Journal: mAbs

Article Title: Targeting IL-7Rα with PNU-159682 antibody–drug conjugates in acute lymphoblastic leukemia: translational implications

doi: 10.1080/19420862.2026.2663639

Figure Lengend Snippet: Generation and characterization of IL-7Rα-targeting antibodies. (a) Schematic workflow of IL-7Rα-specific monoclonal antibodies (mAbs) generation: immunization of IL-7Rα-knockout mice with recombinant human IL-7Rα extracellular domain, hybridoma fusion, and screening/expansion, followed by conversion to human–mouse chimeric IgG. (b) Flow cytometry histograms showing the binding capabilities of in-house clones 577, 2D5, 165, and 24 to IL-7Rα-positive cells compared with a commercial anti-IL-7Rα mAb; secondary-only and unstained controls are included. (c) Competitive binding (epitope binning) assessment between different in-house antibody pairs using flow cytometry. Cells were pre-blocked with an unlabeled antibody and stained with a fluorophore-labelled competitor. The binding ratios were normalized to the non-pre-blocked condition. (d) Surface plasmon resonance analysis of antibody binding to recombinant IL-7Rα.

Article Snippet: His-tagged recombinant human IL-7Rα ectodomain (R&D Systems, Minneapolis, MN, USA, Cat. No. 10758-IR) was captured on the active flow cell to approximately 50 response units (RU).

Techniques: Bioprocessing, Knock-Out, Recombinant, Flow Cytometry, Binding Assay, Clone Assay, Staining, SPR Assay

Journal: mAbs

Article Title: Targeting IL-7Rα with PNU-159682 antibody–drug conjugates in acute lymphoblastic leukemia: translational implications

doi: 10.1080/19420862.2026.2663639

Figure Lengend Snippet: Generation and cytotoxicity assessment of IL-7Rα-targeting ADCs. (a) Internalization kinetics of four IL-7Rα-targeting monoclonal antibodies in IL-7Rα-positive REH cells. Surface-bound antibody levels of the four antibodies at 0, 15, 60, and 240 minutes were determined by flow cytometry and normalized to the signal at minute 0. (b) Schematic representation of the conjugation process for generating IL-7Rα-targeting ADCs. Antibodies were partially reduced with 20 mM 2-mercaptoethylamine (2-MEA) for 0.5 hours at 37°C, followed by conjugation with 10 mM mc–vc–PAB–MMAE for 16 hours at 4°C, yielding an average drug-to-antibody ratio of 3–4. (c) Quantification of IL-7Rα expression (molecules per cell) in three different leukemia cell lines, CCRF-CEM (low), NALM6 (medium), and REH (high), using flow cytometry. (d) Cytotoxicity of free MMAE, isotype control IgG–MMAE, and four IL-7Rα-targeting ADCs in CCRF-CEM, NALM6, and REH cells. Cell viability was assessed using the WST-8 assay 72 hours after each treatment. Data are presented as mean ± SEM; n = 6 technical replicates from a single experiment.

Article Snippet: His-tagged recombinant human IL-7Rα ectodomain (R&D Systems, Minneapolis, MN, USA, Cat. No. 10758-IR) was captured on the active flow cell to approximately 50 response units (RU).

Techniques: Bioprocessing, Flow Cytometry, Conjugation Assay, Expressing, Control

Journal: mAbs

Article Title: Targeting IL-7Rα with PNU-159682 antibody–drug conjugates in acute lymphoblastic leukemia: translational implications

doi: 10.1080/19420862.2026.2663639

Figure Lengend Snippet: In vivo efficacy and biodistribution of IL-7Rα-targeting agents. (a) Schematic representation of the subcutaneous tumor model and treatment schedule with four IL-7Rα-targeting ADCs. (b) Tumor volumes over time for each treatment group. (c) Relative body weight changes during treatment. PBS, phosphate-buffered saline. Lines show mean ± SEM, n = 6–9 per group. (d) Serial in vivo fluorescence imaging of fluorophore-labelled parent anti-IL-7Rα mAbs and an isotype antibody control in a separate tracer-dose cohort (representative animals). (e) Quantification of tumor region-of-interest (ROI) fluorescence; each animal was normalized to its own 5-min post-injection value. NC, negative control. Data are presented as mean ± SEM; n = 3–5 per group. (f) Relative performance of the four anti-IL-7Rα mAbs (577, 2D5, 165, and 24) was compared across five parameters. Binding activity, SPR-derived apparent binding affinity, internalization, and pIC 50 (-log10 IC 50 [M]) and in vivo efficacy were evaluated using the respective ADCs. Ratings were assigned based on the experimental data shown in , using a semi-quantitative scale from “+” (lowest) to “++++” (highest). The scale reflects the relative ranking within each parameter and does not represent absolute quantitative values.

