Immunostimulatory Nanobody-STING Agonist Conjugates to Improve Immunotherapy

Introduction:: While immunotherapy has revolutionized cancer treatment, the tumor microenvironment (TME) limits the efficacy of therapies like immune checkpoint inhibitors and CAR-T cell therapy. To combat the immunosuppressive nature of the TME, our lab has focused on targeting the stimulator of interferon genes (STING) pathway, which can trigger immune-activating cues that increase tumor immunogenicity. Although there are a variety of STING agonists now available, they have suboptimal properties for systemic administration, resulting in low antitumor efficacy and/or systemic inflammation with potential cause toxic side effects. To improve the pharmacological properties and tumor selectivity of a diamidobenzimidazole (diABZI) STING agonist we developed an “albumin hitchhiking” strategy that leverages an anti-albumin nanobody to which drugs can be conjugated in a site-selective manner. Albumin is a naturally occurring protein that has been shown to localize to tumors while also increasing the circulation time of drugs that bind to it. Additionally, by generating nanobody fusions, our approach also enables integration of tumor targeting domains. B7H3 is a molecule that is highly expressed in a diversity of solid tumors that also has immunosuppressive function. Here, we characterize anti-albumin, anti-B7H3, and anti-albumin/anti-B7H3 fusion nanobodies conjugated to a diABZI STING agonist as a strategy to increase the efficacy of immunotherapies.


Materials and Methods:: The DNA sequence of the protein being synthesized was combined into a plasmid via the Golden Gate Assembly mix (New England BioLabs). The plasmid containing the protein sequence was transformed into SHuffle® T7 Competent E. coli for protein expression. The expressed protein was purified using histidine to Ni affinity via Ni-NTA columns. The proteins were purified through fast protein liquid chromatography (FPLC) and an azide group was conjugated onto the protein via sortase cleavage. Next, using click chemistry, the protein-azide conjugate was then conjugated to either DBCO-Cy5 or DBCO-diABZI. The steps of each reaction were tested for purity via Electrospray Ionisation Mass Spectrometry (ESI-MS) and SDS-PAGE. To test activity, THP-1 Dual cells and A549 Dual cells were treated with protein conjugates and type I interferon production was quantified using the Quanti-Luc^TM (InvivoGen) assay. In vivo experiments were conducted on C57BL/6 mice which were inoculated with 1,000,000 EMT6 cells subcutaneously. When tumors reached ~75 mm^3, mice were treated intravenously with the protein-Cy5 conjugates and sacrificed at 24 hours at which point organs were dissected and imaged on an IVIS. Lastly, blood was analyzed for Cy5 signal at multiple timepoints via tail vein injections.

Results, Conclusions, and Discussions:: We first characterized the anti-albumin, anti-B7H3, and anti-albumin/anti-B7H3 fusion nanobody using mass spectrometry. Firstly, we observed improved pharmacokinetic properties with increased half-life of approximately 20-fold compared to Cy5 administered alone in C57BL/6 mice (Fig 1A). For initial studies, we selected only the anti-albumin nanobody conjugated to Cy5 to assess pharmacokinetics and biodistribution. The protein conjugate was highly localized to the tumor compared to other internal organs (Fig 1B). Next, the ability to conjugate payloads to nanobodies was tested by conjugating Cy5, a fluorescent molecule, to each nanobody and confirmed using SDS-PAGE as well as mass spectrometry (Fig 1C-F). Next, the activity of the protein conjugates when linked to diABZI was tested in vitro on A549D and THP1D (IFN-ß reporter lines), which showed increased expression of interferon-beta (IFN-ß), an inflammatory marker (Fig 1G-H). Current in vitro studies are underway to determine the immune response to the protein conjugate in tumor cells that express B7H3. This can further be extended to include cases where the TME components express B7H3 to open a pro-inflammatory window. Immediate future work includes developing a tumor mouse model to determine the in vivo efficacy of the conjugate system. Taken together, this data indicates potential to improve efficacy of already existing therapies.

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