Mechanistic and pharmacodynamic studies of DuoBody-CD3x5T4 in preclinical tumor models.

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.

CXCR4, but not CXCR3, drives CD8+ T-cell entry into and migration through the murine bone marrow.

The BM serves as a blood-forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T-cell migration to and localization inside the BM. As BM accumulates many CXCR3-expressing memory CD8+ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8+ T cells in BM, is critically important for homing of all CD8+ T-cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8+ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM-1+VCAM-1+BP-1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL-7 and IL-15. We therefore conclude that CXCR4 is not only crucial for entry of CD8+ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.

A novel in vitro model for the assessment of postnatal myonuclear accretion.

BACKGROUND: Due to the post-mitotic nature of myonuclei, postnatal myogenesis is essential for skeletal muscle growth, repair, and regeneration. This process is facilitated by satellite cells through proliferation, differentiation, and subsequent fusion with a pre-existing muscle fiber (i.e., myonuclear accretion). Current knowledge of myogenesis is primarily based on the in vitro formation of syncytia from myoblasts, which represents aspects of developmental myogenesis, but may incompletely portray postnatal myogenesis. Therefore, we aimed to develop an in vitro model that better reflects postnatal myogenesis, to study the cell intrinsic and extrinsic processes and signaling involved in the regulation of postnatal myogenesis. METHODS: Proliferating C2C12 myoblasts were trypsinized and co-cultured for 3 days with 5 days differentiated C2C12 myotubes. Postnatal myonuclear accretion was visually assessed by live cell time-lapse imaging and cell tracing by cell labeling with Vybrant® DiD and DiO. Furthermore, a Cre/LoxP-based cell system was developed to semi-quantitatively assess in vitro postnatal myonuclear accretion by the conditional expression of luciferase upon myoblast-myotube fusion. Luciferase activity was assessed luminometrically and corrected for total protein content. RESULTS: Live cell time-lapse imaging, staining-based cell tracing, and recombination-dependent luciferase activity, showed the occurrence of postnatal myonuclear accretion in vitro. Treatment of co-cultures with the myogenic factor IGF-I (p < 0.001) and the cytokines IL-13 (p < 0.05) and IL-4 (p < 0.001) increased postnatal myonuclear accretion, while the myogenic inhibitors cytochalasin D (p < 0.001), myostatin (p < 0.05), and TNFα (p < 0.001) decreased postnatal myonuclear accretion. Furthermore, postnatal myonuclear accretion was increased upon recovery from electrical pulse stimulation-induced fiber damage (p < 0.001) and LY29004-induced atrophy (p < 0.001). Moreover, cell type-specific siRNA-mediated knockdown of myomaker in myoblasts (p < 0.001), but not in myotubes, decreased postnatal myonuclear accretion. CONCLUSIONS: We developed a physiologically relevant, sensitive, high-throughput cell system for semi-quantitative assessment of in vitro postnatal myonuclear accretion, which can be used to mimic physiological myogenesis triggers, and can distinguish the cell type-specific roles of signals and responses in the regulation of postnatal myogenesis. As such, this method is suitable for both basal and translational research on the regulation of postnatal myogenesis, and will improve our understanding of muscle pathologies that result from impaired satellite cell number or function.