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Structure–Activity Relationship of Antibody–Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody–siRNA Conjugates for Drug Development
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Structure–Activity Relationship of Antibody–Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody–siRNA Conjugates for Drug Development
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  • Michael Cochran
    Michael Cochran
    Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
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Journal of Medicinal Chemistry

Cite this: J. Med. Chem. 2024, 67, 17, 14852–14867
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https://doi.org/10.1021/acs.jmedchem.4c00802
Published August 28, 2024

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Abstract

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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.

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Introduction

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Antibodies and oligonucleotides, two powerful classes of molecules, have diverse applications in biology and medicine. As highly specific proteins, antibodies recognize and bind to a wide range of targets. Their proven effectiveness as receptor antagonists and as delivery vehicles for small molecules makes them potent therapeutics. (1−4)
Oligonucleotides exert their therapeutic effect through multiple mechanisms, including inducing the degradation of mRNA or promoting splice modulation. (5−9) Over the last 25 years, more than 16 oligonucleotide therapeutics have been approved for multiple therapeutic indications, including homozygous familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, hereditary transthyretin-mediated amyloidosis, familial chylomicronemia syndrome, acute hepatic porphyria, and primary hyperoxaluria. (10)
Small interfering ribonucleic acids (siRNAs) are 20–25 nucleotide-long, double-stranded RNA molecules containing a complementary sense and antisense strand. When inside the cell, these duplexes load into the Ago2 protein. Following this, the sense strand of the duplex siRNA is cleaved, and its fragments ejected, leaving behind a functional RNA-induced silencing complex (RISC) that comprises the antisense strand and Ago2. The RISC then uses the antisense strand to recognize and bind to complementary mRNA sequences within the cell. Once fully hybridized with the mRNA, Ago2 cleaves the mRNA between target bases 10 and 11, resulting in mRNA degradation and reduced gene expression. siRNAs are proven therapeutic agents, especially in diseases caused by the overexpression of certain genes or the presence of harmful genetic mutations. By selectively targeting specific disease-related RNA, siRNA-based therapies hold promise for treating a wide range of conditions, including certain types of cancer, viral infections, and genetic disorders. (5−9)
siRNA therapeutics face three main challenges: selectivity, stability, and delivery. Selectivity is primarily tackled through bioinformatics or chemical modifications, which can help predict the efficacy of candidate oligonucleotides and ensure they are designed to minimize off-target effects. (11) Stability is addressed by chemical modifications, such as phosphorothioate linkages or 2′-O-methyl modifications, which protect the oligonucleotides from degradation by nucleases in the bloodstream and other tissues. Delivery is an issue because different tissue types can present unique barriers, requiring specific targeted delivery strategies dependent on the target tissue. siRNA delivery to the liver is achieved with lipid nanoparticle formulations and conjugation of ligands such as N-acetylgalactosamine (GalNAc), which has a high affinity for asialoglycoprotein receptors (ASGPRs) that are predominantly expressed on hepatic parenchymal cells. Also, intrathecal delivery of siRNA modified with C16, or 2′-O-hexadecyl has produced potent and durable knockdown (KD) in the central nervous system (CNS). (12) However, there remains a significant unmet need for technologies that can effectively deliver oligonucleotides systemically to other tissues. (13) Owing to their inherent specificity for cell surface receptors and their demonstrated capability to transport small molecules to a variety of tissues, antibodies have emerged as promising vehicles for oligonucleotide delivery. Antibody–oligonucleotide conjugates (AOCs) present a promising solution for overcoming the challenges associated with oligonucleotide delivery.
Previous research by different groups has explored the use of antibody-mediated targeted delivery of oligonucleotides, yielding varying degrees of success. Seminal work by Cuellar et al. (2015) showed that siRNA oligonucleotides could be conjugated to thiol-containing monoclonal antibodies (mAbs), resulting in active, stable conjugates in vitro. (14) They showed that the mAb-siRNA conjugates retained a similar binding affinity and gene-silencing capacity to that of their constituent mAb or siRNA. However, when tested in mouse tumor xenograft studies, their activity was modest. Sela et al. (2023) recently reported on a new method for delivering antisense-oligonucleotides (ASOs) to the CNS for treating neurological disorders. (15) They conjugated the ASO to an antibody that could cross the blood–brain barrier. The study found promising in vitro activity and in vivo pharmacokinetics (PK) behavior, suggesting potential for future ASO-based CNS drugs. Of particular interest is the targeted delivery of various oligonucleotides to muscle for the treatment of muscle disorders via a transferrin receptor 1 (TfR1)-mediated approach. (16) Recent publications, such as the work by Malecova et al. (2023), have demonstrated effective delivery of diverse oligonucleotides to muscle tissue. (17)
While other studies have demonstrated delivery of oligonucleotides conjugated to mAbs and antigen-binding fragments, there is currently a lack of information on comprehensive SARs of antibody–siRNA conjugates. (16,18−20) This publication will provide a comprehensive SAR of antibody–siRNA conjugates, including optimal linker selection, points of conjugation on the antibody and siRNA, bioconjugation strategies to link an antibody and siRNA oligonucleotide, and the impact of siRNA oligonucleotide-to-antibody ratios on activity. The AOC approach offers a flexible strategy to increase the potential delivery of oligonucleotides to target tissues.

AOC Naming Convention

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In the rapidly evolving field of AOCs, the need for a clear, standardized naming convention is paramount because a multitude of variables exist, such as the type of antibody, its target, the linker used, and the siRNA conjugated. Our naming convention, detailed below, offers a systematic approach using specific symbols and abbreviations to denote each component of an AOC.
Take the following conjugates as examples: αhEGFR-Cys-MCC-siKRAS, αmTfR1-Cys-MCC-siMstn, αmTfR1-Cys-PDP-siMstn (for disulfide linkers). Here, “α” means “anti” to indicate that the antibody binds to EGFR (epidermal growth factor receptor) or TfR1. “h” or “m” denote whether the antibody is human (h) or mouse (m) specific. “Cys” or “Lys” represent the amino acids cysteine (Cys) or lysine (Lys) that the linker is attached to on the antibody. “MCC” or “PDP” are the linkers, 4-(N-maleimidomethyl)cyclohexane-1-carboxyamide and 3-(2-Pyridyldithio)propionamide. The “siKRAS” or “siMstn” are the siRNAs, targeting KRAS mRNA and Mstn mRNA, respectively, that are conjugated to the antibody. For example, “αhEGFR-Cys-MCC-siKRAS” would be an anti-EGFR antibody that is specific to humans, with an MCC linker attached to a cysteine residue, and conjugated to an siRNA targeting KRAS.
In our naming convention for linkers, we use the linker name, rather than the reagent used in the reaction. This is because during the reaction, a part of the reagent is removed. For instance, instead of using “SMCC” (the reagent) as the name of the linker, we simply use “MCC”. This approach ensures that the names we use are representative of the actual structure of the AOCs. However, in the Experimental Section, we use the full reagent name when discussing the linker reaction with the siRNA; for example, SMCC (reagent) versus MCC (linker).

Results and Discussion

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We first describe a general overview of the AOC synthesis used to generate antibody–siRNA conjugates. AOCs can be generated using any antibody isotype and siRNA with a conjugation handle, using cysteine, lysine or Asn297 conjugation methods and their corresponding linkers. The conditions and yields may vary, based on the constructs being used, but the general scheme remains the same.
We then detail the results of bioconjugation reaction and purification steps that reduce the risk of product aggregation and yield a highly pure AOC product. We then describe our investigations into antibody–siRNA conjugate SARs. We evaluated siRNA chemical modifications, siRNA conjugation locations on the mAb, cleavable versus noncleavable linkers, linker location on the siRNA, the addition of polyethylene glycol (PEG), and antibody isotypes.
Table S1 describes the antibody target, linker, siRNA sequence and chemical modification pattern for each antibody–siRNA conjugate. Furthermore, Table S2 provides a comprehensive list of the siRNA conjugation components, detailing the siRNA conjugation handles, linker reagent structures, linker-siRNA structures, antibody reactive groups, and final antibody–siRNA conjugate structures.

AOC Synthesis

General Synthetic Scheme for Antibody–siRNA Conjugate Generation

Scheme 1 outlines the general synthetic scheme we use to generate antibody–siRNA conjugates. For cysteine-conjugated AOCs, we first couple a linker-maleimide to the amino group at the 5′ end of the sense strand of a duplex siRNA using N-hydroxysuccinimide (NHS) chemistry. (21,22) This generates an intermediate siRNA-linker-maleimide (Scheme 1a).

Scheme 1

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
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We then partially reduce the interchain disulfide of the antibody with tris(2-carboxyethyl)phosphine (TCEP) (Scheme 1b). This means the disulfide bonds between the cysteine residues in the antibody are broken, exposing the cysteine residue thiol groups, which can then be used for conjugation with the oligonucleotide. The antibody is then conjugated to the siRNA-linker-maleimide. The resulting intermediate conjugate is processed through anion exchange chromatography, dehydroascorbic acid (DHAA) oxidation, N-ethylmaleimide (NEM) capping, and buffer exchange. This results in an antibody–siRNA conjugate with a drug-to-antibody ratio of 1 (DAR1). Scheme 1b depicts random cysteine conjugation. In this paper, we will also discuss site-specific cysteine conjugation.
In addition to the cysteine conjugation shown in Scheme 1b, AOCs can be synthesized via Asn297 (Scheme 1c) and lysine (Scheme 1d) conjugations. (23−25) For synthesis via Asn297, we modify the N-linked glycans at Asn297 enzymatically to azide-activate the antibody on each heavy chain. A bifunctional dibenzocyclooctyne (DBCO)-PEG-trans-cyclooctene (TCO) linker is added to the azide conjugation handle on the antibody to generate an antibody with a TCO conjugation handle at the Asn297 location. In a separate reaction, methyltetrazine-siRNA is generated by conjugating methyltetrazine NHS ester to the siRNA amine conjugation handle. A TCO-methyltetrazine click chemistry is performed between the functionalized antibody and siRNA to generate an AOC with a DAR of 1. For synthesis via lysine, the antibody is activated using the noncleavable linker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); the NHS ester of SMCC reacts with the amino groups of the lysine residues attaching reactive maleimide groups to the lysine side chain. The maleimide-activated antibody is then reacted with the thiol-containing siRNA and the resulting intermediate conjugate is then processed through anion exchange chromatography and buffer exchange, resulting in a DAR1 antibody–siRNA conjugate.
Figure 1 shows a typical structure of an siRNA molecule conjugated with a 4-(N-maleimidomethyl)cyclohexane-1-carboxyamide (MCC) linker through a C6 amine linker. This paper describes AOCs generated with siRNAs conjugated at the 5′ and 3′ termini of the sense strand. There are potential advantages of each of these strategies, including using the steric bulk of the conjugation to protect against 3′ nucleases as well as preventing unwanted loading of the sense strand into RISC by conjugating to the 5′ end. (26)

Figure 1

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.

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Throughout this paper, AOC dose levels are based on the siRNA component of the AOC. Calculations to convert siRNA to mAb or AOC concentrations are provided in the Experimental Section. A 1 mg/kg siRNA dose is equivalent to a 10.6 mg/kg mAb dose, and an 11.6 mg/kg DAR1 AOC dose. While the molecular weight of the siRNA and AOC may change with sequence, modifications, and linkers, the impact of these changes is less than 5% of the total MW and does not significantly impact these ratios.

Optimization of Disulfide Bond Reduction and Capping in Cys Conjugation Method

The cysteine DAR 1 AOCs generated by partially reducing the interchain disulfide bonds of an antibody have 1–7 unreacted thiols depending upon the number of reduced disulfides in an antibody molecule. These free cysteines could lead to aggregates, disulfide scrambling, and impact the conjugate stability due to a fewer number of disulfide bonds holding the conjugate together. To preemptively solve these potential challenges, we used oxidation with DHAA to reform the reduced excess disulfide bonds, then a capping of the remaining one cysteine of the isolated DAR1 AOC using NEM alkylation to form a thioether.
We evaluated the number of free cysteines on a DAR1 conjugate (Figure 2). After TCEP reduction, siRNA conjugation, and purification using strong anion exchange chromatography (SAX), DAR1 αhEGFR-siRNA AOCs (αhEGFR-Cys-MCC-siAR) without DHAA oxidation or NEM capping had more than one free cysteine available, indicating that some DAR1 conjugate molecules contain at least one other reduced disulfide bond. This isolated DAR1 AOC was split into three treatment groups: no treatment (DAR1), five equivalents of NEM to cap unreacted cysteine (DAR1 + NEM), and 10 equiv of oxidizing DHAA (DAR1 + DHAA). NEM capping blocked nearly all free cysteines (0.13 free cysteines per antibody) of DAR1 AOC, while DHAA oxidation left one free cysteine available for potential conjugation (0.89 free cysteines per antibody). AOCs with a DAR of 1 were at least 95% pure by strong anion exchange chromatography and size exclusion chromatography (SEC). The results suggest that after oxidation with DHAA, the αhEGFR hIgG1 DAR1 AOC used in this experiment will have approximately one free cysteine available to conjugate other functional groups (such as peptides or fluorescent dyes) or that free cysteine must be capped by alkylation to prevent AOC dimer formation in storage conditions (data not shown). These results are specific to the antibody used in this experiment. It is expected that each antibody will have its own degree of reduction after TCEP treatment. However, the presence of free cysteines and the requirement for alkylation to reduce dimer formation have been observed for DAR1 AOCs made with two other hIgG1 antibodies (data not shown).

Figure 2

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).

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siRNA Chemical Modifications

The susceptibility of the siRNA internucleotide phosphodiester bond to degradation by endonucleases or exonucleases upon systemic administration hinders the accumulation of intact therapeutic siRNA in target tissues. It is important for siRNAs to be stable in circulation as well as in tissues to show not only initial target mRNA KD, but also long duration of action after deposition in the target tissues. Chemically modifying siRNAs, such as replacement of 2′–OH with a 2′-deoxy-2′-fluoro (2′-F), 2′-O-methyl (2′-OMe), or 2′-O-methoxyethyl (2′-MOE) group, as well as substitutions of certain internucleotide phosphates with phosphorothioates (P═S) is a widely used strategy to improve siRNA stability in circulation as well as intracellular environments. (27,28) One commonly used modification pattern includes two P═S at each terminus of the antisense strand, two P═S at the 5′ end of the sense strand, and either one or no P═S on the 3′ end of the sense strand, which is frequently used for GalNAc conjugates with a 3′-pyrrolidine linker. (10,29) We tested the same pattern with a C6 amine linker as an AOC and found it to be either less active or to have low durability with AOCs in vivo, as shown in Figure 3. We presume this is due to nuclease-mediated cleavage of siRNA from the unprotected end of the sense strand in mice.

Figure 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.

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To improve the stability and activity of siRNAs on AOCs in muscle tissue, we explored combinations of sugar modifications including locked nucleic acid (LNA) and unlocked nucleic acid (UNA), which are known to improve siRNA stability in biological matrices, and phosphorothioate patterns. We used AOCs consisting of an antimouse TfR1 mAb (αmTfR1) conjugated to siRNAs targeting the myostatin (Mstn) gene with the MCC linker (αmTfR1-Cys-MCC-siMstn). The modification pattern of each sense strand is shown in Figure 3. Each sense strand was duplexed with the same antisense strand (vpUusUfsAoUoUoAfUoUoUoGoUoUoCoUfUoUfGoCoCosUosUo) containing an unlocked 2′-O-methoxymethyl uridine base with a vinylphosphonate at the 5′ end (Scheme S1). A 5′-phosphate is required for loading into the mid-domain of the Argonaut2 protein; however, natural phosphorylation of the siRNA by Clp1 kinase can be impacted by chemical modifications to the siRNA. 5′-vinylphosphonates, metabolically stable 5′-phosphate mimics, which have been shown to improve tissue accumulation and efficacy of conjugated siRNAs in vivo, (30,31) were included in this experiment to reduce the potential impact of new modifications on activity. The unlocked sugar with 2′-O-methoxyethyl is believed to provide flexibility to load into the mid-domain of Argonaut2 along with steric bulk to protect against nucleases.
We administered 1 mg/kg (siRNA) doses to mice at Day 0, then sacrificed four animals from each treatment at each time point: 24 h, and 2, 4, 6, and 8 weeks. We measured Mstn mRNA expression and siRNA concentrations in the gastrocnemius muscle at each time point. Among the sugar modifications, UNA was found to be more stable than 2′-O-methyl, while LNA was more stable than UNA. This trend is true for siRNAs with no P═S on the termini of the sense strand (groups 1, 2, 4, and 6), as well as siRNAs that have one P═S at each end of the sense strand (groups 3, 5, and 7). The 5′ end of the sense strand may be the most vulnerable to degradation in the absence of P═S groups, which can be seen by the reduced KD detected at Weeks 2 and 4. In the absence of P═S, the addition of 2 UNA or LNA (groups 4 and 6) at each termini of the sense strand improved KD at Weeks 2 and 4 compared with the 2′-O-methyl (group 2); however, by Weeks 6 and 8 it is clear that UNA and LNA in the absence of P═S are not sufficient due to the loss of KD. With all three sugar modifications (2′-O-methyl, UNA, and LNA) adding a single P═S to each side of the sense strand improves activity from Weeks 4 through 8, indicating that these more stable modifications can lead to longer durations of silencing. The two most active and stable modification patterns were found to be two P═S at each terminus of the sense and antisense strands as well as the pattern with two LNAs and one P═S at each terminus of the sense and two P═S at each terminus of the antisense strand; both maintain better than 80% KD over 8 weeks while all other patterns begin to show reduced KD by Week 8 (Figure 3). As demonstrated by Brown et al. (2020), stable siRNAs can remain in acidic compartments of the cell and act as a depot that results in continuous RISC loading beyond 2 weeks and sustained KD, despite decreasing siRNA concentrations in the tissue. (32) Although the data are shown only for one antibody and siRNA conjugate, several other AOCs with different siRNA sequences and antibodies showed similar stability patterns in vivo. These findings highlight the importance of carefully designing and selecting chemical modification patterns to optimize siRNA stability and activity for therapeutic applications.

siRNA Conjugation to mAb Via Cysteine, Lysine, and Sugar Locations

Once we identified the optimal siRNA chemical modification pattern for in vivo delivery and long duration of action, we next evaluated the differences in various conjugation positions on the antibody, including cysteine, lysine, and sugar locations. We assessed differences in plasma PK in mice for antibody–siRNA conjugates comprised of an αhEGFR mAb, which does not cross-react with mouse EGFR, conjugated to an siRNA targeting KRAS (siKRAS) at different sites on the antibody. The conjugation methods included random cysteine (TCEP reduction of disulfide followed by conjugation with siRNA-maleimide), random lysine (by nonselective addition of a TCO linker to lysine side chains), and glycan conjugation at Asn297 (sugar functionalization with TCO linker) (Figure 4a). AOCs were evaluated at a dose level of 0.5 mg/kg (siRNA). The full siRNA sequences for test articles evaluated in this experiment and throughout the manuscript are listed in Table S1.

