Differential expression of CCR8 in tumors versus normal tissue allows specific depletion of tumor-infiltrating T regulatory cells by GS-1811, a novel Fc-optimized anti-CCR8 antibody

Abstract

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

Keywords: CCR8; PD-1 resistance; T regulatory cells; cancer immunotherapy; treg depletion.

Figure 1.

CCR8 expression is highly restricted to tumor-infiltrating T regulatory cells in human tumors of different lineage. a) Schematic of the Treg bioinformatic screen used to identify putative targets for Treg depletion. b) t-SNE plots displaying single cell RNAseq gene expression profiles from 43 patients (HCC n=9, HNSCC n=18, melanoma n=19) including tumors, blood, LN and normal tissue that were combined and characterized by cell marker genes, as indicated. c) Gene expression analysis on bulk RNA-seq (TCGA) from primary tumor samples (red bars) and adjacent normal tissues (gray bars). The degree of statistical significance between tumor and adjacent normal samples is indicated below each pair of boxplots with tumor types (***: p<0.0001; **: p<0.01; *: p<0.05 from unpaired, one-tailed Mann-Whitney U tests). d) Flow cytometry analysis of peripheral blood mononuclear cells (PBMC) from healthy donors and indicated freshly dissociated human tumor samples. FoxP3 staining was used to identify T cell populations within CD45/CD3/CD4+ cells, and CCR8 expression was evaluated in Treg (FoxP3+, red histograms) and non-Treg (Tconv, FoxP3-, blue histograms) cells. Isotype control histograms are shown in black for total CD3+ T cells. Healthy PBMC data is representative of 3 independent donors. e) Percentage of CCR8+ cells (left) and median density of CCR8 molecules per cell (right) in Tconv (blue), Treg (red) and CD8 T cells (black) in freshly dissociated human tumor samples (Tumor) or normal adjacent tissue (NAT). Indications include breast, head and neck, lung, ovarian, colon, and bladder cancer. T cell populations were identified as in (d). Each data point represents a single sample. Error bars represent standard deviation from the average. The degree of statistical significance between populations indicated on top of the graphs (****: p<0.0001; **: p<0.01; *: p<0.05; ns: no significant difference).

Figure 2.

Anti-mouse CCR8 antibody treatment results in robust antitumor efficacy in mouse tumor models and synergizes with PD-1 blockade. Tumor growth analysis of cohorts of mice bearing MC38 (a), Pan02 (b), CT26 (c) or MBT-2 (d) tumors and treated with the indicated antibodies. Mice were randomized into treatment groups when average tumor volume was approximately 100mm3, except in (b) where average tumor volume at randomization was either 100mm³ or 250 mm³. Day0 indicates day of first dose. Tumor growth curves show average +/- SEM; CR corresponds to the number of complete responses in each group.

Figure 3.

Depletion of CCR8+ tumor Tregs is required for antitumor efficacy of anti-mouse CCR8 Ab in the MC38 model. a) Tumor growth (left) and survival (right) analysis of cohorts of C57BL/6 mice bearing MC38 tumors and treated with the indicated antibodies. Mice were randomized into treatment groups when average tumor volume was approximately 100mm3. Day0 indicates day of first dose (left) or tumor inoculation (right). Tumor growth curves show average +/- SEM; CR corresponds to the number of complete responses in each group. Survival p values were obtained with a pairwise Log-rank (Mantel-Cox) test (****: p<0.0001). b) Flow cytometry analysis on tumor immune infiltrate and spleens from MC38 tumor-bearing mice treated with either one (Day 3) or two (Day 7) doses of the indicated antibodies. Days refer to time after first dose. Graphs show the percentages of Tregs (CD4+CD25+FoxP3+) out of live CD45+CD3+ cells in the tumor (right) and spleen (left). c) IHC analysis of FoxP3+ cells in MC38 tumors from mice treated with the indicated antibodies as in (b). Representative images are shown on the left and summary graphs on the right. In (b) and (c) data is presented as average +/-SEM. ANOVA was used to compare the three treatment arms, followed by Tukey's multiple comparison post hoc test to evaluate differences between the individual treatment arms. p values are shown for statistically significant differences (*: p=0.01 to 0.05; **: p=0.001 to 0.01; ***: p<0.001).