Article Snippet: His-tagged recombinant human IL-7Rα ectodomain (R&D Systems, Minneapolis, MN, USA, Cat. No. 10758-IR) was captured on the active flow cell to approximately 50 response units (RU).

Techniques: In Vivo, Saline, Fluorescence, Imaging, Control, Injection, Negative Control, Binding Assay, Activity Assay, Derivative Assay

Journal: mAbs

Article Title: Targeting IL-7Rα with PNU-159682 antibody–drug conjugates in acute lymphoblastic leukemia: translational implications

doi: 10.1080/19420862.2026.2663639

Figure Lengend Snippet: Enhanced anti-tumor activity of IL-7Rα-targeting ADCs with novel payload PNU-159682. (a) Schematic representation of the conjugation process for generating PNU-159682-linked ADCs. Antibodies were partially reduced with 20 mM 2-mercaptoethylamine (2-MEA) for 0.5 hours at 37 °C, followed by conjugation with 10 mM Mal–PEG4–VC–PAB–DMEA–PNU-159682 for 16 hours at 4 °C, yielding a drug-to-antibody ratio of 3–4. (b) In vitro cytotoxicity of 577-PNU, 577-MMAE, isotype control IgG–PNU, and free PNU-159682 in NALM6 cells. Cell viability was measured using the WST-8 assay 72 hours after treatment. Data are shown as mean ± SEM. (c) Comparison of IC 50 values between 577-MMAE and 577-PNU in NALM6 cells, calculated from nine independent experiments performed on separate days; IC 50 values analyzed after log10 transformation; paired t-test (two-tailed), p < 0.0001; geometric mean ratio (MMAE/PNU) = 85.3 (95% CI 57.7–126.0). (d) In vivo anti-tumor efficacy of each treatment in NALM6 xenografts (subcutaneous model). Mice were treated with a single dose of 577-MMAE (10 mg/kg; n = 4), 577-PNU (0.5 mg/kg; n = 5), isotype control IgG–PNU (0.5 mg/kg; n = 4), free PNU-159682 (17 µg/kg, the dose of PNU equal to 0.5 mg/kg 577-PNU; n = 3), or phosphate-buffered saline (PBS) vehicle ( n = 5). Tumor volumes were measured twice weekly. (e) Complete response (CR) rate on day 28 following treatment with 577-MMAE (10 mg/kg; n = 4) or 577-PNU (0.5 mg/kg; n = 5). Two-sided Fisher’s exact test comparing groups: p = 0.0476. (f) Relative body weight change (%) during treatment. Data are presented as mean ± SEM; n = 3–5 per group. * p < 0.05; **** p < 0.0001.

Article Snippet: His-tagged recombinant human IL-7Rα ectodomain (R&D Systems, Minneapolis, MN, USA, Cat. No. 10758-IR) was captured on the active flow cell to approximately 50 response units (RU).

Techniques: Activity Assay, Conjugation Assay, In Vitro, Control, Comparison, Transformation Assay, Two Tailed Test, In Vivo, Saline

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: IL4 and IL7 kineTACs enable cell type–specific VEGF internalization in cell lines. ( A ) mRNA normalized TPM of IL4Rα from Human Protein Atlas for Daudi and SiHa cell lines. ( B ) 24 h VEGF-647 internalization at 25 nM as a dose response of IL4-Bevacizumab in Daudi and SiHa cell lines. Curves are three-parameter nonlinear regressions, with dotted lines indicating bell-shaped curve fits. Means ± SEM of three biological replicates are shown. ( C ) mRNA normalized TPM of IL7R from Human protein atlas for Daudi and SiHa cell lines. ( D ) 24 h VEGF-647 internalization at 25 nM as a dose response of IL7-Bevacizumab in Daudi and SiHa cell lines. Curves are best fits for three-parameter nonlinear regressions. Mean values ± SEM are from three biological replicates. ( E ) Diagram of coculture experiments. Daudi cells (gray) and SiHa cells (purple) are coincubated with VEGF-647 and either IL4-Bevacizumab kineTAC (green) or IL7-Bevacizumab kineTAC (blue). Cell type–specific receptor expression allows for cell-specific internalization of VEGF using the respective kineTAC. ( F ) Representative flow cytometry data from coculture experiment. Daudi and SiHa cells in equal amounts were incubated for 24 h with 25 nM VEGF-647, and 0.3 nM IL4-Bevacizumab or 10 nM IL7-Bevacizumab. Gates show thresholds for VEGF positivity, defined as approximately 1% of total cells for VEGF-647 only. Top row: FITC + Daudi GFP . Bottom row: FITC − SiHa. Percentages in gate are displayed. ( G ) Fold change in VEGF-647 median fluorescence intensity in the coculture Daudi and SiHa experiment from F . Mean fold change over 25 nM VEGF-647 alone ± SEM is presented. The asterisk represents a discovery (q < 1%) using the false discovery rate to correct for multiple comparisons. ( H ) Coculture VEGF-647 percent positivity (same gate as in F ) as a dose response of IL4-Bevacizumab. Mean values and ± SEM are from three biological replicates. Curves are three-parameter nonlinear regressions, with dotted lines indicating bell-shaped curve fits. ( I ) same as in H , but for IL7-Bevacizumab.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Expressing, Flow Cytometry, Incubation, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A cytokine receptor–targeting chimera toolbox for expanding extracellular targeted protein degradation