Figure 4

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.

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Lysine and glycan conjugation of siRNA exhibited increased plasma clearance [46 and 11% lower area under the curve (AUC)], respectively, compared to cysteine conjugation (Figure 4a; SEM n = 4). The reduced plasma exposure of the lysine-conjugated siRNA is likely attributed to the greater heterogeneity and lower in vivo stability of lysine conjugates. While glycan conjugation is well tolerated for many antibody–drug conjugates (ADCs), it is not clear if the small reduction in plasma exposure of the glycan-conjugated AOC is the result of linker stability or the location of the siRNA on the antibody.
We also investigated the impact of random cysteine conjugation versus site-specific cysteine engineering on plasma clearance, since some locations of cysteine on the antibody show improved plasma PK of ADCs. (33) For random cysteine conjugation, we used αhTfR1-Cys-MCC-siMstn, while for site-specific cysteine engineering we used αhTfR1-K188C-MCC-siMstn, where the cysteine was engineered at position lysine 188 of the antibody light chain. No significant differences in plasma clearance were observed when conjugating siRNA to a random cysteine (Figure 4b).
While these data indicate cysteine conjugates may have advantages over glycan and lysine conjugates, additional studies are required to elucidate differences between PK, tissue delivery, and efficacy. Based on our data, additional results described herein will focus on random cysteine conjugation. Random cysteine conjugation was selected based on the relative ease of conjugate synthesis from most antibodies without the necessity of molecular engineering, less heterogeneity, clinical evaluation as shown by numerous ADCs, and manufacturability.

Drug to Antibody Ratio

We investigated the impact of DAR on AOC plasma clearance by assessing AOCs comprised of αhEGFR mAb conjugated to one, two, or three siRNAs targeting DMPK (αhEGFR-Cys-MCC-siDMPK). An antibody that does not bind to a target in mouse was selected to show the impact of DAR on plasma clearance in the absence of target-mediated drug deposition. We observed clear trends of faster plasma clearance as a function of increasing DAR (Figure 5a). Figure 5b shows the clearance rate of DAR2 is nearly five times greater than the clearance rate of DAR1 (0.101 vs 0.019 mL/h) calculated from 0 to 96 h. To determine if increasing DAR impacts delivery to tissue or activity in muscle, αmTfR1-Cys-MCC-siMstn conjugates with a DAR of 1 and 2 were tested in mice. The rapid clearance from plasma observed with DAR2 is likely caused by greater nonproductive uptake by the liver. This rapid uptake by the liver leads to lower delivery and activity in the muscle (Figure S1). AOCs with a higher DAR may have altered physicochemical properties, such as increased size and significantly greater negative charge density, which can affect their interactions with plasma proteins and clearance mechanisms. Examples of the increased size and charge density of DAR2 conjugates include a left shift on SEC (Figure S2) and a right shift on strong anion exchange (Figure S9a). Protein interaction data are not available for these conjugates but binding of oligonucleotides has been observed by others. (34,35)

Figure 5

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).

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Oligonucleotides themselves have been observed to interact strongly with plasma and cell surface proteins and it is possible that increasing the DAR may increase the avidity of these interactions. (36−38) A similar phenomenon of rapid DAR2 clearance with ASO-antibody conjugates (ASOs are single strands and typically fully phosphorothioated oligonucleotides) has been observed by us (unpublished) and others. (15) However, phosphorodiamidate morpholino oligonucleotide (PMO)-antibody conjugates (charge-neutral oligonucleotides) with up to DAR9.7 have no impact on plasma PK, (39) suggesting the charge and/or phosphorothioate content in siRNA and ASO DAR2 AOCs is driving clearance. Based on these data, we selected DAR1 for further evaluation throughout this paper.

Cysteine Conjugates with Cleavable Versus Non-cleavable Linkers

After selecting random cysteine conjugation as the conjugation strategy for antibody–siRNA conjugates, we focused on evaluating the impact of linker on plasma clearance. We evaluated AOCs using various cleavable and noncleavable linkers to assess their impact on in vivo stability as measured by plasma PK. To attain ideal pharmacokinetic profiles for active siRNA and desired effectiveness, it is necessary to regulate both the stability of antibody-conjugates and the release of siRNA. Favorable systemic stability is sought to safeguard linker-siRNA from hydrolysis while circulating. Simultaneously, efficient cleavage of linkers and release of siRNAs are crucial for accumulating a satisfactory quantity of active siRNA and meeting efficacy end points. Understanding these nuances is crucial for optimizing the design and functionality of antibody–siRNA conjugates. Table S2 summarizes the structures of the linkers. We assessed cleavable linkers including reducible disulfide (SS) linkers SS(methyl) and SS(gem-dimethyl) which exhibit increasing steric hindrance around the disulfide bond. Additionally, we evaluated a cathepsin-cleavable valine-citrulline (VC) linker, 3-(2-pyridyldithio)propionate (PDP) and methyl-(2-pyridyldithio)toluene (MPT). Noncleavable linkers included MCC, m-maleimidobenzoyl (MB), 4-((4-(cyanoethynyl)benzoyl)oxy) (CB), and bismaleimide (BisMal).
The impact of these linkers on AOC plasma PK was tested on conjugates with a 5000 Da MW PEG (PEG5k) attached to the sense strand at the end opposite of antibody conjugation. The benefits of PEGylation for AOC stability are included in a subsequent section. We conducted mouse PK studies with AOCs comprised of an αhEGFR antibody conjugated to siEGFR-PEG5k (PEG5k conjugated to the 3′ end of siEGFR by a stable thioether). We included a stable MCC linker between the mAb and siRNA (αhEGFR-Cys-MCC-siEGFR-PEG5k) and compared it with an enzyme-cleavable VC linker (αhEGFR-Cys-VC-siEGFR-PEG5k). Further iterations of these AOCs were synthesized to evaluate the impact of a cleavable SS linker between the siRNA and PEG5k (αhEGFR-Cys-siEGFR-SS-PEG5k and αhEGFR-VC-siEGFR-SS-PEG5k). We measured the plasma PK of these AOCs over time (Figure 6a,b) and observed no significant differences in clearance of the siRNA. However, our stem-loop assay for the detection of the antisense strand is insensitive to the presence of a PEG group attached to the sense strand, meaning the stability of the PEG5k group attached via a disulfide bond remains unclear. Furthermore, it is possible that a large PEG at the end of the siRNA could influence linker stability, for instance by limiting enzyme accessibility to the VC linker. However, considering the radius of gyration for PEG5k (∼3 nm) and the length of a 21-nucleotide siRNA duplex (∼7.5 nm), it is unlikely that PEG attached to the 3′ end of the sense strand would have a significant impact on the stability of the linker at the 5′ end. Further studies are needed to fully elucidate the impact of these modifications on the PK of AOCs.

Figure 6

Figure 6. (a,b) Mouse PK of AOCs containing cleavable linkers.

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Further mouse PK studies were conducted with AOCs comprised of αhEGFR mAb conjugated to siKRAS via different cleavable disulfide linkers (PDP, MPT, SS[methyl], and SS[gem-dimethyl]) between the mAb and siRNA. (40) These AOCs each contained a PEG5k conjugated directly to the siKRAS. We dosed the AOCs at 0.5 mg/kg (siRNA) (Figure 6b). These results demonstrate that the linker stability matters for AOCs, and that increasing steric bulk around the disulfide (SS) linker is required to maintain plasma stability of these cleavable linkers. Incorporating the gem-dimethyl group into the disulfide linker reduced the clearance by more than 40 times (0.951 to 0.02 mL/h for PDP vs SS[gem-dimethyl] as shown in Table S3), suggesting that increased bulk slows down disulfide cleavage in circulation. Similar results have been observed with ADCs demonstrating the impact of disulfide linker length and steric hindrance on PK and efficacy. (41,42) The rest of this manuscript will focus on noncleavable (stable) linkers other than the data shown in Figure 9.

Further Exploration of Non-cleavable Linkers (MCC, CB, MB, and BisMal)

αhEGFR-siKRAS AOC In Vitro Stability

To compare the in vitro stability of different noncleavable linkers, we conjugated a human immunoglobulin (Ig) G1 antibody targeting EGFR (αhEGFR) to KRAS siRNA using four different noncleavable linkers (MCC, CB, MB, and BisMal). We tested the linker stability by observing siRNA release from the antibody after incubating it at 37 °C in PBS with 0.5 mM glutathione (Figure S3). Free antibody was measured by using strong-anion exchange high-performance liquid chromatography to indicate the loss of KRAS siRNA. Our results showed that αhEGFR-siKRAS AOCs containing CB, MB, and BisMal linkers were more stable than the MCC AOC in vitro, matching expectations from the literature. (43)

Linker Impact on mTfR1 Antibody-Based siRNA Delivery to Muscles and Other Tissues

We synthesized and evaluated αmTfR1 antibody–siHprt conjugates via four different linkers (MCC, CB, MB, and BisMal) to understand the impact of in vitro linker stability differences on the AOC plasma PK, siRNA tissue delivery and target mRNA KD efficacy in a mouse PK and Hprt mRNA KD study. Despite differences in in vitro stability, we observed nearly identical plasma clearance profiles, as well as siRNA concentration and mRNA reduction in skeletal muscle, for all four conjugates (Figure 7). While others have shown that cytotoxic ADCs targeting tumors have improved activity when using stabilized linkers, (44) our data show that for αmTfR1 conjugates, there is no improvement in plasma exposure, tissue delivery, or KD when using linkers more stable than MCC. This result is best explained by Figure 7a showing greater than 80% of the dosed material is no longer in plasma after 4 h. This rapid target-mediated drug disposition is expected for a highly expressed receptor like TfR1 and results in the majority of the AOC being taken into the tissue before any differences in linker stability could have an impact. It would be intriguing to explore whether these linker patterns persist with AOCs featuring extended plasma residence.

Figure 7

Figure 7. (a–c) PK/PD studies with αmTfR1–siHprt AOCs containing four different noncleavable linkers.

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Linker Location on the siRNA

The spatial arrangement of the linker on the siRNA molecule could play a pivotal role in determining the stability of the siRNA itself. This influence is likely attributed to heightened steric hindrance, where the size and the proximity of the antibody creates obstacles to enzymes that can affect the stability of the siRNA as part of the conjugate as well as after release inside the cell. Understanding the impact of linker placement on siRNA stability is crucial for optimizing the design and performance of antibody–siRNA conjugates, especially in the context of therapeutic applications where stability is a critical factor for efficacy and duration of action. AOCs were synthesized to evaluate the effect of linker locations at the 3′ or 5′ end of the siRNA sense strand on the target mRNA KD in different tissue types. The AOCs we evaluated were comprised of an αmTfR1 antibody conjugated via an MCC linker to an siHprt at either the 3′ or 5′ end: αmTfR1-Cys-MCC-N-5′-siHprt and αmTfR1-Cys-MCC-N-3′-siHprt. We measured the KD of these AOCs in four tissues: muscle, heart, liver, and lung (Table S1 and Figure S4). These results demonstrate that the siRNA could be linked at either the 3′ or 5′ end of the sense strand without an impact on KD in the four tissues assessed. This also indicates that any remnant of the linker or the antibody at the 3′ or 5′ end of the siRNA sense strand does not impact RISC loading of duplex siRNA. These data show that the concentration of siRNA in tissue (Figure S4e) does not necessarily correlate with activity when comparing across tissues. While 20 nM concentrations in muscle generate approximately 70% KD, greater than 200 nM concentrations can be measured in the liver without achieving the same amount of activity. These data suggest that AOC activity can be impacted by siRNA delivery as well as cell type and, potentially, mechanism of siRNA internalization.
We synthesized additional AOCs to further evaluate the impact of linker positions in the middle of the siRNA sense strand. αhEGFR antibody was lysine conjugated to siKRAS with bicyclo[6.1.0]nonyne (BCN) linkers at siRNA positions 7, 13, or 17 on the sense strand off a 2′ amine (Table S1 and Figure S5). Although we hypothesized that conjugating to the backbone of the siRNA would enhance protection against potential nucleases by antibody steric interference, we observed no impact on plasma PK based on the siRNA backbone linker location.
The impact of siRNA conjugation position on in vivo potency was tested in both liver and muscle with two different targeting antibodies. To assess the impact of siRNA conjugation positions on KD in the liver, we synthesized AOCs containing antimouse ASGPR mAb (αmASGPR) conjugated to siHprt via a BisMal linker at three different positions: 5′ end versus positions 8 or 14 of the sense strand via 2′ primary amine (Figure 8). We measured percent Hprt mRNA expression in the liver. The αmASGPR-Cys-BisMal-siHprt (5′) demonstrated higher KD versus backbone positions 8 or 14 on the sense strand at lower doses, indicating potential interference in siRNA RISC loading with a linker attached at 2′ locations. It is important to note that while conjugates at 2′ positions 8 and 14 were less active at lower doses, they were able to achieve the same maximum KD as the 5′ conjugate when the dose was increased to 2 mg/kg.

Figure 8

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.

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Additional AOCs were synthesized to evaluate the impact of siRNA conjugation sites on KD in the gastrocnemius. These AOCs were comprised of an αmTfR1 conjugated to siHprt with either a stable MCC or cleavable VC linker (Figure 9a). The MCC linker was evaluated at the 5′ end as well as positions 8 and 14 on the sense strand via a 2′ primary amine. Better gastrocnemius KD was observed for the MCC linker at the 5′ end versus the backbone positions tested for the αmTfR1–siHprt AOCs at 0.2 mg/kg (p < 0.05), once again indicating the potential impact on the RISC loading of the released siRNA with linkers at positions 8 and 14 on the sense strand. An enzyme-cleavable VC linker was then evaluated at position 8 of the sense strand via a 2′ primary amine. The αmTfR1-Cys-VC-siHprt (2′pos8 sense strand) AOC was synthesized to test the hypothesis that the self-immolating enzyme-cleavable linker would have the greatest impact at the center of the sense strand because of the potential for the noncleavable linker to have more steric bulk and prevent RISC loading. We based this hypothesis on in vitro research with similar modifications. (12) However, the AOC with the cleavable VC linker at position 8 of the sense strand showed lower activity compared with a noncleavable MCC linker 5′ end conjugation at lower doses, indicating that propyl amine at 2′ of position 8 of the siRNA sense strand is unlikely to be an optimal 2′ substitution for RISC loading and target gene KD in muscle or liver. The VC linker cleavage kinetics for antibody–siRNA conjugates are not known. It is conceivable that the VC linker at position 8 could be more hindered than at the 5′ end resulting in slower release of siRNA. The Hprt mRNA KD reaches saturation beyond a dose of 1 mg/kg, yet the concentration of siRNA in tissues continues to rise (Figure 9b), indicating that receptor capacity is not the limiting factor. Our hypothesis to explain the absence of a dose response beyond 1 mg/kg, despite higher siRNA concentrations in tissues, is that Hprt is expressed in both myotubes and other cell types (such as fibroblasts and endothelial cells), whereas siRNA delivery primarily targets muscle cells.

Figure 9

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.

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We recognize the extensive work done around internucleotide triester, 2′-linker, and nucleobase conjugation approaches that identified SAR trends for GalNAc and other ligand conjugates. (45) Our study did not delve into these aspects; therefore, we caution against overgeneralization of our findings and recommend further studies to comprehensively understand the impact of siRNA conjugation sites on KD in different tissues and under varying conditions.

Impact of PEG on AOC Activity

PEG increases solubility, reduces nonspecific protein binding, provides protection from enzymes, and reduces clearance of various molecules such as proteins. (46) To assess the impact of PEG on the plasma PK of an AOC, we evaluated a set of AOCs with PEG on the inside versus the outside of siRNA (i.e., mAb-PEG5k-siRNA vs mAb-siRNA-PEG5k). The delivery and KD of PEG AOCs in mice with lymph node carcinoma of the prostate (LNCaP) xenograft were also assessed. These mice were treated with AOCs comprised of antihuman prostate-specific membrane antigen antibody (αhPSMA) conjugated to EGFR siRNA (siEGFR). The PEG was placed either between the mAb and siRNA or on the outside of the siRNA: αhPSMA-Cys-MCC-siEGFR, αhPSMA-Cys-MCC-siEGFR-PEG5k, and αhPSMA-Cys-PEG5k-siEGFR (Table S1 and Figure S6). These AOCs were designed to test the hypothesis that placing PEG on the outside of the siRNA would protect it from nucleases and increase its stability. Observations showed no significant change in plasma PK based on the size or placement of the PEG. However, placing PEG on the outside led to more KD and higher tissue concentration in the tumor, indicating a potential increase in the intracellular stability of siRNA. Furthermore, the placement of PEG5k externally is likely to have an impact on the hydrophilicity of the conjugate. This could influence the tumor-to-liver ratio and subsequent mRNA reduction. In addition, PEG might shield some of the siRNA charge, which could further affect its interaction with cellular components and its overall effectiveness. These concepts are well described in ADCs. (46)
To further evaluate the potential impact of PEGylation on siRNA in the muscle, AOCs were synthesized using an αmTfR1 antibody conjugated at the cysteine via an MCC linker to an siCtnnb1 sense strand with and without PEG5k on the outside (Figure S7). Tissue PK data of these αmTfR1–siCtnnb1 AOCs suggest that PEG5k attached to the 3′ end of siCtnnb1 sense strand (while conjugating the 5′ end to the antibody) increases the amount of siRNA detected in gastrocnemius as well as Ctnnb1 mRNA KD. Specifically, a 1 mg/kg dose with PEG achieved the same tissue concentration as the 3 mg/kg dose without PEG. Moreover, the KD at 1 mg/kg with PEG was comparable to the KD at 3 mg/kg without PEG.
Our data indicate that including PEG in the AOC architecture could help with siRNA tissue PK, likely resulting in increased gene KD efficacy. Further research is needed to understand the optimal size of the PEG needed in these AOCs.

