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Summary We developed an RNA sequencing-based pipeline to discover differentially expressed cell surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. Additionally, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2 directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.

While the aqueous solubility for bilayer phospholipids is less than 10(-10) M--keeping lipid membranes at essentially constant mass, single chain surfactants can have a significant aqueous solubility. Thus, in surfactant solutions, both monomer and micelles can interact with a lipid bilayer, and the mass and composition of the bilayer can be changed in seconds. These changes in composition are expected to have direct consequences on bilayer structure and material properties. We have found that the exchange of surfactants like lysolecithin can be described in terms of a kinetic model in which monomer and micelles are transported to the membrane from bulk solution. Molecular transport is considered at the membrane interfaces and across the midplane between the two monolayers of the bilayer. Using micropipet manipulation, single vesicles were transferred into lysolecithin solutions, and the measurement of vesicle area change gave a direct measure of lysolecithin uptake. Transfer back to lysolecithin-free media resulted in desorption. The rates of uptake and desorption could therefore be measured at controlled levels of membrane stress. With increasing lysolecithin concentration in the bulk phase, the amount of lysolecithin in the membrane reached saturation at approximately 3 mol% for concentrations below the critical micelle concentration (CMC) and at > 30 mol% for concentrations above the CMC. When convective transport was used to deliver lysolecithin, uptake occurred via a double exponential: initial uptake into the outer monolayer was fast (approximately 0.2 sec-1); transfer across the bilayer midplane was much slower (0.0019 sec-1).

Human leukocyte chemoattractant receptors activate chemotactic and cytotoxic pathways to varying degrees and also activate different G-proteins depending on the receptor and the cell-type. To determine the relationship between G-protein usage and the biological and biochemical responses activated, receptors for the chemoattractants formyl peptides (FR), platelet-activating factor (PAFR), and leukotriene B 4 (BLTR) were transfected into RBL-2H3 cells. Pertussis toxin (Ptx) served as a G␣ i inhibitor. These receptors were chosen to represent the spectrum of G i usage as Ptx had differential effects on their ability to induce calcium mobilization, phosphoinositide hydrolysis, and exocytosis with complete inhibition of all responses by FR, intermediate effects on BLTR, and little effect on PAFR. Ptx did not affect ligand-induced phosphorylation of PAFR and BLTR but inhibited phosphorylation of FR. In contrast, chemotaxis to formylmethionylleucylphenylalanine, leukotriene B 4 , and platelet-activating factor was completely blocked by Ptx. Wortmannin, a phosphotidylinositol 3-kinase inhibitor, also completely blocked ligand-induced chemotaxis by all receptors but did not affect calcium mobilization or phosphoinositide hydrolysis; however, it partially blocked the exocytosis response to formylmethionylleucylphenylalanine and the platelet-activating factor. Membrane ruffling and pseudopod extension via the BLTR was also completely inhibited by both Ptx and wortmannin. These data suggest that of the chemoattractant receptors studied, G-protein usage varies with FR being totally dependent on G i , whereas BLTR and PAFR utilize both G i and a Ptxinsensitive G-protein. Both Ptx-sensitive and -insensitive G-protein usage can mediate the activation of phospholipase C, mobilization of intracellular calcium, and exocytosis by chemoattractant receptors. Chemotaxis, however, had an absolute requirement for a G i -mediated pathway.Migration of leukocytes to sites of inflammation is mediated via the activation of G-protein-coupled chemoattractant receptors (1, 2). Chemoattractants at low concentrations elicit shape change, pseudopod extension, and chemotaxis, and at higher doses; many of them also trigger degranulation and generation of superoxide anions (1, 3). Pathways leading to these activities have been shown to have different dose requirements, kinetics and regulation (4, 5), but the role of G-protein usage remains unknown.Formylpeptides (fMLP), 1 platelet-activating factor (PAF), and leukotriene B 4 (LTB 4 ) are potent chemoattractants for neutrophils and to varying degrees also activate exocytosis and generation of superoxide anions (3,4,6). These activities are mediated through G-protein-coupled receptors (FR, PAFR, and BLTR) (2, 7). G-protein usage of chemoattractant receptors was known to be different depending on cell types (1, 8 -10). Previous studies in RBL cells indicated that FR activated G i , whereas PAFR utilized both G i and a Ptx-insensitive G-protein to activate phosphoinositide hydrolysis, calcium mobilizatio...