Bioconjugate Chemistry最新文献

Disialoganglioside GD2 (GD2) is highly expressed in several tumors. While anti-GD2 antibodies and GD2-based antibody–drug conjugates (ADCs) have shown some therapeutic efficacy, their activity requires enhancement. Elastase, abundant in tumor microenvironment, has substrates that can serve as ADC linkers. Here, we constructed an anti-GD2 ADC by conjugating the anti-GD2 antibody Hu3F8 to the payload monomethyl auristatin E (MMAE) via an elastase-cleavable tetrapeptide linker (BMOA). Hu3F8 was produced using a 293F expression system, and its specific binding to GD2-positive cells was confirmed. The cytotoxicity of the ADCs was evaluated in both GD2-positive and GD2-negative tumor cell lines with or without elastase and its antitumor effects were investigated. It showed GD2-dependent cytotoxicity, with a significantly decreased IC₅₀ in GD2-positive cells upon elastase addition, while no cytotoxicity was observed in GD2-negative cells. Additionally, Hu3F8-BMOA-MMAE induced significant cell cycle arrest and apoptosis in GD2-positive cells, with stronger effects in the presence of elastase. In M21 melanoma mouse models, Hu3F8-BMOA-MMAE achieved an obvious tumor inhibition rate compared to the control. These findings demonstrate that utilizing a neutrophil elastase tetrapeptide substrate as a linker in anti-GD2 ADCs is a potential strategy for the therapy of GD2-positive tumors.

The tumor microenvironment (TME) exhibits metabolic dysfunction characterized by lactate (LA) accumulation, which leads to tumor progression, angiogenesis, and therapy resistance. Targeting LA metabolism through lactate oxidase (LOX) converting LA to pyruvate and hydrogen peroxide (H₂O₂) under aerobic conditions is a promising therapeutic strategy. However, LOX activity is limited by TME hypoxia. To overcome this limitation, we developed an integrated nanotheranostic system based on hollow MnO₂ nanoparticles loaded with LOX and cinnamaldehyde (CA) and modified with hyaluronic acid (HA) for tumor-targeted delivery (denoted as MCLH). In the acidic TME, MCLH decomposes to release LOX and CA, while MnO₂ reacts with endogenous H₂O₂ to generate O₂. The resulting O₂ maintains LOX-mediated LA oxidation, thereby providing additional H₂O₂ to promote further O₂ production, establishing a self-sustaining cycle that continuously consumes lactate. Meanwhile, CA depletes glutathione via Michael addition, disrupting redox homeostasis and enhancing H₂O₂ accumulation to increase oxidative stress. The released manganese ions (Mn²⁺) also enable magnetic resonance imaging (MRI) contrast for real-time monitoring. These cascading effects collectively achieve the synergistic regulation of LA metabolism and oxidative damage, providing an effective strategy for tumor treatment.

Various natural drugs such as glycyrrhizic acid (GA), hesperidin (Hes), and baicalin (Bai) exhibit anti-SARS-CoV-2 potential but suffer from poor water solubility, short half-life, and low binding capacity to viral targets. Hence, a dual-targeted, long-circulating, ROS-responsive, and broad-spectrum antiviral inhibitor (mPAGHB) is designed through a strategy of “polymer–drug linkage”. The mPAGHB suppresses SARS-CoV-2 through two “lines of defense”, including extracellular inhibition of the “spike protein-angiotensin converting enzyme 2 (ACE2)” process and intracellular inhibition of the enzymatic activity of major protease. It exhibits higher affinity for spike protein (K_D = 4.95 × 10^–2 μM), excellent inhibition of M^pro activity (76.27 ± 5.94% inhibition), and improved inhibition for pseudovirus of SARS-CoV-2 and Omicron variants in cellular and animal assays. In addition, as an efficient generic strategy, the “polymer–drug linkage” strategy facilitates the rapid construction of antiviral inhibitors based on natural active ingredients for the outbreaks of viral public infections.

