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R&D Systems murine cd25

<t>CD25</t> (IL-2Rα) and NKG2D were immobilized to individual flow cells of a sensor chip by primary amine chemistry. Serial dilutions of IL-2, mutIL-2, and OMCPmutIL-2 were injected across each flow cell and association and dissociation phases were measured. Renditioned graphic model of protein binding (left) with experimental binding to CD25 (middle) and NKG2D (right) for wild type human IL-2 (A), mutant human (R38A, F42K) IL-2 (B), and OMCPmutIL-2 (C). Experimental binding curves are shown for a representative experiment. (D) For binding of CD25:wild-type IL-2 and OMCPmutIL-2:NKG2D experimental curves were fitted using ANABEL software and the observed binding constant (Kobs) was measured for each concentration. The plot of Kobs vs concentration is shown with the slope as the association rate (Kon) and y-axis intercept as the dissociation rate (Koff).

Murine Cd25, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 91 stars, based on 6 article reviews

murine cd25 - by Bioz Stars, 2026-03

91/100 stars

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1) Product Images from "Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy"

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.2000926

CD25 (IL-2Rα) and NKG2D were immobilized to individual flow cells of a sensor chip by primary amine chemistry. Serial dilutions of IL-2, mutIL-2, and OMCPmutIL-2 were injected across each flow cell and association and dissociation phases were measured. Renditioned graphic model of protein binding (left) with experimental binding to CD25 (middle) and NKG2D (right) for wild type human IL-2 (A), mutant human (R38A, F42K) IL-2 (B), and OMCPmutIL-2 (C). Experimental binding curves are shown for a representative experiment. (D) For binding of CD25:wild-type IL-2 and OMCPmutIL-2:NKG2D experimental curves were fitted using ANABEL software and the observed binding constant (Kobs) was measured for each concentration. The plot of Kobs vs concentration is shown with the slope as the association rate (Kon) and y-axis intercept as the dissociation rate (Koff).

Figure Legend Snippet: CD25 (IL-2Rα) and NKG2D were immobilized to individual flow cells of a sensor chip by primary amine chemistry. Serial dilutions of IL-2, mutIL-2, and OMCPmutIL-2 were injected across each flow cell and association and dissociation phases were measured. Renditioned graphic model of protein binding (left) with experimental binding to CD25 (middle) and NKG2D (right) for wild type human IL-2 (A), mutant human (R38A, F42K) IL-2 (B), and OMCPmutIL-2 (C). Experimental binding curves are shown for a representative experiment. (D) For binding of CD25:wild-type IL-2 and OMCPmutIL-2:NKG2D experimental curves were fitted using ANABEL software and the observed binding constant (Kobs) was measured for each concentration. The plot of Kobs vs concentration is shown with the slope as the association rate (Kon) and y-axis intercept as the dissociation rate (Koff).

Techniques Used: Injection, Protein Binding, Binding Assay, Mutagenesis, Software, Concentration Assay

(A) Fold TIL expansion of murine B16ova -resident leucocytes. (B) Surface expression of NKG2D and IL-2R alpha chain on mouse antigen specific CD8+ T cells compared to those with ovalbumin non-reactive TCR. (C) Fold TIL expansion of Human melanoma -derived TILS in either wild-type IL-2 (blue) or OMCPmutIL-2 (red) (C). *p<.05; **p<.01, ***p<.001, NS p>.05

Figure Legend Snippet: (A) Fold TIL expansion of murine B16ova -resident leucocytes. (B) Surface expression of NKG2D and IL-2R alpha chain on mouse antigen specific CD8+ T cells compared to those with ovalbumin non-reactive TCR. (C) Fold TIL expansion of Human melanoma -derived TILS in either wild-type IL-2 (blue) or OMCPmutIL-2 (red) (C). *p<.05; **p<.01, ***p<.001, NS p>.05

Techniques Used: Expressing, Derivative Assay

Image Search Results

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Figure Lengend Snippet: CD25 (IL-2Rα) and NKG2D were immobilized to individual flow cells of a sensor chip by primary amine chemistry. Serial dilutions of IL-2, mutIL-2, and OMCPmutIL-2 were injected across each flow cell and association and dissociation phases were measured. Renditioned graphic model of protein binding (left) with experimental binding to CD25 (middle) and NKG2D (right) for wild type human IL-2 (A), mutant human (R38A, F42K) IL-2 (B), and OMCPmutIL-2 (C). Experimental binding curves are shown for a representative experiment. (D) For binding of CD25:wild-type IL-2 and OMCPmutIL-2:NKG2D experimental curves were fitted using ANABEL software and the observed binding constant (Kobs) was measured for each concentration. The plot of Kobs vs concentration is shown with the slope as the association rate (Kon) and y-axis intercept as the dissociation rate (Koff).

