WO2025238253A1 - Antibody-oligonucleotide conjugates - Google Patents

Abstract

The invention concerns homogenous antibody-oligonucleotide conjugates (AOCs) having structure (1): Ab–[ (Z)_y1 – L^D – (D)_x ]_z (1), wherein Ab is an antibody, Z is a connecting group obtainable by reaction between two click probes, x is 1, 2, 3 or 4, y1 is 1 or 2, z is 2 or 4, L^D is an heterobifunctional (x + y1)- valent linker; and D is an oligonucleotide. The AOCs are homogenous, readily prepared, and effective in targeted delivery of the oligonucleotide to a cell of interest with high efficacy. The AOCs according to the invention have an improved therapeutic window and improved efficacy over conventional AOCs. Hence, the AOCs are effective in the treatment of disorders like muscular dystrophy. In a first aspect, the invention concerns the use of novel linkers for the efficient preparation of AOCs. In a second aspect, the invention concerns the medical use of AOCs for the treatment of neuromuscular disorders. In a third aspect, the invention concerns specific AOCs that are particularly suitable in treatment. In a fourth aspect, the invention concerns the use of ultrafast click chemistry in the preparation of AOCs.

Description

Antibody-oligonucleotide conjugates

Field of the invention

[0001] The present invention is in the field of medicine. More specifically, the present invention relates to antibody-conjugates with an oligonucleotide payload, that are homogenous and do not require genetic antibody modification. Such antibody-conjugates can be applied for more effective treatment of diseases, in particular muscular dystrophy.

Background

[0002] Antibody-drug conjugates (ADC), considered as one of the major classes of targeted therapy, are comprised of an antibody to which is attached a pharmaceutical agent. The antibodies (also known as binding agents or ligands) can be small protein formats (scFv’s, Fab fragments, DARPins, Affibodies, etc.) but are generally monoclonal antibodies (mAbs) of IgG type which have been selected based on their high selectivity and affinity for a given antigen, their long circulating half-lives, and little to no immunogenicity. Thus, mAbs as ligands for a carefully selected biological receptor provide an ideal targeting platform for selective delivery of pharmaceutical drugs. For example, a monoclonal antibody known to bind selectively with a specific cancer-associated antigen can be used for delivery of a chemically conjugated cytotoxic agent to the tumour, via binding, internalization, intracellular processing and finally release of active catabolite. The cytotoxic agent may be small molecule toxin, a protein toxin or other formats, like oligonucleotides. As a result, the tumour cells can be selectively eradicated, while sparing normal cells which have not been targeted by the antibody. Similarly, chemical conjugation of an antibacterial drug (antibiotic) to an antibody can be applied for treatment of bacterial infections, while conjugates of anti-inflammatory drugs are under investigation forthe treatment of autoimmune diseases and for example attachment of an oligonucleotide to an antibody is a potential promising approach for the treatment of neuromuscular diseases. Hence, the concept of targeted delivery of an active pharmaceutical drug to a specific cellular location of choice is a powerful approach for the treatment of a wide range of diseases, with many beneficial aspects versus systemic delivery of the same drug.

[0003] ADCs are prepared by conjugation of a linker-drug to a protein, a process known as bioconjugation. Many technologies are known for bioconjugation, as summarized in G.T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd Ed. 2013, incorporated by reference. Conceptually, the method the preparation of an ADC by bioconjugation entails the reaction of x number of reactive moieties F present on the antibody with a complementary reactive moiety Q present on the pharmaceutical drug (the payload), see Figure 1 .

[0004] Typically, a chemical linker is present between Q and the payload. This linker needs to possess a number of key attributes, including the requirement to be stable in plasma after drug administration for an extended period of time. A stable linker enables localization of the ADC to the projected site or cells in the body and prevents premature release of the payload in circulation, which would indiscriminately induce undesired biological response of all kinds, thereby lowering the therapeutic index of the ADC. Upon internalization, the ADC should be processed such that the payload is effectively released so it can exert its mode-of-action inside the cell. The linker can also contain a spacer element. There are two families of linkers, non-cleavable and cleavable. Non-cleavable linkers consist of a chain of atoms between the antibody and the payload, which is fully stable under physiological conditions, irrespective of which organ or biological compartment the antibody-drug conjugate resides in. As a consequence, liberation of the payload from an ADC with a non-cleavable linker relies on the complete (lysosomal) degradation of the antibody after internalization of the ADC into a cell. As a result of this degradation, the payload will be released, still carrying the linker, as well as a peptide fragment and/or the amino acid from the antibody the linker was originally attached to. Cleavable linkers utilize an inherent property of a cell or a cellular compartment for selective release of the payload from the ADC, which generally leaves no trace of linker after processing. For cleavable linkers, there are three commonly used mechanisms: (1) susceptibility to specific enzymes, (2) pH-sensitivity, and (3) sensitivity to redox state of a cell (or its microenvironment). The cleavable linker may also contain a self-immolative unit, for example based on a para-aminobenzyl alcohol group or para-hydroxybenzyl alcohol and derivatives and/or analogues thereof or a cyclization linker based on for example 1 ,2-diaminoethane carbamate derivatives. A linker may also contain an additional element, often referred to as spacer or stretcher unit, to connect the linker with a reactive group for attachment to the antibody via a reactive moiety F present on the antibody.

[0005] The reactive moiety F can be naturally present in the antibody, for example the reactive moiety can be the side chain of lysine or cysteine, which can be employed for acylation (lysine side chain) or alkylation (cysteine side chain).

[0006] Acylation of the e-amino group in a lysine side-chain is typically achieved by subjecting the protein to a reagent based on an activated ester or activated carbonate derivative, for example SMCC is applied for the manufacturing of Kadcyla®. Based on the fact that a given antibody may contain 60- 90 occurrences of lysine, of which the vast majority will display reactivity to the acylating agent, careful titration of the acylating agent is required which nevertheless results in a highly heterogeneous mixture of conjugation with only an average drug loading based on stochastic distribution of drugs attached to the antibody. For example, Kadcyla® has an average drug-to-antibody ratio (DAR) of approximately four but in fact consists of a mixture of components with DAR 0-12. Addition of a larger quantity of acylating agents will lead to a higher average DAR, for example DAR 6 or DAR8 can be achieved, based on a stochastic distribution containing even higher DAR species (i.e. >DAR 12).

[0007] Various reagents are known for alkylation of the thiol group in cysteine side-chain, see Figure 2. Amongst the cysteine alkylation strategies, the vast majority is based on the use of maleimide reagents, as is for example applied in the manufacturing of Adcetris®, Polivy® and Padcev®. Besides standard maleimide reagents, a range of maleimide variants are also applied for more stable cysteine conjugation, as for example demonstrated by James Christie et al., J. Contr. Rel. 2015, 220, 660-670 and Lyon et al., Nat. Biotechnol. 2014, 32, 1059-1062, both incorporated by reference. Other approaches for cysteine alkylation involve for example nucleophilic substitution of haloacetamides (typically bromoacetamide or iodoacetamide), see for example Alley et al., Bioconj. Chem. 2008, 19, 759-765, incorporated by reference, or various approaches based on nucleophilic addition on unsaturated bonds, such as reaction with acrylate reagents, see for example Bernardim et al., Nat. Commun. 2016, 7, 13128 and Ariyasu et al., Bioconj. Chem. 2017, 28, 897-902, both incorporated by reference, reaction with phosphonamidates, see for example Kasper et al., Angew. Chem. Int. Ed. 2019, 58, 11625-11630, incorporated by reference, reaction with allenamides, see for example Abbas et al., Angew. Chem. Int. Ed. 2014, 53, 7491-7494, incorporated by reference, reaction with cyanoethynyl reagents, see for example Kolodych et al., Bioconj. Chem. 2015, 26, 197-200, incorporated by reference, reaction with vinylsulfones, see for example Gil de Montes et al., Chem. Sci. 2019, 10, 4515- 4522, incorporated by reference, or reaction with vinylpyridines, see for example Seki et al, Chem. Sci., 2021 , 12, 9060-9068 and https://iksuda.com/science/permalink/ (accessed July 26th, 2020).

[0008] In terms of DAR, similar to lysine conjugation this is controlled by titration of alkylating reagent for reaction with free cysteine side-chains (liberated by reduction of interchain disulfides with for example TCEP or DTT). The final DAR is typically an average number comprised of a stochastic mixture of different components, again similar to lysine conjugation. However, a few notable differences can be noted: (a) the different DAR species typically consist of a multitude of 2 (i.e. 2, 4, 6, 8) and (b) the maximum DAR that can be achieved is 8 (if all liberated interchain cysteine side-chains have reacted). This also means that by comprehensive alkylation of all interchain cysteine side-chains, a homogeneous DAR8 ADC can be achieved. This is by far the most common method to generate DAR8 ADCs and likely the only method employed for any clinical ADC with DAR8. It must be noted that such approach cannot generate homogeneous DAR6 ADC or ADCs with DAR>8, unless specific cysteines are engineered out or added into the antibody sequence by recombinant DNA technology. It must also be noted that any method involving a reduction step may lead to antibody degradation (reduction of additional disulfide bonds) or fragment scrambling (due to exchange of light chains for example).

[0009] An alternative approach to antibody conjugation to interchain disulfide bridges involves the use of a cysteine cross-linking reagent, i.e. a reagent that will react with two cysteine side-chains concurrently. Examples of such cross-linking agents are bis-sulfone reagents, see for example Balan et al., Bioconj. Chem. 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceutics 2015, 12, 1872-1879, both incorporated by reference, mono- or bis-bromomaleimides, see for example Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269, both incorporated by reference, bis-maleimide reagents, see for example WO2014114207, bis(phenylthio)maleimides, see for example Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261- 7269 and Aubrey et al., Bioconj. Chem. 2018, 29, 3516-3521 , both incorporated by reference, bis- bromopyridazinediones, see for example Robinson et al., RSC Advances 2017, 7, 9073-9077, incorporated by reference, bis(halomethyl)benzenes, see for example Ramos-Tomillero et al., Bioconj. Chem. 2018, 29, 1199-1208, incorporated by reference or other bis(halomethyl)aromatics, see for example WO2013173391 . Typically, ADCs prepared by cross-linking of cysteines have a drug-to- antibody loading of four (DAR4), which is achieved by complete alkylation of all cysteine side-chains liberated by reduction.

[0010] Another useful technology for conjugation to a cysteine side chain is by means of formation of a novel disulfide bond, by treatment of a liberated cysteine side-chain with thiolating agent (i.e. a non- symmetrical disulfide bond of which one thiol is part of a good leaving group), leading to a bioactivatable connection that has been utilized for reversibly connecting protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see for example Pillow et al., Chem. Sci. 2017, 8, 366-370, incorporated by reference). Similar to cysteine alkylation, the average DAR of such ADCs can be tailored to around 2-8 in case native interchain disulfide bonds are reduced.

[0011] Besides conjugation to the side chains of the naturally present amino acids lysine or cysteine, a range of other conjugation technologies has been explored based on a two-stage strategy involving (a) introduction on a novel reactive group F, followed by (b) reaction with another complementary reactive group Q. For example, a method can be used to introduce a given number of reactive moieties F onto an antibody, which can be two, four or eight, see Figure 3.

[0012] An example of an unnatural reactive functionality F that can be employed for bioconjugation of linker-drugs is the oxime group, suitable for oxime ligation or the azido group, suitable for click chemistry conjugation. The oxime can be installed in the antibody by genetic encoding of a non-natural amino acid, e.g. p-acetophenylalanine, as for example demonstrated by Axup et al. Proc. Nat. Acad. Sci. 2012, 109, 16101-16106, incorporated by reference, or by enzymatic alkylation of a cysteine present in a CAAX sequence with a prenyl group containing a remote keto group, as for example disclosed in WO2012153193. The azide can be installed in the antibody by genetic encoding of p- azidomethylphenylalanine or p-azidophenylalanine, as for example demonstrated by Axup et al. Proc. Nat. Acad. Sci. 2012, 109, 16101-16106, incorporated by reference. Similarly, Zimmerman et al., Bioconj. Chem. 2014, 25, 351-361 , incorporated by reference have employed a cell-free protein synthesis method to introduce p-azidomethylphenylalanine (AzPhe) into monoclonal antibodies for conversion into ADCs by means of metal-free click chemistry. Also, it has also be shown by Nairn et al., Bioconj. Chem. 2012, 23, 2087-2097, incorporated by reference, that a methionine analogue like azidohomoalanine (Aha) can be introduced into protein by means of auxotrophic bacteria and further converted into protein conjugates by means of click chemistry. Finally, genetic encoding of aliphatic azides in recombinant proteins using a pyrrolysyl-tRNA synthetase/tRNACUA pair was shown by Nguyen et al., J. Am. Chem. Soc. 2009, 131 , 8720-8721 , incorporated by reference, and labelling was achieved by click chemistry, either by copper-catalyzed alkyne-azide cycloaddition (CuAAC) or strain- promoted alkyne-azide cycloaddition (SPAAC). Besides, CuAAC and SPAAC, bioconjugation of linkerdrugs to antibodies (and other biomolecules such as glycans, nucleic acids) can be achieved by a range of other metal-free click chemistries, see e.g. Nguyen and Prescher, Nature Rev. Chem. 2020, 4, 476- 489, incorporated by reference. For example, oxidation of a specific tyrosine in a protein can give an ortho-quinone, which readily undergoes cycloaddition with strained alkenes (e.g. TCO) or strained alkynes, see e.g. Bruins et al., Chem. Eur. J. 2017, 24, 4749-4756, incorporated by reference. Besides cyclooctyne, certain cycloheptynes are also suitable for metal-free click chemistry, as reported by Wetering et al. Chem. Sci. 2020, 11 , 901 1-9016, incorporated by reference. A tetrazine moiety can also be introduced into a protein or a glycan by various means, for example by genetic encoding or chemical acylation, and may also undergo cycloaddition with cyclic alkenes and alkynes. A list of pairs of functional groups F and Q for metal-free click chemistry is provided in Figure 4.

[0013] In a SPAAC bioconjugation, the linker-drug is functionalized with a cyclic alkyne and the cycloaddition with azido-modified antibody is driven by relief of ring-strain. Conversely, the linker-drug can be functionalized with azide and the antibody with cyclic alkyne. Various strained alkynes suitable for metal-free click chemistry are indicated in Figure 5. [0014] A method of increasing popularity in the field of ADCs is based on enzymatic installation of a non-natural functionality F. For example, Lhospice et al., Mol. Pharmaceut. 2015, 12, 1863-1871 , incorporated by reference, employ the bacterial enzyme transglutaminase (BTG or TGase) for installation of an azide moiety onto an antibody. A genetic method based on C-terminal TGase- mediated azide introduction followed by conversion in ADC with metal-free click chemistry was reported by Cheng et al., Mol. Cancer Therap. 2018, 17, 2665-2675, incorporated by reference.

[0015] It has been shown in WO2014065661 , by van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, Verkade et al., Antibodies 2018, 7, 12, and Wijdeven at al. MAbs 2022, 14, 2078466, all incorporated by reference, that enzymatic remodelling of the native antibody glycan at N297 enables introduction of an azido-modified sugar, suitable for attachment of cytotoxic payload using metal-free click chemistry, see Figure 6. Similarly, the enzymatic glycan remodelling protocol can also be employed to install a free thiol group on an antibody for conjugation based on any of the methods described above for cysteine conjugation.

[0016] Although most of the ADCs have cytotoxic payloads, other payloads are also known. In particular, ADCs having an oligonucleotide payload are known in the art and are referred to as antibody- oligonucleotide conjugates (AOC).

[0017] Oligonucleotide therapies without an antibody are well known in the prior art and these therapies can be used to treat a wide variety of diseases, see for example Evers et al. Advanced drug delivery reviews, 2015, 87 90-103.

[0018] Conjugating an oligonucleotide to an antibody has many benefits, such as targeted delivery, reduced clearance. AOCs are known in the prior art, WO2022/212886 describes molecules and pharmaceutical compositions that induce an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion. Also described herein include methods for treating a disease or disorder that comprises a molecule or a pharmaceutical composition that induces an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion.

[0019] W02020028831 A1 relates relate to complexes comprising a muscle-targeting agent covalently linked to a molecular payload. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload inhibits activity of ACVR1 . In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.

[0020] There remains a need for improved targeted delivery of anti-sense oligonucleotide therapy.

Summary of the invention

[0021] The inventors developed antibody-oligonucleotide conjugates (AOCs) that are surprisingly effective in the treatment of disorders like muscular dystrophy. The present invention resides on the general finding of homogenous AOCs, wherein the oligonucleotides are connected through the glycan of the antibody, for targeted delivery of the oligonucleotide to the cell of interest with high efficacy. The AOCs according to the invention have an improved therapeutic window and improved efficacy over AOCs known from the art. In particular, the inventors found that unexpected efficacies in terms of exon skipping and dystrophin restoration percentages could be obtained, as well as a prolonged duration of the effect. Furthermore, the inventors have developed a process which enables the production of a new class of homogenous AOCs, i.e. having a DAR at or close to the theoretical DAR with a narrow distribution. Advantageously, the process of the present invention does not require any genetic modification of the antibody and is applicable to any antibody.

[0022] In a first aspect, the invention concerns the use of novel linkers for the efficient preparation of AOCs. In a second aspect, the invention concerns the medical use of AOCs for the treatment of neuromuscular disorders. In a third aspect, the invention concerns specific AOCs that are particularly suitable in treatment. In a fourth aspect, the invention concerns the use of ultrafast click chemistry in the preparation of AOCs. These aspects of the invention are reflected in the following preferred embodiments.

[0023] Firstly, the invention includes An antibody-oligonucleotide conjugate having structure (1): Ab-[ (Z)y1 - L^D - (D)x]z

(1) wherein:

- Ab is an antibody;

- Z is a connecting group obtainable by reaction between two click probes;

- x is 1 , 2, 3 or 4;

- y1 is 1 or 2;

- z is 2 or 4;

- L^D is an heterobifunctional (x + y1)-valent linker; and

- D is an oligonucleotide.

[0024] Secondly, the invention includes the antibody-oligonucleotide conjugate having structure (1) for use in treatment.

[0025] Thirdly, the invention includes the antibody-oligonucleotide conjugate having structure (1) for use in the treatment of a hereditary neuromuscular disorder, preferably wherein the hereditary neuromuscular disorder is selected from Duchenne, myotonic dystrophy, spinal muscular atrophy, homozygous familial hypercholesterolemia and primary hyperoxaluria type 1 .

[0026] Fourthly, the invention includes a process for preparing an antibody-oligonucleotide conjugate according to any one of claims 1 - 9, comprising:

(a) providing a modified antibody having the structure Ab(F¹)_Z2, wherein Ab is an antibody, z2 is 2 or 4, and F¹ is a click probe;

(b) reacting the modified antibody with z2/y1 equivalents of (Q¹)yi - L^A - (F²)_y2, wherein Q¹ is a click probe that is reactive towards F¹, L^A is a heterobifunctional (y1 + y2)-valent linker, y1 is 1 or 2, y2 is 1 , 2, 3 or 4, and F² is a click probe that is not reactive towards Q¹, to obtain an antibody-linker construct of structure Ab[(Z¹)_yi - L^A - (F²)_y2 ]z, wherein z is z2 / y1 , and Z¹ is a connecting group obtained by reaction of F¹ and Q¹;

(c) reacting the antibody-linker construct with z x y2 equivalents of Q² - L^B - D, wherein Q² is a click probe that is reactive towards F², L^B is a bivalent linker and D is an oligonucleotide, to obtain the conjugate of structure Ab [ (Z¹)_yi - L^A - (Z² - L^B - D)_y2 ]_z, wherein Z² is a connecting group obtained by reaction of F² and Q²; or

(a) providing a modified antibody having the structure Ab[ (L⁶) - (F) ]_z, wherein:

- Ab is an antibody;

- L⁶ is -GlcNAc(Fuc)v^(G)j-S-(L⁷)w-, wherein G is a monosaccharide, j is an integer in the range of 0 - 10, S is a sugar or a sugar derivative, GIcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1 , w’ is 0 or 1 and L⁷ is -N(H)C(O)CH₂-, -N(H)C(O)CF₂- or -CH₂-;

- z is 2 or 4;

- F is a click probe,

(d) reacting the modified antibody with z equivalents of (Q)— (L¹)— (L²)_o— (L³)_P— (L⁴)_q— D, wherein:

- Q is a click probe that is reactive towards F;

- L¹, L², L³ and L⁴ are each individually linkers;

- o, p and q are each individually 0 or 1 ; to obtain the conjugate of structure Ab-[(L⁶)-(Z)-(L¹)-(L²)₀-(L³)_P-(L⁴)_q-D ]_z.

[0027] Fifthly, the invention includes the use of ultrafast click chemistry for conjugating an oligonucleotide to an antibody, wherein ultrafast click chemistry is defined as having a reaction rate that is at least 10 times greater than the rate of the click reaction between azide and bicyclononyne.

Detailed description

Definitions

[0028] The verb “to comprise”, and its conjugations, as used in this description and in the claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0029] Reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

[0030] A “linker” is herein defined as a moiety that connects (i.e. covalently links) two or more elements of a compound. A linker may comprise one or more spacer moieties. A spacer-moiety is herein defined as a moiety that spaces (i.e. provides distance between) and covalently links together two (or more) parts of a linker. The linker may be part of e.g. a linker-construct, a linker-conjugate, a linker-payload (e.g. linker-drug) or an antibody-conjugate, as defined below.

[0031] A “self-immolative group” is herein defined as a part of a linker in an antibody-drug conjugate with a function is to conditionally release free drug at the site targeted by the ligand unit. The activatable self-immolative moiety comprises an activatable group (AG) and a self-immolative spacer unit. Upon activation of the activatable group, for example by enzymatic conversion of an amide group to an amino group or by reduction of a disulfide to a free thiol group, a self-immolative reaction sequence is initiated that leads to release of free drug by one or more of various mechanisms, which may involve (temporary) 1 ,6-elimination of a p-aminobenzyl group to a p-quinone methide, optionally with release of carbon dioxide and/or followed by a second cyclization release mechanism. The self-immolative assembly unit can part of the chemical spacer connecting the antibody and the payload (via the functional group). Alternatively, the self-immolative group is not an inherent part of the chemical spacer, but branches off from the chemical spacer connecting the antibody and the payload.

[0032] A “hydrophilic group” or “polar linker” is herein defined as any molecular structure containing one or more polar functional groups that imparts improved polarity, and therefore improved aqueous solubility, to the molecule it is attached to. Preferred hydrophilic groups are selected from a carboxylic acid group, an alcohol group, an ether group, a polyethylene glycol group, an amino group, an ammonium group, a sulfonate group, a phosphate group, an acyl sulfamide group or a carbamoyl sulfamide group. In addition to higher solubility other effects of the hydrophilic group include improved click conjugation efficiency, and, once incorporated into an antibody-drug conjugate: less aggregation, improved pharmacokinetics resulting in higher efficacy and in vivo tolerability.

[0033] The compounds according to the invention may exist in salt form, which are also covered by the present invention. The salt is typically a pharmaceutically acceptable salt, containing a pharmaceutically acceptable anion. The term “salt thereof’ means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt. The term “pharmaceutically accepted” salt means a salt that is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0034] The term “click probe” refers to a functional moiety that is capable of undergoing a click reaction, i.e. two compatible click probes mutually undergo a click reaction such that they are covalently linked in the product. Compatible probes for click reactions are known in the art, and preferably include (cyclic) alkynes and azides. In the context of the present invention, click probe Q in the compound according to the invention is capable of reacting with click probe F on the (modified) protein, such that upon the occurrence of a click reaction, a conjugate is formed wherein the protein is conjugated to the compound according to the invention. Herein, F and Q are compatible click probes. Click reactions are known in the art and typically refer to cycloaddition reactions such as the [4+2] cycloaddition (e.g. Diels-Alder, inverse electron-demand Diels-Alder) and the [3+2] cycloaddition (e.g. 1 ,3-dipolar cycloaddition). In the context of the present invention, the term click reaction may also be referred to as cycloaddition.

[0035] The term “(hetero)alkyl” refers to alkyl groups and heteroalkyl groups. Heteroalkyl groups are alkyl groups wherein one or more carbon units in the alkyl chain (e.g. CH2, CH or C) are replaced by heteroatoms, such as O, S, S(O), S(O)2 or NR⁴. In other words, the alkyl chain is interrupted with one or more elements selected from O, S, S(O), S(O)2 and NR⁴. Such interruptions are distinct from substituents, as they occur within the chain of an alkyl group, whereas substituents are pendant groups, monovalently attached to e.g. a carbon atom of an alkyl chain. In a preferred embodiment, the (hetero)alkyl group is an alkyl group, e.g. ethyl (Et), isopropyl (i-Pr), n-propyl (n-Pr), tert-butyl (t-Bu), isobutyl (i-Bu), n-butyl (n-Bu) or n-pentyl.

[0036] Likewise, the term “(hetero)aryl” refers to aryl groups and heteroaryl groups. Heteroaryl groups are aryl groups wherein one or more carbon units in the ring (e.g. CH) are replaced by heteroatoms, such as O, S, N or NR⁴.

[0037] An “acylsulfamide moiety” is herein defined as a sulfamide moiety (H2NSO2NH2) that is N- acylated or N-carbamoylated on one end of the molecule and N-alkylated (mono or bis) at the other end of the molecule.

[0038] A “domain” may be any region of a protein, generally defined on the basis of sequence homologies and often related to a specific structural or functional entity. The term domain is used in this document to designate either individual Ig-like domains, such as “N-domain” or for groups of consecutive domains, such as “A3-B3 domain”.

[0039] A “coding sequence” or a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0040] The term “glycoprotein” is herein used in its normal scientific meaning and refers to a protein comprising one or more monosaccharide or oligosaccharide chains (“glycans”) covalently bonded to the protein. A glycan may be attached to a hydroxyl group on the protein (O-linked-glycan), e.g. to the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline, or to an amide function on the protein (A/-gly co protein), e.g. asparagine or arginine, or to a carbon on the protein (C- glycoprotein), e.g. tryptophan. A glycoprotein may comprise more than one glycan, may comprise a combination of one or more monosaccharide and one or more oligosaccharide glycans, and may comprise a combination of N-linked, O-linked and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and therefore qualify as glycoprotein. Examples of glycoproteins include PSMA (prostate-specific membrane antigen), CAL (Candida antartica lipase), gp41 , gp120, EPO (erythropoietin), antifreeze protein and antibodies.

[0041] The term “glycan” is herein used in its normal scientific meaning and refers to a monosaccharide or oligosaccharide chain that is linked to a protein. The term glycan thus refers to the carbohydrate-part of a glycoprotein. The glycan is attached to a protein via the C-1 carbon of one sugar, which may be without further substitution (monosaccharide) or may be further substituted at one or more of its hydroxyl groups (oligosaccharide). A naturally occurring glycan typically comprises 1 to about 10 saccharide moieties. However, when a longer saccharide chain is linked to a protein, said saccharide chain is herein also considered a glycan. A glycan of a glycoprotein may be a monosaccharide. Typically, a monosaccharide glycan of a glycoprotein consists of a single N-acetylglucosamine (GIcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently attached to the protein. A glycan may also be an oligosaccharide. An oligosaccharide chain of a glycoprotein may be linear or branched. In an oligosaccharide, the sugar that is directly attached to the protein is called the core sugar. In an oligosaccharide, a sugar that is not directly attached to the protein and is attached to at least two other sugars is called an internal sugar. In an oligosaccharide, a sugar that is not directly attached to the protein but to a single other sugar, i.e. carrying no further sugar substituents at one or more of its other hydroxyl groups, is called the terminal sugar. For the avoidance of doubt, there may exist multiple terminal sugars in an oligosaccharide of a glycoprotein, but only one core sugar. A glycan may be an O-linked glycan, an N-linked glycan or a C-linked glycan. In an O-linked glycan a monosaccharide or oligosaccharide glycan is bonded to an O-atom in an amino acid of the protein, typically via a hydroxyl group of serine (Ser) or threonine (Thr). In an N-linked glycan a monosaccharide or oligosaccharide glycan is bonded to the protein via an N-atom in an amino acid of the protein, typically via an amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). In a C-linked glycan a monosaccharide or oligosaccharide glycan is bonded to a C-atom in an amino acid of the protein, typically to a C-atom of tryptophan (Trp).

[0042] The term “antibody” (AB) is herein used in its normal scientific meaning. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. An antibody is an example of a glycoprotein. The term antibody herein is used in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g. bispecific antibodies), antibody fragments, and double and single chain antibodies. The term “antibody” is herein also meant to include human antibodies, humanized antibodies, chimeric antibodies and antibodies specifically binding cancer antigen. The term “antibody” is meant to include whole antibodies, but also fragments of an antibody, for example an antibody Fab fragment, F(ab’)2, Fv fragment or Fc fragment from a cleaved antibody, a scFv-Fc fragment, a minibody, a diabody or a scFv. Furthermore, the term includes genetically engineered antibodies and derivatives of an antibody. Antibodies, fragments of antibodies and genetically engineered antibodies may be obtained by methods that are known in the art.

[0043] An antibody may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (I) and kappa (k). The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The immunoglobulin can be of any type (e.g. IgG, I g E , IgM, I g D , and IgA), class (e.g. IgG 1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass, or allotype (e.g. human G1 m1 , G1 m2, G m3, non- G1 m1 [that, is any allotype other than G1 m1], G1 m17, G2m23, G3m21 , G3m28, G3m1.1 , G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1 , A2m2, Km1 , Km2 and Km3) of immunoglobulin molecule. Preferred allotypes for administration include a non-G1 m1 allotype (nG1 m1), such as G1 m17,1 , G1 m3, G1 m3.1 , G1 m3.2 or G1 m3.1.2. More preferably, the allotype is selected from the group consisting of the G1 m17,1 or G1 m3 allotype. The antibody may be engineered in the Fc-domain to enhance or nihilate binding to Fc-gamma receptors, as summarized by Saunders et al. Front. Immunol. 2019, 10, doi: 10.3389/fimmu.2019.01296 and Ward et al., Mol. Immunol. 2015, 67, 131-141. For example, the combination of Leu234Ala and Leu235Ala (commonly called LALA mutations) eliminate FcyRlla binding. Elimination of binding to Fc-gamma receptors can also be achieved by mutation of the N297 amino acid to any other amino acid except asparagine, by mutation of the T299 amino acid to any other amino acid except threonine or serine, or by enzymatic Deglycosylation or trimming of the fully glycosylated antibody with for example PNGase F or an endoglycosidase. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin. Each chain contains distinct sequence domains.

[0044] A percentage of “sequence identity” may be determined by comparing the two sequences, optimally aligned over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. A sequence “at least 85% identical to a reference sequence” is a sequence having, on its entire length, 85%, or more, for instance 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of the reference sequence.

[0045] The term “CDR” refers to complementarity-determining region: the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs therefore refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. “CDR”

[0046] The term “monoclonal antibody” or “mAb” as used herein refers to an antibody molecule of a single amino acid sequence, which is directed against a specific antigen, and is not to be construed as requiring production ofthe antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering.

[0047] The term “chimeric antibody” refers to an engineered antibody which, in its broadest sense, contains one or more regions from one antibody and one or more regions from one or more other antibodies. In an embodiment, a chimeric antibody comprises a VH domain and a VL domain of an antibody derived from a non-human animal, in association with a CH domain and a CL domain of another antibody, in an embodiment, a human antibody. As the non-human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. A chimeric antibody may also denote a multispecific antibody having specificity for at least two different antigens.

[0048] The term “humanised antibody” refers to an antibody which is wholly or partially of non-human origin and which has been modified to replace certain amino acids, for instance in the framework regions of the VH and VL domains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most of the time human CH and CL domains. “Fragments” of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.

[0049] The term “multivalent” refers to a linker molecule or linker part of a bigger molecule, with multiple connecting groups. For a linker molecule, these connecting groups are formed by reactive groups capable of covalently attaching to other molecules (e.g. payload or antibody). For a linker part of a bigger molecule, such as a conjugate, the connecting groups are covalent attachment to other parts of the molecule (e.g. payload or antibody). A bivalent linker has two connecting groups, a trivalent linker has three connecting groups, etc. In the context of the current invention, bivalent is also referred to as 2-valent, trivalent is also referred to a 3-valent, etc.

[0050] The term “heterofunctional” refers to a linker molecule or linker part of a bigger molecule with connecting groups that are not identical and have different reactivity or are obtained by different reactivity. Typically, such connecting groups in a heterofunctional molecule are mutually non-reactive. [0051] A “conjugate” is herein defined as a compound wherein an antibody is covalently connected to a payload via a linker. A conjugate comprises one or more antibodies and/or one or more payloads.

[0052] The term “payload” refers to the moiety within a conjugate that is covalently attached to a targeting moiety such as an antibody, but also to the molecule that is released from the conjugate upon uptake of the protein conjugate and/or cleavage of the linker. Payload thus refers to the monovalent moiety having one open end which is covalently attached to the targeting moiety via a linker and also to the molecule that is released therefrom. In the context of the present invention, the payload is denoted with D.

