Antibody-oligonucleotide conjugate

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Schematic structure of an antibody-oligonucleotide conjugate (AOC) Antibody oligonucleotide conjugate.png
Schematic structure of an antibody-oligonucleotide conjugate (AOC)

Antibody-oligonucleotide conjugates or AOCs belong to a class of chimeric molecules combining in their structure two important families of biomolecules: monoclonal antibodies and oligonucleotides. [1]

Contents

Combination of exceptional targeting capabilities of monoclonal antibodies with numerous functional modalities of oligonucleotides has been fruitful for a variety of applications with AOC including imaging, detection and targeted therapeutics. [1] [2] [3]

Cell uptake/internalisation still represents the biggest hurdle towards successful ON therapeutics. A straightforward uptake, like for most small-molecule drugs, is hindered by the polyanionic backbone and the molecular size of ONs. Being adapted from the broad and successful class of Antibody-Drug conjugates, antibodies and antibody analogues are more and more used in research in order to overcome hurdles related to delivery and internalisation of ON therapeutics. By exploiting bioconjugation methodology several conjugates have been obtained.

Development of therapeutic AOCs

The first AOC was reported in 1995 where the lysines of a transferrin-antibody were connected using a SMCC bifunctional linker (NHS ester and maleimide moiety) to radiolabelled and cys-bearing ASOs targeting HIV mRNA. [4] Marcin and his colleagues developed a different construct using the same chemistry, but they utilized siRNA instead of an ASO in 2011. [5] In 2013, MYERS and coworkers then unspecifically labelled an anti-CD19 antibody with N-succinimidyl 3-(2-pyridyl-dithio) propionate to form disulphide bonds with cys-modified ASO targeting the mRNA of oncoprotein E2A–PBX1. [6] Ultimately, they could prove in-vivo antitumour effects which in contrast were not obtained with the single entities. [7] In the same timeframe, several antibodies were exploited for ON delivery in combination with nanoparticles and in non-covalent strategies. [8] [9] [10]

Only recently the first examples for a site-selective conjugation between an ON therapeutic and a mAb was published: in 2015 Genentech exploited the SMCC linker to conjugate siRNA to several engineered mAb based on their proprietary Thiomab technology, which allows site-specific introduction of a cysteine into the antibody sequence[32]. [11] They could prove the functionality of both entities in the construct and by screening different antibodies, they validated their importance for an effective antisense effect. [11] The main obstacle encountered was a limited endosomal escape but ultimately a functional construct which shows antisense effect in-vivo was reported. [11] After development of the SMCC based conjugates, there were two constructs reported in literature based on strain-promoted alkyne-azide cycloadditions: an MXD3 mRNA targeting gapmer (cEt and PS modified) linked to an anti-CD22 antibody targeting preB cells leads to in-vitro apoptosis of targeted cells and in-vivo increased length of mouse survival in xenograft models. Notably, the dose required for the same therapeutic effect was 20 times lower for the developed conjugate (vs. naked mAb). [12] Another reported conjugate, exploiting the same unselective conjugation chemistry, employs an CD44 respectively EphA2 targeting antibody which covalently carries a therapeutically irrelevant “sense-carrier” oligonucleotide. [13] This oligonucleotide base pairs with the actual antisense oligonucleotide (gapmer bearing phosphorothioate linkages and 2’-deoxy-2’-fluoro-beta-D-arabinonucleic acid modifications and a terminal fluorophor) aiming for an increased RNaseH activity. [14] [15] [16]

Antibody Analogue-Oligonucleotide Conjugate

Despite their tremendous potential, ADCs and AOCs suffer from the physical size of the antibody (mAb) entity (150 kDa) which limits solid tumour penetration (at least at low concentrations). Moreover, the site-selective modification of the antibody is hardly achievable: due to the difficult production of mAbs the selective introduction of an unnatural amino acid into the protein is not easily possible. [17]

Thats why there is intensive research to exploit antibody analogues and antibody fragments which retain a high target specificity but combined with a smaller size and a greater possibility of modification. Nanobodies for example are natural single-domain antibodies found in camelids with an average mass of 15kDa. They bear an increased stability, solubility and tissue penetration compared to mAbs. [18] [19] [20]

One conjugate, consisting out of an EGFR Nanobody and a siRNA being combined through maleimide bioconjugation, proves the possibility of successful delivery of ONs by nanobodies. [21]

