Function-Driven Design of a Surrogate Interleukin-2 Receptor Ligand

We developed an artificial signaling system to enable the assembly of cytokine receptor surrogate ligands. A JAK-STAT5 signaling module—comprising the JAK3-specific ?c receptor and a functional IL-2Rß receptor—was stably expressed in a STAT-deficient cell line. Using a combinatorial antibody library, we screened for receptor binders with selective emphasis on IL-2Rß affinity. Among various bispecific scaffolds, the single-chain diabody (scDb) format demonstrated superior potency and efficacy, effectively recapitulating native IL-2 ligand activity by inducing functional dimerization of IL-2Rß and ?c receptors. The lead scDb-2d (also termed J9H4) agonist antibody functioned as a surrogate IL-2 receptor ligand, selectively driving Teff and NK cell proliferation and activation. Overall design: Human NK cells were purified using EasySep™ Human NK Cell Isolation Kit (STEMCELL Technologies, #17955), then the cells were proliferated with irradiated (100 Gy) wild type K562 cells (feeder cell to NK cell ratio of 1:1) adding 20 nM natural cytokine (IL-2, IL-15) or antibody (scDb-2d) for 7 days then stimulation with 20 nM natural cytokine and antibody for another 6 days, natural cytokine and antibody were refreshed every 2 days. Then the cells were washed and rested overnight for another 24h stimulation by 20 nM IL-2/IL-15 or scDb-2d.