The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.

During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx21 (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in sphingosine-1-phosphate receptor 5 (S1P5) transcript levels, and S1P5-deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S1pr5 locus. S1P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S1P1 also plays a role in NK cell egress from LNs. S1P5 is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S1P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S1P in BM egress. In summary, these findings identify S1P5 as a T-bet–induced gene that is required for NK cell egress from LNs and BM.

Lymphocyte egress from lymph nodes (LNs) is dependent on sphingosine-1-phosphate (S1P), but the cellular source of this S1P is not defined. We generated mice that expressed Cre from the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) locus and that showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report that mice with Lyve-1 CRE-mediated ablation of sphingosine kinase (Sphk) 1 and lacking Sphk2 have a loss of S1P in lymph while maintaining normal plasma S1P. In Lyve-1 Cre+ Sphk-deficient mice, lymphocyte egress from LNs and Peyer's patches is blocked. Treatment with pertussis toxin to overcome Gαi-mediated retention signals restores lymphocyte egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial cadherin distribution at cell–cell junctions. Our data provide evidence that lymphatic endothelial cells are an in vivo source of S1P required for lymphocyte egress from LNs and Peyer's patches, and suggest a role for S1P in lymphatic vessel maturation.