Bioconjugate Chemistry最新文献

One-bead-one-compound (OBOC) DNA-encoded library (DEL) enables high-throughput activity-based screening to find novel hits against pharmaceutical targets. Herein, we developed solid-phase and DNA-compatible conditions to implement the three-component Van Leusen reaction (imidazole formation) for OBOC DELs. High-throughput validation was conducted (employing every potential building block) to simultaneously provide the groundwork for potential library synthesis and demonstrate the reagent scope. A total of 251 aldehydes, 380 amines, and 19 tosylmethyl isocyanides (TosMICs) were validated, with 65 aldehydes, 194 amines, and 15 TosMICs meeting the 50% yield cutoff. We take particular interest in aliphatic TosMICs because they yield more drug-like products. Encouragingly, aliphatic TosMICs perform well in solid-phase, but not solution-phase.

Sulfatase 2 (Sulf-2), an extracellular sulfatase that modulates the sulfation pattern of heparan sulfate proteoglycans (HSPGs), serves as a critical biomarker for various pathological conditions. Monitoring Sulf-2 activity in living cell samples provides valuable insights for diagnostic applications and therapeutic evaluation of Sulf-2-related diseases. In this study, we developed a novel plasma-membrane-targeted fluorogenic probe, MAR-S, to visualize the activity of Sulf-2 secreted by living cells. Upon incubation with Sulf-2-containing culture supernatant from pancreatic cancer cells, MAR-S exhibited a significant increase in fluorescence at approximately 540 nm. Notably, MAR-S allowed for time-lapse monitoring of endogenous Sulf-2 activity in living cancer cells overexpressing Sulf-2, demonstrating its potential as a valuable tool for Sulf-2-related cancer diagnostics and therapeutic research.

Azanorbornadienes (ANDs) containing a bromovinyl sulfone are able to accept a first thiol and, in a further stage, fragment upon reaction with a second thiol. This fragmentation has been studied in a collection of differently substituted ANDs. The substitution pattern of the AND influences the rate of the first thiolation and, specially, the further fragmentation. N-pyramidalization of selected ANDs was demonstrated via X-ray diffraction. This structural feature attenuates the resonance effect of N-substituents in the further reactivity of ANDs. A comparison with related oxanorbornadienes is also reported. The installation of a fluorogenic AND onto a single domain Antibody against PD-L1 (PD-L1 sdAb) resulted in a conjugate capable of releasing the corresponding fluorogenic pyrrole in the presence of glutathione (GSH) under physiological conditions. Overall, these scaffolds demonstrate potential to be implemented as new drug delivery systems.

Herein, we describe an optimized method for the generation of “thiolated Q295” site-specific antibody-drug conjugates (ADCs) with drug-to-antibody ratio (DAR) 2 from nonengineered IgG1 antibodies. Traditional ADCs take advantage of the 4 intrachain disulfide residues as the sites of attachment. While operationally simple to prepare, ADCs that rely on attachment to these endogenous cysteine residues suffer from heterogeneity arising from stochastic mixtures of differently loaded species. Our team recently reported a site-specific thiolation method targeting the conserved Q295 residue in deglycosylated antibodies. This approach involves deglycosylation of Q297 (using PNGase F) to eliminate steric hindrance from the N-glycan, followed by introducing a thiol-containing small molecule, cysteamine, at Q295, using microbial transglutaminase (mTGase). Our original method employed a global reduction/reoxidation to liberate the Q295 thiol for conjugation. However, this process was challenging due to competing reoxidation of the newly introduced Q295 thiol. In order to overcome this issue, we systematically explored various reducing agents and conditions, ultimately resulting in a new process that avoids the need for reduction/reoxidation. This resin-supported method, which is suitable for high-throughput synthesis, relies on the selective reduction of the engineered disulfide by sterically hindered phosphine, monosulfonated triphenylphosphine (TPPMS). Relying on this optimized methodology, we studied a small set of tubulysin ADCs showing that the resulting Q295-conjugated ADCs have favorable biophysical and biological properties as compared to traditional stochastic conjugation.

