Multiple tumor types overexpress Nectin-4 and the antibody–drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a “Bicycle Toxin Conjugate” (BTC) consisting of a Nectin-4–binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1–2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.

Antimicrobial peptides (AMPs) are short oligopeptides that can penetrate the bacterial inner and outer membranes. Together with cell-penetrating peptides (CPPs), they are called membrane active peptides; peptides which can translocate across biological membranes. Over the last fifty years, attempts have been made to understand the molecular features that drive the interactions of membranes with membrane active peptides. This review examines the features of a membrane these peptides exploit for translocation, as well as the physicochemical characteristics of membrane active peptides which are important for translocation. Moreover, it presents examples of how these features have been used in recent years to create conjugates consisting of a membrane active peptide, called a “vector”, attached to either a current or novel antibiotic, called a “cargo” or “payload”. In addition, the review discusses what properties may contribute to an ideal peptide vector able to deliver cargoes across the bacterial outer membrane as the rising issue of antimicrobial resistance demands new strategies to be employed to combat this global public health threat.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles’ inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.

Penicillin binding proteins (PBPs) are well validated antimicrobial targets, but the prevalence of β-lactamase driven resistance and, more rarely, target-based mutations, necessitates new classes of PBP-targeting drugs. Here we describe the discovery and optimisation of bicyclic peptide (Bicycle^®) inhibitors of E. coli PBP3 (EcPBP3) using a proprietary phage display platform, and their conjugation to linear antimicrobial peptides to confer outer membrane permeation. These molecules exhibited high-affinity binding to E. coli PBP3 and a viable spectrum of killing activity against clinically relevant species of the Enterobacterales. X-ray crystallography was used to explore the mode of binding to PBP3, enabling increased target affinity and improvement of in vitro stability. These compounds bind to the transpeptidase active site cleft of PBP3 and represent, to our knowledge, a novel non-β-lactam chemical class of high affinity, non-covalent penicillin binding protein inhibitors. This work demonstrates an approach to rapidly find binders to antimicrobial targets, combined with an entry mechanism to provide access to the Gram negative cell.

The GDF15–GFRaL–RET signaling complex is involved in a broad range of disease states, with agonistic action of GDF15 affecting metabolism and body weight control, while inhibition is indicated in cancer and wasting disorders like cachexia. Here, we describe the discovery of the peptide inhibitors of the GDF15–GFRaL protein–protein interaction to prevent RET-induced signaling using both a structure-guided design and a phage display approach. Phage display provided bicyclic peptide hits with high affinity for GFRaL, and these were dimerized to mimic the bidentate interaction of homodimeric GDF15. Guided by structural data, the monomeric peptides were converted into tandem Bicycle molecules with picomolar affinities, similar to that of the endogenous GDF15 ligand. These dimerized protein mimetics inhibited cell signaling in a functional assay and showed improved pharmacokinetic properties compared with their monomeric counterparts. This is the first example of a homodimeric Bicycle molecule inhibiting receptor complex formation, thereby antagonizing the intracellular signaling response.

The lipopolysaccharide (LPS) transport (Lpt) system in Gram-negative bacteria maintains the integrity of the asymmetric bacterial outer membrane (OM). LPS biogenesis systems are essential in most Gram-negative bacteria, with LptDE responsible for the delivery of LPS to the outer leaflet of the OM. As an externally accessible, essential protein, LptDE offers a promising target for inhibitor development without the need for cellular penetration. However, there are no direct inhibitors of E. coli LptDE, and drug discovery is made challenging since it is a membrane target without a conventional active site. Here, the bicycle phage display platform was used in combination with cryogenic-electron microscopy (cryo-EM) and surface plasmon resonance to identify and map bicyclic peptide binders to Shigella flexneri LptDE (SfLptDE). Four distinct epitopes with unique bicycle molecule binding motifs were identified across the SfLptD β-barrel. This method represents a streamlined workflow for the identification and prioritization of hit molecules against LptDE.