Glycosylation of variable immunoglobulin domains

Abstract

The present invention provides nucleotide sequences encoding polypeptides comprising immunoglobulin variable domains with engineered glycosylation acceptor sites. Specifically, the present invention provides immunoglobulin variable domain proteins modified with selected glycans, and specific glycan conjugates thereof. Also provided herein are methods for producing glycosylated immunoglobulin variable domains and glycan conjugates thereof.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present application relates to the field of glycosylation engineering, and more specifically to immunoglobulin domains and glycosylated derivatives thereof. In particular, the present invention provides nucleotide sequences encoding polypeptides comprising immunoglobulin variable domains with engineered glycosylation acceptor sites. Consequently, the present invention provides immunoglobulin variable domain proteins modified with selected glycans, and specific glycan conjugates thereof. Also provided herein are methods for the production of glycosylated immunoglobulin variable domains and glycan conjugates thereof. [Background technology]

[0002] 2. Background of the Invention The field of recombinant antibody technology has developed rapidly in the last two decades, mainly due to the interest in their therapeutic use in humans. The ability to select specific human antibodies by display techniques and improve their affinity, stability, and expression levels by molecular evolution has further boosted the field. Whole antibodies are composite molecules consisting of heavy and light chains. Although the heavy and light chains of isolated antibodies can retain antigen-binding specificity, their affinity and solubility are often reduced.

[0003] However, the paired N-terminal variable domains of the heavy (VH) and light (VL) chains are sufficient for antigen binding. Such antibody fragments can be produced as monovalent antibody fragments (Fab) or as single chain Fvs (scFv), in which the VH and VL domains are linked by a polypeptide linker. The accidental discovery that camelids produce functional antibodies without light chains (Hamers-Casterman et al (1993) Nature 363:446-448) shaped new thinking in the field, because it was subsequently shown that their single N-terminal domains (VHH, also called Nanobodies®) bind antigens without the need for domain pairing. These heavy chain-only antibodies also lack the CH1 domain, which in conventional antibodies is associated with the light chain and, to a lesser degree, interacts with the VH domain.

[0004] Later, such single domain antibodies were also identified in specific cartilaginous fish (Greenberg et al (1995) Nature 374:168-173). These are often designated together with VHH as immunoglobulin single variable domain antibodies (ISVD). ISVDs pose interesting therapeutic possibilities thanks to their small size, high stability, ease of modification by gene fusion, and good production levels in microorganisms. When Nanobodies® are produced in eukaryotic cells, about one tenth of them are glycosylated (see Functional Glycomics, June 11, 2009). However, glycosylation is generally avoided in the production of ISVDs, and therefore glycosylation acceptor sites are mutated, since the presence of glycans can introduce heterogeneity, which can also interfere with folding and antigen recognition. The small size of ISVDs is also a therapeutic disadvantage since they are rapidly cleared from the circulation when administered to a patient.

[0005] On the other hand, the small size of ISVDs offers the opportunity to couple them to half-life extension molecules or to specific drugs (e.g., formation of antibody-drug conjugates) or tracers. Various coupling methods have been described in the art (e.g., applied especially in the field of monoclonal antibody modification), which focus on conjugation via primary amine groups (lysine residues and the N-terminus) or via cysteine ​​by acylation or alkylation, respectively. However, the site control of conjugation is generally low and complete homogeneity is rarely obtained. Glycan-specific conjugation of monoclonal antibodies, as described by Synaffix BV (see, e.g., WO2014065661, WO2015057065, and WO2015057064), offers more uniformity, but this strategy suffers from the fact that the glycans must be prepared in vitro before they are suitable for further chemical coupling.

[0006] It would be desirable to identify specific sites in the ISVDs that can be modified with glycan structures that do not interfere with the binding or folding functions of these ISVDs. This would lead to efficient glycosylation when produced in a suitable production system and would result in a uniform, chemically coupling-ready glucan structure. In the prior art, modification of ISVDs with glycans has been shown to be useful for preventing binding to pre-existing anti-VH autoantibodies (see, for example, WO2016150845). However, no specific design strategy was used for the introduction of glycosylation sites, and their locations were chosen randomly, focusing mainly on the exposed C-terminal region of the ISVD. As a result, the impact of the presence of glycans on the specific properties of the ISVDs, as well as the efficiency of glycosylation, is currently unpredictable and must be evaluated against individual criteria. Summary of the Invention

[0007] SUMMARY OF THE PRESENT APPLICATION An important object of the present application is to provide polypeptides comprising immunoglobulin variable domains (IVDs), where the IVDs have glycosylation acceptor sites present in specific selected regions identified through rational design approaches. The presence of these specific glycosylation acceptor sites in specific regions in the IVDs allows for efficient glycosylation without interfering with the binding affinity of the IVDs with their ligands and without interfering with the folding of the IVDs. They can be recombinantly produced in suitable host cells containing glycans of uniform morphology at specific positions that can be further modified with various components as further described herein.

[0008] Thus, according to a first aspect, there is provided a nucleotide sequence encoding a polypeptide comprising an IVD, wherein the IVD comprises an amino acid sequence comprising four framework regions (FR) and three complementarity determining regions (CDRs) according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(1); or any suitable fragment thereof, wherein the IVD has a glycosylation acceptor site present at any of amino acids 83 to 88 and / or any of amino acids 27 to 40 of the IVD (according to the AHo numbering convention). In a specific aspect, the IVD is an immunoglobulin single variable domain.

[0009] The glycosylation acceptor site of the IVD may be an asparagine residue that can be N-glycosylated. Specifically, the glycosylation acceptor site of the IVD contains a NXT, NXS, NXC, or NXV motif (wherein X can be any amino acid except proline (P)), such that the asparagine residue of the NXT / NXS / NXC / NXV motif is present at any of positions 83-88 and / or any of positions 27-40 of the IVD (according to the AHo numbering convention). In certain embodiments, the IVD has additional glycosylation acceptor sites in the IVD, such as at positions 14 and / or 48 (according to the AHo numbering convention).

[0010] In another aspect, there is provided a polypeptide comprising an IVD encoded by a nucleotide sequence of the invention. According to other aspects, there is provided an expression vector comprising the nucleotide sequence, and a cell comprising the expression vector.

