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Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction
Published on: September 14, 2018
Generation and validation of structurally defined antibody-siRNA conjugates
Alex R Nanna1,2, Alexander V Kel'in3, Christopher Theile3
1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA.
Abstract:
Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody-siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.
Insights
Researchers developed antibody-siRNA conjugates (ARCs) for targeted gene silencing in cancer. This new method enables efficient and specific delivery of RNA interference (RNAi) therapeutics to cancer cells, overcoming key delivery challenges.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Gene silencing via RNA interference (RNAi) shows therapeutic potential for diseases, including cancer.
- Efficient and selective delivery of small interfering RNAs (siRNAs) to cancer cells is a major hurdle for therapeutic application.
- Existing antibody-based siRNA delivery methods face limitations such as non-specific conjugation or complex site-specific strategies.
Purpose of the Study:
- To develop a novel, efficient, and site-specific method for generating antibody-siRNA conjugates (ARCs) for targeted cancer therapy.
- To create structurally defined ARCs using engineered dual variable domain (DVD) antibodies and chemically stabilized siRNA.
- To demonstrate the proof-of-concept for DVD-ARCs in delivering siRNA to multiple myeloma cells for gene knockdown.
Main Methods:
- Engineered dual variable domain (DVD) antibodies with a unique lysine residue were utilized for site-specific conjugation.
- A β-lactam linker-functionalized siRNA was conjugated to the DVD antibody, forming antibody-siRNA conjugates (ARCs) in a 1:2 ratio.
- DVD-ARCs targeting BCMA on multiple myeloma cells were generated to deliver siRNA against CTNNB1.
Main Results:
- A straightforward method for generating structurally defined, site-specific antibody-siRNA conjugates (ARCs) was established.
- The conjugation process was efficient and did not impair the antibody's antigen-binding affinity.
- Low concentrations (10 nM) of BCMA-targeting DVD-ARCs effectively reduced CTNNB1 mRNA and protein levels in multiple myeloma cells.
Conclusions:
- The developed ARC platform offers an efficient and easy-to-assemble approach for site-specific antibody-siRNA conjugation.
- This technology enables precise delivery of RNA interference therapeutics, holding promise for targeted cancer treatments.
- The proof-of-concept study demonstrates the potential of DVD-ARCs for selective gene silencing in cancer cells, such as multiple myeloma.
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