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.

The ERK-cPLA2-ACSL4 axis mediating M2 macrophages ferroptosis impedes mucosal healing in ulcerative colitis.

Ulcerative colitis (UC) is a chronic gastrointestinal disease that can be managed with 5-aminosalicylic acid (5-ASA), the standard treatment for UC. However, the effectiveness of 5-ASA is not always optimal. Our study revealed that despite 5-ASA treatment, cells continued to experience excessive ferroptosis, which may hinder mucosal healing in UC and limit the success of this treatment approach in achieving disease remission. We found that combining 5-ASA with the ferroptosis inhibitor Fer-1 led to a significant inhibition of ferroptosis in macrophages present in the colon tissue, along with an increase in the proportion of M2 macrophages, suggesting that targeting ferroptosis in M2 macrophages could be a potential therapeutic strategy for alleviating UC. Our study also demonstrated that M2 macrophages are more susceptible to ferroptosis compared to M1 macrophages, and this susceptibility is associated with the activated arachidonic acid (AA) metabolism pathway mediated by ERK-cPLA2-ACSL4. Additionally, we found that the expression of cPLA2 gene pla2g4a was increased in the colon of UC patients compared to healthy controls. Furthermore, targeted metabolomics analysis revealed that the combination treatment group, as opposed to the 5-ASA treatment group, exhibited the ability to modulate AA metabolism. Overall, our findings emphasize the importance of addressing macrophage ferroptosis in order to enhance macrophage anti-inflammation, improve mucosal healing, and achieve better therapeutic outcomes for patients with UC.

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.

Ferroptosis: a promising candidate for exosome-mediated regulation in different diseases.

Ferroptosis is a newly discovered form of cell death that is featured in a wide range of diseases. Exosome therapy is a promising therapeutic option that has attracted much attention due to its low immunogenicity, low toxicity, and ability to penetrate biological barriers. In addition, emerging evidence indicates that exosomes possess the ability to modulate the progression of diverse diseases by regulating ferroptosis in damaged cells. Hence, the mechanism by which cell-derived and noncellular-derived exosomes target ferroptosis in different diseases through the system Xc-/GSH/GPX4 axis, NAD(P)H/FSP1/CoQ10 axis, iron metabolism pathway and lipid metabolism pathway associated with ferroptosis, as well as its applications in liver disease, neurological diseases, lung injury, heart injury, cancer and other diseases, are summarized here. Additionally, the role of exosome-regulated ferroptosis as an emerging repair mechanism for damaged tissues and cells is also discussed, and this is expected to be a promising treatment direction for various diseases in the future. Video Abstract.

The C-type lectin COLEC10 is predominantly produced by hepatic stellate cells and involved in the pathogenesis of liver fibrosis.

Hepatic stellate cell is one of the major nonparenchymal cell types in liver. It has been proved the hepatic stellate cells are activated upon liver injury and produce excessive extracellular matrix to induce liver fibrosis. Single-cell RNA sequencing has been introduced to identify the subpopulations and function of hepatic stellate cells for its remarkable resolution of representation of single-cell transcriptome. According to the re-analysis of single-cell RNA sequencing data and pseudotime trajectory inference, we have found the C-type lectins including Colec10 and Colec11 are not produced by hepatocytes but predominantly produced by hepatic stellate cells, especially quiescent ones in the mice livers. In addition, the expression of Colec10 is decreased in the fibrotic livers of CCl4-challenged mice. COLEC10 is also mainly expressed in the hepatic stellate cells of human livers and the expression of COLEC10 is decreased with the progression of liver fibrosis. The bulk RNA sequencing data of the lentivirus transfected LX-2 cells indicates the function of COLEC10 is associated with inflammation, angiogenesis and extracellular matrix alteration. Surprisingly, the in vitro overexpression of COLEC10 in LX-2 cells promotes the mRNA expression of extracellular matrix components including COL1A1, COL1A2 and COL3A1 and the extracellular matrix degradation enzyme MMP2. To further investigate the role of COLEC10 in the pathogenesis of liver fibrosis, the serum concentration of COLEC10 in patients with chronic liver disease and healthy donors is measured. The serum concentration of COLEC10 is elevated in the patients with chronic liver disease compared to the healthy donors and positively correlated with serum concentration of the D-dimer but not the most of liver function markers. Altogether, we conclude that the C-type lectin COLEC10 is predominantly produced by the hepatic stellate cells and involved in the pathogenesis of liver fibrosis.

Selective oxidative protection leads to tissue topological changes orchestrated by macrophage during ulcerative colitis.

