Antibody-Oligonucleotide Conjugates: a Twist to Antibody-Drug Conjugates

Journal of Clinical Medicine

Review Antibody-Oligonucleotide Conjugates: A Twist to Antibody-Drug Conjugates

Julien Dugal-Tessier 1,* , Srinath Thirumalairajan 2 and Nareshkumar Jain 1

1 NJ Bio, 675 US Highway 1, Suite B129, North Brunswick, NJ 08902, USA; [email protected] 2 Seagen, 21717 30th Drive, S.E., Bothell, WA 98021, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-732-202-5664

Abstract: A summary of the key technological advancements in the preparation of antibody– oligonucleotide conjugates (AOCs) and the distinct advantages and disadvantages of AOCs as novel therapeutics are presented. The merits and demerits of the different approaches to conjugating oligonucleotides to antibodies, antibody fragments or other proteins, mainly from the perspective of AOC purification and analytical characterizations, are assessed. The lessons learned from in vitro and in vivo studies, especially the findings related to silencing, trafficking, and cytotoxicity of the conjugates, are also summarized.

Keywords: antibody-oligonucleotide conjugates (AOCs); oligonucleotide; siRNA; precision medicine

1. Introduction Antibody-drug conjugates (ADCs) have evolved to be an important new arsenal in Citation: Dugal-Tessier, J.; the treatment of various cancers due to their preferential delivery of chemotherapeutics to Thirumalairajan, S.; Jain, N. cancer cells [1]. This approach has allowed an improvement of the therapeutic index of Antibody-Oligonucleotide many antibodies. It has similarly aided the utilization of many small molecules that are Conjugates: A Twist to potent and efﬁcacious but possess properties that preclude them from being developed into Antibody-Drug Conjugates. J. Clin. a chemotherapeutic. For example, well-known potent payloads such as calicheamycins, Med. 2021, 10, 838. https:// auristatins and maytansinoids were found to be too toxic to be used as standalone single- doi.org/10.3390/jcm10040838 agent chemotherapeutics but were found to have favorable clinical beneﬁts in liquid

Academic Editor: Jagath (cancers of the blood such as leukemias, lymphomas and myelomas) and solid tumors Reddy Junutula when conjugated to antibodies [2,3]. Antibodies have proven to be ideal delivery agents, in Received: 31 December 2020 general, due to their high specificity, long half-life and low immunogenicity [2]. The field of Accepted: 16 February 2021 ADCs has evolved rapidly in the last decade, resulting in a better understanding of the role Published: 18 February 2021 of target selection, release mechanism, payload potency and the role of each component in the overall activity profile. As more clinical data have become available, dosing levels Publisher’s Note: MDPI stays neutral and toxicity management of ADCs are better understood, leading to an increase in clinical with regard to jurisdictional claims in success [3]. published maps and institutional affil- Although selectivity in an ADC is achieved through the antibody, non-specific toxicity iations. is still observed. If more precise payloads could be used, the synergistic combination could potentially result in a more selective and safer ADC. Oligonucleotides qualify as such a class of precise payloads due to their ability to arrest protein production by homing in on specific genes. The use of small interfering RNA (siRNA) and anti-sense oligonucleotides (ASO)

Copyright: © 2021 by the authors. has rapidly evolved over the past decade [4]. Even though oligonucleotides offer selectivity, Licensee MDPI, Basel, Switzerland. they suffer from challenges such as short serum stability, low membrane permeability, This article is an open access article and lack of tissue selectivity. Antibodies, with their longer half-life, ability to selectively distributed under the terms and deliver therapeutics inside the cells, and targeting properties, make them ideal partners conditions of the Creative Commons for the targeted delivery of oligonucleotides. ADCs suffer from systemic toxicity due to Attribution (CC BY) license (https:// non-selective payloads, but the selectivity of oligonucleotides could enhance the ability of creativecommons.org/licenses/by/ conjugates to only affect target disease cells. Antibody–oligonucleotide conjugates (AOCs) 4.0/).

J. Clin. Med. 2021, 10, 838. https://doi.org/10.3390/jcm10040838 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, x FOR PEER REVIEW 2 of 17

toxicity due to non-selective payloads, but the selectivity of oligonucleotides could en- J. Clin. Med. 2021, 10, 838 2 of 17 hance the ability of conjugates to only affect target disease cells. Antibody–oligonucleotide conjugates (AOCs) combine the high precision of siRNA and ASOs with the deliverability combineof antibodies, the high thus precision synergizing of siRNA the advantages and ASOs of with both thetechnologies. deliverability of antibodies, thus synergizingThe field the of advantages AOCs started of bothinitially technologies. as a means to develop powerful diagnostic tools but has evolved more recently as a targeted therapeutic approach for many diseases [5]. The field of AOCs started initially as a means to develop powerful diagnostic tools The use of AOCs grew rapidly due to their ability to detect antigens with greater sensitiv- but has evolved more recently as a targeted therapeutic approach for many diseases [5]. ity with PCR compared to fluorescent or other colorimetric methods [5]. As the field of The use of AOCs grew rapidly due to their ability to detect antigens with greater sensitivity oligonucleotides matured and selective tissue delivery became an important challenge for with PCR compared to fluorescent or other colorimetric methods [5]. As the field of oligonu- clinical use, AOCs transformed into potential single-agent therapeutics. Both the ADC cleotides matured and selective tissue delivery became an important challenge for clinical and the oligonucleotide field have had tremendous growth in chemistry, conjugation and use, AOCs transformed into potential single-agent therapeutics. Both the ADC and the analytics over the past decade, which has increased the chances of generating a successful oligonucleotide field have had tremendous growth in chemistry, conjugation and analytics AOC. over the past decade, which has increased the chances of generating a successful AOC. We will highlight the development and use of AOCs for the potential treatment of We will highlight the development and use of AOCs for the potential treatment diseases. The focus will be on conjugation methods and the in vitro and in vivo data gen- oferated diseases. from Thethese focus AOCs. will The be review on conjugation will point methods out differences and the andin vitrosimilaritiesand in between vivo data generatedADCs and from AOCs. these While AOCs. some The of review the aspects will point of development out differences and and preparation similarities of betweenADCs ADCsand AOCs and AOCs. might Whileseem related, some of they the aspectsare differ ofent development modalities with and preparationtheir own unique of ADCs chal- and AOCslenges. might seem related, they are different modalities with their own unique challenges. 2. Conjugation of Oligonucleotides to Antibodies 2. Conjugation of Oligonucleotides to Antibodies Conjugation of drug-linkers in ADCs exclusively uses the direct conjugation method. Conjugation of drug-linkers in ADCs exclusively uses the direct conjugation method. Oligonucleotides,Oligonucleotides, on the other hand, have have more more conjugation conjugation methods methods than than typical typical small small molecules.molecules. Figure 11 illustratesillustrates thethe fourfour general methods used used to to prepare prepare AOCs. AOCs. These These methodsmethods involve involve ionicionic interactionsinteractions (Figure(Figure1 1A),A), affinityaffinity bindingbinding (Figure(Figure1 1B),B), direct direct conjuga- conju- tiongation (1C) (1C) and and utilization utilization of theof the double-strand double-strand as as a conjugationa conjugation moiety moiety (Figure (Figure1D). 1D). The The use anduse advantagesand advantages of each of each will will be discussed. be discussed.

A) Ionic Interactions B) Affinity Conjugtaion

Avidin Biotin

C) Direct Conjugation D) Hybridization

O N SH S N O O FigureFigure 1. 1.General General methods methods to to conjugate conjugate oligonucleotidesoligonucleotides by ( A)) electrostatic electrostatic interactions, interactions, (B (B) affinity) afﬁnity between between biotin biotin and and avidin, (C) directly to antibody and (D) using double-strand hybridization. avidin, (C) directly to antibody and (D) using double-strand hybridization.

ConjugatingConjugating oligonucleotidesoligonucleotides toto proteins proteins generates generates different different challenges challenges from from the the more traditionalmore traditional conjugation conjugation of small of molecules.small molecules. Challenges Challenges in the in preparation the preparation of ADCs of ADCs include heterogeneity,include heterogeneity, hydrophobicity hydrophobicity of the drug-linker, of the drug-linker, aggregation aggregation and stability and stability of the of linker.the Thelinker. size The of size the drugof the anddrugits and charge its charge contribution contribution to theto the resultant resultant conjugate conjugate alsoalso have a significanta significant impact impact on on the the preparation preparation ofof anan ADC, but these these challenges challenges are are amplified amplified in in AOCs. For example, the molecular weight (MW) of a typical drug-linker in an ADC is less AOCs. For example, the molecular weight (MW) of a typical drug-linker in an ADC is less than 2 kDa, while an oligonucleotide is larger and can have an MW greater than 10 kDa. This means that the influence of the oligonucleotide on the physical and chemical properties of the conjugated antibody (150 kDa) will be larger than for small molecules. Thus, the analytical and purification methods used in the preparation of ADCs may not translate to AOCs. J. Clin. Med. 2021, 10, 838 3 of 17

