Biology subjects

Beilhartz, G. L.

Publications and source records attributed to Beilhartz, G. L..

7 recordsLinked to original sources

bioRxiv2026

Balancing receptor engagement and payload catalytic activity broadens the therapeutic window of immunotoxins

Immunotoxins (ITs) are potent targeted cancer therapeutics that couple tumor-selective receptor binding with efficient intracellular delivery and cytotoxic enzymatic activity of bacterial toxins. While ITs have shown impressive efficacy in hematologic malignancies, dose-limiting toxicity remains a major barrier to their broader clinical application, particularly in solid tumors. The exceptional potency of immunotoxin payloads contributes to off-tumor toxicity because even limited uptake by receptor-low cells can be sufficient to cause cell death. Here, we systematically investigated how receptor-binding domain (RBD) affinity and avidity interact with catalytic payload activity to determine IT selectivity. Using CD123-, HER3-, and HER2-targeted ITs, we found that increasing receptor-binding affinity or avidity altered potency but provided limited control over the selectivity between receptor-high and receptor-low cells. In contrast, attenuating catalytic activity produced profound gains in selectivity when payload potency was appropriately matched to receptor engagement. The optimal level of attenuation varied with receptor context and toxin scaffold, demonstrating that maximal selectivity emerges from balancing cellular delivery with catalytic activity rather than maximizing either property alone. In vivo, payload attenuation increased the maximum tolerated dose of a HER2-targeted IT by 400-fold and enabled robust antitumor activity well below its toxicity limit, whereas the WT counterpart showed no detectable efficacy at tolerated doses. Together, these findings uncover and establish payload activity as a tunable determinant of immunotoxin selectivity and provide a framework for matching receptor engagement with catalytic potency to develop safer and more broadly applicable targeted toxins.

pharmacology and toxicology↗

bioRxiv2024

De novo design of potent inhibitors of Clostridioides difficile toxin B

Clostridioides difficile is a major cause of secondary disease in hospitals. During infection, C. difficile toxin B drives disease pathology. Here we use deep learning and Rosetta-based approaches to de novo design small proteins that block the entry of TcdB into cells. These molecules have binding affinities and neutralization IC50s in the pM range and are compelling candidates for further clinical development. By directly targeting the toxin rather than the pathogen, these molecules have the advantage of immediate cessation of disease and lower selective pressure for escape compared to conventional antibiotics. As C. difficile infects the colon, the protease and pH resistance of the designed proteins opens the door to oral delivery of engineered biologics. Significance statementC. difficile infection (CDI) is a major public health concern with over half a million cases in the United States annually resulting in 30,000 deaths. Current therapies are inadequate and frequently result in cycles of recurrent infection (rCDI). Progress has been made in the development of anti-toxin mAb therapies that can reduce the rate of rCDI, but these remain unaffordable and out of reach for many patients. Using de novo protein design, we developed small protein inhibitors targeting two independent receptor binding sites on the toxin that drives pathology during CDI. These molecules are high affinity, potently neutralizing and stable in simulated intestinal fluid, making them strong candidates for the clinical development of new CDI therapies.

microbiology↗

bioRxiv2024

Enhanced delivery of protein therapeutics with a diphtheria toxin-like platform that evades pre-existing neutralizing immunity

Targeted intracellular delivery of therapeutic peptides and proteins remains an important but unresolved goal in biotechnology. A promising approach is to engineer bacterial exotoxins that deliver their cytotoxic enzymes into cells and can be engineered to target cancer cells as is the case with immunotoxins. The well-studied diphtheria toxin translocation domain is ideally suited as a delivery platform as it has been shown to be capable of delivering a wide range of macromolecular cargo. Widespread deployment of DT-based therapeutics in humans, however, is complicated by the prevalence of pre-existing anti-DT antibodies from childhood vaccinations that reduce the exposure, efficacy and safety of this important class of protein drugs. Thus, there is a great need for delivery platforms with no pre-existing immunity in humans. Here, we describe the discovery and characterization of a distant diphtheria toxin homolog from the ancient reptile pathogen Austwickia chelonae that we have named Chelona Toxin (CT). We show that CT is comparable to DT structure and function in all respects except that it is not recognized by pre-existing anti-DT antibodies present in human sera. Moreover, we demonstrate that the CT translocase is superior to the DT translocase at delivering therapeutic protein cargo into target cells. These findings highlight CT as a potentially class-enabling new chassis for developing safer and more efficacious immunotoxins and intracellular protein delivery platforms for cancer therapy.

biochemistry↗

bioRxiv2020

Exploiting toxin internalization receptors to enhance delivery of proteins to lysosomes for enzyme replacement therapy

