EP3413916A1 - Antikörperkonjugate mit verbessertem therapeutischem index zur abzielung auf cd30-tumoren und verfahren zur verbesserung des therapeutischen index von antikörperkonjugaten - Google Patents

Antikörperkonjugate mit verbessertem therapeutischem index zur abzielung auf cd30-tumoren und verfahren zur verbesserung des therapeutischen index von antikörperkonjugaten

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Publication number
EP3413916A1
EP3413916A1 EP17703994.8A EP17703994A EP3413916A1 EP 3413916 A1 EP3413916 A1 EP 3413916A1 EP 17703994 A EP17703994 A EP 17703994A EP 3413916 A1 EP3413916 A1 EP 3413916A1
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EP
European Patent Office
Prior art keywords
group
groups
antibody
linker
hetero
Prior art date
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Pending
Application number
EP17703994.8A
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English (en)
French (fr)
Inventor
Sander Sebastiaan Van Berkel
Jorge Merijn Mathieu Verkade
Maria Antonia WIJDEVEN
Ryan HEESBEEN
Petrus Josephus Jacobus Maria VAN DE SANDE
Remon VAN GEEL
Brian Maria Gerardus JANSSEN
Inge Catharina Josephina HURKMANS
Floris Louis Van Delft
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Synaffix BV
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Synaffix BV
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Publication date
Priority claimed from JP2016155927A external-priority patent/JP2017197512A/ja
Application filed by Synaffix BV filed Critical Synaffix BV
Publication of EP3413916A1 publication Critical patent/EP3413916A1/de
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention is in the field of bioconjugation. More specifically, the invention relates to a specific mode of conjugation to prepare bioconjugates that have a beneficial effect on the therapeutic index of the bioconjugate, in particular in the targeting of CD30-expressing tumours. Background of the invention
  • Bioconjugation is the process of linking two or more molecules, of which at least one is a biomolecule.
  • the biomolecule(s) may also be referred to as "biomolecule(s) of interest", the other molecule(s) may also be referred to as “target molecule” or “molecule of interest”.
  • target molecule or “molecule of interest”.
  • the biomolecule of interest (BOI) will consist of a protein (or peptide), a glycan, a nucleic acid (or oligonucleotide), a lipid, a hormone or a natural drug (or fragments or combinations thereof).
  • the other molecule of interest may also be a biomolecule, hence leading to the formation of homo- or heterodimers (or higher oligomers), or the other molecule may possess specific features that are imparted onto the biomolecule of interest by the conjugation process.
  • the modulation of protein structure and function by covalent modification with a chemical probe for detection and/or isolation has evolved as a powerful tool in proteome-based research and biomedical applications. Fluorescent or affinity tagging of proteins is key to studying the trafficking of proteins in their native habitat. Vaccines based on protein-carbohydrate conjugates have gained prominence in the fight against HIV, cancer, malaria and pathogenic bacteria, whereas carbohydrates immobilized on microarrays are instrumental in elucidation of the glycome.
  • Synthetic DNA and RNA oligonucleotides require the introduction of a suitable functionality for diagnostic and therapeutic applications, such as microarray technology, antisense and gene- silencing therapies, nanotechnology and various materials sciences applications.
  • a suitable functionality such as microarray technology, antisense and gene- silencing therapies, nanotechnology and various materials sciences applications.
  • attachment of a cell-penetrating ligand is the most commonly applied strategy to tackle the low internalization rate of ONs encountered during oligonucleotide-based therapeutics (antisense, siRNA).
  • the preparation of oligonucleotide-based microarrays requires the selective immobilization of ONs on a suitable solid surface, e.g. glass.
  • two strategic concepts can be recognized in the field of bioconjugation technology: (a) conjugation based on a functional group already present in the biomolecule of interest, such as for example a thiol, an amine, an alcohol or a hydroxyphenol unit or (b) a two-stage process involving engineering of one (or more) unique reactive groups into a BOI prior to the actual conjugation process.
  • a functional group already present in the biomolecule of interest such as for example a thiol, an amine, an alcohol or a hydroxyphenol unit
  • a two-stage process involving engineering of one (or more) unique reactive groups into a BOI prior to the actual conjugation process.
  • the first approach typically involves a reactive amino acid side-chain in a protein (e.g. cysteine, lysine, serine and tyrosine), or a functional group in a glycan (e.g. amine, aldehyde) or nucleic acid (e.g. purine or pyrimidine functionality or alcohol).
  • a reactive amino acid side-chain in a protein e.g. cysteine, lysine, serine and tyrosine
  • a functional group in a glycan e.g. amine, aldehyde
  • nucleic acid e.g. purine or pyrimidine functionality or alcohol
  • Typical examples of a functional group that may be imparted onto the BOI include (strained) alkyne, (strained) alkene, norbornene, tetrazine, azide, phosphine, nitrile oxide, nitrone, nitrile imine, diazo compound, carbonyl compound, (O-alkyl)hydroxylamine and hydrazine, which may be achieved by either chemical or molecular biology approach.
  • Each of the above functional groups is known to have at least one reaction partner, in many cases involving complete mutual reactivity.
  • cyclooctynes react selectively and exclusively with 1 ,3-dipoles, strained alkenes with tetrazines and phosphines with azides, leading to fully stable covalent bonds.
  • some of the above functional groups have the disadvantage of being highly lipophilic, which may compromise conjugation efficiency, in particular in combination with a lipophilic molecule of interest (see below).
  • the final linking unit between the biomolecule and the other molecule of interest should preferentially also be fully compatible with an aqueous environment in terms of solubility, stability and biocompatibility.
  • a highly lipophilic linker may lead to aggregation (during and/or after conjugation), which may significantly increase reaction times and/or reduce conjugation yields, in particular when the MOI is also of hydrophobic nature.
  • highly lipophilic linker- MOI combination may lead to unspecific binding to surfaces or specific hydrophobic patches on the same or other biomolecules. If the linker is susceptible to aqueous hydrolysis or other water- induced cleavage reactions, the components comprising the original bioconjugate separate by diffusion.
  • linker should be inert to functionalities present in the bioconjugate or any other functionality that may be encountered during application of the bioconjugate, which excludes, amongst others, the use of linkers featuring for example a ketone or aldehyde moiety (may lead to imine formation), an ⁇ , ⁇ -unsaturated carbonyl compound (Michael addition), thioesters or other activated esters (amide bond formation).
  • PEG linkers are highly water soluble, non-toxic, non-antigenic, and lead to negligible or no aggregation. For this reason, a large variety of linear, bifunctional PEG linkers are commercially available from various sources, which can be selectively modified at either end with a (bio)molecule of interest.
  • PEG linkers are the product of a polymerization process of ethylene oxide and are therefore typically obtained as stochastic mixtures of chain length, which can be partly resolved into PEG constructs with an average weight distribution centred around 1 , 2, 4 kDa or more (up to 60 kDa).
  • dPEGs Homogeneous, discrete PEGs
  • dPEGs Homogeneous, discrete PEGs
  • the PEG unit itself imparts particular characteristics onto a biomolecule.
  • protein PEGylation may lead to prolonged residence in vivo, decreased degradation by metabolic enzymes and a reduction or elimination of protein immunogenicity.
  • Several PEGylated proteins have been FDA-approved and are currently on the market.
  • PEG linkers are perfectly suitable for bioconjugation of small and/or water-soluble moieties under aqueous conditions.
  • the polarity of a PEG unit may be insufficient to offset hydrophobicity, leading to significantly reduced reaction rates, lower yields and induced aggregation issues.
  • lengthy PEG linkers and/or significant amounts of organic co-solvents may be required to solubilize the reagents.
  • auristatins E or F maytansinoids, duocarmycins, calicheamicins or pyrrolobenzodiazepines (PBDs), with many others are underway.
  • PBDs pyrrolobenzodiazepines
  • auristatin F all toxic payloads are poorly to non-water- soluble, which necessitates organic co-solvents to achieve successful conjugation, such as 25% dimethylacetamide (DMA) or 50% propylene glycol (PG).
  • DMA dimethylacetamide
  • PG propylene glycol
  • Linkers are known in the art, and disclosed in e.g. WO 2008/070291 , incorporated by reference.
  • WO 2008/070291 discloses a linker for the coupling of targeting agents to anchoring components.
  • the linker contains hydrophilic regions represented by polyethylene glycol (PEG) and an extension lacking chiral centres that is coupled to a targeting agent.
  • PEG polyethylene glycol
  • WO 01/88535 discloses a linker system for surfaces for bioconjugation, in particular a linker system having a novel hydrophilic spacer group.
  • WO 2014/100762 describes compounds with a hydrophilic self- immolative linker, which is cleavable under appropriate conditions and incorporates a hydrophilic group to provide better solubility of the compound.
  • the compounds comprise a drug moiety, a targeting moiety capable of targeting a selected cell population, and a linker which contains an acyl unit, an optional spacer unit for providing distance between the drug moiety and the targeting moiety, a peptide linker which can be cleavable under appropriate conditions, a hydrophilic self- immolative linker, and an optional second self-immolative spacer or cyclization self-elimination linker.
  • the hydrophilic self-immolative linker is e.g. a benzyloxycarbonyl group.
  • the invention relates to a method or use for increasing the therapeutic index of a bioconjugate, i.e. the conjugate of a biomolecule and a target molecule.
  • a bioconjugate prepared via a specific mode of conjugation exhibits a greater therapeutic index compared to the same bioconjugate, i.e. the same biomolecule, the same target molecule (e.g. active substance) and the same biomolecule drug ratio, obtained via a different mode of conjugation.
  • the mode of conjugating a biomolecule to a target molecule is exposed in the linker itself and/or in the attachment point of the linker to the biomolecule.
  • linker and/or attachment point could have an effect on the therapeutic index of a bioconjugate, such as an antibody-drug-conjugate, could not be envisioned based on the current knowledge.
  • linkers are considered inert when it comes to treatment and are solely present as a consequence of the preparation of the bioconjugate. That the selection of a specific mode of conjugation has an effect on the therapeutic index is unprecedented and a breakthrough discovery in the field of bioconjugates, in particular antibody-drug-conjugates.
  • the bioconjugates according to the invention are on one hand more efficacious (therapeutically effective) as the same bioconjugates, i.e. the same biomolecule, the same target molecule (e.g. active substance) and the same biomolecule/target molecule ratio, obtained via a different mode of conjugation, and/or on the other hand exhibit a greater tolerability.
  • This finding has dramatic implications on the treatment of subjects with the bioconjugate according to the invention, as the therapeutic window widens. As a result of the expansion of the therapeutic window, the treatment dosages may be lowered and as a consequence potential, unwanted, side-effects are reduced.
  • the mode of conjugation according to the invention comprises:
  • S(F ) X and x are as defined above; AB represents an antibody; GlcNAc is N- acetylglucosamine; Fuc is fucose; b is 0 or 1 ; and y is 1 , 2, 3 or 4; and
  • linker-conjugate comprising a functional group Q capable of reacting with functional group F and a target molecule D connected to Q via a linker L 2 to obtain the antibody-conjugate wherein linker L comprises S-Z 3 -L 2 and wherein Z 3 is a connecting group resulting from the reaction between Q and F .
  • the mode of conjugation according to the invention ensure that the bioconjugate contains a linker L comprising a group according to formula (1 ) or a salt thereof:
  • a bioconjugate prepared such that it contains a linker L comprising a group according to formula (1 ) or a salt thereof exhibits a greater therapeutic index compared to the same bioconjugate, i.e. the same biomolecule, the same target molecule (e.g. active substance) and the same biomolecule drug ratio, containing a linker without the group according to formula (1 ) present.
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is a further target molecule D, wherein D is optionally connected to N via a spacer moiety.
  • the mode of conjugation is being used to connect a biomolecule B with a target molecule D via a linker L.
  • Conjugation refers to the specific mode of connecting the biomolecule to the target molecule.
  • the bioconjugate according to the invention is represented by formula (A):
  • - B is a biomolecule
  • - L is a linker linking B and D;
  • - B is a biomolecule
  • - L is a linker linking B and D;
  • - a is 0 or 1 ; and - R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S or NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is an additional target molecule D, wherein the target molecule is optionally connected to N via a spacer moiety.
  • the method according to embodiment 1 further comprising a step of administering the bioconjugate to a subject in need thereof.
  • biomolecule is an antibody and the bioconjugate is an antibody-drug-conjugate.
  • target molecule D is an active substance, preferably a cytotoxin.
  • bioconjugate has the formula B-Z 3 -L-D, wherein Z 3 is obtained by the reacting reactive group Q with the functional group F .
  • Z 3 is obtained by the reacting a linker- conjugate having formula Q -L-D, wherein L comprises a group according to formula (1 ) or a salt thereof:
  • - a is independently 0 or 1 ;
  • - b is independently 0 or 1 ;
  • - c is 0 or 1 ;
  • - d is 0 or 1 ;
  • - e is 0 or 1 ;
  • - f is an integer in the range of 1 to 150;
  • - g is 0 or 1 ;
  • - i is 0 or 1 ;
  • - D is a target molecule
  • - Q is a reactive group capable of reacting with a functional group F present on a biomolecule
  • - Z is a connecting group that connects Q or Sp 3 to Sp 2 , O or C(O) or N(R 1 );
  • - Z 2 is a connecting group that connects D or Sp 4 to Sp 1 , N(R 1 ), O or C(O);
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups; or R is D, -[(Sp )b(Z 2 ) e (Sp 4 )iD] or -[(Sp 2 )c(Z)
  • Sp 1 , Sp 2 , Sp 3 and Sp 4 are independently selected from the group consisting of linear or branched C1-C200 alkylene groups, C2-C200 alkenylene groups, C2-C200 alkynylene groups, C3-C200 cydoalkylene groups, C5-C200 cydoalkenylene groups, C8-C200 cydoalkynylene 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, cydoalkylene groups, cydoalkenylene groups, cydoalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally
  • Sp ⁇ Sp 2 , Sp 3 and Sp 4 are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, the alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group consisting of O, S or NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, and whe is according to formula (9a), (9q), (9n), (9o) or (9p), (9t) or (9zh):
  • R 9 is hydrogen, a linear or branched Ci - C12 alkyl group or a C4 - C12 (hetero)aryl group.
  • R 0 is a (thio)ester group
  • R 8 is selected from the group consisting of, optionally substituted, Ci - C12 alkyl groups and C4 - C12 (hetero)aryl groups.
  • Z is selected from hydrogen, methyl and pyridyl.
  • - B is a biomolecule
  • - L is a linker linking B and D;
  • L comprises a group thereof:
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is an additional target molecule D, wherein the target molecule is optionally connected to N via a spacer moiety.
  • the present invention thus concerns in a first aspect a method or use for increasing the therapeutic index of a bioconjugate, wherein the mode of conjugation according to the invention is comprised in or used to prepare the bioconjugate.
  • the mode of conjugation comprises steps (i) and (ii) as defined herein.
  • the mode of conjugation comprises the step of preparing the bioconjugate of formula (A) such that linker L as defined above is comprised in the bioconjugate.
  • the method or use according to the invention further comprises administering the bioconjugate to a subject in need thereof.
  • the invention according to the first aspect can also be worded as the use of the mode of conjugation as defined above in a bioconjugate for increasing the therapeutic index of the bioconjugate, or to the use of linker L as defined above in a bioconjugate for increasing the therapeutic index of the bioconjugate.
  • the present invention concerns the treatment of a subject in need thereof, comprising the administration of the bioconjugate according to the invention.
  • the bioconjugate is administered in a therapeutically effective dose.
  • administration may occur less frequent as in treatment with conventional bioconjugates and/or in a lower dose.
  • administration may occur more frequent as in treatment with conventional bioconjugates and/or in a higher dose.
  • Administration may be in a single dose or may e.g. occur 1 - 4 times a month, preferably 1 - 2 times a month, more preferable administration occurs once every 3 or 4 weeks, most preferably every 4 weeks.
  • the dose of the bioconjugate according to the invention may depend on many factors and optimal doses can be determined by the skilled person via routine experimentation.
  • the bioconjugate is typically administered in a dose of 0.01 - 50 mg/kg body weight of the subject, more accurately 0.03 - 25 mg/kg or most accurately 0.05 - 10 mg/kg, or alternatively 0.1 - 25 mg/kg or 0.5 - 10 mg/kg.
  • the administration of the bioconjugate according to the invention is at a dose that is lower than the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the dose is at most 99-90%, more preferably at most 89- 60%, even more preferable 59-30%, most preferably at most 29-10% of the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • the administration of the bioconjugate according to the invention is at a dose that is higher than the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the dose is at most 10-29%, more preferably at most 30-59%, even more preferable 60-89%, most preferably at most 90-99% of the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • the administration of the bioconjugate according to the invention is at a dose that is lower than the ED50 of the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the dose is at most 99-90%, more preferably at most 89-60%, even more preferable 59-30%, most preferably at most 29-10% of the ED50 of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • the administration of the bioconjugate according to the invention is at a dose that is higher than the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the dose is at most a factor 1.1 - 1.49 higher, more preferably at most a factor 1 .5 - 1.99 higher, even more preferable a factor 2 - 4.99 higher, most preferably at most a factor 5 - 10 higher of the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • the preparation of the bioconjugate typically comprises the step of reacting linker-conjugate having the formula Q -L-D, wherein L and D are as defined above and Q is a reactive group capable of reacting with a functional group F ⁇ with a biomolecule having the formula B-F , wherein B is as defined above and F is a functional group capable of reacting with Q .
  • Q and F react to form a connecting group Z 3 , which is located in the bioconjugate according to formula (A) in the spacer moiety between B and L.
  • Figure 1 describes the general concept of conjugation of biomolecules: a biomolecule of interest (BOI) containing one or more functional groups F is incubated with (excess of) a target molecule D (also referred to as molecule of interest or MOI) covalently attached to a reactive group Q via a specific linker.
  • a chemical reaction between F and Q takes place, thereby forming a bioconjugate comprising a covalent connection between the BOI and the MOI.
  • the BOI may e.g. be a peptide/protein, a glycan or a nucleic acid.
  • Figure 2 shows several structures of derivatives of UDP sugars of galactosamine, which may be modified with e.g. a 3-mercaptopropionyl group (11a), an azidoacetyl group (11 b), or an azidodifluoroacetyl group (11c) at the 2-position, or with an azido azidoacetyl group at the 6- position of N-acetyl galactosamine (11d).
  • a 3-mercaptopropionyl group 11a
  • an azidoacetyl group 11 b
  • an azidodifluoroacetyl group 11c
  • Figure 3 schematically displays how any of the UDP-sugars 11a-d may be attached to a glycoprotein comprising a GlcNAc moiety 12 (e.g. a monoclonal antibody the glycan of which is trimmed by an endoglycosidase) under the action of a galactosyltransferase mutant or a GalNAc- transferase, thereby generating a ⁇ -glycosidic 1-4 linkage between a GalNAc derivative and GlcNAc (compounds 13a-d, respectively).
  • a GlcNAc moiety 12 e.g. a monoclonal antibody the glycan of which is trimmed by an endoglycosidase
  • Figure 4 shows how a modified antibody 13a-d may undergo a bioconjugation process by means of nucleophilic addition to maleimide (as for 3-mercaptopropionyl-galactosamine-modified 13a leading to thioether conjugate 14, or for conjugation to a engineered cysteine residue leading to thioether conjugate 17) or upon strain-promoted cycloaddition with a cyclooctyne reagent (as for 13b, 13c or 13d leading to triazoles 15a, 15b or 16, respectively).
  • maleimide as for 3-mercaptopropionyl-galactosamine-modified 13a leading to thioether conjugate 14, or for conjugation to a engineered cysteine residue leading to thioether conjugate 17
  • a cyclooctyne reagent as for 13b, 13c or 13d leading to triazoles 15a, 15b or 16, respectively.
  • Figure 5 shows a representative set of functional groups (F ) in a biomolecule, either naturally present or introduced by engineering, which upon reaction with reactive group Q lead to connecting group Z 3 .
  • Functional group F may also be artificially introduced (engineered) into a biomolecule at any position of choice.
  • FIG. 6 shows preferred bioconjugates according to the invention.
  • Conjugates 52-57 and 59 are prepared and the therapeutic index thereof investigated in the examples.
  • Conjugates 52-57 are conjugated to brentuximab as antibody and conjugate 59 to iratumumab.
  • Figures 7A-F depict the results of the tolerability studies of Example 38 for control antibody- conjugate Adcetris (Fig. 7A) and antibody-conjugates according to the invention 57 (Fig. 7B), 56 (Fig. 7C), 52 (Fig. 7D), 54 (Fig. 7E), 53 (Fig. 7F).
  • Percentage body weight change ( ⁇ BW), based on 100 % on the start of treatment (day 1 ), over time is depicted. Reductions in body weight indicate that the conjugate is not tolerated at the specific dose.
  • C vehicle treated.
  • Figure 9 shows the regression of the drug antibody ratio (DAR) over time, for control antibody- conjugate Adcetris and for antibody-conjugates according to the invention 56 and 57, corresponding to Example 39.
  • the antibody-conjugates according to the invention have a theoretical DAR of 4 which hardly decreases over time, whereas the control antibody-conjugate starts with a slightly higher DAR which quickly decreases below the DAR of the antibody- conjugates according to the invention.
  • the conjugation reaction involves on the one hand the biomolecule (BOI) containing a functional group F , and on the other hand the target molecule (MOI) containing a reactive group Q , or a "linker-conjugate" as defined herein, wherein Q reacts with F to form a connecting group that joins the BOI and the MOI in a bioconjugate.
  • reactive group Q is joined via a linker to the MOI, said linker comprising the sulfamide moiety according to formula (1 ).
  • Reactive group Q may be attached to either ends of the moiety of formula (1 ), in which case the MOI is attached to the opposite end of the moiety of formula (1 ).
  • reactive group Q is attached to the moiety of formula (1 ) via the carbonyl end and the MOI is attached via the sulfamide end of the moiety of formula (1 ).
  • reactive group Q is attached to the moiety of formula (1 ) via the sulfamide end and the MOI is attached via the carbonyl end of the moiety of formula (1 ).
  • 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”.
  • the compounds disclosed in this description and in the claims may comprise one or more asymmetric centres, and different diastereomers and/or enantiomers may exist of the compounds.
  • the description of any compound in this description and in the claims is meant to include all diastereomers, and mixtures thereof, unless stated otherwise.
  • any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise.
  • the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer.
  • the compounds may occur in different tautomeric forms.
  • the compounds according to the invention are meant to include all tautomeric forms, unless stated otherwise.
  • the structure of a compound is depicted as a specific tautomer, it is to be understood that the invention of the present application is not limited to that specific tautomer.
  • the compounds disclosed in this description and in the claims may exist as cis and trans isomers. Unless stated otherwise, the description of any compound in the description and in the claims is meant to include both the individual cis and the individual trans isomer of a compound, as well as mixtures thereof. As an example, when the structure of a compound is depicted as a cis isomer, it is to be understood that the corresponding trans isomer or mixtures of the cis and trans isomer are not excluded from the invention of the present application. When the structure of a compound is depicted as a specific cis or trans isomer, it is to be understood that the invention of the present application is not limited to that specific cis or trans isomer.
  • Unsubstituted alkyl groups have the general formula C n H2n+i and may be linear or branched. Optionally, the alkyl groups are substituted by one or more substituents further specified in this document. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1- dodecyl, etc.
  • a cycloalkyl group is a cyclic alkyl group.
  • Unsubstituted cycloalkyl groups comprise at least three carbon atoms and have the general formula C n H2n-i .
