Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The structure and characterization of CD25 aptamer. ( A ) The secondary structure of the CD25 aptamer was estimated by RNAstructure software v6.4 of Mathews Lab. The sequence of CD25 aptamer is shown with modifications indicated (Z, 5-[ N -(1-naphthylmethyl)carboxamide]-2′-deoxyuridine; N me , 2′- O -methyl nucleosides). ( B ) The binding affinity of CD25 aptamer to the CD25 recombinant protein was determined by the BLI method. Ni-NTA probes were immobilized with His-tag CD25 protein, followed by incubation with the aptamer 125 (green), 250 (yellow), or 500 nM (red). The binding signal over time is shown. Kd is expressed as mean ± SD. ( C ) The cells were stained with biotin-aptamer combined with NeutrAvidin DyLight 650 or APC-conjugated anti-CD25 monoclonal antibody (mAb), and then the specificity of the antibody and the aptamer to the cells was examined by flow cytometry (control for aptamer: DyLight 650 only; control for antibody: not stained).

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Software, Sequencing, Binding Assay, Recombinant, Incubation, Staining, Flow Cytometry, Control

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The CD25 aptamer specifically binds and internalizes into CD25-positive cells. ( A ) The cell internalization of Cy-5-labeled CD25 aptamer (red) was visualized for 0, 1, and 4 h using confocal fluorescence microscopy using CD25-positive Karpas299 and CD25-negative Daudi cell lines. The nuclei were stained with DAPI (blue). ( B ) The rate of internalized CD25 aptamer was determined using the MFI value of flow cytometry analysis at 0 to 240 min. ( C ) Cellular trafficking of the CD25 aptamer. Fluorescence microscopy visualized the lysosomal delivery of pHrodo-labeled CD25 aptamer (red) for up to 4 h.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Labeling, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: Effects of the CD25 aptamer on CD25/IL-2 signaling. ( A ) A competitive binding assay was performed by adding biotinylated IL-2 proteins to 96-well plates coated with CD25 proteins, in the presence or absence of the CD25 aptamer. ( B , C ) Karpas299 cells were pre-treated with the CD25 aptamer for 30 min, followed by stimulation with IL-2 for 15 min. The levels of pSTAT5 protein and TGF-β mRNA were analyzed by Western blotting and quantitative RT-PCR, respectively. ( D , E ) HuT78 cells were treated with IL-2 in the presence of either the CD25 aptamer or the anti-CD25 antibody Daclizumab. The expression of pSTAT5 was then assessed by Western blot analysis. ( F ) HuT78 cells were pre-treated with the indicated concentrations of the CD25 aptamer, stimulated with IL-2, and the secretion of IL-4 was measured as described in the Materials and Methods. Results are expressed as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: Competitive Binding Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vitro cytotoxicity of CD25 aptamer–MMAE conjugates. Karpas299 and Daudi Cells were treated with CD25-ApDC MMAE1 ( A ) or CD25-ApDC MMAE3 ( B ) for 3 days, after which cell viability was assessed, as described in the Materials and Methods. ( C ) Karpas299 and HuT78 cells were co-cultured at a 1:1 ratio for 24 h, stained with anti-CD4 and anti-CD25 antibodies, and analyzed by flow cytometry. The co-cultured cells were subsequently incubated with 45 nM CD25-ApDC MMAE3 for 24, 48, or 72 h, and analyzed again using flow cytometry. ( D ) Cells were treated with increasing concentrations of MMAE or CD25-ApDC MMAE3 for 24 h. Western blot analysis of total PRAP, cleaved PARP, total caspase-3, and cleaved caspase-3 was performed. ( E ) The cell cycle was analyzed using flow cytometry after staining with PI. Results are expressed as mean ±SD. * p < 0.05, *** p < 0.001.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: In Vitro, Cell Culture, Staining, Flow Cytometry, Incubation, Western Blot

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vivo antitumor efficacy of CD25 aptamer–MMAE conjugates in xenograft models. Tumor growth curves were generated by measuring tumor volumes in Karpas299 tumor-bearing mice following intravenous administration of CD25 aptamer–MMAE conjugates when tumors reached an average volume of 150 mm 3 . ( A ) Red arrows indicate the time points of injection with CD25-ApDC MMAE1 at doses of 1, 2, or 4 mg/kg. ( B ) Mice were treated either four times with 4 mg/kg (red arrows) or twice with 12 mg/kg (green arrows). ( C ) Tumor-bearing mice received a single dose of 0.4, 0.8, or 1.6 mg/kg, or were administered doses three times (once per week) with 0.8 or 1.6 mg/kg CD25-ApDC MMAE3 . Data are the mean tumor volume ±SE of eight animals per group. ( D ) NOD/SCID mice were systemically inoculated with Karpas299 cells and treated intravenously with the indicated dose of CD25-ApDC MMAE1 or CD25-ApDC MMAE3 twice per week for 3 weeks. Kaplan–Meier survival curves show the percentage of survival for each group, with statistical comparison performed using log-rank tests.

Article Snippet: A 5 μg/mL recombinant human CD25 His-tagged protein solution (R&D Systems, Minneapolis, MN, USA) was immobilized onto Ni-NTA probes (Gator Bio).

