Search

Summary: Antibody-oligonucleotide conjugates (AOCs) are a fast-expanding modality for targeted delivery of therapeutic oligonucleotides to tissues. Here, we present a protocol to generate, purify, and analyze AOCs from off-the-shelf antibodies. We describe steps to conjugate single/double-stranded oligonucleotides bearing amine handles to linkers and, then, to antibodies using well-established chemistry. In addition, we provide details regarding the purification techniques and analytical methods suitable for AOC. This protocol can be applied for several purposes where AOC is a modality of interest.For complete details on the use and execution of this protocol, please refer to Rady et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Double-stranded RNAs (dsRNA)-based strategies appeared as promising therapies to induce an inflammation in the tumor microenvironment. However, currently described systems generally lack active targeting of tissues, and their clinical translation is thus limited to intratumoral injection. Herein, we developed an antibody-siRNA-5′triphosphate conjugate with multiple modes of action, combining cell surface EphA2-specific internalization, leading to a simultaneous gene silencing and activation of the receptor retinoic acid-inducible gene I (RIG-I). Recognition of cytosolic siRNA-5′triphosphate by RIG-I triggers the expression of interferons and pro-inflammatory cytokines, inducing an inflammation of the tumor environment and activating neighboring immune cells. In addition, these RIG-I-specific effects synergized with siRNA-mediated PLK1 silencing to promote cancer cell death by apoptosis. Altogether, such immune-stimulating antibody-RNA conjugate opens a novel modality to overcome some limitations encountered by dsRNA molecules currently in clinical trials.