Figure 4.

Depletion of CCR8+ tumor Tregs induces CD8 infiltration, proinflammatory responses and immunological memory. a) Flow cytometry analysis on tumor immune infiltrate from MC38 tumor-bearing mice treated with either one (Day 3) or two (Day 7) doses of the indicated antibodies. Days refer to time after first dose. Graphs show the percentages of CD8+ T cells out of live CD45+CD3+ cells (right) and the ratio of CD8/Treg cells (left) in the tumors. b) IHC analysis of CD8+ cells in MC38 tumors from mice treated with the indicated antibodies as in (a). Representative images from the Day7 group are shown on top, summary graphs on the bottom. In (a) and (b) data is presented as average +/-SEM. ANOVA was used to compare the three treatment arms, followed by Tukey's multiple comparison post hoc test to evaluate differences between the individual treatment arms. p values are shown for statistically significant differences (*: p=0.01 to 0.05; **: p=0.001 to 0.01; ***: p<0.001). c) NanoString gene expression analysis on MC38 tumors from mice treated with a single dose of anti-mouse CCR8 Ab in the mIgG2a or mIgG1 format or with isotype control; n=4 per group, evaluated at day 3 after dosing. Top: volcano plot showing differential gene expression between anti-mouse CCR8 mIgG2a and isotype groups; bottom: genes with greater than one log fold-change in expression in anti-CCR8 mIgG2a compared to anti-CCR8 mIgG1 and isotype samples. d)Tumor growth upon rechallenge in MC38 tumor-bearing mice that were fully cured by anti-mouse CCR8 antibody treatment (red) or in naïve mice as control (black). Mice were inoculated with MC38 and B16-F10 tumor cells on contralateral flanks. Curves represent average +/- SEM.

Figure 5.

Human anti-CCR8 mAb GS-1811 binds specifically to human CCR8 and antagonizes its activity. a) Specific binding of GS-1811 to CHO-S cells expressing hCCR8, hCCR4 or to control parental cell line as determined by flow cytometry. Each data point represents average of three replicates ± SD. b) Antagonistic activity of GS-1811 in the presence of human CCL-1, evaluated by the PathHunter eXpress human CCR8 CHO-K1 ßArrestin GPCR Assay. Each data point represents average of three replicates ± SD. c) Summary of GS-1811 on-cell affinity as measured by MSD assay. Mean dissociation constant (KD) and standard deviation (SD) were calculated from 3 independent experiments. d) Flow cytometry analysis showing GS-1811 binding to different T cell populations from healthy PBMCs or human primary colon tumor samples; left: representative histograms; right: percentage of CCR8+ cells in Tconv (blue), Treg (red) and CD8 T cells (black) in 8 tumor samples. T cell subsets were identified as in Figure 1. Error bars represent standard deviation from the average. The degree of statistical significance between populations indicated on top of the graph (****: p<0.0001).

Figure 6.

GS-1811 specifically mediates depletion of cells expressing CCR8 at densities comparable to tumor-infiltrating Tregs. a) In vitro ADCC assay with a fixed concentration (1μg/mL) of afucosylated or fucosylated (Fuc) versions of GS-1811 mAb against CHO cell lines expressing increasing densities of human CCR8 in presence of freshly isolated NK cells from healthy human donors (NK : target cell ratio, 5 : 1). Data is from one representative NK donor; error bars represent SD from technical triplicates. The dotted vertical lines represent average CCR8 densities in normal peripheral and tumor Tregs. b) ADCC activity measured against target cells expressing 2,500 CCR8 molecules/cell, with increasing concentrations of afucosylated or fucosylated (Fuc) chimeric version of GS-1811. One representative NK donor is shown in the graph. EC50 values (in μg/mL) from 10 independent donors are reported in the table on the bottom. c) GS-1811 ADCC activity on Hut78 cells endogenously expressing human CCR8. The curve is from one representative NK donor; error bars represent SD from technical triplicates. d) GS-1811 ADCC activity on TILs from primary human tumor samples in the presence of added NK cells from healthy donors. Tregs were identified as CD4+FoxP3+ out of live CD45+CD3+ cells. Data from one representative NK cell donor; error bars represent SD from technical duplicates.