Applicability of siRNA Bioconjugation across Multiple Antibody Isotypes

We can apply siRNA bioconjugation to a variety of antibody isotypes, including human (h) IgG1, hIgG2, hIgG4, rat IgG2a, and rabbit IgG. The structure of the antibody and conditions used for partial reduction determine the conjugation efficiency and DAR distribution of random cysteine conjugation of siRNA. Each antibody isotype requires different ratios of reagents to maximize DAR1, and IgG4 predominantly gives DAR2 (Table 1). We evaluated antibodies for conjugation, including antimouse TfR1, anti-PSMA, anti-EGFR, and antimouse ASGPR. Interestingly, for hIgG4, the use of a monofunctional versus bifunctional linker (MCC, BisMal) influenced the ratio of DAR1 and DAR ≥ 2. This linker-dependent change in DAR distribution may be due to the Fab arm exchange observed in IgG4 antibodies and the ability of the BisMal linker to stabilize the DAR1 structure. While DAR1 yields may vary greatly across antibody isotypes, we can obtain sufficient yields of pure DAR1 for preclinical research. The observed variation in DAR distribution between antibody isotypes for AOCs is consistent with the differences observed with ADCs generated with different isotypes. (47) This correlation is anticipated, given the similar conditions used for partial reduction of the antibodies. However, AOCs present a unique challenge as they require the isolation of only DAR1. This is further complicated by the high MW and negative charge of siRNAs which creates steric and charge repulsion that can slow reactions for less exposed positions on the antibody.
Table 1. Summary of Reaction Conditions and DAR Distributions for Various mAb Isotypes (%)
mAb isotypeTCEP eq(linker) siRNA eqDAR0 (%)DAR1 (%)DAR ≥ 2 (%)isolated DAR 1 yield (%)
hIgG12(MCC) 125571846
hIgG26(MCC) 127403334
hIgG41.75(MCC) 1.2547223116
hIgG41.75(BisMal) 1.2546312325
Rat IgG2a4(MCC) 1.1538412136
Rabbit IgG4(MCC) 1.126413335

Conclusions

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AOCs are complex bioconjugates and the data herein illustrate a comprehensive SAR evaluation of siRNA AOCs. This evaluation has allowed us to optimize siRNA chemistries, select linkers, identify points of conjugation on the antibody and siRNA, develop strategies to conjugate an antibody to an siRNA oligonucleotide, and understand the impact of oligonucleotide-to-antibody ratios on AOC activity. Our findings show the importance of selecting appropriate siRNA chemical modifications to improve tissue delivery in vivo and achieve higher target gene KD in tissues of interest. Our platform can accommodate various points of conjugation on the antibody, offering flexibility to choose between cysteine, site-specific cysteine, and sugar conjugations. Initial studies indicate that lysine conjugates are not optimal for AOCs, but additional studies are needed to understand the differences. The siRNA-to-antibody ratio is limited to 1, likely due to high charge of each siRNA and the potential phosphorothioate-mediated nonspecific binding of DAR ≥ 2 AOCs. The AOC platform can accommodate most noncleavable linker chemistries when conjugated on both ends of the sense strand, indicating that remnants of linker attached to the released siRNA sense strand, after AOC digestion in lysosomes, may not interfere with Ago2 protein binding and RISC loading. We found no significant differences in plasma PK, siRNA tissue delivery, and target gene KD between noncleavable linkers like MCC, BisMal, CB, and MB with rapidly internalized αmTfR1 AOCs. Additional evaluation with AOCs that show longer plasma PK are needed to tease out any differences between these noncleavable linkers. Results from our evaluation of cleavable linkers demonstrate that AOCs with less sterically hindered disulfide linkers like PDP, MPT and SS(methyl) are less stable than more sterically hindered disulfide SS(gem-dimethyl). The reduced plasma clearance of AOCs correlated with the increasing steric hindrance around the disulfide bond. The VC linker offered no significant improvement over stable MCC or BisMal linkers, indicating the tolerance for remnant linker attached to siRNA for Ago2 binding and RISC loading. The exploration of conjugation positions, other than 5′ or 3′ end positions on the sense strand, on both strands of siRNA also revealed that the internal conjugation locations show lower activity than the 5′ end conjugation. This is likely due to interference of siRNA duplex interaction with either Ago2 protein or RISC loading. Addition of 5000 molecular weight PEG on the other side of siRNA in an AOC, by sandwiching siRNA between antibody and PEG, showed approximately three-fold improved tissue delivery to the targeted tumor cells with αhPSMA AOC and a five-fold increase in muscle with αmTfR1 AOC. The increased delivery of siRNA resulted in higher target gene KD. PEG is likely providing steric shielding to nucleases and may reduce nonspecific protein binding of AOC.
Overall, our SAR study provides important insights into the design and optimization of siRNA AOCs, identifying key factors that can influence PK, tissue delivery, and efficacy of these complex bioconjugates. These findings are consistent across multiple antibody targets and siRNAs, suggesting these design principles can be applied to the entire AOC class. By understanding the SAR of AOCs, researchers can continue to develop new and improved therapeutics for a range of diseases and conditions.

Experimental Section

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siRNA Synthesis

The oligonucleotide siRNA sequences were assembled on solid phase using well-described and standard phosphoramidite methodology. Single strands were synthesized on a MerMade 12 synthesizer (BioAutomation) with phosphoramidite monomers (2′OMe, 2′F, LNAs, inverted abasic, C6NH2, and C6SSC6dT) and universal controlled pore glass solid support purchased from commercial sources. The vinylphosphonate UNA-MOE-U phosphoramidite monomer was synthesized in-house as per the synthesis shown in Scheme S1. (48) All phosphoramidites were used at a concentration of 100 mM in 100% anhydrous acetonitrile utilizing a 2.5 molar excess of ETT activator in acetonitrile with 2 × 6 min coupling time. Oxidation of phosphite linkages was achieved using 0.02 M I2 in 9:1 acetonitrile/H2O for 60 s. Sulfurization was achieved with 0.05 M DDTT (3-dimethylaminomethylene amino-3H-1,2,4-dithiazole-5-thione) in 60/40 pyridine/acetonitrile for 6 min per cycle. Capping was performed with tetrahydrofuran (THF)/pyridine/acetic anhydride for 60 s and 16% methyl imidazole in THF for 60 s per cycle. Deblocking was performed with 3% trichloroacetic acid in acetonitrile for 2 × 40 s per cycle. Cleavage of siRNA from solid support and deprotection of all protecting groups was accomplished using a 1:1 ratio of 28% aqueous ammonia and 40% methylamine in methanol for 2 h at room temperature. Crude oligonucleotides were purified using strong anion exchange with phosphate buffers (pH 7.2) containing sodium bromide; the desired fractions were chosen based on LCMS analysis, then pooled and desalted. The identification and purity of the single strands were determined by LCMS and RP-HPLC followed by lyophilization. The single strands were then dissolved in H2O and equal molar amounts of antisense and sense strand were annealed by heating to 65 °C for 5 min and cooling to room temperature to form the final duplex, which was analyzed by anion exchange chromatography.

Characterization of Oligonucleotides

Single-strand purities were measured by ion-pair RP-HPLC using an XBridge C18 OST column (Waters, Milford MA; 2.1 × 50 mm, 2.5 μm) with a gradient from 6 to 35% mobile phase B over 15 min at a flow rate of 0.25 mL/min and column temperature of 65 °C. Mobile phase A consisted of 100 mM 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), 16.3 mM triethylamine (TEA), and 1% methanol. Mobile phase B consisted of 100 mM HFIP, 16.3 mM TEA, and 95% methanol. All single strands were at least 85% pure. Single-strand identities were confirmed by mass spectrometry (MS), on LCQ Deca XP + Ion Trap MS (Thermo Finnigan, San Jose, CA), used in line with ion-pair reversed-phase HPLC. The MS was run in negative ion mode with a capillary temperature of 315 °C and voltage of −100 V. Data analysis was performed with ProMass deconvolution software (Novatia, Newtown, PA). Duplex purity was measured by anion exchange chromatography using a DNAPac-200 column (Thermo Fisher, Waltham, MA; 4 × 250 mm) with a gradient of 15–58% mobile phase B over 9 min with a flow rate of 1 mL/min. Mobile phase A contained 20 mM sodium phosphate buffer and 20% ethanol; mobile phase B consisted of mobile phase A plus 800 mM NaClO4. All duplexes were at least 90% pure.

siRNA-PEG Synthesis

The siRNA sense strands used for conjugation were synthesized with a C6 amino or C6 sulfur linker at the 5′- or 3′-end, or 2′-internal modifications (C3 amino). siRNAs with 3′ sense strand C6SSC6dT modifications were dissolved in 100 mM phosphate buffer pH 7.4 then reduced with 20 eq TCEP at room temperature. Excess TCEP was removed using ultrafiltration into 20 mM phosphate buffer with 150 mM sodium chloride then added to 3 equiv of maleimide-PEG5k or 5 equiv of orthopyridyl disulfide PEG5k (Laysan Bio, Arab AL) for a reducible linkage. Resulting siRNA-PEG was purified using the strong anion exchange chromatography method described for AOCs below. The desired fractions were then isolated using ultrafiltration with 3 kDa molecular weight cutoff (MWCO). siRNA-PEG purity was measured by anion exchange chromatography using an Agilent 1200 with a Thermo Fisher ProPac SAX-10 BioLC column (250 × 4 mm) at ambient temperature. siRNA-PEG was separated from unconjugated siRNA and excess PEG with a gradient of 10–60% mobile phase B over 8 min and flow rate at 0.75 mL/min, monitoring at 260 nm. Mobile phase A was 10 mM Tris Base pH 7.2 with 20% ethanol, mobile phase B contained A plus 1.5 M sodium chloride. All siRNA-PEG purities were greater than 95%.

Linker-siRNA Generation

SiRNA duplex in 50 mM phosphate buffer at a concentration of 100 mg/mL was pH adjusted with sodium hydroxide to pH 7.4. The siRNA was then diluted with dimethyl sulfoxide (DMSO) to achieve a final DMSO concentration of 50% once the linker was added. Linker (10 equiv to the siRNA) dissolved in DMSO was added to the siRNA and mixed at room temperature for 30 min. The progress of the reaction was monitored by MS, and the absence of a sense strand indicated the completion of the conjugation. Excess linker was removed using ultrafiltration with a 3 kDa MWCO. Linker-siRNA purity was not determined and was used in situ. Disulfide linkers used were succinimidyl 3-(2-pyridyldithio)propionate (SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene (SMPT), SS[methyl] (2,5-dioxopyrrolidin-1-yl 4-(pyridine-2-yldisulfanyl)pentanoate), and SS[gem-dimethyl] 2,5-dioxopyrrolidin-1-yl 4-methyl-4-((5-nitropyridin-2-yl)disulfanyl)pentanoate (Annova Chem, San Diego, CA). Maleimide linkers used were NHS-PEG5k-maleimide (Laysan Bio, Arab, AL), maleimidocaproyl-l-valine-l-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (MC-Val-Cit–PAB-PNP) (Broadpharm, San Diego, CA), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Thermo Fisher, Waltham, MA), bis-maleimide-dPEG4-TFP ester (BisMal) (Quanta Biodesign, Plain City, OH), sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), and MB-N-hydroxysuccinimide ester (Sigma, St. Louis, MO). Click linkers used were TCO-PEG4-NHS, NHS-PEG1-Azide (Broadpharm, San Diego, CA), and methyltetrazine-PEG4-NHS (Sigma, St. Louis, MO).

Cysteine, Lysine, and Glycan Conjugations

Cysteine-conjugated AOCs were generated using a standard random cysteine conjugation method. The interchain disulfide bonds of the antibody were partially reduced with TCEP (Thermo Fisher, Waltham, MA) at 37 °C for 2–4 h prior to conjugation with a maleimide linker-oligonucleotide (molar equivalents of TCEP and siRNA-linker required for conjugation to each antibody isotype are listed in Table 1). Lysine-conjugated AOCs were generated using a standard random lysine conjugation method (with the degree of conjugation relative to the antibody). The antibody at 10 mg/mL in PBS (pH 7.4) was incubated with 2 equiv relative to antibody of NHS-linker (TCO, maleimide, azide) at room temperature for 90 min. Excess linker was removed using 50 kDa MWCO ultrafiltration prior to conjugation with 1.75 equiv of a functionalized (tetrazine, sulfhydryl, BCN)-oligonucleotide. Glycan-conjugated AOCs were generated using Asn297 TCO modified antibody from Fisher Scientific, using SiteClick and conjugated with a methyl tetrazine-oligonucleotide. All reaction mixtures after the conjugation were purified on fast protein liquid chromatography using a preparative anion exchange purification method to isolate the DAR1 antibody–siRNA conjugate.

Preparative Anion Exchange Purification

SAX purification was performed on an ÄKTA Pure system with a Sartobind Q (Sartorius, Göttingen, Germany; Nano 3 mL with 8 mm bed height) at ambient temperature with a gradient of 20–40% mobile phase B over 20 column volumes and a flow rate of 3 mL/min, monitoring at ultraviolet (UV) 220, 260, and 280 nm. Mobile phase A was 20 mM sodium phosphate buffer pH 7.2; mobile phase B contained A plus 1.5 M sodium chloride (Figure S8).

Overview of Cysteine Conjugation Using NEM Capping with or without DHAA Oxidation

AOCs (αhEGFR-siKRAS) were generated via cysteine conjugation. Cysteine conjugates were made by partially reducing antibody interchain disulfide bonds with TCEP to react with maleimide on the siRNA. On human IgG1 antibodies, the primary location of siRNA conjugation is on the cysteine of the heavy chain previously forming the heavy chain–light chain disulfide. (47) During this process, some cysteine remained unreacted.

Overview of Free Cysteine Determination

The average number of free cysteines on DAR1 AOCs was estimated by quantifying the amount of fluorescein-5-maleimide (AnaSpec, Fremont, CA) able to react with the antibody relative to a standard curve measured by SEC-HPLC. For each replicate, 0.25 mg of mAb or AOC in PBS at 5 mg/mL was incubated with 5 mol equiv of fluorescein-5-maleimide (5 μL at 0.715 mg/mL in DMSO) at room temperature for 1 h protected from light (5 equiv was selected based on optimization experiments to determine the amount of fluorescein-5-maleimide needed to saturate the free cysteine reaction on mAb reduced with 1.5 EQ of TCEP). After a 3× dilution, 30 μL of each sample was tested by SEC-HPLC (Yarra SEC-3000 column, 1 mL/min flow rate, mobile phase: phosphate buffer +300 mM NaCl) the absorbance at 495 nm was measured and the AUCs for peaks corresponding to the antibody or DAR1 AOC were calculated and compared with a standard curve of fluorescein-5-maleimide to quantify the number of cysteine available for reaction.

Purity Statement

AOCs with a DAR of 1 are at least 95% pure by strong anion exchange chromatography and SEC. The single-strand oligonucleotides used to make the duplex siRNAs for conjugation were at least 85% pure by the appropriate HPLC methods and identified by MS.

Animal Studies

All animal procedures were approved by the Institutional Animal Care and Use Committee of Explora BioLabs in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. Mice were housed in ventilated cages under a 12 h light–dark cycle and fed ad libitum with standard rodent chow. All in vivo experiments were conducted using four mice per group, each weighing ∼24 g.
In vivo studies in wild-type mice were performed in female Hsd/ICR (Envigo) mice. All the dosing was based on the siRNA weight, not the total conjugate (1 mg/kg = 1 mg siRNA/kg). Mice were dosed intravenously at 5 mL/kg. At end of study, mice were euthanized and tissues removed for analysis. For the assessment of delivery to tumors, male Crl:SHO-PrkdcscidHrhr (Charles River) mice were inoculated in the right flank subcutaneously with 7 million LNCaP cells in a mixture of 0.2 mL total of RPMI 1640 with matrigel (1:1). Mice were randomly grouped when tumors reached 200 mm3 and given an intravenous dose of the test article at 5 mL/kg. At end of study, or when tumors reached 2000 mm3, mice were euthanized and tissues removed for analysis.

Pharmacodynamic Evaluation

Tissue was processed for RNA extraction using the Zymo Direct-zol-96 RNA purification kit (Zymo Research Corporation, Irvine, CA). Once RNA quality was confirmed, cDNA was generated from purified RNA and used in quantitative reverse transcription polymerase chain reaction analysis for gene expression for each of the targets of interests. (49) The reference mRNA transcripts for Ppib were used in ΔΔCt calculations for tissue samples. (50)
To evaluate siRNA concentrations in plasma or tissues, custom stem-loop-RT-qPCR assays were developed as described previously. (51) For each siRNA, a specific stem-loop-RT-qPCR assay was designed to quantify the antisense strand, using custom DNA forward, reverse, and reverse transcription primers (Integrated DNA Technologies, Coralville, IA) and a custom Taqman probe (Thermo Fisher, Waltham, MA). Noncompartmental analysis was performed on individual mouse plasma total siRNA concentration versus time data to estimate PK parameters for AUC and clearance using Phoenix WinNonlin v8.3 (Certara, Princeton, NJ). An intravenous bolus administration model (Plasma 200–202) was used and AUC was calculated using the “linear-up log-down” calculation method.

AOC Characterization

The isolated DAR1 antibody–siRNA conjugate was then characterized by SAX, SEC, reduced capillary gel electrophoresis (RCGE), UV, and MS.

Analytical Anion Exchange Chromatography

After bioconjugation, the DAR distribution of the AOC reaction mixture was analyzed by SAX. SAX analysis was performed on an Agilent 1200 with Thermo Fisher ProPac SAX-10 BioLC column (250 × 4 mm) at ambient temperature with a gradient of 10–60% mobile phase B over 8 min and flow rate at 0.75 mL/min, monitoring at UV 220, 260, and 280 nm. Mobile phase A was 10 mM Tris Base pH 7.2 with 20% ethanol; mobile phase B contained A plus 1.5 M sodium chloride. DAR1 was then isolated by column chromatography (Figure S9a). The DAR1 final product was then characterized by SAX [same method as described above; UV (Figure S9b)], SEC (Figure S9c), RCGE (Figure S9d), and MS (Figure S9e).

UV Spectroscopy

AOC concentrations were determined using the absorbance at 280 nm and an extinction coefficient of 2.28 L·g–1·cm–1 for DAR1 and 3.43 L·g–1·cm–1 for DAR2. Absorbance ratios at 260 nm/280 nm were also measured to confirm DAR. The extinction coefficient used for DAR1 and DAR2 AOCs was calculated using the UV absorbance at 280 nM and the BCA measurement of antibody concentration (compared to a standard curve with the same unconjugated antibody) of more than 20 different purified conjugates. The values were within 13 and 9% (DAR1 and DAR2) of the values calculated using the mAb and siRNA extinction coefficients and the formula below. The concentration of the mAb portion of an AOC was used to calculate siRNA concentration and total AOC concentration using the siRNA MW of 14,100 Da and antibody MW of 150,000 Da. A 1 mg/kg siRNA dose corresponds to 10.6 mg/kg mAb dose and 11.6 mg/kg AOC dose.
[mAb]=A280smplε280,mAb+DARε280,siRNA
SiRNA single strand concentrations were determined by using the absorbance of each strand at 260 nM and an extinction coefficient estimated using the nearest neighbor method. Duplex siRNA concentrations were calculated based on the known amount of each single strand used and the appropriate dilutions. When needed, duplex extinction coefficients were estimated using the formula below including a correction factor of 0.8 to account for the hypochromic effect of duplexed nucleic acids.
ε260,siRNA=(ε260,antisensestrand+ε260,sensestrand)×0.8

Size Exclusion

SEC analysis was performed on an Agilent 1200 with a Yarra 3 μm SEC-300 column (300 × 7.8 mm) at ambient temperature with an isocratic flow of 55 mM potassium phosphate monobasic 62 mM sodium phosphate dibasic 100 mM sodium sulfate with 0.05% sodium azide pH 6.7 buffer with a flow rate of 1 mL/min for 15 min, monitoring at UV 260 and 280 nm.

Reduced Capillary Electrophoresis

Reduced capillary electrophoresis-sodium dodecyl sulfate was performed on ProteinSimple Maurice Plus instrument using Maurice CE-SDS PLUS cartridges according to manual instructions. Samples were prepared to 1 mg/mL by protein concentration. Internal standard and 2-mercaptoethanol were added, then heated to 70 °C for 10 min. Reduced samples were run with a sample load of 20 s at 4600 V, and separation for 25 min at 5750 V.

MS

Intact MS was performed by Thermo Fisher with a Q Exactive BioPharma. 5 μg of sample was loaded onto an Acquity UPLC protein BEH SEC column (4.5 × 100 mm, 1.7 μm) and eluted with 50 mM ammonium acetate at a flow rate of 0.3 mL/min and a column temperature of 55 °C. MS was run with a spray voltage of 4.2 kV and an ion transfer tube temperature of 320 °C.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00802.

  • Please see siRNA AOC Supplement. siRNA sequence and chemical modifications for evaluated test articles; siRNA conjugation comprehensive guide; noncompartmental analysis of plasma PK data in Figure 5b; plasma PK, tissue concentration, and mRNA expression of αmTfR1-Cys-MCC-siMstn conjugates; DAR1 versus DAR2 SEC comparison; in vitro stability of αhEGFR-siKRAS AOCs; KD and tissue concentration of αmTfR1-siHprt AOCs; plasma PK of αhEGFR-siKRAS AOCs; tumor delivery and KD of αhPSMA-siEGFR AOCs; PK of αmTfR1-siCtnnb1 AOCs; purification of antibody–siRNA conjugation reaction mixture by SAX; purification and characterization of DAR1 AOC; UNA vinylphosphonate (vpUq) amidite synthesis (PDF)

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Author Information

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  • Corresponding Author
    • Venkata Ramana Doppalapudi - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States;  Email: [email protected]
  • Authors
    • Michael Cochran - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
    • Danny Arias - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
    • Rob Burke - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States;  Present Address: Seawolf Therapeutics, One Sansome Street Suite 3630, San Francisco, CA 94104, USA
    • David Chu - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States;  Present Address: Capstan Therapeutics, 9880 Campus Point Drive, San Diego, CA 92121
    • Gulin Erdogan - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
    • Michael Hood - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
    • Philip Kovach - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
    • Hae Won Kwon - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
    • Yanling Chen - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
    • Michael Moon - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States;  Present Address: Amgen Inc., 750 Gateway Blvd., Suite 100, South San Francisco, California 94080
    • Christopher D. Miller - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States;  Present Address: California North state University College of Medicine, 9700 W Taron Dr, Elk Grove, CA 95757
    • Hanhua Huang - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
    • Arthur Levin - Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United States
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare the following competing financial interest(s): At the time this research was conducted, all authors were employees of Avidity Biosciences and may have equity in the company.

Acknowledgments

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Authors acknowledge John Manhard for generating antibodies with site-specific cysteine conjugation handles. Thanks to Baorui Lin and Zhanlei Wei of WuXi TIDES for their work on the synthesis and scale-up of vp-UNA-3′-phosphoramidite. Special thanks to Son Lam and Han Cho for technical review of the manuscript. The authors would like to thank Gemma Hall, DPhil, and Megan Hotard Jarrell, MS, from Lighthouse Medical Communications, New York, NY, USA, for providing medical writing support. Medical writing support was funded by Avidity Biosciences, San Diego, CA, USA, in accordance with Good Publication Practice (GPP3) guidelines.

