Journal: Nature cancer

Article Title: CD25-T reg -depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

doi: 10.1038/s43018-020-00133-0

Figure Lengend Snippet: (A and B) Stem cell humanized female NOG mice bearing an established s.c. BxPC-3 tumor were injected i.v. with vehicle, RG6292 [10, 1, 0.1 and 0.01 mg/kg]. Splenocytes, blood lymphocytes and tumor infiltrating lymphocytes were isolated 1, 4, 7 and 14 days after injection and evaluated for depletion of Tregs (huCD45 + , huCD3 + , huCD4 + , huCD25 + , huFoxP3 + ) and activation of CD8 + T cells (huCD45 + , huCD3 + , huCD8 + GranzymeB + ). (A) Representative dot plots and histograms showing the decrease of FoxP3 and the increase of Granzyme B expression on intra-tumoral CD4 + and CD8 + T cells 14 days after treatment with RG6292 compared to vehicle-injected animals. (B) Systemic and dose-dependent decrease in Tregs and an increase in activated CD8 + T cells, respectively, was only evident after administration of RG6292. Each symbol represents the mean of 4 animals, and error bars indicate the SD. Statistical analysis (2 way ANOVA, Dunnet’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01,) of RG6292 treated groups against the vehicle group is indicated. Actual p-values are summarized in tabular form in the (C and D) The toxicity and pharmacokinetic/pharmacodynamic (PK/PD) relationship of RG6292 was evaluated in cynomolgus monkeys following Q2W (C) or weekly (D) Repeated IV administrations during 4 (D) and 2 weeks (C) , respectively. Shown is the mean of three animals (male and female) +/- SD. (D) The only drug-related finding after application of supra-pharmacological doses (here 100 mg/kg/dose; Q2W) in the 4-week toxicity study, was an exacerbation of ulcerative/erosive rhinitis at 30 and 100 mg/kg shown in the right image (left image of nasal cavity from an unaffected animal for comparison), characterized by (partial) loss of epithelium (arrow) accompanied by inflammatory cell infiltrates (asterisk) in the nasal cavities of individual animals. Reversibility of the nasal lesions was demonstrated. The 4-week GLP toxicity study was conducted according to regulatory guidelines once.

Article Snippet: For humanization, mice were injected with Busulfan (15 mg/kg) followed 24 hours later by an injection of human CD34 + cord blood cells (1*10 5 per mouse; STEMCELL Technologies) as described earlier .

Techniques: Injection, Isolation, Activation Assay, Expressing, Comparison

Journal: Nature cancer

Article Title: CD25-T reg -depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity

doi: 10.1038/s43018-020-00133-0

Figure Lengend Snippet: Stem cell humanized female NOG mice bearing an established s.c. BxPC-3 tumor were injected i.p. with vehicle, RG6292 [4 mg/kg] or Ipilimumab [10 mg/kg]. After 72 hrs, splenocytes, blood lymphocytes and tumor infiltrating lymphocytes were isolated and evaluated for counts of activated CD8 + T cells (huCD45 + , huCD3 + , huCD8 + huCTLA-4 + ) and Tregs (huCD45 + , huCD3 + , huCD4 + , huFoxP3 + ) as well as for markers of recent T cell activation. (A) Ipilimumab as well as RG6292 decreased the intratumoral Treg counts. An increase of intratumoral activated CD8 + T cell count was only evident after administration of RG6292. Normalized counts were plotted for the respective treatment groups. Each symbol represents one animal (n=5 mice), CD8 and Treg cells are connected for the same animals (B) Intratumoral CD8 + T cells after RG6292 treatment were highly activated and had increased levels of HLA-DR, PD-1 and CTLA-4 (MFI as well as % of positive cells). Each symbol represents one animal (n=5 mice). The box and whiskers plots show minima and maxima and the median. Statistical analysis of RG6292 and Ipilimumab treated groups against vehicle group is indicated. Data was analyzed using 2-way ANOVA, Dunnet’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (p-value between RG6292 and Ipilimumab was 0.0001 for CTLA4 MFI on CD8 T cells and 0.0008 for HLA-DR on CD8 T cells. (C) Representative FACS plots showing CD25 expression versus FoxP3 expression in CD4 + T cells and PD-1 expression versus CTLA-4 expression in CD8 + T cells for vehicle, RG6292 and Ipilimumab treated animals.

Article Snippet: For humanization, mice were injected with Busulfan (15 mg/kg) followed 24 hours later by an injection of human CD34 + cord blood cells (1*10 5 per mouse; STEMCELL Technologies) as described earlier .

Techniques: Injection, Isolation, Activation Assay, Cell Counting, Expressing

Journal: BMC Cancer

Article Title: 3- O -Acetyloleanolic acid inhibits VEGF-A-induced lymphangiogenesis and lymph node metastasis in an oral cancer sentinel lymph node animal model

doi: 10.1186/s12885-018-4630-0

Figure Lengend Snippet: Effects of 3AOA on expressions of VEGFR-1 and VEGFR-2, and activation of VEGFR-1, VEGFR-2 and lymphangiogenesis related downstream signaling factors in rhVEGF-A-treated HLMECs. a-b Expression levels of VEGFR-1 and -2 proteins were determined using Western blot analysis. Amounts of VEGFR-1 and -2 obtained in three independent experiments were quantified and represented as a bar diagram. Levels of VEGFR-1 and -2 in 3AOA- and rhVEGF-A-untreated cells were estimated as 100%. c-d , Cell lysates were immunoprecipitated with anti-phospho-Tyr (anti-p-Tyr). The level of phosphorylated VEGFR-1 and -2 in immunoprecipitates was detected using Western blot analysis with anti-VEGFR-1 and anti-VEGFR-2. Phosphorylation levels of VEGFR-1 and -2 obtained in three independent experiments were quantified and represented as a bar diagram. Phosphorylation levels of VEGFR-1 and -2 in 3AOA- and rhVEGF-A-untreated cells were estimated as 100%. e , HLMECs were serum starved for 6 h, then were treated with different concentrations of 3AOA (0, 2.5, 5 μM) in the presence of rhVEGF-A (20 ng/mL) for 60 min. The phosphorylation levels of FAK, PI3K, AKT, and ERK1/2 were determined using Western blot analysis with anti-p-FAK, anti-p-PI3K, anti-p-AKT, and anti-p-ERK1/2. Data are presented as a mean ± S.D. of three independent experiments ( * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Immunoprecipitated proteins were subjected to SDS-PAGE (6%) and Western blotting using mouse anti-VEGFR-1 and goat anti-VEGFR-2 antibodies (Santa Cruz Biotech.

Techniques: Activation Assay, Expressing, Western Blot, Immunoprecipitation