Tocilizumab, netakimab, and baricitinib in patients with mild-to-moderate COVID-19: An observational study.

OBJECTIVE: The aim of the study was to assess inflammatory markers and clinical outcomes in adult patients admitted to hospital with mild-to-moderate COVID-19 and treated with a combination of standard-of-care (SOC) and targeted immunosuppressive therapy including anti-IL-17A (netakimab), anti-IL-6R (tocilizumab), or JAK1/JAK2 inhibitor (baricitinib) or with a standard-of-care therapy alone. METHODS: The observational cohort study included 154 adults hospitalized between February and August, 2020 with RT-PCR-confirmed SARS-CoV-2 with National Early Warning Score2 (NEWS2) < 7 and C-reactive protein (CRP) levels ≤ 140 mg/L on the day of the start of the therapy or observation. Patients were divided into the following groups: I) 4 mg baricitinib, 1 or 2 times a day for an average of 5 days (n = 38); II) 120 mg netakimab, one dose (n = 48); III) 400 mg tocilizumab, one dose (n = 34), IV) SOC only: hydroxychloroquine, antiviral, antibacterial, anticoagulant, and dexamethasone (n = 34). RESULTS: CRP levels significantly decreased after 72 h in the tocilizumab (p = 1 x 10-5) and netakimab (p = 8 x 10-4) groups and remained low after 120 h. The effect was stronger with tocilizumab compared to other groups (p = 0.028). A significant decrease in lactate dehydrogenase (LDH) levels was observed 72 h after netakimab therapy (p = 0.029). NEWS2 scores significantly improved 72 h after tocilizumab (p = 6.8 x 10-5) and netakimab (p = 0.01) therapy, and 120 h after the start of tocilizumab (p = 8.6 x 10-5), netakimab (p = 0.001), or baricitinib (p = 4.6 x 10-4) therapy, but not in the SOC group. Blood neutrophil counts (p = 6.4 x 10-4) and neutrophil-to-lymphocyte ratios (p = 0.006) significantly increased 72 h after netakimab therapy and remained high after 120 h. The percentage of patients discharged 5-7 days after the start of therapy was higher in the tocilizumab (44.1%) and netakimab (41.7%) groups than in the baricitinib (31.6%) and SOC (23.5%) groups. Compared to SOC (3 of the 34; 8.8%), mortality was lower in netakimab (0 of the 48; 0%, RR = 0.1 (95% CI: 0.0054 to 1.91)), tocilizumab (0 of the 34; 0%, RR = 0.14 (95% CI: 0.0077 to 2.67)), and baricitinib (1 of the 38; 2.6%, RR = 0.3 (95% CI: 0.033 to 2.73)) groups. CONCLUSION: In hospitalized patients with mild-to-moderate COVID-19, the combination of SOC with anti-IL-17A or anti-IL-6R therapy were superior or comparable to the combination with JAK1/JAK2 inhibitor, and all three were superior to SOC alone. Whereas previous studies did not demonstrate significant benefit of anti-IL-17A therapy for severe COVID-19, our data suggest that such therapy could be a rational choice for mild-to-moderate disease, considering the generally high safety profile of IL-17A blockers. The significant increase in blood neutrophil count in the netakimab group may reflect efflux of neutrophils from inflamed tissues. We therefore hypothesize that neutrophil count and neutrophil-to-lymphocyte ratio could serve as markers of therapeutic efficiency for IL-17A-blocking antibodies in the context of active inflammation.

Pinpointing the tumor-specific T cells via TCR clusters.

Adoptive cell transfer (ACT) is a promising approach to cancer immunotherapy, but its efficiency fundamentally depends on the extent of tumor-specific T cell enrichment within the graft. This can be estimated via activation with identifiable neoantigens, tumor-associated antigens (TAAs), or living or lysed tumor cells, but these approaches remain laborious, time-consuming, and functionally limited, hampering clinical development of ACT. Here, we demonstrate that homology cluster analysis of T cell receptor (TCR) repertoires efficiently identifies tumor-reactive TCRs allowing to: (1) detect their presence within the pool of tumor-infiltrating lymphocytes (TILs); (2) optimize TIL culturing conditions, with IL-2low/IL-21/anti-PD-1 combination showing increased efficiency; (3) investigate surface marker-based enrichment for tumor-targeting T cells in freshly isolated TILs (enrichment confirmed for CD4+ and CD8+ PD-1+/CD39+ subsets), or re-stimulated TILs (informs on enrichment in 4-1BB-sorted cells). We believe that this approach to the rapid assessment of tumor-specific TCR enrichment should accelerate T cell therapy development.

Accounting for B-cell Behavior and Sampling Bias Predicts Anti-PD-L1 Response in Bladder Cancer.

