T cell-redirecting antibody for treatment of solid tumors via targeting mesothelin.

T cell engaging bispecific antibodies (TCBs) have recently become significant in cancer treatment. In this study we developed MSLN490, a novel TCB designed to target mesothelin (MSLN), a glycosylphosphatidylinositol (GPI)-linked glycoprotein highly expressed in various cancers, and evaluated its efficacy against solid tumors. CDR walking and phage display techniques were used to improve affinity of the parental antibody M912, resulting in a pool of antibodies with different affinities to MSLN. From this pool, various bispecific antibodies (BsAbs) were assembled. Notably, MSLN490 with its IgG-[L]-scFv structure displayed remarkable anti-tumor activity against MSLN-expressing tumors (EC50: 0.16 pM in HT-29-hMSLN cells). Furthermore, MSLN490 remained effective even in the presence of non-membrane-anchored MSLN (soluble MSLN). Moreover, the anti-tumor activity of MSLN490 was enhanced when combined with either Atezolizumab or TAA × CD28 BsAbs. Notably, a synergistic effect was observed between MSLN490 and paclitaxel, as paclitaxel disrupted the immunosuppressive microenvironment within solid tumors, enhancing immune cells infiltration and improved anti-tumor efficacy. Overall, MSLN490 exhibits robust anti-tumor activity, resilience to soluble MSLN interference, and enhanced anti-tumor effects when combined with other therapies, offering a promising future for the treatment of a variety of solid tumors. This study provides a strong foundation for further exploration of MSLN490's clinical potential.

Long-term passaging of pseudo-typed SARS-CoV-2 reveals the breadth of monoclonal and bispecific antibody cocktails.

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses challenges to the effectiveness of neutralizing antibodies. Rational design of antibody cocktails is a realizable approach addressing viral immune evasion. However, evaluating the breadth of antibody cocktails is essential for understanding the development potential. Here, based on a replication competent vesicular stomatitis virus model that incorporates the spike of SARS-CoV-2 (VSV-SARS-CoV-2), we evaluated the breadth of a number of antibody cocktails consisting of monoclonal antibodies and bispecific antibodies by long-term passaging the virus in the presence of the cocktails. Results from over two-month passaging of the virus showed that 9E12 + 10D4 + 2G1 and 7B9-9D11 + 2G1 from these cocktails were highly resistant to random mutation, and there was no breakthrough after 30 rounds of passaging. As a control, antibody REGN10933 was broken through in the third passage. Next generation sequencing was performed and several critical mutations related to viral evasion were identified. These mutations caused a decrease in neutralization efficiency, but the reduced replication rate and ACE2 susceptibility of the mutant virus suggested that they might not have the potential to become epidemic strains. The 9E12 + 10D4 + 2G1 and 7B9-9D11 + 2G1 cocktails that picked from the VSV-SARS-CoV-2 system efficiently neutralized all current variants of concern and variants of interest including the most recent variants Delta and Omicron, as well as SARS-CoV-1. Our results highlight the feasibility of using the VSV-SARS-CoV-2 system to develop SARS-CoV-2 antibody cocktails and provide a reference for the clinical selection of therapeutic strategies to address the mutational escape of SARS-CoV-2.

Identification of Sirtuin 3, a mitochondrial protein deacetylase, as a new contributor to tamoxifen resistance in breast cancer cells.

The current study reports a previously unappreciated role of Sirtuin 3 (SIRT3), a mitochondrial protein deacetylase, in altering sensitivity of breast cancer cells to tamoxifen (Tam), a commonly used anti-estrogen agent. We showed that SIRT3 was significantly up-regulated at both mRNA and protein levels in the Tam-resistance human breast cancer cell line MTR-3, which was derived from MCF-7 line by continuous selective culture in the presence of 1µM of Tam for two years. We further demonstrated that SIRT3 was rapidly up-regulated in the sensitive MCF-7 cells following exposure to Tam. Transfection of MCF-7 cells with a SIRT3 expression plasmid decreased cellular sensitivity to Tam and blocked the Tam-induced apoptosis. Furthermore, silencing of SIRT3 expression in MTR-3 cells sensitized the resistant cells to Tam and enhanced apoptotic cell death. MTR-3 cells with silencing of SIRT3 expression showed increases in the mitochondrial content of ERß, ROS level and apoptosis. These results not only uncovered a new role for SIRT3 in cancer but also identified this mitochondrial protein deacetylase as a previously unrecognized factor that participates in regulation of Tam sensitivity in breast cancer cells. Thus, SIRT3 might be considered as a potential target for overcoming Tam resistance in treatment of breast cancer.

Immune mechanisms of Concanavalin A model of autoimmune hepatitis.

As a chronic inflammatory disease of the liver, the pathogenic mechanisms of autoimmune hepatitis (AIH) have not yet been elucidated, with prognosis and diagnosis remaining unsatisfied. Currently the only viable treatments of AIH are immunosuppressant application and liver transplantation. It is considered that lack of good animal AIH models is the main reason for the shortage of a simple and efficient cure. The Concanavalin A (Con A) model is a typical and well established model for investigating T-cell and macrophage dependent liver injury in mice, which closely mimics the pathogenesis mechanisms and pathological changes of patients, and is regarded as the best experimental model for AIH research so far. In this paper we elucidated the pathogenic mechanisms of AIH and the evolution of relative animal models. We go on to further focus on Con A-induced liver injury from the point of immunological mechanisms and the change of cytokine levels. Finally, we manifested the clinical significance of the AIH animal models and the challenges they would meet during their future development.

