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Pan-T cell targeting by CD3-based T cell engagers has brought program-shift treatment and management of blood tumors. However, these modalities have been shown to provoke all types of T cells leading to cytokine storm syndrome, and activate Treg cells. Thus, modulating and potentiating the antitumor responses of a specific T cell subset was encouraged. We initially found that high purity of mucosa-associated invariant T (MAIT) cells could be expanded by the combination of plate-immobilized Vα7.2 mAb (Clone 3C10) and IL2 plus IL15. Then, we generated a novel anti-Vα7.2 TCR bsAb, Vα7.2 x PD-L1, to invoke the anti-tumor potency of these expanded MAIT cells. Furthermore, our data have demonstrated that Vα7.2 x PD-L1 could mediate the cell-to-cell conjunction between MAIT cell and tumor cell line, selectively elicit the activation, cytokine production, degranulation, and cytotoxicity of the expanded MAIT cells in the presence of target cell only. Collectively, this proof-of-concept study provides a new tool to explore the clinical potential of MAIT cells in fighting against PD-L1 positive solid tumors and suggests additional encouragement in designing novel T cell engagers targeting TCR alpha chain specific innate-like T cells subsets, other than pan CD3+ T cells. • We provide a simple protocol for expanding MAIT cells from human PBMCs. • We generate a Vα7.2 TCR-specific bsAb, Vα7.2 X PD-L1 on the Y-body platform. • Vα7.2 X PD-L1 redirects the expanded MAIT cells to kill PD-L1 positive tumor cells of various origins. [ABSTRACT FROM AUTHOR]

A key component of severe COVID-19 is a "cytokine storm" i.e., the excessive expression of unneeded cytokines. Previous studies suggest that SARS-CoV-2 proteins can induce macrophages to secrete pro-inflammatory cytokines; a process that may involve Toll-like receptors (TLRs). Glycogen synthase kinase-3 (GSK-3) has been implicated in TLR signal transduction and a selective GSK-3 inhibitor, termed COB-187, dramatically attenuates cytokine expression induced by the TLR ligand lipopolysaccharide (LPS). In the present study, we provide evidence that the SARS-CoV-2 spike protein (S) and the S2 subunit (S2) induce production of CXCL10 (a chemokine elevated in severe COVID-19) by a human macrophage cell line. Further, we report that two clinically relevant GSK-3 inhibitors and COB-187 attenuate S and S2 protein-induced CXCL10 production. Combined, our observations provide impetus for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19. • Cytokine storms, the overexpression of unneeded cytokines, are a key aspect of severe COVID-19. • CXCL10, a chemokine, has been shown to be elevated in severe COVID-19. • We provide evidence that the SARS-CoV-2 spike protein induces expression of CXCL10 by a human macrophage cell line. • Two clinically relevant GSK-3 inhibitors, and GSK-3 inhibitor COB-187, attenuate spike protein-induced CXCL10 expression. • These findings provide impetus for exploring GSK-3 inhibitors as potential therapeutics for severe COVID-19. [ABSTRACT FROM AUTHOR]

The interaction of the multifunctional cytokine interleukin (IL)-6 and its receptor (IL-6R) is involved in various diseases, including not only autoimmune diseases such as rheumatoid arthritis but also cancer and cytokine storms in coronavirus disease 2019 (COVID-19). In this study, systematic evolution of ligands by exponential enrichment (SELEX) against human IL-6R from mRNA-displayed unnatural peptide library ribosomally initiated and cyclized with m -(chloromethyl)benzoic acid (mClPh) incorporated by genetic code expansion (sense suppression) was performed using the PURE (Protein synthesis Using Recombinant Elements) system. A novel 13-mer unnatural mClPh-cyclized peptide that binds to the extracellular domain of IL-6R was discovered from an extremely diverse random peptide library. In vitro affinity maturation of IL-6R-binding unnatural mClPh-cyclized peptide from focused libraries was performed, identifying two IL-6R-binding unnatural mClPh-cyclized peptides by next-generation sequencing. Because cyclization can increase the protease resistance of peptides, novel IL-6R-binding mClPh-cyclized peptides discovered in this study have the potential to be used for a variety of research, therapeutic, and diagnostic applications involving IL-6/IL-6R signaling. • m-(Chloromethyl)benzoic acid was ribosomally incorporated by genetic code expansion. • mRNA-displayed peptide library was cyclized with m-(chloromethyl)benzoic acid. • Systematic Evolution of Ligands by EXponential enrichment (SELEX) was performed. • A novel IL-6R-binding unnatural thioether-cyclized peptide was discovered. • In vitro affinity maturation of IL-6R-binding unnatural cyclic peptide was performed. [ABSTRACT FROM AUTHOR]

