Page 124 - Haematologica Vol. 109 - July 2024

ARTICLE - Cytotoxic reprogramming for BiTE immunotherapy M. Casey et al.
Figure 4. Priming by IL-21 augments granule-dependent myeloma-killing activity. (A) Isolated CD8 T cells were stimulated with anti-CD3/CD28 beads in the presence or absence of recombinant interleukin-21 (rIL-21), followed by maintenance for 2 days without anti-CD3/CD28 beads. After the washout of rIL-21, these effector CD8 T cells were used for assays. Schematic illustrates the preparation of control and IL-21-primed CD8 T cells. (B) CD8 T cells were co-cultured with target myeloma cells expressing luciferase at a 2:1 effector to target cell ratio (E: T ratio) for 4 hours. Graphs showing target cytotoxicity with different concen- trations of T-cell-engaging bispecific antibody targeting B-cell maturation antigen (anti-BCMA T-BsAb) (N=7). (C) CD8 T cells were co-cultured with RPMI8226 cells at a 3:1 E: T ratio in the presence of anti-BCMA T-BsAb (0.1 μg/mL). Graphs showing the impact of anti-CD226 blocking monoclonal antibody (mAb, 5 μg/mL) (left), anti-interferon-γ (IFN-γ) neutralizing mAb (5 μg/mL) (middle), or pretreatment with a perforin inhibitor (Perforin-I, 50 nM) (right) on target cytotoxicity (N=5-7). (D) Representative plots (left) and graphs (right) showing frequencies of CD107a+ cells in CD8 T cells after co-culture with RPMI8226 myeloma cells for indi- cated periods in the presence of anti-BCMA T-BsAb (N=7). (E, F) CTV-labeled CD8 T cells were co-cultured with RPMI8226-GFP myeloma cells for 15 minutes at a 1:1 E: T ratio in the presence of anti-BCMA T-BsAb. Representative confocal images showing polarization of the microtubule-organizing center (MTOC) to the immunological synapse (IS) with 3D-reconstituted images (E). Scale bars indicate 10 μm. Graphs showing the distance between the MTOC and IS (right), with each symbol representing 1 donor (N=4) (F). Data are shown as mean ± standard error of mean. Results were pooled from 2 experiments. Differences were tested for statistical significance using a paired t test (B, D), a repeated measures ANOVA with a post hoc Holm-Sidak’s multiple com- parisons test (C), and a Mann-Whitney U test (F). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Perforin-I: perforin inhibitor; GFP: green fluorescent protein; CTV: CellTraceTM Violet.
CD4 T cells expressing CD226 and granzyme B (Figure 5E, F). These results support that rIL-21 treatment can enhance the cytotoxic lymphocyte program in the myeloma BM.
We next asked whether rIL-21 can enhance the efficacy of anti-BCMA T-BsAb therapy in the Vk14451 myeloma pre- clinical model (Figure 6A). Consistent with in vitro results, pretreatment with rIL-21 did not enhance T-BsAb-mediated IFN-γ release, while the combination approach significantly increased plasma levels of granzyme B (Figure 6B). Strikingly, the combination therapy reduced serum paraprotein levels (Figure 6C), and significantly prolonged survival compared to T-BsAb monotherapy (Figure 6D). Together, IL-21 priming is a potential approach to enhance the efficacy of T-BsAb against multiple myeloma.
Treatment with IL-21 modulates effector functions in primary bone marrow CD8 T cells from patients with multiple myeloma
Exhausted CD8 T cells are frequently observed in BM, com- pared to peripheral blood in patients with multiple myeloma.35 Thus, we addressed whether rIL-21 treatment could augment cytotoxic effector phenotypes in myeloma BM T cells. To this end, patient-derived BM mononuclear cells were cultured in the presence of T-BsAb with or without rIL-21 for 3 days (Figure 7A). In line with our results from PBMC, treatment with rIL-21 significantly upregulated CD226 in T-BsAb-stim- ulated myeloma BM CD8 T cells, while the upregulation of CD69 and CD28 was mainly driven by T-BsAb (Figure 7B-D). Importantly, rIL-21 treatment markedly enhanced the re- lease of granzyme B and perforin, but not pro-inflammatory cytokines in T-BsAb-stimulated myeloma BM mononuclear cells (Figure 7E). Taken together, IL-21 can augment cytotoxic effector functions in patient-derived BM CD8 T cells.
Discussion
In this study, we demonstrated that harnessing the cy-
totoxic granule exocytosis pathway through IL-21 can aug- ment T-BsAb-mediated anti-myeloma effects in vitro and in vivo. Unlike other immunostimulatory cytokines, IL-21 treatment did not enhance the release of IFN-γ and other pro-inflammatory cytokines, indicating that IL-21 priming preferentially harnesses cytotoxic effector functions, and can augment granule-dependent myeloma-killing activities by T-BsAb therapy.
Recent results from single-cell immune landscape profiling revealed an accumulation of terminally differentiated, func- tionally impaired T cells in the myeloma BM, especially in patients with relapsed/refractory multiple myeloma.9,36 Since the effector mechanism of T-BsAb therapy largely depends on endogenous T cells, functional impairment of T cells may limit T-BsAb efficacy against multiple myeloma. Indeed, Friedrich et al. recently demonstrated that the presence of exhausted-like CD8 T cell clones was associated with treatment failure in patients treated with anti-BCMA T-BsAb therapy,7 providing a rationale for reprogramming dysfunc- tional CD8 T cells to achieve better clinical responses. Over the past few years, new approaches have been developed to improve the functionality of CAR T cells during ex vivo manufacturing.37,38 By contrast, to improve the efficacy of T-BsAb therapy, systemic therapy will be inevitable to rein- vigorate endogenous T cells in the myeloma BM. However, the optimal therapeutic approach remains to be established due to the risk of CRS exacerbation. In this context, results from this study reveal IL-21 as an ideal partner for T-BsAb therapy, providing important translational implications. We showed that treatment with rIL-21 did not enhance IFN-γ release both in vitro and in vivo. Given that IFN-γ is a key driver for CRS and MAS/HLH,18,19,39 but dispensable for elimi- nating malignant B cells by immunotherapy,18,23 avoiding the excess release of IFN-γ will be an important consideration for T-BsAb therapy against hematological malignancies. Of note, conflicting results have been reported on whether or not IL-21 can induce Th1 responses and IFN-γ production, possibly due to context-dependent effects of IL-21 on T-cell
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