Antibody-Oligonucleotide Conjugation – Chemistry, Biology and Therapeutics

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Introduction
Nucleic acid therapeutics (NATs) have emerged as a promising new class of therapeutics for treating genetic and epigenetic disorders. These artificially synthesised single or double stranded nucleic acids can bind to target sequences, interfere with RNA regulating machinery in physiological and pathological processes and eventually regulate the expression of target genes. Examples of these drugs include small interfering RNAs (siRNAs) and anti-sense oligonucleotides (ASOs). Currently, NATs used in clinical trials often encounter significant hurdles, including targeted delivery to specific tissues (1).

 

To improve the delivery of oligonucleotide therapeutics, various methods have been explored. These include chemical structure modification of native ASOs and siRNAs or encapsulation within lipid nanoparticles or virus-derived vectors. Conjugating moieties on to NATs has also emerged as a novel and promising approach. Tested conjugates include lipids, peptides, and N-acetylgalactosamine (GalNAc) (2).
Antibody-oligonucleotide conjugates (AOC) are a relatively new member of this family, (3) that can improve cell-specific uptake, half-life and the in vivo efficacy of the NAT after being administered, compared with non-conjugated oligonucleotides (4). The aim of this review is to provide an overview to both oligonucleotide and antibody research communities about the potential benefits of combining the target specificity of nucleic acid therapeutics (NATs) with the tissue specificity of antibody-based therapies for developing innovative and advanced therapeutic approaches for both rare diseases and more prevalent pathologies. We also highlight the advancement of cell model systems, such as three-dimensional (3D) cultures which serve as valuable tools for new conjugates evaluation, with the added potential of using patient-derived cells for NAT testing.

 

Conjugation Components
The optimal antibody being conjugated to the active chemical moieties should have relatively efficient internalization, tolerable immunogenicity and satisfying plasma half-life. However, too much affinity between the antibodies and the tissue specific receptors can also lead to ‘trapping’ of the conjugates close to the blood vessels, limiting their penetration to the more distal tissue sites (5).

 

Conjugation Methods
The methods used to prepare AOCs can be broadly divided into two approaches: noncovalent and covalent (Figure 1). Both approaches have their advantages and disadvantages. The main advantage of the noncovalent approach is its general applicability, without requiring purification steps and thus requires low amounts of antibody. However, although the AOCs obtained by noncovalent approaches are useful for analytical applications and screening, they are not usually appropriate for therapeutic applications, for which stable bonds between the partners are required. On the other hand, the covalent approach provides stable linkages in AOCs and affords well defined structures. However, as a result, larger amounts of starting materials are required for conjugation since purification is necessary. Classical methods for development of antibody conjugates utilize the lysine or cysteine residues present on native antibodies for conjugation. These techniques usually furnish heterogeneous mixtures consisting of conjugate species with varying ratios of oligonucleotide and antibody. Another drawback could be the loss of activity of the resultant conjugate due to the use of unselective chemical reagents. In order to afford homogeneous AOCs, research is focused on protein engineering and enzymatic approaches; however most of them are not applicable to native antibodies and require expensive protein engineering approaches.

Noncovalent AOCs consist of antibody and oligonucleotides noncovalently attached by a special linker that can interface between them. Some important examples include streptavidin−biotin and protein A/G−Fc mAb universal adapters (6). The advantage of avidin-based conjugation is the in vivo stability of the resultant conjugates due to the strong interactions between a biotin-labelled oligonucleotide and avidin. Niemeyer et al. conjugated thiol-modified DNA to maleimide-modified streptavidin to produce streptavidin-DNA conjugates (7). Similarly, Pardridge et al prepared antibody-siRNA conjugates using this technique, which showed gene inhibition in luciferase gene expression in intra-cranial brain cancer in vivo (8). In another approach neutravidin was used as a linker for the AOC. An IgG containing a cysteine was connected through neutravidin linkage and using a biotin tag, protamine was conjugated to the antibody (9). This AOC exhibited increased in vivo efficacy with the directed siRNA and it was used for selectively knocking down genes in specific tissues. Su et al (10) developed another analogue containing a furin cleavage site and HA2 fragment between the scFv (anti-PSMA) and protamine domains, which are responsible for endosomal cleavage and escape. This proved to be beneficial for siRNA delivery in vivo. Classical methods have also been employed to chemically link protamine with antibodies and the conjugates have been utilized successfully for the targeted delivery of siRNA in vivo. The AOC was prepared by first maleimide-modifying the protamine sulfate with SMCC, followed by a click reaction with the cysteine residues of partially reduced anti-EGFR mAb to afford the anti-EGFR mAb-protamine adduct (11). Similarly, a SMCC-modified protamine was used to prepare an antibody-siRNA conjugate which inhibited TRIM24, that is overexpressed in prostate cancer (12).

