CD25-targeted antibody-drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

Abstract

Background: Regulatory T cells (T_regs) contribute to an immunosuppressive tumor microenvironment. They play an important role in the establishment and progression of tumors with high T_regs infiltration and present a major obstacle to tumor eradication by immunotherapies. Numerous strategies have been attempted to deplete or block T_regs, although their success has been limited.

Methods: A CD25-targeted, pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) was investigated for its ability to deplete T_regs and induce antitumor immunity. Antitumor activity of CD25-ADC either alone or in combination with an anti-programmed cell death protein 1 (PD-1) antibody was evaluated in CD25-negative syngeneic models that exhibit tumor infiltration of CD25-expressing T_regs, and its pharmacodynamics and pharmacokinetics were assessed.

Results: Single low doses of CD25-ADC resulted in potent and durable antitumor activity in established syngeneic solid tumor models and the combination of a suboptimal dose was synergistic with PD-1 blockade. Tumor eradication by the CD25-targeted ADC was CD8+ T cell-dependent and CD25-ADC induced protective immunity. Importantly, while CD25-ADC mediated a significant and sustained intratumoral T_regs depletion, accompanied by a concomitant increase in the number of activated and proliferating tumor-infiltrating CD8+ T effector cells, systemic T_regs depletion was transient, alleviating concerns of potential autoimmune side effects.

Conclusions: This study shows that a PBD dimer-based, CD25-targeted ADC is able to deplete T_regs and eradicate established tumors via antitumor immunity. This represents a novel approach to efficiently deplete T_regs via a very potent DNA damaging toxin known to induce immunogenic cell death. Moreover, this study provides proof of concept for a completely new application of ADCs as immunotherapeutic agents, as the main mode of action relies on the ADC directly targeting immune cells, rather than tumor cells. These strong preclinical data warrant the clinical evaluation of camidanlumab tesirine (ADCT-301), a PBD-based ADC targeting human CD25, either alone or in combination with checkpoint inhibitors in solid tumors with known T_regs infiltration. A phase I trial (NCT03621982) of camidanlumab tesirine in patients with selected advanced solid tumors is ongoing.

Keywords: immunotherapy.

Figure 1

Structure and in vitro characterization of CD25-ADC. (A) Structure and (B) in vitro characterization of CD25-ADC. (i) ELISA showing binding of anti-CD25 antibody PC61 to mouse recombinant CD25. (ii–iv) Flow cytometry measurement of PC61 and isotype-control antibody binding to Yac-1, MC38 and CT26 cells. (v–vii) Yac-1, MC38 and CT26 cells’ viability after exposure to CD25-ADC and isotype-ADC (and the naked pyrrolobenzodiazepine-dimer SG3199 in MC38 and CT26 cell lines). MFI, median fluorescence intensity; PABA, para amino benzoic acid.

Figure 2

In vivo antitumor activity of CD25-ADC in the s.c. MC38 syngeneic model. Treatment with (i) vehicle, (ii) anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8), (iii) non-binding ADC (1 mg/kg, single dose on day 1) alone or (iv) in combination with anti-PD-1 antibody, (v–vii) CD25-ADC (0.1, 0.5, and 1 mg/kg single dose on day 1) alone or (viii–x) in combination with anti-PD-1 antibody, started at a group mean tumor volume of 103 mm³. Data are shown as tumor volumes (mm³) over time for each individual mouse (10 mice/group). (xi) Survival of mice shown in i–x. Lines for G4, G5, G8 and G9 are overlapping.

Figure 3

In vivo antitumor activity of CD25-ADC in the s.c. CT26 syngeneic model. Treatment with (i) vehicle, (ii) anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8), (iii) isotype-ADC (1 mg/kg, single dose on day 1) alone or (iv) in combination with anti-PD-1 antibody, (v–vii) CD25-ADC (0.1, 0.5, and 1 mg/kg single dose on day 1) alone or (viii–x) in combination with anti-PD-1 antibody, started at a group mean tumor volume of 110 mm³. Data are shown as tumor volumes (mm³) over time for each individual mouse (10 mice/group). (xi) Survival of mice shown in i–x.

Figure 4

Role of CD8+ T_eff cells in CD25-ADC antitumor activity in the MC38 syngeneic model. Depletion of CD8+ T_eff cells significantly reduces the antitumor activity of CD25-ADC. CD25-ADC was administered intraperitoneally (i.p.) at a group mean tumor volume of 89 mm³ as a single dose on day 1 at 0.5 mg/kg alone or in combination with anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8). Anti-CD8 T-cell depleting antibody (10 mg/kg) was injected i.p. on days 0, 5, 8, and 13. Data are shown as mean tumor volumes (mm³) ± SEM over time (n=10/group).

Figure 5

Intratumoral T-cell immunophenotype analysis in MC38-bearing mice. (A) Absolute quantification of intratumoral T_regs, CD8+ T cells and CD8+/T_reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. (B) Percentage of CD69+, Ki67+ and IFNγ+ tumor-infiltrating CD8+ T cells. Tumors were processed at the indicated times (days post CD25-ADC dose). Horizontal bars represent median value. Statistical differences between treatment groups were calculated using JMP 15 by the Dunn method for joint ranking. Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01. IFN, interferon.

Figure 6

Circulating and thymic T-cell immunophenotype analysis in MC38-bearing mice. (A) Absolute quantification of circulating T_regs, CD8+ T cells and CD8+/T_reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. Blood was processed at the indicated times (days post CD25-ADC dose). (B) Absolute quantification of thymic T_reg cells and CD8+/T_reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. Thymus was processed at the indicated times (days post CD25-ADC dose). Statistical differences between treatment groups were calculated using JMP 15 by the Dunn method for joint ranking. Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01.

Figure 7

T-cell dynamic study in non-tumor-bearing mice. Effect of CD25-ADC on the percentage of T_regs and T_eff levels in non-tumor-bearing mice. Female C57BL/6 mice were injected i.p. with vehicle, CD25-ADC (0.5 mg/kg), or isotype control ADC (0.5 mg/kg) on day 0. (A) Spleen, (B) lymph node, and (C) thymus were collected 4 hours post dose, and 6, 13, and 20 days post dose for T-cell immune profiling. Levels of T_regs, CD8+ T, and conventional CD4+ T cells in spleen, lymph nodes, and thymus are presented as % of CD45 cells±SEM over time.