Fig. 1: Design, synthesis and in vitro characterization of an anti-albumin nanobody for site-selective conjugation of STING agonists.

a, Scheme depicting the concept of an albumin-hitchhiking nanobody–STING agonist conjugate for cancer immunotherapy. Anti-albumin nanobodies conjugated to STING agonists bind to circulating albumin in situ, resulting in improved pharmacokinetics and increased biodistribution to tumour sites that stimulates antitumour innate and adaptive immune responses. b, Computational model of the anti-albumin nanobody (nAlb) binding at domain IIB of HSA. c, ITC traces (top) and binding isotherms (bottom) of nAlb binding to human and mouse serum albumin at pH 7.5 with calculated dissociation constant (K_d). d, Reaction scheme for generating molecularly homogeneous nAlb conjugates through site-selective enzymatic ligation of an amine-PEG₃-azide followed by conjugation of agonist or dye cargo through strain-promoted azide-alkyne cycloaddition (SPAAC). e, Structure of diABZI STING agonist conjugated to a DBCO-PEG₁₁ handle for ligation to azide-functionalized nanobodies via SPAAC. f,g, ESI–MS (f) and SDS–PAGE (g) showing nanobody conjugate purity and molecular weight (see Source Data for uncropped gel in ref. ⁹⁰). h,i, Dose–response curves in A549-Dual (n = 3) (h) and THP1-Dual type I interferon reporter cell lines (n = 3) (i) with estimated EC₅₀ values indicated in the legends; RLU, relative light unit. j, qPCR analysis of gene expression in mouse BMDMs treated in vitro with 0.25 µM of free diABZI or nAlb–diABZI conjugate (n = 3). P values determined by one-way ANOVA with Dunnett’s multiple comparison test with groups compared to PBS. Replicates are biological, and data are shown as mean ± s.e.m. Panel a created with BioRender.com.