Extracellular vesicles comprising biomolecule-conjugates

Abstract

The present invention is in the field of medicine. More specifically, the present invention relates to extracellular vesicles, namely lipid nanoparticles that are conjugated to a biomolecule, namely an antibody or a nanobody, using a click reaction. Such extracellular vesicles can be applied for more effective treatment of diseases, in particular in vivo CAR T therapy, cardiac fibrosis and lysosomal storage diseases (LSD).

Description

Extracellular vesicles comprising biomolecule-conjugatesField of the invention

[0001] The present invention is in the field of medicine. More specifically, the present invention relates to extracellular vesicles such as lipid nanoparticles that are conjugated to a biomolecule using a click reaction. Such extracellular vesicles can be applied for more effective treatment of diseases, in particular in vivo CAR T therapy, cardiac fibrosis and lysosomal storage diseases (LSD).Background of the invention

[0002] Lipid nano particles (LNP) are vessels to deliver therapeutic agents into a cell. While they had been known much longer, LNPs reached center stage during the COVID pandemic, when Pfizer and Moderna successfully developed a nucleoside-modified mRNA lipid nanoparticle vaccine against COVID-19. Other medicines using LNPs as delivery system are also known such as Doxil. Doxil is a PEGylated nanoliposome comprising the cytotoxic agent doxorubicin and is used to treat cancer.

[0003] A problem associated with LNPs is that they accumulate in the liver after intravenous administration which can give rise to toxic side effects. Thus, LNPs are in general not effective at treating a specific organ besides the liver.

[0004] Targeted delivery of drugs is known. 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 halflives, 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 for the 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.

[0005] 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 of 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).

[0006] 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. 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.

[0007] 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).

[0008] 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).

[0009] Various reagents are known for alkylation of the thiol group in cysteine side-chain. 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).

[0010] 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).

[0011] 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 crosslinking 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.

[0012] 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 bioactivatableconnection 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.

[0013] 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

[0014] 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 linker-drugs 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 o / Yho-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.

[0015] 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.

[0016] A method of increasing popularity in the field of ADCs is based on enzymatic installation of a nonnatural 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.

[0017] 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. 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.

[0018] Although most of the ADCs have cytotoxic payloads, other payloads are also known, such as for example immunomodulatory payloads (STING, TLR agonists) and protein degraders (PROTACs). In particular, ADCs having an oligonucleotide payload are known in the art and are referred to as antibody- oligonucleotide conjugates (AOC).

[0019] 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.

[0020] Conjugating an oligonucleotide to an antibody has many benefits, such as targeted delivery, reduced clearance. AOCs are known in the prior art, for example WO2022 / 212886 describes molecules and pharmaceutical compositions and associated methods for treating a disease by inducing an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion.

[0021] W02020028831 A1 relates 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.

[0022] However, conjugating an oligonucleotide directly to an antibody only allows delivery of short strands of genetic material, but not for a longer strands of genetic material encoding for an entire protein. Therefore, there is a need to use an extracellular vesicle as a vehicle for delivering payloads.

[0023] Targeted gene delivery of extracellular vesicles has been described in the prior art, see for example Lee et al. Strategies for targeted gene delivery using lipid nanoparticles and cell-derived nanovesicles,2023, 5(15), 3834-3856. However, there remains a need of targeting extracellular vesicles with improved characteristics.Summary of the invention

[0024] A first aspect of the invention relates to an extracellular vesicle comprising a cavity encapsulated by a lipid membrane and at least one conjugate having structure B-L-V, wherein:• B is a biomolecule;• L represents a linker comprising a connecting group Z obtainable by a strain promoted click reaction;• V represents a hydrophobic moiety that is embedded in the membrane.

[0025] A second aspect of the invention relates to a process for producing the extracellular vesicle according to the invention, said process comprising:(l a) providing a biomolecule having structure B [ LB(F)x ]z, wherein F is a click probe, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(l b) reacting B(LB(F)x)z with a linker-payload construct Q-LA-V, wherein Q is a click probe that is reactive towards F, LBis a linker and V is a hydrophobic moiety, to obtain a conjugate of structure B [LB(Z-LA-V)x ]z;(l c) providing an extracellular vesicle; and(1 d) incorporating B [LB(Z-LA-V)X]z into the membrane of the extracellular vesicle, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane; or(2c) providing an extracellular vesicle;(2d) incorporating linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, into the membrane of the extracellular vesicle to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(2a) providing a biomolecule having structure B [ LB(F)X]z, wherein F is a click probe that is reactive towards Q, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(2b) reacting B[LB(F)x]z with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle; or(3c) assembling an extracellular vesicle in the presence of linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(3a) providing a biomolecule having structure B [ LB(F)X]z, wherein F is a click probe that is reactive towards Q, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(3b) reacting B[LB(F)x]z with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle.

[0026] A third aspect of the invention relates to a pharmaceutical composition comprising the extracellular vesicle according to the invention.

[0027] A fourth aspect of the invention relates to medical treatment, in particular, treatment of cancer or a genetic disorder, in particular in vivo CAR therapy, cardiac fibrosis and lysosomal storage diseases (LSD).List of embodiments

[0028] The present invention may be defined according to the following list of embodiments:1. An extracellular vesicle comprising a cavity encapsulated by a lipid membrane and at least one conjugate having structure B-L-V, wherein:• B is a biomolecule;• L represents a linker comprising a connecting group Z obtainable by a strain promoted click reaction;• V represents a hydrophobic moiety that is embedded in the membrane.2. An extracellular vesicle comprising a cavity encapsulated by a lipid membrane and at least one conjugate having structure B-L-V. wherein:• B is a biomolecule;• L represents a linker comprising a connecting group Z selected from 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;• V represents a hydrophobic moiety that is embedded in the membrane.3. The extracellular vesicle according to embodiment 1 or 2, wherein the extracellular vesicle is selected from an exosome, a nanovesicle, an apoptotic body, a microvesicle, a lysosome, an endosome, an enveloped virus, a liposome, a lipid nanoparticle, a micelle, a multilamellar structure, a revesiculated vesicle, or an extruded cell, preferably wherein the extracellular vesicle is a lipid nanoparticle selected from a solid lipid nanoparticle (SLN), liquid lipid nanoparticle, nanostructured lipid carrier (NLC), hybrid- lipid polymeric nanoparticle, more preferably the extracellular vesicle is a liquid lipid nanoparticle.4. The extracellular vesicle according to any one of the preceding embodiments, wherein the extracellular vesicle has a diameter in the range of 10-1000 nm, preferably in the range of 20-250 nm, more preferably in the range of 50-150 nm.5. The extracellular vesicle according to any one of the preceding embodiments, wherein the cavity comprises an active compound and / or a stabilizing lipid, preferably wherein• the active compound is a cytotoxin or a nucleic acid, preferably a nucleic acid selected from plasmid, 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 (dsRNA), single stranded DNA(ssDNA), double stranded DNA, plasmids, ribozymes, aptamer, Gapmer, and triplex forming oligonucleotides, and• the stabilizing lipid is selected from cholesterol, cationic lipids, ionizable lipid, squaramide and combinations thereof, preferably the stabilizing lipid is a mixture of ionizable lipids and cholesterol.6. The extracellular vesicle according to any one of the preceding embodiments, wherein the membrane comprises stealthing lipids selected from PEGylated lipids, XTEN peptide lipids, PAS peptide lipids and polysarcosine lipids, preferably PEGylated lipids comprising PEG chains having an average length (AVG) of 5-200 repeating monomeric PEG units, preferably 10-120 repeating monomeric PEG units, more preferably 20-80 repeating monomeric PEG units, most preferably 40-50 monomeric PEG units, and wherein at least 80 % of the PEGylated chains have a length in the range of AVG -2 to AVG +2, more preferably 99.5-100 % of the PEGylated chains have a length of AVG.7. The extracellular vesicle according to any one of the preceding embodiments, wherein V is a lipid, preferably selected from phospholipid, sphingolipid or glycerolipid, more preferably wherein V is according to (V17):wherein the wavy line indicates the connection to L, and each HT is a hydrophobic tail individually selected from a saturated or unsaturated hydrocarbon chain from 5 to 29 carbon atoms long, preferably 11 to 23 carbon atoms long, more preferably 15 to 19 carbon atoms long, most preferably 17 carbon atoms long.8. The extracellular vesicle according to any one of the preceding embodiments wherein L has structure LA-Z-LB, whereinLAis a linker connecting V to Z;LBis a linker connecting B to Z; and and Z has structure (Z1):wherein the bond depicted as - is a single bond or a double bond;R15is a substituent independently selected from the group consisting of hydrogen, halogen,-OR16, -NO2, -CN, -S(O)216, -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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- Y2is C(R31)2, O, S, S<+)R31, S(O)R31, S(O)=NR31or NR31, wherein S<+> is a cationic sulphur atom counterbalanced by B< >, wherein B< > is an anion, and wherein each R31individually is R15or a connection with V, 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 is an integer in the range 8 - 16;- ring Z is the moiety obtainable by a strain promoted click reaction, and is selected from a triazole, a cyclohexene, a cyclohexadiene, a [2.2.2]-bicyclooctadiene, a [2.2.2]-bicyclooctene, an isoxazoline, an isoxazolidine, a pyrazole, a pyrazoline, a pyridine, a pyridazine, a phenyl, a diazacycloalkane or an oxazacycloalkane, which may optionally be substituted and comprises the connection to LB.9. The extracellular vesicle according to any one of the preceding embodiments, wherein biomolecule B is selected from a monoclonal antibody, polyclonal antibody, dimer, multimer, multispecific antibody (e.g. bispecific antibody), antibody fragment, and double and single chain antibodies, preferably B is a monoclonal antibody.10. The extracellular vesicle according to embodiment 8, wherein LAand / or LBcomprises a branching moiety, preferably wherein:• LBis (L11)(L12)BM(L13), wherein L11and L12are linkers connecting the same B to BM, and L13is a linker connecting BM to Z, and BM is a branching moiety; and / or. LAis -(W)c-(A)d-(B)e-(A)f-(W)g-(Str)h-(W)i- (A)j-(B)k-(A)i-(W)m-, wherein:- d = 0 or 1 , preferably d =0;- f = 0 or 1 , preferably f =0;- j = 0 or 1 , preferably j =0;- I = 0 or 1 , preferably I =0;- c = 0 or 1 , preferably c =1- g = 0 or 1 , preferably g =1 ;- i = 0 or 1 , preferably i = 1 ; m = 0 or 1 , preferably m =1 ; h = 0 or 1 , preferably h =1 ;- e = an integer in the range of 0-10, preferably, e = 0, 1 , 2, 3, 4, 5 or 6, most preferably e = 1 , 2, 3, or 4; k = an integer in the range of 0-10, preferably, k = 0, 1 , 2, 3, 4, 5 or 6, most preferably k = 1 , 2, 3, or 4;- A is sulfamide group according to structure (23):- B is -CH2-;- 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;- Str is a stretching moiety selected from a PEG chain or a peptide.11 . The extracellular vesicle according to any one of the preceding embodiments, wherein:• the extracellular vesicle is a lipid nanoparticle, wherein the cavity contains an active compound and / or a stabilizing lipid and the membrane comprises PEGylated lipids and / or polysarcosine lipids;• the biomolecule B is a trimmed antibody;• the connecting group Z comprises a 1 ,2,3-triazole;• the linker L is connected to the C-terminus or the glycan of the antibody, wherein: o the connection to the C-terminus is preferably obtainable by tubulin tyrosine ligase, peptide asparaginyl ligase, sortase or transglutaminase conjugation, more preferably sortase or transglutaminase; o the glycan has structure L6, wherein L6is -GlcNAc(Fuc)^ [ S-(L7)W]yi— , wherein y1 is 1 , 2, 3 or 4, S is a sugar or a sugar derivative, GIcNAc is N- acetylglucosamine and Fuc is fucose, w is 0 or 1 , w’ is 1 or 2, L7is a bond, -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-, and preferably y1 = 1.12. The extracellular vesicle according to any one of the preceding embodiments, wherein linker L is defined as LA-Z-LB, wherein• LAis a linker connecting V to Z;• LBis a linker connecting B to Z; and wherein Z-LBis according to *-Z2-LB2-(Z1-LB1-**)2, wherein• Z1and Z2are each a connecting group obtainable by a strain promoted click reaction;* indicates the connection to LA; ** indicates the connection to B LB1and LB2are linkers. The extracellular vesicle according to any one of the preceding embodiments, wherein L comprises a PEG chain of 5-200 monomeric PEG units and optionally a branching moiety connected to a second Z1or Z2moiety as defined in embodiment 12. A process of producing the extracellular vesicle according to any one of the preceding embodiments, comprising:(l a) providing a biomolecule having structure B [ LB(F)x ]z, wherein F is a click probe, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(l b) reacting B(LB(F)x)z with a linker-payload construct Q-LA-V, wherein Q is a click probe that is reactive towards F, LBis a linker and V is a hydrophobic moiety, to obtain a conjugate of structure B [LB(Z-LA-V)x ]z;(l c) providing an extracellular vesicle, wherein the cavity thereof optionally comprises an active compound and / or a stabilizing lipid; and(ld) incorporating B [LB(Z-LA-V)X]z into the membrane of the extracellular vesicle, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane; or(2c) providing an extracellular vesicle, wherein the cavity thereof optionally comprises an active compound and / or a stabilizing lipid;(2d) incorporating linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, into the membrane of the extracellular vesicle to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(2a) providing a biomolecule having structure B [ LB(F)X]z, wherein F is a click probe that is reactive towards Q, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(2b) reacting B[LB(F)x]z with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle; or(3c) assembling an extracellular vesicle in the presence of linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, and optionally an active compound and / or a stabilizing lipid, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane, and optionally the cavity thereof comprises said active compound and / or a stabilizing lipid;(3a) providing a biomolecule having structure B [ LB(F)X]z, wherein F is a click probe that is reactive towards Q, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(3b) reacting B[LB(F)x]z with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle. The process according to embodiment 13, wherein the biomolecule B [ LB(F)x ]z in step (a) is obtained by reacting B-[LB1-(F1)a]b with (Q1)yi-LB1(F2)y2, wherein a, b, y1 and y2 are integers in the range of 1 -4, wherein F1and Q1are click probes reactive to each other, F2and Q2are click probes reactive to each other but F2and Q1are not reactive to each other, and wherein LB1and LB2are linkers, and wherein in step (b) Q-LA-V is Q2-LA-V. The process according to embodiment 14 or 15, wherein the extracellular vesicle is a lipid nanoparticle and is provided in step 1 (c), 2(c) or 3(c) by mixing an aqueous buffer comprising an active compound and / or a stabilizing lipid, preferably a nucleic acid, with an ethanol solution comprising membrane lipids, optionally linker-payload construct Q-LA-V in the case of step (3c), and optionally stabilizing lipids. The process according to any one of embodiments 14 - 16, wherein click probe F is selected from the group consisting of azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, ortho- quinone, dioxothiophene and sydnone, more preferably F is according to any one of structures (F1)- (F10):wherein:- the wavy bond represents the connection to LB, where for (F3), (F4), (F8) and (F9), 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 R32is 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, most preferably F is according to structure (F1) or (F8), and / or wherein click probe Q comprises a (hetero)cycloalkyn moiety, preferably wherein click probe Q is selected from the group consisting of (Q21) - (Q38), (Q38a) - (Q38d) and (Q44) - (Q56):(Q49) (Q50) (Q51)(Q52) (Q53) (Q54) (Q55) (Q56) wherein- B<-> is an anion and B<+> is a cation;- R35is selected from the group consisting of hydrogen, C1-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(R14)2, S and NR14wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups;- R group(s) on Si in (Q50) and (Q51) are alkyl or aryl.18. A pharmaceutical composition comprising the extracellular vesicle according to any one of embodiments 1-13 and optionally a pharmaceutically acceptable carrier or excipient.19. The extracellular vesicle according to any one of embodiments 1-13 or the pharmaceutical composition according to embodiment 18 for use in medical treatment.20. The extracellular vesicle according to any one of embodiments 1-13 or the pharmaceutical composition according to embodiment 18 for use the treatment of cancer or a genetic disorder.21. The extracellular vesicle or pharmaceutical composition for use according to embodiment 18 or 19, wherein the treatment is an in vivo CAR therapy.Detailed descriptionDefinitions

[0029] 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.

[0030] 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”.

[0031] 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.

[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 or extracellular vesicle antibody 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 elementsselected 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 / -glycoprotein), 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 (prostatespecific 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 monosaccharideglycan 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, IgE, IgM, IgD, and IgA), class (e.g. lgG1 , 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 of the 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 a biomolecule is covalently connected to a payload via a linker. A conjugate comprises one or more biomolecules and / or one or more payloads. In the context of the present invention, the payload is hydrophobic moiety V, and the biomolecule is preferably an antibody.

[0052] 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 drugcandidates 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).

[0053] 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.

[0054] 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.

[0055] 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 hydrophilic moiety V. Hence, in the present application, the term DAR refers to the ratio between hydrophilic moiety V and the antibody, not to the ratio of the active compounds within the cavity and the amount of antibodies per encapsulated vesicle.

[0056] 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 linkerpayload constructs each containing one payload V, the resulting conjugate has a theoretical DAR of 4. In the preparation of conjugates, typically a distribution of conjugates with varying amount of pay load attached is formed, and the average DAR of the obtained product will deviate from the theoretical DAR.General structure of the extracellular vesicle

[0057] The invention concerns extracellular vesicles comprising a cavity encapsulated by a lipid membrane and at least one conjugate having structure B-L-V, wherein:B is a biomolecule;- V represents a hydrophobic moiety that is embedded in the membrane;L represents a linker comprising a connecting group Z obtainable by a strain promoted click reaction.

[0058] Extracellular vesicles are well known in the art. Extracellular vesicles refers to a cell-derived vesicle or a synthetic analogue comprising a membrane that encloses an internal space, i.e. cavity. The purposeof the extracellular vesicle is to carry an active ingredient into a cell. Extracellular vesicles are typically membrane-bound vesicles that have a smaller diameter than ordinary cells. Typically, extracellular vesicles have a diameter in the range of 10-1000 nm, preferably in the range of 20-250 nm, more preferably in the range of 50-150 nm.

[0059] The extracellular vesicles of the invention comprise a cavity encapsulated by a lipid membrane. The lipid membrane may be a monolayer, a bilayer or a trilayer. Preferably, the lipid membrane is selected from a monolayer or a bilayer. Lipid monolayers, bilayer and trilayers are well known in the art. The cavity contains the active compound. The active compound may be encapsulated by another lipid membrane within the extracellular vesicle. In a preferred embodiment, the extracellular vesicles comprise an outer lipid monolayer and multiple internal lipid monolayers encapsulating the active compounds.

[0060] The extracellular vesicles of the present invention are preferably selected from an exosome, a nanovesicle, an apoptotic body, a microvesicle, a lysosome, an endosome, an enveloped virus, a liposome, a lipid nanoparticle, a micelle, a multilamellar structure, a revesiculated vesicle, or an extruded cell, preferably wherein the extracellular vesicle is a lipid nanoparticle selected from a solid lipid nanoparticle (SLN), liquid lipid nanoparticle, nanostructured lipid carrier (NLC), hybrid-lipid polymeric nanoparticle, more preferably the extracellular vesicle is a liquid lipid nano particle.

[0061] The extracellular vesicle according to the invention comprises at least one B-L-V conjugate, preferably the extracellular vesicle comprises between 2 and 200 B-L-V conjugates, more preferably the extracellular vesicle comprises between 5 and 150 B-L-V conjugates, even more preferably the extracellular vesicle comprises between 20 and 120, most preferably the extracellular vesicle comprises between 40 and 100 B-L-V conjugates. As will be understood by the skilled person, the extracellular vesicle according to the invention is typically obtained as a mixture, wherein each individual vesicle may have a different number of B-L-V conjugates embedded in the membrane. The preferred ranges defined above thus apply to the average amount of B-L-V conjugates per extracellular vesicle. There may thus be extracellular vesicles present within the mixture with fewer or more B-L-V conjugates embedded in the membrane.

[0062] Preferably, the lipid fraction of the extracellular vesicle comprises:1-50 mol% of helper lipids, more preferably 5-15 mol%, more preferably 8-12 mol%;- 0-25 mol% of stealthing lipids, more preferably 0.5-5 mol%, more preferably 1-2 mol%;- 0- 60 mol% of sterol lipids, preferably 30-50 mol%, more preferably 35-45 mol%;- 0-80 mol% of ionizable lipids, preferably 35-70 mol%, more preferably 45-55 mol%;- 0.001-5 mol% of conjugate B-L-V, preferably 0.01-0.5 mol%, more preferably 0.05-0.2 mol% Herein it is preferred, the sum of helper lipids, stealthing lipids, sterol lipids, ionizable lipids and conjugate B-L-V comprises 50-100 mol% of the lipid fraction, preferably 70 -99.99 mol%, more preferably 80-99.5 mol%, most preferably 90-99 mol%.Lipids

[0063] The extracellular vesicle according to the invention comprises a lipid membrane, which acts as abarrier between the cavity (and its content) and the environment. The lipid membrane typically contains lipids having a hydrophilic head and a hydrophobic tail. As the extracellular vesicles are suitable to be used in vivo, i.e. an aqueous environment, the hydrophilic head of the outermost lipids are on the outside of the vesicle and the hydrophobic tail on the inside. In the embodiment in which the lipid membrane is a monolayer, the cavity typically contains a hydrophobic medium. In the embodiment in which the lipid membrane is a bilayer, the cavity typically contains a hydrophilic medium. In the embodiment, in which the outer lipid membrane is a monolayer but the cavity contains smaller vesicles with lipid monolayers, the content of the cavity outside the smaller vesicles is hydrophobic, but the content inside the smaller vesicles is hydrophilic. Such configurations of extracellular vesicles and the lipids needed to form these are well known in the art. The extracellular vesicle according to the invention is distinguished from conventional extracellular vesicles by the presence of conjugate B-L-V, wherein moiety V is embedded with the lipid membrane and biomolecule B typically protrudes out of the membrane of the extracellular vesicle. Hence, conjugate B-L-V also classifies as a lipid that forms the membrane of the extracellular vesicle. In other words, the main aspect of the invention can preferably be defined as: An extracellular vesicle comprising a cavity encapsulated by a lipid membrane, wherein the lipid membrane comprises at least one lipid having structure: B-L-V, wherein:B is a biomolecule;L represents a linker comprising a connecting group Z obtainable by a strain promoted click reaction;- V represents a hydrophobic moiety that is embedded in the membrane.

