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

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Structure and in vitro characterization of CD25-ADC. (A) Structure and (B) in vitro characterization of CD25-ADC. (i) ELISA showing binding of anti-CD25 antibody PC61 to mouse recombinant CD25. (ii–iv) Flow cytometry measurement of PC61 and isotype-control antibody binding to Yac-1, MC38 and CT26 cells. (v–vii) Yac-1, MC38 and CT26 cells’ viability after exposure to CD25-ADC and isotype-ADC (and the naked pyrrolobenzodiazepine-dimer SG3199 in MC38 and CT26 cell lines). MFI, median fluorescence intensity; PABA, para amino benzoic acid.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Flow Cytometry, Control, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: In vivo antitumor activity of CD25-ADC in the s.c. MC38 syngeneic model. Treatment with (i) vehicle, (ii) anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8), (iii) non-binding ADC (1 mg/kg, single dose on day 1) alone or (iv) in combination with anti-PD-1 antibody, (v–vii) CD25-ADC (0.1, 0.5, and 1 mg/kg single dose on day 1) alone or (viii–x) in combination with anti-PD-1 antibody, started at a group mean tumor volume of 103 mm 3 . Data are shown as tumor volumes (mm 3 ) over time for each individual mouse (10 mice/group). (xi) Survival of mice shown in i–x. Lines for G4, G5, G8 and G9 are overlapping.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vivo, Activity Assay, Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: In vivo antitumor activity of CD25-ADC in the s.c. CT26 syngeneic model. Treatment with (i) vehicle, (ii) anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8), (iii) isotype-ADC (1 mg/kg, single dose on day 1) alone or (iv) in combination with anti-PD-1 antibody, (v–vii) CD25-ADC (0.1, 0.5, and 1 mg/kg single dose on day 1) alone or (viii–x) in combination with anti-PD-1 antibody, started at a group mean tumor volume of 110 mm 3 . Data are shown as tumor volumes (mm 3 ) over time for each individual mouse (10 mice/group). (xi) Survival of mice shown in i–x.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vivo, Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Role of CD8+ T eff cells in CD25-ADC antitumor activity in the MC38 syngeneic model. Depletion of CD8+ T eff cells significantly reduces the antitumor activity of CD25-ADC. CD25-ADC was administered intraperitoneally (i.p.) at a group mean tumor volume of 89 mm 3 as a single dose on day 1 at 0.5 mg/kg alone or in combination with anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8). Anti-CD8 T-cell depleting antibody (10 mg/kg) was injected i.p. on days 0, 5, 8, and 13. Data are shown as mean tumor volumes (mm 3 ) ± SEM over time (n=10/group).

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Activity Assay, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Intratumoral T-cell immunophenotype analysis in MC38-bearing mice. (A) Absolute quantification of intratumoral T regs , CD8+ T cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. (B) Percentage of CD69+, Ki67+ and IFNγ+ tumor-infiltrating CD8+ T cells. Tumors were processed at the indicated times (days post CD25-ADC dose). Horizontal bars represent median value. Statistical differences between treatment groups were calculated using JMP 15 by the Dunn method for joint ranking. Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01. IFN, interferon.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Quantitative Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Circulating and thymic T-cell immunophenotype analysis in MC38-bearing mice. (A) Absolute quantification of circulating T regs , CD8+ T cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. Blood was processed at the indicated times (days post CD25-ADC dose). (B) Absolute quantification of thymic T reg cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. Thymus was processed at the indicated times (days post CD25-ADC dose). Statistical differences between treatment groups were calculated using JMP 15 by the Dunn method for joint ranking. Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Quantitative Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: T-cell dynamic study in non-tumor-bearing mice. Effect of CD25-ADC on the percentage of T regs and T eff levels in non-tumor-bearing mice. Female C57BL/6 mice were injected i.p. with vehicle, CD25-ADC (0.5 mg/kg), or isotype control ADC (0.5 mg/kg) on day 0. (A) Spleen, (B) lymph node, and (C) thymus were collected 4 hours post dose, and 6, 13, and 20 days post dose for T-cell immune profiling. Levels of T regs , CD8+ T, and conventional CD4+ T cells in spleen, lymph nodes, and thymus are presented as % of CD45 cells±SEM over time.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Injection, Control

