James A. Ernst

Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.

Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies

β-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.

The Soluble Wnt Receptor Frizzled8CRD-hFc Inhibits the Growth of Teratocarcinomas In vivo

Wnt signaling is important for normal cell proliferation and differentiation, and mutations in pathway components are associated with human cancers. Recent studies suggest that altered wnt ligand/receptor interactions might also contribute to human tumorigenesis. Therefore, agents that antagonize wnt signaling at the extracellular level would be attractive therapeutics for these cancers. We have generated a soluble wnt receptor comprising the Frizzled8 cysteine-rich domain (CRD) fused to the human Fc domain (F8CRDhFc) that exhibits favorable pharmacologic properties in vivo. Potent antitumor efficacy was shown using the mouse mammary tumor virus-Wnt1 tumor model under dosing conditions that did not produce detectable toxicity in regenerating tissue compartments. In vitro, F8CRDhFc inhibited autocrine wnt signaling in the teratoma cell lines PA-1, NTera-2, Tera-2, and NCCIT. In vivo, systemic administration of F8CRDhFc significantly retarded the growth of tumor xenografts derived from two of these cell lines, PA-1 and NTera-2. Pharmacodynamic markers of wnt signaling, identified by gene expression analysis of cultured teratoma cells, were also modulated in the tumor xenografts following treatment with F8CRDhFc. Additionally, these markers could be used as indicators of treatment efficacy and might also be useful in identifying patients that would benefit from the therapeutic agent. This is the first report showing the efficacy of a soluble wnt receptor as an antitumor agent and suggests that further development of wnt antagonists will have utility in treating human cancer.

CCBE1 Is Essential for Mammalian Lymphatic Vascular Development and Enhances the Lymphangiogenic Effect of Vascular Endothelial Growth Factor-C In Vivo

Rationale: Collagen- and calcium-binding EGF domains 1 (CCBE1) has been associated with Hennekam syndrome, in which patients have lymphedema, lymphangiectasias, and other cardiovascular anomalies. Insight into the molecular role of CCBE1 is completely lacking, and mouse models for the disease do not exist. Objective: CCBE1 deficient mice were generated to understand the function of CCBE1 in cardiovascular development, and CCBE1 recombinant protein was used in both in vivo and in vitro settings to gain insight into the molecular function of CCBE1. Methods and Results: Phenotypic analysis of murine Ccbe1 mutant embryos showed a complete lack of definitive lymphatic structures, even though Prox1+ lymphatic endothelial cells get specified within the cardinal vein. Mutant mice die prenatally. Proximity ligation assays indicate that vascular endothelial growth factor receptor 3 activation appears unaltered in mutants. Human CCBE1 protein binds to components of the extracellular matrix in vitro, and CCBE1 protein strongly enhances vascular endothelial growth factor-C–mediated lymphangiogenesis in a corneal micropocket assay. Conclusions: Our data identify CCBE1 as a factor critically required for budding and migration of Prox-1+ lymphatic endothelial cells from the cardinal vein. CCBE1 probably exerts these effects through binding to components of the extracellular matrix. CCBE1 has little lymphangiogenic effect on its own but dramatically enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo. Thus, our data suggest CCBE1 to be essential but not sufficient for lymphangiogenesis.

Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates

Bispecific antibodies engineered to both bind to the primate transferrin receptor and inhibit β-secretase are taken up by the nonhuman primate brain and reduce brain β-amyloid. A Two-Pronged Approach for Central Nervous System Therapeutics The brain has been considered off-limits to antibody therapies because of the blood-brain barrier (BBB), which protects the brain from circulating toxins while selectively transporting essential molecules into the brain. Efforts to use natural transport mechanisms to deliver antibody therapies into the brain have been successful in rodents. Whether a similar approach can be used in primates, including humans, remains unknown. Using bispecific antibodies with one arm binding to the transferrin receptor and the other to an Alzheimer’s disease drug target, we show that therapeutic antibodies can effectively and safely cross the BBB and enter the primate brain, thus paving the way for antibody therapeutics to treat central nervous system diseases in humans. Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target β-secretase (BACE1) can cross the BBB and reduce brain amyloid-β (Aβ) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain Aβ in a TfR affinity–dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced Aβ both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain.

Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

The safety of therapeutic bispecific antibodies that use TfR for delivery to the brain can be improved by reducing affinity for TfR and eliminating antibody effector function. Averting Roadblocks En Route to the Brain The blood-brain barrier represents a formidable blockade preventing therapeutic antibody delivery into the brain. Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting therapeutic antibody uptake into the brain. Although TfR can act as a molecular lift to promote brain uptake, little is known about the safety ramifications of this approach. Building on a pair of studies published in Science Translational Medicine, Couch and colleagues now report that when mice were dosed with therapeutic TfR antibodies, the animals showed acute clinical reactions and a reduction in immature red blood cells, known as reticulocytes. TfR bispecific antibodies engineered to lack Fc interactions with immune cells eliminated adverse acute clinical reactions and reduced reticulocyte loss; the extent of reticulocyte loss was also influenced by binding to TfR and interaction with the complement cascade. Because reticulocytes express high levels of TfR, other cell types that express high levels of TfR were also investigated. The authors observed, for example, that the blood-brain barrier remained completely intact after TfR antibodies were administered to mice, despite the high expression of TfR in brain endothelial cells. Finally, multiple doses of TfR/BACE1 bispecific antibodies reduced amyloid-β, a toxic protein implicated in Alzheimer’s disease, with minimal sustained toxicity. Investigation of monkey and human TfR levels in circulating reticulocytes suggested that loss of these cells may be less likely to occur in primates than in mice. The translational implications of these discoveries suggest that the blood-brain barrier is not the only obstacle to surmount on the way to the brain, at least when using TfR as a molecular lift. Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.

Molecular mechanism of the synaptotagmin–SNARE interaction in Ca2+-triggered vesicle fusion

In neurons, SNAREs, synaptotagmin and other factors catalyze Ca2+-triggered fusion of vesicles with the plasma membrane. The molecular mechanism of this process, especially the interaction between synaptotagmin and SNAREs, remains an enigma. Here we characterized this interaction by single-molecule fluorescence microscopy and crystallography. The two rigid Ca2+-binding domains of synaptotagmin 3 (Syt3) undergo large relative motions in solution. Interaction with SNARE complex amplifies a particular state of the two domains that is further enhanced by Ca2+. This state is represented by the first SNARE-induced Ca2+-bound crystal structure of a synaptotagmin fragment containing both domains. The arrangement of the Ca2+-binding loops of this structure of Syt3 matches that of SNARE-bound Syt1, suggesting a conserved feature of synaptotagmins. The loops resemble the membrane-interacting loops of certain viral fusion proteins in the postfusion state, suggesting unexpected similarities between both fusion systems.

Regulation of Epithelial Branching Morphogenesis and Cancer Cell Growth of the Prostate by Wnt Signaling

Although Wnt signaling has been shown to be important for embryonic morphogenesis and cancer pathogenesis of several tissues, its role in prostatic development and tumorigenesis is not well understood. Here we show that Wnt signaling regulated prostatic epithelial branching morphogenesis and luminal epithelial cell differentiation in developing rat prostate organ cultures. Specifically, Wnt signaling regulated the proliferation of prostate epithelial progenitor cells. Assessment of the expression levels of a Wnt pathway transcriptional target gene, Axin2, showed that the Wnt pathway was activated in the developing prostate, but was down-regulated in the adult. Castration resulted in an upregulation of Axin2 whereas androgen replacement resulted in a down regulation of Axin2. Such dynamic changes of Wnt activity was also confirmed in a BAT-gal transgenic mouse line in which β-galactosidase reporter is expressed under the control of β-catenin/T cell factor responsive elements. Furthermore, we evaluated the role of Wnt signaling in prostate tumorigenesis. Axin2 expression was found upregulated in the majority of human prostate cancer cell lines examined. Moreover, addition of a Wnt pathway inhibitor, Dickkopf 1 (DKK1), into the culture medium significantly inhibited prostate cancer cell growth and migration. These findings suggest that Wnt signaling regulates prostatic epithelial ductal branching morphogenesis by influencing cell proliferation, and highlights a role for Wnt pathway activation in prostatic cancer progression.

Discovery of Novel Blood-Brain Barrier Targets to Enhance Brain Uptake of Therapeutic Antibodies

The blood-brain barrier (BBB) poses a major challenge for developing effective antibody therapies forxa0neurological diseases. Using transcriptomic and proteomic profiling, we searched for proteins in mouse brain endothelial cells (BECs) that could potentially be exploited to transport antibodies across the BBB. Due to their limited protein abundance, neither antibodies against literature-identified targets nor BBB-enriched proteins identified by microarray facilitated significant antibody brain uptake. Using proteomic analysis of isolated mouse BECs, we identified multiple highly expressed proteins, including basigin, Glut1, and CD98hc. Antibodies to each of these targets were significantly enriched in the brain after administration inxa0vivo. In particular, antibodies against CD98hc showed robust accumulation in brain after systemic dosing, and a significant pharmacodynamic response as measured by brain Aβ reduction. The discovery of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain expands the current approaches available for enhancing brain uptake of therapeutic antibodies.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.