Matthew Edwin Call

Current cell and gene therapies lack clinically practical mechanisms to selectively promote or suppress therapeutic cells in vivo, a limitation that is particularly acute in patients requiring ongoing immunosuppression. This includes gene therapy for immune dysregulation syndromes, and antigen-specific or chimeric antigen receptor (CAR) T-cell therapy for patients requiring immunosuppression (eg, transplant recipients), in which both pathogenic and therapeutic cells may be suppressed. Here, we develop a multiplex prime-editing platform that converts commonly used immunosuppressive drugs into tools for in vivo control of T-cell therapies via defined, pathway-specific drug resistance. Focusing initially on gene therapy, prime editing efficiently edited loci of multiple pathogenic variants associated with immune dysregulation in primary human T-cells and corrected the HAVCR2 driver mutation in T cells from multiple patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL). Comprehensive genomic, transcriptional, immunophenotypic, and clonal analyses demonstrated minimal off-target perturbation. Multiplexed gene correction and drug-resistance editing of T-cells from patients with SPTCL enabled selective in vivo expansion of corrected cells under immunosuppressive pressure in humanized mouse models and exhibited retained sensitivity to alternative agents permitting rapid in vivo suppression. Extending this approach, prime-edited, drug-resistant antigen-specific and CAR T cells retained effector function despite pharmacologic immunosuppression, demonstrating the generalizability of this platform to diverse cellular therapies. Together, these findings establish multiplex prime editing as a promising preclinical framework for generating drug-controllable T-cell therapies, enabling selective in vivo modulation in settings in which immunosuppression cannot be withdrawn.

Tumor necrosis factor (TNF) is a pivotal inflammatory cytokine, and it predominantly signals by binding to TNF receptor 1 (TNFR1), a type I single-spanning transmembrane protein that is thought to exist primarily as monomers and dimers. The binding of trimeric ligands induces the formation of signaling-competent trimers and higher-order oligomers that lead to full activation of downstream NF-κB and MAPK signaling pathways. Several TNFR superfamily members, including TNFR1, can form trimeric structures through their transmembrane domains (TMDs). For Fas and DR5, these structures support ligand-induced activation, and ectodomain (ECD) interactions prevent ligand-independent signaling. To explore the structures’ role in TNFR1 activation, we replaced the native TNFR1 TMDs with natural and de novo designed versions that formed stable and specific structures of defined monomeric or oligomeric states. We found that none of these TMD variant receptors displayed defects in TNF-induced signaling in mouse fibroblasts, but some showed increased autoactivation in the absence of ligand, particularly where the engineered TMD sequence prevented self-association. Autoactivation depended on intracellular death domain interactions and was exacerbated by a disease-associated mutation but was unaffected by mutation in the preligand assembly domain. Our results demonstrate that, unlike for other TNFR family members, no specific oligomeric TMD structure is required for normal, ligand-induced activation of TNFR1, but self-association through the native TMD may instead act together with ECD interactions to help suppress ligand-free autoactivation.

Immune receptors are modular sensors that detect molecular changes indicating infection, cell/tissue damage, oncogenic transformation, or exposure to allergens, and the signals they transmit initiate and regulate immune responses. These transmembrane receptors are often composed of one or more single-spanning membrane proteins and are modular in two important aspects. First, many immune receptors assemble from ligand-binding modules and signaling modules made up of separate proteins that come together during biosynthesis or upon activation through specific transmembrane domain (TMD) interactions. Second, the assembled receptor complexes comprise extracellular modules (structured protein domains) that interact with their environments and intracellular modules (often intrinsically unstructured domains) that read out these interactions biochemically. These are connected by and must communicate through the receptor TMDs. Here we review the molecular principles and structural motifs that organize and regulate immune receptors in the lipid bilayer with a view to understanding both how they are built and what roles the molecular interactions with and within the membrane play in their structures and functions. We focus primarily on examples where biochemical and structural data reveal highly specific interactions and show how these represent solutions that appear repeatedly across receptor families and can be exploited in synthetic biology applications.

