Targeted protein degradation has emerged as a new paradigm to manipulate cellular proteostasis. Proteolysis-targeting chimeras (PROTACs) are bifunctional small molecules that recruit an E3 ligase to a target protein of interest, promoting its ubiquitination and subsequent degradation. Here, we report the development of antibody-based PROTACs (AbTACs), fully recombinant bispecific antibodies that recruit membrane-bound E3 ligases for the degradation of cell-surface proteins. We show that an AbTAC can induce the lysosomal degradation of programmed death-ligand 1 by recruitment of the membrane-bound E3 ligase RNF43. AbTACs represent a new archetype within the PROTAC field to target cell-surface proteins with fully recombinant biological molecules.

Targeted degradation of cell surface and extracellular proteins via lysosomal delivery is an important means to modulate extracellular biology. However, these approaches have limitations due to lack of modularity, ease of development, restricted tissue targeting and applicability to both cell surface and extracellular proteins. We describe a lysosomal degradation strategy, termed cytokine receptor-targeting chimeras (KineTACs), that addresses these limitations. KineTACs are fully genetically encoded bispecific antibodies consisting of a cytokine arm, which binds its cognate cytokine receptor, and a target-binding arm for the protein of interest. We show that KineTACs containing the cytokine CXCL12 can use the decoy recycling receptor, CXCR7, to target a variety of target proteins to the lysosome for degradation. Additional KineTACs were designed to harness other CXCR7-targeting cytokines, CXCL11 and vMIPII, and the interleukin-2 (IL-2) receptor-targeting cytokine IL-2. Thus, KineTACs represent a general, modular, selective and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and cell surface targets with broad or tissue-specific distribution.

A serious public health crisis is currently unfolding due to the SARS-CoV-2 pandemic. SARS-CoV-2 viral entry depends on an interaction between the receptor binding domain of the trimeric viral Spike protein (Spike-RBD) and the dimeric human angiotensin converting enzyme 2 (ACE2) receptor. While it is clear that strategies to block the Spike/ACE2 interaction are promising as anti-SARS-CoV-2 therapeutics, our current understanding is insufficient for the rational design of maximally effective therapeutic molecules. Here, we investigated the mechanism of Spike/ACE2 interaction by characterizing the binding affinity and kinetics of different multimeric forms of recombinant ACE2 and Spike-RBD domain. We also engineered ACE2 into a split Nanoluciferase-based reporter system to probe the conformational landscape of Spike-RBDs in the context of the Spike trimer. Interestingly, a dimeric form of ACE2, but not monomeric ACE2, binds with high affinity to Spike and blocks viral entry in pseudotyped virus and live SARS-CoV-2 virus neutralization assays. We show that dimeric ACE2 interacts with an RBD on Spike with limited intra-Spike avidity, which nonetheless contributes to the affinity of this interaction.Additionally, we demonstrate that a proportion of Spike can simultaneously interact with multiple ACE2 dimers, indicating that more than one RBD domain in a Spike trimer can adopt an ACE2accessible "up" conformation. Our findings have significant implications on the design strategies of therapeutic molecules that block the Spike/ACE2 interaction. The constructs we describe are freely available to the research community as molecular tools to further our understanding of SARS-CoV-2 biology. Introduction:In late 2019, a novel, pathogenic coronavirus (SARS-CoV-2) entered the human population and has since spread throughout the world. The number of people suffering from the associated disease (COVID-19) continues to rise, increasing the need for effective therapeutic interventions. SARS-CoV-1 and SARS-CoV-2 Spike proteins are highly homologous (~76% sequence identity). Similar to SARS-CoV-1, the interaction between the SARS-CoV-2 Spike protein and the angiotensinconverting enzyme 2 (ACE2) on human cells is critical for viral entry into host cells (Gralinski & Menachery, 2020;Tai et al., 2020;Wu et al., 2020). SARS-CoV-2 Spike is an obligate trimer, while ACE2 presents as a dimer on the cell surface (Chen, Liu, & Guo, 2020). Several highresolution structures of SARS-CoV-2 Spike receptor binding domain (Spike-RBD) bound to ACE2 have been published (Lan et al., 2020;Yan et al., 2020). However, as of this writing, structures of SARS-CoV-2 Spike trimer in complex with either the dimeric or monomeric form of ACE2 have not been reported, resulting in an incomplete understanding of the nature of this interaction.Structural studies of trimeric SARS-CoV-2 and SARS-CoV-1 Spike protein demonstrate that each of the Spike-RBDs, as in other coronaviruses, can undergo hinge-like movements to transition between "up" or "down" conformations. The...