  • the cycloalkyl groups are substituted by one or more substituents further specified in this document. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • An alkenyl group comprises one or more carbon-carbon double bonds, and may be linear or branched. Unsubstituted alkenyl groups comprising one C-C double bond have the general formula C n H2n-i . Unsubstituted alkenyl groups comprising two C-C double bonds have the general formula C n H2n-3.
  • An alkenyl group may comprise a terminal carbon-carbon double bond and/or an internal carbon-carbon double bond.
  • a terminal alkenyl group is an alkenyl group wherein a carbon-carbon double bond is located at a terminal position of a carbon chain.
  • An alkenyl group may also comprise two or more carbon-carbon double bonds.
  • alkenyl group examples include ethenyl, propenyl, isopropenyl, t-butenyl, 1 ,3-butadienyl, 1 ,3-pentadienyl, etc.
  • an alkenyl group may optionally be substituted with one or more, independently selected, substituents as defined below.
  • an alkenyl group may optionally be interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S.
  • An alkynyl group comprises one or more carbon-carbon triple bonds, and may be linear or branched. Unsubstituted alkynyl groups comprising one C-C triple bond have the general formula CnH2n-3.
  • An alkynyl group may comprise a terminal carbon-carbon triple bond and/or an internal carbon-carbon triple bond.
  • a terminal alkynyl group is an alkynyl group wherein a carbon-carbon triple bond is located at a terminal position of a carbon chain.
  • An alkynyl group may also comprise two or more carbon-carbon triple bonds. Unless stated otherwise, an alkynyl group may optionally be substituted with one or more, independently selected, substituents as defined below.
  • alkynyl group examples include ethynyl, propynyl, isopropynyl, t-butynyl, etc. Unless stated otherwise, an alkynyl group may optionally be interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S.
  • An aryl group comprises six to twelve carbon atoms and may include monocyclic and bicyclic structures.
  • the aryl group may be substituted by one or more substituents further specified in this document.
  • Examples of aryl groups are phenyl and naphthyl.
  • Arylalkyl groups and alkylaryl groups comprise at least seven carbon atoms and may include monocyclic and bicyclic structures.
  • the arylalkyl groups and alkylaryl may be substituted by one or more substituents further specified in this document.
  • An arylalkyl group is for example benzyl.
  • An alkylaryl group is for example 4-i-butylphenyl.
  • Heteroaryl groups comprise at least two carbon atoms (i.e. at least C2) and one or more heteroatoms N, O, P or S.
  • a heteroaryl group may have a monocyclic or a bicyclic structure.
  • the heteroaryl group may be substituted by one or more substituents further specified in this document.
  • heteroaryl groups examples include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl.
  • Heteroarylalkyl groups and alkylheteroaryl groups comprise at least three carbon atoms (i.e. at least C3) and may include monocyclic and bicyclic structures.
  • the heteroaryl groups may be substituted by one or more substituents further specified in this document.
  • an aryl group is denoted as a (hetero)aryl group, the notation is meant to include an aryl group and a heteroaryl group.
  • an alkyl(hetero)aryl group is meant to include an alkylaryl group and a alkylheteroaryl group
  • (hetero)arylalkyl is meant to include an arylalkyl group and a heteroarylalkyl group.
  • a C2 - C24 (hetero)aryl group is thus to be interpreted as including a C2 - C24 heteroaryl group and a Ce - C24 aryl group.
  • a C3 - C24 alkyl(hetero)aryl group is meant to include a C7 - C24 alkylaryl group and a C3 - C24 alkylheteroaryl group
  • a C3 - C24 (hetero)arylalkyl is meant to include a C7 - C24 arylalkyl group and a C3 - C24 heteroarylalkyl group.
  • a cycloalkynyl group is a cyclic alkynyl group.
  • An unsubstituted cycloalkynyl group comprising one triple bond has the general formula Cnhbn-s.
  • a cycloalkynyl group is substituted by one or more substituents further specified in this document.
  • An example of a cycloalkynyl group is cyclooctynyl.
  • a heterocycloalkynyl group is a cycloalkynyl group interrupted by heteroatoms selected from the group of oxygen, nitrogen and sulphur.
  • a heterocycloalkynyl group is substituted by one or more substituents further specified in this document.
  • An example of a heterocycloalkynyl group is azacyclooctynyl.
  • a (hetero)aryl group comprises an aryl group and a heteroaryl group.
  • An alkyl(hetero)aryl group comprises an alkylaryl group and an alkyl heteroaryl group.
  • a (hetero)arylalkyl group comprises a arylalkyl group and a heteroarylalkyl groups.
  • a (hetero)alkynyl group comprises an alkynyl group and a heteroalkynyl group.
  • a (hetero)cycloalkynyl group comprises an cycloalkynyl group and a heterocycloalkynyl group.
  • a (hetero)cycloalkyne compound is herein defined as a compound comprising a (hetero)cycloalkynyl group.
  • fused (hetero)cycloalkyne compounds i.e. (hetero)cycloalkyne compounds wherein a second ring structure is fused, i.e. annulated, to the (hetero)cycloalkynyl group.
  • a fused (hetero)cyclooctyne compound a cycloalkyl (e.g. a cyclopropyl) or an arene (e.g. benzene) may be annulated to the (hetero)cyclooctynyl group.
  • the triple bond of the (hetero)cyclooctynyl group in a fused (hetero)cyclooctyne compound may be located on either one of the three possible locations, i.e. on the 2, 3 or 4 position of the cyclooctyne moiety (numbering according to "lUPAC Nomenclature of Organic Chemistry", Rule A31.2).
  • the description of any fused (hetero)cyclooctyne compound in this description and in the claims is meant to include all three individual regioisomers of the cyclooctyne moiety.
  • alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, (hetero)aryl groups, (hetero)arylalkyl groups, alkyl(hetero)aryl groups, alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, (hetero)arylene groups, alkyl(hetero)arylene groups, (hetero)arylalkylene groups, (hetero)arylalkenylene groups, (hetero)arylalkynylene groups, alkenyl groups, alkoxy groups, alkenyloxy groups, (hetero)aryloxy groups, alkynyloxy groups and cycloalkyloxy groups may be substituted with one or more substituents independently selected from the group consisting of Ci - Ci2 alkyl groups, C2 - C12 alkenyl groups, C
  • sugar is herein used to indicate a monosaccharide, for example glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc).
  • sugar derivative is herein used to indicate a derivative of a monosaccharide sugar, i.e. a monosaccharide sugar comprising substituents and/or functional groups. Examples of a sugar derivative include amino sugars and sugar acids, e.g.
  • glucosamine (GlcNhb), galactosamine (GalNhb) N-acetylglucosamine (GlcNAc), 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).
  • a sugar derivative also include compounds herein denoted as S(F ) X , wherein S is a sugar or a sugar derivative, and wherein S comprises x functional groups F .
  • a core /V-acetylglucosamine substituent (core-GlcNAc substituent) or core /V-acetylglucosamine moiety is herein defined as a GlcNAc that is bonded via C1 to an antibody, preferably via an N- glycosidic bond to the amide nitrogen atom in the side chain of an asparagine amino acid of the antibody.
  • the core-GlcNAc substituent may be present at a native glycosylation site of an antibody, but it may also be introduced on a different site on the antibody.
  • a core-/V- acetylglucosamine substituent is a monosaccharide substituent, or if said core-GlcNAc substituent is fucosylated, a disaccharide core- GlcNAc-(D l-6-Fuc) substituent, further referred to as GlcNAc(Fuc).
  • a "core-GlcNAc substituent" is not to be confused with a "core-GlcNAc”.
  • a core-GlcNAc is herein defined as the inner GlcNAc that is part of a poly- or an oligosaccharide comprising more than two saccharides, i.e. the GlcNAc via which the poly- or oligosaccharide is bonded to an antibody.
  • An antibody comprising a core-N-acetylglucosamine substituent as defined herein is thus an antibody, comprising a monosaccharide core-GlcNAc substituent as defined above, or if said core-GlcNAc substituent is fucosylated, a disaccharide core-GlcNAc(Fuc) substituent. If a core- GlcNAc substituent or the GlcNAc in a GlcNAc-S(F ) x substituent is fucosylated, fucose is most commonly linked a-1 ,6 to 06 of the core-GlcNAc substituent.
  • a fucosylated core-GlcNAc substituent is denoted core-GlcNAc(Fuc)
  • a fucosylated GlcNAc-S(F ) x substituent is denoted GlcNAc(Fuc)-S(F )x.
  • nucleotide is herein used in its normal scientific meaning.
  • nucleotide refers to a molecule that is composed of a nucleobase, a five-carbon sugar (either ribose or 2- deoxyribose), and one, two or three phosphate groups. Without the phosphate group, the nucleobase and sugar compose a nucleoside.
  • a nucleotide can thus also be called a nucleoside monophosphate, a nucleoside diphosphate or a nucleoside triphosphate.
  • the nucleobase may be adenine, guanine, cytosine, uracil or thymine.
  • nucleotide examples include uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP).
  • UDP uridine diphosphate
  • GDP guanosine diphosphate
  • TDP thymidine diphosphate
  • CDP cytidine diphosphate
  • CMP cytidine monophosphate
  • protein is herein used in its normal scientific meaning.
  • polypeptides comprising about 10 or more amino acids are considered proteins.
  • a protein may comprise natural, but also unnatural amino acids.
  • 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.
  • 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
  • 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.
  • glycoproteins include PSMA (prostate-specific membrane antigen), CAL (Candida antartica lipase), gp41 , gp120, EPO (erythropoietin), antifreeze protein and antibodies.
  • 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.
  • a monosaccharide glycan of a glycoprotein consists of a single N-acetylglucosamine (GlcNAc), 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.
  • the sugar that is directly attached to the protein is called the core sugar.
  • a sugar that is not directly attached to the protein and is attached to at least two other sugars is called an internal sugar.
  • 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.
  • a glycan may be an O-linked glycan, an N-linked glycan or a C-linked glycan.
  • 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).
  • 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).
  • 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).
  • the term "antibody” 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.
  • antibody is herein also meant to include human antibodies, humanized antibodies, chimeric antibodies and antibodies specifically binding cancer antigen.
  • antibody is meant to include whole antibodies, but also antigen-binding 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, a bispecific antibody or a scFv.
  • 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.
  • Typical examples of antibodies include, amongst others, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, tositumomab-1131 , cetuximab, ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab and brentuximab.
  • the antibody comprising a core-N- acetylglucosamine substituent is a monoclonal antibody (mAb).
  • said antibody is selected from the group consisting of IgA, IgD, IgE, IgG and IgM antibodies. More preferably, said antibody is an IgG antibody, and most preferably said antibody is an lgG1 antibody.
  • the antibody preferably comprises one or more, more preferably one, core-GlcNAc substituent on each heavy chain, said core- GlcNAc substituent being optionally fucosylated.
  • Said whole antibody thus preferably comprises two or more, preferably two, optionally fucosylated, core-GlcNAc substituents.
  • the antibody preferably comprises one or more core-GlcNAc substituent, which is optionally fucosylated.
  • said core-GlcNAc substituent may be situated anywhere on the antibody, provided that said substituent does not hinder the antigen-binding site of the antibody.
  • said core /V-acetylglucosamine substituent is present at a native A/-glycosylation site of the antibody.
  • a linker is herein defined as a moiety that connects two or more elements of a compound.
  • a bioconjugate a biomolecule and a target molecule are covalently connected to each other via a linker; in the linker-conjugate a reactive group Q is covalently connected to a target molecule via a linker; in a linker-construct a reactive group Q is covalently connected to a reactive group Q 2 via a linker.
  • 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, the linker-conjugate or a bioconjugate, as defined below.
  • a bioconjugate is herein defined as a compound wherein a biomolecule is covalently connected to a target molecule via a linker.
  • a bioconjugate comprises one or more biomolecules and/or one or more target molecules.
  • the linker may comprise one or more spacer moieties.
  • An antibody- conjugate refers to a bioconjugate wherein the biomolecule is an antibody.
  • a biomolecule is herein defined as any molecule that can be isolated from nature or any molecule composed of smaller molecular building blocks that are the constituents of macromolecular structures derived from nature, in particular nucleic acids, proteins, glycans and lipids.
  • the biomolecule may also be referred to as biomolecule of interest (BOI).
  • BOI biomolecule of interest
  • examples of a biomolecule include an enzyme, a (non-catalytic) protein, a polypeptide, a peptide, an amino acid, an oligonucleotide, a monosaccharide, an oligosaccharide, a polysaccharide, a glycan, a lipid and a hormone.
  • a target molecule also referred to as a molecule of interest (MOI) is herein defined as molecular structure possessing a desired property that is imparted onto the biomolecule upon conjugation.
  • 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.
  • 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.
  • salt means a salt that is acceptable for administration to a patient, such as a mammal (salts with counter-ions 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.
  • sulfamide linker refers to a linker comprising a sulfamide group, more particularly an acylsulfamide group [-C(0)-N(H)-S(0)2-N(R 1 )-] and/or a carbamoyl sulfamide group [-O-C(O)-
  • the term "therapeutic index" 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).
  • 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, 1 1 , 751-761 . At an early development stage, the clinical Tl of a drug candidate is often not yet known.
  • 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).
  • 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.
  • the therapeutic effect of a bioconjugate e.g. an ADC
  • the efficacy refers to the ability of the ADC to provide a beneficial effect.
  • the tolerability of said ADC in a rodent safety study can also be a measure of the therapeutic effect.
  • 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.
  • the "mode of conjugation” refers to the process that is used to conjugate a target molecule D to a biomolecule B, in particular an antibody AB, as well as to the structural features of the resulting bioconjugate, in particular of the linker that connects the target molecule to the biomolecule, that are a direct consequence of the process of conjugation.
  • the mode of conjugation refers to a process for conjugation a target molecule to a biomolecule, in particular an antibody.
  • the mode of conjugation refers to structural features of the linker and/or to the attachment point of the linker to the biomolecule that are a direct consequence of the process for conjugation a target molecule to a biomolecule, in particular an antibody.
  • the mode of conjugation comprises at least one of "core- GlcNAc functionalization” and “sulfamide linkage” as defined further below.
  • the mode of conjugation comprises both the “core-GlcNAc functionalization” and “sulfamide linkage” as defined further below.
  • the mode of conjugation according to the invention is referred to as "core- GlcNAc functionalization", which refers to a process comprising:
  • S(F ) X and x are as defined above; AB represents an antibody; GlcNAc is N- acetylglucosamine; Fuc is fucose; b is 0 or 1 ; and y is 1 , 2, 3 or 4; and
  • linker-conjugate comprising a functional group Q capable of reacting with functional group F and a target molecule D connected to Q via a linker L 2 to obtain the antibody-conjugate wherein linker L comprises S-Z 3 -L 2 and wherein Z 3 is a connecting group resulting from the reaction between Q and F .
  • the antibody is conjugated via a glycan that is trimmed to a core- GlcNAc residue (optionally substituted with a fucose).
  • This residue is functionalized with a sugar derivative S(F ) X , comprising 1 or 2 functional groups F , which are subsequently reacted with a functional group Q present on a linker-conjugate comprising target molecule D.
  • the structural feature of the resulting linker L that links the antibody with the target molecule, that are a direct consequence of the conjugation process include:
  • the point of attachment of the linker L to the antibody AB is at a specific amino acid residue which is glycosylated in the naturally occurring antibody or is an artificially introduced glycosylation site, by mutation of specific amino acid residues in the antibody.
  • the point of attachment of the linker to the antibody can be specifically selected, which affords a highly predictable target molecule to antibody ratio (or DAR: "drug antibody ratio").
  • the linker L is conjugated to a core-GlcNAc moiety of the antibody, and has the general structure -S-(M) PP -Z 3 -L 2 (D) r .
  • Z 3 is a connecting group that is obtained by the reaction between Q and F . Options for Q , F and Z 3 are known to the skilled person and discussed in further detail below.
  • Z 3 is connected via linker L 2 to at least one target molecule D (i.e. r > 1 ).
  • linker L in particular linker L 2 , comprises the group according to formula (1 ) or a salt thereof as defined for mode of conjugation referred to "sulfamide linkage". It has been found that when the mode of conjugation according to the present embodiment is combined with the "sulfamide linkage" mode of conjugation, the best results were obtained in terms of improving the therapeutic index of the resulting bioconjugates.
  • the therapeutic index of antibody-conjugates having the mode of conjugation according to the present embodiment have an improved therapeutic index over antibody-conjugates not having the mode of conjugation according to the present embodiment.
  • the use of the mode of conjugation according to the present invention is distinct from a process of preparing an antibody-conjugate, wherein the mode of conjugation is used to prepare the antibody-conjugate.
  • the mode of conjugation is used to prepare the antibody-conjugate.
  • the mode of conjugation comprises the step of preparing the bioconjugate of formula (A):
  • - B is a biomolecule
  • - L is a linker linking B and D;
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S or NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is an additional target molecule D, wherein the target molecule is optionally connected to N via a spacer moiety.
  • a glycoprotein comprising 1 - 4 core /V-acetylglucosamine moieties is contacted with a compound of the formula S(F )x-P in the presence of a catalyst, wherein S(F ) X is a sugar derivative comprising x functional groups F capable of reacting with a functional group Q , x is 1 or 2 and P is a nucleoside mono- or diphosphate, and wherein the catalyst is capable of transferring the S(F ) X moiety to the core-GlcNAc moiety.
  • the glycoprotein is typically an antibody, such as an antibody that has been trimmed trimmed to a core-GlcNAc residue as described further below.
  • Step (i) affords a modified antibody according to Formula (24):
  • S(F ) X and x are as defined above; AB represents an antibody; GlcNAc is N- acetylglucosamine; Fuc is fucose; b is 0 or 1 ; and y is 1 , 2, 3 or 4.
  • the antibody comprising a core-GlcNAc substituent, wherein said core- GlcNAc substituent is optionally fucosylated is of the Formula (21 ), wherein AB represents an antibody, GlcNAc is /V-acetylglucosamine, Fuc is fucose, b is 0 or 1 and y is 1 to 4, preferably y is 1 or 2.
  • 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.
  • 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, preferably of EndoS, EndoA, EndoF, EndoM, EndoD, EndoH and EndoSH enzymes and/or a combination thereof, the selection of which depends on the nature of the glycan.
  • EndoSH is further defined below in the fourth aspect of the present invention.
  • the endoglycosidase is EndoS, EndoS49, EndoF, EndoH, EndoSH or a combination thereof, more preferably EndoS, EndoS49, EndoF, EndoSH or a combination thereof.
  • the endoglycosidase is EndoS, EndoF or a combination thereof.
  • the endoglycosidase is EndoS.
  • the endoglycosidase is EndoS49.
  • the endoglycosidase is EndoSH. Herien, EndoF typically refers to one of EndoFI , EndoF2 and EndoF3.
  • y 2 or 4
  • y 1 or 2.
  • the antibody AB is capable of targeting tumours that express an antigen selected from Axl, 5T4 (TPBG), av-integrin/ITGAV, BCMA, C4.4a, cadherin-6 (CDH6), CA-IX, CD19, CD19b, CD22, CD25, CD30, CD33, CD37, CD40, CD43, CD56, CD70, CD74, CD79b, CD123, CD352, c-KIT (CD1 17), CD138/SDC1 , CEACAM5 (CD66e), Cripto, CS1 , DLL3, EFNA4, EGFR, EGFRvlll, Endothelin B Receptor (ETBR), ENPP3 (AGS-16), EpCAM, EphA2, FGFR2, FGFR3, FLT3, FOLR1 (folate receptor a), gpNMB, guanyl cyclase C (GCC), HER2 (Erb- B2), HER3 (Erb-
  • the antibody is capable of targeting tumours that express an antigen selected from 5T4 (TPBG), av-integrin/ITGAV, BCMA, C4.4a, CA-IX, CD19, CD19b, CD22, CD25, CD30, CD33, CD37, CD40, CD56, CD70, CD74, CD79b, c- KIT (CD1 17), CD138/SDC1 , CEACAM5 (CD66e), Cripto, CS1 , DLL3, EFNA4, EGFR, EGFRvlll, Endothelin B Receptor (ETBR), ENPP3 (AGS-16), EpCAM, EphA2, FGFR2, FGFR3, FOLR1 (folate receptor a), gpNMB, guanyl cyclase C (GCC), HER2, Erb-B2, Lamp-1 , Lewis Y antigen, LIV-1 (SLC39A6, ZIP6), Mesothelin (MSLN), MUC1 (CA6,
  • the antibody AB is capable of targeting CD30-expressing tumours, more preferably the antibody AB is selected from the group consisting of from Ki-2, Ki-2, Ki-4, Ki- 6, Ki-7, HRS-1 , HRS-4, Ber-H8, Ber-H2, 5F1 1 (MDX-060, iratumumab), Ki-1 , Ki-5, M67, Ki-3, M44, HeFi-1 , AC10, cAC10 (brentuximab) and functional analogues thereof. More preferably, the antibody AB capable of targeting CD30-expressing tumours is iratumumab or brentuximab, most preferably brentuximab.
  • S(F )x is defined as a sugar derivative comprising x functional groups F , wherein x is 1 or 2 and F is a functional group capable of reacting with Q present on the linker-conjugate to form a connecting moiety Z 3 .
  • the sugar derivative S(F ) X may comprise 1 or 2 functional groups F .
  • each functional group F is independently selected, i.e. one S(F ) X may comprise different functional groups F .
  • x 1.
  • x 2.
  • Sugar derivative S(F ) X is derived from a sugar or a sugar derivative S, e.g. an amino sugar or an otherwise derivatized sugar.
  • sugars and sugar derivatives examples include galactose (Gal), mannose (Man), glucose (Glc), glucuronic acid (GlucA) and fucose (Fuc).
  • An amino sugar is a sugar wherein a hydroxyl (OH) group is replaced by an amino group and examples include N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc).
  • examples of an otherwise derivatized sugar include glucuronic acid (GlucA) and N-acetylneuraminic acid (sialic acid).
  • Sugar derivative S(F ) X is preferably derived from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (GlucA), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), preferably from the group consisting of GlcNAc, Glc, Gal and GalNAc. More preferably S(F ) X is derived from Gal or GalNAc, and most preferably S(F ) X is derived from GalNAc.
  • the 1 or 2 functional groups F in S(F ) X may be linked to the sugar or sugar derivative S in several ways.
  • the 1 or 2 functional groups F may be bonded to C2, C3, C4 and/or C6 of the sugar or sugar derivative, instead of a hydroxyl (OH) group. It should be noted that, since fucose lacks an OH-group on C6, if F is bonded to C6 of Fuc, then F takes the place of an H-atom. When F is an azido group, it is preferred that F is bonded to C2, C4 or C6.
  • the one or more azide substituent in S(F ) X may be bonded to C2, C3, C4 or C6 of the sugar or sugar derivative S, instead of a hydroxyl (OH) group or, in case of 6-azidofucose (6- AzFuc), instead of a hydrogen atom.
  • the N-acetyl substituent of an amino sugar derivative may be substituted by an azidoacetyl substituent.
  • S(F ) X is selected from the group consisting of 2-azidoacetamidogalactose (GalNAz), 2-azidodifluoroacetamido-2-deoxy-galactose (F2-GalNAz), 6-azido-6-deoxygalactose (6-AzGal), 6-azido-6-deoxy-2-acetamidogalactose (6-AzGalNAc or 6-N3-GalNAc), 4-azido-4-deoxy-2- acetamidogalactose (4-AzGalNAc), 6-azido-6-deoxy-2-azidoacetamidogalactose (6-AzGalNAz), 2-azidoacetamidoglucose (GlcNAz), 6-azido-6-deoxyglucose (6-AzGlc), 6-azido-6-deoxy-2- acetamidoglucose (Gl
  • S(F )x-P wherein F is an azido group are shown below.
