hek blue il 2 reporter system  (InvivoGen)

Journal: bioRxiv

Article Title: A Dynamically Regulated Designed IL-2 for Tumor-localized Signaling

doi: 10.64898/2026.09.18.752703

Figure Lengend Snippet: a, Structure of WT IL-2 model, based on Protein Data Bank (PDB) ID 1M47 (cartoon representation), bound to IL-2Rαβγ from PDB ID 2B5I; IL-2Rα (CD25) shown in gray (cartoon representation), IL-2Rβ (CD122) and IL-2Rγ (CD132) shown in light gray (surface representation). Rosetta Design was used to replace the regions in red with short, structured loops (light blue) that reconnect α-helices H1-H4, generating relooped designs R1.1-38. b, IL-2Rβγ signaling activity (HEK-Blue reporter assay) and expression level from Expi293 supernatants diluted 1:400; designs R1.1-38 normalized to WT IL-2 levels. c, Optimization of design R1.1 by extending IL-2Rγ-interacting helices and redesigning the H2-H3 loop (“Round 2 optimization”) to produce design R2.1, which was further optimized by mutating two IL-2Rβ-interacting residues, R88N and I99L, to produce design R2.2. d, Binding affinity to IL-2Rβγ-Fc and IL-2Rα as measured by biolayer interferometry (BLI): design R2.2 binds IL-2Rβγ with a K D close to that of WT IL-2 (0.80 nM and 0.44 nM, respectively), consistent with previously reported measurements for WT IL-2 , . The purified proteins measured here contain STII tags; NB = no binding. e, Circular dichroism (CD) wavelength scan at 25℃ ( left ) and thermal melt monitoring helical signal at 208 nm ( right ). f, STAT5 phosphorylation (pSTAT5) in resting T cell subtypes after treatment with the indicated designed cytokines or WT IL-2. NC = not calculated.

Article Snippet: The activity of relooped IL-2 molecules in Expi293 supernatants (undiluted, and diluted 1:10, 1:50, 1:100, 1:200, and 1:400) was assayed on HEK-Blue IL2R cells (Invivogen) according to the manufacturer’s instructions.

Techniques: Activity Assay, Reporter Assay, Expressing, Binding Assay, Purification, Circular Dichroism, Phospho-proteomics

Journal: bioRxiv

Article Title: A Dynamically Regulated Designed IL-2 for Tumor-localized Signaling

doi: 10.64898/2026.09.18.752703

Figure Lengend Snippet: Designs were expressed in Expi293 cell and supernatants collected and evaluated for binding to IL-2Rα by ELISA ( top , normalized to WT IL-2) and IL-2Rβγ signaling activity by HEK-Blue assay ( bottom , levels normalized to that of WT IL-2). NTC is a non-transfected control.

Article Snippet: The activity of relooped IL-2 molecules in Expi293 supernatants (undiluted, and diluted 1:10, 1:50, 1:100, 1:200, and 1:400) was assayed on HEK-Blue IL2R cells (Invivogen) according to the manufacturer’s instructions.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Transfection, Control

Journal: Science Advances

Article Title: Engineered probiotic Bifidobacterium for tumor-targeted pancreatic cancer therapy

doi: 10.1126/sciadv.adz1388

Figure Lengend Snippet: ( A ) Schematic illustrating the BifidoSumIL-2 platform. BifidoSumIL-2 exerts antitumor effects by activating the innate immune system through a stimulator of interferon genes (STING)–dependent pathway and selectively promoting T eff cell activation over T reg cells. Additionally, BifidoSumIL-2 is combined with chemotherapy, radiation, and immunotherapy to further enhance its antitumor efficacy. ( B ) Plasmid construction for SumIL-2 secretion. Bifidobacterium constitutive promoter (P tuf ) was placed upstream of the SumIL-2 gene, with or without a secretion signal peptide derived from a hypothetical protein (BLON_RS02330) into the pJL21 plasmid. ( C ) The concentration of SumIL-2 (ng per ml) in the culture supernatant was measured using enzyme-linked immunosorbent assay (ELISA) after 8 hours of growth in MRS medium ( n = 4 per group). N.D., not detected. Limit of detection, 15 pg/ml. ( D ) Bioactivity of recombinant hIL-2 and BifidoSumIL-2 (Bl.SumIL-2) supernatants. Data were fitted to a three-parameter dose-response curve [hIL-2, coefficient of determination ( R 2 ) = 0.9669; BifidoSumIL-2, R 2 = 0. 9860] to determine the median effective concentration (EC 50 ) for hIL-2 (36.38 pg/ml; black dotted line) and for BifidoSumIL-2 supernatant (62.19 pg/ml; red dotted line). ( E to H ) C57BL/6 mice were subcutaneously injected with 1 × 10 6 MC38 (F), B16F1 (G), or KPC cells (H) on day 0 and treated with PBS (intravenous, 200 μl), hIL-2 (intraperitoneal, 10 μg), wild-type B. longum (Bl; intravenous, 1 × 10 7 CFU), or BifidoSumIL-2 (Bl.SumIL-2; intravenous, 1 × 10 7 CFU) on days 14, 16, and 18 (F); days 11, 13, and 15 (G); or days 12, 14, and 16 (H) ( n = 3 to 4 per group). Mean tumor growth curves for the tumor-bearing mice. Two-way analysis of variance (ANOVA) tests were used to analyze the tumor growth. Data are presented as means ± SEM. * P < 0.05; ** P < 0.01. One of the two representative experiments is shown. ip, intraperitoneal; iv, intravenous; sc, subcutaneous.