[0011] Recombinant cells may, according to specific embodiments, be higher eukaryotic cells, such as mammalian or plant cells, lower eukaryotic cells, such as filamentous fungal or yeast cells, or, under certain conditions, prokaryotic cells. Of particular relevance are glyco-engineered cells, in particular glyco-engineered lower eukaryotic cells.

[0012] More specifically, higher eukaryotic cells according to the present invention are vertebrate cells, especially mammalian cells, examples of which include, but are not limited to, CHO cells or HEK293 cells (e.g., HEK293S cells). Using these cells, IVDs modified with glycans at specific, rationally selected sites can be produced. Glycoengineered cells are particularly advantageous in that they are preferred for the production of IVDs modified with specifically desired glycans and / or uniform glycans. This uniform glycosylation profile is highly desirable as it results in a product whose properties are well-predictable.

[0013] Moreover, the above-described cells are useful for the production of IVDs directly modified with GlcNAc, LacNAc, or sialyl-LacNAc glycans that are favorable for conjugation in the cells. Moreover, the employment of these cells leads to IVDs with a uniform glycosylation profile. Thus, a specific advantage over conventional approaches is obtained from the high uniformity of the obtained products. This is in contrast to conventional approaches that typically require in vitro enzymatic treatment of heterogeneous glycans to provide GlcNAc, Gal, or Sia residues as starting points for further modification. Besides the high cost, in vitro enzymatic treatment may carry the risk of incomplete treatment and thus a heterogeneous product. Another conventional approach is based on the direct treatment of heterogeneously glycosylated proteins, resulting in a product that again lacks uniformity.

[0014] According to a particular embodiment, the polypeptide according to the invention comprises a glycosylated IVD. The glycosylation may according to a particular embodiment comprise one or more glycans with terminal GlcNAc, GalNAc, galactose, sialic acid, glucose, glucosamine, galactosamine, bacillosamine, mannose or mannose-6-P sugars, or chemically modified monosaccharides such as GalNAz, GlcNAz, or azido-sialic acid, present on one or more glycans.

[0015] According to another particular embodiment, the glycosylation consists of one or more glycans selected from the list consisting of GlcNAc, LacNAc (=GlcNAc-Gal), sialyl-LacNAc, Man5GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, complex glycans, hybrid glycans and GlcNAz, GlcNAc-GalNAz and LacNAc-azido-sialic acid (for carbohydrate nomenclature see Alan D. McNaught (1996) Pure & Appl. Chem. Vol. 68, No 10, 1919-2008). IVDs modified at certain positions with the above-mentioned glycans are particularly useful for glycan-specific conjugation. In particular, a glycosylation profile consisting of GlcNAc, LacNAc or sialyl-LacNAc is advantageous for site-specific conjugation.

[0016] In a particular embodiment, an IVD conjugate is provided, comprising a polypeptide according to the present invention and a conjugated moiety conjugated to a glycan. An IVD modified with glycans at a rationally selected position is an ideal starting point for glycan-based conjugation. Linking a moiety to the glycan present on the IVD, for example, allows the production of an IVD conjugate, in which the ratio of the IVD and the conjugated moiety is well-defined.

[0017] Even more advantageous is an IVD modified with a uniform glycan, which allows particularly efficient conjugation. Conjugation can be performed either chemically (e.g., using periodate oxidation of glycan components, followed by conjugation via methods known in the art (e.g., oxime ligation, hydrazone ligation, etc.) or via reductive amination) or enzymatically (e.g., using galactose oxidase to oxidize galactose, followed by conjugation via oxime ligation, hydrazone ligation, or via reductive amination). Alternatively, tagged glycan residues may be incorporated to allow for subsequent conjugation reactions (e.g., incorporation of GalNAz into the glycan chain, using mutant galactosyltransferase, followed by conjugation via click chemistry).

[0018] The conjugated moiety may include a half-life extending moiety, a therapeutic agent, a detection unit, or a targeting moiety. The opportunities of using glycans on an IVD according to the present invention as a bio-orthogonal handle for conjugation to drugs, tracers, etc. via glycan conjugation methodologies are not limited to the examples described herein.

[0019] Also provided in the present application are methods for producing polypeptides comprising an IVD and IVD-conjugates as described herein. In particular, a method is provided for producing a polypeptide comprising an IVD according to the invention in a suitable cell, comprising the following steps: - Providing suitable cells - introducing into said cell an expression vector comprising a nucleotide sequence encoding a polypeptide according to the invention - expressing a polypeptide comprising the IVD under suitable conditions; and - isolating a polypeptide comprising an IVD The method may further comprise the step of linking the conjugated moiety to the polypeptide.

[0020] The present invention also relates to a composition comprising a polypeptide comprising an IVD or a conjugate thereof.Preferably, the composition is a pharmaceutical composition.Even more preferably, the composition further comprises at least one pharma- ceutically acceptable carrier, diluent, excipient, or adjuvant, and optionally comprises one or more polypeptides of the present invention. [Brief description of the drawings]

[0021] Brief description of the figure [Figure 1] Figure 1: Tertiary structure (left) and secondary structure topology (right) of a representative ISVD, here a nanobody (chain B from PDB entry id: 3K74). Several virtual regions were determined for N-linked glycosylation sequon introduction (9 regions outlined in black in the right panel). [Diagram 2] Figure 2: Ten specific sites (indicated with an X) selected for introduction of N-linked glycosylation sequons in the reference nanobody PDB id: 3K74 (numbering in the figure refers to the aHo numbering scheme; for alternative (Kabat) numbering see Figure 9).

[0022] [Diagram 3] Figure 3: Amino acid alignment of nanobody GBP with nanobody 3K74. [Figure 4] Figure 4: Specific sites selected for the introduction of N-linked glycosylation signatures in GBP nanobodies are depicted (numbering in the figure refers to the aHo numbering scheme; for alternative (Kabat) numbering see Figures 9 or 11). N-glycosylation efficiency was estimated based on His-specific Western blot or mass spectrometry data. [Diagram 5]Figure 5: Coomassie and anti-HIS Western blots (AHo numbering) of nanobody GBP and the GBP-R86N variant expressed in Pichia pastoris wild-type (WT), GlycoSwitchM5 (GSM5), and GlycoDelete (GD) strains.

[0023] [Figure 6] Figure 6: Coomassie and anti-HIS Western blot (or equivalent terminology His6-specific Western blot) analysis of N-glycotag variants of GBP expressed in Pichia pastoris wild-type (WT) and GlycoSwitchM5 (GSM5). Samples were either mock-treated or digested with PNGaseF to remove N-glycans. [Figure 7] Figure 7: Coomassie and anti-HIS WB analysis of nine "glycovariants" of GBP expressed in Pichia GlycoSwitchM5 (GSM5). Samples were either mock treated or digested with PNGaseF to remove N-glycans. White boxes indicate the increase in molecular weight due to the addition of N- (8 amino acids) or C- (22 amino acids) terminal glycan tag amino acid sequences.

[0024] [Figure 8] Figure 8: ESI-QTOF MS analysis of nanobody GBP-R86N produced in Pichia pastoris GlycoDelete (GD) strain. The N-glycosylated variant is detected at 13699 Da, whereas the non-glycosylated fraction is detected at 13496 Da. [Figure 9] Figure 9: Sequence alignment of nanobodies Nb 41, F-VHH-4, and F-VHH-L66 with nanobodies GBP and 3K74. [Figure 10]Figure 10: Coomassie and anti-HIS WB analysis of Nb41 and Nb41-K86N variants expressed in Pichia pastoris wild type (WT), GlycoSwitchM5 (GSM5), and GlycoDelete (GD). Samples were either mock treated or digested with PNGaseF to remove N-glycans.

[0025] [Figure 11] Figure 11: Overview of nanobody GBP glycosylation sites. [Figure 12] Figure 12: Melting curves of GBP glycan variants. Nanobodies produced in Pichia pastoris GlycoSwitchM5 (GSM5) (Man5GlcNAc2 glycan), purified via IMAC and SEC, and eluted in either phosphate buffered saline (PBS, top) or HEPES buffered saline (HBS, bottom). Thermal shift assay using SYPRO Orange dye in a qPCR machine. [Figure 13] Figure 13: GBP N-glycosylation variants (except GBP-P48N-K50T) bind to their antigen GFP with similar characteristics as unmodified GBP-WT. Nanobodies were produced in Pichia pastoris GlycoSwitchM5 (GSM5, Man5GlcNAc2 glycan), purified via IMAC and SEC, and eluted in either phosphate-buffered saline (PBS) or HEPES-buffered saline (HBS). Biolayer interferometry (BLI) was performed with 100 nM biotinylated Avitag-GFP immobilized on a streptavidin chip. Affinity for two-fold serial dilutions of GBP glycan variants (8–0.125 nM) was measured at 25 °C in a 96-well format. Summary affinity parameters are shown above and were calculated by ForteBio Data Analysis 9.0 (error bars indicate SEM).

[0026] [Figure 14] Figure 14: Neutralization assay for RSV-specific VHH, F-VHH-L66. F-VHH-L66 was produced in both wild-type (wt) Pichia pastoris and GlycoSwitchM5 (GSM5) and Glycodelete (GD) strains. [Figure 15] Figure 15: Partial sequence alignment of nanobody GBP with glycan variants in region 27–40 (top row) and region 83–88 (bottom row).

[0027] [Figure 16] Figure 16: N-glycans can be introduced at virtually any position in two selected regions in nanobody GBP, but with variable site occupancy. GBP glycovariants were expressed in Pichia pastoris GlycoSwitchM5 (GSM5). Supernatants of all recombinant variants were harvested, treated with PNGaseF or mock treated, and assayed for the presence of N-glycosylation on 14-20% gradient SDS-PAGE, followed by Bio-Rad TGX stain-free detection (top) and His-tag specific Western blot analysis (bottom). Supernatant samples of wild-type GBP, glycan variants in each selected region (GBP-P30A-V31N-R33T in region 27-40, and GBP-D84N-A85T in region 83-88), and glycan variants containing an artificial insert (GBP-33-39-longinsert) were analyzed by intact protein mass spectrometry (bottom right). The mass of the latter three was confirmed to be 1216 Da, with Man5GlcNAc2 glycan modifications present as shown diagrammatically below. Normalized spectra are shown in normalized length (NL), 2.27E3 for GBP-WT, 4.29E3 for GBP-D83N-A85T, 7.64E2 for GBP-P30T-V31N-R33T, and 2.14E3 for GBP-33-39-longinsert. SS: single disulfide bond, pyroQ: N-terminal pyroglutamine residue, M5: Man5GlcNAc2 glycan modification.

[0028] [Figure 17] FIG. 17: Schematic diagram of LacNAc-based periodate oxidation followed by oxime ligation. [Figure 18] FIG. 18: Schematic diagram of sialyl-LacNAc-based periodate oxidation followed by oxime ligation. [Figure 19] FIG. 19: Schematic schematic of GAO-based LacNAc oxidation followed by oxime ligation. [Figure 20] FIG. 20: Schematic diagram of GAO-F2-based GlcNAc oxidation followed by oxime ligation.

[0029] [Figure 21] FIG. 21: Schematic scheme for the chemo-enzymatic coupling of an azide-modified form of GalNAc (GalNAz) to a single GlcNAc N-glycan, followed by click chemistry reaction of the azide with a strained alkyne. [Figure 22] FIG. 22: Schematic scheme for the chemoenzymatic coupling of an azide-modified form of Sia (AzSia) to LacNAc N-glycans followed by click chemistry reaction of the azide with a strained alkyne. [Figure 23] Figure 23: Coomassie and anti-HIS Western blot analysis of nanobody F-VHH-4 variants (upper panel) and F-VHH-L66 variants (lower panel) produced in Pichia pastoris wild type (NRRLY11430), GlycoSwitchM5 strain, and GlycoDelete strain. For Coomassie analysis, supernatants of three different clones were tested for each nanobody-Pichia strain combination; for Western blot analysis, supernatants of one representative clone were analyzed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description The present invention will be described with respect to specific embodiments and with reference to certain drawings, but the present invention is not limited thereto, but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. The drawings described are schematic only and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. When the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. When an indefinite or definite item is used in reference to a singular noun, for example "a" or "an", "the", this includes the plural of that noun unless specifically stated otherwise.