[Differential expression of MAPK-pathway genes during liver regeneration of mouse.].

To investigate the different expression profiles of MAPK pathway genes and their corresponding functions during liver regeneration, we used a CCl4 induced mouse liver regeneration model in this study. Mouse was injected with CCl4 in the abdominal cavity to cause damage in the liver and followed by liver histology examination and measurement of serum ALT levels in blood sample collected at 0, 0.5, 1.5, 4.5, and 7 d after CCl4 injection. Differentially expressed genes in the MAPK pathway during liver regeneration were analyzed using mouse cDNA microarray method (Affymetrix). The results obtained were further subjected to hierarchical clustering study and were validated with real-time PCR. Microarray hybridization identified 31 out of the 93 MAPK pathway component genes, which have significantly altered their expression levels during liver regeneration. Among them, both up- and down-regulated genes were classified into various groups according to clustering studies and functional analysis. At the initial stage of liver regeneration, the number of up-regulated genes was greater than the down-regulated genes, while at the late stage the situation was reversed. Our results suggest that MAPK pathway might play different regulatory roles in responding to different stages of liver regeneration.

Protein-protein interaction map is a key gateway into liver regeneration.

Recent studies indicate that the process of liver regeneration involves multiple signaling pathways and a variety of genes, cytokines and growth factors. Protein-protein interactions (PPIs) play a role in nearly all events that take place within the cell and PPI maps should be helpful in further understanding the process of liver regeneration. In this review, we discuss recent progress in understanding the PPIs that occur during liver regeneration especially those in the transforming growth factor beta signaling pathways. We believe the use of large-scale PPI maps for integrating the information already known about the liver regeneration is a useful approach in understanding liver regeneration from the standpoint of systems biology.

Protective effect of recombinant human IL-1Ra on CCl4-induced acute liver injury in mice.

AIM: To evaluate the effects of positive regulation of recombinant human interleukin 1 receptor antagonist (rhIL-1Ra) on hepatic tissue recovery in acute liver injury in mice induced by carbon tetrachloride (CCl(4)). METHODS: Acute liver damage was induced by injecting 8-wk-old mice with CCl(4) 1 mL/kg (1:3 dilution in corn oil) intraperitoneally (ip). Survival after liver failure was assessed by injecting 8-wk-old mice with a lethal dose of CCl(4) 2.6 mL/kg (1:1 dilution in corn oil) ip. Mice were subcutaneously injected with 1 mg/kg recombinant human IL-1Ra twice a day after CCl(4) treatment for 5 d. Serum alanine amino transferase (ALT) and aspartate aminotransferase (AST) levels were determined with a commercial assay kit. Serum IL-1beta, IL-1Ra levels were measured by enzyme-linked immunosorbent assay kit. Quantitative real-time polymerase chain reaction was used to determine liver IL-1beta, IL-1Ra and IL-6 expression during CCl(4)-induced acute liver injury. Liver sections were stained with hematoxylin-eosin. A histology-injury grading system was used to evaluate the degree of necrosis after acute liver injury. Proliferating cell nuclear antigen (PCNA) staining was used to evaluate the role of rhIL-1Ra in promoting hepatocyte proliferation. RESULTS: Quantitative analysis showed a higher level of IL-6 mRNA expression and reduced serum AST and ALT levels in the livers of the rhIL-1Ra-treated group at the early phase of CCl(4)-induced acute liver injury. Histological examination indicated a decrease in centrilobular necrotic areas in mice treated with rhIL-1Ra, and a novel role of rhIL-1Ra in promoting hepatocyte proliferation was also supported by an increase of PCNA staining. All these results, accompanied by a strong survival benefit in rhIL-1Ra-treated vs PBS-treated groups, demonstrated that rhIL-1Ra administration ameliorated the histological damage and accelerated the regeneration and recovery process of the liver. CONCLUSION: rhIL-1Ra could be further developed as a novel therapeutic agent for the treatment of acute liver injury because of its ability to reduce hepatocellular damage and facilitate liver regeneration.

A protein-protein interaction network of transcription factors acting during liver cell proliferation.

Liver regeneration is a complex process that involves a multitude of cellular functions, including primarily cell proliferation, apoptosis, inflammation, and metabolism. A number of signaling pathways that control these processes have been identified, and cross communication between them by direct protein-protein interactions has been shown to be crucial in orchestrating liver regeneration. Previously, we have identified a group of transcription factors capable of regulating liver cell growth and that may be involved in liver cancer development. The expression of some of their mouse counterpart genes was altered dramatically after liver injury and regeneration induced by CCl(4) in mice. In an effort to elucidate the molecular basis for liver regeneration through protein-protein interactions (PPI), a matrix mating Y2H approach was produced to generate a PPI network between a set of 32 regulatory proteins. Sixty-four interactions were identified, including 4 that had been identified previously. Ten of the interactions were further confirmed with GST pull-down and coimmunoprecipitation assays. Information provided by this PPI network may shed further light on the molecular mechanisms that regulate liver regeneration at the protein interaction level and ultimately identify regulatory factors that may serve as candidate drug targets for the treatment of liver diseases.