Structure–Activity Relationship of Antibody–Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody–siRNA Conjugates for Drug DevelopmentClick to copy article linkArticle link copied!
- Michael CochranMichael CochranAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Michael Cochran
- Danny AriasDanny AriasAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Danny Arias
- Rob BurkeRob BurkeAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Rob Burke
- David ChuDavid ChuAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by David Chu
- Gulin ErdoganGulin ErdoganAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Gulin Erdogan
- Michael HoodMichael HoodAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Michael Hood
- Philip KovachPhilip KovachAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Philip Kovach
- Hae Won KwonHae Won KwonAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Hae Won Kwon
- Yanling ChenYanling ChenAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Yanling Chen
- Michael MoonMichael MoonAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Michael Moon
- Christopher D. MillerChristopher D. MillerAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Christopher D. Miller
- Hanhua HuangHanhua HuangAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Hanhua Huang
- Arthur LevinArthur LevinAvidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Arthur Levin
- Venkata Ramana Doppalapudi*Venkata Ramana Doppalapudi*Email: [email protected]Avidity Biosciences, Inc., 10578 Science Center Drive Suite 125. San Diego, California 92121, United StatesMore by Venkata Ramana Doppalapudi
Abstract
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0011.gif)
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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You are free to share(copy and redistribute) this article in any medium or format within the parameters below:
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Attribution (BY): Credit must be given to the creator.
Non-Commercial (NC): Only non-commercial uses of the work are permitted.
No Derivatives (ND): Derivative works may be created for non-commercial purposes, but sharing is prohibited.
*Disclaimer
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
Non-Commercial (NC): Only non-commercial uses of the work are permitted.
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Attribution (BY): Credit must be given to the creator.
Non-Commercial (NC): Only non-commercial uses of the work are permitted.
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Introduction
AOC Naming Convention
Results and Discussion
AOC Synthesis
General Synthetic Scheme for Antibody–siRNA Conjugate Generation
Scheme 1
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0010.gif)
Figure 1
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0001.gif)
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.
Optimization of Disulfide Bond Reduction and Capping in Cys Conjugation Method
Figure 2
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0002.gif)
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).
siRNA Chemical Modifications
Figure 3
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0003.gif)
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.
siRNA Conjugation to mAb Via Cysteine, Lysine, and Sugar Locations
Figure 4
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0004.gif)
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.
Drug to Antibody Ratio
Figure 5
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0005.gif)
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).
Cysteine Conjugates with Cleavable Versus Non-cleavable Linkers
Figure 6
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0006.gif)
Figure 6. (a,b) Mouse PK of AOCs containing cleavable linkers.
Further Exploration of Non-cleavable Linkers (MCC, CB, MB, and BisMal)
αhEGFR-siKRAS AOC In Vitro Stability
Linker Impact on mTfR1 Antibody-Based siRNA Delivery to Muscles and Other Tissues
Figure 7
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0007.gif)
Figure 7. (a–c) PK/PD studies with αmTfR1–siHprt AOCs containing four different noncleavable linkers.
Linker Location on the siRNA
Figure 8
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0008.gif)
Figure 8. (a,b) Liver KD analysis of αmASGPR–siHprt AOCs containing BisMal linkers to siRNA at the 5′ end and positions (pos) 8 or 14 of the sense strand at 96 h postdose.
Figure 9
![](/cms/10.1021/acs.jmedchem.4c00802/asset/images/medium/jm4c00802_0009.gif)
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.