[0053] Herein, the term “therapeutic index” (Tl) has the conventional meaning well known to a person skilled in the art, and refers to the ratio of the dose of drug that is toxic (i.e. causes adverse effects at an incidence or severity not compatible with the targeted indication) for 50% of the population (TD50) divided by the dose that leads to the desired pharmacological effect in 50% of the population (effective dose or ED50). Hence, Tl = TD50 / ED50. The therapeutic index may be determined by clinical trials or for example by plasma exposure tests. See also Muller, et al. Nature Reviews Drug Discovery 2012, 11 , 751-761. At an early development stage, the clinical Tl of a drug candidate is often not yet known. However, understanding the preliminary Tl of a drug candidate is of utmost importance as early as possible, since Tl is an important indicator of the probability of the successful development of a drug. Recognizing drug candidates with potentially suboptimal Tl at earliest possible stage helps to initiate mitigation or potentially re-deploy resources. At this early stage, Tl is typically defined as the quantitative ratio between safety (maximum tolerated dose in mouse or rat) and efficacy (minimal effective dose in a mouse xenograft).

[0054] Herein, the term “therapeutic efficacy” denotes the capacity of a substance to achieve a certain therapeutic effect, e.g. reduction in tumour volume. Therapeutic effects can be measured determining the extent in which a substance can achieve the desired effect, typically in comparison with another substance under the same circumstances. A suitable measure for the therapeutic efficacy is the ED50 value, which may for example be determined during clinical trials or by plasma exposure tests. In case of preclinical therapeutic efficacy determination, the therapeutic effect of a bioconjugate (e.g. an ADC), can be validated by patient-derived tumour xenografts in mice in which case the efficacy refers to the ability of the ADC to provide a beneficial effect. Alternatively the tolerability of said ADC in a rodent safety study can also be a measure of the therapeutic effect.

[0055] Herein, the term “tolerability” refers to the maximum dose of a specific substance that does not cause adverse effects at an incidence or severity not compatible with the targeted indication. A suitable measure for the tolerability for a specific substance is the TD50 value, which may for example be determined during clinical trials or by plasma exposure tests.

[0056] The term “DAR” refers to drug to antibody ratio. In the art, the term “drug” in DAR is used for any payload and not only for drug molecules. In the context of the present application, the “drug” in DAR refers to the oligonucleotide moiety (D). A single payload D may comprise multiple oligonucleotides, e.g. double stranded oligonucleotides. Hence, in the present application, the term DAR refers to the ratio between payload D and the antibody specifically, which may or may not be equal to the oligonucleotide to antibody ratio.

[0057] The term “theoretical DAR” refers to the theoretical DAR of a conjugate molecule. For example, when four connection sites are reacted with four equivalents (or more in case an excess is used) of linker-payload constructs each containing one payload D, the resulting conjugate has a theoretical DAR of 4. In the preparation of conjugates, typically a distribution of conjugates with varying amount of payload attached is formed., and the average DAR of the obtained product will deviate from the theoretical DAR.

The invention

[0058] The inventors have for the first time been able to prepare homogenous antibody-oligonucleotide conjugates (AOCs), which are conjugated via the glycan of an antibody, and which exhibit improved in vitro and in vivo characteristics for treatment of hereditary diseases. The conjugates according to the invention are homogeneous, i.e. have a DAR at or close to the theoretical DAR with a narrow distribution, and do not require any genetic modification of the antibody. In a first aspect, the invention concerns the use of novel linkers for the efficient preparation of AOCs. In a second aspect, the invention concerns the medical use of AOCs for the treatment of neuromuscular disorders. In a third aspect, the invention concerns specific AOCs that are particularly suitable in treatment. In a fourth aspect, the invention concerns the use of ultrafast click chemistry in the preparation of AOCs. [0059] Also contemplated within the present invention are salts, preferably pharmaceutically acceptable salts, of the conjugates according to the invention.

[0060] The conjugates according to the invention are ideally suited for treatment, in particular for the treatment of neuromuscular disorders, immune disorders, infections and cancer. Therefore, the invention also concerns a method for targeting a cell expressing a specific extracellular receptor, comprising contacting the conjugate according to the invention with cells that may possibly express the extracellular receptor, wherein the antibody specifically targets the extracellular receptor. Likewise, the invention also concerns a method for the treatment of neuromuscular disorders, immune disorders, infections or cancer, comprising administering to a subject in need thereof the conjugate according to the invention.

[0061] The invention according to the present aspect further concerns a pharmaceutical composition comprising the conjugate according to the second aspect of the invention and a pharmaceutically acceptable carrier.

[0062] The inventors have developed a modular, non-genetic preparation method for such conjugates, involving a few simple steps and starting from any antibody (see Figure 7). These steps are (a) enzymatic remodeling of the glycan to give an antibody functionalized with two or four click probes (e.g. an azide) per antibody, (b) a click reaction (e.g. a strain-promoted azide-alkyne cycloaddition) with a multivalent, bifunctional reagent comprising one click probe reactive towards the remodelled antibody (e.g. a cyclic alkyne) and at least two click probes that are not reactive towards the other click probes (e.g. tetrazines), and (c) a separate click reaction (e.g. an inverse electron-demand Diels-Alder cycloaddition) of the click probes with branched linker-drug constructs comprising one click probe reactive towards the previously non-reactive click probes (e.g. cyclic alkyne or strained alkene), connected to one or more payloads preferably connected through a cleavable linker. The resulting conjugates are rapidly generated with high homogeneity, close to theoretical DAR values and with surprising stability. Moreover, the native glycosylation sites of the antibody are used for site-specific conjugation, such that without the need for genetic engineering of the antibody, conjugates with high DAR can be obtained. In addition, HIC profiles, as well as in vitro and in vivo efficacy and tolerability studies of the resulting ADCs indicate small relative retention time and therefore show high potential in the treatment of cancer.

[0063] Here below, the AOC according to the invention is first defined. The structural features of the conjugate according to the invention also apply to the process for preparing the conjugate according to the invention and the uses and methods of the invention. As the skilled person will appreciate, the structural features of the conjugates according to the invention applies to all aspects of the invention. The skilled person understands that any structural feature that is unchanged in the conjugation reaction is defined equally for the intermediates as well as the final conjugates according to the invention. In the conjugation reactions, only reactive moieties F and Q are transformed into connecting groups Z. As will be understood by the skilled person, the definition of the chemical moieties, as well as their preferred embodiments, apply to all aspects of the invention. Conjugate of general structure (1)

[0064] The invention concerns conjugates of general structure (1):

Ab-[ (Z)_yi - L^D - (D)x]z

(I) wherein:

- Ab is an antibody;

- Z is a connecting group obtainable by reaction between two click probes;

- x is 1 , 2, 3 or 4;

- y1 is 1 or 2;

- z is 2 or 4;

- L^D is an heterobifunctional (x + y1)-valent linker; and

- D is an oligonucleotide.

[0065] AOCs according to the invention preferably have structure (11) or (12) as defined here below. In a first aspect, the invention concerns the use of novel linkers for the efficient preparation of AOCs, wherein the AOCs preferably have structure (11). In a second aspect, the invention concerns the medical use of AOCs for the treatment of neuromuscular disorders, wherein the AOCs preferably have structure (11) or (12). In a third aspect, the invention concerns specific AOCs that are particularly suitable in treatment, wherein the AOCs preferably have structure (11) or (12). In a fourth aspect, the invention concerns the use of ultrafast click chemistry in the preparation of AOCs wherein the AOCs preferably have structure (11) or (12). Herein, the nature of any connecting group Z is obtained by ultrafast click chemistry. In case the AOC is according to structure (11), preferably Z² is obtained by ultrafast click chemistry.

[0066] Thus, in a first preferred embodiment, the conjugate according to the invention has general structure (1), wherein Z = Z¹ and L^D = L^A(Z²-L^B)_y2. Such conjugates are represented by general structure (11).

Ab-[ (Z¹)y1 - L^A ( Z² - L^B - D )y₂ ]z

(I I)

[0067] According to the present embodiment, an intermediate spacer L^A is used to connect the payloads D to the antibody Ab. As such, two connecting groups are present in the AOC, identified as Z¹ and Z², wherein the definition of Z applies to both Z¹ and Z² independently. L^A is a linkerthat connects y¹ occurrences of Z¹ to y2 occurrences of Z², and L^B is a linker that connects Z² to D, wherein in the structure of the conjugate linker L^A occurs z times and linker L^B occurs z x y2 times.

[0068] Herein, y2 may be 1 or 2. In other words, linker L^A may have one (y2 = 1) or two (y2 = 2) points of attachment to the antibody. In case y2 = 1 , the AOC according to the invention may also be represented by structure (21). In case y2 = 2, the AOC according to the invention may also be represented by structure (22). Ab-[ Z¹ - L^A ( Z² - L^B - D )_y2]z

(21)

[0069] AOCs according to the present aspect typically are DAR 1 , DAR 2, DAR 4 or DAR 8, and have y1 = 1 or 2, y2 = 2 and z = 2 or 4. In one preferred embodiment, the AOC is DAR 1 and has structure (22), wherein z = 1 and y2 = 1 (corresponding to structure (33)). In one preferred embodiment, the AOC is DAR 2 and has structure (21), wherein z = 2 and y2 = 1 (corresponding to structure (34)). In one preferred embodiment, the AOC is DAR 4 and has structure (21), wherein z = 4, y1 = 1 and y2 = 1 (corresponding to structure (35)), or z = 2, y1 = 1 and y2 = 2 (corresponding to structure (36)). In one preferred embodiment, the AOC is DAR 8 and has structure (21), wherein z = 4, y1 = 1 and y2 = 1 , or z = 2, y1 = 1 and y2 = 2 (corresponding to structure (37)).

(36) (37)

[0070] Preferred AOCs according to the present embodiment are DAR 1 , DAR 2 or DAR 4, more preferably DAR 4. Alternatively, preferred AOCs according to the present embodiment have structure (33), (36) or (37), most preferably structure (36).

[0071] This embodiment is particularly preferred in the first aspect of the invention, wherein improved linkers are used in the conjugates according to the invention. The inventors have found that AOCs with such linkers, with intermediate spacer L^A, outperform conventional AOCs.

[0072] In a further preferred embodiment, the conjugate according to the invention has general structure (1), wherein y1 = 1 and L^D = (L¹)— (L²)_o— (L³)_P— (L⁴)_q. Such conjugates are represented by general structure (12).

Ab-[(L⁶)-(Z)-(L¹)-(L²)₀-(L³)_P-(L⁴)_q-D ]_z

(12)

[0073] Herein, L¹, L², L³ and L⁴ are each individually linkers to together connect the antibody Ab, via connecting group Z, with payloads D, and o, p and q are each individually 0 or 1. Furthermore, L⁶ represents (part of) the glycan of the antibody, and is further defined below. AOCs according to the present aspect typically are DAR 2 or DAR 4, and have z = 2 or 4. Most preferably they are DAR 2 and have z = 2.

[0074] Integer y1 defines the number of connective groups Z¹ are present within one branch. y1 may be 1 or 2, preferably y1 is 1 . In case y1 is 1 , the linker L^D has a single connection point to the antibody Ab, whereas if y1 is 2, the linker L^D has two connection points to the antibody Ab.

[0075] Integer y2 defines the number of connective groups Z² are present within one branch. y2 may be 1 , 2, 3 or 4. Preferably, y2 is 1 or 2, most preferably y2 is 2. In case y2 is 1 , the linker L^A is bivalent (or linear), i.e. connects one Q² or Z² per linker to the antibody. In case y2 is 1 , the linker L^A is trivalent (or branched), i.e. connects two Q² or Z² per linker to the antibody.

[0076] Integer z defines the number of branches containing payload(s) that are connected to a single antibody. Since a branch may have one (y1 = 1) or two (y1 = 2) connection points to the antibody, the integer z does not correspond to the amount of connection points per antibody (which is referred to as z2). Integer z is z2 / y1 . z may be 1 , 2 or 4, preferably z is 1 or 2, most preferably z is 2.

[0077] Integer z2 defines the number of connection points per antibody. Since a branch may have one (y1 = 1) or two (y1 = 2) connection points to the antibody, the integer z2 does not correspond to the amount of branches per antibody (which is referred to as z). Integer z2 is z x y1. z2 may be 2 or 4, preferably z2 is 2.

[0078] Integer x defines the number of payloads connected to linker L^D in a single branch of the conjugate, and corresponds to y2 for AOCs of structure (11) and to 1 for AOCs of structure (12). In other words, x may be 1 , 2, 3 or 4.

[0079] The various structural elements of the AOCs according to the invention are further defined here below.

The antibody Ab

[0080] Ab is an antibody. Antibodies are known in the art and include IgA, IgD, IgE, IgG, IgM, Fab, VHH, scFv, diabody, minibody, affibody, affylin, affimers, atrimers, fynomer, Cys-knot, DARPin, adnectin/centryin, knottin, anticalin, FN3, Kunitz domain, OBody, bicyclic peptides and tricyclic peptides. Preferably, the antibody is a monoclonal antibody, more preferably selected from the group consisting of IgA, IgD, IgE, IgG and IgM antibodies. Even more preferably Ab is an IgG antibody. The IgG antibody may be of any IgG isotype. The antibody may be any IgG isotype, e.g. lgG1 , lgG2, Igl3 or lgG4. Preferably Ab is a full-length antibody, but Ab may also be a Fc fragment.

[0081] The antibody Ab is typically specific for an extracellular receptor on a specific cell. The AOCs of the present invention are suitable for treating muscular diseases, and the antibody is thus preferably specific for an extracellular receptor on a muscle cell or a protein associated with muscular cells, more preferably the antibody is selected from anti-myosin antibodies, anti-transferrin receptor antibodies, anti-insulin receptor antibody (CD220), anti-insulin-like growth factor receptor (IGF1 -R/ CD221), anti- Glucose transporter 4 (GLUT4), anti-clathrin antibody, anti-caveolin antibody, lysosomal associated membrane protein 1 (LAMP1), lysosomal associated membrane protein 1 (LAMP3/CD63), anti- hemojuvelin antibody, anti-Duchenne muscular dystrophy peptide, anti- myosin Hb antibody, anti- CD98hc antibodies, antibodies that recognize muscle-specific kinase (MuSK) or a myogenic precursor protein, preferably the myogenic precursor protein is selected from alpha-Sarcoglycan, beta- Sarcoglycan, Calpain Inhibitors, Creatine Kinase MM/CKMM, elF5A, Enolase 2/Neuron-specific Enolase, epsilon-Sarcoglycan, FABP3/H-FABP, GDF-8/Myostatin, GDF-ll/GDF-8, Integrin alpha 7, Integrin alpha 7 beta 1 , Integrin beta 1/CD29, MCAM/CD146, MyoD, Myogenin, Myosin Light Chain Kinase Inhibitors, NCAM-1/CD56, and Troponin I.

[0082] The ACCs of the present invention are also suitable for targeting immune cells, and the antibody is thus preferably specific for an extracellular receptor on an immune cell. Antibodies known to bind T cells are known in the art, highlighted by Martin et al., Clin. Immunol. 2013, 148, 136-147 and Rossi et al., Int. Immunol. 2008, 20, 1247-1258, both incorporated by reference, for example OKT3, UCHT3, BMA031 and humanized versions thereof. Antibodies known to bind to V-/9V62 T cells are also known, see for example de Bruin et al., J. Immunol. 2017, 198, 308-317, incorporated by reference. In a preferred embodiment, the antibody targets an immune cell, preferably a T cell, an NK cell, a monocyte, a macrophage or a granulocyte. More preferably, the antibody is:

- specific for a cellular receptor on a T cell, preferably wherein the cellular receptor on a T cell is selected from the group consisting of CD3, CD28, CD137, CD134, CD27, V-/9V62 and ICOS; or

- specific for a cellular receptor on an NK cell, preferably wherein the cellular receptor on a NK cell is selected from the group consisting of CD16, CD56, CD335, CD336, CD337, CD28, NKG2A, NKG2D, KIR, DNAM-1 and CD161 ; or

- specific for a cellular receptor on a monocyte or a macrophage, preferably wherein the cellular receptor on the monocyte or macrophage is CD64; or

- specific for a cellular receptor on a granulocyte, preferably wherein the cellular receptor on the granulocyte is CD89.

[0083] The AOCs of the present invention are also suitable for treating neurological diseases, such as Huntington, and the antibody is thus preferably specific for an extracellular receptor on a neuron . Suitable receptors in the context of this embodiment include the transferrin receptor, the insulin receptor, the low-density lipoprotein receptor family and the diphtheria toxin receptor. An example of a suitable antibody is the anti-transferrin receptor antibody RI7217.

[0084] The AOCs of the present invention are also suitable for treating cancer, and the antibody is thus preferably specific for an extracellular receptor on a tumour cell, preferably wherein the extracellular receptor on the tumour cell is selected from the group consisting of 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1 , av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1 B, Brevican, c- KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138/syndecan-1 , CD166, CD19, CD20, CD203c, CD205, CD21 , CD22, CD228, CD25, CD30, CD324, CD33, CD37, CD38, CD45, CD46, CD48a, CD56, CD70, CD71 , CD72, CD74, CD79a, CD79b, CEACAM5, claudin-18.2, claudin- 6, CLEC12A, CLL-1 , Cripto, CRIPTO, CS1 , CXCR5, DLK-1 , DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1 , FcRH2, FcRH5, FGFR2, fibronectin, FLT3, folate receptor alpha, Gal-3BP, GD3, GDNF-Ra1 , GEDA, GFRA1 , Globo H, gpNMB, GPR172A, GPR19, GPR54, guanyl cyclase C, HER2, HER3, HLA-DOB, IGF-1 R, IL13R, IL20Ra, Lewis Y, LGR5, LIV-1 , LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA/B, MOSPD2, MPF, MSG783, MUC1 , MUC16, NaPi2b, NCA, nectin-4, Notch3, P-cadherin, P2X5, PD-L1 , PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1 , ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1 , STEAP2, TAG72, TENB2, TF, TIM-1 , TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinases (RTK), tenascin.

[0085] Part of the antibody may be a linker L⁶ that connects the click probe F or connecting group Z to the peptide part of the cell-binding agent. Herein, L⁶ represents (part of) the glycan of the antibody. Preferably, the connecting group Z is connected to the antibody Ab via a glycan of Ab. Such conjugates are represented by general structure (3).

Ab-[(L6) - (Z)y1 - LD- (D)x]z

(3) wherein:

- L⁶ is -GlcNAc(Fuc)w-(G)j-S-(L⁷)_w-, wherein G is a monosaccharide, j is an integer in the range of 0 - 10, S is a sugar or a sugar derivative, GIcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1 , w’ is 0 or 1 and L⁷ is -N(H)C(O)CH₂-, -N(H)C(O)CF₂- or -CH₂-.

[0086] For the conjugates according to structure (12), and preferred embodiments thereof, linker L⁶ is present. For the conjugates according to structure (11), and preferred embodiments thereof, linker L⁶ is optionally present.

Linker L⁶

[0087] Linker L⁶ is preferably present, wherein reactive group F may be introduced at a specific position of the antibody. This is for example the case for conjugation via an artificially introduced reactive group F¹, such as for example using transglutaminase, using sortase or by enzymatic glycan modification (e.g. glycosyltransferase or a-1 ,3-mannosyl-glycoprotein-2-p-N-acetylglucosaminyl-transferase). For example, a modified sugar residue S(F) or S(F)₂ may be introduced at the glycan, extending the glycan with one monosaccharide residue S, which introduces one or two reactive groups F on each glycan of an antibody. In a most preferred embodiment, conjugation occurs via the glycan of the antibody, i.e. linker L⁶ is present. The site of conjugation is preferably a glycosylation site at the heavy chain of the antibody.

[0088] All recombinant antibodies, generated in mammalian host systems, contain the conserved N- glycosylation site at the asparagine residue at or close to position 297 of the heavy chain (Kabat numbering), which is modified by a glycan of the complex type. This naturally occurring glycosylation site of antibodies is preferably used, but other glycosylation sites, including artificially introduced ones, may also be used for the connection of linker L⁶. Thus, in a preferred embodiment, L⁶ is connected to an amino acid of the antibody which is located at a position in the range of 250 - 350 of the heavy chain, preferably in the range of 280 - 310 of the heavy chain, more preferably in the range of 295 - 300 of the heavy chain, most preferably at position 297 of the heavy chain. Using this conserved glycosylation position of the antibody, the obtained conjugates are formed as symmetrical dimers, wherein each half antibody contains one F, and both click probes F will form an arm with one or more payloads D. Some antibodies may have a second glycosylation site per half antibody, which is not used as conjugation site in the embodiment where z = 2. The skilled person is able to perform the enzymatic conversions in such a way that only the main glycosylation site is utilized for conjugation. Alternatively, the skilled person is able to perform the enzymatic conversion in such a way that also the second glycosylation site is utilized for conjugation, thereby doubling the DAR of the antibody-drug conjugate. In this embodiment, z = 4 and each half antibody contains two occurrences of F, and all four click probes F will form an arm with one or more payloads.

[0089] L⁶ is a linker that connects Ab to F or Z, and is represented by -GlcNAc(Fuc) W— (G)j-S-(L⁷)w-, wherein G is a monosaccharide, j is an integer in the range of 0 - 10, S is a sugar or a sugar derivative, GIcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1 , w’ is 0 or 1 and L⁷ is -N(H)C(O)CH2-, - N(H)C(O)CF2- or -CH2-. Typically, L⁶ is at least partly formed by the glycan of an antibody.

[0090] The -GlcNAc(Fuc)v^(G)j- moiety of L⁶ is formed from the glycan of the antibody. Hence, the - GlcNAc(Fuc)v^(G)j- moiety typically originates from the original antibody. Fuc is typically bound to GIcNAc via an a-1 ,6-glycosidic bond. Normally, antibodies may (w = 1) or may not be fucosylated (w = 0). In the context of the present invention, the presence of a fucosyl moiety is irrelevant, and similar effects are obtained with fucosylated (w = 1) and non-fucosylated (w = 0) antibody conjugates. The GIcNAc residue may also be referred to as the core-GIcNAc residue and is the monosaccharide that is directly attached to the peptide part of the antibody.

[0091] S may be directly connected to the core-GlcNAc(Fuc)_w moiety, i.e. j = 0, meaning that the remainder of the glycan is removed from the core-GlcNAc(Fuc)_w moiety before S is attached. Such trimming of glycans is well-known in the art and can be achieved by the action of an endoglycosidase. Alternatively, there are one or more monosaccharide residues present in between the core- GlcNAc(Fuc)_w moiety and S, i.e. j is an integer in the range of 1 - 10, preferably j = 1 - 5. In one preferred embodiment, (G)j is an oligosaccharide fraction comprising j monosaccharide residues G, wherein j is an integer in the range of 2 - 5. In another preferred embodiment, (G)j is a monosaccharide fraction comprising j monosaccharide residues G, wherein j is 1 . (G)j is connected to the GIcNAc moiety of GlcNAc(Fuc)_w, typically via a p-1 ,4 bond. In a preferred embodiment, j is 0, 1 , 3, 4 or 5, more preferably, j is 0 or 1 , most preferably j is 0.

[0092] Although any monosaccharide that may be present in a glycan may be employed as G, each G is preferably individually selected from the group consisting of galactose, glucose, N- acetylgalactosamine, A/-acetylglucosamine, mannose and A/-acetylneuraminic acid. More preferred options for G are galactose, A/-acetylglucosamine, mannose. In case j = 1 , it is preferred that G = galactose and S = A/-acetylneuraminic acid.

[0093] When j is in the range of 3 - 10, (G)j may be linear or branched. Preferred examples of branched oligosaccharides (G)j are (a), (b), (c), (d), (e), (f), (h) and (h) as shown below.

Man Man — GIcNAc

Man — GIcNAc Man — GIcNAc a b Man — GIcNAc Man — GIcNAc

[0094] In case (G)j is present with j > 2, it is preferred that it ends in GIcNAc or Gal, preferably GIcNAc. In other words, the monosaccharide residue directly connected to S is preferably GIcNAc or Gal. The presence of a GIcNAc moiety facilitates the synthesis of the functionalized antibody, as monosaccharide derivative S can readily be introduced by glycosyltransfer onto a terminal GIcNAc residue. The presence of a Gal moiety facilitates the synthesis of the functionalized antibody, as monosaccharide derivative S sialic acid can readily be introduced by sialyltransferase onto a terminal Gal residue. In the above preferred embodiments for (G)j, having structure (a) - (h), moiety S may be connected to any of the terminal GIcNAc residues, i.e. not the one with the wavy bond, which is connected to the core GIcNAc residue on the antibody.

[0095] In a preferred embodiment in which z is 4, (G)j is a branched oligosaccharide, and the -S-(L⁷)_W- group is present on two branches. Suitable options for (G)j include structures (b) and (h).

[0096] Antibodies and antibody conjugates having j = 0 or 1 show no or significantly reduced binding to Fc-gamma receptors, while antibodies and antibody conjugates having j in the range of 4 - 10 do bind to Fc-gamma receptors. Thus, by selecting a certain value for j, the desired extent of binding to Fc-gamma receptors can be obtained. It is thus preferred that j = 0, 1 , 4, 5, 6, 7, 8, 9 or 10, more preferably j = 0, 1 , 4 or 5, most preferably the antibody is trimmed and j = 0.

[0097] S is a sugar or sugar derivative. The term “sugar derivative” is herein used to indicate a derivative of a monosaccharide sugar, i.e. a monosaccharide sugar comprising substituents and/or functional groups. Suitable examples for S include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugars and sugar acids, e.g. glucosamine (GIcNFh), galactosamine (GaINFh) N- acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referred to as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA). Preferably, S is selected from Gal, GalNAc and NeuNAc. In an especially preferred embodiment, S is GalNAc. [0098] Connecting group Z or reactive group F may be attached directly to S, or there may be a linker L⁷ present in between S and Z or F. Thus, L⁷ may be present (w’ = 1 or 2) or absent (w’ = 0). Typically, each moiety Z may be connected to S via a linker L⁷. Preferably, L⁷ is absent and each connecting moiety Z is directly attached to S. If present, L⁷ may be selected from -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-. In a preferred embodiment, w’ = 0 or 1 , most preferably w’ = 0.

[0099] In one embodiment, the conjugates according to the invention contain two connecting groups Z¹, which are formed during a click reaction wherein the antibody of structure Ab(F¹)_Z2 and a heterobifunctional (y1 + y2)-valent linker construct of structure of (Q¹)yi - L^A - (F²)_y2. In a first click reaction, F¹ and Q¹ react to form a covalent connection between the antibody and z2 (z x yi) moieties F² by forming connecting groups Z¹.

Connecting group Z

[0100] Z is a connecting group. The term “connecting group’ refers to a structural element connecting one part of the conjugate and another part of the same conjugate. Connecting group Z results from a reaction, here between Q and F, connecting one part of the conjugate with another part of the same conjugate. In the present invention, the connecting group(s) Z connect antibody Ab with the oligonucleotide payloads D. The conjugates according to the invention may contain one distinct connecting group Z, for example as in structure (12). Herein, the conjugate contains z connecting groups Z, but all of the same type. Alternatively, the conjugates according to the invention may contain two distinct connecting groups Z¹ and Z², which are individually selected, for example as in structure (11). Herein, the conjugate contains y1 x z connecting groups Z¹ and y2 x z connecting groups Z², but all Z¹ are of the same type and all Z² are of the same type. Z¹ is formed by a click reaction between Q¹ and F¹. Likewise, Z² is formed by a click reaction between Q² and F². Herein, the definition of Z and preferred embodiments thereof also apply to Z¹ and Z². Likewise, the definition of Q and preferred embodiments thereof also apply to Q¹ and Q², and the definition of F and preferred embodiments thereof also apply to F¹ and F²

[0101] As will be understood by the person skilled in the art, the exact nature of the connecting group depends on the exact structure of the click probes Q and F. The skilled person is aware of complementary click probes which are reactive towards each other and form suitable reaction partners Q¹/F¹ and Q²/F². For example, when F comprises or is an alkynyl group, complementary groups Q include azido groups. For example, when F comprises or is an azido group, complementary groups Q include alkynyl groups. For example, when F comprises or is a cyclopropenyl group, a trans-cyclooctene group, a cycloheptyne or a cyclooctyne group, complementary groups Q include tetrazinyl groups. In these particular cases, Z is only an intermediate structure and will expel N2, thereby generating a dihydropyridazine (from the reaction with alkene) or pyridazine (from the reaction with alkyne) as shown in Figure 4.

[0102] Connecting groups Z are obtained by a cycloaddition reaction, preferably wherein the cycloaddition is a [4+2] cycloaddition or a 1 ,3-dipolar cycloaddition. Conjugation reactions via cycloadditions are known to the skilled person, and the skilled person is capable of selecting appropriate reaction partners F and Q, and will understand the nature of the resulting connecting group Z. Preferred cycloadditions are a [4+2]-cycloaddition (e.g. a Diels-Alder reaction) or a [3+2]-cycloaddition (e.g. a 1 ,3- dipolar cycloaddition). Preferably, the cycloaddition is the Diels-Alder reaction or the 1 ,3-dipolar cycloaddition. The preferred Diels-Alder reaction is the inverse electron-demand Diels-Alder cycloaddition. In another preferred embodiment, the 1 ,3-dipolar cycloaddition is used, more preferably the alkyne-azide cycloaddition. Cycloadditions, such as Diels-Alder reactions and 1 ,3-dipolar cycloadditions are known in the art, and the skilled person knows how to perform them.

[0103] Preferably, Z contains a moiety selected from the group consisting of a triazole, a cyclohexene, a cyclohexadiene, a [2.2.2]-bicyclooctadiene, a [2.2.2]-bicyclooctene, an isoxazoline, an isoxazolidine, a pyrazoline, a piperazine or a pyridazine, more preferably a triazole, an isoxazoline or pyridazine. Triazole moieties are especially preferred to be present in Z. In one embodiment, Z comprises a (hetero)cycloalkene moiety, i.e. formed from Q comprising a (hetero)cycloalkyne moiety. In an alternative embodiment, Z comprises a (hetero)cycloalkane moiety, i.e. formed from Q comprising a (hetero)cycloalkene moiety. Herein, aromatic rings such as a triazole ring are considered a heterocycloalkane ring, since it is formed by reaction of an alkyne moiety and an azide moiety.

[0104] In an especially preferred embodiment, connecting group Z is obtained by an ultrafast click reaction. Ultrafast click and preferred embodiments thereof are further defined below. In one embodiment, Z¹ and/or Z² is obtained by ultrafast click, more preferably at least Z² is obtained by ultrafast click.

[0105] In a preferred embodiment, Z has the structure (Z1):

Herein, the bond depicted as - is a single bond or a double bond. Furthermore:

- ring Z is obtained by a cycloaddition, preferably ring Z is selected from (Za) - (Zu), preferably from

(Za) - (Zm) defined below, wherein the carbon atoms labelled with ** correspond to the two carbon atoms of the bond depicted as - of (Z1) to which ring Z is fused;

- the wavy bond labelled with * is connected to Ab;

- R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, -NO2, -CN, -S(O)2R¹⁶, -S(O)₃<->, CI - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups and wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R¹⁵ may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R¹⁶ is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups; - Y² is C(R³¹)₂, O, S, S⁽⁺⁾R³¹ , S(O)R³¹, S(O)=NR³¹ or NR³¹, wherein S⁽⁺⁾ is a cationic sulphur atom counterbalanced by B< ), wherein B< > is an anion, and wherein each R³¹ individually is R¹⁵ or a connection with D, connected via L;

- u is 0, 1 , 2, 3, 4 or 5; - u’ is 0, 1 , 2, 3, 4 or 5, wherein u + u’ = 0, 1 , 2, 3, 4, 5, 6, 7 or 8;

- v = an integer in the range 8 - 16;

- Ring Z is formed by the cycloaddition, and is preferably selected from (Za) - (Zm).

[0106] In a preferred embodiment, u + u’ = 0, 4, 5, 6, 7 or 8, more preferably 0, 4 or 5. In case the bond depicted as - is a double bond, it is preferred that u + u’ = 4, 5, 6, 7 or 8, more preferably u + u’ = 4 or 5. In case the bond depicted as - is a single bond, it is preferred that u + u’ = 0 or 5. Preferably, the wavy bond labelled with * is connected to CB, optionally via L⁶, and the wavy bond labelled with ** is connected to L.

[0107] It is especially preferred that Z comprises a (hetero)cycloalkene moiety, i.e. the bond depicted as - is a double bond. In a preferred embodiment, Z is selected from the structures (Z2) - (Z20c), preferably selected from the structures (Z2) - (Z20), depicted here below:

(Z11) (Z12) (Z13) (Z14) (Z15)

(Z20a) (Z20b) (Z20c)

[0108] Herein, the connection to L is depicted with the wavy bond. B<^_) is an anion, preferably a pharmaceutically acceptable anion. B⁽⁺⁾ is a cation, preferably a pharmaceutically acceptable cation.

[0109] R³⁶ is an halogen selected from fluor, chlorine, bromine and iodine, preferably R³⁶ is fluor. Y⁴ is a heteroatom, preferably Y⁴ is O or NH. R³⁵ is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, Si, S and NR¹⁴ wherein R¹⁴ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably R³⁵ is selected from H, C5H11, CH₃, CH2CH3, CH2OH or CH2OTBS.

[0110] Ring Z is formed by the cycloaddition reaction, and preferably is a triazole, a cyclohexene, a cyclohexadiene, a [2.2.2]-bicyclooctadiene, a [2.2.2]-bicyclooctene, an isoxazoline, an isoxazolidine, a pyrazoline or a piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have the structure selected from (Za) - (Zm) depicted below, wherein the carbon atoms labelled with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring of (Z2) - (Z20), to which ring Z is fused. Since the connecting group Z is formed by reaction with a (hetero)cycloalkyne in the context of the present embodiment, the bond depicted above as - is a double bond.

