An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Non-nutritive sweeteners possess a bacteriostatic effect and alter gut microbiota in mice.

Non-nutritive sweeteners (NNSs) are widely used in various food products and soft drinks. There is growing evidence that NNSs contribute to metabolic dysfunction and can affect body weight, glucose tolerance, appetite, and taste sensitivity. Several NNSs have also been shown to have major impacts on bacterial growth both in vitro and in vivo. Here we studied the effects of various NNSs on the growth of the intestinal bacterium, E. coli, as well as the gut bacterial phyla Bacteroidetes and Firmicutes, the balance between which is associated with gut health. We found that the synthetic sweeteners acesulfame potassium, saccharin and sucralose all exerted strong bacteriostatic effects. We found that rebaudioside A, the active ingredient in the natural NNS stevia, also had similar bacteriostatic properties, and the bacteriostatic effects of NNSs varied among different Escherichia coli strains. In mice fed a chow diet, sucralose increased Firmicutes, and we observed a synergistic effect on Firmicutes when sucralose was provided in the context of a high-fat diet. In summary, our data show that NNSs have direct bacteriostatic effects and can change the intestinal microbiota in vivo.

Sucralose Promotes Food Intake through NPY and a Neuronal Fasting Response.

Non-nutritive sweeteners like sucralose are consumed by billions of people. While animal and human studies have demonstrated a link between synthetic sweetener consumption and metabolic dysregulation, the mechanisms responsible remain unknown. Here we use a diet supplemented with sucralose to investigate the long-term effects of sweet/energy imbalance. In flies, chronic sweet/energy imbalance promoted hyperactivity, insomnia, glucose intolerance, enhanced sweet taste perception, and a sustained increase in food and calories consumed, effects that are reversed upon sucralose removal. Mechanistically, this response was mapped to the ancient insulin, catecholamine, and NPF/NPY systems and the energy sensor AMPK, which together comprise a novel neuronal starvation response pathway. Interestingly, chronic sweet/energy imbalance promoted increased food intake in mammals as well, and this also occurs through an NPY-dependent mechanism. Together, our data show that chronic consumption of a sweet/energy imbalanced diet triggers a conserved neuronal fasting response and increases the motivation to eat.

Not on the menu: autophagy-independent clearance of prions.

A common feature of neurodegenerative diseases is the accumulation of disease-specific, aggregated protein species in the nervous system. Transmissible spongiform encephalopathies are universally fatal neurodegenerative diseases involving the transconformation and aggregation of prion proteins. At the cellular level macroautophagy has been identified as an efficient pathway for the clearance of these toxic protein aggregates. Hence, recent research has focused on the pharmacological manipulation of autophagy as a potential treatment for neurodegenerative diseases. Independent of their effects on the estrogen receptor, tamoxifen and its metabolite 4-hydroxytamoxifen are well known inducers of autophagy. However, we recently reported that the ability of 4-hydroxytamoxifen to clear prion infection is independent of autophagy. In contrast, we provide a model whereby perturbation of cholesterol metabolism, and not autophagy, is the main mechanism whereby 4-hydroxytamoxifen is able to exert its anti-prion effects. Thus, while tamoxifen, a widely available pharmaceutical, may have applications in prion therapy, prions may also represent a special case and may require different pharmacological interventions than other proteinopathies.

4-hydroxytamoxifen leads to PrPSc clearance by conveying both PrPC and PrPSc to lysosomes independently of autophagy.

Prion diseases are fatal neurodegenerative disorders involving the abnormal folding of a native cellular protein, named PrP(C), to a malconformed aggregation-prone state, enriched in beta sheet secondary structure, denoted PrP(Sc). Recently, autophagy has garnered considerable attention as a cellular process with the potential to counteract neurodegenerative diseases of protein aggregation such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Stimulation of autophagy by chemical compounds has also been shown to reduce PrP(Sc) in infected neuronal cells and prolong survival times in mouse models. Consistent with previous reports, we demonstrate that autophagic flux is increased in chronically infected cells. However, in contrast to recent findings we show that autophagy does not cause a reduction in scrapie burden. We report that in infected neuronal cells different compounds known to stimulate autophagy are ineffective in increasing autophagic flux and in reducing PrP(Sc). We further demonstrate that tamoxifen and its metabolite 4-hydroxytamoxifen lead to prion degradation in an autophagy-independent manner by diverting the trafficking of both PrP and cholesterol to lysosomes. Our data indicate that tamoxifen, a well-characterized, widely available pharmaceutical, may have applications in the therapy of prion diseases.

Prions hijack tunnelling nanotubes for intercellular spread.