Impact of PEG on AOC Activity
Applicability of siRNA Bioconjugation across Multiple Antibody Isotypes
mAb isotype | TCEP eq | (linker) siRNA eq | DAR0 (%) | DAR1 (%) | DAR ≥ 2 (%) | isolated DAR 1 yield (%) |
---|---|---|---|---|---|---|
hIgG1 | 2 | (MCC) 1 | 25 | 57 | 18 | 46 |
hIgG2 | 6 | (MCC) 1 | 27 | 40 | 33 | 34 |
hIgG4 | 1.75 | (MCC) 1.25 | 47 | 22 | 31 | 16 |
hIgG4 | 1.75 | (BisMal) 1.25 | 46 | 31 | 23 | 25 |
Rat IgG2a | 4 | (MCC) 1.15 | 38 | 41 | 21 | 36 |
Rabbit IgG | 4 | (MCC) 1.1 | 26 | 41 | 33 | 35 |
Conclusions
Experimental Section
siRNA Synthesis
Characterization of Oligonucleotides
siRNA-PEG Synthesis
Linker-siRNA Generation
Cysteine, Lysine, and Glycan Conjugations
Preparative Anion Exchange Purification
Overview of Cysteine Conjugation Using NEM Capping with or without DHAA Oxidation
Overview of Free Cysteine Determination
Purity Statement
Animal Studies
Pharmacodynamic Evaluation
AOC Characterization
Analytical Anion Exchange Chromatography
UV Spectroscopy
Size Exclusion
Reduced Capillary Electrophoresis
MS
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)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
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.
% 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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- 9Hu, B.; Zhong, L.; Weng, Y.; Peng, L.; Huang, Y.; Zhao, Y.; Liang, X.-J. Therapeutic siRNA: State of the Art. Signal Transduct. Target. Ther. 2020, 5 (1), 101, DOI: 10.1038/s41392-020-0207-xGoogle Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38nit1ajtw%253D%253D&md5=9688b0f424af54cc42999c96e75ee90dTherapeutic siRNA: state of the artHu Bo; Weng Yuhua; Huang Yuanyu; Zhong Liping; Zhao Yongxiang; Peng Ling; Liang Xing-JieSignal 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.
- 10Egli, M.; Manoharan, M. Chemistry, Structure and Function of Approved Oligonucleotide Therapeutics. Nucleic Acids Res. 2023, 51 (6), 2529– 2573, DOI: 10.1093/nar/gkad067Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhsV2lsL%252FJ&md5=e897407f82b379bf7796781afd43bc5eChemistry, structure and function of approved oligonucleotide therapeuticsEgli, Martin; Manoharan, MuthiahNucleic 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.
- 11Janas, 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/s41467-018-02989-4Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mrjt1eqtA%253D%253D&md5=0bb4a8142d0353305e7b13b76fd886d9Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicityJanas 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 VasantNature 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.
- 12Brown, 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-xGoogle Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVGgsbzM&md5=ef7076014388a2adaf3bd4adb0c39db6Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugatesBrown, 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, VasantNature 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.
- 13Roberts, 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/s41573-020-0075-7Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFOlsbzM&md5=369f924e752c5e88372ddab12cae819aAdvances in oligonucleotide drug deliveryRoberts, 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.
- 14Cuellar, 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/gku1362Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtlCju78%253D&md5=64d667608939d34735cf88b833dad663Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB-siRNA conjugatesCuellar, 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.
- 15Sela, 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.3c00393Google ScholarThere is no corresponding record for this reference.
- 16Sugo, 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.2016.06.036Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFahtLnM&md5=c7d18e154441247f59ddf48181e90879Development of antibody-siRNA conjugate targeted to cardiac and skeletal musclesSugo, 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, HirokazuJournal 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.
- 17Malecova, 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/gkad415Google ScholarThere is no corresponding record for this reference.
- 18Cao, 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.2022.100128Google ScholarThere is no corresponding record for this reference.
- 19Chernikov, 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.2019.00444Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVGks7fK&md5=db0d5eb9dd7598ab67fbd63942c9e422Current development of siRNA bioconjugates: from research to the clinicChernikov, 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.
- 20Tai, W. Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery. Molecules 2019, 24 (12), 2211, DOI: 10.3390/molecules24122211Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVWqsr3J&md5=1c6ab17f5ce67dcd65554a82cd3ef49fCurrent aspects of siRNA bioconjugate for in vitro and in vivo deliveryTai, WanyiMolecules (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.
- 21Sun, 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/bc050201yGoogle Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpsleqsbw%253D&md5=27abd49b337ef306d04282533f31cecdReduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody DisulfidesSun, 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.
- 22Shen, 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.2108Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVGqtLg%253D&md5=c647fc5bdfe93dfdabb15ca40934837fConjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugatesShen, 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.
- 23Zhou, 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/bc400505qGoogle Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXisFKitbw%253D&md5=ea91284e451dab447573ca146bdf2a6cSite-Specific Antibody-Drug Conjugation through GlycoengineeringZhou, 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.
