Figure 5. GARP-specific antibody has therapeutic value against preclinical model of breast cancer.

(A) Surface staining of pre-B cells stably expressing human GARP (pre-B-hGARP) by 4D3 GARP antibody. Grey histogram represents staining with isotype control antibody. (B) pre-B-hGARP cells were incubated without or with human LTGFβ (huLTGF-β), in the presence of GARP antibodies or Isotype control antibody. Cells were then stained for cell surface hLTGF-β, in order to determine the activity of the antibody to block the binding of hLTGF-β to GARP. (C) 4T1-EV (empty vector) cells and 4T1-GARP (human GARP overexpression cells) was stained with 4D3 or a commercial anti-GARP antibody (G14D9), followed by flow cytometry. To determine if G14D9 blocks staining by 4D3 antibody, cells were pre-incubated with 1 or 5 μg/ml G14D9 antibody, followed by 4D3 antibody (1 μg/ml) and appropriate fluorochrome-labeled secondary antibody, and flow cytometric analysis. (D) 4T1-GARP cells were seeded in triplicates in a 96-well plate (2,000 cells per well) with 10 μg/ml 4D3 or mouse IgG followed by MTT assay kinetically. (E) BALB/c mice were injected with 5×10⁵ 4T1-hGARP mammary tumors orthotopically in the 4^th mammary fat pad, followed by IP injection of 200 μg/mouse of 4D3 GARP antibody every three days. The primary tumor growth kinetics was measured three times a week. Significance is indicated at various time points along the tumor curves. (F) Four weeks after tumor injection, mice were sacrificed. Lungs were isolated and the numbers of visible metastatic nodules were counted. (G) Spleens were harvested and percentage of Treg (FoxP3⁺CD25⁺CD4⁺) cells in the antibody treated mice versus Isotype antibody groups were determined by flow cytometry. (H) BALB/c mice were injected with 5×10⁵ 4T1-hGARP mammary tumors orthotopically in the 4^th mammary fat pad, followed by one dose of cyclophosphamide (CY) and IP injection of 200 μg/mouse 4D3 GARP antibody every three days. The primary tumor growth kinetics was measured three times a week. Significance is indicated at various time points along the tumor curves. (I) Five weeks after tumor injection, mice were sacrificed, tumors were excised and weighted. (J) Lungs were isolated and numbers of visible metastatic nodules were counted. *P<0.05; **P<0.01; ***P<0.001. Significance was determined by two-tailed Student’s t-test. Results are representative of two independent experiments.