Supplementary Figure S4 from Overexpression of an Engineered SERPINB9 Enhances Allogeneic T-cell Persistence and Efficacy

Supplementary Figure S4. CD30.CAR with 4-1BB spacer (CD30.bb) EBVSTs expressing different forms of SB9 had similar CD30.bb expression and T cell characteristics. (A, left) SDS-PAGE analysis of SB9 expression from a representative donor, where the top band (∼62 kDa) indicates the pre-formed GzmB and SB9 complexes in the cytosol and the bottom band (∼43 kDa) indicates the functional SB9 monomer. Transduction was carried out in the presence of IL-7 (10 ng/mL) and either low (10 ng/mL) or high (100 ng/mL) IL-15 concentration, as denoted by ‘L’ and ‘H’ respectively. (A, right) Densitometry analysis of SB9 monomer normalized to CD30.bb EBVST control and calnexin as a loading control under condition ‘L’ (IL-7 and IL-15 at 10 ng/mL). (B, left) CD30.bb expression on EBVSTs expressing different variants of SB9, measured by flow cytometry analysis of a representative donor. Pooled data of CD30.bb expression (%; B, middle) and CD30.bb expression (geometric mean fluorescence intensity, MFI; B, right) on CD30.bb EBVSTs, measured by flow cytometry analysis of n = 4 donors, where bars denote median ± SD. (C) Background subtracted percentage of CD3-positive T cells producing IFNγ and/or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry, where the bars represent the mean ± SD (n = 4 donors). Background was determined by measuring percentage of CD3-positive T cells producing IFNγ and/or TNFα in response to HIV peptides. D-H, Flow cytometry analysis of CD4/CD8 subsets (%) (D), memory T cell subsets (%), where CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA (E), CD39 expression (%) (F), CD25 expression (%) (G, left) and geometric mean fluorescence intensity (MFI) (G, right), and PD-1, TIM-3, LAG-3 expression (H), of CD30.bb EBVSTs from a representative donor.