 

Classical chemical methods are mainly applied in case of covalent approaches for conjugating the oligonucleotide to antibodies. The lysine, arginine, reduced cysteine, aspartic or glutamic acid residues of the antibody are usually used for the conjugation by utilising their free amine , thiol and carboxylic acid functionalities respectively. The bifunctional chemical linker is selected based on the accessibility of the reactive groups on the antibody as well as the availability of functional groups on the conjugating partner which is the oligonucleotide. Some widely used conjugation techniques for synthesising AOCs using bifunctional linkers have been described here.
In the case of amine-to-thiol coupling, either the oligonucleotide can be first converted into a thiol analogue by reacting an amino-modified substrate with commercially available linkers containing functional groups that are reactive towards amines and simultaneously deliver thiol functionality, such as N-succinimidyl S-acetylthioacetate (SATA) (13).

 

Alternatively, the thiol-modified oligonucleotide can be directly prepared on an automated oligonucleotide synthesizer using standard solid-phase phosphoramidite chemistry protocol. On the other hand, the antibody can be converted into a maleimide-modified moiety by amide coupling techniques like NHS-ester reaction. The two partners are then conjugated at a basic pH and usually gel filtration is performed to obtain the purified conjugate. Direct conjugations can be advantageous due to their universal applicability and lack of requirements for re-engineering. However, one of the limitations of the maleimide-modified AOCs are their poor stability in thiol-containing environment such as intercellular compartments, human plasma etc, due to thiol exchange via retro-Michael reaction (14). This can be overcome by using self-hydrolysable maleimides which result in self-stabilized conjugates via a sacrificial succinimide ring opening (15). A complementary approach of antibody thiolation is using SATA to install protected thiol groups on to the amine residues of the antibody. The latent thiol groups can consequently be deprotected by hydroxylamine to generate the free thiols for conjugation. The conjugation reaction can be performed by adding hydroxylamine into the SATA-modified antibody followed by addition of the active oligonucleotide partner which is maleimide-modified using SMCC (16). Free thiols can also be generated by partially reducing the interchain disulfide bonds in antibodies (17). Thiol-reactive siRNAs have been conjugated to reduced Fab targeted towards cardiac and skeletal muscles (18). The site-specific cysteine engineered antibody, Thiomab™, developed by Genentech, was conjugated to 21-mer oligonucleotides using SMCC type linkers to improve serum stability (19). The internalization and gene silencing activities of these AOCs were explored using fluorescent-labelled oligonucleotides.

Another widely used robust direct conjugation method for AOC synthesis is the click chemistry approach, because of the orthogonality of this method and its compatibility with oligonucleotides. Strain-promoted alkyne−azide coupling (SPAAC) was among the first “click” reactions to be applied to bioconjugation, leading to stoichiometric linkage between two conjugating partners. The advantage of SPAAC over classical azide−alkyne click reaction is that it does not require the use of copper, which might be toxic in vivo, hence enhancing the applicability of SPAAC. Several methods are available for introducing strained alkynes or azides into the antibody. The reverse approach has also been successfully used for preparation of targeted anticancer agents (20), wherein the antibody has been modified with azide and conjugated with a DBCO-modified oligonucleotide. Click chemistry has also been utilized to afford AOCs using modified 2’-deoxy-2’-fluoro-β-D-arabinonucleic acid oligonucleotides to generate conjugates (21), with enhanced serum stabilities.

