Journal: Clinical and Experimental Medicine

Article Title: Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody

doi: 10.1007/s10238-024-01362-8

Figure Lengend Snippet: CCR8 is predominantly expressed in activated tumor-infiltrating Tregs. A Expression of CCR8 across a variety of human cancer and immune cell types as determined by gene expression analysis of single-cell RNA-Seq datasets. Rows indicate cancer data sets and corresponding indications and columns denote immune cell types. Blue to red colors indicate the average expression of CCR8 across all cells assigned to a cell type. White squares indicate the absence of a cell type from a given dataset. NSCLC, non-small cell lung carcinoma; MEL, melanoma; HCC, hepatocellular carcinoma; ENC, endometrial carcinoma; CRC, colorectal cancer BRC, breast cancer; DC, dendritic cell; MAIT, mucosal-associated invariant T cell; NK, natural killer cell. B Differential gene expression between activated Tregs and resting Tregs or other immune cell types. X- and Y-axes represent linear expression fold changes. Highlighted genes were further analyzed in C by flow cytometry. C Comparison of surface expression of CCR8 protein and other common Treg receptors as measured by flow cytometry. Human PBMCs were activated using anti-CD3/anti-CD28 stimulation for 6 days and gated for Treg (CD4+CD25+FoxP3+CD127 low ), as well as CD8+ and CD4+ effector T cell populations ( n = 2 donors). In contrast to the other markers, CCR8 expression was largely limited to the activated Treg population

Article Snippet: Treg cells were isolated from human PBMCs of healthy donors using magnetic-based EasySepTM Human CD4+ CD127 low CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) and subsequently expanded and activated in vitro by treatment with IL-2 (130–097-745, Miltenyi Biotec) and CD3/CD28 antibodies containing TransACT (130–128-758, Miltenyi Biotec) to induce CCR8 expression.

Techniques: Expressing, Gene Expression, RNA Sequencing, Flow Cytometry, Comparison

Journal: Clinical and Experimental Medicine

Article Title: Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody

doi: 10.1007/s10238-024-01362-8

Figure Lengend Snippet: Robust preclinical antitumor activity of anti-mouse CCR8 antibodies is mediated via Fc-mediated effector functions of antibodies inducing ADCC and ADCP. A ADCC activity of anti-mouse CCR8 mIgG2a, hIgG1, and N297A-aglycosylated hIgG1 antibody variants and respective non-binding isotype controls, in co-culture of mouse CCR8 expressing HEK293 target cells and primary mouse NK cells as effector cells, at effector/target (E:T) cell ratio of 10:1 ( n = 2). Cytotoxicity was determined by measuring target cell apoptosis induction relative to the no-antibody control at the 20-h co-culture time point. B ADCP activity (real-time) of anti-mouse CCR8 mIgG2a, hIgG1, and N297A-aglycosylated hIgG1 antibody variants and respective non-binding isotype controls, in co-cultures of mouse CCR8 expressing HEK293 target cells and mouse M2 macrophages as effector cells, at effector/target (E:T) cell ratio of 4:1 ( n = 3). The percentage of phagocytosed target cells was determined by measuring phagocytosis relative to no-antibody control at 4-h co-culture time point. C Tumor growth inhibition in mice bearing CT26 tumor and treated with the anti-mouse CCR8 mIgG2a, hIgG1, and N297A-aglycosylated hIgG1 antibody variants, and the respective non-binding isotype controls (all at 10 mg/kg, Q3/4D, i.p., n = 10 mice/group). Black arrows indicate antibody treatment days (days 7, 11, 15, and 18) relative to tumor inoculation. Treatment with anti-mouse CCR8 mIgG2a and hIgG1 antibodies resulted in treatment/control (T/C) values of 0.11 ( p < 0.001) and 0.02 ( p < 0.001), respectively. T/C: treatment/control ratio calculated from mean tumor volumes at study end. Statistical analysis was performed using an ANOVA model with contrasts. *** p < 0.001 compared with the respective isotype controls. Q3/4D: every third or fourth day (twice weekly), i.p.: intraperitoneally. D-E Flow cytometric analysis of D intratumoral Tregs (CD45+CD4+CD25+FoxP3+) and E CD8+ T cells in CT26 tumor lysates from study described above ( C ). Tumors were collected 24 h after the second treatment dose ( n = 6 mice/group). Statistical analysis was performed using an ANOVA model with contrasts. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the respective isotype control. F Calculated ratio of intratumor CD8 + T cells to Tregs (CD45+CD4+CD25+FoxP3+) as calculated based on data in (D) and (E), 24 h after second antibody treatment ( n = 6 mice/group)

Article Snippet: Treg cells were isolated from human PBMCs of healthy donors using magnetic-based EasySepTM Human CD4+ CD127 low CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) and subsequently expanded and activated in vitro by treatment with IL-2 (130–097-745, Miltenyi Biotec) and CD3/CD28 antibodies containing TransACT (130–128-758, Miltenyi Biotec) to induce CCR8 expression.