Oligonucleotides with their negatively charged phosphate backbones are water- soluble, which can contrast with some small molecules found in ADCs. The negative charge that imparts solubility is also a challenge once conjugated to an antibody as it significantly changes the overall charge of the antibody. Conventional drug-linkers are usually neutral or have fewer than two charges/drug-linker and are optimized to not significantly change the properties of the parent antibody once conjugated. Even if the ADC has mea- surable differences by imaged capillary isoelectric focusing (icIEF) and ion-exchange (IEX) chromatography compared with the antibody, the resultant conjugate behaves much like the parent antibody and can be analyzed and purified as such. However, oligonucleotides are inherently negatively charged (with over 20 negative charges) and will dominate the overall charge profile of the AOC. Thus, the analysis and characterization of an AOC is anything but conventional or straightforward. For example, while most mass-spectrometry (MS)-based methods used in the characterization of ADCs rely on detection using the positive mode, AOCs with their many negative charges are almost invisible under those detection conditions. Another main difference in the characterization of ADCs and AOCs is how the drug/oligonucleotide-to-antibody ratio can be calculated. Typical standard drug to antibody ratio (DAR) characterization for ADCs involves reverse-phase HPLC and/or hydrophobic interaction chromatography (HIC), which rely on the hydrophobicity of the drug-linker. These chromatographic methods are not transferable to the determination of oligonucleotide to antibody ratios (OAR). Methods used to determine OAR will be discussed in the individual conjugation method sections. With an ADC, the conjugate behaves much like an antibody and not like a small molecule; with an AOC, the conjugate behaves both like an antibody and an oligonucleotide. Because of such differences, most recent advances in the purification and analytics of ADCs will not transfer to AOCs. This creates new challenges and the need for novel approaches for the purification and characterization of AOCs.

2.1. Conjugation Using Ionic Interactions The addition of linkable groups to oligonucleotides is not trivial and can result in the synthesis of uncommon or expensive nucleotides [6]. However, the negative charge of the oligonucleotide backbone presents an attractive avenue for the preparation of conjugates. To this end, initial fusion or modification of an antibody with a multi-cationic moiety such as protamine or polyarginine allows for a general conjugation process [7,8]. The negative charge of the oligonucleotide backbone and positive charge of the protamine binds the oligonucleotide and protein strongly via ionic interactions. This method is applicable to many types of oligonucleotides since it does not require any chemical modification. Protamine is the most used multi-cationic group since it is an endogenous protein found in sperm and has been used to delay the activity of insulin [9]. The advantage of this method is its simplicity and flexibility in allowing the conjugation of different oligonucleotide sequences and technology with one polycationic protein. This could be useful for evaluating different gene knockdowns with the same antibody and measuring their activity in cells. Another advantage of ionic conjugation is that once the oligonucleotide enters cells, the polycationic complex acts as a lysosomal escape agent. In the lysosome, the polycationic complex acts as a proton sponge, and chloride ions diffuse inside to compensate for the charge imbalance causing osmotic swelling, which results in a leakier membrane [10]. This lysosomal escape is important, since oligonucleotides are not very membrane-permeable, and this allows for more oligonucleotides to enter the cytosol [10]. However, the main drawback of this method is that the interactions are ionic and thus, reversible. The conjugate could potentially be unstable, especially under changing pH or salt concentration. Another drawback of ionic interactions is the difficulty in determining the OAR, which is usually accomplished by fluorescence or PCR amplification. The first example of antibody-mediated delivery of siRNA was published in 2005 by the Liberman group [7]. In this work, they prepared an antibody-protamine fusion J. Clin. Med. 2021, 10, x FOR PEER REVIEW 4 of 17

are ionic and thus, reversible. The conjugate could potentially be unstable, especially under changing pH or salt concentration. Another drawback of ionic interactions is the difficulty in determining the OAR, which is usually accomplished by fluorescence or PCR J. Clin. Med. 2021, 10, 838 4 of 17 amplification. The first example of antibody-mediated delivery of siRNA was published in 2005 by proteinthe Liberman and used group it to [7]. deliver In this siRNA work, against they pr theepared HIV-1 an capsidantibody-protamine gene gag. Using fusion a fluorescein protein isothiocyanateand used it to deliver (FITC)-labeled siRNA against oligonucleotide, the HIV-1 capsid it was gene observed gag. Using that each a fluorescein protamine isothi- could bindocyanate around (FITC)-labeled six siRNA molecules.oligonucleotide, The antibodyit was observed fragment that targeted each protamine the HIV-1 could envelope, bind whicharound resulted six siRNA in themolecules. inhibition The of antibody HIV replication fragment intargeted only HIV-infected the HIV-1 envelope, primary which T-cells inresulted vitro. Since in the there inhibition are no ofin HIV vivo replicationmodels for in HIV,only aHIV-infected cancer cell lineprimary (B16) T-cells was transfected in vitro. withSince the there HIV are envelope, no in vivo and models siRNA for inhibiting HIV, a cancer oncogenes cell line such (B16) as was c-myc, transfected MDM2 andwith VEGFthe HIV envelope, and siRNA inhibiting oncogenes such as c-myc, MDM2 and VEGF were were prepared. The power of protamine conjugation was clearly exemplified, as three prepared. The power of protamine conjugation was clearly exemplified, as three different different constructs could be generated rapidly using this conjugation method. Using a constructs could be generated rapidly using this conjugation method. Using a fluorescent- fluorescent-labeled oligonucleotide, it was found that a trastuzumab-based AOC was labeled oligonucleotide, it was found that a trastuzumab-based AOC was stable, and the stable, and the siRNA was selectively delivered to ErbB2-expressing cell lines. siRNA was selectively delivered to ErbB2-expressing cell lines. Following up on this discovery, a subsequent report used a human protamine fusion Following up on this discovery, a subsequent report used a human protamine fusion protein to silence genes in cells. The approach was to use AOCs to transfect cell lines, protein to silence genes in cells. The approach was to use AOCs to transfect cell lines, which was otherwise difficult to accomplish. One target of interest was the lymphocyte- which was otherwise difficult to accomplish. One target of interest was the lymphocyte- function-associated antigen-1 (LFA-1), where selected genes could be silenced or modified function-associated antigen-1 (LFA-1), where selected genes could be silenced or modified in primary lymphocytes, monocytes and dendritic cells [11]. The internalization capacity in primary lymphocytes, monocytes and dendritic cells [11]. The internalization capacity of antibodies allowed gene silencing in hard-to-transfect cells in vitro. The AOC was also of antibodies allowed gene silencing in hard-to-transfect cells in vitro. The AOC was also ableable to to direct direct siRNAsiRNA inin LFA-1 engrafted lungs lungs of of SCID SCID mice mice as as an an inin vivo vivo proofproof of ofconcept. concept. AA protamine protamine anti-EGFRanti-EGFR antibody was was prepared prepared by by the the Baumer Baumer group, group, who who showed showed thatthat an an siRNAsiRNA towards towards KRAS KRAS showed showed activity activity both both in vitroin vitro andand in vivoin vivo (Figure(Figure 2) [12].2)[ Of12 ]. Ofnote, note, KRAS KRAS has has been been a notoriously a notoriously difficult difficult oncogene oncogene to inhibit to inhibit using standard using standard small-mol- small- moleculeecule approaches approaches [12]. [12 The]. The Baumer Baumer group group compared compared protamine protamine linked linked to toboth both cysteine cysteine andand lysine lysine versus versus an an antibody antibody fusion fusion construct. construct. The The activity activity was was similar similar between between the the fusion fu- antibodysion antibody and the and chemically the chemically modified modified ones, ones, but thebut linkerthe linker modified modified approach approach was was more flexiblemore flexible [13]. [13].

N O O N O SO3H O O O Sulfo-SMCC N H N O Protamine O

O N S H N O O Reduction Protamine Conjugate

O N S H N O O AOC FigureFigure 2. 2.Protamine Protamine conjugatedconjugated using Sulfo-SMCC. Sulfo-SMCC.

Similarly, a SMCC-protamine antibody-siRNA conjugate was reported to inhibit TRIM24, which is overexpressed in prostate cancer [14]. An antibody towards prostate- speciﬁc membrane antigen (PSMA) was used to show selective internalization of FAM- siRNA [14]. The PSMA AOC showed potent activity in vitro and in vivo compared to negative controls. In another approach that utilized ionic interaction conjugation, lysine residues on an antibody were initially derivatized with Traut’s reagent and further elaborated into a disulﬁde polyarginine conjugation handhold (Figure3). A STAT-3 siRNA sequence was J. Clin. Med. 2021, 10, x FOR PEER REVIEW 5 of 17 J. Clin. Med. 2021, 10, x FOR PEER REVIEW 5 of 17

Similarly, a SMCC-protamine antibody-siRNA conjugate was reported to inhibit Similarly, a SMCC-protamine antibody-siRNA conjugate was reported to inhibit TRIM24, which is overexpressed in prostate cancer [14]. An antibody towards prostate- TRIM24, which is overexpressed in prostate cancer [14]. An antibody towards prostate- specific membrane antigen (PSMA) was used to show selective internalization of FAM- specific membrane antigen (PSMA) was used to show selective internalization of FAM- siRNA [14]. The PSMA AOC showed potent activity in vitro and in vivo compared to siRNA [14]. The PSMA AOC showed potent activity in vitro and in vivo compared to negative controls. J. Clin. Med. 2021, 10, 838 negative controls. 5 of 17 In another approach that utilized ionic interaction conjugation, lysine residues on an In another approach that utilized ionic interaction conjugation, lysine residues on an antibody were initially derivatized with Traut’s reagent and further elaborated into a di- antibody were initially derivatized with Traut’s reagent and further elaborated into a disulfide polyarginine conjugation handhold (Figure 3). A STAT-3 siRNA sequence was de- deliveredsulfide polyarginine using an AOC conjugation targeting handhold the Lewis-Y (Figure antigen 3). A STAT-3 (Figure siRNA3)[ 15]. sequence The polyarginine was delivered using an AOC targeting the Lewis-Y antigen (Figure 3) [15]. The polyarginine con- constructlivered using delivered an AOC siRNA targeting specifically the Lewis-Y to cancer antigen cells (Figure (A431) 3) that [15]. overexpressed The polyarginine the con- Lewis- struct delivered siRNA specifically to cancer cells (A431) that overexpressed the Lewis-Y Ystruct antigen delivered and resulted siRNA specifically in a 70% knockdown to cancer cells of (A431) STAT-3. that In overexpressed comparison, athe conjugate Lewis-Y in antigen and resulted in a 70% knockdown of STAT-3. In comparison, a conjugate in which whichantigen the and oligonucleotide resulted in a 70% was knockdown covalently of linked STAT-3. to the In comparison, antibody was a conjugate also prepared in which using the oligonucleotide was covalently linked to the antibody was also prepared using disul- disulfidethe oligonucleotide chemistry was without covalently polyarginine. linked to Interestingly,the antibody was the covalentalso prepared conjugate using wasdisul- not fide chemistry without polyarginine. Interestingly, the covalent conjugate was not effica- fide chemistry without polyarginine. Interestingly, the covalent conjugate was not effica- efficaciouscious on its on own its own and and required required the theaddition addition of ofchloroquine, chloroquine, which which promoted promoted lysosomal lysosomal cious on its own and required the addition of chloroquine, which promoted lysosomal escape.escape. ItIt was speculated speculated that that the the superior superior activity activity of the of thepolyarginine polyarginine conjugate conjugate was be- was escape. It was speculated that the superior activity of the polyarginine conjugate was be- becausecause it ithelped helped the the oligonucleotide oligonucleotide escape escape the the endosomes. endosomes. This This work work demonstrates demonstrates how how cause it helped the oligonucleotide escape the endosomes. This work demonstrates how ionicionic conjugation conjugation cancan benefitbenefit the activity of of the the AOC AOC by by promoting promoting endosomal endosomal escape. escape. ionic conjugation can benefit the activity of the AOC by promoting endosomal escape. H N NH H N NH NH NO O 2 2 NH NO S O NH 2 2 S N 2 S H NH2 N S NH2 N O -Cl+H2N S H N NH O 9 -Cl+H2N S 2 (Npys)-Cys-r9 9 NH2 NH (Npys)-Cys-r9 NH NH NH2 NH SH NH S S Cys-r9 NH S S Cys-r9 HN SH HN S S Cys-r 9 HN S S Cys-r HN HN 9 HN AOC AOC FigureFigure 3.3. PolyargininePolyarginine conjugation. Figure 3. Polyarginine conjugation. TheThe Schultz Schultz group group followed followed a similara similar strategy strategy to createto create a polyarginine a polyarginine copolymer copolymer using The Schultz group followed a similar strategy to create a polyarginine copolymer RAFTusing polymerizationRAFT polymerization (Figure (Figure4)[ 16]. 4) One [16]. end One of end the of polymer the polymer contained contained a hydroxylamine, a hydrox- using RAFT polymerization (Figure 4) [16]. One end of the polymer contained a hydrox- whichylamine, formed which an formed oxime an bond oxime by bond condensation by condensation with thewith non-natural the non-natural amino amino acid acid para- ylamine, which formed an oxime bond by condensation with the non-natural amino acid acetylphenylalaninepara-acetylphenylalanine (pAF). (pAF). The cationicThe cationic polymer polymer could could then then react react with with the the negatively nega- para-acetylphenylalanine (pAF). The cationic polymer could then react with the nega- chargedtively charged backbone backbone of oligonucleotides. of oligonucleotides. In this In example,this example, anFITC-labeled an FITC-labeled oligonucleotide oligonucle- tively charged backbone of oligonucleotides. In this example, an FITC-labeled oligonucle- wasotide used was to used show to targeted show targeted delivery delivery to cancer to cellscancer using cells a using trastuzumab-modified a trastuzumab-modified antibody. otide was used to show targeted delivery to cancer cells using a trastuzumab-modified antibody. The knockdown activity of the AOC was measured by a PCR assay of mRNA Theantibody. knockdown The knockdown activity of activity the AOC of the was AOC measured was measured by a PCR by assay a PCR of assay mRNA of mRNA levels of levels of GAPDH, the siRNA target. GAPDH,levels of GAPDH, the siRNA the target. siRNA target. NH NH HN NH2 HN NH2

HN O O HN O O O O O * H2N N N O O H O H 22 55* H2N N 3 N H H HN22 55O 3 HN O

O N O N O O N O N O

AOC AOC Figure 4. Polyarginine copolymer conjugation. FigureFigure 4.4. PolyargininePolyarginine copolymer conjugation. conjugation. Using electrostatic interactions to conjugate oligonucleotides is an ingenious and Using electrostatic interactions to conjugate oligonucleotides is an ingenious and generalizableUsing electrostatic method that interactions is flexible to across conjugate different oligonucleotides types of oligonucleotides. is an ingenious An and inter- gen- generalizable method that is flexible across different types of oligonucleotides. An inter- eralizableesting advantage method of that ionic is ﬂexible conjugation across is differentthat the polycations types of oligonucleotides. help the oligonucleotide An interesting es- esting advantage of ionic conjugation is that the polycations help the oligonucleotide es- advantagecape the endosomes of ionic conjugation or lysosomes. isthat Characterizati the polycationson of these help conjugates the oligonucleotide with respect escape to the the endosomescape the endosomes or lysosomes. or lysosomes. Characterization Characterizati of theseon of conjugates these conjugates with respect with respect to the numberto the of oligonucleotides attached is usually accomplished by using PCR or a ﬂuorescent-labeled oligonucleotide. However, more comprehensive studies are needed to gain further insight into the in vivo stability, rate of release and ADME properties.

2.2. Avidin-Based Conjugation In a similar approach, AOCs have also been successfully generated using the strong interactions between a biotin-labeled oligonucleotide and avidin (Figure1B) [ 17]. A conjugate generated using this technique showed gene inhibition which was observed by transfecting human 293 cells with a luciferase gene. An antibody to the human insulin receptor showed that maximal inhibition of 90% was achieved after 48 h, and no inhibition J. Clin. Med. 2021, 10, 838 6 of 17

was observed after 7 days by measuring luciferase activity. The advantage of avidin-based conjugation is the in vivo stability of the resultant conjugates. While polycationic complexes can aggregate in vivo due to changes in saline concentration, biotin-complexes are much more resistant to such effects [18]. Another approach used neutravidin as a linkable group for antibodies [19]. A podocyte- directed sheep divalent IgG was linked with neutravidin through cysteine, and protamine was conjugated to the antibody using a biotin tag [19]. An AOC was prepared using electrostatic interactions as described in Section 2.1. This AOC had increased in vivo efficacy found only with the directed siRNA but not with a random siRNA control. The goal of the study was to show that the antibody could be used to knockdown genes selectively in specific tissues. The future value of this approach would be to knockdown genes in specific tissues without generating the systemic effects of a knockdown mice model. Since it is expensive and time-consuming to develop transgenic mice, delivery of siRNA using antibodies offers a more general, easier approach to model disease states. Although this is an efficient and practical method to conjugate, it still involves chemical modification of the antibody and the oligonucleotide or protamine with either biotin or streptavidin. Even if the bond between streptavidin and biotin is strong and efficient, this is a hybrid method that involves steps as described in Section 2.1 as well as those in the direct conjugation method described in the next section. Although it offers a stable bond, this method of generating AOCs is not as general as protamine and usually requires as many steps as other conjugation methods.

2.3. Direct Conjugation The direct conjugation method is more analogous to ADCs in which a linkable group is added to the oligonucleotide and conjugated directly to a lysine, cysteine, or an engineered amino acid of the antibody. This method allows the use of the same linker versatility found in ADCs such as cleavable, disulfide, and non-cleavable linkers [20]. This method also has the advantage that linkers are smaller and have less impact on the overall conjugate, compared to the larger protamine or avidin complexes. On the other hand, each oligonucleotide construct must contain a linker handhold and be chemically modified to attach the linker. The linker technology must be compatible and stable in the presence of DNA or RNA and their double-strand annealing process. The linker in the siRNA is usually incorporated into the sense strand rather than the anti-sense strand. The reasoning behind selecting the sense strand is that the anti-sense strand is the one used in the RNA-induced silencing complex (RISC) that results in gene silencing [21]. Modification of the sense-strand removes the chances that the residual linker group or the modified nucleotide will interfere with the siRNA binding to RISC. AOCs prepared by the direct conjugation method do not incorporate lysosomal escape agents compared to conjugates prepared using the ionic method discussed in Section 2.1. As a result, this could potentially lead to a slow escape of the oligonucleotide from the lysosome and thus, could impact the activity of the AOC. A powerful method to control DAR in ADCs is the introduction of an engineered site-specific cysteine on the antibody, which improves the overall properties of the ADC in vivo [22]. The site-specific cysteine engineered antibodies developed by Genentech are named Thiomab™. Analogous to ADCs, various Thiomab™ antibodies were conjugated to 21-mer oligonucleotides using SMCC and SPDB type linkers (Figure5)[ 23]. The addition of the oligonucleotides added ~1/10th (for OAR =1) or ~1/6th (for OAR = 2) of the antibody weight and thus enabled the determination of OAR by MS. The AOCs were generated with oligonucleotides that contained modified nucleosides to improve serum stability. Using a fluorescent-labeled sequence, the internalization of the AOCs to the target cells was observed. Interestingly, only certain conjugates exhibited gene silencing activity, though they all internalized. J. Clin. Med. 2021, 10, x FOR PEER REVIEW 7 of 17

J. Clin. Med. 2021, 10, x FOR PEER REVIEW 7 of 17

in vivo [22]. The site-specific cysteine engineered antibodies developed by Genentech are namedin vivo Thiomab™. [22]. The site-specific Analogous cysteine to ADCs, engi variouneereds antibodiesThiomab™ developed antibodies by were Genentech conjugated are tonamed 21-mer Thiomab™. oligonucleotides Analogous using to SMCC ADCs, andvariou SPDBs Thiomab™ type linkers antibodies (Figure were 5) [23]. conjugated The addi- tionto 21-mer of the oligonucleotidesoligonucleotides using added SMCC ~1/10th and (for SPDB OAR type =1) linkers or ~1/6th (Figure (for 5) OAR[23]. The = 2) addi- of the tion of the oligonucleotides added ~1/10th (for OAR =1) or ~1/6th (for OAR = 2) of the antibody weight and thus enabled the determination of OAR by MS. The AOCs were gen- antibody weight and thus enabled the determination of OAR by MS. The AOCs were generated with oligonucleotides that contained modified nucleosides to improve serum sta- erated with oligonucleotides that contained modified nucleosides to improve serum stability. Using a fluorescent-labeled sequence, the internalization of the AOCs to the target bility. Using a fluorescent-labeled sequence, the internalization of the AOCs to the target cells was observed. Interestingly, only certain conjugates exhibited gene silencing activity, J. Clin. Med. 2021, 10, 838 cells was observed. Interestingly, only certain conjugates exhibited gene silencing activity,7 of 17 though they all internalized. though they all internalized.

O O O N O O N O O N O NO N H O N NH O O N SMCC OO O SMCC O O O H N 2 H N or or O 2 O O O OO S S N N S O N N S S O O N S S N O O N S HN SPDBSPDB O H N N S S H H N N O O O O oror

O O HSHS SHSH S S S S N N H H FigureFigureFigure 5. 5. DirectDirect Direct conjugate conjugateconjugate method method to oligonucleotide amine. amine.

UsingUsingUsing the thethe direct directdirect conjugation conjugation method method withwith with aa a variety variety variety of of of different different different linker linker types, types, the the Sugo Sugo Sugo

groupgroupgroup was waswas able able to to targettarget skeletalskeletal and cardiaccardiacrdiac muscle muscle with withwith siRNA siRNAsiRNA (Figure (Figure(Figure 6)6 )[6) [24]. [24].24].The The amountamountamount of ofof siRNA siRNA siRNA present present present waswas was determined determined usinusin usinggg real-time real-time real-time polymerase polymerase polymerase chain chain reaction chain reaction reaction (RT- (RT- PCR).(RT-PCR).PCR). The The stoichiometry The stoichiometry stoichiometry ofof siRNAsiRNA of siRNA load load(OAR) (OAR) variedvaried varied between between between 1.0 1.0 to to 1.02.2 2.2 toper per 2.2 Fab’. Fab’. per Fab’.

H H2N N H H2N N O O H O H O O N O N O H N H N H O H N H N O N N O N O O N O N P O H P O H H OO N N OO N O O O O -O O -O O P O O O P O O O -O O O -O O non-cleavable maleimide cleavable maleimide non-cleavable maleimide cleavable maleimide O N H O O O H O N S N N S O N O P O H S P O H S N O N S OO H -O O -O NO S NO O P O S P O S N O H -O O -O O O Direct Disulfide Linker Maleimide Disulfide Linker Direct Disulfide Linker Maleimide Disulfide Linker Figure 6. Conjugation through phosphate backbone. Figure 6. ConjugationConjugation through through phosphate backbone. Another method of direct conjugation uses the DBCO-azide click or otherwise called the Anotherstrain-promotedAnother method method azide-alkyne of of direct direct conjugation conjugation cycloaddition uses uses (SPAAC)the the DBCO-azide DBCO-azide reaction clickto generate or otherwise otherwise conjugates called thetheas strain-promotedshown strain-promoted in Figure 7.azide-alkyne azide-alkyne The advantages cycloaddition cycloaddition of this method (SPAAC) (SPAAC) are that reaction reaction one batch to to generate generate of antibody conjugates conjugates with asasthe shown shown same inlinker-antibody in Figure Figure 77.. The ratio advantages can be functionalized of thisthis methodmethod and areare different thatthat oneone oligonucleotides batchbatch ofof antibodyantibody can withwithbe thetheconjugated same same linker-antibody linker-antibody orthogonally. ratio Varying ratio can can bethe be functionalized equivale functionalizednts of andDBCO and different differentand/or oligonucleotidesazide oligonucleotides modified oligo- can can be conjugatedbenucleotide conjugated orthogonally.resulted orthogonally. in different Varying Varying higher-molec the equivale the equivalentsular-weightnts of DBCO of species DBCO and/or (OAR), and/or azide as expected.modified azide modified Fastoligo- nucleotideoligonucleotideprotein liquid resulted chromatography resulted in different in different higher-molec (FPLC) higher-molecular-weight of Cyular-weight5-modified oligonucleotide species species (OAR), (OAR), was as expected. used as expected. to de- Fast proteinFasttermine protein liquid the average liquid chromatography chromatography OAR, which (FPLC) was (FPLC)found of Cy to5-modified of be Cy5-modified 1.2 when oligonucleotide four oligonucleotideequivalents was of theused was azide- to used de- terminetofunctionalized determine the average the oligonucleotide average OAR, OAR,which were which was used found was [25]. foundto beThe 1.2 to reverse bewhen 1.2 four whenreaction equivalents four in equivalentswhich of the the azide azide- of the functionalizedazide-functionalizedwas conjugated oligonucleotide to the oligonucleotide antibody, were and used a were DBCO-modified [25]. used The [25 ].reverse The oligonucleotide reverse reaction reaction in waswhich inused, whichthe wasazide the wasazidealso conjugated shown was conjugated to be to successful the toantibody, the [26]. antibody, AOCsand a andgeneDBCO-modified arated DBCO-modified using click oligonucleotide chemistry oligonucleotide have was used used, was modi- used,was alsowasfied shownalso 2’-deoxy-2’-fluoro- shown to be tosuccessful be successfulβ-D -arabinonucleic[26]. AOCs [26]. AOCs gene acidrated generated (FANA) using click using oligonucleotides chemistry click chemistry have to usedgenerate have modi- used more serum stable conjugates [27]. The click method of direct conjugation is often fol- fiedmodified 2’-deoxy-2’-fluoro- 2’-deoxy-2’-fluoro-β-D-arabinonucleicβ-D-arabinonucleic acid acid (FANA) (FANA) oligonucleotides oligonucleotides to to generate generate moremore serum stablestable conjugatesconjugates [ 27[27].]. The The click click method method of directof direct conjugation conjugation is often is often followed fol- in the preparation of AOCs that are used in diagnostics [28]. Due to the orthogonality of click chemistry, and the compatibility with the oligonucleotide, this is a robust and popular method for conjugation. The hydrophobicity of DBCO and BCN reagents does not pose a challenge due to the water solubility of the oligonucleotide.

J. Clin. Med. 2021, 10, x FOR PEER REVIEW 8 of 17 J. Clin. Med. 2021, 10, x FOR PEER REVIEW 8 of 17 J. Clin. Med. 2021, 10, x FOR PEER REVIEW 8 of 17

lowed in the preparation of AOCs that are used in diagnostics [28]. Due to the orthogo- lowed in the preparation of AOCs that are used in diagnostics [28]. Due to the orthogonality of click chemistry, and the compatibility with the oligonucleotide, this is a robust nalitylowed of in click the preparationchemistry, andof AOCs the compatibility that are used with in diagnostics the oligonucleotide, [28]. Due thisto the is orthogo-a robust J. Clin. Med. 2021, 10, 838 and popular method for conjugation. The hydrophobicity of DBCO and BCN reagents8 of 17 andnality popular of click method chemistry, for conjugation.and the compatibility The hydrophobicity with the oligonucleotide, of DBCO and thisBCN is reagentsa robust does not pose a challenge due to the water solubility of the oligonucleotide. doesand popularnot pose methoda challenge for dueconjugation. to the water The solubility hydrophobicity of the oligonucleotide. of DBCO and BCN reagents does not pose a challenge due to the water solubility of the oligonucleotide.

