Bioconjugate Chemistry最新文献

Chimeric antigen receptor-T cell (CAR-T) therapy shows promise for treating hematologic malignancies but faces limitations including insufficient CAR-T cell activation. We developed a novel tetramer CAR-T (tCAR-T) system targeting CD19, which is overexpressed in hematologic malignancies. We created a tetrameric antibody scaffold by linking anti-CD19 scFv to traptavidin, then engineered T cells expressing CD8 hinge, CD8 transmembrane, costimulatory domains (4-1BB, CD3ζ), and YFP, followed by biotinylation using biotin ligase. The tCAR-T system was assembled via biotin–avidin interaction. This tetrameric arrangement significantly enhanced antibody avidity, promoting stronger antigen engagement and demonstrating potent cytotoxicity against CD19⁺ B-cell lymphoblast lines including Raji, Nalm-6, and Ramos. Notably, tCAR-T exhibited superior antitumor activity compared to Kymriah, with enhanced cytokine release and improved target cell elimination. This innovative approach improves cancer treatment efficacy through enhanced binding avidity and modular targeting flexibility, demonstrating that tCAR-T can overcome limitations of conventional CAR-T therapy and highlighting its potential to improve therapeutic outcomes for cancer patients.

The Tn antigen is an attractive target for cancer vaccine development. In this study, we report the efficacies of dendrimer-based multivalent Tn antigens to elicit profound antibody generation in mice, in a subtype-specific manner. Multivalency of the Tn antigen is achieved by utilizing poly(ether imine) (PETIM) dendrimers of first-, second-, and third-generations, leading to tetra-, octa-, and hexadecavalent Tn antigen moieties at their peripheries through squarate ester linkage. The in vivo toxicities of the multivalent Tn antigens on mice show that these antigens are well-tolerated, with no signs of toxicity. The efficacies of the multivalent Tn epitopes to generate antigen-specific antibodies are assessed by periodic immunization of mice and assessment of the antibody production through ELISA analysis. The studies show that all three multivalent Tn epitopes express significantly stronger IgG antibody responses compared to the conventional Tn-BSA conjugate. Among the epitopes, the third-generation glycoconjugate induces the highest antibody titers, as adjudged through quantitation of antibody titer which occurs at a 1:204800 dilution. Sera from mice immunized with the hexadecavalent Tn antigen are tested on MCF-7 and Jurkat cells using FITC-labeled secondary antibodies. The antibodies show strong and selective binding to MCF-7 and Jurkat cells, whereas noncancerous HEK293 cells lack the binding to the Tn antigen-specific antibody. These results provide direct evidence of the correlation of antigen valencies, the efficacies of the antibody production, and the specificities.

Hapten immunogenicity and affinity of the antihapten response represent critical characteristics in the assessment of vaccine efficacy and quality of antibody production. The phenomenon is influenced by a multitude of factors, including the inherent characteristics of the hapten itself, the manner in which the hapten is expressed within the hapten-carrier conjugate structure, and the properties that the carrier exhibits. The preceding studies, which involved the comparison of protein carriers, did not yet resolve the role of the carrier size. The present paper examines the influence of carrier protein size on hapten immunogenicity and the affinity of the resulting antihapten antibodies. A series of novel metronidazole derivatives, designed as model haptens, were synthesized to prepare conjugates with mono-, di-, tri-, and tetramers of bovine serum albumin (BSA). The prepared immunogen constructs were designed to be equivalent in terms of hapten loading and presentation. The only distinguishing factor between the constructs was their varying carrier sizes. This approach aimed to assess the impact of carrier size in a murine immunization model. The results demonstrate that administration of even BSA dimer or larger conjugates resulted in a substantial augmentation in antihapten antibody titer and expedited generation and maturation. Thus, an enlarged protein carrier in comparison with conventional conjugate enhanced the immunogenicity of the hapten and the affinity of the antihapten antibodies produced. The study hypothesizes that using larger protein carriers could be a valuable strategy for developing vaccine candidates, particularly for weakly immunogenic haptens.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are two of the most prevalent neurodegenerative disorders, both characterized by abnormal protein folding. Amyloid-β (Aβ) and α-synuclein (α-syn) are often associated with these diseases, which might occur simultaneously in later stages. In this study, we developed a dual-target fluorescent probe, DiFboron-6, designed to specifically target Aβ and α-syn aggregates. DiFboron-6 exhibited excellent fluorescence properties, with 59-fold and 49-fold increases in fluorescence intensity upon binding to Aβ and α-syn aggregates, respectively. The probe demonstrated strong binding affinity to both proteins, with dissociation constants of Kd_Aβ = 12.4 nM and Kd_α-syn = 174 nM. Mouse brain slice staining and in vitro experiments further confirmed that DiFboron-6 could clearly label both protein plaques and effectively cross the blood–brain barrier (BBB). DiFboron-6 has been shown to effectively detect both Aβ and α-syn aggregates, thereby serving as a dual-target detection tool. Its unique structure offers a promising foundation for the development of future dual-target probes.

