Engineering Nanobody-Drug Conjugates for Logic-Gated Immunotherapy

The stimulator of interferon genes (STING) pathway is an emerging and clinically relevant target for cancer immunotherapy. The innate pathway functions to detect cytosolic DNA through the activation of cyclic GMP-AMP Synthase (cGAS). Activated cGAS generates cGAMP, which then activates ER-resident protein STING, leading to the downstream production of immunostimulatory molecules. Natural cyclic dinucleotide agonists suffer from rapid clearance and poor stability, whereas powerful synthetic diamidobenzimidizole (diABZI) agonists can cause immune-related adverse effects when administered systemically. To combat these delivery problems, we developed STING-activating nanobody conjugates that target specific cell populations. We have previously shown that macrophages constitute at least 20% of tumor volume in a murine breast cancer model. Macrophages within the tumor microenvironment, also called tumor-associated macrophages (TAMs), are divided into two classes: M1 and M2. M1 macrophages are proinflammatory, drive increased immune infiltration in solid tumors, and have high CD80 expression. M2 macrophages are anti-inflammatory, promote tumor growth, and have high CD206 expression. CD206, also known as the macrophage mannose receptor (MMR), is involved in pathogen recognition and resolving inflammation. Studies have shown that STING activation can reprogram M2 TAMs to M1 TAMs. By targeting M2 TAMs with a diABZI STING agonist conjugated to an anti-MMR nanobody, we propose that we can elicit the reprogramming of M2 TAMs while avoiding the systemic inflammatory effects of free STING agonists.

To test our hypothesis, we generated an anti-MMR nanobody by transforming a plasmid vector containing the nanobody DNA sequence, an enzymatic conjugation tag, and a 6xHistidine affinity tag into E. coli. We grew and induced large bacterial cultures, then lysed the cells by sonication. The lysate was centrifuged, then purified by nickel affinity chromatography and an endotoxin-removal column. Endotoxin-free protein was then purified using size exclusion chromatography. An azide-containing linker was conjugated to the nanobody using an engineered sortase enzyme. A DBCO-functionalized diABZI STING agonist was then covalently conjugated to the nanobody through click chemistry. Through ELISA, we found micromolar binding affinity for murine MMR, which is unusually poor for nanobodies. However, we showed increased STING activation in vitro in M2 polarized versus M1 polarized RAW264-Dual murine macrophages. Future work involves testing the ability of anti-MMR-diABZI conjugates to reprogram polarized bone marrow-derived macrophages in vitro and assessing the biodistribution in vivo in tumor-bearing mice. We aim to assess the therapeutic potential of reprogramming TAMs with STING agonists and display the modular ability of nanobodies to target immune cell populations.