KDM5 Family Demethylase Inhibitor KDOAM-25 Reduces Entry of SARS-CoV-2 Pseudotyped Viral Particles into Cells.

We studied the effect of KDM5 family demethylase inhibitors (JIB-04, PBIT, and KDOAM-25) on the penetration of SARS-CoV-2 pseudotyped viruses into differentiated Caco-2 cells and HEK293T cells with ACE2 hyperexpression. The above drugs were not cytotoxic. Only KDOAM-25 significantly reduced virus entry into the cells. The expression of ACE2 mRNA in Caco-2 significantly increased, while TMPRSS2 expression did not significantly change under these conditions. In differentiated Caco-2 cells, KDOAM-25 did not affect the expression of BRCA1, CDH1, TP53, SNAI1, VIM, and UGCG genes, for which an association with knockdown or overexpression of KDM5 demethylases or with the action of demethylase inhibitors had previously been shown. In undifferentiated Caco-2 cells, the expression of BRCA1, SNAI1, VIM, and CDH1 was significantly increased under the action of KDOAM-25.

Barnase-barstar Specific Interaction Regulates Car-T Cells Cytotoxic Activity toward Malignancy.

The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.

Engineered Removal of PD-1 From the Surface of CD19 CAR-T Cells Results in Increased Activation and Diminished Survival.

CAR-T cell therapy is the most advanced way to treat therapy resistant hematologic cancers, in particular B cell lymphomas and leukemias, with high efficiency. Donor T cells equipped ex vivo with chimeric receptor recognize target tumor cells and kill them using lytic granules. CAR-T cells that recognize CD19 marker of B cells (CD19 CAR-T) are considered the gold standard of CAR-T therapy and are approved by FDA. But in some cases, CD19 CAR-T cell therapy fails due to immune suppressive microenvironment. It is shown that tumor cells upregulate expression of PD-L1 surface molecule that binds and increases level and signal provided by PD-1 receptor on the surface of therapeutic CAR-T cells. Induction of this negative signaling results in functional impairment of cytotoxic program in CAR-T cells. Multiple attempts were made to block PD-1 signaling by reducing binding or surface level of PD-1 in CAR-T cells by various means. In this study we co-expressed CD19-CAR with PD-1-specific VHH domain of anti-PD-1 nanobody to block PD-1/PD-L1 signaling in CD19 CAR-T cells. Unexpectedly, despite increased activation of CAR-T cells with low level of PD-1, these T cells had reduced survival and diminished cytotoxicity. Functional impairment caused by disrupted PD-1 signaling was accompanied by faster maturation and upregulation of exhaustion marker TIGIT in CAR-T cells. We conclude that PD-1 in addition to its direct negative effect on CAR-induced signaling is required for attenuation of strong stimulation leading to cell death and functional exhaustion. These observations suggest that PD-1 downregulation should not be considered as the way to improve the quality of therapeutic CAR-T cells.

Molecular Approaches to Safe and Controlled Engineered T-cell Therapy.

Chimeric antigen receptor-modified T-cell therapy (CAR-T therapy) is one of the fastest developing areas of immuno-oncology. Over the past decade, it has revolutionized the cell therapy modality and expedited its pace of development, from optimization of the structure of chimeric antigen receptors and animal model experiments to successful clinical application. The initial designs of the CAR configuration focused on increasing T-cell activation, cytotoxicity, and persistence. However, the first attempts to treat patients with CAR T cells have demonstrated the need for increased safety and controlled activation of genetically modified T cells. Herein, we summarize the different molecular approaches to engineering chimeric antigen receptors for reducing the potential clinical risks of T-cell therapy.

Directed Change in TNFα Specificity to Create DR5 Antagonists.

Death receptor 5 (DR5) is a promising target for antitumor therapy due to its high expression on different tumor cells. Resistance of various tumor cells against TRAIL, a natural ligand for the death receptors, reduces its therapeutic potential and prompts the search for novel agonists at these receptors. Previous screening across the combinatorial peptide library yielded a peptide sequence KVVLTHR that specifically binds DR5. Incorporation of this sequence into TNFα resulted in binding DR5 with mutant protein TNFα-mut and appearance of cytotoxicity against lymphoma cells.