human igg1 fc domain r d systems  (R&D Systems)

Journal: Science immunology

Article Title: Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy.

doi: 10.1126/sciimmunol.add8005

Figure Lengend Snippet: Fig. 1. IgG scaffolds of the FDA-approved PD-L1 mAbs do not engage beneficial FcγR pathways. (A) Binding ELISA of avelumab or atezolizumab to plate-bound recombinant human (left) and mouse (right) PD-L1 protein. Data are shown from one of three independent experiments with similar results. (B) PD-1/L1–blocking activity of avelumab and atezolizumab. ELISA-based assay to determine binding of human (left) and mouse (right) soluble PD-1 protein to plate-bound recombinant human (left) and mouse (right) PD-L1 protein in the presence of increasing concentrations of the indicated anti–PD-L1 mAbs. Data shown are from one of two independent experiments with similar results. (C) IgG scaffolds of anti–PD-L1 mAbs avelumab, atezolizumab, and durvalumab with glycosylation at asparagine (Asn) 297. Mutation to alanine (Aln) results in deglycosylation. Glycan resi- dues are as follows: core glycan in dash: GlcNac (blue rectangle); mannose (green circle); non-core: fucose (red triangle); galactose (yellow circle); sialic acid (purple diamond). Image was created with Biorender.com. (D) huFcγR mice were inoculated with MC38 tumor cells and treated with IgG1 and IgG1-N297A variants (100 μg) of avelumab and atezolizumab as indicated. Data are graphed as means ± SEM and show one experiment representative of three repeats. One-way ANOVAwith Tukey’s post hoc test. *P ≤0.05. n = 9 or 10. (E) Flow cytometry analysis of immune cell composition in MC38 tumors dissected from tumor-bearing huFcγR mice 8 days after initi- ation of treatment with anti–PD-L1 Fc variants. Data are graphed as bar plots with means ± SEM and represent one of two independent experiments. One-way ANOVA with Tukey’s post hoc test. *P ≤0.05. n = 5. Gating strategy and individual mice are shown in figs. S1 (G to I).

Article Snippet: For the generation of the human IgG1 afucosylated Fc domain variant, 200 μM 2-deoxy-2-fluoro-L-fucose (Biosynth Carbosynth) was added 1 day after transfection.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Activity Assay, Glycoproteomics, Mutagenesis, Flow Cytometry

Journal: Science immunology

Article Title: Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy.

doi: 10.1126/sciimmunol.add8005

Figure Lengend Snippet: Fig. 2. FcγRIIB is an inhibitory checkpoint of antitumor activity by anti–PD-L1 IgG1. (A) dLNs, spleens, and MC38 tumors were dis- sected 7 days after tumor inoculation in huFcγR mice and analyzed by flow cytometry. Gating strategy is shown in fig. S2 (A to C). ΔMFIs of huFcγRIIB are shown. Each dot represents an individual mouse (n = 6), and data are graphed as means ± SEM. Kruskal- Wallis with Dunn’s post hoc test or one-way ANOVA with Tukey’s post hoc test was performed on the basis of normality. *P ≤0.05, **P ≤0.01, ***P ≤0.001. (B) huFcγR mice were inoculated with MC38 tumor cells and treated with avelumab alone or in combination with huFcγRIIB blocker (clone 2B6). Left: Tumor pro- gression over time. Data are graphed as means ± SEM. One-way ANOVA with Tukey’s post hoc test. **P ≤0.01, ****P < 0.0001. Middle: Individual mice on day 21 after treatment onset. Data are graphed as means ± SEM. Mann-Whitney test. *P ≤0.05. Right: Survival over time. Log-rank test with Bonferroni corrected threshold. **P ≤0.01, ***P ≤0.001. Data shown are from one experiment represen- tative of two independent repeats, n = 10 or 11. (C) CD16A+/+CD16B+/+

Article Snippet: For the generation of the human IgG1 afucosylated Fc domain variant, 200 μM 2-deoxy-2-fluoro-L-fucose (Biosynth Carbosynth) was added 1 day after transfection.

Techniques: Activity Assay, Flow Cytometry, MANN-WHITNEY

Journal: Science immunology

Article Title: Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy.

doi: 10.1126/sciimmunol.add8005

Figure Lengend Snippet: Fig. 3. Fc afucosylation of anti–PD-L1 mediates FcγR-dependent increased antitumor activity. (A) IgG1 and aFuc-IgG variants of avelumab were subjected to mass spectrometry analysis to evaluate the Fc glycan composition. Pie charts represent the distribution of the different glycoforms shown on the right. Glycan residues are as follows: GlcNac (blue rectangular); mannose (green circle); fucose (red triangle); galactose (yellow circle); sialic acid (purple diamond). Image was created with Biorender.com. (B) Binding ELISA of anti–PD-L1 IgG1, IgG1-N297A, and aFuc-IgG1 avelumab to plate-bound recombinant huFcγR proteins. Data are graphed as means ± SEM and show one experiment representative of two independent repeats. (C) huFcγR mice were inoculated with MC38 tumor cells and treated with IgG1 or aFuc-IgG1 avelumab. Left: Tumor progression over time. Kruskal-Wallis with Dunn’s post hoc test. *P ≤0.05, ***P ≤0.001, ****P < 0.0001. Right: Individual mice on day 17 after treatment initiation. Unpaired, two-tailed Student’s t test. *P ≤0.05. Data are graphed as means ± SEM. n = 20 or 21. (D) huFcγR KO mice were inoculated with MC38 tumor cells and treated with IgG1 or aFuc-IgG1 avelumab. Left: Tumor progression over time. Kruskal-Wallis with Dunn’s post hoc test. ***P ≤0.001. Right: Individual mice on day 17 after treatment initiation. Mann-Whitney test. ns: not significant. Data are graphed as means ± SEM; n = 14 to 16. (E) huFcγR mice were inoculated with MC38 tumor cells and treated with IgG1 or aFuc-IgG1 avelumab in combination with huFcγRIIB blocker (clone 2B6). Tumor progression over time is shown. Data are graphed as means ± SEM. n = 26.