Another example consists out of an anti-CD71 Fab fragment which was conjugated to a maleimide bearing siRNA (itself having 2’OMe/2’F modifications and phosphorothioate linkages). Several (cleavable and uncleavable) linkers between the maleimide moiety and the siRNA were screened revealing only a small influence on silencing efficacy (uncleavable linkers leading to the best results). To play out the small size of the Fab fragment, subcutaneous administration was investigated in mouse models leading to equivalent silencing results compared to intravenous administration. By comparison with other mAb-siRNA conjugates the authors even speculate that endosomal escape is largely facilitated by the smaller size of the Fab (vs. mAb). [22]

Moreover, Nanobody-ON conjugates are intensively used for imaging purposes exploiting the small nanobody size to reduce imaging displacement. [23] [24]

See also

Related Research Articles

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small fragments of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression, or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

<span class="mw-page-title-main">Small interfering RNA</span> Biomolecule

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20–24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.

<span class="mw-page-title-main">Morpholino</span> Chemical compound

A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule used in molecular biology to modify gene expression. Its molecular structure contains DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to small specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Morpholinos are used as research tools for reverse genetics by knocking down gene function.

Antisense therapy is a form of treatment that uses antisense oligonucleotides (ASOs) to target messenger RNA (mRNA). ASOs are capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA. Several ASOs have been approved in the United States, the European Union, and elsewhere.

<span class="mw-page-title-main">Antisense RNA</span>

Antisense RNA (asRNA), also referred to as antisense transcript, natural antisense transcript (NAT) or antisense oligonucleotide, is a single stranded RNA that is complementary to a protein coding messenger RNA (mRNA) with which it hybridizes, and thereby blocks its translation into protein. The asRNAs have been found in both prokaryotes and eukaryotes, and can be classified into short and long non-coding RNAs (ncRNAs). The primary function of asRNA is regulating gene expression. asRNAs may also be produced synthetically and have found wide spread use as research tools for gene knockdown. They may also have therapeutic applications.

<span class="mw-page-title-main">Aptamer</span> Oligonucleotide or peptide molecules that bind specific targets

Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities, with variable levels of off-target binding and are sometimes classified as chemical antibodies. Aptamers and antibodies can be used in many of the same applications, but the nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. This difference can make aptamers a better choice than antibodies for some purposes.

<span class="mw-page-title-main">Single-domain antibody</span> Antibody fragment

A single-domain antibody (sdAb), also known as a Nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments and single-chain variable fragments.

<span class="mw-page-title-main">Maleimide</span> Chemical compound

Maleimide is a chemical compound with the formula H2C2(CO)2NH (see diagram). This unsaturated imide is an important building block in organic synthesis. The name is a contraction of maleic acid and imide, the -C(O)NHC(O)- functional group. Maleimides also describes a class of derivatives of the parent maleimide where the NH group is replaced with alkyl or aryl groups such as a methyl or phenyl, respectively. The substituent can also be a small molecule (such as biotin, a fluorescent dye, an oligosaccharide, or a nucleic acid), a reactive group, or a synthetic polymer such as polyethylene glycol. Human hemoglobin chemically modified with maleimide-polyethylene glycol is a blood substitute called MP4.

Therapeutic gene modulation refers to the practice of altering the expression of a gene at one of various stages, with a view to alleviate some form of ailment. It differs from gene therapy in that gene modulation seeks to alter the expression of an endogenous gene whereas gene therapy concerns the introduction of a gene whose product aids the recipient directly.

Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular intake and uptake of molecules ranging from nanosize particles to small chemical compounds to large fragments of DNA. The "cargo" is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions.

Magnetofection is a transfection method that uses magnetic fields to concentrate particles containing vectors to target cells in the body. Magnetofection has been adapted to a variety of vectors, including nucleic acids, non-viral transfection systems, and viruses. This method offers advantages such as high transfection efficiency and biocompatibility which are balanced with limitations.

<span class="mw-page-title-main">Therapeutic Targets Database</span> Database of protein targets in drug design

Therapeutic Target Database (TTD) is a pharmaceutical and medical repository constructed by the Innovative Drug Research and Bioinformatics Group (IDRB) at Zhejiang University, China and the Bioinformatics and Drug Design Group at the National University of Singapore. It provides information about known and explored therapeutic protein and nucleic acid targets, the targeted disease, pathway information and the corresponding drugs directed at each of these targets. Detailed knowledge about target function, sequence, 3D structure, ligand binding properties, enzyme nomenclature and drug structure, therapeutic class, and clinical development status. TTD is freely accessible without any login requirement at https://idrblab.org/ttd/.

