Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic antibodies in advanced Sézary syndrome

Abstract

Sézary syndrome (SS) is a rare form of cutaneous T-cell lymphoma often refractory to treatment. SS is defined as adenopathy, erythroderma with high numbers of atypical T cells. This offers an opportunity for new interventions and perhaps antibody-based therapeutic by virtue of its high expression of the TNFR2 oncogene on the tumor cells and on T-regulatory cells (T_regs). Potent human-directed TNFR2 antagonistic antibodies have been created that preferentially target the TNFR2 oncogene and tumor-infiltrating TNFR2⁺ T_regs. Here we test the therapeutic potential of TNFR2 antagonists on freshly isolated lymphocytes from patients with Stage IVA SS and from healthy controls. SS patients were on a variety of end-stage multi-drug therapies. Baseline burden T_reg/T effector (T_eff) ratios and the responsiveness of tumor and infiltrating T_regs to TNFR2 antibody killing was studied. We show dose-escalating concentrations of a dominant TNFR2 antagonistic antibody killed TNFR2⁺ SS tumor cells and thus restored CD26^- subpopulations of lymphocyte cell numbers to normal. The abundant TNFR2⁺ T_regs of SS subjects are also killed with TNFR2 antagonism. Beneficial and rapid expansion of T_eff was observed. The combination of T_reg inhibition and T_eff expansion brought the high T_reg/T_eff ratio to normal. Our findings suggest a marked responsiveness of SS tumor cells and T_regs, to targeting with TNFR2 antagonistic antibodies. These results show TNFR2 antibodies are potent and efficacious in vitro.

Fig. 1

Sézary syndrome patients exhibit characteristically high levels of CD26⁻ cells and T_regs with increased TNFR2 expression. a Percentage of CD26⁻ cells from freshly isolated peripheral human CD4⁺ cells of Sézary syndrome patients and healthy controls (n = 1 Subjects E, H, I; n = 2 Subjects A, D, C; n = 3 Subjects B, FG, and n = 10 Controls). b Proportion of CD26^+/− and CD7^+/− lymphocytes. c. Proportion of lymphocyte populations (CD26⁻, T_reg, and T_eff) and TNFR2 expression of all patients except samples from subjects with tumor cells > 90% of CD4+ (Subjects E, I) and controls (n = 11 Patients, n = 11 Controls). d Individual histograms showing the massive amounts of TNFR2 expression on either tumor containing CD26⁻ cells or T_reg, cells in a characteristic Sezary syndrome subject (pink) compared to a control subject (blue). Data are mean ± SEM, underlined asterisks indicate significant difference between patient and controls determined by Z test (95% CI)

Fig. 2

TNRF2⁺ CD26⁻ cells are reduced in response to treatment with TNFR2 antagonist. a Proportion of TNRF2⁺ CD26⁻ cells from Sézary syndrome patients (n = 15) after treatment with IL-2 (200 U/ml) and TNFR2 antagonist (0–125 µg/ml) for 48 to 72 h. b Proportion of TNRF2⁺ CD26⁻ cells from healthy controls (n = 11) treated as described in a. c Relative change in TNFR2⁺ CD26⁻ cells in patients and controls as described in a. d Relative change in TNFR2⁺ CD26⁻ cells in patients on various treatment regimens (Investigative Therapy A (n = 4), Investigative Therapy B (n = 3), methotrexate (n = 2), Control (n = 11). Data are mean ± SEM and asterisks indicates significant difference from baseline (TNFR2 antagonist 0 µg/ml (a, b) or 0.1 µg/ml (c)) (t test p < 0.05), and underlined asterisks indicate significant difference in patients versus controls (Z test 95% CI)

Fig. 3

TCR Vbeta clonal CD4+ cells of Sézary syndrome patients are inhibited in a dose-dependent manner by treatment with the TNFR2 antagonist antibody. a. Proportion of CD4+ TCR Vb-specific cells in patient versus control for Subject C (left) and Subject E (right). b. Relative change in the proportion of CD4+ Vb-specific clonal cells for Subject C (left) and Subject E (right). c. Relative change in the proportion of TNFR2+ Vb-specific cells for Subject C (left) and Subject E (Right) (bottom). Data are from a single representative experiment

Fig. 4

TNFR2 antagonist inhibits Tregs in Sézary syndrome and healthy control CD4+ cell culture. a. Proportion of Treg cells from patients (n = 15) after incubation of freshly isolated CD4+ cells with IL-2 and TNFR2 antagonist (0–125 µg/ml) for 48–72 hrs. b. Proportion of Treg cells from controls (n = 11) as described in (a). c. Relative change in Treg of patients and controls after culture with TNFR2 antagonist as described in (a). d. Comparison of baseline proportions of Treg from patients and controls treated with IL-2 alone or with TNFR2 agonist (12.5 µg/ml). Data are mean ± SEM, stars (*) indicate significant difference (T-test p<0.05) from baseline (TNFR2 antagonist 0 µg/ml (B) or 0.1 µg/ml (C)), and underlined stars (*) indicate significant difference in patients versus controls (Z-test 95%CI)

Fig. 5

TNFR2 antagonist enables continuous dose-dependent T_eff expansion in Sézary syndrome patient samples. a Proportion of T_eff cells from patients (n = 18 samples) after incubation of freshly isolated CD4⁺ cells with IL-2 and TNFR2 antagonist (0–125 µg/ml) for 48–72 h. b Proportion of T_eff cells from controls (n = 11) as described in a. c Relative change in T_eff of patients and controls after culture with TNFR2 antagonist as described in a. d Comparison of baseline proportions of T_eff from patients and controls. Data are mean ± SEM, asterisks indicate significant difference from baseline (TNFR2 antagonist 0.1 µg/ml; t test p < 0.05), and underlined asterisks indicate significant difference in patients versus controls (Z test 95%CI)

Fig. 6

TNFR2 antagonism corrects T_reg/T_eff ratio and reduces TNFR2⁺ tumor cells in Sézary syndrome patient samples regardless of underlying therapy. a Ratio of T_reg/T_eff in patients (n = 15 samples) and controls (n = 11 samples) after incubation of freshly isolated CD4⁺ cells with IL-2 and TNFR2 antagonist (0–125 µg/ml) for 48–72 h. b Ratio of T_reg/T_eff after TNFR2 antagonist treatment (0 and 12.5 µg/ml) as describe in a of early and late longitudinal samples from patients on various clinical treatment regimens. c Proportion of CD26⁻ of early and late patient samples at baseline (TNFR2 0 µg/ml). d Relative change in proportion of TNFR2⁺ CD26⁻ in samples treated with TNFR2 antagonist (12.5 µg/ml). Data are mean ± SEM and ns indicates no significant difference between patients and controls (t test p < 0.05)