Bio-orthogonal Glycan Imaging of Culture Cells and Whole Animal C. elegans with Expansion Microscopy.

Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O -glycans throughout the entirety of the Caenorhabditis elegans model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix. A flexible strategy is demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, we resolve metabolically labelled O -glycans on the gut microvilli and other nanoscale anatomical features using our ExM reagents and optimized protocols. We use transmission electron microscopy images of C. elegans nano-anatomy as ground truth data to validate the fidelity and isotropy of gel expansion. We construct whole organism maps of C. elegans O -glycosylation in the first larval stage and identify O -glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans , we provide validated ExM protocols for nanoscale imaging of metabolically labelled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labelled biomolecules at enhanced resolutions with ExM.

Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization.

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA "breathing" on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

Substrate Design Enables Heterobifunctional, Dual "Click" Antibody Modification via Microbial Transglutaminase.

Site-specific modification of native antibodies has proven advantageous, as it enhances the properties of antibody-based bioconjugates without the need to manipulate the genetic code. However, native antibody modification is typically limited to strategies that introduce a single functional handle. In this work, we addressed this limitation by designing heterobifunctional substrates for microbial transglutaminase (MTG) that contain both azide and methyltetrazine "click" handles. Structure-conjugation relationships for these substrates were evaluated using the Her2-targeted antibody trastuzumab. Förster resonance energy transfer (FRET) was used to demonstrate that these chemical handles are mutually orthogonal. This orthogonality was leveraged for the one-pot synthesis of a bifunctional antibody-drug conjugate (ADC). This ADC, containing a maytansine-derived payload and a hydrophobicity-masking polyethylene glycol (PEG) side chain, demonstrated potent in vitro activity in SKOV3 cells. These studies establish the dual "click" approach as a powerful technique in the toolbox for native antibody modification.

Dual Site-Specific Antibody Conjugates for Sequential and Orthogonal Cargo Release.

Antibody-drug conjugates utilize the antigen specificity of antibodies and the potency of chemotherapeutic and antibiotic drugs for targeted therapy. However, as cancers and bacteria evolve to resist the action of drugs, innovative controlled release methods must be engineered to deliver multidrug cocktails. In this work, we engineer lipoate-acid ligase A (LplA) acceptor peptide (LAP) tags into the constant heavy and light chain of a humanized Her2 targeted antibody, trastuzumab. These engineered LAP tags, along with the glutamine 295 (Q295) residue in the heavy chain, were used to generate orthogonally cleavable site-specific antibody conjugates via a one-pot chemoenzymatic ligation with microbial transglutaminase (mTG) and LplA. We demonstrate orthogonal cargo release from these dual-labeled antibody bioconjugates via matrix metalloproteinase-2 and cathepsin-B-mediated bond cleavage. To the best of our knowledge, this is the first demonstration of temporal control on dual-labeled antibody conjugates, and we believe this platform will allow for sequential release and cooperative drug combinations on a single antibody bioconjugate.

Antibody-Mediated Endocytosis of Polysialic Acid Enables Intracellular Delivery and Cytotoxicity of a Glycan-Directed Antibody-Drug Conjugate.

The specific targeting of differentially expressed glycans in malignant cells has emerged as an attractive anticancer strategy. One such target is the oncodevelopmental antigen polysialic acid (polySia), a polymer of α2,8-linked sialic acid residues that is largely absent during postnatal development but is re-expressed during progression of several malignant human tumors, including small-cell and non-small cell lung carcinomas, glioma, neuroblastoma, and pancreatic carcinoma. In these cancers, expression of polySia correlates with tumor progression and poor prognosis and appears to modulate cancer cell adhesion, invasiveness, and metastasis. To evaluate the potential of PolySia as a target for anticancer therapy, we developed a chimeric human polySia-specific mAb that retained low nanomolar (nmol/L) target affinity and exhibited exquisite selectivity for polySia structures. The engineered chimeric mAb recognized several polySia-positive tumor cell lines in vitro and induced rapid endocytosis of polySia antigens. To determine whether this internalization could be exploited for delivery of conjugated cytotoxic drugs, we generated an antibody-drug conjugate (ADC) by covalently linking the chimeric human mAb to the tubulin-binding maytansinoid DM1 using a bioorthogonal chemical reaction scheme. The resulting polySia-directed ADC demonstrated potent target-dependent cytotoxicity against polySia-positive tumor cells in vitro. Collectively, these results establish polySia as a valid cell-surface, cancer-specific target for glycan-directed ADC and contribute to a growing body of evidence that the tumor glycocalyx is a promising target for synthetic immunotherapies. SIGNIFICANCE: These findings describe a glycan-specific antibody-drug conjugate that establishes polySia as a viable cell surface target within the tumor glycocalyx.

Antibacterial isoamphipathic oligomers highlight the importance of multimeric lipid aggregation for antibacterial potency.