[0064] Typically, lipids have the following structure: V-HH, wherein V represents a hydrophobic moiety and HH an hydrophilic head. For the conjugate B-L-V, in a non-limiting example, HH can be defined as B-L.

[0065] More preferably, lipids can be depicted as (HT)I-2-X-(LX)O-I-HH, whereinHT is a hydrophobic tail- X is selected from (-C(0)0(CH2)O-I)2CH2OP(0)(OH)0-, (CO-0-(CH2)O-I)2-CH-NH-, NH, N HH is a hydrophilic head;Lxis an optional linker, preferably -CH2CH2NHC(O)-.

[0066] The lipid membrane may comprise several types of lipids, such as helper lipids, stealthing lipids, stabilizing lipids. These lipids preferably are all according to general structure: V-HH. Helper lipids typically include phospholipids, sphingolipids, glycolipids and fatty acids, Preferably, at least phospholipids are present. Stabilizing lipids typically include ionizable lipids, sterol lipids, cationic lipids, squaramides and combinations thereof. The lipid membrane preferably comprises a mixture of different lipids. Typically, the outer lipid membrane comprises a mixture of hydrophilic moiety V, phospholipids, stealthing lipids, stabilizing lipids

[0067] Helper lipids are responsible for the formation of the lipid layer and typically form the main bulk of the outer lipid membrane. The helper lipids are typically neutral lipids. In an especially preferred embodiment, the helper lipid is preferably a phospholipid. Phospholipids are the typical main constituent ofthe outer lipid membrane and are preferably depicted by structure (V1):

[0068] Herein, HH is preferably selected from hydroxyalkyl, ethyltrimethylammonium, ethylamine, o- serine, inositol, saccharide, oligosaccharide, more preferably from ethylamine and ethyltri methylammonium and Ci-Ce hydroxyalkyl. Each HT is individually a saturated or unsaturated hydrocarbon chain being 5-50 carbon atoms long, preferably 11-29 carbon atoms long, more preferably 11 -23 carbon atoms long, most preferably each HT is a saturated hydrocarbon of 17 carbon atoms, most preferably a -(CH2)ieCH3 group.

[0069] Alternatively, the helper lipids are preferably selected from phosphatidylcholine (PC)), cephalins, sphingolipids, glycoglycerolipids, phosphatidylamine selected from: 1 ,2-dilauroyl-L-phosphatidyl- ethanolamine (DLPE), 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) 1 ,2-Diphytanoyl-sn- glycero-3-phosphoethanolamine (DPhPE) 1 ,3-Dipalmitoyl-sn-glycero-2-phosphoethanolamine (1 ,3-DPPE) 1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1 ,3-POPE), Biotin-Phosphatidylethanolamine, 1 ,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1 ,2-Distearoyl-sn-glycero-3-phosphoethanol- amine (DSPE), and Dipalmitoylphosphatidylethanolamine (DPPE), and combinations thereof.

[0070] The stealthing lipids help stabilizing the extracellular vesicle under aqueous conditions and especially in vivo. In particular, the stealthing lipids help to avoid aggregation and increases in vivo circulation time. The stealthing lipid typically include PEGylated lipid, XTEN peptide lipid, polysarcosine (pSAR)peptide lipid, PAS peptide lipid, herein a stealthing lipid is thus preferably according to V-HH, wherein HH comprises a PEG chain, XTEN peptide, polysarcosine peptide or a PAS peptide.

[0071] More preferably, the stealthing lipid is according to any one structure (V2)-(V6):T(V2) (V3) (V4) (V5) (V6)

[0072] Herein, each HT is individually a saturated or unsaturated hydrocarbon chain from 5-50 carbonatoms long, preferably 11 -29 carbon atoms long, more preferably 11 -23 carbon atoms long, most preferably each HT is a saturated hydrocarbon of 17 carbon atoms long, most preferably a -(CH2)ieCH3 group. Hydrocarbon chains may be branched or linear and saturated or unsaturated, the hydrocarbon chains are preferably linear and saturated. HH preferably comprises PEG, XTEN, pSAR or PAS peptide.

[0073] In a preferred embodiment, the stealthing lipid is PEGylated lipid. Preferably, a PEGylated lipid selected from DMG-PEG, PEG-cDMA, 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1 ,2- dimyristyloxy-propylamine; PEG-cDSA, 3-N-(-methoxy poly(ethylene glycol)2000)carbamoyl-1 ,2- distearyloxy-propylamine, PEG-Amine, DSPE-PEG, and combinations thereof.

[0074] In an especially preferred embodiment, the PEGylated lipid is according to structure V-PEG-CAP, more preferably, the PEGylated lipid is according to any one of structures (V7)-(V11):(V7) (V8) (V9) (V10) (V11)

[0075] Herein, HT is defined as above, PEG is a polyethylene glycol chain, and CAP represents a hydrogen, hydrophilic group, ionizable group or a small aliphatic group. Preferably CAP is selected from methyl, ethyl, n-propyl, iso-propyl, amine, hydroxyalkyl. Preferably, PEG has a length of 5-200 of monomeric units, more preferably 10-150, even more preferably 40-50.

[0076] In the art it is known to use a mixture of PEGylated lipids having a different PEG length and is encompassed by the invention. The present invention also includes extracellular vesicles with monodisperse PEGylated lipids. Monodisperse PEG chains are known in the art, but not used in the field of extracellular vesicles. The use of monodisperse PEGylated lipids in extracellular vesicles has the advantage that more stable LNPs can be generated, a more precise biodistribution and enhanced intracellular uptake can be achieved. Preferably, the extracellular vesicle comprises PEGylated lipids having monodisperse PEG chains, wherein monodisperse PEG chains are defined as PEG chains having an average length (AVG) of 5-200 repeating monomeric PEG units, preferably 10-120 monomeric PEG units, more preferably 20-80 monomeric PEG units, most preferably 40-50 monomeric PEG units and wherein at least 80 %, preferably 90 %, more preferably 95 %, of the PEG chains have a length in the range of AVG -2 to AVG +2. In other words, it is preferred that the PEGylated lipids have approximately the same length and a very narrow distribution in length. More preferably, monodisperse PEG chains are defined as having at least 80% of the PEG chains with a length in the range of AVG-1 to AVG +1 , even more preferablyat least 99.5-100 % of the PEG chains have a length of AVG.

[0077] PEG is a non-natural polymer and it is known that a large percentage of the population produces anti-PEG antibodies which potentially facilitates the clearance of the extracellular vesicles. Thus, in another preferred embodiment, the stealthing lipid is a non-PEGylated lipid. Preferably, the non-PEGylated stealthing lipid is a peptide attached to a lipid. More preferably, the non-PEGylated stealthing lipid is according to V-PEP-CAP, even more preferably, the stealthing lipid is according to any one of structure (V12)-(V16):

[0078] Herein, the preferred embodiments of HT are the same as for (V7)-(V11), PEP is peptide sequence, optionally comprising unnatural amino acids, and CAP represents a hydrogen, hydrophilic group, ionizable group or a small aliphatic group. Preferably CAP is selected from methyl, ethyl, n-propyl, iso-propyl, amine, hydroxyalkyl. Preferably, the peptide is selected from polysarcosine (pSAR), XTEN peptide and PAS (Pro- Ala-Ser).

[0079] Thus, in a preferred embodiment the extracellular vesicle comprises pSAR lipids. pSAR modified extracellular vesicles are known in the art and have been described in Wilhelmny et al. “Polysarcosine- Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy”, Pharmaceutics, 2023, 15, 2068, incorporated by reference. Thus, in this preferred embodiment PEP is according to structure (P1)

[0080] Herein, x is an integer in the range of 10-200, preferably 15-150, more preferably 20-100, most preferably 25-75.

[0081] In another preferred embodiment the stealthing lipid comprises a XTEN peptide. XTEN is known in the art and often used as a mask moiety for increasing the circulation time of a drug or masking an antibody. In particular, XTEN is an unstructured polypeptide which acts like PEG, but is less immunogenic, is biodegradable and can be produced by a microorganism. XTEN is described in EP3406347, incorporatedby reference. XTEN peptides are typically made of 4, 5 or 6 types of amino acids selected from glycine, alanine, serine, threonine, glutamate or proline and is arranged in substantially non-repetitive sequences. Preferably, the XTEN sequence consists of 10-200 amino acids, more preferably 20-150, most preferably 35-100. In this preferred embodiment, the stealthing lipid is preferably according to any one of structures (V12)-(V16) and PEP has a sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100% to following sequence (SEQ ID NO 1): GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG, or a sub fragment thereof.

[0082] Alternatively, PEP has a sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100% to the following sequence (SEQ ID NO 2): GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAP, or a sub fragment thereof.

[0083] In a preferred embodiment, PEP is an XTEN peptide comprising a sequence selected from GGSPAGSCTSP (SEQ ID NO 3), GASASCAPSTG (SEQ ID NO 4), TAEAAGCGTAEAA (SEQ ID NO 5), GPEPTCPAPSG (SEQ ID NO 6) and combinations thereof.

[0084] In another preferred embodiment, the stealthing lipid is a (PAS)x modified lipid, also known as PASylation. PASylation is known in the art and has been described in Gebauer M, Skerra A. “Prospects of PASylation® forthe design of protein and peptide therapeutics with extended half-life and enhanced action.” Bioorg Med Chem. 2018 Jun 1 ;26(10):2882-2887. PAS peptides are peptides made of the small L-amino acids proline, alanine and / or serine. Hence, it is preferred that the stealthing lipid is according to any one of (V12)-(V16) wherein PEP consists of proline, alanine and / or serine residues. More preferably, PEP comprises a 1-10 repeating units of ASPAAPAPASPAAPAPSAPA (SEQ ID NO 7),AAPASPAPAAPSAPAPAAPS (SEQ ID NO 8), APSSPSPSAPSSPSPASPSS (SEQ ID NO 9),SAPSSPSPSAPSSPSPASPS (SEQ ID NO 10), SSPSAPSPSSPASPSPSSPA (SEQ ID NO 1 1),AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO 12), ASAAAPAAASAAASAPSAAA (SEQ ID NO 13), AAPAAPAPAAPAAPAPAAPA (SEQ ID NO 14), AAPAAAPAPAAPAAPAPAAP (SEQ ID NO 15),AAAPAAAPAAAPAAAPAAAP (SEQ ID NO 16), AAPAAPAAPAAPAAPAAPAAPAAP (SEQ ID NO 17), APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO 18), AAAPAAPAAPPAAAAPAAPAAPPA (SEQ ID NO 19) or APAPAPAPAPAPAPAPAPAP (SEQ ID NO 20), including multimer(s) of these sequences.

[0085] The extracellular vesicle may comprise ionizable lipids. Typically, the cavity comprises ionizable lipids, but the ionizable lipid may also be present in the outer membrane as well. In a preferred embodiment, the extracellular vesicle is a lipid nanoparticle, wherein the ionizable lipids in the cavity encapsulate the active compound(s). The ionizable lipids are lipid molecules that are neutral at a physiological pH, but become an anion, cation or zwitterion at a different pH. It is believed that ionizable lipids may facilitate endosomal escape due to the ionization of these lipids at the low pH of the endosome. Furthermore, the ionizable lipids help to stabilize the endosomal vesicle, in particular the ionizable lipid can help to stabilize the active compound. In a preferred embodiment, the active compound is a negatively charged nucleic acid and encapsulated by ionizable lipids within the cavity.

[0086] In a preferred embodiment, the extracellular vesicle comprises ionizable lipids, more preferably these ionizable lipids are cationic lipids selected from ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,1- diyl) bis(2-hexyldecanoate)), SM-102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}octanoate), DLinDMA, DLin-MC3-DMA, DLin-KC2-DMA, N,N-dimethyl-N',N'- di[(9Z,12Z)-octadeca-9,12-dien-1-yl]ethane-1 ,2-diamine, Di-oleyl-succinyl-serinyl-tobramycin, Di-oleyl- adipyl-tobramycin, Di-oleyl-suberyl-tobramycin, Di-oleyl-sebacyl-tobramycin, Di-oleyl-dithioglycolyl- tobramycin, monocationic lipid N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane (DOTAP), BCAT O-(2R-1 ,2-di-O-(1 'Z, 9'Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate, BGSC (Bis- guanidinium-spermidine-cholesterol), BGTC (Bis-guanidinium-tren-cholesterol), CDAN (N'-cholesteryl oxycarbony 1-3,7-diazanonane-1 ,9-diamine), CHDTAEA (Cholesteryl hemidithiodiglycolyl tris(amino(ethyl)amine), DCAT (0-(1 ,2-di-O-(9'Z-octadecanyl)-glycerol)-3-N-(bis-2-aminoethyl)- carbamate), DC-Chol (3p[N — (N', N'-dimethylaminoethane)-carbamoyl] cholesterol), DLKD (O,O'-Dilauryl N-lysylaspartate), DMKD (O,O'-Dimyristyl N-lysylaspartate), DOG (Diolcylglycerol, DOGS (Dioctadecylamidoglycylspermine), DOGSDSO (1 ,2-Dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine), DOPC (1 ,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE (1 ,2-Dioleoyl-sn-glycerol-3- phosphoethanolamine, DOSN (Dioleyl succinyl ethylthioneomycin), DOSP (Dioleyl succinyl paromomycin), DOST (Dioleyl succinyl tobramycin), 1 ,2-Uiolcoyl-3-trimethyl ammoniopropane, DOTMA (N'[1 -(2,3- Dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DPPES (Di-palmitoyl phosphatidyl ethanolamidosperminc), DDAB and DODAP, or any combination thereof. More preferably, the cationic lipid is selected from DLinDMA, DLin-MC3-DMA, DLin-KC2-DMA, Di-oleyl-succinyl-serinyl-tobramycin, Di-oleyl- adipyl-tobramycin, Di-oleyl-suberyl-tobramycin, Di-oleyl-sebacyl-tobramycin, N,N-dimethyl-N',N'-di[(9Z,12Z)-octadeca-9,12-dien-1-yl] ethane-1 ,2-diamine and Di-oleyl-dithioglycolyl-tobramycin, or any combination thereof.

[0087] In another preferred embodiment, the ionizable lipid is a cationic lipid according structure (V5), wherein each HT is individually selected from an optionally branched alkyl chain of 5-50 carbon atoms, preferably 1 1-29 carbon atoms, more preferably 13-23 carbon atoms, and wherein the alkyl chain may be interrupted by an ester, ether, amine, amide, sulfamide, sulfonamide or thioether moiety, preferably an ester moiety, and wherein HH is a hydrophilic moiety, preferably a hydroxyalkyl, more preferably a Ci-Ce hydroxy alkyl.

[0088] The extracellular vesicle preferably comprises a sterol. Sterols are mainly hydrophobic, therefore sterols may be present in the lipid membrane. In case the lipid membrane is a monolayer or a trilayer, sterol can also be present in the cavity as in these extracellular vesicles the cavity is hydrophobic except for the optional encapsulated active compounds within this cavity as described above.

[0089] The sterol is preferably selected from cholesterol, vitamin D3, vitamin D2, calcipotriol, stigmasterol, p-Sitosterol, Betulin, Lupeol, Ursolic acid, oleanolic acid and combinations thereof. In an especially preferred embodiment, the sterol is cholesterol.

[0090] Hydrophobic moiety V anchors B-L-V in the membrane of the extracellular vesicle. Hydrophobic moiety V is anchored to the lipid membrane by van der Waals forces, i.e. London Dispersion, between V and the lipid membrane and the lack of polar interactions between V and water. Typically, hydrophobic moiety V is a lipid. In a preferred embodiment, hydrophobic moiety V is represented by structure (V7a) or (V7b):(V7a) (V7b)Herein:- the wavy bond represents the connection to L;- HT is a hydrophobic tail, preferably a saturated or unsaturated hydrocarbon chain from 5-29 carbon atoms long;- X is a hydrophilic moiety selected from -O-P(O)(OX1)-O-, -NH- and -N(HT)-, wherein X1is H or a pharmaceutically acceptable cation;- L8is a linker having 1 - 10 building blocks selected from (hetero)aryl, CH2, CH=CH, C=C, C(O), NR13, O, S, S(O), S(O)2, PR13and 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;- R13is selected from 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 andCs - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR14wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or two occurrences of R13are connected to form a cyclic structure;- w is 0 or 1 .

[0091] HT, X, L8, R13and w are already defined for the PEGylated lipids, which equally applies here. In an especially preferred embodiment, the structure of HT, X, L8and w for the lipid-conjugate is the same as for the PEGylated lipid.

[0092] In a further preferred embodiment, V is according to any one of structures (V17)-(V21):(V17) (V18) (V19) (V20) (V21)

[0093] Herein, HT is a hydrophobic tail, typically individually selected from an optionally branched alkyl chain of 5-50 carbon atoms, preferably 1 1 -29 carbon atoms, more preferably 13-23 carbon atoms, particularly 17 carbon atoms, most preferably a -(CH2)ieCH3 group, and wherein the alkyl chain may be interrupted by an ester, ether, amine, amide, sulfamide, sulfonamide or thioether moiety, preferably an ester moiety.

[0094] In an especially preferred embodiment, the stealthing lipid is according to V-HH and V is identical to V in B-L-V. In, other words, it is preferred that if the stealthing lipid is according to (V2)-(V6), V is according to the corresponding (V17)-(V21), wherein the individual HT moieties in (V17)-(V21) are identical to (V2)- (V6). More preferably, V is according to (V19) and the stealthing lipid is according to (V3)

[0095] The stealthing lipid may also be a remnant of the precursor to B-L-V that did not react in a click reaction, in particular step (b) of the process according to the invention as defined below. In other words, it is especially preferred that the stealthing lipid is V-LA-Q, wherein LApreferably comprises a PEG or PEP fragment.

[0096] In an alternative preferred embodiment, the stealthing lipids in the lipid membrane are a mixture of V-LA-Q and non-click functionalized lipids, preferably a PEGylated lipid or a peptide lipid, more preferably a lipid according to (V7)-(V16).

[0097] In another preferred embodiment, virtually all the V-LA-Q moieties have reacted with F-LB-B and the stealthing lipid composition essentially consists of stealthing lipids without click probe Q.Cavity

[0098] The extracellular vesicle according to the invention contains a cavity encapsulated by a lipid membrane. The cavity may also be referred to as the core of the extracellular vesicle, and the lipid membrane as the shell of the extracellular vesicle. As the extracellular vesicle is suitable for in vivo applications, and thus stable in aqueous solutions, the exact nature of the cavity is dependent on the type of lipid membrane and the active compound that may be encapsulated within the cavity. If the active compound is hydrophilic and the lipid membrane is a lipid bilayer, the cavity is preferably hydrophilic. If the active compound is hydrophilic and the lipid membrane is a lipid monolayer, the cavity is preferably hydrophobic but with hydrophilic regions comprising the active compound. Such a hydrophilic region preferably concerns an active compound encapsulated by ionizable lipids.

[0099] The hydrophilic region of the cavity may comprise water and pharmaceutical acceptable salts, in particular sodium chloride.

[0100] The cavity may also comprise polymers or hydrocolloids. In a preferred embodiment, the extracellular vesicle is a solid lipid nanoparticle, a nanostructure lipid carrier or a hybrid-lipid polymeric nanoparticle and the cavity comprises a polymer. More preferably, the polymer is selected from polysorbate, poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylysine, polyglutamic acid, most preferably the polymer is tween 80.Active compound

[0101] The extracellular vesicle according to the invention preferably comprises an active compound encapsulated within its cavity or core. Typically, the active compound provides the pharmacological effect of the extracellular vesicle. The extracellular vesicle ensures that the active compound is delivered to a targeted cell and that the pharmacological effects are targeted at that specific cell. Furthermore, encapsulating the active compound in an extracellular vesicle increases the longevity of the active compound in vivo and enhances cellular uptake. In particular, the extracellular vesicle helps to promote lysosomal and / or endosomal escape. Extracellular vesicles containing an active compound encapsulated within its cavity or core are known in the art. Preferably, the active compound is defined as a compound that is biologically and / or pharmaceutically active, more preferably the active compound is selected from a drug or a prodrug, a diagnostic agent, a protein, a peptide, an amino acid, a glycan, a lipid, a vitamin, a steroid, a nucleotide, a nucleoside, a polynucleotide, RNA, DNA or combination thereof. More preferably, the active compound is a nucleotide or a cytotoxic agent, even more preferably a nucleotide. The active compound may also be a mixture of different physiologically active compounds such as a CRISPR / CAS system.

[0102] In an especially preferred embodiment, the active compound is a nucleic acid selected from plasmid, 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 (dsRNA), single stranded DNA (ssDNA), double stranded DNA, plasmids,ribozymes, aptamer, Gapmer, and triplex forming oligonucleotides, more preferably the nucleotide is a mRNA. Delivery of mRNA via an extracellular vesicle is highly effective as mRNA is highly unstable extracellular and not able to effectively transfer the cellular membrane.

[0103] In a preferred embodiment, the active compound is mRNA including synthetic variants thereof, preferably the active compounds translates to a protein or manipulates the genetic expression in a cell. In a preferred embodiment, the active compound is mRNA translating for a protein.

[0104] Preferably, the extracellular vesicle is an LNP having a lipid monolayer and said LNP comprising 1-20 strands of nucleotides each decoding for a protein, preferably 4-12 strands of nucleotides, and wherein each nucleotide is encapsulated by an internal lipid monolayer. In another preferred embodiment, the active compound is a protein such as an enzyme, a fusion protein or an antibody.

[0105] In an especially preferred embodiment, the active compound is one or more components of a CRISPR / CAS system, preferably a CRISPR / CAS nuclease, or a nucleic acid encoding the CRISPR / Cas nuclease, and / or a guide RNA. In some embodiments, the payload is one or more components of a CRISPR-Cas9, CRISPR-Casl2a, or CRISPR-Casl2b system.

[0106] In another preferred embodiment, the active compound is a cytotoxic agent. Cytotoxic agents are known in the art, it is especially well known to attach a cytotoxic agent to an antibody to ensure targeted delivery. Encapsulating the cytotoxic agent in an extracellular vesicle instead of attaching a cytotoxic agent covalently to an antibody has the benefit that it enhances endosomal escape, in particular for larger compounds and / or charged compounds. Furthermore, hydrophobic compounds that are not encapsulated may be prone to aggregation which can be overcome by the extracellular vesicle of the present invention.

[0107] In yet another embodiment, the active compound is a degrader such as proteolysis-targeting chimeras (PROTAC).Biomolecule B

[0108] B is the moiety that is responsible for the targeting effect of the extracellular vesicle according to the invention. B typically has a very high affinity to a moiety on a targeted object such as a specific receptor of a specific cell. Preferably, B is selected from an aptamer, a protein, a polypeptide, peptide, a glycan, a nucleic acid, oligonucleotide, a polysaccharide, an oligosaccharide, an enzyme, a hormone. In a preferred embodiment, biomolecule B comprises or is a glycan, such as a glycoprotein, glycolipid or a glycan. The presence of a glycan allows for site-specific conjugation with high stability and homogeneity of the conjugate. It is especially preferred that biomolecule B is a glycoprotein, most preferably an antibody.

[0109] In an especially preferred embodiment, the biomolecule is an antibody or a fragment thereof. Antibodies are well known in the art and include IgA, IgD, IgE, IgG, IgM, Fab, VHH, scFv, diabody, minibody, nanobody, 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. Theantibody may be any IgG isotype, e.g. lgG1 , lgG2, Igl3 or lgG4. Preferably B is a full-length antibody, but B may also be a Fc fragment. In an especially preferred embodiment, B is a nanobody.

[0110] The antibody B is typically specific for an extracellular receptor on a specific cell. In a preferred embodiment, the extracellular vesicles 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, antitransferrin 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.

[0111] In another preferred embodiment, the extracellular vesicles of the present invention are 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, CD4, CD5, CD7, CD8 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; or- specific for a cellular receptor on a hematopoietic stem cell, preferably wherein the receptor is CD45 or CD117; or- specific for a cellular receptor in stroma, preferably wherein the receptor is FAP, LRRC15, VEGF, CD105, TEM1 , TEM8, uPAR (CD87), tenascin, CAIX.

[0112] In another embodiment, the extracellular vesicles 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, insulin-like growth factor-1 receptor (IGF1 R), CD98hc and the diphtheria toxin receptor. An example of a suitable antibody is the anti-transferrin receptor antibody RI7217.

[0113] In yet another preferred embodiment, the extracellular vesicles 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, Serna 5b, SLITRK6, SSTR2, STEAP1 , STEAP2, TAG72, TENB2, TF, TIM-1 , TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinases (RTK), tenascin.

[0114] Part of the antibody may be a linker L6that connects the click probe F or connecting group Z to the peptide part of the cell-binding agent. Herein, L6represents (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).B-[(L6- Z)y - LD- (V)x]z(3) wherein:- L6is -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 L7is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-.- y, x, and z are individually integers in the range of 1 -4;- LDis a linker connecting Z with V. Herein, L6, Z and LDform together linker L.Linker L6

[0115] Linker L6is 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 tubulin tyrosine ligase, using peptide asparaginyl ligase, 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 L6is present. The site of conjugation is preferably a glycosylation site at the heavy chain of the antibody.

[0116] 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, L6is 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 V. 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.

[0117] L6is 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 L7is -N(H)C(O)CH2-, - N(H)C(O)CF2- or -CH2- Typically, L6is at least partly formed by the glycan of an antibody.

[0118] The -GlcNAc(Fuc)w-(G)j- moiety of L6is 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.

[0119] S may be directly connected to the core-GlcNAc(Fuc)wmoiety, i.e. j = 0, meaning that the remainder of the glycan is removed from the core-GlcNAc(Fuc)wmoiety 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)wmoiety 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.

[0120] 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, A / -acetylgalactosamine, N- acetylglucosamine, mannose and A / -acetylneuraminic acid. More preferred options for G are galactose, N- acetylglucosamine, mannose. In case j = 1 , it is preferred that G = galactose and S = A / -acetylneuraminic acid.

[0121] 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.ManMan Man — GIcNAc

[0122] 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.

[0123] 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).

[0124] 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.

[0125] 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.

[0126] Connecting group Z or reactive group F may be attached directly to S, or there may be a linker L7present in between S and Z or F. Thus, L7may be present (w’ = 1 or 2) or absent (w’ = 0). Typically, each moiety Z may be connected to S via a linker L7. Preferably, L7is absent and each connecting moiety Z is directly attached to S. If present, L7may 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.

[0127] In one embodiment, the conjugates according to the invention contain two connecting groups Z, i.e. Z1and Z2, 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- LA- (F2)y2. In a first click reaction, F1and Q1react to form a covalent connection between the antibody and y2 x (z2 / y1) moieties F2by forming connecting groups Z1.

[0128] An extracellular vesicle conjugated to a trimmed antibody is particularly effective of delivering the active compound to the targeted cell which increases efficacy and reduces off site effects. Especially, as the extracellular vesicle promotes endosomal and / or lysosomal escape, immune cells with Fc receptors such as macrophages, megakaryocites, thrombocytes are in particular at risk of off-target effects when an extracellular vesicle is connected to an antibody with Fc gamma receptor affinity. In other words, the useof a trimmed antibody as biomolecule greatly enhances the therapeutic window and / or efficacy.

[0129] Thus, in a preferred embodiment, the extracellular vesicle is a lipid nanoparticle, wherein the cavity contains an active compound and / or a stabilizing lipids and the membrane comprises stealthing lipids, the biomolecule B is a trimmed antibody; the connecting group Z preferably comprises a 1 ,2,3-triazole; the linker L is connected to the C-terminus or the glycan of the antibody, wherein:- the connection to the C-terminus is preferably obtainable by tubulin tyrosine ligase, peptide asparaginyl ligase, sortase or transglutaminase, more preferably sortase or translgutaminase conjugation;- the connection to the glycan has structure B(L6)x, wherein L6is -GlcNAc(Fuc)^ [ S-(L7)W]yi— , wherein y1 is 1 , 2, 3 or 4, S is a sugar or a sugar derivative, GIcNAc is N- acetylglucosamine and Fuc is fucose, w is 0 or 1 , w’ is 1 or 2, L7is a bond, -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-, and preferably y1 = 1.Connecting group Z

[0130] 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 biomolecule B with the hydrophilic moiety V. The conjugates according to the invention may contain one distinct connecting group Z. 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 Z1and Z2, which are individually selected, for example as in structure. Likewise, Z2is formed by a click reaction between Q2and F2. Herein, the definition of Z and preferred embodiments thereof also apply to Z1and Z2. Likewise, the definition of Q and preferred embodiments thereof also apply to Q1and Q2, and the definition of F and preferred embodiments thereof also apply to F1and F2

[0131] 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 / F1and 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.

[0132] 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 knownto 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.

[0133] 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.

[0134] 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, Z1and / or Z2is obtained by ultrafast click, more preferably at least Z2is obtained by ultrafast click.

[0135] 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) - (Zv) 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;- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24alky l(hetero)ary I groups and C7 - C24 (hetero)arylalkyl groups;- Y2is C(R31)2, O, S, S<+)R31, S(O)R31, S(O)=NR31or NR31, wherein S<+> is a cationic sulphur atom counterbalanced by B( ), wherein B( )is an anion, and wherein each R31individually is R15or a connection with V, 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.

[0136] 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.

[0137] Preferably, the wavy bond labelled with * is connected to B, optionally via L6, and the wavy bond labelled with ** is connected to the remainder of L. 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), depicted here below:(Z20a) (Z20b) (Z20c)

[0138] Herein, the connection to L is depicted with the wavy bond. p (-) is an anion, preferably a pharmaceutically acceptable anion. p(+)is a cation, preferably a pharmaceutically acceptable cation. RingZ 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, pyrazole, pyridine, phenyl, pyridazine, diazacycloalkane. (1 ,4-diazacyclohexane = piperazine, 1 ,3-diazacyclohexane or 1 ,3-diazacycloheptane, oxazacycloalkane or a piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have the structure selected from (Za) - (Zn) 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.

[0139] R36is a halogen selected from fluoro, chloro, bromo and iodo, preferably R36is fluoro. Y4is a heteroatom, preferably Y4is O or NH. R35is 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)arylalkylroups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, Si, S and NR14wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably R35is selected from H, C5H11, CH3, CH2CH3, CH2OH or CH2OTBS.

[0140] 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.

[0141] Herein, R29is 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 R33is H or C1-4 alkyl. Preferably, R29is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl. It was found that R29is 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 R29is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R29is hydrogen.

[0142] Herein, the R41group is preferably 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 NR32whereinR32is independently selected from the group consisting of hydrogen and Ci - C alkyl groups. In a particular preferred embodiment, R41is L10XR4, wherein L10is a linker of structure (C(R3)2)z, wherein z is 2 or 3, and each R3is individually selected from H and C1-4 alkyl, wherein two occurrences of R3may be joined together to form a C3-6 (hetero)cycloalkyl group, R4is selected from H and C1-4 alkyl, X is S, O or NH. Herein, each R42group is preferably selected from H and C1-4 alkyl, preferably R42is H.

[0143] In case R41is L10XR4, (Zh) may rearrange into (Zn), thus L10is a linker of structure (C(R3)2)z, wherein z is 2 or 3, and each R3is individually selected from H and C1-4 alkyl, wherein two occurrences of R3may be joined together to form a C3-6 (hetero)cycloalkyl group, R4is selected from H and C1-4 alkyl, X is S, O or NH. This rearrangement is described in WO2024 / 218164 and is incorporated by reference.

[0144] 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).

[0145] In a further preferred embodiment, Z is selected from the structures (Z21) - (Z38d), depicted here below:(Z35) (Z36) (Z37) (Z38) (Z38a).(Z38b) (Z38c) (Z38d)

[0146] 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), p(~> is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from structures (Za) - (Zm), as defined above. R35and R36are as defined above for (Z20a) - (Z20c).

[0147] R35is selected from the group consisting of hydrogen, C1-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(R14)2, S and NR14wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups.

[0148] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety or a (hetero)cycloheptene moiety, preferably according to structure (Z8), (Z26), (Z27), (Z28), (Z37), (Z38a), (Z38b) or (Z38c) which are optionally substituted. Each of these preferred options for Z are further defined here below.

[0149] 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.

[0150] 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.

[0151] 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 Y1is O or NR11, wherein R11is 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 notfurther substituted. Most preferably, Z is according to structure (Z43), defined further below.

[0152] In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group and is according to structure (Z37).Herein:- the bond labelled with * is connected to B and the wavy bond labelled with ** is connected to L;- R15is independently selected from the group consisting of hydrogen, halogen, -OR16, -NO2, -CN, -S(O)2R16, -S(O)3H,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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- R18is 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;- R19is 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 R19is a second occurrence ofZ (or Q) or V connected via a spacer moiety; and- I is an integer in the range 0 to 10.

[0154] In a preferred embodiment of the group according to structure (Z42), R15is independently selected from the group consisting of hydrogen, halogen, -OR16, Ci - Ce alkyl groups, C5 - Ce (hetero)aryl groups,wherein R16is hydrogen or Ci - Ce alkyl, more preferably R15is independently selected from the group consisting of hydrogen and Ci - Ce alkyl, most preferably all R15are H. In a preferred embodiment of the group according to structure (Z42), R18is independently selected from the group consisting of hydrogen, Ci - Ce alkyl groups, most preferably both R18are H. In a preferred embodiment of the group according to structure (Z42), R19is H. In a preferred embodiment of the group according to structure (Z42), I is 0 or 1 , more preferably I is 1

[0155] 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 B and the wavy bond labelled with ** is connected to L;- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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.

[0156] In a preferred embodiment of the group according to structure (Z43), R15is independently selected from the group consisting of hydrogen, halogen, -OR16, -S(O)3( ), Ci - Ce alkyl groups, C5 - Ce (hetero)aryl groups, wherein R16is hydrogen or Ci - Ce alkyl, more preferably R15is 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.

[0157] In an especially preferred embodiment, Z comprises a heterocycloheptenyl group and is according to structure (Z37) or (Z38a), wherein ring Z is a triazole.

[0158] 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, Y3is selected from C(R23)2, NR23or O, wherein each R23is 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 (Z52), (Z53),

[0159] 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 (Zo) - (Zv), 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 B. 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.(Zs) (Zt) (Zu) (Zv)

[0160] Herein, R29is 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 R33is H or C1-4 alkyl. Preferably, R29is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl. It was found that R29is hydrogen gave optimal results in reactivity in the cycloaddition reaction, especially in case ring (Zv) is formed. Thus, in a preferred embodiment, ring Z is (Zv) wherein R29is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R29is hydrogen.

[0161] R41is preferably 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 NR32wherein R32is independently selected from the group consisting of hydrogen and Ci - C alkyl groups. More preferably, R41is L10XR4, wherein L10is a linker of structure (C(R3)2)z, wherein z is 2 or 3, and each R3is individually selected from H and C1-4 alkyl, wherein two occurrences of R3may be joined together to form a C3-6 (hetero)cycloalkyl group, R4is selected from H and C1-4 alkyl, X is S, O or N. In an alternative preferredembodiment R41is methyl. Herein, each R42group is preferably selected from H and C1-4 alkyl, preferably R42is H.

[0162] In case Z comprises a (hetero)cycloalkane moiety, it is preferred that ring Z is selected from (Zo), (Zs), (Zr) or (Zv), most preferably ring Z is according to structure (Zv).

[0163] In a preferred embodiment, connection group Z comprise a moiety selected from (Z1) - (Z56), wherein ring Z is selected from (Za) - (Zv).

[0164] In case the extracellular vesicle according to the invention contains two distinct connecting groups Z1and Z2, these typically differ, as Z1is formed by reaction of Q1and F1, whereas Z2is formed by reaction of Q2and F2. Herein, F2is reactive towards Q2but not towards Q1, such that Q1and Q2should differ. Alternatively, Q1and F2are the same click probes and then F1may be the same as Q2which results in a Z1that is the same as Z2.

[0165] In a preferred embodiment, F1is azide and Q1is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F2is tetrazine or nitrone and Q2is bicyclononyne or cycloalkene, such as a trans- cyclooctene or a cyclopropene. More preferably, F1is azide and Q1is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F2is tetrazine and Q2is bicyclononyne.

[0166] Herein, the reaction of F1and Q1will preferably form a connecting group Z1according to structure (Z5), (Z6), (Z7), (Z11), (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 F2and Q2will preferably form a connecting group Z2according 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 (Zv), preferably according to structure (Zl) or (Zv), most preferably according to structure (Zl). Alternatively, connecting group Z2is according to structure (Z8), wherein ring Z is according to structure (Zl), preferably according to structure (Z29), more preferably according to structure (Z42).

[0167] In an alternative preferred embodiment, F1is a nitrone and Q1is a (hetero)cycloalkyne, while F2is a (hetero)cycloalkyne and Q2is a tetrazine.

[0168] Herein, the reaction of F1and Q2will form a connecting group Z1according to structure (Z2)-(Z56) wherein ring Z is according to structure (Zd), (Zh) or (Zn), preferably according to (Z26), (Z29) or (z37), most preferably according to (Z29). Herein, the reaction of F2and Q2 will preferably form a connecting group Z2according 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 (Zl).

[0169] In yet an alternative preferred embodiment, F1is an o / Yho-quinone and Q1is a (hetero)cycloalkyne, while F2is a (hetero)cycloalkyne and Q2is a tetrazine.

[0170] Herein, the reaction of F1and Q2will form a connecting group Z1according to structure (Z2)-(Z56) wherein ring Z is according to structure (Zi) or (Zm), preferably according to (Z26), (Z29) or (z37), mostpreferably according to (Z29). Herein, the reaction of F2and Q2 will preferably form a connecting group Z2according 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 (Zl).Linker L

[0171] Linker L connects hydrophilic moiety V to biomolecule B and comprises connecting moiety Z. As the lipid membrane typically comprises a stealthing lipid and such stealthing lipid provides much steric bulk, linker L is preferably a long chain to ensure that biomolecule B is sufficiently exposed to bind to a target. Thus, in an especially preferred embodiment, linker L comprises one or more PEG or peptide moieties to provide sufficient length. More preferably, linker L comprises at least one PEG or peptide fragment of 5- 200 repeating units, more preferably 5-120, even more preferably 40-50 repeating units. Preferably, linker L comprises a monodisperse PEG chain as defined above.

[0172] A single biomolecule B is preferably connected to 1 - 4 occurrences of V, more preferably 1 or 2 occurrences of V, most preferably 1 occurrence of V.

[0173] Typically, linker L may be represented as LB-Z-LA, wherein LBis connected to B and LAis connected to V. Linker LAand LBare individually present. In a preferred embodiment, B is connected to two instances of V. An additional hydrophobic moiety V increases the attachment to the lipid membrane, but increases the risk of aggregation as the two moieties V can potentially be integrated into lipid membranes from different extracellular vesicles. In this embodiment the hydrophilic moiety biomolecule conjugate preferably has structure (C1) or (C2):(C1) (C2)

[0174] In another preferred embodiment, B is connected to one instance of V. This embodiment is especially preferred as this prevents aggregation between lipid nanoparticles, as B can only be connected to one lipid membrane. In this embodiment the hydrophilic moiety biomolecule conjugate is preferably according to structure (C3) or (C4):B-LB-Z-LAV(C4)

[0175] In yet another preferred embodiment, two instances of B are connected to one instance of V. Such a structure has the advantage that the biomolecule per extracellular vesicle ratio is increased which enhances the targeting capabilities. In this embodiment the hydrophilic moiety biomolecule conjugate ispreferably according to structure (C5):B,LB-Z - LA-VB(C5)

[0176] In an especially preferred embodiment, linker L comprises two types of connecting moieties Z, Z1and Z2, and linker L may thus be represented as LB1-Z1-LB2-Z2-LA. This embodiment is especially preferred as the extracellular vesicles with two types of connecting moieties Z1and Z2are more homogenous. In this embodiment is particularly preferred that the hydrophilic moiety biomolecule conjugate is selected from any one of structures (C6)-(10):B - LB1- Z1- LB2— Z2- LA- V

[0177] In an especially preferred embodiment, biomolecule B is an antibody and said biomolecule is conjugated to a single hydrophobic moiety V. This embodiment has the advantage that stability is greatly enhanced as an antibody with only one V cannot crosslink multiple extracellular vesicles.

[0178] In this embodiment, it is preferred that the conjugate is according to (C8) and is obtainable by reacting B(-LB1-F1)2 with (Q1-)2LB2-F2to obtain B(-LB1-Z1-)2LB2-F2and reacting this with Q1-LA-V to obtain B(-LB1-Z1-)2LB2-Z2-LA-V. Preferably, F1is an azide and the conjugate is according to (C8a):

[0179] Herein:- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- Y2is C(R31)2, O, S, S(+)R31, S(O)R31, S(O)=NR31or NR31, wherein S(+)is a cationic sulphur atom counterbalanced by B( ), wherein B( )is an anion, and wherein each R31individually is R15or a connection with V, 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.

[0180] In this embodiment, it is preferred that F1is azide, Q1is DBCO, F2is tetrazine and Q2is BCN. In another preferred embodiment, the conjugate is according to (C8), wherein F1is azide, Q1and F2are both BCN and Q2is azide or tetrazine.

[0181] In an alternative preferred embodiment, the conjugate is according to (C8a) and F2is a strained alkyne or alkene, preferably a strained alkyne and Q2is tetrazine. Accordingly, the conjugate is preferably according to (C8b):

[0182] Herein, - represents a single or a double bond, preferably a double bond.

[0183] In a particular preferred embodiment, the conjugate is according to (C8b), wherein Q1and F2are BCN and Q2is tetrazine, more preferably LB1is glycan L6. Accordingly, in this preferred embodiment, the conjugate is according t(C8c).

[0184] Alternatively, the conjugate (C8) is obtainable by reacting Q2-LA-V with (Q1-)2LB2-F2) to obtain (Q1- )2LB2-Z2-LA-V) and subsequently reacting this with B(-LB1-F1)2 to obtain B(-LB1-Z1-)2LB2-Z2-LA-V. In this embodiment, it is preferred that Q2is tetrazine, F2is BCN, Q1is DBCO and F1is azide or that Q2is BCN, F2is tetrazine, Q1is DBCO and F1is azide.

[0185] Other methods of connecting a single payload to an antibody are known in the art. For instance, it is known to reduce the interchain disulfides of an antibody and subsequently protect Cys229 with zinc. The unprotected non-coordinated cysteines are then reoxidized. Deprotecting the zinc group from Cys229 cysteines results in an antibody having two reactive Cys229 residues. Those reactive Cys229 residues can subsequently be used as anchoring point to prepare an antibody with a single payload, e.g. by using a bis- maleimide linker. This process has been described in detail in Dattler D., Schlimpen F., Semenchenko C., Koniev O. & Kolodych S. Site-specific conjugation on Cys229 of the hinge region of native antibodies improves pharmacological properties of ADCs, and is incorporated by reference. In a preferred embodiment, the conjugate is obtainable by this method. In other words, the conjugate is according to (C4) or C8, wherein LBis connected to the two Cys229 residues.

[0186] In another preferred embodiment, the conjugate is according to (C1) or (C4) wherein the LBmoieties are connected to the C termini of an antibody. A process to introduce a click moiety at the C terminus of anantibody is well known, for instance with the use of transglutaminase, sortase, tubulin tyrosine ligase or peptide asparaginyl ligase, and has been described in for example WO2021174091 A1.

[0187] In an alternative preferred embodiment, the conjugate is according to (C7) or (C8), wherein the LB1are connected to C termini of an antibody.

[0188] In an alternative preferred embodiment, the conjugate is according (C6) or (C8), wherein LB1are connected to the N termini of an antibody or LB1is connected to the N-terminus of a nanobody. Surprisingly, N-terminal conjugation on the nanobody gave conjugates and targeting extracellular vesicles with excellent targeting properties. The N-terminus of a nanobody is associated with the antigen-binding region, which is not disrupted by N-terminal conjugation. Further, N-terminal conjugated conjugates with a relative short linker also gave excellent targeting properties, while the N-terminus of a nanobody is positioned more within the cavity of the nanobody, compared to the C-terminus.

[0189] In an especially preferred embodiment, the conjugate is according to (C6) or (C8). More preferably, the conjugate is according to (C6) or (C8), wherein the intermediate linker prior to conjugation is homofunctional, in other words it is preferred that (C6) was obtained by using an intermediate linker Q1-LB- F2, wherein Q1is equal to F2and (C8) was obtained by intermediate linker (Q1)2-LB-F2, wherein Q1is also equal to F2. In this embodiment, it is especially preferred that Q1and F2are (hetero)cycloalkynes or (hetero)cyloalkenes, more preferably Q1and F2are (hetero)cycloalkynes, most preferably Q1and F2are BCN.

