DFO-SC16.56. The initial phase I included patients with small-cell lung cancer (n=3) that showed high tumoral uptake of the diagnostic tracer without any major adverse events. The expansion cohort included NEPC patients that received a single infusion of [ 89 Zr]Zr-DFO-SC16.56 at the same activity (1-2 mCi) and mass dose (2-3 mg) as in the initial cohort followed by a single PET-CT scan 3–5 days later. Retrospectively collected tumor biopsy samples were assessed for DLL3 by immunohistochemistry (IHC). DLL3 positive expression was defined as ≥ 5% weak (1+) IHC staining intensity. The primary endpoint of the expansion cohort was to determine the correlation between tumor uptake of [ 89 Zr]Zr-DFO-SC16.56 with expression of DLL3 as determined by IHC. Results: Between April 2023, and October 2024, 11 men with NEPC were enrolled, with a median age of 60 years (range 51–80). A single immunoPET/CT scan on day 3–5 post-administration could delineate DLL3-avid tumors in 6 (55%) of 11 patients. A range in tumoral uptake was observed within individual patients as well as the entire cohort, with a wide range in maximum standardized uptake value (from 0·4 to 75·5). Tumoral uptake by [ 89 Zr]Zr-DFO-SC16.56 was associated with protein expressionin 8 (80%) of 10 patients who had DLL3 IHC performed.None of the patients had any adverse events noted, and no clinically meaningful changes were observed in vital signs or laboratory parameters post injection. Conclusions: DLL3 PETCT imaging of patients with NEPC is safe and feasible. These results show the potential utility of [ 89 Zr]Zr-DFO-SC16.56 for non-invasive in vivo detection of NEPC patients. Clinical trial information: 19-292 . Characteristics of patients included in the study. Age DLL3 avid SUV max of highest DLL3-avid lesion (anatomic location of most avid lesion) Blood uptake from day 3–5 images, SUV mean Biopsy location (immunohistochemistry H-score) Patient 1 71 No 2·2 (liver) 3·7 Liver (0) Patient 2 58 Yes 75·5 (liver) 2·4 Liver (220) Patient 3 57 No 0·4 (Sacrum) 0·7 Prostate (0) Patient 4 60 Yes 11·3 (Humerus) 1·3 Liver (190) Patient 5 80 Yes 39·9 (liver) 5·8 Pleurae (280) Patient 6 62 Yes 10·4 (Sacrum) 7·1 Liver (0) Patient 7 51 No 6·7 (lymph node) 6·5 Lymph node (170) Patient 8 60 Yes 36·4 (liver) 6·3 Liver (230) Patient 9 55 Yes 20·3 (liver) 7·0 R Liver (170) Patient 10 74 No 3·9 (vertebrae) 7·5 Liver (NA) Patient 11 65 No 1·7 (vertebrae) 5·4 Lymph node (0) NA=not applicable.

Purpose:Neuroendocrine prostate cancer (NEPC) is an aggressive form of prostate cancer with poor prognosis and limited treatment options. As NEPC aberrantly expresses delta-like ligand 3 (DLL3), the activity of tarlatamab, a bispecific T-cell engager that directs cytotoxic T cells to DLL3-positive (DLL3+) cells, was evaluated in the DeLLpro-300 study (NCT04702737).Patients and Methods:This was a phase 1b, open-label study evaluating tarlatamab monotherapy in patients with metastatic de novo or treatment-emergent NEPC defined by histologic, genomic, or IHC criteria. Tarlatamab was administered intravenously every 2 weeks at a dose of 100 mg with a 1-mg step dose. The primary objective was safety, and a secondary objective was objective response rate (ORR) per RECIST v.1.1; DLL3 expression was retrospectively assessed by IHC.Results:Forty patients were enrolled (DLL3+ tumors, n = 18; DLL3− tumors, n = 14; and DLL3 unknown tumors, n = 8). The most common treatment-related adverse events were cytokine release syndrome (82.5%), dysgeusia (42.5%), and decreased appetite (40.0%). Cytokine release syndrome was predominantly of low grade (grade 1/2/3/4+, 62.5%/15%/5%/0%), occurred exclusively in cycle 1, and was transient in duration (median duration, 3 days). The ORR was 10.5% [95% confidence interval (CI), 2.9–24.8]; the median duration of response was 7.3 months in the overall cohort. Patients with DLL3+ tumors (vs. patients with DLL3−/DLL3 unknown tumors) achieved a higher ORR [22.2% (95% CI, 6.4–47.6) vs. 0% (95% CI, 0–15.4)] and radiographic progression-free survival rate at 6 months [27.7% (95% CI, 8.7–50.9) vs. 0%].Conclusions:The DeLLpro-300 study provides preliminary evidence for the safety and antitumor activity of tarlatamab in DLL3+ NEPC.