Ulcerative colitis is a chronic inflammatory bowel disorder with cellular heterogeneity. To understand the composition and spatial changes of the ulcerative colitis ecosystem, here we use imaging mass cytometry and single-cell RNA sequencing to depict the single-cell landscape of the human colon ecosystem. We find tissue topological changes featured with macrophage disappearance reaction in the ulcerative colitis region, occurring only for tissue-resident macrophages. Reactive oxygen species levels are higher in the ulcerative colitis region, but reactive oxygen species scavenging enzyme SOD2 is barely detected in resident macrophages, resulting in distinct reactive oxygen species vulnerability for inflammatory macrophages and resident macrophages. Inflammatory macrophages replace resident macrophages and cause a spatial shift of TNF production during ulcerative colitis via a cytokine production network formed with T and B cells. Our study suggests components of a mechanism for the observed macrophage disappearance reaction of resident macrophages, providing mechanistic hints for macrophage disappearance reaction in other inflammation or infection situations.

Breaking through the therapeutic ceiling of inflammatory bowel disease: Dual-targeted therapies.

Emerging biologics and small-molecule drugs have changed the clinical status quo of inflammatory bowel disease (IBD). However, current treatments remain at a standstill in terms of response and remission in many cases. Accumulating evidence indicates that dual-targeted therapy (DTT) could be promising in overcoming the existing ceiling of IBD treatment. However, data on the efficacy and safety of DTT on Crohn's disease and ulcerative colitis are still limited or insufficient. Moreover, there is a lack of studies delineating the mechanisms of DTT. Given that various targeted drugs have different targets among the extensive redundant inflammatory networks, DTT could result in various outcomes. In this review, we have summarized the current data on the safety, effectiveness, and clinical development status of novel targeted drugs related to refractory IBD, and have explored the mechanism of action of therapy. We have categorized therapeutic agents into "Therapeutic Agents Targeting Cellular Signaling Pathways" and "Therapeutic Agents Targeting Leukocyte Trafficking" based on the different therapeutic targets, and also by classifying therapeutic agents targeting the cellular signaling pathways into "JAK-dependent" and "JAK-independent," and placed the existing drug combinations into 3 categories based on their mechanisms, namely, overlapping, synergistic, and complementary effects. Lastly, we have proposed the possible mechanisms of DTT to conceive a theoretical framework for clinical decision-making and further drug development and research from an IBD standpoint.

Correlation of Macrophages with Inflammatory Reaction in Ulcerative Colitis and Influence of Curcumin on Macrophage Chemotaxis.

Objective: Our study aimed to elucidate the correlation of macrophage (mø) with the inflammatory reaction in ulcerative colitis (UC) and the influence of curcumin (Cur) on mø chemotaxis in mice with UC. Methods: A total of 49 patients with UC (research group; RG) admitted between June 2020 and October 2021 and 56 healthy individuals (control group; CG) who visited concurrently were selected as the study participants. The peripheral blood mononuclear cells (PBMCs) were analyzed, and M1-type/M2-type mø and inflammatory factors (IFs) interleukin (IL)-1, IL-6, IL-10, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-ß) were detected. In addition, 15 BALB/c mice were purchased and divided into the normal group fed normally, the UC model group established with sodium dextran sulfate (DSS) and the Cur group induced by DSS + Cur feeding. Colon tissue mø was collected from mice to measure mø activity via CCK-8 and to quantify levels of IFs and chemokine CCL2 by polymer chain reaction (PCR)c and Western blotting. Results: The RG had a higher percentage of peripheral blood M1-type mø and a lower percentage of M2-type mø and M1/M2 mø ratio than the CG (P < .05). In the RG, IL-1, IL-6 and TNF-α all increased and were inversely correlated with the ratio of M1/M2 mø, while IL-10 and TGF-ß decreased, with a positive connection with the M1/M2 mø ratio. In the UC model mice, mø activity increased, but the apoptosis rate decreased. mø activity was lower in the Cur group than in the model and normal groups; mø apoptosis in the Cur group was higher than in the model group but lower than in the normal group. In addition, proIFs increased and anti-IFs decreased in the model group, and Cur also ameliorated this process. Finally, CCL2 and MCP-1 levels in the model group were also increased, while those in the Cur group were lower compared with the model group. Conclusion: In UC, the M1/M2 mø ratio is severely misadjusted, activation of M1-type mø is enhanced and pro-IFs are released in large quantities. Cur can ameliorate the abnormal activation of mø in mice with UC, inhibit mø chemotaxis and alleviate the inflammatory reaction, which may make it a new option for UC treatment in the future.

Genetic mutation and tumor microbiota determine heterogenicity of tumor immune signature: Evidence from gastric and colorectal synchronous cancers.