Article Snippet: Surface plasmon resonance (SPR) A ProteOn XPR36 instrument (BioRad) was used to determine the kinetics of interaction of WT IL-2, mutIL-2, and OMCPmutIL-2 for murine CD25 (#2438-RM, R&D systems) and murine NKG2D-Fc (#139-NK-050, R&D systems).

Techniques: Injection, Protein Binding, Binding Assay, Mutagenesis, Software, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Figure Lengend Snippet: (A) Fold TIL expansion of murine B16ova -resident leucocytes. (B) Surface expression of NKG2D and IL-2R alpha chain on mouse antigen specific CD8+ T cells compared to those with ovalbumin non-reactive TCR. (C) Fold TIL expansion of Human melanoma -derived TILS in either wild-type IL-2 (blue) or OMCPmutIL-2 (red) (C). *p<.05; **p<.01, ***p<.001, NS p>.05

Techniques: Expressing, Derivative Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Techniques: Injection, Protein Binding, Binding Assay, Mutagenesis, Software, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Techniques: Expressing, Derivative Assay

(A) Number of TIL leucocytes transferred into CD45.1 congenic tumor-bearing recipients after expansion in either wild-type IL-2 or OMCPmutIL-2. Flow cytometric analysis performed 24 hours after transfer. (B)Immunohistochemical analysis of B16ova tumors in wild-type C57Bl/6 mice injected with 20×106 TILs from C57Bl/6EGFP mice expanded for 2 weeks in either wild-type IL-2 or OMCPmutIL-2. Histologic evaluation performed 120 hours after transfer. (C) Relative expression, by median fluorescence intensity, of LFA-1, CD49a or CXCR3 on TIL-resident CD8+ T cells, NK cells or gamma delta T cells after 2-week expansion in IL-2 (blue) or OMCPmutIL-2 (red). (D) Relative expression, by median fluorescence intensity, of LFA-1 on TIL-resident CD8+ T cells, NK cells or γδ T cells after either MDSC or Treg depletion prior to expansion in IL-2 (blue) or OMCPmutIL-2 (red). *p<.05; **p<.01, ***p<.001, NS p>.05

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Figure Lengend Snippet: (A) Number of TIL leucocytes transferred into CD45.1 congenic tumor-bearing recipients after expansion in either wild-type IL-2 or OMCPmutIL-2. Flow cytometric analysis performed 24 hours after transfer. (B)Immunohistochemical analysis of B16ova tumors in wild-type C57Bl/6 mice injected with 20×106 TILs from C57Bl/6EGFP mice expanded for 2 weeks in either wild-type IL-2 or OMCPmutIL-2. Histologic evaluation performed 120 hours after transfer. (C) Relative expression, by median fluorescence intensity, of LFA-1, CD49a or CXCR3 on TIL-resident CD8+ T cells, NK cells or gamma delta T cells after 2-week expansion in IL-2 (blue) or OMCPmutIL-2 (red). (D) Relative expression, by median fluorescence intensity, of LFA-1 on TIL-resident CD8+ T cells, NK cells or γδ T cells after either MDSC or Treg depletion prior to expansion in IL-2 (blue) or OMCPmutIL-2 (red). *p<.05; **p<.01, ***p<.001, NS p>.05

Techniques: Immunohistochemical staining, Injection, Expressing, Fluorescence

(A) NKG2D expression in CD8+ TILs. (B) Expansion of NKG2Dlow or NKG2Dhigh CD8+ T cells in OMCPmutIL-2 or wild-type IL-2. (C) Expression of NKG2D (top), perforin (middle), and FasLigand (bottom) on flow cytometrically sorted NKG2Dlow CD8+ T cells immediately post sort (black line bar open circles), after 7 days of culture in wild-type IL-2 (blue bar, open circles) or OMCPmutIL-2 (red bar, open circles). ***p<.001

