(B) The samples transduced with an LV dose of MOI 10 were analyzed 4?days and 10?days post transduction to confirm stable transgene expression

(B) The samples transduced with an LV dose of MOI 10 were analyzed 4?days and 10?days post transduction to confirm stable transgene expression. or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10?days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future. cultivation of B-ALL cells is difficult and mainly supporting cell survival with minimal or even lack LY341495 of proliferation.15,16 The cultivation conditions for malignant cells were chosen based on established protocols and were confirmed to enable survival of the cells for the duration of the experiment LY341495 (Figure?S6A).17 In line with published studies, malignant B cells were transduced at higher levels than B cells of healthy donorseven when a 4-fold lower LV dose was applied (Figure?S6B).17, 18, 19, 20 Transduction levels are similar to results published by Biagi and colleagues,17 who reported a transduction level of 4.4%C21% using a comparable LV dose. In general, transduction efficiency is dependent on multiple factors (e.g., expression of respective surface LY341495 receptors, cell cycle, and proliferation, as well as cellular restriction factors).21 To identify the cause of enhanced transduction efficiency of the malignant cells compared to the healthy B cells, we analyzed differences in proliferation capacity by using cell trace dye (Figure?S6A). As expected, a lack of proliferation of the B-ALL cells was observed. In contrast, activated healthy B cells readily proliferate under the chosen cultivation conditions, excluding different proliferative capacity as potential cause of increased transduction efficiency. In 2014, LDLR was identified as the main receptor of VSV-G, although related family members of LDLR may be used as well.12,13 Stimulation via the B cell receptor (BCR) on B cells was shown to only marginally enhance expression of LDLR, which is therefore suspected to cause Rabbit polyclonal to JAKMIP1 low transduction levels with VSV-G LV.13,22 For this reason, we hypothesized that higher expression levels of LDLR on malignant B cells were causing the enhanced transduction efficiency. However, LDLR was not expressed by the malignant cells, as measured by flow cytometry (Figure?S6C). The presence of functional LV as measured by transducing activity was confirmed by analyzing control transductions containing raltegravir, an HIV integrase inhibitor, blocking transgene integration into the host cell genome and LV-mediated gene expression only.23 Functional LVs promote raltegravir-dependent, stable expression at high levels. In contrast, exosomes cause transient, highly variable protein transfer (i.e., pseudotransduction) in a raltegravir-independent manner. Next, we aimed to resemble the cellular composition of a directly transduced leukapheresis sample of a B-ALL patient by transducing a mixture containing 30% malignant B cells and 70% unstimulated PBMCs of a healthy donor (Figure?4A). Malignant B cells (CD19+/CD20?) could be distinguished from residual non-malignant B cells (CD19+/CD20+) solely by analyzing CD19 and CD20 surface expression (Figure?4B; Figure?S7). The use of GFP-encoding LVs displaying CARs had multiple advantages: (1) Since GFP is expressed instead of CAR, CD19 antigen is not masked by the CAR, which still allows identification of the cocultured malignant cells by flow cytometry. (2) Applying GFP-encoding LVs further avoids generation of CAR-expressing T?cells, which would induce lysis of the malignant B cells. (3) Potential differences in transduction efficiency levels caused by different transfer vector constructs are excluded. LV stocks were titrated by transduction of SupT1 cells with serially diluted LVs and afterwards tested on activated T?cells in advance to confirm that the identical LV dose was applied (Figure?S8A). Unbound LVs were removed by three successive washing LY341495 steps 1.5?h post transduction. The transduction efficiency was assessed 4?days later by flow cytometry. Malignant B cells were transduced at 8-fold higher levels when LVs displaying anti-CD19 CARs were applied as compared to LVs displaying anti-CD318 CAR or no CAR. Also, for anti-CD20 CARs displaying LVs, a tendency of increased transduction LY341495 efficiency was detectable, pointing to malignant cells potentially expressing CD20 at low levels (Figures 4C and 4D). Alternatively, residual.