Antibody–drug conjugates (ADCs) are an emerging class of molecules for cancer therapy. An ADC consists of an antibody that is attached to a toxic payload via a linker molecule. Once the ADC is internalized into the cancer cell, the payload is released inside the cell, which leads to tumor cell death. Most approved ADC molecules make use of enzymatically cleavable linker structures. The kinetics of antibody internalization, linker cleavage, and payload release are evident for the mode of action of ADCs in vitro and in vivo. We have previously described the generation of the tool molecule TORCH (Turn On after Release by Cathepsin) for studying ADC kinetics by analyzing increasing fluorescence. The molecular TORCH is a fluorophore–quencher molecule that is separated by a valin–citrullin (VC) linker. The VC linker is cleaved by the protease cathepsin B. We previously demonstrated the in vitro proof of principle with the molecular TORCH. These studies strongly facilitated ADC research and the analysis of internalization and release kinetics. Here, we show an improved design of the TORCH molecule, also named flexTORCH, overcoming challenges in synthesis, conjugation, and flexibility of design. The flexTORCH molecule enables modular and flexible assembling. For this, different TORCH linker–quencher modules and the fluorophore were equipped with functional groups for orthogonal click chemistry. This study shows the feasibility of flexTORCH synthesis, its stepwise conjugation to trastuzumab, and the in vitro proof of principle. For showcasing the flexibility of the flexTORCH, four different constructs were produced, including VC-PABC (para-aminobenzyl carbamate), ß-glucuronide-PABC, AAN-PABC, and AAN linker that represent recognition patterns for cathepsin B, ß-glucuronidase, and legumain.

The transmembrane integrin, very late antigen-4 (VLA-4), which is a critical integrin involved in promoting tumor progression, angiogenesis, and metastasis, is overexpressed in metastatic melanoma. The peptidomimetic LLP2A has a high binding affinity to VLA-4 and is used as a radiopharmaceutical targeting agent for imaging and therapy. Previous studies demonstrated that the albumin-binding compound, [¹⁷⁷Lu]Lu-DOTAGA-pIBA-PEG₄-LLP2A, significantly improved tumor retention and blood circulation time but resulted in lower tumor-to-nontumor tissue ratios compared to the nonalbumin-binding compound, [¹⁷⁷Lu]Lu-DOTAGA-PEG₄-LLP2A. To streamline the synthesis of VLA-4 targeting molecules as therapeutic agents and allow a modular approach, we investigated three click chemistry linkers for preparing DOTAGA-pIBA-PEG₄-LLP2A analogues: [¹⁷⁷Lu]Lu-DOTAGA-pIBA-TCO-tetrazine-PEG₄-LLP2A ([¹⁷⁷Lu]Lu-1), [¹⁷⁷Lu]Lu-DOTAGA-pIBA-BCN-azide-PEG₄-LLP2A ([¹⁷⁷Lu]Lu-2), and [¹⁷⁷Lu]Lu-DOTAGA-pIBA-DBCO-azide-PEG₄-LLP2A ([¹⁷⁷Lu]Lu-3). Determining the click linkage that provides optimal synthesis ease and pharmacokinetics will allow us to readily produce additional VLA-4 targeting radiopharmaceuticals. Saturation binding assays demonstrated high binding affinity of [¹⁷⁷Lu]Lu-1, [¹⁷⁷Lu]Lu-2, and [¹⁷⁷Lu]Lu-3 to VLA-4 in B16F10 cells, with K_d = 1.2 ± 0.2, 0.8 ± 0.4, and 1.6 ± 0.5 nM, respectively. Biodistribution of [¹⁷⁷Lu]Lu-1 showed peak tumor uptake at 24 h (12.2 ± 0.7%IA/g) and retention to 96 h (9.5 ± 1.7%IA/g), while [¹⁷⁷Lu]Lu-2 peaked at 48 h (13.5 ± 2.2%IA/g) and gradually decreased (9.93 ± 3.3%IA/g at 96 h). [¹⁷⁷Lu]Lu-3 peaked at 48 h (16.9 ± 3.9%IA/g) and was retained to 96 h (14.8 ± 3.8%IA/g). Compared with [¹⁷⁷Lu]Lu-1 and [¹⁷⁷Lu]Lu-3, [¹⁷⁷Lu]Lu-2 cleared more rapidly from normal tissues. [¹⁷⁷Lu]Lu-2 showed higher tumor-to-kidney ratios compared to [¹⁷⁷Lu]Lu-1 at all time points and higher tumor-to-liver ratios up to 96 h. [¹⁷⁷Lu]Lu-2 also showed higher tumor-to-liver ratios compared to [¹⁷⁷Lu]Lu-3 up to 48 h. The tumor can be clearly visualized with all compounds using SPECT/CT. The BCN click linkage ([¹⁷⁷Lu]Lu-2) will be applied in future compounds with other targeting ligands, radionuclides, albumin binders, and chelators.