  • F is a keto group
  • F is bonded to C2 instead of the OH-group of S.
  • F may be bonded to the N-atom of an amino sugar derivative, preferably a 2-amino sugar derivative.
  • the sugar derivative then comprises an -NC(0)R 36 substituent.
  • R 36 is preferably a C2 - C24 alkyl group, optionally substituted. More preferably, R 36 is an ethyl group.
  • S(F ) X is selected from the group consisting of 2- deoxy-(2-oxopropyl)galactose (2-ketoGal), 2-N-propionylgalactosamine (2-N-propionylGalNAc), 2- N-(4-oxopentanoyl)galactosamine (2-N-LevGal) and 2-N-butyrylgalactosamine (2-N- butyrylGalNAc), more preferably 2-ketoGalNAc and 2-N-propionylGalNAc.
  • Examples of S(F ))cP wherein F is a keto group are shown below.
  • F is an alkynyl group, preferably a terminal alkynyl group or a (hetero)cycloalkynyl group, it is preferred that said alkynyl group is present on a 2-amino sugar derivative.
  • An example of S(F ) X wherein F is an alkynyl group is 2-(but-3-yonic acid amido)-2-deoxy-galactose.
  • An example of S(F ))cP wherein F is an alkynyl group is shown below.
  • F is selected from the group consisting of an azido group, a keto group and an alkynyl group.
  • An azido group is an azide functional group -N3.
  • a keto group is a -[C(R 37 )2]oC(0)R 36 group, wherein R 36 is a methyl group or an optionally substituted C2 - C24 alkyl group, R 37 is independently selected from the group consisting of hydrogen, halogen and R 36 , and o is 0 - 24, preferably 0 - 10, and more preferably 0, 1 , 2, 3, 4, 5 or 6.
  • R 37 is hydrogen.
  • An alkynyl group is preferably a terminal alkynyl group or a (hetero)cycloalkynyl group as defined above.
  • the alkynyl group is a -[C(R 37 )2]oC ⁇ C-R 37 group, wherein R 37 and o are as defined above; R 37 is preferably hydrogen.
  • F is an azide or an alkyne moiety. Most preferably, F is an azido group (-N3).
  • F is an azide moiety
  • Q is an (cyclo)alkyne moiety
  • Z 3 is a triazole moiety.
  • S(F )x-P is selected from the group consisting of GalNAz-UDP (25), 6-AzGal-UDP (26), 6-AzGalNAc-UDP (6-azido-6-deoxy-N-acetylgalactosamine-UDP) (27), 4-AzGalNAz-UDP, 6- AzGalNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP and 2-(but-3-yonic acid amido)-2-deoxy- galactose-UDP (28).
  • S(F )x-P is GalNAz-UDP (25) or 6-AzGalNAc-UDP (27).
  • Suitable catalyst that are capable of transferring the S(F ) X moiety to the core-GlcNAc moiety are known in the art.
  • a suitable catalyst is a catalyst wherefore the specific sugar derivative nucleotide S(F )x-P in that specific process is a substrate. More specifically, the catalyst catalyzes the formation of a (1 ,4)-glycosidic bond .
  • the catalyst is selected from the group of galactosyltransferases and N-acetylgalactosaminyltransferases, more preferably from the group of j 8(1 ,4)-N-acetylgalactosaminyltransferases (GalNAcT) and j 8(1 ,4)-galactosyltransferases (GalT), most preferably from the group of j 8(1 ,4)-N-acetylgalactosaminyltransferases having a mutant catalytic domain.
  • the catalyst is a wild-type galactosyltransferase or N-acetylgalactosaminyl- transferase, preferably a N-acetylgalactosaminyltransferase.
  • the catalyst is a mutant galactosyltransferase or N-acetylgalactosaminyltransferases, preferably a mutant N-acetylgalactosaminyltransferase. Mutant enzymes described in WO 2016/022027 and PCT/EP2016/059194 (WO 2016/170186)are especially preferred.
  • sugar derivative S(F ) X is linked to the core-GlcNAc substituent in step (i), irrespective of whether said GlcNAc is fucosylated or not.
  • Step (i) 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.
  • 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.
  • the buffer solution is phosphate-buffered saline (PBS) or tris buffer.
  • Step (i) 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 (i) 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 (i) is performed at a pH in the range of about 7 to about 8.
  • step (ii) the modified antibody is reacted with a linker-conjugate comprising a functional group Q capable of reacting with functional group F and a target molecule D connected to Q via a linker L 2 to obtain the antibody-conjugate wherein linker L comprises S-Z 3 -L 2 and wherein Z 3 is a connecting group resulting from the reaction between Q and F .
  • linker L comprises S-Z 3 -L 2 and wherein Z 3 is a connecting group resulting from the reaction between Q and F .
  • linker-conjugates and preferred embodiments thereof are defined further below.
  • the linker L 2 preferably comprises the group according to formula (1 ) or a salt thereof, and said linker is further defined below.
  • Complementary functional groups Q for the functional group F on the modified antibody are known in the art.
  • reactive group Q and functional group F are capable of reacting in a bioorthogonal reaction, as those reactions do not interfere with the biomolecules present during this reaction.
  • Bioorthogonal reactions and functional groups suitable therein are known to the skilled person, for example from Gong and Pan, Tetrahedron Lett. 2015, 56, 2123-2132, and include Staudinger ligations and copper-free Click chemistry.
  • Q is selected from the group consisting of 1 ,3-dipoles, alkynes, (hetero)cyclooctynes, cyclooctenes, tetrazines, ketones, aldehydes, alkoxyamines, hydrazines and triphenylphosphine.
  • F is an azido group
  • linking of the azide-modified antibody and the linker-conjugate preferably takes place via a cycloaddition reaction.
  • Functional group Q is then preferably selected from the group consisting of alkynyl groups, preferably terminal alkynyl groups, and (hetero)cycloalkynyl groups.
  • linking of the keto-modified antibody with the linker-conjugate preferably takes place via selective conjugation with hydroxylamine derivatives or hydrazines, resulting in respectively oximes or hydrazones.
  • Functional group Q is then preferably a primary amino group, e.g. an -NH2 group, an aminooxy group, e.g. -O-NH2, or a hydrazinyl group, e.g. -N(H)NH2.
  • the linker-conjugate is then preferably H 2 N-L 2 (D , H 2 N-0-L 2 (D or H 2 N-N(H)-L 2 (D respectively.
  • linking of the alkyne-modified antibody with the linker-conjugate preferably takes place via a cycloaddition reaction, preferably a 1 ,3-dipolar cycloaddition.
  • Functional group Q is then preferably a 1 ,3-dipole, such as an azide, a nitrone or a nitrile oxide.
  • the linker-conjugate is then preferably N 3 -L 2 (D) r .
  • an azide on the azide-modified antibody according to the invention reacts with an alkynyl group, preferably a terminal alkynyl group, or a (hetero)cycloalkynyl group of the linker-conjugate via a cycloaddition reaction.
  • This cycloaddition reaction of a molecule comprising an azide with a molecule comprising a terminal alkynyl group or a (hetero)cycloalkynyl group is one of the reactions that is known in the art as "click chemistry".
  • the linker-conjugate comprises a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group.
  • a suitable catalyst preferably a Cu(l) catalyst.
  • the linker-conjugate comprises a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group.
  • the (hetero)cycloalkynyl is a strained (hetero)cycloalkynyl group
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • Strained (hetero)cycloalkynyl groups are known in the art and are described in more detail below.
  • step (ii) comprises reacting a modified antibody with a linker-conjugate, wherein said linker-conjugate comprises a (hetero)cycloalkynyl group and one or more molecules of interest, wherein said modified antibody is an antibody comprising a GlcNAc- S(F )x substituent, wherein GlcNAc is an N-acetylglucosamine, wherein S(F ) X is a sugar derivative comprising x functional groups F wherein F is an azido group and x is 1 or 2, wherein said GlcNAc-S(F )x substituent is bonded to the antibody via C1 of the N-acetylglucosamine of said GlcNAc-S(F )x substituent, and wherein said GlcNAc is optionally fucosylated.
  • said linker-conjugate comprises a (hetero)cycloalkynyl group and one or more molecules of interest
  • said modified antibody is an antibody comprising
  • said (hetero)cycloalkynyl group is a strained (hetero)cycloalkynyl group.
  • Target molecule D may be selected from the group consisting of an active substance, a reporter molecule, a polymer, a solid surface, a hydrogel, a nanoparticle, a microparticle and a biomolecule.
  • active substance relates to a pharmacological and/or biological substance, i.e. a substance that is biologically and/or pharmaceutically active, for example a drug, a prodrug, a diagnostic agent, a protein, a peptide, a polypeptide, a peptide tag, an amino acid, a glycan, a lipid, a vitamin, a steroid, a nucleotide, a nucleoside, a polynucleotide, RNA or DNA.
  • a pharmacological and/or biological substance i.e. a substance that is biologically and/or pharmaceutically active, for example a drug, a prodrug, a diagnostic agent, a protein, a peptide, a polypeptide, a peptide tag, an amino acid, a glycan, a lipid, a vitamin, a steroid, a nucleotide, a nucleoside, a polynucleotide
  • peptide tags include cell-penetrating peptides like human lactoferrin or polyarginine.
  • An example of a glycan is oligomannose.
  • An example of an amino acid is lysine.
  • the active substance is preferably selected from the group consisting of drugs and prodrugs. More preferably, the active substance is selected from the group consisting of pharmaceutically active compounds, in particular low to medium molecular weight compounds (e.g. about 200 to about 2500 Da, preferably about 300 to about 1750 Da). In a further preferred embodiment, the active substance is selected from the group consisting of cytotoxins, antiviral agents, antibacterials agents, peptides and oligonucleotides.
  • cytotoxins examples include colchicine, vinca alkaloids, anthracyclines, camptothecins, doxorubicin, daunorubicin, taxanes, calicheamycins, tubulysins, irinotecans, enediynes, an inhibitory peptide, amanitin, deBouganin, duocarmycins, maytansines, auristatins, indolinobenzodiazepines or pyrrolobenzodiazepines (PBDs).
  • PBDs pyrrolobenzodiazepines
  • preferred active substances include vinca alkaloids, anthracyclines, camptothecins, taxanes, tubulysins, enediynes, duocarmycins, maytansines, auristatins, indolinobenzodiazepines and pyrrolobenzodiazepines, in particular vinca alkaloids, anthracyclines, camptothecins, taxanes, tubulysins, enediynes, maytansines, pyrrolobenzodiazepines and auristatins.
  • reporter molecule refers to a molecule whose presence is readily detected, for example a diagnostic agent, a dye, a fluorophore, a radioactive isotope label, a contrast agent, a magnetic resonance imaging agent or a mass label.
  • fluorophores also referred to as fluorescent probes
  • fluorescent probes A wide variety of fluorophores, also referred to as fluorescent probes, is known to a person skilled in the art.
  • fluorophores are described in more detail in e.g. G.T. Hermanson, "Bioconjugate Techniques", Elsevier, 3 rd Ed. 2013, Chapter 10: “Fluorescent probes", p. 395 - 463, incorporated by reference.
  • fluorophore include all kinds of Alexa Fluor (e.g. Alexa Fluor 555), cyanine dyes (e.g.
  • Cy3 or Cy5 and cyanine dye derivatives, coumarin derivatives, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, boron dipyrromethene derivatives, pyrene derivatives, naphthalimide derivatives, phycobiliprotein derivatives (e.g. allophycocyanin), chromomycin, lanthanide chelates and quantum dot nanocrystals.
  • cyanine dye derivatives coumarin derivatives, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, boron dipyrromethene derivatives, pyrene derivatives, naphthalimide derivatives, phycobiliprotein derivatives (e.g. allophycocyanin), chromomycin, lanthanide chelates and quantum dot nanocrystals.
  • preferred fluorophores include cyanine dyes, coumarin derivatives, fluorescein and derivatives thereof, pyrene derivatives, naphthalimide derivatives, chromomycin, lanthanide chelates and quantum dot nanocrystals, in particular coumarin derivatives, fluorescein, pyrene derivatives and chromomycin.
  • radioactive isotope label examples include 99m Tc, ⁇ , 4m ln, 5 ln, 8 F, 4 C, 64 Cu, 3 ⁇ , 25 l, 2 3 l, 2 2 Bi, 88 Y, 90 Y, 67 Cu, 86 Rh, 88 Rh, 66 Ga, 67 Ga and 0 B, which is optionally connected via a chelating moiety such as e.g.
  • DTPA diethylenetriaminepentaacetic anhydride
  • DOTA diethylenetriaminepentaacetic anhydride
  • DOTA diethylenetriaminepentaacetic anhydride
  • DOTA diethylenetriaminepentaacetic anhydride
  • DOTA diethylenetriaminepentaacetic anhydride
  • DOTA diethylenetriaminepentaacetic anhydride
  • DOTA dioxododecane-/V,/V',/V" /V'"-tetraacetic acid
  • NOTA 1,4,7-triazacyclononane ⁇ , ⁇ ', ⁇ "- triacetic acid
  • TETA 1,4,8, 1 1-tetraazacyclotetradecane-A/,A/',A/" A/"-tetraacetic acid
  • DTTA N 1 - (p-isothiocyanatobenzy -diethylenetriamine-A/ ⁇ A/ 2 , A/ 3 ,A/ 3 -tetraacetic acid
  • Isotopic labelling techniques are known to a person skilled in the art, and are described in more detail in e.g. G.T. Hermanson, “Bioconjugate Techniques", Elsevier, 3 rd Ed. 2013, Chapter 12: “Isotopic labelling techniques", p. 507 - 534, incorporated by reference.
  • Polymers suitable for use as a target molecule D in the compound according to the invention are known to a person skilled in the art, and several examples are described in more detail in e.g. G.T. Hermanson, "Bioconjugate Techniques", Elsevier, 3 rd Ed. 2013, Chapter 18: “PEGylation and synthetic polymer modification", p. 787 - 838, incorporated by reference.
  • target molecule D is a polymer
  • target molecule D is preferably independently selected from the group consisting of a polyethylene glycol (PEG), a polyethylene oxide (PEO), a polypropylene glycol (PPG), a polypropylene oxide (PPO), a 1 ,xx-diaminoalkane polymer (wherein xx is the number of carbon atoms in the alkane, and preferably xx is an integer in the range of 2 to 200, preferably 2 to 10), a (poly)ethylene glycol diamine (e.g. 1 ,8-diamino-3,6-dioxaoctane and equivalents comprising longer ethylene glycol chains), a polysaccharide (e.g.
  • dextran a poly(amino acid) (e.g. a poly(L- lysine)) and a polyvinyl alcohol).
  • a poly(amino acid) e.g. a poly(L- lysine)
  • a polyvinyl alcohol e.g. a poly(L- lysine)
  • preferred polymers include a 1 ,xx-diaminoalkane polymer and polyvinyl alcohol).
  • Solid surfaces suitable for use as a target molecule D are known to a person skilled in the art.
  • a solid surface is for example a functional surface (e.g. a surface of a nanomaterial, a carbon nanotube, a fullerene or a virus capsid), a metal surface (e.g. a titanium, gold, silver, copper, nickel, tin, rhodium or zinc surface), a metal alloy surface (wherein the alloy is from e.g.
  • target molecule D is a solid surface, it is preferred that D is independently selected from the group consisting of a functional surface or a polymer surface.
  • Hydrogels are known to the person skilled in the art. Hydrogels are water-swollen networks, formed by cross-links between the polymeric constituents. See for example A. S. Hoffman, Adv. Drug Delivery Rev. 2012, 64, 18, incorporated by reference.
  • the target molecule is a hydrogel, it is preferred that the hydrogel is composed of poly(ethylene)glycol (PEG) as the polymeric basis.
  • Micro- and nanoparticles suitable for use as a target molecule D are known to a person skilled in the art.
  • a variety of suitable micro- and nanoparticles is described in e.g. G.T. Hermanson, "Bioconjugate Techniques", Elsevier, 3 rd Ed. 2013, Chapter 14: “Microparticles and nanoparticles", p. 549 - 587, incorporated by reference.
  • the micro- or nanoparticles may be of any shape, e.g. spheres, rods, tubes, cubes, triangles and cones.
  • the micro- or nanoparticles are of a spherical shape.
  • the chemical composition of the micro- and nanoparticles may vary.
  • the micro- or nanoparticle is for example a polymeric micro- or nanoparticle, a silica micro- or nanoparticle or a gold micro- or nanoparticle.
  • the polymer is preferably polystyrene or a copolymer of styrene (e.g.
  • the surface of the micro- or nanoparticles is modified, e.g. with detergents, by graft polymerization of secondary polymers or by covalent attachment of another polymer or of spacer moieties, etc.
  • Target molecule D may also be a biomolecule. Biomolecules, and preferred embodiments thereof, are described in more detail below. When target molecule D is a biomolecule, it is preferred that the biomolecule is selected from the group consisting of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids and monosaccharides. Preferred options for D are described further below for the antibody-conjugate according to the third aspect. The bioconjugates in the context of the present invention may contain more than one target molecule D, which may be the same or different.
  • r 2.
  • those target molecules are different, more preferably they both are active substances, more preferably anti-cancer agents, most preferably cytotoxins.
  • the bioconjugate of the present invention comprises two distinct target molecules, preferably two distinct active substances, more preferably two distinct anti-cancer agents, most preferably two distinct cytotoxins.
  • the linker-conjugate comprises a (hetero)cycloalkynyl group.
  • said linker-conjugate has the Formula 31 ):
  • - L 2 is a linker as defined herein;
  • - D is a target molecule
  • - r is 1 - 20;
  • R 3 is independently selected from the group consisting of hydrogen, halogen, -OR 35 , -NO2, -CN, -S(0)2R 35 , 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 R 3 may be linked together to form an annelated cycloalkyl or an annelated (hetero)arene substituent, and wherein R 35 is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 -
  • - aa is 0, 1 , 2, 3, 4, 5, 6, 7 or 8.
  • said linker-con ugate has the Formula (31 b):
  • - L 2 is a linker as defined herein;
  • - D is a target molecule
  • - r is 1 - 20;
  • R 3 is independently selected from the group consisting of hydrogen, halogen, -OR 35 , -NO2, -CN, -S(0)2R 35 , 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 R 3 may be linked together to form an annelated cycloalkyl or an annelated (hetero)arene substituent, and wherein R 35 is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 -
  • - X is C(R 3 )2, O, S or NR 32 , wherein R 32 is R 3 or L 2 (D , and wherein L 2 , D and r are as defined above;
  • - aa is 0, 1 , 2, 3, 4, 5, 6, 7 or 8;
  • - aa' is 0,1 , 2, 3, 4, 5, 6, 7 or 8;
  • aa + aa' is 4, 5, 6 or 7, more preferably aa + aa' is 4, 5 or 6 and most preferably aa + aa' is 5.
  • said linker-conjugate has the Formula (31c):
  • - L 2 is a linker as defined herein;
  • - 1 is 0 - 10. In a preferred embodiment, if q is 1 then X is C(R 3 )2, O, S or NR 3 .
  • a is 5, i.e. said (hetero)cycloalkynyl group is preferably a (hetero)cyclooctyne group.
  • X is C(R 32 )2 or NR 32 . When X is C(R 32 )2 it is preferred that R 32 is hydrogen. When X is NR 32 , it is preferred that R 32 is L 2 (D) r . In yet another preferred embodiment, r is 1 to 10, more preferably, r is 1 , 2, 3, 4, 5, 6 7 or 8, more preferably r is 1 , 2, 3, 4, 5 or 6, most preferably r is 1 , 2, 3 or 4.
  • the L 2 (D) r substituent may be present on a C-atom in said (hetero)cycloalkynyl group, or, in case of a heterocycloalkynyl group, on the heteroatom of said heterocycloalkynyl group.
  • the (hetero)cycloalkynyl group comprises substituents, e.g. an annelated cycloalkyl
  • the L 2 (D) r substituent may also be present on said substituents.
  • the methods to connect a linker L 2 to a (hetero)cycloalkynyl group on the one end and to a target molecule on the other end, in order to obtain a linker-conjugate, depend on the exact the nature of the linker, the (hetero)cycloalkynyl group and the target molecule. Suitable methods are known in the art.
  • the linker-conjugate comprises a (hetero)cyclooctyne group, more preferably a strained (hetero)cyclooctyne group.
  • Suitable (hetero)cycloalkynyl moieties are known in the art.
  • DIFO, DIF02 and DIF03 are disclosed in US 2009/0068738, incorporated by reference.
  • DIBO is disclosed in WO 2009/067663, incorporated by reference.
  • DIBO may optionally be sulphated (S-DIBO) as disclosed in J. Am. Chem. Soc. 2012, 134, 5381 .
  • BARAC is disclosed in J. Am. Chem. Soc. 2010, 132, 3688 - 3690 and US 201 1/0207147, all incorporated by reference.
  • linker-conjugates comprising a (hetero)cyclooctyne group are shown below.
  • DIBAC also known as ADIBO or DBCO
  • BCN cyclooctyne moieties that are known in the art. DIBAC is disclosed in Chem. Commun. 2010, 46, 97 - 99, incorporated by reference. BCN is disclosed in WO 201 1/136645, incorporated by reference.
  • said linker-conjugate has the Formula (32), (33), (34), (35) or (36). In another preferred embodiment said linker-conjugate has the Formula (37):
  • - Y is O, S or NR 32 , wherein R 32 is as defined above;
  • R 33 is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, C6 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups;
  • R 34 is selected from the group consisting of hydrogen, Y-L 2 (D) r , -(CH2)nn-Y-L 2 (D) r , halogen, Ci - C24 alkyl groups, C6 - 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; and
  • - nn is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • R 3 is hydrogen.
  • R 33 is hydrogen.
  • n is 1 or 2.
  • R 34 is hydrogen, Y-L 2 (D) r or -(CH2)nn-Y-L 2 (D) r .
  • R 32 is hydrogen or L 2 (D) r .
  • the linker-conjugate has the Formula 38:
  • said linker-conjugate has the Formula (39):
  • said linker-conjugate has the Formula (35):
  • pp and the nature of M depend on the azide-substituted sugar or sugar derivative S(F )x that is present in the azide-modified antibody according to the invention that is linked to a linker-conjugate. If an azide in S(F ) X is present on the C2, C3, or C4 position of the sugar or the sugar derivative (instead of a sugar OH-group), then pp is 0. If the S(F ) X is an azidoacetamido- sugar derivative, S(F ) X is e.g. GalNAz or GlcNAz, then pp is 1 and M is -N(H)C(0)CH 2 -. If the azide in S(F ) X is present on the C6 position of the sugar or the sugar derivative, then pp is 0 and M is absent.
  • Linkers also referred to as linking units, are well known in the art.
  • L is linked to a target molecule as well as to a functional group Q .
  • L 2 may 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.
  • 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 and NR 35 , wherein R 35 is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 - C24 (hetero)arylalkyl groups.
  • heteroatom is O.
  • suitable linking units include (poly)ethylene glycol diamines (e.g. 1 ,8-diamino-3,6-dioxaoctane or equivalents comprising longer ethylene glycol chains), polyethylene glycol or polyethylene oxide chains, polypropylene glycol or polypropylene oxide chains and 1 , ⁇ -diaminoalkanes wherein xx is the number of carbon atoms in the alkane.
  • cleavable linkers Another class of suitable linkers comprises cleavable linkers.
