Bioconjugate Chemistry最新文献

Antibody-drug conjugates (ADCs) are modern biopharmaceuticals that combine the therapeutic effects of small-molecule drugs with the outstanding selectivity of monoclonal antibodies (mAbs). Since their introduction in the biomedical field, research has focused on elucidating the structure, stability, and mode of action of ADCs. Nevertheless, standard characterization methods for ADCs heavily rely on disruptive techniques like mass spectrometry in a non-physiological environment. Here, we present an NMR approach combining ¹H-¹³C ALSOFAST-HMQC and T₂-edited ¹H CPMG experiments, which together provide information on: i. the fingerprint and higher-ordered structure (HOS) of mAbs and ADCs and ii. the properties of the bound linker-payload fragment. In this study, we chose Trastuzumab as a well-known mAb and a Remdesivir-derived fragment as a linker-payload model system to validate our approach.

The plasma membrane is central to numerous cellular processes, yet long-term fluorescence imaging has been limited by insufficient probe retention. Here, we report a rational design of naphthalimide-based, phospholipid-mimicking probes that combine hydrophobic alkyl chains with ionic head groups to achieve strong membrane affinity. Among the series, a zwitterionic derivative, Betaine-DNap, exhibited selective and prolonged plasma membrane staining, maintaining localization for extended time-lapse imaging. Structural analyses revealed that molecular length comparable to phospholipids, zwitterionic electrostatics, and balanced hydrophobicity are key parameters for stable retention. Using Betaine-DNap, we visualized plasma membrane dynamics including endocytosis and oxidative stress-induced vesicle release, processes previously difficult to monitor due to probe instability. This work provides a generalizable design principle for long-term plasma membrane probes and establishes Betaine-DNap as a practical tool for studying membrane physiology in living cells.

Aldehyde-based bioconjugation offers attractive alternatives to N-hydroxysuccinimide (NHS) esters for the selective modification of lysine and N-terminal amines, but most existing methods require auxiliary reagents or suffer from poor stability. We introduce 2-(bromoalkyl)benzaldehydes as a versatile class of reagents that react directly with primary amines in aqueous media without catalysts or reducing agents. The imine of the ortho-bromomethylbenzaldehyde undergoes rapid intramolecular cyclization to form isoindoles, enabling tandem coupling to maleimides through a Diels-Alder reaction and thereby labeling of proteins and oligonucleotides, albeit with limited stability in buffer. In contrast, ortho-bromoethyl analogues form stable isoquinolinium intermediates and the reagents exhibit markedly improved aqueous stability compared to NHS esters. Incorporation of alkyne or azide handles further allows CuAAC and SPAAC coupling, including a one-pot copper-free DNA-antibody conjugation. This modular platform enables mild, efficient, and durable labeling of proteins, oligonucleotides, and antibodies, providing a reagent-economical and broadly applicable strategy for bioconjugation in targeted therapeutics, molecular imaging, and nanoscale engineering.

CD105 (endoglin) is a proliferation-associated transmembrane glycoprotein selectively expressed on activated endothelial cells in tumor neovasculature and serves as an attractive biomarker for imaging tumor angiogenesis. Here, we report the development of a novel CD105-targeted PET tracer, ⁶⁸Ga-DOTA-CDP, based on a high-affinity peptide (KD = 13.5 nM) identified from a combinatorial library. The radiotracer was obtained with high radiochemical purity (>97%), excellent stability in phosphate-buffered saline and fetal bovine serum, and favorable hydrophilicity. In vitro confocal imaging and flow cytometry demonstrated specific binding of CDP to CD105-positive HUVECs with minimal uptake in CD105-negative cells. Micro-PET imaging in multiple tumor-bearing mouse models, including 4T1, A549, H1975, MDA-MB-231, and JIMT-1 xenografts, enabled rapid tumor visualization at early time points following injection. Tracer uptake was significantly higher in CD105-high tumors compared with CD105-low tumors, with the highest accumulation observed in the triple-negative breast cancer model MDA-MB-231. Biodistribution studies revealed predominant renal clearance, low hepatic uptake, and favorable tumor-to-background ratios. Blocking experiments with excess unlabeled peptide markedly reduced tumor uptake, confirming receptor-mediated targeting. Immunohistochemical analysis further validated heterogeneous CD105 expression in tumor neovasculature and demonstrated a positive correlation between CD105 expression levels and PET-derived tumor uptake. Overall, ⁶⁸Ga-DOTA-CDP shows promise as a peptide-based PET tracer for noninvasive tumor angiogenesis imaging.