- 24Li, 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.201402606Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXos1aks7w%253D&md5=d59a17169fb0891a63e88960285c4f55Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditionsLi, Xiuru; Fang, Tao; Boons, Geert-JanAngewandte 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.
- 25Brun, 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 .Google ScholarThere is no corresponding record for this reference.
- 26Kumar, 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/C9CC00977AGoogle ScholarThere is no corresponding record for this reference.
- 27Allerson, 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/jm049167jGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXkvFSqsA%253D%253D&md5=911025d60f203ffcbae4272177c3e5fbFully 2'-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNAAllerson, Charles R.; Sioufi, Namir; Jarres, Russell; Prakash, Thazha P.; Naik, Nishant; Berdeja, Andres; Wanders, Lisa; Griffey, Richard H.; Swayze, Eric E.; Bhat, BalkrishenJournal 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.
- 28Foster, 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.2017.12.021Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXktlShtr0%253D&md5=7c2ceb9a19cb1e6b065d8fe323395e34Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA ConjugatesFoster, 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, StuartMolecular 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.
- 29Nair, 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/ja505986aGoogle Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVSju7vJ&md5=d47cb552873ffdaa46ed17d8d1fb4116Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene SilencingNair, 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, MuthiahJournal 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.
- 30Haraszti, 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/gkx507Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1aitrw%253D&md5=8eeadc06cccdfd34e67ff0bba78cd19a5'-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivoHaraszti, Reka A.; Roux, Loic; Coles, Andrew H.; Turanov, Anton A.; Alterman, Julia F.; Echeverria, Dimas; Godinho, Bruno M. D. C.; Aronin, Neil; Khvorova, AnastasiaNucleic 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.
- 31Parmar, 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.201600130Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmvVGhtr0%253D&md5=8ac8d86ab8ccc85fa82309b5fa5699725'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc ConjugatesParmar, 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, VasantChemBioChem (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.
- 32Brown, 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/gkaa670Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXovFWlsLw%253D&md5=a5a507cbd7957015406b0ae21997ff10Investigating the pharmacodynamic durability of GalNAc-siRNA conjugatesBrown, 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, VasantNucleic 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.
- 33Strop, 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.2013.01.010Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtFWht74%253D&md5=0b419848b5fd4dfa4863a29de0555cf4Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug ConjugatesStrop, 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, ArvindChemistry & 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.
- 34Miller, 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/gkw112Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28jjvVOlsg%253D%253D&md5=a2fbbf2b53b6c766ddbff71c50f6a225Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liverMiller Colton M; Blank Emma E; Egger Andrew W; Kellar Brianna M; Harris Edward N; Donner Aaron J; Ostergaard Michael E; Seth Punit PNucleic 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.
- 35Lehot, 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-wGoogle ScholarThere is no corresponding record for this reference.
- 36Rocca, 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-2019-0151Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlOhtrrF&md5=5bb187baea97e216a982f45966b8da54Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNARocca, Carrie; Dennin, Sean; Gu, Yongli; Kim, Joohwan; Chigas, Samantha; Najarian, Diana; Chong, Saeho; Gutierrez, Shannon; Butler, James; Charisse, Klaus; Robbie, Gabriel; Xu, Yuanxin; Brown, KirkBioanalysis (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.
- 37Liang, 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/gkv143Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFaitbvN&md5=c63e07ea7719c6e39f74433f0850c77aIdentification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkagesLiang, 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.
- 38Gaus, 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.8b00126Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmtFCntrY%253D&md5=aee84e23d41d0dce12c811a82e0e3c53Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance ReceptorGaus, 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.
- 39Structure−Activity Relationship of Antibody−Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody−Phosphorodiamidate Morpholino Oligomer Conjugates for Drug Development, Manuscript in Preparation.Google ScholarThere is no corresponding record for this reference.
- 40Dosio, 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/toxins3070848Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVShtLnO&md5=c6b576cc671529e9aa360173a9338487Immunotoxins and anticancer drug conjugate assemblies: the role of the linkage between componentsDosio, Franco; Brusa, Paola; Cattel, LuigiToxins (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.
- 41Su, 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.7b00785Google ScholarThere is no corresponding record for this reference.