[0111] Herein, R²⁹ is selected from hydrogen, C1-6 alkyl, aryl, C(O)-Ci-6 alkyl, C(O)-aryl, C(O)-O-Ci-6 alkyl, C(O)-O-aryl, C(O)-NR³³-CI-6 alkyl and C(O)-NR³³-aryl, wherein R³³ is H or C1-4 alkyl. Preferably, R²⁹ is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl. It was found that R²⁹ is hydrogen gave optimal results in reactivity in the cycloaddition reaction, especially in case ring (Zl) is formed. Thus, in a preferred embodiment, ring Z is (Zl) wherein R²⁹ is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R²⁹ is hydrogen.

[0112] Isoxazoline ring (Zh) may rearrange to (Zh’) in case R¹ is L¹⁰XR³⁶. For isoxazoline (Zh) and rearrangement product (Zh’), the following applies: - R¹ is H, C1-6 alkyl or L¹⁰XR³⁸;

- L¹⁰ is a linker of structure (C(R³⁷)2)z, wherein z is 2 or 3, and each R³⁷ is individually selected from H and C1-4 alkyl, wherein two occurrences of R³⁷ may be joined together to form a C3-6 (hetero)cycloalkyl group;

- R³⁸ is selected from H and C1-4 alkyl; - X is S, O or NH,

- R² is selected from H and C1-4 alkyl, preferably R² is H. [0113] Preferably, R¹ is H or methyl and R¹ is H. Preferred embodiments are defined for nitrone reactive group (F3a) below, which equally apply to isoxazoline (Zh) and rearrangement product (Zh’).

[0114] In case Z comprises a (hetero)cycloalkene moiety, it is preferred that ring Z is selected from (Za), (Zj), (Zk) or (Zl), more preferably ring Z is according to structure (Za) or (Zl). [0115] In a further preferred embodiment, Z is selected from the structures (Z21) - (Z38d), preferably selected from the structures (Z21) - (Z38), depicted here below:

(Z38b) (Z38c) (Z38d).

[0116] Herein, the connection to the linker is depicted with the wavy bond. Structure (Z29) can be in endo or exo configuration, preferably it is in endo configuration. In structure (Z28), B⁽⁺⁾ is a cation, preferably a pharmaceutically acceptable cation. In structure (Z38), B^(_) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from structures (Za) - (Zm), as defined above. R³⁵ and R³⁶ are as defined above for (Z20a) - (Z20c).

[0117] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety or a (hetero)cycloheptene moiety, preferably according to structure (Z8), (Z26), (Z27), (Z28) , (Z37) or (Z38a), which are optionally substituted. Each of these preferred options for Z are further defined here below.

[0118] Thus, in a preferred embodiment, Z comprises a heterocycloheptene moiety according to structure (Z37), which is optionally substituted. Preferably, the heterocycloheptene moiety according to structure (Z37) is not substituted.

[0119] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety according to structure (Z8), more preferably according to (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is not substituted. In the context of the present embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z39) as shown below, wherein V is (CH2)I and I is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, I is 0, 1 , 2, 3 or 4, more preferably I is 0, 1 or 2 and most preferably I is 0 or 1 . In the context of group (Z39), I is most preferably 1 . Most preferably, Z is according to structure (Z42), defined further below.

[0120] In an alternative preferred embodiment, Z comprises a (hetero)cyclooctene moiety according to structure (Z26), (Z27) or (Z28), which is optionally substituted. In the context of the present embodiment, Z preferably comprises a (hetero)cyclooctene moiety according to structure (Z40) or (Z41) as shown below, wherein Y¹ is O or NR¹¹, wherein R¹¹ is independently selected from the group consisting of hydrogen, a linear or branched Ci - C12 alkyl group or a C4- C12 (hetero)aryl group. The aromatic rings in (Z40) are optionally O-sulfated at one or more positions, whereas the rings of (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moiety according to structure (Z40) or (Z41) is not further substituted. Most preferably, Z is according to structure (Z43), defined further below.

[0121] In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group and is according to structure (Z37).

(Z37) (Z39) (Z40) (Z41)

[0122] In an especially preferred embodiment, Z comprises a cyclooctenyl group and is according to structure (Z42):

Herein:

- the bond labelled with * is connected to CB and the wavy bond labelled with ** is connected to L;

- R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, -NO2, -CN, -S(O)2R¹⁶, -S(O)3^H,CI - C24 alkyl groups, C5 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups and wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R¹⁵ may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R¹⁶ is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups;

- R¹⁸ is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups;

- R¹⁹ is selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups, the alkyl groups optionally being interrupted by one of more hetero-atoms selected from the group consisting of O, N and S, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are independently optionally substituted, or R¹⁹ is a second occurrence of Z (or Q) or D connected via a spacer moiety; and

- I is an integer in the range 0 to 10.

[0123] In a preferred embodiment of the group according to structure (Z42), R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, Ci - Ce alkyl groups, C5 - Ce (hetero)aryl groups, wherein R¹⁶ is hydrogen or Ci - Ce alkyl, more preferably R¹⁵ is independently selected from the group consisting of hydrogen and Ci - Ce alkyl, most preferably all R¹⁵ are H. In a preferred embodiment of the group according to structure (Z42), R¹⁸ is independently selected from the group consisting of hydrogen, Ci - Ce alkyl groups, most preferably both R¹⁸ are H. In a preferred embodiment of the group according to structure (Z42), R¹⁹ is H. In a preferred embodiment of the group according to structure (Z42), I is 0 or 1 , more preferably I is 1 .

[0124] In an especially preferred embodiment, Z comprises a (hetero)cyclooctenyl group and is according to structure (Z43):

Herein:

- the bond labelled with * is connected to CB and the wavy bond labelled with ** is connected to L;

- R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, -NO2, -CN, -S(O)2R¹⁶, -S(O)₃(->, CI - C24 alkyl groups, C5 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups and wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R¹⁵ may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R¹⁶ is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups;

- Y is N or CR¹⁵;

- a carbon atom in the fused aromatic rings may be replaced by a nitrogen atom, as in (Z6a) - (Z6d), preferably wherein Y is CR¹⁵.

[0125] In a preferred embodiment of the group according to structure (Z43), R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, -S(O)3^{( )}, Ci - Ce alkyl groups, C5 - Ce (hetero)aryl groups, wherein R¹⁶ is hydrogen or Ci - Ce alkyl, more preferably R¹⁵ is independently selected from the group consisting of hydrogen and -S(O)3^{( )}. In a preferred embodiment of the group according to structure (Z43), Y is N or CH, more preferably Y = N.

[0126] In an especially preferred embodiment, Z comprises a heterocycloheptenyl group and is according to structure (Z37) or (Z38a), wherein ring Z is a triazole.

(Z37) (Z38a)

[0127] In an alternative preferred embodiment, connecting group Z comprises a (hetero)cycloalkane moiety, i.e. the bond depicted as - is a single bond. The (hetero)cycloalkane group may also be referred to as a heterocycloalkyl group or a cycloalkyl group, preferably a cycloalkyl group, wherein the (hetero)cycloalkyl group is optionally substituted. Preferably, the (hetero)cycloalkyl group is a (hetero)cyclopropyl group, a (hetero)cyclobutyl group, a norbornyl group, a norbornenyl group, a (hetero)cycloheptyl group, a (hetero)cyclooctyl group, which may all optionally be substituted.

Especially preferred are (hetero)cyclopropyl groups, (hetero)cycloheptyl group or (hetero)cyclooctyl groups, wherein the (hetero)cyclopropyl group, the (hetero)cycloheptyl group or the (hetero)cyclooctyl group is optionally substituted. Preferably, Z comprises a cyclopropyl moiety according to structure (Z44), a hetereocyclobutane moiety according to structure (Z45), a norbornane or norbornene group according to structure (Z46), a (hetero)cycloheptyl moiety according to structure (Z47) or a

(hetero)cyclooctyl moiety according to structure (Z48). Herein, Y³ is selected from C(R²³)2, NR²³ or O, wherein each R²³ is individually hydrogen, Ci - Ce alkyl or is connected to L, optionally via a spacer, and the bond labelled - is a single or double bond. In a further preferred embodiment, the cyclopropyl group is according to structure (Z49). In another preferred embodiment, the

(hetero)cycloheptane group is according to structure (Z50) or (Z51). In another preferred embodiment, the (hetero)cyclooctane group is according to structure

[0128] Herein, the R group(s) on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably Ci-Ce alkyl.

Ring Z is formed during the cycloaddition reaction and is typically selected from structures (Zn) - (Zu), wherein the carbon atoms labelled with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring of (Z44) - (Z56) to which ring Z is fused, and the carbon a carbon labelled with * is connected to CB. Since the connecting group Z is formed by reaction with a (hetero)cycloalkene in the context of the present embodiment, the bound depicted above as - is a single bond.

(Zn) (Zo) (Zp) (Zq)

[0129] Herein, R²⁹ is selected from hydrogen, C1-6 alkyl, aryl, C(O)-Ci-6 alkyl, C(O)-aryl, C(O)-O-Ci-6 alkyl, C(O)-O-aryl, C(O)-NR³³-CI-6 alkyl and C(O)-NR³³-aryl, wherein R³³ is H or C1-4 alkyl. Preferably, R²⁹ is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl. It was found that R²⁹ is hydrogen gave optimal results in reactivity in the cycloaddition reaction, especially in case ring (Zu) is formed. Thus, in a preferred embodiment, ring Z is (Zu) wherein R²⁹ is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R²⁹ is hydrogen.

[0130] In case Z comprises a (hetero)cycloalkane moiety, it is preferred that ring Z is selected from (Zn), (Zs), (Zt) or (Zu), most preferably ring Z is according to structure (Zu).

[0131] In a preferred embodiment, connection group Z comprise a moiety selected from (Z1) - (Z56), wherein ring Z is selected from (Za) - (Zu).

[0132] In case the AOC according to the invention contains two distinct connecting groups Z¹ and Z², these typically differ, as Z¹ is formed by reaction of Q¹ and F¹, whereas Z² is formed by reaction of Q² and F². Herein, F² is reactive towards Q² but not towards Q¹, such that Q¹ and Q² should differ.

[0133] In a preferred embodiment, F¹ is azide and Q¹ is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F² is tetrazine or nitrone and Q² is bicyclononyne or cycloalkene, such as a frans-cyclooctene or a cyclopropene. More preferably, F¹ is azide and Q¹ is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F² is tetrazine and Q² is bicyclononyne.

[0134] Herein, the reaction of F¹ and Q¹ will preferably form a connecting group Z¹ according to structure (Z5), (Z6), (Z7), (Z1 1), (Z17), (Z18), (Z19) or (Z19a), wherein ring Z is according to structure (Za), preferably according to structure (Z26), (Z27), (Z28), (Z32), (Z37), (Z38) or (Z38a), more preferably according to structure (Z40), (Z41) or (Z43), or according to structure (Z37) or (Z43). Herein, the reaction of F² and Q² will preferably form a connecting group Z² according to structure (Z8), (Z44), (Z47), (Z48), (Z49), (Z54), (Z55) or (Z56), more preferably according to structure (Z29), (Z48) or (Z49), most preferably according to structure (Z42). Herein, ring Z is according to structure (Zd), (Zl), (Zq) or (Zu), preferably according to structure (Zl) or (Zu), most preferably according to structure (Zl). Alternatively, connecting group Z² is according to structure (Z8), wherein ring Z is according to structure (Zl), preferably according to structure (Z29), more preferably according to structure (Z42). Linker L^A

[0135] Linker L^A connects payload D, via linker L^B and connecting groups Z², with Ab via connecting group Z¹ (in the conjugates according to the invention) or connects reactive group F² with reactive group Q¹ (in the linker). Linkers are known in the art and may be cleavable or non-cleavable. Linker L^A preferably is not cleavable linker, while linker L^B preferably is cleavable. Preferably, linker L^A is present, i.e. the AOC is according to structure (11). The inventors found that the AOCs, especially DAR 4 AOCs, are readily prepared with intermediate spacer (11), and the resulting AOCs have excellent therapeutic properties as demonstrated in the examples.

[0136] Linker L^A is connected to y1 occurrences of Z¹ (or Q¹) and y2 occurrences of Z² (or F²). Thus, the valency of linker L^A is y1 + y2 or “(y1 + y2)-valent”. Herein, “(y1 + y2)-valent” refers to the number of connecting points, being either a reactive group F or Q (before reaction) or a connecting group Z (after reaction). For example, in case y1 = 1 and y2 = 1 , the linker is connected to one Z¹/Q¹ and one Z²/F² and the linker has a valency of 1 + 1 = 2. Thus, when y1 = 1 and y2 = 1 , the linker L^A is bivalent. Likewise, in case y1 = 1 and y2 = 1 , the linker is connected to one Z¹/Q¹ and two Z²/F² and the linker has a valency of 1 + 2 = 3. Thus, when y1 = 1 and y2 = 2, the linker L^A is trivalent. Thus, in case y1 = 1 and y2 = 3, the linker is connected to one Z¹/Q¹ and three Z²/F² and the linker has a valency of 1 + 3 = 4. Thus, when y1 = 1 and y2 = 3, the linker L^A is tetravalent. Thus, in case y1 = 2 and y2 = 1 , the linker is connected to one Z¹/Q¹ and four Z²/F² and the linker has a valency of 2 + 1 = 1 . Thus, when y1 = 2 and y2 = 1 , the linker L^A is trivalent. Preferably, linker L^A is bivalent or trivalent.

[0137] Linker L^A may be referred to as “heterobifunctional”, which means that is contains two different functionalities, referring to the chemically different connectivities to Z¹/Q¹ and Z²/F².

[0138] In a preferred embodiment, L^A is a heterobifunctional linker of structure

(L¹¹)y1-BM-(L¹²)y2 wherein:

- each L¹¹ is connected to Q¹ or Z¹, and each L¹² is connected to F² or Z², wherein L¹¹ and L¹² each individually consist of one or more building blocks selected from C(R¹³)2, C(R¹³)=C(R¹³), C=C, aryl, C(O), NR¹³, O, S, S(O), S(O)₂, PR¹³ and P(O)R¹³, preferably the building blocks are selected from CH₂, CH=CH, Ph, C(O), NR¹³, O, S, S(O) and S(O)₂,

- each R¹³ is as defined below for structure (23), preferably R¹³ is H;

- BM is a branching moiety, which is present if y1 + y2 = 3 or higher. The branching moiety is further defined below;

- y1 is 1 or 2, preferably y1 = 1 ;

- y2 is 1 , 2, 3 or 4, preferably y2 is 1 or 2.

[0139] In a more preferred embodiment, L^A is a heterobifunctional bivalent linker of structure

(L¹¹)-(L¹²) wherein:

- L¹¹ is connected to Q¹ or Z¹, and L¹² is connected to F² or Z², wherein L¹¹ and L¹² each individually consist of one or more building blocks selected from C(R¹³)2, C(R¹³)=C(R¹³), C=C, aryl, C(O), NR¹³, O, S, S(O), S(O)₂, PR¹³ and P(O)R¹³, preferably the building blocks are selected from CH2, CH=CH, Ph, C(O), NR¹³, O, S, S(O) and S(O)₂, and - each R¹³ is as defined below for structure (23), preferably R¹³ is H.

[0140] In an alternative more preferred embodiment, L^A is a heterobifunctional trivalent linker of structure

(L¹¹)-BM(L¹²)(L¹³) wherein:

- L¹¹ is connected to Q¹ or Z¹ , L¹² is connected to F² or Z², and L¹³ is connected to Q¹ or Z¹ or to F² or Z², wherein L¹¹, L¹² and L¹³ each individually consist of one or more building blocks selected from C(R¹³)₂, C(R¹³)=C(R¹³), CEC, aryl, C(O), NR¹³, O, S, S(O), S(O)₂, PR¹³ and P(O)R¹³, preferably the building blocks are selected from CH₂, CH=CH, C(O), Ph, NR¹³, O, S, S(O) and S(O)₂, and

- each R¹³ is as defined below for structure (23), preferably R¹³ is H; and

- BM is a branching moiety. The branching moiety is further defined below.

[0141] In case linker L^A is a heterobifunctional trivalent linker with two connectivities to Q¹ or Z¹, it is preferred that both L¹¹ and L¹³ are identical. In case linker L^A is a heterobifunctional trivalent linker with two connectivities to Q² or Z², it is preferred that both L¹² and L¹³ are identical.

[0142] L¹¹, L¹² and L¹³ may for example be selected from the group consisting of linear or branched Ci-C₂oo alkylene groups, C₂-C₂oo alkenylene groups, C₂-C₂oo alkynylene groups, C3-C₂oo cycloalkylene groups, C5-C₂oo cycloalkenylene groups, Ca-C₂oo cycloalkynylene groups, C7-C₂oo alkylarylene groups, C7-C₂oo arylalkylene groups, Ca-C₂oo arylalkenylene groups, Cg-C₂oo arylalkynylene groups. Optionally the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups may be substituted, and optionally said groups may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably being selected from the group consisting of O, S(O)_y and NR²¹, wherein y’ is 0, 1 or 2, preferably y’ = 2, and R²¹ is independently selected from the group consisting of hydrogen, halogen, Ci - C₂4 alkyl groups, Ce - C₂4 (hetero)aryl groups, C7 - C₂4 alkyl(hetero)aryl groups and C7 - C₂4 (hetero)arylalkyl groups. In one embodiment, the optional substituents may be selected from polar groups, such as oxo groups, (poly)ethylene glycol diamines, (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, carboxylic acid groups, carbonate groups, carbamate groups, cyclodextrins, crown ethers, saccharides (e.g. monosaccharides, oligosaccharides), phosphates or esters thereof, phosphonic acid or ester, phosphinic acid or ester, sulfoxides, sulfones, sulfonic acid or ester, sulfinic acid, or sulfenic acid.

[0143] In the context of the present invention, a linker construct of structure (Q¹)yi - L^A- (F²)_y2 reacts with Ab(F¹)_z2, thereby performing z2 click reactions between F¹ and Q¹ per antibody Ab, and thus forming z2 connecting groups Z¹ per antibody Ab. Herein, z2 equals z x y1 . Preferably, y1 = 1 and thus z2 = z. Alternatively, y1 = 2 and thus z2 = 2 * z. Upon completion of the reaction, an antibody-linker construct is formed containing z x y2 click probes F² per antibody. The antibody-linker construct thus formed has a structure Ab [ (Z¹)_yi - L^A (F²)_y2 ]_z. The branching moiety BM

[0144] A “branching moiety” in the context of the present invention refers to a moiety that is embedded in a linker connecting three moieties. In other words, the branching moiety comprises at least three bonds to other moieties.

[0145] Any moiety that contains at least three bonds to other moieties is suitable as branching moiety in the context of the present invention. Suitable branching moieties include a carbon atom (BM-1), a nitrogen atom (BM-3), a phosphorus atom (phosphine (BM-5) and phosphine oxide (BM-6)), aromatic rings such as a phenyl ring (e.g. BM-7) or a pyridyl ring (e.g. BM-9), a (hetero)cycle (e.g. BM-11 and BM-12) and polycyclic moieties (e.g. BM-13, BM-14 and BM-15). Preferably, BM is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cycle or a polycyclic moiety, more preferably BM is a carbon atom or a nitrogen atom. In case BM is a nitrogen atom, the branching nitrogen may be part of an amide group, for example with the connection to Z¹ or Q¹ via the C=O group and the connection to two occurrences of Z² or F² via the two substituents on nitrogen. In case BM is a carbon atom, the carbon atom is preferably part of a trivalent amino acid, such as lysine, aspartic acid or glutamic acid.

[0146] Suitable branching moieties BM are selected from structures (BM-1) to (BM-15) depicted here below, wherein the three branches, i.e. bonds to other moieties as defined above, are indicated by * (a bond labelled with *).

BM-13 BM-14 BM-15 [0147] In (BM-1), one of the branches labelled with * may be a single or a double bond, indicated with 2^^. In (BM-11) to (BM-15), the following applies:

- each of n, p, q and q is individually an integer in the range of 0 - 5, preferably 0 or 1 , most preferably 1 ;

- each of W¹, W² and W³ is independently selected from C(R²¹)_W and N;

- each of W⁴, W⁵ and W⁶ is independently selected from C(R²¹)_w+i, N(R²²)_W, O and S;

- each - represents a single or double bond;

- w is 0 or 1 or 2, preferably 0 or 1 ;

- each R²¹ is independently selected from the group consisting of hydrogen, OH, Ci - C24 alkyl groups, Ci - C24 alkoxy groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, wherein the Ci - C24 alkyl groups, Ci - C24 alkoxy groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR³ wherein R³ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups; and

- each R²² is independently selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, wherein the Ci - C24 alkyl groups, Ci - C24 alkoxy groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR³ wherein R³ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups.

[0148] The skilled person appreciates that the values ofw and the bond order of the bonds represented by - are interdependent. Thus, whenever an occurrence of W is bonded to an endocyclic double bond, w = 1 for that occurrence of W, while whenever an occurrence of W is bonded to two endocyclic single bonds, w = 0 for that occurrence of W. For BM-12, at least one of 0 and p is not 0.

[0149] Representative examples of branching moieties according to structure (BM-11) and (BM-12) include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, aziridine, azetidine, diazetidine, oxetane, thietane, pyrrolidine, dihydropyrrolyl, tetrahydrofuranyl, di hydrofuranyl, thiolany I, imidazolinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, piperidinyl, oxanyl, thianyl, piperazinyl, morpholino, thiomorpholino, dioxanyl, trioxanyl, dithyanyl, trithianyl, azepanyl, oxepanyl and thiepanyl. Preferred cyclic moieties for use as branching moiety include cyclopropenyl, cyclohexyl, oxanyl (tetrahydropyran) and dioxanyl. The substitution pattern of the three branches determines whether the branching moiety is of structure (BM-11) or of structure (BM-12).

[0150] Representative examples of branching moieties according to structure (BM-13) to (BM-15) include decalin, tetralin, dialin, naphthalene, indene, indane, isoindene, indole, isoindole, indoline, isoindoline, and the like. [0151] In a preferred embodiment, BM is a carbon atom. In case the carbon atom is according to structure (BM-1) and has all four bonds to distinct moieties, the carbon atom is chiral. The stereochemistry of the carbon atom is not crucial for the present invention, and may be S or R. The same holds for the phosphine (BM-6). Most preferably, the carbon atom is according to structure (BM- 1). One of the branches indicated with * in the carbon atom according to structure (BM-1) may be a double bond, in which case the carbon atom may be part of an alkene or imine. In case BM is a carbon atom, the carbon atom may be part of a larger functional group, such as an acetal, a ketal, a hemiketal, an orthoester, an orthocarbonate ester, an amino acid and the like. Preferred amino acids in this respect are Asp, Gly, Lys and iGlu. This also holds in case BM is a nitrogen or phosphorus atom, in which case it may be part of an amide, an imide, an imine, a phosphine oxide (as in BM-6) or a phosphotriester.

[0152] In a preferred embodiment, BM is a phenyl ring. Most preferably, the phenyl ring is according to structure (BM-7). The substitution pattern of the phenyl ring may be of any regiochemistry, such as 1 ,2,3-substituted phenyl rings, 1 ,2,4-substituted phenyl rings, or 1 ,3,5-substituted phenyl rings. To allow optimal flexibility and conformational freedom, it is preferred that the phenyl ring is according to structure (BM-7), most preferably the phenyl ring is 1 ,3,5-substituted. The same holds for the pyridine ring of (BM-9).

[0153] In the preferred structures for BM defined above, BM typically contains three connection points. The skilled person is capable to determine corresponding BMs with four or five connection points. For example (BM-1) and (BM-7) - (BM-15) would also be suitable as BM with more connection points. Alternatively, the linker may contain more than one BM to create four or five connection points.

[0154] In a preferred embodiment, the branching moiety BM is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cycle or a polycyclic moiety, more preferably a carbon atom or a nitrogen atom. Most preferably, BM is a nitrogen atom, preferably a nitrogen atom part of an amide.

Preferred linkers L^A

[0155] Especially preferred linkers L^A have the structure -(L¹¹)-BM(L¹²-)(L¹³-), wherein L¹¹ is according to any one of (i) - (x):

(i) *-C(O)-(CH₂)a-C(O)-**, wherein the bond labelled * is connected to Q¹, Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, wherein a’ is an integer in the range 1 - 5, preferably a - 3. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

(ii) *-NH-C(O)-(CH₂)a-**, wherein the bond labelled * is connected to Q¹, Z¹ , Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, wherein a’ is an integer in the range 0 - 5, preferably a - 0. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

(iii) *=N-C(O)-(CH₂)a-**, wherein the double bond labelled * is connected to Q¹, Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, wherein a’ is an integer in the range 0 - 5, preferably a - 0. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N. (iv) *-NH-C(0)-(CH2)a-C(0)-**, wherein the bond labelled * is connected to Q¹, Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, wherein a’ is an integer in the range 1 - 5, preferably a - 3. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

(v) *=N-C(O)-(CH2)a-C(O)-**, wherein the double bond labelled * is connected to Q¹, Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, wherein a’ is an integer in the range 1 - 5, preferably a - 3. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

(vi) *-C(O)-**, wherein the bond labelled * is connected to Q¹, Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, wherein it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

(vii) *-(23)-(CH₂CH₂O)_a-(C(O))_a ■■-**, wherein the bond labelled * is connected to Q¹, Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, and wherein (23) refers to a group according to structure (23). Further, a’ is an integer in the range 0 - 5, preferably a - 1 - 4, most preferably a’ = 2, and a” is 0 or 1 , preferably a” is 1. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

(viii) *-(23)-(CH2CH2O)_a-(23)-**, wherein the bond labelled * is connected to Q¹, Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, and wherein (23) refers to a group according to structure (23). Further, a’ is an integer in the range 0 - 5, preferably a - 1 - 4, most preferably a’ = 2. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

(ix) *-C(O)-NH-(CH2CH2O)_a-C(O)-**, wherein the bond labelled * is connected to Q¹, Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, wherein a’ is an integer in the range 1 - 5, preferably a - 3. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

(x) *-C(O)-NH-(CH2CH2O)_a-O-C(O)-NH-**, wherein the bond labelled * is connected to Q¹ , Z¹, Q² or Z², preferably to Q¹ or Z¹, and the bond labelled ** is connected to BM, wherein a’ is an integer in the range 1 - 5, preferably a - 3. Further, it is preferred that BM is a nitrogen atom or carbon atom. Most preferably, BM = N.

[0156] Especially preferred linkers L^A have the structure -(L¹¹)-BM(L¹²-)(L¹³-), wherein L¹² and L¹³ are according to any one of (xi) - (xiv):

(xi) *-(CH2)_a-O-C(O)-NH)-(CH2CH₂O)_a -CH2-CH2-N-C(O)-**, wherein the bond labelled * is connected to BM and the bond labelled ** is connected to Q¹, Z¹, F² or Z², preferably to F² or Z², wherein a’ is an integer in the range 0 - 4, preferably a’ = 2, and a” is an integer in the range 0 - 4, preferably a” = 2. Further, it is preferred that BM is a nitrogen atom.

(xii) *-(CH2CH2O)_a-CH2-CH2-C(O)-NH-(CH2)_a -Ph-**, wherein the bond labelled * is connected to BM and the bond labelled ** is connected to Q¹, Z¹, F² or Z², preferably to F² or Z², wherein a’ is an integer in the range 0 - 5, preferably a’ = 2, and a” is an integer in the range 0 - 4, preferably a” is 1 , 2 or 3. Preferably, Ph is 1 ,4-Ph. Further, it is preferred that BM is a nitrogen atom. (xiii) *-(CH2CH₂O)_a-CH2-CH2-C(O)-NH-(CH2)a -NH-C(O)-Ph-**, wherein the bond labelled * is connected to BM and the bond labelled ** is connected to Q¹, Z¹, F² or Z², preferably to F² or Z², wherein a’ is an integer in the range 0 - 5, preferably a’ = 2, and a” is an integer in the range 0 - 4, preferably a” is 2 or 3. Preferably, Ph is 1 ,4-Ph. Further, it is preferred that BM is a nitrogen atom.

(xiv) *-(CH2CH₂O)a-CH2-CH2-C(O)-NH-(CH2)a ”-O-Ph-**, wherein the bond labelled * is connected to BM and the bond labelled ** is connected to Q¹, Z¹, F² or Z², preferably to F² or Z², wherein a’ is an integer in the range 0 - 5, preferably a’ = 2, and a” is an integer in the range 0 - 4, preferably a” is 2 or 3. Preferably, Ph is 1 ,4-Ph. Further, it is preferred that BM is a nitrogen atom.

[0157] Preferably, linkers L¹² and L¹³ both have the same structure. In an especially preferred embodiment of L^A, linker L¹¹ is according to any one of (i) - (x) and linkers L¹² and L¹³ are according to any one of (xi) - (xiv).

[0158] Such preferred linkers L^A can be employed in the linker-construct according to the invention, in the conjugate according to the invention and in the intermediate antibody-linker construct. In the context of this preferred embodiment, Q¹ is preferably according to (Q26) or (Q37) and F² is preferably according to (F8a), wherein R²⁹ is hydrogen or methyl. Alternatively, it is preferred that Z¹ is preferably according to (Z26) or (Z37), wherein ring Z is according to (Za), and Z² is preferably according to (Z29), wherein ring Z is according to (Zj), wherein R²⁹ is hydrogen or methyl.

Linker L^B

[0159] Linker L^B connects payload D with Ab, via connecting groups Z² and linker L^A (in the conjugates according to the invention) or connects payload D with reactive group Q² (in the payload-linker construct). Linkers are known in the art and may be cleavable or non-cleavable. Linker L^B preferably is a cleavable linker, while linker L^A preferably is not cleavable. Linker L^B is connected to one occurrence of Z² (or Q²) and one occurrence of D. In other words, the payload-linker construct contains 1 payload D. Thus, the valency of linker L^B is 1 + 1 or bivalent. Linker L^B may be referred to as “hetereobifunctional”, which means that is contains two different functionalities, referring to the chemically different connectivities to Z²/Q² and D.

[0160] In a preferred embodiment, L^B is a heterobifunctional linker of structure

— (L¹)— (L²)_o— (L³)_P— (L⁴)_q— wherein:

- L¹ is connected to Q² or Z², and L⁴ is connected to D;

- L¹, L², L³ and L⁴ are each individually linkers that together link Q² or Z² to D;

- o, p and q are each individually 0 or 1 , preferably wherein o = p = 1 .

[0161] In the context of the present invention, a payload-linker construct of structure Q² - L^B - D reacts with an antibody-linker construct of structure Ab [ (Z¹)_yi - L^A (F²)_y2 ]z, thereby performing z x y2 click reactions between F² and Q² per antibody Ab, and thus forming z x y2 connecting groups Z² per antibody Ab. Upon completion of the reaction, an antibody-conjugate is formed containing z x y2 payloads D per antibody. The antibody-conjugate thus formed has a structure Ab [ (Z¹) - L^A - (Z² - L^B - D)_y2 ]z. [0162] L¹, L², L³ and L⁴ are linkers or linking units and each of o, p and q are individually 0 or 1 , preferably wherein o = p = 1 . In a preferred embodiment, at least linkers L¹ and L² are present (i.e. o = 1 ; p = 0 or 1 ; q = 0 or 1), more preferably linkers L¹, L² and L³ are present (i.e. o = 1 ; p = 1 ; q = 0 or 1). In one embodiment, L¹, L², L³ and L⁴ each individually consist of one or more building blocks selected from (hetero)aryl, CH₂, CH=CH, C^C, C(O), NR¹³, O, S, S(O), S(O)₂, PR¹³ and P(O)R¹³, preferably the building blocks are selected from aryl, CH₂, CH=CH, C(O), NR¹³, O, S, S(O) and S(O)₂. Preferred embodiments for each of L¹ , L², L³ and L⁴ are provided below.

[0163] In a preferred embodiment, linker L^B has structure (28):

[0164] In the context of structure (28):

- i is an integer in the range of 0 - 10, preferably in the range of 1 - 5;

- j is 0 or 1 , preferably j = 1 ;

- k is an integer in the range of 1 - 100, preferably in the range of 1 - 20, more preferably in the range of 2 - 5.

Linker L¹

[0165] L¹ may for example be selected from the group consisting of linear or branched Ci-C₂oo alkylene groups, C₂-C₂oo alkenylene groups, C₂-C₂oo alkynylene groups, C3-C₂oo cycloalkylene groups, C5-C₂oo cycloalkenylene groups, Ca-C₂oo cycloalkynylene groups, C?-C₂oo alkylarylene groups, C?-C₂oo arylalkylene groups, Ca-C₂oo arylalkenylene groups, Cg-C₂oo arylalkynylene groups. Optionally the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups may be substituted, and optionally said groups may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably being selected from the group consisting of O, S(O)_y and NR²¹, wherein y’ is 0, 1 or 2, preferably y’ = 2, and R²¹ is independently selected from the group consisting of hydrogen, halogen, Ci - C₂4 alkyl groups, Ce - C₂4 (hetero)aryl groups, C7 - C₂4 alkyl(hetero)aryl groups and C7 - C₂4 (hetero)arylalkyl groups. In one embodiment, the optional substituents may be selected from polar groups, such as oxo groups, (poly)ethylene glycol diamines, (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, carboxylic acid groups, carbonate groups, carbamate groups, cyclodextrins, crown ethers, saccharides (e.g. monosaccharides, oligosaccharides), phosphates or esters thereof, phosphonic acid or ester, phosphinic acid or ester, sulfoxides, sulfones, sulfonic acid or ester, sulfinic acid, or sulfenic acid.

