Switchable targeting of solid tumors by BsCAR T cells.

The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2+ ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.

Clinical and Genetic Characteristics of Pediatric Patients with Hypophosphatasia in the Russian Population.

(1) Hypophosphatasia (HPP) is a rare inherited disease caused by mutations (pathogenic variants) in the ALPL gene which encodes tissue-nonspecific alkaline phosphatase (TNSALP). HPP is characterized by impaired bone mineral metabolism due to the low enzymatic activity of TNSALP. Knowledge about the structure of the gene and the features and functions of various ALPL gene variants, taking into account population specificity, gives an understanding of the hereditary nature of the disease, and contributes to the diagnosis, prevention, and treatment of the disease. The purpose of the study was to describe the spectrum and analyze the functional features of the ALPL gene variants, considering various HPP subtypes and clinical symptoms in Russian children. (2) From 2014−2021, the study included the blood samples obtained from 1612 patients with reduced alkaline phosphatase activity. The patients underwent an examination with an assessment of their clinical symptoms and biochemical levels of TNSALP. DNA was isolated from dried blood spots (DBSs) or blood from the patients to search for mutations in the exons of the ALPL gene using Sanger sequencing. The PCR products were sequenced using a reagent BigDye Terminator 3.1 kit (Applied Biosystems). Statistical analysis was performed using the GraphPad Prism 8.01 software. (3) The most common clinical symptoms in Russian patients with HPP and two of its variants (n = 22) were bone disorders (75%), hypomyotonia (50%), and respiratory failure (50%). The heterozygous carriage of the causal variants of the ALPL gene was detected in 225 patients. A total of 2 variants were found in 27 patients. In this group (n = 27), we identified 28 unique variants of the ALPL gene, of which 75.0% were missense, 17.9% were frameshift, 3.6% were splicing variants, and 3.6% were duplications. A total of 39.3% (11/28) of the variants were pathogenic, with two variants being probably pathogenic, and 15 variants had unknown clinical significance (VUS). Among the VUS group, 28.6% of the variants (7/28) were discovered by us for the first time. The most common variants were c.571G > A (p.Glu191Lys) and c.1171del (Arg391Valfs*12), with frequencies of 48.2% (13/28) and 11% (3/28), respectively. It was found that the frequency of nonsense variants of the ALPL gene was higher (p < 0.0001) in patients with the perinatal form compared to the infantile and childhood forms of HPP. Additionally, the number of homozygotes in patients with the perinatal form exceeded (p < 0.01) the frequencies of these genotypes in children with infantile and childhood forms of HPP. On the contrary, the frequencies of the compound-heterozygous and heterozygous genotypes were higher (p < 0.01) in patients with infantile childhood HPP than in perinatal HPP. In the perinatal form, residual TNSALP activity was lower (p < 0.0005) in comparison to the infantile and childhood (p < 0.05) forms of HPP. At the same time, patients with the heterozygous and compound-heterozygous genotypes (mainly missense variants) of the ALPL gene had greater residual activity (of the TNSALP protein) regarding those homozygous patients who were carriers of the nonsense variants (deletions and duplications) of the ALPL gene. Residual TNSALP activity was lower (p < 0.0001) in patients with pathogenic variants encoding the amino acids from the active site and the calcium and crown domains in comparison with the nonspecific region of the protein.

Next-Generation Sequencing-Based Monitoring of Intestinal Bacteria and Bacteriophages Following Fecal Microbiota Transplantation in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) and acute graft-versus-host disease (GVHD) are associated with persistent intestinal dysfunction preceded by gut bacterial dysbiosis. There are limited data on intestinal bacteriophages in these conditions. The aim of the present work was to detect associations between dominant intestinal bacteria by means of 16S rRNA gene sequencing, and some clinically significant viruses detected with a customized primer panel for NGS-based study. The clinical group included patients with Crohn's disease (IBD, n = 9), or GVHD (n = 6) subjected to fecal microbiota transplantation (FMT) from healthy donors. The stool specimens were taken initially, and 5 times post-FMT until day 120. Using NGS approach, we have found a higher abundance of Proteobacterota phylum in GVHD, especially, at later terms post-FMT. Moreover, we found an early increase of Klebsiella and E. coli/Shigella abundance in GVHD, along with decreased relative content of Faecalibacterium. Upon evaluation of intestinal phageome, the relative amount of Caudoviricetes class was higher in GVHD. A significant correlation was found between Proteobacteria and Caudoviricetes, thus suggesting their association during the post-FMT period. Moreover, the relative amounts of five Caudoviricetes phage species showed distinct correlations with Klebsiella and Enterococcus ratios at different terms of FMT. In conclusion, parallel use of 16S rRNA gene sequencing and targeted NGS viral panel is a feasible and useful option for tracing specific viral strains in fecal microbiota. The developed array of viral primers may be extended to detect other phages infecting the clinically relevant bacteria.