 

Conjugation of oligonucleotides to antibodies is often achieved by hydrazone bond formation. In this approach, the antibody is reacted with succinmidyl 4-hydrozinonicotinate acetone hydrazine (S-HyNic) and the oligonucleotide is treated with succinmidyl 4-formylbenzoate. Both these analogues are purified and then reacted with each other to generate the corresponding AOC with a hydrazone bond. Hnatowich et al used this method to generate AOCs with phosphorodiamidate Morpholino oligomers (PMOs), for cancer therapy (22). This method has been used successfully for targeted payload delivery (23).

 

Hybridization conjugation is an approach used for the preparation of AOCs with double-strand oligonucleotides. The length of the oligonucleotide plays a key role in case of the hybridization approach, since an oligonucleotide that is too short would lead to instability of the duplex in plasma and if it is too long, it could lead to the formation of secondary structures (24). An example of this approach was to use Cetuximab as a targeting agent for the delivery of doxorubicin, by intercalation into the oligonucleotide double-strand (25). In this case, the length of the oligonucleotide sequence determined the amount of doxorubicin that can be carried by the AOC. Although for the hybridization approach, the kinetics are faster in comparison to click chemistry; the cost of having a linker-payload attached to an oligonucleotide makes it less favourable for therapeuticapplications.

 

The above conjugation methods have been successfully used to conjugate antibodies to oligonucleotides allowing for characterization and testing of these conjugates. However, synthesis, purification and analysis of AOCs are still a challenge, and novel methods are being developed to address these challenges. Enzymatic approaches have also been utilized to generate AOCs which are not covered in this review; however, these can be found in the review by Zhang et al (26).

 

Preclinical evaluation of novel conjugates
Traditionally nuclease stability, affinity towards the target sequence and solubility are the main parameters for novel NAT chemistry evaluation (27). In vitro and in vivo models are still the mainstream testing platforms for AOCs with 3D in vitro models also emerging as valuable tools to evaluate certain aspects of AOCs, such as tissue distribution and specificity. 3D models have played a crucial role in the successful translation of Sepofarsen into clinical trials, to treat a form of inherited retinal disease (28).

 

Preclinical Potency Evaluation

 

Transcriptional and translational read-outs
Typically, the first metric to be considered is the on-target potency of the conjugated oligonucleotides by investigating mRNA and protein expression profiles of cells or tissues after treatment.(29). The gold standards for evaluating AOC activity are polymerase chain reaction (PCR) assays, including quantitative PCR (qPCR) and digital droplet PCR (ddPCR). These are highly sensitive assays which require careful design of primers and the avoidance of assay artefacts. Western blotting provides information on the protein expression level, although limitations include the limited availability of highly specific antibodies (30).

 

Intracellular trafficking imaging
Imaging-based techniques have also been developed to assess AOCs, with many studies focusing on intracellular trafficking pathways (31) (Figure 2). There has been evidence that AOCs labelled with fluorophores co-localise with lysosomal proteins such as LAMP2 (4). Observations also suggest that the successful internalisation of AOCs involve endosomal pathways, in contrast to small molecule conjugates. This is possibly because small molecules can still be passively internalised by cell membrane permeability. Endosomal escape is a limiting factor for the success of tested AOCs. Other energy-consuming pathways, such as caveolae-mediated endocytosis, have also been explored with conjugated molecules. In these cases, key molecule such as caveolin-1 are implicated in pathological processes such as oncogenesis. Therefore, interfering with these molecules not only provides a route for therapeutics to enter cells, but also a way to increase the sensitivity of cells to the therapy (32).

Other functional assays and direct binding assays
To evaluate protein-protein interactions between AOCs and target epitopes the binding to certain receptors on the cell surface can be assessed using approaches such as co-immunopreciptation pull-down assays (33). Further evaluation of direct binding events can be made by using enzyme-linked immunosorbent assay (ELISAs) employing specialised antibodies to detect AOCs and their binding to target epitopes (34). Other binding assays in living cells include time resolved analysis, and competition assay (35).