Journal: Journal of Cancer Immunology

Article Title: Negative Feedback Expansion of Tregs Caused by Endogenous IL-2 Limits the Activity of IL-2-based Therapies

doi: 10.33696/cancerimmunol.5.074

Figure Lengend Snippet: Figure 1. AU-007 binds to endogenous IL-2 and breaks the negative feedback loop in human PBMCs. A-E: naive hPBMCs were treated once at day 0 with either 1uM AU-007 (red) or with an isotype control antibody (blue). No exogenous IL-2 was added. The culture was monitored for 7 days, and immune cell subpopulations were analyzed daily by flow cytometry. Values were normalized to untreated samples (UNT) at each day. AU-007 completely inhibits Tregs expansion (A) and significantly increases Teffs:Tregs ratio (B), without hindering NKs (C). AU-007 downregulates the suppressive markers of CD4+Treg from panel A, as defined by a significant reduction in mean fluorescence intensity (MFI) of CD25 and FoxP3 (D-E). F-K: Total hPBMCs were stimulated for 24h with anti-CD3/anti-CD28 (stimulation only, green) or stimulated with anti-CD3/anti-CD28 in the presence of 200nM of AU-007 mAb (red) or with 200nM of isotype control mAb (blue). No exogenous IL-2 was added. Immune cells subpopulations were analyzed by flow cytometry. AU-007 inhibits Tregs without hindering effector cells and NKs (F-I). AU-007 downregulates the suppressive markers of CD4+Treg from panel G, as defined by a significant reduction in MFI of CD25 and FoxP3 (J-K).

Article Snippet: epitopes To test whether the CD25 epitope is blocked in the nonalpha-IL-2 format, a biotinylated human CD25 (AVI10305-050, R&D systems) was captured on a streptavidin-coated chip (BR100531, Cytiva) and 200 nM of human IL-2 (RKP60568, Reprokine) or non-alpha-IL-2 (IL-2 conjugated to CD25 subunit) were injected for 60 seconds.

Techniques: Control, Flow Cytometry, Fluorescence

Journal: Journal of Cancer Immunology

Article Title: Negative Feedback Expansion of Tregs Caused by Endogenous IL-2 Limits the Activity of IL-2-based Therapies

doi: 10.33696/cancerimmunol.5.074

Figure Lengend Snippet: Figure 2. AU-007 can capture and redirect endogenous IL-2 to break the auto-inhibitory loop in hPBMCs while HD IL-2 or naIL-2 cannot. AU-007 promotes the expansion of NKs and CD8 T-cells while completely inhibiting the expansion of regulatory T-cells. A-E: naive hPBMCs were treated once on day 0 with 1nM of naIL-2 (purple) or with HD IL-2 (1nM) combined with 1uM of isotype control Ab (black) or with either 1uM AU-007 (red) or 10uM AU-007 (turquoise). The culture was monitored for 7 days, and immune cell subpopulations were analyzed daily by flow cytometry. Values were normalized to untreated samples (UNT) at each day. While naIL-2 expands NKs similarly to AU-007 it fails to inhibit Tregs expansion, while AU-007 completely inhibits Tregs expansion in culture (A) and significantly increases the Teffs:Tregs ratio (B), without hindering NKs (C). AU-007 downregulates the suppressive markers of CD4+Treg from panel A, as defined by a significant reduction in MFI of CD25 and FoxP3 (D-E). F-H: AU-007 rescues activated lymphocyte viability decreased by treatment with HD IL-2. hPBMCs culture was stimulated once with anti-CD3/anti-CD28 Abs with or without 10uM of AU-007. 3 days post-stimulation all samples were given HD IL-2 (1nM) and were monitored daily for cell viability using flow cytometry.

Article Snippet: epitopes To test whether the CD25 epitope is blocked in the nonalpha-IL-2 format, a biotinylated human CD25 (AVI10305-050, R&D systems) was captured on a streptavidin-coated chip (BR100531, Cytiva) and 200 nM of human IL-2 (RKP60568, Reprokine) or non-alpha-IL-2 (IL-2 conjugated to CD25 subunit) were injected for 60 seconds.