Antibody-drug conjugates (ADCs) are a promising and emerging class of biotherapeutics that combine the targeting precision of monoclonal antibodies (mAbs) with the cytotoxic potency of small-molecule drugs. Their manufacturing, however, requires conjugation of the mAb with the pertinent small molecule drug, a step typically is inefficient, incurring wastage of both the mAb and the drug. In addition, ADC manufacturing is challenged by precise control of drug-to-antibody ratio (DAR), aggregation, safe handling of cytotoxic payloads, among other factors. In this work, we present a novel approach for continuous conjugation of the mAb and the drug, by utilizing a coiled flow inverter reactor (CFIR) to facilitate the thiol-maleimide conjugation at interchain cysteines residues in the mAb. As an initial step, the process parameters for reducing thiol groups and the conjugation steps were screened, followed by optimization of the significant parameters (concentration of mAb and drug/linker payload, reaction duration, and temperature) using design of experiments (DoE) methodology. The performance of the CFIR was then compared to that of traditional batch conjugation. We demonstrate that the CFIR offers 64.40% higher productivity, 70% lower cost of production, and a safer alternative to the traditional batch conjugation, while producing clinically relevant DAR. This work illustrates the potential of continuous processing to transform ADC manufacturing by enabling more efficient, scalable, and sustainable production platforms.

Lipid nanoparticles (LNPs) have emerged as a promising nonviral nucleic acid delivery platform for clinical use. To expand LNPs as a treatment option in nonhepatic-based diseases, LNPs surface coated with targeting moieties produce a precise modular delivery method that can bind to and be internalized by specific receptors expressed on target cells. This study showcases the tetrazine-trans-cyclooctene inverse electron-demand Diels-Alder click reaction and directly compares its performance with the widely employed thiol-maleimide conjugation. We also compare direct mixing and micelle mixing insertion methods under different conditions to determine the optimal formulation to produce targeted LNPs. For thiol-maleimide chemistry, monoclonal antibody cetuximab was modified with N-succinimidyl S-acetylthioacetate, followed by reaction with either 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀-maleimide) directly or in preformed DSPE-PEG₂₀₀₀-maleimide: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG₂₀₀₀) micelles. For clickable tetrazine chemistry, cetuximab was modified with 2,5-dioxo-1-pyrrolidinyl 5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate, followed by reaction with either 1,2-Distearoyl-sn-glycero-3-PE-polyethylene glycol-2000- trans-cyclooctene (DSPE-PEG₂₀₀₀-TCO) directly or preformed DSPE-PEG₂₀₀₀-TCO:DMG-PEG₂₀₀₀ micelles. LNPs were prepared by mixing lipids in ethanol and mRNA (10 mM citrate buffer, pH 3.5) at a ratio of 3:1 (v/v). Insertion was carried out by combining either the direct conjugate solution or the micelle solution with the LNPs and mixing at 60 °C for 1 h. Only the micelle mixing method produced stable particles with mRNA encapsulation above 80%. The LNPs were found to be stable up to 3 weeks (stored at 4 °C) as indicated by the RiboGreen assay and particle sizes. Targeted LNPs displayed relatively weak and neutral zeta potential. In vitro studies revealed enhanced cellular uptake of Green Fluorescent Protein (GFP) mRNA by targeted LNPs with similar transfection rates with thiol-maleimide and tetrazine-TCO chemistries showing around 97% GFP⁺ cells, compared to less than 30% in control groups at 6 h. Both targeted LNP formulations showed a significant rise in mean fluorescence intensity, achieving at least a 3-fold increase over the controls.

The design and synthesis of macromolecules through controlled branching via hyperbranch polymerization have recently attracted attention from the scientific community due to their diverse biomedical applications, including diagnosis and drug delivery. This article highlights the development of a naphthalene-derived, photoswitchable, self-assembling hyperbranch polymer (PNap-Azo) in an aqueous medium, in which the azo bond serves as a stimulus-responsive feature in the presence of an overexpressed enzyme in a hypoxic microenvironment. PNap-Azo has a significant biological impact in hypoxic environments due to an azo bond (N═N), which transforms from a spherical to a twisted-rod shape upon cis-trans isomerization. Due to its amphiphilic nature and extensive hydrogen bonding, it exhibits an excellent twisted-rod morphology, enhancing the polymer's therapeutic efficacy in a hypoxic tumor environment. Therefore, PNap-Azo can detect and monitor azoreductase activity in a hypoxic environment through turn-on fluorescence responses, which are verified both spectroscopically and biologically in live cell lines. Our synthesized polymer, PNap-Azo, can monitor azoreductase activity in real time, potentially preventing complications before the condition worsens.