- 42Su, 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.2021.687926Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVOisr7F&md5=e780af6c3ef469a24cce09481290cc77Linker design impacts antibody-drug conjugate pharmacokinetics and efficacy via modulating the stability and payload release efficiencySu, Dian; Zhang, DongluFrontiers 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.
- 43Kolodych, 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/bc500610gGoogle Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVWgu7w%253D&md5=1b3c24d458a79fe4c9d5b30a606c9248CBTF: New Amine-to-Thiol Coupling Reagent for Preparation of Antibody Conjugates with Increased Plasma StabilityKolodych, Sergii; Koniev, Oleksandr; Baatarkhuu, Zoljargal; Bonnefoy, Jean-Yves; Debaene, Francois; Cianferani, Sarah; Van Dorsselaer, Alain; Wagner, AlainBioconjugate 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.
- 44Lyon, 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.2968Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFKms7jN&md5=96c3b19b856cc9c685cbdc704a842797Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugatesLyon, 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.
- 45Meade, 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.3078Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFKlsrnF&md5=9ec4778a5e210e35dca76dea456ec5d5Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modificationsMeade, 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.
- 46Burke, 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-16-0343Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsF2hug%253D%253D&md5=94f2bf9dba796252569277f466e9be33Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug ConjugatesBurke, 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.
- 47Wiggins, 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.24338Google ScholarThere is no corresponding record for this reference.
- 48Prakash, 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/gkv162Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFChtLrO&md5=e0cb4f7ecbd323fea8f0d4d761fa102aIdentification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activityPrakash, 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.
- 49Green, 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.prot095042Google ScholarThere is no corresponding record for this reference.
- 50Livak, 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.2001.1262Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtFelt7s%253D&md5=f849383250ea43f7380ed540db8a64b1Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT methodLivak, 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.
- 51Chen, C. Real-Time Quantification of microRNAs by Stem-Loop RT-PCR. Nucleic Acids Res. 2005, 33 (20), e179 DOI: 10.1093/nar/gni178Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlSju77P&md5=9783e014d1b81ac6421a9c1cb8f3e95aReal-time quantification of microRNAs by stem-loop RT-PCRChen, 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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Abstract
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 ConjugationFigure 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.
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).
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.
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.
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).
Figure 6
Figure 6. (a,b) Mouse PK of AOCs containing cleavable linkers.
Figure 7
Figure 7. (a–c) PK/PD studies with αmTfR1–siHprt AOCs containing four different noncleavable linkers.
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.
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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- 1Arslan, F. B.; Ozturk, K.; Calis, S. Antibody-Mediated Drug Delivery. Int. J. Pharm. 2021, 596, 120268, DOI: 10.1016/j.ijpharm.2021.1202681https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXis1OrtLY%253D&md5=77e8f0bc939f1bca28de5314eec33dfaAntibody-mediated drug deliveryArslan, Fatma Betul; Ozturk Atar, Kivilcim; Calis, SemaInternational Journal of Pharmaceutics (Amsterdam, Netherlands) (2021), 596 (), 120268CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)A review. Passive and active targeted nanoparticulate delivery systems show promise to compensate for lacking properties of conventional therapy such as side effects, insufficient efficiency and accumulation of the drug at target site, poor pharmacokinetic properties etc. For active targeting, phys. or covalently conjugated ligands, including monoclonal antibodies and their fragments, are consistently used and researched for targeting delivery systems or drugs to their target site. Currently, there are several FDA approved actively targeted antibody-drug conjugates, whereas no active targeted delivery system is in clin. use at present. However, efforts to successfully formulate actively targeted delivery systems continue. The scope of this review will be the use of monoclonal antibodies and their fragments as targeting ligands. General information about targeted delivery and antibodies will be given at the first half of the review. As for the second half, fragmentation of antibodies and conjugation approaches will be explained. Monoclonal antibodies and their fragments as targeting ligands and approaches for conjugating these ligands to nanoparticulate delivery systems and drugs will be the main focus of this review, polyclonal antibodies will not be included.