Cationic charge and hydrophobicity have long been understood to drive the potency and selectivity of antimicrobial peptides (AMPs). However, these properties alone struggle to guide broad success in vivo, where AMPs must differentiate bacterial and mammalian cells, while avoiding complex barriers. New parameters describing the biophysical processes of membrane disruption could provide new opportunities for antimicrobial optimization. In this work, we utilize oligothioetheramides (oligoTEAs) to explore the membrane-targeting mechanism of oligomers, which have the same cationic charge and hydrophobicity, yet show a unique ~ 10-fold difference in antibacterial potency. Solution-phase characterization reveals little difference in structure and dynamics. However, fluorescence microscopy of oligomer-treated Staphylococcus aureus mimetic membranes shows multimeric lipid aggregation that correlates with biological activity and helps establish a framework for the kinetic mechanism of action. Surface plasmon resonance supports the kinetic framework and supports lipid aggregation as a driver of antimicrobial function.

Sequence-Defined Backbone Modifications Regulate Antibacterial Activity of OligoTEAs.

In response to the urgent need for new antibiotic development strategies, antimicrobial peptides (AMPs) and other synthetic polymers are being actively investigated as promising alternatives to traditional antibiotics. Although most AMPs display lytic activity against several types of bacteria, they have poor toxicology profiles and are susceptible to proteolysis in vivo. While many synthetic variants have been created to mimic AMPs by tuning the hydrophobic to cationic ratio of the side-chain groups, few have decoupled the effects of charge from hydrophobicity in discrete systems, and none have investigated the effect of backbone hydrophobicity. We recently developed a rapid and efficient approach for the assembly of synthetic sequence-defined oligothioetheramides (oligoTEAs) that are resistant to protease activity. Our oligoTEA assembly scheme allows direct access to the oligomer backbone, which enables precise tuning of oligoTEA hydrophobicity while keeping charge constant. In this study, we synthesized a new class of antibacterial oligoTEAs (AOTs) with precise control over backbone hydrophobicity and composition. Our studies suggest that AOTs lyse cells via membrane permeabilization and that hydrophobicity and macromolecular conformation are key properties that regulate AOT activity. Some of our AOTs show highly promising antibacterial activity (MIC ∼ 0.5-5 µM) against clinically relevant pathogens in the presence of serum, with little to no toxicity against RBCs and HEK293 cells. Taken together, our data identify design parameters and criteria that may be useful for assembling the next generation of potent and selective AOTs.

Sequence-defined bioactive macrocycles via an acid-catalysed cascade reaction.

Synthetic macrocycles derived from sequence-defined oligomers are a unique structural class whose ring size, sequence and structure can be tuned via precise organization of the primary sequence. Similar to peptides and other peptidomimetics, these well-defined synthetic macromolecules become pharmacologically relevant when bioactive side chains are incorporated into their primary sequence. In this article, we report the synthesis of oligothioetheramide (oligoTEA) macrocycles via a one-pot acid-catalysed cascade reaction. The versatility of the cyclization chemistry and modularity of the assembly process was demonstrated via the synthesis of >20 diverse oligoTEA macrocycles. Structural characterization via NMR spectroscopy revealed the presence of conformational isomers, which enabled the determination of local chain dynamics within the macromolecular structure. Finally, we demonstrate the biological activity of oligoTEA macrocycles designed to mimic facially amphiphilic antimicrobial peptides. The preliminary results indicate that macrocyclic oligoTEAs with just two-to-three cationic charge centres can elicit potent antibacterial activity against Gram-positive and Gram-negative bacteria.

Persistent enhancement of bacterial motility increases tumor penetration.

Motile bacteria can overcome the transport limitations that hinder many cancer therapies. Active bacteria can penetrate through tissue to deliver treatment to resistant tumor regions. Bacterial therapy has had limited success, however, because this motility is heterogeneous, and within a population many individuals are non-motile. In human trials, heterogeneity led to poor dispersion and incomplete tumor colonization. To address these problems, a swarm-plate selection method was developed to increase swimming velocity. Video microscopy was used to measure the velocity distribution of selected bacteria and a microfluidic tumor-on-a-chip device was used to measure penetration through tumor cell masses. Selection on swarm plates increased average velocity fourfold, from 4.9 to 18.7 µm/s (P < 0.05) and decreased the number of non-motile individuals from 51% to 3% (P < 0.05). The selected phenotype was both robust and stable. Repeating the selection process consistently increased velocity and eliminated non-motile individuals. When selected strains were cryopreserved and subcultured for 30.1 doublings, the high-motility phenotype was preserved. In the microfluidic device, selected Salmonella penetrated deeper into cell masses than unselected controls. By 10 h after inoculation, control bacteria accumulated in the front 30% of cell masses, closest to the flow channel. In contrast, selected Salmonella accumulated in the back 30% of cell masses, farthest from the channel. Selection increased the average penetration distance from 150 to 400 µm (P < 0.05). This technique provides a simple and rapid method to generate high-motility Salmonella that has increased penetration and potential for greater tumor dispersion and clinical efficacy.