  • Cleavable linkers are well known in the art. For example Shabat ef a/. , Soft Matter 2012, 6, 1073, incorporated by reference herein, discloses cleavable linkers comprising self-immolative moieties that are released upon a biological trigger, e.g. an enzymatic cleavage or an oxidation event.
  • cleavable linkers are peptide-linkers that are cleaved upon specific recognition by a protease, e.g.
  • cathepsin plasmin or metalloproteases, or glycoside-based linkers that are cleaved upon specific recognition by a glycosidase, e.g. glucuronidase, or nitroaromatics that are reduced in oxygen- poor, hypoxic areas.
  • a glycosidase e.g. glucuronidase
  • nitroaromatics that are reduced in oxygen- poor, hypoxic areas.
  • Step (ii) is preferably performed at a temperature in the range of about 20 to about 50 °C, more preferably in the range of about 25 to about 45 °C, even more preferably in the range of about 30 to about 40 °C, and most preferably in the range of about 32 to about 37 °C.
  • Step (ii) 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.
  • step (ii) is performed at a pH in the range of about 7 to about 8.
  • Step (ii) is preferably performed in water. More preferably, said water is purified water, even more preferably ultrapure water or Type I water as defined according to ISO 3696. Suitable water is for example milliQ® water. Said water is preferably buffered, for example with phosphate-buffered saline or tris. Suitable buffers are known to a person skilled in the art. In a preferred embodiment, step (ii) is performed in milliQ water which is buffered with phosphate-buffered saline or tris.
  • reaction of step (ii) is a(n) (cyclo)alkyne-azide conjugation to from a connecting moiety Z 3 that is represented by (10e), (10i), (10g), (10j) or (10k), preferably by (10e), (10i), (10g), most preferably by (10g), as represented by:
  • cycle A is a 7-10-membered (hetero)cyclic moiety.
  • Connecting moieties (10e), (10j) and (10k) may exist in either one of the possible two regioisomers.
  • bioconjugate that comprises or that is obtained by the present mode of conjugation is preferably represented by Formula (40) or (40b):
  • - AB is an antibody
  • S is a sugar or a sugar derivative
  • GlcNAc is N-acetylglucosamine
  • R 3 is independently selected from the group consisting of hydrogen, halogen, -OR 35 , -NO2, -CN, -S(0)2R 35 , Ci - C24 alkyl groups, C6 - 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 R 3 may be linked together to form an annelated cycloalkyl or an annelated (hetero)arene substituent, and wherein R 35 is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, C6 - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C
  • - X is C(R 3 ) 2 , O, S or NR 32 , wherein R 32 is R 3 or L 2 (D , wherein L 2 is a linker, and D is as defined in claim 1 ;
  • - r is 1 - 20;
  • - aa is 0, 1 , 2, 3, 4, 5, 6, 7 or 8;
  • - aa' is 0,1 , 2, 3, 4, 5, 6, 7 or 8;
  • - b is 0 or 1 ;
  • - M is -N(H)C(0)CH 2 - -N(H)C(0)CF 2 -, -CH 2 -, -CF 2 - or a 1 ,4-phenylene containing 0 - 4 fluorine substituents, preferably 2 fluorine substituents which are preferably positioned on C2 and C6 or on C3 and C5 of the phenylene;
  • the antibody-conjugate according to the invention is of the Formula (41 ):
  • AB, L 2 , D, Y, S, M, x, y, b, pp, R 32 , GlcNAc, R 3 , R 33 , R 34 , nn and r are as defined above and wherein said N-acetylglucosamine is optionally fucosylated (b is 0 or 1 ).
  • R 3 , R 33 and R 34 are hydrogen and nn is 1 or 2, and in an even more preferred embodiment x is 1.
  • N- acetylglucosamine is optionally fucosylated (b is 0 or 1 ); or according to regioisomer (42b):
  • the antibody-conjugate is of the Formula (35b):
  • the antibody-conjugate is of the Formula (40c):
  • AB, L 2 , D, S, b, pp, x, y, M and GlcNAc are as defined above, and wherein said N- acetylglucosamine is optionally fucosylated.
  • the antibody-conjugate is of the Formula (40d): wherein AB, L 2 , D, S, b, pp, x, y, M and GlcNAc are as defined above, wherein I is 0 - 10 and wherein said N-acetylglucosamine is optionally fucosylated.
  • the mode of conjugation according to the invention is referred to as "sulfamide linkage", which refers to the presence of a specific linker L which links the biomolecule B and the target molecule D. All said about the linker L in the context of the present embodiment preferably also applies to the linker, in particular linker L 2 , according to the embodiment on core- GlcNAc functionalization as mode of conjugation.
  • the linker L comprises a group according to formula (1 ) or a salt thereof:
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is a further target molecule D, wherein D is optionally connected to N via a spacer moiety.
  • linker L according to the invention comprises a group according to formula (1 ) wherein a is 0, or a salt thereof.
  • linker L thus comprises a group according to formula (2) or a salt thereof:
  • linker L according to the invention comprises a group according to formula (1 ) wherein a is 1 , or a salt thereof.
  • linker L thus comprises a group according to formula (3) or a salt thereof:
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is a further target molecule D, wherein D is optionally connected to N via a spacer moiety;
  • R is hydrogen or a Ci - C20 alkyl group, more preferably R is hydrogen or a Ci - C16 alkyl group, even more preferably R is hydrogen or a Ci - C10 alkyl group, wherein the alkyl group is optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 , preferably O, wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups.
  • R is hydrogen.
  • R is a Ci - C20 alkyl group, more preferably a Ci - C16 alkyl group, even more preferably a Ci - C10 alkyl group, wherein the alkyl group is optionally interrupted by one or more O-atoms, and wherein the alkyl group is optionally substituted with an - OH group, preferably a terminal -OH group.
  • R is a (poly)ethyleneglycol chain comprising a terminal -OH group.
  • R is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl and i- propyl, and even more preferably from the group consisting of hydrogen, methyl and ethyl. Yet even more preferably R is hydrogen or methyl, and most preferably R is hydrogen.
  • R is a further target molecule D.
  • D is connected to N via one or more spacer-moieties.
  • the spacer-moiety if present, is defined as a moiety that spaces, i.e. provides a certain distance between, and covalently links D and N.
  • Target molecule D and preferred embodiments thereof are defined in more detail above.
  • the group of formula (1 ) can be introduced in one of three options.
  • the linker L comprising the group according to formula (1 ) or a salt thereof may be present in the linker-conjugate represented by Q -D, wherein L is the spacer between Q and D.
  • the linker L comprising the group according to formula (1 ) or a salt thereof may be present in the biomolecule represented by B-F , wherein L is the spacer between B and F .
  • the group according to formula (1 ) or a salt thereof may be formed during the conjugation reaction itself. In the latter option, Q and F are selected as such that their reaction product, i.e.
  • connecting group Z 3 contains or is the group according to formula (1 ) or a salt thereof.
  • the group according to formula (1 ) or a salt thereof is introduced according to the first or second of the above mentioned options, most preferably according to the first option.
  • the positive effect on solubility and absence of in-process aggregation, as recited above improve the efficiency of the conjugation reaction.
  • the group according to formula (1 ) or a salt thereof is present in the linker-conjugate, even hydrophobic drugs can readily be subjected to the conjugation reaction.
  • the linker-conjugate is represented by Q -D, preferably by Q -L-D, wherein D is a target molecule, L is a linker linking Q and D as further defined above, Q is a reactive group capable of reacting with functional group F on the biomolecule and each occurrence of "-" is independently a bond or a spacer moiety. In one embodiment, "-" is a spacer moiety as defined herein. In one embodiment, "-" is a bond, typically a covalent bond.
  • the linker-conjugate is a compound wherein a target molecule is covalently connected to a reactive group Q , preferably via a linker or spacer, most preferably via linker L as defined above. The linker-conjugate may be obtained via reaction of a reactive group Q 2 present on a linker-construct with a reactive group present on a target molecule.
  • the group according to formula (1 ), or the salt thereof is situated in between Q and D.
  • reactive group Q is covalently bonded to a first end of the group according to formula (1 )
  • target molecule D is covalently bonded to a second end of the group according to formula (1 ).
  • first end and second end both refer to either the carbonyl or carboxy end of the group according to formula (1 ) or to the sulfamide end of the group according to formula (1 ), but logically not to the same end.
  • the linker-conjugate according to the invention may comprise more than one target molecule D, e.g. two, three, four, five, etc. Consequently, the linker-conjugate may thus comprise more than one "second end".
  • the linker-conjugate may comprise more than one reactive group Q , i.e. the linker-conjugate may comprise more than one first end.
  • the groups Q may be the same or different, and when more than one target molecule D is present the target molecule D may be the same or different.
  • the linker-conjugate according to the invention may therefore also be denoted as (Q ) y Sp(D) z , wherein y' is an integer in the range of 1 to 10 and z is an integer in the range of 1 to 10.
  • y' is an integer in the range of 1 to 10
  • z is an integer in the range of 1 to 10.
  • - y' is an integer in the range of 1 to 10;
  • - z is an integer in the range of 1 to 10;
  • - Q is a reactive group capable of reacting with a functional group F present on a biomolecule
  • - Sp is a spacer moiety, wherein a spacer moiety is defined as a moiety that spaces (i.e. provides a certain distance between) and covalently links reactive group Q and target molecule D, preferably wherein said spacer moiety is linker L as defined above, and thus comprises a group according to formula (1 ) or a salt thereof.
  • y' is 1 , 2, 3 or 4, more preferably y' is 1 or 2 and most preferably, y' is 1.
  • z is 1 , 2, 3, 4, 5 or 6, more preferably z is 1 , 2, 3 or 4, even more preferably z is 1 , 2 or 3, yet even more preferably z is 1 or 2 and most preferably z is 1. More preferably, y' is 1 or 2, preferably 1 , and z is 1 , 2, 3 or 4, yet even more preferably y' is 1 or 2, preferably 1 , and z is 1 , 2 or 3, yet even more preferably y' is 1 or 2, preferably 1 , and z is 1 or 2, and most preferably y' is 1 and z is 1.
  • the linker-conjugate is according to the formula Q Sp(D)4, Q Sp(D)3, Q Sp(D) 2 or Q SpD.
  • D is preferably an "active substance” or "pharmaceutically active substance", and refers to a pharmacological and/or biological substance, i.e. a substance that is biologically and/or pharmaceutically active, for example a drug, a prodrug, a diagnostic agent.
  • the active substance is selected from the group consisting of drugs and prodrugs. More preferably, the active substance is a pharmaceutically active compounds, in particular low to medium molecular weight compounds (e.g. about 200 to about 2500 Da, preferably about 300 to about 1750 Da).
  • the active substance is selected from the group consisting of cytotoxins, antiviral agents, antibacterials agents, peptides and oligonucleotides.
  • cytotoxins include colchicine, vinca alkaloids, anthracyclines, camptothecins, doxorubicin, daunorubicin, taxanes, calicheamycins, tubulysins, irinotecans, enediynes, an inhibitory peptide, amanitin, deBouganin, duocarmycins, maytansines, auristatins or pyrrolobenzodiazepines (PBDs).
  • PBDs pyrrolobenzodiazepines
  • Preferred active substances include vinca alkaloids, anthracyclines, camptothecins, taxanes, tubulysins, amanitin, duocarmycins, maytansines, auristatins and pyrrolobenzodiazepines, in particular vinca alkaloids, anthracyclines, camptothecins, taxanes, tubulysins, amanitin, maytansines and auristatins.
  • the linker-conjugate comprises a reactive group Q that is capable of reacting with a functional group F present on a biomolecule.
  • Functional groups are known to a person skilled in the art and may be defined as any molecular entity that imparts a specific property onto the molecule harbouring it.
  • a functional group in a biomolecule may constitute an amino group, a thiol group, a carboxylic acid, an alcohol group, a carbonyl group, a phosphate group, or an aromatic group.
  • the functional group in the biomolecule may be naturally present or may be placed in the biomolecule by a specific technique, for example a (bio)chemical or a genetic technique.
  • the functional group that is placed in the biomolecule may be a functional group that is naturally present in nature, or may be a functional group that is prepared by chemical synthesis, for example an azide, a terminal alkyne or a phosphine moiety.
  • the term "reactive group” may refer to a certain group that comprises a functional group, but also to a functional group itself.
  • a cyclooctynyl group is a reactive group comprising a functional group, namely a C- C triple bond.
  • an N-maleimidyl group is a reactive group, comprising a C-C double bond as a functional group.
  • a functional group for example an azido functional group, a thiol functional group or an amino functional group, may herein also be referred to as a reactive group.
  • the linker-conjugate may comprise more than one reactive group Q .
  • the linker-conjugate comprises two or more reactive groups Q , the reactive groups Q may differ from each other.
  • the linker-conjugate comprises one reactive group Q .
  • Reactive group Q that is present in the linker-conjugate is able to react with a functional group F that is present in a biomolecule to form connecting group Z 3 .
  • reactive group Q needs to be complementary to a functional group F present in a biomolecule.
  • a reactive group is denoted as "complementary" to a functional group when said reactive group reacts with said functional group selectively to form connecting group Z 3 , optionally in the presence of other functional groups.
  • Complementary reactive and functional groups are known to a person skilled in the art, and are described in more detail below.
  • reactive group Q is selected from the group consisting of, optionally substituted, N-maleimidyl groups, halogenated N-alkylamido groups, sulfonyloxy N-alkylamido groups, ester groups, carbonate groups, sulfonyl halide groups, thiol groups or derivatives thereof, alkenyl groups, alkynyl groups, (hetero)cycloalkynyl groups, bicyclo[6.1.0]non-4-yn-9-yl] groups, cycloalkenyl groups, tetrazinyl groups, azido groups, phosphine groups, nitrile oxide groups, nitrone groups, nitrile imine groups, diazo groups, ketone groups, (O-alkyl)hydroxylamino groups, hydrazine groups, halogenated N-maleimidyl groups, 1 , 1-bis(sulfonylmethyl)methylcarbonyl groups or elimination derivative
  • Q is an N-maleimidyl group.
  • Q is preferably unsubstituted.
  • Q is thus preferably according to formula (9a), as shown below.
  • a preferred example of such a maleimidyl group is 2,3-diaminopropionic acid (DPR) maleimidyl, which may be connected to the remainder of the linker-conjugate through the carboxylic acid moiety.
  • DPR 2,3-diaminopropionic acid
  • Q is a halogenated N-alkylamido group.
  • Q is according to formula (9b), as shown below, wherein k is an integer in the range of 1 to 10 and R 4 is selected from the group consisting of -CI, -Br and -I.
  • k is 1 , 2, 3 or 4, more preferably k is 1 or 2 and most preferably k is 1 .
  • R 4 is -I or -Br. More preferably, k is 1 or 2 and R 4 is -I or -Br, and most preferably k is 1 and R 4 is -I or Br.
  • Q is a sulfonyloxy N-alkylamido group.
  • Q is a sulfonyloxy N-alkylamido group
  • Q is according to formula (9b), as shown below, wherein k is an integer in the range of 1 to 10 and R 4 is selected from the group consisting of -O-mesyl, -O-phenylsulfonyl and -O-tosyl.
  • k is 1 , 2, 3 or 4, more preferably k is 1 or 2, even more preferably k is 1.
  • R 4 is selected from the group consisting of -O-mesyl, -O-phenylsulfonyl and -O-tosyl.
  • Q is an ester group.
  • the ester group is an activated ester group.
  • Activated ester groups are known to the person skilled in the art.
  • An activated ester group is herein defined as an ester group comprising a good leaving group, wherein the ester carbonyl group is bonded to said good leaving group.
  • Good leaving groups are known to the person skilled in the art.
  • the activated ester is according to formula (9c), as shown below, wherein R 5 is selected from the group consisting of -N-succinimidyl (NHS), -N-sulfo-succinimidyl (sulfo-NHS), -(4-nitrophenyl), -pentafluorophenyl or -tetrafluorophenyl (TFP).
  • R 5 is selected from the group consisting of -N-succinimidyl (NHS), -N-sulfo-succinimidyl (sulfo-NHS), -(4-nitrophenyl), -pentafluorophenyl or -tetrafluorophenyl (TFP).
  • Q is a carbonate group.
  • the carbonate group is an activated carbonate group.
  • Activated carbonate groups are known to a person skilled in the art.
  • An activated carbonate group is herein defined as a carbonate group comprising a good leaving group, wherein the carbonate carbonyl group is bonded to said good leaving group.
  • the carbonate group is according to formula (9d), as shown below, wherein R 7 is selected from the group consisting of -N- succinimidyl, -N-sulfo-succinimidyl, -(4-nitrophenyl), -pentafluorophenyl or -tetrafluorophenyl.
  • Q is a sulfonyl halide group according to formula (9e) as shown below, wherein X is selected from the group consisting of F, CI, Br and I.
  • X is CI or Br, more preferably CI.
  • Q is a thiol group (9f), or a derivative or a precursor of a thiol group.
  • a thiol group may also be referred to as a mercapto group.
  • the thiol derivative is preferably according to formula (9g), (9h) or (9zb) as shown below, wherein R 8 is an, optionally substituted, Ci - C12 alkyl group or a C2 - C12 (hetero)aryl group, V is O or S and R 6 is an, optionally substituted, Ci - C12 alkyl group.
  • R 8 is an, optionally substituted, Ci - C6 alkyl group or a C2 - C6 (hetero)aryl group, and even more preferably R 8 is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl or phenyl. Even more preferably, R 8 is methyl or phenyl, most preferably methyl.
  • R 6 is an optionally substituted Ci - C6 alkyl group, and even more preferably R 6 is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl, most preferably methyl.
  • Q is a thiol-derivative according to formula (9g) or (9zb)
  • Q is reacted with a reactive group F on a biomolecule
  • said thiol- derivative is converted to a thiol group during the process.
  • Q is according to formula (9h)
  • Q is -SC(0)OR 8 or -SC(S)OR 8 , preferably SC(0)OR 8 , wherein R 8 , and preferred embodiments thereof, are as defined above.
  • Q is an alkenyl group, wherein the alkenyl group is linear or branched, and wherein the alkenyl group is optionally substituted.
  • the alkenyl group may be a terminal or an internal alkenyl group.
  • the alkenyl group may comprise more than one C-C double bond, and if so, preferably comprises two C-C double bonds.
  • the alkenyl group is a dienyl group, it is further preferred that the two C-C double bonds are separated by one C-C single bond (i.e. it is preferred that the dienyl group is a conjugated dienyl group).
  • said alkenyl group is a C2 - C24 alkenyl group, more preferably a C2 - C12 alkenyl group, and even more preferably a C2 - C6 alkenyl group. It is further preferred that the alkenyl group is a terminal alkenyl group. More preferably, the alkenyl group is according to formula (9i) as shown below, wherein I is an integer in the range of 0 to 10, preferably in the range of 0 to 6, and p is an integer in the range of 0 to 10, preferably 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 .
  • p is 0, 1 , 2, 3 or 4, more preferably p is 0, 1 or 2 and most preferably p is 0 or 1 . It is particularly preferred that p is 0 and I is 0 or 1 , or that p is 1 and I is 0 or 1.
  • Q is an alkynyl group, wherein the alkynyl group is linear or branched, and wherein the alkynyl group is optionally substituted.
  • the alkynyl group may be a terminal or an internal alkynyl group.
  • said alkynyl group is a C2 - C24 alkynyl group, more preferably a C2 - C12 alkynyl group, and even more preferably a C2 - C6 alkynyl group. It is further preferred that the alkynyl group is a terminal alkynyl group.
  • the alkynyl group is according to formula (9j) as shown below, wherein 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 a further preferred embodiment, the alkynyl group is according to formula (9j) wherein I is 3.
  • Q is a cycloalkenyl group.
  • the cycloalkenyl group is optionally substituted.
  • said cycloalkenyl group is a C3 - C24 cycloalkenyl group, more preferably a C3 - C12 cycloalkenyl group, and even more preferably a C3 - Cs cycloalkenyl group.
  • the cycloalkenyl group is a irans-cycloalkenyl group, more preferably a trans- cyclooctenyl group (also referred to as a TCO group) and most preferably a irans-cyclooctenyl group according to formula (9zi) or (9zj) as shown below.
  • the cycloalkenyl group is a cyclopropenyl group, wherein the cyclopropenyl group is optionally substituted.
  • the cycloalkenyl group is a norbornenyl group, an oxanorbornenyl group, a norbornadienyl group or an oxanorbornadienyl group, wherein the norbornenyl group, oxanorbornenyl group, norbornadienyl group or an oxanorbornadienyl group is optionally substituted.
  • the cycloalkenyl group is according to formula (9k), (91), (9m) or (9zc) as shown below, wherein T is CH2 or O, R 9 is independently selected from the group consisting of hydrogen, a linear or branched Ci - C12 alkyl group or a C4 - C12 (hetero)aryl group, and R 9 is selected from the group consisting of hydrogen and fluorinated hydrocarbons.
  • R 9 is independently hydrogen or a Ci - C6 alkyl group, more preferably R 9 is independently hydrogen or a Ci - C4 alkyl group.
  • R 9 is independently hydrogen or methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Yet even more preferably R 9 is independently hydrogen or methyl.
  • R 9 is selected from the group of hydrogen and -CF3, -C2F5, -C3F7 and -C4F9, more preferably hydrogen and -CF3.
  • the cycloalkenyl group is according to formula (9k), wherein one R 9 is hydrogen and the other R9 is a methyl group.
  • the cycloalkenyl group is according to formula (9I), wherein both R 9 are hydrogen.
  • the cycloalkenyl group is a norbornenyl (T is CH2) or an oxanorbornenyl (T is O) group according to formula (9m), or a norbornadienyl (T is CH2) or an oxanorbornadienyl (T is O) group according to formula (9zc), wherein R 9 is hydrogen and R 9 is hydrogen or -CF3, preferably -CF3.
  • Q is a (hetero)cycloalkynyl group.
  • the (hetero)cycloalkynyl group is optionally substituted.
  • the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, i.e. a heterocyclooctynyl group or a cyclooctynyl group, wherein the (hetero)cyclooctynyl group is optionally substituted.
  • the (hetero)cyclooctynyl group is substituted with one or more halogen atoms, preferably fluorine atoms, more preferably the (hetero)cyclooctynyl group is substituted with one fluorine atom, as in mono-fluoro-cyclooctcyne (MFCO).
  • MFCO mono-fluoro-cyclooctcyne
  • the mono-fluoro-cyclooctcyne group is according to formula (9zo).
  • the (hetero)cyclooctynyl group is according to formula (9n), also referred to as a DIBO group, (9o), also referred to as a DIBAC group or (9p), also referred to as a BARAC group, or (9zk), also referred to as a COMBO group, all as shown below, wherein U is O or NR 9 , and preferred embodiments of R 9 are as defined above.
  • the aromatic rings in (9n) are optionally O-sulfonylated at one or more positions, whereas the rings of (9o) and (9p) may be halogenated at one or more positions.
  • U is preferably O.
  • the nitrogen atom attached to R in compound (4b) is the nitrogen atom in the ring of the heterocycloalkyne group such as the nitrogen atom in (9o).
  • c, d and g are 0 in compound (4b) and R and Q , together with the nitrogen atom they are attached to, form a heterocycloalkyne group, preferably a heterocyclooctyne group, most preferably the heterocyclooctyne group according to formula (9o) or (9p).
  • the carbonyl moiety of (9o) is replaced by the sulfonyl group of the group according to formula (1 ).
  • the nitrogen atom to which R is attached is the same atom as the atom designated as U in formula (9n).
