Page 120 - Haematologica Vol. 109 - July 2024

ARTICLE - Cytotoxic reprogramming for BiTE immunotherapy M. Casey et al. AB
CD
EF
Figure 1. Functional screening of immunostimulatory cytokines to determine an ideal combination partner for T-cell-engaging bispecific antibody therapy. (A) Schematic illustrates the screening of immunostimulatory cytokines. Peripheral blood mononu- clear cells (PBMC, 4×105) from healthy donors (HD, N=5) and patients with multiple myeloma (MM, N=5) were co-cultured with JJN-3 myeloma cells (1×105) in the presence of T-cell-engaging bispecific antibody targeting B-cell maturation antigen (anti-BC- MA T-BsAb, 0.1 μg/mL) and each of following recombinant immunostimulatory cytokines: recombinant interleukin-2 (rIL-2) (500 U/mL), rIL-7, rIL-12, rIL-15, rIL-18 and rIL-21 (50 ng/mL). (B-F) Individual graphs showing the levels of granzyme B (B), perforin (C), interferon-γ (IFN-γ) (D), tumor necrosis factor-α (TNF-α) (E), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (F) in culture supernatants 3 days after stimulation, determined by enzyme-linked immunosorbant assay (ELISA). Data are shown as fold changes, relative to T-BsAb stimulation without cytokine treatment. Results from 1 experiment are shown, and differenc- es were tested for statistical significance using a Friedman test with a Dunn’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
S1B; Online Supplementary Table S4), supporting the cy- totoxic granule pathway as a key target of IL-21. Indeed, genes encoding cytotoxic granules (GZMB, GZMH, NKG7, and GNLY) were significantly increased in IL-21-primed CD8 T cells (Figure 3D; Online Supplementary Table S2). Together, IL-21 transcriptionally induces the cytotoxic effector pro- gram in CD8 T cells.
IL-21 priming augments cytotoxic granule exocytosis and myeloma-killing activity by T-cell-engaging bispecific antibody
The results from transcriptome profiling strongly suggest that IL-21 priming could enhance cytotoxic granule-depen- dent tumor-killing activities. Thus, we assessed T-BsAb-me- diated cytotoxic activities of CD8 T cells primed with or without rIL-21 (Figure 4A). Indeed, IL-21 priming markedly augmented T-BsAb-mediated cytotoxicity against three
Haematologica | 109 July 2024
2134