[0031] Furthermore, the terms first, second, third, etc. in the description and claims are used to distinguish between like elements and are not necessarily used to describe an order or chronology. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the aspects of the invention described herein are capable of operating in orders other than those described or illustrated herein.

[0032] The following terms or definitions are provided solely to aid in the understanding of the present invention. Unless otherwise defined herein, all terms used herein have the same meaning to those skilled in the art of the present invention. Practitioners are specifically directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016) for definitions and terms in the art. The definitions provided herein should not be construed to have a scope less than that understood by those skilled in the art.

[0033] As used herein, the term "nucleotide sequence" refers to any polymeric form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length. Nucleotide sequences may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting examples of nucleotide sequences include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleotide sequences may be linear or circular.

[0034] As used herein, the term "polypeptide" refers to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Polypeptide sequences can be depicted with just the single letter (or one-letter) amino acid code or the three letter amino acid code as depicted herein below:

[0035] [Table 1]

[0036] The term "immunoglobulin domain" as used herein refers to a globular region of an antibody chain (such as, for example, a chain of a conventional four-chain antibody or a chain of a heavy-chain antibody) or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by retaining the immunoglobulin fold characteristic of antibody molecules, consisting of a two-layer sandwich of about seven antiparallel beta strands arranged in two beta sheets and optionally stabilized by conserved disulfide bonds.

[0037] The term "immunoglobulin variable domain", as used herein, refers to an immunoglobulin domain consisting essentially of four "framework regions", referred to in the art and herein below as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", interrupted by three "complementarity determining regions" or "CDRs", referred to in the art and herein below as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Thus, the general structure or sequence of an immunoglobulin variable domain can be shown as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain(s) that confers specificity for an antigen to the antibody by carrying the antigen binding site.

[0038] The term "immunoglobulin single variable domain" (abbreviated as "ISVD") is equivalent to the term "single variable domain" and defines a molecule in which an antigen-binding site resides on and is formed by a single immunoglobulin domain. Apart from "conventional" immunoglobulins or fragments thereof, this defines an immunoglobulin single variable domain in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen-binding site. That is, a total of six CDRs will be involved in forming the antigen-binding site.

[0039] In view of the above definition, the antigen-binding domain of a conventional four-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, an F(ab')2 fragment, an Fv fragment (such as a disulfide-linked Fv or scFv fragment), or of a bispecific antibody derived from such a conventional four-chain antibody (all known in the art) would not normally be considered as an immunoglobulin single variable domain, since in these cases binding to the respective epitope of an antigen is usually not effected by one (single) immunoglobulin domain, but by a pair of (associated) immunoglobulin domains (such as a light and heavy chain variable domain), i.e. a VH-VL pair of immunoglobulin domains (which bind jointly to the respective epitope of the antigen).

[0040] In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain. Thus, the antigen-binding site of an immunoglobulin single variable domain is formed by only three CDRs.

[0041] Thus, a single variable domain may be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, so long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that consists essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0042] In one embodiment of the invention, the immunoglobulin single variable domain is a heavy chain variable domain sequence (e.g., a VH sequence); more specifically, the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a traditional four-chain antibody, or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0043] For example, an immunoglobulin single variable domain may be a (single) domain antibody (or a suitable amino acid sequence for use as a (single) domain antibody), a "dAb" or dAb (or a suitable amino acid sequence for use as a dAb), or a Nanobody (as defined herein, including but not limited to VHHs); other single variable domains, or any suitable fragment of any one of these.

[0044] In particular, the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx NV] For a general description of Nanobodies, see further below as well as the prior art cited herein (such as, for example, the description in WO 08 / 020079 (page 16)).

[0045] "VHH domain" also refers to VHH, V H Also known as H domains, VHH antibody fragments, and VHH antibodies, were first described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., of "antibodies lacking light chains"; Hamers-Casterman et al (1993) Nature 363: 446-448). The term "VHH domain" refers to these variable domains as distinct from the heavy chain variable domains present in conventional four-chain antibodies (which are referred to herein as "V H The light chain variable domain present in conventional four-chain antibodies (referred to herein as a "VH domain") is LThe VL domain was chosen to distinguish it from the VL domains (also referred to as "VL domains" or "VL domains").

[0046] For further description of VHHs and Nanobodies, see the review by Muyldermans (review in Molecular Biotechnology 74: 277-302, 2001) as well as the following patent applications which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079, and WO 96 / 34103 by Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231, and WO 02 / 48193 by Unilever; WO WO 03 / 050531 by Algonomics NV and Ablynx NV; WO 01 / 90190 by the National Research Council of Canada; WO 03 / 025020 (=EP 1433793) by the Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO See WO 06 / 079372, WO 06 / 122786, WO 06 / 122787, and WO 06 / 122825, as well as further published patent applications by Ablynx NV.

[0047] See also the further prior art mentioned in these applications, in particular the list of references mentioned on pages 41-43 of International Application WO 06 / 040153. These lists and references are incorporated herein by reference. As described in these references, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) may be characterized, in particular, by the presence of one or more "Hallmark residues" in one or more of the framework sequences. Further description of Nanobodies, including humanization and / or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives, or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences), and various modifications that increase the half-life of Nanobodies and their preparations, can be found, for example, in WO 08 / 101985 and WO 08 / 142164. For a further general description of Nanobodies, see the prior art cited herein (eg, the description at page 16 of WO 08 / 020079).

[0048] "Domain antibodies", also known as "Dabs", "Domain Antibodies" and "dAbs" (the terms "Domain Antibodies" and "dAbs" are used as trademarks by the GlaxoSmithKline family of companies), are described, for example, in EP 0368684, Ward et al. (Nature 341: 544-546, 1989), Holt et al. (Tends in Biotechnology 21: 484-490, 2003) and WO 03 / 002609, as well as, for example, WO 04 / 068820, WO 06 / 030220, WO 06 / 003388 and other published patent applications by Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammalian, especially human, four-chain antibodies. To bind to an epitope as a single antigen-binding domain, i.e. without pairing with a VL or VH domain, specific selection for such antigen-binding properties is required, e.g. by using a library of human single VH or VL domain sequences. Domain antibodies, like VHHs, have a molecular weight of approximately 13 kDa to approximately 16 kDa and, if derived from fully human sequences, do not require humanization, e.g. for therapeutic use in humans.

