Differential Expression of Inducible Caspase-9 (iC9) in Allogeneic T Cells Allows Selective Depletion of Activated T Cells Following Exposure to Rimiducid and Permits In Vivo Allodepletion
Authors: Zhou Xiaoou , Lu An , Sharp Kelly L , Harris Margie , Anumula Madhavi , Bayle J. , Spencer David M. , Foster Aaron , Shaw Joanne 
Published: October 2019

Abstract
Introduction: BPX-501 is an allogeneic product consisting of T cells modified to express the inducible caspase-9 (iC9) safety switch, which can provide virus and tumor-specific immunity following stem cell transplant. In instances of graft-versus-host disease (GvHD), activation of iC9 with rimiducid leads to rapid killing of alloreactive T cells and resolution of GvHD. However, gene-modified T cells re-expand in the host. Here we evaluate the relationship between transgene expression and sensitivity to rimiducid to understand differential apoptosis in patients treated with allogeneic, iC9-modified T cells.
Methods: The safety switch system consists of a bicistronic vector encoding a mutated FKBP12 binding protein linked to caspase-9 and truncated CD19 (ΔCD19) to allow selection of gene-modified T cells (SFG-iC9-ΔCD19). Exposure to rimiducid dimerizes iC9 resulting in apoptosis of gene-modified T cells. To evaluate the effect of transgene expression levels to the sensitivity of rimiducid-induced apoptosis, BPX-501 T cells were sorted into 3 equal populations based on the intensity of CD19 staining (CD19high, CD19med and CD19low). Phenotyping and functional assays (i.e., apoptosis) were performed by flow cytometry, qPCR and Western blot before and after T cell reactivation using anti-CD3/anti-CD28 antibodies. In vivo studies were performed by i.v. injection of control or gene-modified T cells co-expressing luciferase into NSG mice, followed by i.p. injection of a titrated dose of rimiducid (0.001 to 1 mg/kg), control drug (temsirolimus; 1 mg/kg) or vehicle. Bioluminescent imaging and flow cytometry were subsequently performed to assess in vivo depletion following iC9 activation.
Results: Purity of BPX-501 cells after transduction and CD19 selection was 95%. Sorting BPX-501 cells based on CD19 mean fluorescence intensity (MFI) resulted in a CD19 MFI of 73, 46 and 22 for CD19 high, medium and low sorted populations, respectively. There was no significant difference in the percentage of CD8 and CD4 compartments among these cells, however, iC9-ΔCD19low expressing cells contained less terminal effector memory cells and more naïve-like cells than iC9-ΔCD19high (28±6% vs 39±8% (TEMRA), and 57±9% vs 42±12% (Naïve), respectively, P<0.05). In a 4-hour apoptosis assay, the killing efficiency was significantly diminished in iC9-ΔCD19low compared to unsorted or iC9-ΔCD19high cells (P<0.05, P<0.001, respectively). iC9-ΔCD19low cells also expressed less caspase-9 protein as measured by Western blot correlating to decreased rimiducid sensitivity. Animal studies showed a dose dependent decrease in the MFI of CD19 even in mice treated with 0.001 mg/kg of rimiducid demonstrating preferential killing of iC9-ΔCD19high T cells, and sparing of iC9-ΔCD19low T cells. However, because iC9-ΔCD19 transgene expression is regulated by the retroviral LTR promoter and sensitive to the activation state of the T cell, we measured the activation status following TCR cross-linking using CD25, CD69 and PD-1 markers, and transgene levels using CD19. While unstimulated iC9-ΔCD19 showed differential killing based on transgene expression (86%, 76% and 50% for high, medium and low iC9-ΔCD19, respectively), reactivation increased transgene MFI and apoptosis in all fractions to over 90% when exposed to rimiducid, confirming the relationship of T cell activation with transgene expression.
Summary: In vivo depletion of T cells with iC9 is dependent on the level of transgene expression, which is regulated by the activation state of the T cell. Highly activated alloreactive T cells express higher levels of iC9 which makes them more sensitive to rimiducid-induced apoptosis, and serves to selectively deplete GvHD-causing T cells while sparing T cells with other specificities.

Disclosures
Zhou: Bellicum Pharmaceuticals: Employment, Equity Ownership. Lu:Bellicum Pharmaceuticals: Employment, Equity Ownership. Sharp:Bellicum Pharmaceuticals: Employment, Equity Ownership. Harris:Bellicum Pharmaceuticals: Employment, Equity Ownership. Anumula:Bellicum Pharmaceuticals: Employment, Equity Ownership. Bayle:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Foster:Bellicum: Employment, Equity Ownership. Shaw:Bellicum Pharmaceuticals: Employment, Equity Ownership.