Fig. 4: Design, synthesis and testing of bivalent nanobody–STING agonist conjugate for albumin hitchhiking and targeting of PD-L1.

a, Scheme for the cloning, expression and bioconjugation of small molecule cargo to generate the AP–diABZI conjugate. b,c, SDS–PAGE (b) and ESI–MS (c) confirming the purity and molecular weight of AP conjugates (see Source Data for uncropped gels in ref. ⁹⁰). d,e, Dose–response curves for indicated nanobody–diABZI conjugate in A549-Dual (n = 3) (d) and THP1-Dual type I interferon reporter cell lines (n = 3) (e) with estimated EC₅₀ values indicated in the legends. f, qPCR analysis of genes associated with STING activation in BMDMs in response to treatment at discrete time points with indicated agonist at 0.25 µM (n = 3). g,h, Dose–response curve for nAlb–Cy5 and AP–Cy5 conjugate intracellular uptake and surface binding at 37 °C and 4 °C as measured by flow cytometry in B16.F10 cells (n = 2 at 4 °C and n = 3 at 37 °C) (g) and EMT6 cells (n = 3) (h). i, MFI for nAlb–Cy5 and AP–Cy5 conjugate surface binding at 2 µM compared to PBS (0 µM) for EMT6 WT and EMT6 PD-L1 KO cell lines at 37 °C (n = 3). KO, knock-out; WT, wild type. j, Pharmacokinetics of indicated nanobody–Cy5 conjugate in healthy Balb/c female mice (n = 4 for nPD-L1–Cy5; n = 5 for all other groups). Elimination phase half-life and AUC are indicated in the legend. k, Representative IVIS fluorescence images of excised tumours and major organs (left) and quantification of average radiant efficiencies (right) of tumours and major organs 48 h after administration of nPD-L1–Cy5 and AP–Cy5 in mice with EMT6 breast tumours (n = 4). P values determined by repeated measures ANOVA with Dunnett’s multiple comparison test for tumour compared to indicated tissue. l, Comparison of Cy5 radiant efficiencies in tumour tissue 48 h following administration of indicated nanobody–Cy5 conjugate (n = 6 for PBS and nAlb–Cy5; n = 4 for AP–Cy5; n = 3 for nPD-L1–Cy5). P values determined by one-way ANOVA with post hoc Tukey’s correction for multiple comparisons with comparisons between all groups and PBS and between nAlb–Cy5 and AP–Cy5 as indicated. m, Representative IVIS fluorescence images of excised tumours and major organs (left) and quantification of average radiant efficiencies (right) of tumours and major organs 48 h after administration of AP–Cy5 in mice with wild-type EMT6 (WT) and PD-L1 knock-out EMT6 (PD-L1 KO) breast tumours (n = 5). P values determined by repeated measures ANOVA with Dunnett’s multiple comparison test for WT versus PD-L1 KO groups. Replicates are biological, and data are shown as mean ± s.e.m. Panel a created with BioRender.com.