Mesothelin (MSLN) is overexpressed in various malignancies, making it a promising target for molecular imaging and therapeutic strategies. Anti-MSLN VH-Fc fusion proteins show high tumor uptake as compared with monoclonal antibodies; however, elevated accumulation in Fc-rich organs (liver, spleen) can compromise tumor-to-background ratios and limit clinical applicability. To overcome this, we developed Fc mutant anti-MSLN VH-Fc fusion proteins incorporating G236R/L328R (GRLR) and L234A/L235A/P329G (LALAPG) mutations to eliminate FcγRs interactions. Engineered mutants exhibited high purity (>95%), retained strong MSLN binding (KD 2.2–3.7 nM), and effectively silenced FcγR binding by ex vivo and in vivo analyses. Following zirconium-89 radiolabeling, PET imaging was conducted across multiple xenograft models with varying MSLN expression. In HCT116 xenografts, [⁸⁹Zr]Zr-2A10-VH-Fc_LALAPG demonstrated substantially higher uptake (13.0 ± 0.1%ID/g at 120 h p.i.) than [⁸⁹Zr]Zr-2A10-VH-Fc_WT (4.2 ± 0.6%ID/g), while substantially reducing liver (LALAPG: 4.3 ± 0.6%ID/g vs WT: 19.8 ± 2.8%ID/g) and spleen (LALAPG: 9.3 ± 0.1%ID/g vs WT: 95.0 ± 39.3%ID/g) uptake. Biodistribution studies in additional xenograft models confirmed a high specific uptake for [⁸⁹Zr]Zr-2A10-VH-Fc_LALAPG in tumors with moderate to high MSLN expression. Notably for the mutants, females exhibited higher renal retention than males, indicating sex-dependent pharmacokinetics. These findings highlight Fc-engineered VH-Fc fusion proteins, particularly the LALAPG, as promising agents with enhanced tumor specificity, improved pharmacokinetics, and significantly reduced off-target uptake, supporting their use in PET imaging-guided therapeutic applications.

The landscape of nucleic acid modification technologies is rapidly evolving, with chemoselective postsynthetic labeling strategies emerging as indispensable chemical tools for generating functionalized oligonucleotides (ONs). These methods greatly rely on reactions between groups such as amine/thiol or clickable handles like azide/alkyne with cognate reaction partners. However, achieving precise covalent labeling in the presence of competing functionalities and preserving ON integrity, particularly in RNA, presents significant challenges. Here, we present an innovative chemoenzymatic platform for DNA and RNA labeling that leverages the unique chemoselective reactivity of terminal glycine-modified (Gly-tag) nucleotide analogs with o-substituted benzaldehyde substrates equipped with strategic hydrogen bond acceptors. The Gly-tagged nucleotide analogs (d^GTTP and ^GUTP) serve as excellent substrates for DNA and RNA polymerases and terminal uridylyl transferase, thereby allowing the incorporation of the reactive Gly-tag at desired positions into DNA and RNA ONs. Notably, o-substituted benzaldehyde substrates, bearing a proximal oxyacetamide moiety, facilitate efficient postenzymatic conjugation, enabling site-selective installation of functionalities including affinity tags, fluorescent probes and clickable groups with good yields and remarkable selectivity. Taken together, this chemoenzymatic methodology represents a new toolkit for late-stage ON labeling, opening up new avenues for advancing nucleic acid applications in diagnostics and biotechnology.

Dual amylin and calcitonin receptor agonists (DACRAs) offer a promising strategy for treating obesity and related metabolic disorders but are limited by aggregation and the short half-lives of native peptides. Here, we report the design of a long-acting and stapled DACRA via a streamlined on-resin Ugi macrocyclization strategy based on a salmon calcitonin template. The lead candidate UDA-6 exhibited potent and balanced activation of AMY₃R and CTR, with enhanced helical stability and favorable pharmacokinetics properties supporting once-weekly dosing. In diet-induced obese rats, UDA-6 elicited substantial weight loss and improved metabolic and hepatic parameters. Combination therapy of UDA-6 with semaglutide or tirzepatide yielded synergistic efficacies, achieving up to 41% vehicle-adjusted body-weight reduction and near-normalized liver lipid profiles. These findings establish UDA-6 as a potent and durable DACRA and highlight Ugi macrocyclization as a versatile platform for the long-acting peptide drug design.

DNA bioconjugates integrate the programmable recognition and structural precision of nucleic acids with the diverse properties of (bio)polymers, enabling functional architectures across sensing, biomedicine, and nanotechnology. While versatile conjugation chemistry (e.g., click reaction) is available, linking DNA oligonucleotides with synthetic polymers and biomacromolecules is hampered by steric shielding and length-dependent masking of reactive sites. Here, we report a facile freezing strategy that exploits ice confinement to drive highly efficient, template-free coupling of DNA oligonucleotides with diverse (bio)polymers. Our freezing strategy enables near-quantitative (>90%) coupling of oligonucleotides with synthetic polymers (polyethylene glycol) and biopolymers (bovine serum albumin and anti-PD-L1) with preserved cell targeting/uptake capability. Moreover, the accelerated template-free ligation allows us to obtain ssDNA with architectures previously challenging, such as long strands (>150 nt), strands with inverted orientations (5′–5′, 3′–3′), and branched structures. By unifying multiple conjugation chemistries under biocompatible, low-temperature conditions, our results establish freezing as a general strategy for constructing DNA-(bio)polymer conjugates, with broad implications for analytical chemistry, chemical biology, nanotechnology, and precision medicine.

