Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells

Reprogramming of NK cells with a chimeric antigen receptor (CAR) proved an effective strategy to increase NK cell reactivity and recognition specificity toward tumor cells. To enhance the cytotoxicity of NK cells against CD138-positive multiple myeloma (MM) cells, we generated genetically modified NK-92MI cells carrying a CAR that consists of an anti-CD138 single-chain variable fragment (scFv) fused to the CD3ζ chain as a signaling moiety. The genetic modification through a lentiviral vector did not affect the intrinsic cytolytic activity of NK-92MI toward human erythroleukemic cell line K562 cells or CD138-negative targets. However, these retargeted NK-92MI (NK-92MI-scFv) displayed markedly enhanced cytotoxicity against CD138-positive human MM cell lines (RPMI8226, U266 and NCI-H929) and primary MM cells at various effector-to-target ratios (E:T) as compared to the empty vector-transfected NK-92MI (NK-92MI-mock). In line with the enhanced cytotoxicity of NK-92MI-scFv, significant elevations in the secretion of granzyme B, interferon-γ and proportion of CD107a expression were also found in NK-92MI-scFv in response to CD138-positive targets compared with NK-92MI-mock. Most importantly, the enhancement in the cytotoxicity of NK-92MI-scFv did not attenuate with 10Gy-irradiation that sufficiently blocked cell proliferation. Moreover, the irradiated NK-92MI-scFv exerted definitely intensified anti-tumor activity toward CD138-positive MM cells than NK-92MI-mock in the xenograft NOD-SCID mouse model. This study provides the rationale and feasibility for adoptive immunotherapy with CD138-specific CAR-modified NK cells in CD138-positive plasmacytic malignancies, which potentially further improves remission quality and prolongs the remission duration of patients with MM after upfront chemotherapy.     

Development of a novel, physiologically relevant cytotoxicity model: Application to the study of chemotherapeutic damage to mesenchymal stromal cells

There is an increasing need for development of physiologically relevant in-vitro models for testing toxicity, however determining toxic effects of agents which undergo extensive hepatic metabolism can be particularly challenging. If a source of such metabolic enzymes is inadequate within a model system, toxicity from prodrugs may be grossly underestimated. Conversely, the vast majority of agents are detoxified by the liver, consequently toxicity from such agents may be overestimated.In this study we describe the development of a novel in-vitro model, which could be adapted for any toxicology setting. The model utilises HepG2 liver spheroids as a source of metabolic enzymes, which have been shown to more closely resemble human liver than traditional monolayer cultures. A co-culture model has been developed enabling the effect of any metabolised agent on another cell type to be assessed. This has been optimised to enable the study of damaging effects of chemotherapy on mesenchymal stem cells (MSC), the supportive stem cells of the bone marrow. Several optimisation steps were undertaken, including determining optimal culture conditions, confirmation of hepatic P450 enzyme activity and ensuring physiologically relevant doses of chemotherapeutic agents were appropriate for use within the model. The developed model was subsequently validated using several chemotherapeutic agents, both prodrugs and active drugs, with resulting MSC damage closely resembling effects seen in patients following chemotherapy.Minimal modifications would enable this novel co-culture model to be utilised as a general toxicity model, contributing to the drive to reduce animal safety testing and enabling physiologically relevant in-vitro study.     

High efficacy of anti DBL4ɛ-VAR2CSA antibodies in inhibition of CSA-binding Plasmodium falciparum-infected erythrocytes from pregnant women

Malaria during pregnancy is a major cause of intra-uterine growth-retardation and infant death in sub-Saharan Africa. Ideally, this could be prevented by a vaccine delivered before the first pregnancy. Antibodies against domain DBL4? from VAR2CSA has been shown to inhibit adhesion of laboratory isolates to the placental receptor chondroitin sulfate A. In this study, the binding inhibitory efficacy of IgG elicited by two different DBL4? recombinant proteins was tested on a panel of fresh clinical isolates from pregnant women living in Benin and Tanzania. The most promising recombinant protein elicited antibodies with similar efficacy as pooled plasma from immune multi-gravid African women.     