 

Preclinical Specificity and Toxicity evaluation

 

Gene Specificity
Despite the specificity of Watson-Crick base pairing, oligonucleotide therapeutics can still exhibit sequence-dependent or sequence-independent off-target effects, leading to toxicity or unwanted side effects. However, there are also situations where payload cytotoxicity is desired, such as evaluating anti-cancer conjugates. Although serving different purposes, cytotoxic anti-cancer conjugate development can still provide some inspiration for unwanted cytotoxicity evaluation for other oligonucleotide therapeutics (36). Cytotoxicity can be evaluated by analysing metabolic activities of living cells or using metabolites as markers for dead cells (37). Potential side effects associated with conjugated antibodies are dependent on a given target and dependent on both the antibody and drug chosen. So far, tolerance issues have been reported for mouse monoclonal antibodies and plant-derived proteins (38).

 

Targeting tissue specificity to reduce toxicity
Oligonucleotide therapeutics can induce hepatotoxicity by entering the liver through the bloodstream, regardless of their hepatic or non-hepatic target. A classic strategy for tissue-specific delivery involves the use of GalNAc-conjugated oligonucleotides (3), (35). In addition, oligonucleotide-conjugates can also accumulate in kidneys, particularly in renal tubular cells, causing renal toxicity (39). Therefore, strategies to increase uptake in other tissue types are likely to improve AON safety profiles.

 

Indeed, such extrahepatic tissue targeting has begun to show some success. One example is the use of transferrin as a receptor; Sugo et al have successfully targeted muscle tissue with siRNA conjugated with anti-CD71 Fab’ fragment via intravenous administration (18). There are also considerable efforts leveraging transferrin system to achieve central nervous system delivery through systemic administration (39).

 

Clinical translation
There has been a strong history of personalised medicine in the clinical translation of oligonucleotide therapeutics. Accordingly, scientific guidelines have been published with respect to in vitro studies at the early stages of preclinical evaluation, taking into consideration the clinical relevance of the tissue being targeted, the difficulty level of the genetic and cellular techniques involved, and the genetic context of the genes involved in the case (29). These guidelines should also be considered while developing AOCs for an expedited translational pathway.

 

Perspectives from clinical trials
The success of clinical trials is closely related to the patho-physiological relevance of the pre-clinical models applied during the approval process. False positive results will waste large numbers of resources, while false negative results will lead to missing valuable targets and hits. Preclinical studies can also learn from the methodologies of clinical studies. Toxicity management is one good example: for instance, learning from phase I and phase II clinical studies to design immunogenicity assays and strategies (37). Such early clinical studies are valuable for dosing level estimation. Furthermore pharmacokinetic and pharmacodynamic studies of conjugates need especially careful interpretation due to the complex and combined structures of the conjugates (40). It is highly promising that AOC drug candidates have been progressed into multiple independent clinical trials, as detailed in Table 1 (41).

 

Immediate responses translate into long-term effects
One advantage of oligonucleotide therapeutics is the potential for repeat dosing. This can translate an immediate effect into weeks or even months of efficacy, providing long-lastingalleviation of symptoms. To evaluate these effects, conventional models are based on animals with genetic or epigenetic manipulation and/or backgrounds. However, more recently we have witnessed the significant improvement and development of 3D in vitro models, which can be a useful alternative to animal models. Some examples include organoids, such as retinal organoids (42) and cerebral organoids (43), (44), (45). Another classic 3D culture tool is multicellular spheroids that can be grown to containorganised structures with heterogeneous cell populations. Recently, studies have shown spheroids as important and versatile screening tools in oligonucleotide research, particularly in oncology to treat cancers with unmet needs such as pancreatic cancer (46). We predict that 3D in vitro models will further facilitate the translation of potential candidates into clinical applications. For instance, spheroids have been helpful in demonstrating the penetration efficiency of oligonucleotides (46). In addition, as indicated in Figure 3 (lower panels), a fundamental advantage of using 3D in vitro models is that they can be derived from an individual patient. This may be essential in the case of rare and ultra-rare disease, where the AOC candidate will be designed against a specific pathogenic mutation; thus, the best model system for AOC screening is, by nature, the patient’s cells themselves (42, 43).