Protein grafting techniques that enable conjugation of diverse compounds to target proteins endow them with new functions. Sortase-mediated ligation (SML) is one such method in which sortases catalyze transpeptidation between a substrate protein or peptide bearing a cell wall sorting signal (CWSS) and a nucleophile containing an N-terminal triglycine motif. In this study, we identified novel sortase Es suitable for SML by combining in silico enzyme screening with ancestral sequence reconstruction (ASR). Eight ancestral sortases Es (1AcSE-8AcSE) were designed, of which four (1AcSE, 2AcSE, 3AcSE, and 8AcSE) were expressed in soluble form and exhibited moderate thermal stability. HPLC analysis using Ac-YNL(A/P)ETGA and GGGKY peptides revealed that 2AcSE displayed no activity toward Ac-YNLPETGA, suggesting its unique substrate specificity. Structural analysis of 2AcSE indicated that differences in Loop A, which corresponds with the β3-β4 loop, may contribute to its specificity. We designed ΔAcSE5, a variant of a previously characterized high-activity AcSE5, by replacing its Loop A (residues 56'-61', GEAPLK) with the shorter TG motif from 2AcSE. ΔAcSE5 exhibited improved specificity for Ac-YNLAETGA over Ac-YNLPETGA, with a trade-off in activity and reduced byproduct formation during conjugation of a shark antibody to GGG-Venus. These findings demonstrate that database-driven screening and structure-function analysis of AcSEs can guide the design of novel sortase E variants optimized for SML applications.

Radioimmunotherapy (RIT) delivers radionuclides to tumors through antibody-based targeting. However, radiolabeling with high-linear energy transfer (LET) isotopes like Lu-177 can induce aggregation, reducing antibody stability and bioactivity. In this study, we evaluated [¹⁷⁷Lu]Lu-DOTA-Trastuzumab, a HER2-targeting radioimmunoconjugate, to assess how conjugation chemistry, radiolabeling conditions, and purification methods affect aggregation and therapeutic performance. Dynamic light scattering (DLS) was used to monitor aggregation throughout formulation. Higher chelator-to-antibody ratios and increased radioactivity caused significant aggregation, which was not detected by conventional SEC-HPLC or radio-TLC quality control methods. Compared to the diagnostic isotope Cu-64, the therapeutic isotope Lu-177 exhibited markedly greater aggregation, reflecting the stronger radiolytic stress from high LET isotopes. Aggregation inversely correlated with HER2-specific uptake and tumor accumulation in vivo. Centrifugal membrane filtration removed aggregates more effectively than PD-10 columns. In HER2-positive mouse models, aggregate-free [¹⁷⁷Lu]Lu-DOTA-Trastuzumab achieved higher tumor accumulation and resulted in ∼65% tumor growth inhibition by day 21. These findings demonstrate that DLS is a sensitive and practical quality control tool. Given the greater aggregation risk in therapeutic radiolabeling, aggregate monitoring is especially critical for ensuring the safety, consistency, and effectiveness of radioimmunotherapy agents.

The development of synthetic transcription factors (TFs) that generate functional outputs in response to specific stimuli holds significant promise for modulating key cellular processes in both basic research and biomedical applications. Here, we rationally designed synthetic TFs bearing reversible modifications that mimic post-translational modifications regulatory mechanisms. By combining native chemical ligation (NCL) with palladium-mediated C-S cross-coupling, we synthesized a caged Max variant in which key residues (e.g., Lys31/57) were masked with o-nitroveratryloxycarbonyl groups. While the preparation of photoreactive proteins is generally incompatible with traditional NCL-desulfurization approaches, our strategy highlights the power of integrating total synthesis with late-stage transformations to access novel photoreactive proteins. Remarkably, whereas the engineered caged Max displayed a pronounced reduction in DNA-binding activity, potent binding to the enhancer box was rapidly restored upon site-selective unmasking of Lys31/57. The caged Max can be efficiently activated on-demand within minutes by simple in situ photolysis, enabling precise modulation of its DNA-binding activity. Our approach provides an effective means for producing and activating TF proteins, paving the way for light-responsive TF analogs with on-demand control across diverse applications.