O O O O NO N N O O N O O N N O N O O O O N3 O N N O N O N O N 3 N NN NH O N N N 2 O N N3 O N N NH2 NH NH NHS-DBCO H O N Azide-Oligo H O N N NH2 NHS-DBCO N O Azide-Oligo N O H H NHS-DBCO O Azide-Oligo O Figure 7. Azide-alkyne click chemistry based conjugation. FigureFigure 7.7. Azide-alkyneAzide-alkyne click chemistry chemistry based based conjugation. conjugation. Figure 7. Azide-alkyne click chemistry based conjugation. Conjugation of oligonucleotides can also use the same site-specific conjugation tech- ConjugationConjugation of oligonucleotides can can also also use use the the same same site-specific site-speciﬁc conjugation conjugation tech- tech- nology used in the generation of homogeneous ADCs. Thus, an AOC with an OAR of 2 nologynologyConjugation usedused in in the the of generation generation oligonucleotides of of homogeneous homogeneous can also use ADCs. ADCs. the same Thus, Thus, site-specific an an AOC AOC with with conjugation an an OAR OAR of tech- of 2 was2 was prepared using site-specific conjugation by the addition of an azide-containing spacer preparedwasnology prepared used using in using site-speciﬁc the generationsite-specific conjugation of conjugation homogeneous by the by addition the ADCs. addition ofThus, an of azide-containing an azAOCide-containing with an spacerOAR spacer of and 2 a and a transglutaminase compatible linker (Figure 8) [29]. The addition of a DBCO func- transglutaminaseandwas aprepared transglutaminase using compatible site-specific compatible linker conjugation (Figurelinker (Figure8 )[by29 the]. 8) addition The [29]. addition The of anaddition ofazide-containing a DBCO of a DBCO functionalized spacer functionalized oligonucleotide could then undergo a click reaction to generate a homogeneous oligonucleotidetionalizedand a transglutaminase oligonucleotide could then compatible could undergo then alinker undergo click (Figure reaction a click 8) to reaction[29]. generate The to addition generate a homogeneous of a homogeneousa DBCO AOC func- with AOC with OAR of 2 by measuring MW shift by SDS-PAGE. OARAOCtionalized ofwith 2 by oligonucleotideOAR measuring of 2 by measuring MW could shift then byMW SDS-PAGE.undergo shift by a SDS-PAGE.click reaction to generate a homogeneous

AOCO with OAR ofO 2 by measuringO MW shift by SDS-PAGE. O H O H O a) H2N O HN HN N N NH a) H2N O O N O N O 2 NH 6 H NH H NH a) NHAc O O 2 H2N H O6 N OHH N NHAc N O N O NH2 H OHH NHAc 6 O O Transglutamidase OH Transglutamidase N3 O N3 O O O O O Transglutamidase H H b) HNO O O N O N O O N3 H H b) HN O N O N O N N O NH2 NH H NH H NH OOH b) OAR 2 NHAc 6 O O 2 OH HN H O N OHH N OAR 2 NHAc N 6 O N O NH2 H OHH OH N OAR 2 NHAc 6 O O N OH O N N O N N N N O N N O N N N O N O

Figure 8. Transglutaminase-click chemistry. FigureFigure 8. Transglutaminase-click chemistry. chemistry. Figure 8. Transglutaminase-click chemistry. Another interesting method for direct conjugation uses a disuccinimidyl linker (Fig- AnotherAnother interestinginteresting method method for for direct direct conjugation conjugation uses uses a disuccinimidyla disuccinimidyl linker linker(Figure (Fig-9). ure 9).Another Even though interesting the linkermethod used for isdirect homobifunctional, conjugation uses when a disuccinimidyl an excess of the linker linker (Fig- is Evenure 9). though Even though the linker the usedlinker is used homobifunctional, is homobifunctional, when when an excess an excess of the of linker the linker is added is ureadded 9). toEven an amine-modifiedthough the linker oligonucleotide, used is homobifunctional, the major product when anis oneexcess linker/oligonucle- of the linker is toadded an amine-modified to an amine-modified oligonucleotide, oligonucleotide, the major the major product product is one is one linker/oligonucleotide. linker/oligonucle- addedotide. The to an intermediate amine-modified linker-derivatized oligonucleotide, oligonucleotide the major product is conjugated is one linker/oligonucle- directly to the ly- Theotide. intermediate The intermediate linker-derivatized linker-derivatized oligonucleotide oligonucleotide is conjugated is conjugated directly directly to the to the lysine ly- of otide.sine of The antibodies intermediate [6]. The linker-derivatized modification of oligonucleotidethe antibody is determinedis conjugated by directly SDS-PAGE to the and ly- antibodiessine of antibodies [6]. The [6]. modification The modification of the antibodyof the antibody is determined is determined by SDS-PAGE by SDS-PAGE and MALDI- and sineMALDI-TOF. of antibodies The [6].main The advantage modification of this of methodthe antibody is that is it determined is relatively by inexpensive SDS-PAGE com- and TOF.MALDI-TOF. The main The advantage main advantage of this method of this method is that itis isthat relatively it is relatively inexpensive inexpensive compared com- to MALDI-TOF.pared to other The direct main conjugation advantage methods. of this method Amine-containing is that it is relatively DNA sequences inexpensive are easier com- otherpared direct to other conjugation direct conjugation methods. methods. Amine-containing Amine-containing DNA sequences DNA sequences are easier are toeasier access paredto access to othercommercially direct conjugation than thio- methods.or azide-mo Amine-containingdified sequences. DNA The sequencesauthors determined are easier commerciallyto access commercially than thio- than or azide-modified thio- or azide-mo sequences.dified sequences. The authors The determined authors determined that it is the tothat access it is the commercially most cost-efficient than thio- and or rapid azide-mo methoddified to generatesequences. AOCs The andauthors only determined requires 10 mostthat it cost-efficient is the most cost-efficient and rapid method and rapid to generate method AOCs to generate and only AOCs requires and only 10 µ requiresg of antibody. 10 thatµg of it antibody.is the most This cost-efficient method was and used rapid to methodgenerate to AOCs generate for use AOCs in diagnostics. and only requires 10 Thisµg of method antibody. was This used method to generate was used AOCs to generate for use in AOCs diagnostics. for use in diagnostics. µg of antibody. This method was used to generate AOCs for use in diagnostics.

O O O O N O O O NH2 OO N N O O O NH2 O O N O O NH O N OO O O O 2 O O O O N O O O NO O O H2N O O O N N N N O 6O H O O H2N O O O N NH 6 NH commercially available N 6 H H 6 H H2N O O N commercially available 6 N N O H H 6 H commercially available Figure 9. Disuccinimidyl linker. Figure 9. Disuccinimidyl linker. FigureFigure 9.9. Disuccinimidyl linker. linker.

Using a non-natural pAF group, an AOC with trastuzumab was generated [30]. This AOC was used as a PCR primer to detect the presence of Her2+ samples with greater

precision and sensitivity. This showcases the versatility of the orthogonal oxime linker chemistry as a protein labeling tool. Recently, a site-speciﬁc lysine conjugation has been described in which a beta-lactam can react selectively to an H38C2 antibody as shown in Figure 10[ 31]. This hidden lysine J. Clin. Med. 2021, 10, x FOR PEER REVIEW 9 of 17

Using a non-natural pAF group, an AOC with trastuzumab was generated [30]. This AOC was used as a PCR primer to detect the presence of Her2+ samples with greater precision and sensitivity. This showcases the versatility of the orthogonal oxime linker J. Clin. Med. 2021, 10, 838 chemistry as a protein labeling tool. 9 of 17 Recently, a site-specific lysine conjugation has been described in which a beta-lactam can react selectively to an H38C2 antibody as shown in Figure 10 [31]. This hidden lysine has aa lowerlower pKa pKa (6.0) (6.0) since since it isit inis anin accessiblean accessible hydrophobic hydrophobic pocket, pocket, which which in turn in increases turn in- bycreases orders by of orders magnitude of magnitude its reactivity its reactivity towards towards electrophiles electrophiles [32]. A targeting [32]. A targeting extra variable extra domainvariable (Her2domain or (Her2 BCMA) or BCMA) is added is to added the H38C2 to the antibody,H38C2 antibody, which generateswhich generates the AOCs. the TheAOCs. conjugation The conjugation was achieved was achieved in good in yields good and yields resulted and resulted in a conjugate in a conjugate with an OARwith an of 2OAR using of SDS-PAGE.2 using SDS-PAGE. Although Although no cytotoxicity no cytoto wasxicity exhibited was exhibited by this conjugate, by this conjugate, silencing wassilencing observed was observed in vitro. in vitro.

NH H OH O 2 O N Linker-siRNA N 5' 3' O N O O O P O H H O O O N N NH -O O O O O- O O O O P O 5' 3' N HO O O N O O H H N N O O O

Linker-siRNA FigureFigure 10.10. Beta-Lactam conjugation.conjugation.

Finally, non-engineerednon-engineered chemical site-specificsite-specific conjugation technology has been recently reported using Fc affinityaffinity [[33].33]. An advantage ofof thisthis technologytechnology isis thatthat itit generatesgenerates homogeneous DARDAR usingusing standard standard chemistries chemistries without without the the need need for for antibody antibody engineering, engineer- therebying, thereby simplifying simplifying the analytics the analytics [34]. [34]. This Th approachis approach was appliedwas applied to AOCs to AOCs and could and could help simplifyhelp simplify the conjugation the conjugation and analyticsand analytics [35]. [35]. To summarize, thethe linkerlinker andand antibodyantibody engineeringengineering usedused inin ADCsADCs cancan applyapply toto AOCs when using the direct conjugationconjugation method.method. The direct conjugation method seems to offeroffer the greatest flexibility,flexibility, butbut judiciousjudicious choicechoice ofof linkerlinker compatibilitycompatibility andand positionposition onon thethe oligonucleotide are still required. We expectexpect thatthat the direct conjugation willwill be a preferred method to prepare AOCsAOCs goinggoing forward.forward. 2.4. DNA Origami or Hybridization Conjugation 2.4. DNA Origami or Hybridization Conjugation AOCs with double-strand oligonucleotides could be obtained by a hybridization AOCs with double-strand oligonucleotides could be obtained by a hybridization ap- approach. A single strand oligonucleotide is first conjugated to an antibody and a comple- proach. A single strand oligonucleotide is first conjugated to an antibody and a complementary strand is hybridized to form a double-strand AOC. The kinetics of double-strand mentary strand is hybridized to form a double-strand AOC. The kinetics of double-strand hybridization is estimated to be >106 M·s-1, which is orders of magnitude faster than click hybridization is estimated to be >106 M·s-1, which is orders of magnitude faster than click chemistry and other conjugation methods [36,37]. The rate and specificity of hybridization chemistry and other conjugation methods [36,37]. The rate and specificity of hybridization allows one to build defined structures and incorporate chemotherapeutics. One example wasallows to useone cetuximabto build defined as a targeting structures agent and forincorporate the delivery chemotherapeutics. of doxorubicin, whichOne example can be deliveredwas to use to cetuximab cells by intercalation as a targeting into agent the oligonucleotide for the delivery double-strand of doxorubicin, (Figure which 11 can)[38 be]. Thisdelivered strategy to cells allowed by intercalation the incorporation into the of oligonucleotide up to 480 molecules double-strand of doxorubicin/structure (Figure 11) [38]. withThis threestrategy targeting allowed antibodies. the incorporation of up to 480 molecules of doxorubicin/structure with Thisthree approach targeting hasantibodies. been modified where the AOC is a carrier for a cytotoxin like an ADC (Figure 12A). A double-strand oligonucleotide was used to intercalate about eight doxorubicin per DNA molecule, which significantly increased the delivery of doxorubicin to cells [39]. In another instance, an AOC containing a single-strand oligonucleotide was formed using a direct attachment method, and separately, a complementary strain was attached with an ADC drug-linker and hybridized (Figure 12B). Two examples of this approach have been documented, and both have used trastuzumab as the targeting antibody [40,41]. In one example, the first oligonucleotide strand was conjugated to the

antibody using interchain disulﬁdes to give a variety of OAR, which was controlled by adjusting TCEP equivalents used during the reduction step. The complementary strain of DNA was modiﬁed with a thiol or an amine to conjugate MC-Val-Cit-PAB-MMAE, SMCC- J. Clin. Med. 2021, 10, x FOR PEER REVIEW 10 of 17

J. Clin. Med. 2021, 10, 838 10 of 17

DM1 or DM1 directly. AOCs with OAR ranging from 1.9 to 4.6 measured by SDS-PAGE and IEX chromatographyO were obtained and evaluated [40]. In vitro activity increased withNH OAR, and theN activity observed was comparable to T-DM1. In another example, the O single strand oligonucleotide was conjugated using a two-step click conjugation process O through lysine [41]. The complementary strand wasDoxorubicin conjugated to MC-Val-Cit-PAB-MMAE by having a cysteine residue at the 3’ to add the drug-linker. The OAR distribution varied SH Cetuximab-Sulfo-SMCC HS SH between 0 and 5 with an average of 1.9 measured by native MS. The in vitro assay data indicated selective activity towards target positive cells but was around 100-fold less potent

TH than trastuzumab conjugated directlyTHC3 to the linker-payload (Val-Cit-PAB-MMAE).THDC3 The author suggested that this could be due to the negative charge of the oligonucleotide, which Figure 11. Doxorubicin intercalation into a 3D structures. can slow internationalization of the AOC. TheThis length approach of the has oligonucleotide been modified iswhere very the important AOC is fora carrier the hybridization for a cytotoxin approach. like an If theADC oligonucleotide (Figure 12A). sequenceA double-strand is too short, oligonucleotide the duplex was will used not beto stableintercalate enough about in eight plasma, anddoxorubicin if it is too per long, DNA it can molecule, form secondary which significantly structures increased [42]. In the delivery case of intercalation of doxorubicin with doxorubicin,to cells [39]. theIn another length ofinstance, the sequence an AOC determines containing the a single-strand amount of doxorubicin oligonucleotide that was can be carriedformed by using the AOC. a direct Even attachment though the method, kinetics and are separately, higher than a forcomplementary click chemistry, strain the was cost of havingattached a linker-payload with an ADC attacheddrug-linker to anand oligonucleotide hybridized (Figure does 12B). not seem Two to examples favor conjugation of this J. Clin. Med. 2021, 10, x FOR PEER REVIEW 10 of 17 throughapproach hybridization. have been documented, This hybridization and both techniquehave used istrastuzumab very powerful as the for targeting diagnostic anti- use, butbody it seems [40,41]. more In one challenging example, the for first AOCs oligon targeteducleotide towards strand disease. was conjugated to the antibody using interchain disulfides to give a variety of OAR, which was controlled by adjusting TCEP equivalents used during the reduction step. The complementary strain of DNA was modified with a thiol or an amine to conjugate MC-Val-Cit-PAB-MMAE, SMCC-DM1 Oor DM1 directly. AOCs with OAR ranging from 1.9 to 4.6 measured by SDS-

PAGENH and IEX Nchromatography were obtained and evaluated [40]. In vitro activity increased with OAR, andO the activity observed was comparable to T-DM1. In another example,O the single strand oligonucleotide was conjugated using a two-step click conjuga- Doxorubicin tion process through lysine [41]. The complementary strand was conjugated to MC-Val- SH Cetuximab-Sulfo-SMCCCit-PAB-MMAE by having a cysteine residue at the 3’ to add the drug-linker. The OAR HS SH distribution varied between 0 and 5 with an average of 1.9 measured by native MS. The in vitro assay data indicated selective activity towards target positive cells but was around TH THC THDC 100-fold less potent than trastuzumab3 conjugated directly to the linker-payload3 (Val-Cit- PAB-MMAE).FigureFigure 11.11. DoxorubicinDoxorubicin The author intercalation suggested intothat into a athis 3D 3D structures.co structures.uld be due to the negative charge of the oligonucleotide, which can slow internationalization of the AOC. This approach has been modified where the AOC is a carrier for a cytotoxin like an A ADC (Figure 12A). A double-strand oligonucleotide was used to intercalate about eight

doxorubicinDoxorubicin per DNA molecule, which significantly increased the delivery of doxorubicin to cells [39]. In another instance, an AOC containing a single-strand oligonucleotide was formed using a direct attachment method, and separately, a complementary strain was attached with an ADC drug-linker and hybridized (Figure 12B). Two examples of this

B approach have been documented, and both have used trastuzumab as the targeting anti- O body [40,41]. In one example, the first oligonucleotide strand was conjugated to the anti- F body using interchainBCN disulfides to give a variety of OAR, which was controlled by ad- N3 justing TCEP equivalents used during the reduction step. The complementary strain of NH2 NH NH N N 3 N DNAO was modified with a thiol orO an amineN to conjugate MC-Val-Cit-PAB-MMAE, SMCC-DM1 or DM1 directly. AOCs with OAR ranging from 1.9 to 4.6 measured by SDS- PAGE and IEX chromatography were obtained and evaluated [40]. In vitro activityMMAE in- CN creased with OAR, and the activity observed was comparable to T-DM1. In another ex- APN-VC-MMAE SH ample,O the single strand oligonucleotideS was conjugated using a two-step click conjuga- VC-MMAE O NC N tion process through lysine [41]. The complementary strand was conjugated to MC-Val- N N Cit-PAB-MMAE by having a cysteine residue at the 3’ to add the drug-linker. The OAR APN-VC-MMAE distribution varied between 0 and 5 with an average of 1.9 measured by native MS. The FigureFigure 12. 12.(A ()A Intercalation) Intercalationin vitro assay of of doxorubicin doxorubicin data indicated andand ( (selectiveBB)) hybridization activity of of towards the the drug-linker drug-linker target carryingpositive carrying strand. cells strand. but was around 100-fold less potent than trastuzumab conjugated directly to the linker-payload (Val-Cit- PAB-MMAE). The author suggested that this could be due to the negative charge of the oligonucleotide, which can slow internationalization of the AOC.