doi: 10.1073/pnas.2524129123

Figure Lengend Snippet: IL4 and IL7 kineTACs enable cell type–specific VEGF internalization in primary lymphocytes and PBMCs. ( A ) Diagram depicting tissue-specific expression of potential kineTAC receptors. ( B ) tSNE analysis of IL4Rα expression from scRNA seq of two PBMC donors, obtained from the Immune Cell Atlas ( 57 ). Notice the prominent naïve B cell cluster of high IL4Rα expressors (green). ( C ) tSNE analysis of IL7R expression from the same dataset. Notice primarily T cell restricted expression of IL7R (blue). ( D ) Immune Cell Atlas cluster annotations for primary cell lineages, colored by lineage as in legend to the right. ( E ) Raw VEGF-647 median fluorescence intensities across two donors for isolated CD19+ cells from four technical replicates. Cells were incubated with 25 nM VEGF-647 and 10 nM of indicated kineTAC or isotype. ( F ) same as E , but using CD3+ T-cells. ( G and H ) VEGF-647 with PBMC components. 10 nM of the indicated kineTAC or isotype was added to 25 nM VEGF-647 along with 200 k cells/well PBMCs and allowed to internalize for 24 h. Percent VEGF positive was defined by the no VEGF-647 condition ( SI Appendix , Fig. S6 ). Analyzed by repeated measures donor-paired one-way ANOVA, with P values for Dunnett’s correction for multiple comparisons to the kineTAC of interest (IL4-Beva for B cells, IL7-Beva for T cells) (* P < 0.05, ** P < 0.01). ( G ) Analysis of B cell gate and h analysis of T cell gate.

Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, Cat# 4267S, 1:1,000), rabbit anti-human PD-1 (Cell Signaling Technology, Cat# D4W2J, 1:1,000), mouse anti-human IL7R (R&D Systems, Cat# MAB306, 1:1,000), rabbit anti-human phospho NF-κB p65 Ser 536 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human NF-κB p65 (Cell Signaling Technology, Cat#93H1, 1:1,000), mouse anti-human β-actin (Cell Signaling Technology, Cat# 8H10D10, 1:1,000), and mouse anti-human β-tubulin (Cell Signaling Technology, Cat# DM1a, 1:1,000).

Techniques: Expressing, Fluorescence, Isolation, Incubation

Journal: Chemical research in toxicology

Article Title: Induction of Thymus Atrophy and Disruption of Thymocyte Development by Fipronil through Dysregulation of IL-7-Associated Genes.

doi: 10.1021/acs.chemrestox.4c00060

Figure Lengend Snippet: Figure 6. Fipronil significantly decreased mRNA expression of Lyl1, SOX13, SCF, IL-7R, and GABPα in ConA-stimulated thymocytes. The total RNA of thymocytes (6 × 106 cells/mL) harvested from different treatment groups stimulated by ConA was extracted to detect the mRNA expression of Lyl1, SOX13, SCF, and IL-7 receptors by qPCR. The expression level of HPRT was used as the control for semiquantification. The expression level of HPRT was used as the control for semiquantification. Results were expressed as the mean ± SEM pooled from four independent experiments with technological duplication in each group (N = 20/group). *p < 0.05 was significant compared to the VH group.

Article Snippet: After being blocked with EveryBlot Blocking Buffer at room temperature, the PVDF membranes were incubated overnight at 4 °C with primary antibodies against IL-7 (ThermoFisher), IL-7R (Origene), SCF (ThermoFisher), c-KIT, FOXO3A, GABPA (Genetex, Hsinchu, Taiwan), LYL1 (ThermoFisher), or β-actin (Genetex).

Techniques: Expressing, Control