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