An engineering strategy to target activated EGFR with CAR T cells.

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast, CAR T cell treatment of solid tumors is associated with several challenges, in particular the expression of most tumor-associated antigens at lower levels in vital organs, resulting in on-target/off-tumor toxicities. Thus, innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands, we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show, in several experimental systems, that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers.

An antisteatosis response regulated by oleic acid through lipid droplet-mediated ERAD enhancement.

Although excessive lipid accumulation is a hallmark of obesity-related pathologies, some lipids are beneficial. Oleic acid (OA), the most abundant monounsaturated fatty acid (FA), promotes health and longevity. Here, we show that OA benefits Caenorhabditis elegans by activating the endoplasmic reticulum (ER)-resident transcription factor SKN-1A (Nrf1/NFE2L1) in a lipid homeostasis response. SKN-1A/Nrf1 is cleared from the ER by the ER-associated degradation (ERAD) machinery and stabilized when proteasome activity is low and canonically maintains proteasome homeostasis. Unexpectedly, OA increases nuclear SKN-1A levels independently of proteasome activity, through lipid droplet-dependent enhancement of ERAD. In turn, SKN-1A reduces steatosis by reshaping the lipid metabolism transcriptome and mediates longevity from OA provided through endogenous accumulation, reduced H3K4 trimethylation, or dietary supplementation. Our findings reveal an unexpected mechanism of FA signal transduction, as well as a lipid homeostasis pathway that provides strategies for opposing steatosis and aging, and may mediate some benefits of the OA-rich Mediterranean diet.

Identification of Activating Mutations in the Transmembrane and Extracellular Domains of EGFR.

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer, most notably non-small-cell lung cancer and glioblastoma. While many frequently occurring EGFR mutations are known to confer constitutive EGFR activation, the situation is less clear for rarely detected variants. In fact, more than 1000 distinct EGFR mutations are listed in the Catalogue of Somatic Mutations in Cancer (COSMIC), but for most of them, the functional consequence is unknown. To identify additional, previously unknown activating mutations in EGFR, we screened a randomly mutated EGFR library for constitutive EGFR phosphorylation using a recently developed high-throughput approach termed PhosphoFlowSeq. Enrichment of the well-known activating mutations S768I, T790M, and L858R validated the experimental approach. Importantly, we also identified the activating mutations S442I and L658Q located in the extracellular and transmembrane domains of EGFR, respectively. To the best of our knowledge, neither S442I nor L658Q has been associated with an activating phenotype before. However, both have been detected in cancer samples. Interestingly, molecular dynamics (MD) simulations suggest that the L658Q mutation located in the hydrophobic transmembrane region forms intermolecular hydrogen bonds, thereby promoting EGFR dimerization and activation. Based on these findings, we screened the COSMIC database for additional hydrophilic mutations in the EGFR transmembrane region and indeed detected moderate constitutive activation of EGFR-G652R. Together, this study demonstrates that unbiased screening for activating mutations in EGFR not only yields well-established substitutions located in the kinase domain but also activating mutations in other regions of EGFR, including the extracellular and transmembrane domains.

Affinity and Stability Analysis of Yeast Displayed Proteins.

Yeast surface display is a powerful protein engineering technology that is extensively used to improve various properties of proteins, including affinity, specificity, and stability or even to add novel functions (usually ligand binding). Apart from its robustness and versatility as an engineering tool, yeast display offers a further critical advantage: Once the selection campaign is finished, usually resulting in an oligoclonal pool, these enriched protein variants can be analyzed individually on the surface of yeast without the need for any sub-cloning, soluble expression, and purification. Here, we provide detailed protocols for determining both the affinity and the thermal stability of yeast displayed proteins. In addition, we discuss the advantages, challenges, and potential pitfalls associated with affinity and stability analysis using yeast surface display.

Engineering Strategies to Overcome the Stability-Function Trade-Off in Proteins.

In addition to its biological function, the stability of a protein is a major determinant for its applicability. Unfortunately, engineering proteins for improved functionality usually results in destabilization of the protein. This so-called stability-function trade-off can be explained by the simple fact that the generation of a novel protein function─or the improvement of an existing one─necessitates the insertion of mutations, i.e., deviations from the evolutionarily optimized wild-type sequence. In fact, it was demonstrated that gain-of-function mutations are not more destabilizing than other random mutations. The stability-function trade-off is a universal phenomenon during protein evolution that has been observed with completely different types of proteins, including enzymes, antibodies, and engineered binding scaffolds. In this review, we discuss three types of strategies that have been successfully deployed to overcome this omnipresent obstacle in protein engineering approaches: (i) using highly stable parental proteins, (ii) minimizing the extent of destabilization during functional engineering (by library optimization and/or coselection for stability and function), and (iii) repairing damaged mutants through stability engineering. The implementation of these strategies in protein engineering campaigns will facilitate the efficient generation of protein variants that are not only functional but also stable and therefore better-suited for subsequent applications.

PhosphoFlowSeq - A High-throughput Kinase Activity Assay for Screening Drug Resistance Mutations in EGFR.

Drug resistance poses a major challenge for targeted cancer therapy. To be able to functionally screen large randomly mutated target gene libraries for drug resistance mutations, we developed a biochemically defined high-throughput assay termed PhosphoFlowSeq. Instead of selecting for proliferation or resistance to apoptosis, PhosphoFlowSeq directly analyzes the enzymatic activities of randomly mutated kinases, thereby reducing the dependency on the signaling network in the host cell. Moreover, simultaneous analysis of expression levels enables compensation for expression-based biases on a single cell level. Using EGFR and its kinase inhibitor erlotinib as a model system, we demonstrate that the clinically most relevant resistance mutation T790M is reproducibly detected at high frequencies after four independent PhosphoFlowSeq selection experiments. Moreover, upon decreasing the selection pressure, also mutations which only confer weak resistance were identified, including T854A and L792H. We expect that PhosphoFlowSeq will be a valuable tool for the prediction and functional screening of drug resistance mutations in kinases.

Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor.

Disabled 1 (Dab1) is an adapter protein for very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) and an integral component of the Reelin pathway which orchestrates neuronal layering during embryonic brain development. Activation of Dab1 is induced by binding of Reelin to ApoER2 and VLDLR and phosphorylation of Dab1 mediated by Src family kinases. Here we show that Dab1 also acts as an adaptor for epidermal growth factor receptor (EGFR) and can be phosphorylated by epidermal growth factor (EGF) binding to EGFR. Phosphorylation of Dab1 depends on the kinase activity of EGFR constituting a signal pathway independent of Reelin and its receptors.