When it comes to DLL3, everyone thinks of ROVA-T, an ADC drug that has disappointed AbbVie's $5.8 billion expectation of DLL3. AbbVie's collapse also made the popularity of DLL3 targeting** drop for a while, but Amgen's DLL3 CD3 bispecific antibody tarlatamab data released at the 2023 ESMO is eye-catching. On December 13 of the same year, Amgen also announced that the FDA's BLA application for tarlatamab had been accepted and granted priority review. As soon as the news was released, the popularity of targeting DLL3** continued to rise. So what exactly is DLL3? At present, which other pharmaceutical companies have laid out the best pipelines targeting DLL3?
Delta-like ligand 3 (DLL3) is a member of the delta-like ligands (DLLS) family. DLLS is a single-shot transmembrane protein attached to the cell surface, and in addition to DLL3, there are two members of this family, DLL1 and DLL4. The genes encoding DLL1, DLL3, and DLL4 are located on chromosome 6., respectively2 and 15q151 on [1]. Human DLL3 protein is composed of 619 amino acids, which is characterized by an N-terminal conserved DSL (delta, serrate, lag2) domain composed of 40 amino acids, 6 EGF-like repeats, and a transmembrane domain, among which the SDL domain is highly conserved in the ligand family and is an essential structure for binding to the Notch receptor. As shown in Figure 1, DLL1 and DLL4 have 8 EGF-like repeats, and DLL1 and DLL4 also have a PDZ-binding motif at the C-terminus, which DLL3 lacks [2]. DLL3 is highly conserved in the evolutionary process, and the homology of human and mouse DLL3 protein sequences can reach 82%.
figure 1. the structure of dlls [1]
DLL3 RNA is mainly expressed in the brain, endocrine tissue, and blood. DLL3 alone is hardly expressed in normal tissues. DLL3 was found to be highly expressed in approximately 80% of small cell lung cancers (SCLCs) and other neuroendocrine tumors. The differential expression of DLL3 in normal and tumor cells makes it an attractive tumor-selective target.
DLL3 is an inhibitory ligand for the Notch signaling pathway[3] and plays a key role in Notch signaling. The Notch pathway is a highly conserved cell-to-cell signaling pathway that involves multiple processes of growth and development, such as differentiation of pluripotent progenitor cells, apoptosis, cell proliferation, and formation of cell boundaries.
In general, the Notch signaling pathway regulates cell fate through local cell-cell interactions. There are four NOTCH receptors (NOTCH1, NOTCH2, NOTCH3, NOTCH4) and five NOTCH ligands (DLL and Jagged 1 2) in mammals. The Notch receptor is cleaved into 2 fragments by the Furin protease (S1 cleaved) in the Golgi apparatus and transported to the cell surface to form transmembrane heterodimers. When interacting with a Notch ligand on adjacent cells, the Notch receptor undergoes two more cleavages, first by ADam1017 catalyzing peptide bond cleavage (S2 cleavage) near the extracellular membrane, and then by cleavage by the -secretase complex (S3 cleavage) close to the intracellular membrane, thereby releasing the Notch intracellular domain (NICD). The released nicd then enters the nucleus and binds to the DNA-binding protein CSL (CBF-1) to form the NICD CSL transcriptional activation complex. This complex then recruits MAML (Mastermind-likeProtein) and activates genes targeted by transcriptional notch, including the family of transcriptional repressors such as HES, HEY, and HERP [4] [5].
DLLS are modified with O-fucose in the Golgi apparatus to increase or decrease DLL-Notch signaling before being transported to the cell membrane. After DLL on the cell membrane binds to EGF, it homeopathically activates the Notch signal through endocytosis. DLLs that are endocytosed are then degraded by proteases or lysosomes, or are transported to the surface of the cell membrane after circulation. The degradation or recycling of DLLs after endocytosis is controlled by the ubiquitinated DLL intracellular domain (DICD). In addition to activating the NOTCH signal, DLLS can also suppress the NOTCH signal in a variety of ways.
figure 2. notch ligands and notch signaling [6]
Going back to DLL3, unlike other mammalian NOTCH family members, DLL3 localizes to the Golgi apparatus and cytoplasmic vesicles during normal development, interacting with the unprocessed full-length NOTCH1 and DLL1, preventing them from localizing on the cell surface, thereby inhibiting NOTCH signaling.
