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

Foldamers of β-peptides : conformational preference of peptides formed by rigid building blocks : The first MI-IR spectra of a triamide nanosystem

To determine local chirality driven conformational preferences of small aminocyclobutane-1-carboxylic acid derivatives, X-(ACBA) n -Y, their matrix-isolation IR spectra were recorded and analyzed. For the very first time model systems of this kind were deposited in a frozen (~10 K) noble gas matrix to reduce line width and thus, the recorded sharp vibrational lines were analyzed in details. For cis-(S,R)-1 monomer two “zigzag” conformers composed of either a six or an eight-membered H-bonded pseudo ring was identified. For trans-(S,S)-2 stereoisomer a zigzag of an eight-membered pseudo ring and a helical building unit were determined. Both findings are fully consistent with our computational results, even though the relative conformational ratios were found to vary with respect to measurements. For the dimers (S,R,S,S)-3 and (S,S,S,R)-4 as many as four different cis,trans and three different trans,cis conformers were localized in their matrix-isolation IR (MI-IR) spectra. These foldamers not only agree with the previous computational and NMR results, but also unambiguously show for the first time the presence of a structure made of a cis,trans conformer which links a “zigzag” and a helical foldamer via a bifurcated H-bond. The present work underlines the importance of MI-IR spectroscopy, applied for the first time for triamides to analyze the conformational pool of small biomolecules. We have shown that the local chirality of a β-amino acid can fully control its backbone folding preferences. Unlike proteogenic α-peptides, β- and especially (ACBA) n type oligopeptides could thus be used to rationally design and influence foldamer’s structural preferences

Molecular Monitoring of RUNX1-RUNX1T1 Transcript Level in Acute Myeloblastic Leukemias on Treatment

Background. The current approach to treatment of acute myeloblastic leukemia (AML) includes the achievement of maximum tumor reduction and, therefore, eradication of a leukemic clone. The goal of the therapy is to achieve undetectable levels of the target gene, except an isolated molecular rearrangement of RUNX1-RUNX1T1. Aim. To estimate the dynamics of the RUNX1-RUNX1T1 level and relevant clinical manifestations during the monitoring of various stages of the program therapy and after its completion. Methods. The article presents a description of 10 cases of AML with isolated RUNX1-RUNX1T1 expression (n = 4) and the expression in combination with different molecular and cytogenetic abnormalities (n = 6). In addition, a long-term monitoring of the gene expression by quantitative determination of RUNX1-RUNX1T1 using a real-time PCR was presented. Results. The incidence of relapses in a group with a decreased RUNX1-RUNX1T1 expression level of >2 log is 75 % as compared to patients with a less significant reduction of the transcript level (with the relapse incidence equal to 0 %) (p = 0.05). The increase of the RUNX1-RUNX1T1 level against the background of bone marrow remission by more than 1 log coincided with a bone marrow relapse within 5–18 weeks. In addition, long-term persistence of a certain transcript level after the completion of a program therapy without relapse is possible. Conclusion. The study analyzed possible molecular background of different clinical outcomes of long-term persistence of the RUNX1-RUNX1T1 transcript that might lead to an individualized approach to AML patients