Article Snippet: HEK-Blue IL-2 reporter cells were purchased from InvivoGen and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), penicillin-streptomycin (Pen-Strep; 100 U/ml; Gibco), normocin (100 μg/ml; InvivoGen), and puromycin (1 μg/ml; InvivoGen).

Techniques: Activation Assay, Plasmid Preparation, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, R-Squared, Injection, Analysis

Journal: Scientific Reports

Article Title: CD25 genetic ablation on lymphoid cell lines to obtain models of stimulation through the Interleukin-2 beta/gamma receptor

doi: 10.1038/s41598-026-61486-7

Figure Lengend Snippet: Assessment of the differential biological activity of IL-2 muteins with disrupted CD25-binding interface on cells displaying either trimeric or dimeric IL-2 receptor (IL-2R). Fc-fused recombinant versions of two IL-2-derived muteins (NA and 3x), with disrupted CD25 binding interface, were tested ( a ). Distinctive amino acid replacements in each mutein are shown between parentheses. A similarly formatted reference protein comprising IL-2 was included. The alignment shows the sequence of IL-2 segment 38–62. Short lines indicate conservation of the original residues in the muteins, while replacing amino acids are displayed at mutated positions. The ability of the fusion proteins to induce proliferation of cells having the trimeric IL-2R was evaluated using wild-type (wt) mouse CTLL2 ( b ) and human Kit225 cells ( d ). Their capacity to induce proliferation of their CD25-KO counterparts, which display the dimeric beta/gamma IL-2R, was assessed in parallel ( c, e ). Cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and IL-2-derived NA mutein at 1 µg/mL, and washed with FBS-supplemented RPMI to remove the mutein before performing proliferation assays. In the case of Kit225 cells (both wt and CD25-KO), a starvation step of 18 h in FBS-supplemented RPMI was required before going ahead. For the proliferation assays, 20 000 cells were seeded in each well of 96-well plates and grown for 48 h (mouse cells) or 72 h (human cells) in the presence of different concentrations of the fusion proteins. IL-6/Fc was included as negative control protein. Alamar Blue reagent was added to the wells and incubated during 18 h (mouse cells) or 6 h (human cells) before reading the absorbances at 540 and 630 nm. The difference between both absorbance values, as a function of the cytokine moiety concentration, was used to build the dose-response curves. Graphs show the mean value and SD of two individual data points for each concentration. In order to measure the differences in biological activity, effective concentrations required to achieve half-maximal proliferation (EC50) were estimated from each curve, and specific activity values (U/mg) were calculated as the inverse of the cytokine dose (mg) necessary to achieve half-maximal response in 1 mL ( f ).

Article Snippet: HEK-Blue IL-2 cells were generated by InvivoGen through transfection with the human IL-2R alpha, beta and gamma genes, and a Stat5-inducible reporter gene.