[0049] It should also be noted that some single variable domains may be derived from certain sharks (eg the so-called "IgNAR domains", see, for example, WO 05 / 18629).

[0050] Thus, within the meaning of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from non-human sources, preferably from camelids, preferably camelid heavy chain antibodies. These may be humanized as described above. Moreover, the term includes polypeptides derived from non-camelid sources, for example from mice or humans, that have been "camelized", as described, for example, by Davies and Riechmann (FEBS 339: 285-290, 1994; Biotechnol. 13: 475-479, 1995; Prot. Eng. 9: 531-537, 1996) and by Riechmann and Muyldermans (J. Immunol. Methods 231: 25-38, 1999).

[0051] For the numbering of the amino acid residues of the IVD, various numbering schemes may be applied. For example, the numbering may be performed according to the AHo numbering scheme for all heavy chain variable domains (VH) and light chain variable domains (VL) given by Honegger, A. and Plueckthun, A. (J.Mol.Biol. 309, 2001) as applied to VHH domains from camelids. Alternative methods for numbering the amino acid residues of VH domains, which may also be applied in a similar manner to VHH domains, are known in the art. For example, the delineation of the FR and CDR sequences may be done by using the Kabat numbering system as applied to VHH domains from camelids in the article by Riechmann, L. and Muyldermans, S., 231(1-2), J Immunol Methods. 1999. The determination of the CDR regions may also be done according to various methods.

[0052] In the CDR determination according to Kabat, FR1 of the VHH comprises amino acid residues at positions 1-30, CDR1 of the VHH comprises amino acid residues at positions 31-35, FR2 of the VHH comprises amino acids at positions 36-49, CDR2 of the VHH comprises amino acid residues at positions 50-65, FR3 of the VHH comprises amino acid residues at positions 66-94, CDR3 of the VHH comprises amino acid residues at positions 95-102 and FR4 of the VHH comprises amino acid residues at positions 103-113. An overview of the two different numbering schemes applied to the particular nanobodies used in the examples section is depicted in Figures 9 or 11. However, in the present description and claims, the numbering according to AHo as described above will be followed.

[0053] -V H It should be noted that for domains and for VHH domains, as is well known in the art - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by Kabat or AHo numbering (i.e. one or more positions according to Kabat or AHo numbering may not be occupied in the actual sequence or the actual sequence may contain more amino acid residues than the number considered by Kabat or AHo numbering). This means that in general the numbering according to Kabat or AHo may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in VH and VHH domains will most likely be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0054] Immunoglobulin single variable domains, such as domain antibodies and Nanobodies (including VHH domains), may be subjected to humanization. In particular, humanized immunoglobulin single variable domains, such as Nanobodies (including VHH domains), may be immunoglobulin single variable domains as generally defined in the previous paragraph, but in which there is at least one amino acid residue (in particular at least one framework residue) which is and / or corresponds to a humanizing substitution (as defined herein).

[0055] Potentially useful humanization substitutions include the naturally occurring V HH The sequences of the framework regions of the sequences are compared with one or more closely related human V H By comparing the V sequence with the corresponding framework sequence, one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can then be identified. HH sequence (in any manner known per se, as further described herein), resulting in a humanized V HH The sequences can be tested for affinity to the target, for stability, for ease and level of expression, and / or for other desired properties. Thus, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled artisan based on the present disclosure herein. Also based on the previous description, immunoglobulin single variable domains such as Nanobodies (including VHH domains) (among the framework regions) can be partially humanized or fully humanized.

[0056] Immunoglobulin single variable domains such as domain antibodies and Nanobodies (including VHH domains and humanized VHH domains) can also be subjected to affinity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs, which alterations result in an improved affinity of the resulting immunoglobulin single variable domain for its respective antigen compared to the respective parent molecule. Affinity matured immunoglobulin single variable domain molecules of the invention can be prepared by the methods described, for example, by Marks et al. (Biotechnology 10:779-783, 1992), Barbas, et al. (Proc. Nat. Acad. Sci, USA 91: 3809-3813, 1994), Shier et al. (Gene 169: 147-155, 1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J. Immunol. 154: 3310-9, 1995), Hawkins et al. (J. MoI. Biol. 226: 889 896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1999), and others. They may be prepared by methods known in the art, such as those described by John D. Wilson, J. Clin. Pharmacol. 2001, 14:1311-1323 (1996).

[0057] The process of designing / selecting and / or preparing a polypeptide starting from an immunoglobulin single variable domain (such as a domain antibody or Nanobody) is also referred to herein as "formatting" said immunoglobulin single variable domain; an immunoglobulin single variable domain that constitutes part of a polypeptide is said to be "formatted" or to be "in the format" of said polypeptide. Examples of ways in which immunoglobulin single variable domains can be formatted and examples of such formats will be clear to the skilled person based on the present disclosure herein; such formatted immunoglobulin single variable domains form a further aspect of the present invention.

[0058] The term "glycosylation acceptor site" refers to a position within the IVD that can be N- or O-glycosylated. N-linked glycans are typically attached to asparagine (Asn), while O-linked glycans are generally linked to the hydroxyl oxygen of a serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chain.

[0059] The "NXT", "NXS", "NXC" or "NXV" motif refers to the consensus sequence Asn-Xaa-Thr / Ser or Asn-Xaa-Cys / Val, where Xaa can be any amino acid except proline (Shrimal, S. and Gilmore, R., J Cell Sci. 126(23), 2013, Sun, S. and Zhang, H., Anal. Chem. 87 (24), 2015). It is well known in the art that potential N-glycosylation acceptor sites are specific for the consensus sequence Asn-Xaa-Thr / Ser or Asn-Xaa-Cys / Val. It has been shown in the art that the presence of proline between Asn and Thr / Ser leads to inefficient N-glycosylation. In a specific aspect, the N-linked glycosylation acceptor site of an IVD or ISVD according to the invention is extended with an aromatic residue, such as a natural or modified aromatic amino acid residue, such as phenylalanine (F), tyrosine (Y), histidine (H) or tryptophan (W).