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retargeting Interleukin-2 Signaling to NKG2D-Expressing Tumor Infiltrating Leukocytes Improves Adoptive Transfer Immunotherapy

doi: 10.4049/jimmunol.2000926

Figure Lengend Snippet: (A) NKG2D expression in CD8+ TILs. (B) Expansion of NKG2Dlow or NKG2Dhigh CD8+ T cells in OMCPmutIL-2 or wild-type IL-2. (C) Expression of NKG2D (top), perforin (middle), and FasLigand (bottom) on flow cytometrically sorted NKG2Dlow CD8+ T cells immediately post sort (black line bar open circles), after 7 days of culture in wild-type IL-2 (blue bar, open circles) or OMCPmutIL-2 (red bar, open circles). ***p<.001

Techniques: Expressing

C57BL6 mice were injected with 500,000 MCA205 tumor cells. Once tumors were palpable, on day 5, mice were injected IP with αCD25 PC61 /αCD25 NIB /αCD25 NIB + αIL2 (200μg). Tumors and LN were harvested on day 12 post-tumor inoculation and processed as described in . (A) Representative FACS plots showing expression of FoxP3 versus CD25 in CD4 + T cells. (B) Graph showing % FoxP3 + cells of total CD4 + cells. (C) Absolute number of Tregs shown as number of Tregs/g of tumor. P-value=0.0086 for No Tx vs αCD25 PC61 and p=0.0204 for No Tx vs aCD25 NIB group. (D) Ratio of effector T cells over Tregs. On the left, p value for CD8/Treg ratio between No Tx and aCD25 NIB = 0.0003. On the right, p value for NK/Treg ratio between No Tx and αCD25 NIB =0.0170. (E) Representative FACS plots showing Granzyme B expression versus Ki67 expression in CD8, CD4 effectors and NK cells. (F) Graph showing percentage of Granzyme B + cells in different effector subsets. In the CD8 subset, p-value between no Tx and αCD25NIB group=0.0452 and 0.0012 for No Tx vs αCD25 NIB + αIL2 group. For the CD4 eff group, p value for No tx vs αCD25 PC61 =0.0221, between No tx and αCD25 NIB =0.0059, between No tx and αCD25 NIB +αIL2=0.0276. For the NK subset, p value between the no tx and αCD25 NIB group=0.0232. (G) Graph showing the Mean Fluorescence Intensity of Granzyme B of the effector cells plotted in (F) . In the CD8 subset, p value for no tx vs αCD25 PC61 =0.0222, and 0.0190 for No tx vs αCD25 NIB + αIL2 group. For the CD4 eff subset, p value between No tx and αCD25NIB=0.0112. For the NK subset, p-value for No tx vs αCD25 NIB =0.0057 and between αCD25 PC61 vs αCD25 NIB =0.0295. All quantification plots: mean, 1-way ANOVA, Tukey’s multiple comparison test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

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: C57BL6 mice were injected with 500,000 MCA205 tumor cells. Once tumors were palpable, on day 5, mice were injected IP with αCD25 PC61 /αCD25 NIB /αCD25 NIB + αIL2 (200μg). Tumors and LN were harvested on day 12 post-tumor inoculation and processed as described in . (A) Representative FACS plots showing expression of FoxP3 versus CD25 in CD4 + T cells. (B) Graph showing % FoxP3 + cells of total CD4 + cells. (C) Absolute number of Tregs shown as number of Tregs/g of tumor. P-value=0.0086 for No Tx vs αCD25 PC61 and p=0.0204 for No Tx vs aCD25 NIB group. (D) Ratio of effector T cells over Tregs. On the left, p value for CD8/Treg ratio between No Tx and aCD25 NIB = 0.0003. On the right, p value for NK/Treg ratio between No Tx and αCD25 NIB =0.0170. (E) Representative FACS plots showing Granzyme B expression versus Ki67 expression in CD8, CD4 effectors and NK cells. (F) Graph showing percentage of Granzyme B + cells in different effector subsets. In the CD8 subset, p-value between no Tx and αCD25NIB group=0.0452 and 0.0012 for No Tx vs αCD25 NIB + αIL2 group. For the CD4 eff group, p value for No tx vs αCD25 PC61 =0.0221, between No tx and αCD25 NIB =0.0059, between No tx and αCD25 NIB +αIL2=0.0276. For the NK subset, p value between the no tx and αCD25 NIB group=0.0232. (G) Graph showing the Mean Fluorescence Intensity of Granzyme B of the effector cells plotted in (F) . In the CD8 subset, p value for no tx vs αCD25 PC61 =0.0222, and 0.0190 for No tx vs αCD25 NIB + αIL2 group. For the CD4 eff subset, p value between No tx and αCD25NIB=0.0112. For the NK subset, p-value for No tx vs αCD25 NIB =0.0057 and between αCD25 PC61 vs αCD25 NIB =0.0295. All quantification plots: mean, 1-way ANOVA, Tukey’s multiple comparison test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Measurement of apparent Kd of binding surrogate murine antibodies αCD25 NIB and αCD25 PC61 to recombinant mouse CD25 (Sino Biological 50292-M08H) was determined using the dextran-based carboxymethylated sensor chip CM5.

