A single-chain fab derived drug conjugate for HER2 specific delivery.

Despite the development of antibody-drug conjugates, the fragment Fab-based drug conjugates offer some unique capabilities in terms of safety, clearance, penetration and others. Current methods for preparing Fab drug conjugates are limited by the availability and stability of Fab proteins, leaving reports on this rare. Here, we found that a single-chain scaffold of Fab enables stabilization of the paired structure and supports high-yield expression in bacteria cytoplasm. Furthermore, we conjugated anti-neoplastic agent SN38 to the C-terminus by sortase A ligation and generated a homogenous Fab conjugate with the drug-to-Fab ratio of 1. The resulting anti-HER2 Fab-SN38 conjugate demonstrated potent and antigen-dependent cell-killing ability with the aid of its special cathepsin-triggered cyclization-promoted release mechanism. In vivo, Fab-SN38 can prevent growths of HER2-positive tumors in athymic mice and be well tolerated to the treatment at 7 mg/kg per dose. Anti-tumor activity, high dose tolerance and penetration advantage observed in this study would merit Fab conjugate investigation in target chemotherapy.

Mono-amino acid linkers enable highly potent small molecule-drug conjugates by conditional release.

The endosome cleavable linkers have been widely employed by antibody-drug conjugates and small molecule-drug conjugates (SMDCs) to control the accurate release of payloads. An effective linker should provide stability in systemic circulation but efficient payload release at its targeted tumor sites. This conflicting requirement always leads to linker design with increasing structural complexity. Balance of the effectiveness and structural complexity presents a linker design challenge. Here, we explored the possibility of mono-amino acid as so far the simplest cleavable linker (X-linker) for SMDC-based auristatin delivery. Within a diverse set of X-linkers, the SMDCs differed widely in bioactivity, with one (Asn-linker) having significantly improved potency (IC50 = 0.1 nM) and fast response to endosomal cathepsin B cleavage. Notably, this SMDC, once grafted with effector protein fragment crystallizable (Fc), demonstrated a profound in vivo therapeutic effect in aspects of targetability, circulation half-life (t1/2 = 73 h), stability, and anti-tumor efficacy. On the basis of these results, we believe that this mono-amino acid linker, together with the new SMDC-Fc scaffold, has significant potential in targeted delivery application.

Transcriptomics-based investigation of molecular mechanisms underlying synergistic antimicrobial effects of AgNPs and Domiphen on the human fungal pathogen Aspergillus fumigatus.

Critically ill patients have higher risk of serious fungal infections, such as invasive aspergillosis (IA) which is mainly caused by the human fungal pathogen Aspergillus fumigatus. Triazole drugs are the primary therapeutic agents for the first-line treatment of IA, which could easily cause drug resistance problems. Here, we assess the potential of AgNPs synthesized with Artemisia argyi leaf extract and domiphen as new antifungal agents to produce synergistic antimicrobial effects on Aspergillus fumigatus, and dissect possible molecular mechanisms of action. Plate inoculation assays combined with drug susceptibility test and cytotoxicity test showed that the combination of AgNPs and domiphen has synergistic antimicrobial effects on A. fumigatus with low cytotoxicity. Gene Ontology (GO) enrichment analysis showed that AgNPs and domiphen inhibit the growth of A. fumigatus by suppressing nitrate assimilation, and purine nucleobase metabolic process and amino acid transmembrane transport, respectively. When the two drugs are combined, AgNPs has epistatic effects on domiphen. Moreover, the combination of AgNPs and domiphen primarily influence secondary metabolites biosynthesis, steroid biosynthesis and nucleotide sugar metabolism of A. fumigatus via Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, protein-protein interactions (PPI) analysis combined with validation experiments showed that the combination of AgNPs and domiphen could enhance the expression of copper transporter and inhibit nitrogen source metabolism. In addition, the synergistic antimicrobial effects could be enhanced or eliminated depending on exogenous addition of copper and nitrogen source, respectively. Taken together, the results of this study provide a theoretical basis and a new strategy for the treatment of IA.

In vivo therapeutic effects of small molecule-drug conjugates enhanced by Fc grafting.

Small molecule-drug conjugate (SMDC) shows great potential as a new class of targeted chemotherapeutic agents to tackle cancer. However, its in vivo therapeutic effect is compromised by its poor pharmacokinetic parameters. Herein we describe an approach that enables the precise conjugation of SMDC on N-terminus of the Fc protein to produce a SMDC-Fc bioconjugate (Fc1070) with superior specificity, affinity and potency to tumor cells. In vivo, Fc1070 exhibited an antibody-like pharmacokinetic profile with a long circulation half-life (t1/2 = 79 h) and pro-liver clearance pathway, that is distinct from the parent SMDC (t1/2 = 0.5 h and renal clearance). Intravenous injection of Fc1070 can eliminate the tumor with a single dosing of 7 mg/kg. Coupled with a predefined ligand toolbox, this approach allows the fast generation of other SMDC bioconjugates on demand, thus extending the format easily to other tumor targets. This may provide a general approach for the development of SMDC with enhanced therapeutic properties.

Tribo-rheology and kinetics of soymilk gelation with different types of milk proteins.

The effect of different types of milk proteins (Whey protein isolate [WPI], milk protein concentrate [MPC] and sodium caseinate [NaCas]) on gelation behavior of soymilk was investigated. WPI caused the most positive effect on the gelation of soymilk by reducing gelation time, increasing gel strength, and enhancing water-holding capacity. The gelation rate at different stages suggested that WPI enhanced soymilk gelation mainly during the acidification, while MPC and NaCas enhanced the gelation during cooling. Introducing low proportion of milk proteins effectively reduced the average particle size of the gels, consequently reducing the friction at mixed regime, while high proportions of milk proteins resulted in boundary regime and high friction during mixed regime, due to the high gel firmness. No dramatic change on the "secondary structure" of the gel was caused by introducing MPC or NaCas; while more strands and network were formed in the "primary structure," especially with NaCas.

Comparison of rheological, tribological, and microstructural properties of soymilk gels acidified with glucono-δ-lactone or culture.

The physical properties of soymilk gels acidified with glucono-δ-lactone (GDL) or cultures with varying exopolysaccharide (EPS) production capabilities (EXPRESS CN < YFL-903) were studied. The acidification time and final pH were controlled in all samples. Earlier gelation and higher gelation pH were induced using GDL. The rheological properties and size distribution of GDL gel were closer to those of the EXPRESS CN gel than YFL-903 gel. Better lubrication property in the mixed regime was found in culture acidified gels, with YFL-903 gel showing the best results. Firmness, adhesiveness and water holding capacity were higher in culture- induced gels than in the GDL gel. The microstructures of the gels were found to be similar, but the subtle stranded structure of the GDL gel seemed denser than the culture-induced gel. The results suggest that both acidification rate and EPS production should be considered when comparing GDL and culture-induced soymilk gels.