- 2Awwad, S.; Angkawinitwong, U. Overview of Antibody Drug Delivery. Pharmaceutics 2018, 10 (3), 83, DOI: 10.3390/pharmaceutics100300832https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVOjtL7P&md5=af00e0ff0da5be5810242c38b1923874Overview of antibody drug deliveryAwwad, Sahar; Angkawinitwong, UkritPharmaceutics (2018), 10 (3), 83CODEN: PHARK5; ISSN:1999-4923. (MDPI AG)Monoclonal antibodies (mAbs) are one of the most important classes of therapeutic proteins, which are used to treat a wide no. of diseases (e.g., oncol., inflammation and autoimmune diseases). Monoclonal antibody technologies are continuing to evolve to develop medicines with increasingly improved safety profiles, with the identification of new drug targets being one key barrier for new antibody development. There are many opportunities for developing antibody formulations for better patient compliance, cost savings and lifecycle management, e.g., s.c. formulations. However, mAb-based medicines also have limitations that impact their clin. use; the most prominent challenges are their short pharmacokinetic properties and stability issues during manufg., transport and storage that can lead to aggregation and protein denaturation. The development of long acting protein formulations must maintain protein stability and be able to deliver a large enough dose over a prolonged period. Many strategies are being pursued to improve the formulation and dosage forms of antibodies to improve efficacy and to increase the range of applications for the clin. use of mAbs.
- 3Dovgan, I.; Koniev, O.; Kolodych, S.; Wagner, A. Antibody-Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents. Bioconjugate Chem. 2019, 30 (10), 2483– 2501, DOI: 10.1021/acs.bioconjchem.9b003063https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsVaisb3O&md5=7d2eb85c30db0d24aae7aea1dd357543Antibody-Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection AgentsDovgan, Igor; Koniev, Oleksandr; Kolodych, Sergii; Wagner, AlainBioconjugate Chemistry (2019), 30 (10), 2483-2501CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)A review. Antibody-oligonucleotide conjugates (AOCs) are a novel class of synthetic chimeric biomols. that has been continually gaining traction in different fields of modern biotechnol. This is mainly due to the unique combination of the properties of their two constituents: exceptional targeting abilities and antibody biodistribution profiles, in addn. to an extensive scope of oligonucleotide functional and structural roles. Combining these two classes of biomols. in one chimeric construct has therefore become an important milestone in the development of numerous biotechnol. applications, including imaging (DNA-PAINT), detection (PLA, PEA) and therapeutics (targeted siRNA/antisense delivery). Numerous synthetic approaches have been developed to access AOCs ranging from stochastic chem. bioconjugation to site-specific conjugation with reactive handles, introduced into antibody sequences through protein engineering. This review gives a general overview of the current status of AOC applications with a specific emphasis on the synthetic methods used for their prepn. The reported synthetic techniques are discussed in terms of their practical aspects and limitations. The importance of the development of novel methods for the facile generation of AOCs possessing a defined constitution is highlighted as a priority in AOC research to ensure the advance of their new applications.
- 4Ovacik, M.; Lin, K. Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early Development. Clin. Transl. Sci. 2018, 11 (6), 540– 552, DOI: 10.1111/cts.125674https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MbkvVCgtg%253D%253D&md5=f729adbab0590f304c69771e53768425Tutorial on Monoclonal Antibody Pharmacokinetics and Its Considerations in Early DevelopmentOvacik Meric; Lin KedanClinical and translational science (2018), 11 (6), 540-552 ISSN:.The tutorial introduces the readers to the fundamentals of antibody pharmacokinetics (PK) in the context of drug development. Topics covered include an overview of antibody development, PK characteristics, and the application of antibody PK/pharmacodynamics (PD) in research and development decision-making. We also discuss the general considerations for planning a nonclinical PK program and describe the types of PK studies that should be performed during early development of monoclonal antibodies.
- 5Chery, J. RNA Therapeutics: RNAi and Antisense Mechanisms and Clinical Applications. Postdoc J. 2016, 4 (7), 35– 50, DOI: 10.14304/SURYA.JPR.V4N7.5There is no corresponding record for this reference.
- 6Levin, A. A. Treating Disease at the RNA Level with Oligonucleotides. N. Engl. J. Med. 2019, 380 (1), 57– 70, DOI: 10.1056/NEJMra17053466https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cnnt1yhtg%253D%253D&md5=68c0689485dda6b76d6e6e688fa670dfTreating Disease at the RNA Level with OligonucleotidesLevin Arthur AThe New England journal of medicine (2019), 380 (1), 57-70 ISSN:.There is no expanded citation for this reference.