In variant Creutzfeldt-Jakob disease, prions (PrP(Sc)) enter the body with contaminated foodstuffs and can spread from the intestinal entry site to the central nervous system (CNS) by intercellular transfer from the lymphoid system to the peripheral nervous system (PNS). Although several means and different cell types have been proposed to have a role, the mechanism of cell-to-cell spreading remains elusive. Tunnelling nanotubes (TNTs) have been identified between cells, both in vitro and in vivo, and may represent a conserved means of cell-to-cell communication. Here we show that TNTs allow transfer of exogenous and endogenous PrP(Sc) between infected and naive neuronal CAD cells. Significantly, transfer of endogenous PrP(Sc) aggregates was detected exclusively when cells chronically infected with the 139A mouse prion strain were connected to mouse CAD cells by means of TNTs, identifying TNTs as an efficient route for PrP(Sc) spreading in neuronal cells. In addition, we detected the transfer of labelled PrP(Sc) from bone marrow-derived dendritic cells to primary neurons connected through TNTs. Because dendritic cells can interact with peripheral neurons in lymphoid organs, TNT-mediated intercellular transfer would allow neurons to transport prions retrogradely to the CNS. We therefore propose that TNTs are involved in the spreading of PrP(Sc) within neurons in the CNS and from the peripheral site of entry to the PNS by neuroimmune interactions with dendritic cells.

Distinct regions within the erlins are required for oligomerization and association with high molecular weight complexes.

The group of stomatin/prohibitin/flotillin/HflK/C (SPFH) domain-containing proteins comprise members of diverse subcellular localization and function. Association with detergent-resistant membranes (DRMs) and the propensity to form oligomers are two common properties of SPFH domain proteins and likely important for the function of these proteins. Our laboratory recently discovered two novel members of this protein group, which, based on their endoplasmic reticulum (ER) localization and association with DRMs, were named ER lipid raft-associated protein (erlin)-1 and -2. Here we characterized erlin oligomerization and identified domains within the erlins responsible for oligomerization and DRM association. Using co-immunoprecipitation and sucrose density gradient centrifugation approaches on endogenous and ectopically expressed erlin proteins, we found that they formed homo- and hetero-oligomers and were part of large multimeric complexes. These properties were independent of their DRM association. By analyzing truncation and point mutants of erlin-2 we discovered that interaction between erlin monomers (oligomerization) and association with high molecular weight complexes require distinct regions within the protein. Although oligomerization and DRM association were mediated by a region immediately downstream of the SPFH domain (residues 228-300), integration into high molecular weight complexes was absolutely dependent on a phenylalanine residue C-terminal of this region (Phe-305), which lies within a short stretch of hydrophobic residues. Our data demonstrate that lower order oligomerization and incorporation into multimeric complexes are two separate biochemical properties of the erlins, because they are mediated by distinct regions.

The SPFH domain-containing proteins: more than lipid raft markers.

Membrane microdomains with distinct lipid compositions, called lipid rafts, represent a potential mechanism for compartmentalizing cellular functions within the plane of biological membranes. SPFH domain-containing proteins are found in lipid raft microdomains in diverse cellular membranes. The functions of these proteins are just beginning to be elucidated. Recent advances in the understanding of structural features and their roles within lipid rafts include a potential function for SPFH proteins in the formation of membrane microdomains and lipid raft-associated processes, such as endocytosis and mechanosensation.

Erlin-1 and erlin-2 are novel members of the prohibitin family of proteins that define lipid-raft-like domains of the ER.

Our laboratory was interested in characterizing the molecular composition of non-caveolar lipid rafts. Thus, we generated monoclonal antibodies to lipid raft proteins of human myelomonocytic cells. Two of these proteins, KE04p and C8orf2, were found to be highly enriched in the detergent-insoluble, buoyant fraction of sucrose gradients in a cholesterol-dependent manner. They contain an evolutionarily conserved domain placing them in the prohibitin family of proteins. In contrast to other family members, these two proteins localized to the ER. Furthermore, the extreme N-termini of KE04p and C8orf2 were found to be sufficient for heterologous targeting of GFP to the ER in the absence of classical ER retrieval motifs. We also demonstrate that all prohibitin family members rely on sequences in their extreme N-termini for their distinctive subcellular distributions including the mitochondria, plasma membrane and Golgi vesicles. Owing to their subcellular localization and their presence in lipid rafts, we have named KE04p and C8orf2, ER lipid raft protein (erlin)-1 and erlin-2, respectively. Interestingly, the ER contains relatively low levels of cholesterol and sphingolipids compared with other organelles. Thus, our data support the existence of lipid-raft-like domains within the membranes of the ER.