Lysosomal storage diseases are a group of over 70 inherited genetic diseases caused by a defect or deficiency in a lysosomal protein. Enzyme replacement therapy, in which a functional copy of the defective enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases; however, the inefficient uptake of recombinant enzymes into cells and tissues via the low-affinity mannose-6-phosphate receptor prohibits broader utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endo-lysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that the targeted TPP1 chimera binds with high affinity to target cells and is delivered into lysosomes with much greater efficiency than TPP1 alone. Further, we demonstrate efficient and durable uptake of the chimera in vivo following intracerebroventricular injection in mice lacking TPP1. Targeting the highly efficient diphtheria toxin internalization pathway represents a novel approach for improving the efficacy and utility of enzyme replacement therapy for treating lysosomal storage diseases.

pharmacology and toxicology↗

bioRxiv2019

Inhibition of tumor growth by a novel engineered chimeric toxin that cleaves activated mutant and wild-type RAS

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small molecule approaches has proven to be a major challenge. Here we describe the development of a novel pan-RAS biologic inhibitor comprised of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Further, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS. Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising new approach to developing RAS therapeutics against a broad array of cancers. SignificanceRAS oncoproteins have long been considered among the most elusive drug targets in cancer research. At issue is the lack of accessible drug binding sites and the extreme affinity for its GTP substrate. Covalent inhibitors against the KRAS G12C mutant have shown early clinical promise, however, targeting the other oncogenic RAS mutants across three RAS isoforms has proven challenging. Inhibition of activated wild-type RAS in the absence of canonical RAS mutations is also highly desirable in certain tumors. Here, we demonstrate delivery of an extremely potent pan-RAS and RAP1 cleaving enzyme in therapeutic quantities to specific receptor-bearing cells in vitro and in vivo. We aim to advance this approach to engineer the first targeted pan-RAS inhibitor for cancer therapy. One Sentence SummaryEngineered chimeric toxin halts tumor growth in vivo via RAS cleavage

cancer biology↗

bioRxiv2019

Identification of the C. sordellii lethal toxin receptor elucidates principles of receptor specificity in clostridial toxins

Clostridium sordellii lethal toxin (TcsL) is responsible for an almost invariably lethal toxic shock syndrome associated with gynecological C. sordellii infections. Here, using CRISPR/Cas9 screening, we identify semaphorins SEMA6A and SEMA6B as the cellular receptors for TcsL and demonstrate that soluble extracellular SEMA6A can protect mice from TcsL-induced edema. A 3.3 [A] cryo-EM structure shows that TcsL binds SEMA6A with the same region that the highly related C. difficile TcdB toxin uses to bind structurally unrelated Frizzled receptors. Remarkably, reciprocal mutations in this evolutionarily divergent surface are sufficient to switch receptor specificity between the toxins. Our findings establish semaphorins as physiologically relevant receptors for TcsL, and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

molecular biology↗

bioRxiv2019

bioPROTACs as versatile modulators of intracellular therapeutic targets: Application to proliferating cell nuclear antigen (PCNA)

Targeted degradation approaches have recently generated much excitement as a paradigm shift to address human disease in unprecedented ways. Amongst these, small molecule based approaches such as Proteolysis targeting chimeras (PROTACs) have attracted the lions share of attention due to their potential to tackle historically intractable targets and achieve greater potency, efficacy, and specificity over traditional small molecule inhibitors. Despite their promise, the identification of high-affinity ligands that can serve as starting points for PROTAC strategies remains challenging. As a complementary approach, we describe herein a class of intracellular biologics termed bioPROTACs. The substrate binding component of these fusion proteins consists of a peptide or an antibody-mimetic which allows for an unprecedented diversity of protein targets that can be addressed. The high-affinity binder is linked directly to an E3 ubiquitin ligase to harness the power of targeted degradation. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. Indeed, 9 out of 16 binder-E3 combinations tested resulted in greater than 70% target clearance. Through a systematic approach, we then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA), a sliding DNA clamp with critical roles in DNA replication and repair. The bioPROTAC, termed Con1-SPOP, elicited rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Compared to RNAi-based approaches which typically take days to manifest, PCNA knockdown using Con1-SPOP was evident within 4 h. The advantage of degradation versus stoichiometric inhibition was also clearly demonstrated with bioPROTAC strategies. Combining superior pharmacological inhibition and relative ease of development, bioPROTACs are powerful tools for interrogating the degradability of a substrate, for guiding the identification of the fittest E3 ligase, for studying the functional consequences associated with target protein down-regulation, and potentially for making therapeutic impacts.

cell biology↗