Abbreviations

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% ID

percent of injected dose

ADC

antibody–drug conjugate

Ago2

Argonaute2

AOC

antibody–oligonucleotide conjugate

ASGPR

asialoglycoprotein receptor

ASO

antisense oligonucleotide

BCN

bicyclo[6.1.0]nonyne

BisMal

bismaleimide

CBTF

sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate

DAR

drug-to-antibody ratio

DHAA

dehydroascorbic acid

EGFR

epidermal growth factor receptor

Eq

equivalent

GalNAc

N-acetylgalactosamine

HFIP

1,1,1,3,3,3-hexafluoroisopropanol

HPLC

high-performance liquid chromatography

KD

knockdown

LNA

locked nucleic acid

LNCaP

lymph node carcinoma of the prostate

mAb

monoclonal antibody

MBS

m-maleimidobenzoyl-N-hydroxysuccinimide ester

MS

mass spectrometry

NEM

N-ethylmaleimide

NHS

N-hydroxysuccinimide

P═S

phosphorothioates

PBS

phosphate buffered saline

PEG

polyethylene glycol

PSMA

prostate-specific membrane antigen

RCGE

reduced capillary gel electrophoresis

RISC

RNA-induced silencing complex

RT-qPCR

quantitative reverse transcription polymerase chain reaction

SAR

structure–activity relationship

SAX

strong anion exchange chromatography

SEC

size exclusion chromatography

siRNA

small interfering RNA

SMCC

succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

SMPT

4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene

SPDP

succinimidyl 3-(2-pyridyldithio)propionate

SS

disulfide

TCEP

tris (2-carboxyethyl) phosphine

TEA

triethylamine

TfR1

transferrin receptor 1

UNA

unlocked nucleic acid

VC

valine-citrulline

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    Nature Biotechnology (2022), 40 (10), 1500-1508CODEN: NABIF9; ISSN:1087-0156. (Nature Portfolio)
    Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solns. are needed to target addnl. organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 mo. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclin. efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiol. and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.
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  13. 13
    Roberts, T. C.; Langer, R.; Wood, M. J. A. Advances in Oligonucleotide Drug Delivery. Nat. Rev. Drug Discovery 2020, 19 (10), 673– 694,  DOI: 10.1038/s41573-020-0075-7
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    13
    Advances in oligonucleotide drug delivery
    Roberts, Thomas C.; Langer, Robert; Wood, Matthew J. A.
    Nature Reviews Drug Discovery (2020), 19 (10), 673-694CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)
    A review. Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degrdn. by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these mols. have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technol. advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches -including chem. modification, bioconjugation and the use of nanocarriers -which aim to address the delivery challenge.
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  14. 14
    Cuellar, T. L.; Barnes, D.; Nelson, C.; Tanguay, J.; Yu, S.-F.; Wen, X.; Scales, S. J.; Gesch, J.; Davis, D.; van Brabant Smith, A.; Leake, D.; Vandlen, R.; Siebel, C. W. Systematic Evaluation of Antibody-Mediated siRNA Delivery Using an Industrial Platform of THIOMAB-siRNA Conjugates. Nucleic Acids Res. 2015, 43 (2), 1189– 1203,  DOI: 10.1093/nar/gku1362
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    14
    Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB-siRNA conjugates
    Cuellar, Trinna L.; Barnes, Dwight; Nelson, Christopher; Tanguay, Joshua; Yu, Shang-Fan; Wen, Xiaohui; Scales, Suzie J.; Gesch, Julie; Davis, David; van Brabant Smith, Anja; Leake, Devin; Vandlen, Richard; Siebel, Christian W.
    Nucleic Acids Research (2015), 43 (2), 1189-1203CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible soln. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, the authors built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. The authors report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technol., the authors generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. The authors identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only obsd. using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. The authors' broad characterization of multiple parameters using therapeutic-grade conjugate technol. provides a thorough assessment of this delivery technol., highlighting both examples of success as well as remaining challenges.
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  15. 15
    Sela, T.; Mansø, M.; Siegel, M.; Marban-Doran, C.; Ducret, A.; Niewöhner, J.; Ravn, J.; Martin, R. E.; Sommer, A.; Lohmann, S.; Krippendorff, B.-F.; Ladefoged, M.; Indlekofer, A.; Quaiser, T.; Bueddefeld, F.; Koller, E.; Mohamed, M. Y.; Oelschlaegel, T.; Gothelf, K. V.; Hofer, K.; Schumacher, F. F. Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties. Bioconjugate Chem. 2023, 34 (11), 2096– 2111,  DOI: 10.1021/acs.bioconjchem.3c00393
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    There is no corresponding record for this reference.
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    Sugo, T.; Terada, M.; Oikawa, T.; Miyata, K.; Nishimura, S.; Kenjo, E.; Ogasawara-Shimizu, M.; Makita, Y.; Imaichi, S.; Murata, S.; Otake, K.; Kikuchi, K.; Teratani, M.; Masuda, Y.; Kamei, T.; Takagahara, S.; Ikeda, S.; Ohtaki, T.; Matsumoto, H. Development of Antibody-siRNA Conjugate Targeted to Cardiac and Skeletal Muscles. J. Controlled Release 2016, 237, 1– 13,  DOI: 10.1016/j.jconrel.2016.06.036
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    16
    Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles
    Sugo, Tsukasa; Terada, Michiko; Oikawa, Tatsuo; Miyata, Kenichi; Nishimura, Satoshi; Kenjo, Eriya; Ogasawara-Shimizu, Mari; Makita, Yukimasa; Imaichi, Sachiko; Murata, Shumpei; Otake, Kentaro; Kikuchi, Kuniko; Teratani, Mika; Masuda, Yasushi; Kamei, Takayuki; Takagahara, Shuichi; Ikeda, Shota; Ohtaki, Tetsuya; Matsumoto, Hirokazu
    Journal of Controlled Release (2016), 237 (), 1-13CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after i.v. administration in normal mice. In particular, 1 μg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected i.m. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by i.m. injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.
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  17. 17
    Malecova, B.; Burke, R. S.; Cochran, M.; Hood, M. D.; Johns, R.; Kovach, P. R.; Doppalapudi, V. R.; Erdogan, G.; Arias, J. D.; Darimont, B.; Miller, C. D.; Huang, H.; Geall, A.; Younis, H. S.; Levin, A. A. Targeted Tissue Delivery of RNA Therapeutics Using Antibody-Oligonucleotide Conjugates (AOCs). Nucleic Acids Res. 2023, 51 (12), 5901– 5910,  DOI: 10.1093/nar/gkad415
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    There is no corresponding record for this reference.
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    Cao, W.; Li, R.; Pei, X.; Chai, M.; Sun, L.; Huang, Y.; Wang, J.; Barth, S.; Yu, F.; He, H. Antibody-siRNA Conjugates (ARC): Emerging siRNA Drug Formulation. Med. Drug Discovery 2022, 15, 100128,  DOI: 10.1016/j.medidd.2022.100128
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    Chernikov, I. V.; Vlassov, V. V.; Chernolovskaya, E. L. Current Development of siRNA Bioconjugates: From Research to the Clinic. Front. Pharmacol 2019, 10, 444,  DOI: 10.3389/fphar.2019.00444
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    Current development of siRNA bioconjugates: from research to the clinic
    Chernikov, Ivan V.; Vlassov, Valentin V.; Chernolovskaya, Elena L.
    Frontiers in Pharmacology (2019), 10 (), 444CODEN: FPRHAU; ISSN:1663-9812. (Frontiers Media S.A.)
    A review. Small interfering RNAs (siRNAs) acting via RNA interference mechanisms are able to recognize a homologous mRNA sequence in the cell and induce its degrdn. The main problems in the development of siRNA-based drugs for therapeutic use are the low efficiency of siRNA delivery to target cells and the degrdn. of siRNAs by nucleases in biol. fluids. Various approaches have been proposed to solve the problem of siRNA delivery in vivo (e.g., viruses, cationic lipids, polymers, nanoparticles), but all have limitations for therapeutic use. One of the most promising approaches to solve the problem of siRNA delivery to target cells is bioconjugation; i.e., the covalent connection of siRNAs with biogenic mols. (lipophilic mols., antibodies, aptamers, ligands, peptides, or polymers). Bioconjugates are "ideal nanoparticles" since they do not need a pos. charge to form complexes, are less toxic, and are less effectively recognized by components of the immune system because of their small size. This review is focused on strategies and principles for constructing siRNA bioconjugates for in vivo use.
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    Tai, W. Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery. Molecules 2019, 24 (12), 2211,  DOI: 10.3390/molecules24122211
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    20
    Current aspects of siRNA bioconjugate for in vitro and in vivo delivery
    Tai, Wanyi
    Molecules (2019), 24 (12), 2211CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    A review. Studies on siRNA delivery have seen intense growth in the past decades since siRNA has emerged as a new class of gene therapeutics for the treatment of various diseases. siRNA bioconjugate, as one of the major delivery strategies, offers the potential to enhance and broaden pharmacol. properties of siRNA, while minimizing the heterogeneity and stability-correlated toxicol. This review summarizes the recent developments of siRNA bioconjugate, including the conjugation with antibody, peptide, aptamer, small chem., lipidoid, cell-penetrating peptide polymer, and nanoparticle. These siRNA bioconjugate, either administrated alone or formulated with other agents, could significantly improve pharmacokinetic behavior, enhance the biol. half-life, and increase the targetability while maintaining sufficient gene silencing activity, with a concomitant improvement of the therapeutic outcomes and diminishment of adverse effects. This review emphasizes the delivery application of these siRNA bioconjugates, esp. the conjugation strategy that control the integrity, stability and release of siRNA bioconjugates. The limitations conferred by these conjugation strategies have also been covered.
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  21. 21
    Sun, M. M. C.; Beam, K. S.; Cerveny, C. G.; Hamblett, K. J.; Blackmore, R. S.; Torgov, M. Y.; Handley, F. G. M.; Ihle, N. C.; Senter, P. D.; Alley, S. C. Reduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides. Bioconjugate Chem. 2005, 16 (5), 1282– 1290,  DOI: 10.1021/bc050201y
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    Reduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides
    Sun, Michael M. C.; Beam, Kevin S.; Cerveny, Charles G.; Hamblett, Kevin J.; Blackmore, Richard S.; Torgov, Michael Y.; Handley, Felicia G. M.; Ihle, Nathan C.; Senter, Peter D.; Alley, Stephen C.
    Bioconjugate Chemistry (2005), 16 (5), 1282-1290CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Site-specific conjugation of small mols. and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biol. properties. We describe for the first time chem. redn. and oxidn. methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purifn. with hydrophobic interaction chromatog. followed by anal. using reversed-phase HPLC and capillary electrophoresis. These anal. methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential redn. of heavy-light chain disulfides, while reoxidn. of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60-90%, allowing for control of the distribution of mol. species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.
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  22. 22
    Shen, B.-Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.; Yu, S.-F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.; Eigenbrot, C.; Nguyen, T.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel, D.; Spencer, S. D.; Khawli, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.; Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R. Conjugation Site Modulates the in Vivo Stability and Therapeutic Activity of Antibody-Drug Conjugates. Nat. Biotechnol. 2012, 30 (2), 184– 189,  DOI: 10.1038/nbt.2108
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    22
    Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates
    Shen, Ben-Quan; Xu, Keyang; Liu, Luna; Raab, Helga; Bhakta, Sunil; Kenrick, Margaret; Parsons-Reponte, Kathryn L.; Tien, Janet; Yu, Shang-Fan; Mai, Elaine; Li, Dongwei; Tibbitts, Jay; Baudys, Jakub; Saad, Ola M.; Scales, Suzie J.; McDonald, Paul J.; Hass, Philip E.; Eigenbrot, Charles; Nguyen, Trung; Solis, Willy A.; Fuji, Reina N.; Flagella, Kelly M.; Patel, Darshana; Spencer, Susan D.; Khawli, Leslie A.; Ebens, Allen; Wong, Wai Lee; Vandlen, Richard; Kaur, Surinder; Sliwkowski, Mark X.; Scheller, Richard H.; Polakis, Paul; Junutula, Jagath R.
    Nature Biotechnology (2012), 30 (2), 184-189CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a pos. charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected pos. by succinimide ring hydrolysis and neg. by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chem. and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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  23. 23
    Zhou, Q.; Stefano, J. E.; Manning, C.; Kyazike, J.; Chen, B.; Gianolio, D. A.; Park, A.; Busch, M.; Bird, J.; Zheng, X.; Simonds-Mannes, H.; Kim, J.; Gregory, R. C.; Miller, R. J.; Brondyk, W. H.; Dhal, P. K.; Pan, C. Q. Site-Specific Antibody-Drug Conjugation through Glycoengineering. Bioconjugate Chem. 2014, 25 (3), 510– 520,  DOI: 10.1021/bc400505q
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    23
    Site-Specific Antibody-Drug Conjugation through Glycoengineering
    Zhou, Qun; Stefano, James E.; Manning, Charlene; Kyazike, Josephine; Chen, Bo; Gianolio, Diego A.; Park, Anna; Busch, Michelle; Bird, Julie; Zheng, Xiaoyang; Simonds-Mannes, Helene; Kim, Jennifer; Gregory, Rick C.; Miller, Robert J.; Brondyk, William H.; Dhal, Pradeep K.; Pan, Clark Q.
    Bioconjugate Chemistry (2014), 25 (3), 510-520CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Antibody-drug conjugates (ADCs) have been proven clin. to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prep. ADCs by targeting primary amines or hinge disulfides have a no. of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidn. of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatog. and LC-MS analyses revealed the incorporation of ∼1.6 cytotoxic agents per antibody mol., approximating the no. of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-pos. tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-pos. tumor xenograft model. These findings suggest that enzymic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chem. process to ensure reproducible drug loading.
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  24. 24
    Li, X.; Fang, T.; Boons, G. Preparation of Well-Defined Antibody-Drug Conjugates through Glycan Remodeling and Strain-Promoted Azide-Alkyne Cycloadditions. Angew. Chem., Int. Ed. 2014, 53 (28), 7179– 7182,  DOI: 10.1002/anie.201402606
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    24
    Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions
    Li, Xiuru; Fang, Tao; Boons, Geert-Jan
    Angewandte Chemie, International Edition (2014), 53 (28), 7179-7182CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Antibody-drug conjugates hold considerable promise as anticancer agents, however, producing them remains a challenge and there is a need for mild, broadly applicable, site-specific conjugation methods that yield homogenous products. It was envisaged that enzymic remodeling of the oligosaccharides of an antibody would enable the introduction of reactive groups that can be exploited for the site-specific attachment of cytotoxic drugs. This is based on the observation that glycosyltransferases often tolerate chem. modifications in their sugar nucleotide substrates, thus allowing the installation of reactive functionalities. An azide was incorporated because this functional group is virtually absent in biol. systems and can be reacted by strain-promoted alkyne-azide cycloaddn. This method, which does not require genetic engineering, was used to produce an anti-CD22 antibody modified with doxorubicin to selectively target and kill lymphoma cells.
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  25. 25
    Brun, M.-P.; Gauzy-Lazo, L. Protocols for Lysine Conjugation. In Antibody-Drug Conjugates; Ducry, L., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2013; Vol. 1045, pp 173– 187. DOI: 10.1007/978-1-62703-541-5_10 .
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    There is no corresponding record for this reference.
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    Kumar, P.; Parmar, R. G.; Brown, C. R.; Willoughby, J. L. S.; Foster, D. J.; Babu, I. R.; Schofield, S.; Jadhav, V.; Charisse, K.; Nair, J. K.; Rajeev, K. G.; Maier, M. A.; Egli, M.; Manoharan, M. 5′-Morpholino Modification of the Sense Strand of an siRNA Makes It a More Effective Passenger. Chem. Commun. 2019, 55 (35), 5139– 5142,  DOI: 10.1039/C9CC00977A
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    Allerson, C. R.; Sioufi, N.; Jarres, R.; Prakash, T. P.; Naik, N.; Berdeja, A.; Wanders, L.; Griffey, R. H.; Swayze, E. E.; Bhat, B. Fully 2‘-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNA. J. Med. Chem. 2005, 48 (4), 901– 904,  DOI: 10.1021/jm049167j
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    27
    Fully 2'-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNA
    Allerson, Charles R.; Sioufi, Namir; Jarres, Russell; Prakash, Thazha P.; Naik, Nishant; Berdeja, Andres; Wanders, Lisa; Griffey, Richard H.; Swayze, Eric E.; Bhat, Balkrishen
    Journal of Medicinal Chemistry (2005), 48 (4), 901-904CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-Me and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.
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  28. 28
    Foster, D. J.; Brown, C. R.; Shaikh, S.; Trapp, C.; Schlegel, M. K.; Qian, K.; Sehgal, A.; Rajeev, K. G.; Jadhav, V.; Manoharan, M.; Kuchimanchi, S.; Maier, M. A.; Milstein, S. Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates. Mol. Ther. 2018, 26 (3), 708– 717,  DOI: 10.1016/j.ymthe.2017.12.021
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    Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates
    Foster, Donald J.; Brown, Christopher R.; Shaikh, Sarfraz; Trapp, Casey; Schlegel, Mark K.; Qian, Kun; Sehgal, Alfica; Rajeev, Kallanthottathil G.; Jadhav, Vasant; Manoharan, Muthiah; Kuchimanchi, Satya; Maier, Martin A.; Milstein, Stuart
    Molecular Therapy (2018), 26 (3), 708-717CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)
    Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chem. modified small interfering RNA (siRNA) designs, including the recently described Enhanced Stabilization Chem. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after s.c. administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chem., optimizing the positioning of 2'-deoxy-2'-fluoro and 2'-O-Me ribosugar modifications across both strands of the double-stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2'-deoxy-2'-fluoro content that yield significantly improved potency and duration in preclin. species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.