<div>Abstract<p>Neuroendocrine prostate cancer (NEPC) is an aggressive variant of prostate cancer with limited therapeutic options. Delta-like ligand 3 (DLL3) is a cell-surface protein and therapeutic target expressed in the vast majority of NEPC tumors. The DLL3-targeted T cell–activating construct MK-6070 (formerly called HPN328) binds to both DLL3 on tumor cells and CD3 on T cells, as well as serum albumin to extend half-life. A phase I/II trial of MK-6070 is currently underway, which includes an NEPC cohort (NCT04471727). In this study, we report the preclinical activity of MK-6070 in prostate cancer models, showing high specificity and antitumor activity in DLL3-expressing NEPC models both <i>in vitro</i> and <i>in vivo</i>, with T-cell activation and tumor infiltration of T cells after treatment. MK-6070 also demonstrates antitumor activity in mixed tumors, affecting DLL3-negative prostate cancer cells after engagement with surrounding DLL3-expressing tumor cells, supporting a potential bystander effect. Overall, these data demonstrate the promising activity of MK-6070 in NEPC preclinical models including heterogeneous tumors, supporting the clinical development of MK-6070.</p></div>

Neuroendocrine prostate cancer (NEPC) is an aggressive variant of prostate cancer with limited therapeutic options. Delta-like ligand 3 (DLL3) is a cell-surface protein and therapeutic target expressed in the vast majority of NEPC tumors. The DLL3-targeted T cell-activating construct MK-6070 (formerly called HPN328) binds to both DLL3 on tumor cells and CD3 on T cells, as well as serum albumin to extend half-life. A phase I/II trial of MK-6070 is currently underway, which includes an NEPC cohort (NCT04471727). In this study, we report the preclinical activity of MK-6070 in prostate cancer models, showing high specificity and antitumor activity in DLL3-expressing NEPC models both in vitro and in vivo, with T-cell activation and tumor infiltration of T cells after treatment. MK-6070 also demonstrates antitumor activity in mixed tumors, affecting DLL3-negative prostate cancer cells after engagement with surrounding DLL3-expressing tumor cells, supporting a potential bystander effect. Overall, these data demonstrate the promising activity of MK-6070 in NEPC preclinical models including heterogeneous tumors, supporting the clinical development of MK-6070.

Delta-like ligand 3 (DLL3) is a tumor-selective cell surface protein upregulated in high-grade neuroendocrine tumors, including small-cell lung cancer (SCLC) and neuroendocrine prostate cancer (NEPC). Here, we report on the development of anti-DLL3 radioimmunoconjugates for use as either a diagnostic imaging tracer based on the positron-emitter zirconium-89 (89Zr) or a therapeutic agent utilizing the beta-emitter lutetium-177 (177Lu). To begin, we generated a panel of human monoclonal antibodies targeting human DLL3 by immunizing transgenic mice engineered with a human immunoglobulin repertoire. The panel was extensively screened to identify high-affinity internalizing monoclonal antibodies (mAbs) recognizing a diversity of DLL3 epitopes. Select mAbs were reformatted as fully human Fab-arm exchange-deficient IgG4 to reduce effector function and then produced by recombinant methods. A pilot immunoPET study was performed in athymic female nude mice bearing human NCI-H82 SCLC tumors to nominate a lead candidate. ImmunoPET identified [89Zr]Zr-DFO-TDI-Y-010 as the top-performing diagnostic tracer, with excellent in vivo biodistribution and tumor-to-background-organ ratios consistently >4. Therapeutic studies with [177Lu]Lu-CHX-A"-DTPA-TDI-Y-010 demonstrated strong antitumor effects, significantly improving (P <0.05) overall survival compared with the benchmark clone [177Lu]Lu-CHX-A"-DTPA-SC16.56 in two SCLC tumor models (NCI-H82 and Lu149) and achieving comparable overall survival in a NEPC tumor model.

Real-world experience in treatment outcome and genomic insights for metastatic prostate neuroendocrine carcinoma

Therapeutic approaches targeting proteins on the surface of cancer cells have emerged as an important strategy for precision oncology. To capitalize on the potential impact of drugs targeting surface proteins, detailed knowledge about the expression patterns of the target proteins in tumor tissues is required. In castration-resistant prostate cancer (CRPC), agents targeting prostate-specific membrane antigen (PSMA) have demonstrated clinical activity. However, PSMA expression is lost in a significant number of CRPC tumors. The identification of additional cell surface targets is necessary to develop new therapeutic approaches. Here, we performed a comprehensive analysis of the expression heterogeneity and co-expression patterns of trophoblast cell-surface antigen 2 (TROP2), delta-like ligand 3 (DLL3), and carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in CRPC samples from a rapid autopsy cohort. We show that DLL3 and CEACAM5 exhibit the highest expression in neuroendocrine prostate cancer (NEPC), while TROP2 is expressed across different CRPC molecular subtypes, except for NEPC. We further demonstrated that AR alterations were associated with higher expression of PSMA and TROP2. Conversely, PSMA and TROP2 expression was lower in RB1-altered tumors. In addition to genomic alterations, we show a tight correlation between epigenetic states, particularly histone H3 lysine 27 methylation (H3K27me3) at the transcriptional start site and gene body of TACSTD2 (encoding TROP2), DLL3, and CEACAM5, and their respective protein expression in CRPC patient-derived xenografts. Collectively, these findings provide insights into patterns and determinants of expression of TROP2, DLL3, and CEACAM5 with implications for the clinical development of cell surface targeting agents in CRPC.

Neuroendocrine prostate cancer (NEPC) is a rare and aggressive subtype of prostate cancer, presenting significant diagnostic and therapeutic challenges. NEPC arises in two distinct forms: de novo NEPC, affecting less than 2% of cases, and treatment-emergent NEPC (t-NEPC), which develops in up to 30% of patients with castration-resistant prostate cancer (CRPC). This aggressive variant is characterized by androgen receptor (AR)-independent growth, resistance to conventional hormone therapies, rapid progression, and frequent visceral metastases. Diagnosis of NEPC remains challenging due to the absence of prostate-specific antigen (PSA) elevation, limitations in tissue sampling, and reliance on advanced imaging techniques like fluorodeoxyglucose-positron emission tomography (FDG-PET). Current treatments rely on platinum-based chemotherapy, but outcomes remain poor, with median survival under 17 months. Emerging therapies focus on molecular alterations, including phosphatase and tensin homlog (PTEN) loss, brest cancer gene 1 (BRCA1), brest cancer gene 2 (BRCA2) mutations, and delta-like ligand 3 (DLL3) expression. Despite advances in understanding NEPC biology, effective treatments remain limited, underscoring the urgent need for novel therapeutic strategies to improve patients’ survival and quality of life.

Cell-surface targets for prostate cancer therapeutics.

TPS2700 Background: Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is aberrantly expressed on the surface of tumor cells, in particular on those with neuroendocrine differentiation. Tarlatamab is a bispecific T cell engager that binds to DLL3 and CD3 to promote T cell killing of DLL3-expressing cells. Prior studies of tarlatamab have demonstrated encouraging antitumor activity and manageable toxicity in patients with small cell lung cancer (SCLC; DeLLphi-301) and neuroendocrine prostate cancer (NEPC; DeLLpro-300). Meanwhile, DLL3 has been reported to be highly expressed in multiple tumor types, including in many neuroendocrine neoplasms (NENs) other than SCLC and NEPC. The role of anti-DLL3 therapies in these cancers has not been established. Methods: This is a phase 2, multicenter, open-label, basket study designed to evaluate the efficacy of tarlatamab in patients with DLL3-expressing cancers. Key inclusion criteria include presence of advanced stage disease with progression following ≥1 prior line of therapy and positive tumor DLL3 expression by immunohistochemistry (Ventana SP347 assay). Patients with de novo SCLC or NEPC are excluded, but all other tumor types and NENs are eligible, including large cell neuroendocrine carcinoma and SCLC transformed from previously treated NSCLC. Tarlatamab will be administered at an initial step-up dose (1 mg on D1 and 10 mg on D8 and D15 of cycle 1) followed by 10 mg every 2 weeks. Treatment will continue until unacceptable toxicity, progressive disease, or withdrawal of consent. The study will follow a Simon's two-stage design: in Stage 1, 10 patients with tumor DLL3 expression ≥25% will be enrolled, and the study will be stopped if ≤1 patient achieves an objective response; otherwise, an additional 19 patients with tumor DLL3 expression ≥1% will be enrolled for Stage 2. The primary endpoint is the objective response rate. Secondary endpoints include safety, progression free survival, duration of response, and overall survival. Exploratory studies will evaluate correlation of antitumor activity with tissue and blood-based biomarkers, such as DLL3 expression on tumor and liquid biopsies. This study is currently enrolling patients through the University of California Lung Cancer Consortium (UCLCC). Clinical trial information: NCT06788938 .