Phosphorylation is a prevalent post-translational modification that controls many signaling pathways by regulating protein–protein interactions. Traditionally, these interactions are studied with chemically synthesized phosphopeptides, which are often expensive and prone to dephosphorylation, or in vitro kinase reactions, which often give incomplete or off-target phosphorylation. Here, using genetic code expansion with amber-codon-directed incorporation of a nonhydrolyzable phosphoserine analog (nhpS) that is autonomously produced in E. coli, we developed a technology to produce PermaPhosPeptides and validated its utility by obtaining functional 12-mer fragments of the SARS-CoV-2 nucleocapsid protein (N) from Wuhan and later coronavirus variants. PermaPhosPeptides are phosphatase-proof and accurately mimic authentic phosphopeptides in being recognized by pS/pT-binding 14–3–3 proteins, exhibiting an average K_D difference at pH 7–8 with respect to the Wuhan phosphopeptide of only 9%, as measured by fluorescence polarization. At pH 5.5, K_D for the 14–3–3 complex with PermaPhosPeptide increases by 68% compared with the phosphopeptide but remains in the low micromolar range despite the charge −1 of the nhpS-group, indicating that stereochemistry of the target group is a more critical driver for 14–3–3 recognition than its precise charge. Furthermore, PermaPhosPeptides revealed consistent effects of N mutations on binding affinities for the seven human 14–3–3 isoforms, indicating specificity and sensitivity of the interaction. Given the modular genetic encoding system used, PermaPhosPeptide technology is scalable and adaptable, in principle enabling production of almost any phosphopeptide in a permanently phosphorylated form for studies by low- and high-throughput methods.

Despite decades of research, the successful clinical translation of intravenously administered nanomedicines is underwhelming. One significant barrier to progress is the opsonization and rapid clearance from the bloodstream due to protein corona formation as an innate immune response. Biocompatible nanoparticle coatings that act as a barrier between the nanomaterial and the physiological environment are being continuously explored to elevate delivery success. However, the formation and composition of protein coronae, especially across species, are still poorly understood, which hinders the progress of translation from preclinical animal models to human applications. Here, we use quantitative protein assays, LC-MS proteomics, and machine learning to catalog the protein coronae from human and mouse serum formed on poly(lactic-co-glygolic) acid (PLGA) nanoparticles and explore the impact of a large library of coatings comprised of cholinium fatty acid–based ionic liquids (ILs) from 4- to 10-carbon chains with varying degrees of unsaturation. We discover that the species matters, with vast changes in the coronae being observed, depending on the source of the serum sample. Additionally, even very small changes in the ionic liquid anion structure result in the formation of diverse hard coronae. We identify several ILs that show enriched dysopsonins and depleted opsonins relative to serum that are promising candidates for future development as therapeutics or in biosensing.

CRISPR–Cas12a is a versatile biosensing platform that detects sequence-specific DNA or RNA targets via a CRISPR RNA (crRNA) guide. While Cas12a’s specificity is dictated by its crRNA, chemical modifications within the crRNA can influence nuclease performance. Here, we examined the effects of two well-known RNA modifications, N⁶-methyladenosine (m⁶A) and 5-methylcytosine (m⁵C), introduced into the different positions of the guide region of a crRNA. Melting temperature (T_m) analysis showed that m⁶A had a minimal impact on RNA–DNA duplex stability. In contrast, the incorporation of m⁵C residues stabilized the duplex. Using a fluorescence recovery assay, we found that both modifications preserved Cas12a’s nuclease activity, indicating that small thermodynamic shifts in duplex formation are insufficient to disrupt its catalytic function. Despite the greater T_m increase with m⁵C, m⁶A incorporation led to a faster fluorescence recovery rate than that with m⁵C.