N,N-bis(cyclohexanol)amine aryl esters inhibit P-glycoprotein as transport substrates

P-Glycoprotein (Pgp) inhibition by three sets of four isomers of N,N-bis(cyclohexanol)amine aryl esters was assessed on rhodamine 123 (R123) efflux in human MDR1-gene transfected mouse T-lymphoma L5178 cells and on Sf9 ATPase activity. The most active compounds inhibited Pgp with IC₅₀ values much lower than those of either cyclosporin A (CSA) or GF120918. As to R123 efflux inhibition, the role of the bond present in the second aryl moiety appeared important since the triple bond derivatives (3a–d) were the most powerful as compared to the double bond (2a–d) and the single bond (1a–d) counterparts. Concentration–inhibition curves of 2c and 3d exhibited a biphasic behaviour suggesting the existence of two binding sites in the recognition domain of Pgp. Persistence of inhibition by these compounds resulted to be intermediate between that caused by CSA and GF120918. R123 exhibited positive interaction with CSA, 1d, 1c, 2d, 2c and 3c, the concentration–inhibition curves being shifted leftward when R123 concentration was increased, while it exhibited negative interaction with 3d and no effect with GF120918. Sf9 ATPase activity was stimulated in an increasing order of potency by 2c, 3c, 2d, CSA, epirubicin and 3d. In a decreasing order of potency 3d, 2c, GF120918, CSA, 2d and 3c inhibited at sub-nanomolar concentrations epirubicin-stimulated ATPase activity. In conclusion, isomeric geometry and restriction of molecular flexibility of N,N-bis(cyclohexanol)amine aryl esters were crucial for their presentation to and inhibition of Pgp as transport substrates, R123 and epirubicin cooperating with them to this inhibition.     

Rapid optimized flow cytometric crossmatch (FCXM) assays: The Halifax and Halifaster protocols

The flow cytometric crossmatch (FCXM) assay, which detects the presence of donor specific HLA antibodies in patient sera, is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time, we developed and validated two modified crossmatch procedures, namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time?>60% and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform, reduced wash times, increased serum to cell suspension volume ratio, shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification, employing methods that improve lymphocyte purity compared to density gradient centrifugation (96?±?2.63% vs 69?±?19.06%), reduce cell isolation time (by?～40%) and conserve FCXM assay reagents. Importantly, linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R² of 0.98–0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally, a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7% and 96.8% specificity and sensitivity, respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care.     

Significant IgG subclass heterogeneity in HLA-specific antibodies: Implications for pathogenicity,prognosis, and the rejection response

IgG subclasses differ in their ability to fix complement and bind Fc receptors. This study describes a detailed analysis of the distribution of HLA-specific IgG subclasses in order to define how this varies in sensitised waiting-list patients. We found significant variation in the level, presence and combinations of each HLA-specific IgG subclass between and within individuals and this is influenced by the type of sensitising event. Graft failure in particular provokes higher levels of IgG1 (vs transfusion, p = 0.071 and pregnancy, p = 0.042), IgG2 (vs transfusion, p = 0.001 and pregnancy, p = 0.016), and IgG4 (vs transfusion, p = 0.052). Both graft failure and pregnancy tend to stimulate multiple IgG subclass responses against HLA, whereas transfusion stimulated antibodies are dominated by responses limited to IgG1 (p = 0.033) and have a low incidence of IgG4 (p = 0.046). In marked contrast, IgG4 characterised nearly all HLA DQ-specific antibodies stimulated by graft rejection (p = 0.006). Such widely varying IgG subclass heterogeneity is likely to be due to underlying immunological processes dependent on the route of sensitisation. This diversity, which implies functional variation, may help explain why HLA-specific antibodies are an obstacle to transplantation in some circumstances but not others. The subclass association with rejection has potential as a biomarker for chronic rejection.