Techniques: Control, Flow Cytometry

Journal: Journal of Cancer Immunology

Article Title: Negative Feedback Expansion of Tregs Caused by Endogenous IL-2 Limits the Activity of IL-2-based Therapies

doi: 10.33696/cancerimmunol.5.074

Figure Lengend Snippet: Figure 3. IL-2 negative feedback loop caused by endogenous IL-2 limits the activity of modified IL-2-based therapies. A. Schematic representation of IL-2 role as an immunomodulator in homeostasis and inflammation. B. Exogenous administration of modified IL-2 with bias selectivity to dimer-expressing cells promotes the expansion of CD25 negative (CD25-) effector cells yet is undermined by the endogenous IL-2 that pushes the system back to homeostasis. C. AU-007 captures and redirects endogenous IL-2, allowing it to expand CD25 negative (CD25-) effector cells while breaking the auto-inhibitory loop and expanding the inflammation & immune stimulation stage.

Article Snippet: epitopes To test whether the CD25 epitope is blocked in the nonalpha-IL-2 format, a biotinylated human CD25 (AVI10305-050, R&D systems) was captured on a streptavidin-coated chip (BR100531, Cytiva) and 200 nM of human IL-2 (RKP60568, Reprokine) or non-alpha-IL-2 (IL-2 conjugated to CD25 subunit) were injected for 60 seconds.

Techniques: Activity Assay, Modification, Expressing

Journal: mAbs

Article Title: The CD25-binding antibody Daclizumab High-Yield Process has a distinct glycosylation pattern and reduced antibody-dependent cell-mediated cytotoxicity in comparison to Zenapax®

doi: 10.1080/19420862.2016.1207031

Figure Lengend Snippet: Zenapax and DAC HYP interaction with CD25. Differences in glycosylation do not affect direct binding of Zenapax and DAC HYP to CD25 as determined by Biacore and inhibition of IL-2 dependent proliferation of KIT225/K6 cells in vitro.

Article Snippet: Recombinant CD25 (R&D Systems, Cat. No. 223-2ACF) was injected at various concentrations in duplicate and reference surface for 2 minutes using an automated method.

Techniques: Glycoproteomics, Binding Assay, Inhibition

Journal: Science translational medicine

Article Title: Targeted antibody and cytokine cancer immunotherapies through collagen affinity

doi: 10.1126/scitranslmed.aau3259

Figure Lengend Snippet: (A) Schematic of conjugation of a collagen-binding domain (CBD), the recombinant VWF A3 domain, to checkpoint inhibitor (CPI) antibody, resulting in affinity for collagen. CBD-fused IL-2 was recombinantly expressed, with the CBD on the N-terminus of IL-2 using a (GGGS)2 linker. (B) Dissociation constants (KD values) of CBD- and unmodified aPD-L1, αCTLA4, and IL-2 against collagen type I and collagen type III, recombinant mouse (rm)CTLA4, rmPD-L1, and/or rmIL-2Ra were measured by ELISA. N.D.= not determined because of low signals. Graphs of concentrations vs signals are shown in fig. S2. (C) 5 × 105 MMTV-PyMT cells were inoculated in the mammary fat pad. When the tumor volume reached 500 mm3, 300 μg of DyLight 800-labeled CBD was injected i.v.. A pie chart represents the biodistribution of CBD protein 48 hr after injection as determined by fluorescence analysis of each organ (n = 4). (D) Intratumoral imaging was performed on MMTV-PyMT tumors when they reached 200 mm3 by injecting 100 μg of DyLight 594-labeled CBD-αPD-L1 or (E) 100 μg of DyLight 594-labeled αPD-L1 i.v. 30 min after injection. The tumor was then harvested, and fluorescence was analyzed by microscopy. Top panels: images of whole tumors, scale bar = 500 μm. Bottom panels: images of enlarged yellow squares within upper panels, scale bar = 50 μm. Representative images of 2 tumors each. (F, G) Binding of (F) CBD-IL-2 or (G) unmodified IL-2 to human melanoma cryosections was imaged by fluorescence microscopy. Scale bar = 100 μm. Two experimental replicates. Statistical analyses were done using ANOVA with Tukey’s test. **p < 0.01.

Article Snippet: 96-well ELISA plates were coated with 10 μg/mL collagen I (EMD Millipore), collagen III (EMD Millipore), or 1 μg/mL recombinant mouse IL-2Ra (SinoBiological) in PBS for 1 hour at 37°C, followed by blocking with 2% BSA in PBS-T for 1 hour at RT.

Techniques: Conjugation Assay, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling, Injection, Fluorescence, Imaging, Microscopy