Drug-escape, where a target evolves to escape inhibition from a drug, has the potential to lead to cross-resistance where drugs that are structurally related or share similar binding mechanisms all become less effective. PLpro inhibitors are currently under development and many emerging PLpro inhibitors are derived from GRL0617, a repurposed SARS-CoV PLpro inhibitor with moderate activity against SARS-CoV-2. Two leading derivatives, PF-07957472 and Jun12682, demonstrate low nanomolar activity and display activity in mice. WEHI-P8 is structurally distinct but binds to a similar pocket adjacent to the active site as GRL0617-like compounds. Using deep mutational scanning, we assessed the potential for PLpro to develop resistance to PF-07957472, Jun12682, and WEHI-P8. PF-07957472 and Jun12682 exhibited largely overlapping escape mutations due to their shared scaffold and binding modes, whereas WEHI-P8 resistance mutations were distinct. These findings underscore the importance of developing structurally diverse inhibitors to minimize resistance risks and ensure that viral mutations against one compound do not compromise the efficacy of others.

Deep mutational scanning (DMS), a high-throughput method leveraging next-generation sequencing, has been crucial in mapping the functional landscapes of key severe acquired respiratory syndrome-coronavirus 2 (SARS-CoV-2) proteins. By systematically assessing thousands of amino acid changes, DMS provides a framework to understand Angiotensin-converting enzyme 2 (ACE2) binding and immune evasion by the spike protein, mechanisms and drug escape potential of the main and papain-like viral proteases and has highlighted areas of concern in the nucleocapsid protein that may affect most currently available rapid antigen testing kits. Each application has required the design of bespoke assays in eukaryotic (yeast and mammalian) cell models, providing an exemplar for the application of this technique to future pandemics. This minireview examines how DMS has predicted key evolutionary changes in SARS-CoV-2 and affected our understanding of SARS-CoV-2 biology, specifically highlighting their relevance for therapeutics development.

Bacteriophage contaminations pose substantial risks to biomolecular production pipelines, and their resolution is especially difficult when the identity of the offending agent is unknown. We recently experienced an outbreak of Escherichia coli culture lysis in our Melbourne-based structural biology labs that halted protein production despite our use of T1-resistant ( TonA / FhuA -disrupted) strains. Genetic analysis of the isolated phage yielded a 45,053 bp genome showing 80–90% identity with multiple Rtp-like siphophages, and transmission electron microscopy images were consistent with this classification. Further analysis revealed that our isolate was nearly identical to a highly virulent lytic coliphage MSK, recently isolated in Hangzhou, China, whose host receptor has not been determined. Sequence and structural modelling analysis of its putative receptor-binding protein suggested that its terminal receptor was likely to be LptD, an essential outer membrane protein involved in lipopolysaccharide transport. Based on a recent report of spontaneously arising mutations that blocked infection by other LptD-dependent bacteriophages, we designed a targeted genomic LptD loop deletion that successfully generated resistance to vB_EcoS_OzMSK in E. coli BL21(DE3) without apparent detriment to fitness. Here, we report a CRISPR-based, single-plasmid solution that will benefit other labs or facilities experiencing challenges due to LptD-dependent lytic phage outbreaks.

B cells surveil the body for foreign matter using their surface-expressed B cell antigen receptor (BCR), a tetrameric complex comprising a membrane-tethered antibody (mIg) that binds antigens and a signaling dimer (CD79AB) that conveys this interaction to the B cell. Recent cryogenic electron microscopy (cryo-EM) structures of IgM and IgG isotype BCRs provide the first complete views of their architecture, revealing that the largest interaction surfaces between the mIg and CD79AB are in their transmembrane domains (TMDs). These structures support decades of biochemical work interrogating the requirements for assembly of a functional BCR and provide the basis for explaining the effects of mutations. Here we report a focused saturating mutagenesis to comprehensively characterize the nature of the interactions in the mIg TMD that are required for BCR surface expression. We examined the effects of 600 single-amino-acid changes simultaneously in a pooled competition assay and quantified their effects by next-generation sequencing. Our deep mutational scanning results reflect a feature-rich TMD sequence, with some positions completely intolerant to mutation and others requiring specific biochemical properties such as charge, polarity or hydrophobicity, emphasizing the high value of saturating mutagenesis over, for example, alanine scanning. The data agree closely with published mutagenesis and the cryo-EM structures, while also highlighting several positions and surfaces that have not previously been characterized or have effects that are difficult to rationalize purely based on structure. This unbiased and complete mutagenesis dataset serves as a reference and framework for informed hypothesis testing, design of therapeutics to regulate BCR surface expression and to annotate patient mutations.

Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.

The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR’s homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane.

De novo-designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.