Article Snippet: For the generation of the human IgG1 afucosylated Fc domain variant, 200 μM 2-deoxy-2-fluoro-L-fucose (Biosynth Carbosynth) was added 1 day after transfection.

Techniques: Activity Assay, Mass Spectrometry, Glycoproteomics, Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Two Tailed Test, MANN-WHITNEY

Journal: Science immunology

Article Title: Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy.

doi: 10.1126/sciimmunol.add8005

Figure Lengend Snippet: Fig. 4. Afucosylated avelumab decreases frequencies of PD-L1+ myeloid cell subsets in the TME. (A) Flow cytometry analysis of Ly6G Ly6C MDSC percentages in MC38 tumors dissected from huFcγR mice 8 days after IgG1 and aFuc-IgG1 avelumab treatment initiation. Each dot represents an individual mouse average from two technical repeats, and data are graphed as a violin plot. One-way ANOVAwith Tukey’s post hoc test was performed. *P ≤0.05, **P ≤0.01. n = 6. (B) Flow cytometry analysis of PD-L1+ nonimmune cells (CD45−), PD-L1+ total immune cells (CD45+), and PD-L1+ myeloid cells in MC38 tumors dissected from tumor-bearing huFcγR mice 8 days after IgG1 and aFuc-IgG1 avelumab treatment initiation. Each dot represents an individual mouse, and data are graphed as violin plots. Kruskal-Wallis test or one-way ANOVAwith Tukey’s post hoc test was performed on the basis of normality. *P ≤0.05, ***P ≤0.001. n = 12 or 13. (C) MFIs of PD-L1 are shown for different cells in the TME of MC38 dissected from tumor-bearing huFcγR mice 8 days after inoculation. Each dot represents an individual mouse, and data are graphed as means ± SEM; n = 13.

Article Snippet: For the generation of the human IgG1 afucosylated Fc domain variant, 200 μM 2-deoxy-2-fluoro-L-fucose (Biosynth Carbosynth) was added 1 day after transfection.

Techniques: Flow Cytometry

Journal: Science immunology

Article Title: Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy.

doi: 10.1126/sciimmunol.add8005

Figure Lengend Snippet: Fig. 5. FcγR-dependent TME immune modulation after treatment with afucosylated avelumab. Flow cytometry analysis of (A) absolute immune cell numbers in MC38 tumors, (B) absolute lymphocyte numbers in MC38, and (C) lymphocyte composition in dLNs dissected from tumor-bearing huFcγR mice 8 days after IgG1 or aFuc- IgG1 avelumab treatment initiation. Each dot represents an individual mouse, and data are graphed as a violin plot. (D) Flow cytometry analysis of relative frequencies of immune cell populations in MC38 tumors dissected from tumor-bearing huFcγR mice (left) or FcγR KO mice (right) 8 days after IgG1 and aFuc-IgG1 avelumab treatment initiation. Data are graphed as bar plots with means ± SEM. For all plots, one-way ANOVA with Tukey’s post hoc test was performed. *P ≤0.05, **P ≤0.01, ***P ≤0.001. n = 5 to 7.

Article Snippet: For the generation of the human IgG1 afucosylated Fc domain variant, 200 μM 2-deoxy-2-fluoro-L-fucose (Biosynth Carbosynth) was added 1 day after transfection.

Techniques: Flow Cytometry

Journal: Science immunology

Article Title: Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy.

doi: 10.1126/sciimmunol.add8005

Figure Lengend Snippet: Fig. 6. Neutrophils mediate the superior antitumor activity of afucosylated avelumab. (A) Number of DEGs (y axis) by cell type (x axis). IgG1 is shown in red, and IgG1-aFuc is shown in blue. (B) Log-normalized gene expression of PD-L1 on neutrophils in MC38 tumors dissected from tumor-bearing huFcγR mice 8 days after IgG1 and aFuc-IgG1 avelumab treatment initiation. *P ≤0.05, Wilcoxon rank sum test. (C) Highly variable genes in neutrophils in MC38 tumors dissected from tumor-bearing huFcγR mice 8 days after IgG1 and aFuc-IgG1 avelumab treatment initiation. Graphs are color-coded for z-score gene expression. (D) GSEA performed on neutrophils in MC38 tumors dissected from tumor-bearing huFcγR mice 8 days after IgG1 and aFuc-IgG1 avelumab treatment initiation. Graphs are color-coded for normalized enrich- ment score (NES); bubble size depicts −log10 false discovery rate–adjusted P value. For (A) to (D), n = 3 to 5. (E) Tumor challenge and treatment scheme for neutrophil depletion experiment. (F and G) huFcγR mice were inoculated with MC38 tumor cells and treated with aFuc-IgG1 (F) or IgG1 avelumab (G) in combination with anti-Gr1 or IC. Left: Tumor progression over time. Right: Individual mice on day 11 after treatment onset. Data are graphed as means ± SEM. Mann-Whitney test. *P ≤0.05. n = 15. i.v., intravenous; s.c., subcutaneous; i.p., intraperitoneal.

Article Snippet: For the generation of the human IgG1 afucosylated Fc domain variant, 200 μM 2-deoxy-2-fluoro-L-fucose (Biosynth Carbosynth) was added 1 day after transfection.

Techniques: Activity Assay, Gene Expression, MANN-WHITNEY