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  29. 29
    Nair, J. K.; Willoughby, J. L. S.; Chan, A.; Charisse, K.; Alam, M. R.; Wang, Q.; Hoekstra, M.; Kandasamy, P.; Kel’in, A. V.; Milstein, S.; Taneja, N.; O’Shea, J.; Shaikh, S.; Zhang, L.; Van Der Sluis, R. J.; Jung, M. E.; Akinc, A.; Hutabarat, R.; Kuchimanchi, S.; Fitzgerald, K.; Zimmermann, T.; Van Berkel, T. J. C.; Maier, M. A.; Rajeev, K. G.; Manoharan, M. Multivalent N -Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing. J. Am. Chem. Soc. 2014, 136 (49), 16958– 16961,  DOI: 10.1021/ja505986a
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    29
    Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing
    Nair, Jayaprakash K.; Willoughby, Jennifer L. S.; Chan, Amy; Charisse, Klaus; Alam, Md. Rowshon; Wang, Qianfan; Hoekstra, Menno; Kandasamy, Pachamuthu; Kel'in, Alexander V.; Milstein, Stuart; Taneja, Nate; O'Shea, Jonathan; Shaikh, Sarfraz; Zhang, Ligang; van der Sluis, Ronald J.; Jung, Michael E.; Akinc, Akin; Hutabarat, Renta; Kuchimanchi, Satya; Fitzgerald, Kevin; Zimmermann, Tracy; van Berkel, Theo J. C.; Maier, Martin A.; Rajeev, Kallanthottathil G.; Manoharan, Muthiah
    Journal of the American Chemical Society (2014), 136 (49), 16958-16961CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of std. oligonucleotides. S.c. (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chem. achieved a 5-fold improvement in efficacy over the parent design in vivo with a median ED (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose vols. of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 mo with no adverse effects in rodents. The optimally chem. modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.
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    Haraszti, R. A.; Roux, L.; Coles, A. H.; Turanov, A. A.; Alterman, J. F.; Echeverria, D.; Godinho, B. M. D. C.; Aronin, N.; Khvorova, A. 5′-Vinylphosphonate Improves Tissue Accumulation and Efficacy of Conjugated siRNAs in Vivo. Nucleic Acids Res. 2017, 45 (13), 7581– 7592,  DOI: 10.1093/nar/gkx507
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    5'-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo
    Haraszti, Reka A.; Roux, Loic; Coles, Andrew H.; Turanov, Anton A.; Alterman, Julia F.; Echeverria, Dimas; Godinho, Bruno M. D. C.; Aronin, Neil; Khvorova, Anastasia
    Nucleic Acids Research (2017), 45 (13), 7581-7592CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    5'-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5'-vinylphosphonate confers novel properties to siRNAs. Specifically, 5'-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5'-to-3' exonucleases. Delivery of conjugated siRNAs requires extensive chem. modifications to achieve stability in vivo. Because chem. modified siRNAs are poor substrates for phosphorylation by kinases, and 5'-phosphate is required for loading into RNA-induced silencing complex, the synthetic addn. of a 5'-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5'-phosphate is removed within 2 h. The 5'-phosphate mimic 5'-(E)-vinylphosphonate stabilizes the 5' end of the guide strand by protecting it from phosphatases and 5'-to-3' exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5'-(E)- vinylphosphonate stabilizes 5' phosphate, thereby enabling systemic delivery to and silencing in kidney and heart.
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  31. 31
    Parmar, R.; Willoughby, J. L. S.; Liu, J.; Foster, D. J.; Brigham, B.; Theile, C. S.; Charisse, K.; Akinc, A.; Guidry, E.; Pei, Y.; Strapps, W.; Cancilla, M.; Stanton, M. G.; Rajeev, K. G.; Sepp-Lorenzino, L.; Manoharan, M.; Meyers, R.; Maier, M. A.; Jadhav, V. 5′-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates. ChemBioChem 2016, 17 (11), 985– 989,  DOI: 10.1002/cbic.201600130
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    31
    5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates
    Parmar, Rubina; Willoughby, Jennifer L. S.; Liu, Jingxuan; Foster, Donald J.; Brigham, Benjamin; Theile, Christopher S.; Charisse, Klaus; Akinc, Akin; Guidry, Erin; Pei, Yi; Strapps, Walter; Cancilla, Mark; Stanton, Matthew G.; Rajeev, Kallanthottathil G.; Sepp-Lorenzino, Laura; Manoharan, Muthiah; Meyers, Rachel; Maier, Martin A.; Jadhav, Vasant
    ChemBioChem (2016), 17 (11), 985-989CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be crit. for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chem. modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.
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  32. 32
    Brown, C. R.; Gupta, S.; Qin, J.; Racie, T.; He, G.; Lentini, S.; Malone, R.; Yu, M.; Matsuda, S.; Shulga-Morskaya, S.; Nair, A. V.; Theile, C. S.; Schmidt, K.; Shahraz, A.; Goel, V.; Parmar, R. G.; Zlatev, I.; Schlegel, M. K.; Nair, J. K.; Jayaraman, M.; Manoharan, M.; Brown, D.; Maier, M. A.; Jadhav, V. Investigating the Pharmacodynamic Durability of GalNAc-siRNA Conjugates. Nucleic Acids Res. 2020, 48 (21), 11827– 11844,  DOI: 10.1093/nar/gkaa670
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    32
    Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates
    Brown, Christopher R.; Gupta, Swati; Qin, June; Racie, Timothy; He, Guo; Lentini, Scott; Malone, Ryan; Yu, Mikyung; Matsuda, Shigeo; Shulga-Morskaya, Svetlana; Nair, Anil V.; Theile, Christopher S.; Schmidt, Karyn; Shahraz, Azar; Goel, Varun; Parmar, Rubina G.; Zlatev, Ivan; Schlegel, Mark K.; Nair, Jayaprakash K.; Jayaraman, Muthusamy; Manoharan, Muthiah; Brown, Dennis; Maier, Martin A.; Jadhav, Vasant
    Nucleic Acids Research (2020), 48 (21), 11827-11844CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclin. species and humans. Here, we investigated the underlying biol. supporting this extended duration of pharmacol. activity. We found that siRNA accumulation and stability in acidic intracellular compartments is crit. for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity obsd. in vivo.
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  33. 33
    Strop, P.; Liu, S.-H.; Dorywalska, M.; Delaria, K.; Dushin, R. G.; Tran, T.-T.; Ho, W.-H.; Farias, S.; Casas, M. G.; Abdiche, Y.; Zhou, D.; Chandrasekaran, R.; Samain, C.; Loo, C.; Rossi, A.; Rickert, M.; Krimm, S.; Wong, T.; Chin, S. M.; Yu, J.; Dilley, J.; Chaparro-Riggers, J.; Filzen, G. F.; O’Donnell, C. J.; Wang, F.; Myers, J. S.; Pons, J.; Shelton, D. L.; Rajpal, A. Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates. Chem. Biol. 2013, 20 (2), 161– 167,  DOI: 10.1016/j.chembiol.2013.01.010
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    33
    Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates
    Strop, Pavel; Liu, Shu-Hui; Dorywalska, Magdalena; Delaria, Kathy; Dushin, Russell G.; Tran, Thomas-Toan; Ho, Wei-Hsien; Farias, Santiago; Casas, Meritxell Galindo; Abdiche, Yasmina; Zhou, Dahui; Chandrasekaran, Ramalakshmi; Samain, Caroline; Loo, Carole; Rossi, Andrea; Rickert, Mathias; Krimm, Stellanie; Wong, Teresa; Chin, Sherman Michael; Yu, Jessica; Dilley, Jeanette; Chaparro-Riggers, Javier; Filzen, Gary F.; O'Donnell, Christopher J.; Wang, Fang; Myers, Jeremy S.; Pons, Jaume; Shelton, David L.; Rajpal, Arvind
    Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 161-167CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compds. at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chem. instability, as was obsd. with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.
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    Miller, C. M.; Donner, A. J.; Blank, E. E.; Egger, A. W.; Kellar, B. M.; Østergaard, M. E.; Seth, P. P.; Harris, E. N. Stabilin-1 and Stabilin-2 Are Specific Receptors for the Cellular Internalization of Phosphorothioate-Modified Antisense Oligonucleotides (ASOs) in the Liver. Nucleic Acids Res. 2016, 44 (6), 2782– 2794,  DOI: 10.1093/nar/gkw112
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    Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver
    Miller Colton M; Blank Emma E; Egger Andrew W; Kellar Brianna M; Harris Edward N; Donner Aaron J; Ostergaard Michael E; Seth Punit P
    Nucleic acids research (2016), 44 (6), 2782-94 ISSN:.
    Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro®, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties.
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  35. 35
    Lehot, V.; Kuhn, I.; Nothisen, M.; Erb, S.; Kolodych, S.; Cianférani, S.; Chaubet, G.; Wagner, A. Non-Specific Interactions of Antibody-Oligonucleotide Conjugates with Living Cells. Sci. Rep. 2021, 11 (1), 5881,  DOI: 10.1038/s41598-021-85352-w
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  36. 36
    Rocca, C.; Dennin, S.; Gu, Y.; Kim, J.; Chigas, S.; Najarian, D.; Chong, S.; Gutierrez, S.; Butler, J.; Charisse, K.; Robbie, G.; Xu, Y.; Brown, K. Evaluation of Electrophoretic Mobility Shift Assay as a Method to Determine Plasma Protein Binding of siRNA. Bioanalysis 2019, 11 (21), 1927– 1939,  DOI: 10.4155/bio-2019-0151
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    Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNA
    Rocca, Carrie; Dennin, Sean; Gu, Yongli; Kim, Joohwan; Chigas, Samantha; Najarian, Diana; Chong, Saeho; Gutierrez, Shannon; Butler, James; Charisse, Klaus; Robbie, Gabriel; Xu, Yuanxin; Brown, Kirk
    Bioanalysis (2019), 11 (21), 1927-1939CODEN: BIOAB4; ISSN:1757-6180. (Future Science Ltd.)
    Aim: The electrophoretic mobility shift assay (EMSA) was evaluated as an alternative to ultrafiltration (UF) to assess plasma protein binding (PPB) of small interfering RNAs (siRNA) and antisense oligonucleotides (ASO). Results & methodol.: EMSA anal. showed that PPB depended on siRNA and plasma concn. Conversely, when analyzed by ultrafiltration, siRNA bound the filtration device nonspecifically and PPB remained >98% across physiol. relevant siRNA concns. Using EMSA, siRNA exhibited charge-based interactions with plasma proteins, while ASO remained highly bound to plasma proteins or albumin in the presence of 500 mM salt. Conclusion: PPB characteristics of siRNA and ASO can be distinguished using EMSA. Characterization of siRNA PPB by EMSA enhances our knowledge of siRNA absorption, distribution, metab. and excretion and advanced development of RNA interference therapeutics.
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    Liang, X.; Sun, H.; Shen, W.; Crooke, S. T. Identification and Characterization of Intracellular Proteins That Bind Oligonucleotides with Phosphorothioate Linkages. Nucleic Acids Res. 2015, 43 (5), 2927– 2945,  DOI: 10.1093/nar/gkv143
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    37
    Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages
    Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T.
    Nucleic Acids Research (2015), 43 (5), 2927-2945CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Although the RNase H-dependent mechanism of inhibition of gene expression by chem. modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and redn. in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms.
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    Gaus, H.; Miller, C. M.; Seth, P. P.; Harris, E. N. Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance Receptor. Biochemistry 2018, 57 (14), 2061– 2064,  DOI: 10.1021/acs.biochem.8b00126
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    38
    Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance Receptor
    Gaus, Hans; Miller, Colton M.; Seth, Punit P.; Harris, Edward N.
    Biochemistry (2018), 57 (14), 2061-2064CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The Stabilin receptors are systemic clearance receptors for some classes of chem. modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarization-based assay. The tested ASOs varied in their backbone compn., modification of the ribose 2' position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5-10-5 pattern of flanking 2'-O-methoxyethyl modifications was then used to test the effects of pH and salt concn. on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissocn. and fit within the biol. parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors.
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    Structure−Activity Relationship of Antibody−Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody−Phosphorodiamidate Morpholino Oligomer Conjugates for Drug Development, Manuscript in Preparation.
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    There is no corresponding record for this reference.
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    Dosio, F.; Brusa, P.; Cattel, L. Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components. Toxins 2011, 3 (7), 848– 883,  DOI: 10.3390/toxins3070848
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    Immunotoxins and anticancer drug conjugate assemblies: the role of the linkage between components
    Dosio, Franco; Brusa, Paola; Cattel, Luigi
    Toxins (2011), 3 (), 848-883CODEN: TOXIB7; ISSN:2072-6651. (MDPI AG)
    A review. Immunotoxins and antibody-drug conjugates are protein-based drugs combining a target-specific binding domain with a cytotoxic domain. Such compds. are potentially therapeutic against diseases including cancer, and several clin. trials have shown encouraging results. Although the targeted elimination of malignant cells is an elegant concept, there are numerous practical challenges that limit conjugates' therapeutic use, including inefficient cellular uptake, low cytotoxicity, and off-target effects. During the prepn. of immunoconjugates by chem. synthesis, the choice of the hinge component joining the two building blocks is of paramount importance: the conjugate must remain stable in vivo but must afford efficient release of the toxic moiety when the target is reached. Vast efforts have been made, and the present article reviews strategies employed in developing immunoconjugates, focusing on the evolution of chem. linkers.
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    Su, D.; Kozak, K. R.; Sadowsky, J.; Yu, S.-F.; Fourie-O’Donohue, A.; Nelson, C.; Vandlen, R.; Ohri, R.; Liu, L.; Ng, C.; He, J.; Davis, H.; Lau, J.; Del Rosario, G.; Cosino, E.; Cruz-Chuh, J. D.; Ma, Y.; Zhang, D.; Darwish, M.; Cai, W.; Chen, C.; Zhou, H.; Lu, J.; Liu, Y.; Kaur, S.; Xu, K.; Pillow, T. H. Modulating Antibody-Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification. Bioconjugate Chem. 2018, 29 (4), 1155– 1167,  DOI: 10.1021/acs.bioconjchem.7b00785
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    Su, D.; Zhang, D. Linker Design Impacts Antibody-Drug Conjugate Pharmacokinetics and Efficacy via Modulating the Stability and Payload Release Efficiency. Front. Pharmacol 2021, 12, 687926,  DOI: 10.3389/fphar.2021.687926
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    Linker design impacts antibody-drug conjugate pharmacokinetics and efficacy via modulating the stability and payload release efficiency
    Su, Dian; Zhang, Donglu
    Frontiers in Pharmacology (2021), 12 (), 687926CODEN: FPRHAU; ISSN:1663-9812. (Frontiers Media S.A.)
    The development of antibody-drug conjugates (ADCs) has significantly been advanced in the past decade given the improvement of payloads, linkers and conjugation methods. In particular, linker design plays a crit. role in modulating ADC stability in the systemic circulation and payload release efficiency in the tumors, which thus affects ADC pharmacokinetic (PK), efficacy and toxicity profiles. Previously, we have investigated key linker parameters such as conjugation chem. (e.g., maleimide vs. disulfide), linker length and linker steric hindrance and their impacts on PK and efficacy profiles. Herein, we discuss our perspectives on development of integrated strategies for linker design to achieve a balance between ADC stability and payload release efficiency for desired efficacy in antigen-expressing xenograft models. The strategies have been successfully applied to the design of site-specific THIOMABTM antibody-drug conjugates (TDCs) with different payloads. We also propose to conduct dose fractionation studies to gain guidance for optimal dosing regimens of ADCs in pre-clin. models.
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  43. 43
    Kolodych, S.; Koniev, O.; Baatarkhuu, Z.; Bonnefoy, J.-Y.; Debaene, F.; Cianférani, S.; Van Dorsselaer, A.; Wagner, A. CBTF: New Amine-to-Thiol Coupling Reagent for Preparation of Antibody Conjugates with Increased Plasma Stability. Bioconjugate Chem. 2015, 26 (2), 197– 200,  DOI: 10.1021/bc500610g
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    CBTF: New Amine-to-Thiol Coupling Reagent for Preparation of Antibody Conjugates with Increased Plasma Stability
    Kolodych, Sergii; Koniev, Oleksandr; Baatarkhuu, Zoljargal; Bonnefoy, Jean-Yves; Debaene, Francois; Cianferani, Sarah; Van Dorsselaer, Alain; Wagner, Alain
    Bioconjugate Chemistry (2015), 26 (2), 197-200CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Amine-to-thiol coupling is the most common route for the prepn. of antibody-drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the prepn. of remarkably stable conjugates. A series of antibody-dye conjugates have been prepd. using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates.
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  44. 44
    Lyon, R. P.; Setter, J. R.; Bovee, T. D.; Doronina, S. O.; Hunter, J. H.; Anderson, M. E.; Balasubramanian, C. L.; Duniho, S. M.; Leiske, C. I.; Li, F.; Senter, P. D. Self-Hydrolyzing Maleimides Improve the Stability and Pharmacological Properties of Antibody-Drug Conjugates. Nat. Biotechnol. 2014, 32 (10), 1059– 1062,  DOI: 10.1038/nbt.2968
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    Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates
    Lyon, Robert P.; Setter, Jocelyn R.; Bovee, Tim D.; Doronina, Svetlana O.; Hunter, Joshua H.; Anderson, Martha E.; Balasubramanian, Cindy L.; Duniho, Steven M.; Leiske, Chris I.; Li, Fu; Senter, Peter D.
    Nature Biotechnology (2014), 32 (10), 1059-1062CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    Many antibody-drug conjugates (ADCs) are unstable in vivo because they are formed from maleimide-contg. components conjugated to reactive thiols. These thiosuccinimide linkages undergo two competing reactions in plasma: elimination of the maleimide through a retro-Michael reaction, which results in loss of drug-linker from the ADC, and hydrolysis of the thiosuccinimide ring, which results in a deriv. that is resistant to the elimination reaction. In an effort to create linker technologies with improved stability characteristics, we used diaminopropionic acid (DPR) to prep. a drug-linker incorporating a basic amino group adjacent to the maleimide, positioned to provide intramol. catalysis of thiosuccinimide ring hydrolysis. This basic group induces the thiosuccinimide to undergo rapid hydrolysis at neutral pH and room temp. Once hydrolyzed, the drug-linker is no longer subject to maleimide elimination reactions, preventing nonspecific deconjugation. In vivo studies demonstrate that the increased stability characteristics can lead to improved ADC antitumor activity and reduced neutropenia.
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Cite this: J. Med. Chem. 2024, 67, 17, 14852–14867
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  • Abstract