Cancer immunotherapy is predominantly based on T cell-centric approaches. At the same time, the adaptive immune response in the tumor environment also includes clonally produced immunoglobulins and clonal effector/memory B cells that participate in antigen-specific decisions through their interactions with T cells. Here, we investigated the role of infiltrating B cells in bladder cancer via patient dataset analysis of intratumoral immunoglobulin repertoires. We showed that the IgG1/IgA ratio is a prognostic indicator for several subtypes of bladder cancer and for the whole IMVigor210 anti-PD-L1 immunotherapy study cohort. A high IgG1/IgA ratio associated with the prominence of a cytotoxic gene signature, T-cell receptor signaling, and IL21-mediated signaling. Immunoglobulin repertoire analysis indicated that effector B-cell function, rather than clonally produced antibodies, was involved in antitumor responses. From the T-cell side, we normalized a cytotoxic signature against the extent of immune cell infiltration to neutralize the artificial sampling-based variability in immune gene expression. Resulting metrics reflected proportion of cytotoxic cells among tumor-infiltrating immune cells and improved prediction of anti-PD-L1 responses. At the same time, the IgG1/IgA ratio remained an independent prognostic factor. Integration of the B-cell, natural killer cell, and T-cell signatures allowed for the most accurate prediction of anti-PD-L1 therapy responses. On the basis of these findings, we developed a predictor called PRedIctive MolecUlar Signature (PRIMUS), which outperformed PD-L1 expression scores and known gene signatures. Overall, PRIMUS allows for reliable identification of responders among patients with muscle-invasive urothelial carcinoma, including the subcohort with the low-infiltrated "desert" tumor phenotype.

Measuring Intratumoral Heterogeneity of Immune Repertoires.

There is considerable clinical and fundamental value in measuring the clonal heterogeneity of T and B cell expansions in tumors and tumor-associated lymphoid structures-along with the associated heterogeneity of the tumor neoantigen landscape-but such analyses remain challenging to perform. Here, we propose a straightforward approach to analyze the heterogeneity of immune repertoires between different tissue sections in a quantitative and controlled way, based on a beta-binomial noise model trained on control replicates obtained at the level of single-cell suspensions. This approach allows to identify local clonal expansions with high accuracy. We reveal in situ proliferation of clonal T cells in a mouse model of melanoma, and analyze heterogeneity of immunoglobulin repertoires between sections of a metastatically-infiltrated lymph node in human melanoma and primary human colon tumor. On the latter example, we demonstrate the importance of training the noise model on datasets with depth and content that is comparable to the samples being studied. Altogether, we describe here the crucial basic instrumentarium needed to facilitate proper experimental setup planning in the rapidly evolving field of intratumoral immune repertoires, from the wet lab to bioinformatics analysis.

Sol-gel derived boehmite nanostructures is a versatile nanoplatform for biomedical applications.

Alumina is one of the most promising carriers for drug delivery due to the long history of its usage as a vaccine adjuvant. Sol-gel synthesis provides excellent conditions for entrapment of biomolecules within an inorganic cage providing stabilization of proteins under the extremal conditions. In this paper, we show in vitro investigation of monodisperse alumina xerogel nanocontainers (AXNCs) using bovine serum albumin as a model protein entrapped in sol-gel alumina building blocks. Particularly, dose and cell-type dependent cytotoxicity in HeLa and A549 cancer cell lines were employed as well as investigation of antibacterial effect and stability of AXNCs in different biological media. It was shown, that the release of entrapped protein could be provided only in low pH buffer (as in cancer cell cytoplasm). This property could be applied for anticancer drug development. We also discovered boehmite nanoparticles effect on horizontal gene transfer and observed the appearance of antibiotic resistance by means of exchanging of the corresponding plasmid between two different E. coli strains. The present work may help to understand better the influence of AXNCs on various biological systems, such as prokaryotic and eukaryotic cells, and the activity of AXNCs in different biological media.

Towards DNA Nanomachines for Cancer Treatment: Achieving Selective and Efficient Cleavage of Folded RNA.

Despite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme-based DNA machine that can i) recognize the sequence of a cancer biomarker with high selectivity, ii) tightly bind a structured fragment of a housekeeping gene mRNA, and iii) cleave it with efficiency greater than that of a traditional DZ-based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.

Nanostructured Layer-by-Layer Polyelectrolyte Containers to Switch Biofilm Fluorescence.

The development of stimuli-responsive nanocontainers is an issue of utmost importance for many applications such as targeted drug delivery, regulation of the cell and tissue behavior, making bacteria have useful functions and here converting light. The present work shows a new contribution to the design of polyelectrolyte (PE) containers based on surface modified mesoporous titania particles with deposited Ag nanoparticles to achieve chemical light upconversion via biofilms. The PE shell allows slowing down the kinetics of a release of loaded l-arabinose and switching the bacteria luminescence in a certain time. The hybrid TiO2/Ag/PE containers activated at 980 nm (IR) illumination demonstrate 10 times faster release of l-arabinose as opposed to non-activated containers. Fast IR-released l-arabinose switch bacteria fluorescence which we monitor at 510 nm. The approach described herein can be used in many applications where the target and delayed switching and light upconversion are required.

LFA-1 integrin antibodies inhibit leukocyte α4ß1-mediated adhesion by intracellular signaling.

Binding of intercellular adhesion molecule-1 to the ß2-integrin leukocyte function associated antigen-1 (LFA-1) is known to induce cross-talk to the α4ß1 integrin. Using different LFA-1 monoclonal antibodies, we have been able to study the requirement and mechanism of action for the cross-talk in considerable detail. LFA-1-activating antibodies and those inhibitory antibodies that signal to α4ß1 induce phosphorylation of Thr-758 on the ß2-chain, which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the ß1-chain of α4ß1 and loss of binding to its ligand vascular cell adhesion molecule-1. The results show that with LFA-1 antibodies, we can activate LFA-1 and inhibit α4ß1, inhibit both LFA-1 and α4ß1, inhibit LFA-1 but not α4ß1, or not affect LFA-1 or α4ß1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications.