Serial Activation of the Inducible Caspase 9 Safety Switch After Human Stem Cell Transplantation

Activation of the inducible caspase 9 (iC9) safety gene by a dimerizing drug (chemical inducer of dimerization (CID) AP1903) effectively resolves the symptoms and signs of graft-versus-host disease (GvHD) in haploidentical stem cell transplant (HSCT) recipients. However, after CID treatment, 1% of iC9-T cells remain and can regrow over time; although these resurgent T cells do not cause recurrent GvHD, it remains unclear whether repeat CID treatments are a safe and feasible way to further deplete residual gene-modified T cells should any other adverse effects associated with them occur. Here, we report a patient who received an infusion of haploidentical iC9-T cells after HSCT and subsequently received three treatments with AP1903. There was a mild (grade 2) and transient pancytopenia following each AP1903 administration but no non-hematological toxicity. Ninety five percent of circulating iC9-T cells (CD3+CD19+) were eliminated after the first AP1903 treatment. Three months later, the residual cells had expanded more than eightfold and had a lower level of iC9 expression. Each repeated AP1903 administration eliminated a diminishing percentage of the residual repopulating cells, but elimination could be enhanced by T-cell activation. These data support the safety and efficiency of repeated CID treatments for persistent or recurring toxicity from T-cell therapies

Clonal Dynamics In Vivo of Virus Integration Sites of T Cells Expressing a Safety Switch

Safety switches are becoming relevant for the clinical translation of T-cell-based immunotherapies. In patients receiving an allogeneic hematopoietic stem cell transplant, the inducible caspase-9 gene (iC9) safety switch expressed by donor-derived T lymphocytes efficiently controls acute graft versus host disease (GvHD). However, in vivo elimination of iC9-T cells by the chemical inducer of dimerization (CID) that activates the iC9 protein is incomplete. To study this effect, we characterized the clonal diversity and dynamics of vector insertion sites (VIS) in iC9-T cells pre- and post-CID administration in four patients who developed GvHD. We identified 3,203 VIS among four patients and followed their in vivo clonal dynamics up to 161 days post-CID. VIS were categorized by their proximity to host genome elements, gene associations, and cis-modulatory relationship to mapped promoters. We found that VIS are preferentially located near open chromatin and promoter regions; furthermore, there was no evidence for selection bias among VIS surviving the CID treatment. The majority of iC9-T cells with high normalized VIS copy number at the time of GvHD onset were eliminated by CID, while iC9-T cells detectable post-CID generally have low normalized VIS copy number. We propose that suboptimal iC9 transgene expression is responsible for the incomplete elimination of iC9-T cells and illustrate here by simple model how cis-modulatory influences of local genome context and T-cell receptor activation status at time of CID treatment contribute to stochastic sparing of iC9-T cells

Institutional Risk of Russian Public Sector Organizations Management

The Government of the Russian Federation has been overhauling a public sector of economy since 2010. One of the transformation’s goals is a gradual reduction of public spending on the public-sector organizations, and simultaneous increase in efficiency of their economic activity and in the quality of rendered public services. After the federal law on the legal status of public (municipal) institutions improvement came into force, the operating mechanisms and internal institutional links of public sector organizations have been changed, including full participation of public sector organizations in market relations. For instance, budgetary funds are allocated on contractual basis in form of government tasks. However, the significant improvements have not been achieved. Experts have a common ground in reasons of organizational fails. Among them are poor quality of planning, goal setting, and incorrect setting of development indicators' target values

Global Gene Expression Analysis of Reactive Stroma in Prostate Cancer

Purpose: Marked reactive stroma formation, designated as grade 3 reactive stroma, is associated with poor outcome in clinically localized prostate cancer. To understand the biological processes and signaling mechanisms underlying the formation of such reactive stroma, we carried out microarray gene expression analysis of laser-captured reactive stroma and matched normal stroma

Notch and TGFβ Form a Reciprocal Positive Regulatory Loop that Suppresses Murine Prostate Basal Stem/Progenitor Cell Activity

SummaryThe role of Notch signaling in the maintenance of adult murine prostate epithelial homeostasis remains unclear. We found that Notch ligands are mainly expressed within the basal cell lineage, while active Notch signaling is detected in both the prostate basal and luminal cell lineages. Disrupting the canonical Notch effector Rbp-j impairs the differentiation of prostate basal stem cells and increases their proliferation in vitro and in vivo, but does not affect luminal cell biology. Conversely, ectopic Notch activation in adult prostates results in a decrease in basal cell number and luminal cell hyperproliferation. TGFβ dominates over Notch signaling and overrides Notch ablation-induced proliferation of prostate basal cells. However, Notch confers sensitivity and positive feedback by upregulating a plethora of TGFβ signaling components including TgfβR1. These findings reveal crucial roles of the self-enforced positive reciprocal regulatory loop between TGFβ and Notch in maintaining prostate basal stem cell dormancy

Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent

Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB costimulation in CARs may enable T cells to resist this functional exhaustion; however, the potential ramifications of tonic 4-1BB signaling in CAR T cells remain unclear. Here, we found that tonic CAR-derived 4-1BB signaling can produce toxicity in T cells via continuous TRAF2-dependent activation of the nuclear factor κB (NF-κB) pathway and augmented FAS-dependent cell death. This mechanism was amplified in a non-self-inactivating gammaretroviral vector through positive feedback on the long terminal repeat (LTR) promoter, further enhancing CAR expression and tonic signaling. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic signaling and improved T cell expansion and anti-tumor function. These studies illuminate the interaction between tonic CAR signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design