Techniques: Activity Assay, Binding Assay, Recombinant, Derivative Assay, Sequencing, Negative Control, Incubation, Concentration Assay

Journal: Scientific Reports

Article Title: CD25 genetic ablation on lymphoid cell lines to obtain models of stimulation through the Interleukin-2 beta/gamma receptor

doi: 10.1038/s41598-026-61486-7

Figure Lengend Snippet: Assessment of super-agonism of IL-2 muteins with increased affinity for CD122 on cells displaying the dimeric IL-2 receptor (IL-2R). A panel of Fc-fused recombinant versions of IL-2-derived muteins, containing mutations that enhance the interaction with CD122, was tested ( a ). Distinctive amino acid replacements in each mutein are shown between parentheses. A similarly formatted reference protein comprising IL-2 was included. The alignment shows the sequence of IL-2 segment 80–92. Short lines indicate conservation of the original residues in the muteins, while replacing amino acids are displayed at mutated positions. The ability of the fusion proteins to induce proliferation of cells having the trimeric IL-2R was evaluated using wild-type (wt) mouse CTLL2 ( b ) and human Kit225 cells ( d ). The capacity of fusion proteins to induce proliferation of their CD25-KO counterparts, which display the dimeric beta/gamma IL-2R, was assessed in parallel ( c , e ). Cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and IL-2-derived NA mutein at 1 µg/mL, and washed with FBS-supplemented RPMI to remove the mutein before performing proliferation assays. In the case of Kit225 cells (both wt and CD25-KO), a starvation step of 18 h in FBS-supplemented RPMI was required before going ahead. For the proliferation assays, 20 000 cells were seeded in each well of 96-well plates and grown for 48 h (mouse cells) or 72 h (human cells) in the presence of different concentrations of the fusion proteins. IL-6/Fc was included as negative control protein. Alamar Blue reagent was added to the wells and incubated during 18 h (mouse cells) or 6 h (human cells) before reading the absorbances at 540 and 630 nm. The difference between both absorbance values, as a function of the cytokine moiety concentration, was used to build the dose-response curves. Graphs show the mean value and SD of two individual data points for each concentration. In order to measure the differences in biological activity, effective concentrations required to achieve half-maximal proliferation (EC50) were estimated from each curve, and specific activity values (U/mg) were calculated as the inverse of the cytokine dose (mg) necessary to achieve half-maximal response in 1 mL ( g ).

Article Snippet: HEK-Blue IL-2 cells were generated by InvivoGen through transfection with the human IL-2R alpha, beta and gamma genes, and a Stat5-inducible reporter gene.

Techniques: Recombinant, Derivative Assay, Sequencing, Negative Control, Incubation, Concentration Assay, Activity Assay

Journal: Scientific Reports

Article Title: CD25 genetic ablation on lymphoid cell lines to obtain models of stimulation through the Interleukin-2 beta/gamma receptor

doi: 10.1038/s41598-026-61486-7

Figure Lengend Snippet: Evaluation of the ability of IL-2-derived muteins with mutations in the binding interface with gamma IL-2R subunit to antagonize IL-2-mediated signaling. Two Fc-fused variants were tested. RETR variant displayed disrupting replacements at the CD132 binding interface. H9-RETR combined these mutations with changes that increase the affinity for CD122 ( a ). Distinctive amino acid replacements in each mutein are shown between parentheses. A similarly formatted reference protein comprising IL-2 was included. The alignment shows the sequence of IL-2 segments 18–22, 80–92 and 126–130, targeted across the variants. Short lines indicate conservation of the original residues in the muteins, while replacing amino acids are displayed at mutated positions. The inability of the mutated fusion proteins to induce lymphoid cell proliferation was shown on wild-type (wt) mouse CTLL2 and human Kit225 cells, and on their CD25-KO versions ( b ). Cells were maintained in RPMI supplemented with fetal bovine serum (FBS) and IL-2-derived NA mutein , and washed with FBS-supplemented RPMI. In the case of Kit225 cells (both wt and CD25-KO), a starvation step of 18 h in FBS-supplemented RPMI was performed. To assess proliferation, 20 000 cells/well were subsequently seeded in 96-well plates and grown for 48 h (mouse cells) or 72 h (human cells) in the presence of the fusion proteins. IL-6/Fc was included as negative control protein. Alamar Blue reagent was added to the wells and incubated during 18 h (mouse cells) or 6 h (human cells) before reading the absorbances at 540 and 630 nm. The difference between both absorbance values was used as a proliferation indicator. Blocking capacity of fusion proteins comprising inactive cytokines was similarly assessed on wild-type mouse CTLL-2 ( c ) and human Kit225 ( e ), and their CD25-KO versions ( d , f ). Washed cells were grown in the presence of a fixed (proliferation-inducing) concentration of IL-2/Fc, and variable concentrations of the blocking fusion proteins. The difference between absorbance values at 540 nm and 630 nm, as a function of the blocking cytokine moiety concentration, was used to build the inhibition curves. IL-6/Fc was included as a negative control competitor. Graphics show the mean value and SD of two individual data points from each concentration.

