Figure 5.

PRAME TCR-T cells kill OVCA cells in vitro and demonstrate in vivo killing potential in an established MM model. (A, B) Purified PRAME TCR-T cells were tested for cytotoxic capacity in a 6-hour ⁵¹Cr-release assay at E:T ratio 10:1 against (A) primary OVCA patient samples and OVCA cell lines, and (B) PRAME negative cells (Raji and imDCs), or target HLA negative cells (COV362.4). Except for COV362.4, all target cells expressed the target HLA alleles, either wildtype or Td. COV318 and OVCAR-3 were Td with A24, Raji cells were Td with A2, A24 or B7. Primary malignant ascites patient sample OVCA-L23 (wildtype HLA-A2) was either passage 0 (included for TCR DSK3 and HSS3) or passage 10 Td with A24 or B7 (included for TCR 16.3C1 and 8.10C4). imDCs were isolated from PBMCs of a A2+, A24+ and B7+ donor. Percentage relative PRAME expression is depicted, as determined by qPCR. Cytotoxic capacity of PRAME TCR- and CMV TCR-T cells were compared using a paired t-test (two-sided). Mean and SD of technical triplicates are depicted for four donors tested in two independent experiments. (C) NSG mice engrafted with 2*10⁶ U266 MM cells Td with Luc2 luciferase. Mice were i.v. treated with 5*10⁶ PRAME or CMV TCR-T cells 14 days after tumor infusion. Mean and SD of tumor outgrowth (average radiance measured by bioluminescence imaging) over time on the ventral side are depicted. N=6 for PRAME TCR-T cells and n=4 for CMV TCR-T cells. Tumor outgrowth in mice treated with PRAME or CMV-TCR T cells was compared for each time point using two-way ANOVA on log-transformed data, followed by Bonferroni post-hoc analysis. Only significant results are depicted. (ANOVA, analysis of variance, E:T, effector:target ratio; ns, not significant; imDCs, immature dendritic cells; MM, multiple myeloma; OVCA, primary ovarian carcinoma sample; Td, transduced). Meaning of the * are listed in the M&M. Significance levels are indicated as p <.05 *, p <.01 **, p <.001 ***, and p <.0001 ****. ns, not significant.