Both colorectal and gastric cancer are lethal solid-tumor malignancies, leading to the majority of cancer-associated deaths worldwide. Although colorectal cancer (CRC) and gastric cancer (GC) share many similarities, the prognosis and drug response of CRC and GC are different. However, determinants for such differences have not been elucidated. To avoid genetic background variance, we performed multi-omics analysis, including single-cell RNA sequencing, whole-exome sequencing, and microbiome sequencing, to dissect the tumor immune signature of synchronous primary tumors of GC and CRC. We found that cellular components of juxta-tumoral sites were quite similar, while tumoral cellular components were specific to the tumoral sites. In addition, the mutational landscape and microbiome contributed to the distinct TME cellular components. Overall, we found that different prognoses and drug responses of GC and CRC were mainly due to the distinct TME determined by mutational landscape and microbiome components.

Modeling spatial interaction networks of the gut microbiota.

How the gut microbiota is organized across space is postulated to influence microbial succession and its mutualistic relationships with the host. The lack of dynamic or perturbed abundance data poses considerable challenges for characterizing the spatial pattern of microbial interactions. We integrate allometric scaling theory, evolutionary game theory, and prey-predator theory into a unified framework under which quasi-dynamic microbial networks can be inferred from static abundance data. We illustrate that such networks can capture the full properties of microbial interactions, including causality, the sign of the causality, strength, and feedback loop, and are dynamically adaptive along spatial gradients, and context-specific, characterizing variability between individuals and within the same individual across time and space. We design and conduct a gut microbiota study to validate the model, characterizing key spatial determinants of the microbial differences between ulcerative colitis and healthy controls. Our model provides a sophisticated means of unraveling a complete atlas of how microbial interactions vary across space and quantifying causal relationships between such spatial variability and change in health state.

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.

Positive Interventional Effect of Engineered Butyrate-Producing Bacteria on Metabolic Disorders and Intestinal Flora Disruption in Obese Mice.

The substantially increased prevalence of obesity and obesity-related diseases has generated considerable concern. Currently, synthetic biological strategies have played an essential role in preventing and treating chronic diseases such as obesity. A growing number of symbiotic bacteria used as vectors for genetic engineering have been applied to create living therapeutics. In this study, using Bacillus subtilis as a cellular chassis, we constructed the engineered butyrate-producing strain BsS-RS06551 with a butyrate yield of 1.5 g/liter. A mouse model of obesity induced by a high-fat diet (HFD) was established to study the long-term intervention effects of this butyrate-producing bacteria on obesity. Combined with phenotypic assay results, we found that BsS-RS06551 could effectively retard body weight gain induced by a high-fat diet and visceral fat accumulation of mice, whereas it could improve glucose tolerance and insulin tolerance, reducing liver damage. We explored the BsS-RS06551 mechanism of action on host function and changes in intestinal flora by integrating multiple omics profiling, including untargeted metabolomics and metagenomics. The results showed that 24 major differential metabolites were involved in the metabolic regulation of BsS-RS06551 to prevent obesity in mice, including bile acid metabolism, branch chain amino acids, aromatic amino acids, and other metabolic pathways. Continuous ingestion of BsS-RS06551 could regulate gut microbiota composition and structure and enhance intestinal flora metabolic function abundance, which was closely related to host interactions. Our results demonstrated that engineered butyrate-producing bacteria had potential as an effective strategy to prevent obesity. IMPORTANCE Obesity is a chronic metabolic disease with an imbalance between energy intake and energy expenditure, and obesity-related metabolic diseases have become increasingly common. There is an urgent need to develop effective interventions for the prevention and treatment of obesity. This study showed that long-term consumption of BsS-RS06551 had a significant inhibitory effect on obesity induced by a high-fat diet and was more potent in inhibiting obesity than prebiotic inulin. In addition, this study showed a beneficial effect on host glucose, lipid metabolism, and gut microbe composition. Considering its colonization potential, this engineered bacteria provided a new strategy for the effective and convenient treatment of obesity in the long term.

Lactic Acid-Producing Probiotic Saccharomyces cerevisiae Attenuates Ulcerative Colitis via Suppressing Macrophage Pyroptosis and Modulating Gut Microbiota.