Doxorubicin

B O

F BCN N3

NH2 NH NH N N 3 N O O N

MMAE CN

APN-VC-MMAE SH O S VC-MMAE O NC N N N APN-VC-MMAE Figure 12. (A) Intercalation of doxorubicin and (B) hybridization of the drug-linker carrying strand.

J. Clin. Med. 2021, 10, 838 11 of 17

2.5. Summary All four methods of conjugation have yielded AOCs that could be characterized and tested in vitro and some in vivo. Most AOCs currently have OARs less than 4, but like ADCs, this could change over time. The overarching observation is that current methods used to determine OAR and distribution still pose a challenge for AOCs. The ability to attach a ﬂuorescent probe to the oligonucleotide does allow measurement of OAR as an average, but without further separation of the species, their distribution is unknown. PCR is a powerful method to determine OAR by how much oligonucleotide is present, but distribution is also unknown. Due to the size and charge of the oligonucleotide, SDS-PAGE and IEX have been used to separate species and determine a distribution. Mass-spectrometry has been used for OAR determination, especially with homogeneous conjugation. General analytical methods for characterization are still a challenge, and as the ﬁeld matures, these will surely become more routine.

3. In Vitro Data This section will cover the use of in vitro data showing gene silencing, cytotoxicity, internalization kinetics, and processing of AOCs. If AOCs follow a similar pattern to ADCs, there could be little correlation between in vitro and in vivo results. Nonetheless, these in vitro studies can help shed some light on the nuances between conjugates. In vitro testing is necessary to understand parameters that play an important role in the in vivo activity of AOCs. When it comes to in vitro mechanisms and controls, oligonucleotides do have some advantages compared to ADCs and other therapeutics. For example, a negative control can be made by just having a random oligonucleotide sequence of the same length and type. The addition of a ﬂuorophore on the sense or anti-sense strand allows for the simultaneous tracking of the therapeutic and measurement of its activity with the same construct. In short, one well-designed AOC could give different levels of information, from cytotoxicity to stability and ﬁnally to imaging with one molecule.

3.1. Evaluating Gene Inhibition The efficacy of an AOC will depend on its ability to inhibit protein production. The ability of the AOC to knockdown a gene can be performed by measuring mRNA [23], Western blotting or incorporating a green fluorescent protein (GFP) into the system. In combination with a fluorophore, the gene silencing activity and trafficking could be cor- related together. A study by Xia et al. showed that by following luciferase activity, the highest-level gene silencing was observed at 48 h, and its silencing activity disappeared at day 7 [17]. Most studies measure gene silencing activity between 24 h and 72 h, since that is typically the time for maximal gene inhibition. At this time, it is not clear what minimum level of inhibition is needed for activity and if it correlates to in vivo data, but measuring gene inhibition appears to be important in vitro assay.

3.2. Internalization and Trafficking The internalization and tracking of AOCs can be accomplished by labeling the oligonucleotide with a fluorophore or other tracking agent (Figure 13). Internalization of AOCs is generally observed for most targets, but escape from the lysosome seems more variable. Studies of different targets and silencing activity found that not all targets lead to silencing even when they are internalized [23]. Investigation by confocal microscopy showed strong co-localization of the AOC and LAMP2, which indicates processing through the endosomal pathway. It was postulated that differences in processing and/or escape from the lysosome can change the activity significantly between targets [23]. This reinforces the notion that some targets might require protamine or other lysosomal escape agents to increase the activity of the AOC. From in vitro studies, lysosomal escape seems to be an important consideration when developing an AOC and a major consideration in the selection of the target and construct design. J. Clin. Med. 2021, 10, x FOR PEER REVIEW 12 of 17

is generally observed for most targets, but escape from the lysosome seems more variable. Studies of different targets and silencing activity found that not all targets lead to silencing even when they are internalized [23]. Investigation by confocal microscopy showed strong co-localization of the AOC and LAMP2, which indicates processing through the endosomal pathway. It was postulated that differences in processing and/or escape from the lysosome can change the activity significantly between targets [23]. This reinforces the notion that some targets might require protamine or other lysosomal escape agents to increase the activity of the AOC. From in vitro studies, lysosomal escape seems to be an J. Clin. Med. 2021, 10, 838 important consideration when developing an AOC and a major consideration in 12the of se- 17 lection of the target and construct design.

Fluorophore FigureFigure 13.13. FluorophoreFluorophore containingcontaining AOC.AOC.

3.3.3.3. CytotoxicityCytotoxicity CytotoxicityCytotoxicity isis aa usefuluseful measurementmeasurement inin oncologyoncology forfor thethe abilityability ofof thethe constructconstruct toto arrestarrest division and and kill kill cells. cells. Since Since AOCs AOCs are are being being investigated investigated in genetic in genetic diseases diseases as well as wellas in as oncology, in oncology, cytotoxicity cytotoxicity assays assays might might not be not the be best the bestmeasurement measurement of activity. of activity. To put To put this into context, the IC of AOCs are generally in the nanomolar range, compared this into context, the IC50 of50 AOCs are generally in the nanomolar range, compared to toADCs, ADCs, which which are are mostly mostly in the in the picomolar picomolar range. range. This This difference difference in activity in activity could could be due be dueto the to lack the lackof lysosomal of lysosomal escape, escape, trafficking trafficking differences differences or stability. or stability. The target The target of the ofoligo- the oligonucleotide could also be important; some cells may be more sensitive to the silencing nucleotide could also be important; some cells may be more sensitive to the silencing of a of a gene than others. No matter the reason, the selection of the antibody target will have gene than others. No matter the reason, the selection of the antibody target will have to to be matched with the activity of the AOC. be matched with the activity of the AOC. Cytotoxicity is an important measurement for AOCs that deliver cytotoxic agents. Cytotoxicity is an important measurement for AOCs that deliver cytotoxic agents. A A doxorubicin intercalated cetuximab AOC showed 2.7 to 7 times more potent activity doxorubicin intercalated cetuximab AOC showed 2.7 to 7 times more potent activity to- towards EGFR+ cell lines than free doxorubicin [39]. An in vitro cytotoxicity assay for wards EGFR+ cell lines than free doxorubicin [39]. An in vitro cytotoxicity assay for a Her2 a Her2 targeting oligonucleotide using DM1 and MMAE showed similar activity and targeting oligonucleotide using DM1 and MMAE showed similar activity and specificity specificity compared with conventional ADCs [40]. An experiment with a trastuzumab compared with conventional ADCs [40]. An experiment with a trastuzumab MMAE con- MMAE containing AOC showed single-digit nanomolar activity but was still about 10 taining AOC showed single-digit nanomolar activity but was still about 10 times less po- times less potent than the comparable ADC [41]. In these two examples, the antibodies were tent than the comparable ADC [41]. In these two examples, the antibodies were different, different, but both targeted Her2-expressing cell lines and contained different cleavable MMAEbut both oligonucleotide targeted Her2-expressing constructs. cell This line illustratess and contained that even different when cleavable releasing MMAE the same ol- MMAEigonucleotide molecule, constructs. with the This same illustrates release mechanism,that even when the releasing oligonucleotide the same chemistry MMAE mol- can impactecule, with the activity. the same Regardless, release mechanism, the activity the of theseoligonucleotide oligo-conjugates chemistry is on can par impact with early the ADCactivity. work Regardless, and remains the promising.activity of these oligo-conjugates is on par with early ADC work and remains promising. 3.4. Stability 3.4. StabilityThe question of stability in blood is an important consideration due to the short half-lifeThe of question oligonucleotides of stability alone in blood in plasma is an important [41]. A study consideration of DNA-based due AOCto the tripledshort half- the half-lifelife of oligonucleotides (5.7 days) compared alone with in plasma oligonucleotide [41]. A study alone of (1.9 DNA-based days) in human AOC tripled plasma the [41 half-]. A cetuximablife (5.7 days) oligonucleotide compared with stabilized oligonucleotid to protaminee alone showed (1.9 stabilitydays) in afterhuman 22 hplasma in serum [41]. [12 A]. Earlycetuximabin vitro oligonucleotidestability studies stabilized indicate to that protamine the oligonucleotide showed stability portion after of the22 h AOC in serum does show[12]. Early enough in vitro stability stability to be dosedstudies in indicate vivo and that have the pharmacological oligonucleotide activity.portion of the AOC does show enough stability to be dosed in vivo and have pharmacological activity. 3.5. Summary Overall, the work presently published indicates that AOCs do offer a stability advantage over oligonucleotides alone, but more stable modified nucleotides are still necessary.

AOCs seem to follow a similar internalization to the lysosome pathway as ADCs. A challenge for AOCs is the endosomal/lysosomal escape of the active oligonucleotide. AOC cannot simply rely on membrane permeability to enter the cytosol, unlike small molecules. The maximum gene silencing seems to occur between 24 h to 72 h in vitro. Current data show the ability of AOCs to inhibit cell growth by the delivery of either siRNA or cytotoxic agents. Overall, AOCs show promising results in vitro, but the concentrations required for activity are higher than their ADC counterparts. With promising in vitro data, the translation to in vivo models will be discussed next. J. Clin. Med. 2021, 10, 838 13 of 17

4. In Vivo Data Conjugates have shown in vitro silencing, but without in vivo data, it is hard to know how much gene silencing is required for activity and if enough of the oligonucleotide can be delivered in xenograft models. AOCs have the same challenges as many other therapeutics in that even if silencing of protein is observed, it is unknown if that will lead to in vivo efficacy due to redundant systems in the cell or low levels of delivery. Thus, assessing the activity of the oligonucleotides alone is difficult. The selection of the antibody, the gene being silenced and the trafficking of targets will all be crucial for in vivo success.