The emerging field of glycoRNAs, RNA molecules covalently modified with glycans, challenges the long-held belief that glycosylation is exclusive to proteins and lipids. The discovery of 3-(3-amino-3-carboxypropyl) uridine (acp3U) as a specific N-glycan attachment site has been a major breakthrough, establishing glycoRNA as a structurally defined and functionally relevant biopolymer. This new function of acp3U suggests its crucial regulatory node that correlates translation with other cellular processes, such as immune modulation and cell signaling. The presence of glycoRNAs on the cell surface and their interaction with immune receptors imply their involvement in cell-to-cell communication. Furthermore, studies have begun to associate altered glycoRNA patterns with conditions like cancer and inflammation, opening up possibilities for diagnostic and therapeutic applications. Despite the rapid progress in this field, several key challenges remain, including the inherent bias of current detection methods, the difficulty of isolating pure glycoRNA samples from complex cellular mixtures, and the largely unknown mechanisms of specific glycan linkages. Future research must focus on developing unbiased and sensitive analytical technologies to accurately map these modification patterns at a single-nucleotide resolution. This review summarizes the chemical and enzymatic mechanisms of RNA glycosylation sites, highlights its potential functional roles in cells, and outlines future research aimed at uncovering its full biological and therapeutic potential.

In vitro transcription (IVT) using T7 RNA polymerase is a key step in mRNA synthesis for therapeutic applications. However, the generation of double-stranded RNA (dsRNA) byproducts during IVT─primarily due to RNA rebinding and self-priming─triggers innate immune responses and reduces translation efficiency. Here, we present a simple and effective strategy to minimize dsRNA formation during IVT by incorporating PEGylated graphene oxide (PEG-GO). Graphene oxide (GO) preferentially binds single-stranded nucleic acids, but its use is limited by protein adsorption and low solubility in Mg²⁺-containing buffers. PEG modification improves GO’s dispersibility and reduces protein binding, allowing selective sequestration of nascent RNA without inhibiting T7 RNA polymerase activity. The addition of PEG-GO to the IVT reaction reduced the dsRNA content by over 75% while maintaining RNA yield and accelerating transcription kinetics. Moreover, mRNA synthesized in the presence of PEG-GO showed enhanced protein expression and reduced interferon-β secretion in transfected cells, comparable to post-IVT-purified mRNA. Our work demonstrates PEG-GO as a practical additive for improving the quality and scalability of IVT-based mRNA production.

The scarcity of a simple, cost-effective, and green method for the immobilization of enzymes severely hampers their application. Herein, a versatile and mild xylanase and lichenase bienzyme (XLBE) immobilization strategy including biofunctionalization of the magnetic particles, enzyme-free purification, and spontaneous covalent bridging based on SpyCatcher “Click Biology” was proposed. Only biocompatible tannic acid (TA), Fe³⁺, and elastin-like polypeptide-SpyCatcher were fed. The biomodified magnetic particles exhibited excellent stability with a loss of only 3.51% EC after 1 h of incubation at pH 7.5. Then, they were applied to immobilize SpyTag fused XLBE directly from the crude solution at a loading of 12.5 mg/g. The retention of XLBE and the xylanase activity were as high as 87.73% and 82.77%, respectively. The half-lives of the immobilized xylanase increased by 1535.75% (50 °C) compared to those of the free xylanase. The immobilized XLBE showed excellent reusability, retaining 70.15% (xylanase) and 78.81% (lichenase) of the initial activity after 8 cycles of recycling. They also showed superior catalytic performance with 202.25% improvement in green production of total reducing sugar and 30.77% improvement in juice clarification. Moreover, the versatility of the immobilization strategy was also demonstrated on inorganic carriers such as silicon dioxide and carbon nanotubes. This innovative all-in-one strategy avoids too many chemical reagents for surface modification and omits the complex enzyme prepurification process for immobilization, which will shed light on the green biocatalytic applications based on time-effective and low-byproduct surface functionalization strategies.