Regulatory T cells hamper the efficacy of T-cell-engaging bispecific antibody therapy

Abstract

T-cell-engaging bispecific antibodies (T-BsAb) have produced impressive clinical responses in patients with relapsed/refractory B-cell malignancies, although treatment failure remains a major clinical challenge. Growing evidence suggests that a complex interplay between immune cells and tumor cells is implicated in the mechanism of action and therefore, understanding immune regulatory mechanisms might provide a clue for how to improve the efficacy of T-BsAb therapy. Here, we investigated the functional impact of regulatory T (Treg) cells on anti-tumor immunity elicited by T-BsAb therapy. In a preclinical model of myeloma, the activation and expansion of Treg cells in the bone marrow were observed in response to anti-B-cell maturation antigen (BCMA) T-BsAb therapy. T-BsAb triggered the generation of induced Treg cells from human conventional CD4 cells after co-culture with tumor cells. Moreover, T-BsAb directly activated freshly isolated circulating Treg cells, leading to the production of interleukin-10 and inhibition of T-BsAb-mediated CD8 T-cell responses. The activation of Treg cells was also seen in bone marrow samples from myeloma patients after ex vivo treatment with T-BsAb, further supporting that T-BsAb have an impact on Treg homeostasis. Importantly, transient ablation of Treg cells in combination with T-BsAb therapy dramatically improved effector lymphocyte activities and disease control in the preclinical myeloma model, leading to prolonged survival. Together, this information suggests that therapy-induced activation of Treg cells critically regulates anti-tumor immunity elicited by T-BsAb therapy, with important implications for improving the efficacy of such treatment.

Figure 1.

T-cell-engaging bispecific antibody therapy triggers the activation of regulatory T cells in myeloma bone marrow. (A) C57BL6 wild-type mice were challenged with Vk14451 myeloma cells, and treated with anti-mouse B-cell maturation antigen T-cell engaging bispecific antibody. The schematic illustrates the experimental design. (B) Representative plots and graphs showing frequencies of enhanced green fluorescent protein-positive tumor cells in the bone marrow (BM) (N=9 per group), pooled from two experiments. (C) t-distributed stochastic neighbor embedding plots showing the density of events of indicated subsets in BM TCRβ-positive cells from one experiment (N=4 per group). (D, E) Graphs showing numbers of indicated immune cells (D) and the CD8/Treg ratio (E) in the myeloma BM (N=9 per group), pooled from two experiments. (F) Histograms and graphs showing expression levels of PD-1, Tigit and CD38 on CD8 T cells and Treg cells in the myeloma BM. Representative results from two experiments are shown (N=4-5). Numbers indicate mean fluorescence intensity. Data are shown as mean ± standard error of mean. Differences were tested for statistical significance, using a Mann-Whitney U test (B, D, and E) and Student t test (F). *P<0.05, **P<0.01, ****P<0.0001. WT: wild-type; T-BsAb: T-cell-engaging bispecific antibody; BM: bone marrow; SSC; side scatter; EGFP: enhanced green fluorescent protein; t-SNE: t-distributed stochastic neighbor embedding; Treg: regulatory T cells; FMO: fluorescence minus one; MFI: mean fluorescence intensity.

Figure 2.

In vitro differentiation of induced regulatory T cells by T-cell-engaging bispecific antibody therapy. (A) Peripheral blood mononuclear cells were co-cultured with JJN-3 myeloma cells in the presence of anti-human B-cell maturation antigen T-cell-engaging bispecific antibody (T-BsAb). A schematic illustrating the experimental design. (B) Representative flow cytometry plots showing the frequency of CD4 T cells expressing FOXP3 and CD25 at the indicated time points (left). Representative histograms showing expression levels of CTLA-4 and ICOS in CD25⁺FOXP3⁺CD4 T cells (right). (C, D) Box and whisker plots showing the frequency of FOXP3⁺CD25⁺ cells in CD4 T cells (C), and the CD8/FOXP3⁺CD25⁺CD4 ratio (D) at indicated time points after treatment with T-BsAb. Pooled results from two experiments are shown (N=8). (E, F) Isolated CD4 T cells were stimulated with T-BsAb or anti-CD3/CD28 beads for 4 days. Subsequently, T-BsAb-mediated induced regulatory T cells and activated CD4 T cells were co-cultured with CellTrace™ Violet-labeled CD8 T cells to test immunosuppressive activities. A schematic illustrating the experimental design (E). Representative histograms and graphs showing CD8 T-cell proliferation at the indicated CD4/CD8 ratios 3 days after stimulation (F). Data are shown as mean ± standard error of mean, pooled from two experiments (N=6). Differences were tested for statistical significance using repeated measures analysis of variance with a post-hoc Tukey multiple comparisons test (C, D) and a paired t test (F). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. hBCMA; human B-cell maturation antigen; PBMC: peripheral blood mononuclear cells; iTreg; induced regulatory T cells; CTV: CellTrace™ Violet.

Figure 3.

