Impact of bortezomib on T-cell subsets and immune checkpoints under bispecific antibody therapy in multiple myeloma

Abstract

Abstract Background The combination of proteasome inhibitors (PI) and bispecific antibodies (bsAb) has demonstrated both synergistic and potentially antagonistic effects in the treatment of multiple myeloma (MM). While PIs may enhance bsAb-mediated cytotoxicity by increasing surface expression of target antigens, emerging evidence suggests that PIs may also promote immune evasion through up-regulation of immune checkpoint molecules. In this study, we aimed to dissect the immunological consequences of this combination by investigating the impact of the most commonly used PI bortezomib (BTZ) on T-cell subset composition and function in the context of bsAb therapy in MM. Methods Peripheral blood samples and clinical data from 104 pre-treated MM patients were prospectively collected and T-cell subsets were analyzed by flow cytometry. In vitro functional assays were conducted for BTZ, teclistamab (Tec) and talquetamab (Tal) using the PI-resistant MM cell line L363 and healthy donor T-cells. Proteasome subunit activity (β1, β2, β5) in T-cells was visualized with activity-based probes. MM cell viability was assessed, and cytokine concentrations were measured via ELISA in culture supernatants. Results Peripheral blood samples of 104 patients with pre-treated MM were analyzed. Median age of our cohort was 68 years (range 43-89) and patients were pre-treated with a median of 2 lines of therapy (range 1-8). Overall, 89 (85.6%), 27 (25.9%) and 13 (12.5%) patients had received PI, bsAb and chimeric antigen receptor modified (CAR) T-cell therapy, respectively, and 63 (60.6%) patients had prior high-dose melphalan and autologous stem cell transplant (ASCT). At sampling, 76 (73.1%) patients received ongoing anti-MM therapy while the remaining 28 (26.9%) patients were in a therapy-free interval. In a multivariate generalized linear model, prior Tal therapy was related with an increased proportion (%) of PD-1+ T-cells (β=3.55, P=0.03) while Tec positively correlated with increased frequencies of LAG-3+ (β=1.07, P=0.04) and TIM-3+ (β=9.16, P<0.001) T-cells. Moreover, exposure to BTZ led to a decrease of CD8+ LAG-3+ T-cells (β=-6.63, P=0.03). Since time between last treatment and sampling did not correlate with frequencies of different T-cell subsets in our study, these results suggest that anti-MM therapies including bsAb and BTZ may lead to persistent changes in circulating T-cell subsets in MM patients, potentially influencing the efficacy of T-cell based immunotherapies. Next, we performed functional assays to investigate whether the addition of BTZ to a bsAb modulates T-cell subsets and alters immune checkpoint expression on T-cells. Healthy donor T-cells were pre-treated with BTZ for 72 hours prior to co-culture to exclude potential confounding effects from reduced T-cell viability. A significant toxicity to healthy T-cells was observed upon treatment with 100 nM BTZ, resulting in >30% reduction in T-cell viability and significant inhibition (>50%) of proteasome subunits (β1, β2, and β5) after 72 hours. Co-culture of PI-resistant L363 cells with donor-derived T-cells plus Tal or Tec led to a significant increase in MM cell viability in the presence of BTZ (Tal: 2-fold increase, P=0.006; Tec: 5-fold increase, P<0.001). This finding was consistent with a marked reduction in cytokine production by T-cells, including IFN-γ (0.3-fold, P<0.001) and IL-6 (0.6-fold, P=0.03) in Tec with vs. without BTZ co-cultures. Phenotypic analysis of T-cell subsets revealed that BTZ significantly increased the CD4/CD8 ratio (Tal: P=0.005; Tec: P<0.001) and the frequency of effector memory T cells re-expressing CD45RA (Temra) (Tal and Tec: P<0.001), while reducing the proportion of central memory T cells (Tcm) (Tal and Tec: P<0.001). Additionally, we observed a significant decrease in the expression of the immune checkpoint markers PD-1, LAG-3 and TIM-3 on T-cells following BTZ co-treatment (PD-1: Tal and Tec, P<0.001; LAG-3: Tal P=0.03; Tec P<0.001; TIM-3: Tal P=0.006; Tec P<0.001). Our results suggest that the addition of BTZ to bsAbs may alter the T-cell activation by bsAb, characterized by decreased cytokine production, shift in T-cell subsets and lower expression of activation markers on T-cell, e.g. immune checkpoints. Conclusion Taken together, our findings provide the first evidence that BTZ may impair T-cell activation by bsAb in MM in vitro, thereby potentially diminishing the anti-tumor efficacy of bsAb.