Article Snippet: HEK-Blue IL-2 cells were generated by InvivoGen through transfection with the human IL-2R alpha, beta and gamma genes, and a Stat5-inducible reporter gene.

Techniques: Derivative Assay, Binding Assay, Variant Assay, Sequencing, Negative Control, Incubation, Blocking Assay, Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: CD25 genetic ablation on lymphoid cell lines to obtain models of stimulation through the Interleukin-2 beta/gamma receptor

doi: 10.1038/s41598-026-61486-7

Figure Lengend Snippet: Common problems faced during in vitro proliferation assays based on IL-2 dependent cell lines. Wild-type (wt) CTLL-2 and CD25-KO CTLL-2 cells (from mice), as well as human wt Kit225 and CD25-KO Kit225 were used. Cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and IL-2-derived NA mutein at 1 µg/mL for maintenance, and washed with FBS-supplemented RPMI to remove the mutein before performing proliferation assays. In the case of Kit225 cells (boh wt and CD25-KO), a starvation step of 18 h in FBS-supplemented RPMI was required before going ahead. For the proliferation assays, 20 000 cells were seeded in each well of 96-well plates and grown for 48 h (mouse cells) or 72 h (human cells) in the presence of different concentrations of the fusion proteins. IL-6/Fc was included as negative control protein. Alamar Blue reagent was added to the wells and incubated during 18 h (mouse cells) or 6 h (human cells) before reading the absorbances at 540 and 630 nm. The difference between both absorbance values, as a function of the cytokine moiety concentration, was used to build the dose-response curves. Graphs show the media and SD of two individual data points for each concentration. Low sensitivity of CD25-KO CTLL-2 cells resulted in the need of high initial concentrations (in the µg/mL range) and low dilution factors (ideally 1/2) to obtain dose-response curves ( a ). The complete sigmoidal curve was not obtained for most agonists. In the case of wt and CD25-KO Kit 225 cells, starvation periods in FBS-supplemented media below 16 h caused intense cytokine-independent proliferation (b). Prolonged incubation of Kit225 cells (either wt or CD25-KO) with Alamar Blue reagent (longer than 6 h) produced indistinguishable color signals in all wells, not dependent on proliferation rates ( c ). Even though assays based on Kit-225 cells were prone to exhibit high basal cytokine-independent signals, careful control of incubation times and conditions allowed the detection of specific IL-2 activity over this background (d). The use of wells located in the borders of multi-well plates resulted in high non-specific signals and unacceptable variability ( e ).

Article Snippet: HEK-Blue IL-2 cells were generated by InvivoGen through transfection with the human IL-2R alpha, beta and gamma genes, and a Stat5-inducible reporter gene.

Techniques: In Vitro, Derivative Assay, Negative Control, Incubation, Concentration Assay, Produced, Control, Activity Assay

Journal: Scientific Reports

Article Title: CD25 genetic ablation on lymphoid cell lines to obtain models of stimulation through the Interleukin-2 beta/gamma receptor

doi: 10.1038/s41598-026-61486-7

Figure Lengend Snippet: Assessment of the ability of recombinant fusion proteins comprising IL-2-derived muteins to induce Stat5 phosphorylation in cells displaying either the dimeric or trimeric IL-2 receptor (IL-2-R). The set of tested recombinant proteins included an alpha non-binder (NA/Fc), a beta super-binder (SB834/Fc) and and a gamma non-binder unable to induce signaling (RETR/Fc), as well as the control protein IL-2/Fc. The ability of the proteins to induce Stat5 phosphorylation on cells having the trimeric IL-2R was evaluated using wild-type (wt) mouse CTLL2 ( a ) and human Kit225 cells ( c ). Their capacity to induce phosphorylation in their CD25-KO counterparts, which display the dimeric beta/gamma IL-2R, was assessed in parallel ( b, d ). Cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and IL-2-derived NA mutein at 1 µg/mL, and washed three times with RPMI 1640. A starvation of 6 h in culture without any IL-2 source was performed before going ahead. 2.5 × 10 5 cells/well were seeded in U-bottom 96-well plates, and incubated during only 15 min in the presence of different concentrations of the recombinant fusion proteins under study. Untreated cells were used to assess phosphorylation background levels. Supernatants were removed by centrifugation and the cells were fixed and permeabilized with cold 90% methanol during at least 20 min. Methanol was removed by centrifugation, and the cells were washed and stained with either PE-conjugated anti-pSTAT5 or isotype control antibody during 30 min at 4 °C. After flow cytometry analysis, dose-response curves were built by plotting the percentage of pStat5 + cells against cytokine concentration. Graphs show the mean value and SD of two individual data points for each concentration. EC50 values were estimated as the cytokine concentrations required to reach half-maximal percentage of pStat5 + cells ( e ). For treatments with RETR/Fc (unable to induce phosphorylation) EC50 could not be determined (ND).