Induced T regulatory cells preferentially form aggregates around tumor cells. (A) CellTrace™ Violet-labeled CD8 T cells, PKH26-labeled T-cell-engaging bispecific antibody (T-BsAb)-mediated induced regulatory T cells (iTreg) and green fluorescent protein (GFP)-expressing RPMI8226 myeloma cells were co-cultured in a 1:1:1 ratio in the presence of anti-B-cell maturation antigen T-BsAb (0.1 µg/mL). The schematic illustrates the experimental design. (B, C) Representative histograms (B) and graphs (C) showing conjugate formation with GFP⁺ myeloma cells by CD8 T cells or iTreg cells. Data are shown as mean ± standard error of mean, pooled from two experiments (N=6). Differences were tested for statistical significance using a paired t-test. **P<0.01. (D) Confocal images showing conjugate formation 30 min after co-culture (scale bar: 10 µm). Representative images from six donors are shown. CTV: CellTrace™ Violet.

Figure 4.

T-cell-engaging bispecific antibody-activated regulatory T cells negatively regulate CD8 T-cell responses. (A) Representative flow cytometry plots showing purity of CD25⁺CD127^low regulatory cells (Treg cells) isolated from peripheral blood mononuclear cells. (B) Freshly isolated human Treg cells were co-cultured with RPMI8226 myeloma cells expressing green fluorescent protein in the presence or absence of anti-B-cell maturation antigen (BCMA) T-cell-engaging bispecific antibody (T-BsAb). Representative confocal images and graphs showing accumulation of LFA-1 and F-actin between Treg cells and multiple myeloma cells after stimulation by anti-BCMA T-BsAb (scale bar: 10 µm). Images were processed using MetaMorph software. Representative results from two experiments are shown. (C) CD25⁺CD127^low Treg cells (2×10⁵) were co-cultured with JJN-3 myeloma cells (1×10⁵) with indicated concentrations of anti-BCMA T-BsAb for 3 days. The graphs show levels of interleukin-10 in culture supernatants. Data are shown as mean ± standard error of mean (N=4). (D) CD4⁺CD25⁺CD127^low Treg cells and CD8 T cells were co-cultured with JJN-3 myeloma cells at different Treg/CD8 ratios in the presence of anti-BCMA T-BsAb for 3 days. The schematic illustrates the experimental design. (E, F) Representative histograms showing CD8 T-cell proliferation at indicated Treg/CD8 ratios (E, left). Individual graphs showing the suppressive effect of Treg cells on CD8 T-cell proliferation (E, right). Individual graphs showing interferon-g production at different Treg/CD8 ratios (F). Pooled results from three experiments are shown (N=8). Differences were tested for statistical significance using a repeated measures analysis of variance with a post-hoc Dunnet multiple comparisons test (C) and Tukey multiple comparisons test (E, F). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DIC: differential interference contrast; GFP: green fluorescent protein; LFA-1: lymphocyte function-associated antigen 1; MFI: mean fluorescent intensity; CTV: CellTrace™ Violet; IL: interleukin; IFN: interferon.

Figure 5.

Transient ablation of regulatory T cells improves the efficacy of anti-B-cell maturation T-cell-engaging bispecific antibody therapy. (A) C57BL6 Foxp3^DTR mice were challenged with Vk14451 multiple myeloma cells, and treated with diphtheria toxin in combination with anti-mouse B-cell maturation antigen T-cell-engaging bispecific antibodies (T-BsAb). The schematic illustrates the the experimental design. (B) Tumor-bearing mice were given the indicated treatment. Box and whisker plots showing levels of interferon-γ and granzyme B in plasma 6 h after treatment (N=6-7 per group). (C) Representative flow cytometry plots and box and whisker plots showing frequencies of interferon-^γ+ CD8 T cells in the myeloma bone marrow 24 h after treatment (N=6-7 per group). (D) Kaplan-Meier survival curves of mice after the indicated treatment (N=7-11 per group). Data are pooled from two independent experiments. Differences were tested for statistical significance using two-way analysis of variance with a post-hoc Tukey multiple comparisons test (B, C) and a Mantel-Cox test (D). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DTR: diphtheria toxin receptor; DT: diphtheria toxin; T-BsAb: T-cell-engaging bispecific antibody; BM: bone marrow; IFN: interferon.

Figure 6.

T-cell-engaging bispecific antibody-mediated activation of regulatory T cells in primary bone marrow samples from patients with multiple myeloma. (A) Bone marrow mononuclear cells (1×10⁶ cells) from newly diagnosed patients with multiple myeloma were stimulated with anti-human B-cell maturation antigen T-cell-engaging bispecific antibody (T-BsAb). The schematic illustrates the the experimental design. (B, C) Representative flow cytometry plots showing frequencies of CD4 T cells expressing FOXP3 and CD25 4 days after treatment (B). Graphs showing the CD8/FOXP3⁺CD25⁺CD4 ratio 4 days after treatment (C). (D, E) Representative histograms (D) and graphs (E) showing expression levels of CTLA-4 and ICOS in FOXP3⁺CD25⁺CD4 T cells (D). Numbers indicate mean fluorescence intensity. Results from one experiment (N=5) are shown. Differences were tested for statistical significance using a paired t-test. **P<0.01, ***P<0.001. (F) A graphical summary of the results. T-BsAb therapy triggers (i) differentiation of induced regulatory T cells from CD4 T cells, and (ii) activation of regulatory T cells, leading to suppression of CD8 T-cell responses. BM MNC: bone marrow mononuclear cells; MFI: mean fluorescence intensity; Treg: regulatory T cells; iTreg: induced regulatory T cells.