  • Q is an, optionally substituted, bicyclo[6.1.0]non-4-yn-9-yl] group, also referred to as a BCN group.
  • the bicyclo[6.1.0]non-4-yn-9-yl] group is according to formula (9q) as shown below.
  • Q is a conjugated (hetero)diene group capable of reacting in a Diels-Alder reaction.
  • Preferred (hetero)diene groups include optionally substituted tetrazinyl groups, optionally substituted 1 ,2-quinone groups and optionally substituted triazine groups. More preferably, said tetrazinyl group is according to formula (9r), as shown below, wherein R 9 is selected from the group consisting of hydrogen, a linear or branched Ci - C12 alkyl group or a C4 - C12 (hetero)aryl group.
  • R 9 is hydrogen, a Ci - Ce alkyl group or a C4 - C10 (hetero)aryl group, more preferably R 9 is hydrogen, a Ci - C4 alkyl group or a C4 - C6 (hetero)aryl group. Even more preferably R 9 is hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t- butyl or pyridyl. Yet even more preferably R 9 is hydrogen, methyl or pyridyl. More preferably, said 1 ,2-quinone group is according to formula (9zl) or (9zm).
  • Said triazine group may be any regioisomer. More preferably, said triazine group is a 1 ,2,3-triazine group or a 1 ,2,4-triazine group, which may be attached via any possible location, such as indicated in formula (9zn). The 1 ,2,3-triazine is most preferred as triazine group.
  • Q is an azido group according to formula (9s) as shown below.
  • Q is an, optionally substituted, triarylphosphine group that is suitable to undergo a Staudinger ligation reaction.
  • the phosphine group is according to forumula (9t) as shown below, wherein R 0 is a (thio)ester group.
  • R 0 is a (thio)ester group
  • R 0 is -C(0)-V-R 11 , wherein V is O or S and R is a Ci - C12 alkyl group.
  • R is a Ci - C6 alkyl group, more preferably a Ci - C4 alkyl group.
  • R is a methyl group.
  • Q is a nitrile oxide group according to formula (9u) as shown below.
  • Q is a nitrone group.
  • the nitrone group is according to formula (9v) as shown below, wherein R 2 is selected from the group consisting of linear or branched Ci - C12 alkyl groups and C6 - C12 aryl groups.
  • R 2 is a Ci - C6 alkyl group, more preferably R 2 is a Ci - C4 alkyl group.
  • R 2 is methyl, ethyl, n-propyl, i- propyl, n-butyl, s-butyl or t-butyl. Yet even more preferably R 2 is methyl.
  • Q is a nitrile imine group.
  • the nitrile imine group is according to formula (9w) or (9zd) as shown below, wherein R 3 is selected from the group consisting of linear or branched Ci - C12 alkyl groups and C6 - C12 aryl groups.
  • R 3 is a Ci - C6 alkyl group, more preferably R 3 is a Ci - C4 alkyl group.
  • R 3 is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Yet even more preferably R 3 is methyl.
  • Q is a diazo group.
  • the diazo group is according to formula (9x) as shown below, wherein R 4 is selected from the group consisting of hydrogen or a carbonyl derivative. More preferably, R 4 is hydrogen.
  • Q is a ketone group. More preferably, the ketone group is according to formula (9y) as shown below, wherein R 5 is selected from the group consisting of linear or branched Ci - C12 alkyl groups and C6 - C12 aryl groups.
  • R 5 is a Ci - C6 alkyl group, more preferably R 5 is a Ci - C4 alkyl group.
  • R 5 is methyl, ethyl, n- propyl, i-propyl, n-butyl, s-butyl or t-butyl. Yet even more preferably R 5 is methyl.
  • Q is an (O-alkyl)hydroxylamino group. More preferably, the (O- alkyl)hydroxylamino group is according to formula (9z) as shown below.
  • Q is a hydrazine group.
  • the hydrazine group is according to formula (9za) as shown below.
  • Q is a halogenated N-maleimidyl group or a sulfonylated N- maleimidyl group.
  • Q is preferably according to formula (9ze) as shown below, wherein R 6 is independently selected from the group consisting of hydrogen F, CI, Br, I -SR 8a and -OS(0)2R 8b , wherein R 8a is an optionally substituted C4 - C12 (hetero)aryl groups, preferably phenyl or pyrydyl, and R 8b is selected from the group consisting of, optionally substituted, Ci - C12 alkyl groups and C4 - C12 (hetero)aryl groups, preferably tolyl or methyl, and with the proviso that at least one R 6 is not hydrogen.
  • R 6 is halogen (i.e. when R 6 is F, CI, Br or I), it is preferred that R 6 is Br.
  • the halogenated N-maleimidyl group is halogentated 2,3-diaminopropionic acid (DPR) maleimidyl, which may be connected to the remainder of the linker-conjugate through the carboxylic acid moiety.
  • DPR 2,3-diaminopropionic acid
  • Q is a carbonyl halide group according to formula (9zf) as shown below, wherein X is selected from the group consisting of F, CI, Br and I.
  • X is CI or Br, and most preferably, X is CI.
  • Q is an allenamide group according to formula (9zg).
  • Q is a 1 , 1-bis(sulfonylmethyl)methylcarbonyl group according to formula (9zh), or an elimination derivative thereof, wherein R 8 is selected from the group consisting of, optionally substituted, Ci - C12 alkyl groups and C4 - C12 (hetero)aryl groups. More preferably, R 8 is an, optionally substituted, Ci - C6 alkyl group or a C4 - C6 (hetero)aryl group, and most preferably a phenyl group.
  • conjugation is accomplished via a cycloaddition, such as a Diels-Alder reaction or a 1 ,3-dipolar cycloaddition, preferably the 1 ,3-dipolar cycloaddition.
  • the reactive group Q (as well as F on the biomolecule) is selected from groups reactive in a cycloaddition reaction.
  • reactive groups Q and F are complementary, i.e. they are capable of reacting with each other in a cycloaddition reaction, the obtained cyclic moiety being connecting group Z 3 .
  • one of F and Q is a diene and the other of F and Q is a dienophile.
  • Hetero-Diels-Alder reactions with N- and O-containing dienes are known to a person skilled in the art. Any diene known in the art to be suitable for Diels-Alder reactions may be used as reactive group Q or F . Preferred dienes include tetrazines as described above, 1 ,2- quinones as described above and triazines as described above. Although any dienophile known in the art to be suitable for Diels-Alder reactions may be used as reactive groups Q or F ⁇ the dienophile is preferably an alkene or alkyne group as described above, most preferably an alkyne group.
  • F is the diene and Q is the dienophile.
  • Q is a dienophile
  • Q is or comprises an alkynyl group
  • F is a diene, preferably a tetrazine, 1 ,2-quinone or triazine group.
  • one of F and Q is a 1 ,3-dipole and the other of F and Q is a dipolarophile.
  • Any 1 ,3-dipole known in the art to be suitable for 1 ,3-dipolar cycloadditions may be used as reactive group Q or F .
  • Preferred 1 ,3-dipoles include azido groups, nitrone groups, nitrile oxide groups, nitrile imine groups and diazo groups.
  • the dipolarophile is preferably an alkene or alkyne group, most preferably an alkyne group.
  • F is the 1 ,3-dipole
  • Q is the dipolarophile.
  • Q is a dipolarophile
  • Q is or comprises an alkynyl group
  • F is a 1 ,3-dipole, preferably an azido group.
  • Q is selected from dipolarophiles and dienophiles.
  • Q is an alkene or an alkyne group.
  • Q comprises an alkyne group, preferably selected from the alkynyl group as described above, the cycloalkenyl group as described above, the (hetero)cycloalkynyl group as described above and a bicyclo[6.1.0]non-4-yn-9-yl] group, more preferably Q is selected from the formulae (9j), (9n), (9o), (9p), (9q), (9zk) and (9zo) as defined above and depicted above, such as selected from the formulae (9j), (9n), (9o), (9p), (9q) and (9zk), more preferably selected from the formulae (9n), (9o), (9p), (9q) and (9zk) or from the formulae (9j), (9n), (9q) and (9zo).
  • Q is a bicyclo[6.1.0]non-4-yn-9-yl] group, preferably of formula (9q).
  • These groups are known to be highly effective in the conjugation with azido-functionlized biomolecules as described herein, and when the sulfamide linker according to the invention is employed in such linker-conjugates, any aggregation is beneficially reduced to a minimum.
  • Q is capable of reacting with a reactive group F that is present on a biomolecule.
  • Complementary reactive groups F for reactive group Q are known to a person skilled in the art, and are described in more detail below. Some representative examples of reaction between F and Q and their corresponding products comprising connecting group Z 3 are depicted in Figure 5.
  • D and Q are covalently attached in the linker-conjugate according to the invention, preferably via linker L as defined above.
  • Covalent attachment of D to the linker may occur for example via reaction of a functional group F 2 present on D with a reactive group Q 2 present on the linker.
  • Suitable organic reactions for the attachment of D to a linker are known to a person skilled in the art, as are functional groups F 2 that are complementary to a reactive group Q 2 . Consequently, D may be attached to the linker via a connecting group Z.
  • connecting group refers to the structural element connecting one part of a compound and another part of the same compound.
  • the nature of a connecting group depends on the type of organic reaction with which the connection between the parts of said compound was obtained.
  • R-C(0)-OH is reacted with the amino group of H 2 N-R' to form R-C(0)-N(H)-R'
  • R is connected to R' via connecting group Z
  • Z may be represented by the group -C(0)-N(H)-.
  • Reactive group Q may be attached to the linker in a similar manner. Consequently, Q may be attached to the spacer-moiety via a connecting group Z.
  • the linker-conjugate is a compound according to the formula:
  • - y' is an integer in the range of 1 to 10;
  • - z is an integer in the range of 1 to 10;
  • - Q is a reactive group capable of reacting with a functional group F present on a biomolecule
  • - D is a target molecule
  • - Sp is a spacer moiety, wherein a spacer moiety is defined as a moiety that spaces (i.e. provides a certain distance between) and covalently links Q and D;
  • spacer moiety is linker L, and thus comprises a group according to formula (1 ) or a salt thereof, wherein the group according to formula (1 ) is as defined above.
  • a in the group according to formula (1 ) is 0. In another preferred embodiment, a in the group according to formula (1 ) is 1.
  • Preferred embodiments for y' and z are as defined above for (Q ) y Sp(D) z . It is further preferred that the compound is according to the formula Q (Z w )Sp(Z x )(D) 4 , Q (Z w )Sp(Z x )(D) 3 , Q (Z w )Sp(Z x )(D) 2 or Q (Z w )Sp(Z x )D, more preferably Q (Z w )Sp(Z x )(D) 2 or Q (Z w )Sp(Z x )D and most preferably Q (Z w )Sp(Z x )D, wherein Z w and Z x are as defined above.
  • the linker-conjugate is compound according to formula (4a) or (4b), or a salt thereof:
  • - a is independently 0 or 1 ;
  • - b is independently 0 or 1 ;
  • - c is 0 or 1 ;
  • - d is 0 or 1 ;
  • - e is 0 or 1 ;
  • - f is an integer in the range of 1 to 150;
  • - g is 0 or 1 ;
  • - i is 0 or 1 ;
  • - D is a target molecule
  • - Q is a reactive group capable of reacting with a functional group F present on a biomolecule
  • - Z is a connecting group that connects Q or Sp 3 to Sp 2 , O or C(O) or N(R 1 );
  • - Z 2 is a connecting group that connects D or Sp 4 to Sp 1 , N(R 1 ), O or C(O); and - R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3- C24 cycloalkyl groups, Ci - C24 (hetero)aryl groups, Ci - C24 alkyl(hetero)aryl groups and Ci - 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 NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups; or
  • D is D, -[(Sp )b(Z 2 )e(Sp 4 )iD] or -[(Sp 2 )c(Z ) d (Sp 3 ) g Q 1 ], wherein D is a further target molecule and Sp ⁇ Sp 2 , Sp 3 , Sp 4 , Z , Z 2 , Q , b, c, d, e, g and i are as defined above.
  • a is 1 in the compound according to formula (4a) or (4b). In another preferred embodiment, a is 0 in the compound according to formula (4a) or (4b).
  • Z is a connecting group that connects Q or Sp 3 to Sp 2 , O or C(O) or N(R 1 ), and Z 2 is a connecting group that connects D or Sp 4 to Sp 1 , N(R 1 ), O or C(O).
  • connecting group refers to a structural element connecting one part of a compound and another part of the same compound.
  • connecting group Z when present (i.e. when d is 1 ), connects Q (optionally via a spacer moiety Sp 3 ) to the O-atom or the C(O) group of the compound according to formula (4a), optionally via a spacer moiety Sp 2 . More particularly, when Z is present (i.e. d is 1 ), and when Sp 3 and Sp 2 are absent (i.e. g is 0 and c is 0), Z connects Q to the O-atom (a is 1 ) or to the C(O) group (a is 0) of the linker-conjugate according to formula (4a). When Z is present (i.e. when d is 1 ), Sp 3 is present (i.e.
  • connecting group Z when present (i.e. when d is 1 ), connects Q (optionally via a spacer moiety Sp 3 ) to the N-atom of the N(R 1 ) group in the linker- conjugate according to formula (4b), optionally via a spacer moiety Sp 2 . More particularly, when Z is present (i.e. d is 1 ), and when Sp 3 and Sp 2 are absent (i.e. g is 0 and c is 0), Z connects Q to the N-atom of the N(R 1 ) group of the linker-conjugate according to formula (4b). When Z is present (i.e. when d is 1 ), Sp 3 is present (i.e.
  • Z connects Q to spacer moiety Sp 2 of the linker-conjugate according to formula (4b).
  • Q is attached directly to the O-atom (when a is 1 ) or to the C(O) group (when a is 0) of the linker-conjugate according to formula (4a).
  • connecting group Z 2 when present (i.e. when e is 1 ), connects D (optionally via a spacer moiety Sp 4 ) to the N-atom of the N(R 1 ) group in the linker- conjugate according to formula (4a), optionally via a spacer moiety Sp 1 . More particularly, when Z 2 is present (i.e. e is 1 ), and when Sp 1 and Sp 4 are absent (i.e. b is 0 and i is 0), Z 2 connects D to the N-atom of the N(R 1 ) group of the linker-conjugate according to formula (4a). When Z 2 is present (i.e. when e is 1 ), Sp 4 is present (i.e.
  • Z 2 connects spacer moiety Sp 4 to the N-atom of the N(R 1 ) group of the linker-conjugate according to formula (4a).
  • Z 2 connects spacer moiety Sp 1 to spacer moiety Sp 4 of the linker-conjugate according to formula (4a).
  • Z 2 connects D to spacer moiety Sp 1 of the linker-conjugate according to formula (4a).
  • connecting group depends on the type of organic reaction with which the connection between the specific parts of said compound was obtained.
  • a large number of organic reactions are available for connecting a reactive group Q to a spacer moiety, and for connecting a target molecule to a spacer-moety. Consequently, there is a large variety of connecting groups Z and Z 2 .
  • Sp ⁇ Sp 2 , Sp 3 and Sp 4 are spacer-moieties.
  • Sp 1 , Sp 2 , Sp 3 and Sp 4 may be, independently, absent or present (b, c, g and i are, independently, 0 or 1 ).
  • Sp 1 , if present, may be different from Sp 2 , if present, from Sp 3 and/or from Sp 4 , if present.
  • Spacer-moieties are known to a person skilled in the art.
  • suitable spacer-moieties include (poly)ethylene glycol diamines (e.g. 1 ,8-diamino-3,6-dioxaoctane or equivalents comprising longer ethylene glycol chains), polyethylene glycol chains or polyethylene oxide chains, polypropylene glycol chains or polypropylene oxide chains and 1 ,xx-diaminoalkanes wherein xx is the number of carbon atoms in the alkane.
  • cleavable spacer-moieties comprises cleavable spacer-moieties, or cleavable linkers.
  • Cleavable linkers are well known in the art. For example Shabat et ai , Soft Matter 2012, 6, 1073, incorporated by reference herein, discloses cleavable linkers comprising self-immolative moieties that are released upon a biological trigger, e.g. an enzymatic cleavage or an oxidation event.
  • suitable cleavable linkers are disulfide-linkers that are cleaved upon reduction, peptide-linkers that are cleaved upon specific recognition by a protease, e.g.
  • suitable cleavable spacer-moieties also include spacer moieties comprising a specific, cleavable, sequence of amino acids. Examples include e.g. spacer-moieties comprising a Val-Ala (valine-alanine) or Val-Cit (valine-citrulline) moiety.
  • spacer moieties Sp 1 , Sp 2 , Sp 3 and/or Sp 4 if present, comprise a sequence of amino acids.
  • Spacer- moieties comprising a sequence of amino acids are known in the art, and may also be referred to as peptide linkers. Examples include spacer-moieties comprising a Val-Cit moiety, e.g. Val-Cit- PABC, Val-Cit-PABC, Fmoc-Val-Cit-PABC, etc.
  • a Val-Cit-PABC moiety is employed in the linker-conjugate.
  • spacer moieties Sp 1 , Sp 2 , Sp 3 and Sp 4 are independently selected from the group consisting of linear or branched C1-C200 alkylene groups, C2-C200 alkenylene groups, C2-C200 alkynylene groups, C3-C200 cycloalkylene groups, C5-C200 cycloalkenylene groups, C8-C200 cycloalkynylene groups, C7-C200 alkylarylene groups, C7-C200 arylalkylene groups, C8-C200 arylalkenylene groups and C9-C200 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkeny
  • 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.
  • spacer moieties Sp ⁇ Sp 2 , Sp 3 and Sp 4 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, C8-C100 cycloalkynylene groups, C7-C100 alkylarylene groups, C7-C100 arylalkylene groups, Cs-doo arylalkenylene groups and C9-C100 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally
  • spacer moieties Sp 1 , Sp 2 , Sp 3 and Sp 4 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 substitute
  • spacer moieties Sp 1 , Sp 2 , Sp 3 and Sp 4 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
  • 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 NR 3 , preferably O, wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably hydrogen or methyl.
  • spacer moieties Sp ⁇ Sp 2 , Sp 3 and Sp 4 are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, the alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C2 - C24 alkenyl groups, C2 - C24 alkynyl groups and C3 - C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted.
  • the alkylene groups are unsubstituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR 3 , preferably O and/or S, wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably hydrogen or methyl.
  • Preferred spacer moieties Sp 1 , Sp 2 , Sp 3 and Sp 4 thus include -(CH2)n-, -(CH2CH2)n-, -(CH 2 CH 2 0)n-, -(OCH 2 CH 2 )n-, -(CH 2 CH20)nCH 2 CH2-, -CH 2 CH2(OCH 2 CH2)n-, -(CH2CH 2 CH 2 0)n-, -(OCH2CH 2 CH2)n-, -(CH2CH2CH20)nCH2CH 2 CH2- and -CH2CH2CH2(OCH2CH 2 CH2)n-, wherein n 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
  • Sp 1 , Sp 2 , Sp 3 and Sp 4 are independently selected, Sp 1 , if present, may be different from Sp 2 , if present, from Sp 3 and/or from Sp 4 , if present.
  • Reactive groups Q are described in more detail above.
  • reactive group Q is selected from the group consisting of, optionally substituted, N-maleimidyl groups, halogenated N-alkylamido groups, sulfonyloxy N- alkylamido groups, ester groups, carbonate groups, sulfonyl halide groups, thiol groups or derivatives thereof, alkenyl groups, alkynyl groups, (hetero)cycloalkynyl groups, bicyclo[6.1.0]non- 4-yn-9-yl] groups, cycloalkenyl groups, tetrazinyl groups, azido groups, phosphine groups, nitrile oxide groups, nitrone groups, nitrile imine groups, diazo groups, ketone groups, (O- alkyl)hydroxylamino groups, hydrazine groups, halogenated N-maleimi
  • Q is according to formula (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9I), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj) or (9zk), wherein (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9I), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zk), wherein (9a
  • Q is according to formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9zo) or (9r).
  • Q is according to formula (9a), (9j), (9n), (9o), (9q), (9p), (9t), (9zh), (9zo) or (9s)
  • Q is according to formula (9a), (9q), (9n), (9o), (9p), (9t), (9zo) or (9zh), and preferred embodiments thereof, as defined above.
  • Target molecule D and preferred embodiments for target molecule D in the linker-conjugate according to formula (4a) and (4b) are as defined above.
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is D, -[(Sp ) b (Z 2 ) e (Sp 4 )iD] or -[(Sp 2 )
  • R is hydrogen or a Ci - C20 alkyl group, more preferably R is hydrogen or a Ci - C16 alkyl group, even more preferably R is hydrogen or a Ci - C10 alkyl group, wherein the alkyl group is optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 , preferably O, wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups.
  • R is hydrogen.
  • R is a Ci - C20 alkyl group, more preferably a Ci - C16 alkyl group, even more preferably a Ci - C10 alkyl group, wherein the alkyl group is optionally interrupted by one or more O-atoms, and wherein the alkyl group is optionally substituted with an -OH group, preferably a terminal -OH group.
  • R is a polyethyleneglycol chain comprising a terminal -OH group.
  • R is a Ci - C12 alkyl group, more preferably a Ci - C6 alkyl group, even more preferably a Ci - C4 alkyl group, and yet even more preferably R is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t- butyl.
  • R is a further target molecule D, -[(Sp )b(Z 2 ) e (Sp 4 )iD] or -[(Sp 2 )c(Z )d(Sp 3 )gQ 1 ], wherein D is a target molecule and Sp 1 , Sp 2 , Sp 3 , Sp 4 , Z ⁇ Z 2 , Q ⁇ b, c, d, e, g and i are as defined above.
  • the linker-conjugate is according to formula (4a).
  • linker-conjugate (4a) comprises two target molecules D, which may be the same or different.
  • R is -[(Sp )b(Z 2 ) e (Sp 4 )iD]
  • Sp 1 , b, Z 2 , e, Sp 4 , i and D in -[(Sp ) b (Z 2 ) e (Sp 4 )iD] may be the same or different from Sp 1 , b, Z 2 , e, Sp 4 , i and D in -[(Sp )b(Z 2 ) e (Sp 4 )iD] that is attached to the N-atom of N(R 1 ).
  • both -[(Sp )b(Z 2 )e(Sp 4 )iD] and -[(Sp )b(Z 2 ) e (Sp 4 )iD] on the N-atom of N(R 1 ) are the same.
  • linker-conjugate (4b) comprises two target molecules Q , which may be the same or different.
  • -[(Sp 2 )c(Z )d(Sp 3 )gQ 1 ] groups on the N-atom of N(R 1 ) are the same.
  • f is an integer in the range of 1 to 150.
  • the linker-conjugate may thus comprise more than one group according to formula (1 ), the group according to formula (1 ) being as defined above.
  • more than one group according to formula (1 ) is present, i.e. when f is 2 or more, then a, b, Sp 1 and R are independently selected.
  • each a is independently 0 or 1
  • each b is independently 0 or 1
  • each Sp 1 may be the same or different and each R may be the same or different.
  • f is an integer in the range of 1 to 100, preferably in the range of 1 to 50, more preferably in the range of 1 to 25, and even more preferably in the range of 1 to 15. More preferably, f is 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, even more preferably f is 1 , 2, 3, 4, 5, 6, 7 or 8, yet even more preferably f is 1 , 2, 3, 4, 5 or 6, yet even more preferably f is 1 , 2, 3 or 4, and most preferably f is 1 in this embodiment.
