Page 119 - Haematologica Vol. 109 - July 2024

ARTICLE - Cytotoxic reprogramming for BiTE immunotherapy M. Casey et al.
In vivo treatment
C57BL/6 wild-type mice were bred and maintained in house. Mice were intravenously challenged with 2×106 Vk14451 cells expressing EGFP. Tumor-bearing mice with paraprotein- emia were intraperitoneally (i.p.) treated with recombinant murine IL-21 (100 μg, i.p.), according to a dosing schedule reported previously.27,28 In some experiments, tumor-bearing mice were treated with anti-mouse BCMA T-BsAb (25 μg, i.p.) either alone or in combination with rIL-21. Mice were monitored over time for clinical endpoints. All experiments were approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee (P3533).
Statistical analysis
Statistical differences were tested with GraphPad Prism 9 software. For comparisons between two groups, a Mann-Whitney U test or paired t test was used. For multiple comparisons, a Friedman test, a repeated measures ANO- VA, or Kruskal-Wallis test was used, followed by indicated post hoc tests. Differences in survival were evaluated with a Mantel-Cox test. P<0.05 was considered to be statistically significant (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).
Results
Functional screening of immunostimulatory cytokines to determine an ideal combination partner for T-cell- engaging bispecific antibody therapy
We sought to identify an immunostimulatory cytokine regulating the cytotoxic granule pathway with negligible impacts on the production of IFN-γ. To this end, PBMC from five healthy donors and five patients with newly di- agnosed multiple myeloma were co-cultured with JJN-3 myeloma cells in the presence of anti-BCMA T-BsAb and one of the following immunostimulatory cytokines: rIL-2, rIL-7, rIL-12, rIL-15, rIL-18, or rIL-21 (Figure 1A). After 3 days of co-culture, levels of effector molecules in culture su- pernatants were determined. The release of granzyme B was enhanced by the addition of rIL-12, rIL-18, or rIL-21, whereas a significant increase in perforin levels was only seen by rIL-21 (Figure 1B, C). Consistent with the ability to drive type 1 immune responses,29 both rIL-12 and rIL-18 dramatically augmented the release of IFN-γ (Figure 1D). However, notably, treatment with rIL-21 showed a negligible impact on IFN-γ production (Figure 1D). Moreover, treatment with rIL-21 did not increase levels of TNF-α and GM-CSF (Figure 1E, F). These results suggest that rIL-21 might be a potential adjuvant for enhancing the cytotoxic granule pathway, but not pro-inflammatory responses.
Dose-dependent effects of IL-21 on CD8 T cells stimulated with T-cell-engaging bispecific antibody
In order to validate the results from the immunostimula- tory cytokine screening, we examined the dose-dependent
effects of rIL-21 on T-BsAb-induced cytokine production from eight different healthy donor-derived PBMC. Indeed, treatment with rIL-21, at 20 ng/mL or higher, significantly increased the release of granzyme B and perforin without increasing levels of pro-inflammatory cytokines IFN-γ, TNF, and GM-CSF (Figure 2A). Intriguingly, levels of IFN-γ and GM-CSF were modestly but significantly reduced in the presence of rIL-21 (Figure 2A), further supporting the differential impact of IL-21 on T-BsAb-induced release of cytotoxic granules and pro-inflammatory cytokines. Next, we addressed whether rIL-21 could modulate the activation of CD8 T cells. The upregulation of an early acti- vation marker CD69 was detectable on CD8 T cells 5 hours after T-BsAb stimulation, especially at high concentrations of T-BsAb (Figure 2B). Frequencies of CD69-expressing CD8 T cells significantly increased in the presence of rIL-21, even at suboptimal concentrations of T-BsAb, suggesting that treatment with rIL-21 lowered the activation thresh- old in CD8 T cells (Figure 2B). By contrast, treatment with rIL-21 showed negligible impacts on T-BsAb-mediated proliferation (Figure 2C). Intriguingly, treatment with rIL- 21 upregulates the expression levels of CD226 and CD28, key receptors for tumor recognition and co-stimulation, respectively (Figure 2D).30-32 Together, these results suggest that IL-21 modulates selective effector functions, rather than broadly stimulates CD8 T cells.
Treatment with IL-21 transcriptionally regulates the cytotoxic granule pathway in CD8 T cells stimulated with T-cell-engaging bispecific antibody
It is appreciated that inflammatory cytokines act as a third signal for functional maturation of CD8 T cells.29 In order to obtain a comprehensive profiling of effector programs regulated by IL-21, we performed bulk RNA sequencing (RNA-seq) of T-BsAb-stimulated CD8 T cells from six healthy donors in the absence (control) or presence of rIL-21 using a paired design (Figure 3A; Online Supplementary Figure 1A; Online Supplementary Table S1). Principal components analysis (PCA) revealed samples clustered by donor along principal component (PC) 1 and PC2, whilst PC2 versus PC3 separated control CD8 T cells and IL-21-primed CD8 T cells (Figure 3B). Differential expression analysis between IL-21- primed and control CD8 T cells identified 2,609 differen- tially expressed genes, of which 1,280 were upregulated, and 1,329 were downregulated (false discovery rate <0.05; Online Supplementary Table S2). Reactome pathway anal- ysis of the genes upregulated in IL-21-primed CD8 T cells revealed enrichment of neutrophil degranulation (granule exocytosis) as well as Rho and other GTPase pathways (Figure 3C; Online Supplementary Table S3), key signaling pathways for cytoskeletal reorganization in cytotoxic T cells.33 By contrast, pro-inflammatory pathways such as IFN, TNF, and IL-1 signaling pathways were enriched based on Reactome pathway analysis of the genes downregulated in IL-21-primed CD8 T cells (Online Supplementary Figure
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