Baricitinib is a Janus kinase (JAK) 1 and 2 inhibitor approved for treating rheumatoid arthritis (RA). The JAK/STAT system is essential in the intracellular signaling of different cytokines and in the activation process of the monocyte lineage. This study verifies the effects of baricitinib on STAT phosphorylation in monocytes of RA patients and evaluates the correlation between STAT phosphorylation and response to therapy. We evaluated the disease activity of patients (DAS28CRP) at baseline (T0) and after 4 and 12 weeks (T1–T3) of treatment with baricitinib, dividing them into responders (n = 7) and non-responders (n = 7) based on the reduction of DAS28CRP between T0 and T1 of at least 1.2 points. Through flow cytometry, STAT1 phosphorylation was analyzed at T0/T1/T3 in monocytes, at basal conditions and after IL2, IFNα, and IL6 stimulation. We showed that monocyte frequency decreased from T0 to T1 only in responders. Regarding the phosphorylation of STAT1, we observed a tendency for higher basal pSTAT1 in monocytes of non-responder patients and, after 4 weeks, a significant reduction of cytokine-induced pSTAT1 in monocytes of responders compared with non-responders. The single IFNα stimulation only partially recapitulated the differences in STAT1 phosphorylation between the two patient subgroups. Finally, responders showed an increased IFN signature at baseline compared with non-responders. These results may suggest that monocyte frequency and STAT1 phosphorylation in circulating monocytes could represent early markers of response to baricitinib therapy.

Baricitinib therapy response in rheumatoid arthritis patients associates to STAT1 phosphorylation in monocytes

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) neutralizing antibodies are shown to be effective therapeutics for providing coronavirus disease 2019 (COVID‐19) protection. However, recurrent variants arise and facilitate significant escape from current antibody therapeutics. Bispecific antibodies (bsAbs) represent a unique platform to increase antibody breadth and to reduce neutralization escape. Herein, a novel immunoglobulin G–variable domains of heavy‐chain‐only antibody (IgG–VHH) format bsAb derived from a potent human antibody R15‐F7 and a humanized nanobody P14‐F8‐35 are rationally engineered. The resulting bsAb SYZJ001 efficiently neutralizes wild‐type SARS‐CoV‐2 as well as the alpha, beta, gamma, and delta variants, with superior efficacy to its parental antibodies. Cryo‐electron microscopy structural analysis reveals that R15‐F7 and P14‐F8‐35 bind to nonoverlapping epitopes within the RBD and sterically hindered ACE2 receptor binding. Most importantly, SYZJ001 shows potent prophylactic and therapeutic efficacy against SARS‐CoV‐2 in three established mouse models. Collectively, the current results demonstrate that the novel bsAb format is feasible and effective, suggesting great potential as an inspiring antiviral strategy.

An Engineered IgG–VHH Bispecific Antibody against SARS‐CoV‐2 and Its Variants

Nanobodies with cross-neutralizing activity provide prominent therapeutic efficacy in mild and severe COVID-19 rodent models.

Simple Summary Understanding the effects of the tumor microenvironment is an essential step to advance treatments for cancer, but it is also one that is problematic to reproduce in vitro or study in vivo. When such approaches are difficult to implement, in silico methods are often able to help surmount these barriers. Network models of biological systems focus on the interactions of components (such as proteins or genes) and use available data about the parts of a system to predict emergent qualities. We focused our study on macrophages and how their environment affects their polarization. Thus, we built a Boolean control network model of the early response events going on in macrophages by collating information from a manual search of the literature that interprets the changes in the inner state of the cell. We used this model to simulate combinatorial treatment options that target multiple targets at the same time and have synergistic effects on macrophage polarization. Abstract Background: The function and polarization of macrophages has a significant impact on the outcome of many diseases. Targeting tumor-associated macrophages (TAMs) is among the greatest challenges to solve because of the low in vitro reproducibility of the heterogeneous tumor microenvironment (TME). To create a more comprehensive model and to understand the inner workings of the macrophage and its dependence on extracellular signals driving polarization, we propose an in silico approach. Methods: A Boolean control network was built based on systematic manual curation of the scientific literature to model the early response events of macrophages by connecting extracellular signals (input) with gene transcription (output). The network consists of 106 nodes, classified as 9 input, 75 inner and 22 output nodes, that are connected by 217 edges. The direction and polarity of edges were manually verified and only included in the model if the literature plainly supported these parameters. Single or combinatory inhibitions were simulated mimicking therapeutic interventions, and output patterns were analyzed to interpret changes in polarization and cell function. Results: We show that inhibiting a single target is inadequate to modify an established polarization, and that in combination therapy, inhibiting numerous targets with individually small effects is frequently required. Our findings show the importance of JAK1, JAK3 and STAT6, and to a lesser extent STK4, Sp1 and Tyk2, in establishing an M1-like pro-inflammatory polarization, and NFAT5 in creating an anti-inflammatory M2-like phenotype. Conclusions: Here, we demonstrate a protein–protein interaction (PPI) network modeling the intracellular signalization driving macrophage polarization, offering the possibility of therapeutic repolarization and demonstrating evidence for multi-target methods.

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Effective Reversal of Macrophage Polarization by Inhibitory Combinations Predicted by a Boolean Protein–Protein Interaction Model

The camelid-derived single chain antibody (sdAb), also termed VHH or nanobody, is a unique, functional heavy (H)-chain antibody (HCAb). In contrast to conventional antibodies, sdAb is a unique antibody fragment consisting of a heavy-chain variable domain. It lacks light chains and a first constant domain (CH1). With a small molecular weight of only 12~15 kDa, sdAb has a similar antigen-binding affinity to conventional Abs but a higher solubility, which exerts unique advantages for the recognition and binding of functional, versatile, target-specific antigen fragments. In recent decades, with their unique structural and functional features, nanobodies have been considered promising agents and alternatives to traditional monoclonal antibodies. As a new generation of nano-biological tools, natural and synthetic nanobodies have been used in many fields of biomedicine, including biomolecular materials, biological research, medical diagnosis and immune therapies. This article briefly overviews the biomolecular structure, biochemical properties, immune acquisition and phage library construction of nanobodies and comprehensively reviews their applications in medical research. It is expected that this review will provide a reference for the further exploration and unveiling of nanobody properties and function, as well as a bright future for the development of drugs and therapeutic methods based on nanobodies.

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Xiaochuan Xiong

Papers

An Engineered IgG–VHH Bispecific Antibody against SARS‐CoV‐2 and Its Variants

Tofacitinib enhances IGF1 via inhibiting STAT6 transcriptionally activated-miR-425-5p to ameliorate inflammation in RA-FLS