Article Snippet: HEK-Blue IL-2 cells were generated by InvivoGen through transfection with the human IL-2R alpha, beta and gamma genes, and a Stat5-inducible reporter gene.

Techniques: Recombinant, Derivative Assay, Phospho-proteomics, Control, Incubation, Centrifugation, Staining, Flow Cytometry, Concentration Assay

Journal: Nature Communications

Article Title: Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting

doi: 10.1038/s41467-026-75024-6

Figure Lengend Snippet: a Schematic overview of the VHH selection strategy against human IL-2 receptor subunits CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ). b – d Anti-CD25 ( b ), anti-CD122 ( c ), and anti-CD132 ( d ) VHH clones plotted by CDR3 sequence family versus binding capacity (dissociation rate k off ) to their recombinant targets. Colored dots indicate clones selected for further study. The dissociation rate constant ( k off ) was obtained by SPR off-rate screening, where periplasmic extracts containing soluble VHHs were injected over CM5 chips with immobilized IL-2R subunits, and k off was derived by fitting the dissociation phase of the sensorgrams after the association step (buffer wash only). The principle and analysis were the same for all IL-2R subunits (IL-2Rα, IL-2Rβ, IL-2Rγ); differences were limited to assay setup details such as the specific chip, immobilized ligand, pH for coupling, and regeneration conditions optimized for each subunit. e Domain architecture of VHH-hFc fusion constructs used for biochemical and cellular assays.

Article Snippet: The potency of bispecific and trispecific anti-IL-2R antibodies to induce IL-2 signaling was analyzed by determining the level of STAT5 phosphorylated by the HEK-Blue IL-2 recombinant cell line (Invivogen, #hkb-il2) in the presence of the antibodies.

Techniques: Selection, Clone Assay, Sequencing, Binding Assay, Recombinant, Injection, Derivative Assay, Construct

Journal: Nature Communications

Article Title: Engineering trispecific IL-2 receptor agonistic antibodies through geometry optimization for enhanced Treg targeting

doi: 10.1038/s41467-026-75024-6

Figure Lengend Snippet: a Fc-fused IL-2 mutein (V91K) and formats of the trispecific 2:1 anti-CD25:anti-CD122/CD132 antibodies used in the study. Recombinant human IL-2 and the parental trispecific αβγVHH-48 were used as reference agonists. αVHH in blue, βVHH in green, γVHH in yellow. b – d Dose-dependent STAT5 activity of native IL-2, selected trispecific 2:1 anti-CD25:anti-CD122/CD132 antibodies and the Fc-fused IL-2 mutein (V91K) in human PBMC subsets. b Results expressed as percentage of pSTAT5 + cells. c Difference in area under the curve (AUC) between Treg and non-Treg cell populations. d Results expressed as pSTAT5 mean fluorescence intensity (MFI). Dose-response curves were generated from experimental data using a three-parameter logistic (3PL) fit. Data are from two donors and shown as mean of both data points. IL-2Rα (CD25) expression levels for the PBMC donors used in these assays are shown in Fig. and Supplementary Fig. . e Purified Treg cell expansion was assessed by measuring absolute cell counts by flow cytometry after 7 days of culture. Gating strategies for Fig. 5b–d are shown in Supplementary Fig. , and for Fig. 5e in Supplementary Fig. . Data are from three donors, mean ± s.e.m.

Article Snippet: The potency of bispecific and trispecific anti-IL-2R antibodies to induce IL-2 signaling was analyzed by determining the level of STAT5 phosphorylated by the HEK-Blue IL-2 recombinant cell line (Invivogen, #hkb-il2) in the presence of the antibodies.

Techniques: Recombinant, Activity Assay, Fluorescence, Generated, Expressing, Purification, Flow Cytometry