Structure-Activity Relationship of Antibody-Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody-siRNA Conjugates for Drug Development

Abstract

Antibody-oligonucleotide conjugates are a promising class of therapeutics for extrahepatic delivery of small interfering ribonucleic acids (siRNAs). These conjugates can be optimized for improved delivery and mRNA knockdown (KD) through understanding of structure-activity relationships. In this study, we systematically examined factors including antibody isotype, siRNA chemistry, linkers, conjugation chemistry, PEGylation, and drug-to-antibody ratios (DARs) for their impact on bioconjugation, pharmacokinetics (PK), siRNA delivery, and bioactivity. Conjugation site (cysteine, lysine, and Asn297 glycan) and DAR proved critical for optimal conjugate PK and siRNA delivery. SiRNA chemistry including 2' sugar modifications and positioning of phosphorothioates were found to be critical for delivery and duration of action. By utilizing cleavable and noncleavable linkers, we demonstrated the impact of linkers on PK and mRNA KD. To achieve optimal properties of antibody-siRNA conjugates, a careful selection of siRNA chemistry, DAR, conjugation sites, linkers, and antibody isotype is necessary.

Scheme 1. (a) Synthetic Scheme for Conjugating an siRNA with a Linker; (b) Antibody–siRNA Conjugate Synthesis via Interchain Disulfide (Cysteine) Conjugation; (c) Antibody–siRNA Conjugate Synthesis via Asn297 Conjugation; (d) Antibody–siRNA Conjugate Synthesis via Lysine Conjugation

Figure 1

Typical structure of an siRNA molecule. MCC linker: a heterobifunctional cross-linker that contains two reactive groups: an NHS ester and a maleimide. The NHS ester reacts with primary amines, while the maleimide reacts with sulfhydryl groups. This allows the MCC to form stable bonds with both amine- and sulfhydryl-containing molecules. C6 amino: a linker between the MCC and the siRNA. It provides a sufficient distance between the siRNA and the MCC to minimize any potential steric hindrance or interference with the siRNA’s function. Sense strand 5′ end: the 5′ end of the sense strand of the siRNA. The 5′ end is where the MCC is attached via the C6 amine linker. Sense strand 3′ end: the 3′ end of the sense strand of the siRNA. Antisense strand: designed to be complementary to the target mRNA sequence. It guides the RISC to the target mRNA. Phosphodiester and phosphorothioate bonds: the bonds that connect the nucleotides in the siRNA. Phosphodiester bonds are the standard bonds in RNA, while phosphorothioate bonds are modified versions that contain a sulfur atom, which can provide increased stability to the siRNA.

Figure 2

Evaluation of free cysteines available for conjugation on an αhEGFR-Cys-MCC-siAR DAR1 AOC following treatments with TCEP, NEM, and DHAA (n = 3).

Figure 3

Impact of siRNA chemical modifications on siRNA stability and activity in targeting Mstn through evaluation of tissue siRNA concentration (TC) and percent mRNA remaining relative to phosphate buffered saline (PBS) in gastrocnemius muscle of mice treated with αmTfR1-Cys-MCC-siMstn AOCs. ^aAll sense strands were duplexed with the antisense strand: vpUusUfsAoUoUoAfUoUoUoGoUoUoCoUfUoUfGoCoCosUosUo. Abbreviations: X refers to bases evaluated (AUGC): Xo = 2’O-methyl, Xf = 2’Fluoro, Xb = LNA, Xu = UNA-2’MOE, s = phosphorothioate, vp = vinylphosphonate.

Figure 4

(a) Plasma PK analysis of αhEGFR-siKRAS AOCs conjugated to the mAb cysteine, lysine, and Asn297 as measured by percent of injected dose (% ID) versus time (h). (b) Mouse plasma PK of DAR1 αhTfR1-siMstn AOCs generated via random cysteine versus cysteine engineered at position 188 of the light chain.

Figure 5

(a) Mouse plasma PK of AOCs (αhEGFR-Cys-MCC-siDMPK) comprised of αhEGFR mAb conjugated to one, two, or three siRNAs targeting DMPK mRNA. (b) Noncompartmental analysis of plasma PK data (0–96 h).

Figure 6

(a,b) Mouse PK of AOCs containing cleavable linkers.

Figure 7

(a–c) PK/PD studies with αmTfR1–siHprt AOCs containing four different noncleavable linkers.

Figure 8

(a,b) Liver KD analysis of αmASGPR–siHprt AOCs containing BisMal linkers to siRNA at the 5′ end and positions (pos) 8 or 14 of the sense strand at 96 h postdose.

Figure 9

(a) Expression of Hprt in muscle and (b) siRNA tissue concentration after treatment with αmTfR1–siHprt AOCs with MCC and VC linkers at various positions on the siHprt at 96 h postdose.