[0166] In a preferred embodiment, linker L¹ contains a polar group, which may also be present in the chain of L¹. Such a polar group may be selected from (poly)ethylene glycol diamines (e.g. 1 ,8-diamino- 3,6-dioxaoctane or equivalents comprising longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains and 1 ,x’- diaminoalkanes (wherein x’ is the number of carbon atoms in the alkane, preferably x’ = 1 - 10), -(O)_a- C(O)-NH-S(O)2-NR¹³- (as further defined below, see structure (23)), -C(S(O)3^(_))-> -C(C(O)₂(-))-, -S(O)₂-, -P(O)₂<-)-, -O(CH₂CH₂O)t-, -NR³⁰(CH2CH₂NR³⁰)t-, and the following two structures:

[0167] For the polar groups defined here above, it is irrelevant which end is connected to Z¹ and which end to (L²)_o.

[0168] The polar group may also contain an amino acid, preferably selected from Arg, Glu, Asp, Ser and Thr. Herein, R¹³ is further defined below for structure (23). t is an integer in the range of integer in the range of 0 - 15, preferably 1 - 10, more preferably 2 - 5, most preferably t = 2 or 4. Each R³⁰ is individually H, C1-12 alkyl, C1-12 aryl, C1-12 alkaryl or C1-12 aralkyl. Linker L¹ may contain more than one such polar group, such as at least two polar groups. The polar group may also be present in a branch of linker L¹, which branches off a branching moiety as defined elsewhere. In the context of L¹, a nitrogen or carbon atom is preferably used as branching moiety. It is especially preferred to have a - O(CH2CH2O)t- polar group present in a branch.

[0169] In a preferred embodiment, Linker L¹ is or comprises a sulfamide group, preferably a sulfamide group according to structure (23): o o o X(O)a^N' A H _R13

(23)

[0170] The wavy lines represent the connection to the remainder of the compound, typically to Q² or Z² and to L², L³, L⁴ or D. Preferably, the (O)_aC(O) moiety is connected to Q² or Z² and the NR¹³ moiety to L², L³, L⁴ or D, preferably to L².

[0171] In structure (23), a = 0 or 1 , preferably a = 1 , and R¹³ is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR¹⁴ wherein R¹⁴ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. Alternatively, R¹³ is connected to elsewhere in the linker, optionally via a spacer moiety, to form a cyclic structure. For example, R¹³ may be connected to the linker via a CH2CH2 spacer moiety to form a piperazinyl ring, where the connection to D is via the second nitrogen of the piperazinyl ring.

[0172] In a preferred embodiment, R¹³ is hydrogen, a Ci - C20 alkyl group, preferably a Ci— C16 alkyl group, more preferably a Ci - C10 alkyl group, or connected to elsewhere in the linker optionally via a spacer moiety. Herein, the alkyl group is optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR¹⁴, preferably O, wherein R¹⁴ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. In another preferred embodiment, R¹³ is a Ci - C20 alkyl group, more preferably a Ci -C16 alkyl group, even more preferably a Ci - C10 alkyl group, wherein the alkyl group is optionally interrupted by one or more O-atoms, and wherein the alkyl group is optionally substituted with an -OH group, preferably a terminal -OH group. In this embodiment it is further preferred that R¹³ is a (poly)ethylene glycol chain comprising a terminal -OH group. In another preferred embodiment, R¹³ is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i- propyl, n-butyl, s-butyl and t-butyl, or connected to elsewhere in the linker optionally via a spacer moiety, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl and i-propyl, or connected to elsewhere in the linker optionally via a spacer moiety, and even more preferably from the group consisting of hydrogen, methyl and ethyl, or connected to elsewhere in the linker optionally via a spacer moiety. Yet even more preferably, R¹³ is hydrogen or connected to elsewhere in the linker optionally via a spacer moiety, and most preferably R¹³ is hydrogen.

[0173] In a preferred embodiment, L¹ is according to structure (24):

[0174] Herein, a and R¹³ are as defined above, Sp¹ and Sp² are independently spacer moieties and b and c are independently 0 or 1 . Preferably, b = 0 or 1 and c = 1 , more preferably b = 0 and c = 1 . In one embodiment, spacers Sp¹ and Sp² are independently selected from the group consisting of linear or branched C1-C200 alkylene groups, C2-C200 alkenylene groups, C2-C200 alkynylene groups, C3-C200 cycloalkylene groups, C5-C200 cycloalkenylene groups, C8-C200 cycloalkynylene groups, C7-C200 alkylarylene groups, C7-C200 arylalkylene groups, C8-C200 arylalkenylene groups and C9-C200 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR¹⁶, wherein R¹⁶ is independently selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C2- C24 alkenyl groups, C2- C24 alkynyl groups and C3 - C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted. When the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, it is preferred that said groups are interrupted by one or more O-atoms, and/or by one or more S-S groups.

[0175] More preferably, spacer moieties Sp¹ and Sp², if present, are independently selected from the group consisting of linear or branched C1-C100 alkylene groups, C2-C100 alkenylene groups, C2-C100 alkynylene groups, C3-C100 cycloalkylene groups, C5-C100 cycloalkenylene groups, Cs-Cioo cycloalkynylene groups, C7-C100 alkylarylene groups, C7-C100 arylalkylene groups, Cs-C-ioo arylalkenylene groups and C9-C100 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR¹⁶, wherein R¹⁶ is independently selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C2- C24 alkenyl groups, C2- C24 alkynyl groups and C3- C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted.

[0176] Even more preferably, spacer moieties Sp¹ and Sp², if present, are independently selected from the group consisting of linear or branched C1-C50 alkylene groups, C2-C50 alkenylene groups, C2-C50 alkynylene groups, C3-C50 cycloalkylene groups, C5-C50 cycloalkenylene groups, Cs-Cso cycloalkynylene groups, C7-C50 alkylarylene groups, C7-C50 arylalkylene groups, Cs-Cso arylalkenylene groups and C9-C50 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR¹⁶, wherein R¹⁶ is independently selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C2 - C24 alkenyl groups, C2- C24 alkynyl groups and C3- C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted.

[0177] Yet even more preferably, spacer moieties Sp¹ and Sp², if present, are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, C2-C20 alkenylene groups, C2- C20 alkynylene groups, C3-C20 cycloalkylene groups, C5-C20 cycloalkenylene groups, C8-C20 cycloalkynylene groups, C7-C20 alkylarylene groups, C7-C20 arylalkylene groups, C8-C20 arylalkenylene groups and C9-C20 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR¹⁶, wherein R¹⁶ is independently selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C2 - C24 alkenyl groups, C2- C24 alkynyl groups and C3- C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted.

[0178] In these preferred embodiments it is further preferred that the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are unsubstituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR¹⁶, preferably O, wherein R¹⁶ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably hydrogen or methyl.

[0179] Most preferably, spacer moieties Sp¹ and Sp², if present, are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, the alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR¹⁶, wherein R¹⁶ is independently selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C2- C24 alkenyl groups, C2- C24 alkynyl groups and C3- C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted. In this embodiment, it is further preferred that the alkylene groups are unsubstituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR¹⁶, preferably O and/or S-S, wherein R¹⁶ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably hydrogen or methyl.

[0180] Preferred spacer moieties Sp¹ and Sp² thus include -(CH2)r-, -(CH2CH2)r-, -(CH2CH2O)_r-, -(OCH₂CH₂)r-, -(CH₂CH2O)rCH₂CH2-, -CH₂CH2(OCH₂CH2)r-, -(CH2CH₂CH₂O)r-, -(OCH₂CH₂CH2)r-, -(CH₂CH2CH2O)rCH2CH₂CH2- and -CH2CH2CH2(OCH2CH2CH2)r-, wherein r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20 and yet even more preferably in the range of 1 to 15. More preferably n is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1 , 2, 3, 4, 5, 6, 7 or 8, even more preferably 1 , 2, 3, 4, 5 or 6, yet even more preferably 1 , 2, 3 or 4.

[0181] Alternatively, preferred linkers L¹ may be represented by — (\ZV)k— (A)d— (B)_e— (A)r— (C(O))_g— , wherein:

- d = 0 or 1 , preferably d = 1 ;

- e = an integer in the range 0 - 10, preferably e = 0, 1 , 2, 3, 4, 5 or 6, preferably an integer in the range 1 - 10, most preferably e = 1 , 2, 3 or 4;

- f = 0 or 1 , preferably f = 0;

- wherein d + e + f is at least 1 , preferably in the range 1 - 5; and preferably wherein d + f is at least

1 , preferably d + f = 1 .

- g = 0 or 1 , preferably g = 1 ;

- k = 0 or 1 , preferably k = 1 ;

- A is a sulfamide group according to structure (23);

- B is a -CH2-CH2-O- or a -O-CH2-CH2- moiety, or (B)_e is a -(CH2-CH2-O)_ei-CH2-CH2- or a - (CH₂-CH2-O)_ei-CH₂- moiety, wherein e1 is defined the same way as e;

- W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-

-C(O)(CH₂)mC(O)-, -C(O)(CH₂)mC(O)NH- or -(4-Ph)CH₂NHC(O)(CH₂)mC(O)NH-, preferably wherein W is -OC(O)NH-, -C(O)(CH2)mC(O)NH- or -C(O)NH-, and wherein m is an integer in the range 0 - 10, preferably m = 0, 1 , 2, 3, 4, 5 or 6, most preferably m = 2 or 3;

- preferably wherein L¹ is connected to Q via (W)k and to L², L³ or D, preferably to L², via (C(O))_g, preferably via C(O).

[0182] In the context of the present embodiment, the wavy lines in structure (23) represent the connection to the adjacent groups such as (W)k, (B)_e and (C(O))_g. It is preferred that A is according to structure (23), wherein a = 1 and R¹³ = H or a Ci - C20 alkyl group, more preferably R¹³ = H or methyl, most preferably R¹³ = H.

[0183] Preferred linkers L¹ have structure — (W)k— (A)d— (B)_e— (A)r— (C(O))_g— , wherein:

(a) k = 0; d = 1 ; g = 1 ; f = 0; B = -CH2-CH2-O-; e = 1 , 2, 3 or 4, preferably e = 2.

(b) k = 1 ; W = -C(O)(CH₂)mC(O)NH-; m = 2; d = 0; (B)_e = -(CH2-CH2-O)_ei-CH2-CH₂-; f = 0; g = 1 ; e1 = 1 , 2, 3 or 4, preferably e = 1 . (c) k = 1 ; W = -OC(O)NH-; d = 0; B = -CH2-CH2-O-; g = 1 ; f = 0; e = 1 , 2, 3 or 4, preferably e = 2.

(d) k = 1 ; W = -C(O)(CH₂)mC(O)NH-; m = 2; d = 0; (B)_e = -(CH2-CH2-O)_ei-CH2-CH₂-; f = 0; g = 1 ; e1 = 1 , 2, 3 or 4, preferably e1 = 4.

(e) k = 1 ; W = -OC(O)NH-; d = 0; (B)_e = -(CH2-CH2-O)_ei-CH2-CH₂-; g = 1 ; f = 0; e1 = 1 , 2, 3 or 4, preferably e1 = 4.

(f) k = 1 ; W = -(4-Ph)CH₂NHC(O)(CH₂)mC(O)NH-, m = 3; d = 0; (B)_e = -(CH₂-CH2-O)_ei-CH₂- CH2-; g = 1 ; f = 0; e1 = 1 , 2, 3 or 4, preferably e1 = 4.

(g) k = 0; d = 0; g = 1 ; f = 0; B = -CH2-CH2-O-; e = 1 , 2, 3 or 4, preferably e = 2.

(h) k = 1 ; W = -C(O)NH-; d = 0; g = 1 ; f = 0; B = -CH2-CH2-O-; e = 1 , 2, 3 or 4, preferably e = 2. [0184] Herein, it is preferred that when d and/or f = 1 , than a = 1 and R¹³ = H. Most preferred is the linker is structure (a).

Linker L²

[0185] Linker L² is a peptide spacer. Linker L² is either absent (0 = 0) or present (0 = 1). Preferably, linker L² is present and 0 = 1 . The combination of a peptide spacer L² and a cleavable linker L³ is well- known in the art. Linker L² functions as recognition and cleave site for cleaving-enzymes, more preferably the peptides are recognized by specific cleaving enzymes. This allows for cleavage at specific environments in which these cleaving enzymes are expressed such as specific tumours. Since different peptide sequences are cleaved by different enzymes, the L² group also allows customizing the conjugate for specific treatments. The peptide sequences may be cleaved by intracellular enzymes and/or extracellular enzymes. Typically, linker L² contains a cleavable site for a protease, preferably for a mammalian protease. In therapeutic applications, the protease cleavage site can be cleaved by a protease that is present, typically overexpressed, near or at the target cells, for example mascles cells, cancer cells, infected cells or pathogens. These proteases may be extracellular enzymes produced by the target cells, or intracellular enzymes that are leaked outside of the target cells. Typically, the cleavable peptide linker is specifically cleaved by proteases present in the microenvironment of the target cell. Such proteases are normally overexpressed in the target microenvironment.

[0186] The peptide spacer may also be defined by (NH-CR¹⁷-CO)_n, wherein R¹⁷ represents an amino acid side chain as known in the art. Also covered within this definition is proline, which has R¹⁷ joined with the nitrogen atom to form a cyclic moiety. Herein, the amino acid may be a natural or a synthetic amino acid. Preferably, the amino acid(s) are all in their L-configuration. n is an integer in the range of 1 - 5, preferably in the range of 2 - 4. Thus, the peptide spacer contains 1 - 5 amino acids. Preferably, the peptide is a dipeptide (n = 2), tripeptide (n = 3) or tetrapeptide (n = 4), most preferably the peptide spacer is a dipeptide. R¹⁷ represents the amino acid side chain, preferably selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine and citrulline. Preferred amino acid side chains are those of Vai, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, lie, Trp, Glu, Asp and Asn, more preferably from the side chains of Vai, Cit, Ala, Glu and Lys. Alternatively worded, R¹⁷ is preferably selected from CH3 (Ala), CH₂CH(CH₃)2 (Leu), CH2CH₂CH₂NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), CH2CH₂CH₂NHC(O)CH3 (AcLys), CH2CH₂CH₂NHC(=NH)NH2 (Arg), CH₂Ph (Phe), CH(CH₃)₂ (Vai), CH(CH₃)CH₂CH₃ (lie), CH₂C(O)NH₂ (Asn), CH₂CH₂C(O)OH (Glu), CH₂C(O)OH (Asp) and CH₂(1 H- indol-3-yl) (Trp). Especially preferred embodiments of R¹⁷ are CH₃ (Ala), CH2CH₂CH₂NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), CH₂CH₂C(O)OH (Glu) and CH(CH₃)₂ (Vai). Most preferably, R¹⁷ is CH₃ (Ala), CH2CH₂CH₂NHC(O)NH2 (Cit), CH2CH2CH2CH2NH2 (Lys), or CH(CH₃)₂ (Vai).

[0187] Although any peptide spacer may be used, preferably the peptide spacer is selected from Val- Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Glu-Gly-Cit, Glu-Gly-Val, Asp-Val-Cit, iGlu-Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, lle-Cit, Trp-Cit, Asn-Asn, Ala-Ala-Asn, Ala-Asn, Asn-Ala, Phe-Phe, Gly, Gly-Gly, Gly- Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID No: 19), Leu-Gly, Tyr-Gly, Ala-Gly, Pro-Gly, Phe-Gly, Phe-Gly, Ser- Gly, Gly-Phe-Gly, Gly-Gly-Phe-Gly (SEQ ID No: 20), Gly-Phe-Gly-Gly (SEQ ID No: 21), Phe-Gly-Gly- Gly (SEQ ID No: 22), Gly-Gly-Gly-Phe (SEQ ID No: 23), Phe-Phe-Gly-Gly (SEQ ID No: 24), Gly-Gly- Phe-Phe (SEQ ID No: 25), Gly-Gly-Gly-Phe-Gly (SEQ ID No: 26) and Lys, more preferably Val-Cit, Val- Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, even more preferably Glu-Val-Ala, Glu-Gly-Cit, Val-Cit, Val-Ala, Asn-Asn, Ala-Ala-Asn, Asn-Ala most preferably Glu-Val-Ala, Glu-Gly-Cit, Val-Cit, Val-Ala or Asn-Ala. Herein, AcLys is e-N-acetyllysine and iGlu is isoglutamate. In one embodiment, L² = Val-Cit. In another embodiment, L² = Val-Ala. In another embodiment, L² = Asn-Ala. In another embodiment, L² = Glu-Gly-Cit. In another embodiment, L² = Glu-Val-Ala.

[0188] In one embodiment, the amino acid side chain R¹⁷ is substituted with a polar group, preferably selected from oxo groups, (poly)ethylene glycol diamines, (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, carboxylic acid groups, carbonate groups, carbamate groups, cyclodextrins, crown ethers, saccharides (e.g. monosaccharides, oligosaccharides), phosphates or esters thereof, phosphonic acid or ester, phosphinic acid or ester, sulfoxides, sulfones, sulfonic acid or ester, sulfinic acid, or sulfenic acid.

[0189] In an especially preferred embodiment, L² comprises the peptide spacer represented by general structure (25), preferably L² is represented by general structure (25):

[0190] Herein, R¹⁷ is as defined above, preferably R¹⁷ is CH₃ (Ala) or CH₂CH2CH₂NHC(O)NH2 (Cit). The wavy lines indicate the connection to (L¹)_n and (L³)_P, preferably L² according to structure (25) is connected to (L¹)_n via NH and to (L³)_P via C(O).

Linker L³

[0191] Linker L³ is a self-cleavable spacer, also referred to as self-immolative spacer. Linker L³ is either absent (p = 0) or present (p = 1). Preferably, linker L³ is present and p = 1. Cleavage of L² results in 1 ,6-p elimination in linker L³, resulting in decarboxylation and the release of the payload, D. This advantageously allows for increased probability of release of the payload in regions wherein enzymes -M- that are able to cleave L² are overexpressed. In addition, release of a payload can induce bystander killing which is advantageous for tumours in which not all cancer cells have overexpression of the targeted receptor. Hence, in this embodiment, it is preferred that both linker L² and L³ are present (o = p = 1).

[0192] Preferably, L³ is para-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (26):

[0193] Herein, the wavy lines indicate the connection to L¹ or L², and to L⁴ or D. Typically, the PABC derivative is connected via NH to L¹ or L², preferably to L², and via OC(O) to L⁴ or D.

[0194] Ring A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Suitable 5-membered rings are oxazole, thiazole and furan. Suitable 6-membered rings are phenyl and pyridyl. Ring A may be substituted with a substituent selected from halogen, X²R⁴, N(R⁴)2, C1-4 alkyl and NO2. Herein, X² and R⁴ are as defined above, including preferred embodiments thereof. In a preferred embodiment, the optional substituent is selected from F, Cl, Br, OH, OR⁴, SH, NH2, Et, Me and NO2. In an especially preferred embodiment, ring A comprises 0 - 2 substituents, more preferably 0 or 1 substituent, most preferably ring A is not substituted. In a preferred embodiment, ring A is 1 ,4-phenyl, 1 ,2-phenyl, 2,5-pyridyl or 3 ,6-py ridy I . Most preferably, A is 1 ,4-phenyl. [0195] R²¹ is selected from H, R²⁶, C(O)OH and C(O)R²⁶, wherein R²⁶ is Ci - C24 (hetero)alkyl groups, C3 - C10 (hetero)cycloalkyl groups, C2 - C10 (hetero)aryl groups, C3 - C10 alkyl(hetero)aryl groups and C3 - C10 (hetero)arylalkyl groups, which are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR²⁸ wherein R²⁸ is independently selected from the group consisting of hydrogen and Ci - C alkyl groups. Preferably, R²⁶ is C3 - C10 (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably a polyethylene glycol or a polypropylene glycol, more preferably -(CH2CH2O)_SH or -(CH2CH2CH2O)_SH. The polyalkylene glycol is most preferably a polyethylene glycol, preferably -(CH2CH2O)_SH, wherein s is an integer in the range 1 - 10, preferably 1 - 5, most preferably s = 1 , 2, 3 or 4. More preferably, R²¹ is H or C(O)R²⁶, wherein R²⁶ = 4- methyl-piperazine or morpholine. Most preferably, R²¹ is H.

[0196] In an alternative embodiment, linker L³ is present (p = 1) and linker L² is not (0 = 0) and L³ is a para-glucuronide-meta-amide-benzyloxycarbonyl derivative, preferably a glucuronide derivative according to structure (27):

[0197] Herein, the wavy lines indicate the connection to L¹, and to L⁴ or D. Typically, the glucuronide derivative is connected via NH to L¹, and via (O)CO to L⁴ or D. Ring A and R²¹ are defined as for the PABC derivative according to structure (26). Preferably, ring A a 6-membered aromatic or heteroaromatic ring, such as oxazole, thiazole, furan, phenyl and pyridyl. In a preferred embodiment, ring A is 1 ,3,4-phenyl, 2,4,5-pyridyl or 2,5,6-pyridyl. Most preferably, A is 1 ,3,4-phenyl. Preferably, R²¹ is H or C(O)R²⁶, wherein R²⁶ = 4-methyl-piperazine or morpholine. Most preferably, R²¹ is H.

[0198] Linker L³ according to structure (27) is cleavable by p-glucuronidase, similar to the mechanism in PABC, which results in self-immolation of the para-hydroxybenzyloxy group, decarboxylation and the release of the payload. An ADC comprising the glucuronide derivative according to structure (27) is especially useful for treating cancers having an overexpression of p-glucuronidase. B-glucuronidase concentrations in many solid tumours, including lung, breast, and gastrointestinal cancers, as well as in the tumour microenvironment, are reported to be higher than those in normal tissues, and the enzyme is not found in the general circulation. Thus, it is preferred that the conjugates according to the invention comprising the glucuronide derivative according to structure (27) are used to treat patients suffering from lung, breast, and gastrointestinal cancers.

Linker L⁴

[0199] Linker L⁴ is either absent (q = 0) or present (q = 1). Preferably, linker L⁴ is present and q = 1. Linker L⁴ is selected from:

- an aminoalkanoic acid spacer according to the structure - NR²²-(Cx-alkylene)-C(O)-, wherein x is an integer in the range 1 - 20 and R²² is H or Ci - C4 alkyl;

- an ethyleneglycol spacer according to the structure -NR²²-(CH2-CH2-O)e6-(CH2)e7-C(O)-, wherein e6 is an integer in the range 1 - 10, el is an integer in the range 1 - 3 and R²² is H or Ci - C4 alky; and

- an diamine spacer according to the structure - NR²²-(Cx-alkylene)-NR²²-(C(O))h-, wherein h is 0 or 1 , x is an integer in the range 1 - 20 and R²² is H or Ci - C4 alkyl.

[0200] Linker L⁴ may be an aminoalkanoic acid spacer, i.e. -NR²²-(C_x-alkylene)-C(O)-, wherein x is an integer in the range 1 to 20, preferably 1 - 10, most preferably 1 - 6. Herein, the aminoalkanoic acid spacer is typically connected to L³ via the nitrogen atom and to D via the carbonyl moiety. Preferred linkers L⁴ are selected from 6-aminohexanoic acid (Ahx, x = 5), p-alanine (x = 2) and glycine (Gly, x = 1), even more preferably 6-aminohexanoic acid or glycine. In one embodiment, L⁴ = 6-aminohexanoic acid. In one embodiment, L⁴ = glycine. Herein, R²² is H or Ci - C4 alkyl, preferably R²² is H or methyl, most preferably R²² is H.

[0201] Alternatively, linker L⁴ may be an ethyleneglycol spacer according to the structure -NR²²-(CH2- CH2- O)e6- (CH2)e7- (C(O)- , wherein e6 is an integer in the range 1 - 10, preferably e6 is in the range 2 - 6, and el is an integer in the range 1 - 3, preferably el is 2. Herein, R²² is H or Ci - C4 alkyl, preferably R²² is H or methyl, most preferably R²² is H.

[0202] Alternatively, linker L⁴ may be a diamine spacer according to the structure - NR²²-(C_X- alkylene)-NR²²-(C(O))h-, wherein h is 0 or 1 , x is an integer in the range 1 - 20, preferably an integer in the range 2 - 6, even more preferably x = 2 or 5, most preferably x = 2. R²² is H or Ci - C4 alkyl. Herein, R²² is H or Ci - C4 alkyl, preferably R²² is H or methyl, most preferably R²² is methyl. Herein, h is preferably 1 , in which case linker L⁴ is especially suited for conjugation via a phenolic hydroxyl group present on payload D.

Payload D

[0203] D, also referred to in the art as the “payload”, represents the compound that is or is to be connected to antibody Ab. Payload molecules are well-known in the art, especially in the field of antibody-drug conjugates, as the moiety that is covalently attached to the antibody and that is released therefrom upon uptake of the conjugate and/or cleavage of the linker. In the context of the present invention, the payload is an oligonucleotide.

[0204] In a preferred embodiment, oligonucleotide payload D has structure D¹-D²-D³, wherein D² is represents the active oligonucleotide component, and D¹ and D³ independently represent oligonucleotide modifiers, which are optionally present. Herein, the oligonucleotide is connected through the remainder of the AOC through D¹. Oligonucleotide modifiers are known in the art and can be present at the 3’-terminus and/or the 5’-terminus. Oligonucleotide modifiers may be used to imbue specific characteristics on the oligonucleotide. Preferably, the oligonucleotide modifier remains attached to D² upon cleavage of linker L^B. Conveniently, the oligonucleotide modifier is used as spacer to covalently attach the oligonucleotide D² to the remainder of the AOC, typically for the synthesis of Q²- L^B-D or (Q)-(L¹)-(L²)₀-(L³)p-(L⁴)q-D, when D is coupled via D¹ to L^B or (L⁴)_q. Thus, the oligonucleotide modifier typically comprises a handle for conjugation to the remainder of the AOC. in one embodiment, D¹ comprises a phosphate moiety, a phosphoramidite moiety or a phosphorodiamidate moiety.

[0205] D² is the pharmaceutically active oligonucleotide. All defined herein for “the oligonucleotide” or for “oligonucleotide D”, including preferred embodiments thereof, equally applies to D² in case payload D has structure D¹-D²-D³. Medical treatments with oligonucleotides are known in the art. Oligonucleotides are known to the skilled person and may be selected from small interfering RNA (siRNA), messenger RNA (mRNA), DNA, anti-sense oligonucleotide (ASO), microRNA (miRNA), single-guide RNA (sgRNA), double stranded RNA (dsRNA), single stranded RNA (ssRNA), double stranded RNA (dsDNA), single stranded DNA (ssDNA), ribozymes, aptamer, Gapmer, and triplex forming oligonucleotides. In the present invention, the oligonucleotide is preferably ASO or siRNA. The double-helical structure of DNA is well known in the art. Anti-sense DNA has the same structure except that only one strain is present. Similarly, natural RNA is a single strain of nucleotides. The oligonucleotide may have the following structure:

[0206] Herein, for DNA the oxygen atom between parentheses is absent and NB is a nucleobase selected from adenine, thymine, guanine and cytosine, while for RNA the oxygen atom between parentheses is present and NB is a nucleobase selected from cytosine, guanine, adenine and uracil.

[0207] The present invention encompasses oligonucleotides such as present in nature and described above, but also synthetic variants. In principle, any sequence of nucleotides is a type of oligonucleotide and is encompassed by the present invention. The skilled person is familiar with synthetic variants of oligonucleotides which are also known in the art as xeno nucleic acids. In synthetic variants, the phosphate backbone and/or the ribose backbone may be substituted with a different chemical moiety. In a preferred embodiment, the oligonucleotide is a synthetic oligonucleotide selected from 2’-OMe-PS (2’-O-methylphosphorothioate), PMO (morpholino phosphorodiamidate), 2’-OMOE-PS (2’-O- methoxyethylphosphorothioate), PNA (peptide nucleic acid), tcDNA (tricyclic DNA), LNA (locked nucleic acid), HNA (1 ,5-anhydrohexitol nucleic acid), CeNA (cyclohexene nucleic acid), LceNA (locked cyclohexene nucleic acid),TNA (threose nucleic acid), GNA (glycol nucleic acid), FANA (fluoroarabino nucleic acid), 2’MOE (2’-O-methoxyethyl), S-cEt (2'-0-ethyl), and combinations thereof. Preferably the synthetic oligonucleotides are according to the structures below, wherein NB represents a nucleobase, preferably a nucleobase selected from adenine, thymine, guanine and cytosine and uracil, more preferably a nucleobase selected from adenine, thymine, guanine and cytosine (DNA-type) or a nucleobase selected from adenine, uracil, guanine and cytosine (RNA-type). Oligonucleotide D² is not limited to one specific type of oligonucleotide, but may contain a combination. For example, D² could be a combination of RNA and DNA, such as Gapmer. Moreover, the sequence in oligonucleotide D² may be interrupted with one or more oligonucleotide modifiers. Such internal modifiers are known and convenient for conjugation to a phosphate group within the oligonucleotide sequence. In one embodiment, D² does not contain an oligonucleotide modifier, or even the entire oligonucleotide D does not contain an oligonucleotide modifier.

[0208] Preferably synthetic oligonucleotides are selected from the following structures:

[0209] Synthetic oligonucleotide have the advantage that they are less recognizable by nucleases which ensures that they remain longer pharmaceutically active and make them more likely to escape the endosome and/or lysosome. Thus, it is preferred that the oligonucleotide is a synthetic oligonucleotide, more preferably PMO or 2’-OMePS.

[0210] In a preferred embodiment, oligonucleotide D is a single strain of oligonucleotide. In another embodiment, the oligonucleotide is a complex of at least two strains of oligonucleotides hold together by inter-molecular forces. In this embodiment, it is preferred that the oligonucleotide comprises a sense strand and an antisense strand, wherein either the sense strand or antisense strand is covalently connected to the antibody Ab. Preferably, the oligonucleotide is single stranded, more preferably an antisense.

[0211] In a preferred embodiment, oligonucleotide D is not transcribed or translated in the cell, but interfere with the translation or transcription of oligo- and/or polynucleotides in the cell. Therefore, it is preferred that the oligonucleotide does not encode for an entire protein, but has a relatively short nucleobase sequence. Preferably, the oligonucleotide comprises 1 - 200 nucleobases, more preferably 5 - 100 nucleobases, even more preferably 10 - 50 nucleobases, most preferably 13 - 30 nucleobases. [0212] The skilled person Is familiar with the mode of action of these types of oligonucleotides. In one preferred embodiment, the oligonucleotide is used for splicing, e.g. exon skipping or exon inclusion. Splicing is well established in the art, and is useful for correcting mRNA in a cell of a person suffering from genetic disorder, or at least ensuring that the targeted mRNA is translated into a functionable protein. Splicing is thus particularly useful for correcting a frameshift-mutation. Exon skipping is a type of splicing which induces the pre-mRNA splicing machinery to skip a specific exon. Duchenne, facioscapulohumeral muscular dystrophy and Spinocerebellar Ataxias are examples of disorders that may be treated with exon skipping. Alternatively, the oligonucleotide invokes exon inclusion which prevents the pre-mRNA splicing machinery from skipping specific exons and can be used to treat diseases such as spinal muscular atrophy (SMA) or Menkes disease. Such use of oligonucleotides is known in the art and available to the skilled person.

[0213] In another preferred embodiment, the oligonucleotide induces mRNA degradation of a targeted sequence. In a preferred embodiment, the oligonucleotide is a gapmer which binds to the targeted sequence and induces cleavage by RNase H. In an alternative preferred embodiment, the oligonucleotide is a siRNA which can be incorporated into the RNA induced silencing complex (RISC) and thereby activates the RISC to degrade the targeted mRNA. These two preferred embodiments are useful for blocking the production of a certain type of protein and can be used to treat diseases such as ALS, Huntington, Alzheimer, Creutzfeldt-Jacob.

[0214] The oligonucleotide may also have a different purpose than interfering with the translation or transcription in a cell. In one embodiment, the oligonucleotide may have a high affinity and specificity to targets other than nucleotides, such as when the oligonucleotide is an aptamer. Such an antibodyaptamer conjugate may be an alternative to a bispecific antibody. Bispecific antibodies are known to the skilled person and are described in for instance WO 2021/144315. In this embodiment, the mode of action can be outside the cell, the antibody may bind for instance to an immune cell receptor such as to PD-L1 and the aptamer to a cancer cell, prion, bacteria or virus.

[0215] Alternatively, the oligonucleotide is used to prevent the undesired interactions between a polynucleotide in a cell and another molecule such as a protein. Persons suffering from myotonic dystrophy type 1 have a mutation that results in a noncoding CUG repeat that binds to proteins and thereby hampers their function. The oligonucleotide may bind to this CUG repeat and can thereby prevent or reduce adhesion of the CUG repeat to the protein, which therefore can retain their function. [0216] Alternatively, the oligonucleotide payload D is a cytotoxic oligonucleotide. The use of cytotoxins as payloads is well known in the context of antibody-drug conjugates (ADCs). Herein, the oligonucleotide acts a cytotoxic drug of an ADC. Such ADCs are well-known to be suitable in the treatment of cancer. Thus, in case the AOC according to the invention contains a cytotoxic oligonucleotide as payload, it is preferred that the antibody targets a tumour cell. Preferred anti-cancer targets are further defined above.

[0217] The oligonucleotide is not limited to any one of these modes of action. The skilled person is familiar with oligonucleotide therapies and knows which disorders may be treated with a certain oligonucleotide, and which oligonucleotides would be suitable for the disorder in suit. Hence, the skilled person is able to select the appropriate oligonucleotide and antibody of the AOC according to the invention for use in the treatment of the appropriate disorder, and to use the process according to the invention to prepare the AOC with the desired therapeutic effect.

[0218] Preferred oligonucleotides are those that are known to mitigate the effects of an hereditary disease, in particular a neuromuscular disorder. The oligonucleotide payload may also be defined as capable of exon skipping In an especially preferred embodiment, the oligonucleotide comprises a sequence having at least 70 % sequence identity with the sequence identified by any one of SEQ ID No: 2 - 5, preferably 2 or 4. Preferably, the sequence identity is at least 80 %, such as at least 85 %, more preferably at least 90 %, such as at least 95 % or even at least 99 %, most preferably the oligonucleotide has the sequence identified by any one of SEQ ID No: 2 - 5, preferably 2 or 4.

Process for synthesizing the conjugate according to general structure (1)

[0221] In a further aspect, the present invention relates to a process for the preparation of the AOCs according to the invention.

[0222] In a first preferred embodiment, the process is for preparing an AOC of structure (11)

Ab-[ (Z¹)y1 - L^A ( Z² - L^B - D )y₂]z

(11) and comprises:

(a) providing a modified antibody having the structure Ab(F¹)_Z2, wherein Ab is an antibody, z2 is 2 or 4, and F¹ is a click probe;

(b) reacting the modified antibody with z2/y1 equivalents of (Q¹)yi - L^A - (F²)_y2, wherein Q¹ is a click probe that is reactive towards F¹, L^A is a heterobifunctional (y1 + y2)-valent linker, y1 is 1 or 2, y2 is 1 , 2, 3 or 4, and F² is a click probe that is not reactive towards Q¹, to obtain an antibody-linker construct of structure Ab[(Z¹)_yi - L^A - (F²)_y2 ]z, wherein z is z2 / y1 , and Z¹ is a connecting group obtained by reaction of F¹ and Q¹;

(c) reacting the antibody-linker construct with z x y2 equivalents of Q² - L^B - D, wherein Q² is a click probe that is reactive towards F², L^B is a bivalent linker and D is an oligonucleotide, to obtain the conjugate of structure Ab [ (Z¹)_yi - L^A - (Z² - L^B - D)_y2 ]z, wherein Z² is a connecting group obtained by reaction of F² and Q².

[0223] In a second preferred embodiment, the process is for preparing an AOC of structure (12) Ab-[(L⁶)-(Z)-(L¹)-(L²)₀-(L³)_P-(L⁴)_q-D ]_z

(12) and comprises:

(a) providing a modified antibody having the structure Ab[ (L⁶) - (F) ]_z, wherein: - Ab is an antibody;

- L⁶ is -GlcNAc(Fuc)w-(G)j-S-(L⁷)_w-, wherein G is a monosaccharide, j is an integer in the range of 0 - 10, S is a sugar or a sugar derivative, GIcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1 , w’ is 0 or 1 and L⁷ is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH₂-;

- z is 2 or 4;

- F is a click probe,

(d) reacting the modified antibody with z equivalents of (Q)— (L¹)— (L²)_o— (L³)_P— (L⁴)_q— D, wherein:

- Q is a click probe that is reactive towards F;

- L¹, L², L³ and L⁴ are each individually linkers;

- o, p and q are each individually 0 or 1 ; to obtain the conjugate of structure Ab-[(L⁶)-(Z)-(L¹)-(L²)₀-(L³)_P-(L⁴)_q-D ]_z.

[0224] The reactions performed in steps (b), (c) and (d) are click reactions, wherein click probes F react with click probes Q to form connecting groups Z. This conjugation technique is known to the skilled person. The present reactions occur under conditions such that Q is reacted with F to form covalent connections. In the process according to the invention, Q reacts with F, forming a covalent connection between the antibody and the payloads. Complementary reactive groups Q and reactive groups F are known to the skilled person and are described in more detail below.

[0225] Thus, the reactions in the process according to the invention, conjugation is accomplished via a cycloaddition. Preferred cycloadditions are a (4+2)-cycloaddition (e.g. a Diels-Alder reaction) or a [3+2]-cycloaddition (e.g. a 1 ,3-dipolar cycloaddition). The reaction of step (b) and (d) is preferably a [3+2]-cycloaddition, more preferably a 1 ,3-dipolar cycloaddition. The 1 ,3-dipolar cycloaddition is preferably the alkyne-azide cycloaddition, and most preferably wherein Q is or comprises an alkyne group and F is an azido group. The reaction of step (c) is a [4+2]-cycloaddition, preferably a Diels-Alder reaction. The preferred Diels-Alder reaction is the inverse-electron demand Diels-Alder cycloaddition. Cycloadditions, such as Diels-Alder reactions and 1 ,3-dipolar cycloadditions are known in the art, and the skilled person knowns how to perform them.

[0226] In case the AOC according to the invention has DAR 1 according to structure (33)

(33), the process typically comprises:

(a) providing a modified antibody having the structure Ab(F¹)_Z2, wherein Ab is an antibody, z2 is 2, and F¹ is a click probe;

(b) reacting the modified antibody with one equivalent of (Q¹)_yi - L^A- (F²)_y2, wherein Q¹ is a click probe that is reactive towards F¹, L^A is a heterobifunctional trivalent linker, y1 is 2, y2 is 1 , and F² is a click probe that is not reactive towards Q¹, to obtain an antibody-linker construct of structure Ab[(Z¹)_yi - L^A - (F²)y2 ]z, wherein z is 1 , and Z¹ is a connecting group obtained by reaction of F¹ and Q¹;

(c) reacting the antibody-linker construct with one equivalent of Q²-L^B-D, wherein Q² is a click probe that is reactive towards F², L^B is a bivalent linker and D is an oligonucleotide, to obtain the conjugate of structure (33), wherein Z² is a connecting group obtained by reaction of F² and Q².

[0227] In case the AOC according to the invention has DAR 2 according to structure (34)

_ZI__LA__Z2__LB__D

Ab

Z¹-L^A-Z²-L^B-D

(34), the process typically comprises:

(a) providing a modified antibody having the structure Ab(F¹)_Z2, wherein Ab is an antibody, z2 is 2, and F¹ is a click probe;

(b) reacting the modified antibody with one equivalent of (Q¹)_yi - L^A- (F²)_y2, wherein Q¹ is a click probe that is reactive towards F¹, L^A is a heterobifunctional bivalent linker, y1 is 1 , y2 is 1 , and F² is a click probe that is not reactive towards Q¹, to obtain an antibody-linker construct of structure Ab[(Z¹)_yi - L^A - (F²)y2 ]z, wherein z is 2, and Z¹ is a connecting group obtained by reaction of F¹ and Q¹;

(c) reacting the antibody-linker construct with one equivalent of Q² - L^B - D, wherein Q² is a click probe that is reactive towards F², L^B is a bivalent linker and D is an oligonucleotide, to obtain the conjugate of structure (34), wherein Z² is a connecting group obtained by reaction of F² and Q².

[0228] In case the AOC according to the invention has DAR 4 according to structure (35)

(35), the process typically comprises:

(a) providing a modified antibody having the structure Ab(F¹)_Z2, wherein Ab is an antibody, z2 is 4, and F¹ is a click probe;

(b) reacting the modified antibody with one equivalent of (Q¹)_yi - L^A- (F²)_y2, wherein Q¹ is a click probe that is reactive towards F¹, L^A is a heterobifunctional bivalent linker, y1 is 1 , y2 is 1 , and F² is a click probe that is not reactive towards Q¹, to obtain an antibody-linker construct of structure Ab[(Z¹)_yi - L^A - (F²)y2 ]z, wherein z is 4, and Z¹ is a connecting group obtained by reaction of F¹ and Q¹;

(c) reacting the antibody-linker construct with one equivalent of Q² - L^B - D, wherein Q² is a click probe that is reactive towards F², L^B is a bivalent linker and D is an oligonucleotide, to obtain the conjugate of structure (35), wherein Z² is a connecting group obtained by reaction of F² and Q².

[0229] In case the AOC according to the invention has DAR 4 according to structure (36)

(36), the process typically comprises:

(a) providing a modified antibody having the structure Ab(F¹)_Z2, wherein Ab is an antibody, z2 is 2, and F¹ is a click probe;

(b) reacting the modified antibody with one equivalent of (Q¹)_yi - L^A- (F²)_y2, wherein Q¹ is a click probe that is reactive towards F¹, L^A is a heterobifunctional trivalent linker, y1 is 1 , y2 is 2, and F² is a click probe that is not reactive towards Q¹, to obtain an antibody-linker construct of structure Ab[(Z¹)_yi - L^A - (F²)_y2 ]z, wherein z is 2, and Z¹ is a connecting group obtained by reaction of F¹ and Q¹;

(c) reacting the antibody-linker construct with one equivalent of Q² - L^B - D, wherein Q² is a click probe that is reactive towards F², L^B is a bivalent linker and D is an oligonucleotide, to obtain the conjugate of structure (36), wherein Z² is a connecting group obtained by reaction of F² and Q².

Step (a)

[0230] In step (a), an antibody having the structure Ab(F)_Z2, in some embodiments denoted as Ab(F¹)_Z2, is provided. Typically, two or four click probes F are chemically or enzymatically introduced onto the antibody. Preferably, z = 2 and two click probes are introduced onto the antibody. Such methods are known in the art, and any such method is encompassed by the present invention. Preferred modification methods are described here below. In a preferred embodiment, click probe F is introduced at the glycan, more preferably via an S(F) moiety connected to the core-GIcNAc as further described here below.

[0231] In a preferred embodiment, an antibody comprising two or four, preferably two, core N- acetylglucosamine moieties is contacted with a compound of the formula S(F)-P in the presence of a catalyst, wherein S(F) is a sugar derivative comprising two reactive groups F capable of reacting with a reactive group Q, and P is a nucleoside mono- or diphosphate, and wherein the catalyst is capable of transferring the S(F) moiety to the core-GIcNAc moiety. Herein, the antibody is typically an antibody that has been trimmed to a core-GIcNAc residue as described further below.

[0232] The starting material, i.e. the antibody comprising two or four, preferably two, core-GIcNAc substituents, is known in the art and can be prepared by methods known by the skilled person. In one embodiment, the process according to the invention further comprises the deglycosylation of an antibody glycan having a core N-acetylglucosamine, in the presence of an endoglycosidase, in order to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein said core N- acetylglucosamine and said core N-acetylglucosamine substituent are optionally fucosylated. Depending on the nature of the glycan, a suitable endoglycosidase may be selected. The endoglycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT and EndoSH and/or a combination thereof, the selection of which depends on the nature of the glycan. EndoSH is described in PCT/EP2017/052792, see Examples 1 - 3, and SEQ ID No: 1 , which is incorporated by reference herein.

[0233] Structural feature S is defined above for the conjugate according to the invention, which equally applies to the present aspect. Compounds of the formula S(F)-P, wherein a nucleoside monophosphate or a nucleoside diphosphate P is linked to a sugar derivative S(F), are known in the art. For example Wang et al., Chem. Eur. J. 2010, 16, 13343-13345, Piller et al., ACS Chem. Biol. 2012, 7, 753, Piller et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459-5462 and WO 2009/102820, all incorporated by reference herein, disclose a number of compounds S(F)-P and their syntheses. In a preferred embodiment nucleoside mono- or diphosphate P in S(F)-P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP), more preferably P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP) and cytidine diphosphate (CDP), most preferably P = UDP. Preferably, S(F)-P is selected from the group consisting of GalNAz- UDP, F2-GalNAz-UDP (/V-(azidodifluoro)acetyl-galactosamine), 6-AzGal-UDP, 6-AzGalNAc-UDP (6- azido-6-deoxy-N-acetylgalactosamine-UDP), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, GIcNAz-UDP, 6- AzGIc-UDP, 6-AzGlcNAz-UDP and 2-(but-3-yonic acid amido)-2-deoxy-galactose-UDP. Most preferably, S(F)-P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0234] Suitable catalyst that are capable of transferring the S(F) moiety to the core-GIcNAc moiety are known in the art. A suitable catalyst is a catalyst wherefore the specific sugar derivative nucleotide S(F)- P in that specific process is a substrate. More specifically, the catalyst catalyses the formation of a P(1 ,4)-glycosidic bond. Preferably, the catalyst is selected from the group of galactosyltransferases and A/-acetylgalactosaminyltransferases, more preferably from the group of P(1 ,4)-N-acetylgalactosaminyl- transferases (GalNAcT) and P(1 ,4)-galactosyltransferases (GalT), most preferably from the group of P(1 ,4)-N-acetylgalactosaminyltransferases having a mutant catalytic domain. Suitable catalysts and mutants thereof are disclosed in WO 2014/065661 , WO 2016/022027 and WO 2016/170186, all incorporated herein by reference. In one embodiment, the catalyst is a wild-type galactosyltransferase or A/-acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or A/-acetylgalactosaminyl- transferases, preferably a mutant N-acetylgalactosaminyltransferase. Mutant enzymes described in WO 2016/022027 and WO 2016/170186 are especially preferred. These galactosyltransferase (mutant) enzyme catalysts are able to recognize internal sugars and sugar derivatives as an acceptor. Thus, sugar derivative S(F) is linked to the core-GIcNAc substituent in step (a), irrespective of whether said GIcNAc is fucosy lated or not.

[0235] Alternatively, such transfer of an S(F) moiety to a GIcNAc moiety occurs at an non-trimmed glycan, i.e. at a terminal GIcNAc moiety of the glycan. This modification step is well known in the art. Complex glycans may have one or two terminal GIcNAc moieties, which are suitable acceptors of the transfer of S(F). In this embodiment, if two GIcNAc moieties are functionalized with S(F), z2 is 2, if only one GIcNAc moiety is functionalized with S(F), z2 is 2.

[0236] Alternative methods of modifying the glycan with click probe F known in the art are also encompassed by the present invention. It is known to introduce a biantennary glycan having an a2,6- sialic acid structure by first trimming the antibody and then using an endo-p-N-acetylglucosaminidase to introduce the biantennary glycan as described for example in WO2024/053574. Suitable endo-p-N- acetylglucosaminidase includes Endo-M, Endo-Om, Endo-CC, or Endo-Rp and Endo-M mutants, Endo- Om mutants, Endo-CC mutants, or Endo-Rp with reduced hydrolysis activity. In this embodiment, if the both branches of the biantennary glycan comprise a F moiety z2 is 4, if only one branch comprises a F moiety z2 is 2.

[0237] It is also known to introduce a secondary modified fucose comprising a F moiety to the core- GIcNAc, as has been described in for example WO2022/037665. In this embodiment, a fucosyltransferase is typically used to introduce a fucosyl moiety comprising a F group onto the core- GIcNAc. In the context of the aspect, the antibody may be trimmed or non-trimmed.

[0238] Alternative methods of modifying antibodies with F on different locations than the glycan, to obtain homogeneous modified antibodies Ab(F)_Z2 are known to the skilled person and also encompassed by the present invention. For instance, it is known to use a ligase such as sortase to introduce peptide that has been modified with a F group to the C-terminus of the heavy chain of an antibody, as described in WO2013/003555. It is further known to transform a tyrosine residue into an ortho-quinone group by oxidizing the tyrosine residue with a oxidizing agent as described in WO2022/108452. In this embodiment, F is an ortho-quinone and part of a side group of an amino acid in the antibody, and the antibody is preferably trimmed to expose a tyrosine residue. Other amino acid modifications known in the art include the modification of cysteine or lysine residues with linkers comprising F moieties.

[0239] Step (a) is preferably performed in a suitable buffer solution, such as for example phosphate, buffered saline (e.g. phosphate-buffered saline, tris-buffered saline), citrate, HEPES, tris and glycine. Suitable buffers are known in the art. Preferably, the buffer solution is phosphate-buffered saline (PBS) or tris buffer. Step (a) is preferably performed at a temperature in the range of about 4 to about 50 °C, more preferably in the range of about 10 to about 45 °C, even more preferably in the range of about 20 to about 40 °C, and most preferably in the range of about 30 to about 37 °C. Step (a) is preferably performed a pH in the range of about 5 to about 9, preferably in the range of about 5.5 to about 8.5, more preferably in the range of about 6 to about 8. Most preferably, step (a) is performed at a pH in the range of about 7 to about 8.

Step (b)

[0240] In step (b), the modified antibody Ab(F¹)_Z2 is reacted with a linker construct (Q¹)_yi-L^A-(F²)_y2, comprising a reactive group Q¹ capable of reacting with reactive group F¹ , to obtain an antibody-linker construct, containing connecting group Z¹ resulting from the reaction between Q¹ and F¹. Such reaction occurs under condition such that reactive group Q¹ is reacted with the reactive group F¹ of the antibody to covalently link the antibody to the linker-construct. In step (b), the reaction occurs with z2/y1 equivalents of (Q¹)_yi-L^A-(F²)_y2, although more equivalents of (Q¹)_yi-L^A- F²)_y2 may be present in the reaction mixture in order to ensure complete reaction. The skilled person is able to determine the optimal reaction conditions and stoichiometry of the reactants in order to obtain an optimal yield.

[0241] In a preferred embodiment, in step (b) an azide on an azide-modified antibody reacts with a benzoannulated or tetramethylated (hetero)cycloalkyne group, preferably wherein Q¹ is according to structure (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19) or (Q19a), preferably according to structure (Q26), (Q27), (Q28), (Q32), (Q37), (Q38) or (Q38a), more preferably according to structure (Q40), (Q41) or (Q43) or according to structure (Q37) or (Q43), via a cycloaddition reaction. Using such benzoannulated or tetramethylated (hetero)cycloalkyne groups, the presence of a catalyst is not required, and the cycloaddition reaction may even occur spontaneously by a reaction called strain- promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in the art as “metal- free click chemistry”.

Step (c)

[0242] In step (c), the modified antibody-linker construct Ab[(Z¹)_yi-L^A-(F²)_y2]z is reacted with a payloadlinker, comprising a reactive group Q² capable of reacting with reactive group F², to obtain an antibodyconjugate, containing connecting group Z² resulting from the reaction between Q² and F². Such reaction occurs under condition such that reactive group Q² is reacted with the reactive group F² to covalently link the antibody to the payloads. Step (c) may also be referred to as the conjugation reaction. In step (c), the reaction occurs with z x y2 equivalents of Q²-L^B-D, although more equivalents of Q²-L^B-D may be present in the reaction mixture in order to ensure complete reaction. The skilled person is able to determine the optimal reaction conditions and stoichiometry of the reactants in order to obtain an optimal yield.

[0243] In step (c), the conjugate is formed by covalently connecting one or more payloads to an antibody. Thus, in this step the DAR of the conjugate is established. The conjugation step of the present invention is especially effective, as it affords conjugates with an average DAR close to the theoretical value. As such, the conjugates according to the present invention exhibit high homogeneity. The inventors further found that a negative charge at the antibody-side of the conjugation reaction, i.e. in Ab[(Z¹)_yi-L^A-(F²)_y2]z, typically within L^A, negatively affects the conjugation reaction, such that lower DAR values may be obtained. However, the process according to the present invention is able to deal with such negatively charged modified antibody-linker constructs and to afford conjugates with unprecedented DAR values. A negatively charge in L^A is present at the process conditions of step (c) and may for example come from deprotonation of a carboxylic acid group or sulfamide group (e.g. according to structure (23)). Thus, in one embodiment, linker L^A is not negatively charged, and preferably does not comprises a carboxylic acid group and a sulfamide group. In an alternative embodiment, linker L^A is negatively charged, and preferably comprises a carboxylic acid group and/or a sulfamide group.

[0244] In a preferred embodiment, step (c) employs ultrafast click chemistry as defined below. Especially preferred is the reaction between a 1 ,2,4,5-tetrazine on the antibody-linker construct with a bicyclononyne group, preferably wherein Q¹ is according to structure (Q8), more preferably according to structure (Q29), most preferably according to structure (Q42), via a cycloaddition reaction. Using such bicyclononyne groups, the presence of a catalyst is not required, and the cycloaddition reaction may even occur spontaneously by a reaction called strain-promoted cycloaddition. This is one of the reactions known in the art as “metal-free click chemistry” Step (d)

[0245] In step (b), the modified antibody Ab[(L⁶)-(F)]_z, is reacted with a payload-construct (Q)-(L¹)- (L²)₀-(L³)p-(L⁴)q-D, comprising a reactive group Q capable of reacting with reactive group F, to obtain an antibody-conjugate, containing connecting group Z resulting from the reaction between Q and F. Such reaction occurs under condition such that reactive group Q is reacted with the reactive group F of the antibody to covalently link the antibody to the payload-construct. In step (d), the reaction occurs with z equivalents of (Q)— (L¹)— (L²)_o— (L³)_P— (L⁴)_q— D, although more equivalents of (Q)-(L¹)-(L²)_O-(L³)_P- (L⁴)_q— D may be present in the reaction mixture in order to ensure complete reaction. The skilled person is able to determine the optimal reaction conditions and stoichiometry of the reactants in order to obtain an optimal yield.

[0246] In step (d), the conjugate is formed by covalently connecting one or more payloads to an antibody. Thus, in this step the DAR of the conjugate is established. The conjugation step of the present invention is especially effective, as it affords conjugates with an average DAR close to the theoretical value. As such, the conjugates according to the present invention exhibit high homogeneity. Since step (d), when performed, is the only click reaction that needs to be performed, there is no requirement that Q² is not reactive towards F¹, and therefore there are no constraints on the structures of Q and F, and the corresponding connecting group Z. Hence, the preferred embodiments for Q and F, and the corresponding connecting group Z, as defined for steps (b) and (c) both apply to step (d).

Reactive moiety Q

[0247] Reactive moieties Q, in some embodiments referred to as Q¹ or Q², are click probes. In the context of the present invention, Q refers to Q¹ and Q². In the context of the present invention, the term “reactive moiety” may refer to a chemical moiety that comprises a reactive group, but also to a reactive group itself. For example, a cyclooctynyl group is a reactive group comprising a reactive group, namely a C-C triple bond. However, a reactive group, for example an azido reactive group, may herein also be referred to as a reactive moiety.

[0248] Q is reactive towards and complementary to F. Herein, a reactive group is denoted as “complementary” to a reactive group when said reactive group reacts with said reactive group selectively, optionally in the presence of other functional groups. Complementary reactive click probes are known to a person skilled in the art, and are described in more detail below. The exact nature of Q, and F, depends on the type of click reaction that is employed. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an o/Yho-quinone, a dioxothiophene, a sydnone, an alkene moiety and an alkyne moiety. Preferably, click probe Q comprises or is an alkene moiety or an alkyne moiety, more preferably wherein the alkene is a (hetero)cycloalkene and/or the alkyne is a terminal alkyne or a (hetero)cycloalkyne.

[0249] In an especially preferred embodiment, Q comprises a cyclic (hetero)alkyne moiety. The alkynyl group may also be referred to as a (hetero)cycloalkynyl group, i.e. a heterocycloalkynyl group or a cycloalkynyl group, wherein the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctynyl group, a (hetero)cyclononynyl group or a (hetero)cyclodecynyl group. Herein, the (hetero)cycloalkynes may optionally be substituted. Preferably, the (hetero)cycloalkynyl group is an optionally substituted (hetero)cycloheptynyl group or an optionally substituted (hetero)cyclooctynyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, wherein the (hetero)cyclooctynyl group is optionally substituted.

[0250] In an especially preferred embodiment, Q comprises a (hetero)cycloalkynyl or (hetero)cycloalkenyl group and is according to structure (Q1):

Herein:

- the bond depicted as - is a double bond or a triple bond;

- R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, -NO2, -CN, -S(O)2R¹⁶, -S(O)₃<->, CI - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups and wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R¹⁵ may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R¹⁶ is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups;

- Y² is C(R³¹)₂, O, S, S⁽⁺⁾R³¹ , S(O)R³¹, S(O)=NR³¹ or NR³¹, wherein S⁽⁺⁾ is a cationic sulphur atom counterbalanced by B^{( )}, wherein B^{( )} is an anion, and wherein each R³¹ individually is R¹⁵ or a connection with the linker;

- u is 0, 1 , 2, 3, 4 or 5;

- u’ is 0, 1 , 2, 3, 4 or 5, wherein u + u’ = 0, 1 , 2, 3, 4, 5, 6, 7 or 8;

- v = an integer in the range 0 - 16;

[0251] Typically, v = (u + u’) x 2 (when the connection to the linker, depicted by the wavy bond, is via Y²) or [(u + u’) x 2] - 1 (when the connection to the linker, depicted by the wavy bond, is via one of the carbon atoms of u and u’).

[0252] In a preferred embodiment of structure (Q1), reactive group Q comprises a (hetero)cycloalkynyl group and is according to structure (Q1 a):

Herein, - R¹⁵ and Y² are as defined above

- u is 0, 1 , 2, 3, 4 or 5;

- u’ is 0, 1 , 2, 3, 4 or 5, wherein u + u’ = 4, 5, 6, 7 or 8;

- v = an integer in the range 8 - 16. [0253] In a preferred embodiment, u + u’ = 4, 5 or 6, more preferably u + u’ = 5. In a preferred embodiment, v = 8, 9 or 10, more preferably v = 9 or 10, most preferably v = 10.

[0254] In a preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2) - (Q20c), preferably from the group consisting of (Q2) - (Q20), depicted here below.

(Q20) (Q20a) (Q20b) (Q20c)

[0255] Herein, the connection to the linker (e.g. L^A, L^B or L¹), depicted with the wavy bond, may be to any available carbon or nitrogen atom of Q. The nitrogen atom of (Q10), (Q13), (Q14) and (Q15) may bear the connection to the linker, or may contain a hydrogen atom or be optionally functionalized. B^(_) is an anion, which is preferably selected from <->OTf, CI<->, Br<-> or |(->, most preferably B(~> is (~>OTf. B<⁺> is a cation, preferably a pharmaceutically acceptable cation. In the conjugation reaction, B(~> does not need to be a pharmaceutically acceptable anion, since B(~> will exchange with the anions present in the reaction mixture anyway. In case (Q19) is used for Q, the negatively charged counter-ion is preferably pharmaceutically acceptable upon isolation of the conjugate according to the invention, such that the conjugate is readily useable as medicament. R³⁶ is an halogen selected from fluor, chlorine, bromine and iodine, preferably R³⁶ is fluor. Y⁴ is a heteroatom, preferably Y⁴ is O or NH. R³⁵ is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, Si, S and NR¹⁴ wherein R¹⁴ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably R³⁵ is selected from H, C5H11, CH3, CH2CH3, CH2OH or CH₂OTBS.

[0256] In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21) - (Q38a) depicted here below.

(Q35) (Q36) (Q37) (Q38) (Q38a)

(Q38b) (Q38c) (Q38d)

[0257] In structure (Q28), B<⁺> is a cation, preferably a pharmaceutically acceptable cation. In structure (Q38), B(-> is an anion, which is preferably selected from <->OTf, CI<->, Br<-> or |(->, most preferably B(~> is (~>OTf. Groups R³⁵ and R³⁶ on (Q38b), (Q38c) and (Q38d) are defined above for (Q20a) - (Q20c), which equally applies here.

[0258] In a preferred embodiment, Q comprises a (hetero)cyclooctyne moiety or a (hetero)cycloheptyne moiety, preferably according to structure (Q8), (Q26), (Q27), (Q28) , (Q37) or (Q38a), which are optionally substituted. Each of these preferred options for Q are further defined here below.

[0259] Thus, in a preferred embodiment, Q comprises a heterocycloheptyne moiety according to structure (Q37), also referred to as a TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety according to structure (Q37) is not substituted.

[0260] In an alternative preferred embodiment, Q comprises a cyclooctyne moiety according to structure (Q8), more preferably according to (Q29), also referred to as a bicyclo[6.1 .0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctyne moiety according to structure (Q8) or (Q29) is not substituted. In the context of the present embodiment, Q preferably is a (hetero)cyclooctyne moiety according to structure (Q39) as shown below, wherein V is (CH2)I and I is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, I is 0, 1 , 2, 3 or 4, more preferably I is 0, 1 or 2 and most preferably I is 0 or 1. In the context of group (Q39), I is most preferably 1 . Most preferably, Q is according to structure (Q42), defined further below.

[0261] In an alternative preferred embodiment, Q comprises a (hetero)cyclooctyne moiety according to structure (Q26), (Q27) or (Q28), also referred to as a DIBO, DIBAC, DBCO or ADIBO group, which are optionally substituted. In the context of the present embodiment, Q preferably is a (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) as shown below, wherein Y¹ is O or NR¹¹, wherein R¹¹ is independently selected from the group consisting of hydrogen, a linear or branched Ci - C12 alkyl group or a C4 - C12 (hetero)aryl group. The aromatic rings in (Q40) are optionally O- sulfonylated at one or more positions, whereas the rings of (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) is not further substituted. Most preferably, Q is according to structure (Q43), defined further below.

[0262] In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group and is according to structure (Q37).

[0263] In an especially preferred embodiment, Q comprises a cyclooctynyl group and is according to structure (Q42):

Herein:

- R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, -NO2, -CN, -S(O)2R¹⁶, -S(O)3^H,CI - C24 alkyl groups, C5 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups and wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R¹⁵ may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R¹⁶ is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups;

- R¹⁸ is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups;

- R¹⁹ is selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups, the alkyl groups optionally being interrupted by one of more hetero-atoms selected from the group consisting of O, N and S, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are independently optionally substituted, or R¹⁹ forms a second connection of trivalent linker L^A, wherein BM is the carbon atom to which R¹⁹ is attached; and

- I is an integer in the range 0 to 10.

[0264] In a preferred embodiment of the reactive group according to structure (Q42), R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, Ci - Ce alkyl groups, C5 - Ce (hetero)aryl groups, wherein R¹⁶ is hydrogen or Ci - Ce alkyl, more preferably R¹⁵ is independently selected from the group consisting of hydrogen and Ci - Ce alkyl, most preferably all R¹⁵ are H. In a preferred embodiment of the reactive group according to structure (Q42), R¹⁸ is independently selected from the group consisting of hydrogen, Ci - Ce alkyl groups, most preferably both R¹⁸ are H. In a preferred embodiment of the reactive group according to structure (Q42), R¹⁹ is H.

In a preferred embodiment of the reactive group according to structure (Q42), I is 0 or 1 , more preferably I is 1.

[0265] In an especially preferred embodiment, Q comprises a (hetero)cyclooctynyl group and is according to structure (Q43):

Herein:

- R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, -NO2, -CN, -S(O)2R¹⁶, -S(O)₃(->, CI - C24 alkyl groups, C5 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups and wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R¹⁵ may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R¹⁶ is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups;

- Y is N or CR¹⁵;

- a carbon atom in the fused aromatic rings may be replaced by a nitrogen atom, as in (Q6a) - (Q6d), preferably wherein Y is CR¹⁵.

[0266] In a preferred embodiment of the reactive group according to structure (Q43), R¹⁵ is independently selected from the group consisting of hydrogen, halogen, -OR¹⁶, -S(O)3^{( )}, Ci - Ce alkyl groups, C5 - Ce (hetero)aryl groups, wherein R¹⁶ is hydrogen or Ci - Ce alkyl, more preferably R¹⁵ is independently selected from the group consisting of hydrogen and -S(O)3^{( )}. In a preferred embodiment of the reactive group according to structure (Q43), Y is N or CH, more preferably Y = N.

[0267] In an alternative preferred embodiment, Q comprises a cyclic alkene moiety. The alkenyl group Q may also be referred to as a (hetero)cycloalkenyl group, i.e. a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, wherein the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornene group, a norbornadiene group, a frans-(hetero)cycloheptenyl group, a frans-(hetero)cyclooctenyl group, a frans-(hetero)cyclononenyl group or a trans- (hetero)cyclodecenyl group, which may all optionally be substituted. Especially preferred are (hetero)cyclopropenyl groups, frans-(hetero)cycloheptenyl group or frans-(hetero)cyclooctenyl groups, wherein the (hetero)cyclopropenyl group, the frans-(hetero)cycloheptenyl group or the trans- (hetero)cyclooctenyl group is optionally substituted. Preferably, Q comprises a cyclopropenyl moiety according to structure (Q44), a hetereocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a frans-(hetero)cycloheptenyl moiety according to structure (Q47) or a frans-(hetero)cyclooctenyl moiety according to structure (Q48). Herein, Y³ is selected from C(R²³)2, NR²³ or O, wherein each R²³ is individually hydrogen, Ci - Ce alkyl or is connected to the linker, optionally via a spacer, and the bond labelled - is a single or double bond.

In a further preferred embodiment, the cyclopropenyl group is according to structure (Q49). In another preferred embodiment, the frans-(hetero)cycloheptene group is according to structure (Q50) or (Q51). In another preferred embodiment, the frans-(hetero)cyclooctene group is according to structure (Q52), (Q53), (Q54), (Q55) or (Q56).

(Q52) (Q53) (Q54) (Q55) (Q56)

[0268] Herein, the R group(s) on Si in (Q50) and (Q51) are typically alkyl or aryl, preferably Ci-Ce alkyl. [0269] In a preferred embodiment, click probes Q comprise a moiety selected from (Q1) - (Q56), more preferably is a moiety selected from (Q1) - (Q56).

[0270] In the present invention, the exact structure of Q¹ and Q² should differ, as Q¹ is not reactive towards F², whereas Q² is reactive towards F².

[0271] In a preferred embodiment, F¹ is azide and Q¹ is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F² is tetrazine or nitrone and Q² is bicyclononyne or cycloalkene, such as a frans-cyclooctene or a cyclopropene. More preferably, F¹ is azide and Q¹ is an benzoannulated or tetramethylated (hetero)cycloalkyne, while F² is tetrazine and Q² is bicyclononyne. Herein, Q¹ is preferably according to structure (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19) or (Q19a), more preferably according to structure (Q26), (Q27), (Q28), (Q32), (Q37), (Q38) or (Q38a), most preferably according to structure (Q40), (Q41) or (Q43) or according to structure (Q37) or (Q43). Herein, Q² is preferably according to structure (Q8), (Q44), (Q47), (Q48), (Q49), (Q54), (Q55) or (Q56), more preferably according to structure (Q29), (Q48) or (Q49), most preferably according to structure (Q42). Alternatively, Q² is preferably according to structure (Q8), more preferably according to structure (Q29), most preferably according to structure (Q42).

Reactive moiety F

[0272] Reactive moieties F, in some embodiments referred to as F¹ or F², are click probes. In the context of the present invention, F refers to F¹ and F². F is reactive towards and complementary to Q. Herein, a reactive group is denoted as “complementary” to a reactive group when said reactive group reacts with said reactive group selectively, optionally in the presence of other functional groups.

Complementary reactive click probes are known to a person skilled in the art, and are described in more detail below. The exact nature of Q, and F, depends on the type of click reaction that is employed. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an ortho-quinone, a dioxothiophene, a sydnone, an alkene moiety and an alkyne moiety. Preferably, click probe F comprises or is an azide moiety, a nitrone moiety or a tetrazine moiety.

[0273] F is reactive towards Q in the conjugation reaction defined below, preferably wherein the conjugation reaction is a cycloaddition or a nucleophilic reaction. As the skilled person will understand, the options for F are the same as those for Q, provided that F and Q are reactive towards each other. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an ortho-quinone, a dioxothiophene, a sydnone, an alkene moiety and an alkyne moiety. Preferably, the click probe comprises or is an azide, a tetrazine, a triazine, a nitrone, a nitrile oxide, a nitrile imine, a diazo compound, an ortho-quinone, a dioxothiophene or a sydnone, most preferably an azide.

[0274] The reactive group F on the antibody are typically introduced by a specific technique, for example a (bio)chemical or a genetic technique. The reactive group that is placed in the antibody is prepared by chemical synthesis, for example an azide or a terminal alkyne. Methods of preparing modified antibodies are known in the art, e.g. from WO 2014/065661 , WO 2016/170186 and WO 2016/053107, which are incorporated herein by reference. From the same documents, the conjugation reaction between the modified antibody and a linker-toxin-construct is known to the skilled person.

[0275] Preferably, F is a click probe reactive towards a (hetero)cycloalkene and/or a (hetero)cycloalkyne, and is typically selected from the group consisting of azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, dioxothiophene sydnone iminosydnone, catechol, ortho-quinone and tetrazole. Preferred structures for the reactive group are structures (F1) - (F11) depicted here below.

[0276] Herein, the wavy bond represents the connection to Ab or L^A. For (F3), (F4), (F8), (F9) and (F1 1), the payload can be connected to any one of the wavy bonds. The other wavy bond may then be connected to an R group selected from hydrogen, Ci - C24 alkyl groups, C2 - C24 acyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups, C3 - C24 (hetero)arylalkyl groups and Ci - C24 sulfonyl groups, each of which (except hydrogen) may optionally be substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR³² wherein R³² is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. The skilled person understands which R groups may be applied for each of the groups F. For example, the R group connected to the nitrogen atom of (F3) may be selected from alkyl and aryl, and the R group connected to the carbon atom of (F3) may be selected from hydrogen, alkyl, aryl, acyl and sulfonyl. Likewise, the R group connected to the nitrogen atom of (F7) may be selected from alkyl and aryl. Preferably, the reactive moiety F is selected from azides or tetrazines.

[0277] In an especially preferred embodiment, F is a tetrazine according to structure (F8a):

[0278] Herein, R²⁹ is selected from hydrogen, C1-6 alkyl, aryl, C(O)-Ci-6 alkyl, C(O)-aryl, C(O)-O-Ci-6 alkyl, C(O)-O-aryl, C(O)-NR³³-CI-6 alkyl and C(O)-NR³³-aryl, wherein R³³ is H or C1-4 alkyl. Preferably, R²⁹ is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl. It was found that R²⁹ is hydrogen gave optimal results in reactivity in the cycloaddition reaction. Thus, in a preferred embodiment, ring F, in particular F², is (F8a) wherein R²⁹ is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R²⁹ is hydrogen or methyl, most preferably R²⁹ is methyl.

[0279] In another especially preferred embodiment, F is a nitrone according to structure (F3a).

[0280] R¹ is L¹⁰XR⁴. Herein, X is a heteroatom having a lone pair which is capable of capturing the imine intermediate by reacting with the imine carbon atom. In case this reaction forms a 5- or 6- membered ring, this capture of imine intermediate is efficient and will stop the rearrangement reaction. Hence, L¹⁰ should be a linker of two or three carbon atoms.

[0281] More specifically, L¹⁰ is a linker of structure (C(R³⁹)2)z, wherein z is 2 or 3. Each R³⁹ is individually selected from H and C1-4 alkyl. Alternatively, two occurrences of R³⁹ may be joined together to form an oxo group or a C3-6 (hetero)cycloalkyl group. Preferably, L¹⁰ is a linker of structure CH2- C(R³⁹)₂ or CH₂-CH₂-C(R³⁹)2, more preferably L¹⁰ = CH2-C(R³⁹)2. In case two occurrences of R³⁹ are joined together to form a spiro-connected ring, it is preferred that such a ring is a C3 - Ce ring, preferably a C4 or C5 ring.

[0282] Preferred embodiments of L¹⁰ are (L¹⁰A) - (L¹⁰P):

[0283] Herein, the wavy bond labelled with * is connected to the nitrogen atom of the nitrone group and the wavy bond labelled with * is connected to XR⁴. Ring (L) is spiro connected to the backbone atoms of L¹⁰. Ring (L) is preferably a cyclobutyl ring or a cyclopentyl ring, most preferably a cyclobutyl ring. Especially preferred are (L¹⁰A), (L¹⁰B), (L¹⁰C) and (L¹⁰G), wherein ring L is a cyclobutyl ring.

[0284] X is S, O or NH. Most preferably, X is O. R⁴ is selected from H and C1-4 alkyl. Typically, R⁴ is H when X is O or NH, and R⁴ is H or C1-4 alkyl when X is S. Thus, XR⁴ is typically selected from OH, NH2, SH and S-C1-4 alkyl. In a preferred embodiment, XR⁴ is SH, OH, NH2, most preferably XR⁴ is OH.

[0285] The nature of R^2a and R^2b is not crucial for the present invention, and any suitable substituent for a nitrone compound can be used. R^2a and R^2b may be the same or different, typically they are different. Each of R^2a and R^2b may correspond to R² as comprised in ring (Zh) and (Zh’), defined above. In case R^2a and R^2b are different, the configuration of the double bound between the nitrogen atom and carbon atom of the nitrone group may either be in E-configuration of in Z-configuration. The exact configuration has no influence on the working of the present invention. Herein, the connection to AB or D is typically via R^2a or R^2b.

[0286] Typically, R^2a is selected from H and Ci - Ce (cyclo)alkyl. In a preferred embodiment, R^2a is selected from H and Ci - C5 (cyclo)alkyl, more preferably R^2a is H, Me or Et, most preferably R^2a is H. [0287] Typically, R^2b is selected from the group consisting of Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, which may optionally be substituted and which may optionally be interrupted by one or more heteroatoms selected from O, S and NR¹⁴, wherein R¹⁴ is independently selected from the group consisting of hydrogen and Ci - C alkyl groups. Alternatively, R^2a and R^2b are joined to form a (hetero)cyclic moiety. In an alternative embodiment, R^2b is L(D)_r, wherein r is an integer in the range of 1 - 10, and L is a linker covalently connecting D with the nitrone group. In a further alternative embodiment, R^2b is L⁶AB, wherein L⁶ is a linker covalently connecting AB with the nitrone group. Preferred embodiments of antibody AB, payload D, linkers L and L⁶ and integer r are defined elsewhere.

[0288] In a preferred embodiment, R^2b is hydrogen, a Ci - C20 alkyl group, preferably a Ci— C16 alkyl group, more preferably a Ci - C10 alkyl group, L(D)_r or L⁶AB. Herein, the alkyl group is optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR¹⁴, preferably O, wherein R¹⁴ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. In another preferred embodiment, R^2b is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, L(D)_r or L⁶AB, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, L(D)_r or L⁶AB, and even more preferably from the group consisting of hydrogen, methyl, ethyl, L(D)_r or L⁶AB.

[0289] It is especially preferred that the nitrone compound according to the invention is used in the preparation of a bioconjugate, wherein antibody AB is covalently connected to a payload D. Hence, it is especially preferred that R^2b is L(D)_r or L⁶AB. In one embodiment, R^2b is L(D)_r and the nitrone compound is to be coupled with a (hetero)cycloalkyne compound comprising a antibody AB. In one embodiment, R^2b is L⁶B and the nitrone compound is to be coupled with a (hetero)cycloalkyne compound comprising a payload D. It is especially preferred that the nitrone is coupled to the payload and thus that R^2b is L(D)_r.

[0290] In a preferred embodiment, click probes F are selected from the group consisting of azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, dioxothiophene sydnone iminosydnone, catechol, ortho-quinone and tetrazole. Note that catechol in situ oxidizes to an ortho- quinone group, which is reactive as click probe. Likewise, the term “tetrazine” also encompasses “hydrotetrazine”, a known precursor that forms tetrazine upon in situ oxidation. Such precursors of click probes, which in situ form reactive groups, are also covered in the present invention. Preferred click probes F are selected from the group consisting of azide, tetrazine, tetrazole, o/Yho-quinone and nitrone, more preferably from azide, tetrazine and nitrone. In a preferred embodiment, F¹ is an azide or nitrone, and F² is iminosydnone, catechol, which forms a o/Yho-quinone group in situ, tetrazine or tetrazole. More preferably, F² is iminosydnone according to structure (F7), catechol, which forms structure (F10) in situ, tetrazine according to structure (F8) or tetrazole according to structure (F11). Even more preferably, F¹ is an azide according to structure (F1) or nitrone according to structure (F3) and F² is a tetrazine according to structure (F8a) or tetrazole according to structure (F1 1). Most preferably, F¹ is an azide according to structure (F1) and F² is a tetrazine according to structure (F8a).

Application

[0291] The conjugates of the present invention have a high homogeneity and DAR value close to the theoretical DAR value. The conjugates of the present invention are further characterized by a high stability, a low tendency to aggregate and excellent therapeutic efficacy and tolerability. The conjugates of the present invention are therefore especially suitable in delivering an oligonucleotide to a cell in the need thereof. Therefore, the conjugates are especially suitable for treating diseases that can be treated with oligonucleotide treatment, for example disorders wherein specific cells produce erroneous RNA that can be corrected and/or mitigated with oligonucleotides. The conjugates of the invention exhibit unexpectedly high extent of exon skipping and dystrophin restoration, as evidenced in Figures 16 - 18, especially at the higher dosages. The exon skipping and dystrophin restoration percentages at 30 mg/kg (for DAR4 conjugates) and 60 mg/kg (for DAR2 conjugates) in Figures 16 - 18 are unprecedented in the art (see e.g. Cochran, Michael, et al. "Structure-Activity Relationship of Antibody-Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody-siRNA Conjugates for Drug Development." Journal of medicinal chemistry 67.17 (2024): 14852-14867 and Desjardins, Cody A., et al. "Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1 -targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice." Nucleic Acids Research 50.20 (2022): 1 1401-11414, both incorporated by reference). Moreover, the conjugates of the invention exhibit a prolonged duration of the therapeutic effect, such that the benefits are observed for a prolonged period after administration of the conjugate.

[0292] In that light, the invention further concerns a method for treatment, comprising administering to a subject in need thereof the AOC according to the invention. In the method according to this aspect, the antibody-conjugate is typically administered in a therapeutically effective dose. The present aspect of the invention can also be worded as a conjugate according to the invention for use in treatment. In other words, this aspect concerns the use of a conjugate according to the invention for the preparation of a medicament or pharmaceutical composition for use in treatment. In the present context, treatment is envisioned to encompass treating, imaging, diagnosing and prevention.

[0293] In that light, the invention further concerns a method for the treatment of hereditary diseases, also known as genetic disorders, such as hereditary neuromuscular disease, comprising administering to a subject in need thereof the AOC according to the invention. The subject in need thereof is typically a patient suffering from the hereditary disease. The use of oligonucleotides is well-known in the field of the treatment of hereditary diseases, and the conjugates according to the invention are especially suited in this respect. In the method according to this embodiment, the AOC is typically administered in a therapeutically effective dose. The present aspect of the invention can also be worded as an AOC according to the invention for use in the treatment of hereditary disease. In other words, this aspect concerns the use of an AOC according to the invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of hereditary disease. The hereditary disease may be a neuromuscular disease or a neurological disease, preferably as further defined below. Most preferred is the treatment of neuromuscular diseases. In a preferred embodiment, the hereditary disease is selected from Duchenne, myotonic dystrophy, spinal muscular atrophy, homozygous familial hypercholesterolemia, primary hyperoxaluria type 1. ACCs of structure (11) and (12) are ideally suited for the treatment of hereditary diseases selected from Duchenne, myotonic dystrophy, spinal muscular atrophy, homozygous familial hypercholesterolemia, primary hyperoxaluria type 1. Thus, in one embodiment, the AOC has structure (11) or (12).

[0294] More specifically, the invention further concerns a method for the treatment of a neuromuscular disease, preferably a hereditary neuromuscular disease. Preferably, the neuromuscular disease is selected from Adult Pompe, Becker muscular disease (BMD), Centronuclear myopathy (CNM), congenital myasthenic syndromes, congenital muscular dystrophies (e.g. merosin deficiency, Ullrich, dystroglycanopathy, integrin deficiency and rigid spine), distal muscular dystrophies (e.g. Miyoshi, Nonaka, Welander, Markesbery, Laing), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDS), Facioscapulohumeral muscular, dystrophy (FSHD), Familial hypertrophic cardiomyopathy, Fibrodysplasia Ossificans Progressiva (FOP), Friedreich’s ataxia (FRDA), Inclusion body myopathy 2, Laing distal myopathy, laminopathies, Limb girdle muscular dystrophy (LGMD), Myofibrillar myopathy, Myotonia congenita (autosomal dominant form, Thomsen Disease), Myotonic dystrophy type l/ll, non-dystrophic myotonia (including Becker's myotonia and paramyotonia congenita), oculopharyngeal muscular dystrophy (OPMD) and periodic paralysis. More preferably, the neuromuscular disease is selected from Adult Pompe, Becker muscular disease (BMD), Centronuclear myopathy (CNM), congenital myasthenic syndromes, Duchenne muscular dystrophy (DMD), Emery- Dreifuss muscular dystrophy (EDS), Facioscapulohumeral muscular, dystrophy (FSHD), Familial hypertrophic cardiomyopathy, Fibrodysplasia Ossificans Progressiva (FOP), Friedreich’s ataxia (FRDA), Inclusion body myopathy 2, Laing distal myopathy, laminopathies, Myofibrillar myopathy, Myotonia congenita (autosomal dominant form, Thomsen Disease), Myotonic dystrophy type l/ll, oculopharyngeal muscular dystrophy (OPMD). In a further preferred embodiment, the disease is a muscular dystrophy selected from BMD, DMD, EDS, FSHD, LGMD, OPMD, congenital muscular dystrophies and distal muscular dystrophies or a Myotonic dystrophy or a non-dystrophic myotonia. In that light, the invention further concerns a method for the treatment of neuromuscular disease, comprising administering to a subject in need thereof the AOC according to the invention. The subject in need thereof is typically a patient suffering from the neuromuscular disease.

[0295] Alternatively, the AOC according to the invention is used to treat a neurological disease selected from adult motor neuron diseases, Alzheimer’s disease, Parkison’s disease, hereditary dystonia, epilepsy, a pain disorder, glycogen synthesis disorder, neurodegeneration, small fiber neuropathy, nociceptionrleated phenotype, Alexander disease, Angelman Syndrome, retinitis, pigmentosa, isolated macular dystrophy, multiple sclerosis (MS), spinocerebellar ataxia (SCA); frontotemporal dementia (FTD); motor neuron disease; Dravet syndrome; Batten disease; GM1 gangliosidosis; Niemann-Pick Type A; metachromatic leukodystrophy; Krabbe disease; Tay-Sachs; Sandhoff disease; Gaucher disease, type II or III; or Rett syndrome, Creutzfeldt-Jakob, Menkes disease, Spinocerebellar Ataxias, infantile spinal muscular atrophy, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington, juvenile spinal muscular atrophy, Frontotemporal dementia (FTD), autoimmune motor neuropathy with multifocal conductor block, Spinal muscular atrophy (SMA), paralysis due to stroke or spinal cord injury, or skeletal immobilization due to trauma. In that light, the invention further concerns a method for the treatment of neurological disease, comprising administering to a subject in need thereof the AOC according to the invention. The subject in need thereof is typically a patient suffering from the neurological disease.

[0296] The use of oligonucleotides is well-known in the field of the treatment of neuromuscular and neurological diseases, and the conjugates according to the invention are especially suited in this respect. In the method according to this embodiment, the AOC is typically administered in a therapeutically effective dose. The present aspect of the invention can also be worded as an AOC according to the invention for use in the treatment of a neuromuscular or neurological disease. In other words, this aspect concerns the use of an AOC according to the invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of a neuromuscular or neurological disease. AOCs of structure (11) and (12) are ideally suited for the treatment of neuromuscular diseases. Thus, in one embodiment, the AOC has structure (11) or (12).

[0297] In another preferred embodiment, the AOC according to the invention can be used to sabotage a cell by interfering with processes crucial to the viability of a cell, such as the production of crucial proteins. In this embodiment it is preferred that treatment concerns the treatment of cancer or an infection, preferably cancer. Alternatively, the method of treatment concerns cancer and the oligonucleotides interfere with the production of receptor proteins so that the receptor proteins are modified and more easily recognized by the immune system. The use of such cytotoxic oligonucleotides in the treatment of cancer or infection is known in the art, and the conjugates according to the invention are especially suited in this respect. In the method according to this embodiment, the AOC is typically administered in a therapeutically effective dose. The present aspect of the invention can also be worded as an AOC according to the invention for use in the treatment of cancer or infection. In other words, this aspect concerns the use of an AOC according to the invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of cancer or infection.

[0298] This aspect of the present invention may also be worded as a method for delivering oligonucleotides to a cell expressing a specific extracellular receptor, comprising contacting the AOC according to the invention with cells that may possibly express the extracellular receptor, and wherein the antibody specifically targets the extracellular receptor. The method according to this aspect is also suitable to determine whether the cells are expressing the desired extracellular receptor. These cells may be present in a subject, in which case the method comprises administering to a subject in need thereof the AOC according to the invention. Alternatively, the method occurs ex vivo or in vitro. In a preferred embodiment, the cells that may possibly express the extracellular receptor are cells that express the extracellular receptor.

[0299] The skilled person understands that the choice of oligonucleotide moiety D, including the specific sequence, and antibody Ab is dependent on the application of the AOC. Preferred targets for the antibody Ab for specific applications are defined above.

[0300] The medical uses and methods defined herein benefit from administration at a relative high dose. The inventors found that especially at such higher doses, unprecedented exon skipping and dystrophin restoration percentages were obtained. The best results were obtained at a dose of 30 mg/kg (for DAR4 AOC) and 60 mg/kg (for DAR2 AOC), corresponding to a theoretical dose of 120 mg/kg for a hypothetical DAR1 AOC. Such a dose in mice corresponds to a human dose of about 10 mg/kg for a hypothetical DAR1 AOC, and to about 5 mg/kg for a DAR2 AOC. Thus, in a preferred embodiment, the therapeutic effective dose is at least 1 mg/kg, more preferably at least 2 mg/kg, even more preferably in the range of 3 - 100 mg/kg, even more preferably in the range of 4 - 20 mg/kg, most preferably in the range of 4.5 - 10 mg/kg. Herein, the AOC dose to be administered (in mg) concerns DAR2 AOCs, meaning that the dose is divided by 2 for a DAR4 AOC and divided by 4 for a DAR8 AOC. These doses are determined based on body weight (in kg) of the subject. The doses recited here are particularly preferred in case the subject is a human.

[0301] In this light, the invention also concerns a pharmaceutical composition comprising the conjugate according to the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition typically contains the conjugate according to the invention in a pharmaceutically effective dose.

[0302] The invention further concerns the use of the conjugation technology of the AOCs according to the invention for improving the efficacy of an AOC, in particular wherein the improved efficacy involves an improvement in exon skipping and/or dystrophin restoration. Alternatively or additionally, the improved efficacy involves an extended duration of the therapeutic effect of the AOC. Such extended duration typically manifests as an improved limb strength beyond week 12 after administration of the AOC, such as during week 13 - 20 or even during week 14 - 16 after administration. The improved efficacy may also involve an improved limb strength. The effects on improved grip strength are preferably obtained at the higher doses as defined above. In the context of the present embodiment, the conjugation technology of the AOCs according to the invention in particular refers to the click conjugation defined by connecting group Z, preferably Z¹ and Z² in combination with heterobifunctional (y1 + y2)-valent linker L^A and bivalent linker L^B, and preferably in combination with the glycan conjugation defined by L⁶. These structural motives of the AOCs according to the invention contribute to the improved efficacy according to the present embodiment.

Use of ultrafast Click

[0303] In all aspects of the invention, it is preferred that the click reaction for conjugation, as in step (c) or (d), is performed by ultrafast click. Although ultrafast click is a preferred embodiment for all aspects of the present invention, it is the crucial element of the fourth aspect as defined above, wherein the invention concerns the use of ultrafast click chemistry in the preparation of AOCs. Thus, the invention also concerns the use of ultrafast click chemistry for conjugating an oligonucleotide to an antibody in the preparation of an antibody-oligonucleotide conjugate (AOC). By using an ultrafast click reaction for the conjugation step between the oligonucleotide moiety D and the modified antibody, the conjugation step is very efficient, requiring only a near stochiometric amount of linker-payload construct comprising the oligonucleotide. Herein, the linker-payload construct typically has structure Q²-L^B-D or Q-(L¹)- (L²)₀-(L³)p-(L⁴)q-D, as defined herein, and the ultrafast click chemistry occurs between Q² and F² or between Q and F, also as defined herein. Hence, in the context of the present embodiment, Q (or Q²) and F (or F²) are reaction partners in an ultrafast click reaction.

[0304] Herein, the click reaction or click chemistry is copper-free or strain-promoted. Click reactions are known in the art and refer to cycloaddition reactions such as the [4+2] cycloaddition (e.g. Diels- Alder, inverse electron-demand Diels-Alder) and the [3+2] cycloaddition (e.g. 1 ,3-dipolar cycloaddition). In the context of the present invention, the term click reaction may also be referred to as cycloaddition. Preferably, the click reaction is an inverse electron-demand Diels-Alder or a 1 ,3-dipolar cycloaddition. [0305] Ultrafast click chemistry is defined as a click reaction having a reaction rate greater than the rate of the click reaction between azide and bicyclononyne (BCN, according to structure (Q29)), preferably a reaction rate at least 10 times greater, more preferably at least 100 times greater, even more preferably at least 10³ times greater, even more preferably at least 10⁴ times greater, most preferably at least 10⁶ times greater. Herein, the reaction rate of the ultrafast click reaction is determined at the same conditions and with the same substituents as the click reaction between azide and BCN. In absolute amounts, the reaction rate of the ultrafast click reaction is at least 2 x 10³ L/mol s, preferably at least 5 x 10³ L/mol s, or even at least 1 x 10⁴ L/mol s. In one embodiment, the reaction rate is determined in aqueous solution at neutral pH and ambient temperature and pressure, such as pH in the range of 5 - 9, temperature in the range of 15 - 40 °C and pressure in the range of 0.8 - 1 .2 bar. Also, for proper comparison with the reaction between azide and bicyclononyne, the concentration of both click probes (Q and F) should be the same, as click reactions typically have second order rate constants.

[0306] The skilled person is able to determine whether a click reaction is an ultrafast click reaction by comparing the reaction rate of a reaction between a first molecule comprising azide and a second molecule comprising BCN, and the reaction between the same molecules except that the azide moiety and/or BCN moiety is substituted with a different click probe under the same reaction conditions. Alternatively, the skilled person is also able use a model such as DFT or coupled cluster to calculate the activation energy and use that to determine the respective reaction rates, and whether a click reaction classifies as ultrafast click.

[0307] In a preferred embodiment, the ultrafast click reaction is between a click probe F (or F²) selected from tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, o/Yho-quinone, dioxothiophene or sydnone and a click probe Q (or Q²) selected from (hetero)cycloalkene and (hetero)cycloalkyne. In an alternative preferred embodiment, the ultrafast click reaction is between a click probe F (or F²) selected from 1 ,3-dipoles and a click probe Q (or Q²) being TMTHSI, preferably according to structure (Q37). Especially preferred ultrafast click reactions are:

(i) between click probe F (or F²) being tetrazine and click probe Q (or Q²) being bicyclononyne, preferably wherein the tetrazine is according to structure (F8a) and the bicyclononyne according to structure (Q8), more preferably according to structure (Q29), most preferably according to structure (Q42);

(ii) between click probe F (or F²) being tetrazine and click probe Q (or Q²) being frans-bicyclononene, preferably wherein the tetrazine is according to structure (F8a) and the frans-bicyclononene according to structure (Q55);

(iii) between click probe F (or F²) being sydnone and click probe Q (or Q²) being bicyclononyne, preferably wherein the sydnone is according to structure (F7) and the bicyclononyne according to structure (Q8), more preferably according to structure (Q29), most preferably according to structure (Q42);

(iv) between click probe F (or F²) being azide and click probe Q (or Q²) being TMTHSI, preferably wherein the TMTHSI according to structure (Q37);

(v) between click probe F (or F²) being nitrone and click probe Q (or Q²) being (hetero)cycloalkyne, preferably wherein the nitrone is according to structure (F3) and the (hetero)cycloalkyne according to structure (Q1), more preferably according to structure selected from (Q21) - (Q38d), even more preferably according to structure selected from (Q21) - (Q38d), most preferably according to structure (Q29);

(vi) between click probe F (or F²) being tetrazole and click probe Q (or Q²) being (hetero)cycloalkyne, preferably wherein the tetrazole is according to structure (F11) and the (hetero)cycloalkyne according to structure (Q1), more preferably according to structure selected from (Q21) - (Q38d), even more preferably according to structure selected from (Q21) - (Q38a), most preferably according to structure (Q29).

[0308] In an especially preferred embodiment, the ultrafast click reaction is according to option (i), (ii), (v) or (vi) defined above, more preferably according to option (i), (ii) or (vi), most preferably according to option (i). Second order rate constants of 4 x 10⁴ L/mol s have been reported for bicyclononyne/tetrazole click reactions, and of 10⁵ to 10⁶ L/mol s for bicyclononyne/tetrazine click reactions (see e.g. Oliviera et al. Chem. Soc. Rev. 2017, 46, 4895; Kondengadan, Acta Pharmaceutica Sinica B, 2023, 13(5), 1990). Such high rate constants are unprecedented in the art of click chemistry, and offer several benefits as discussed below.

[0309] The inventors have for the first time used ultrafast click chemistry to conjugate oligonucleotides to antibodies, and found that this conjugation reaction occurs especially efficient. As such, the amount of linker-payload construct comprising the oligonucleotide that is needed for complete conjugation is minimized. Normally, in conjugation reaction the linker-payload construct is used in (large) excess, to ensure complete reaction with the modified antibody, as antibodies are expensive, difficult to make and instable, and therefore used in low amounts to minimize any waste of unreacted antibody. However, in case of oligonucleotide payloads, the payload is also expensive, difficult to make and instable, and therefore preferably also used in minimal amounts. Regular conjugation reactions do not provide sufficiently complete reaction when low amounts of modified antibody and low amounts of linkerpayload construct are used. The inventors found that such conjugation reactions are efficiently performed using ultrafast click chemistry.

[0310] Especially beneficial results have been obtained when ultrafast click chemistry is used for conjugation in combination with the use of linkers L^A and L^B (i.e. as in structure (11)). As such, AOCs with DAR4 could be readily obtained. Thus, in an especially preferred embodiment, the conjugation reaction employs ultrafast click chemistry and the AOC is according to structure (11), and more preferably the AOC has DAR4.

[0311] To reflect the improved reaction at lower amounts of both modified antibody and linker-payload construct, it is preferred that the excess of linker-payload construct is less than 100 mol% of the modified antibody, based on number of click probes. Typically, the linker-payload construct has one click probe Q and the modified antibody contains z x y2 click probes F. Hence, the excess of linker-payload construct is typically determined per mol modified antibody x z x y2. Preferably the excess of linkerpayload construct is less than 50 mol%, more preferably less than 25 mol%, even more preferably less than 15 mol%, most preferably less than 10 mol%. This stoichiometry between linker-payload construct and modified antibody applies to the conjugation reaction, and is typically employed in step (c) or (d) of the process according to the invention. [0312] A further benefit of the ultrafast click chemistry resides in the shorter time needed for the conjugation reaction to occur. Reaction times could be reduced from overnight reaction of typically 18 hours to below 30 minutes to reach completion. Thus, in a preferred embodiment, the duration of the conjugation reaction is in the range of 10 min - 2 h, preferably 15 min - 1 h, more preferably 20 - 45 min. In a preferred embodiment, these reaction times apply to step (c) or (d) of the process according to the invention.

Description of the figures

[0313] Figure 1 shows the general scheme for preparation of antibody-drug conjugates by reaction of a monoclonal antibody (in most cases a symmetrical dimer) containing an x number of functionalities F. By incubation of antibody-(F)x with excess of a linker-drug construct (Q-spacer-linker-payload) a conjugate is obtained by reaction of F with Q, forming connecting group Z.

[0314] Figure 2 depicts a range of reagents suitable for reaction with cysteine side-chains. Reagents may be monoalkylation type (A) or may be a cross-linker (B) for reaction with two cysteine side-chains. [0315] Figure 3 shows the general process for non-genetic conversion of a monoclonal antibody (mAb) into an antibody containing probes for click conjugation (F). The click probe may be on various positions in the antibody, depending on the technology employed. For example, the antibody may be converted into an antibody containing two click probes (structure on the left) or four click probes (bottom structure) or eight probes (structure on the right) for click conjugation.

[0316] Figure 4 shows a representative (but not comprehensive) set of functional groups (F) that can be introduced into an antibody by engineering, by chemical modification, or by enzymatic means, which upon metal-free click reaction with a complementary reactive group Q lead to connecting group Z. Functional group F may be artificially introduced (engineered) into an antibody at any position of choice. Some functional groups F (e.g. nitrile oxide, quinone), may besides strained alkynes also react with strained alkenes, which as an example is depicted for triazine or tetrazine (bottom line). The pyridine or pyridazine connecting group is the product of the rearrangement of the tetrazabicyclo[2.2.2]octane connecting group, formed upon reaction of triazine or tetrazine with alkyne (but not alkene), respectively, with loss of N2. Connecting groups Z depicted in Figure 4 are preferred connecting groups to be used in the present invention.

[0317] Figure 5 shows cyclic alkynes suitable for metal-free click chemistry, and preferred embodiments for reactive moiety Q. The list is not comprehensive, for example alkynes can be further activated by fluorination, by substitution of the aromatic rings or by introduction of heteroatoms in the aromatic ring.

[0318] Figure 6 depicts a specific example of site-specific conjugation of a payload based on glycan remodeling of a full-length IgG followed by azide-cyclooctyne click chemistry. The IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of azido-sugar onto the core GIcNAc liberated by endoglycosidase. In the next step, the azido-remodeled IgG is subjected to an oligonucleotide, which has been modified with a single cyclooctyne for metal-free click chemistry (SPAAC), leading to an antibody-oligonucleotide conjugate. It is also depicted that the cyclooctyne-oligonucleotide construct will have a specific spacer between cyclooctyne and oligonucleotide, which enables tailoring of IgG- oligonucleotide distance or impart other properties onto the resulting conjugate.

[0319] Figure 7 describes the two-stage process as applied herein in the formation of conjugates according to structure (11), whereby a glycan-remodeled antibody with functionality F¹ is reacted with a bifunctional linker Q¹-L-(F²)₂-4 (wherein the click probes Q¹ and F² are mutually non-reactive), thereby undergoing a metal-free click chemistry reaction to form bond Z¹. The bifunctional linker may comprise 2 to 4 occurrences of F² (/.e. trivalent, tetravalent or pentavalent linker). In the second step, the antibody containing the reactive group F² is reacted with click probe Q², which is part of the linker-oligonucleotide. [0320] Figure 8 depicts three examples of a bifunctional linker Q¹-L-(F²)2-4that fulfill the condition that Q¹ and F² are not mutually reactive, where Q¹ is either DBCO (as in A) or TMTHSI (as in B and C) and F² is a tetrazine analogue (as in A-C).

[0321] Figure 9 shows two examples of a bifunctional linker Q¹-L-(F²)₂-4 that fulfill the condition that Q¹ and F² are not mutually reactive, where Q¹ is either TMTHSI (as in D) or DBCO (as in E), the tetrazine is a alkylmethyltetrazine (as in D) or a phenyltetrazine (as in E) and where the linker is either tetravalent (as in D) or pentavalent (as in E).