[0190] In an especially preferred embodiment, the conjugate is according to (C6) and B is a nanobody or sdAb. In this embodiment, it is preferred that Z1is obtained from a SPOCQ (strain promoted oxidation controlled cyclooctyne-1 ,2 quinone) reaction or a SPANC (strain promoted alkyne nitrone cycloaddition) reaction and Z2is obtained from cycloalkyne tetrazine reaction or a cycloalkene tetrazine reaction, preferably a cycloalkyne tetrazine reaction.

[0191] In an especially preferred embodiment, Z1is obtained by a SPANC reaction and Z1comprises (Zd), (Zh) or (Zn). More preferably, the conjugate is according to (C6a), (C6b) or (C6c):

[0192] Herein:- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- Y2is C(R31)2, O, S, S<+)R31, S(O)R31, S(O)=NR31or NR31, wherein S<+> is a cationic sulphur atom counterbalanced by B< >, wherein B< > is an anion, and wherein each R31individually is R15or a connection with V, 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;- L10is a linker of structure (C(R3)2)z, wherein z is 2 or 3, and each R3is individually selected from H and C1-4 alkyl, wherein two occurrences of R3may be joined together to form a C3-6 (hetero)cycloalkyl group;- X is S, O or N;- each R42group is preferably selected from H and C1-4 alkyl, preferably R42is H.- R41is preferably 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 NR32wherein R32is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups; more preferably R41is L10XR4, wherein L10is a linker of structure (C(R3)2)z, wherein z is 2 or 3, and each R3is individually selected from H and C1-4 alkyl, wherein two occurrences of R3may be joined together to form a C3-6 (hetero)cycloalkyl group, R4is selected from H and C1-4 alkyl, X is S, O or N.

[0193] In the context of Z1being obtainable by SPANC, the conjugate is more preferably according to any one of structures (C6d)-(C6f):(C6d) (C6e)

[0194] Herein, the - represents a single or a double bond, preferably a double bond, and R29is 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 R33is H or C1-4 alkyl, more preferably R29is hydrogen or methyl.

[0195] In the context of Z1being obtainable by SPANC, the conjugate is even more preferably a conjugate according to any one of structures (C6g)-(C6i):

[0196] In this embodiment, LB1is any linker that may connect the nitrone (F1) or the (reconfigured) isoxazoline (Z1) to B, F1is obtained by oxidation of the N-terminal serine group as described in WO 2024 / 218164 and Temming, Rinske P., et al. "N-terminal dual protein functionalization by strain-promoted alkyne-nitrone cycloaddition." Organic & Biomolecular Chemistry 11.17 (2013): 2772-2779, both incorporated by reference. Herein, LB1can be considered to be (CO) as the nitrone group according to (F3) is separated from the N-terminus of the remaining amino acids by a C(O) moiety. Surprisingly, despite the linker being connected to the N-terminus of the antibody or nanobody which is associated with the antigenbinding region, the inventors found excellent targeting capabilities of extracellular vesicles bound to the N- terminus of an antibody.

[0197] In an alternative preferred embodiment, Z1is obtained by a SPOCQ reaction, more preferably Z1is according to (Zi) or (Zm). More preferably, the conjugate is according to structure (C6j) or (C6k):

[0198] Herein:- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- Y2is C(R31)2, O, S, S(+)R31, S(O)R31, S(O)=NR31or NR31, wherein S(+)is a cationic sulphur atom counterbalanced by B< >, wherein B< > is an anion, and wherein each R31individually is R15or a connection with V, 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.

[0199] In the context of Z1being obtainable by SPOCQ, the conjugate is more preferably according to structure (C6I) or (C6m):

[0200] In this embodiment, it is especially preferred that the conjugate is according to structure (C6n) or (C60):

[0201] In this embodiment, LB1is any linker that may connect the o / Yho-quinone (F1) or SPOCQ-adduct (Z2) to B. Preferably, LB1is absent and the o / Yho-quinone group was formed by the oxidation of tyrosine group. Oxidation of the tyrosine group has been described in WO 2022 / 108452 and is incorporated by reference. Preferably, in case B is a full antibody, the tyrosine group is located Y296 or Y300.

[0202] Excellent results in terms of cell targeting and mRNA incorporation into the cell are obtained with conjugates that have been prepared via a SPANO or a SPOCQ conjugation reaction. Also, the extent of protein aggregation and LNP cluster formation during prolonged storage in serum was lowest for these conjugates. Hence, these two conjugation techniques are most preferred in the context of the present invention.

[0203] In a preferred embodiment, linker LA, linker LB, Linker LB1or linker LB2comprises a branching moiety BM. Thus, in a preferred embodiment, linker LA, linker LB, linker LB1and / or linker LB2, preferably linker LB2, is according to structure(L1 1)y1- BM- (L12)y2 wherein:- L11and L12each 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, PR13and 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 R13is as defined below for structure (23), preferably R13is 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.

[0204] In a preferred embodiment, linker LA, linker LBand / or linker LB2are according to structure (L11)-(L12) wherein:L11and L12each 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, PR13and 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 R13is as defined below for structure (23), preferably R13is H;L11and L12may individually be absent, preferably at least L11is present.

[0205] L11, L12and L13may for example be selected from the group consisting of linear or branched Ci- 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, C9-C200 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)yand NR21, wherein y’ is 0, 1 or 2, preferably y’ = 2, and R21is 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.

[0206] It is especially preferred that the hydrophilic moiety biomolecule conjugate is according to (C8) and LB2is according to (L11)2-BM-(L12).Branching moiety BM

[0207] 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.

[0208] 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 Z1or Q1via the C=O group and the connection to two occurrences of Z2or F2via 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.

[0209] 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 *).

[0210] In (BM-1), one of the branches labelled with * may be a single or a double bond, indicated with — — . 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, W2and WP is independently selected from C(R21)Wand N;- each of W4, W5and W6is 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 R21is 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 NR3wherein R3is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups; and- each R22is 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 NR3wherein R3is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups.

[0211] The skilled person appreciates that the values of w 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.

[0212] 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, tetra hydrofuranyl, di hydrofuranyl, thiolanyl, 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).

[0213] 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.

[0214] 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.

[0215] 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 optimalflexibility 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).

[0216] 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.

[0217] 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 LA

[0218] Linker LAconnects V to Z. Linker LAmay be absent, in this embodiment connecting moiety Z is directly attached to V. Preferably, Linker LAis present. In one preferred embodiment, Linker LAconsist 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, PR13and 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.

[0219] LAmay for example be 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, C9-C200 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)yand NR21, wherein y’ is 0, 1 or 2, preferably y’ = 2, and R21is 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.

[0220] In a preferred embodiment, linker LAcontains 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’-diammoalkanes (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:

[0221] For the polar groups defined here above, it is irrelevant which end is connected to Z and which end to V.

[0222] The polar group may also contain an amino acid, preferably selected from Arg, Glu, Asp, Ser and Thr. Herein, R13is 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 R30is individually H, Ci- 12 alkyl, C1-12 aryl, C1-12 alkaryl or C1-12 aralkyl. Linker L1may 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.

[0223] In a preferred embodiment, Linker LAis or comprises a sulfamide group, preferably a sulfamide group according to structure (23):

[0224] The wavy lines represent the connection to the remainder of the compound.

[0225] In structure (23), a = 0 or 1 , preferably a = 1 , and R13is 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 NR14wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. Alternatively, R13is connected to elsewhere in the linker, optionally via a spacer moiety, to form a cyclic structure. For example, R13may be connected to the linker via a CH2CH2 spacer moiety to form a piperazinyl ring, where the connection to V is via the second nitrogen of the piperazinyl ring.

[0226] In a preferred embodiment, R13is 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 moreheteroatoms selected from O, S and NR14, preferably O, wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. In another preferred embodiment, R13is 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 R13is a (poly)ethylene glycol chain comprising a terminal -OH group. In another preferred embodiment, R13is 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, R13is hydrogen or connected to elsewhere in the linker optionally via a spacer moiety, and most preferably R13is hydrogen.

[0227] In a preferred embodiment, LAis according to structure (24):

[0228] Herein, a and R13are as defined above, Sp1and Sp2are 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 Sp1and Sp2are 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 R16is 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.

[0229] More preferably, spacer moieties Sp1and 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-Cioo arylalkenylene groups and C9-C100arylalkynylene 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 R16is 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.

[0230] Even more preferably, spacer moieties Sp1and 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 R16is 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.

[0231] Yet even more preferably, spacer moieties Sp1and 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 R16is 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.

[0232] 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 R16is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably hydrogen or methyl.

[0233] Most preferably, spacer moieties Sp1and Sp2, if present, are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, the alkylene groups being optionally substitutedand optionally interrupted by one or more heteroatoms selected from the group of O, S and NR16, wherein R16is 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 R16is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably hydrogen or methyl.

[0234] Preferred spacer moieties Sp1and Sp2thus 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.

[0235] Typically, LAcomprises a stretching moiety and bridging moieties, wherein the bridging moieties connect V to the stretching moiety and connect the stretching moiety to Z. Preferably, the stretching moiety is selected from a PEG moiety or a peptide moiety. In a preferred embodiment, the stretching moiety in linker LAis the same as the modification in the stealthing lipids, e.g. in case the stealthing lipid is a PEGylated lipid, it is preferred that the stretching moiety is a PEG moiety. More preferably, the length of the PEG in the PEGylated lipid is equal to the length of PEG stretching moiety. In an especially preferred embodiment, the stretching moiety is a monodisperse PEG chain.

[0236] The bridging moieties connect V to the stretching moiety and the stretching moiety to Z. In an alternative preferred embodiment, the stretching moiety is absent and a bridging moiety connecting to V to Z is present. Typically, the bridging moiety comprises small alkane chains, amides, esters, carbamates, sulfamides and combinations thereof. In the embodiment in which the stretching moiety is a PEG moiety, it is preferred that the terminal oxygen atom of the PEG moiety forms a carbamate with a terminal amide of the bridging moiety or connected to a small alkane moiety of the bridging moiety and that the terminal carbon atom is connected to the nitrogen of a terminal amide in a bridging moiety.

[0237] In a preferred embodiment, linker LAmay be represented by -(W)c-(A)d-(B)e-(A)f-(W)g-(Str)h- (W)i- (A)j-(B)k-(A)i-(W)m-, wherein:- d = 0 or 1 , preferably d =0;- f = 0 or 1 , preferably f =0;- j = 0 or 1 , preferably j =0;- I = 0 or 1 , preferably I =0;- c = 0 or 1 , preferably c =1- g = 0 or 1 , preferably g =1 ;- i = 0 or 1 , preferably i = 1 ;m 0 or 1 , preferably m =1 ; h = 0 or 1 , preferably h =1 ;- e = an integer in the range of 0-10, preferably, e = 0, 1 , 2, 3, 4, 5 or 6, most preferably e = 1 , 2, 3, or 4; k = an integer in the range of 0-10, preferably, k = 0, 1 , 2, 3, 4, 5 or 6, most preferably k = 1 , 2, 3, or 4;- A is sulfamide group according to structure (23):- B is -CH2-;- 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;- Str is a stretching moiety selected from a PEG chain and a peptide.

[0238] Thus, in a preferred embodiment Str is according to structure (S1) or (S2):Herein:- x is an integer in the range of 0-200, preferably 5-200, more preferably 5-120, even more preferably 40-50;R17is a side chain of an amino acid;R16is H or methyl; orR16and R17together form a (CH2)3 bridge, i.e. the structure represents proline;- the wavy lines indicate a connection to the remainder of the linker (e.g. the bridging moieties), Z or

[0239] Preferably, the amino acid(s) are all in their L-configuration. In an especially preferred embodiment, Str is an XTEN peptide, wherein the R17are individually H, CH3, CH2OH, CHOHCH3, CH2CH2COOH and R16is H, or R17and R16from a (CH2)3 bridge. In an especially, preferred embodiment the XTEN peptide comprises GGSPAGSCTSP (SEQ ID NO 3), GASASCAPSTG (SEQ ID NO 4), TAEAAGCGTAEAA (SEQ ID NO 5), GPEPTCPAPSG (SEQ ID NO 6) and combinations thereof. More, preferably Str is a peptide having a sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100% to following sequence (SEQ ID NO 1): GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG, or a sub fragment thereof.

[0240] Alternatively, Str is a peptide having a sequence identity of at least 90%, preferably at least 95%, more preferably at least 99%, most preferably 100% to the following sequence (SEQ ID NO 2): GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAP, or a sub fragment thereof.

[0241] In another preferred embodiment, Str is polysarcosine and R16is methyl and R17is H.

[0242] In another preferred embodiment, Str is a PAS peptide, wherein R16is H and R17is methyl or CH2OH, or R16and R17together from a (CH2)3 bridge. More preferably, preferably, PEP comprises a 1 -10 repeating units of ASPAAPAPASPAAPAPSAPA (SEQ ID NO 7), AAPASPAPAAPSAPAPAAPS (SEQ ID NO 8), APSSPSPSAPSSPSPASPSS (SEQ ID NO 9), SAPSSPSPSAPSSPSPASPS (SEQ ID NO 10), SSPSAPSPSSPASPSPSSPA (SEQ ID NO 1 1), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO 12), ASAAAPAAASAAASAPSAAA (SEQ ID NO 13), AAPAAPAPAAPAAPAPAAPA (SEQ ID NO 14), AAPAAAPAPAAPAAPAPAAP (SEQ ID NO 15), AAAPAAAPAAAPAAAPAAAP (SEQ ID NO 16), AAPAAPAAPAAPAAPAAPAAPAAP (SEQ ID NO 17), APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO 18), AAAPAAPAAPPAAAAPAAPAAPPA (SEQ ID NO 19) or APAPAPAPAPAPAPAPAPAP (SEQ ID NO 20), including multimer(s) of these sequences.Preferred linkers LB

[0243] Linker LBrepresents the connection between biomolecule B and Z. In a preferred embodiment, biomolecule B is an antibody and LBis L6. In a preferred embodiment, LBcomprises an additional connecting moiety and may be represented as -LB1-Z1-LB2-, wherein LB1is connected to B and LB2is connected to Z, in this embodiment referred to as Z2. In other words, LB1is an optional linker that connects click probe F1(prior to conjugation) or connecting group Z1(post conjugation) to biomolecule B. In a preferred embodiment, B is an antibody and LB1is a glycan L6, especially if F1is N3 (F1). In an especially preferred embodiment, biomolecule B is an antibody or fragment thereof, and a N-terminal serine group was oxidized to a nitrone(F3), herein linker LB1can be considered to be the C(O) group that remains from the serine group that is connected to next N-terminal amino acid. Likewise, in an alternative preferred embodiment B is antibody or fragment that is functionalized with an o / Yho-quinone (F10) by oxidizing a tyrosine moiety, herein LB1can be considered to be the methylene moiety remaining from the tyrosine group (or alternatively defined methylene can be considered to be part of B, and LB1is absent). It is known in the art to modify biomolecules with click probe F (or F1), and the skilled person understands which linkers LB1may be used to connect a click probe to a biomolecule.

[0244] LB2is the linker connecting Z1and Z2, and may also be referred to as “intermediate linker”. Linker LB2is connected to y1 occurrences of Z1(or Q1) and y2 occurrences of Z2(or F2). Thus, the valency of linker LB2is 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 / Q1and one Z2 / F2and the linker has a valency of 1 + 1 = 2. Thus, when y1 = 1 and y2 = 1 , the linker LB2is bivalent. Likewise, in case y1 = 1 and y2 = 1 , the linker is connected to one Z1 / Q1and two Z2 / F2and the linker has a valency of 1 + 2 = 3. Thus, when y1 = 1 and y2 = 2, the linker LB2is trivalent. Thus, in case y1 = 1 and y2 = 3, the linker is connected to one Z1 / Q1and three Z2 / F2and the linker has a valency of 1 + 3 = 4. Thus, when y1 = 1 and y2 = 3, the linker LB2is tetravalent. Thus, in case y1 = 2 and y2 = 1 , the linker is connected to one Z1 / Q1and four Z2 / F2and the linker has a valency of 2 + 1 = 1. Thus, when y1 = 2 and y2 = 1 , the linker LAis trivalent. Preferably, linker LB2is bivalent or trivalent.

[0245] Linker LB2may be referred to as “heterobifunctional”, which means that is contains two different functionalities, referring to the chemically different connectivities to Z1 / Q1and Z2 / F2.

[0246] In a particular preferred embodiment, linker LB2prior to conjugation, i.e. (Q1)yi-LB2-(F2)2 is “homofunctional”, which means that it contains the same chemical reactive groups, in other words it is preferred that Q1is equal to F2, more preferably Q1= F2= a (hetero)cycloalkyne or a (hetero)cycloalkene, even more preferably Q1= F2= a (hetero)cycloalkyne, most preferably Q1= F2= BCN. This has the advantage that (Q1)yi-LB2-(F2)2 is more easily synthesized, leading to an overall facile synthesis route for preparing targeting extracellular vesicles. In this embodiment, F1and Q2may also be the same which means Z1is the same as Z2, or F1and Q2are not the same, which means Z1is not equal to Z2. Herein, it is preferred that Z1is not equal to Z2.

[0247] In a preferred embodiment, LB2is a heterobifunctional linker of structure(L11)yi-BM-(L12)y2wherein:- each L11is connected to Q1or Z1, and each L12is connected to F2or Z2, wherein L11and L12each 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, PR13and 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 R13is as defined below for structure (23), preferably R13is 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.

[0248] In a more preferred embodiment, LB2is a heterobifunctional bivalent linker of structure(L11)-(L12) wherein:- L11is connected to Q1or Z1, and L12is connected to F2or Z2, wherein L11and L12each 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, PR13and 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 R13is as defined below for structure (23), preferably R13is H.

[0249] In an alternative more preferred embodiment, LB2is a heterobifunctional trivalent linker of structure (L11)-BM(L12)(L13) wherein:- L11is connected to Q1or Z1, L12is connected to F2or Z2, and L13is connected to Q1or Z1or to F2or Z2, wherein L11, L12and L13each 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, PR13and 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 R13is as defined below for structure (23), preferably R13is H; and- BM is a branching moiety. The branching moiety is further defined below.

[0250] In case linker LB2is a heterobifunctional trivalent linker with two connectivities to Q1or Z1, it is preferred that both L11and L13are identical. In case linker LB2is a heterobifunctional trivalent linker with two connectivities to Q2or Z2, it is preferred that both L12and L13are identical.

[0251] L11, L12and L13may for example be 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, C9-C200 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)yand NR21, wherein y’ is 0, 1 or 2, preferably y’ = 2, and R21is 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.

[0252] In the context of the present invention, a linker construct of structure (Q1)yi - LA- (F2)y2 reacts with B(F1)Z2, thereby performing z2 click reactions between F1and Q1per B, and thus forming z2 connecting groups Z1per B. Herein, z2 equals z x y1 . Preferably, y1 = 1 and thus z2 = z. Alternatively, y1 = 2 and thus z2 = 2 x z. Upon completion of the reaction, an antibody-linker construct is formed containing z x y2 click probes F2per B. The antibody-linker construct thus formed has a structure B [ (Z1)yi- LA(F2)y2 ]z.

[0253] In a preferred embodiment LB2comprises a Str moiety as defined above.

[0254] Preferably, L comprises a Str moiety as defined above. More preferably, the Str moiety is a PEG chain according to structure (S1). In an especially preferred embodiment, Str in the different B-L-V conjugates are monodisperse PEG chains.

[0255] In an especially preferred embodiment, both extracellular vesicle comprises both PEGylated lipids and as well as the conjugates comprise monodisperse PEG chains as defined above. In a preferred embodiment, the AVG of the PEG chains in the conjugate is equal to the AVG of the PEG chains in the PEGylated lipid.

[0256] In an alternative preferred embodiment, the ratio of the AVG of the PEG chain in the conjugate to the AVG of the PEG chain in the PEGylated lipid is 1 :1.1 - 1 : 5, preferably, 1 :1 .5 - 1 :3, more preferably 1 :1 .45 - 1 : 1 .55. In this embodiment it is especially preferred that that B is an antibody or fragment thereof, and that linker L is connected to the C terminus of the antibody.

[0257] In an alternative preferred embodiment, the ratio of the AVG of the PEG chain in the PEGylated lipid to the AVG of the PEG chain in the conjugate is 1 :1 .1 - 1 : 5, preferably, 1 :1 .5 - 1 :3, more preferably 1 :1.45 - 1 : 1.55. In this embodiment, it is especially preferred that B is an antibody and that linker L is connected to the glycan of the antibody.Process for synthesising the extracellular vesicle

[0258] In a further aspect, the present invention relates to a process for the preparation of the extracellular vesicles according to the invention. The inventors have developed three approaches towards the synthesis of these extracellular vesicles, which are versatile and high yielding. In the process according to the invention, a biomolecule having structure B [ LB(F)x ]z is provided in step (a), the moiety F is reacted with a linker-payload construct Q-LA-V in step (b), an extracellular vesicle is provided in step (c), optionally in the presence of moiety V, or optionally the moiety is V is embedded within the extracellular vesicle in separate step (d).

[0259] More in particular, the process according to the invention is according to one of the following three options (1), (2) or (3):(1 a) providing a biomolecule having structure B [ LB(F)x ]z, wherein F is a click probe, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(l b) reacting B(LB(F)x)z with a linker-payload construct Q-LA-V, wherein Q is a click probe that is reactive towards F, LBis a linker and V is a hydrophobic moiety, to obtain a conjugate of structure B [LB(Z-LA- V)x ]z;(l c) providing an extracellular vesicle; and(ld) incorporating B [LB(Z-LA-V)X]z into the membrane of the extracellular vesicle, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane; or(2c) providing an extracellular vesicle;(2d) incorporating linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, into the membrane of the extracellular vesicle to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(2a) providing a biomolecule having structure B [ LB(F)X]z, wherein F is a click probe that is reactive towards Q, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(2b) reacting B[LB(F)x]z with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle; or(3c) assembling an extracellular vesicle in the presence of linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(3a) providing a biomolecule having structure B [ LB(F)X]z, wherein F is a click probe that is reactive towards Q, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(3b) reacting B[LB(F)x]z with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle.Step (a)

[0260] In step (a), a biomolecule having structure B [ LB(F)X]z is provided, wherein biomolecule B comprises x x z click probes F, wherein x is 1 , 2, 3 or 4, and z is 1 or 2. Linker LBconnects x occurrences of click probe F to the biomolecule B, and each biomolecule B comprises z branches of LB(F)X. The skilled person is capable of provided biomolecules comprising click probes, e.g. from WO 2014 / 065661 , WO2014 / 177771 , W02020188061.

[0261] In a preferred embodiment, biomolecule B is 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.

[0262] The starting material, i.e. the antibody comprising two or four, preferably two, core-GIcNAcsubstituents, 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 WO2017 / 137459, see Examples 1 - 3, and SEQ ID No: 1 , which is incorporated by reference herein.

[0263] 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-AzGlc-UDP, 6- AzGIcNAz-UDP and 2-(but-3-yonic acid amido)-2-deoxy-galactose-UDP. Most preferably, S(F)-P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0264] 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 N- 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 N- acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or A / -acetylgalactosaminyltransferases, preferably a mutant N-acetylgalactosaminyltransferase. Mutant enzymes described in WO 2016 / 022027and 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 fucosylated or not.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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 or transglutaminase 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 or WG2020 / 188061 .

[0269] It is further known to transform a tyrosine residue into an o / Yho-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.

[0270] Alternatively, a serine group is oxidized into a nitrone as described in Ning, Xinghai, et al. "Protein modification by strain-promoted alkyne-nitrone cycloaddition." Angewandte Chemie (International ed. in English) 49.17 (2010): 3065. Typically this concerns a N-terminal serine group, as the nitrone is obtained via an aldehyde intermediate that requires a primary amine group. However, a serine amino acid that is connected to for instance a branched lysine is also suitable, as that will also have a primary amine group. Preferably, the serine group is on the N-terminus.

[0271] 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.

[0272] In an especially preferred embodiment, step (a) encompasses step (ai) and step (aii), wherein step (ai) is the modification of a biomolecule with a click moiety as described above and step (aii) concerns reacting the click modified biomolecule with a linker (Q1)yi-LB2-(F2)y2, wherein Q1and F2are click probes non-reactive towards each other, and y1 and y2 are integers in the range of 1 -4, preferably integers in the range of 1-2.

[0273] In this embodiment, the modified biomolecule obtained from step (ai) can be described as B [ LB1(F1)a]b Herein, a and b are integers in the range of 1 -4, preferably in the range of 1 -2. The value of a, b, y1 and y2 determines the structure of the conjugate and ultimately the amount of V per biomolecule. In the embodiment, in which step ai concerns transferring a S(F1) moiety to the core-GIcNAc moiety a =1 and b=2. Reacting that with linker (Q1)yi-LB2-(F2)y2, wherein y1 =2 and y2=1 results in a biomolecule having structure B[(-LB1-Z1-)2LB1-F2] which can also be described as B[LBF]. Alternatively, B [ LB1(F1)]2 can also be reacted with (Q1)yi-LB2-(F2)y2, wherein y1 =1 and y2 =1 , which results in a biomolecule having structure B[- LBi-Zi-LB2-F2]2, i.e. B[LB-F]2. Consequently, the ratio between y1 and y2 determines, compared to B[LB1(F1)a]b, whether the intermediate linker introduces additional click probes, i.e. y2 / y1 > 1 , reduces the amount of click probes y2 / y1 < 1 or the amount of click probes remains the same. The ratio between B and F2is defined by a x b x y2 / y1 .

[0274] The reactions performed in steps (aii) are click reactions, wherein click probes F1react with click probes Q1to form connecting groups Z1. This conjugation technique is known to the skilled person. The present reactions occur under conditions such that Q1is reacted with F1to form covalent connections. Herein, F2does not react with Q1as that would mean that the intermediate linker is inherently unstable. Preferably, F2does not react with F1. In an alternative, preferred embodiment, F2is equal to Q1. In the process according to the invention, Q1reacts with F1, forming a covalent connection between the biomolecule and F2. Complementary reactive groups Q1and reactive groups F1are known to the skilled person and are described in more detail below.

[0275] The skilled person is able to determine the optimal reaction conditions and stoichiometry of the reactants in order to obtain an optimal yield.

[0276] In a preferred embodiment, in step (aii) an azide on an azide-modified antibody reacts with a benzoannulated or tetramethylated (hetero)cycloalkyne group, preferably wherein Q1is according to structure (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19) or (Q19a), preferablyaccording to structure (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), (Q38a), (Q38b) or (Q38c), 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”.

[0277] In another preferred embodiment, in step (aii) a nitrone on a nitrone modified antibody reacts with a (hetero)cycloalkyne group or (hetero)cycloalkyne group, preferably wherein Q1is according to structure (Q2)-(Q56), preferably according to structure (Q26)-(Q29), (Q38a), (Q38b) or (Q38c), more preferably according to structure (Q40)-(Q43) or according to structure (Q37), (Q42) or (Q43), via a cycloaddition reaction. By using these (hetero)cycloalkyne or (hetero)cycloalkene groups, the presence of a catalyst is not required, and the cycloaddition reaction may even occur spontaneously by a reaction called strain- promoted nitrone-alkyne cycloaddition (SPANC). This is one of the reactions known in the art as “metal- free click chemistry”.

[0278] In yet another preferred embodiment, in step (aii) an o / Yho-quinone on an o / Yho-quinone modified antibody reacts with a (hetero)cycloalkyne group or (hetero)cycloalkyne group, preferably wherein Q1is according to structure (Q2)-(Q56), preferably according to structure (Q26)-(Q29), (Q38a), (Q38b) or (Q38c), more preferably according to structure (Q40)-(Q43) or according to structure (Q37), (Q42) or (Q43), via a cycloaddition reaction. By using these (hetero)cycloalkyne or (hetero)cycloalkene groups, the presence of a catalyst is not required, and the cycloaddition reaction may even occur spontaneously by a reaction called strain-promoted oxidation-controlled cyclooctyne-1 ,2-quinone cycloaddition (SPOCQ). This is one of the reactions known in the art as “metal-free click chemistry”.Step (b)

[0279] In step (b), the click probe F is reacted with a linker-payload construct Q-LA-V, wherein Q is a click probe that is reactive towards F and V is a hydrophobic moiety. Linker LBconnects Q to V. Step (b) may occur with the free linker-payload construct or with the linker-payload construct having moiety V embedded within the membrane of the extracellular vesicle. The reaction of step (b) can be performed without problem for both situations. Even if the linker-payload construct is embedded within the membrane, moiety Q sticks out of the membrane and is available for a click reaction with moiety F.

[0280] In step (b), a click reaction is performed. Herein 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 B and V. Complementary reactive groups Q and reactive groups F are known to the skilled person and are described in more detail below.

[0281] Thus, the click 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).

[0282] 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 Q1is 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), (Q38a), (Q38b) or (Q38c), 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”.

[0283] In a preferred embodiment of step (b), the modified antibody-linker construct B[-(LB1-(Z1)a)b-(LB2- (F2)y2)c]d; is reacted with a hydrophilic moiety V-linker, comprising a reactive group Q2capable of reacting with reactive group F2, to obtain an antibody-conjugate, containing connecting group Z2resulting from the reaction between Q2and F2. Such reaction occurs under condition such that reactive group Q2is reacted with the reactive group F2to covalently link the antibody to the payloads. Step (b) may also be referred to as the conjugation reaction. In step (b), the reaction occurs with y2 x c x d equivalents of Q2-LA-V, although more equivalents of Q2-LA-V 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.

[0284] In step (b), the conjugate is formed by covalently connecting one or more hydrophilic moieties V 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.

[0285] 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 Q1is 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 (c)

[0286] In step (c), an extracellular vesicle is provided. The skilled person is well aware how to prepare extracellular vesicles, and any suitable method can be used in step (c). In one embodiment, the extracellular vesicle is assembled in the absence of linker-payload construct Q-LA-V, and moiety V is separately incorporated into the membrane of the extracellular vesicle in step (d). Alternatively, step (c) is performed in the presence of linker-payload construct Q-LA-V, in which case moiety V is embedded within themembrane of the extracellular vesicle during the assembly of step (c) and step (d) does not need to be performed.

[0287] In an especially preferred embodiment, the extracellular vesicle is a lipid nanoparticle and is provided in step (c) by mixing an aqueous buffer comprising an active substance, preferably a nucleic acid, with an ethanol solution comprising membrane lipids, optionally linker-payload construct Q-LA-V in the case of step (3c), and optionally stabilizing lipids.

[0288] In a preferred embodiment, step (c) encompasses step (ci) and (cii). Herein, step (ci) is the assembly of the extracellular vesicle in the presence of linker-payload construct Q1-LA-V. Step (cii) concerns reacting the embedded Q1-LA-V with Q1yi-LB2-F2y2 to obtain Q1yi-LB2-(Z2-LA-V)y2. In an alternative preferred embodiment, step (ci) is present but step (cii) is absent.Step (d)

[0289] In step (d), moiety V is embedded within the membrane of the extracellular vesicle. In one embodiment, step (d) occurs with the free linker-payload construct Q-LA-V, to provide an extracellular vesicle comprising click probes Q connected to the membrane. Alternatively, step (d) occurs with the conjugate of B with V, having structure B [LB(Z-LA-V)x ]z, i.e. after the click reaction of step (b). The embedding of step (d) can be performed without problem for both situations. Even if moiety Q already reacted with the biomolecule having structure B [ LB(F)x ]z, moiety V is available for embedding within the membrane of the extracellular vesicle.In a preferred embodiment, step (d) encompasses step (di) and step (dii). Herein step (i) involves incorporating linker-payload construct Q2-LA-V, into the membrane of the extracellular vesicle to obtain an extracellular vesicle wherein moiety V is embedded in the membrane. Step (2dii) is reacting the embedded linker-payload construct Q2-LA- V with of (Q1)yi-LB2-(F2)y2 to obtain (Q1)yi-LB2-(Z2-LA-V)y2in an alternative preferred embodiment, step (di) is present but step (dii) is absent.Preferred processes

[0290] As described above, in a preferred embodiment, the process involves the use of an intermediate linker.

[0291] Thus, in a preferred embodiment, the process is according to the first option and comprises:(1 ai) providing a biomolecule having structure B [ LB1(F1)a]b;(1 aii) reacting B [ LB1(F1)a]b with c equivalence of (Q1)yi-LB2-(F2)y2 to provide B[-(LB1-(Z1)a)b-(LB2-(F2)y2)c]d;(l b) reacting B[-(LB1-(Z1)a)b-(LB2-(F2)y2)c]d; with y2 * c * d equivalence of linker-payload construct Q2- LA-V, to obtain a conjugate of structure B[-(LB1-(Z1)a)b-(LB2- (Z2-LA-V)y2)c]d;(l c) providing an extracellular vesicle; and(1 d) incorporating B[-(LB1-(Z1)a)b-(LB2- (Z2-LA-V)y2)c]d into the membrane of the extracellular vesicle, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;

[0292] In this preferred embodiment, LB(F)Xis thus LB1-((Z1)yi-LB2-(F2)y2)c, wherein x is equal to y2*c.

[0293] In another preferred embodiment, the process is according to the second option and comprises the following steps:(2c) providing an extracellular vesicle;(2di) incorporating linker-payload construct Q2-LA-V, into the membrane of the extracellular vesicle to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(2dii) reacting the embedded linker-payload construct Q2-LA- V with of (Q1)yi-LB2-(F2)y2 to obtain (Q1)yi- [_B2_(Z2_LA_V)y2(2a) providing a biomolecule having structure B [ LB1(F1)a]b, wherein F1is a click probe that is reactive towards Q1, LB1is a linker linking biomolecule B to a occurrences of F1, a is 1 , 2, 3 or 4, and b is 1 or 2;(2b) reacting B [ LB1(F1)a]b with linker-payload construct (Q1)yi-LB2-(Z2-LA-V)y2 embedded in the membrane of the extracellular vesicle to obtain conjugate B[-(LB1-(Z1)a)b-(LB2- (Z2-LA-V)y2)c]d embedded in the membrane of the extracellular vesicle, or(2c) providing an extracellular vesicle;(2d) incorporating linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, into the membrane of the extracellular vesicle to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(2ai) providing a biomolecule having structure B [ LB1(F1)a]b, ;(2aii) reacting B [ LB1(F1)a]bwith (Q1)yi-LB2-(F2)y2to provide B[-(LB1-(Z1)a)b-(LB2-(F2)y2)c]d;(2b) reacting B[-(LB1-(Z1)a)b-(LB2-(F2)y2)c]d with of linker-payload construct Q2-LA-V embedded in the membrane of the extracellular vesicle.

[0294] In another preferred embodiment, the process is according to the third option and comprises the following steps:(3c) assembling an extracellular vesicle in the presence of linker-payload construct Q-LA- V to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(3ai) providing a biomolecule having structure B [ LB1(F1)a]b;(3aii) reacting B [ LB1(F1)a]bwith (Q1)yi-LB2-(F2)y2to provide B[-(LB1-(Z1)a)b-(LB2-(F2)y2)c]d;(3b) reacting B[-(LB1-(Z1)a)b-(LB2-(F2)y2)c]d with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle, or(3ci) assembling an extracellular vesicle in the presence of linker-payload construct Q2-LA- V, wherein Q2is a click probe, and V is a hydrophobic moiety, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(3cii) reacting the embedded linker-payload construct Q2-LA- V with (Q1)yi-LB2-(F2)y2 to obtain (Q1)yi-LB2- (Z2-LA-V)y2 embedded in the membrane;(3a) providing a biomolecule having structure B [ LB1(F1)a]b;(3b) reacting B [ LB1(F1)a]b with the linker-payload construct (Q1)yi-LB2-(Z2-LA-V)y2 embedded in the membrane of the extracellular vesicle.Reactive moiety Q

[0295] Reactive moieties Q, in some embodiments referred to as Q1or Q2, are click probes. In the context of the present invention, Q refers to Q1and 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.

[0296] 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 ortho-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.

[0297] 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.

[0298] 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;- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- Y2is C(R31)2, O, S, S<+)R31, S(O)R31, S(O)=NR31or NR31, wherein S<+> is a cationic sulphur atom counterbalanced by B< >, wherein B< > is an anion, and wherein each R31individually is R15or 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;

[0299] 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’).

[0300] In a preferred embodiment of structure (Q1), reactive group Q comprises a (hetero)cycloalkynyl group and is according to structure (Q1 a):Herein,- R15and Y2are 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.

[0301] 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.

[0302] 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)

[0303] Herein, the connection to the linker (e.g. LA, LBor 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, p is an anion, which is preferably selected from <->OTf, CI(->, Br(~> or |(->, most preferably p is <->OTf. p(+)is a cation, preferably a pharmaceutically acceptable cation. In the conjugation reaction, p does not need to be a pharmaceutically acceptable anion, since p 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 readilyuseable as medicament. R36is an halogen selected from fluoro, chloro, bromo and iodo, preferably R36is fluoro. Y4is a heteroatom, preferably Y4is O or NH. R35is 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 NR14wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably R35is selected from H, C5H11, CH3, CH2CH3, CH2OH or CH2OTBS.

[0304] In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21) - (Q38a) depicted here below.(Q38b) (Q38c) (Q38d)

[0305] Herein, p is an ion, p is an anion, which is preferably selected from <->OTf, Cl<-), Br<-) or |(->, most preferably p is <->OTf, and p<+> is a cation. Groups R35and R36on (Q38b), (Q38c) and (Q38d) are defined above for (Q20a) - (Q20c), which equally applies here.

[0306] Herein, R35is selected from the group consisting of hydrogen, C1-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(R14)2, S and NR14wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups.

[0307] 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.

[0308] 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.

[0309] 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.

[0310] 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 Y1is O or NR11, wherein R11is 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.

[0311] In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group and is according to structure (Q37).

[0312] In an especially preferred embodiment, Q comprises a cyclooctynyl group and is according to structure (Q42):Herein:- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- R18is 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;- R19is 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 R19forms a second connection of trivalent linker LA, wherein BM is the carbon atom to which R19is attached; and- I is an integer in the range 0 to 10.

[0313] In a preferred embodiment of the reactive group according to structure (Q42), R15is independently selected from the group consisting of hydrogen, halogen, -OR16, Ci - Ce alkyl groups, C5 - Ce (hetero)aryl groups, wherein R16is hydrogen or Ci - Ce alkyl, more preferably R15is independently selected from the group consisting of hydrogen and Ci - Ce alkyl, most preferably all R15are H. In a preferred embodiment of the reactive group according to structure (Q42), R18is independently selected from the group consisting of hydrogen, Ci - Ce alkyl groups, most preferably both R18are H. In a preferred embodiment of the reactivegroup according to structure (Q42), R19is H. In a preferred embodiment of the reactive group according to structure (Q42), I is 0 or 1 , more preferably I is 1 .

[0314] In an especially preferred embodiment, Q comprises a (hetero)cyclooctynyl group and is according to structure (Q43):Herein:- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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.

[0315] In a preferred embodiment of the reactive group according to structure (Q43), R15is independently selected from the group consisting of hydrogen, halogen, -OR16, -S(O)3( ), Ci - Ce alkyl groups, C5 - Ce (hetero)aryl groups, wherein R16is hydrogen or Ci - Ce alkyl, more preferably R15is 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.

[0316] 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 trans- (hetero)cyclooctenyl group, a frans-(hetero)cyclononenyl group or a frans-(hetero)cyclodecenyl group, which may all optionally be substituted. Especially preferred are (hetero)cyclopropenyl groups, trans- (hetero)cycloheptenyl group or frans-(hetero)cyclooctenyl groups, wherein the (hetero)cyclopropenyl group, the frans-(hetero)cycloheptenyl group or the frans-(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 accordingto structure (Q46), a frans-(hetero)cycloheptenyl moiety according to structure (Q47) or a trans- (hetero)cyclooctenyl moiety according to structure (Q48). Herein, Y3is selected from C(R23)2, NR23or O, wherein each R23is 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 trans- (hetero)cyclooctene group is according to structure (Q52), (Q53), (Q54), (Q55) or (Q56).(Q52) (Q53) (Q54) (Q55) (Q56)

[0317] Herein, the R group(s) on Si in (Q50) and (Q51) are typically alkyl or aryl, preferably Ci-Ce alkyl.

[0318] In a preferred embodiment, click probes Q comprise a moiety selected from (Q1) - (Q56), more preferably is a moiety selected from (Q1) - (Q56).

[0319] In the present invention, the exact structure of Q1and Q2should preferably differ, as Q1is preferably not reactive towards F2, whereas Q2is reactive towards F2.

[0320] In a preferred embodiment, F1is azide and Q1is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F2is tetrazine or nitrone and Q2is bicyclononyne or cycloalkene, such as a trans- cyclooctene or a cyclopropene. More preferably, F1is azide and Q1is an benzoannulated or tetramethylated (hetero)cycloalkyne, while F2is tetrazine and Q2is bicyclononyne. Herein, Q1is 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, Q2is 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, Q2is preferably according to structure (Q8), more preferably according to structure (Q29), most preferably according to structure (Q42).

[0321] In another preferred embodiment, F1is a nitrone and Q1is a (hetero)cycloalkene or (hetero)cycloalkene, while F2is a (hetero)cycloalkene or (hetero)cycloalkene and Q2is a tetrazine, more preferably both Q1and F2are a (hetero)cycloalkyne, more preferably both Q1and F2are bicyclononyne.

[0322] In another preferred embodiment, F1is a ortho-quinone and Q1is a (hetero)cycloalkene or (hetero)cycloalkene, while F2is a (hetero)cycloalkene or (hetero)cycloalkene and Q2is a tetrazine, more preferably both Q1and F2are a (hetero)cycloalkyne, more preferably both Q1and F2are bicyclononyne.Reactive moiety F

[0323] Reactive moieties F, in some embodiments referred to as F1or F2, are click probes. In the context of the present invention, F refers to F1and 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, an ortho-quinone moiety or a tetrazine moiety.

[0324] 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.

[0325] 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.

[0326] 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) - (F10) depicted here below.

[0327] Herein, the wavy bond represents the connection to B or LA. For (F3), (F4), (F8) and (F9), 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 NR32wherein R32is 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.

[0328] In a preferred embodiment, (F3) is according to structure (F3a) or (F3b):R42R420° ©(F3a) (F3b)

[0329] Herein, the wavy bond represent the connection to B, R41is preferably 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 NR32wherein R32is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. More preferably, R41is L10XR4, wherein L10is a linker of structure (C(R3)2)z, wherein z is 2 or 3, and each R3is individually selected from H and C1-4 alkyl, wherein two occurrences of R3may be joined together to form a C3-6 (hetero)cycloalkyl group, R4is selected from H and C1-4 alkyl, X is S, O or N. In an alternative preferred embodiment R41is methyl or hydrogen. Herein, each R42group is preferably selected from H and C1-4 alkyl, preferably R42is H.

[0330] Preferred embodiments of L10are (L10A) - (L10P):

[0331] Herein, the wavy bonds labelled with * and ** are connected to the nitrogen atom of the nitrone and to XR4. Preferably, 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.

[0332] X is S, O or NH. Most preferably, X is O. R4is selected from H and C1-4 alkyl. Typically, R4is H when X is O or NH, and R4is H or C1-4 alkyl when X is S. Thus, XR4is typically selected from OH, NH2, SH and S-C1-4 alkyl. In a preferred embodiment, XR4is SH, OH, NH2, most preferably XR4is OH.

[0333] The nature of R42is not crucial for the present invention, and any suitable substituent for a nitrone compound can be used. The R42groups may be the same or different, typically they are different. Each of R42may correspond to R42as comprised in ring (Zh) and (Zn), defined above. In case, the R42are 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.

[0334] Typically, R42is selected from H and Ci - Ce (cyclo)alkyl. In a preferred embodiment, R42is selected from H and Ci - C5 (cyclo)alkyl, more preferably R2ais H, Me or Et, most preferably R2ais H.

[0335] Alternatively, R42is 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)arylgroups 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 R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. Alternatively, the R42groups of (F3b) are joined to form a (hetero)cyclic moiety.

[0336] In a preferred embodiment, R42is hydrogen, a Ci - C20 alkyl group, preferably a Ci— C16 alkyl group, more preferably a Ci - C10 alkyl group. Herein, the alkyl group is optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR14, preferably O, wherein R14is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups. In another preferred embodiment, R42is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl or i-propyl, and even more preferably from the group consisting of hydrogen, methyl or ethyl.

[0337] In a preferred embodiment, (F4) is obtained from a photolytic cleavage of

[0338] In an especially preferred embodiment, F is a tetrazine according to structure (F8a):N=NH / hR29N-N(F8a)

[0339] Herein, R29is 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 R33is H or C1-4 alkyl. Preferably, R29is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl. It was found that R29is hydrogen gave optimal results in reactivity in the cycloaddition reaction. Thus, in a preferred embodiment, ring F, in particular F2, is (F8a) wherein R29is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl and pyrimidinyl, more preferably R29is hydrogen or methyl, most preferably R29is methyl.

[0340] 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, ortho-quinone and nitrone, more preferably from azide, tetrazine and nitrone. In a preferred embodiment, F1is an azide or nitrone, and F2is iminosydnone, catechol, which forms a ortho-quinone group in situ, tetrazine or tetrazole. More preferably, F2is iminosydnone according to structure (F7), catechol, which forms structure (F10) in situ, tetrazine accordingto structure (F8) or tetrazole according to structure (F11). Even more preferably, F1is an azide according to structure (F1) or nitrone according to structure (F3), more preferably a nitrone according to (F3a), and F2is a tetrazine according to structure (F8a) or tetrazole according to structure (F11). Most preferably, F1is an azide according to structure (F1) and F2is a tetrazine according to structure (F8a).

[0341] Especially preferred molecules V-LA-Q are structures (V22)-(V25), wherein n is an integer in the range of 1-100, preferably 20-50, more preferably n = 24 or 45, preferably monodisperse as described above:

[0342] Especially preferred embodiments of intermediate linker Q1yi-LB2-F2y2 are structures (T1)-(T3), wherein n is an integer in the range of 1 -100, preferably 20-50 more preferably n =24 or 48, preferably structures (V22)-(V23) and (V25) have a monodisperse PEG chain as described above:

[0343] It is especially preferred that (V22) is used in the process according to the first or second option without the use of an intermediate linker. It is especially preferred that (V23) is used in the process according to the first or second option with the use of intermediate linker (T2). In another preferred embodiment, the process is according to the third option, wherein in step (3c) (V24) is used and in step (bii) intermediate linker (T1) is used. In an especially preferred embodiment, the intermediate linker (T3) is used in combination with (V25).Ultrafast Click chemistry

[0344] In all aspects of the invention, it is preferred that the click reaction between F2and Q2is performed by ultrafast click. In some particular preferred embodiments, the click reaction between F1and Q1is also performed by ultrafast click.

[0345] 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.

[0346] 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 103times greater, even more preferably at least 104times greater, most preferably at least 106times 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 103L / mol s, preferably at least 5 x 103L / mol s, or even at least 1 x 104L / 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.

[0347] 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.

[0348] 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) - (Q38a), 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) - (Q38a), most preferably according to structure (Q29);(vii) between click probe F (or F2) being o / Yho-quinone and click probe Q (or Q2) being (hetero)cycloalkyne, preferably wherein the o / Yho-quinone is according to structure (F10) and the (hetero)cycloalkyne according to (Q1), more preferably according to structure selected from (Q21) - (Q38a), most preferably according to structure (Q29).

[0349] 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 104L / mol s have been reported for bicyclononyne / tetrazole click reactions, and of 105to 106L / 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.

[0350] The inventors found that such conjugation reactions are efficiently performed using ultrafast click chemistry.

[0351] To reflect the improved reaction at lower amounts of both modified biomolecule 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 linker-payload 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%.

[0352] 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. The reducedreaction time is of great benefit for the present invention as that reduces the risk that the extracellular vesicles degrade or become contaminated during the conjugation step.Application

[0353] The extracellular vesicles of the present invention have a high homogeneity. The extracellular vesicles of the present invention are further characterized by a high stability, a low tendency to aggregate, excellent therapeutic efficacy and tolerability, improved targeting of the target cells and increased delivery and uptake of an active compound within the extracellular vesicles by the target cells. Hence, the extracellular vesicle according to the present invention is ideally suitable for medical applications as defined below. Hence, the extracellular vesicles of the present invention may be used for one or more of: reducing the tendency to aggregate, improving the therapeutic efficacy and tolerability, improving targeting of target cells, increasing delivery of an active compound within the extracellular vesicles to target cells and increasing uptake of an active compound within the extracellular vesicles by target cells. Such reduction is achieved over extracellular vesicles which are not targeted and over targeted extracellular vesicles which are prepared with a different conjugation technique. The invention thus further concerns a pharmaceutical composition comprising the extracellular vesicle according to the present invention and optionally a pharmaceutically acceptable carrier or excipient.

[0354] In that light, the invention further concerns a method for in vivo chimeric antigen receptor (CAR) therapy. CAR therapy is known in the art, in particular CAR T cell therapy which involves genetic modification of the patient’s T lymphocyte. Upon binding to their targets, CARs induce intracellular signalling that results in antigen-specific killing of the target cell and simultaneous proliferation of the CAR T cell. Thus, the extracellular vesicles described herein may be used to target and modify immune cells. In an especially preferred embodiment, the extracellular vesicles may be used to modify T cells. Preferably, T- cells may include any subpopulation of T-cells, e.g., CD4+, CD8+, gamma-delta, naive T cells, stem cell memory T cells, central memory T cells, or a mixture of subpopulations. In some embodiments, the extracellular vesicles may be used to deliver or modify a T-cell receptor (TCR) in a T cell. In an especially preferred embodiment, the extracellular vesicles may be used to deliver at least one chimeric antigen receptor (CAR) to T-cells. For instance, in specific embodiments, the extracellular vesicles can be used to deliver mRNA encoding a CAR to T-cells. In some embodiments, the extracellular vesicles may be used to deliver at least one CAR to natural killer (NK) cells. In some embodiments, the extracellular vesicles can be used to deliver at least one CAR to natural killer T (NKT) cells. In some embodiments, the extracellular vesicles may be used to deliver at least one CAR to a progenitor cell, e.g., a progenitor cell of T, NK, or NKT cells. In some embodiments, cells modified with at least one CAR (e.g., CAR-T cells, CAR-NK cells, CAR-NKT cells), or a combination of cells modified with at least one CAR (e.g., a mixture of CAR-NK / T cells) are used to treat a condition as identified in the targetable landscape of CAR therapies in MacKay, et al. Nat Biotechnol 38, 233-244 (2020), incorporated by reference herein in its entirety. In some embodiments, the immune cells comprise a CAR specific to a tumor or a pathogen antigen selected froma group consisting of AChR (fetal acetylcholine receptor), ADGRE2, AFP (alpha fetoprotein), BAFF-R, BCMA, CAIX (carbonic anhydrase IX), CCR1 , CCR4, CEA (carcinoembryonic antigen), CD3, CD5, CD8, CD7, CD10, CD13, CD14, CD15, CD19, CD20, CD22, CD30, CD33, CLLI, CD34, CD38, CD41 , CD44, CD49f, CD56, CD61 , CD64, CD68, CD70,CD74, CD99,CD117, CD123, CD133, CD138, CD44v6, CD267, CD269, CDS, CLEC12A, CS1 , EGP-2 (epithelial glycoprotein-2), EGP-40 (epithelial glycoprotein-40), EGFR(HERI), EGFR-VIII, EpCAM (epithelial cell adhesion molecule), EphA2, ERBB2 (HER2, human epidermal growth factor receptor 2), ERBB3, ERBB4, FBP (folate-binding protein), Flt3 receptor, folate receptor-a, GD2 (ganglioside G2), GD3 (ganglioside G3), GPC3 (glypican-3), GPI00, hTERT (human telomerase reverse transcriptase), ICAM-1 , integrin B7, interleukin 6 receptor, IL13Ra2 (interleukin- 13 receptor 30 subunit alpha-2), kappa-light chain, KDR (kinase insert domain receptor), LeY (Lewis Y), LI CAM (LI cell adhesion molecule), LILRB2 (leukocyte immunoglobulin like receptor B2), MARTI, MAGE-A1 (melanoma associated antigen Al), MAGE- A3, MSLN (mesothelin), MUC16 (mucin 16), MUCI (mucin I), KG2D ligands, NY-ESO-1 (cancer-testis antigen), PRI (proteinase 3), TRBCI, TRBC2, TFM-3, TACI, tyrosinase, survivin, hTERT, oncofetal antigen (h5T4), p53, PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), hRORI, TAG-72 (tumor- associated glycoprotein 72), VEGF-R2 (vascular endothelial growth factor R2), WT-1 (Wilms tumor protein), and antigens of HIV (human immunodeficiency virus), hepatitis B, hepatitis C, CMV (cytomegalovirus), EBV (Epstein-Barr virus), HPV (human papilloma virus).

[0355] In some embodiments, an extracellular vesicle as described herein is administered to an immune cell, e.g., a T-cell, NK cell, NKT cell, or progenitor cell ex vivo or in vitro to deliver a therapeutic payload (e.g., a gene modifying system) and then the cells are delivered to a patient. In some embodiments, immune cells, e.g., T-cells, NK cells, NKT cells, or progenitor cells are modified ex vivo or in vitro and then delivered to a patient. In some embodiments, a nucleic acid (e.g., DNA or RNA, such as mRNA) is delivered by one of the methods mentioned herein, and immune cells, e.g., T-cells, NK cells, NKT cells, or progenitor cells are modified in vivo in the patient. In some embodiments the patient is a human patient such as a human patient in need of such treatment.

[0356] In certain embodiments, biomolecule B is a T-cell targeting moiety, for example, an antibody, Fab fragment or ScFv, that binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CDS, CD28, CD137, CD45, T-cell receptor (TCR)p,TCR-a, TCR-a / p, TCR-y / 5, PD1 , CTLA4, fl M3. LAG3, CD 18, IL-2 receptor, CDI la, TLR2, TLR4, TLR5, IL-7 receptor, or IL-15 receptor.

[0357] In some embodiments, an extracellular vesicle as described herein is administered to an HSC (e.g., a LT-HSC) or a HSC progenitor ex vivo or in vitro to deliver a therapeutic payload (e.g., a gene modifying system) and then the cells are delivered to a patient. In some embodiments, an extracellular vesicle as described herein is administered to an HSC (e.g., a LT-HSC) or a HSC progenitor in vivo to deliver a therapeutic payload (e.g., a gene modifying system). In some embodiments, HSCs (e.g., LT-HSCs) or HSC progenitor cells are modified ex vivo or in vitro and then delivered to a patient. In some embodiments, HSCs (e.g., LT-HSCs) or HSC progenitor cells are modified in vivo in the patient. In some embodiments the patientis a human patient such as a human patient in need of such treatment.

[0358] In certain embodiments, the targeting moiety is a HSC targeting moiety, for example, an antibody. Fab fragment or ScFv, that binds to an HSC antigen selected from CD90 and CD1 17.

[0359] 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 extracellular vesicle according to the invention. The subject in need thereof is typically a patient suffering from the 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. The use of nucleotides 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 extracellular vesicle is typically administered in a therapeutically effective dose. The present aspect of the invention can also be worded as an extracellular vesicle according to the invention for use in the treatment of hereditary disease. In other words, this aspect concerns the use of an extracellular according to the invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of hereditary disease. In a preferred embodiment, the hereditary disease is selected from Duchenne muscular dystrophy, myotonic dystrophy, facioscapulohumeral muscular dystrophy (FSHD), spinal muscular atrophy, homozygous familial hypercholesterolemia, primary hyperoxaluria type 1 or lysosomal storage disease.

[0360] 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), myotonia, Bethlem myopathy, 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, 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, limb gridle muscular dystrophy, 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 extracellular 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, nociception related 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 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 extracellular vesicle according to the invention. The subject in need thereof is typically a patient suffering from the neuromuscular disease. The use of nucleotides is well-known in the field of the treatment of neuromuscular diseases, and the conjugates according to the invention are especially suited in this respect. In the method according to this embodiment, the extracellular vesicle is typically administered in a therapeutically effective dose. The present aspect of the invention can also be worded as an extracellular vesicle according to the invention for use in the treatment of neuromuscular disease.

[0361] In another preferred embodiment, the extracellular vesicle is used to treat a lung disease, preferably selected from alpha-1 antirypsin deficiency, cystic fibrosis, primary ciliary dyskinesia, primary pulmonary hypertension I, surfactant protein B deficiency.

[0362] In another preferred embodiment, the extracellular vesicle according to the invention can be used to treat a skin disease, preferably the skin disease is selected from epidermolysis Bullosa Dystrophica Recessive, Epidermolysis Bullosa Junctional, Epidermolytic Ichtyois, Hailey-Hailey disease, Lemellar Ichtoyosis, Nonbullous Congenital Ichtyosiform Erythroderma, Netherton syndrome.

[0363] In another preferred embodiment, the extracellular vesicle is used to treat a liver disease, preferably a liver disease selected from Acute intermittent porphyria, Alagi lie syndrome, Alpha-1 antitrypsin deficiency, Carbamyol phosphate synthetase I deficiency, Citrullinemia I, Crigler-Najjar, Fabry, Familial chylomicronemia syndrome, Gaucher, GSD1 a, GSD IV, Heme A, Heme B, HoFH, Methylmalonic acidemia, MPS II, MPS III, MPS IV, MSUD, OTC Deficiency, Polycystic Liver Disease, Pompe, Primary Hyperoxaluria I, Progressive familial intrahepatic cholestasis types 1 -3, Propionic acidemia, Wilson’s Disease.

[0364] In another preferred embodiment, the extracellular vesicle of the invention is used to treat hematopoietic stem cell disease (HCS), preferably the disease is selected from adrenoleukodystrophy (CALD), Alpha-mannosidosis, Fanconi anemia, Gaucher disease, Globoid cell leukodystrophy (Krabbe disease), Hemophagocytic lymphohistiocytosis, Malignant infantile osteopetrosis-autosomal recessive ostepetrosis, Metachromatic leukodystrophy, MPS 1 S (Scheie syndrome), MPS2, MPS7, Mucolipidosis II, Niemann-Pick disease A and B, Niemann-Pict disease C, Pompe disease, Sickle cell disease (SCD), Tay Sachs, Thalassemia.

[0365] In another preferred embodiment, the extracellular vesicle of the invention can be used to treat a kidney disease, preferably the kidney disease is selected from congenital nephrotic syndrome, cystinosis.

[0366] In another preferred embodiment, the extracellular vesicle according to the invention can be usedas vaccine, preferably the vaccine is against a virus, a bacterium, a parasite or a cancer type.

[0367] In another preferred embodiment, the extracellular vesicle 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. In this embodiment, it is especially preferred that the active compound is a cytotoxic agent. Alternatively, the method of treatment concerns cancer and the active compound is a nucleotide which interferes with the production of receptor proteins so that the receptor proteins are modified and more easily recognized by the immune system or translates into such proteins. In the method according to this embodiment, the extracellular vesicle is typically administered in a therapeutically effective dose. The present aspect of the invention can also be worded as an extracellular vesicle according to the invention for use in the treatment of cancer or infection. In other words, this aspect concerns the use of an extracellular vesicle according to the invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of cancer or infection.Description of the figures

[0368] Figure 1 presents an overview of readouts from the characterization of tLNP3 by Nano Flow Cytometry. Fig 1 a: Separation of particles into no-low conjugated LNP (grey) and ligand positive tLNPs (black) was performed based on a fluorescence threshold gating (FITC-A signal ~ 100). FITC-A corresponds to the area of the fluorescence burst detected in the FITC channel. SS-A corresponds to the area of the burst in the side-scattering channel. Fig 1 b: Based on the calibration by the QC beads and the size standard beads, the concentration and size distribution of ligand positive tLNPs was obtained. Figl c: Calibration with the Equivalent number of Reference Fluorophore (ERF) standard beads enables the calculation of the ligand number distribution per tLNP (black; right peak) and LNP (gray; left peak) as well as Fig 1 d: The ligand density per 100 nm2 for tLNP (black; right peak) and no-low conjugated LNP (gray; left peak).

[0369] Figure 2 depicts the size of stability samples (tLNP2, tLNP8, tLNP9) incubated in either PBS supplemented with 10% sucrose (Fig. 2A), PBS supplemented with 10% sucrose and 5 mM glutathione (Fig. 2B), or PBS supplemented with 10% sucrose and 10% mouse serum (Fig. 2C).

[0370] Figure 3 depicts the relative luminescence unit (RLU) of stability samples collected after 0, 2, 4, or 6h incubation, subsequently transfection (80 ng) of seeded SK-BR-3 cells (20.000 cells / well) for 24h, followed by luciferase readout of samples incubated in either PBS supplemented with 10% sucrose (Fig. 3A), PBS supplemented with 10% sucrose and 5 mM glutathione (Fig. 3B), or PBS supplemented with 10% sucrose and 10% mouse serum (Fig. 3C).

[0371] Figure 4 depicts the ligand positive (%) of stability samples, measured by NanoFCM, after Oh, 24h, and 72h of incubation in either PBS supplemented with 10% sucrose (Fig. 4A), or PBS supplemented with 10% sucrose and 5 mM glutathione (Fig. 4B).

[0372] Figure 5 shows the dose-response curves in NCI-N87 cells. Normalized RLU percentage from NCI- N87 cells transfected (24h) with a concentration series of LNP1 (EC50 = 258.7 ng / mL), tLNP2 (EC50 = 162.7 ng / mL), tLNP7 (EC50 = 198.6 ng / mL), or tLNP9 (EC50 = 111.9 ng / mL).

[0373] Figure 6 depicts the relative luminescence unit (RLU) signal from tumor region quantification luminescence imaging in NCI-N87 tumor-bearing mice 6h after dosing LNPs / tLNPs (7 pg based on encapsulated flue mRNA was injected).Examples

[0374] The invention is illustrated by the following examples.1. General reagents and analytical conditions1. 1. Materials and general procedures for chemical synthesis

[0375] Chemicals were purchased from commonly used suppliers (Sigma-Aldrich, Acros, Alfa Aesar, Fluorochem, Apollo Scientific, Ltd and TCI) and were used without further purification. PEGylated compounds were obtained from Broadpharm, Iris biotech, and nanosoft biotechnology. Triphenylphosphine-3,3’,3”-trisulfonic acid trisodium salt (TPPTS) and mushroom Tyrosinase were purchased from Sigma-Aldrich. Gly-Gly-Gly-PEGs-BCN was purchased from Conju-Probe. T300 Andracon Recombinant Microbial Transglutaminase was obtained from Zedira. N-hydroxy-glycine was purchased from Santa Cruz Biotechnology. Methyltetrazine-NHS ester and methyltetrazine-propylamine HCI were purchased from Broadpharm. Reduced L-Glutathione and 5 / 6-carboxylfluorescein succinimidyl ester were purchased from ThermoFisher Scientific. (2,5-dioxopyrrolidin-1-yl) 2-bromoacetate was purchased from Combi-Blocks.

[0376] Solvents (including dry solvents) for chemical transformations, work-up and chromatography were purchased from Aldrich (Dorset, UK) at HPLC grade, and used without further distillation. Silica gel 60 F254 analytical thin layer chromatography (TLC) plates were from Merck (Darmstadt, Germany) and visualized under UV light, with potassium permanganate stain. Chromatographic purifications were performed using prepacked columns (Silicycle) in combination with a Teledyne LABS’ CombiFlash NextGen system. Reversed phase HPLC purifications were performed using a Teledyne LABS' CombiFlash NextGen system equipped with either a Waters Xbridge C18 column (5 pm OBD, 30 x 100 mm, PNI 86002982) or a Sylicycle C8 reversed phase column. Deuterated solvents used for NMR spectroscopy were obtained from Cambridge Isotope Laboratories.1.2. General procedure for analytical RP-UPLC

[0377] 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 Polyphenyl column, 450 A, 2.7 pm, 2.1 x 150 mm(Waters) with a column temperature of 70 °C. Absorbance of eluted peaks was measured at 215 nm followed by automated integration (MassLynx Waters) to determine reaction conversion.

[0378] For (modified) nanobodies: Prior to RP-UPLC analysis, nanobody was diluted to 1 mg / mL. During RP-UPLC, a linear gradient was applied in 11 minutes from 15 to 90% acetonitrile in 0.1 % TFA and water.

[0379] For (modified) monoclonal antibodies - DTT treated sample: Prior to RP-UPLC analysis, IgG (10 pL, 1 mg / mL in TBS pH 7.4) was added to 2.5 pL 200 mM DTT, TBS pH 7.5 (37.5 pL) and incubated for 15 minutes at 37 °C. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 pL). During RP-UPLC, a linear gradient was applied in 11 minutes from 30 to 90% acetonitrile in 0.1 % TFA and water.1.3. General procedure for mass spectral analysis

[0380] Analysis was done on a Xevo G2-XS QTof Quadrupole Time-of-Flight Mass Spectrometry system (ESI-QTOF) combined with a UPLC system (Aquity series, Waters). On the UPLC system a bioZen ™ 3.6 pm Intact XB-C8, LC column 50 x 2.1 mm. Deconvoluted spectra were obtained using Magtran software.

[0381] For (modified) nanobodies: Prior to mass spectral analysis, nanobody was diluted to 1 mg / mL.

[0382] For (modified) monoclonal antibodies: Prior to mass spectral analysis, IgG was treated with IdeS, which allows analysis of the Fc / 2 fragment. For analysis of both light and heavy chain, a solution of 20 pg (modified) IgG was incubated for 5 minutes at 37 °C with 100 mM DTT in a total volume of 4 pL. For analysis of the Fc / 2 fragment, a solution of 20 pg (modified) IgG was incubated for 1 hour at 37 °C with IdeS / Fabricator™ (1.25 U / pL) in phosphate-buffered saline (PBS) pH 7.4 in a total volume of 20 pL. Samples were 10x diluted before injection in the UPLC-MS system.1.4. General procedure for analytical SEC analysis

[0383] HPLC-SEC analysis was performed on an Agilent 1100 series (Hewlett Packard) equipped with a Xbridge BEH200A (3.5pm, 7.8x300 mm, PN186007640 Waters) column. The sample (5pL, 1 mg / mL) was injected and measured with 0.5 mL / min isocratic method (100 mM sodium phosphate (NaHPO4 / Na2PO4), 200 mM NaCI, pH 6.8, containing 10% isopropanol) for 25 minutes with a column temperature at RT.2. Chemical synthesis of conjugation probes with BCN click handleExample 1. Synthesis of compound BL1BL1

[0384] Preparation of [(1 R,8S)-9-bicyclo[6.1.0]non-4-ynyl]methyl (4-nitrophenyl) carbonate (BCN-OPNP) from endo-9-Hydroxymethylbicyclo[6.1 .0]non-4-yne (BCN-OH) has been described in Example 1. Synthesis of compound 102 in patent WO 2021 / 144314, incorporated herein by reference.

