Engineering Nanobody-Drug Conjugates for Cancer 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 a STING-activating nanobody conjugate targeting Integrin Alpha M, also known as CD11b, a cell surface receptor present on myeloid cells. The STING pathway is active in myeloid cells so targeting CD11b allows for the specific delivery of drugs to this cell population.

To test our hypothesis, we generated a CD11b-targeting 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. Initial production revealed the sequence, derived from literature, did not express in our production and purification system. Site-directed mutagenesis was performed to eliminate histidine residues in the sequence, resulting in strong expression. Computational modeling provided estimates of the effects of each mutation on folding. Binding was confirmed by flow cytometry with RAW macrophages. An azide-containing linker was then conjugated to the nanobody using an engineered sortase enzyme. A DBCO-functionalized diABZI STING agonist or Cy5 dye was then covalently conjugated to the nanobody through click chemistry. The binding affinity was determined by biolayer interferometry. Future work involves performing dose-response studies with the agonist-conjugated nanobody and in vivo biodistribution and pharmacokinetic studies using mice.

As CD11b has been shown to internalize slowly, a cleavable linker is necessary o ensure internalization of the drug. A urokinase-cleavable linker was added to the nanobody conjugates after a literature search revealed high levels of urokinase expression in breast tumors and glioblastoma. Cleavage was confirmed in vitro using purified enzyme. Future work involves dose response studies in vitro compared to the previously developed conjugates and characterizing the biodistribution and pharmacokinetics.