  • f is an integer in the range of 2 to 150, preferably in the range of 2 to 100, more preferably in the range of 2 to 50, more preferably in the range of 2 to 25, and even more preferably in the range of 2 to 15. More preferably, f is 2, 3, 4, 5, 6, 7, 8, 9 or 10, even more preferably f is 2, 3, 4, 5, 6, 7 or 8, yet even more preferably f is 2, 3, 4, 5 or 6, yet even more preferably f is 2, 3 or 4, and most preferably f is 2 in this embodiment.
  • a is 0 in the compound according to formula (4a) or (4b).
  • the linker-conjugate may therefore also be a compound according to formula (6a) or (6b),
  • a is 1 in the compound according to formula (4a) or (4b).
  • the linker-conjugate may therefore also be a compound according to formula (7a) or (7b),
  • a, b, c, d, e, f, g, D, Q , Sp 1 , Sp 2 , Sp 3 , Z , Z 2 and R , and their preferred embodiments, are as defined above for (4a) and (4b).
  • a is 0.
  • a is 1.
  • linker-conjugate particularly a linker-conjugate according to formula (4a), (4b), (4c), (4d), (6a), (6b), (7a) or (7b), Sp ⁇ Sp 2 Sp 3 and Sp 4 , if present, are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, the alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group consisting of O, S and NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, and Q is according to formula (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9I), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9ze), (9a), (9a
  • Q is according to formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s) (9t), (9zh), (9zo) or (9r).
  • Q is according to formula (9a), (9j), (9n), (9o), (9p), (9q), (9t), (9zh), (9zo) or (9s)
  • Q is according to formula (9a), (9q), (9n), (9p), (9t), (9zh), (9zo) or (9o), and preferred embodiments thereof, as defined above.
  • Linker L as preferably comprised in the linker-conjugate according to formula (4a), (4b), (4c), (4d), (6a), (6b), (7a) or (7b) as defined above, linker as defined above may be represented by formula (8a) and (8b), respectively:
  • spacer-moieties (8a) and (8b) may depend on e.g. the nature of reactive groups Q and D in the linker-conjugate, the synthetic method to prepare the linker-conjugate (e.g. the nature of complementary functional group F 2 on a target molecule), the nature of a bioconjugate that is prepared using the linker- conjugate (e.g. the nature of complementary functional group F on the biomolecule).
  • Q is for example a cyclooctynyl group according to formula (9n), (9o), (9p), (9q) or (9zk) as defined above, then preferably Sp 3 is present (g is 1 ).
  • the linker-conjugate was prepared via reaction of a reactive group Q 2 that is a cyclooctynyl group according to formula (9n), (9o), (9p), (9q) or (9zk) with an azido functional group F 2 , then preferably Sp 4 is present (i is 1 ).
  • At least one of Sp 1 , Sp 2 , Sp 3 and Sp 4 is present, i.e. at least one of b, c, g, and i is not 0. In another preferred embodiment, at least one of Sp 1 and Sp 4 and at least one of Sp 2 and Sp 3 are present.
  • linker-moiety (8a) and (8b) also hold for the linker-conjugate when comprised in the bioconjugates according to the invention as described in more detail below.
  • Sp 1 , Sp 2 , Sp 3 and Sp 4 are as defined above.
  • the biomolecule is represented by B-F , wherein B is a biomolecule and F is a functional group capable of reacting with reactive group Q on the linker-conjugate and "-" is a bond or a spacer moiety.
  • the biomolecule is a modified antibody represented by formula (24), wherein F is a functional group capable of reacting with reactive group Q on the linker-conjugate.
  • the modified antibody represented by formula (24) and preferred embodiments thereof are defined in detail above.
  • biomolecule is a spacer moiety as defined herein.
  • “-” is a bond, typically a covalent bond.
  • the biomolecule may also be referred to as "biomolecule of interest” (BOI).
  • the biomolecule may be a biomolecule as naturally occurring, wherein functional group F is a already present in the biomolecule of interest, such as for example a thiol, an amine, an alcohol or a hydroxyphenol unit. Conjugation with the linker-conjugate then occurs via the first approach as defined above.
  • the biomolecule may be a modified biomolecule, wherein functional group F is specifically incorporated into the biomolecule of interest and conjugation with the linker-conjugate occurs via this engineered functionality, i.e. the two-stage approach of bioconjugation as defined above.
  • modification of biomolecules to incorporate a specific functionality is known, e.g. from WO 2014/065661 , incorporated herein by reference in its entirety.
  • biomolecule B is preferably selected from the group consisting of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids and monosaccharides. More preferably, biomolecule B is selected from the group consisting of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides and enzymes. More preferably, biomolecule B is selected from the group consisting of proteins, including glycoproteins and antibodies, polypeptides, peptides and glycans. Most preferably, biomolecule B is an antibody or an antigen-binding fragment thereof.
  • Functional group F is capable of reacting with reactive group Q on the linker-conjugate to form a connecting group Z 3 .
  • functional group F is capable of reacting with a complementary reactive group Q .
  • Functional groups F that are complementary to reactive groups Q are described in more detail below.
  • a reactive group Q that is present in the linker-conjugate is typically reacted with functional group F .
  • More than one functional group F may be present in the biomolecule. When two or more functional groups are present, said groups may be the same or different.
  • the biomolecule comprises two or more functional groups F, which may be the same or different, and two or more functional groups react with a complementary reactive group Q of a linker-conjugate.
  • a biomolecule comprising two functional groups F, i.e. F and F 2 may react with two linker-conjugates comprising a functional group Q , which may be the same or different, to form a bioconjugate.
  • Examples of a functional group F in a biomolecule comprise an amino group, a thiol group, a carboxylic acid, an alcohol group, a carbonyl group, a phosphate group, or an aromatic group.
  • the functional group in the biomolecule may be naturally present or may be placed in the biomolecule by a specific technique, for example a (bio)chemical or a genetic technique.
  • the functional group that is placed in the biomolecule may be a functional group that is naturally present in nature, or may be a functional group that is prepared by chemical synthesis, for example an azide, a terminal alkyne, a cyclopropene moiety or a phosphine moiety.
  • F is group capable of reacting in a cycloaddition, such as a diene, a dienophile, a 1 ,3-dipole or a dipolarophile, preferably F is selected from a 1 ,3-dipole (typically an azido group, nitrone group, nitrile oxide group, nitrile imine group or diazo group) or a dipolarophile (typically an alkenyl or alkynyl group).
  • a 1 ,3-dipole typically an azido group, nitrone group, nitrile oxide group, nitrile imine group or diazo group
  • a dipolarophile typically an alkenyl or alkynyl group
  • F is a 1 ,3- dipole when Q is a dipolarophile and F is a dipolarophile when Q is a 1 ,3-dipole, or F is a diene when Q is a dienophile and F is a dienophile when Q is a diene.
  • F is a 1 ,3- dipole, preferably F is or comprises an azido group.
  • Figure 2 shows several structures of derivatives of UDP sugars of galactosamine, which may be modified with e.g. a thiopropionyl group (11a), an azidoacetyl group (11 b), or an azidodifluoroacetyl group (11c) at the 2-position, or with an azido group at the 6-position of N- acetyl galactosamine (11d).
  • functional group F is a thiopropionyl group, an azidoacetyl group, or an azidodifluoroacetyl group.
  • Figure 3 schematically displays how any of the UDP-sugars 11a-d may be attached to a glycoprotein comprising a GlcNAc moiety 12 (e.g. a monoclonal antibody the glycan of which is trimmed by an endoglycosidase) under the action of a galactosyltransferase mutant or a GalNAc- transferase, thereby generating a ⁇ -glycosidic 1-4 linkage between a GalNAc derivative and GlcNAc (compounds 13a-d, respectively).
  • a GlcNAc moiety 12 e.g. a monoclonal antibody the glycan of which is trimmed by an endoglycosidase
  • Preferred examples of naturally present functional groups F include a thiol group and an amino group.
  • Preferred examples of a functional group that is prepared by chemical synthesis for incorporation into the biomolecule include a ketone group, a terminal alkyne group, an azide group, a cyclo(hetero)alkyne group, a cyclopropene group, or a tetrazine group.
  • complementary reactive groups Q and functional groups F are known to a person skilled in the art, and several suitable combinations of Q and F are described above, and shown in Figure 5.
  • a list of complementary groups Q and F is disclosed in in Table 3.1 , pages 230 - 232 of Chapter 3 of G.T. Hermanson, "Bioconjugate Techniques", Elsevier, 3 rd Ed. 2013 (ISBN :978-0-12-382239-0), and the content of this Table is expressly incorporated by reference herein.
  • a bioconjugate is herein defined as a compound wherein a biomolecule is covalently connected to a target molecule D via a linker.
  • a bioconjugate comprises one or more biomolecules and/or one or more target molecules.
  • the linker may comprise one or more spacer moieties.
  • the bioconjugate according to the invention is conveniently prepared by the process for preparation of a bioconjugate according to the invention, wherein the linker-conjugate comprising reactive group Q is conjugated to a biomolecule comprising functional group F . In this conjugation reaction, groups Q and F react with each other to form a connecting group Z 3 which connects the target molecule D with the biomolecule B.
  • the linker- conjugate and the biomolecule thus equally apply to the bioconjugate according to the invention, except for all said for Q and F , wherein the bioconjugate according to the invention contains the reaction product of Q and F , i.e. connecting group Z 3 .
  • the invention also concerns the bioconjugates, preferably the antibody-conjugates, described herein.
  • the bioconjugate according to the invention has formula (A):
  • - B is a biomolecule, preferably an antibody AB;
  • - L is a linker linking B and D;
  • the bioconjugate is obtainable by the mode of conjugation defined as "core-GlcNAc functionalization", i.e. by steps (i) and (ii) as defined above.
  • the bioconjugate according to formula (A) has a linker L comprising a group according to formula (1 ) or a salt thereof:
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24
  • (hetero)arylalkyl groups the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups; or R is a target molecule D, wherein D is optionally connected to N via a spacer moiety.
  • "-" is a spacer moiety as defined herein. In one embodiment, "-" is a bond, typically a covalent bond.
  • the bioconjugate is presented by B-Z 3 -L-D, wherein B, L, D and "-" are as defined above and Z 3 is a connecting group which is obtainable by reaction of Q with F .
  • Z 3 is obtainable by a cycloaddition, preferably a 1 ,3-dipolar cycloaddition reaction, most preferably Z 3 is a 1 ,2,3-triazole ring, which is located in a spacer moiety, preferably the spacer moiety between B and L, most preferably between B and the carbonyl or carboxyl end of the group according to formula (1 ).
  • the bioconjugate according to the invention comprises a salt of the group according to formula (1 ), the salt is preferably a pharmaceutically acceptable salt.
  • the bioconjugate according to the invention may comprise more than one target molecule. Similarly, the bioconjugate may comprise more than one biomolecule.
  • Biomolecule B and target molecule D are described in more detail above.
  • Preferred embodiments for D in the bioconjugate according to the invention correspond to preferred embodiments of D in the linker-conjugate according to the invention as were described in more detail above.
  • Preferred embodiments for the linker (8a) or (8b) in the bioconjugate according to the invention correspond to preferred embodiments of the linker in the linker-conjugate, as were described in more detail above.
  • Preferred embodiments for B in the bioconjugate according to the invention correspond to preferred embodiments of B in the biomolecule according to the invention as were described in more detail above.
  • the bioconjugate according to the invention may also be defined as a bioconjugate wherein a biomolecule is conjugated to a target molecule via a spacer-moiety, wherein the spacer-moiety comprises a group according to formula (1 ), or a salt thereof, wherein the group according to formula (1 ) is as defined above.
  • the bioconjugate according to the invention may also be denoted as (B) y Sp(D) z , wherein y' is an integer in the range of 1 to 10 and z is an integer in the range of 1 to 10.
  • the invention thus also relates to a bioconjugate according to the formula:
  • - y' is an integer in the range of 1 to 10;
  • - z is an integer in the range of 1 to 10;
  • - B is a biomolecule
  • - D is a target molecule
  • - Sp is a spacer moiety, wherein a spacer moiety is defined as a moiety that spaces (i.e. provides a certain distance between) and covalently links biomolecule B and target molecule D; and wherein said spacer moiety comprises a group according to formula (1 ) or a salt thereof, wherein the group according to formula (1 ) is as defined above.
  • said spacer moiety further comprises a moiety that is obtainable by a cycloaddition, preferably a 1 ,3-dipolar cycloaddition reaction, most preferably a 1 ,2,3-triazole ring, which is located between B and said group according to formula (1 ).
  • y' is 1 , 2, 3 or 4, more preferably y' is 1 or 2 and most preferably, y' is 1.
  • z is 1 , 2, 3, 4, 5 or 6, more preferably z is 1 , 2, 3 or 4, even more preferably z is 1 , 2 or 3, yet even more preferably z is 1 or 2 and most preferably z is 1.
  • the bioconjugate is according to the formula BSp(D)4, BSp(D)3, BSp(D)2 or BSpD.
  • the bioconjugate according to the invention comprises a group according to formula (1 ) as defined above, or a salt thereof.
  • the bioconjugate comprises a group according to formula (1 ) wherein a is 0, or a salt thereof.
  • the bioconjugate thus comprises a group according to formula (2) or a salt thereof, wherein (2) is as defined above.
  • the bioconjugate comprises a group according to formula (1 ) wherein a is 1 , or a salt thereof.
  • the bioconjugate thus comprises a group according to formula (3) or a salt thereof, wherein (3) is as defined above.
  • R spacer moiety Sp, as well as preferred embodiments of R and Sp, are as defined above for the linker-conjugate according to the invention.
  • the bioconjugate is according to formula (5a) or (5b), or a salt thereof:
  • - h is 0 or 1 ;
  • - Z 3 is a connecting group that connects B to Sp 3 , Z , Sp 2 , O or C(O);
  • - B is a biomolecule.
  • h is 1.
  • biomolecule B Preferred embodiments of biomolecule B are as defined above.
  • the bioconjugate according to the invention is a salt of (5a) or (5b), the salt is preferably a pharmaceutically acceptable salt.
  • Z 3 is a connecting group.
  • connecting group herein refers to the structural element connecting one part of a compound and another part of the same compound.
  • a bioconjugate is prepared via reaction of a reactive group Q present in the linker- conjugate with a functional group F present in a biomolecule.
  • connecting group Z 3 depends on the type of organic reaction that was used to establish the connection between the biomolecule and the linker- conjugate. In other words, the nature of Z 3 depends on the nature of reactive group Q of the linker-conjugate and the nature of functional group F in the biomolecule.
  • complementary groups Q include N-maleimidyl groups and alkenyl groups, and the corresponding connecting groups Z 3 are as shown in Figure 5.
  • complementary groups Q also include allenamide groups.
  • complementary groups Q include ketone groups and activated ester groups, and the corresponding connecting groups Z 3 are as shown in Figure 5.
  • complementary groups Q include (O- alkyl)hydroxylamino groups and hydrazine groups, and the corresponding connecting groups Z 3 are as shown in Figure 5.
  • complementary groups Q include azido groups, and the corresponding connecting group Z 3 is as shown in Figure 5.
  • complementary groups Q include alkynyl groups, and the corresponding connecting group Z 3 is as shown in Figure 5.
  • complementary groups Q include tetrazinyl groups, and the corresponding connecting group Z 3 is as shown in Figure 5.
  • Z 3 is only an intermediate structure and will expel N2, thereby generating a dihydropyridazine (from the reaction with alkene) or pyridazine (from the reaction with alkyne).
  • At least one of Z 3 , Sp 3 , Z and Sp 2 is present, i.e. at least one of h, g, d and c is not 0. It is also preferred that at least one of Sp 1 , Z 2 and Sp 4 is present, i.e. that at least one of b, e and i is not 0. More preferably, at least one of Z 3 , Sp 3 , Z and Sp 2 is present and at least one of Sp 1 , Z 2 and Sp 4 is present, i.e. it is preferred that at least one of b, e and i is not 0 and at least one of h, g, d and c is not 0.
  • the bioconjugate according to the invention is typically obtained by a process for the preparation of a bioconjugate as defined herein.
  • any method of preparing the bioconjugate can be used as long as the obtained bioconjugate comprises linker L as defined herein.
  • the group according to formula (1 ) may be present in linker L between B and Z 3 , i.e. it originates form the biomolecule, or between Z 3 and D, i.e. it originates from the linker-conjugate, or Z 3 is or comprises the group according to formula (1 ), i.e.
  • the group according to formula (1 ) is formed upon conjugation.
  • the group according to formula (1 ) is present in linker L between B and Z 3 or between Z 3 and D, most preferably, the group according to formula (1 ) is present in linker L between Z 3 and D.
  • the exact mode of conjugation including the nature of Q and F have a great flexibility in the context of the present invention. Many techniques for conjugating BOIs to MOIs are known to a person skilled in the art and can be used in the context of the present invention.
  • the conjugation method comprises steps (i) and (ii) as defined above.
  • the mode of conjugation is selected from any of the conjugation modes depicted in Figure 5, i.e.
  • a connecting moiety Z 3 that may be represented as (10a) or (10b)
  • amino-(activated) carboxylic acid conjugation wherein the (activated) carboxylic acid is represented by -C(0)X, wherein X is a leaving group
  • ketone-hydrazino conjugation preferably acetyl-hydrazino conjugation
  • Y NH
  • ketone-oxyamino conjugation preferably acetyl-oxyamino conjugation
  • Y O, alkyne-azide conjugation
  • the bioconjugate according to the invention is typically prepared by a process comprising the step of reacting a reactive group Q of the linker-conjugate as defined herein with a functional group F of the biomolecule, also referred to as a biomolecule.
  • a biomolecule of interest (BOI) comprising one or more functional groups F is incubated with (excess of) a target molecule D (also referred to as molecule of interest or MOI), covalently attached to a reactive group Q via a specific linker.
  • the BOI may e.g. be a peptide/protein, a glycan or a nucleic acid.
  • the bioconjugation reaction typically comprises the step of reacting a reactive group Q of the linker-conjugate with a functional group F of a the biomolecule, wherein a bioconjugate of formula (A) is formed, wherein linker L comprises a group according to formula (1 ) or a salt thereof:
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci
  • R is a further target molecule D, which is optionally connected to N via a spacer moiety.
  • the bioconjugate is prepared via a cycloaddition, such as a (4+2)- cycloaddition (e.g. a Diels-Alder reaction) or a (3+2)-cycloaddition (e.g. a 1 ,3-dipolar cycloaddition).
  • a cycloaddition such as a (4+2)- cycloaddition (e.g. a Diels-Alder reaction) or a (3+2)-cycloaddition (e.g. a 1 ,3-dipolar cycloaddition).
  • the conjugation is a Diels-Alder reaction or a 1 ,3-dipolar cycloaddition.
  • the preferred Diels-Alder reaction is the inverse-electron demand Diels-Alder cycloaddition.
  • the 1 ,3-dipolar cycloaddition is used, more preferably the alkyne- azide cycloaddition, and most preferably wherein Q is or comprises an alkyne group and F is an azido group.
  • Cycloadditions such as Diels-Alder reactions and 1 ,3-dipolar cycloadditions are known in the art, and the skilled person knowns how to perform them.
  • a is 0 in the group according to formula (1 ).
  • the linker-conjugate thus comprises a group according to formula (2), as defined above.
  • a is 1 in the group according to formula (1 ).
  • the linker-conjugate thus comprises a group according to formula (3), as defined above.
  • Biomolecules are described in more detail above.
  • the biomolecule is selected from the group consisting of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids and monosaccharides.
  • biomolecule B is selected from the group consisting of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides and enzymes.
  • biomolecule B is selected from the group consisting of proteins, including glycoproteins and antibodies, polypeptides, peptides and glycans.
  • B is an antibody or an antigen-binding fragment thereof.
  • reactive group Q is selected from the group consisting of, optionally substituted, N-maleimidyl groups, halogenated N- alkylamido groups, sulfonyloxy N-alkylamido groups, ester groups, carbonate groups, sulfonyl halide groups, thiol groups or derivatives thereof, alkenyl groups, alkynyl groups, (hetero)cycloalkynyl groups, bicyclo[6.1.0]non-4-yn-9-yl] groups, cycloalkenyl groups, tetrazinyl groups, azido groups, phosphine groups, nitrile oxide groups, nitrone groups, nitrile imine groups, diazo groups, ketone groups, (O-alkyl)hydroxylamino groups, hydrazine groups, halogenated N- maleimidyl groups, 1 , 1-bis(sulfonyl
  • Q is according to formula (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9I), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj) or (9zk), wherein (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9I), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zk), wherein (9a
  • Q is according to formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9ze), (9zh), (9zo) or (9r). Even more preferably, Q is according to formula (9a), (9j), (9n), (9o), (9p), (9q), (9t), (9ze), (9zh), (9zo) or (9s), and most preferably, Q is according to formula (9a), (9p),(9q), (9n), (9t), (9ze), (9zh), (9zo) or (9o), and preferred embodiments thereof, as defined above.
  • Q comprises an alkyne group, preferably selected from the alkynyl group as described above, the cycloalkenyl group as described above, the (hetero)cycloalkynyl group as described above and a bicyclo[6.1.0]non-4-yn-9-yl] group, more preferably Q is selected from the formulae (9j), (9n), (9o), (9p), (9q), (9zo) and (9zk), as defined above. Most preferably, Q is a bicyclo[6.1.0]non-4-yn-9-yl] group, preferably of formula (9q).
  • the linker- conjugate is according to formula (4a) or (4b), or a salt thereof:
  • - a is independently 0 or 1 ;
  • - b is independently 0 or 1 ;
  • - c is 0 or 1 ;
  • - d is 0 or 1
  • - e is 0 or 1 ;
  • - f is an integer in the range of 1 to 150;
  • - g is 0 or 1 ;
  • - i is 0 or 1 ;
  • - D is a target molecule
  • - Q is a reactive group capable of reacting with a functional group F present on a biomolecule
  • - Z is a connecting group that connects Q or Sp 3 to Sp 2 , O or C(O) or N(R 1 );
  • - Z 2 is a connecting group that connects D or Sp 4 to Sp 1 , N(R 1 ), O or C(O);
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cydoalkyi 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 cydoalkyi 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 NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C 4 alkyl groups; or R is D, -[(Sp )b(Z 2 ) e (Sp 4 )iD] or -[(Sp 2 )c(Z ) d (Sp 3 ) g Q 1 ], wherein D is a target molecule and Sp 1 , Sp 2 , Sp 3 , Sp 4 , Z , Z 2 , Q , b, c, d, e, g and i are as defined above.
  • Sp 1 , Sp 2 , Sp 3 and Sp 4 are, independently, spacer moieties, in other words, Sp 1 , Sp 2 , Sp 3 and Sp 4 may differ from each other.
  • Sp 1 , Sp 2 , Sp 3 and Sp 4 may be present or absent (b, c, g and i are, independently, 0 or 1 ). However, it is preferred that at least one of Sp 1 , Sp 2 , Sp 3 and Sp 4 is present, i.e. it is preferred that at least one of b, c, g and i is not 0.
  • Sp 1 , Sp 2 , Sp 3 and Sp 4 are independently selected from the group consisting of linear or branched C1-C200 alkylene groups, C2-C200 alkenylene groups, C2-C200 alkynylene groups, C3-C200 cycloalkylene groups, C5-C200 cycloalkenylene groups, C8-C200 cycloalkynylene groups, C7-C200 alkylarylene groups, C7-C200 arylalkylene groups, C8-C200 arylalkenylene groups and C9-C200 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected
  • 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.
  • spacer moieties Sp ⁇ Sp 2 , Sp 3 and Sp 4 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, C8-C100 cycloalkynylene groups, C7-C100 alkylarylene groups, C7-C100 arylalkylene groups, Cs-doo arylalkenylene groups and C9-C100 arylalkynylene groups, the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups being optionally substituted and optionally
  • spacer moieties Sp 1 , Sp 2 , Sp 3 and Sp 4 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 substitute
  • spacer moieties Sp 1 , Sp 2 , Sp 3 and Sp 4 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
  • 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 NR 3 , preferably O, wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably hydrogen or methyl.
  • spacer moieties Sp ⁇ Sp 2 , Sp 3 and Sp 4 are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, the alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C2 - C24 alkenyl groups, C2 - C24 alkynyl groups and C3 - C24 cycloalkyl groups, the alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups being optionally substituted.
  • the alkylene groups are unsubstituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR 3 , preferably O and/or S-S, wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, preferably hydrogen or methyl.
  • Particularly preferred spacer moieties Sp 1 , Sp 2 , Sp 3 and Sp 4 include -(CH2)n-, -(CH2CH2)n-, -(CH 2 CH 2 0)n-, -(OCH 2 CH 2 )n-, -(CH 2 CH20)nCH 2 CH2-, -CH 2 CH2(OCH 2 CH2)n-, -(CH2CH 2 CH 2 0)n-, -(OCH2CH 2 CH2)n-, -(CH2CH2CH20)nCH2CH 2 CH2- and -CH2CH2CH2(OCH2CH 2 CH2)n-, wherein n 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
  • spacer moieties Sp 1 , Sp 2 , Sp 3 and/or Sp 4 if present, comprise a sequence of amino acids.
  • Spacer-moieties comprising a sequence of amino acids are known in the art, and may also be referred to as peptide linkers. Examples include spacer-moieties comprising a Val-Ala moiety or a Val-Cit moiety, e.g. Val-Cit-PABC, Val-Cit- PABC, Fmoc-Val-Cit-PABC, etc.
  • Z and Z 2 are connecting groups.
  • Sp ⁇ Sp 2 , Sp 3 and Sp 4 are independently selected from the group consisting of linear or branched C1-C20 alkylene groups, the alkylene groups being optionally substituted and optionally interrupted by one or more heteroatoms selected from the group consisting of O, S and NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, and wherein Q is according to formula (9a), (9j), (9p), (9q), (9n), (9t), (9ze), (9zh), (9zo) or (9o):
  • - 1 is an integer in the range 0 - 10;
  • R 0 is a (thio)ester group
  • R 8 is selected from the group consisting of, optionally substituted, Ci - C12 alkyl groups and C4 - C12 (hetero)aryl groups.
  • FIG. 4 shows how a modified antibody 13a-d may undergo a bioconjugation process by means of nucleophilic addition with maleimide (as for 3-mercaptopropionyl-galactosamine-modified 13a leading to thioether conjugate 14, or for conjugation to an engineered cysteine residue leading to thioether conjugate 17) or upon strain-promoted cycloaddition with a cyclooctyne reagent (as for 13b, 13c or 13d, leading to triazoles 15a, 15b or 16, respectively).
  • maleimide as for 3-mercaptopropionyl-galactosamine-modified 13a leading to thioether conjugate 14, or for conjugation to an engineered cysteine residue leading to thioether conjugate 17
  • a cyclooctyne reagent as for 13b, 13c or 13d, leading to triazoles 15a, 15b or 16, respectively.
  • a further advantages of the process for the preparation of a bioconjugate as described herein, and of the linker-conjugates and sulfamide linker according to the invention is that conjugation efficiency increases in case a sulfamide linker is used instead of a typical polyethylene glycol (PEG) spacer.
  • An additional advantage of a sulfamide group, in particular of an acylsulfamide or a carbamoylsulfamide group, is its high polarity, which imparts a positive effect on the solubility of a linker comprising such group, and on the construct as a whole, before, during and after conjugation.
  • conjugation with linker-conjugate containing the sulfamide linker according to the invention are particularly suited to conjugate hydrophobic target compounds to a biomolecule.
  • the high polarity of the sulfamides also has a positive impact in case hydrophobic moieties are conjugated to a biomolecule of interest, which is known to require large amounts of organic co-solvent during conjugation and/or induce aggregation of the bioconjugate.
  • High levels of co-solvent up to 25% of DMF or even 50% of DMA, propylene glycol, or DMSO may induce protein denaturation during the conjugation process and/or may require special equipment in the manufacturing process.
  • the problem of aggregation associated with the hydrophobic linking moieties in bioconjugates is efficiently solved by using the sulfamide linker according to the invention in the spacer between the target molecule and the reactive group Q in the linker-conjugate in the formation of the bioconjugate.
  • An additional advantage of a sulfamide linker according the invention, and its use in bioconjugation processes, is its ease of synthesis and high yields.
  • PCT/NL2015/050697 (WO 2016/053107), in particular to Tables 1 - 3, Figures 1 1 - 14, 23 and 24, and Examples 57, 58, 60 and 61 therein. These Tables, Figures and Examples of PCT/NL2015/050697 (WO 2016/053107) are incorporated herein.
  • the invention thus concerns in a first aspect the use of a mode of conjugation comprising at least one of "core-GlcNAc functionalization” and "sulfamide linkage", as defined above, for increasing the therapeutic index of a bioconjugate.
  • the invention according to the present aspect can also be worded as a method for increasing the therapeutic index of a bioconjugate.
  • the mode of conjugation is being used to connect a biomolecule B with a target molecule D via a linker L, wherein the mode of conjugation comprises:
  • S(F ) X and x are as defined above; AB represents an antibody; GlcNAc is N- acetylglucosamine; Fuc is fucose; b is 0 or 1 ; and y is 1 , 2, 3 or 4; and
  • linker-conjugate comprising a functional group Q capable of reacting with functional group F and a target molecule D connected to Q via a linker L 2 to obtain the antibody-conjugate wherein linker L comprises S-Z 3 -L 2 and wherein Z 3 is a connecting group resulting from the reaction between Q and F .
  • the biomolecule is preferably an antibody
  • the bioconjugate is preferably an antibody- conjugate.
  • the therapeutic index is increased compared to a bioconjugate which does not comprise or is obtainable by the mode of conjugation according to the invention.
  • the therapeutic index is increased compared to a bioconjugate not obtainable by steps (i) and (ii) as defined above, or - in other words - not containing the structural feature of the resulting linker L that links the antibody with the target molecule, that are a direct consequence of the conjugation process.
  • the therapeutic index is increased compared to a bioconjugate of formula (A), wherein linker L does not comprise a group according to formula (1 ) or a salt thereof.
  • the increased therapeutic index could solely be attributed to the mode of conjugation according to the invention.
  • the increased therapeutic index is preferably an increased therapeutic index in the treatment of cancer, or alternatively in targeting of CD30-expressing tumours.
  • the method according to the first aspect of the invention may also be worded as a method for increasing the therapeutic index of a bioconjugate, comprising the step of providing a bioconjugate having the mode of conjugation according to the invention.
  • the mode of conjugation according to the invention has an effect on both aspects of the therapeutic index: (a) on the therapeutic efficacy and (b) on the tolerability.
  • the present use or method for increasing the therapeutic index is preferably for (a) increasing the therapeutic efficacy, and/or (b) increasing the tolerability of a bioconjugate of formula (A).
  • the bioconjugate is an antibody-conjugate and the present use or method is for increasing the therapeutic index of an antibody-conjugate, preferably for (a) increasing the therapeutic efficacy of the antibody- conjugate, and/or (b) increasing the tolerability of the antibody-conjugate.
  • the present method or use is for increasing the therapeutic efficacy of the bioconjugate, preferably the antibody-conjugate. In one embodiment, the present method or use is for increasing the tolerability of the bioconjugate, preferably the antibody-conjugate.
  • the use or method according to the first aspect is for increasing the therapeutic efficacy of a bioconjugate of formula (A).
  • “increasing the therapeutic efficacy” can also be worded as “lowering the effective dose”, “lowering the ED50 value” or “increasing the protective index”.
  • the method according to the first aspect is for increasing the tolerability of a bioconjugate of formula (A).
  • “increasing the tolerability” can also be worded as “increasing the maximum tolerated dose (MTD)", “increasing the TD50 value", “increasing the safety” or “reducing the toxicity”.
  • the method according to the first aspect is for (a) increasing the therapeutic efficacy and (b) increasing the tolerability of a bioconjugate of formula (A).
  • the method according to the first aspect is largely non-medical.
  • the method is a non-medical or a non-therapeutic method for increasing the therapeutic index of a bioconjugate.
  • the first aspect of the invention can also be worded as a mode of conjugation for use in improving the therapeutic index (therapeutic efficacy and/or tolerability) of a bioconjugate, wherein the mode of conjugation is as defined above.
  • the present aspect is worded as a the mode of conjugation according to the "core-GlcNAc functionalization" as defined above for use in improving the therapeutic efficacy of a bioconjugate of formula (A), wherein L and (A) are as defined above.
  • the present aspect is worded as a mode of conjugation for use in improving the therapeutic index (therapeutic efficacy and/or tolerability) of a bioconjugate, preferably an antibody-conjugate.
  • the first aspect concerns the use of a mode of conjugation for the preparation of a bioconjugate, preferably an antibody-conjugate, for improving the therapeutic index (therapeutic efficacy and/or tolerability) of the bioconjugate.
  • the invention according to the first aspect can also be worded as the use of a mode of conjugation in a bioconjugate, preferably an antibody-conjugate, or in the preparation of a bioconjugate, preferably an antibody-conjugate, for increasing the therapeutic index (therapeutic efficacy and/or tolerability) of the bioconjugate.
  • the use as defined herein may be referred to as non-medical or non-therapeutic use.
  • the method, use or mode of conjugation for use according to the first aspect of the invention further comprises the administration of the bioconjugate according to the invention to a subject in need thereof, suitably a patient suffering from a disorder associated with CD30 expression, e.g.
  • lymphoma selected from lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin lymphoma (NHL), anaplastic large-cell lymphoma (ALCL), large B-cell lymphoma, paediatric lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukaemia, such as acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and mast cell leukaemia, germ cell cancer, graft-versus-host disease (GvHD) and lupus, in particular systemic lupus erythematosus (SLE).
  • HL Hodgkin's lymphoma
  • NHL non-Hodgkin lymphoma
  • ALCL anaplastic large-cell lymphoma
  • large B-cell lymphoma paediatric lymphoma
  • the subject is a cancer patient, more suitably a patient suffering from CD30-expressing tumours.
  • bioconjugates such as antibody- drug-conjugates
  • the bioconjugates according to the invention are especially suited in this respect.
  • the bioconjugate is administered in a therapeutically effective dose.
  • Administration may be in a single dose or may e.g. occur 1 - 4 times a month, preferably 1 - 2 times a month.
  • administration occurs once every 3 or 4 weeks, most preferably every 4 weeks.
  • administration may occur less frequent as would be the case during treatment with conventional bioconjugates.
  • the dose of the bioconjugate according to the invention may depend on many factors and the optimal dosing regime can be determined by the skilled person via routine experimentation.
  • the bioconjugate is typically administered in a dose of 0.01 - 50 mg/kg body weight of the subject, more accurately 0.03 - 25 mg/kg or most accurately 0.05 - 10 mg/kg, or alternatively 0.1 - 25 mg/kg or 0.5 - 10 mg/kg. In one embodiment, administration occurs via intravenous injection.
  • the invention concerns in a second aspect a method for targeting CD30-expressing cells, comprising the administration of the bioconjugate according to the invention.
  • CD30-expressing cells may also be referred to as CD30-expressing tumour cells.
  • the subject in need thereof is most preferably a cancer patient.
  • bioconjugates such as antibody-drug-conjugates, is well-known in the field of cancer treatment, and the bioconjugates according to the invention are especially suited in this respect.
  • the method as described is typically suited for the treatment of cancer.
  • the bioconjugate according to the invention is described a great detail above, which equally applies to the bioconjugate used in the second aspect of the invention.
  • the second aspect of the invention can also be worded as a bioconjugate according to the invention for use in targeting CD30-expressing cells in a subject in need thereof.
  • the second aspect concerns the use of a according to the invention for the preparation of a medicament for use in the targeting CD30-expressing cells in a subject in need thereof.
  • the targeting of CD30-expressing cells includes one or more of treating, imaging, diagnosing, preventing the proliferation of, containing and reducing CD30- expressing cells, in particular CD30-expressing tumours. Most preferably, the present aspect is for the treatment of CD30-expressing tumours.
  • the present aspect concerns a method for the treatment of a subject in need thereof.
  • the second aspect of the invention can also be worded as a bioconjugate according to the invention for use in the treatment of a subject in need thereof, preferably for the treatment of cancer.
  • the second aspect concerns the use of a bioconjugate according to the invention for the preparation of a medicament for use in the treatment of a subject in need thereof, preferably for use in the treatment of cancer.
  • the subject suitably suffers from a disorder selected form lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin lymphoma (NHL), anaplastic large- cell lymphoma (ALCL), large B-cell lymphoma, paediatric lymphoma, T-cell lymphoma and enteropathy-associated T-cell lymphoma (EATL), leukaemia, such as acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and mast cell leukaemia, germ cell cancer, graft- versus-host disease (GvHD) and lupus, in particular systemic lupus erythematosus (SLE).
  • the disorder is cancer, most suitably lymphoma, such as Hodgkin's lymphoma (HL).
  • target molecule D is an anti-cancer agent, preferably a cytotoxin.
  • the bioconjugate is typically administered in a therapeutically effective dose.
  • Administration may be in a single dose or may e.g. occur 1 - 4 times a month, preferably 1 - 2 times a month.
  • administration occurs once every 3 or 4 weeks, most preferably every 4 weeks.
  • the dose of the bioconjugate according to the invention may depend on many factors and the optimal dosing regime can be determined by the skilled person via routine experimentation.
  • the bioconjugate is typically administered in a dose of 0.01 - 50 mg/kg body weight of the subject, more accurately 0.03 - 25 mg/kg or most accurately 0.05 - 10 mg/kg, or alternatively 0.1 - 25 mg/kg or 0.5 - 10 mg/kg. In one embodiment, administration occurs via intravenous injection.
  • the administration of the bioconjugate according to the invention is at a dose that is lower than the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the dose is at most 99-90%, more preferably at most 89-60%, even more preferable at most 59-30%, most preferably at most 29-10% of the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • the administration of the bioconjugate according to the invention occurs less frequent as administration would occur for the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the number of administration events is at most 75%, more preferably at most 50% of the number of administration events of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • administration may occur in a higher dose as in treatment with conventional bioconjugates.
  • the administration of the bioconjugate according to the invention is at a dose that is higher than the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the dose is at most 25-50%, more preferably at most 50-75%, most preferably at most 75-100% of the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • the administration of the bioconjugate according to the invention is at a dose that is lower than the ED50 of the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the dose is at most 99-90%, more preferably at most 89-60%, even more preferable at most 59-30%, most preferably at most 29-10% of the ED50 of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • the administration of the bioconjugate according to the invention occurs less frequent as administration would occur for the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the number of administration events is at most 75%, more preferably at most 50% of the number of administration events of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • administration may occur in a higher dose as in treatment with conventional bioconjugates.
  • the administration of the bioconjugate according to the invention is at a dose that is higher than the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention, preferably the dose is at most a factor 1.1 - 1.49 higher, more preferably at most a factor 1 .5 - 1.99 higher, even more preferable a factor 2 - 4.99 higher, most preferably at most a factor 5 - 10 higher of the TD50 of the same bioconjugate but not comprising the mode of conjugation according to the invention.
  • the use or method or conjugation mode for use according to the present aspect is a bioconjugate for use in the treatment of a subject in need thereof, wherein the bioconjugate is represented by formula (A):
  • - B is a biomolecule
  • - L is a linker linking B and D;
  • L comprises a group according to formula (1 ) or a salt thereof:
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is an additional target molecule D, wherein the target molecule is optionally connected to N via a spacer moiety.
  • the invention pertains to antibody-conjugates which are particularly suitable in targeting CD30-expressing tumours.
  • the antibody-conjugates according to the invention comprise an antibody AB connected to a target molecule D via a linker L, wherein the antibody-conjugate comprises or is obtainable by the mode of conjugation according to the invention.
  • the antibody-conjugates according to the invention are obtainable by:
  • S(F ) X and x are as defined above; AB represents an antibody; GlcNAc is N- acetylglucosamine; Fuc is fucose; b is 0 or 1 ; and y is 1 , 2, 3 or 4; and
  • linker-conjugate comprising a functional group Q capable of reacting with functional group F and a target molecule D connected to Q via a linker L 2 to obtain the antibody-conjugate wherein linker L comprises S-Z 3 -L 2 and wherein Z 3 is a connecting group resulting from the reaction between Q and F .
  • antibody AB is capable of targeting CD30-expressing tumours and target molecule D is selected from the group consisting consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vinca alkaloids, maytansinoids, calicheamycins and enediynes, duocarmycins, tubulysins, amatoxins, dolastatins and auristatins, pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof.
  • target molecule D is a cytotoxin.
  • target molecule D is selected from the group consisting of anthracyclines, maytansinoids, calicheamycins and enediynes, duocarmycins, tubulysins, dolastatins and auristatins, pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, more preferably from the group consisting of anthracyclines, maytansinoids, dolastatins and auristatins, pyrrolobenzodiazepine dimers.
  • the antibody-conjugate according to the present aspect is according to formula (A) and preferably linker L contains the group according to formula (1 ) or a salt thereof, wherein (A) and (1 ) are as defined above.
  • S(F ) X is 6-azido- 6-deoxy-A/-acetylgalactosamine.
  • the antibody AB capable of targeting CD30-expressing tumours is selected from the group consisting of from Ki-2, Ki-2, Ki-4, Ki-6, Ki-7, HRS-1 , HRS-4, Ber-H8, Ber- H2, 5F1 1 (MDX-060, iratumumab), Ki-1 , Ki-5, M67, Ki-3, M44, HeFi-1 , AC10, cAC10 (brentuximab) and functional analogues thereof. More preferably, the antibody AB capable of targeting CD30-expressing tumours is iratumumab or brentuximab, most preferably brentuximab.
  • the antibody AB is iratumumab or brentuximab, most preferably brentuximab
  • the antibody-conjugate according is represented by Formula (40) or (40b):
  • R 3 is independently selected from the group consisting of hydrogen, halogen, -OR 35 , -NO2, -CN, -S(0)2R 35 , 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 R 3 may be linked together to form an annelated cycloalkyl or an annelated (hetero)arene substituent, and wherein R 35 is independently selected from the group consisting of hydrogen, halogen, Ci - C24 alkyl groups, Ce - C24 (hetero)aryl groups, C7 - C24 alkyl(hetero)aryl groups and C7 -
  • - X is C(R 3 )2, O, S or NR 32 , wherein R 32 is R 3 or L 3 (D , wherein L 3 is a linker, and D is as defined in claim 1 ;
  • - r is 1 - 20;
  • - aa is 0,1 , 2, 3, 4, 5, 6, 7 or 8;
  • - aa' is 0,1 , 2, 3, 4, 5, 6, 7 or 8;
  • - b is 0 or 1 ;
  • - pp is 0 or 1 ;
  • - M is -N(H)C(0)CH 2 -, -N(H)C(0)CF 2 -, -CH2-, -CF 2 - or a 1 ,4-phenylene containing 0 - 4 fluorine substituents, preferably 2 fluorine substituents which are preferably positioned on C2 and C6 or on C3 and C5 of the phenylene;
  • - Fuc is fucose.
  • the antibody is according to any one of Formulae (41 ), (42), (42b), (35b), (40c) and (40d) as defined above.
  • the antibody-conjugate according to the present aspect is a bioconjugate represented by formula (A):
  • - B is a biomolecule
  • - L is a linker linking B and D;
  • L comprises a group according to formula (1 ) or a salt thereof:
  • R is selected from the group consisting of hydrogen, Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups, the Ci - C24 alkyl groups, C3 - C24 cycloalkyl groups, C2 - C24 (hetero)aryl groups, C3 - C24 alkyl(hetero)aryl groups and C3 - C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups, or R is an additional target molecule D, wherein the target molecule is optionally connected to N via a spacer moiety.
  • conjugates (I) - (VII) are listed here below as conjugates (I) - (VII).
  • the antibody-conjugate according to the present aspect is selected from the conjugates defined here below as (I) - (VII), more preferably selected from the conjugates defined here below as (IV) - (VII).
  • the antibody-conjugate according to the present aspect is not a conjugate defined here below as (I) - (III), preferably not a conjugate defined here below as (I) - (VII).
  • (9q) is represented by:
  • the antibody-conjugates according to the present aspect have an improved therapeutic index compared to known antibody-conjugates, wherein the therapeutic index is preferably for the treatment of CD30-expressing tumours.
  • the improved therapeutic index may take the form of an improved therapeutic efficacy and/or an improved tolerability.
  • the antibody- conjugates according to the present aspect have an improved therapeutic efficacy compared to known antibody-conjugates for the treatment of CD30-expressing tumours.
  • the antibody-conjugates according to the present aspect have an improved tolerability compared to known antibody-conjugates for the treatment of CD30-expressing tumours.
  • the antibody-conjugates according to the invention outperform the known antibody-conjugates also in other aspects.
  • the inventors have found that the present antibody-conjugates exhibit an increased stability (i.e. they exhibit less degradation over time).
  • the inventors have also found that the present antibody-conjugates exhibit decreased aggregation issues (i.e. they exhibit less aggregation over time).
  • the present antibody-conjugates are a marked improvement over prior art antibody-conjugates.
  • the invention thus also concerns the use of the mode of conjugation as defined herein for improving stability of a bioconjugate, typically an antibody-conjugate.
  • the invention thus also concerns the use of the mode of conjugation as defined herein for decreasing aggregation of an bioconjugate, typically an antibody-conjugate.
  • the invention concerns a fusion enzyme comprising two endoglycosidases.
  • the two endoglycosidases EndoS and EndoH are connected via a linker, preferably a -(Gly4Ser)3-(His)6-(Gly4Ser)3- linker.
  • the fusion enzyme according to the invention as also referred to as EndoSH.
  • the enzyme according to the invention has at least 50% sequence identity with SEQ ID NO: 1 , preferably at least 70%, more preferably at least 80% sequence identity with SEQ ID NO: 1 , such as at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1.
  • the enzyme of the invention having the above indicated sequence identity to SEQ ID NO: 1 , has EndoS and EndoH activity. Most preferably, the enzyme according to the invention has 100% sequence identity with SEQ ID NO: 1.
  • fusion enzymes of EndoS and EndoH wherein the linker is replaced by another suitable linker known in the art, wherein said linker may be a rigid, or flexible.
  • said linker is a flexible linker allowing the adjacent protein domains to move relative freely to one another.
  • said flexible linker is composed of amino residues like glycine, serine, histidine and/or alanine and has a length of 3 to 59 amino acid residues, preferably 10 to 45 or 15 to 40 amino acid residues, such as 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues, or 20 to 38, 25 to 37 or 30 to 36 amino acid residues.
  • the fusion enzyme is covalently linked to, or comprises, a tag for ease of purification and or detection as known in the art, such as an Fc-tag, FLAG-tag, poly(His)- tag, HA-tag and Myc-tag.
  • Trimming of glycoproteins is known in the art, from e.g. WO 2007/133855 or WO 2014/065661 .
  • the enzyme according to the invention exhibits both EndoS and EndoH activity, and is capable of trimming glycans on glycoproteins (such as antibodies) at the core-GlcNAc unit, leaving only the core-GlcNAc residue on the glycoprotein (EndoS activity) as well as well as splitting off high- manose glycans (EndoH activity).
