Linker Design Principles for the Precision Targeting of Oncogenic G-Quadruplex DNA with G4-Ligand-Conjugated OligonucleotidesAbrahamsson, Berner, Golebiewska-Pikula
et alBioconjug Chem (2025)
Abstract: G-quadruplex (G4) DNA structures are noncanonical secondary structures found in key regulatory regions of the genome, including oncogenic promoters and telomeres. Small molecules, known as G4 ligands, capable of stabilizing G4s hold promise as chemical probes and therapeutic agents. Nevertheless, achieving precise specificity for individual G4 structures within the human genome remains a significant challenge. To address this, we expand upon G4-ligand-conjugated oligonucleotides (GL-Os), a modular platform combining the stabilizing properties of G4-ligands with the sequence specificity of guide DNA oligonucleotides. Central to this strategy is the linker that bridges the G4 ligand and the guide oligonucleotide. In this study, we develop multiple conjugation strategies for the GL-Os that enabled a systematic investigation of the linker in both chemical composition and length, enabling a thorough assessment of their impact on targeting oncogenic G4 DNA. Biophysical, biochemical, and computational evaluations revealed GL-Os with optimized linkers that exhibited enhanced binding to target G4s, even under thermal or structural stress. Notably, longer linkers broadened the range of targetable sequences without introducing steric hindrance, thereby enhancing the platform's applicability across diverse genomic contexts. These findings establish GL-Os as a robust and versatile tool for the selective targeting of individual G4s. By facilitating precise investigations of G4 biology, this work provides a foundation for advancing G4-targeted therapeutic strategies and exploring their role in disease contexts.
Development of highly bioactive long-acting recombinant porcine FSH for batch production management of sowsZhang, Shi, Zhong
et alSci Rep (2025) 15 (1), 4775
Abstract: To improve the economic benefits, exogenous hormones are used to control follicular development and synchronize ovulation in the batch flow management of gilts and weaned sows. Equine chorionic gonadotropin (eCG) is widely used to stimulate follicular development in both experimental and farm animals. Despite its effectiveness, several side effects have been found, including the occurrence of follicular cysts, follicular premature luteinization, and increased follicular atresia. As eCG is a heterologous protein, the generation of antibodies has been found in the superovulation of cattle. Moreover, the extraction of eCG from pregnant mare serum raises concerns regarding animal welfare, as well as potential risks for disease transmission. Follicle stimulating hormone (FSH) controls the follicular growth and maturation in the porcine ovary under physiological conditions. In the current study, we developed a novel long-acting recombinant porcine FSH (rpFSH-pFc) consisting of porcine FSH and porcine fragment crystallizable (Fc) via (G4S)3 linker by using the available protein fusion technology to control the follicular development and maturation. The results of pharmacokinetic studies indicated that rpFSH-pFc exhibited a prolonged half-life in both rats and sows. The efficacy of rpFSH-pFc was confirmed by cAMP level evaluation and germinal vesicle breakdown (GVBD) analysis in vitro. Through ovarian weight gain, superovulation and fertility testing assays, our results revealed that a single rpFSH-pFc treatment could effectively promote follicular development and maturation in vivo. Meanwhile, the mRNA expression levels of the target genes associated with follicular development, maturation and ovulation were significantly up-regulated after rpFSH-pFc treatment. Taken together, our results revealed that rpFSH-pFc could bind to the FSH receptor, stimulate follicular growth and development in female mice, and possess a prolonged half-life in both rats and sows. These characteristics suggest that rpFSH-pFc may be an ideal candidate for promoting follicular growth and development in livestock production.© 2025. The Author(s).
Discovery and investigation of the truncation of the (GGGGS)n linker and its effect on the productivity of bispecific antibodies expressed in mammalian cellsFang, Chen, Sun
et alBioprocess Biosyst Eng (2025) 48 (1), 159-170
Abstract: Protein engineering is a powerful tool for designing or modifying therapeutic proteins for enhanced efficacy, increased safety, reduced immunogenicity, and improved delivery. Fusion proteins are an important group of therapeutic compounds that often require an ideal linker to combine diverse domains to fulfill the desired function. GGGGS [(G4S)n] linkers are commonly used during the engineering of proteins because of their flexibility and resistance to proteases. However, unexpected truncation was observed in the linker of a bispecific antibody, which presented challenges in terms of production and quality. In this work, a bispecific antibody containing 5*G4S was investigated, and the truncation position of the linkers was confirmed. Our investigation revealed that codon optimization, which can overcome the negative influence of a high repetition rate and high GC content in the (G4S)n linker, may reduce the truncation rate from 5-10% to 1-5%. Moreover, the probability of truncation when a shortened 3* or 4*G4S linker was used was much lower than that when a 5*G4S linker was used in mammalian cells. In the case of expressing a bispecific antibody, the bioactivity and purity of the product containing a shorter G4S linker were further investigated and are discussed.© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Linker-specific monoclonal antibodies present a simple and reliable detection method for scFv-based CAR NK cellsSchindler, Ruppel, Müller
et alMol Ther Methods Clin Dev (2024) 32 (3), 101328
Abstract: Chimeric antigen receptor (CAR) T cell therapies have demonstrated significant successes in treating cancer. Currently, there are six approved CAR T cell products available on the market that target different malignancies of the B cell lineage. However, to overcome the limitations of CAR T cell therapies, other immune cells are being investigated for CAR-based cell therapies. CAR natural killer (NK) cells can be applied as allogeneic cell therapy, providing an economical, safe, and efficient alternative to autologous CAR T cells. To improve CAR research and future in-patient monitoring of cell therapeutics, a simple, reliable, and versatile CAR detection reagent is crucial. As most existing CARs contain a single-chain variable fragment (scFv) with either a Whitlow or a G4S linker site, linker-specific monoclonal antibodies (mAbs) can detect a broad range of CARs. This study demonstrates that these linker-specific mAbs can detect different CAR NK cells in vitro, spiked in whole blood, and within patient-derived tumor spheroids with high specificity and sensitivity, providing an effective and almost universal alternative for scFv-based CAR detection. Additionally, we confirm that linker-specific antibodies can be used for functional testing and enrichment of CAR NK cells, thereby providing a useful research tool to fast-track the development of novel CAR-based therapies.© 2024 The Author(s).
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