<span class="mw-page-title-main">Antibody–drug conjugate</span> Class of biopharmaceutical drugs

Antibody–drug conjugates or ADCs are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. As of 2019, some 56 pharmaceutical companies were developing ADCs.

Photoimmunotherapy (PIT) is an oncological treatment that combines photodynamic therapy of tumor with immunotherapy treatment. Combining photodynamic therapy with immunotherapy enhances the immunostimulating response and has synergistic effects for metastatic cancer treatment.

RNA therapeutics are a new class of medications based on ribonucleic acid (RNA). Research has been working on clinical use since the 1990s, with significant success in cancer therapy in the early 2010s. In 2020 and 2021, mRNA vaccines have been developed globally for use in combating the coronavirus disease. The Pfizer–BioNTech COVID-19 vaccine was the first mRNA vaccine approved by a medicines regulator, followed by the Moderna COVID-19 vaccine, and others.

Gapmers are short DNA antisense oligonucleotide structures with RNA-like segments on both sides of the sequence. These linear pieces of genetic information are designed to hybridize to a target piece of RNA and silence the gene through the induction of RNase H cleavage. Binding of the gapmer to the target has a higher affinity due to the modified RNA flanking regions, as well as resistance to degradation by nucleases. Gapmers are currently being developed as therapeutics for a variety of cancers, viruses, and other chronic genetic disorders.

ncRNA therapy

A majority of the human genome is made up of non-protein coding DNA. It infers that such sequences are not commonly employed to encode for a protein. However, even though these regions do not code for protein, they have other functions and carry necessary regulatory information.They can be classified based on the size of the ncRNA. Small noncoding RNA is usually categorized as being under 200 bp in length, whereas long noncoding RNA is greater than 200bp. In addition, they can be categorized by their function within the cell; Infrastructural and Regulatory ncRNAs. Infrastructural ncRNAs seem to have a housekeeping role in translation and splicing and include species such as rRNA, tRNA, snRNA.Regulatory ncRNAs are involved in the modification of other RNAs.

<span class="mw-page-title-main">Intracellular delivery</span> Scientific research area

Intracellular delivery is the process of introducing external materials into living cells. Materials that are delivered into cells include nucleic acids, proteins, peptides, impermeable small molecules, synthetic nanomaterials, organelles, and micron-scale tracers, devices and objects. Such molecules and materials can be used to investigate cellular behavior, engineer cell operations or correct a pathological function.