Small cell lung cancer (SCLC) is a poorly differentiated neuroendocrine tumor, which is the most aggressive type of lung cancer, and the progression of SCLC is closely related to the Notch signaling pathway, which regulates many basic processes necessary for normal development, and this pathway inactivation mutation can induce neuroendocrine differentiation of non-neuroendocrine tumor cells or tumor precursor cells. DLL3 is the only inhibitory ligand of the Notch signaling pathway, which is highly expressed in 85% of SCLC but not in normal lung tissue. These results suggest that DLL3 may be involved in neuroendocrine tumorigenesis, but its mechanism of action is unclear. In addition, DLL3 is a downstream target of achaete-scute homolog 1 (ASCL1) and plays a key role in neuroendocrine cell differentiation and SCLC growth. In a study, Furuta M et al. [7] demonstrated that DLL3 overexpression promotes the growth of SCLC cells and enhances their ability to migrate and invade. Deng et al. also found that DLL3 enhanced tumor growth in mice by promoting Akt protein phosphorylation and inhibiting the Notch receptor by promoting cell proliferation and reducing apoptosis in mouse Lewis lung cancer (LLC) cells [8].
In addition to SCLC, DLL3 is also highly upregulated and aberrantly expressed on the cell surface in large cell neuroendocrine lung cancer (LCNEC), certain other neuroendocrine carcinomas, and prostate cancers, making it a potential target.
Currently, there are a total of 16 DLL3-targeting programs, of which 15 are in the clinical stage and 1 is in the preclinical stage. **Varies, including antibody-drug conjugates (ADCs), CAR-T and antibody drugs.
4.1 dll3-adc
rova-tRovalpituzumab tesirine, the full name of which is rovalpituzumab tesirine, is an ADC drug targeting DLL3. The ADC was originally developed by Stemcentrx, which Abbvie acquired in 2016 for an upfront payment of $5.8 billion, and then continued the clinical development of ROVA-T. The latest development is the termination of development, targeting the indication of SCLC. ROVA-T is a DLL3-specific humanized monoclonal antibody conjugated to pyrrolodiazine (PBD) dimer toxin via a cleavable dipeptide linker. ROVA-T triggers receptor-mediated endocytosis by binding to cell surface DLL3. Endocytosis causes the rova-t-DLL3 complex to internalize into the cell and then fuse with lysosomes. The valine-alanine linker of ROVA-T is cleaved by lysosome-associated cathepsin, and PBD is released from the complex into the cytoplasm. The PDB in the cytoplasm then enters the nucleus, inserts between the DNA in a site-specific manner, causes DNA damage, and ultimately induces cell death through apoptosis [9].
figure 3. mechanism of action of rova-t [10]
ROVA-T has conducted at least 10 clinical trials, including two Phase 3 clinical trials. In 2016, the first-in-human study of ROVA-T produced an objective response rate (ORR) of 18% and a 1-year survival rate of 36% in second- and third-line** SCLC patients. Based on this result, ABBVIE directly skipped the Phase II clinical study and conducted a Phase III study, but unfortunately both Phase III studies were terminated early because they did not meet the pre-specified interim primary endpoint of PFS and/or OS. Based on the analysis of the results and other factors, ABBVIE announced the interruption of the development of ROVA-T.
fz-ad005It is the third next-generation ADC product by Fudan Zhangjiang using its Linker-Drug platform (BB05 platform). The ADC product is conjugated with a recombinant human-mouse chimeric anti-DLL3 monoclonal antibody and a topoisomerase I inhibitor through BB05, which was approved for clinical trial on December 22, 2023, and is intended to be developed for advanced solid tumors, including but not limited to small cell lung cancer, large cell neuroendocrine cancer, prostate cancer, etc.
