Preclinical Targeting of Aggressive T Cell Malignancies Using Anti-CD5 Chimeric Antigen

Preclinical targeting of aggressive T cell malignancies using anti-CD5 chimeric antigen receptor

SUPPLEMENTARY METHODS AND MATERIALS

CAR construct design, transduction and characterization

The CD5-specific CAR (pRSC.SFFV.CD5.3G) consists of an extracellular scFv domain directed against the target protein CD5, and an intracellular tandem signaling domain made up of a CD28 domain upstream 4-1BB and CD3ζ signaling domains. Production of lentiviral vector and subsequent NK-92 transduction and detection were performed as described previously10 and CD5CAR NK-92 cells were sorted via FACS for avid expression. The sorted cells retained stable expression of around 25-30% CAR during 3 months of expansion.

Co-cultures, antibodies, and gating schemes

Co-cultures were carried out at ratios of 2:1 and 5:1 (200,000 and 500,000 effector cells to 100,000 target cells respectively) in 1 ml of 2.5% serum NK-92-cell culture media without IL-2. Dosage dependent experiments were performed at lower ratios from 0.25:1 (25,000 effector cells to 100,000 target cells), to 0.5:1 to 1:1. After 24 hours, remaining live cells were harvested and stained with mouse anti-human CD56 and CD5 antibodies for cell line and T-cell depletion assays. Combinations of mouse anti-human CD3, CD7, CD19, and CD34 were additionally used to stain SPT-1, PT4, MCL (JeKo and L3-G), and T-ALL patient samples. CD56 single positives denote NK-92 cells, and CD5 generally denotes the target cell populations. For target cell phenotypes refer to Supplementary Figures 1 and 3. All cells were washed with FACS buffer, resuspended in 2% formalin, and analyzed by flow cytometry.

Xenogeneic mouse model and statistics

Male 12-week-old NSG mice (NOD.Cg-Prkdcsid Il2rgtm1Wjl/SzJ) were purchased from the Jackson Laboratory (Bar Harbor, ME) and used under a Stony Brook University IACUC-approved protocol. NSG mice were irradiated with a sublethal (2.0 Gy) dose of gamma irradiation (N=6 for mouse model #1 [MM1] and N=8 for mouse model #2 [MM2]). Next day (Day 1), mice were intravenously (IV) injected with 1.0 x106Jurkat cells that had been stably transduced to express luciferase, in order to cause a measurable IV tumor to form. On day 4, 15 x 106 CD5CAR or control NK-92 cells were injected during the window of NK cell life expectancy and concluding by 14 for MM1. For MM2, 10 x 106 cells were injected during the window concluding by day 11. Two additional low doses consisting of 5 x 106 cells were administered through day 21 to observe tumor suppression maintenance. On days 6, 11, 15, and 21 (mouse model #1) and days 3, 6, 11, and 14 (mouse model #2) mice were injected intraperitoneally (IP) and analyzed as described previously10. Xenogeneic model sample sizes were estimated using 2-sample, 2-sided equality power analysis (90% power and <5% significance) without blinding. Unpaired student t-tests were used to determine significance of tumor size area and light intensity. Survival curves were constructed using the Kaplan-Meier method and statistical analyses of survival was performed using a log-rank (Mantel-Cox) test with P <0.05 considered significant. Statistical analyses were performed using GraphPad Prism 6 software. Variance was determined to be similar between the treatment and control group prior to unpaired student t-tests.