The Telomeric Repeats of HHV-6A Do Not Determine the Chromosome into Which the Virus Is Integrated.

Human herpes virus 6A (HHV-6A) is able to integrate into the telomeric and subtelomeric regions of human chromosomes representing chromosomally integrated HHV-6A (ciHHV-6A). The integration starts from the right direct repeat (DRR) region. It has been shown experimentally that perfect telomeric repeats (pTMR) in the DRR region are required for the integration, while the absence of the imperfect telomeric repeats (impTMR) only slightly reduces the frequency of HHV-6 integration cases. The aim of this study was to determine whether telomeric repeats within DRR may define the chromosome into which the HHV-6A integrates. We analysed 66 HHV-6A genomes obtained from public databases. Insertion and deletion patterns of DRR regions were examined. We also compared TMR within the herpes virus DRR and human chromosome sequences retrieved from the Telomere-to-Telomere consortium. Our results show that telomeric repeats in DRR in circulating and ciHHV-6A have an affinity for all human chromosomes studied and thus do not define a chromosome for integration.

Identification of Genetic Risk Factors of Severe COVID-19 Using Extensive Phenotypic Data: A Proof-of-Concept Study in a Cohort of Russian Patients.

The COVID-19 pandemic has drawn the attention of many researchers to the interaction between pathogen and host genomes. Over the last two years, numerous studies have been conducted to identify the genetic risk factors that predict COVID-19 severity and outcome. However, such an analysis might be complicated in cohorts of limited size and/or in case of limited breadth of genome coverage. In this work, we tried to circumvent these challenges by searching for candidate genes and genetic variants associated with a variety of quantitative and binary traits in a cohort of 840 COVID-19 patients from Russia. While we found no gene- or pathway-level associations with the disease severity and outcome, we discovered eleven independent candidate loci associated with quantitative traits in COVID-19 patients. Out of these, the most significant associations correspond to rs1651553 in MYH14p = 1.4 × 10-7), rs11243705 in SETX (p = 8.2 × 10-6), and rs16885 in ATXN1 (p = 1.3 × 10-5). One of the identified variants, rs33985936 in SCN11A, was successfully replicated in an independent study, and three of the variants were found to be associated with blood-related quantitative traits according to the UK Biobank data (rs33985936 in SCN11A, rs16885 in ATXN1, and rs4747194 in CDH23). Moreover, we show that a risk score based on these variants can predict the severity and outcome of hospitalization in our cohort of patients. Given these findings, we believe that our work may serve as proof-of-concept study demonstrating the utility of quantitative traits and extensive phenotyping for identification of genetic risk factors of severe COVID-19.

Analysis of the Spectrum of ACE2 Variation Suggests a Possible Influence of Rare and Common Variants on Susceptibility to COVID-19 and Severity of Outcome.

OBJECTIVES: In March 2020, the World Health Organization declared that an infectious respiratory disease caused by a new severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, causing coronavirus disease 2019 (COVID-19)] became a pandemic. In our study, we have analyzed a large publicly available dataset, the Genome Aggregation Database (gnomAD), as well as a cohort of 37 Russian patients with COVID-19 to assess the influence of different classes of genetic variants in the angiotensin-converting enzyme-2 (ACE2) gene on the susceptibility to COVID-19 and the severity of disease outcome. RESULTS: We demonstrate that the European populations slightly differ in alternative allele frequencies at the 2,754 variant sites in ACE2 identified in the gnomAD database. We find that the Southern European population has a lower frequency of missense variants and slightly higher frequency of regulatory variants. However, we found no statistical support for the significance of these differences. We also show that the Russian population is similar to other European populations when comparing the frequencies of the ACE2 variants. Evaluation of the effect of various classes of ACE2 variants on COVID-19 outcome in a cohort of Russian patients showed that common missense and regulatory variants do not explain the differences in disease severity. At the same time, we find several rare ACE2 variants (including rs146598386, rs73195521, rs755766792, and others) that are likely to affect the outcome of COVID-19. Our results demonstrate that the spectrum of genetic variants in ACE2 may partially explain the differences in severity of the COVID-19 outcome.

Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma.

We report the development of a novel platform to enhance the efficacy and safety of follicular lymphoma (FL) treatment. Since lymphoma is a clonal malignancy of a diversity system, every tumor has a different antibody on its cell surface. Combinatorial autocrine-based selection is used to rapidly identify specific ligands for these B cell receptors on the surface of FL tumor cells. The selected ligands are used in a chimeric antigen receptor T cell (CAR-T) format for redirection of human cytotoxic T lymphocytes. Essentially, the format is the inverse of the usual CAR-T protocol. Instead of being a guide molecule, the antibody itself is the target. Thus, these studies raise the possibility of personalized treatment of lymphomas using a private antibody binding ligand that can be obtained in a few weeks.