yl212ZL-1310, also known as ZL-1310, is a DLL3 ADC product developed by the Tamlin technology platform with independent intellectual property rights of Yilian Biotech. The Tmalin platform is a novel antibody-drug conjugate platform technology characterized by the use of the tumor microenvironment to overcome the challenges of current ADC drugs. Different from traditional intracellular lysis, YL212 has a dual mechanism of extracellular and intracellular cleavage, and is a candidate molecule based on novel ADC technology. On April 27, 2023, Zai Lab and Yilian Biopharma entered into a strategic collaboration and global licensing agreement for YL212. On December 1, 2023, CDE officially announced that Zai Lab's clinical application for ZL-1310 for injection was accepted. It is currently in Phase I clinical trial for the indication of cancer.
4.2 DLL3 bispecific antibodies
amg 757, also known as tarlatamab, is a bispecific antibody targeting DLL3 and CD3 developed by Amgen. In December 2023, the BLA application was accepted by the FDA and granted priority review status for the treatment of second-line small cell lung cancer, which is expected to become the world's first CD3 DLL3 bispecific antibody. Tarlatamab is a bispecific T cell engager (TCE) with dual affinity for DLL3 on tumor cells and CD3 on T cells. This dual binding binds tumor cells to autologous T cells, triggers the formation of immune synapses and T cell activation, and initiates a polyclonal T cell response characterized by CD3 aggregation, T cell proliferation, and release of porogenic granzyme and perforin. This sequence of events culminates in tumor cell apoptosis and amplification of T cell responses.
The tarlatamab molecule consists of two single-chain variable fragments (SCFVs) and includes a stable, effector-free functional FC domain to increase half-life. Currently, there are six ongoing clinical studies evaluating tarlatamab.
figure 4. the structure of action of tarlatamab [10]
qls31904, independently developed by Qilu Pharmaceutical, is a TCE bispecific antibody targeting DLL3 CD3. The drug includes an anti-DLL3-specific Fab fragment, an anti-CD3 SCFV fragment, and a modified Fc region to support heterodimers. At present, it is in clinical phase I and is intended to be used for advanced solid tumors such as small cell lung cancer.
figure 5. the structure of action of qls31904 [10]
bi764532, developed by Boehringer Ingelheim, is also a TCE bispecific antibody with an IgG backbone targeting DLL3 CD3. In October 2023, BI 764532 received Fast Track Designation from the FDA, ** for extensive-stage small cell lung cancer (SCLC) and advanced or metastatic extrapulmonary neuroendocrine carcinoma (NEC). Currently, BI764532 is in clinical trial.
figure 6. the structure of action of bi764532 [10]
pt-217, developed by Phanes Therapeutics, is a bispecific antibody with a native IgG structure targeting DLL3 and CD47. PT217 can directly kill tumor cells through the ADCP activity of macrophages and the ADCC activity of NK cells, and expand the tumor killing range by simultaneously targeting DLL3 and CD47 overexpressed on the surface of tumor cells. In addition, PT217 is expected to induce tumor neoantigen presentation by directing tumor cells into phagocytic antigen-presenting cells (APCs) and indirectly activate T cell killing of tumor cells with low or no DLL3 expression by recognizing tumor neoantigens, thereby stimulating the adaptive immune system. PT217's anti-CD47 single arm is highly differentiated and has been shown in preclinical models to maintain strong CD47-binding activity to tumor cells while minimally binding to human erythrocytes.
PT217 was approved by the U.S. Food and Drug Administration (FDA) for a multi-center Phase I trial in the U.S. in 2022 (clinical registration number NCT05652686), and in 2022, it received orphan drug designation from the FDA for the treatment of **small cell lung cancer.
4.3 DLL3 triantibodies
hpn328is a trispecific antibody constructed by Harpoon using its proprietary Tri-Specific T Cell Activation Constructs (TriTAC) platform, consisting of three humanized antibody-derived domains: the N-terminal domain that binds DLL3 on tumor cells, the intermediate domain that binds human serum albumin (for half-life extension), and the C-terminal domain that binds CD3. HPN328 is currently in a Phase 1 2 clinical trial to evaluate the safety, tolerability, and pharmacokinetics of HPN328 as a monotherapy** in patients with advanced cancer harboring DLL3 expression. On January 9, 2024, Merck & Co., Inc. (MSD) announced that it would invest about 6US$800 million acquisition of Harpoon Therapeutics. Since then, Merck will have a pipeline of T cell engagers** in development, including HPN328, as it expands its oncology pipeline.
figure 7. the structure of action of hpn328 [10]
zg006, developed by Zelgen Pharmaceuticals, is a trispecific antibody against CD3 and two different DLL3 epitopes (CD3 DLL3 DLL3). Preclinical studies have shown that ZG006 has a significant tumor suppressive effect in mouse tumor models, resulting in complete tumor regression in mice. In non-human primates, ZG006 showed good safety characteristics such as low toxicity. Currently, ZG006 is in a Phase 1 2 clinical trial for the indication of SCLC.
4.4 dll3 car-t
amg 119, a CAR-T cell** developed by Amgen, includes a domain encoding anti-DLL3, a CD28 and 4-1BB costimulatory domain, and a CD3 intracellular domain. In December 2019, the drug was granted orphan drug designation by the FDA. In September 2023, the clinical ** profile and PK characteristics of AMG119 in patients with small cell lung cancer were published in the journal J Clin Pharmacol, and the results showed that AMG119 exhibited strong cell expansion capacity, good cell persistence and good responsiveness, and was clinically safe and well tolerated at the test dose with no dose-limiting toxicity (DLT). Currently, the drug is in phase I clinical trials.
allo-213, is an allogeneic CAR-T targeting DLL3 developed by Allogene, which has not yet been initiated into clinical trials.
lb-2102It is a CAR-T **LB2102 that targets DLL3 biepitope developed by Nanjing Legend and uses VHH antibody technology and "armed" CAR-T technology to overcome tumor microenvironment inhibitory factors. It carries two VHH antibody fragments that recognize DLL3 and a transmembrane protein that can be activated by signals in the tumor microenvironment. In June 2023, it was granted orphan drug designation by the FDA for **SCLC. In November of the same year, Novartis signed an exclusive global license agreement with Legend Biotech for LB2102.
4.5 dll3 car-nk
dll3 car-nk cellsIt is a DLL3 CAR-NK** developed by Tianjin Cancer Hospital, including anti-DLL3 SCFV domain, NKG2D transmembrane domain and 2B4-CD3 domain, and is currently recruiting in phase I clinical trials for ** patients with refractory ES-SCLC.
DMA Biotechnology is a biotechnology company focusing on preclinical R&D products and services for druggable targets. DMA now offers a full range of products and services for DLL3 targets. Products include active proteins, reference antibodies, and flow-proofed monoclonal antibodies; Services include antibody customization for multiple genus proteins, antibody humanization, and affinity maturation services. In addition, in order to accelerate the development of DLL3 bio**, Dima has also prepared a DLL3 target single B cell seed bank, which can obtain lead antibody molecules in 28 days at the earliest; At the same time, Dima has also carried out CAR-T or ADC molecule construction and functional validation for some existing DLL3 lead antibody molecules, helping you to develop DLL3 drugs one step faster.
Recombinant active proteins
human dll3 protein, hfc tag (pme100607)
figure 8. the validated data of pme100607. the purity of the protein is greater than 80% as determined by sds-page and coomassie blue staining (left). elisa plate pre-coated by 2 μg/ml (100 μl/well) human dll3 protein, hfc tag (pme100607) can bind anti-dll3(rovalpituzumab biosimilar) mab (bme100068) in a linear range of 0.64–80 ng/ml (right).
Reference antibody
anti-dll3(rovalpituzumab biosimilar) mab (bme100068)
Flow cytometry validation of monoclonal antibodies
anti-dll3 antibody(67e6), igg1 chimeric mab (dmc101089)
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