 

Conclusion
Overall, the cell and organ-specific delivery of oligonucleotides can be facilitated through conjugations to highly specific biologics like antibodies. As a relatively new class of therapeutics, compared with conventional small molecules, oligonucleotide conjugates can benefit by gaining useful insights from the already established field of small molecule conjugates, like those with human serum albumin (HSA) (47). Different conjugation chemistry strategies can be adopted to enhance the translational capabilities eventually moving into clinical applications of the use of AOCs to help alleviate a range of disorders. In contrast to the molecule-centred strategy for small molecule drug development pipeline, oligonucleotide therapeutics focus more on the target genes of the disease, moving towards personalised and precision medicine. This significantly reduces the development time for AOCs alongside reductions in off-target effect, toxicity, and provides a new opportunity for the antibody research community as well as for oligonucleotide researchers. It is believed that the full potential of this family of drugs remains to be revealed by smarter delivery systems (48). From a technical standpoint, the study design must incorporate appropriate controls, such as native and conjugated antibodies and oligonucleotides. Long-term monitoring of PK/PD is essential for systemic administration of repeated dosing. Assays differentiating intact and partial AOCs can also be helpful to understand the complete profiles of the evaluated AOCs’ behaviour in vivo. The numerous ongoing clinical studies highlight the need for caution when interpreting results from rodent models due to significant genetic and physiological differences between humans and rodents. Finally, to fully leverage the potential for rapid AOC development, a tailored pre-clinical data package should be designed and collected.

 

Conflicts of Interest
The authors declare no conflict of interest.

 

Acknowledgements
The Nucleic Acid Therapy Accelerator (NATA) is funded by the Medical Research Council UK (MRC): Grant reference: MC_PC_20061.

 

Figure 1. Schematic of the conjugation methods for the synthesis of Antibody-oligonucleotide conjugates. Non-Covalent conjugation strategies include (a). Ionic interaction (b). Affinity based conjugation using streptavidin and biotin (c). Hybridisation approach. Covalent conjugation strategies include (d). Strain-promoted azide-alkyne cycloaddition (SPAAC) (e). Thiol-ene click reaction approach (f). Hydrazone coupling approach (g). Amide coupling method. (Adapted from Ref (3), (14)).

 

Figure 2. Mechanistic intracellular behaviour of antibody oligonucleotide siRNA conjugates. The antibody arm of the AOC will first engage its target epitope (1). The recruitment of endosomal formation proteins like Rab5, can help internalise and retain the AOC inside the cell (2-3) and facilitate the release of the oligonucleotide in the late endosome (4) followed by endosomal escape (5). Once successfully released into the cytoplasm, the oligonucleotide arm of the AOC will engage its target mRNA allowing mRNA degradation (6) and cessation of protein synthesis (Adapted from Ref (31)).

 

Figure 3. Translational pipelines for the development and clinical use of AOC therapeutic candidates, with the bottom panel showing a potential fast track of N=1 treating rare diseases. The development of AOCs, similar to other drug conjugates, begins with target identification and mutational candidate screens within patients. Once synthesised, the activity and safety profile of AOCs can be identified using preclinical in vitro and in vivo screens. In case of treatment of rare diseases, it is possible that clinical evaluations can commence in a timely manner once preclinical testing is finished through establishment of early and late phase clinical trials alongside the evaluation of safety profiles in humans. Once passing these screens and trials, an AOC can be approved and manufactured for clinical applications. It is noticeable that for rare disease therapy development, the identification of mutation and the relevant clinical procedure can vary from weeks to years, which is a speed limiting step.

 

Table 1. Representative AOCs processed into IND-enabling and clinical trials.

 

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