Sulfamide linkers for use in bioconjugates

#The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 7, 2018, is named 069818-4060_2018-08-07_Sequence_Listing.txt and is 29 KB.

#The present invention is in the field of bioconjugation. The invention relates to novel sulfamide linkers and conjugates thereof, and to methods for the preparation thereof. More particularly, the invention relates to linkers comprising an acylsulfamide group and/or a carbamoyl sulfamide group and to conjugates comprising said linkers. The invention further relates to a process for the preparation of bioconjugates comprising a linker, the linker comprising an acylsulfamide group and/or a carbamoyl sulfamide group.

#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”. Typically 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 (MOI) 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. For example, 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 (ONs) 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. For example, 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). Similarly, the preparation of oligonucleotide-based microarrays requires the selective immobilization of ONs on a suitable solid surface, e.g. glass.

#FIG. 1 describes the general concept of conjugation of biomolecules: a biomolecule of interest (BOI) containing one or more functional groups F1 is incubated with (excess of) a target molecule D (also referred to as molecule of interest or MOI) covalently attached to a reactive group Q1 via a specific linker. In the process of bioconjugation, a chemical reaction between F1 and Q1 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.

#FIG. 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 (11b), or an azidodifluoroacetyl group (11c) at the 2-position, or with an azido group at the 6-position of N-acetyl galactosamine (11 d).

#FIG. 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 β-glycosidic 1-4 linkage between a GalNAc derivative and GlcNAc (compounds 13a-d, respectively).

#The present invention relates to an improved linker that can be used in the field of bioconjugation. The sulfamide linker that has been developed by the inventors has been put to practice in the following aspects of the invention.

#In a first aspect, the invention concerns a linker-conjugate as defined herein, comprising the linker according to the invention, as well as the use of said linker-conjugate for preparing a bioconjugate according to the invention. In a second aspect, the invention concerns a linker-construct as defined herein, comprising the linker according to the invention, as well as the use of said linker-construct for preparing a linker-conjugate according to the invention and a process for preparing the linker-conjugate according to the invention using said linker-construct. In a third aspect, the invention concerns a bioconjugate as defined herein, comprising the linker according to the invention. In a fourth aspect, the invention concerns a process for preparing the bioconjugate according to the invention and the bioconjugate obtainable thereby. In a fifth aspect, the invention concerns the use of the linker according to the invention in bioconjugation for improving conjugation efficiency, reducing aggregation, increasing stability and increasing therapeutic index. In a sixth aspect, the invention concerns the medical use of the bioconjugate according to the invention, in particular for the treatment of cancer.

#Key to all of the above aspects to the invention is the linker according to the invention, which comprises a branching moiety BM in the main chain of the linker and a sulfamide group SG in the side chain of the linker. Herein, “main chain” refers to the chain of atoms that connects target molecule D (or Q1) with biomolecule B (or Q2). The branching moiety is located in the main chain. The “side chain” refers to the chain of atoms that connects branching moiety BM to capping group E, also referred to as the “branch”. The linker according to the invention is further defined below. The inventors surprisingly found that using a sulfamide group within the side chain (branch), in the configuration according to the present invention, provides many advantages when preparing and using bioconjugates such as antibody-drug-conjugates.