Substrate interactions guide cyclase engineering and lasso peptide diversification.

Lasso peptides are a diverse class of naturally occurring, highly stable molecules kinetically trapped in a distinctive [1]rotaxane conformation. How the ATP-dependent lasso cyclase constrains a relatively unstructured substrate peptide into a low entropy product has remained a mystery owing to poor enzyme stability and activity in vitro. In this study, we combined substrate tolerance data with structural predictions, bioinformatic analysis, molecular dynamics simulations and mutational scanning to construct a model for the three-dimensional orientation of the substrate peptide in the lasso cyclase active site.

SGN-CD228A Is an Investigational CD228-Directed Antibody-Drug Conjugate with Potent Antitumor Activity across a Wide Spectrum of Preclinical Solid Tumor Models.

SGN-CD228A is an investigational antibody-drug conjugate (ADC) directed to melanotransferrin (CD228, MELTF, MFI2, p97), a cell-surface protein first identified in melanoma. SGN-CD228A consists of a humanized antibody, hL49, with high specificity and affinity for CD228 that is stably conjugated to 8 molecules of the clinically validated microtubule-disrupting agent monomethyl auristatin E (MMAE) via a novel glucuronide linker. We performed comprehensive IHC studies, which corroborated published RNA sequencing data and confirmed low CD228 expression in normal tissues and high expression in several cancers, including melanoma, squamous non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), colorectal cancer, and pancreatic cancer.

Improving Antibody-Tubulysin Conjugates through Linker Chemistry and Site-Specific Conjugation.

Tubulysins have emerged in recent years as a compelling drug class for delivery to tumor cells via antibodies. The ability of this drug class to exert bystander activity while retaining potency against multidrug-resistant cell lines differentiates them from other microtubule-disrupting agents. Tubulysin M, a synthetic analogue, has proven to be active and well tolerated as an antibody-drug conjugate (ADC) payload, but has the liability of being susceptible to acetate hydrolysis at the C11 position, leading to attenuated potency.

Glucuronide-Linked Antibody-Tubulysin Conjugates Display Activity in MDR and Heterogeneous Tumor Models.

Although antibody-drug conjugates (ADCs) find increasing applications in cancer treatment, or treatment-emergent resistance mechanisms may impair clinical benefit. Two resistance mechanisms that emerge under prolonged exposure include upregulation of transporter proteins that confer multidrug resistance (MDR) and loss of cognate antigen expression. New technologies that circumvent these resistance mechanisms may serve to extend the utility of next-generation ADCs.

Effects of Cationic Proteins on Gold Nanoparticle/Aptamer Assays.

Gold nanoparticles (AuNPs) and aptamers are compelling building blocks for analytical assays with desired attributes of selectivity and sensitivity and may theoretically form the basis of instrument-free color-changing assays for any target against which a DNA aptamer has been selected. However, assays for proteins based on these components may be subject to significant interferences from the interaction of proteins with nanoparticles. We found that for three representative protein/aptamer systems-thrombin, apolipoprotein E, and platelet-derived growth factor-pH-dependent aggregation occurred, even in the absence of the aptamer, to differing extents.

Development of Novel Quaternary Ammonium Linkers for Antibody-Drug Conjugates.

A quaternary ammonium-based drug-linker has been developed to expand the scope of antibody-drug conjugate (ADC) payloads to include tertiary amines, a functional group commonly present in biologically active compounds. The linker strategy was exemplified with a β-glucuronidase-cleavable auristatin E construct. The drug-linker was found to efficiently release free auristatin E (AE) in the presence of β-glucuronidase and provide ADCs that were highly stable in plasma.