Receptor tyrosine kinases (RTKs) play critical roles in physiological and pathological processes, and are important anticancer drug targets. In vitro mechanistic and drug discovery studies of full-length RTKs require protein that is both fully functional and free from contaminating proteins. Here we describe a rapid cell-free and detergent-free co-translation method for producing full-length and functional ERBB2 and EGFR receptor tyrosine kinases supported by water-soluble apolipoprotein A-I based nanolipoprotein particles.

Highlights d De novo Pitx1 expression defines tumor-propagating SCC cells d PITX1 governs a feed-forward circuit that enables TPCrenewal and clonal expansion d PITX1 function represses KLF4-dependent squamous differentiation d Pitx1 expression and function are restricted by KLF4 activity

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

Engineered outer membrane vesicles (OMVs) derived from laboratory strains of bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. As mimics of the bacterial cell surface, OMVs offer a molecularly-defined architecture for programming repetitive, high-density display of heterologous antigens in conformations that elicit strong B and T cell immune responses. However, antigen display on the surface of OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. To address this shortcoming, we created a universal approach called AddVax (avidin-based dock-and-display for vaccine antigen cross (x)-linking) whereby virtually any antigen that is amenable to biotinylation can be linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen receptor (SNARE) comprised of an outer membrane scaffold protein fused to a member of the avidin family. We show that SNARE-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations were injected in wild-type BALB/c mice, strong antigen-specific antibody responses were observed that depended on the physical coupling between the antigen and SNARE-OMV delivery vehicle. Overall, these results demonstrate AddVax as a modular platform for rapid self-assembly of antigen-studded OMVs with the potential to accelerate vaccine generation, respond rapidly to pathogen threats in humans and animals, and simplify vaccine stockpiling.

The mammalian ErbB receptor tyrosine kinase family is critical for the development and maintenance of a variety of tissues. Though not completely understood, the mechanism of ErbB receptor activation involves the binding of ligand to the extracellular domain leading to a conformational change that allows dimerization and phosphorylation to initiate downstream signaling pathways. Mutations, amplification, and aberrant activation of these receptors lead to oncogenesis and tumor progression in several cancer types, including lung, breast, and colon. Current ErbB-targeted therapies include monoclonal antibodies and tyrosine kinase inhibitors. These treatments are initially effective but many tumors develop resistance, necessitating the discovery of a more specific and efficient drug. Studying the ErbB receptors is often difficult because of their large size and poor water solubility. Here we report success in assembling EGFR into nanolipoprotein particles (NLPs) to study activation and inhibition of the correctly-folded, full-length, and active receptor. NLPs are ∼20 nm cell membrane analogs composed of an apolipoprotein surrounding a lipid bilayer. We produced a homogenous population of EGFR-NLPs, by FLAG-purification of EGFR from mammalian cells, which are of the correct size, phosphorylated, and can be quantified. Furthermore, these EGFR-NLPs can be utilized as a novel target in a one-bead-one-compound (OBOC) screen of small molecules and peptides to identify unique therapeutics. Studies of ligand binding, kinase activity, and EGFR structure are ongoing. Future directions are to incorporate disease-relevant EGFR mutations into NLPs. The T790M mutation is of particular interest because it is a treatment-induced mutation observed in half of all non small cell lung cancers and confers resistance to current ErbB-targeted therapies. Citation Format: Tiffany M. Scharadin, Matthew Saldana, Michael Schlein, Steven Hoang-Phou, Denise Trans, Dennis Chang, Wei He, Kit Lam, Kermit L. Carraway, Matthew A. Coleman, Paul T. Henderson. Using NLPs to study EGFR structure, activation, and inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3321. doi:10.1158/1538-7445.AM2014-3321