Cell-type specific potent Wnt signaling blockade by bispecific antibody.

Cell signaling pathways are often shared between normal and diseased cells. How to achieve cell type-specific, potent inhibition of signaling pathways is a major challenge with implications for therapeutic development. Using the Wnt/ß-catenin signaling pathway as a model system, we report here a novel and generally applicable method to achieve cell type-selective signaling blockade. We constructed a bispecific antibody targeting the Wnt co-receptor LRP6 (the effector antigen) and a cell type-associated antigen (the guide antigen) that provides the targeting specificity. We found that the bispecific antibody inhibits Wnt-induced reporter activities with over one hundred-fold enhancement in potency, and in a cell type-selective manner. Potency enhancement is dependent on the expression level of the guide antigen on the target cell surface and the apparent affinity of the anti-guide antibody. Both internalizing and non-internalizing guide antigens can be used, with internalizing bispecific antibody being able to block signaling by all ligands binding to the target receptor due to its removal from the cell surface. It is thus feasible to develop bispecific-based therapeutic strategies that potently and selectively inhibit signaling pathways in a cell type-selective manner, creating opportunity for therapeutic targeting.

Engineering of a miniaturized, robotic clinical laboratory.

The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.

Macropinocytosis Exploitation by Cancers and Cancer Therapeutics.

Macropinocytosis has long been known as a primary method for cellular intake of fluid-phase and membrane-bound bulk cargo. This review seeks to re-examine the latest studies to emphasize how cancers exploit macropinocytosis to further their tumorigenesis, including details in how macropinocytosis can be adapted to serve diverse functions. Furthermore, this review will also cover the latest endeavors in targeting macropinocytosis as an avenue for novel therapeutics.

High-content analysis of antibody phage-display library selection outputs identifies tumor selective macropinocytosis-dependent rapidly internalizing antibodies.

Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364-2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly active macropinocytosis activity was identified as ephrin type-A receptor 2. Antibody-toxin conjugates created using this macropinocytosing IgG were capable of potent and receptor-dependent killing of a panel of EphA2-positive tumor cell lines in vitro. These studies identify novel methods to screen for and validate antibodies capable of receptor-dependent macropinocytosis, allowing further exploration of this highly efficient and tumor-selective internalization pathway for targeted therapy development.

Regulation of the Golgi complex by phospholipid remodeling enzymes.

The mammalian Golgi complex is a highly dynamic organelle consisting of stacks of flattened cisternae with associated coated vesicles and membrane tubules that contribute to cargo import and export, intra-cisternal trafficking, and overall Golgi architecture. At the morphological level, all of these structures are continuously remodeled to carry out these trafficking functions. Recent advances have shown that continual phospholipid remodeling by phospholipase A (PLA) and lysophospholipid acyltransferase (LPAT) enzymes, which deacylate and reacylate Golgi phospholipids, respectively, contributes to this morphological remodeling. Here we review the identification and characterization of four cytoplasmic PLA enzymes and one integral membrane LPAT that participate in the dynamic functional organization of the Golgi complex, and how some of these enzymes are integrated to determine the relative abundance of COPI vesicle and membrane tubule formation. This article is part of a Special Issue entitled Lipids and Vesicular Transport.

The phospholipase A2 enzyme complex PAFAH Ib mediates endosomal membrane tubule formation and trafficking.

Previous studies have shown that membrane tubule-mediated export from endosomal compartments requires a cytoplasmic phospholipase A(2) (PLA(2)) activity. Here we report that the cytoplasmic PLA(2) enzyme complex platelet-activating factor acetylhydrolase (PAFAH) Ib, which consists of α1, α2, and LIS1 subunits, regulates the distribution and function of endosomes. The catalytic subunits α1 and α2 are located on early-sorting endosomes and the central endocytic recycling compartment (ERC) and their overexpression, but not overexpression of their catalytically inactive counterparts, induced endosome membrane tubules. In addition, overexpression α1 and α2 altered normal endocytic trafficking; transferrin was recycled back to the plasma membrane directly from peripheral early-sorting endosomes instead of making an intermediate stop in the ERC. Consistent with these results, small interfering RNA-mediated knockdown of α1 and α2 significantly inhibited the formation of endosome membrane tubules and delayed the recycling of transferrin. In addition, the results agree with previous reports that PAFAH Ib α1 and α2 expression levels affect the distribution of endosomes within the cell through interactions with the dynein regulator LIS1. These studies show that PAFAH Ib regulates endocytic membrane trafficking through novel mechanisms involving both PLA(2) activity and LIS1-dependent dynein function.