- 7Burnett, J. C.; Rossi, J. J. RNA-Based Therapeutics: Current Progress and Future Prospects. Chem. Biol. 2012, 19 (1), 60– 71, DOI: 10.1016/j.chembiol.2011.12.0087https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Cqtbc%253D&md5=6245eb61746cf2d214bd2f042adf996eRNA-Based Therapeutics: Current Progress and Future ProspectsBurnett, John C.; Rossi, John J.Chemistry & Biology (Oxford, United Kingdom) (2012), 19 (1), 60-71CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)A review. Recent advances of biol. drugs have broadened the scope of therapeutic targets for a variety of human diseases. This holds true for dozens of RNA-based therapeutics currently under clin. investigation for diseases ranging from genetic disorders to HIV infection to various cancers. These emerging drugs, which include therapeutic ribozymes, aptamers, and small interfering RNAs (siRNAs), demonstrate the unprecedented versatility of RNA. However, RNA is inherently unstable, potentially immunogenic, and typically requires a delivery vehicle for efficient transport to the targeted cells. These issues have hindered the clin. progress of some RNA-based drugs and have contributed to mixed results in clin. testing. Nevertheless, promising results from recent clin. trials suggest that these barriers may be overcome with improved synthetic delivery carriers and chem. modifications of the RNA therapeutics. This review focuses on the clin. results of siRNA, RNA aptamer, and ribozyme therapeutics and the prospects for future successes.
- 8Crooke, S. T.; Witztum, J. L.; Bennett, C. F.; Baker, B. F. RNA-Targeted Therapeutics. Cell Metab. 2018, 27 (4), 714– 739, DOI: 10.1016/j.cmet.2018.03.0048https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXntVKqsLc%253D&md5=81de41b2b10fcab31787b830932d5b64RNA-Targeted TherapeuticsCrooke, Stanley T.; Witztum, Joseph L.; Bennett, C. Frank; Baker, Brenda F.Cell Metabolism (2018), 27 (4), 714-739CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)RNA-targeted therapies represent a platform for drug discovery involving chem. modified oligonucleotides, a wide range of cellular RNAs, and a novel target-binding motif, Watson-Crick base pairing. Numerous hurdles considered by many to be impassable have been overcome. Today, four RNA-targeted therapies are approved for com. use for indications as diverse as Spinal Muscular Atrophy (SMA) and redn. of low-d. lipoprotein cholesterol (LDL-C) and by routes of administration including s.c., intravitreal, and intrathecal delivery. The technol. is efficient and supports approaching "undruggable" targets. Three addnl. agents are progressing through registration, and more are in clin. development, representing several chem. and structural classes. Moreover, progress in understanding the mol. mechanisms by which these drugs work has led to steadily better clin. performance and a wide range of mechanisms that may be exploited for therapeutic purposes. Here we summarize the progress, future challenges, and opportunities for this drug discovery platform.
- 9Hu, B.; Zhong, L.; Weng, Y.; Peng, L.; Huang, Y.; Zhao, Y.; Liang, X.-J. Therapeutic siRNA: State of the Art. Signal Transduct. Target. Ther. 2020, 5 (1), 101, DOI: 10.1038/s41392-020-0207-x9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38nit1ajtw%253D%253D&md5=9688b0f424af54cc42999c96e75ee90dTherapeutic siRNA: state of the artHu Bo; Weng Yuhua; Huang Yuanyu; Zhong Liping; Zhao Yongxiang; Peng Ling; Liang Xing-JieSignal 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.
- 10Egli, M.; Manoharan, M. Chemistry, Structure and Function of Approved Oligonucleotide Therapeutics. Nucleic Acids Res. 2023, 51 (6), 2529– 2573, DOI: 10.1093/nar/gkad06710https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3sXhsV2lsL%252FJ&md5=e897407f82b379bf7796781afd43bc5eChemistry, structure and function of approved oligonucleotide therapeuticsEgli, Martin; Manoharan, MuthiahNucleic 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.
