Generation and validation of structurally defined antibody–siRNA conjugates

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.

Site-selective modification strategies in antibody–drug conjugates

“…14A). [216][217][218] This anti-HER2 DAR 2 ADC demonstrated exquisite in vitro and in vivo activity and selectivity. Based on these results, anti-CD138 and anti-CD79b ADCs were also generated from DVD antibodies.…”

Antibody-Oligonucleotide Conjugates: A Twist to Antibody-Drug Conjugates

“…Recently, a site-specific lysine conjugation has been described in which a beta-lactam can react selectively to an H38C2 antibody as shown in Figure 10 [ 31 ]. This hidden lysine has a lower pKa (6.0) since it is in an accessible hydrophobic pocket, which in turn increases by orders of magnitude its reactivity towards electrophiles [ 32 ].…”

“…A study looking specifically at the stability and PK of AOCs has recently been published by the Rader group along with Alnylam. This study looked at the PK profile of the AOC and the naked mAb [ 31 ]. The sense strand of the oligonucleotide was biotinylated, which allowed the capture of the conjugated antibody with streptavidin.…”

Viral Mimicry as a Design Template for Nucleic Acid Nanocarriers

“…Their use in directing nucleic acid carriers has been demonstrated in several studies (Palanca-Wessels et al, 2011;Ngamcherdtrakul et al, 2015;Moffett et al, 2017;Huggins et al, 2019;Nanna et al, 2020). They can be either directly conjugated to the nucleic acid (Huggins et al, 2019;Nanna et al, 2020) or to the vector (Palanca-Wessels et al, 2011;Ngamcherdtrakul et al, 2015;Moffett et al, 2017). Antibody-RNA conjugates (ARCs) are promising in that they overcome possible limitations of nanoparticle-based formulations such as poor diffusivity, toxicity, and Glu-or His-rich peptides undergo aciddriven conformational change to alphahelical structure, leading to pore formation pDNA entrapped in gelatin-silica nanoparticles modified with fusogenic peptides, or nanobiomimetic carrier composed of targeting and fusogenic peptides by which DNA is condensed.…”