DataSheet_1_Geometric parameters that affect the behavior of logic-gated CAR T cells.pdf

Clinical applications of CAR-T cells are limited by the scarcity of tumor-specific targets and are often afflicted with the same on-target/off-tumor toxicities that plague other cancer treatments. A new promising strategy to enforce tumor selectivity is the use of logic-gated, two-receptor systems. One well-described application is termed Tmod™, which originally utilized a blocking inhibitory receptor directed towards HLA-I target antigens to create a protective NOT gate. Here we show that the function of Tmod blockers targeting non-HLA-I antigens is dependent on the height of the blocker antigen and is generally compatible with small, membrane-proximal targets. We compensate for this apparent limitation by incorporating modular hinge units to artificially extend or retract the ligand-binding domains relative to the effector cell surface, thereby modulating Tmod activator and blocker function. By accounting for structural differences between activator and blocker targets, we developed a set of simple geometric parameters for Tmod receptor design that enables targeting of blocker antigens beyond HLA-I, thereby broadening the applications of logic-gated cell therapies.</p

Phamacokinetic and pharmacodynamic properties of compound A <i>in vivo</i>.

<p>Compound A was administered to athymic nude mice by oral gavage. At the indicated time points, blood was collected and plasma levels of compound A (left panel) as well as S1P (right panel) were determined. Compound levels were corrected for binding to murine plasma, and concentrations of free compound A is depicted in this graph. *P<0.05 compared to vehicle.</p

Effects of SPHK inhibition on cell viability.

<p>The human melanoma cell line WM266.4 (panel A) and the human glioblastoma cell line LN229 (panel B) were treated for 72 h with the indicated concentrations of compound A (left panel) and compound B (right panel). Viability was assessed after 72 h.</p

Inhibition of human and murine SPHK activity in biochemical assays.

<p>Inhibition of human and murine SPHK activity in biochemical assays.</p

Reduction of SPHK expression by RNAi in a panel of cancer cell lines.

<p>Multiple cancer cell lines were transfected in a high-throughput format with libraries of siRNA containing multiple triggers for each gene, and cell viability was determined 96 or 120 hours after transfection. The statistical significance of the observed effects was calculated (see materials and methods) and expressed as a p value. Polo like kinase 1 (PLK1) served as a positive control. Each symbol represents the result of one siRNA screen. Most cell lines were tested several times using different transfection conditions. Symbols to the left of the dashed line (p<0.05) indicate a statistically significant effect of gene knockdown on cell viability in a given experiment.</p

siRNA experiments in A375 cells.

<p>SPHK1 (panel A) and SPHK2 (panel B) as well as the cytotoxic controls PLK1 and POLR2A were targeted with numerous siRNAs in the melanoma cell line A375. Each vertical line represents the effects of an individual siRNA transfection on relative cell viability, with negative values representing cell killing. Statistical significance was calculated as described in Materials and Methods.</p

Correlation between SPHK inhibition and cell viability.

<p>A panel of 18 compounds structurally related to compounds A and B was tested in biochemical hSPHK1 assays (inflection point IC₅₀s plotted on x-axis) and 72 h viability assays in WM266.4 cells (inflection point IC₅₀s plotted on y-axis).</p

Cellular activity of literature compound SKII.

<p>The left panels shows levels of endogenous S1P 24 hours after compound treatment, the right panel depicts the result of 72 h viability assays performed in parallel in the human melanoma cell line LOX. The IC₅₀s reflect the inflection point of the titration curve.</p

Effects of SPHK inhibition on cell viability in 72 h assays (the reported IC₅₀ values are the inflection points of the titration curves).

<p>Effects of SPHK inhibition on cell viability in 72 h assays (the reported IC₅₀ values are the inflection points of the titration curves).</p

Inhibition of VEGF-induced vascular permeability in mice treated with Compound A.

<p>HEK 293 cells transfected with murine VEGF or vector were mixed with Matrigel and injected s.c. into female C57Bl/6 mice. A single dose of compound A was given by oral gavage 24 hours after implantation of cells. At various time points after administration, vascular permeability in the skin overlying the Matrigel plug was measured by quantifying the extravasation of Evans blue dye. Columns, relative Evans blue units (n = 5) per group; bars, SE *, P<0.0001, significant difference from VEGF plus vehicle-injected control mice.</p