Naturally split inteins have found widespread use in chemical biology due to their ability to drive the ligation of separately expressed polypeptides through a process termed protein trans-splicing (PTS). In this study, we harness PTS by rendering association of split intein fragments conditional upon the presence of a user-defined protease. We show that these intein “zymogens” can be used to create protein sensors and actuators that respond to the presence of various stimuli, including bacterial pathogens, viral infections, and light. We also show that this design strategy is compatible with several orthogonal split intein pairs, thereby opening the way to the creation of multiplexed sensor systems.

Numerous neutralizing antibodies that target SARS-CoV-2 have been reported, and most directly block binding of the viral Spike receptor-binding domain (RBD) to angiotensin-converting enzyme II (ACE2). Here, we deliberately exploit non-neutralizing RBD antibodies, showing they can dramatically assist in neutralization when linked to neutralizing binders. We identified antigen-binding fragments (Fabs) by phage display that bind RBD, but do not block ACE2 or neutralize virus as IgGs. When these nonneutralizing Fabs were assembled into bispecific VH/Fab IgGs with a neutralizing VH domain, we observed a ~ 25-fold potency improvement in neutralizing SARS-CoV-2 compared to the mono-specific bi-valent VH-Fc alone or the cocktail of the VH-Fc and IgG. This effect was epitope-dependent, reflecting the unique geometry of the bispecific antibody toward Spike. Our results show that a bispecific antibody that combines both neutralizing and non-neutralizing epitopes on Spike-RBD is a promising and rapid engineering strategy to improve the potency of SARS-CoV-2 antibodies.

Split inteins associate to trigger protein splicing in trans, a post-translational modification in which protein sequences fused to the intein pair are ligated together in a traceless manner. Recently, a family of naturally split inteins has been identified that is split at a noncanonical location in the primary sequence. These atypically split inteins show considerable promise in protein engineering applications; however, the mechanism by which they associate is unclear and must be different from that of previously characterized canonically split inteins due to unique topological restrictions. Here, we use a consensus design strategy to generate an atypical split intein pair (Cat) that has greatly improved activity and is amenable to detailed biochemical and biophysical analysis. Guided by the solution structure of Cat, we show that the association of the fragments involves a disorder-to-order structural transition driven by hydrophobic interactions. This molecular recognition mechanism satisfies the topological constraints of the intein fold and, importantly, ensures that premature chemistry does not occur prior to fragment complementation. Our data lead a common blueprint for split intein complementation in which localized structural rearrangements are used to drive folding and regulate protein-splicing activity.

Targeted protein degradation is a promising therapeutic strategy capable of overcoming the limitations of traditional occupancy-based inhibitors. By ablating all of the associated functions of a protein at once, the event-driven pharmacology of degrader technologies has recently enabled the targeting of proteins that have been historically deemed "undruggable". Most degradation strategies utilize the ubiquitin− proteasome system to mediate intracellular target degradation and are thus limited to targeting proteins with cytoplasmic domains. While some of these strategies, such as PROTACs, have shown great promise, there is a need for new modalities that can be applied to specifically target cell surface proteins. We previously described the development of an antibody-based PROTAC (AbTAC) that utilizes genetically encoded IgG bispecific antibody scaffolds to bring the cell surface E3-ligase RNF43 into the proximity of a membrane protein of interest (POI) to mediate its degradation. Here, we employ rational protein engineering strategies to interrogate and optimize the properties necessary for efficient degradation of two therapeutically important membrane proteins, PD-L1 and EGFR. We develop multiple antibodies to RNF43 and show that the specific antibody binding epitopes on RNF43 and the POI are more important than the affinities of the AbTAC antibodies. We further expand the available repertoire of E3 ligases by co-opting the E3-ligase ZNRF3 to degrade both PD-L1 and EGFR and show similar importance of epitope for degradation efficiency. Importantly, we show that both RNF43 and ZNRF3 AbTACs do not potentiate unwanted WNT signaling. Lastly, we find that these AbTACs can be even further improved by exploring various dual-binding and IgG scaffolds that range in flexibility, valency, and orientation of the binding arms. These structure−activity and mechanistic studies provide a roadmap for optimizing the development of AbTACs, thereby greatly expanding their utility for targeted cell surface protein degradation.

Naturally split inteins drive the ligation of separately expressed polypeptides through a process called protein trans splicing (PTS). The ability to control PTS, so-called conditional protein splicing (CPS), has led to the development of tools to modulate protein structure and function at the post-translational level. CPS applications that utilize proximity as a trigger are especially intriguing as they afford the possibility to activate proteins in both a temporal and spatially targeted manner. In this study, we present the first proximity triggered CPS method that utilizes a naturally split fast splicing intein, Npu. We show that this method is amenable to diverse proximity triggers and capable of reconstituting and locally activating the acetyltransferase p300 in mammalian cells. This technology opens up a range of possibilities for the use of proximity triggered CPS.