Anti-GPRC5D/CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma.

Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein-coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.

ARF1 and ARF4 regulate recycling endosomal morphology and retrograde transport from endosomes to the Golgi apparatus.

Small GTPases of the ADP-ribosylation factor (ARF) family, except for ARF6, mainly localize to the Golgi apparatus, where they trigger formation of coated carrier vesicles. We recently showed that class I ARFs (ARF1 and ARF3) localize to recycling endosomes, as well as to the Golgi, and are redundantly required for recycling of endocytosed transferrin. On the other hand, the roles of class II ARFs (ARF4 and ARF5) are not yet fully understood, and the complementary or overlapping functions of class I and class II ARFs have been poorly characterized. In this study, we find that simultaneous depletion of ARF1 and ARF4 induces extensive tubulation of recycling endosomes. Moreover, the depletion of ARF1 and ARF4 inhibits retrograde transport of TGN38 and mannose-6-phosphate receptor from early/recycling endosomes to the trans-Golgi network (TGN) but does not affect the endocytic/recycling pathway of transferrin receptor or inhibit retrograde transport of CD4-furin from late endosomes to the TGN. These observations indicate that the ARF1+ARF4 and ARF1+ARF3 pairs are both required for integrity of recycling endosomes but are involved in distinct transport pathways: the former pair regulates retrograde transport from endosomes to the TGN, whereas the latter is required for the transferrin recycling pathway from endosomes to the plasma membrane.

ARF1 and ARF3 are required for the integrity of recycling endosomes and the recycling pathway.

Small GTPases ARF1 and ARF3 localize mainly to the Golgi apparatus, where they trigger formation of coated carrier vesicles. We previously showed that BIG2, a guanine nucleotide exchange factor specific for ARF1 and ARF3, localizes not only to the trans-Golgi network (TGN) but also to recycling endosomes, where it is involved in regulating the integrity of recycling endosomes. However, it is not yet clear whether ARF1 and ARF3 act downstream of BIG2 to ensure endosome integrity. In this study, we show that EGFP-tagged ARF1 and ARF3 localize to endosomal compartments containing endocytosed transferrin. We further demonstrate that simultaneous depletion of ARF1 and ARF3 induces tubulation of recycling endosomal compartments positive for transferrin receptor, Rab4, and Rab11, but does not significantly affect the integrity of the Golgi apparatus or early or late endosomes. Moreover, the simultaneous depletion of ARF1 and ARF3 suppresses recycling of transferrin but does not affect either its endocytosis or the retrograde transport of TGN38 from early/recycling endosomes to the TGN. In addition, depletion of ARF1 and ARF3 does not affect retrograde transport of CD4-furin from late endosomes to the TGN, or of endocytosed EGF from late endosomes to lysosomes. These results indicate that ARF1 and ARF3 are redundantly required for the integrity of recycling endosomes, and that they regulate transferrin recycling from endosomes to the plasma membrane, but not retrograde transport from endosomal compartments to the TGN.

GBF1-Arf-COPI-ArfGAP-mediated Golgi-to-ER transport involved in regulation of lipid homeostasis.

Eukaryotic cells store neutral lipids and cholesteryl esters in cytoplasmic lipid droplets (LDs), which are generated from the endoplasmic reticulum (ER). Accumulating lines of evidence have indicated that Golgi-to-ER-retrograde transport mediated by COPI-coated vesicles under the control of Arf small GTPases is implicated in LD formation and utilization. However, the detailed mechanism underlying the regulation of lipid homeostasis by COPI-dependent transport has been poorly understood. Here we show that LD deposition and the cellular triacylglycerol content are significantly increased by siRNA-mediated depletion of not only ß-COP (a subunit of the COPI coat complex) but also GBF1 (a guanine nucleotide exchange factor for Arfs), Arf4 and Arf5 (class II Arfs), and ArfGAP1-ArfGAP3 (GTPase-activating proteins for Arfs). Although a previous proteomic study suggested the presence of COPI subunits and Arfs on LDs, we have failed to show that components of the GBF1-Arf-COPI-ArfGAP retrograde transport machinery are directly associated with and closely apposed to LDs. Furthermore, although recent studies suggested that COPI-mediated transport and GBF1 participated in delivery of adipose triglyceride lipase (ATGL) onto the LD surface, we have found that depletion of ß-COP or GBF1 does not affect association of ATGL with LDs or ATGL-mediated lipolysis. On the basis of these results, we propose other mechanisms how the GBF1-Arf-COPI-ArfGAP transport machinery is implicated in the regulation of lipid homeostasis.