Lactic acid, a metabolic by-product of host and intestinal microbiota, has been recovered as an active signal molecule in the immune system. In this study, a lactic acid biosynthesis pathway that directly produces lactic acid from glucose rather than ethanol with high production was reconstructed in Saccharomyces cerevisiae. The engineered S. cerevisiae showed anti-inflammatory activity in dextran sulfate sodium (DSS)-induced mice with improved histological damage, increased mucosal barrier, and decreased intestinal immune response. Lactic acid regulated the macrophage polarization state and inhibited the expression of pro-inflammatory cytokines in vivo and in vitro. Increasing the macrophage monocarboxylic acid transporter-mediated active lactic acid uptake suppressed the excessive activation of the NLRP3 inflammasome and the downstream caspase-1 pathway in macrophages. Moreover, lactic acid promoted histone H3K9 acetylation and histone H3K18 lactylation. Meanwhile, the engineered S. cerevisiae altered the diversity and composition of the intestinal microbiota and changed the abundance of metabolic products in mice with colitis. In conclusion, this study shows that the application of engineered S. cerevisiae attenuated DSS-induced colitis in mice via suppressing macrophage pyroptosis and modulating the intestinal microbiota, which is an effective and safe treatment strategy for ulcerative colitis.

Prenatal Maternal Stress Exacerbates Experimental Colitis of Offspring in Adulthood.

The prevalence of inflammatory bowel disease (IBD) is increasing worldwide and correlates with dysregulated immune response because of gut microbiota dysbiosis. Some adverse early life events influence the establishment of the gut microbiota and act as risk factors for IBD. Prenatal maternal stress (PNMS) induces gut dysbiosis and perturbs the neuroimmune network of offspring. In this study, we aimed to investigate whether PNMS increases the susceptibility of offspring to colitis in adulthood. The related index was assessed during the weaning period and adulthood. We found that PNMS impaired the intestinal epithelial cell proliferation, goblet cell and Paneth cell differentiation, and mucosal barrier function in 3-week-old offspring. PNMS induced low-grade intestinal inflammation, but no signs of microscopic inflammatory changes were observed. Although there was no pronounced difference between the PNMS and control offspring in terms of their overall measures of alpha diversity for the gut microbiota, distinct microbial community changes characterized by increases in Desulfovibrio, Streptococcus, and Enterococcus and decreases in Bifidobacterium and Blautia were induced in the 3-week-old PNMS offspring. Notably, the overgrowth of Desulfovibrio persisted from the weaning period to adulthood, consistent with the results observed using fluorescence in situ hybridization in the colon mucosa. Mechanistically, the fecal microbiota transplantation experiment showed that the gut microbiota from the PNMS group impaired the intestinal barrier function and induced low-grade inflammation. The fecal bacterial solution from the PNMS group was more potent than that from the control group in inducing inflammation and gut barrier disruption in CaCo-2 cells. After treatment with a TNF-α inhibitor (adalimumab), no statistical difference in the indicators of inflammation and intestinal barrier function was observed between the two groups. Finally, exposure to PNMS remarkably increased the values of the histopathological parameters and the inflammatory cytokine production in a mouse model of experimental colitis in adulthood. These findings suggest that PNMS can inhibit intestinal development, impair the barrier function, and cause gut dysbiosis characterized by the persistent overgrowth of Desulfovibrio in the offspring, resulting in exacerbated experimental colitis in adulthood.

Nomogram to predict primary non-response to infliximab in patients with Crohn's disease: a multicenter study.

BACKGROUND: Infliximab (IFX) is effective at inducing and maintaining clinical remission and mucosal healing in patients with Crohn's disease (CD); however, 9%-40% of patients do not respond to primary IFX treatment. This study aimed to construct and validate nomograms to predict IFX response in CD patients. METHODS: A total of 343 patients diagnosed with CD who had received IFX induction from four tertiary centers between September 2008 and September 2019 were enrolled in this study and randomly classified into a training cohort (n = 240) and a validation cohort (n = 103). The primary outcome was primary non-response (PNR) and the secondary outcome was mucosal healing (MH). Nomograms were constructed from the training cohort using multivariate logistic regression. Performance of nomograms was evaluated by area under the receiver-operating characteristic curve (AUC) and calibration curve. The clinical usefulness of nomograms was evaluated by decision-curve analysis. RESULTS: The nomogram for PNR was developed based on four independent predictors: age, C-reactive protein (CRP) at week 2, body mass index, and non-stricturing, non-penetrating behavior (B1). AUC was 0.77 in the training cohort and 0.76 in the validation cohort. The nomogram for MH included four independent factors: baseline Crohn's Disease Endoscopic Index of Severity, CRP at week 2, B1, and disease duration. AUC was 0.79 and 0.72 in the training and validation cohorts, respectively. The two nomograms showed good calibration in both cohorts and were superior to single factors and an existing matrix model. The decision curve indicated the clinical usefulness of the PNR nomogram. CONCLUSIONS: We established and validated nomograms for the prediction of PNR to IFX and MH in CD patients. This graphical tool is easy to use and will assist physicians in therapeutic decision-making.

