Hershman, R. L. | bioRxiv Science

bioRxiv2024

Design, Construction, and Validation of a Yeast-Displayed Chemically Expanded Antibody Library

In vitro display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing O-(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads. Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/596443v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1024b0eorg.highwire.dtl.DTLVardef@18da36corg.highwire.dtl.DTLVardef@1e4397dorg.highwire.dtl.DTLVardef@7a4ad2_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Intracellular Delivery of Antibodies for Selective Cell Signaling Interference

Many intracellular signaling events remain poorly characterized due to a general lack of tools to interfere with "undruggable" targets. Antibodies have the potential to elucidate intracellular mechanisms via targeted disruption of cell signaling cascades because of their ability to bind to a target with high specificity and affinity. However, due to their size and chemical composition, antibodies cannot innately cross the cell membrane, and thus access to the cytosol with these macromolecules has been limited. Here, we describe strategies for accessing the intracellular space with recombinant antibodies mediated by cationic lipid nanoparticles to selectively disrupt intracellular signaling events. To enable such investigations, we first produced a series of antibody constructs, known as scFv-Fcs, containing additional, genetically encoded negative charges located at the C-termini of the constructs. Preparing proteins with negatively charged motifs has previously been shown to enhance intracellular protein delivery with cationic lipids, but usually for the purpose of genome editing or targeted cell death. We started by generating derivatives of scFv-Fc17, an antibody construct previously reported to bind specifically to signal transducer and activator of transcription 3 (STAT3) phosphorylated at Tyr705 (pYSTAT3). We screened a small number of lipids from our combinatorial lipid library with flow cytometry and found that PBA-Q76-O16B facilitated the most efficient delivery of scFv-Fcs under the conditions tested. In HepG2 cells, we observed up to 60.5% delivery efficacy, while in a STAT3-luciferase reporter cell line up to 71.5% delivery efficacy was observed. These results demonstrated the feasibility of accessing the intracellular space with scFv-Fcs. However, we also note that no more than modest changes were observed upon changing the numbers of negative charges in these constructs during delivery. Characterization of the cytotoxicity, size, and encapsulation efficiency of scFv-Fcs with PBA-Q76-O16B revealed that the constructs were generally well-behaved, with addition of differing quantities of negative charge resulting in at most modest effects. Importantly, functional assays monitoring transcriptional activity in luciferase reporter cell lines and HepG2 cells demonstrated significant reduction of gene expression downstream of pYSTAT3 following delivery of scFv-Fc17 constructs. Together, our results establish the use of recombinantly produced antibodies to selectively interfere with cell signaling events driven by a single posttranslational modification. Efficient intracellular delivery of engineered antibodies opens up possibilities for modulation of previously "undruggable" targets, including for potential therapeutic applications.

bioengineering↗