[0385] To a solution of BCN-OPNP (963 mg, 1 eq, 3.1 mmol) in dichloromethane (14 mL) was added 2- [2-(2-aminoethoxy)ethoxy]ethan-1-amine (9.1 g, 8.9 mL, 20 eq, 61 mmol). After stirring for 2h at room temperature, the reaction was diluted with DCM (50 mL) and washed with sat. aq. NaHCO3. The organic layer was dried over anhydrous MgSO4and filtered. The resulting solution was purified by silica gel column chromatography (0-10% (2M NH3in MeOH) in DCM), to yield compound BL1 (546.9 mg, 55.2%). LC-MS (ESI+) calculated for CI7H29N2O+[M+H]+325.2, found 325.4.1H NMR (400 MHz, CDCb) 5 4.12 (d, J = 8.1 Hz, 2H), 3.60 (q, J = 0.9 Hz, 4H), 3.52 (ddd, J = 18.3, 5.6, 4.8 Hz, 4H), 3.36 (q, J = 5.3 Hz, 2H), 2.86 (dd, J = 5.5, 4.9 Hz, 1 H), 2.27-2.12 (m, 6H), 1.63-1.47 (m, 5H), 1.33 (p, J = 8.6 Hz, 1 H), 0.99-0.85 (m, 2H).Example 2. Synthesis of compound BL2

[0386] To a solution of BL1 (117 mg, 45% Wt., 1 eq, 162 pmol) in DCM (6 mL) was added DIPEA (63 mg, 85 pL, 3 eq, 487 pmol) followed by A / -succinimidyl bromoacetate (210 mg, 5.5 eq, 891 pmol). After stirring the mixture for 5h at room temperature, the mixture was purified by silica gel column chromatography (0- 40% acetone in DCM) to yield compound BL2 (36.2 mg, 48.0%) as a yellow oil. LC-MS (ESI ) calculated for Ci9H28BrN3KO5- [M+K-H]- 482.1 , found 481.2.1H NMR (400 MHz, CDCb) 6 6.94 (s, 1 H), 5.19 (s, 1 H), 4.13 (d, J = 8.1 Hz, 2H), 3.87 (s, 2H), 3.61 (s, 10H), 3.37 (q, J = 5.5 Hz, 2H), 2.32 - 2.14 (m, 5H), 1.55 (dq, J = 17.3, 7.2 Hz, 2H), 1 .44-1 .09 (m, 2H), 0.98-0.84 (m, 2H).

[0387] To a solution of 2-[2-(2-aminoethoxy)ethoxy]ethan-1 -amine (53.4 mg, 52.6 pL, 1 eq, 360 pmol) in dichloromethane (1 .6 mL) was added triethylamine (72.9 mg, 100 pL, 2 eq, 721 pmol) followed by BCN- OPNP (250 mg, 2.2 eq, 793 pmol). After stirring for 2.5h at room temperature, the reaction mixture was purified by silica gel column chromatography (0-60% EtOAc in DCM) to yield compound BL3 (141 mg, 78.2%). LC-MS (ESI+) calculated for C28H oN2Na06+[M+Na]+523.3, found 523.5.1H NMR (400 MHz,CDCh) 5 4.15 (d, J = 8.1 Hz, 4H), 3.61 (s, 4H), 3.56 (dd, J = 5.5, 4.6 Hz, 4H), 3.39 (q, J = 5.4 Hz, 4H), 2.35 - 2.16 (m, 12H), 1.58 (d, J = 12.1 Hz, 5H), 1.36 (p, J = 8.6 Hz, 2H), 0.94 (dd, J = 13.1 , 6.5 Hz, 4H).

[0388] Preparation of Bis-BCN-PEG23 has been described in Example 3. Synthesis of compound 105 in patent WO 2021 / 144314, incorporated by reference. To a solution of amino-PEG23-amine (154.4 mg, 1.5 eq, 144 pmol) in dichloromethane (3 mL) was added triethylamine (97.3 mg, 134 pL, 9.8 Eq, 961 pmol). followed by BCN-OPNP (31 mg, 1 eq, 98.3 pmol). After stirring the reaction mixture for 45 minutes at room temperature, the residue was purified by silica gel column chromatography (0-10% methanol, containing 2M NH3, in DCM) to yield compound BL4 (63 mg, 51 %). LC-MS (ESI+) calculated for C7oHi23N2Na2027+[M+2Na-H]+1469.8, found 1469.4.1H NMR (400 MHz, CDCI3) 5 5.29 (s, 2H), 4.14 (d, J = 8.1 Hz, 4H), 3.64 (d, J = 3.3 Hz, 89H), 3.37 (q, J = 5.4 Hz, 5H), 2.37-2.17 (m, 8 H), 1.25 (s, 10H), 1.01-0.80 (m, 8H).3. Chemical synthesis of phospholipid with tetrazine click handle

[0389] To a solution of DSPE-PEG-amine (MW~2000Da, on average 42-48 repeating PEG units) (200.5 mg, 1 eq, 70 pmol) in dichloromethane (0.5 mL) were added DIPEA (18 mg, 24.3 pL, 2 eq, 140 pmol) and methyltetrazine-NHS ester (28 mg, 1 .22 Eq, 85.5 pmol) After stirring for 3h at room temperature in the dark, the mixture was purified by silica gel column chromatography (0-30% MeOH in DCM) to yield compound PLT1 (195 mg, 90.6%).1H NMR (400 MHz, CDCh) 6 8.56 (d, 2H), 7.54 (d, 2H), 6.61 (m, 1 H), 5.19 (m, 3H),4.34 (dd, 1 H), 4.13 (dd, 1 H), 4.04 (m, 2H), 3.95 (m, 4H), 3.81-3.78 (m, 2H), 3.64 (m, 208H), 3.09 (s, 3H), 2.69 (m, 5H), 2.27 (m, 6H), 1.57 (m, 5H), 1.24 (m, 66H), 0.87 (m, 7H).4. Expression and purification of nanobodies4. 1. General procedure for expression of nanobodies

[0390] Nanobodies were expressed in a TurboCHO™ antibody expression system performed by Genscript. Sequences for expression contained the N-terminal leader peptide sequence MGWSCIILFLVATATGVHS (SEQ IS NO 36) for mammalian cells expression, which is cleaved off during expression. Genes were synthesized and the codon-optimized DNA sequence was cloned into a pcDNA3.4 expression vector. Transfection-grade plasmids were prepared and used for expression in 100 mL or 1 L cultures of CHO-S cells for 5 days.4.2. General procedure for nanobody purification

[0391] Cell culture broth was centrifuged, and the supernatant was loaded onto HiTrap Phenyl FF (High sub) by incubating for appropriate time. As binding buffer, a solution of 20 mM Tris, 1 M NaCI, pH 9.0 was used. Cell culture supernatant was spiked with NaCI to a final concentration of 0.5 M. The elution buffer used contains 10 mM Tris, pH 9.0. The column was equilibrated with 5 column volumes of binding buffer and eluted with 5 column volumes of elution buffer. The flowthrough was collected and concentrated to load onto a HiLoad 26 / 600 Superdex 75pg column to purify the protein using PBS, pH 7.2 as elution buffer. The purified protein was analyzed by SDS-PAGE and SEC-HPLC to determine the molecular weight and purity. The endotoxin level of protein was also detected, and the concentration was determined by A280 method. Production yields are reported in Table 1.4.3. Design of nanobody variants

[0392] Nanobody variants were designed consisting of either an N-terminal sequence suitable for chemical modification, ST(G4S)3- (amino acid sequence being identified by SEQ ID NO 30), C-terminal sequence suitable for chemical modification, -C, -(G4S)3C (SEQ ID NO 31), -G4Y (SEQ ID NO 32), -(G4S)2G4Y (SEQ ID NO 33), or C-terminal sequence suitable for enzymatic modification, -GGGSLPETGGHHHHHH (SEQ ID NO 34), -GSGSRPQGF (SEQ ID NO 35). Designed sequences of nanobodies are based on targeting HER2 (receptor tyrosine-protein kinase erbB-2), as recorded under 5my6 > Nanobody 2Rs15d in the PDB (2Rs15d nanobody - reported in D’Huyvetter et al., 2017, Clin Cancer Res, 23, 6616-6628 and published in patent US 2022 / 0323620, SEQ ID NO: 7, par.

[0149] , Table 1).

[0393] Table 1 Expression conditions of purified nanobodies.4.4. VHH sequences

[0394] Sequence of HER2 nanobody 2Rs15d-C (VHH1) (SEQ ID NO: 21):QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSSC

[0395] Sequence of HER2 nanobody 2Rs15d-(G4S)3C (VHH2) (SEQ ID NO: 22):QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSSGGGGSGGGGSGGGGSC

[0396] Sequence of HER2 nanobody 2Rs15d-GGGSLPETGGHHHHHH (VHH3) (SEQ ID NO: 23):QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSSGGGSLPETGGHHHHHH

[0397] Sequence of HER2 nanobody 2Rs15d-GSGSRPQGF (VHH4) (SEQ ID NO: 24):QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSSGSGSRPQGF

[0398] Sequence of HER2 nanobody ST(G4S)3-2Rs15d (VHH5) (SEQ ID NO: 25):STGGGGSGGGGSGGGGSQVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSS

[0399] Sequence of HER2 nanobody 2Rs15d-G4Y (VHH6) (SEQ ID NO: 26):QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSSGGGGY

[0400] Sequence of HER2 nanobody 2Rs15d-(G4S)2G4Y (VHH7) (SEQ ID NO: 27):QVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDTWHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVSSGGGGSGGGGSGGGGY5. Preparation of nanobodies and antibodies conjugates5. 1. BCN-nanobodies by chemical cysteine alkylation conjugation [“Cys”1

[0401] Reduced C-terminal nanobody was prepared by selective reduction of nanobody (VHH1 or VHH2). Nanobodies were selective reduced at the C-terminal cysteine residue by incubation with 50 equivalents TPPTS and addition of 10 mM EDTA at room temperature for 2 hours. The reaction mixture was desalted using a HiPrep 26 / 10 desalting column on a 100F NGC system (Bio-rad) using PBS pH 7.4 as mobile phase.

[0402] A nanobody-BCN conjugate was prepared by C-terminal cysteine alkylation to selective reduced nanobody (NB1 or NB2). Nanobodies were cysteine alkylated by addition of 30 equivalents alkylation probe bromoacetamido-PEG2-BCN (BL2). The reaction was incubated overnight at RT followed by concentration and subsequent purification using a Superdex75 Increase 16 / 600 column (Cytiva) on a 100F NGC system (Bio-Rad) using PBS pH 7.4 as mobile phase.Example 6. Preparation of selective reduced 2Rs15d-C (NB1)

[0403] To a solution of VHH1 (1495 pL, 14.6 mg, 9.79 mg / mL in PBS pH 7.2) was added PBS (130 pL), TPPTS (570 pL, 100 mM in Milli-Q) and EDTA (244 pL, 100 mM in Milli-Q). The reaction (at 6 mg / mL nanobody concentration) was incubated for 2 hours at room temperature. After purification, mass spectral analysis showed one major product (calculated mass 12712 Da, observed mass 12713 Da), corresponding to selective reduced NB1.Example 7. Preparation of selective reduced 2Rs15d-(G4ShC (NB2)

[0404] To a solution of VHH2 (1490 pL, 15 mg, 10.07 mg / mL in PBS pH 7.2) was added PBS (216 pL), TPPTS (544 pL, 100 mM in Milli-Q) and EDTA (250 pL, 100 mM in Milli-Q). The reaction (at 6 mg / mL nanobody concentration) was incubated for 2 hours at room temperature. After purification, mass spectral analysis showed one major product (calculated mass 13657 Da, observed mass 13658 Da), corresponding to selective reduced NB2.Example 8. Preparation of 2Rs15d-C-PEG?-BCN (NB3)

[0405] To NB1 (7927 pL, 13 mg, 1.64 mg / mL in PBS pH 7.4) was added PBS (1173 pL) and bromoacetamido-PEG2-BCN (BL2) (3900 pL; 306 pL from 100 mM stock in DMF combined with 3594 pL propylene glycol). The reaction was set at 1 mg / mL nanobody concentration. Mass spectral analysis of the purified sample showed one single peak (calculated mass 13078 Da, observed mass 13076 Da), corresponding to BCN-conjugated nanobody NB3.Example 9. Preparation of 2Rs15d-(G4ShC-PEG -BCN (NB4)

[0406] To NB2 (9944 pL, 18 mg, 1.81 mg / mL in PBS pH 7.4) was added PBS (2655 pL) and bromoacetamido-PEG2-BCN (BL2) (5400 pL; 395 pL from 100 mM stock in DMF combined with 5005 pL propylene glycol). The reaction was set at 1 mg / mL nanobody concentration. Mass spectral analysis of the purified sample showed one major product (calculated mass 14023 Da, observed mass 14024 Da), corresponding to BCN-conjugated nanobody NB4.5.2. BCN-nanobodies by enzymatic Sortase conjugation [“Sort”]

[0407] A nanobody-BCN conjugate was prepared by C-terminal sortagging using sortase A pentamutant (SEQ ID NO 28). To a solution of nanobody VHH3 was added 40 equivalents of gly-gly-gly-PEGs-BCN, 10 mM CaCL, and 1-mol% sortase A pentamutant. The reaction mixture was incubated overnight at 37 °C. Upon reaction completion, the reaction mixture was purified using a HisTrap HP / HisTrap Excel column, followed by PD-10 desalting column packed with Sephadex G-25 resin (Cytiva) and eluted using PBS on a AKTA pure 25M (Cytiva) system.

[0408] Structure of gly-gly-gly-PEGs-BCN:

[0409] Sequence of Sortase A Pentamutant (SEQ ID NO 28): MHHHHHHKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYP GPATREQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIR NVKPTAVGVLDEQKGKDKQLTLITCDDYNEETGVWETRKIFVATEVKExample 10. Preparation of 2Rs15d-GGGSLPETGGG-PEG,3-BCN (NB5)

[0410] To a solution of VHH3 (3061 pL, 9 mg, 2.94 mg / mL in PBS pH 7.2) was added PBS pH 7.4 (701 pL), gly-gly-gly-PEGs-BCN (253 pL from 100 mM in PBS), CaCL (450 pL, 100mM in MQ) and sortase A pentamutant (35 pL from 182 pM in 50 mM Tris-HCI, 500mM NaCI, 10% Glycerol, pH 8.0). The reaction was set to 2 mg / mL (140 pM nanobody concentration; 1.4 pM sortase enzyme), incubated at 37 °C overnight and further purified upon reaction completion. Mass spectral analysis showed one single peak (calculated mass 13830 Da, observed mass 13831 Da) corresponding to BCN-conjugated nanobody NB5.5.3. BCN-nanobodies by enzymatic Transglutaminase conjugation [“TGase’l

[0411] A nanobody-BCN conjugate was prepared by C-terminal transglutaminase using transglutaminase enzyme (obtained from Zedira). To a solution of nanobody VHH4 was added 40 equivalents of amine- PEG2-BCN (BL1), and 1 U / mL transglutaminase enzyme. The reaction mixture was incubated overnight at 37 °C. Upon reaction completion, the reaction mixture was purified using a Superdex75 Increase 16 / 600 column (Cytiva) on a 100F NGC system (Bio-Rad) using PBS pH 7.4 as mobile phase.Example 11. Preparation of 2Rs15d-GSGSRPQ(GF)-PEG?-BCN (NB6)

[0412] To a solution of VHH4 (1320 pL, 4 mg, 3.03 mg / mL in PBS pH 7.2) was added PBS pH 7.4 (2547 pL), amine-PEG2-BCN (BL1 , 120 pL from 100 mM in PBS), and recombinant microbial transglutaminase enzyme (13 pL from a 312 U / mL solution in 50 mM HEPES pH 7.4). The reaction was set to 1 mg / mL nanobody concentration (1 U / mL enzyme concentration), incubated overnight and further purified upon reaction completion. Mass spectral analysis showed one single peak (calculated mass 13790 Da, observed mass 13790 Da) corresponding to BCN-conjugated nanobody NB6.5.4. BCN-nanobodies by Strain-Promoted Alkyne-Nitrone Cycloaddition conjugation [“SPANC”!

[0413] A nanobody-nitrone intermediate was prepared by N-terminal oxidation of serine, followed by nitrone installation. To a solution of nanobody VHH5 was added 2 equivalents of NalO-4, and the reaction mixture was incubated for 5 minutes at room temperature. To quench the remaining NalO4 in the reaction, 6.6 equivalents of p-methoxybenzene thiol were added. To the quenched reaction was added 160 equivalents p-Anisidine and 160 equivalents N-hydroxy-glycine. The reaction mixture was incubated overnight at room temperature. Upon reaction completion, the reaction mixture was filtered over Amicon Ultra 0.5 mL Centrifugal Filters, 3 kDa MWCO, or Vivaspin® 4 Turbo Centrifugal Concentrator (Sartorius), 3 kDa MWCO, and washed 3 times with PBS.

[0414] To nitrone functionalized nanobody was added 10 equivalents of BCN-PEG2-BCN (BL3) or BCN- PEG23-BCN (BL4). The reaction was incubated overnight at room temperature. Upon reaction completion, the reaction mixture was purified using a Superdex75 Increase 16 / 600 column (Cytiva) on a 100F NGC system (Bio-Rad) using PBS pH 7.4 as mobile phase.

[0415] To a solution of VHH5 (3180 pL, 9 mg, 2.83 mg / mL in PBS pH 7.2) was added PBS (1 189 pL) and NalO4 (131 pL from 10 mM solution in PBS). Reaction concentration was set at 2 mg / mL nanobody concentration. Mass analysis was used to confirm oxidation of serine into corresponding hydrate (observed mass 13747 Da). The reaction was quenched by addition of p-methoxybenzene thiol (654 pL, from 10 mM solution in DMF). To the quenched reaction was added PBS (252 pL), p-anisidine (1047 pL from 100 mM in PBS), and N-hydroxy-glycine (1047 pL from 100 mM in PBS). The reaction was set at 1.2 mg / mL nanobody concentration. After reaction incubation, mass spectral analysis showed the nitrone product (observed mass 13802 Da) corresponding to nitrone-NB7. After concentration, nitrone-NB7 (2996 pL, 8.54 mg, 2.85 mg / mL in PBS pH 7.4) was added PBS (847 pL) and BCN-PEG2-BCN (BL3) (428 pL from 14.5 mM in DMF). The reaction was set at 2 mg / mL nanobody concentration with a maximum of 10% DMF present and incubated overnight at room temperature. After purification, mass spectral analysis showed one product (calculated mass 14302 Da, observed mass 14303 Da) corresponding to BCN-conjugated nanobody NB7.Example 13. Preparation of BCN-PEG23-ST(G4S)3-2Rs15d (NB8)

[0416] To a solution of VHH5 (190 pL, 2 mg, 10.5 mg / mL in PBS pH 7.2) was added PBS (781 pL) and NalO4 (29 pL from 10 mM solution in PBS). Reaction concentration was set at 2 mg / mL nanobody concentration. Mass analysis was used to confirm oxidation of serine into corresponding hydrate (observed mass 13745 Da). The reaction was quenched by addition of p-methoxybenzene thiol (96 pL, from 10 mM solution in DMF). To the quenched reaction was added PBS (106 pL), p-anisidine (233 pL from 100 mM in PBS), and N-hydroxy-glycine (233 pL from 100 mM in PBS). The reaction was set at 1 .2 mg / mL nanobody concentration. After reaction incubation, mass spectral analysis showed the nitrone product (observed mass 13802 Da) corresponding to nitrone-NB8. After concentration, nitrone-NB8 (900 pL, 2 mg, 2.22 mg / mL in PBS pH 7.4) was added BCN-PEG23-BCN (BL4) (100 pL from 14.5 mM in DMF). The reaction was set at 2 mg / mL nanobody concentration with a maximum of 10% DMF present and incubated overnight at room temperature. After purification, mass spectral analysis showed one product (calculated mass 15228 Da, observed mass 15226 Da) corresponding to BCN-conjugated nanobody NB8.5.5. BCN-nanobodies by Strain-Promoted Oxidation-Controlled cyclooctyne-1 ,2-Quinone Cycloaddition conjugation [“SPOCQ”!

[0417] A nanobody-BCN conjugate was prepared by C-terminal SPOCQ using mushroom Tyrosinase (Sigma-Aldrich), >1000 unit / mg. To a solution of nanobody VHH6 or VHH7 was added 10 equivalents of BCN-PEG2-BCN (BL3) or BCN-PEG23-BCN (BL4), and 2.5-mol% of mushroom tyrosinase. The reaction mixture was incubated for 5 hours at room temperature. Upon reaction completion, the reaction mixture was purified using a Superdex75 Increase 16 / 600 column (Cytiva) on a 100F NGC system (Bio-Rad) using PBS pH 7.4 as mobile phase.

[0418] To test the reactivity of tyrosine residues in the HER2 nanobody, a solution of VHH1 (10 pL, 0.1 mg, 9.76 mg / mL) was added PBS (33 pL), BCN-PEG2-BCN (BL3) or BCN-PEG23-BCN (BL4) (5 pL from 13.33 mM solution in DMF), and mushroom Tyrosinase (2.1 pL from 80 pM stock solution for 2.5-mol%, or 8.3 pL from 80 pM stock solution for 10-mol%). The reaction was set at 2 mg / mL nanobody concentration and incubated overnight at room temperature. Mass spectral analysis showed no reaction took place and only unreacted VHH1 was observed (observed mass 12830 Da), indicating that no tyrosine residues are exposed and reactive towards tyrosinase in VHH1 .Example 14. Preparation of 2Rs15d-G4Y-PEG?-BCN (NB9)

[0419] To a solution of VHH6 (963 pL, 3 mg, 3.1 1 mg / mL) was added PBS (1665 pL), BCN-PEG2-BCN (BL3) (300 pL from 7.2 mM solution in DMF), and mushroom Tyrosinase (72 pL from 80 pM stock solution). The reaction was set at 1 mg / mL nanobody concentration and was incubated for 5 hours at room temperature. Mass spectral analysis of purified product showed one single peak (calculated mass 13514 Da, observed mass 13513 Da) corresponding to the BCN-conjugated nanobody NB9.Example 15. Preparation of 2Rs15d-(G4ShG4Y-PEG?-BCN (NB10)

[0420] To a solution of VHH7 (963 pL, 3 mg, 3.1 1 mg / mL) was added PBS (1667 piL), BCN-PEG2-BCN (BL3) (300 pL from 7.2 mM solution in DMF), and mushroom Tyrosinase (69 pL from 80 pM stock solution). The reaction was set at 1 mg / mL nanobody concentration and was incubated for 5 hours at room temperature. Mass spectral analysis of purified product showed one single peak (calculated mass 14145 Da, observed mass 14144 Da) corresponding to the BCN-conjugated nanobody NB10.5.5. BCN-antibodies by GlycoConnect Strain-Promoted Alkyne-Azide Cycloaddition conjugation [“SPAAC”!