Bile Acid-Gut Microbiota Axis in Inflammatory Bowel Disease: From Bench to Bedside.

Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder of the gastrointestinal tract, with increasing prevalence, and its pathogenesis remains unclear. Accumulating evidence suggested that gut microbiota and bile acids play pivotal roles in intestinal homeostasis and inflammation. Patients with IBD exhibit decreased microbial diversity and abnormal microbial composition marked by the depletion of phylum Firmicutes (including bacteria involved in bile acid metabolism) and the enrichment of phylum Proteobacteria. Dysbiosis leads to blocked bile acid transformation. Thus, the concentration of primary and conjugated bile acids is elevated at the expense of secondary bile acids in IBD. In turn, bile acids could modulate the microbial community. Gut dysbiosis and disturbed bile acids impair the gut barrier and immunity. Several therapies, such as diets, probiotics, prebiotics, engineered bacteria, fecal microbiota transplantation and ursodeoxycholic acid, may alleviate IBD by restoring gut microbiota and bile acids. Thus, the bile acid-gut microbiota axis is closely connected with IBD pathogenesis. Regulation of this axis may be a novel option for treating IBD.

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment.

Recombinant antibodies such as nanobodies are progressively demonstrating to be a valid alternative to conventional monoclonal antibodies also for clinical applications. Furthermore, they do not solely represent a substitute for monoclonal antibodies but their unique features allow expanding the applications of biotherapeutics and changes the pattern of disease treatment. Nanobodies possess the double advantage of being small and simple to engineer. This combination has promoted extremely diversified approaches to design nanobody-based constructs suitable for particular applications. Both the format geometry possibilities and the functionalization strategies have been widely explored to provide macromolecules with better efficacy with respect to single nanobodies or their combination. Nanobody multimers and nanobody-derived reagents were developed to image and contrast several cancer diseases and have shown their effectiveness in animal models. Their capacity to block more independent signaling pathways simultaneously is considered a critical advantage to avoid tumor resistance, whereas the mass of these multimeric compounds still remains significantly smaller than that of an IgG, enabling deeper penetration in solid tumors. When applied to CAR-T cell therapy, nanobodies can effectively improve the specificity by targeting multiple epitopes and consequently reduce the side effects. This represents a great potential in treating malignant lymphomas, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma and solid tumors. Apart from cancer treatment, multispecific drugs and imaging reagents built with nanobody blocks have demonstrated their value also for detecting and tackling neurodegenerative, autoimmune, metabolic, and infectious diseases and as antidotes for toxins. In particular, multi-paratopic nanobody-based constructs have been developed recently as drugs for passive immunization against SARS-CoV-2 with the goal of impairing variant survival due to resistance to antibodies targeting single epitopes. Given the enormous research activity in the field, it can be expected that more and more multimeric nanobody molecules will undergo late clinical trials in the next future. Systematic Review Registration.

Therapeutic potential of bicyclol in liver diseases: Lessons from a synthetic drug based on herbal derivative in traditional Chinese medicine.

Bicyclol, an innovative chemical drug with proprietary intellectual property rights in China, is based on derivative of traditional Chinese medicine (TCM) Schisandra chinensis (Wuweizi) of North. Mounting data has proved that bicyclol has therapeutic potential in various pathological conditions in liver. In this narrative review, we provide the first summary of pharmacological activities, pharmacokinetic characteristics and toxicity of bicyclol, and discuss future research perspectives. Our results imply that bicyclol has a wide spectrum of pharmacological properties, including anti-viral, anti-inflammatory, immuno-regulatory, anti-oxidative, antisteatotic, anti-fibrotic, antitumor, cell death regulatory effects and modulation of heat shock proteins. Pharmacokinetic studies have indicated that bicyclol is the main substrate of CYP3A/2E1. Additionally, no obvious drug interactions have been found when bicyclol is administered simultaneously with other prescriptions. Furthermore, the results of chronic toxicity have strongly addressed that bicyclol has no noticeable toxic effects on all biochemical indices and pathological examinations of the main organs. In view of good pharmacological actions and safety, bicyclol is anticipated to be a potential candidate for various liver diseases, including acute liver injury, fulminant hepatitis, non-alcoholic fatty liver disease, fibrosis and hepatocellular carcinoma. Further studies are therefore required to delineate its molecular mechanisms and targets to confer this well-designed drug a far greater potency. We hope that bicyclol-based therapeutics for liver diseases might be broadly used in clinical practice worldwide.