Journal: Oncoimmunology

Article Title: CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy

doi: 10.1080/2162402X.2017.1380142

Figure Lengend Snippet: Structural models of bispecific antibody formats generated in this study and SDS-PAGE analysis. (A) An illustrative representation of the initial antibody and final bispecific antibody format. The format is comprised of IgG-Fc linked to two different Fv domains (CD89/CD20) via 15-amino-acid (G4S)3 linkers. (B) SDS-PAGE analysis of the purification of the bispecific antibody proteins.

Article Snippet: To determine CD89 expression levels on TAMs, M1or M2 macrophages were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD89 antibody (CD89-FITC; Miltenyi Biotech) for 30 min at 4°C and then evaluated by FACS using a FACSVerse system (BD Bioscience).

Techniques: Generated, SDS Page, Purification

Journal: Oncoimmunology

Article Title: CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy

doi: 10.1080/2162402X.2017.1380142

Figure Lengend Snippet: Apparent binding affinities of the Fv components of the bispecific antibody. Increasing concentrations of bispecific antibody were incubated with CD89-positive PMN cell. The binding of both antigens on the CD20 × CD89 molecule was assessed by flow cytometry using (A) A CD20-expressing cell line (Raji cells) and (B) CD89-expressing cell (PMNs). All data are presented as the mean ± SEM (n = 3) from one of three representative experiments.

Article Snippet: To determine CD89 expression levels on TAMs, M1or M2 macrophages were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD89 antibody (CD89-FITC; Miltenyi Biotech) for 30 min at 4°C and then evaluated by FACS using a FACSVerse system (BD Bioscience).

Techniques: Binding Assay, Incubation, Flow Cytometry, Expressing

Journal: Oncoimmunology

Article Title: CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy

doi: 10.1080/2162402X.2017.1380142

Figure Lengend Snippet: ADCC of antibody variants mediated by human or mouse effector cells ex vivo. (A) CD20+ Raji cells were incubated with the anti-CD20 × anti-CD89 molecule or ADCC mutated antibody, together with human PMNs for 4 h. (B) Raji cells were incubated with the anti-CD20 × anti-CD89 molecule or ADCC mutated antibody, together with human PBMC cells for 4 h. Data are the average of three different experiments with three separate donors. (C-D) Raji cells were incubated separately with the isolated CD14+ monocytes, mouse monocyte-depleted PBMC fraction (NK), or PBMCs (E:T = 40:1) from FcαRI Tg mice (C) or wild-type C57BL/6 mice (D) using 1 μg/mL anti-CD20 × anti-CD89 molecule or ADCC mutated antibody. For monocytes group and PBMCs groups, a 20-h ADCC assay was used. For monocyte-depleted PBMCs (NK), a 4-h ADCC assay was used. All data are presented as the mean ± SEM (n = 3) from one of three representative experiments. *CD89-CD20(Not mut) group versus CD20-IgG group. **P < 0.01; ***P < 0.001; ns, not statistically significant by two-way ANOVA.

Article Snippet: To determine CD89 expression levels on TAMs, M1or M2 macrophages were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD89 antibody (CD89-FITC; Miltenyi Biotech) for 30 min at 4°C and then evaluated by FACS using a FACSVerse system (BD Bioscience).

Techniques: Ex Vivo, Incubation, Isolation, ADCC Assay

Journal: Oncoimmunology

Article Title: CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy

doi: 10.1080/2162402X.2017.1380142

Figure Lengend Snippet: Parameter of pharmacokinetics.

Article Snippet: To determine CD89 expression levels on TAMs, M1or M2 macrophages were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD89 antibody (CD89-FITC; Miltenyi Biotech) for 30 min at 4°C and then evaluated by FACS using a FACSVerse system (BD Bioscience).

Techniques: Drug discovery

Journal: Oncoimmunology

Article Title: CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy

doi: 10.1080/2162402X.2017.1380142

Figure Lengend Snippet: Xenograft models of the bispecific antibody in tumor-bearing mice. (A) Effect of the CD89-CD20 bispecific antibody on the growth of Raji Burkitt's lymphoma in SCID mice. (B) WT and (C) FcαRI Tg mice; 1 × 106 LLC-CD20 cells were mixed in solubilized basement membrane matrix and injected subcutaneously into female mice (n = 6–8/group). Antibodies were administered by i.v. injection on days 0, 4, and 8. Animals were monitored for tumor growth. Tumor volumes are plotted as the mean ± standard error. *CD89-CD20 group versus CD20-IgG group. **P < 0.01; ***P < 0.001 by two-way ANOVA.

Article Snippet: To determine CD89 expression levels on TAMs, M1or M2 macrophages were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD89 antibody (CD89-FITC; Miltenyi Biotech) for 30 min at 4°C and then evaluated by FACS using a FACSVerse system (BD Bioscience).

Techniques: Membrane, Injection

Journal: Oncoimmunology

Article Title: CD89-mediated recruitment of macrophages via a bispecific antibody enhances anti-tumor efficacy

doi: 10.1080/2162402X.2017.1380142

Figure Lengend Snippet: Tumor-associated macrophages trigger ADCP in Raji cells with BsAb (CD89-CD20). (A) Flow cytometric analysis of CD89 surface expression on TAMs. CD11b+F4/80+macrophage cells were selected by BD FACs AriaII and TAMs were further purified by microbeads and stained with anti-CD89-FITC. Cells were stained with CD16/32-APC or CD206-APC to identify M1 and M2 macrophages, respectively. The experiment was repeated at least three times, yielding essentially identical results. (B) Representative microscope images showing specific phagocytosis induced by the bispecific antibody. Raji cells (CFSE; green) incubated with macrophages (F4/80-Cy3; red) for 6 hours in the presence of the bispecific antibody (1 μg/ml) demonstrated substantial phagocytosis. (C) Phagocytosis of Raji cells was analyzed and quantified as the percentage of double-positive cells relative to total CFSE-positive cells and F4/80+cells.

Article Snippet: To determine CD89 expression levels on TAMs, M1or M2 macrophages were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD89 antibody (CD89-FITC; Miltenyi Biotech) for 30 min at 4°C and then evaluated by FACS using a FACSVerse system (BD Bioscience).

Techniques: Expressing, Purification, Staining, Microscopy, Incubation