Modifying T cells to attack tumors using engineered chimeric receptors display powerful new therapeutic capabilities. Unfortunately, the effectiveness of therapeutic T cells is limited due to the inherent T cell responses: certain facets of endogenous response programs may be toxic, and the ability to overcome the immunosuppression in TME is deficient. Here we developed a Notch receptor based synNotch T cell platform that is able to response to target tumor cells and selectively lead to CXCL10 production. Further study showed that the administration of synNotch T cells significantly inhibited the tumor growth in a humanized murine model, accompanied by the increased infiltration of CD3 + T cells and elevated level of CXCL10 and IFN-γ in the tumor site. A slightly increased level of CXCL10 and limited IFN-γ were found in the serum in mice received synNotch T cells, suggesting a high security of this treatment. Finally, we demonstrated that CXCL10 is sufficient and indispensable for the synNotch T cells induced anti-tumor effect. This study provided theoretical and experimental bases for the clinical implication of CXCL10 encoding synNotch T cells. • Generation of mesothelin sensing and CXCL10 producing SynNotch T cells. • SynNotch T cells suppress tumor growth in vivo. • SynNotch T cells promote infiltration of T cells in tumor site from treated mice. • CXCL10 is essential for anti-tumor effect without inducing systemic cytokine storm. • Biochemical and Biophysical Research Communications. [ABSTRACT FROM AUTHOR]

With-no-lysine kinase (WNK) plays important roles in regulating electrolyte homeostasis, cell signaling, survival, and proliferation. It has been recently demonstrated that WNK1, a member of the WNK family, modifies the function of immune cells. Here we report that in macrophages, WNK1 has suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses via TGFβ-activated kinase 1 (TAK1)-mediated activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway. We found that WNK1 heterozygous (WNK1 +/− ) mice produced excessive proinflammatory cytokines in an experimental LPS-induced sepsis model, and peritoneal macrophages isolated from WNK1 +/− mice produced higher levels of LPS-induced cytokines and NOS2 expression as canonical proinflammatory M1 macrophage markers. We confirmed that small hairpin RNA (shRNA)-mediated knockdown of WNK1 activated LPS-induced cytokine production and NOS2 expression in RAW 264.7 macrophages. Moreover, we demonstrated that WNK1 knockdown increased the nuclear translocation of NF-κB and activated the p38 and Jun N-terminal kinase (JNK) MAPK signaling pathway and that a TAK1 inhibitor diminished these effects of WNK1 knockdown. These results suggest that WNK1 acts as a physiologic immune modulator via interactions with TAK1. WNK1 may be a therapeutic target against the cytokine storm caused by sepsis. Image 1 • WNK1 is attracting attention as an important immune modulator in immune cells. • WNK1 +/− mice produce excessive proinflammatory cytokines in LPS-induced sepsis model. • WNK1 knockdown promotes TAK1-mediated inflammatory responses in macrophages. • WNK1 may be a therapeutic target against the cytokine storm caused by sepsis. [ABSTRACT FROM AUTHOR]

To detect potential risk of severe cytokine release syndrome, in vitro assay formats with human cells have been developed. The two major testing platforms are a combination of whole blood with aqueous-phase test articles (whole blood cytokine assay, WBCA) and peripheral blood mononuclear cells with solid-phase articles (PBMC assay). Significant induction of cytokines was seen in both assays after treatment with a widely used control agent, TGN1412 or its analog CD28SA, but the WBCA cytokine profile differed from what was expected from clinical experience. In the WBCA, potential risk of CD28SA was detected by elevation of IL-8 whereas IL-2, a key cytokine after stimulation of CD28, was not induced in approximately 40% of donor samples. Therefore, further mechanistic understanding of the different responses in the in vitro assay was needed. In this study of donor samples treated with CD28SA, we compared the induction of cytokines and identified the cytokine-producing cells in the two assays. IL-2 was markedly elevated in all the donors in the PBMC assay but only in 1 of 3 donors in the WBCA. IL-8, the most sensitive biomarker in the WBCA, was produced by monocytes and granulocytes. T cells, the most relevant player in the PBMC assay with CD28SA, did not contribute to the positive response seen in two donors in the WBCA, which suggests that different players caused the positive cytokine responses to CD28SA in the two assays. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies (mAbs) have important roles in treatments for various cancers and inflammatory diseases. Their highly target specificities provide controlled safety profiles. However, therapeutic mAbs commonly pose a risk of the induction of the release of cytokines, which may result in adverse events including infusion reaction and cytokine release syndrome. Several mechanisms are involved in the cytokine releases induced by therapeutic mAbs, and the activation of immune effector cells via Fcγ receptors (FcγRs) is one of the putative mechanisms for most IgG-subclass mAbs. The relationship between cytokine releases and mAbs' Fc functions is not fully understood. Here we developed a simple reporter cell-based assay for estimating the FcγR-mediated activation of human immune effector cells by mAbs. Our use of the cell-based assay to compare Fc-engineered mAbs with different FcγR-activation profiles revealed that the releases of inflammatory cytokines and chemokines from human peripheral blood mononuclear cells (hPBMCs) induced by the mAbs were elevated by treatment with Fc-engineered mAbs with higher FcγR-activation properties. Our results also suggested the involvement of monocytic effector cells in the activation of hPBMCs as sources of released cytokines and chemokines, which may lead to the immune cell-mediated adverse events. Our new reporter cell assay is a promising tool for evaluating and predicting the activation of human immune cells by novel Fc-engineered mAbs. [ABSTRACT FROM AUTHOR]