Achieving a rapid image contrast is a critical attribute of successful imaging biomarkers of T-cell redirecting cancer immunotherapies, as even small shifts in the cluster of differentiation 8 (CD8) expressing T cell populations can be associated with meaningful therapeutic responses. However, T cell imaging agents, such as one-armed (OA) anti-CD8 monoclonal antibodies, are often limited by high renal uptake and poor resolution against the systemic blood signal. Herein we evaluate antibody pretargeting with and without the abrogation of binding to the neonatal Fc receptor (FcRn) as strategies to enhance the tumor contrast of OA antibodies targeting the minimally internalizing receptor, CD8. Single-photon emission computed tomography (SPECT) imaging of indium-111-labeled tracers in a solid CD8-expressing HPB-ALL tumor-bearing mouse model allowed the impact of FcRn binding to be assessed by both targeted and pretargeted imaging methods. We demonstrated that pretargeted imaging resulted in a higher tumor contrast within hours of tracer administration, irrespective of FcRn binding with reduced renal uptake relative to direct targeting. Abrogation of FcRn binding yielded a higher tumor contrast (relative to blood, kidney, and/or liver) at early time points (less than 2 h) for pretargeted imaging, but with reduced tumor enrichment and increased hepatic signal than in the presence of FcRn binding. Our findings demonstrate that pretargeted imaging with an OA anti-CD8 immunoSPECT tracer can overcome potential imaging liabilities associated with this molecule format, such as high renal uptake and poor resolution against the systemic pool.

The failure of chemotherapy to effectively target cancer cells is a major problem in cancer treatment. Herein, an acidic/near-infrared (NIR) dual-triggered drug release nanoplatform, AuNR/Apt-P@DOX, based on aptamer-functionalized gold nanorods (AuNRs) was reported for actively targeted combined chemo-photothermal therapy. The nanoplatform was prepared by functionalizing AuNRs with the PD-L1 aptamer (Apt-P) and loading DOX, which could be triggered to release under weak acidic conditions and NIR stimulation. Meanwhile, the chemotherapy effect coming from DOX and AuNRs played a vital role in photothermal therapy. The MTT results showed that the fatality rate of MCF-7 cancer cells was up to 80% under the targeting effect of Apt-P. Furthermore, the tumors in mice were almost completely cured under the combined action of photothermal and chemotherapy. This targeted combination therapeutic approach could offer novel insights into the advancement of antitumor strategies for clinical translation.

Fibroblast activation protein (FAP)-targeted radioligands have recently emerged as attractive tumor imaging agents. However, the therapeutic applicability of most FAP ligands has been impeded by their short tumor retention. In this study, a tetrazine (Tz)-modified FAPI derivant DOTA-FAPI-Tz was synthesized and radiolabeled with ¹⁷⁷Lu and ⁸⁹Zr to produce ⁸⁹Zr-FAPI-Tz and ¹⁷⁷Lu-FAPI-Tz with high radiochemical purity. Cellular uptake, internalization, efflux, and affinity experiments were performed using the U87MG-FAP cell line (glioma) to evaluate the in vitro FAP-targeting efficacy of the prepared radiotracers. In addition, micro-PET imaging, ex vivo biodistribution, and in vivo anticancer investigations were performed to evaluate the tumor-targeting ability, pharmacokinetic profile, and therapeutic effect of ⁸⁹Zr/¹⁷⁷Lu-FAPI-Tz. The results show that ⁸⁹Zr-FAPI-Tz and ¹⁷⁷Lu-FAPI-Tz demonstrate satisfactory in vitro stability, while ¹⁷⁷Lu-FAPI-Tz has a reduced hydrophilicity compared to ¹⁷⁷Lu-FAPI-04. Consistent with the performance of ¹⁷⁷Lu-FAPI-04, ¹⁷⁷Lu-FAPI-Tz presents rapid and specific FAP-targeting capability but a more significant time-dependent decrease in cellular retention. ⁸⁹Zr-FAPI-Tz and ¹⁷⁷Lu-FAPI-Tz alike display fast tumor localization, showing relatively low radioactivity accumulation in normal organs. Consequently, high-contrast PET images and favorable tumor-to-organ ratios can be obtained. Furthermore, ¹⁷⁷Lu-FAPI-Tz exhibits an effective antitumor efficacy and a satisfactory safety profile in U87MG-FAP tumor-bearing mice. In conclusion, ⁸⁹Zr-FAPI-Tz and ¹⁷⁷Lu-FAPI-Tz are a promising radiopharmaceutical pair for FAP-targeted theranostics, with remarkable tumor accumulation and a favorable pharmacokinetic profile.