Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

Abstract

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology.

Keywords: CD25; Fc gamma receptors; Treg depletion; anti-CD25; anti-PD-1; inhibitory Fc receptor; regulatory T cells; tumor immunotherapy; tumor microenvironment.

Graphical abstract

Figure 1

Anti-CD25-r1-Mediated Depletion of CD25⁺ Regulatory T Cells Is Restricted to Blood and Lymph Nodes (A–C) Mouse LNs and TILs were analyzed by flow cytomery 10 days after MCA205 (n = 10), MC38 (n = 5), B16 (n = 3), or CT26 (n = 3) tumor implantation. (A) CD25 expression on T cell subsets in representative mice. Dotted lines indicate the gate. (B and C) Percentage (B) and MFI (C) of CD25 in each T cell subset. Error bars show standard error of the mean (SEM). p values obtained by two-way analysis of variance (ANOVA). (D–G) Tumor-bearing mice were injected with 200 μg of αCD25-r1, αCD25-m2a, or αCTLA-4 on days 5 and 7 after MCA205 tumor implantation. Blood, LNs, and TILs were harvested and processed on day 9 for flow cytometry analysis. (D) Representative plots showing expression of CD25 (detected with antibody clone 7D4) and FoxP3 in CD3⁺CD4⁺ T cells. Numbers show percentage of cells in each quadrant. (E) MFI of CD25 in CD4⁺FoxP3⁺ Treg cells. (F) Percentage of FoxP3⁺ Treg cells of total CD3⁺CD4⁺ T cells. (G) CD8⁺/Treg cell ratios (n = 10). Experiment was repeated three times.

Figure 2

FcγRIIb Inhibits αCD25-r1-Mediated Treg Cell Depletion in Tumors (A and B) Expression of FcγRs was measured by flow cytometry in leukocytes from blood, spleen, LNs, and MCA205 tumors (TIL) 10 days after tumor implantation. (A) Expression of FcγRs on granulocytes (CD11b⁺Ly6G⁺), conventional dendritic cells (cDCs) (CD11c^hiMHC-II⁺), and monocyte/macrophages (Mono/Mφ) (CD11b⁺Ly6G⁻NK1.1⁻CD11c^lo/neg). Dotted lines indicate the gate, numbers show the percentage of positive cells. (B) Cumulative data of FcγR expression in cell subpopulations (n = 3). Error bars represent SEM; the experiment was repeated three times. (C) Binding affinity of rat IgG1 and mouse IgG2a isotypes to individual mouse FcγRs as determined by surface plasmon resonance (SPR). (D–G) Percentage of CD4⁺FoxP3⁺ Treg cells of total CD4⁺ T cells in TILs of wild-type (WT, n = 5–10), Fcer1g^−/− (n = 10), Fcgr3^−/− (n = 5), Fcgr4^−/− (n = 10), or Fcgr2b^−/− (n = 5) mice treated as in Figures 1D–1G.

Figure 3

Synergistic Effect of Anti-CD25-m2a and Anti-PD-1 Combination Results in Eradication of Established Tumors Tumor-bearing mice were treated with 200 μg of αCD25 on day 5 and 100 μg of αPD-1 on days 6, 9, and 12 after tumor implantation. (A) Growth curves of individual MCA205 tumors, showing the product of three orthogonal tumor diameters. The number of tumor-free survivors is shown in each graph. (B) Survival of mice shown in (A). (C and D) Survival of mice with MC38 or CT26 tumors treated as described above (n = 10 per condition). (E) Percentage of Ki67⁺ cells in tumor-infiltrating CD4⁺FoxP3⁻ and CD8⁺ T cells. (F) CD4⁺FoxP3⁻/CD4⁺FoxP3⁺ and CD8⁺/CD4⁺FoxP3⁺ cell ratios. (G and H) Representative histograms (G) and percentage (H) of IFN-γ-producing CD4⁺ and CD8⁺ TILs in MCA205 tumors determined by intracellular staining after ex vivo re-stimulation with PMA and ionomycin. Graphs show cumulative data of two separate experiments (n = 10).

Figure 4

CD25 Is Highly Expressed on Treg Cell Infiltrating Human Tumors (A) Representative histograms demonstrating CD25 expression on circulating (PBMC) and tumor-infiltrating (TIL) CD8⁺, CD4⁺FoxP3⁻, and CD4⁺FoxP3⁺ T cell subsets. Dotted lines indicate the gate. (B and C) Quantification of CD25 expression (percentage [B] and MFI [C]) on individual T cell subsets in human melanoma (n = 11), NSCLC (n = 9), and RCC (n = 8). Error bars represent SEM; p values obtained by two-way ANOVA. (D) Longitudinal analysis of CD25 expression in human melanoma and RCC lesions prior to (“Baseline”) and during PD-1 blockade (“On therapy”). CD8 staining is displayed in red, FoxP3 in blue, and CD25 in brown. (E) Percentage of CD25 expression on CD8⁺ and FoxP3⁺ T cells at baseline and during PD-1 blockade. Plotted values derive from analysis of 10 ×40 high-power fields per patient at each time point.