[0421] General procedure for enzymatic remodeling of IgG to mAb-(6-N3-GalNAc)2: IgG (15 mg / mL) was incubated with 1 % w / w EndoSH (as described in PCT / EP2017 / 052792, see Examples 1 - 3, and SEQ ID No: 1 , which is incorporated by reference herein), 3% w / w His-TnGalNAcT (as described in PCT / EP2016 / 059194, see Examples 3 and 4, and SEQ ID No: 49, which is incorporated by reference herein), 0.01 % AP (Roche) and UDP 6-Ns-GalNAc (10 eg compared to IgG) in 6 mM MnCL and 20 mM histidine and 150 mM NaCI pH 7.5 for 16 hours at 30 °C. Next, the functionalized IgG was purified using a HiTrap MabSelect sure column. After loading of the reaction mixture, the column was washed with TBS+0.2% triton and TBS. The IgG was eluted with 0.1 M AcOH pH 2.7 and neutralized with 2.5 M Tris- HCI pH 8.8. After three times dialysis to TBS pH 7.5, the IgG was concentrated to 15-20 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius).

[0422] Model mAb-lipid conjugates were prepared by conjugating the mAb-(6-N3-GalNAc)2 with DSPE- PEG-DBCO or the BCN-functionalized mAb with DSPE-PEG-N3 in a SPAAC bioconjugation reaction.

[0423] Structure of UDP 6-Ns-GalNAc and triBCN are depicted below. Preparation of triBCN has been described in Example 24 of WO 2021 / 144314, incorporated by reference.Example 16: Preparation of trastuzumab-tG-Ns-GalNAc)? (AB1)

[0424] According to the general procedure for enzymatic remodeling, trastuzumab (obtained from the pharmacy) was converted to trastuzumab-(6-N3-GalNAc)2 Mass spectral analysis of a sample after IdeS treatment showed one major Fc / 2 product (observed mass 24366 Da, approximately 85% of total Fc / 2 and observed mass for minor -fucose product 24220 Da, approximately 10% of the total Fc / 2), corresponding to the expected product.Example 17: Conjugation of trastuzumab-fG-Ns-GalNAc)? with triBCN to obtain intramolecular conjugate trastuzumab-(BCN) (AB2)

[0425] To a solution of AB1 (1054 pL, 25 mg, 23.72 mg / ml in TBS pH 7.5) was added TBS (2.4 mL) and triBCN (structure above, 21 pL, 40 mM solution in DMF) in propylene glycol (1479 pL). The reaction was incubated overnight at rt. Next the conjugate was purified on a HiTrap 26-10 desalting column (Cytiva), rinsed with 0.2M NaOH and equilibrated with PBS. Lastly the product was concentrated using a Vivaspin Turbo 4 10 kDa MWCO ultrafiltration unit (Sartorius). Mass spectral analysis of the sample after IdeS treatment showed one main Fc / 2 product (observed mass 49797, calculated mass 49796), corresponding to the conjugate AB2, wherein two BCN moieties of triBCN reacted with the two azide moieties of AB1 and one BCN moiety remains unreacted. RP-UPLC analysis of the sample under reducing conditions showed an average conversion of 91 %.Example 18: Conjugation of trastuzumabfS-Ns-GalNAch with DSPE-PEG-DBCO (MW 2000) to obtain conjugate bis-DSPE-PEG functionalized trastuzumab-1 (AB3)

[0426] To a solution of trastuzumab(6-N3-GalNAc)2 (422 pL, 10 mg, 23.72 mg / ml in TBS pH 7.5) was added TBS (3.8 mL) and DSPE-PEG-DBCO (MW 2000, purchased from Broadpharm) (200 pL, 5 mM solution in DMF) in DMF (550 pL). The reaction was incubated overnight at rt. Next the conjugate was purified on a HiLoad 16 / 600 Superdex200 PG column (Cytiva) on an AKTA Pure (Cytiva). Mass spectral analysis of the sample after IdeS treatment showed one main Fc / 2 product (observed mass 27554 Da, other ~ 10 peaks are 44Da apart from each other and hence correspond to the PEG distribution), corresponding to the conjugate AB3. RP-UPLC analysis of the sample under reducing conditions showed an average conversion of 99%.Example 19: Conjugation of BCN-functionalized trastuzumab with DSPE-PEG-N3 (MW 2000) to obtain conjugate bis-DSPE-PEG functionalized trastuzumab-2 (AB4)

[0427] To a solution of BCN-functionalized trastuzumab (304 pL, 5 mg, 16.46 mg / ml in TBS pH 7.5) was added TBS (71 pL) and DSPE-PEG-N3 (MW 2000, purchased from Avanti Polar Lipids) (27 pL, 5 mM solution in DMF) in DMF (48 pL). The reaction was incubated overnight at rt. Next the conjugate was purified on a HiLoad 16 / 600 Superdex200 PG column (Cytiva) on an AKTA Pure (Cytiva). RP-UPLC analysis of the sample under reducing conditions showed an average conversion of 40% corresponding to the conjugate AB4.6. HER2 cell binding of nanobodies

[0428] A nanobody-FITC conjugate was prepared by tetrazine Inverse Electron Demand Diels-Alder (IEDDA) between BCN-nanobody (NB5, NB7, NB8, NB9, NB10) and FITC-MeTz (FT1). To a solution of BCN-nanobody was added 3 equivalents of FITC-MeTz and the reaction mixture was incubated for 3 hours at room temperature. Subsequently, the mixture was filtered over Amicon Ultra 0.5 mL Centrifugal Filters,3 kDa MWCO, and washed 5 times with PBS. Obtained fluorophore-nanobody was measured for concentration with a Nanodrop and the fluorophore per particle (F / P) ratio was calculated. Protein concentration was determined based on A280. FITC has a maximum absorption at 497 nm with an extinction coefficient of 80000 cnr1M-1and a correction factor of 0.35.

[0429] To a solution of mixed isomers 4- and 5-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-2-(6-hydroxy-3- oxo-3H-xanthen-9-yl)benzoic acid (100 mg, 1 equiv., 0.21 mmol) in DCM (2 mL) was added methyltetrazine-propylamine HCI salt (56 mg, 1.1 equiv., 0.23 mmol) and DIPEA (107 pL, 2.9 equiv., 614 pmol). The reaction mixture was protected from light and stirred for 3h at room temperature. The completed reaction was purified by silica gel column chromatography (0-20% MeOH in DCM) to yield FT1 (60.5 mg, 47%) as an orange solid. LCMS (ESI+) calculated for C33H26N5O7+[M+H]+604.5 found 604.3.1H NMR (400 MHz, CD3CN) 5 8.49-8.43 (m, 2H), 8.15 (ddd, J = 8.0, 1.6, 0.6 Hz, 1 H), 7.26 (dd, J = 8.1 , 0.8 Hz, 1 H), 7.19-7.12 (m, 2H), 6.72 (d, J = 2.4 Hz, 2H), 6.63 (s, 1 H), 6.56 (dd, J = 8.7, 2.4 Hz, 2H), 4.23 (t, J = 6.1 Hz, 2H), 3.62 (td, J = 6.6, 4.9 Hz, 2H), 3.27 (s, 2H), 2.97 (s, 3H), 2.58 (s, 4H), 1.39-1.23 (m, 6H).Example 21. Preparation of 2Rs15d-GGGSLPETGGG-PEG^-FITC (NF1)

[0430] To a solution of NB5 (118 pL, 0.3 mg, 2.53 mg / mL) was added PBS (151 pL) and FITC-MeTz (FT1) (6.5 pL from 10 mM stock in DMF added together with 23.5 pL DMF). The reaction was set at 1 mg / mL nanobody concentration and was incubated for 3 hours at room temperature. After spin filtration, mass spectral analysis of purified product showed one single peak (calculated mass 14406 Da, observed mass 14403 Da) corresponding to the FITC-conjugated nanobody NF1. The calculated concentration was 159.1 pM and the F / P ratio was 0.71 .Example 22. Preparation of FITC-PEG?-ST(G4Sh2Rs15d (NF2)

[0431] To a solution of NB7 (94 pL, 0.3 mg, 3.2 mg / mL) was added PBS (176 pL) and FITC-MeTz (FT1) (6.3 pL from 10 mM stock in DMF added together with 23.7 pL DMF). The reaction was set at 1 mg / mL nanobody concentration and was incubated for 3 hours at room temperature. After spin filtration, mass spectral analysis of purified product showed one single peak (calculated mass 14878 Da, observed mass14877 Da) corresponding to the FITC-conjugated nanobody NF2. The calculated concentration was 154.5 pM and the F / P ratio was 0.57.Example 23. Preparation of FITC-PEG?3-ST(G4S)32Rs15d (NFS)

[0432] To a solution of NB8 (78 pL, 0.25 mg, 3.22 mg / mL) was added PBS (147 pL) and FITC-MeTz (FT1) (5.0 pL from 10 mM stock in DMF added together with 20 pL DMF). The reaction was set at 1 mg / mL nanobody concentration and was incubated for 3 hours at room temperature. After spin filtration, mass spectral analysis of purified product showed one single peak (calculated mass 15804 Da, observed mass 15802 Da) corresponding to the FITC-conjugated nanobody NF3. The calculated concentration was 87.3 pM and the F / P ratio was 0.96.Example 24. Preparation of 2Rs15d-G4Y-PEG?-FITC (NF4)

[0433] To a solution of NB9 (36 pL, 0.15 mg, 4.22 mg / mL) was added PBS (99 pL) and FITC-MeTz (FT1) (3.3 pL from 10 mM stock in DMF added together with 11.7 pL DMF). The reaction was set at 1 mg / mL nanobody concentration and was incubated for 3 hours at room temperature. After spin filtration, mass spectral analysis of purified product showed one single peak (calculated mass 14090 Da, observed mass 14088 Da) corresponding to the FITC-conjugated nanobody NF6. The calculated concentration was 79.9 pM and the F / P ratio was 0.60.Example 25. Preparation of 2Rs15d-(G4S)?G4Y-PEG?-FiTC (NF5)

[0434] To a solution of NB10 (35 pL, 0.2 mg, 5.67 mg / mL) was added PBS (145 pL) and FITC-MeTz (FT1) (4.2 pL from 10 mM stock in DMF added together with 15.8 pL DMF). The reaction was set at 1 mg / mL nanobody concentration and was incubated for 3 hours at room temperature. After spin filtration, mass spectral analysis of purified product showed one single peak (calculated mass 14721 Da, observed mass 14719 Da) corresponding to the FITC-conjugated nanobody NF5. The calculated concentration was 145.6 pM and the F / P ratio was 0.59.Example 26. HER2-bindinci of nanobody-FITC; Quantification by flow cytometry

[0435] SK-BR-3 (HER2+++) cells were cultured in RPMI 1640 supplemented with 1 % pen / strep and 10% fetal bovine serum. For Nanobody-FITC binding quantification, 200.000 cells were collected, resuspended in 250 pL FACS flow buffer and cooled to 4 °C. To this, 0.2-0.5 pg of nanobody-FITC (any of NF1-NF5) was added. Typically, a nanobody-FITC of 150 pM (5 pL) was diluted in FACS flow buffer (45 pL) and 5 pL of diluted sample was added to the cell suspension. The mixture was left for 30 minutes at 4 °C in the dark and subsequently centrifuged to replace the supernatant with fresh FACS flow buffer (200-400 pL). Sample was then analyzed with FACS using the following voltage conditions: FSC 200V, SSC 350V, FITC 488V (for standard beads), FITC 300V (for SK-BR-3). For quantification of the number of nanobodies bound per cell, Quantum™ FITC-5 MESF (pre-mixed) beads (Bangs Laboratories, Lot Number 17430) were used.With obtained calibration curve and measured FITC Geometric Mean for the different nanobody-FITC bound to HER2+ cells, the total number of nanobodies bound per cell was calculated (Table 2). NF4 and NF5 showed equal re...

Claims

1. CLAIMS1. An extracellular vesicle comprising a cavity encapsulated by a lipid membrane and at least one conjugate having structure B-L-V, wherein:- B is a biomolecule;- L represents a linker comprising a connecting group Z obtainable by a strain promoted click reaction;- V represents a hydrophobic moiety that is embedded in the membrane.

2. The extracellular vesicle according to claim 1 , wherein B is an antibody or a fragment thereof.

3. The extracellular vesicle according to claim 1 or 2, wherein connecting group Z is formed between a reaction of click probe F selected from (F1), (F3) and (F10) and click probe Q according to (Q1 a):(F1) (F3) (F10) (Q1 a) wherein,- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- Y2is C(R31)2, O, S, S(+)R31, S(O)R31, S(O)=NR31or NR31, wherein S(+)is a cationic sulfur atom counterbalanced by B( ), wherein B( )is an anion, and wherein each R31individually is R15or a connection with V, connected via L;- 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;- the wavy bonds represent the connection to B, for (F3) 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 R32is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups.

4. The extracellular vesicle according to any one of claims 1-3, wherein the conjugate is according to (C6) or (C8):(C6) (C8) wherein LB1, LB2and LAare linkers; Z1and Z2are connecting groups obtainable by strain promoted click reactions.

5. The extracellular vesicle according to claim 4, wherein LB2is according to (L11)-(L12) or (L11)(L12)BM(L13), wherein- L11, L12and L13are linkers 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, C9-C200 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)yand NR21, wherein y’ is 0, 1 or 2, preferably y’ = 2, and R21is 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;- BM is a branching moiety;- LAis -(W)^(A)d-(B)e-(A)f-(W)g-(Str)h-(W)i- (A)j-(B)^(A)i-(W)m-, wherein:- d = 0 or 1 , preferably d =0;- f = 0 or 1 , preferably f =0;- j = 0 or 1 , preferably j =0;- I = 0 or 1 , preferably I =0;- c = 0 or 1 , preferably c =1- g = 0 or 1 , preferably g =1 ;- i = 0 or 1 , preferably i = 1 ;- m = 0 or 1 , preferably m =1 ;- h = 0 or 1 , preferably h =1 ;- e = an integer in the range of 0-10, preferably, e = 0, 1 , 2, 3, 4, 5 or 6, most preferably e = 1 , 2, 3, or 4;- k = an integer in the range of 0-10, preferably, k = 0, 1 , 2, 3, 4, 5 or 6, most preferably k = 1 , 2, 3, or 4;- A is sulfamide group according to structure (23):- B is -CH2-;- 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;- Str is a stretching moiety selected from a PEG chain or a peptide.

6. The extracellular vesicle according to claim 4 or 5, wherein the conjugate is according to (C8), wherein:- LB1is a glycan L6having structure -GlcNAc(Fuc)v^S-(L7)w-, S is a sugar or a sugar derivative, GIcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1 , w’ is 1 or 2, L7is a bond, - N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-;- Z1comprises a 1 ,2,3-triazole.

7. The extracellular vesicle according to claim 6, wherein the conjugate is according to (C8c):(C8c).

8. The extracellular vesicle according to claim 4 or 5, wherein the conjugate is according to (C6), wherein Z1is obtainable by a strain-promoted alkyne-nitrone cycloaddition (SPANC) or strain-promoted oxidation controlled cyclooctyne-1 ,2 quinone (SPOCQ).

9. The extracellular vesicle according to claim 8, wherein the conjugate is according to any one of structures (C6a)-(C6c):wherein,- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- Y2is C(R31)2, O, S, S(+)R31, S(O)R31, S(O)=NR31or NR31, wherein S(+)is a cationic sulphur atom counterbalanced by B< >, wherein B< > is an anion, and wherein each R31individually is R15or a connection with V, connected via L;- L10is a linker of structure (C(R3)2)z, wherein z is 2 or 3, and each R3is individually selected from H and C1-4 alkyl, wherein two occurrences of R3may be joined together to form a C3-6 (hetero)cycloalkyl group;- X is S, O or N;- each R42group is selected from H and C1-4 alkyl, preferably R42is H.- R41is 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 besubstituted and optionally interrupted by one or more heteroatoms selected from O, S and NR32wherein R32is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups; more preferably R41is L10XR4,- R4is selected from H and C1-4 alkyl, X is S, O or N;- 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; preferably the conjugate is according to any one of structures (C6d)-(C6f):wherein, the - represents a single or a double bond, preferably a double bond, and R29is selected from 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-6alkyl and C(O)-NR33-aryl, more preferably the conjugate is according to any one of structures (C6g)-(C6i):(C6i).

10. The extracellular vesicle according to claim 8 or 9, wherein B is an antibody or fragment thereof, preferably a single chain antibody, and LB1is C(O) and connected to the N-terminus of the antibody or fragment thereof.11 The extracellular vesicle according to claim 8, wherein the conjugate is according to structure (C6j) or- R15is 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 R15may be linked together to form an optionally substituted annulated cycloalkyl or an optionally substituted annulated (hetero)arene substituent, and wherein R16is 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;- Y2is C(R31)2, O, S, S(+)R31, S(O)R31, S(O)=NR31or NR31, wherein S(+)is a cationic sulphur atom counterbalanced by B( ), wherein B( )is an anion, and wherein each R31individually is R15or a connection with V, 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; preferably, the conjugate is according to structure (C6I) or (C6m):more preferably, the conjugate is according to (Con) or (Cbo):

12. The extracellular vesicle according to any one of the preceding claims, wherein the conjugate is obtainable by:• reacting a biomolecule B-LB1-F1with Q1-LB1-F2to form B-LB1-Z1-LB2-F2, and reacting B-LB1- Z1-LB2-F2with Q2-LA-V to form B-LB1-Z1-LB2-Z2-LA-V; or• reacting a biomolecule B(-LB1-F1)2 with (Q1-)2LB1-F2to form B(-LB1-Z1-)2LB2-F2, and reacting B(-LB1-Z1-)2LB2-F2with Q2-LA-V to form B(-LB1-Z1-)2LB2-Z2-LA-V; wherein Q1is the same as F2, preferably Q1and F2are BCN.

13. The extracellular vesicle according to any one of the preceding claims, wherein the extracellular vesicle is a lipid nanoparticle (LNP).

14. The extracellular vesicle according to any one of the preceding claims, wherein:- the active compound is a cytotoxin or a nucleic acid, preferably a nucleic acid selected from plasmid, 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 (dsRNA), single stranded DNA (ssDNA), double stranded DNA, plasmids, ribozymes, aptamer, Gapmer, and triplex forming oligonucleotides, and- the stabilizing lipid is selected from cholesterol, cationic lipids, ionizable lipid, squaramide and combinations thereof, preferably the stabilizing lipid is a mixture of ionizable lipids and cholesterol.

15. The extracellular vesicle according to any one of the preceding claims, wherein V is a lipid, preferably selected from phospholipid, sphingolipid or glycerolipid, more preferably wherein V is according to (V17):wherein the wavy line indicates the connection to L, and each HT is a hydrophobic tail individually selected from a saturated or unsaturated hydrocarbon chain from 5 to 29 carbon atoms long,preferably 1 1 to 23 carbon atoms long, more preferably 15 to 19 carbon atoms long, most preferably 17 carbon atoms long.

16. A process of producing the extracellular vesicle according to any one of the preceding claims, comprising:(l a) providing a biomolecule having structure B [ LB(F)x ]z, wherein F is a click probe, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(l b) reacting B(LB(F)x)z with a linker-payload construct Q-LA-V, wherein Q is a click probe that is reactive towards F, LBis a linker and V is a hydrophobic moiety, to obtain a conjugate of structure B [LB(Z-LA-V)x ]z;(l c) providing an extracellular vesicle, wherein the cavity thereof optionally comprises an active compound and / or a stabilizing lipid; and(ld) incorporating B [LB(Z-LA-V)X]z into the membrane of the extracellular vesicle, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane; or(2c) providing an extracellular vesicle, wherein the cavity thereof optionally comprises an active compound and / or a stabilizing lipid;(2d) incorporating linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, into the membrane of the extracellular vesicle to obtain an extracellular vesicle wherein moiety V is embedded in the membrane;(2a) providing a biomolecule having structure B [ LB(F)X]z, wherein F is a click probe that is reactive towards Q, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(2b) reacting B[LB(F)x]z with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle; or(3c) assembling an extracellular vesicle in the presence of linker-payload construct Q-LA- V, wherein Q is a click probe, LBis a linker and V is a hydrophobic moiety, and optionally an active compound and / or a stabilizing lipid, to obtain an extracellular vesicle wherein moiety V is embedded in the membrane, and optionally the cavity thereof comprises said active compound and / or a stabilizing lipid;(3a) providing a biomolecule having structure B [ LB(F)X]z, wherein F is a click probe that is reactive towards Q, LBis a linker linking biomolecule B to x occurrences of F, x is 1 , 2, 3 or 4, and z is 1 or 2;(3b) reacting B[LB(F)x]z with a linker-payload construct Q-LA-V embedded in the membrane of the extracellular vesicle.

17. The process according to claim 16, wherein the biomolecule B [ LB(F)x ]z in step (a) is obtained by reacting B-[LB1-(F1)a]b with (Q1)yi-LB1(F2)y2, wherein a, b, y1 and y2 are integers in the range of 1 -4, wherein F1and Q1are click probes reactive to each other, F2and Q2are click probes reactive to each other but F2and Q1are not reactive to each other, and wherein LB1and LB2are linkers, and wherein in step (b) Q-LA-V is Q2-LA-V.

18. The process according to claim 16, wherein F1is a nitrone or an o / Yho-quinone and Q1is a (hetero)cycloalkene or (hetero)cycloalkyne, F2is a (hetero)cycloalkene or (hetero)cycloalkyne and Q2is a tetrazine, preferably Q1and F2are (hetero)cycloalkyne, more preferably Q1and F2are BCN.

19. A pharmaceutical composition comprising the extracellular vesicle according to any one of claims 1 -15 and optionally a pharmaceutically acceptable carrier or excipient.

20. The extracellular vesicle according to any one of claims 1 -15 or the pharmaceutical composition according to claim 19 for use in medical treatment.

21. The extracellular vesicle according to any one of claims 1 -15 or the pharmaceutical composition according to claim 19 for use in the treatment of cancer or a genetic disorder.

22. The extracellular vesical or pharmaceutical composition for use according to claim 21 , wherein the treatment is an in vivo CAR therapy.

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