Techniques: Injection, Expressing, Fluorescence

For binding experiments with RG6292 and Daclizumab, SU-DHL1 tumor cells (human CD25 + , (A) ) and HSC-F cells (cynomolgus CD25 + , (B) ) were used. To quantify binding of αCD25 NIB , splenocytes were isolated of spleens resected from female C57BL/6-Foxp3tm1Flv/J mice (C) Cells were incubated with indicated serial dilutions of the test antibody detected then by fluorescently labeled 2 nd antibody against human and mouse Fcγ, respectively. Living mouse Treg cells (Aqua - , mRFP + singlets) and tumor cells (Aqua - , singlets), respectively, were gated and the mean fluorescence intensity of the secondary antibody was plotted. EC50 values were calculated by as described in the data analysis section in . Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (D) RG6292 (and the fully fucosylated version RG6292 (FF)) depleted via ADCC in-vitro differentiated Treg cells using purified, IL-2 activated NK cells. Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (E) RG6292 and RG6292 (FF) mediated ADCP of in-vitro differentiated Treg cells when co-cultured with MCSF differentiated macrophages. Flow cytometric analysis was performed to determine percentage of phagocytosis. Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (F) Schematics of binder selection.

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: For binding experiments with RG6292 and Daclizumab, SU-DHL1 tumor cells (human CD25 + , (A) ) and HSC-F cells (cynomolgus CD25 + , (B) ) were used. To quantify binding of αCD25 NIB , splenocytes were isolated of spleens resected from female C57BL/6-Foxp3tm1Flv/J mice (C) Cells were incubated with indicated serial dilutions of the test antibody detected then by fluorescently labeled 2 nd antibody against human and mouse Fcγ, respectively. Living mouse Treg cells (Aqua - , mRFP + singlets) and tumor cells (Aqua - , singlets), respectively, were gated and the mean fluorescence intensity of the secondary antibody was plotted. EC50 values were calculated by as described in the data analysis section in . Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (D) RG6292 (and the fully fucosylated version RG6292 (FF)) depleted via ADCC in-vitro differentiated Treg cells using purified, IL-2 activated NK cells. Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (E) RG6292 and RG6292 (FF) mediated ADCP of in-vitro differentiated Treg cells when co-cultured with MCSF differentiated macrophages. Flow cytometric analysis was performed to determine percentage of phagocytosis. Shown are technical duplicates of one representative experiment out of several independent ones conducted (N>2). (F) Schematics of binder selection.

Techniques: Binding Assay, Isolation, Incubation, Labeling, Fluorescence, In Vitro, Purification, Cell Culture, Selection

