Differential control of human Treg and effector T cells in tumor immunity by Fc-engineered anti-CTLA-4 antibody

Abstract

Anti-CTLA-4 mAb is efficacious in enhancing tumor immunity in humans. CTLA-4 is expressed by conventional T cells upon activation and by naturally occurring FOXP3⁺CD4⁺ Treg cells constitutively, raising a question of how anti-CTLA-4 mAb can differentially control these functionally opposing T cell populations in tumor immunity. Here we show that FOXP3^high potently suppressive effector Treg cells were abundant in melanoma tissues, expressing CTLA-4 at higher levels than tumor-infiltrating CD8⁺ T cells. Upon in vitro tumor-antigen stimulation of peripheral blood mononuclear cells from healthy individuals or melanoma patients, Fc-region-modified anti-CTLA-4 mAb with high antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) activity selectively depleted CTLA-4⁺FOXP3⁺ Treg cells and consequently expanded tumor-antigen-specific CD8⁺T cells. Importantly, the expansion occurred only when antigen stimulation was delayed several days from the antibody treatment to spare CTLA-4⁺ activated effector CD8⁺T cells from mAb-mediated killing. Similarly, in tumor-bearing mice, high-ADCC/ADCP anti-CTLA-4 mAb treatment with delayed tumor-antigen vaccination significantly prolonged their survival and markedly elevated cytokine production by tumor-infiltrating CD8⁺ T cells, whereas antibody treatment concurrent with vaccination did not. Anti-CTLA-4 mAb modified to exhibit a lesser or no Fc-binding activity failed to show such timing-dependent in vitro and in vivo immune enhancement. Thus, high ADCC anti-CTLA-4 mAb is able to selectively deplete effector Treg cells and evoke tumor immunity depending on the CTLA-4-expressing status of effector CD8⁺ T cells. These findings are instrumental in designing cancer immunotherapy with mAbs targeting the molecules commonly expressed by FOXP3⁺ Treg cells and tumor-reactive effector T cells.

Keywords: CTLA-4; FOXP3; antibody-dependent cell-mediated cytotoxicity; cancer immunotherapy; regulatory T cells.

Fig. 1.

Preferential CTLA-4 expression by FOXP3^hi eTreg cells in melanoma tissue. (A and B) Representative plots of CD4⁺ T cell staining (A) and frequencies of each fraction (B). PBMCs of healthy donors (HD, n = 5), and PBMCs and TILs of melanoma patients (n = 11 and 14, respectively) were subjected to direct staining for CD4, CD45RA, and FOXP3. (C and D) Representative plots of CCR7 and CD45RA expression by CD8⁺ T cells (C) and summary of each CD8⁺ T cell subsets (D). (E) CTLA-4–expressing clusters (dotted line) in PBMCs and TILs from melanoma patients by CyTOF analysis. viSNE plots of CD4⁺ and CD8⁺ T cells with heat maps for indicated marker expression levels are shown (n = 2). (F) Surface only or surface and intracellular staining of CTLA-4 in HD PBMC. (G) Surface CTLA-4 expressions by each cellular fraction from TILs and PBMCs of melanoma patients. Means ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 by one-way ANOVA with post hoc Tukey’s HSD test.

Fig. 2.

Concurrent in vitro peptide stimulation with ART-Fc anti–CTLA-4 mAb restricts antigen-specific CD8⁺ T cell responses. (A) Summary of amino acid substitutions made to the Fc portion of anti-human CTLA-4 IgG1 mAb (clone MDX-010, ipilimumab) to generate ART-Fc or silent-Fc mAbs, and their affinities to inhibitory FcγRIIb or activatory FcγRIIIa. Silent-Fc mAb affinity was below the detection limit of BIACORE assay. (B) ADCC activity of various anti–CTLA-4 mAbs against human CTLA-4–expressing CHO cells. Anti–CTLA-4 mAbs (ART, ADCC-enhanced anti–CTLA-4 ART-Fc; IgG1, conventional unmodified anti–CTLA-4 IgG1; Silent, Fc-region-inactivated anti-CTLA-4 silent-Fc) were incubated with CHO cells and HD PBMCs (n = 3) at an E/T ratio of 50:1. Means ± SEM. Asterisks indicate significant differences between each antibody and silent-Fc at respective concentrations for 6-h (black) or 24-h (red) cultures. (C) ADCC activity to each cellular fraction of CD4⁺ and CD8⁺ T cells from PBMCs by ART-Fc, IgG1, or silent-Fc anti–CTLA-4 mAbs (n = 4 or 5). Frequencies of dead cells among CTV prelabeled cells after 24-h culture are indicated. (D) CTLA-4 expression by Fr. II eTreg cells after 9 d of Melan-A peptide stimulation and 1 μg/mL ART-Fc, silent-Fc, or IgG1 anti-CTLA-4 mAb treatment to whole PBMCs (n = 7). (E and F) Surface CTLA-4 expressions by Tet⁺ and Tet⁻ CD8⁺ T cells after indicated peptide stimulations. Representative histograms (E) and summary of mean flouorescence intensity (MFI) (F) (Melan-A, n = 9; CMV, n = 9; Flu, n = 3; ESO-1, n = 4). (G and H) Representative plots and frequencies of Fr. II eTreg cells (G) and antigen-specific CD8⁺ T cells (H) from HDs (n = 6) or melanoma patients (n = 5) after the culture with indicated peptide and 1 μg/mL ART-Fc anti–CTLA-4 mAb as in D. (I) Quantification of the frequency of eTreg cells (Upper) and Tet⁺ CD8⁺ T cells (Lower) after 9 d of Melan-A or CMV antigen stimulation to HD PBMCs with indicated anti-CTLA-4 mAbs in G and H. Means ± SEM. Depicted significance between two groups were calculated using paired t test, one-way ANOVA, or two-way ANOVA with post hoc Tukey’s HSD test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Fig. 3.

