Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

Background A T cell-redirecting bispecific antibody consisting of a tumor-binding unit and a T cell-binding unit is a large group of antibody-based biologics against death-causing cancer diseases. The anti-CD38 × anti-CD3 bispecific antibody (Y150) is potential for treating multiple myeloma (MM). When developing a cell-based reporter gene bioassay to assess the activities of Y150, it was found that the expression of CD38 on the human T lymphocyte cells (Jurkat) caused the nonspecific activation which interfered with the specific T cells activation of mediated by the Y150 and CD38(+) tumor cells. Methods Here we first knocked-out the CD38 expression on Jurkat T cell line by CRISPR-Cas9 technology, then developed a stable monoclonal CD38(−) Jurkat T cell line with an NFAT-RE driving luciferase expressing system. Further based on the CD38(−) Jurkat cell, we developed a reporter gene method to assess the bioactivity of the anti-CD38 × anti-CD3 bsAb. Results Knocking out CD38 expression abolished the nonspecific self-activation of the Jurkat cells. The selected stable monoclonal CD38(−) Jurkat T cell line assured the robustness of the report genes assay for the anti-CD38 × anti-CD3 bsAb. The relative potencies of the Y150 measured by the developed reporter gene assay were correlated with those by the flow-cytometry-based cell cytotoxicity assay and by the ELISA-based binding assay. Conclusions The developed reporter gene assay was Mechanism of Action (MOA)-reflective for the bioactivity of anti-CD38 × anti-CD3 antibody, and suitable for the quality control for the bsAb product.

Perioperative Perfusion of Allografts with Anti-Human T-lymphocyte Globulin Does Not Improve Outcome Post Liver Transplantation—A Randomized Placebo-Controlled Trial

Due to the lack of suitable organs transplant surgeons have to accept unfavorable extended criteria donor (ECD) organs. Recently, we demonstrated that the perfusion of kidney organs with anti-human T-lymphocyte globulin (ATLG) prior to transplantation ameliorates ischemia-reperfusion injury (IRI). Here, we report on the results of perioperative ATLG perfusion in a randomized, single-blinded, placebo-controlled, feasibility trial (RCT) involving 30 liver recipients (LTx). Organs were randomly assigned for perfusion with ATLG/Grafalon® (AP) (n = 16) or saline only (control perfusion = CP) (n = 14) prior to implantation. The primary endpoint was defined as graft function reflected by aspartate transaminase (AST) values at day 7 post-transplantation (post-tx). With respect to the primary endpoint, no significant differences in AST levels were shown in the intervention group at day 7 (AP: 53.0 ± 21.3 mg/dL, CP: 59.7 ± 59.2 mg/dL, p = 0.686). Similarly, exploratory analysis of secondary clinical outcomes (e.g., patient survival) and treatment-specific adverse events revealed no differences between the study groups. Among liver transplant recipients, pre-operative organ perfusion with ATLG did not improve short-term outcomes, compared to those who received placebo perfusion. However, ATLG perfusion of liver grafts was proven to be a safe procedure without the occurrence of relevant adverse events.

Low Ozone Concentrations Affect the Structural and Functional Features of Jurkat T Cells

Autohemotherapy is the most used method to administer O2-O3 systemically. It consists in exposing a limited amount of blood to a gaseous O2-O3 and reinfusing it, thus activating a cascade of biochemical pathways involving plasma and blood cells that gives rise to antioxidant and anti-inflammatory responses. The therapeutic effects strictly depend on the O3 dose; it is therefore necessary to understand the relationship between the O3 concentration and the effects on blood cells involved in antioxidant and immune response. Here we performed a basic study on the effects of the low O3 concentrations used for autohemotherapy on the structural and functional features of the human T-lymphocyte-derived Jurkat cells. Ultrastructural, biomolecular, and bioanalytic techniques were used. Our findings showed that 10, 20, and 30 µg O3 concentrations were able to trigger Nrf2-induced antioxidant response and increase IL-2 secretion. However, viability and proliferation tests as well as ultrastructural observations revealed stress signs after treatment with 20 and 30 µg O3, thus designating 10 µg O3 as the optimal concentration in combining cell safety and efficient antioxidant and immune response in our in vitro system. These data offer novel evidence of the fine regulatory role played by the oxidative stress level in the hormetic response of T lymphocytes to O2-O3 administration.

Biological Properties of New Chiral 2-Methyl-5,6,7,8-tetrahydroquinolin-8-amine-based Compounds

The synthesis of a small library of 8-substituted 2-methyl-5,6,7,8-tetrahydroquinoline derivatives is presented. All the compounds were tested for their antiproliferative activity in non-cancer human dermal microvascular endothelial cells (HMEC-1) and cancer cells: human T-lymphocyte cells (CEM), human cervix carcinoma cells (HeLa), human dermal microvascular endothelial cells (HMEC-1), colorectal adenocarcinoma (HT-29), ovarian carcinoma (A2780), and biphasic mesothelioma (MSTO-211H). Compounds 3a, 5a, and 2b, showing significant IC50 values against the whole panel of the selected cells, were further synthesized and tested as pure enantiomers in order to shed light on how their stereochemistry might impact on the related biological effect. The most active compound (R)-5a was able to affect cell cycle phases and to induce mitochondrial membrane depolarization and cellular ROS production in A2780 cells.

Ligand-Receptor Interactions of Galectin-9 and VISTA Suppress Human T Lymphocyte Cytotoxic Activity

Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.

Characterization of extracellular vesicles and artificial nanoparticles with four orthogonal single-particle analysis platforms

ABSTRACTWe compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). Results were compared with standard EV characterization techniques such as transmission electron microscopy (TEM) and Western blot (WB). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized per MISEV2018 guidelines. Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode and NFCM were able to detect at least two markers on the same particle. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.