- 11Janas, 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/s41467-018-02989-411https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mrjt1eqtA%253D%253D&md5=0bb4a8142d0353305e7b13b76fd886d9Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicityJanas 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 VasantNature 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.
- 12Brown, 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-x12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVGgsbzM&md5=ef7076014388a2adaf3bd4adb0c39db6Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugatesBrown, 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, VasantNature 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.
- 13Roberts, 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/s41573-020-0075-713https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFOlsbzM&md5=369f924e752c5e88372ddab12cae819aAdvances in oligonucleotide drug deliveryRoberts, 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.
- 14Cuellar, 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/gku136214https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtlCju78%253D&md5=64d667608939d34735cf88b833dad663Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB-siRNA conjugatesCuellar, 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.
- 15Sela, 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.3c00393There is no corresponding record for this reference.
- 16Sugo, 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.2016.06.03616https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFahtLnM&md5=c7d18e154441247f59ddf48181e90879Development of antibody-siRNA conjugate targeted to cardiac and skeletal musclesSugo, 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, HirokazuJournal 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.
- 17Malecova, 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/gkad415There is no corresponding record for this reference.
- 18Cao, 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.2022.100128There is no corresponding record for this reference.
- 19Chernikov, 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.2019.0044419https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVGks7fK&md5=db0d5eb9dd7598ab67fbd63942c9e422Current development of siRNA bioconjugates: from research to the clinicChernikov, 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.
- 20Tai, W. Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery. Molecules 2019, 24 (12), 2211, DOI: 10.3390/molecules2412221120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVWqsr3J&md5=1c6ab17f5ce67dcd65554a82cd3ef49fCurrent aspects of siRNA bioconjugate for in vitro and in vivo deliveryTai, WanyiMolecules (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.
- 21Sun, 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/bc050201y21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpsleqsbw%253D&md5=27abd49b337ef306d04282533f31cecdReduction-Alkylation Strategies for the Modification of Specific Monoclonal Antibody DisulfidesSun, 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.
- 22Shen, 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.210822https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVGqtLg%253D&md5=c647fc5bdfe93dfdabb15ca40934837fConjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugatesShen, 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.
- 23Zhou, 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/bc400505q23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXisFKitbw%253D&md5=ea91284e451dab447573ca146bdf2a6cSite-Specific Antibody-Drug Conjugation through GlycoengineeringZhou, 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.
- 24Li, 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.20140260624https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXos1aks7w%253D&md5=d59a17169fb0891a63e88960285c4f55Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditionsLi, Xiuru; Fang, Tao; Boons, Geert-JanAngewandte 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.
- 25Brun, 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.
- 26Kumar, 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/C9CC00977AThere is no corresponding record for this reference.
- 27Allerson, 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/jm049167j27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXkvFSqsA%253D%253D&md5=911025d60f203ffcbae4272177c3e5fbFully 2'-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNAAllerson, Charles R.; Sioufi, Namir; Jarres, Russell; Prakash, Thazha P.; Naik, Nishant; Berdeja, Andres; Wanders, Lisa; Griffey, Richard H.; Swayze, Eric E.; Bhat, BalkrishenJournal 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.
- 28Foster, 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.2017.12.02128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXktlShtr0%253D&md5=7c2ceb9a19cb1e6b065d8fe323395e34Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA ConjugatesFoster, 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, StuartMolecular 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.
- 29Nair, 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/ja505986a29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVSju7vJ&md5=d47cb552873ffdaa46ed17d8d1fb4116Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene SilencingNair, 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, MuthiahJournal 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.
- 30Haraszti, 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/gkx50730https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1aitrw%253D&md5=8eeadc06cccdfd34e67ff0bba78cd19a5'-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivoHaraszti, Reka A.; Roux, Loic; Coles, Andrew H.; Turanov, Anton A.; Alterman, Julia F.; Echeverria, Dimas; Godinho, Bruno M. D. C.; Aronin, Neil; Khvorova, AnastasiaNucleic 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.
- 31Parmar, 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.20160013031https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmvVGhtr0%253D&md5=8ac8d86ab8ccc85fa82309b5fa5699725'-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc ConjugatesParmar, 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, VasantChemBioChem (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.
