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: iScience

Article Title: Translation factor eIF4G2 directs CD8 + T cell lineage commitment by selectively enabling the IL-7 receptor response

doi: 10.1016/j.isci.2026.115313

Figure Lengend Snippet: eIF4G2 specifically sustains surface expression of the IL-7 receptor (A–C) Expression of IL-7Rα (CD127). (A) Representative histogram and (B) quantification of CD127 median fluorescence intensity (MFI) on CD4 + CD8 lo transitional cells ( n = 3, ∗∗ p < 0.01). (C) MFI of CD127 on DP, CD4 SP, and CD8 SP thymocytes ( n = 3, ns p > 0.05, ∗∗∗∗ p < 0.0001). (D–F) Expression of the common γc (CD132). (D) Representative histogram and (E) quantification of CD132 MFI on CD4 + CD8 lo transitional cells ( n = 4, ∗ p < 0.05). ppp(F) MFI of CD132 on DP, CD4 SP, and CD8 SP thymocytes ( n = 3, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (G and H) Expression of IL-4Rα (CD124) expression detection on CD4 + CD8 lo transitional cells. (G) Representative histogram and (H) quantification of CD124 MFI on CD4 + CD8 lo transitional cells ( n = 3, ns p > 0.05). (I and J) Expression of GP130 on CD4 + CD8 lo transitional cells. (I) Representative histogram and (J) quantification of GP130 MFI on CD4 + CD8 lo transitional cells ( n = 3, ∗∗ p < 0.01). (K) Quantification of CD124 MFI on DP, CD4 SP and CD8 SP ( n = 3, ns p > 0.05). (L–N) Expression of IL-2Rα (CD25). (L) Representative histogram and (M) quantification of CD25 MFI on CD4 + CD8 lo transitional cells ( n = 3, ns p > 0.05). (N) MFI of CD25 on DP, CD4 SP, and CD8 SP subsets ( n = 3, ns p > 0.05). Data are representative of at least two independent experiments. Bar graphs show mean ± SEM and unpaired Students’ t test was used to perform the statistical analysis.

Article Snippet: Membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies diluted in blocking buffer: eIF4G2 (CST, 3468S), β-actin (CST, 3700S), eIF4G1 (Proteintech, 15704-1-AP), IL-7Rα (Proteintech, 17626-1-AP), γc (Proteintech, 11409-1-AP), STAT5 (Proteintech, 13179-1-AP), phospho STAT5 (CST, 4322T), STAT6 (Proteintech, 51073-1-AP), mouse phospho STAT6 (CST, 56554S).

Techniques: Expressing, Fluorescence

Journal: iScience

Article Title: Translation factor eIF4G2 directs CD8 + T cell lineage commitment by selectively enabling the IL-7 receptor response

doi: 10.1016/j.isci.2026.115313

Figure Lengend Snippet: eIF4G2 post-transcriptionally sustains γc expression via its mRNA UTRs (A–C) Analysis in primary CD4 + CD8 lo transitional thymocytes. (A) Western blot analysis of γc and IL-7Rα protein levels. (B and C) Quantitative RT-PCR analysis of Il2rg (B) and Il7r (C) mRNA levels ( n = 3, ns p > 0.05, ∗ p < 0.05). (D–H) Mechanistic dissection in 293T cells. (D) Western blot of γc protein in control and EIF4G2 knockdown 293T cells transfected with an IL2RG coding sequence construct containing its native 5′ and 3′ UTRs. (E) Corresponding IL2RG mRNA levels measured by RT-qPCR ( n = 3, ns p > 0.05) . (F and G) Assessment of γc protein stability ( n = 3, ns p > 0.05). (F) Representative western blots and (G) quantification of γc protein levels over time following cycloheximide (CHX) treatment in si-control and si- EIF4G2 293T cells ( n = 3, ns p > 0.05). (H) Western blot of γc protein in si-control and si- EIF4G2 293T cells transfected with an IL2RG CDS construct lacking UTRs. Data are representative of at least two independent experiments. Bar graphs show mean ± SEM and unpaired Students’ t test was used to perform the statistical analysis.

Article Snippet: Membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies diluted in blocking buffer: eIF4G2 (CST, 3468S), β-actin (CST, 3700S), eIF4G1 (Proteintech, 15704-1-AP), IL-7Rα (Proteintech, 17626-1-AP), γc (Proteintech, 11409-1-AP), STAT5 (Proteintech, 13179-1-AP), phospho STAT5 (CST, 4322T), STAT6 (Proteintech, 51073-1-AP), mouse phospho STAT6 (CST, 56554S).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Dissection, Control, Knockdown, Transfection, Sequencing, Construct