Techniques: In Vivo, Generated, Injection, Comparison

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The structure and characterization of CD25 aptamer. ( A ) The secondary structure of the CD25 aptamer was estimated by RNAstructure software v6.4 of Mathews Lab. The sequence of CD25 aptamer is shown with modifications indicated (Z, 5-[ N -(1-naphthylmethyl)carboxamide]-2′-deoxyuridine; N me , 2′- O -methyl nucleosides). ( B ) The binding affinity of CD25 aptamer to the CD25 recombinant protein was determined by the BLI method. Ni-NTA probes were immobilized with His-tag CD25 protein, followed by incubation with the aptamer 125 (green), 250 (yellow), or 500 nM (red). The binding signal over time is shown. Kd is expressed as mean ± SD. ( C ) The cells were stained with biotin-aptamer combined with NeutrAvidin DyLight 650 or APC-conjugated anti-CD25 monoclonal antibody (mAb), and then the specificity of the antibody and the aptamer to the cells was examined by flow cytometry (control for aptamer: DyLight 650 only; control for antibody: not stained).

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: Software, Sequencing, Binding Assay, Recombinant, Incubation, Staining, Flow Cytometry, Control

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: The CD25 aptamer specifically binds and internalizes into CD25-positive cells. ( A ) The cell internalization of Cy-5-labeled CD25 aptamer (red) was visualized for 0, 1, and 4 h using confocal fluorescence microscopy using CD25-positive Karpas299 and CD25-negative Daudi cell lines. The nuclei were stained with DAPI (blue). ( B ) The rate of internalized CD25 aptamer was determined using the MFI value of flow cytometry analysis at 0 to 240 min. ( C ) Cellular trafficking of the CD25 aptamer. Fluorescence microscopy visualized the lysosomal delivery of pHrodo-labeled CD25 aptamer (red) for up to 4 h.

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: Labeling, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: Effects of the CD25 aptamer on CD25/IL-2 signaling. ( A ) A competitive binding assay was performed by adding biotinylated IL-2 proteins to 96-well plates coated with CD25 proteins, in the presence or absence of the CD25 aptamer. ( B , C ) Karpas299 cells were pre-treated with the CD25 aptamer for 30 min, followed by stimulation with IL-2 for 15 min. The levels of pSTAT5 protein and TGF-β mRNA were analyzed by Western blotting and quantitative RT-PCR, respectively. ( D , E ) HuT78 cells were treated with IL-2 in the presence of either the CD25 aptamer or the anti-CD25 antibody Daclizumab. The expression of pSTAT5 was then assessed by Western blot analysis. ( F ) HuT78 cells were pre-treated with the indicated concentrations of the CD25 aptamer, stimulated with IL-2, and the secretion of IL-4 was measured as described in the Materials and Methods. Results are expressed as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: Competitive Binding Assay, Western Blot, Quantitative RT-PCR, Expressing

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vitro cytotoxicity of CD25 aptamer–MMAE conjugates. Karpas299 and Daudi Cells were treated with CD25-ApDC MMAE1 ( A ) or CD25-ApDC MMAE3 ( B ) for 3 days, after which cell viability was assessed, as described in the Materials and Methods. ( C ) Karpas299 and HuT78 cells were co-cultured at a 1:1 ratio for 24 h, stained with anti-CD4 and anti-CD25 antibodies, and analyzed by flow cytometry. The co-cultured cells were subsequently incubated with 45 nM CD25-ApDC MMAE3 for 24, 48, or 72 h, and analyzed again using flow cytometry. ( D ) Cells were treated with increasing concentrations of MMAE or CD25-ApDC MMAE3 for 24 h. Western blot analysis of total PRAP, cleaved PARP, total caspase-3, and cleaved caspase-3 was performed. ( E ) The cell cycle was analyzed using flow cytometry after staining with PI. Results are expressed as mean ±SD. * p < 0.05, *** p < 0.001.

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: In Vitro, Cell Culture, Staining, Flow Cytometry, Incubation, Western Blot

Journal: Pharmaceutics

Article Title: CD25-Targeted Aptamer–Drug Conjugate for the Treatment of CD25-Expressing Hematological Malignancies

doi: 10.3390/pharmaceutics18020217

Figure Lengend Snippet: In vivo antitumor efficacy of CD25 aptamer–MMAE conjugates in xenograft models. Tumor growth curves were generated by measuring tumor volumes in Karpas299 tumor-bearing mice following intravenous administration of CD25 aptamer–MMAE conjugates when tumors reached an average volume of 150 mm 3 . ( A ) Red arrows indicate the time points of injection with CD25-ApDC MMAE1 at doses of 1, 2, or 4 mg/kg. ( B ) Mice were treated either four times with 4 mg/kg (red arrows) or twice with 12 mg/kg (green arrows). ( C ) Tumor-bearing mice received a single dose of 0.4, 0.8, or 1.6 mg/kg, or were administered doses three times (once per week) with 0.8 or 1.6 mg/kg CD25-ApDC MMAE3 . Data are the mean tumor volume ±SE of eight animals per group. ( D ) NOD/SCID mice were systemically inoculated with Karpas299 cells and treated intravenously with the indicated dose of CD25-ApDC MMAE1 or CD25-ApDC MMAE3 twice per week for 3 weeks. Kaplan–Meier survival curves show the percentage of survival for each group, with statistical comparison performed using log-rank tests.

Article Snippet: Recombinant human CD25 protein (R&D Systems) was immobilized on high-binding 96-well ELISA plates (Corning) at a concentration of 0.2 μg/mL in PBS overnight at 4 °C.

Techniques: In Vivo, Generated, Injection, Comparison