Novel bispecific nanobody mitigates experimental intestinal inflammation in mice by targeting TNF-α and IL-23p19 bioactivities.

BACKGROUND: Inflammatory bowel diseases (IBDs) pose significant challenges in terms of treatment non-response, necessitating the development of novel therapeutic approaches. Although biological medicines that target TNF-α (tumour necrosis factor-α) have shown clinical success in some IBD patients, a substantial proportion still fails to respond. METHODS: We designed bispecific nanobodies (BsNbs) with the ability to simultaneously target human macrophage-expressed membrane TNF-α (hmTNF-α) and IL-23. Additionally, we fused the constant region of human IgG1 Fc (hIgG1 Fc) to BsNb to create BsNb-Fc.  Our study encompassed in vitro and in vivo characterization of BsNb and BsNb-Fc. RESULTS: BsNb-Fc exhibited an improved serum half-life, targeting capability and effector function than BsNb. It's demonstrated that BsNb-Fc exhibited superior anti-inflammatory effects compared to the anti-TNF-α mAb (infliximab, IFX) combined with anti-IL-12/IL-23p40 mAb (ustekinumab, UST) by Transwell co-culture assays. Notably, in murine models of acute colitis brought on by 2,4,6-trinitrobenzene sulfonic acid（TNBS) and dextran sulphate sodium (DSS), BsNb-Fc effectively alleviated colitis severity. Additionally, BsNb-Fc outperformed the IFX&UST combination in TNBS-induced colitis, significantly reducing colon inflammation in mice with colitis produced by TNBS and DSS. CONCLUSION: These findings highlight an enhanced efficacy and improved biostability of BsNb-Fc, suggesting its potential as a promising therapeutic option for IBD patients with insufficient response to TNF-α inhibition. KEY POINTS: A bispecific nanobody (BsNb) was created to target TNF-α and IL-23p19, exhibiting high affinity and remarkable stability. BsNb-Fc inhibited the release of cytokines in CD4+T cells during co-culture experiments. BsNb-Fc effectively alleviated colitis severity in mouse model with acute colitis induced by DSS or TNBS, outperforming the IFX&UST combination.

A tailored series of engineered yeasts for the cell-dependent treatment of inflammatory bowel disease by rational butyrate supplementation.

Intestinal microbiota dysbiosis and metabolic disruption are considered essential characteristics in inflammatory bowel disorders (IBD). Reasonable butyrate supplementation can help patients regulate intestinal flora structure and promote mucosal repair. Here, to restore microbiota homeostasis and butyrate levels in the patient's intestines, we modified the genome of Saccharomyces cerevisiae to produce butyrate. We precisely regulated the relevant metabolic pathways to enable the yeast to produce sufficient butyrate in the intestine with uneven oxygen distribution. A series of engineered strains with different butyrate synthesis abilities was constructed to meet the needs of different patients, and the strongest can reach 1.8 g/L title of butyrate. Next, this series of strains was used to co-cultivate with gut microbiota collected from patients with mild-to-moderate ulcerative colitis. After receiving treatment with engineered strains, the gut microbiota and the butyrate content have been regulated to varying degrees depending on the synthetic ability of the strain. The abundance of probiotics such as Bifidobacterium and Lactobacillus increased, while the abundance of harmful bacteria like Candidatus Bacilloplasma decreased. Meanwhile, the series of butyrate-producing yeast significantly improved trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice by restoring butyrate content. Among the series of engineered yeasts, the strain with the second-highest butyrate synthesis ability showed the most significant regulatory and the best therapeutic effect on the gut microbiota from IBD patients and the colitis mouse model. This study confirmed the existence of a therapeutic window for IBD treatment by supplementing butyrate, and it is necessary to restore butyrate levels according to the actual situation of patients to restore intestinal flora.

Affinity maturation of antibody fragments: A review encompassing the development from random approaches to computational rational optimization.

Routinely screened antibody fragments usually require further in vitro maturation to achieve the desired biophysical properties. Blind in vitro strategies can produce improved ligands by introducing random mutations into the original sequences and selecting the resulting clones under more and more stringent conditions. Rational approaches exploit an alternative perspective that aims first at identifying the specific residues potentially involved in the control of biophysical mechanisms, such as affinity or stability, and then to evaluate what mutations could improve those characteristics. The understanding of the antigen-antibody interactions is instrumental to develop this process the reliability of which, consequently, strongly depends on the quality and completeness of the structural information. Recently, methods based on deep learning approaches critically improved the speed and accuracy of model building and are promising tools for accelerating the docking step. Here, we review the features of the available bioinformatic instruments and analyze the reports illustrating the result obtained with their application to optimize antibody fragments, and nanobodies in particular. Finally, the emerging trends and open questions are summarized.

Design of nanobody-based bispecific constructs by in silico affinity maturation and umbrella sampling simulations.

Random mutagenesis is the natural opportunity for proteins to evolve and biotechnologically it has been exploited to create diversity and identify variants with improved characteristics in the mutant pools. Rational mutagenesis based on biophysical assumptions and supported by computational power has been proposed as a faster and more predictable strategy to reach the same aim. In this work we confirm that substantial improvements in terms of both affinity and stability of nanobodies can be obtained by using combinations of algorithms, even for binders with already high affinity and elevated thermal stability. Furthermore, in silico approaches allowed the development of an optimized bispecific construct able to bind simultaneously the two clinically relevant antigens TNF-α and IL-23 and, by means of its enhanced avidity, to inhibit effectively the apoptosis of TNF-α-sensitive L929 cells. The results revealed that salt bridges, hydrogen bonds, aromatic-aromatic and cation-pi interactions had a critical role in increasing affinity. We provided a platform for the construction of high-affinity bispecific constructs based on nanobodies that can have relevant applications for the control of all those biological mechanisms in which more than a single antigen must be targeted to increase the treatment effectiveness and avoid resistance mechanisms.

Evolutionary Insights Into Microbiota Transplantation in Inflammatory Bowel Disease.

The intestinal microbiome plays an essential role in human health and disease status. So far, microbiota transplantation is considered a potential therapeutic approach for treating some chronic diseases, including inflammatory bowel disease (IBD). The diversity of gut microbiota is critical for maintaining resilience, and therefore, transplantation with numerous genetically diverse gut microbiota with metabolic flexibility and functional redundancy can effectively improve gut health than a single probiotic strain supplement. Studies have shown that natural fecal microbiota transplantation or washing microbiota transplantation can alleviate colitis and improve intestinal dysbiosis in IBD patients. However, unexpected adverse reactions caused by the complex and unclear composition of the flora limit its wider application. The evolving strain isolation technology and modifiable pre-existing strains are driving the development of microbiota transplantation. This review summarized the updating clinical and preclinical data of IBD treatments from fecal microbiota transplantation to washing microbiota transplantation, and then to artificial consortium transplantation. In addition, the factors considered for strain combination were reviewed. Furthermore, four types of artificial consortium transplant products were collected to analyze their combination and possible compatibility principles. The perspective on individualized microbiota transplantation was also discussed ultimately.

[Construction of five-level practical teaching system for bioengineering under Emerging Engineering Education background].

Strengthening practical teaching, together with improving innovation ability is one of the key tasks of Emerging Engineering Education. This paper is based on the revision of the training program of bioengineering in School of Chemical Engineering and Technology, Tianjin University, improved the practical teaching system and curriculum content, built a five-level teaching system for basic experiment, comprehensive experiment, course design, scientific research and practical training. In order to cultivate outstanding innovative talents with practical ability and innovative spirit, innovative teaching reform mode is proposed. Furthermore the new thought and new schemes for Emerging Engineering Education are put forward.