[0060] Such modifications are described, namely, in Price, JL et al., Biopolymers. 98(3), 2012, and in Murray, AN et al., Chem Biol. 22(8), 2015. In more specific embodiments, the aromatic residue is located at position -1 (F / Y / H / WNxT / S), -2 (F / Y / H / W-x1-NxT / S), or -3 (F / Y / H / W-x2-x1-NxT / S) relative to an asparagine (N) residue in the N-linked glycosylation sequon (NxT / NxS) (Murray AN et al (2015) Chem. Biol. 22(8):1052-62 and Price JL et al (2012) Biopolymers 98(3):195-211). Such modifications are particularly useful for increasing glycosylation efficiency of N-glycosylation acceptor sites, glycan uniformity, and glycoprotein stability.

[0061] The term "expression vector" as used herein includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenovirus AAV or baculovirus vectors), or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC)). Expression vectors generally contain the desired coding sequence and an appropriate promoter sequence required for expression of the operably linked coding sequence in a specific host organism (e.g., higher eukaryotes, lower eukaryotes, prokaryotes).

[0062] Typically, a vector comprises a nucleotide sequence in which an expressible promoter or regulatory nucleotide sequence is operatively linked to or associated with a nucleotide sequence or DNA region encoding an mRNA such that the regulatory nucleotide sequence can also regulate the transcription or expression of the associated nucleotide sequence. Typically, the regulatory nucleotide sequence or promoter of a vector is not operatively linked to the associated nucleotide sequence as it is found in nature (and thus is heterologous to the coding sequence of the operably linked DNA region).

[0063] The term "operably" or "operably" "linked" as used herein refers to a functional link between an expressible promoter sequence and a DNA region or gene of interest, such that the promoter sequence can initiate transcription of the gene of interest, and the term refers to a functional link between the gene of interest and a transcription termination sequence to ensure proper termination of transcription in eukaryotic cells. An "inducible promoter" refers to a promoter that can be switched "on" or "off" (thereby regulating gene transcription) in response to an external stimulus, such as, but not limited to, temperature, pH, certain nutrients, specific cell signals, and the like. The inducible promoter is used to distinguish it from a "constitutive promoter", which is intended to mean a promoter that is constantly switched "on", i.e., from the constitutive promoter, gene transcription is constitutively active.

[0064] "Glycan" as used herein generally refers to glycosidically linked monosaccharides, oligosaccharides, and polysaccharides. Thus, the carbohydrate moiety of a glycoconjugate, such as a glycoprotein, a glycolipid, or a proteoglycan, is referred to herein as a "glycan." Glycans can be homo- or heteropolymers of monosaccharide residues and can be linear or branched. N-linked glycans can be composed of GalNAc, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides, as also further exemplified herein.

[0065] In eukaryotes, O-linked glycans are assembled one sugar at a time on serine or threonine residues of the peptide chain in the Golgi apparatus. Unlike N-linked glycans, there is no known consensus sequence, but proline residues at either -1 or +3 positions are favored for O-linked glycosylation compared to serine or threonine.

[0066] "Complex N-glycan" as used herein typically refers to a structure having one, two or more (eg, up to six) outer branches, which are most often linked to the inner core structure Man3GlcNAc2. The term "complex N-glycan" is well known to those skilled in the art and has been defined in the literature. Illustratively, complex N-glycans may have at least one branch of GlcNAc or at least two alternating branches, and may also optionally have galactose (Gal) residues that may terminate in various oligosaccharides (but typically will not terminate with a mannose residue). For simplicity, a single GlcNAc, LacNAc, sialyl-LacNAc, or azide-modified versions thereof (and thus lacking the inner core structure Man3GlcNAc2) present on an N-glycosylation site of a glycoprotein is not considered a complex N-glycan.

[0067] "Hypermannosylglycan" refers to an N-glycan that contains more than nine mannose residues. Typically, such hypermannosylglycans are produced in lower eukaryotic cells such as yeast cells, specifically wild-type yeast cells such as wild-type Pichia pastoris. The N-glycans produced in yeast cells such as Pichia pastoris can also be modified mannose-6-phosphate.

[0068] "Higher eukaryotic cells" as used herein refers to eukaryotic cells that are not cells from unicellular organisms. In other words, higher eukaryotic cells are cells from (or derived from, in the case of cell culture) multicellular eukaryotic organisms, such as human cell lines or other mammalian cell lines (e.g., CHO cell lines). Typically, higher eukaryotic cells will not be fungal cells. Specifically, the term generally refers to mammalian cells, human cell lines, and insect cell lines.

[0069] More specifically, the term refers to a vertebrate cell, and even more specifically to a mammalian cell or a human cell. Higher eukaryotic cells, as described herein, will typically be part of a cell culture (e.g., a cell line, such as a HEK cell line or a CHO cell line), although this is not strictly required (e.g., in the case of plant cells, the plant itself may be used to produce recombinant proteins).

[0070] By "lower eukaryotic cells" is intended filamentous fungal cells or yeast cells. Yeast cells may form the species Saccharomyces (e.g., Saccharomyces cerevisiae), Hansenula (e.g., Hansenula polymorpha), Arxula (e.g., Arxula adeninivorans), Yarrowia (e.g., Yarrowia lipolytica), Kluyveromyces (e.g., Kluyveromyces lactis), or Komagataella phaffii (Kurtzman, CP (2009) J Ind Microbiol Biotechnol. 36(11)), although Komagataella phaffii was previously named, and under the old nomenclature Pichia pastoris is better known and is further used herein. According to a particular embodiment, the lower eukaryotic cells are Pichia cells, and in the most particular embodiment, Pichia pastoris cells. In certain embodiments, the filamentous fungal cell is Myceliopthora thermophila (also known as C1 by Dyadic), Aspergillus species (e.g., Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus japonicus), Fusarium species (e.g., Fusarium venenatum), Hypocrea species, and Trichoderma species (e.g., Trichoderma reesei).

[0071] "Prokaryotic cells" typically refer to non-pathogenic prokaryotic organisms, such as bacterial cells, for example, E. coli, Lactococcus species, and Bacillus species.

