Validation and promise of a TCR mimic antibody for cancer immunotherapy of hepatocellular carcinoma.

Monoclonal antibodies are at the vanguard of the most promising cancer treatments. Whereas traditional therapeutic antibodies have been limited to extracellular antigens, T cell receptor mimic (TCRm) antibodies can target intracellular antigens presented by cell surface major histocompatibility complex (MHC) proteins. TCRm antibodies can therefore target a repertoire of otherwise undruggable cancer antigens. However, the consequences of off-target peptide/MHC recognition with engineered T cell therapies are severe, and thus there are significant safety concerns with TCRm antibodies. Here we explored the specificity and safety profile of a new TCRm-based T cell therapy for hepatocellular carcinoma (HCC), a solid tumor for which no effective treatment exists. We targeted an alpha-fetoprotein peptide presented by HLA-A*02 with a highly specific TCRm, which crystallographic structural analysis showed binds directly over the HLA protein and interfaces with the full length of the peptide. We fused the TCRm to the γ and δ subunits of a TCR, producing a signaling AbTCR construct. This was combined with an scFv/CD28 co-stimulatory molecule targeting glypican-3 for increased efficacy towards tumor cells. This AbTCR + co-stimulatory T cell therapy showed potent activity against AFP-positive cancer cell lines in vitro and an in an in vivo model and undetectable activity against AFP-negative cells. In an in-human safety assessment, no significant adverse events or cytokine release syndrome were observed and evidence of efficacy was seen. Remarkably, one patient with metastatic HCC achieved a complete remission after nine months and ultimately qualified for a liver transplant.

SNX5 targets a monoamine transporter to the TGN for assembly into dense core vesicles by AP-3.

The time course of signaling by peptide hormones, neural peptides, and other neuromodulators depends on their storage inside dense core vesicles (DCVs). Adaptor protein 3 (AP-3) assembles the membrane proteins that confer regulated release of DCVs and is thought to promote their trafficking from endosomes directly to maturing DCVs. We now find that regulated monoamine release from DCVs requires sorting nexin 5 (SNX5). Loss of SNX5 disrupts trafficking of the vesicular monoamine transporter (VMAT) to DCVs. The mechanism involves a role for SNX5 in retrograde transport of VMAT from endosomes to the TGN. However, this role for SNX5 conflicts with the proposed function of AP-3 in trafficking from endosomes directly to DCVs. We now identify a transient role for AP-3 at the TGN, where it associates with DCV cargo. Thus, retrograde transport from endosomes by SNX5 enables DCV assembly at the TGN by AP-3, resolving the apparent antagonism. A novel role for AP-3 at the TGN has implications for other organelles that also depend on this adaptor.

Functionally distinct PI 3-kinase pathways regulate myelination in the peripheral nervous system.

The PI 3-kinase (PI 3-K) signaling pathway is essential for Schwann cell myelination. Here we have characterized PI 3-K effectors activated during myelination by probing myelinating cultures and developing nerves with an antibody that recognizes phosphorylated substrates for this pathway. We identified a discrete number of phospho-proteins including the S6 ribosomal protein (S6rp), which is down-regulated at the onset of myelination, and N-myc downstream-regulated gene-1 (NDRG1), which is up-regulated strikingly with myelination. We show that type III Neuregulin1 on the axon is the primary activator of S6rp, an effector of mTORC1. In contrast, laminin-2 in the extracellular matrix (ECM), signaling through the α6ß4 integrin and Sgk1 (serum and glucocorticoid-induced kinase 1), drives phosphorylation of NDRG1 in the Cajal bands of the abaxonal compartment. Unexpectedly, mice deficient in α6ß4 integrin signaling or Sgk1 exhibit hypermyelination during development. These results identify functionally and spatially distinct PI 3-K pathways: an early, pro-myelinating pathway driven by axonal Neuregulin1 and a later-acting, laminin-integrin-dependent pathway that negatively regulates myelination.

microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity.

In both animals and plants, many developmentally important regulatory genes have complementary microRNAs (miRNAs), which suggests that these miRNAs constitute a class of developmental signalling molecules. Leaves of higher plants exhibit a varying degree of asymmetry along the adaxial/abaxial (upper/lower) axis. This asymmetry is specified through the polarized expression of class III homeodomain/leucine zipper (HD-ZIPIII) genes. In Arabidopsis, three such genes, PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV), are expressed throughout the incipient leaf, but become adaxially localized after primordium emergence. Downregulation of the HD-ZIPIII genes allows expression of the KANADI and YABBY genes, which specify abaxial fate. PHB, PHV and REV transcripts contain a complementary site for miRNA165 and miRNA166, which can direct their cleavage in vitro. Here we show that miRNA166 constitutes a highly conserved polarizing signal whose expression pattern spatially defines the expression domain of the maize hd-zipIII family member rolled leaf1 (rld1). Moreover, the progressively expanding expression pattern of miRNA166 during leaf development and its accumulation in phloem suggests that miRNA166 may form a movable signal that emanates from a signalling centre below the incipient leaf.