Predepletion of Treg cells using ART-Fc anti–CTLA-4 mAb enhances self/tumor antigen-specific CD8⁺ T cell responses. (A–D) Antigen-specific CD8⁺ T cell were induced from whole PBMCs of HDs (n = 7) or patients (n = 5) after 5 d of pretreatment with ART-Fc anti–CTLA-4 mAb. (A) Scheme of the experiment and representative FOXP3 and CD45RA staining of CD4⁺ T cells at day −5 and day 0 of the experiment. Summary of Fr. II eTreg frequencies at day 0 (Bottom). (B and C) Representative FOXP3 staining of CD4⁺ T cells (B) and antigen-specific CD8⁺ T cells (C) with or without ART-Fc pretreatment for each peptide after the culture. Summary of eTreg and antigen-specific CD8⁺ T cell frequencies after the culture (Bottom). (D) Summary of Fr. II eTreg and Tet⁺ CD8⁺ T cell frequencies with silent-Fc anti–CTLA-4 mAb pretreatment instead of ART-Fc as in A–C. (E) Expression levels of CD80 and CD86 by Lin-1⁻CD11c⁺HLA-DR⁺ DCs after 5 d of Melan-A stimulation with the pretreatment of indicated anti–CTLA-4 mAbs (n = 6). Numbers on histograms show MFI. (F) Summary of eTreg and Tet⁺CD8⁺ T cell frequencies after pretreatment with indicated anti–CTLA-4 mAbs followed by Melan-A_26–35 stimulation as in A–C. Means ± SEM. Depicted significance between two groups were calculated using paired t test. *P ≤ 0.05.

Fig. 4.

Pretreatment with ADCC/ADCP-enhanced anti–CTLA-4 mAb induces strong antitumor effects in mice. (A) Summary of amino acid substitutions made to the Fc portion of anti-mouse CTLA-4 IgG2a mAb (clone UC10; mIgG2a) to generate ADCC/ADCP-enhanced anti-mouse–CTLA-4 (mEnhanced) or silent-Fc anti-mouse–CTLA-4 (mSilent) mAbs, and their affinities to mouse FcγRIV are shown. mSilent mAb affinity was below the detection limit of BIACORE assay. (B–D) Scheme of the experiment (B). Mice were challenged with 2 × 10⁶ CMS5a fibrosarcoma expressing human NY-ESO-1 on day 0, and left untreated or treated with mEnhanced, mIgG2a, or mSilent anti-mouse–CTLA-4 mAb on day 9. Vaccination of NY-ESO-1_81–88 peptides mixed with CFA was performed on the same day (day 9, concurrent treatment) or 3 d later (day 12, Ab pretreatment). Tumor growth of each mice with bold line indicating the average of each treatment group (C) (n = 8–13 per group). Summary of average tumor growth of each treatment groups (D, Left). Asterisks indicate P values between mEnhanced mAb pretreatment group by two-way ANOVA with Tukey’s multiple comparison test. Summary of survival rate (D, Right) of each treatment groups. P values between the survival of each groups and the survival of vaccine-alone control were calculated by log-rank test. Death event corresponds to tumor length over ≥200 mm or death of the mouse. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Fig. 5.

Ag-specific CD8⁺T cell responses induced in tumor-bearing mice by pretreatment with ADCC/ADCP-enhanced anti–CTLA-4 mAb. (A–G) TILs from Fig. 4 were analyzed 19 d after tumor implantation (n = 4–6 per group). The number of infiltrated T cells per gram of tumor (A). CD8/Treg cell ratio (B). Frequency of Foxp3⁺Treg cells among CD4⁺ T cells (C). Representative plots of IL-2⁺ IFN-γ⁺ CD8⁺ T cells (D). Cytokine production was measured by restimulating TILs with ESO-1_81–88 peptides in vitro for 5 h. Summary of Tet⁺ CD8⁺ T cells in the tumor, and IL-2⁺ IFN-γ⁺ CD8⁺ T cells in D after the restimulation (E). Representative plots (F) and summary (G) of CD44 and CD62L expressions by NY-ESO-1-Tet⁺ CD8⁺ T cells in the tumor of each treatment groups. Data were obtained from three independent experiments. P values between Naïve or EM CD8⁺ T cell subsets of each conditions. Means ± SEM. *P ≤ 0.05, ^†P ≤ 0.05, **P ≤ 0.01, ^††P ≤ 0.01, and ***P ≤ 0.001 by two-way ANOVA with Tukey’s multiple comparison test.