[0072] According to a specific embodiment, the cell of the present invention is a glycoengineered cell. "Glycoengineered cell" refers to a cell genetically modified to express a protein with an altered N-glycan and / or O-glycan structure compared to a wild-type background. Typically, the naturally occurring modifications on glycoproteins are altered by genetic engineering of enzymes involved in the glycosylation pathway. In general, sugar chains in N-linked glycosylation can be divided into three types: high-mannose (typically yeast), complex (typically mammalian) and hybrid glycosylation. Besides, there are various O-glycan patterns, e.g., yeast oligomannosyl glycans, which are different from mucin-type O-glycosylation in mammalian cells.

[0073] All the different types of N- and O-glycosylation are well known to the skilled artisan and defined in the literature. Considerable efforts are directed towards the identification and optimization of strategies for producing glycoproteins with desired N- and / or O-glycosylation patterns and modifying eukaryotic cells known in the art (see, for example, De Pourcq, K. et al., Appl Microbiol Biotechnol. 87(5), 2010). A non-limiting example of such a glycoengineered expression system is described in patent application WO2010015722 and relates to a eukaryotic cell (higher or lower) expressing both an endoglucosaminidase and a target protein, where the recombinant secreted target protein is characterized by a uniform N-glycosylation pattern (in particular one single GlcNAc residue (in lower eukaryotes) or modifications thereof, such as galactose modified with GlcNAc (LacNAc), or sialyl-LacNAc (in mammalian cells).

[0074] Also included are cells that have been genetically engineered to express proteins or glycoproteins with human-like or humanized glycosylation patterns (i.e., complex glycoproteins). This can be accomplished by providing cells, especially lower eukaryotic cells, with inactivated endogenous glycosylation enzymes and / or containing at least one other exogenous nucleic acid sequence encoding at least one enzyme required for complex glycosylation. Endogenous glycosylation enzymes that can be inactivated include alpha-1,6-mannosyltransferase Och1p, Alg3p, alpha-1,3-mannosyltransferases of the Mnn1p family, beta-1,2-mannosyltransferase.

[0075] Enzymes required for complex glycosylation include, but are not limited to: N-acetylglucosaminyltransferase I, N-acetylglucosaminyltransferase II, mannosidase II, galactosyltransferase, fucosyltransferase and sialyltransferase, and enzymes involved in donor sugar nucleotide synthesis or transport. Still other glycoengineered cells, particularly yeast cells, contemplated herein are characterized in that at least one enzyme involved in the production of high mannose structures (high mannose type glycans) is not expressed. The enzyme involved in the production of high mannose structures is typically a mannosyltransferase.

[0076] In particular, alpha-1,6-mannosyltransferase Och1p, Alg3p, alpha-1,3-mannosyltransferase of the Mnn1p family, beta-1,2-mannosyltransferase may not be expressed. Thus, the cell may additionally or alternatively be modified to express one or more enzymes or enzyme activities that allow high yield production of specific N-glycan structures. Such enzymes may be targeted to host cell organelles, where they will have optimal activity, for example, with a signal peptide that is not normally associated with the enzyme. It should be clear that the enzymes and their activities described herein are well known in the art.

[0077] Specifically contemplated herein as "glyco-engineered cells" according to the present invention are cells as described in WO2010015722 and WO2015032899 (further designated herein as GlycoDelete cells, or cells with a GlycoDelete background). Briefly, such cells have been engineered to reduce glycosylation heterogeneity and comprise an expression vector comprising at least a nucleotide sequence encoding an endoglucosaminidase enzyme and a nucleotide sequence encoding a target polypeptide.

[0078] Where heterogeneity in glycosylation originates not only from N-linked sugars but also from O-glycans attached to glycoproteins, it may be desirable to remove these diverse carbohydrate chains from the polypeptides of the invention. This can be achieved by expressing an endoglucosaminidase enzyme in cells with reduced expression and / or activity of endogenous UDP-galactose 4-epimerase (GalE) as described in WO2017005925. The cells described in the latter application are also specifically envisaged as glycomodified cells according to the invention, further described herein as GlycoDoubleDelete cells or cells with a GlycoDoubleDelete background.

[0079] Also specifically referred to herein as "glycoengineered cells" are non-mammalian cells that have been engineered to mimic the human N-glycosylation pathway (i.e., GlycoSwitch®; see also Laukens, B. et al (2015) Methods Mol Biol. 1321 and Jacobs, PP et al. (2009) Nat Protoc. 4(1)).

[0080] An "IVD conjugate" or "ISVD conjugate" is referred to herein as a polypeptide comprising an IVD or ISVD of the invention that is coupled (or conjugated or connected; these are equivalent terms in the art) to a specific moiety, and is further defined herein as a "conjugated moiety." Coupling between the IVD conjugate and the ISVD conjugate can occur via a specific amino acid (e.g., lysine, cysteine) present in the IVD or ISVD. Preferably, coupling occurs via an introduced glycan (e.g., an introduced N-glycan) present in the polypeptide sequence of the IVD or ISVD.

[0081] Glycan-specific conjugation can be performed with glycans present at the introduced glycan site of the IVD or ISVD. In certain cases, the glycan can be further modified in vitro (e.g., trimmed with a specific exoglycosidase enzyme) before coupling to the "conjugated moiety". In addition, coupling can also occur as i) a combination between a specific amino acid present in the IVD or ISVD and the conjugated moiety, and ii) a coupling via an introduced glycan and the conjugated moiety. Conjugation can be performed by any method described in the art, and some non-limiting illustrative embodiments will be outlined in the Examples section.

[0082] As used herein, the term "conjugated moiety" includes agents with a specific biological or specific functional activity (e.g. proteins (e.g. second IVDs or ISVDs), nucleotide sequences, lipids, (other) carbohydrates, polymers, peptides, drug moieties (e.g. cytotoxic drugs), tracers and detection agents). For example, an IVD or ISVD conjugate comprising a polypeptide according to the invention and a conjugated moiety has at least one additional function or property compared to the non-conjugated IVD or ISVD polypeptide of the invention. For example, an IVD or ISVD conjugate comprising a polypeptide of the invention and a conjugated moiety, a cytotoxic drug, results in the formation of a binding polypeptide with drug cytotoxicity as a second function (i.e. in addition to the antigen binding conferred by the IVD or ISVD polypeptide).