Techniques: Activity Assay, Binding Assay, Co-Culture Assay, Expressing, Control, Inhibition

Journal: Clinical and Experimental Medicine

Article Title: Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody

doi: 10.1007/s10238-024-01362-8

Figure Lengend Snippet: Correlation of the intratumoral Treg depletion efficacy with antitumor responses and plasma exposure of anti-mouse CCR8 antibodies. A Correlation between the intratumoral Treg depletion and in vivo antitumor efficacy and anti-mouse CCR8 antibodies (hIgG1 and mIgG2a), across three independent studies using two different mouse tumor models (CT26 and EMT6). Treg depletion efficacy was analyzed by flow cytometry 24 h after second treatment dose and determined in percentages (%) relative to isotype control ( n = 5–6 mice/group). T/C ratio calculated from mean tumor volumes at study end ( n = 10 mice/group). The solid and dashed lines indicate the regression line and the associated 95% confidence intervals, respectively. B Correlation between the plasma exposure of anti-mouse CCR8 mIgG2a antibody and depletion of intratumoral CCR8 + Tregs (CD4+CD25+FoxP3+CCR8+) in EMT6 tumor-bearing mice treated with a single dose of antibody (0.25, 1, or 4 mg/kg; i.p.), as determined on different time points upon treatment (2, 24, 48, 120, 192 and 336 h, n = 5 mice/group/time point)

Article Snippet: Treg cells were isolated from human PBMCs of healthy donors using magnetic-based EasySepTM Human CD4+ CD127 low CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) and subsequently expanded and activated in vitro by treatment with IL-2 (130–097-745, Miltenyi Biotec) and CD3/CD28 antibodies containing TransACT (130–128-758, Miltenyi Biotec) to induce CCR8 expression.

Techniques: Clinical Proteomics, In Vivo, Flow Cytometry, Control

Journal: Clinical and Experimental Medicine

Article Title: Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody

doi: 10.1007/s10238-024-01362-8

Figure Lengend Snippet: Combination of CCR8 + Treg depletion with anti-PD-L1 immune checkpoint blockade results in enhanced antitumor activity. A EMT6 tumor-bearing mice were treated with anti-mouse CCR8 mIgG2a antibody and with the respective non-binding isotype control, and intratumoral IFNγ protein concentration was measured with ELISA on day 19 at study end ( n = 5). ** p < 0.01 indicate statistical significance in comparison to isotype control. B Intratumoral PD-L1 (CD274) expression as determined by RNA-Seq upon treatment of PANC02, Hepa1-6, MBT2, MC38, H22, and CT26 syngeneic tumor-bearing mice with anti-mouse CCR8 mIgG2a and hIgG1 antibody variants and with the respective non-binding isotype controls ( n = 8–10 mice/group). Log2 fold change expressions were 1.375 in PANC02, 1.453 in Hepa1-6, 1.963 in MBT2, 2.669 in MC38, 4.516 in H22 and 5.202 in CT26 model. C Efficacy of anti-mouse CCR8 mIgG2a antibody and anti-PD-L1 antibody as monotherapy and as combination treatment in syngeneic MC38 murine tumor model (also referred as C38). Tumor growth in mice treated with non-binding isotype controls mIgG2a (10 mg/kg, Q3/4Dx5, i.p.) and mIgG1 (3 mg/kg, Q3/4Dx5, i.p.), anti-mouse CCR8 mIgG2a antibody (10 mg/kg, Q3/4Dx5, i.p.), anti-PD-L1 mIgG1 antibody (3 mg/kg, Q3/4Dx5, i.p.), or the combination of anti-mouse CCR8 mIgG2a (10 mg/kg, Q3/4Dx5, i.p.) and anti-PD-L1 mIgG1 antibodies (3 mg/kg, Q3/4Dx5, i.p.). Black arrows indicate treatment days (days 10, 13, 17, 20, and 24) relative to tumor inoculation ( n = 10 mice/group). Treatment with anti-mouse CCR8 mIgG2a and anti-PD-L1 antibodies resulted in T/C values of 0.17 ( p < 0.001) and 0.38 ( p < 0.001), respectively. Combinatorial treatment with anti-mouse CCR8 mIgG2a and anti-PD-L1 antibodies resulted in T/C of 0.02 ( p < 0.001). Statistical analysis was performed using an ANOVA model with contrasts. *** p < 0.001 is significance in comparison to isotype controls, § p < 0.05 in comparison to anti-PD-L1 monotherapy. D Survival of mice described in (C). The treatments started on day 10 and the last treatment doses were given on day 24. Mice were sacrificed individually when they met the predefined termination criteria (tumor area of 220 mm 2 ). The survival times from inoculation to sacrifice are presented as Kaplan–Meier survival plots. Asterisks, hashtags, and section signs indicate statistical significance in comparison to isotype controls, anti-mouse CCR8 monotherapy, and anti-PD-L1 monotherapy, respectively ( # p < 0.05, ***, §§§ p < 0.001; n = 10 mice/group). E Intratumoral Tregs (CD4+CD25+FoxP3+) and F ratio of CD8 + T cells to Tregs were determined by flow cytometry in MC38 tumors of mice treated as described in (C). Tumors were collected 24 h after the second treatment dose on day 14. Asterisks and section signs indicate statistical significance in comparison to isotype control and anti-PD-L1 monotherapy, respectively (*, § p < 0.05, ** p < 0.01, n = 5/group)

Article Snippet: Treg cells were isolated from human PBMCs of healthy donors using magnetic-based EasySepTM Human CD4+ CD127 low CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) and subsequently expanded and activated in vitro by treatment with IL-2 (130–097-745, Miltenyi Biotec) and CD3/CD28 antibodies containing TransACT (130–128-758, Miltenyi Biotec) to induce CCR8 expression.

Techniques: Activity Assay, Binding Assay, Control, Protein Concentration, Enzyme-linked Immunosorbent Assay, Comparison, Expressing, RNA Sequencing, Flow Cytometry