4.1. In Vivo Imaging The ability to add dyes to the oligonucleotide sense and anti-sense strands does allow for in vivo imaging of the AOC and oligonucleotide. Unfortunately, very few studies looking at animal models of the distribution of AOCs have been found. A study performed using a Thiomab-based AOC towards TENB2 and silencing the PPIB gene was dosed 24 mg/kg three times over 3 days. The results showed 33% inhibition of the PPIB gene in mouse tumors [23]. An interesting note was that monitoring of a dye containing AOC showed that only the layers near the vasculature internalized the AOC. The authors speculate that the negative charge of the oligonucleotide repels the negative charge of the membrane, which could prevent penetration through a poorly vascularized tumor. These results also indicate the advantage of labeling the AOCs with a dye on the siRNA sequence to monitor location and activity. The in vivo imaging is an important aspect to investigate further as early results indicate that tumor or tissue penetration is limited. The development of AOCs capable of distributing throughout the entire tumor or tissue could be key in their clinical success.

4.2. Efficacy In general, the gold standard of preclinical data is efficacy in a relevant animal model. No matter how compelling or non-compelling the in vitro data is, programs that show poor in vivo efficacy usually do not progress into development. The limited in vivo data available on AOCs have shown mixed results and will be discussed in more detail below. The reasons for the mixed results are unknown and could include factors such as rescue pathway of inhibition and lack of potency or distribution. This does not mean that the concept of AOCs is flawed; some parameters will need to be optimized in future work. The Lieberman group created an AOC with a cocktail of different gene-silencing siRNAs by ionic conjugation [7]. In mice implanted with a B16 engineered cell line overex- pressing the HIV envelope, an siRNA cocktail to c-myc, VEGF and MDM2 showed in vivo activity. The AOC was dosed to deliver 3 × 80 micrograms of siRNA both intratumorally and intravenously on Days 0, 1 and 3, and the mice were sacrificed after day 9. The tumors were measured daily after 5 days, and at the end of the study, the tumors were collected and weighed. Both injection methods resulted in tumors weighing around 200 mg, while all the controls had weights around 800 mg. This does show the ability of ionic conjugation to deliver multiple siRNA sequences in one construct. In an interesting approach, the Uckun group used bioinformatics to select a gene for inhibition in ALL. An AOC combining a CD19 targeting antibody and an anti-sense oligonucleotide to E2A-PBX1 was prepared using disulfide direct conjugation methodol- ogy [43]. The AOC was microdosed at 93 nmol/kg over 14 days in SCID mice. The mice treated with the AOC doubled their leukemia-free survival time compared to mice dosed with a control. Of note, the model used is resistant to radiotherapy, and this B-lineage ALL is challenging to treat. This a strong example that AOCs can be a general platform in which a gene can be identified, the sequence generated, and the construct tested in vivo without needing to find a small molecule inhibitor. Based on similar reasoning, targets that are difficult to inhibit by small molecules could be rapidly inhibited by siRNA. An AOC using cetuximab and siRNA to KRAS was generated by ionic conjugation. This AOC showed significant tumor growth inhibition J. Clin. Med. 2021, 10, 838 14 of 17

in three KRAS-mutated colon cancer cell lines when dosed at 4 mg/kg twice a week for 3 weeks. The activity was driven by the KRAS-inhibiting AOC as the cetuximab alone and cetuximab with a random oligonucleotide did not show any activity [12]. It is noteworthy that cell line HT29, which is also BRAF-mutated, was resistant to all treatment options. Histological investigation showed that the expression of the proliferation antigen Ki-67 was reduced only in KRAS-silencing groups. This research illustrates that difficult-to-drug targets could be treated using an AOC approach. A research study on using a CD71-targeting AOC developed for the potential treatment of muscle diseases suggested that the AOC could be delivered selectively to the heart and calf muscle [24]. A running test for mice showed that silencing myostatin by intra- muscular injection resulted in a major recovery of running performance. This approach indicates that AOCs could be used specifically for muscular disease and genetic disorders. Delivering oligonucleotides to specific tissues is an important goal that AOC could address. An ASO against MXD3 conjugated to a CD22 antibody showed a significant survival time increase in a Reh xenograft AML model [26]. The animals were dosed twice a week for 3 weeks, and a dose-response could be observed from 0.2 mg/kg to 10 mg/kg. The free antibody was also co-dosed with the free ASO, and a dose response was observed but was significantly less effective than the AOC. Moving to patient-derived samples, a doubling of survival time was observed with doses as low as 0.2 mg/kg, and a greater survival time was noted for the 1 mg/kg dose. This ASO conjugate does look promising since it has a dose response and a clinically relevant dosing schedule. A PSMA AOC that inhibited Trim24 was tested in vivo for efficacy [14]. At a dose of 5 mg/kg five times over 2 weeks, it showed tumor regression compared to a non- binding AOC and PBS controls. Interestingly, the negative random siRNA-control showed significant inhibition, but not as much as the active siRNA AOC. The author speculated that ADCC activity might be an important component of the observed activity, and the targeting PSMA antibody without siRNA was not reported. The role of the siRNA is still to be determined, but this is an encouraging result.

4.3. Pharmacokinetics (PK) A study looking specifically at the stability and PK of AOCs has recently been published by the Rader group along with Alnylam. This study looked at the PK profile of the AOC and the naked mAb [31]. The sense strand of the oligonucleotide was biotinylated, which allowed the capture of the conjugated antibody with streptavidin. The antibody could be captured by standard ELISA methods, and thus, a PK profile of the conjugated and total mAb could be generated. It was reported that the half-life of the AOCs was 9.5 h and 12 h and that of the AOC antibody was 24 h and 37 h. The naked antibody had a half-life of 69 h. The shorter half-life could be due to cleavage of the phosphate bond between the linker and the oligonucleotide. The faster clearance of the total antibody in the AOC versus just the naked antibody could mean that the oligonucleotide also contributes to the removal of the bioconjugate faster due to its size or charge. Analogously, hydrophobic ADCs tend to clear faster [44]. As more PK data are published, a better understanding of the parameters leading to improved in vivo stability will help advance the AOC field.

4.4. Summary Most papers with AOCs do show interesting in vitro data, but in vivo data are scarce. What has been published is promising. AOCs can silence multiple genes simultaneously in an in vivo setting. Using a bioinformatic approach, new targets can be identiﬁed and inhibited in difﬁcult-to-treat models. Intracellular or tissue localization of AOCs can be monitored simultaneously using a dye incorporated within the oligonucleotide strand. In some cases, activity was observed with dosing below 1 mg/kg in xenograft models. There are still many unanswered in vivo questions for AOCs, especially around PK and in vivo stability of the oligonucleotides. Toxicology studies in mice, rats, and cynomolgus monkeys are also missing pieces of the AOC in vivo landscape. Toxicology studies will J. Clin. Med. 2021, 10, 838 15 of 17

be published over time as more AOCs progress through preclinical development. In vivo data indicate that AOCs do have a strong potential to regulate gene expression and could be used as biotherapeutics, but much preclinical validation is required before they enter the clinic.

5. Conclusions and Outlook The field of AOCs started as a diagnostic tool but over the last 10 years has emerged as a novel therapeutic modality. The field is in its infancy, and much more preclinical work is required. As of the writing of this review, we are not aware of any AOCs that have entered the clinic. On the other hand, siRNA conjugated to other targeting moieties have been approved [4]. Based on what has been published, major challenges in the field include analytical characterization, target and disease selection, trafficking and in vivo stability. As more tools become available, knowledge and mechanistic understanding of AOCs will evolve as well. We envision that the landscape of AOCs will change rapidly over the next decade as the fields of ASO and siRNA advance; as new oligonucleotide constructs with improved stability, membrane permeability and lysosomal escape become available; as advances in linker technologies are refined for specific use in AOCs; as instruments and analytical methods are developed for easier measurement of metabolites, PK profiles and AOC characterization; and as target selection for AOCs that traffic directly to the cytosol are developed. These advances will significantly impact the next generation of AOCs. We speculate that with the large effort currently underway within many companies, the landscape will advance by leaps and bounds. The interesting in vitro and in vivo results published appear to justify these large research efforts. AOCs lend themselves to a personalized approach where the oligonucleotide sequence could be tailored for select genetic defects in one tissue, cancer biopsies could lead to individual siRNA cocktails and proteins could be selectively inhibited. AOCs could become an important arsenal in precision medicine, and we look forward to the next 10 years of development.

Author Contributions: J.D.-T. and S.T. writing—original draft preparation, J.D.-T., S.T. and N.J.; writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: We would like to thank Reva Raghupathi for comments and suggestions. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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