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    Scheme 1

    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
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    Figure 1

    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.

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    Figure 2

    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).

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    Figure 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.

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    Figure 4

    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.

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    Figure 5

    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).

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    Figure 6

    Figure 6. (a,b) Mouse PK of AOCs containing cleavable linkers.

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    Figure 7

    Figure 7. (a–c) PK/PD studies with αmTfR1–siHprt AOCs containing four different noncleavable linkers.

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    Figure 8

    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.

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    Figure 9

    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.

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      Hu Bo; Weng Yuhua; Huang Yuanyu; Zhong Liping; Zhao Yongxiang; Peng Ling; Liang Xing-Jie
      Signal transduction and targeted therapy (2020), 5 (1), 101 ISSN:.
      RNA interference (RNAi) is an ancient biological mechanism used to defend against external invasion. It theoretically can silence any disease-related genes in a sequence-specific manner, making small interfering RNA (siRNA) a promising therapeutic modality. After a two-decade journey from its discovery, two approvals of siRNA therapeutics, ONPATTRO(®) (patisiran) and GIVLAARI® (givosiran), have been achieved by Alnylam Pharmaceuticals. Reviewing the long-term pharmaceutical history of human beings, siRNA therapy currently has set up an extraordinary milestone, as it has already changed and will continue to change the treatment and management of human diseases. It can be administered quarterly, even twice-yearly, to achieve therapeutic effects, which is not the case for small molecules and antibodies. The drug development process was extremely hard, aiming to surmount complex obstacles, such as how to efficiently and safely deliver siRNAs to desired tissues and cells and how to enhance the performance of siRNAs with respect to their activity, stability, specificity and potential off-target effects. In this review, the evolution of siRNA chemical modifications and their biomedical performance are comprehensively reviewed. All clinically explored and commercialized siRNA delivery platforms, including the GalNAc (N-acetylgalactosamine)-siRNA conjugate, and their fundamental design principles are thoroughly discussed. The latest progress in siRNA therapeutic development is also summarized. This review provides a comprehensive view and roadmap for general readers working in the field.
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    10. 10
      Egli, M.; Manoharan, M. Chemistry, Structure and Function of Approved Oligonucleotide Therapeutics. Nucleic Acids Res. 2023, 51 (6), 2529– 2573,  DOI: 10.1093/nar/gkad067
      10
      Chemistry, structure and function of approved oligonucleotide therapeutics
      Egli, Martin; Manoharan, Muthiah
      Nucleic Acids Research (2023), 51 (6), 2529-2573CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      A review. Eighteen nucleic acid therapeutics have been approved for treatment of various diseases in the last 25 years. Their modes of action include antisense oligonucleotides (ASOs), splice-switching oligonucleotides (SSOs), RNA interference (RNAi) and an RNA aptamer against a protein. Among the diseases targeted by this new class of drugs are homozygous familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, hereditary transthyretin-mediated amyloidosis, familial chylomicronemia syndrome, acute hepatic porphyria, and primary hyperoxaluria. Chem. modification of DNA and RNA was central to making drugs out of oligonucleotides. Oligonucleotide therapeutics brought to market thus far contain just a handful of first- and second-generation modifications, among them 2'-fluoro-RNA, 2'-O-Me RNA and the phosphorothioates that were introduced over 50 years ago. Two other privileged chemistries are 2'-O-(2-methoxyethyl)-RNA (MOE) and the phosphorodiamidate morpholinos (PMO). Given their importance in imparting oligonucleotides with high target affinity, metabolic stability and favorable pharmacokinetic and -dynamic properties, this article provides a review of these chemistries and their use in nucleic acid therapeutics. Breakthroughs in lipid formulation and GalNAc conjugation of modified oligonucleotides have paved the way to efficient delivery and robust, long-lasting silencing of genes. This review provides an account of the state-of-the-art of targeted oligo delivery to hepatocytes.
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    11. 11
      Janas, M. M.; Schlegel, M. K.; Harbison, C. E.; Yilmaz, V. O.; Jiang, Y.; Parmar, R.; Zlatev, I.; Castoreno, A.; Xu, H.; Shulga-Morskaya, S.; Rajeev, K. G.; Manoharan, M.; Keirstead, N. D.; Maier, M. A.; Jadhav, V. Selection of GalNAc-Conjugated siRNAs with Limited off-Target-Driven Rat Hepatotoxicity. Nat. Commun. 2018, 9 (1), 723,  DOI: 10.1038/s41467-018-02989-4
      11
      Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity
      Janas Maja M; Schlegel Mark K; Harbison Carole E; Yilmaz Vedat O; Jiang Yongfeng; Parmar Rubina; Zlatev Ivan; Castoreno Adam; Xu Huilei; Shulga-Morskaya Svetlana; Rajeev Kallanthottathil G; Manoharan Muthiah; Keirstead Natalie D; Maier Martin A; Jadhav Vasant
      Nature communications (2018), 9 (1), 723 ISSN:.
      Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.
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    12. 12
      Brown, K. M.; Nair, J. K.; Janas, M. M.; Anglero-Rodriguez, Y. I.; Dang, L. T. H.; Peng, H.; Theile, C. S.; Castellanos-Rizaldos, E.; Brown, C.; Foster, D.; Kurz, J.; Allen, J.; Maganti, R.; Li, J.; Matsuda, S.; Stricos, M.; Chickering, T.; Jung, M.; Wassarman, K.; Rollins, J.; Woods, L.; Kelin, A.; Guenther, D. C.; Mobley, M. W.; Petrulis, J.; McDougall, R.; Racie, T.; Bombardier, J.; Cha, D.; Agarwal, S.; Johnson, L.; Jiang, Y.; Lentini, S.; Gilbert, J.; Nguyen, T.; Chigas, S.; LeBlanc, S.; Poreci, U.; Kasper, A.; Rogers, A. B.; Chong, S.; Davis, W.; Sutherland, J. E.; Castoreno, A.; Milstein, S.; Schlegel, M. K.; Zlatev, I.; Charisse, K.; Keating, M.; Manoharan, M.; Fitzgerald, K.; Wu, J.-T.; Maier, M. A.; Jadhav, V. Expanding RNAi Therapeutics to Extrahepatic Tissues with Lipophilic Conjugates. Nat. Biotechnol. 2022, 40 (10), 1500– 1508,  DOI: 10.1038/s41587-022-01334-x
      12
      Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates
      Brown, Kirk M.; Nair, Jayaprakash K.; Janas, Maja M.; Anglero-Rodriguez, Yesseinia I.; Dang, Lan T. H.; Peng, Haiyan; Theile, Christopher S.; Castellanos-Rizaldos, Elena; Brown, Christopher; Foster, Donald; Kurz, Jeffrey; Allen, Jeffrey; Maganti, Rajanikanth; Li, Jing; Matsuda, Shigeo; Stricos, Matthew; Chickering, Tyler; Jung, Michelle; Wassarman, Kelly; Rollins, Jeff; Woods, Lauren; Kelin, Alex; Guenther, Dale C.; Mobley, Melissa W.; Petrulis, John; McDougall, Robin; Racie, Timothy; Bombardier, Jessica; Cha, Diana; Agarwal, Saket; Johnson, Lei; Jiang, Yongfeng; Lentini, Scott; Gilbert, Jason; Nguyen, Tuyen; Chigas, Samantha; LeBlanc, Sarah; Poreci, Urjana; Kasper, Anne; Rogers, Arlin B.; Chong, Saeho; Davis, Wendell; Sutherland, Jessica E.; Castoreno, Adam; Milstein, Stuart; Schlegel, Mark K.; Zlatev, Ivan; Charisse, Klaus; Keating, Mark; Manoharan, Muthiah; Fitzgerald, Kevin; Wu, Jing-Tao; Maier, Martin A.; Jadhav, Vasant
      Nature Biotechnology (2022), 40 (10), 1500-1508CODEN: NABIF9; ISSN:1087-0156. (Nature Portfolio)
      Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solns. are needed to target addnl. organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 mo. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclin. efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiol. and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.
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    13. 13
      Roberts, T. C.; Langer, R.; Wood, M. J. A. Advances in Oligonucleotide Drug Delivery. Nat. Rev. Drug Discovery 2020, 19 (10), 673– 694,  DOI: 10.1038/s41573-020-0075-7
      13
      Advances in oligonucleotide drug delivery
      Roberts, Thomas C.; Langer, Robert; Wood, Matthew J. A.
      Nature Reviews Drug Discovery (2020), 19 (10), 673-694CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)
      A review. Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degrdn. by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these mols. have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technol. advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches -including chem. modification, bioconjugation and the use of nanocarriers -which aim to address the delivery challenge.
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    14. 14
      Cuellar, T. L.; Barnes, D.; Nelson, C.; Tanguay, J.; Yu, S.-F.; Wen, X.; Scales, S. J.; Gesch, J.; Davis, D.; van Brabant Smith, A.; Leake, D.; Vandlen, R.; Siebel, C. W. Systematic Evaluation of Antibody-Mediated siRNA Delivery Using an Industrial Platform of THIOMAB-siRNA Conjugates. Nucleic Acids Res. 2015, 43 (2), 1189– 1203,  DOI: 10.1093/nar/gku1362
      14
      Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB-siRNA conjugates
      Cuellar, Trinna L.; Barnes, Dwight; Nelson, Christopher; Tanguay, Joshua; Yu, Shang-Fan; Wen, Xiaohui; Scales, Suzie J.; Gesch, Julie; Davis, David; van Brabant Smith, Anja; Leake, Devin; Vandlen, Richard; Siebel, Christian W.
      Nucleic Acids Research (2015), 43 (2), 1189-1203CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible soln. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, the authors built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. The authors report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technol., the authors generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. The authors identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only obsd. using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. The authors' broad characterization of multiple parameters using therapeutic-grade conjugate technol. provides a thorough assessment of this delivery technol., highlighting both examples of success as well as remaining challenges.
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    15. 15
      Sela, T.; Mansø, M.; Siegel, M.; Marban-Doran, C.; Ducret, A.; Niewöhner, J.; Ravn, J.; Martin, R. E.; Sommer, A.; Lohmann, S.; Krippendorff, B.-F.; Ladefoged, M.; Indlekofer, A.; Quaiser, T.; Bueddefeld, F.; Koller, E.; Mohamed, M. Y.; Oelschlaegel, T.; Gothelf, K. V.; Hofer, K.; Schumacher, F. F. Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties. Bioconjugate Chem. 2023, 34 (11), 2096– 2111,  DOI: 10.1021/acs.bioconjchem.3c00393
      There is no corresponding record for this reference.
    16. 16
      Sugo, T.; Terada, M.; Oikawa, T.; Miyata, K.; Nishimura, S.; Kenjo, E.; Ogasawara-Shimizu, M.; Makita, Y.; Imaichi, S.; Murata, S.; Otake, K.; Kikuchi, K.; Teratani, M.; Masuda, Y.; Kamei, T.; Takagahara, S.; Ikeda, S.; Ohtaki, T.; Matsumoto, H. Development of Antibody-siRNA Conjugate Targeted to Cardiac and Skeletal Muscles. J. Controlled Release 2016, 237, 1– 13,  DOI: 10.1016/j.jconrel.2016.06.036
      16
      Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles
      Sugo, Tsukasa; Terada, Michiko; Oikawa, Tatsuo; Miyata, Kenichi; Nishimura, Satoshi; Kenjo, Eriya; Ogasawara-Shimizu, Mari; Makita, Yukimasa; Imaichi, Sachiko; Murata, Shumpei; Otake, Kentaro; Kikuchi, Kuniko; Teratani, Mika; Masuda, Yasushi; Kamei, Takayuki; Takagahara, Shuichi; Ikeda, Shota; Ohtaki, Tetsuya; Matsumoto, Hirokazu
      Journal of Controlled Release (2016), 237 (), 1-13CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after i.v. administration in normal mice. In particular, 1 μg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected i.m. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by i.m. injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.
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    17. 17
      Malecova, B.; Burke, R. S.; Cochran, M.; Hood, M. D.; Johns, R.; Kovach, P. R.; Doppalapudi, V. R.; Erdogan, G.; Arias, J. D.; Darimont, B.; Miller, C. D.; Huang, H.; Geall, A.; Younis, H. S.; Levin, A. A. Targeted Tissue Delivery of RNA Therapeutics Using Antibody-Oligonucleotide Conjugates (AOCs). Nucleic Acids Res. 2023, 51 (12), 5901– 5910,  DOI: 10.1093/nar/gkad415
      There is no corresponding record for this reference.
    18. 18
      Cao, W.; Li, R.; Pei, X.; Chai, M.; Sun, L.; Huang, Y.; Wang, J.; Barth, S.; Yu, F.; He, H. Antibody-siRNA Conjugates (ARC): Emerging siRNA Drug Formulation. Med. Drug Discovery 2022, 15, 100128,  DOI: 10.1016/j.medidd.2022.100128
      There is no corresponding record for this reference.
    19. 19
      Chernikov, I. V.; Vlassov, V. V.; Chernolovskaya, E. L. Current Development of siRNA Bioconjugates: From Research to the Clinic. Front. Pharmacol 2019, 10, 444,  DOI: 10.3389/fphar.2019.00444
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      Current development of siRNA bioconjugates: from research to the clinic
      Chernikov, Ivan V.; Vlassov, Valentin V.; Chernolovskaya, Elena L.
      Frontiers in Pharmacology (2019), 10 (), 444CODEN: FPRHAU; ISSN:1663-9812. (Frontiers Media S.A.)
      A review. Small interfering RNAs (siRNAs) acting via RNA interference mechanisms are able to recognize a homologous mRNA sequence in the cell and induce its degrdn. The main problems in the development of siRNA-based drugs for therapeutic use are the low efficiency of siRNA delivery to target cells and the degrdn. of siRNAs by nucleases in biol. fluids. Various approaches have been proposed to solve the problem of siRNA delivery in vivo (e.g., viruses, cationic lipids, polymers, nanoparticles), but all have limitations for therapeutic use. One of the most promising approaches to solve the problem of siRNA delivery to target cells is bioconjugation; i.e., the covalent connection of siRNAs with biogenic mols. (lipophilic mols., antibodies, aptamers, ligands, peptides, or polymers). Bioconjugates are "ideal nanoparticles" since they do not need a pos. charge to form complexes, are less toxic, and are less effectively recognized by components of the immune system because of their small size. This review is focused on strategies and principles for constructing siRNA bioconjugates for in vivo use.
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    20. 20
      Tai, W. Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery. Molecules 2019, 24 (12), 2211,  DOI: 10.3390/molecules24122211
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      Current aspects of siRNA bioconjugate for in vitro and in vivo delivery
      Tai, Wanyi
      Molecules (2019), 24 (12), 2211CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      A review. Studies on siRNA delivery have seen intense growth in the past decades since siRNA has emerged as a new class of gene therapeutics for the treatment of various diseases. siRNA bioconjugate, as one of the major delivery strategies, offers the potential to enhance and broaden pharmacol. properties of siRNA, while minimizing the heterogeneity and stability-correlated toxicol. This review summarizes the recent developments of siRNA bioconjugate, including the conjugation with antibody, peptide, aptamer, small chem., lipidoid, cell-penetrating peptide polymer, and nanoparticle. These siRNA bioconjugate, either administrated alone or formulated with other agents, could significantly improve pharmacokinetic behavior, enhance the biol. half-life, and increase the targetability while maintaining sufficient gene silencing activity, with a concomitant improvement of the therapeutic outcomes and diminishment of adverse effects. This review emphasizes the delivery application of these siRNA bioconjugates, esp. the conjugation strategy that control the integrity, stability and release of siRNA bioconjugates. The limitations conferred by these conjugation strategies have also been covered.
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    21. 21
      Sun, M. M. C.; Beam, K. S.; Cerveny, C. G.; Hamblett, K. J.; Blackmore, R. S.; Torgov, M. Y.; Handley, F. G. M.; Ihle, N. C.; Senter, P. D.; Alley, S. C. Reduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides. Bioconjugate Chem. 2005, 16 (5), 1282– 1290,  DOI: 10.1021/bc050201y
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      Reduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody Disulfides
      Sun, Michael M. C.; Beam, Kevin S.; Cerveny, Charles G.; Hamblett, Kevin J.; Blackmore, Richard S.; Torgov, Michael Y.; Handley, Felicia G. M.; Ihle, Nathan C.; Senter, Peter D.; Alley, Stephen C.
      Bioconjugate Chemistry (2005), 16 (5), 1282-1290CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Site-specific conjugation of small mols. and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biol. properties. We describe for the first time chem. redn. and oxidn. methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purifn. with hydrophobic interaction chromatog. followed by anal. using reversed-phase HPLC and capillary electrophoresis. These anal. methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential redn. of heavy-light chain disulfides, while reoxidn. of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60-90%, allowing for control of the distribution of mol. species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.
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    22. 22
      Shen, B.-Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.; Yu, S.-F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.; Eigenbrot, C.; Nguyen, T.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel, D.; Spencer, S. D.; Khawli, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.; Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R. Conjugation Site Modulates the in Vivo Stability and Therapeutic Activity of Antibody-Drug Conjugates. Nat. Biotechnol. 2012, 30 (2), 184– 189,  DOI: 10.1038/nbt.2108
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      Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates
      Shen, Ben-Quan; Xu, Keyang; Liu, Luna; Raab, Helga; Bhakta, Sunil; Kenrick, Margaret; Parsons-Reponte, Kathryn L.; Tien, Janet; Yu, Shang-Fan; Mai, Elaine; Li, Dongwei; Tibbitts, Jay; Baudys, Jakub; Saad, Ola M.; Scales, Suzie J.; McDonald, Paul J.; Hass, Philip E.; Eigenbrot, Charles; Nguyen, Trung; Solis, Willy A.; Fuji, Reina N.; Flagella, Kelly M.; Patel, Darshana; Spencer, Susan D.; Khawli, Leslie A.; Ebens, Allen; Wong, Wai Lee; Vandlen, Richard; Kaur, Surinder; Sliwkowski, Mark X.; Scheller, Richard H.; Polakis, Paul; Junutula, Jagath R.
      Nature Biotechnology (2012), 30 (2), 184-189CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a pos. charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected pos. by succinimide ring hydrolysis and neg. by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chem. and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
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    23. 23
      Zhou, Q.; Stefano, J. E.; Manning, C.; Kyazike, J.; Chen, B.; Gianolio, D. A.; Park, A.; Busch, M.; Bird, J.; Zheng, X.; Simonds-Mannes, H.; Kim, J.; Gregory, R. C.; Miller, R. J.; Brondyk, W. H.; Dhal, P. K.; Pan, C. Q. Site-Specific Antibody-Drug Conjugation through Glycoengineering. Bioconjugate Chem. 2014, 25 (3), 510– 520,  DOI: 10.1021/bc400505q
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      Site-Specific Antibody-Drug Conjugation through Glycoengineering
      Zhou, Qun; Stefano, James E.; Manning, Charlene; Kyazike, Josephine; Chen, Bo; Gianolio, Diego A.; Park, Anna; Busch, Michelle; Bird, Julie; Zheng, Xiaoyang; Simonds-Mannes, Helene; Kim, Jennifer; Gregory, Rick C.; Miller, Robert J.; Brondyk, William H.; Dhal, Pradeep K.; Pan, Clark Q.
      Bioconjugate Chemistry (2014), 25 (3), 510-520CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Antibody-drug conjugates (ADCs) have been proven clin. to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prep. ADCs by targeting primary amines or hinge disulfides have a no. of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidn. of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatog. and LC-MS analyses revealed the incorporation of ∼1.6 cytotoxic agents per antibody mol., approximating the no. of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-pos. tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-pos. tumor xenograft model. These findings suggest that enzymic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chem. process to ensure reproducible drug loading.
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    24. 24
      Li, X.; Fang, T.; Boons, G. Preparation of Well-Defined Antibody-Drug Conjugates through Glycan Remodeling and Strain-Promoted Azide-Alkyne Cycloadditions. Angew. Chem., Int. Ed. 2014, 53 (28), 7179– 7182,  DOI: 10.1002/anie.201402606
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      Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions
      Li, Xiuru; Fang, Tao; Boons, Geert-Jan
      Angewandte Chemie, International Edition (2014), 53 (28), 7179-7182CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Antibody-drug conjugates hold considerable promise as anticancer agents, however, producing them remains a challenge and there is a need for mild, broadly applicable, site-specific conjugation methods that yield homogenous products. It was envisaged that enzymic remodeling of the oligosaccharides of an antibody would enable the introduction of reactive groups that can be exploited for the site-specific attachment of cytotoxic drugs. This is based on the observation that glycosyltransferases often tolerate chem. modifications in their sugar nucleotide substrates, thus allowing the installation of reactive functionalities. An azide was incorporated because this functional group is virtually absent in biol. systems and can be reacted by strain-promoted alkyne-azide cycloaddn. This method, which does not require genetic engineering, was used to produce an anti-CD22 antibody modified with doxorubicin to selectively target and kill lymphoma cells.
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    25. 25
      Brun, M.-P.; Gauzy-Lazo, L. Protocols for Lysine Conjugation. In Antibody-Drug Conjugates; Ducry, L., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2013; Vol. 1045, pp 173– 187. DOI: 10.1007/978-1-62703-541-5_10 .
      There is no corresponding record for this reference.
    26. 26
      Kumar, P.; Parmar, R. G.; Brown, C. R.; Willoughby, J. L. S.; Foster, D. J.; Babu, I. R.; Schofield, S.; Jadhav, V.; Charisse, K.; Nair, J. K.; Rajeev, K. G.; Maier, M. A.; Egli, M.; Manoharan, M. 5′-Morpholino Modification of the Sense Strand of an siRNA Makes It a More Effective Passenger. Chem. Commun. 2019, 55 (35), 5139– 5142,  DOI: 10.1039/C9CC00977A
      There is no corresponding record for this reference.
    27. 27
      Allerson, C. R.; Sioufi, N.; Jarres, R.; Prakash, T. P.; Naik, N.; Berdeja, A.; Wanders, L.; Griffey, R. H.; Swayze, E. E.; Bhat, B. Fully 2‘-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNA. J. Med. Chem. 2005, 48 (4), 901– 904,  DOI: 10.1021/jm049167j
      27
      Fully 2'-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNA
      Allerson, Charles R.; Sioufi, Namir; Jarres, Russell; Prakash, Thazha P.; Naik, Nishant; Berdeja, Andres; Wanders, Lisa; Griffey, Richard H.; Swayze, Eric E.; Bhat, Balkrishen
      Journal of Medicinal Chemistry (2005), 48 (4), 901-904CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-Me and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.
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    28. 28
      Foster, D. J.; Brown, C. R.; Shaikh, S.; Trapp, C.; Schlegel, M. K.; Qian, K.; Sehgal, A.; Rajeev, K. G.; Jadhav, V.; Manoharan, M.; Kuchimanchi, S.; Maier, M. A.; Milstein, S. Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates. Mol. Ther. 2018, 26 (3), 708– 717,  DOI: 10.1016/j.ymthe.2017.12.021
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      Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates
      Foster, Donald J.; Brown, Christopher R.; Shaikh, Sarfraz; Trapp, Casey; Schlegel, Mark K.; Qian, Kun; Sehgal, Alfica; Rajeev, Kallanthottathil G.; Jadhav, Vasant; Manoharan, Muthiah; Kuchimanchi, Satya; Maier, Martin A.; Milstein, Stuart
      Molecular Therapy (2018), 26 (3), 708-717CODEN: MTOHCK; ISSN:1525-0024. (Cell Press)
      Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chem. modified small interfering RNA (siRNA) designs, including the recently described Enhanced Stabilization Chem. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after s.c. administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chem., optimizing the positioning of 2'-deoxy-2'-fluoro and 2'-O-Me ribosugar modifications across both strands of the double-stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2'-deoxy-2'-fluoro content that yield significantly improved potency and duration in preclin. species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.
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    29. 29
      Nair, J. K.; Willoughby, J. L. S.; Chan, A.; Charisse, K.; Alam, M. R.; Wang, Q.; Hoekstra, M.; Kandasamy, P.; Kel’in, A. V.; Milstein, S.; Taneja, N.; O’Shea, J.; Shaikh, S.; Zhang, L.; Van Der Sluis, R. J.; Jung, M. E.; Akinc, A.; Hutabarat, R.; Kuchimanchi, S.; Fitzgerald, K.; Zimmermann, T.; Van Berkel, T. J. C.; Maier, M. A.; Rajeev, K. G.; Manoharan, M. Multivalent N -Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing. J. Am. Chem. Soc. 2014, 136 (49), 16958– 16961,  DOI: 10.1021/ja505986a
      29
      Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing
      Nair, Jayaprakash K.; Willoughby, Jennifer L. S.; Chan, Amy; Charisse, Klaus; Alam, Md. Rowshon; Wang, Qianfan; Hoekstra, Menno; Kandasamy, Pachamuthu; Kel'in, Alexander V.; Milstein, Stuart; Taneja, Nate; O'Shea, Jonathan; Shaikh, Sarfraz; Zhang, Ligang; van der Sluis, Ronald J.; Jung, Michael E.; Akinc, Akin; Hutabarat, Renta; Kuchimanchi, Satya; Fitzgerald, Kevin; Zimmermann, Tracy; van Berkel, Theo J. C.; Maier, Martin A.; Rajeev, Kallanthottathil G.; Manoharan, Muthiah
      Journal of the American Chemical Society (2014), 136 (49), 16958-16961CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of std. oligonucleotides. S.c. (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chem. achieved a 5-fold improvement in efficacy over the parent design in vivo with a median ED (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose vols. of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 mo with no adverse effects in rodents. The optimally chem. modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.
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    30. 30
      Haraszti, R. A.; Roux, L.; Coles, A. H.; Turanov, A. A.; Alterman, J. F.; Echeverria, D.; Godinho, B. M. D. C.; Aronin, N.; Khvorova, A. 5′-Vinylphosphonate Improves Tissue Accumulation and Efficacy of Conjugated siRNAs in Vivo. Nucleic Acids Res. 2017, 45 (13), 7581– 7592,  DOI: 10.1093/nar/gkx507
      30
      5'-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo
      Haraszti, Reka A.; Roux, Loic; Coles, Andrew H.; Turanov, Anton A.; Alterman, Julia F.; Echeverria, Dimas; Godinho, Bruno M. D. C.; Aronin, Neil; Khvorova, Anastasia
      Nucleic Acids Research (2017), 45 (13), 7581-7592CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      5'-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5'-vinylphosphonate confers novel properties to siRNAs. Specifically, 5'-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5'-to-3' exonucleases. Delivery of conjugated siRNAs requires extensive chem. modifications to achieve stability in vivo. Because chem. modified siRNAs are poor substrates for phosphorylation by kinases, and 5'-phosphate is required for loading into RNA-induced silencing complex, the synthetic addn. of a 5'-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5'-phosphate is removed within 2 h. The 5'-phosphate mimic 5'-(E)-vinylphosphonate stabilizes the 5' end of the guide strand by protecting it from phosphatases and 5'-to-3' exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5'-(E)- vinylphosphonate stabilizes 5' phosphate, thereby enabling systemic delivery to and silencing in kidney and heart.
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    31. 31
      Parmar, R.; Willoughby, J. L. S.; Liu, J.; Foster, D. J.; Brigham, B.; Theile, C. S.; Charisse, K.; Akinc, A.; Guidry, E.; Pei, Y.; Strapps, W.; Cancilla, M.; Stanton, M. G.; Rajeev, K. G.; Sepp-Lorenzino, L.; Manoharan, M.; Meyers, R.; Maier, M. A.; Jadhav, V. 5′-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates. ChemBioChem 2016, 17 (11), 985– 989,  DOI: 10.1002/cbic.201600130
      31
      5'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates
      Parmar, Rubina; Willoughby, Jennifer L. S.; Liu, Jingxuan; Foster, Donald J.; Brigham, Benjamin; Theile, Christopher S.; Charisse, Klaus; Akinc, Akin; Guidry, Erin; Pei, Yi; Strapps, Walter; Cancilla, Mark; Stanton, Matthew G.; Rajeev, Kallanthottathil G.; Sepp-Lorenzino, Laura; Manoharan, Muthiah; Meyers, Rachel; Maier, Martin A.; Jadhav, Vasant
      ChemBioChem (2016), 17 (11), 985-989CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be crit. for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chem. modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.
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    32. 32
      Brown, C. R.; Gupta, S.; Qin, J.; Racie, T.; He, G.; Lentini, S.; Malone, R.; Yu, M.; Matsuda, S.; Shulga-Morskaya, S.; Nair, A. V.; Theile, C. S.; Schmidt, K.; Shahraz, A.; Goel, V.; Parmar, R. G.; Zlatev, I.; Schlegel, M. K.; Nair, J. K.; Jayaraman, M.; Manoharan, M.; Brown, D.; Maier, M. A.; Jadhav, V. Investigating the Pharmacodynamic Durability of GalNAc-siRNA Conjugates. Nucleic Acids Res. 2020, 48 (21), 11827– 11844,  DOI: 10.1093/nar/gkaa670
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      Investigating the pharmacodynamic durability of GalNAc-siRNA conjugates
      Brown, Christopher R.; Gupta, Swati; Qin, June; Racie, Timothy; He, Guo; Lentini, Scott; Malone, Ryan; Yu, Mikyung; Matsuda, Shigeo; Shulga-Morskaya, Svetlana; Nair, Anil V.; Theile, Christopher S.; Schmidt, Karyn; Shahraz, Azar; Goel, Varun; Parmar, Rubina G.; Zlatev, Ivan; Schlegel, Mark K.; Nair, Jayaprakash K.; Jayaraman, Muthusamy; Manoharan, Muthiah; Brown, Dennis; Maier, Martin A.; Jadhav, Vasant
      Nucleic Acids Research (2020), 48 (21), 11827-11844CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclin. species and humans. Here, we investigated the underlying biol. supporting this extended duration of pharmacol. activity. We found that siRNA accumulation and stability in acidic intracellular compartments is crit. for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity obsd. in vivo.
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    33. 33
      Strop, P.; Liu, S.-H.; Dorywalska, M.; Delaria, K.; Dushin, R. G.; Tran, T.-T.; Ho, W.-H.; Farias, S.; Casas, M. G.; Abdiche, Y.; Zhou, D.; Chandrasekaran, R.; Samain, C.; Loo, C.; Rossi, A.; Rickert, M.; Krimm, S.; Wong, T.; Chin, S. M.; Yu, J.; Dilley, J.; Chaparro-Riggers, J.; Filzen, G. F.; O’Donnell, C. J.; Wang, F.; Myers, J. S.; Pons, J.; Shelton, D. L.; Rajpal, A. Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates. Chem. Biol. 2013, 20 (2), 161– 167,  DOI: 10.1016/j.chembiol.2013.01.010
      33
      Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug Conjugates
      Strop, Pavel; Liu, Shu-Hui; Dorywalska, Magdalena; Delaria, Kathy; Dushin, Russell G.; Tran, Thomas-Toan; Ho, Wei-Hsien; Farias, Santiago; Casas, Meritxell Galindo; Abdiche, Yasmina; Zhou, Dahui; Chandrasekaran, Ramalakshmi; Samain, Caroline; Loo, Carole; Rossi, Andrea; Rickert, Mathias; Krimm, Stellanie; Wong, Teresa; Chin, Sherman Michael; Yu, Jessica; Dilley, Jeanette; Chaparro-Riggers, Javier; Filzen, Gary F.; O'Donnell, Christopher J.; Wang, Fang; Myers, Jeremy S.; Pons, Jaume; Shelton, David L.; Rajpal, Arvind
      Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 161-167CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compds. at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chem. instability, as was obsd. with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.
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    34. 34
      Miller, C. M.; Donner, A. J.; Blank, E. E.; Egger, A. W.; Kellar, B. M.; Østergaard, M. E.; Seth, P. P.; Harris, E. N. Stabilin-1 and Stabilin-2 Are Specific Receptors for the Cellular Internalization of Phosphorothioate-Modified Antisense Oligonucleotides (ASOs) in the Liver. Nucleic Acids Res. 2016, 44 (6), 2782– 2794,  DOI: 10.1093/nar/gkw112
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      Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver
      Miller Colton M; Blank Emma E; Egger Andrew W; Kellar Brianna M; Harris Edward N; Donner Aaron J; Ostergaard Michael E; Seth Punit P
      Nucleic acids research (2016), 44 (6), 2782-94 ISSN:.
      Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro®, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties.
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    35. 35
      Lehot, V.; Kuhn, I.; Nothisen, M.; Erb, S.; Kolodych, S.; Cianférani, S.; Chaubet, G.; Wagner, A. Non-Specific Interactions of Antibody-Oligonucleotide Conjugates with Living Cells. Sci. Rep. 2021, 11 (1), 5881,  DOI: 10.1038/s41598-021-85352-w
      There is no corresponding record for this reference.
    36. 36
      Rocca, C.; Dennin, S.; Gu, Y.; Kim, J.; Chigas, S.; Najarian, D.; Chong, S.; Gutierrez, S.; Butler, J.; Charisse, K.; Robbie, G.; Xu, Y.; Brown, K. Evaluation of Electrophoretic Mobility Shift Assay as a Method to Determine Plasma Protein Binding of siRNA. Bioanalysis 2019, 11 (21), 1927– 1939,  DOI: 10.4155/bio-2019-0151
      36
      Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNA
      Rocca, Carrie; Dennin, Sean; Gu, Yongli; Kim, Joohwan; Chigas, Samantha; Najarian, Diana; Chong, Saeho; Gutierrez, Shannon; Butler, James; Charisse, Klaus; Robbie, Gabriel; Xu, Yuanxin; Brown, Kirk
      Bioanalysis (2019), 11 (21), 1927-1939CODEN: BIOAB4; ISSN:1757-6180. (Future Science Ltd.)
      Aim: The electrophoretic mobility shift assay (EMSA) was evaluated as an alternative to ultrafiltration (UF) to assess plasma protein binding (PPB) of small interfering RNAs (siRNA) and antisense oligonucleotides (ASO). Results & methodol.: EMSA anal. showed that PPB depended on siRNA and plasma concn. Conversely, when analyzed by ultrafiltration, siRNA bound the filtration device nonspecifically and PPB remained >98% across physiol. relevant siRNA concns. Using EMSA, siRNA exhibited charge-based interactions with plasma proteins, while ASO remained highly bound to plasma proteins or albumin in the presence of 500 mM salt. Conclusion: PPB characteristics of siRNA and ASO can be distinguished using EMSA. Characterization of siRNA PPB by EMSA enhances our knowledge of siRNA absorption, distribution, metab. and excretion and advanced development of RNA interference therapeutics.
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    37. 37
      Liang, X.; Sun, H.; Shen, W.; Crooke, S. T. Identification and Characterization of Intracellular Proteins That Bind Oligonucleotides with Phosphorothioate Linkages. Nucleic Acids Res. 2015, 43 (5), 2927– 2945,  DOI: 10.1093/nar/gkv143
      37
      Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages
      Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T.
      Nucleic Acids Research (2015), 43 (5), 2927-2945CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Although the RNase H-dependent mechanism of inhibition of gene expression by chem. modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and redn. in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFaitbvN&md5=c63e07ea7719c6e39f74433f0850c77a
    38. 38
      Gaus, H.; Miller, C. M.; Seth, P. P.; Harris, E. N. Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance Receptor. Biochemistry 2018, 57 (14), 2061– 2064,  DOI: 10.1021/acs.biochem.8b00126
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      Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance Receptor
      Gaus, Hans; Miller, Colton M.; Seth, Punit P.; Harris, Edward N.
      Biochemistry (2018), 57 (14), 2061-2064CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The Stabilin receptors are systemic clearance receptors for some classes of chem. modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarization-based assay. The tested ASOs varied in their backbone compn., modification of the ribose 2' position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5-10-5 pattern of flanking 2'-O-methoxyethyl modifications was then used to test the effects of pH and salt concn. on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissocn. and fit within the biol. parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors.
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    39. 39
      Structure−Activity Relationship of Antibody−Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody−Phosphorodiamidate Morpholino Oligomer Conjugates for Drug Development, Manuscript in Preparation.
      There is no corresponding record for this reference.
    40. 40
      Dosio, F.; Brusa, P.; Cattel, L. Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components. Toxins 2011, 3 (7), 848– 883,  DOI: 10.3390/toxins3070848
      40
      Immunotoxins and anticancer drug conjugate assemblies: the role of the linkage between components
      Dosio, Franco; Brusa, Paola; Cattel, Luigi
      Toxins (2011), 3 (), 848-883CODEN: TOXIB7; ISSN:2072-6651. (MDPI AG)
      A review. Immunotoxins and antibody-drug conjugates are protein-based drugs combining a target-specific binding domain with a cytotoxic domain. Such compds. are potentially therapeutic against diseases including cancer, and several clin. trials have shown encouraging results. Although the targeted elimination of malignant cells is an elegant concept, there are numerous practical challenges that limit conjugates' therapeutic use, including inefficient cellular uptake, low cytotoxicity, and off-target effects. During the prepn. of immunoconjugates by chem. synthesis, the choice of the hinge component joining the two building blocks is of paramount importance: the conjugate must remain stable in vivo but must afford efficient release of the toxic moiety when the target is reached. Vast efforts have been made, and the present article reviews strategies employed in developing immunoconjugates, focusing on the evolution of chem. linkers.
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    41. 41
      Su, D.; Kozak, K. R.; Sadowsky, J.; Yu, S.-F.; Fourie-O’Donohue, A.; Nelson, C.; Vandlen, R.; Ohri, R.; Liu, L.; Ng, C.; He, J.; Davis, H.; Lau, J.; Del Rosario, G.; Cosino, E.; Cruz-Chuh, J. D.; Ma, Y.; Zhang, D.; Darwish, M.; Cai, W.; Chen, C.; Zhou, H.; Lu, J.; Liu, Y.; Kaur, S.; Xu, K.; Pillow, T. H. Modulating Antibody-Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification. Bioconjugate Chem. 2018, 29 (4), 1155– 1167,  DOI: 10.1021/acs.bioconjchem.7b00785
      There is no corresponding record for this reference.
    42. 42
      Su, D.; Zhang, D. Linker Design Impacts Antibody-Drug Conjugate Pharmacokinetics and Efficacy via Modulating the Stability and Payload Release Efficiency. Front. Pharmacol 2021, 12, 687926,  DOI: 10.3389/fphar.2021.687926
      42
      Linker design impacts antibody-drug conjugate pharmacokinetics and efficacy via modulating the stability and payload release efficiency
      Su, Dian; Zhang, Donglu
      Frontiers in Pharmacology (2021), 12 (), 687926CODEN: FPRHAU; ISSN:1663-9812. (Frontiers Media S.A.)
      The development of antibody-drug conjugates (ADCs) has significantly been advanced in the past decade given the improvement of payloads, linkers and conjugation methods. In particular, linker design plays a crit. role in modulating ADC stability in the systemic circulation and payload release efficiency in the tumors, which thus affects ADC pharmacokinetic (PK), efficacy and toxicity profiles. Previously, we have investigated key linker parameters such as conjugation chem. (e.g., maleimide vs. disulfide), linker length and linker steric hindrance and their impacts on PK and efficacy profiles. Herein, we discuss our perspectives on development of integrated strategies for linker design to achieve a balance between ADC stability and payload release efficiency for desired efficacy in antigen-expressing xenograft models. The strategies have been successfully applied to the design of site-specific THIOMABTM antibody-drug conjugates (TDCs) with different payloads. We also propose to conduct dose fractionation studies to gain guidance for optimal dosing regimens of ADCs in pre-clin. models.
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    43. 43
      Kolodych, S.; Koniev, O.; Baatarkhuu, Z.; Bonnefoy, J.-Y.; Debaene, F.; Cianférani, S.; Van Dorsselaer, A.; Wagner, A. CBTF: New Amine-to-Thiol Coupling Reagent for Preparation of Antibody Conjugates with Increased Plasma Stability. Bioconjugate Chem. 2015, 26 (2), 197– 200,  DOI: 10.1021/bc500610g
      43
      CBTF: New Amine-to-Thiol Coupling Reagent for Preparation of Antibody Conjugates with Increased Plasma Stability
      Kolodych, Sergii; Koniev, Oleksandr; Baatarkhuu, Zoljargal; Bonnefoy, Jean-Yves; Debaene, Francois; Cianferani, Sarah; Van Dorsselaer, Alain; Wagner, Alain
      Bioconjugate Chemistry (2015), 26 (2), 197-200CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Amine-to-thiol coupling is the most common route for the prepn. of antibody-drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the prepn. of remarkably stable conjugates. A series of antibody-dye conjugates have been prepd. using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates.
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    44. 44
      Lyon, R. P.; Setter, J. R.; Bovee, T. D.; Doronina, S. O.; Hunter, J. H.; Anderson, M. E.; Balasubramanian, C. L.; Duniho, S. M.; Leiske, C. I.; Li, F.; Senter, P. D. Self-Hydrolyzing Maleimides Improve the Stability and Pharmacological Properties of Antibody-Drug Conjugates. Nat. Biotechnol. 2014, 32 (10), 1059– 1062,  DOI: 10.1038/nbt.2968
      44
      Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates
      Lyon, Robert P.; Setter, Jocelyn R.; Bovee, Tim D.; Doronina, Svetlana O.; Hunter, Joshua H.; Anderson, Martha E.; Balasubramanian, Cindy L.; Duniho, Steven M.; Leiske, Chris I.; Li, Fu; Senter, Peter D.
      Nature Biotechnology (2014), 32 (10), 1059-1062CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Many antibody-drug conjugates (ADCs) are unstable in vivo because they are formed from maleimide-contg. components conjugated to reactive thiols. These thiosuccinimide linkages undergo two competing reactions in plasma: elimination of the maleimide through a retro-Michael reaction, which results in loss of drug-linker from the ADC, and hydrolysis of the thiosuccinimide ring, which results in a deriv. that is resistant to the elimination reaction. In an effort to create linker technologies with improved stability characteristics, we used diaminopropionic acid (DPR) to prep. a drug-linker incorporating a basic amino group adjacent to the maleimide, positioned to provide intramol. catalysis of thiosuccinimide ring hydrolysis. This basic group induces the thiosuccinimide to undergo rapid hydrolysis at neutral pH and room temp. Once hydrolyzed, the drug-linker is no longer subject to maleimide elimination reactions, preventing nonspecific deconjugation. In vivo studies demonstrate that the increased stability characteristics can lead to improved ADC antitumor activity and reduced neutropenia.
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    45. 45
      Meade, B. R.; Gogoi, K.; Hamil, A. S.; Palm-Apergi, C.; Berg, A. V. D.; Hagopian, J. C.; Springer, A. D.; Eguchi, A.; Kacsinta, A. D.; Dowdy, C. F.; Presente, A.; Lönn, P.; Kaulich, M.; Yoshioka, N.; Gros, E.; Cui, X.-S.; Dowdy, S. F. Efficient Delivery of RNAi Prodrugs Containing Reversible Charge-Neutralizing Phosphotriester Backbone Modifications. Nat. Biotechnol. 2014, 32 (12), 1256– 1261,  DOI: 10.1038/nbt.3078
      45
      Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications
      Meade, Bryan R.; Gogoi, Khirud; Hamil, Alexander S.; Palm-Apergi, Caroline; Berg, Arjen van den; Hagopian, Jonathan C.; Springer, Aaron D.; Eguchi, Akiko; Kacsinta, Apollo D.; Dowdy, Connor F.; Presente, Asaf; Lonn, Peter; Kaulich, Manuel; Yoshioka, Naohisa; Gros, Edwige; Cui, Xian-Shu; Dowdy, Steven F.
      Nature Biotechnology (2014), 32 (12), 1256-1261CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      RNA interference (RNAi) has great potential to treat human disease. However, in vivo delivery of short interfering RNAs (siRNAs), which are neg. charged double-stranded RNA macromols., remains a major hurdle. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined mol. conjugates. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to pos. influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technol. that will open new avenues for development of RNAi therapeutics.
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    46. 46
      Burke, P. J.; Hamilton, J. Z.; Jeffrey, S. C.; Hunter, J. H.; Doronina, S. O.; Okeley, N. M.; Miyamoto, J. B.; Anderson, M. E.; Stone, I. J.; Ulrich, M. L.; Simmons, J. K.; McKinney, E. E.; Senter, P. D.; Lyon, R. P. Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug Conjugates. Mol. Cancer Ther. 2017, 16 (1), 116– 123,  DOI: 10.1158/1535-7163.MCT-16-0343
      46
      Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug Conjugates
      Burke, Patrick J.; Hamilton, Joseph Z.; Jeffrey, Scott C.; Hunter, Joshua H.; Doronina, Svetlana O.; Okeley, Nicole M.; Miyamoto, Jamie B.; Anderson, Martha E.; Stone, Ivan J.; Ulrich, Michelle L.; Simmons, Jessica K.; McKinney, Erica E.; Senter, Peter D.; Lyon, Robert P.
      Molecular Cancer Therapeutics (2017), 16 (1), 116-123CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      The emergence of antibody-drug conjugates (ADC), such as brentuximab vedotin and ado-trastuzumab emtansine, has led to increased efforts to identify new payloads and develop improved drug-linker technologies. Most antibody payloads impart significant hydrophobicity to the ADC, resulting in accelerated plasma clearance and suboptimal in vivo activity, particularly for conjugates with high drug-to-antibody ratios (DAR). We recently reported on the incorporation of a discrete PEG24 polymer as a side chain in a β-glucuronidase-cleavable monomethylauristatin E (MMAE) linker to provide homogeneous DAR 8 conjugates with decreased plasma clearance and increased antitumor activity in xenograft models relative to a non-PEGylated control. In this work, we optimized the drug-linker by minimizing the size of the PEG side chain and incorporating a self-stabilizing maleimide to prevent payload de-conjugation in vivo. Multiple PEG-glucuronide-MMAE linkers were prepd. with PEG size up to 24 ethylene oxide units, and homogeneous DAR 8 ADCs were evaluated. A clear relationship was obsd. between PEG length and conjugate pharmacol. when tested in vivo. Longer PEG chains resulted in slower clearance, with a threshold length of PEG8 beyond which clearance was not impacted. Conjugates bearing PEG of sufficient length to minimize plasma clearance provided a wider therapeutic window relative to faster clearing conjugates bearing shorter PEGs. A lead PEGylated glucuronide-MMAE linker was identified incorporating a self-stabilizing maleimide and a PEG12 side chain emerged from these efforts, enabling highly potent, homogeneous DAR 8 conjugates and is under consideration for future ADC programs.
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    47. 47
      Wiggins, B.; Liu-Shin, L.; Yamaguchi, H.; Ratnaswamy, G. Characterization of Cysteine-Linked Conjugation Profiles of Immunoglobulin G1 and Immunoglobulin G2 Antibody-Drug Conjugates. J. Pharm. Sci. 2015, 104 (4), 1362– 1372,  DOI: 10.1002/jps.24338
      There is no corresponding record for this reference.
    48. 48
      Prakash, T. P.; Lima, W. F.; Murray, H. M.; Li, W.; Kinberger, G. A.; Chappell, A. E.; Gaus, H.; Seth, P. P.; Bhat, B.; Crooke, S. T.; Swayze, E. E. Identification of Metabolically Stable 5′-Phosphate Analogs That Support Single-Stranded siRNA Activity. Nucleic Acids Res. 2015, 43 (6), 2993– 3011,  DOI: 10.1093/nar/gkv162
      48
      Identification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activity
      Prakash, Thazha P.; Lima, Walt F.; Murray, Heather M.; Li, Wenyu; Kinberger, Garth A.; Chappell, Alfred E.; Gaus, Hans; Seth, Punit P.; Bhat, Balkrishen; Crooke, Stanley T.; Swayze, Eric E.
      Nucleic Acids Research (2015), 43 (6), 2993-3011CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog. In this report we used crystal structure of the 5'-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs contg. various 5'-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was crit. for ss-siRNA activity. Chem. modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacol. effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.
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    49. 49
      Green, M. R.; Sambrook, J. Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Cold Spring Harb. Protoc. 2018, 2018 (10), pdb.prot095042,  DOI: 10.1101/pdb.prot095042
      There is no corresponding record for this reference.
    50. 50
      Livak, K. J.; Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method. Methods 2001, 25 (4), 402– 408,  DOI: 10.1006/meth.2001.1262
      50
      Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method
      Livak, Kenneth J.; Schmittgen, Thomas D.
      Methods (San Diego, CA, United States) (2001), 25 (4), 402-408CODEN: MTHDE9; ISSN:1046-2023. (Academic Press)
      The two most commonly used methods to analyze data from real-time, quant. PCR expts. are abs. quantification and relative quantification. Abs. quantification dets. the input copy no., usually by relating the PCR signal to a std. curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2-ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quant. PCR expts. The purpose of this report is to present the derivation, assumptions, and applications of the 2-ΔΔCT method. In addn., we present the derivation and applications of two variations of the 2-ΔΔCT method that may be useful in the anal. of real-time, quant. PCR data. (c) 2001 Academic Press.
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    51. 51
      Chen, C. Real-Time Quantification of microRNAs by Stem-Loop RT-PCR. Nucleic Acids Res. 2005, 33 (20), e179  DOI: 10.1093/nar/gni178
      51
      Real-time quantification of microRNAs by stem-loop RT-PCR
      Chen, Caifu; Ridzon, Dana A.; Broomer, Adam J.; Zhou, Zhaohui; Lee, Danny H.; Nguyen, Julie T.; Barbisin, Maura; Xu, Nan Lan; Mahuvakar, Vikram R.; Andersen, Mark R.; Lao, Kai Qin; Livak, Kenneth J.; Guegler, Karl J.
      Nucleic Acids Research (2005), 33 (20), e179/1-e179/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR anal. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct anal. of a single cell without nucleic acid purifn. Like std. TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA mols. such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00802.

    • Please see siRNA AOC Supplement. siRNA sequence and chemical modifications for evaluated test articles; siRNA conjugation comprehensive guide; noncompartmental analysis of plasma PK data in Figure 5b; plasma PK, tissue concentration, and mRNA expression of αmTfR1-Cys-MCC-siMstn conjugates; DAR1 versus DAR2 SEC comparison; in vitro stability of αhEGFR-siKRAS AOCs; KD and tissue concentration of αmTfR1-siHprt AOCs; plasma PK of αhEGFR-siKRAS AOCs; tumor delivery and KD of αhPSMA-siEGFR AOCs; PK of αmTfR1-siCtnnb1 AOCs; purification of antibody–siRNA conjugation reaction mixture by SAX; purification and characterization of DAR1 AOC; UNA vinylphosphonate (vpUq) amidite synthesis (PDF)


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