Manufacturing of CD19 Specific CAR T-Cells and Evaluation of their Functional Activity in Vitro

Background. The most promising variant of adoptive immunotherapy of the B-line oncohematological diseases includes the use of cells with the chimeric antigen receptor (CAR T-cells), that showed extraordinary results in clinical studies. Aim. To manufacture CAR T-cells for the clinical use and to study their cytotoxicity in vitro. Methods. Human T-lymphocytes were transduced by the lentiviral vector containing anti-CD19-CAR, RIAD, and GFP genes. The T-cell transduction efficacy was assessed on the basis of GFP protein signal by flow cytometry. Propidium iodide was used to analyse the cell viability. Cytotoxic activity of the manufactured CAR T-cells was studied in the presence of the target cells being directly co-cultivated. Analysis of the number and viability of CAR T-cells and cytokine expression was performed by flow cytometry. Results. The viability of the transduced T-cells and GFP expression reached 91.87 % and 50.87 % respectively. When cultured in the presence of IL-2 and recombinant CD19 (the target antigen), the amount of CAR-T after 120 h of the process was 1.4 times larger compared with the period of 48 h. In the cytotoxic test of co-cultivation CAR-T with the K562-CD19+ cells the percentage of CAR-T increased to 57 % and 84.5 % after 48 h and 120 h of exposure respectively. When cultured with the K562 cells (test line not expressing CD19) the number of CAR T-cells decreased to 36.2 % within 48 h while the number of K562 cells increased to 58.3 %. The viability of target cells in the experimental and control groups was 3.5 % and 36.74 % respectively. Comparison of IL-6 level in the control and experimental groups revealed that the differences are insignificant, as opposed to the level of other cytokines (IFN-γ, IL-2, TNF) which proved to be different in both groups. Conclusion. The present work resulted in the production of anti-CD19 CAR T-cells with adequate viability. The in vitro model demonstrated their cytotoxicity. Manufacturing of CAR T-cells for clinical use is the first step of the development of adoptive immunotherapy in the Russian Federation

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe