The Role of DLL3 in Targeted Therapy for Small Cell Lung Cancer

Notch Receptor and Ligand Family

Mammalian Notch receptor homologs (Notch1 to 4) encode a Notch extracellular domain (NECD) that binds ligands, a transmembrane domain, and a Notch intracellular domain (NICD) that translocates to the nucleus to serve as a transcriptional cofactor. Mammalian NECDs consist of 29 to 36 EGF repeats followed by three Lin–Notch repeats (LNRs). EGF11 and 12 domains alone are sufficient for binding to Notch ligands (Jagged/DLL). All canonical Notch ligands are transmembrane proteins that share a largely similar structure, with an extracellular domain comprised primarily of multiple EGF repeats (6 for DLL3; 8 for DLL1 and DLL4; or 16 for Jagged-1 and Jagged-2), followed by “module at the N-terminus of Notch ligands” (MNNL) domain and by a “Delta/Serrate/Lag-2 (DSL) domain [1].

The non-canonical Notch ligands lack the DSL domain, among these are proteins delta homolog 1 and 2 (DLK1and DLK2) [2]. Some proteins including Contactin-3 and -6 and DNER have been postulated to act as Notch ligands, but confirmation of these observations are still needed [3].

What is DLL3

DLL3's Structure and Function

Delta-like canonical Notch ligand 3 is a structurally divergent DSL family member. Unlike other DSL ligands, DLL3 localizes in the Golgi apparatus and emerges on the cell surface when overexpressed[5]. Delta-like canonical Notch ligand 3 does not bind to Notch receptors, and inactivates Notch signaling in cis[6]. Delta-like canonical Notch ligand 3 also prevents the localization of Notch and/or DLL1 on the cell surface through intracellular retention[7]. Thus, DLL3 is regarded as a cell-autonomous inhibitor of Notch signaling[8]. It is also one of several notch ligands that is a direct downstream target of ASCL1, a transcription factor associated with pulmonary neuroendocrine cell development[9-12]. These findings suggest that DLL3 is related to neuroendocrine tumorigenesis, especially in lung cancer.

Notch Signaling Channel Overview

In mammals, the main components involved in this signaling are four receptors (Notch1–4) and five ligands of the Delta/Serrate/Lag-2 (DSL) family (Delta-like1,3,4 and Jagged 1,2), and the non-canonical ligand such as Delta-like 1 homolog (DLK1) is also involved. Basically, the Notch receptor comprises epidermal growth factor (EGF)-like repeats, a negative regulatory region (NRR), a transmembrane domain (TM), and a nuclear localization sequence (NLS). The DSL ligand contains similar but shorter EGF-like repeats. In the canonical Notch signaling pathway, after synthesized and cleaved by a furine-like convertase (S1 cleavage) in the Golgi apparatus, the Notch receptor is translocated to the cell membrane and binds to the ligands. Then the ligand ubiquitylation enables the clathrin-mediated endocytosis (CME) and the subsequent opening up of the negative regulatory domain (NRR), which induces the following S2 and S3 cleavage. As a result, the released Notch intracellular domain (NICD) is translocated into the nucleus binding to the recombination signal binding protein for immunoglobulin kappa J region and mastermind like transcriptional coactivator (RBPJ/MAML) complex and modulating downstream targets as a transcription factor [13]. Hairy and enhancer of split 1 (HES1) and HES related to YPRW motif 1 (HEY1) are the target genes regulated by NICD and RBPJ/MAML complex. The activation of HES1/HEY1 transcription then inhibits ASCL1, which is a key transcription factor in the NE differentiation of SCLC and an activator of Notch ligand expression, especially DLL3 [14-15].

The Role of Notch Signaling Pathway and DLL3 in SCLC

In a single sample gene set enrichment analysis (ssGSEA), Notch signaling was revealed to be the most significantly enriched gene set among SCLC-relapsed patients with clinical benefit [17]. The prevalence of NOTCH mutations in SCLC was evaluated to be about 25 %− 28 %, leading to a loss of function of Notch signaling [18-19]. Missense mutations were the most common type, and NOTCH1 was the most frequently mutated family member, followed by NOTCH2, NOTCH4 and NOTCH3. Besides, NOTCH mutations seemed to be strictly found in the lung and scarcely found in metastatic biopsy sites such as the liver. Regarding mutations of the NOTCH ligand family, the overall mutation rate was found to be at a rather low status of 4% − 7%, and the members are mutually exclusive (including DLL1,3,4, DLK1, JAG1,2) [20].