This work describes the synthesis and characterization of a new class of spherical nucleic acid (SNA) derived from amphiphilic polymeric micelles (APM-SNA) for the delivery of hydrophobic drugs. Core structures are assembled from amphiphilic copolymers comprising a hydrophobic block (made from hydroxypropyl methacrylate and methacrylates bearing n-butyl, benzyl, or n-hexyl side chains) and a hydrophilic polyethylene glycol block terminated with an azide group. Alkyne-modified oligonucleotides are conjugated to the polymer cores via click chemistry to form APM-SNAs. Cellular uptake of APM-SNAs is dependent on oligonucleotide density, increasing up to 10-fold at the highest densities. Moreover, higher-density APM-SNAs induce up to a six-fold increase in immune activation compared to linear DNA. Increasing the hydrophobicity and pi–pi stacking interactions of the polymer core with benzyl methacrylate enhances cellular uptake relative to APM-SNAs constructed with the least hydrophobic (n-butyl methacrylate) monomers. Additionally, benzyl APM-SNAs show superior drug encapsulation efficiency (67 vs 41%) and extended-release profiles (half-life of 27.8 vs 7.4 h) for the STAT3 inhibitor WP1066 compared to n-butyl APM-SNAs. Oligonucleotide sequence influences the extent of cellular uptake in Caki-1 cells; T20-SNAs exhibit ∼40% greater uptake compared to CpG and G-quadruplex sequences after 24 h. These structure-dependent properties lead to a 40% improvement in WP1066 potency when delivered via T20 benzyl APM-SNAs, reducing the drug’s EC₅₀ from 6.18 to 3.54 μM. Collectively, these results demonstrate that increasing the hydrophobicity of the APM-SNA core and the interactions between the core and the drug enhance drug encapsulation efficiency, prolong release kinetics, and improve therapeutic efficacy.

Cytoreductive surgery is a major procedure to treat advanced-stage epithelial ovarian cancer by removing tumors and nearby tissues to which the cancer may have spread. Current intraoperative procedures largely rely on the surgeon’s discretion, such as naked-eye gross inspection and palpation, which is highly variable. To enhance the precision of fluorescence-guided surgery (FGS) in detecting small peritoneal tumors, we optimized the formulation of CypH-11, a sprayable near-infrared (NIR) tumor-responsive fluorogenic probe. Here, CypH-11 was formulated with the nonionic ethoxylated solubilizer Kolliphor HS 15 and sprayed directly on the exposed abdomen of ovarian tumor-bearing mice. During cytoreductive surgery aided by CypH-11, combined with or without neoadjuvant therapy, we successfully detected and excised submillimeter tumors (<1 mm in diameter) using an FDA-approved NIR video imaging system. Importantly, the overall specificity, which is represented by the true-positive rate for excised tumors, was over 90%, and the false-negative rate was only 2.6%. This fast-acting, easy-to-apply CypH-11 spray can be a powerful tool for real-time FGS, allowing surgeons to visualize and resect tiny tumors without systemic toxicity, thereby achieving a more complete resection of peritoneally disseminated tumors.

Fluorescence-guided surgery (FGS) has emerged as a powerful tool for enhancing a surgeon’s ability to locate and resect all malignant lesions, thereby improving a patient’s probability of survival. While several tumor-targeted near-infrared (NIR) fluorophores have been developed to image selected cancer types, no single tumor-targeted dye has been found to image all cancers. To remedy this deficiency, we have examined the ability of a fibroblast activation protein (FAP)-targeted NIR fluorophore to broad-spectrum solid tumors when combined with pafolacianine (OTL-38, Cytalux). Pafolacianine was selected for this imaging cocktail because it highlights all cells that express either folate receptor α (a receptor that is expressed on ∼40% of human cancers) or folate receptor β (a related receptor expressed on tumor-associated macrophages and myeloid-derived suppressor cells). An FAP-targeted conjugate of the same near-infrared fluorescent dye (S0456) was also selected because it targets myofibroblasts that infiltrate virtually all solid tumors but are essentially absent from healthy tissues. In this paper, we describe the design, synthesis, and characterization of a novel FAP ligand (FAP9) and determine its affinity (K_d ∼ 2 nM) and specificity (>2000-fold over homologous enzymes) for FAP. We then quantify its ability to image many different cancer types in murine tumor models and combine it in different ratios with pafolacianine to determine the ratio that yields the highest total tumor fluorescence and tumor-to-background ratio in seven tumor models. Because the combination of the two fluorescent conjugates invariably images tumors better than either conjugate alone, and since the two-dye combination displays no obvious toxicities, we propose that a cocktail of FAP9-S0456 plus pafolacianine warrants evaluation as a candidate for intraoperative imaging of all human tumors.