References

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  2. ↑ Levin, Arthur A. (2017). "Targeting Therapeutic Oligonucleotides". New England Journal of Medicine. Massachusetts Medical Society. 376 (1): 86–88. doi:10.1056/nejmcibr1613559. ISSN   0028-4793. PMID   28052219. S2CID   33970969.
  3. ↑ Winkler, Johannes (2013). "Oligonucleotide conjugates for therapeutic applications". Therapeutic Delivery. Future Science Ltd. 4 (7): 791–809. doi: 10.4155/tde.13.47 . ISSN   2041-5990. PMC   3787477 . PMID   23883124.
  4. ↑ Walker, Ian; Irwin, William J.; Akhtar, Saghir (1995). "Improved Cellular Delivery of Antisense Oligonucleotides Using Transferrin Receptor Antibody-Oligonucleotide Conjugates". Pharmaceutical Research. 12 (10): 1548–1553. doi:10.1023/a:1016260110049. ISSN   0724-8741. PMID   8584498. S2CID   20247200.
  5. ↑ Ma, Yuelong; Kowolik, Claudia M.; Swiderski, Piotr M.; Kortylewski, Marcin; Yu, Hua; Horne, David A.; Jove, Richard; Caballero, Otavia L.; Simpson, Andrew J. G.; Lee, Fook-Thean; Pillay, Vinochani; Scott, Andrew M. (2011-07-26). "Humanized Lewis-Y Specific Antibody Based Delivery of STAT3 siRNA". ACS Chemical Biology. 6 (9): 962–970. doi:10.1021/cb200176v. ISSN   1554-8929. PMC   3831028 . PMID   21766840.
  6. ↑ Zhang, Ke; Hao, Liangliang; Hurst, Sarah J.; Mirkin, Chad A. (2012-09-28). "Antibody-Linked Spherical Nucleic Acids for Cellular Targeting". Journal of the American Chemical Society. 134 (40): 16488–16491. doi:10.1021/ja306854d. ISSN   0002-7863. PMC   3501255 . PMID   23020598.
  7. ↑ Uckun, Fatih M.; Qazi, Sanjive; Dibirdik, Ilker; Myers, Dorothea E. (2012-09-18). "Rational design of an immunoconjugate for selective knock-down of leukemia-specific E2A–PBX1 fusion gene expression in human Pre-B leukemia". Integrative Biology. 5 (1): 122–132. doi:10.1039/c2ib20114c. ISSN   1757-9708. PMID   22990208.
  8. ↑ Bäumer, Nicole; Appel, Neele; Terheyden, Lisa; Buchholz, Frank; Rossig, Claudia; Müller-Tidow, Carsten; Berdel, Wolfgang E; Bäumer, Sebastian (2015-12-03). "Antibody-coupled siRNA as an efficient method for in vivo mRNA knockdown". Nature Protocols. 11 (1): 22–36. doi:10.1038/nprot.2015.137. ISSN   1754-2189. PMID   26633129. S2CID   33367153.
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  10. ↑ Song, Erwei; Zhu, Pengcheng; Lee, Sang-Kyung; Chowdhury, Dipanjan; Kussman, Steven; Dykxhoorn, Derek M; Feng, Yi; Palliser, Deborah; Weiner, David B; Shankar, Premlata; Marasco, Wayne A; Lieberman, Judy (2005-05-22). "Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors". Nature Biotechnology. 23 (6): 709–717. doi:10.1038/nbt1101. ISSN   1087-0156. PMID   15908939. S2CID   14855642.
  11. 1 2 3 Cuellar, Trinna L.; Barnes, Dwight; Nelson, Christopher; Tanguay, Joshua; Yu, Shang-Fan; Wen, Xiaohui; Scales, Suzie J.; Gesch, Julie; Davis, David; van Brabant Smith, Anja; Leake, Devin; Vandlen, Richard; Siebel, Christian W. (2014-12-30). "Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB–siRNA conjugates". Nucleic Acids Research. 43 (2): 1189–1203. doi:10.1093/nar/gku1362. ISSN   1362-4962. PMC   4333408 . PMID   25550431.
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  16. ↑ Dovgan, Igor; Koniev, Oleksandr; Kolodych, Sergii; Wagner, Alain (2019-07-24). "Antibody–Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents". Bioconjugate Chemistry. 30 (10): 2483–2501. doi:10.1021/acs.bioconjchem.9b00306. ISSN   1043-1802. PMID   31339691. S2CID   198491258.
  17. ↑ Baah, Stephanie; Laws, Mark; Rahman, Khondaker Miraz (2021-05-15). "Antibody–Drug Conjugates—A Tutorial Review". Molecules. 26 (10): 2943. doi: 10.3390/molecules26102943 . ISSN   1420-3049. PMC   8156828 . PMID   34063364.
  18. ↑ Škrlec, Katja; Štrukelj, Borut; Berlec, Aleš (July 2015). "Non-immunoglobulin scaffolds: a focus on their targets". Trends in Biotechnology. 33 (7): 408–418. doi:10.1016/j.tibtech.2015.03.012. ISSN   0167-7799. PMID   25931178.
  19. ↑ Muyldermans, Serge (2021-02-16). "Applications of Nanobodies". Annual Review of Animal Biosciences. 9 (1): 401–421. doi: 10.1146/annurev-animal-021419-083831 . ISSN   2165-8102. PMID   33233943.
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  23. ↑ Hebbrecht, Tim; Liu, Jing; Zwaenepoel, Olivier; Boddin, Gaëlle; Van Leene, Chloé; Decoene, Klaas; Madder, Annemieke; Braeckmans, Kevin; Gettemans, Jan (November 2020). "Nanobody click chemistry for convenient site-specific fluorescent labelling, single step immunocytochemistry and delivery into living cells by photoporation and live cell imaging". New Biotechnology. 59: 33–43. doi:10.1016/j.nbt.2020.05.004. ISSN   1871-6784. PMID   32659511. S2CID   220521390.
  24. ↑ Sograte-Idrissi, Shama; Oleksiievets, Nazar; Isbaner, Sebastian; Eggert-Martinez, Mariana; Enderlein, Jörg; Tsukanov, Roman; Opazo, Felipe (2018-12-19). "Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors". doi:10.1101/500298. PMC   6357156 . Retrieved 2023-03-02.{{cite journal}}: Cite journal requires |journal= (help)
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