- 32Brown, 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/gkaa67032https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXovFWlsLw%253D&md5=a5a507cbd7957015406b0ae21997ff10Investigating the pharmacodynamic durability of GalNAc-siRNA conjugatesBrown, 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, VasantNucleic 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.
- 33Strop, 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.2013.01.01033https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtFWht74%253D&md5=0b419848b5fd4dfa4863a29de0555cf4Location Matters: Site of Conjugation Modulates Stability and Pharmacokinetics of Antibody Drug ConjugatesStrop, 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, ArvindChemistry & 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.
- 34Miller, 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/gkw11234https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28jjvVOlsg%253D%253D&md5=a2fbbf2b53b6c766ddbff71c50f6a225Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liverMiller Colton M; Blank Emma E; Egger Andrew W; Kellar Brianna M; Harris Edward N; Donner Aaron J; Ostergaard Michael E; Seth Punit PNucleic 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.
- 35Lehot, 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-wThere is no corresponding record for this reference.
- 36Rocca, 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-2019-015136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlOhtrrF&md5=5bb187baea97e216a982f45966b8da54Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNARocca, Carrie; Dennin, Sean; Gu, Yongli; Kim, Joohwan; Chigas, Samantha; Najarian, Diana; Chong, Saeho; Gutierrez, Shannon; Butler, James; Charisse, Klaus; Robbie, Gabriel; Xu, Yuanxin; Brown, KirkBioanalysis (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.
- 37Liang, 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/gkv14337https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFaitbvN&md5=c63e07ea7719c6e39f74433f0850c77aIdentification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkagesLiang, 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.
- 38Gaus, 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.8b0012638https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmtFCntrY%253D&md5=aee84e23d41d0dce12c811a82e0e3c53Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin-2 Clearance ReceptorGaus, 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.
- 39Structure−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.
- 40Dosio, 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/toxins307084840https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVShtLnO&md5=c6b576cc671529e9aa360173a9338487Immunotoxins and anticancer drug conjugate assemblies: the role of the linkage between componentsDosio, Franco; Brusa, Paola; Cattel, LuigiToxins (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.
- 41Su, 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.7b00785There is no corresponding record for this reference.
- 42Su, 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.2021.68792642https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVOisr7F&md5=e780af6c3ef469a24cce09481290cc77Linker design impacts antibody-drug conjugate pharmacokinetics and efficacy via modulating the stability and payload release efficiencySu, Dian; Zhang, DongluFrontiers 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.
- 43Kolodych, 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/bc500610g43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVWgu7w%253D&md5=1b3c24d458a79fe4c9d5b30a606c9248CBTF: New Amine-to-Thiol Coupling Reagent for Preparation of Antibody Conjugates with Increased Plasma StabilityKolodych, Sergii; Koniev, Oleksandr; Baatarkhuu, Zoljargal; Bonnefoy, Jean-Yves; Debaene, Francois; Cianferani, Sarah; Van Dorsselaer, Alain; Wagner, AlainBioconjugate 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.
- 44Lyon, 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.296844https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFKms7jN&md5=96c3b19b856cc9c685cbdc704a842797Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugatesLyon, 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.
- 45Meade, 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.307845https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFKlsrnF&md5=9ec4778a5e210e35dca76dea456ec5d5Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modificationsMeade, 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.
- 46Burke, 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-16-034346https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsF2hug%253D%253D&md5=94f2bf9dba796252569277f466e9be33Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug ConjugatesBurke, 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.
- 47Wiggins, 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.24338There is no corresponding record for this reference.
- 48Prakash, 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/gkv16248https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFChtLrO&md5=e0cb4f7ecbd323fea8f0d4d761fa102aIdentification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activityPrakash, 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.
- 49Green, 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.prot095042There is no corresponding record for this reference.
- 50Livak, 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.2001.126250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtFelt7s%253D&md5=f849383250ea43f7380ed540db8a64b1Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT methodLivak, 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.
- 51Chen, C. Real-Time Quantification of microRNAs by Stem-Loop RT-PCR. Nucleic Acids Res. 2005, 33 (20), e179 DOI: 10.1093/nar/gni17851https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlSju77P&md5=9783e014d1b81ac6421a9c1cb8f3e95aReal-time quantification of microRNAs by stem-loop RT-PCRChen, 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
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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