Particularly, analyses from clinical trial populations validated that the inhibitory Notch ligand, DLL3, was expressed in over 75 % of SCLC[21-23], and the majority of SCLC patients had a high DLL3 expression[24-25]. Moreover, high DLL3 expression was validated to inhibit Notch signaling in SCLC, while DLL3-low was found to prevent lateral inhibition in Notch/DLLs interaction [26]. In detail, a successive link was constructed that DLL3-high is associated with Notch-low, HES1-low, ASCL1-high, and NE features, while DLL-low denoted the opposite with less/non-NE features and less hetetrogeneity. Similarly, immunohistochemistry assays revealed that the SCLC-A and SCLC-N subtypes, which harbor high NE gene expression, were associated with a DLL3-high profile, whereas other low-NE subtypes were DLL3-low [27]. To summarize, apart from the high expression on the cell surface, DLL3 possesses an indispensable part in the regulation of Notch signaling. Therefore, DLL3 is perceived as a promising treatment target for SCLC, and the DLL3-specific agents are now being evaluated in numerous preclinical and clinical trials.

Treatment Strategy for DLL3

The broad and specific expression of the Notch inhibitory ligand, DLL3, on SCLC tumor cells renders it an ideal target for treatment. Currently, the DLL3-targeting treatment possesses predominance compared with other therapies affecting Notch signaling. Several different DLL3-targeting modalities are being evaluated, including antibody-drug conjugates (ADC), T-cell engagers, and chimeric antigen receptor (CAR)-T cells.

ADC Therapy

Rovalpituzumab tesirine (Rova-T) is an ADC drug, comprising the humanized anti-DLL3 IgG1 monoclonal antibody SC16, the DNA cross-linking agent SC-DR002 (D6.5), and a protease-cleavable linker that covalently links SC-DR002 to SC16BiTE. SC-DR002 is a cell-cycle independent DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin that could be conveyed into DLL-positive tumor cells to exert tumor-killing function through conjugation with SC16. Although favorable response rate and survival were observed in early phase trials (NCT01901653 [28] and NCT02674568), the phase III trial, Rova-T (TAHOE, NCT03061812) was abrogated because of the inferior OS (6.3 months in Rova-T arm vs. 8.6 months in topotecan arm), and higher rates of adverse events (AEs) [29]. Another phase III study (MERU, NCT03033511) did not exhibit advantageous median OS in the Rova-T dosed patients over the placebo group (8.5 months. vs. 9.8 months), either [30], resulting in the termination of further Rova-T studies.

T cell Engagers

BiTE drugs connect specific antigens on tumor cell surfaces with CD3 on endogenous T cells, redirecting cytotoxic T cells to exert an anti-tumor effect, while preventing extra cytotoxicity on innocent normal cells[31]. AMG757, a DLL3/CD3 BiTE drug, showed evident efficacy in SCLC cell lines and mouse model[32]. Amgen Inc. has launched the open-label, phase I study of AMG757 in Oct. 2017 (DeLLphi300, NCT03319940) in patients with progressed SCLC or recurred following platinum-based chemotherapy[33]. During World Conference on Lung Cancer (WCLC) 2022, Amgen presented the updated data that until June 15, 2022, patients were followed for an average of 8.5 months (0.2–30.7). The established overall response rate (ORR) was 23 %, with 2 complete responses (CRs) and 22 partial responses (PRs). In 37 % of patients, tumor volume was reduced by more than 30%, and the disease control rate was 52 %. The mean duration of patient response to AMG757 therapy was 1.8 months, and the median OS of AMG757 monotherapy was 13.2 months. 56 % of patients experienced cytokine release syndrome (CRS) reactions and 53 % of patients developed treatment-related neurological side effects after treatment, but most of which were limited to grade 1; Neutropenia occurred in 16 % of patients, of which 9% have grade 3 or higher reactions. To summarize, AMG757 has a predictable and manageable safety profile and is eligible to provide promising efficacy and compelling remission persistence in patients with SCLC who have previously received multiple lines of therapy. Based on these results, a phase II study of AMG757 for third-line and subsequent SCLC (NCT05060016) is currently being enrolled.

CAR-T Therapy

CAR-T cells are patient-derived T cells that are genetically modified to express a receptor for the target tumor antigen. Re-administration of CAR-T cells to the patient leads to the specific identification of tumor cells and the subsequent activation and expansion of T cells, which eventually exerts tumor cell killing. AMG119 is a CAR-T-based drug targeting DLL3 on SCLC cells, which has shown robust tumor elimination efficacy both in vitro and in vivo, along with significant cytokine production and proliferation[34]. Therefore, a phase 1 study of AMG 119 (NCT03392064) was initiated as a single administration in relapsed/refractory SCLC patients[35]. However, according to ClinicalTrials.gov, the enrollment is currently on hold due to insufficient active subjects (N = 6) on trial, but may potentially resume. Apart from T cells, CAR-NK-92 cells were also developed to target DLL3 on SCLC cells. Potent in vitro cytotoxicity and cytokine production as well as in vivo tumor regression were attained in the laboratory findings of DLL3-CAR NK-92 cells, broadening the perspective of SCLC treatment strategies[36].

DLL3 Protein

Recombinant Human DLL3 Protein (Tagged)

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