  • both activities of the fusion enzyme function smoothly at a pH around 7 - 8, while monomeric EndoH requires a pH of 6 to operate optimally.
  • the fusion enzyme according to the invention can be prepared by routine techniques in the art, such as introducing an expression vector (e.g. plasmid) comprising the enzyme coding sequence into a host cell (e.g. E. coli) for expression, from which the enzyme can be isolated.
  • an expression vector e.g. plasmid
  • a host cell e.g. E. coli
  • a possible approach for the preparation and purification of the fusion enzyme according to the invention is given in examples 4 - 6, and its functioning is demonstrated in examples 7 and 9, wherein brentuximab and iratumumab are efficiently trimmed in a single step.
  • Sequence identification of fusion protein EndoSH SEQ. ID NO: 1
  • RP-HPLC analysis of reduced monoclonal antibodies Prior to RP-HPLC analysis samples were reduced by incubating a solution of 10 ⁇ g (modified) IgG for 15 minutes at 37 °C with 10 mM DTT and 100 mM Tris pH 8.0 in a total volume of 50 ⁇ _. A solution of 49% ACN, 49% MQ and 2% formic acid (50 ⁇ _) was added to the reduced sample.
  • Mass spectral analysis of monoclonal antibodies Prior to mass spectral analysis, IgGs were either treated with DTT, which allows analysis of both light and heavy chain, or treated with FabricatorTM (commercially available from Genovis, Lund, Sweden), which allows analysis of the Fc/2 fragment.
  • DTT dimethylcellulose
  • FabricatorTM commercially available from Genovis, Lund, Sweden
  • cAC10 was transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) at 5 L scale. The supernatant was purified using a XK 26/20 column packed with 50 mL protein A sepharose. In a single run 5 L supernatant was loaded onto the column followed by washing with at least 10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCI. Retained protein was eluted with 0.1 M Glycine pH 2.7. The eluted cAC10 was immediately neutralized with 1.5 M Tris-HCI pH 8.8 and dialyzed against 25 mM Tris pH 8.0. Next the IgG was concentrated to approximately 20 mg/mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) and stored at -80 °C prior to further use.
  • Vivaspin Turbo 15 ultrafiltration unit Sartorius
  • Example 2 Transient expression and purification of iratumumab
  • Iratumumab was transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) at 125 mL scale. The supernatant was purified using a HiTrap mAbSelect SuRe 5 mL column (GE Healthcare). The supernatant was loaded onto the column followed by washing with at least 10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCI. Retained protein was eluted with 0.1 M Glycine pH 2.7. The eluted product was immediately neutralized with 1.5 M Tris-HCI pH 8.8 and dialyzed against 20 mM Tris pH 7.5.
  • Example 3 Transient expression and purification of His-TnGalNAcT(33-421)
  • His-TnGalNAcT(33-421 ) (identified by SEQ ID NO: 2) was transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) at 5 L scale.
  • the supernatant was purified using a XK 16/20 column packed with 25 mL Ni sepharose excel (GE Healthcare). Each run approximately 1.5L supernatant was loaded onto the column followed by washing with at least 10 column volumes of buffer A (20 mM Tris buffer, 5 mM imidazole, 500 mM NaCI, pH 7.5). Retained protein was eluted with buffer B (20 mM Tris, 500 mM NaCI, 500 mM imidazole, pH 7.5).
  • the buffer of the eluted fractions was exchanged to 25 mM Tris pH 8.0 using a HiPrep H26/10 desalting column (GE Healthcare).
  • the purified protein was concentrated to at least 3 mg/mL using a Vivaspin Turbo 4 ultrafiltration unit (Sartorius) and stored at -80 °C prior to further use.
  • EndoSH EndoS-(G4S)3-(His)6-(G4S)3-EndoH (EndoSH) coding sequence (EndoSH being identified by SEQ ID NO: 1 ) between Ndel-Hindlll sites was obtained from Genscript.
  • the DNA sequence for the EndoSH fusion protein consists of the encoding residues 48-995 of EndoS fused via an /V-terminal linked glycine-serine (GS) linker to EndoH.
  • the glycine- serine (GS) linker comprises a -(G4S)3-(His)6-(G4S)3- format, allowing spacing of the two enzymes and at the same time introducing a IMAC-purification tag.
  • EndoSH fusion protein (identified by SEQ ID NO: 1 ) starts with the transformation of the plasmid (pET22b-EndoSH) into BL21 cells.
  • Next step is the inoculation of 500 mL culture (LB medium + Ampilicin) with BL21 cells. When the OD600 reached 0.7 the cultures were induced with 1 mM IPTG (500 ⁇ _ of 1 M stock solution).
  • the column was first washed with buffer A (20 mM Tris buffer, 20 mM imidazole, 500 mM NaCI, pH 7.5). Retained protein was eluted with buffer B (20 mM Tris, 500 mM NaCI, 250 mM imidazole, pH 7.5, 10 mL). Fractions were analysed by SDS-PAGE on polyacrylamide gels (12%). The fractions that contained purified target protein were combined and the buffer was exchanged against 20 mM Tris pH 7.5 and 150 mM NaCI by dialysis performed overnight at 4 °C. The purified protein was concentrated to at least 2 mg/mL using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore). The product is stored at -80 °C prior to further use.
  • buffer A (20 mM Tris buffer, 20 mM imidazole, 500 mM NaCI, pH 7.5
  • buffer B 20 mM Tris, 500 mM NaCI, 250 mM imid
  • Glycan trimming of cAC10 was performed with fusion protein EndoSH.
  • cAC10 (14.5 mg/mL) was incubated with EndoSH (1 w/w%) in 25 mM Tris pH 7.5 with 150 mM NaCI for approximately 16 hours at 37 °C.
  • EndoSH (1 w/w%) in 25 mM Tris pH 7.5 with 150 mM NaCI for approximately 16 hours at 37 °C.
  • the trimmed IgG was dialyzed against 3 x 1 L of 25 mM Tris- HCI pH 8.0.
  • Substrate 6-N3-GalNAc-UDP (11d) is used for the preparation of the modified biomolecule cAC10-(6-N3-GalNAc)2 13d, suitable as biomolecule in the context of the invention.
  • Biomolecule 13d was purified from the reaction mixture on a HiTrap MabSelect SuRe 5 ml column (GE Healthcare) using an AKTA purifier-10 (GE Healthcare).
  • the eluted IgG was immediately neutralized with 1.5 M Tris-HCI pH 8.8 and dialyzed against PBS pH 7.4.
  • the IgG was concentrated using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore) to a concentration of 23.4 mg/mL.
  • Mass spectral analysis of a fabricator-digested sample showed three peaks of the Fc/2-fragment belonging to one major product (observed mass 24333 Da, approximately 80% of total Fc/2 fragment), corresponding to core 6-N3-GalNAc-GlcNAc(Fuc)-substituted cAC10, and two minor products (observed masses of 24187 and 24461 Da, approximately 5 and 15% of total Fc/2 fragment), corresponding to core 6-N3-GalNAc-GlcNAc-substituted cAC10 and core 6-N3- GalNAc-GlcNAc(Fuc)-substituted cAC10 with C-terminal lysine. Remodeling of iratumumab: Examples 9-10
  • Evitria (Zurich, Switzerland) was performed with fusion protein EndoSH.
  • iratumumab (14.4 mg/mL) was incubated with EndoSH (1 w/w%) in 20 mM Tris pH 7.5 for approximately 16 hours at 37 °C.
  • EndoSH (1 w/w%) in 20 mM Tris pH 7.5 for approximately 16 hours at 37 °C.
  • the trimmed IgG was dialyzed against 3 x 1 L of 25 mM Tris-HCI pH 8.0.
  • Mass spectral analysis of a fabricator-digested sample showed three peaks of the Fc/2-fragment belonging to one major product (observed mass 24104 Da, approximately 85% of total Fc/2 fragment), corresponding to core-GlcNAc(Fuc)-substituted iratumumab , and two minor products (observed masses of 23957 and 24232 Da, approximately 5 and 10% of total Fc/2 fragment), corresponding to core-GlcNAc-substituted iratumumab and core-GlcNAc(Fuc)-substituted iratumumab with C- terminal lysine.
  • Example 10 Glycosyltransfer of 6-N3-GalNAc-UDP to trimmed iratumumab under the action of TnGalNAcT
  • Substrate 6-N3-GalNAc-UDP (11d) is used for the preparation of the modified biomolecule iratumumab-(6-N3-GalNAc)2, suitable as biomolecule in the context of the invention.
  • iratumumab (10 mg/mL), obtained by EndoSH treatment of iratumumab as described above in example 9, was incubated overnight with the substrate 6-N3-GalNAc-UDP (5 mM, commercially available from GlycoHub) and 0.5 mg/mL His-TnGalNAcT(33-421 ) (5 w/w%) in 10 mM MnCk and 20 mM Tris-HCI pH 7.5 at 30 °C. Azide-modified iratumumab was purified from the reaction mixture on a HiTrap MabSelect SuRe 5 ml column (GE Healthcare) using an AKTA purifier-10 (GE Healthcare).
  • the eluted IgG was immediately neutralized with 1.5 M Tris-HCI pH 8.8 and dialyzed against PBS pH 7.4. Next the IgG was concentrated using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore) to a concentration of 25.6 mg/mL.
  • Mass spectral analysis of a fabricator-digested sample showed three peaks of the Fc/2-fragment belonging to one major product (observed mass 24332 Da, approximately 85% of total Fc/2 fragment), corresponding to core 6-N3-GalNAc-GlcNAc(Fuc)-substituted iratumumab, and two minor products (observed masses of 24187 and 24461 Da, approximately 5 and 10% of total Fc/2 fragment), corresponding to core 6-N 3 -GalNAc-GlcNAc-substituted iratumumab and core 6-N 3 -GalNAc-GlcNAc(Fuc)- substituted iratumumab with C-terminal lysine.
  • Chlorosulfonyl isocyanate (CSI; 0.91 mL, 1.48 g, 10 mmol) was added to a cooled (-78°C) solution of ieri-butanol (5.0 mL, 3.88 g, 52 mmol) in Et.20 (50 mL). The reaction mixture was allowed to warm to rt and was concentrated. The residue was suspended in DCM (200 mL) and subsequently Et3N (4.2 mL, 3.0 g, 30 mmol) and 2-(2-aminoethoxy)ethanol (1.0 mL, 1.05 g; 10 mmol) were added. The resulting mixture was stirred for 10 min and concentrated.
  • CSI Chlorosulfonyl isocyanate
  • Val-Cit-PABC-MMAE vc-PABC-MMAE; 13.9 mg; 0.01 1 mmol in DMF (400 ⁇ ) were added Et 3 N (3.4 ⁇ , 2.5 mg, 24.3 ⁇ ) and a solution of BCN-PEG4-OPNP (110, 3.0 mg, 5.6 ⁇ ) in DMF (200 ⁇ ). After 25 min, additional Et 3 N (1.1 ⁇ , 0.80 mg, 7.9 ⁇ ) and BCN- PEG4-OPNP (110, 2.2 mg, 4.1 ⁇ in DMF (33 ⁇ _)) were added.
  • Example 30 Conjugation of cACW with 100 to obtain cAC10-MMAE conjugate 53
  • a bioconjugate according to the invention was prepared by conjugation of compound 100 as linker-conjugate to azide-modified cAC10 as biomolecule.
  • cAC10-(6-N3-GalNAc)2 13d
  • PBS pH 7.4 133 ⁇ _
  • compound 100 27 ⁇ _, 10 mM solution in DMF.
  • the reaction was incubated at rt overnight followed by purification on a Superdex200 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare).
  • Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25844 Da, approximately 80% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment.
  • RP-HPLC analysis of the reduced sample indicated an average DAR of 1.88.
  • a bioconjugate according to the invention was prepared by conjugation of compound 108 as linker-conjugate to azide-modified cAC10 as biomolecule.
  • cAC10-(6-N3-GalNAc)2 13d
  • PBS pH 7.4 133 [it)
  • compound 108 27 ⁇ _, 10 mM solution in DMF.
  • the reaction was incubated at rt overnight followed by purification on a Superdex200 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare).
  • Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25928 Da, approximately 70% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment.
  • RP-HPLC analysis of the reduced sample indicated an average DAR of 1.85.
  • Example 32 Conjugation of cACW with 111 to obtain conjugate cAC10-MMAE 52
  • a bioconjugate according to the invention was prepared by conjugation of compound 111 as linker-conjugate to azide-modified cAC10 as biomolecule.
  • cAC10-(6-N3-GalNAc)2 13d
  • PBS pH 7.4 48.2 ⁇
  • compound 111 1 1 1.8 [it, 4 mM solution in DMF.
  • the reaction was incubated at rt overnight followed by purification on a Superdex200 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare).
  • Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25853 Da, approximately 80% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment.
  • RP-HPLC analysis of the reduced sample indicated an average DAR of 1.88.
  • Example 33 Conjugation of cACW with 67 to obtain conjugate cAC10-MMAD 55
  • a bioconjugate according to the invention was prepared by conjugation of compound 67 as linker- conjugate to azide-modified cAC10 as biomolecule.
  • cAC10-(6-N3-GalNAc)2 13d
  • PBS pH 7.4 57 ⁇
  • compound 67 33 [it, 10 mM solution in DMF.
  • the reaction was incubated at rt overnight followed by purification on a Superdex200 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare).
  • Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25896 Da, approximately 80% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment.
  • RP-HPLC analysis of the reduced sample indicated an average DAR of 1.88.
  • a bioconjugate according to the invention was prepared by conjugation of compound 66 as linker- conjugate to azide-modified cAC10 as biomolecule.
  • cAC10-(6-N3-GalNAc)2 13d
  • propylene glycol 1 1.909 mL
  • compound 66 410.6 ⁇ _, 40 mM solution in DMF
  • the reaction was incubated at rt for approximately 40 hrs.
  • the reaction mixture was dialyzed to PBS pH 7.4 and purified on a HiLoad 26/600 Superdex200 PG (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare).
  • a bioconjugate according to the invention was prepared by conjugation of compound 63 as linker- conjugate to azide-modified cAC10 as biomolecule.
  • cAC10-(6-N3-GalNAc)2 13d
  • PBS pH 7.4 1.0 mL
  • DMF 2.568 mL
  • compound 63 17. ⁇ , 40 mM solution in DMF.
  • the reaction was incubated at rt overnight followed by dialysis and purification on a HiLoad 26/600 Superdex200 PG (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare).
  • Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 27124 Da, approximately 80% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment.
  • RP-HPLC analysis of the reduced sample indicated an average DAR of 3.79.
  • Example 36 Conjugation of iratumumab with 10 to obtain iratumumab 59
  • a bioconjugate according to the invention was prepared by conjugation of compound 100 as linker-conjugate to azide-modified iratumumab as biomolecule.
  • iratumumab(6-N3- GalNAc) 2 189 ⁇ _, 4.8 mg, 25.6 mg/ml in PBS pH 7.4
  • PBS pH 7.4 51 ⁇ _
  • compound 100 80 ⁇ _, 4 mM solution in DMF.
  • the reaction was incubated at rt overnight followed by purification on a Superdex200 10/300 GL (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare).
  • Mass spectral analysis of the fabricator-digested sample showed one major product (observed mass 25853 Da, approximately 80% of total Fc/2 fragment), corresponding to the conjugated Fc/2 fragment.
  • RP-HPLC analysis of the reduced sample indicated an average DAR of 1.89.
  • tumour volume was in the range of 100-150 mm 3
  • groups of eight mice were injected i.v. with a single dose at day 1 of either vehicle (control), Adcetris (A, at 1 mg/kg) and 56 (at 1 mg/kg). Tumours were measured twice weekly for a period of 60 days. The results on tumour volume (mean) are depicted in Figure 8A.
  • tumour volume was in the range of 100-150 mm 3
  • groups of eight mice were injected i.v. with a single dose at day 1 of either vehicle (control), Adcetris (A, at 1 mg/kg), 53 (at 4 mg/kg), 55 (at 2 mg/kg), 55 (at 4 mg/kg), 57 (at 1 mg/kg), and 57 (at 2 mg/kg).
  • Tumours were measured twice weekly for a period of 30 days. The results on tumour volume (median) are depicted in Figure 8B.
  • CR female Wistar rats (2 females per group), 5-6-week-old at the beginning of the experimental phase, obtained from Charles River Laboratories, USA, were treated with 56 or 57 (at 40 mg/kg, 60 mg/kg, 70 mg/kg and 80 mg/kg), or with 52, 53 or 54 (at 80 mg/kg, 120 mg/kg, 140 mg/kg and 160 mg/kg) and compared to Adcetris (at 15 mg/kg, 20 mg/kg and 40 mg/kg).
  • the test items were administered via intravenous (bolus) injection using a microflex infusion set introduced into a tail vein (2 mL/kg at 1 mL/min). One group of animals was treated with vehicle (control). After dosing, all animals were maintained for a 12-day observation period.
  • Human serum (Sigma, H4522-100ml_) was incubated with protein A sepharose (1 mL sepharose/mL serum, commercially available from Repligen) for 1 hour at 4 °C to deplete for IgG.
  • the depleted serum was filter sterilized using a 0.22 ⁇ filter (Millipore), divided into aliquots, snapfrozen and stored at -20 °C until further use (multiple freeze-thawing cycles were avoided).
  • ADCs 56, 57 and Adcetris were added to a final concentration of 0.1 mg/mL and incubated at 37 °C. At pre-set time-point samples (0.5 mL) were taken and stored at -20 °C until further analysis.

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EP17703994.8A 2016-02-08 2017-02-08 Antikörperkonjugate mit verbessertem therapeutischem index zur abzielung auf cd30-tumoren und verfahren zur verbesserung des therapeutischen index von antikörperkonjugaten Pending EP3413916A1 (de)

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EP16154739 2016-02-08
EP16154712 2016-02-08
EP16173599 2016-06-08
JP2016155927A JP2017197512A (ja) 2016-02-08 2016-08-08 Ｃｄ３０腫瘍を標的化するための改善された治療指数を有する新規な抗体−コンジュゲート及び抗体−コンジュゲートの治療指数を改善するための方法
PCT/EP2017/052791 WO2017137458A1 (en) 2016-02-08 2017-02-08 Antibody-conjugates with improved therapeutic index for targeting cd30 tumours and method for improving therapeutic index of antibody-conjugates

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Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2759503T3 (es) 2013-05-02 2020-05-11 Glykos Finland Oy Conjugados de una glicoproteína o un glicano con una carga útil tóxica
JP2019533711A (ja) * 2016-11-14 2019-11-21 ミレニアム ファーマシューティカルズ， インコーポレイテッドＭｉｌｌｅｎｎｉｕｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 非成人のヒトに対する抗ｃｄ３０抗体−薬物コンジュゲートの投与
EP3939616A1 (de) 2017-02-08 2022-01-19 ADC Therapeutics SA Pyrrolobenzodiazepin-antikörper-konjugate
CN110075315B (zh) * 2018-01-26 2023-08-11 上海复旦张江生物医药股份有限公司 一种抗体偶联物及其制备方法和应用
WO2020232638A1 (zh) * 2019-05-21 2020-11-26 上海复旦张江生物医药股份有限公司 一种抗体偶联物及其药物组合物的应用
TW202140075A (zh) 2020-01-09 2021-11-01 美商梅爾莎納醫療公司 具有含肽之連接子的位置特異性抗體－藥物共軛體
NL2026947B1 (en) * 2020-11-20 2022-07-01 Synaffix Bv Tyrosine-based antibody conjugates
CN113234122A (zh) * 2021-05-07 2021-08-10 联宁（苏州）生物制药有限公司 一种用于抗体偶联药物的双臂中间体lnd1037的合成方法
WO2023180484A1 (en) * 2022-03-23 2023-09-28 Synaffix B.V. Antibody-conjugates for targeting of tumours expressing ptk7
WO2023180490A1 (en) * 2022-03-23 2023-09-28 Synaffix B.V. Antibody-conjugates for targeting of tumours expressing nectin-4
WO2023180489A1 (en) * 2022-03-23 2023-09-28 Synaffix B.V. Antibody-conjugates for targeting of tumours expressing carcinoembyronic antigen
EP4344707A1 (de) * 2022-09-29 2024-04-03 Emergence Therapeutics AG Neue anti-nectin-4-antikörper-wirkstoffkonjugate

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1132739B1 (de) 2000-05-16 2001-09-26 BioChip Technologies GmbH Linker-System zum Aktivieren von Oberflächen für die Biokonjugation und Verfahren zu ihrer Verwendung
US8431558B2 (en) 2004-11-01 2013-04-30 The Regents Of The University Of California Compositions and methods for modification of biomolecules
WO2007133855A2 (en) 2006-03-27 2007-11-22 University Of Maryland Biotechnology Institute Glycoprotein synthesis and remodeling by enzymatic transglycosylation
AU2007329793B2 (en) 2006-10-24 2013-01-10 Kereos, Inc. Improved linkers for anchoring targeting ligands
US8133515B2 (en) 2007-11-21 2012-03-13 University Of Georgia Research Foundation, Inc. Alkynes and methods of reacting alkynes with 1,3-dipole-functional compounds
WO2009102820A2 (en) 2008-02-11 2009-08-20 Government Of The U.S A., As Represented By The Secretary, Department Of Health And Human Services Modified sugar substrates and methods of use
US8519122B2 (en) 2010-02-12 2013-08-27 The Regents Of The University Of California Compositions and methods for modification of biomolecules
EP2563753B9 (de) 2010-04-27 2016-07-06 SynAffix B.V. Kondensierte Cyclooctylverbindungen und ihre Verwendung in metallfreien Click-Reaktionen
HUE062385T2 (hu) 2012-10-23 2023-10-28 Synaffix Bv Módosított antitest, antitest-konjugátum és eljárás ezek elõállítására
RU2015129800A (ru) 2012-12-21 2017-01-30 Биоэллаенс К. В. Гидрофильные саморазрушающиеся линкеры и их конъюгаты
ES2759503T3 (es) * 2013-05-02 2020-05-11 Glykos Finland Oy Conjugados de una glicoproteína o un glicano con una carga útil tóxica
CN105814213B (zh) * 2013-10-14 2021-05-04 西纳福克斯股份有限公司 经修饰的糖蛋白、蛋白质-缀合物及其制备方法
US9987373B2 (en) * 2013-10-14 2018-06-05 Synaffix B.V. Modified glycoprotein, protein-conjugate and process for the preparation thereof
WO2015057065A1 (en) * 2013-10-14 2015-04-23 Synaffix B.V. Glycoengineered antibody, antibody-conjugate and methods for their preparation
EP3929301A1 (de) * 2013-10-14 2021-12-29 SynAffix B.V. Durch glyco-engineering hergestellter antikörper, antikörperkonjugat und verfahren zu deren herstellung
WO2016022027A1 (en) 2014-08-04 2016-02-11 Synaffix B.V. Process for the modification of a glycoprotein using a βeta-(1,4)-n-acetylgalactosaminyltransferase or a mutant thereof
WO2016053107A1 (en) 2014-10-03 2016-04-07 Synaffix B.V. Sulfamide linker, conjugates thereof, and methods of preparation
EP3134520B1 (de) 2015-04-23 2017-12-20 Synaffix B.V. Verfahren zur modifikation eines glykoproteins mittels einer glykosyltransferase, die beta-(1,4)-n-acetylgalactosaminyltransferase ist oder davon abgeleitet ist

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