(A) RG6292 did not block IL-2 signaling in a pSTAT5 assay using human PBMCs. Daclizumab was used as blocking control, human IgG1 isotype or untreated samples as reference control. Cells were incubated with 10 μg/ml antibody followed by 10 U/ml IL-2 for 10 min. pSTAT5 was analyzed on CD4 + , CD8 + and Treg cells. Shown is the mean of technical duplicates with SD. (B) RG6292 did not negatively impact Granzyme B induction and proliferation of αCD3/αCD28 activated Pan T cells, other than addition of antibodies either neutralizing IL-2 (αIL-2nAB) or blocking binding of IL2 to CD25 (Daclizumab). Antibodies were tested for 72 hours at 10 μg/ml. Shown is the mean of technical triplicates with SD. (C) The Fab fragment of RG6292 binds CD25 opposite of the IL2-CD25 interaction site. Shown is the superposition of the Fab RG6292 - CD25 structure with the IL2 quaternary signaling complex structure (PDB accession code 2B5I) . The overlay was prepared using all atoms of CD25. IL2Rβ and IL2Rγ of the quaternary complex are shown as white transparent surface whereas IL2 is highlighted in magenta and CD25 in yellow/ red. The Fab light and heavy chains of RG6292 are colored in cyan and blue, respectively. The close-up section provides a detailed view onto the epitope and paratope area with residues labeled contributing to the interface. Further details are provided in the and . (D) RG6292 mediated mostly depletion of CD25 high expressing regulatory T cells and not that of activated CD8 and CD4 effector T cells when present during polyclonal activation (αCD3 coated beads) of human PBMC. The counts of activated regulatory and non-regulatory CD4 + and CD8 + T cells within the PBMC sample were quantified on day 3 by flow cytometry and lysis by PBMC endogenous FcR+ cells was calculated. Shown is the mean of technical triplicates with SEM. (E) CD25 staining was performed to confirm target expression on Treg cells (CD4 + FoxP3 + CFSE high), activated CD4 (CD4 + FoxP3 - CD69 + ) and CD8 T cells (CD8 + CD69 + ) shown in (D) . (F and G) Graphs showing the killing of FoxP3 + CD25 + cells within human tumors (frozen dissociated tumor cells obtained from Conversant Bio) supplemented with allogeneic NK cells mediated by RG6292 (and the fully fucosylated (FF) or Fc silent version of RG6292). Shown is the mean of technical duplicates with SD. All quantification plots: 2-way ANOVA, Sidak’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

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) RG6292 did not block IL-2 signaling in a pSTAT5 assay using human PBMCs. Daclizumab was used as blocking control, human IgG1 isotype or untreated samples as reference control. Cells were incubated with 10 μg/ml antibody followed by 10 U/ml IL-2 for 10 min. pSTAT5 was analyzed on CD4 + , CD8 + and Treg cells. Shown is the mean of technical duplicates with SD. (B) RG6292 did not negatively impact Granzyme B induction and proliferation of αCD3/αCD28 activated Pan T cells, other than addition of antibodies either neutralizing IL-2 (αIL-2nAB) or blocking binding of IL2 to CD25 (Daclizumab). Antibodies were tested for 72 hours at 10 μg/ml. Shown is the mean of technical triplicates with SD. (C) The Fab fragment of RG6292 binds CD25 opposite of the IL2-CD25 interaction site. Shown is the superposition of the Fab RG6292 - CD25 structure with the IL2 quaternary signaling complex structure (PDB accession code 2B5I) . The overlay was prepared using all atoms of CD25. IL2Rβ and IL2Rγ of the quaternary complex are shown as white transparent surface whereas IL2 is highlighted in magenta and CD25 in yellow/ red. The Fab light and heavy chains of RG6292 are colored in cyan and blue, respectively. The close-up section provides a detailed view onto the epitope and paratope area with residues labeled contributing to the interface. Further details are provided in the and . (D) RG6292 mediated mostly depletion of CD25 high expressing regulatory T cells and not that of activated CD8 and CD4 effector T cells when present during polyclonal activation (αCD3 coated beads) of human PBMC. The counts of activated regulatory and non-regulatory CD4 + and CD8 + T cells within the PBMC sample were quantified on day 3 by flow cytometry and lysis by PBMC endogenous FcR+ cells was calculated. Shown is the mean of technical triplicates with SEM. (E) CD25 staining was performed to confirm target expression on Treg cells (CD4 + FoxP3 + CFSE high), activated CD4 (CD4 + FoxP3 - CD69 + ) and CD8 T cells (CD8 + CD69 + ) shown in (D) . (F and G) Graphs showing the killing of FoxP3 + CD25 + cells within human tumors (frozen dissociated tumor cells obtained from Conversant Bio) supplemented with allogeneic NK cells mediated by RG6292 (and the fully fucosylated (FF) or Fc silent version of RG6292). Shown is the mean of technical duplicates with SD. All quantification plots: 2-way ANOVA, Sidak’s multiple comparisons test (ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Techniques: Blocking Assay, Incubation, Binding Assay, Labeling, Expressing, Activation Assay, Flow Cytometry, Lysis, Staining

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.

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.

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

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.

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

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.

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.

Techniques: In Vivo, Activity Assay, Binding Assay

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.

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.

Techniques: In Vivo, Activity Assay

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).

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).

Techniques: Activity Assay, Injection

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.

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.

Techniques: Quantitative Proteomics

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.

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.

Techniques: Quantitative Proteomics

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.

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.

Techniques: Injection, Control

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