[0322] Figure 10 depicts an example of a bifunctional linker Q¹-L-(F²)2 (F), where Q¹ is DBCO and F² is an iminosydnone variant known to react with DBCO extremely slowly (k < 0.001 M ¹s-¹).

[0323] Figure 1 1 shows three examples of a bifunctional linker Q¹-L-(F²)2, whereby F² is a latent click- reactive group (/.e. not reactive as such but requiring chemical or enzymatic conversion to a reactive click probe), such as phenol or catechol in compound G (can be converted into o/Yho-quinone upon treatment with tyrosinase or NalO4, respectively), such as serine in compound H (can be converted into a nitrone upon treatment with NalO4, then N-methylhydroxylamine), such as tetrazole in compound I (can be converted into nitrile imine upon treatment with 200 nm light) or such as dihydrotetrazine in compound M (can be converted into tetrazine upon treatment with horseradish peroxidase or 660 nm light).

[0324] Figure 12 shows the RP-UPLC spectrum of intact chR17-(PMO)4 (A), chR17-(PMO)2 (B) and chR17-(6-N₃-GalNAc)₂.

[0325] Figure 13 shows the SE-UPLC spectrum of chR17-(PMO)₄ (A) and chR17-(PMO)₂ (B).

[0326] Figure 14 shows the HIC-HPLC spectrum of intact chR17-(PMO)4 (A), chR17-(PMO)₂ (B) and chR17-(6-N₃-GalNAc)₂.

[0327] Figure 15 shows exon 23 skip analysis of C2C12 mouse myoblasts treated with unconjugated PMO (A), chR17-(PMO)₂ (B) and chR17-(PMO)4 (C) at a concentration range of 4 nM to 1 pM.

[0328] Figure 16 shows quantification of in vitro exon 23 skipping data in C2C12 mouse myoblasts. Exon 23 skipping is achieved at significantly lower concentrations by DAR2 and DAR4 AOCs compared to free PMO, particularly when taking drug loading into account.

[0329] Figure 17 shows in vivo exon 23 skipping data in mdx mouse model in different muscle tissues. Significantly higher degrees of exon 23 skipping are achieved by DAR2 and DAR4 AOCs compared to free PMO across a wide range of muscle tissues. [0330] Figure 18 shows in vivo dystrophin restoration data in mdx mouse model in different muscle tissues. Significantly higher degrees of dystrophin restoration are achieved by DAR2 and DAR4 AOCs compared to free PMO across a wide range of muscle tissues.

[0331] Figure 19 shows the degree of MBNL1 splice correction by OKT9-(Repeat Blocker^ in DM1 human myoblasts at 200 nM. A decrease in MBNL1 exon 5 inclusion is observed for OKT9-(Repeat Blocker>2 relative to the untreated and negative control groups (control oligo and control mAb AOCs). A similar degree of splice correction compared to PF14-DMPK gapmer nanoparticles (positive control) is observed.

[0332] Figure 20 shows the effect of OKT9-(Repeat Blocker^ on the number of nuclear foci in DM1 human myoblasts at 200 nM. A decrease in number of nuclear foci is observed for OKT9-(Repeat Blocker>2 relative to the untreated and negative control groups (control oligo and control mAb AOCs). A similar reduction in nuclear foci is shown compared to PF14-DMPK gapmer nanoparticles (positive control).

[0333] Figure 21 shows in vivo exon 23 skipping data in mdx mouse model in different muscle tissues. A dose response is observed were higher degrees of exon 23 skipping are observed at higher doses of AOC across a range of muscle tissues.

[0334] Figure 22 shows in vivo dystrophin restoration data (% of WT) in mdx mouse model in different muscle tissues. A dose response is observed were higher degrees of dystrophin restoration are observed at higher doses of AOC across a range of muscle tissues.

[0335] Figure 23 shows the average body weight (g) of WT mice and mdx mice in the different cohorts average time (weeks). A steady increase in body weight is observed over time indicating that the treatments are well tolerated.

[0336] Figure 24 shows the maximum hanging time (sec) achieved in a two limb hanging test by vehicle treated WT and mdx+ mice and AOC treated mdx+ mice over time (weeks). Vehicle treated WT mice achieved a significantly increased hanging time over vehicle treated mdx+ mice showcasing the compromised muscle function of mdx+ mice. Group B showed a restoration in hanging time of the mdx+ mice to WT levels upon treatment that decreased over time. Group C and D showed improvements in hanging time compared to group A.

[0337] Figure 25 shows the maximum hanging time (sec) achieved in a four limbs hanging test by vehicle treated WT and mdx+ mice and AOC treated mdx+ mice over time (weeks). Vehicle treated WT mice achieved a significantly increased hanging time over vehicle treated mdx+ mice showcasing the compromised muscle function of mdx+ mice. Group B showed a restoration in hanging time of the mdx+ mice to WT levels upon treatment. Group C and D showed improvements hanging time compared to group A.

[0338] Figure 26 shows the grip strength normalized to bodyweight (g/g) achieved in a force grip strength test by vehicle treated WT and mdx+ mice and AOC treated mdx+ mice over time (weeks). Vehicle treated WT mice achieved a significantly increased grip strength over vehicle treated mdx+ mice showcasing the compromised muscle function of mdx+ mice. Group B showed a restoration in grip strength of the mdx+ mice to WT levels upon treatment. Group C and D showed improvements grip strength compared to group A. Examples

[0339] The invention is illustrated by the following examples.

General analytics and materials

[0340] Antisense Oligonucleotide and Cell Penetrating Peptide Sequences

A phosphorodiamidate morpholino oligomer (PMO) with the sequence 5’-amine- GGCCAAACCTCGGCTTACCTGAAAT-3’ (PMO) [SEQ ID NO: 1] with a primary amine modification at the 5’ end of the molecule for conjugation was custom-made and purchased from Gene Tools, LLC. For the phosphorothioate antisense oligonucleotides (PS ASO) sequences that follow below: Bold underline, 2’-O-Me; non-bold non-underline, DNA; *, phosphorothioate (PS) linkage:

- A PS ASO oligonucleotide with the sequence 5’-Amine-C6-C*A*G*C*A*G*C*A*G*C*A*G*C*A*G- 3’ (Repeat Blocker) [SEQ ID NO: 2] with a C6-NH2 modification at the 5’ end of the molecule for conjugation was custom-made and purchased from Eurogentec S.A.

- A PS ASO oligonucleotide with the sequence 5’-Amine-C6-G*A*C*G*A*C*G*A*C*G*A*C*G*A*C- 3’ (Control Blocker) [SEQ ID NO: 3] with a C6-NH2 modification at the 5’ end of the molecule for conjugation was custom-made and purchased from Eurogentec S.A.

- A gapmer oligonucleotide with the sequence 5’-C*G*G G*C*G*G*T*T*G*T*G*A*A*C*U*G*G*C- 3’ (DMPK Gapmer) [SEQ ID NO: 4] was custom-made and purchased from Integrated DNA Technologies, INC.

- A gapmer oligonucleotide with the sequence 5’-G*A*C*G C*G*A*C*G*A*C*G*A*C*G*A*C-3’ (Control Gapmer) [SEQ ID NO: 5] was custom-made and purchased from Integrated DNA Technologies, INC.

- The stearylated cell-penetrating peptide (CPP), PepFect14, with the sequence Stearyl- AGYLLGKLLOOLAAAALOOLL-NH2 [SEQ ID NO: 6] (where O is ornithine and -NH₂ is a C-terminal amidation) was purchased from PepScan.

Table 1. Overview of PCR primers used.

[0341] General procedure for mass spectral analysis: Prior to mass spectral analysis, IgG was treated with fabricator (commercially available via Genovis), which allows analysis of the Fc/2 fragment, or treated with DTT, which allows analysis of the HC. For analysis of the Fc/2 fragment, a solution of 10 pg (modified) IgG was incubated for 1 hour at 37 °C with IdeS/Fabricator™ (1.25 U/pL) in PBS Ph 7.4 in a total volume of 10 pL. Samples were diluted to 100 pL using TBS pH 7.5. For analysis of the HC, a solution of 10 pg (modified) IgG was incubated for 15 minutes at 37 °C with DTT (10 mM final concentration) in TBS pH 7.5 in a total volume of 50 pL. Reactions were quenched by addition of 50 pL ACN:MQ:formic acid (49:49:2). MS analysis is performed on JEOL AccuTOF LC-plus JMS-T100LP system (ESI-TOF) combined with a HPLC system (Agilent 1100 series, Hewlett Packard). On the HPLC system a MassPREP™ On-line Desalting Cartridge (Waters P/N 186002785) is installed. Deconvoluted spectra were obtained using Magtran software.

[0342] General procedure for analytical RP-UPLC (DTT treated samples): Prior to RP-UPLC analysis, IgG (10 pL, 1 mg/mL in PBS pH 7.4) was added to 12.5 mM DTT, 100 mM TrisHCI pH 8.0 (40 pL) and incubated for 15 minutes at 37 °C. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 pL). RP-UPLC analysis was performed on a Waters Acquity UPLC-SQD. The sample (5 pL) was injected with 0.4 mL/min onto Bioresolve RP mAb 2.1*150 mm 2.7 pm (Waters) with a column temperature of 70 °C. A linear gradient was applied in 9 minutes from 30 to 54% acetonitrile in 0.1 % TFA and water. Absorbance of eluted peaks was measured at 215 nm followed by automated integration (MassLynx, Waters) to determine reaction conversion.

[0343] General procedure for analytical SE-UPLC: UPLC-SEC analysis was performed on a Waters Acquity UPLC using an ACQUITY UPLC Protein BEH SEC Column (200 A, 1.7 pm, 4.6 mm X 150 mm). The sample was diluted to 1 mg/mL in PBS, 2 pL was injected and measured with 0.3 mL/min isocratic method (0.1 M sodium phosphate buffer pH 6.9 (NaHPO4/Na2PO4) containing 10% isopropanol) for 6 minutes.

[0344] General procedure for analytical HIC-HPLC: Samples were diluted to 1 mg/mL in PBS. HIC analysis is performed on an Agilent 1200 series using a TSKgel® Butyl-NPR HPLC Column (3.5 cm x 4.6 mm, 2.5 pm). 10 pL sample is injected at a flow rate of 0.5 mL/min using a gradient starting from 100% buffer A (2 M ammonium sulfate in 50 mM potassium phosphate pH 6.0) to 100% buffer B (50 mM potassium phosphate pH 6.0 + 20% isopropanol) in 20 minutes.

Examples 1 - 5: Synthesis of click probe and linker-oliqonucleotides

Example 1: Preparation of compound 3

[0345] To a solution of compound 1 (10.0 mg, 1 Eq, 12.2 pmol) in dry DMF (850 pL) was added compound 2 (10.3 mg, 3 Eq, 36.6 pmol), followed by triethylamine (6.18 mg, 8.51 pL, 5 Eq, 61.1 pmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by prep-HPLC; (30% 100%, MeCN/Water + 10 mM NH4HCO3, runtime 21 minutes, Column Xbridge prep C18 5 pm OBD, 30x100 mm) to afford compound 3 (5 mg, 4.7 pmol, 38%) as a pink solid. LCMS

(ESI+) calculated for C57H67N12C (M+H)⁺ 1079.5, found 1079.9. Example 2: Preparation of compound 6 and 7

[0346] Compound 4 (625 mg, 1 Eq, 2.22 mmol) was dissolved in DMF (5 mL) and to this was added bis(4-nitrophenyl) carbonate (811 mg, 1.2 Eq, 2.67 mmol) and triethylamine (674 mg, 929 pL, 3 Eq,

6.66 mmol). The reaction mixture was stirred for 18 hours, after which compound 5 (1.01 g, 1.2 Eq,

2.67 mmol) and additional triethylamine (674 mg, 929 pL, 3 Eq, 6.66 mmol) were added. Stirring was continued for an additional 6 hours. The reaction mixture was concentrated in vacuo and the resulting crude was purified by flash column chromatography over silicagel (0 — 40% MeOH/DCM) to afford compound 6 (850 mg, 1.2 mmol, 53 %, 95% Purity) as a white foam. LCMS (ESI+) calculated for C₃4H₅IN6O₉ ⁺ (M+H)⁺ 687.4, found 687.7.

[0347] Compound 6 (400 mg, 1 Eq, 582 pmol) was dissolved in DMF (4.00 mL) and to this bis(perfluorophenyl) carbonate (459 mg, 2 Eq, 1.16 mmol) and triethylamine (177 mg, 244 pL, 3 Eq, 1 .75 mmol) were added. The resulting solution was stirred at room temperature for 2 hours after which it was diluted with DCM (10 ml) and was purified by flash column chromatography over silicagel (0 — 10% MeOH/DCM) to afford compound 7 (422 mg, 0.45 mmol, 78 %, 96% Purity) as a colorless solid. LCMS (ESI+) calculated for C4iH₅oF₅N60n⁺ (M+H)⁺ 897.3, found 897.7.

Example 3: Preparation of compound 8

[0348] To a stock solution of PMO in 90% DMF and 10% H2O (34.9 mg, 213 pL, 18.8 mmolar, 1 Eq, 4.00 pmol) was added triethylamine (6.07 mg, 8.36 pL, 15 Eq, 60.0 pmol), followed by a stock solution of compound 7 in DMF (7.17 mg, 80.0 pL, 100 mmolar, 2 Eq, 8.00 pmol). The reaction mixture was left to stand at room temperature for 2 hour after which it was purified by prep-HPLC; (5% — 95%, MeCN/Water + 10 mM NH4HCO3, runtime 12 minutes, Column Xbridge prep C18 5 pm OBD, 30x100 mm). The product containing fractions were combined, analyzed by UV-vis (e = 259210 M“¹cm^-1) and lyophilized to afford compound 8 (29.7 mg, 3.15 pmol, 78.8 %, 100% Purity) as a white solid. LCMS (ESI+) calculated for (M+7H)⁺⁷ (9434+7)/7 = 1348.7 found 1348.7.

[0349] To a stock solution of Repeat Blocker (3.82 mg, 35.0 pL, 20 mmolar, 1 Eq, 0.700 pmol) was added triethylamine (1.06 mg, 1.46 pL, 15 Eq, 10.5 pmol), followed by a stock solution of compound 7 in DMF (1 .26 mg, 14.0 pL, 100 mmolar, 2 Eq, 1 .40 pmol). The reaction mixture was left to stand at room temperature for 1 hour after which it was purified by prep-HPLC; (5% —>■ 95%, MeCN/Water + 10 mM NH4HCO3, runtime 12 minutes, Column Xbridge prep C18 5 pm OBD, 30x100 mm). The product containing fractions were combined, analyzed by UV-vis (e = 147800 M^-1cm^-1) and lyophilized to afford compound 9 (1.26 mg, 0.206 pmol, 29.4 %, 100% Purity)as a white solid. LCMS (ESI-) calculated for (M-6H)^-6 (6166-6)/6 = 1026.7, found 1026.2.

Example 5: Preparation of compound 10

[0350] To a stock solution of Control Blocker in DMF and 10% H2O (3.82 mg, 35.0 pL, 20 mmolar, 1 Eq, .700 pmol) was added triethylamine (1.06 mg, 1.46 pL, 15 Eq, 10.5 pmol), followed by a stock solution of compound 7 in DMF (1.26 mg, 14.0 pL, 100 mmolar, 2.00 Eq, 1.40 pmol). The reaction mixture was left to stand at room temperature for 1 hour after which it was purified by prep-HPLC; (5%

95%, MeCN/Water + 10 mM NH4HCO3, runtime 12 minutes, Column Xbridge prep C18 5 pm OBD, 30x100 mm). The product containing fractions were combined, analyzed by UV-vis (e = 150400 M“¹cm^-1) and lyophilized to afford compound 10 (3.55 mg, 0.575 pmol, 82 %, 100% Purity) as a white solid. LCMS (ESI-) calculated for (M-6H)-⁶ (6166-6)/6 = 1026.7, found 1026.1.

Examples 6 - 15: Conjugation of linker-oliqonucleotides to (modified) monoclonal antibodies

Example 6: Transient expression ofchR17 in CHO

[0351] chR17 is an chimeric antibody targeting murine transferrin (TfR1), consisting of a LC sequence being identified by SEQ ID NO: 7, and a HC sequence being identified by SEQ ID NO: 8. chR17 was transiently expressed in CHO KI cells by Evitria (Zurich, Switzerland) at 1500 mL scale. The antibody was purified using two HiTrap MabSelect Sure 5 mL columns connected in series. After loading of the supernatant the column was washed with TBS pH 7.5 for 10 CV. The IgG was eluted with 0.1 M sodium acetate pH 3.0 and neutralized with 2.5 M Tris-HCI pH 7.2. Afterthree times dialysis to 20 mM Histidine- HCI, 150 mM NaCI, pH 7.5 the IgG was concentrated to 20.6 mg/mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) and obtained in a final yield of 94 mg. Mass spectral analysis of the sample after DTT treatment showed one major HC product (observed mass of 51145 Da, approximately 60% of total HC), corresponding to HC without C-terminal lysine and G0F glycoform, and one HC product (observed mass of 51307 Da, approximately 30% of total HC), corresponding to HC without C-terminal lysine and G1 F glycoform.

Example 7: Enzymatic remodeling of chR17 to chR17(6-N3-GalNAc)2

[0352] chR17 (4.53 mL, 94 mg, 20.6 mg/mL in 20 mM Histidine-HCI, 150 mM NaCI, pH 7.5) was incubated at a final concentration of 15 mg/mL with EndoSH (1 % w/w), as described in PCT/EP2017/052792, His-TnGalNAcT, as described in PCT/EP2016/059194 (3% w/w), alkaline phosphatase (commercially available from Roche, 0.01 % w/w) and UDP-6-N3-GalNAc (10 eq compared to IgG), prepared according to PCT/EP2016/059194, in 20 mM Histidine-HCI pH 7.5 150 mM NaCI pH 7.5 and 6 mM MnCI2 for 16 hours at 30 °C. Next, the functionalized IgG was purified using a HiTrap MabSelect Sure 5 mL column. After loading of the reaction mixture the column was washed with TBS + 0.2% Triton for 10 CV followed by TBS pH 7.5 for 10 CV. The IgG was eluted with 0.1 M sodium acetate pH 3.0 and neutralized with 2.5 M Tris-HCI pH 7.2. After three times dialysis to PBS pH 7.4, the IgG was concentrated to 23.6 mg/mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectral analysis of the sample after DTT treatment showed one major HC product (observed mass of 50280 Da, approximately 80% of total HC), corresponding to the 6-N3-GalNAc-GlcNAc(Fuc)-substituted HC without C-terminal lysine, and one minor HC product (observed mass of 50409 Da, approximately 15% of total HC), corresponding to the 6-N3-GalNAc-GlcNAc(Fuc)-substituted HC with C-terminal lysine, and one minor HC product (observed mass of 50136 Da, approximately 5% of total HC), corresponding to the 6-N3-GalNAc-GlcNAc-substituted HC without C-terminal lysine. See Figure 12C and 14C for RP-UPLC and HIC-HPLC analysis respectively.

Example 8: Preparation of chR17-(tetrazine)4

[0353] To a solution of chR17-(6-N3-GalNAc)2 (707 pL, 15 mg, 21 .2 mg/mL in PBS pH 7.4) was added TBS pH 7.5 (343 pL), PG (390 pL) and bistetrazine compound 3 (60 pL, 10 mM stock solution in DMF) in propylene glycol (1 .08 mL, 50% of final volume). The reaction was incubated overnight at rt. To remove the excess of bistetrazine compound 3 , two HiTrap 5 mL desalting columns (Cytiva) were connected in series. The columns were rinsed with 0.2M NaOH and equilibrated with PBS pH 7.4 before the injection and elution of chR17-(tetrazine)4. Subsequently the product was concentrated to 25.0 mg/mL using a Vivaspin Turbo 4 10 kDa MWCO ultrafiltration unit (Sartorius). Mass spectral analysis of the sample after DTT treatment showed one major HC product (observed mass of 51363 Da, approximately 80% of total HC), corresponding to the desired product with 3 conjugated to the HC leaving two reactive tetrazines per HC available for further conjugation.

Example 9: Enzymatic remodeling of palivizumab to palivizumab(6-N3-GalNAc)2

[0354] Palivizumab (Synagis), obtained from the pharmacy, was diluted in 20 mM Histidine-HCI, 150 mM NaCI, pH 7.5 to a concentration of 26.52 mg/mL. Palivizumab (2.60 mL, 69 mg, 26.52 mg/mL in 20 mM Histidine-HCI, 150 mM NaCI, pH 7.5) was incubated at a final concentration of 15 mg/mL with EndoSH (1 % w/w), as described in PCT/EP2017/052792, His-TnGalNAcT, as described in PCT/EP2016/059194 (3% w/w), alkaline phosphatase (commercially available from Roche, 0.01 % w/w) and UDP-6-N3-GalNAc (10 eq compared to IgG), prepared according to PCT/EP2016/059194, in 20 mM Histidine-HCI pH 7.5 150 mM NaCI pH 7.5 and 6 mM MnCI2 for 16 hours at 30 °C. Next, the functionalized IgG was purified using a HiTrap MabSelect Sure 5 mL column. After loading of the reaction mixture the column was washed with TBS + 0.2% Triton for 10 CV followed by TBS pH 7.5 for 10 CV. The IgG was eluted with 0.1 M sodium acetate pH 3.0 and neutralized with 2.5 M Tris-HCI pH 7.2. After three times dialysis to TBS pH 7.5, the IgG was concentrated to 21 .83 mg/mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 24365 Da, approximately 90% of total Fc/2 fragment), corresponding to the 6-N3-GalNAc-GlcNAc(Fuc)-substituted Fc/2 fragment. Example 10: Enzymatic remodeling of OKT9 to OKT9(6-N3-GalNAc)2

[0355] OKT9, an antibody targeting human transferrin (TfR1), was obtained from BioXCell. OKT9 contained a second glycosylation site which could potentially be labelled during enzymatic remodeling. To avoid incorporation of azides on this undesired position, terminal GIcNAc residues were first blocked using GalT(Y289Y) in combination with UDP-galactose. OKT9 was dialyzed to 20 mM Histidine-HCI, 150 mM NaCI, pH 7.5 followed by concentration to 26.52 mg/mL. To block terminal GIcNAc residues, OKT9 (4.19 mL, 96 mg, 26.52 mg/mL in 20 mM Histidine-HCI, 150 mM NaCI, pH 7.5) was incubated at a final concentration of 15 mg/mL with GalT(Y289F) (2.5% w/w), alkaline phosphatase (commercially available from Roche, 0.01 % w/w) and UDP-galactose (50 eq compared to IgG) in 20 mM Histidine- HCI pH 7.5 150 mM NaCI pH 7.5 and 6 mM MnCI2 for 16 hours at 30 °C. Next, the blocked OKT9 was dialyzed three times to 20 mM Histidine-HCI, 150 mM NaCI, pH 7.5 to remove excess UDP-galactose. The blocked OKT9 (3.80 mL, 88 mg, 23.13 mg/mL in 20 mM Histidine-HCI, 150 mM NaCI, pH 7.5) was incubated at a final concentration of 15 mg/mL with EndoSH (1 % w/w), as described in PCT/EP2017/052792, His-TnGalNAcT, as described in PCT/EP2016/059194 (3% w/w), alkaline phosphatase (commercially available from Roche, 0.01 % w/w) and UDP-6-N3-GalNAc (20 eq compared to IgG), prepared according to PCT/EP2016/059194, in 20 mM Histidine-HCI pH 7.5 150 mM NaCI pH 7.5 and 6 mM MnCI2 for 16 hours at 30 °C. Next, the functionalized IgG was purified using two HiTrap MabSelect Sure 5 mL columns connected in series. After loading of the reaction mixture the column was washed with TBS + 0.2% Triton for 10 CV followed by TBS pH 7.5 for 10 CV. The IgG was eluted with 0.1 M sodium acetate pH 3.0 and neutralized with 2.5 M Tris-HCI pH 7.2. After three times dialysis to TBS pH 7.5, the IgG was concentrated to 18.87 mg/mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius). Mass spectral analysis of the reduced sample showed one major product (observed mass 51454 Da, approximately 50% of total HC), corresponding to the 6-Ns-GalNAc- GlcNAc(Fuc)-substituted HC.

Example 11: Conjugation of chR17(6-N3-GalNAc)2 with compound 8 to obtain conjugate chR17-(PMO)2 with DAR2

[0356] chR17-(6-N3-GalNAc)2 (1687 pL, 30 mg, 17.78 mg/ml in PBS pH 7.4) was added to a solution of PBS pH 7.4 (32 pL) and compound 8 (281 pL, 2.85 mM solution in MQ). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 16/600 GL (Cytiva) on a 100F NGC system (Bio-Rad). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 33798 Da, approximately 80% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment. See Figure 12B, 13B and 14B for RP-UPLC, SE-UPLC and HIC-HPLC analysis respectively.

Example 12: Conjugation of chR17(tetrazine)4 with compound 8 to obtain conjugate chR17-(PMO)4 with DAR4

[0357] chR17(tetrazine)4 (5920 pL, 100 mg, 16.8 mg/ml in PBS pH 7.4) was added to a solution of PBS pH 7.4 (3435 pL) and compound 8 (607 pL, 5.03 mM solution in MQ). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 16/600 GL (Cytiva) on a 100F NGC system (Bio-Rad). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 44255 Da, approximately 60% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 35490 Da, approximately 15% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment with fragmentation of one of the vc-PABC linkers during MS analysis. See Figure 12A, 13A and 14A for RP-UPLC, SE-UPLC and HIC-HPLC analysis respectively.

Example 13: Conjugation of OKT9(6-N3-GalNAc)2 with compound 9 to obtain conjugate OKT9-(Repeat Blocker)2 with DAR2

[0358] OKT9(6-N3-GalNAc)2 (159 pL, 3 mg, 18.87 mg/ml in PBS pH 7.4) was added to a solution of PBS pH 7.4 (1 pL) and compound 9 (40 pL, 2.01 mM solution in MQ). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (Cytiva) on a 100F NGC system (Bio-Rad). Mass spectral analysis of the reduced sample showed one major product (observed mass 57623 Da, approximately 50% of total HC), corresponding to the conjugated HC.

Example 14: Conjugation of OKT9(6-N3-GalNAc)2 with compound 10 to obtain conjugate OKT9- (Control Blocker)2 with DAR2

[0359] OKT9(6-N3-GalNAc)2 (159 pL, 3 mg, 18.87 mg/ml in PBS pH 7.4) was added to a solution of PBS pH 7.4 (19 pL) and compound 10 (22 pL, 3.67 mM solution in MQ). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (Cytiva) on a 100F NGC system (Bio-Rad). Mass spectral analysis of the reduced sample showed one major product (observed mass 57624 Da, approximately 50% of total HC), corresponding to the conjugated HC.

[0360] Palivizumab(6-N3-GalNAc)2 (196 pL, 5 mg, 25.56 mg/ml in PBS pH 7.4) was added to a solution of PBS pH 7.4 (94 pL) and compound 9 (44 pL, 3.77 mM solution in MQ). The reaction was incubated at rt overnight followed by purification on a Superdex200 Increase 10/300 GL (Cytiva) on a 100F NGC system (Bio-Rad). Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 30528 Da, approximately 70% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment, and one minor product (observed mass 25033 Da, approximately 30% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment with fragmentation of the vc-PABC linkers during MS analysis.

Example 16-19: Assessment of AOC efficacy in C2C12 myotubes - Proliferation and differentiation of myoblast cultures

Example 16: Collagen coating of well plates for muscle cell culturing

[0361] Vitrogen Collagen (30X = 3 mg/ml) was diluted 1 to 30 with distilled water. The collagen solution was added to the plate covering the entire area, followed by incubation at 37°C for at least 60 min. The collagen solution was removed and the plates were dried for 30 minutes. Finally, the plates were rinsed twice with HBSS to neutralize the pH of collagen solution.

Example 17: Initiation of the cell cultures

[0362] Mouse C2C12 cells stored in liquid nitrogen were thawed in a 37°C water bath. The cell suspension was diluted in a 50 ml tube in 10 ml proliferation medium (424 ml Dulbecco's medium (without phenol red) with 10% FBS (50 ml), 1 % P/S (5 ml), 2% Glutamax (10 ml) and 1 % glucose (11 ml) (all from Gibco-BRL)). The cells were centrifuged for 10 min. at 1200 rpm. The cell pellet was resuspended in proliferation medium (6 ml per T25, 15 ml per T75, or 35 ml per T200 flask). The cells were plated in collagen-coated flasks and incubated at 37°C and 10 % CO2.

Example 18: Splitting of the cells

[0363] After the cells were cultured to confluency, the proliferation medium was removed and the cells were rinsed twice with HBSS followed by addition of Trypsin/EDTA (2 ml per T25, 6 ml per T75, or 10 ml per T200 flask). When all cells were detached, trypsin was inhibited with twice the volume proliferation medium and the cells were collected into a 50 ml tube. The cells were centrifuged for 10 min at 1200 rpm. The supernatant was removed and the cells were resuspended in the appropriate volume of proliferation medium.

Example 19: Differentiation of myoblasts to myotube cultures

[0364] The cells were plated in collagen-coated 6-wells plates (3 ml proliferation medium per well), the cells were cultured to confluency (-100,000 cells). Proliferation medium was removed and the cells were rinsed twice with HBSS. 3 ml of fusion medium (464 ml Dulbecco's medium (without phenol red) with 2% FBS (10 ml), 1 % P/S (5 ml), 2% Glutamax (10 ml) and 1 % glucose (1 1 ml) (all from Gibco- BRL)) was added per well. The cells were allowed to fuse and differentiate into myotubes over a period of 7 to 14 days.

Example 20-22: Assessment of AOC efficacy in C2C12 myotubes - In vitro efficacy study of AOCs in C2C12 myotubes

Example 20: Gymnosis of cultured cells with free PMO, DAR2 and DAR4 AOC

[0365] Stock solutions of PMO, chR17-(PMO)2 or chR17-(PMO>4 were diluted via serial dilution to a concentration range of 4 nM to 1 pM with differentiation medium (464 ml Dulbecco's medium (without phenol red, Invitrogen, 11880) with 2% FBS (10 ml), 2% Glutamax (10 ml), 1 % glucose (11 ml) (all from Gibco-BRL) and antibiotics (5 ml pen/strep (C2C12)). The cells were washed twice followed by addition of solutions at a concentration range of 4 nM to 1 pM of PMO, chR17-(PMO>2 or chR17-(PMO)4to their respective wells. The cells were incubated for 3 days at 37°C and 10 % CO2 after which they were harvested. Example 21: RNA isolation from cultured cells using TRIsure

[0366] The medium was removed from the cells and rinsed twice with HBSS. TRIsure (500 pl) was added per well. The cell lysate was pipetted up and down until a homogeneous solution was obtained. The lysate was transferred into 1 .5 mL Eppendorf tubes. Chloroform (100 pL (1/5 volume)) was added and the tubes were shaken vigorously for 15 sec. After incubating on ice for 5 min, the Eppendorf tubes were centrifuged at 13 000 rpm for 15 min. at 4°C to separate the phases. The upper aqueous phase (-250 pL) was transferred to a new tube and an equal volume of isopropanol was added. To induce precipitation the tubes were incubated for at least 30 min on ice (or overnight at 4°C). After centrifuging at 13 000 rpm for 10 min. at 4°C, the supernatant was removed and RNA pellet was washed with 200 pL 70% ethanol. After centrifuging at 13,000 rpm for 10 min. at 4°C, the supernatant was removed and the pellet was briefly dried dry to air (-5 min.). The pellet was dissolved in 100 pl DEPC-H2O and stored at -80 °C.

Example 22: RT-PCR analysis of dystrophin for in vitro cell cultures using Promega RTase

[0367] The following priming premix was prepared by mixing the isolated RNA (0.1 pg/pL) with H2O, reverse primer m26R (1 mM) and dNTP mix (1 mM). The samples were incubated at 70°C for 5 min and chilled on ice for at least 1 min. The following reaction premix was prepared by mixing 5x reaction buffer (Promega, M5313, 4 pL), rRNasin (40u/pL, 0,5 pL), Promega M-MLV Reverse Transcriptase (200U/pl, 1 pL) and H2O (4.5 pL). 10 pL of reaction premix was added to the 10 pl priming premix and gently mixed by pipetting. The mixture was incubated as follows: 60 min at 42°C, 10 min at 70°C (to terminate reaction).

[0368] The following PCR mixture was prepared by mixing 10x supertaq PCR buffer (2,5 pl), dNTPs (10mM, 0,5 pl), forward primer m20F (l Opmol/pl, 1 pl), reverse primer m26R (l Opmol/pl, 1 pl), Taq DNA polymerase (5U/pl, 0,125 pl) and H2O (16,875 pl). 22 pl of PCR mixture was aliquoted per PCR-tube, and 3 pl cDNA sample was added. PCR program (in a 25 pl volume) was run as follows: 5 min 94°C, 20 cycles: 40 sec 94°C - 40 sec 60°C - 80 sec 72°C, 7 min 72°C and cool down to 22°C.

[0369] For nested PCR the following PCR mixture was prepared by mixing 10x supertaq PCR buffer (5 pl), dNTPs (10mM, 1 pl), forward primer m21 F (10pmol/pl, 2 pl), reverse primer m24R (10pmol/pl, 2 pl), Taq DNA polymerase (5U/pl, 0,25 pl) and H2O (38,25 pl). 48.5 pl of PCR mixture was aliquoted per PCR-tube, and 1 .5 pl of PCR sample was. PCR program (in a 50 pl volume) was run as follows: 5 min 94°C, 32 cycles: 40 sec 94°C - 40 sec 60°C - 60 sec 72°C, 7 min 72°C and cool down to 22°C. The PCR products were analysed by electrophoresis on a 2% agarose gel (see Figure 15) and were quantified with Lab-on-a-chip (Agilent DNA-1000 chip) or by Femto Pulse (see Figure 16).

Example 23-26: Assessment of AOC efficacy in vivo with mdx mouse model for Duchenne muscular dystrophy

Example 23: In vivo efficacy study of AOC in mdx mouse model

[0370] At the age of 5 weeks mdx mice received one i.v. injection of 100 pL containing either vehicle (PBS) or 30 mg/kg chR17-(PMO)₄ or 60 mg/kg chR17-(PMO)₂ or 100 mg/kg PMO or 100 mg/kg chR17- (PM0)4) via intra venous tail injection. Four weeks after the injection (day 28) the mice were sacrificed by cervical dislocation. Tissues were extracted from the mice to check for exon skipping levels and to measure dystrophin protein levels. The tissues extracted were: gastrocnemius, triceps, diaphragm and heart. Muscles were snap frozen into liquid nitrogen.

Example 24: RNA isolation from muscles using TRIsure

[0371] The muscles were maintained in a frozen state using liquid nitrogen. TRIsure (600 pL) was added to Magnalyzer beads tube with cross sections. After centrifuging at 13 000 rpm, the tissue was disrupted for 20 sec on 7000 rpm. After cooling down the samples on ice and the disrupting was repeated until the tissue was homogenized. The solution was transferred to a fresh tube, and per 600 pL of TRIsure 0.1 ml of chloroform was added. The mixture was shaken vigorously (by hand) for 15 sec and put on ice for 5 min. After centrifuging at 13 000 rpm for 15 min. at 4°C to separate phases, the upper aqueous phase was transferred gently without disrupting the interphase to a new tube. An equal volume of 100% isopropanol was added to the aqueous phase and mixed gently by pipetting. To induce precipitation the sample was incubated at least 30 min on ice or at 4 °C overnight. After centrifuging at 13 000 rpm for 10 min. at 4°C, the supernatant was removed carefully and RNA pellet was washed with 200 pl 70% ethanol. After centrifuging at 13,000 rpm for 15 min. at 4°C, the supernatant was carefully removed and the pellet was briefly dried to air. The RNA was dissolved in 100 pl DEPC-H2O and stored at -80°C until use.

Example 25: RT-PCR analysis of dystrophin formouse muscles

[0372] A priming premix was prepared by mixing 8 pl RNA sample (400 ng) with random hexamer N6 (40ng/pl, 1 pl), dNTP mix (10mM, 1 pl). A RT-mixture was prepared by mixing 5x reaction buffer (4 pl), rRNAsin (40u/pl, 0,5 pl), Bioscript Rtase (200U/pl, 1 pl) and Rnase free water (4.5 pl). 10 pl of RT- mixture was added to 10 pl of priming premix and mixed gently. Incubation was performed as follows: 10 min at 25°C, 1 hour at 42°C, 10 min 70°C and cooled down to 4 °C. The cDNA was stored at -20°C, or immediately used for PCR.

[0373] A PCR-mixture was prepared by mixing the following 10x supertaq PCR buffer (5 pl), dNTPs (10mM, 1 pl), M22F (10pmol/pl, 2 pl), M24R (10pmol/pl, 2 pl), Taq DNA polymerase (5U/pl, 0,25 pl), H2O (38,25 pl). 48,5 pl of PCR mixture was aliquoted per PCR-tube and 1 ,5 pl cDNA sample was added. PCR program (in a 50 pl volume) was run as follows: 5 min 94°C, 30 cycles: 30” at 94°C - 30” at 60°C - 30” at 72°C, 7 min 72°C, cool down to 22°C. The PCR products were analysed by electrophoresis on a 1.5% agarose gel and were quantified by Femto Pulse (see Figure 17).

Example 26: Western blot protocol for dystrophin in tissue

[0374] The frozen muscle tissue was put in Zirconium Beads Pre-Filled Tubes. Protein isolation buffer (-0.5-1 ml, a mixture of 4 ml 1.25 M Tris-HCI pH 6.8 buffer, 40 ml 25% SDS, 6 ml H2O) was added. The tissue was ground in the Magnalyzer (Roche) for 20 seconds at speed 7000. This was repeated until the tissue was homogenized, with cooling of tissue between steps. To remove of the foam caused by the SDS, the vials were centrifuged for +/-1 minute. The solution was transferred into a clean 1 .5 ml Eppendorf tube. The samples were heated at 95°C for 10 minutes. After aliquoting, the protein concentration was determined with a BCA kit. The aliquots were stored at -80°C.

[0375] 30 pg of total protein of the samples was prepared in 30 pl total volume with Laemmli buffer. 2p I of (blue) p-mercaptoethanol solution was added to each sample. The samples were heated at 95°C for 5 minutes and quickly spun before use.

30 pL of sample was applied per lane of a gel (Criterion XT 3 - 8% Tris-Acetate Gel) and ran at with 1x XT Tricine (pH 8.2) buffer for 1 hour at 75 V followed by 1 hour at 150 V. The gel was blotted on a nitrocellulose membrane using a Trans-Blot Turbo system at 2.5A for 10 minutes. The membrane was incubated in the presence of 5% blocking buffer with gentle agitation for 1 hour at RT, followed by washing three for 15 minutes with 1x TBST. The primary antibodies for dystrophin (1 : 2000, Ab154168) and a-actinin (1 :1000, Ab72592 OR 66895-1-lg, Table 1) were diluted in Takara Immuno booster 1 (total volume ~3.5 ml) and incubated overnight at 4°C (cold room), on a tube roller. The primary antibody was poured off and washed 3 times in 1x TBST for 15 minutes. The secondary antibody was diluted in Takara Immuno Booster 2: IRDye 800CW donkey-anti-rabbit 1 :5000 for Ab154168; 1 :10,000 for Ab72592 IRDye 680RD donkey-anti-mouse, 1 :10,000 for 66895-1-lg. After incubating for 1 hour at 4°C (cold room) on a tube roller, the secondary antibodies were poured off and washed twice in 1x TBST for 20 minutes and once 20 minutes in 1x TBS. Quantification of dystrophin protein expression was performed by Image-Analysis using alpha-actinin as loading control in the Odyssey software (see Figure 18).

Example 27-30: Assessment of /n vitro AOC efficacy in DM1 myoblasts

Example 27: Preparation of cell culture

[0376] Immortalized human DM1 myoblasts with an expanded repeat of (CTG)13/2600 were derived from primary myoblasts from a DM1 patient (kindly provided by Dr. D. Furling and Dr. V. Mouly). Myoblasts were grown in proliferation medium consisting of a 1 :1 mix of Skeletal Muscle Cell Growth Medium (PromoCell, Heidelberg, Germany) with 1x GlutaMAX (Gibco; Thermo Fisher Scientific, Landsmeer, the Netherlands) and Ham’s F-10 Nutrient Mix with GlutaMAX (Gibco), supplemented with 20% (v/v) HyClone Bovine Growth Serum Supplemented Calf (GE Healthcare, South Logan, UT). All tissue culture vessels were coated with 0.1 % gelatin (Sigma G2500) in Milli-Q for at least 30 min prior to cell seeding. Cells were incubated at 37°C in a humidified incubator with 7.5% CO2.

Example 28: Nanoparticle formation, transfection and AOC incubation

[0377] Nanoparticles comprising of PepFect14 with DMPK Gapmer or Control Gapmer were formed at a charge (N/P) ratio of 3. This corresponds to a molar ratio of CPP:ASO of 9:1 . Peptide and ASOs were diluted to 20x the final concentration, after which they were mixed by simultaneous pipetting against the wall of a PCR tube with the pipette tips in close contact. Nanoparticles were then allowed to stabilize at room temperature for approximately 1 h to afford PF14-DMPK Gapmer and PF14- Control Gapmer nanoparticles respectively. The following constructs: PF14-DMPK Gapmer, PF14- Control Gapmer, OKT9-(Repeat Blocker^, OKT9-(Control Blocker^ and palivizumab-(Repeat Blocker)2 were pre-diluted to 1x the final concentration in proliferation medium for incubation with cells. Medium on cells was replaced with proliferation medium containing nanoparticles or AOCs. The final concentration of ASOs and AOCs (based on the amount of antibody) was 200 nM. After 72 h of incubation, cells were washed once with PBS, followed by RNA isolation or immediate fixation for microscopy.

Example 29: Determination of M BN L1 splice correction through RNA isolation and analysis of RT-PCR products

[0378] Cells were seeded at a density of 20,000 cells per well in 12-well plates (containing 10 mm coverslips) one day prior to the start of the experiment. At 72 h after the start of incubation with nanoparticles or AOCs, RNA was isolated from 12-well plates using the Aurum total RNA mini kit (BioRad, Veenendaal, the Netherlands) according to the manufacturer’s instructions, including DNase treatment. As an additional step to ensure shearing of genomic DNA, lysates were pulled through a 0.5 mm syringe 15x prior to the addition of ethanol. In general, 200 ng RNA (or the maximum volume of 15 pL in case of low RNA yield) was used for cDNA synthesis using the iScript cDNA synthesis kit (BioRad), according to the manufacturer’s instructions.

[0379] To assess alternative splicing, PCRs were performed using Q5 High-Fidelity DNA Polymerase (New England BioLabs, Leiden, the Netherlands). The primer set is listed in Table 1. An annealing temperature of 71 °C was used for MBNL1 . PCR mixes consisted of 1x Q5 reaction buffer, 0.2 mM dNTPs (Invitrogen), 0.5 pM of forward and reverse primer each, 0.4 U Q5 High-Fidelity DNA polymerase and 2 pL 5x diluted cDNA in a total volume of 20 pL. NTC and NRTs were again included in each PCR run to detect possible contaminations. The following program was run on the T100 Thermal Cycler (BioRad): 30 s 98 °C, 30x (10 s 98 °C, 30 s 71 °C, 30 s 72 °C), 2 min 72 °C and « 4 °C. PCR products were analyzed on the QIAxcel Advanced capillary electrophoresis system (QIAGEN) using the DNA High Resolution Kit, along with the 15-600 bp alignment marker and 25-500 bp size marker. The amplicon of interest (MBNL1 exon 5 inclusion) was normalized to all splice variant amplicons in each sample. To determine the relative abundance of the different splice isoforms in each PCR sample, the peak calling function of the QIAxcel ScreenGel software (QIAGEN) was used. The molarity of the DM1 -dominant isoform (MBNL1 exon 5 inclusion) was divided by the sum of the molarities of all isoforms and expressed as a percentage (see Figure 19).

Example 30: Quantification of nuclear foci through confocal microscopy

[0380] Cells were seeded on a 10 mm coverslip in 12-well plates at a density of 20,000 cells per well one day prior to the start of the experiment. At 72 h after start of incubation with nanoparticles or AOCs, cells on coverslips in 12-well plates were fixed using 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Cells were washed three times with ice-cold ethanol and stored at 4 °C under ethanol until further handling. For RNA FISH, the protocol as specified by Stellaris (LGC Biosearch Technologies, Petaluma, CA) was followed. In brief, cells were washed using Stellaris Wash Buffer A and incubated overnight at 37 °C with 10 ng/mL TYE-563 labeled (CAG)6C LNA probe and 125 nM of the Quasar-670 labeled DMPK probe set consisting of 48 different 18- to 20-nucleotide DNA probes spaced along the transcript (Stellaris). Both probes were diluted in hybridization buffer. Cells were then washed and nuclei were counterstained using 1 mg/mL DAPI in wash buffer. Images were acquired using a Zeiss LSM880 Laser Scanning Confocal Microscope (Zeiss, Oberkochen, Germany), using a 63x 1.4 NA oil immersion objective. Frame sequential z stacks were obtained in which fluorescence was excited at 405 nm (DAPI), 514 nm (TYE-563), and 633 nm (Quasar-670) and emission light collected between 410 and 585 nm (DAPI), 538-680 (TYE-563), and 638-754 nm (Quasar-670).

[0381] Microscopy images were processed using FIJI software. In brief, slices from z stacks were combined in maximum intensity projections. Nuclear masks were then created and nuclear foci were counted using either the ‘3D Objects Counter’ plugin (with a size filter of 15-27581040 voxels) or the ‘Find Maxima’ function (see Figure 20).

Example 31-34: Dose finding study of AOC in vivo in the mdx mouse model for Duchenne muscular dystrophy

Example 31: Dose finding study of AOC in the mdx mouse model for Duchenne muscular dystrophy [0382] At the age of 5 weeks cohorts of two male and two female mdx mice received one i.v. injection of 100 pL containing either vehicle (PBS) or 60 mg/kg chR17 or 30 mg/kg chR17-(PMO)4 or 10 mg/kg chR17-(PMO)₄ or 3 mg/kg chR17-(PMO)₄ or 60 mg/kg chR17-(PMO)₂ or 20 mg/kg chR17-(PMO)₂ or 6 mg/kg chR17-(PMO)₂) via intravenous tail injection. Four weeks after the injection (day 28) the mice were sacrificed by cervical dislocation. Tissues were extracted from the mice to check for exon skipping levels and to measure dystrophin protein levels. The tissues extracted were: gastrocnemius, diaphragm and heart. Muscles were snap frozen into liquid nitrogen.

Example 32: RNA isolation from muscles using TRIsure

[0383] RNA isolation was performed based on the protocol described in Example 24.

Example 33: RT-PCR analysis of dystrophin formouse muscles

[0384] Exon-skipping levels were determined based on the protocol described in Example 25. The results are depicted in Figure 21 .

Example 34: Western blot protocol for dystrophin in tissue

[0385] Quantification of dystrophin protein expression was performed according to the protocol described in Example 26. The results are depicted in Figure 22.

Examples 35-36: Synthesis and conjugation of maleimide linker-oliqonucleotide

Example 35: Preparation of compound 12

[0386] To a solution of compound 5 (94.6 mg, 1 .0 Eq, 249 pmol) in DMF (5.00 mL), was added SMCC (100 mg, 1.2 Eq, 299 pmol) followed by triethylamine (75.7 mg, 104 pL, 3 Eq, 748 pmol). After stirring for 1 hours, DCM was added to the reaction mixture. The resulting precipitate was collected by filtration to afford benzyl alcohol intermediate (143 mg, 239 pmol, 95.8%) as a white solid. LCMS (ESI-) calculated for CsoF iNeO?- (M-H)“ 597.3, found 597.2.

[0387] Benzyl alcohol intermediate (142 mg, 1 Eq, 237 pmol) was dissolved in anhydrous DMF (4.00 mL). To this solution (PFP)2O (234 mg, 2.5 Eq, 593 pmol) was added followed after one minute of stirring by triethylamine (72.0 mg, 99.2 pL, 3 Eq, 712 pmol). After stirring the resulting yellow solution for 5 hours, additional (PFP)2<D (234 mg, 2.5 Eq, 593 pmol) was added to the reaction mixture. After 42 hours, the reaction mixture was diluted with DCM (10 ml) and was purified by flash column chromatography over silicagel (0 —>■ 50% MeOH/DCM) to afford compound 11 (68 mg, 66 pmol, 28%, 78% purity) as a colorless solid. LCMS (ESI+) calculated for Cs/F^FsNeOg* (M+H)⁺ 809.3, found 809.6. [0388] To a stock solution of PMO in 90% DMF and 10% H2O (1.74 mg, 10.0 pL, 20 mmolar, 1 Eq, .200 pmol) was added triethylamine (304 pg, 418 nL, 15 Eq, 3.00 pmol), followed by a stock solution of compound 11 (324 pg, 4.00 pL, 100 mmolar, 2 Eq, 0.400 pmol) in DMF. MQ (1 pL) was added to facilitate solubilization of PMO. The reaction mixture was left to stand at room temperature for 18 hours after which it was purified by prep-HPLC; (5% 95%, MeCN/Water + 10 mM NH4HCO3, runtime 12 minutes, Column Xbridge prep C18 5 pm OBD, 30x100 mm). The product containing fractions were combined, analyzed by UV-vis (e = 259210 M“¹cm^-1) and lyophilized to afford compound 12 (MCC- PMO) (802 pg, 0.0858 pmol, 43%, 87% purity) as a white solid. LCMS (ESI+) calculated for (M+8H)⁺⁸ (9345+8)/8 = 1169.1 found 1169.4. Example 36: Reduction and Conjugation of native chR17 with compound 12 (MCC-PMO) to obtain conjugate chR17-(MCC-PMO)4 with DAR4

[0389] Native chR17 (3141 pL, 50 mg, 15.92 mg/mL in PBS+10 mM EDTA pH 7.4) was added to a solution of PBS +10 mM EDTA (1786 pL) and TCEP (73.3 pL, 10 mM, 2.2 equiv.). The mixture was incubated for 90 minutes at 37°C. Subsequently, the conjugation was performed by adding compound 12 (MCC-PMO) (276 pL, 4.8 mM solution in 88% DMF) and incubating it for 90 minutes at 37°C. Subsequently purification was performed on a Superdex200 Increase 16/600 GL (Cytiva) on a 100F NGC system (Bio-Rad). RP-UPLC analysis of the reduced conjugate showed an average DAR of 3.59. ). Mass spectral analysis of the reduced sample showed the corresponding masses for conjugated LC and HC fragments (observed masses for LC-1 , HC-1 , HC-2 and HC-3, respectively; 35057 Da, 59401 Da, 68746 Da, 78092 Da.

Example 37: Stability study of AOCs at physiological conditions

[0390] Samples were diluted to 1 mg/mL in PBS pH 7.4 and stored at 37°C for 21 days. At designated time points (t = 0, 1 , 3, 7, 14 and 21 days) a sample was taken and analyzed using SE-HPLC and RP- UPLC. Relative DAR compared to t = 0 is measured by RP-UPLC (DTT reduced) and listed in table 2. Monomer levels determined by SE-HPLC measurement are shown in table 3.

Table 2. DAR level over time (days) in PBS (% from DAR at t=0).

Table 3. Monomer level (%) over time (days) in PBS.

38 - 41 : Functional assessment of AOC in vivo in the mdx mouse model for Duchenne Example 38: In vivo functional assessment study of AOC in the mdx mouse model for Duchenne muscular dystrophy

[0391] At the age of 5 weeks cohorts of 20 male mdx mice received one i.v. injection of 150 pL containing either vehicle (PBS, A) or 30 mg/kg chR17-(PMO)4 (B) or 10 mg/kg chR17-(PMO)4 (C) or 10 mg/kg chR17-(MCC-PMO)4 (D) via intravenous tail injection. Additionally, at the age of 5 weeks a control group of WT mice received one i.v. injection of 150 pL of vehicle (PBS, A) via intravenous tail injection. Every second week from the injection time, the animals performed functional tests: wire- and four-limb hanging tests and force grip strength test (8 complete sets in total, e.g. week 2,4,6,8,10,12,14,16). Additionally, body weight was recorded every second week (See Figure 23).

[0392] At the week 2,4,8,12,17 four animals from every group (A-D) mice will be sacrificed by isoflurane overdose followed by cervical dislocation. Tissues will then be extracted from the mice to check for exon skipping levels and measure dystrophin protein levels. Tissues to be extracted are: gastrocnemius, heart and diaphragm.

Example 39: Functional assessment by two limb hanging test

[0393] The mice from every group (A-D) were suspended above a metal wire located 42 cm above a cage with soft beddings. After the mouse grasped the wire with its forelimbs, mice were allowed to pull up and use hindlimbs and tail; once the wire was released by the mouse, the hanging time was recorded. The test was completed after a hanging time of 600 s is achieved or after three sessions. The maximum hanging time achieved was used for analysis (See Figure 24 and Table 4).

Table 4. Mean (seconds) and Standard error of mean (SEM, seconds) of the maximum hanging time achieved in a two limb hanging test by a certain number of mice (N) in each cohort over time. Example 40: Functional assessment by four limbs hanging test

[0394] The mice from every group (A-D) were placed on a grid (38 x 38 cm), which was turned upside down, 82 cm above a cage filled with soft bedding. The hanging test was completed after a hanging time of 600 s is achieved or after three sessions. The maximum hanging time achieved was used for analysis. (See Figure 25 and Table 5).

Table 5. Mean (seconds) and Standard error of mean (SEM, seconds) of the maximum hanging time achieved in a four limbs hanging test by a certain number of mice (N) in each cohort over time.

Example 41: Functional assessment by force grip strength test

[0395] The grip strength of the fore limbs of the mice from every group (A-D) was assessed using a triangle attached to an isometric force transducer (Columbus Instruments, Columbus, USA). The force transducer records the maximum force that is required to break the mouse’s grip from the mesh surface.

In total five strength measurements were recorded. The highest value was normalized to body weight (See Figure 26 and Table 6). Table 6. Mean (g force/g bodyweight) and Standard error of mean (SEM, g force/g bodyweight) of the maximum force achieved in a wire hanging test by a certain number of mice (N) in each cohort over time.

Sequences:

Sequence identification ofchR17 light chain (SEQ ID NO: 7):

DVQMTQSPYNLAASPGESISISCKASKSISKYLAWYQQKPGKANKLLIYDGSTLQSGIPSRFSGSGSG

TNFTLTIRSLEPEDFGLYYCQQHDESPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN

NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

Sequence identification ofchR17 heavy chain (SEQ ID NO: 8):

EVQLVESGGGLVQSGRSLKLSCVASGFTFSSYGMNWIRQAPGKGLEWVAYISSAGKYIYYAETMKG

RFTISRDNARNTLYLQMTSLRSENTALYYCARLGTGSEGHYWYFDFWGPGTMVTVSSASTKGPSVF

PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC

VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK

ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

Claims

1 . An antibody-oligonucleotide conjugate having structure (1):

Ab-[ (Z)_yi - L^D - (D)x]z

(I) wherein:

- Ab is an antibody;

- Z is a connecting group obtainable by reaction between two click probes;

- x is 1 , 2, 3 or 4;

- y1 is 1 or 2;

- z is 2 or 4;

- L^D is an heterobifunctional (x + y1)-valent linker; and

- D is an oligonucleotide.

2. The antibody-oligonucleotide conjugate according to claim 1 , wherein conjugation occurs through the glycan of the antibody and the antibody-oligonucleotide conjugate has structure (3):

Ab-[ (L⁶ - Z)_yi - L^D- (D)x]z

(3) wherein L⁶ is -GlcNAc(Fuc)v^(G)j-S-(L⁷)w-, wherein:

- GIcNAc is N-acetylglucosamine;

- Fuc is fucose;

- w is 0 or 1 ;

- G is a monosaccharide;

- j is an integer in the range of 0 - 10;

- S is a sugar or a sugar derivative;

- w’ is 0 or 1 ; and

- L⁷ is -N(H)C(O)CH₂- -N(H)C(O)CF₂- or -CH₂-.

3. The antibody-oligonucleotide conjugate according to claim 1 or 2, having structure (11):

Ab-[ (Z¹)y1 - L^A ( Z² - L^B - D )y₂ ]z

(I I) wherein:

- Z¹ and Z² are connecting groups obtainable by reaction between two click probes;

- y2 is 1 , 2, 3 or 4;

- L^A is an heterobifunctional (y1 + y2)-valent linker; and

- L^B is bivalent linker.

4. The antibody-oligonucleotide conjugate according to claim 1 or 2, having structure (12):

Ab-[(L⁶)-(Z)-(L¹)-(L²)₀-(L³)_P-(L⁴)_q-D ]_z

(12) wherein:

L¹, L², L³ and L⁴ are each individually linkers; o, p and q are each individually 0 or 1 ; and L⁶ is as defined in claim 2.

5. The antibody-oligonucleotide conjugate according to claim 3, having a structure selected from (33)

- (37):

(36) (37).

6. The antibody-oligonucleotide conjugate according to any one of the preceding claims, wherein Z, preferably Z² as defined in claim 3 or 4, is obtainable by ultrafast click reaction, wherein ultrafast click reaction is defined as having a reaction rate that is at least 10 times greater than the rate of the click reaction between azide and bicyclononyne.

7. The antibody-oligonucleotide conjugate according to any one of the preceding claims, wherein linker L^B has structure

-(L¹)-[(L²)o-(L³)p-(L⁴)_q- and wherein:

- L¹ is connected to Q² or Z², and L⁴ is connected to D;

- L¹, L², L³ and L⁴ are each individually linkers that together link Q² or Z² to D;

- o, p and q are each individually 0 or 1 , preferably wherein o = p = 1 ; and preferably wherein:

(a) linker L¹ is represented by:

-(W A)d-(B)e-(A)f-(C(O))_s- wherein:

- d = 0 or 1 ;

- e = an integer in the range 0 - 10;

- f = 0 or 1 ;

- wherein d + e + f is at least 1 ;

- g = 0 or 1 ;

- k = 0 or 1 ;

- A is a sulfamide group according to structure (23);

- wherein a = 0 or 1 , and R¹³ is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, Cs - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR¹⁴ wherein R¹⁴ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R¹³ is D connected to N via a spacer moiety;

- B is a -CH2-CH2-O- or a -O-CH2-CH2- moiety, or (B)_e is a -(CH2-CH2-O)_ei-CH2-CH2- or a -(CH₂-CH2-O)_ei-CH₂- moiety, wherein e1 is defined the same way as e;

- W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O- -C(O)(CH₂)mC(O)-, -C(O)(CH₂)mC(O)NH- or -(4-Ph)CH₂NHC(O)(CH₂)mC(O)NH-, wherein m is an integer in the range 0 - 10; and/or

(b) linker L² is a peptide spacer, preferably comprising 1 - 5 amino acids, more preferably a dipeptide, tripeptide or tetrapeptide spacer; and/or

(c) linker L³ is a self-immolative spacer, preferably:

- a para-aminobenzyloxycarbonyl (PABC) derivative according to structure (26):

- a glucuronide derivative according to structure (27): wherein

- R²¹ is H, R²⁶ or C(O) R²⁶, wherein R²⁶ is Ci - C24 (hetero)alkyl groups, C3 - C10 (hetero)cycloalkyl groups, C2 - C10 (hetero)aryl groups, C3 - C10 alkyl(hetero)aryl groups and C3 - C10 (hetero)arylalkyl groups, which are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR²⁸ wherein R²⁸ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably wherein R²¹ is H or C(O)R²⁶, wherein R²⁶ = 4-N-methyl-piperazine or morpholine, most preferably wherein R²¹ is H;

- ring A is a 5- or 6-membered aromatic or heteroaromatic ring; and/or

(d) linker L⁴ is a spacer selected from:

- an aminoalkanoic acid spacer according to the structure - NR²²-(Cx-alkylene)-C(O)-, wherein x is an integer in the range 1 - 20 and R²² is H or Ci - C4 alkyl; or - an ethyleneglycol spacer according to the structure -NR²²-(CH2-CH2-O)e6-(CH2)e7-C(O)-, wherein e6 is an integer in the range 1 - 10, el is an integer in the range 1 - 3 and R²² is H or Ci - C4 alkyl; or

- a diamine spacer according to the structure -NR²²-(Cx-alkylene)-NR²²-(C(O))h-, wherein h is 0 or 1 , x is an integer in the range 1 - 10 and R²² is H or Ci - C4 alkyl.

8. The antibody-oligonucleotide conjugate according to any one of the preceding claims, wherein linker L^A has structure

(L^{1 1})y1-BM-(L¹²)y2 wherein:

- each L¹¹ is connected to Q¹ or Z¹, and each L¹² is connected to F² or Z², wherein L¹¹ and L¹² each individually consist of one or more building blocks selected from C(R¹³)2, C(R¹³)=C(R¹³), C=C, aryl, C(O), NR¹³, O, S, S(O), S(O)₂, PR¹³ and P(O)R¹³,

- each R¹³ is as defined below for structure (23);

- BM is a branching moiety, which is present if y1 + y2 = 3 or higher.

9. The antibody-oligonucleotide conjugate according to any one of the preceding claims, wherein the oligonucleotide D is selected from RNA, DNA, 2’-OMe-PS (2’-O-methylphosphorothioate), PMO (morpholino phosphorodiamidate), 2’-OMOE-PS (2’-O-methoxyethylphosphorothioate), PNA (peptide nucleic acid), tcDNA (tricyclic DNA), LNA (locked nucleic acid), HNA (1 ,5-anhydrohexitol nucleic acid), CeNA (cyclohexene nucleic acid), LceNA (locked cyclohexene nucleic acid),TNA (threose nucleic acid), GNA (glycol nucleic acid), FANA (fluoroarabino nucleic acid), 2’MOE (2’-O- methoxyethyl), S-cEt (2'-O-ethyl), and combinations thereof.

10. The antibody-oligonucleotide conjugate as defined in any one of claims 1 - 9, for use in treatment.

11. The antibody-oligonucleotide conjugate as defined in any one of claims 1 - 9, for use in the treatment of a hereditary neuromuscular disorder, preferably wherein the hereditary neuromuscular disorder is selected from Duchenne, myotonic dystrophy, spinal muscular atrophy, homozygous familial hypercholesterolemia and primary hyperoxaluria type 1 .

12. A process for preparing an antibody-oligonucleotide conjugate according to any one of claims 1 - 9, comprising:

(a) providing a modified antibody having the structure Ab(F¹)_Z2, wherein Ab is an antibody, z2 is 2 or 4, and F¹ is a click probe;

(b) reacting the modified antibody with z2/y1 equivalents of (Q¹)yi - L^A- (F²)_y2, wherein Q¹ is a click probe that is reactive towards F¹, L^A is a heterobifunctional (y1 + y2)-valent linker, y1 is 1 or 2, y2 is 1 , 2, 3 or 4, and F² is a click probe that is not reactive towards Q¹, to obtain an antibody-linker construct of structure Ab[(Z¹)_yi - L^A - (F²)_y2 ]z, wherein z is z2 / y1 , and Z¹ is a connecting group obtained by reaction of F¹ and Q¹;

(c) reacting the antibody-linker construct with z x y2 equivalents of Q² - L^B - D, wherein Q² is a click probe that is reactive towards F², L^B is a bivalent linker and D is an oligonucleotide, to obtain the conjugate of structure Ab [ (Z¹)_yi - L^A - (Z² - L^B - D)_y2 ]z, wherein Z² is a connecting group obtained by reaction of F² and Q²; (a) providing a modified antibody having the structure Ab[ (L⁶) - (F) ]_z, wherein:

- Ab is an antibody;

- L⁶ is -GlcNAc(Fuc)w-(G)j-S-(L⁷)_w-, wherein G is a monosaccharide, j is an integer in the range of 0 - 10, S is a sugar or a sugar derivative, GIcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1 , w’ is 0 or 1 and L⁷ is -N(H)C(O)CH2-, -N(H)C(O)CF2- or

-CH₂-;

- z is 2 or 4;

- F is a click probe,

(d) reacting the modified antibody with z equivalents of (Q)-(L¹)-(L²)₀-(L³)_P-(L⁴)_q-D, wherein: - Q is a click probe that is reactive towards F;

- L¹, L², L³ and L⁴ are each individually linkers;

- o, p and q are each individually 0 or 1 ; to obtain the conjugate of structure Ab-[(L⁶)-(Z)-(L¹)-(L²)₀-(L³)_P-(L⁴)_q-D ]_z.

13. The process according to claim 12, wherein Q, Q¹ and Q² are individually click probes comprising a (hetero)cycloalkyne moiety or a (hetero)cyclo-E-alkene moiety, preferably wherein the click probes are selected from the group consisting of (Q2) - (Q20c) according to the following structures:

(Q11) (Q12) (Q13) (Q14) (Q15)

(Q20) (Q20a) (Q20b) (Q20c) wherein:

- the wavy bond represents the connection to L¹ ,L^A or L^B, and is connected to any available carbon or nitrogen atom;

- the nitrogen atom of (Q10), (Q13), (Q14) and (Q15) may bear the connection to L^A or L^B, or contains a hydrogen atom or is substituted;

- B^(_) is an anion;

- B⁽⁺⁾ is a cation;

- R³⁵ is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, Si, S and NR¹⁴ wherein R¹⁴ is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups;

- R³⁶ is an halogen selected from fluor, chlorine, bromine and iodine;

- Y⁴ is a heteroatom; and/or

- F, F¹ and F² are individually click probes selected from the group consisting of azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, dioxothiophene, sydnone, iminosydnone, catechol and tetrazole, preferably wherein the click probes are selected from the group consisting of (F1) - (F11) according to the following structures: wherein:

- the wavy bond represents the connection to Ab or L^A, where for (F3), (F4), (F8), (F9) and (F11), the connection can be via any one of the wavy bonds, and the other wavy bond may then be connected to a group selected from hydrogen, Ci - C24 alkyl groups, C2 - C24 acyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups, C3 - C24 (hetero)arylalkyl groups and Ci - C24 sulfonyl groups, which may optionally be substituted and optionally be interrupted by one or more heteroatoms selected from O, S and NR³², wherein R³² is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups; - the R group connected to the nitrogen atom of (F7) is selected from alkyl and aryl.

14. The process according to claim 13, wherein Q² and F² are reactive in an ultrafast click reaction, wherein ultrafast click reaction is defined as having a reaction rate that is at least 10 times greater than the rate of the click reaction between azide and bicyclononyne.

15. Use of ultrafast click chemistry for conjugating an oligonucleotide to an antibody, wherein ultrafast click chemistry is defined as having a reaction rate that is at least 10 times greater than the rate of the click reaction between azide and bicyclononyne.