[0083] In another alternative example, conjugation of a second binding polypeptide to the IVD or ISVD polypeptide of the invention may confer additional binding properties. In one embodiment, when the conjugated moiety is a genetically encoded therapeutic or diagnostic protein or nucleotide sequence, the conjugated moiety may be synthesized or expressed by either peptide synthesis or recombinant DNA methods well known in the art. In another aspect, when the conjugated moiety is a non-genetically encoded peptide, e.g., a drug moiety, the conjugated moiety may be artificially synthesized or purified from a natural source.

[0084] The present invention aims to provide polypeptides comprising an IVD or ISVD which have glycosylation acceptor sites present in certain regions, in particular in regions which allow efficient glycosylation, which glycosylation does not interfere with the binding and folding of the IVD or ISVD, making them more amenable for further uses, e.g. for the production of IVD or ISVD conjugates.

[0085] The present invention provides a nucleotide sequence encoding a polypeptide, which comprises an antibody mimetic, wherein the antibody mimetic has at least one artificially modified N-glycosylation site introduced at any possible position, and wherein the glycosylation of the glycosylation acceptor site consists of one or more glycans selected from the group consisting of GlcNAc, LacNAc and sialyl-LacNAc.

[0086] In another alternative embodiment, the present invention provides a polypeptide, which comprises an antibody mimetic, wherein the antibody mimetic has at least one artificially modified N-glycosylation site introduced at any possible position, and wherein the glycosylation of the glycosylation acceptor site consists of one or more glycans selected from the group consisting of GlcNAc, LacNAc and sialyl-LacNAc.

[0087] In another alternative embodiment, the present invention provides a conjugate comprising a polypeptide, the polypeptide comprising an antibody mimetic, wherein the antibody mimetic has at least one artificially modified N-glycosylation site introduced at any possible position, and wherein the glycosylation of the glycosylation acceptor site comprises one or more glycans selected from the group consisting of GlcNAc, LacNAc and sialyl-LacNAc, and a conjugated moiety coupled to the glycan.

[0088] The term "antibody mimetic" as used herein refers to artificial (poly-)peptides that specifically bind to antigens like antibodies, but are structurally unrelated to antibodies. They are usually significantly smaller than antibodies, with molecular weights of about 3-20 kDa. Non-limiting examples of antibody mimetics include abdurins, adnectins, affibodies, affilins, affimers, alphabodies, affitins, anticalins, avimers, DARPins, fynomers, Kunits domain peptides, monobodies, Z domain of protein A, gamma B crystallins, ubiquitin, cystatin, Sulfolobus acidocaldarius, lipocalin, A domain of a membrane receptor, ankyrin repeat motif, SH3 domain of Fyn, Kunitz domain of a protease inhibitor, fibronectin type III domain 10, 3- or 4-helix bundle protein, armadillo repeat domain, leucine-rich repeat domain, PDZ domain, SUMO or SUMO-like domain, immunoglobulin-like domain, phosphotyrosine binding domain, pleckstrin homology domain, src homology 2 domain, or synthetic peptide ligands, e.g. from a (random) peptide library.

[0089] Other examples of antigen-binding proteins also include synthetic binding proteins, more specifically monobodies (see, for example, Sha et al., 2017. Protein Science. 26:910-924 for a review). Monobodies are synthetic proteins built on fibronectin type III domains. Monobodies have been isolated that bind with high affinity to a diverse array of targets, including extracellular domains of receptors, kinases, steroid hormone receptors, and modular protein domains (Koide, 2012).

[0090] In another alternative embodiment, the present invention provides an ISVD polypeptide, the polypeptide having at least one artificially modified N-glycosylation site introduced at any possible position in the polypeptide sequence, and wherein the glycosylation of the glycosylation acceptor site consists of one or more glycans selected from the group consisting of GlcNAc, LacNAc and sialyl-LacNAc.

[0091] In another alternative embodiment, the present invention provides a conjugate comprising an ISVD polypeptide, the polypeptide having at least one artificially modified N-glycosylation site introduced at any possible position in its sequence, and wherein the glycosylation of the glycosylation acceptor site consists of one or more glycans selected from the group consisting of GlcNAc, LacNAc and sialyl-LacNAc, and a conjugated moiety coupled to the glycan.

[0092] In another alternative embodiment, the present invention provides a nucleotide sequence encoding a polypeptide comprising an IVD, wherein the IVD has a glycosylation acceptor site present at any of amino acids 83 to 88 and / or any of amino acids 27 to 40 of the IVD (according to the AHo numbering convention). For ease of understanding, an IVD having a glycosylation acceptor site present at any of amino acids 83 to 88 and / or any of amino acids 27 to 40 of the IVD (according to the AHo numbering convention) is further designated herein as an "IVD of the invention" or an "ISVD of the invention".

[0093] In another alternative embodiment, the invention provides a nucleotide sequence encoding a polypeptide comprising an IVD, wherein the IVD has a glycosylation acceptor site located at any of amino acids 83 to 88 of the IVD (according to the AHo numbering convention). In another alternative embodiment, the invention provides a nucleotide sequence encoding a polypeptide comprising an IVD, wherein the IVD has a glycosylation acceptor site located at any of amino acids 27 to 40 of the IVD (according to the AHo numbering convention). In another alternative embodiment, the invention provides a nucleotide sequence encoding a polypeptide comprising an IVD, wherein the IVD has a glycosylation acceptor site located at any of amino acids 83 to 88 and any of amino acids 27 to 40 of the IVD (according to the AHo numbering convention).

[0094] The glycosylation acceptor sites may (but are not necessarily) modified with N- or O-linked glycans. It is specifically contemplated herein that the present invention is not limited to N-glycosylation. The present disclosure provides a means to employ both N- and O-glycosylation.

[0095] In an embodiment according to the invention, a nucleotide sequence is provided encoding a polypeptide comprising an IVD, wherein the IVD comprises any of amino acids 84 to 88, 85 to 88, 86 to 88, 87 and 88, 83 to 87, 84 to 87, 85 to 87, 83, 84, 85, 86, 87, 88, 83 to 86, 84 to 86, 85 and 86, 83 to 85, 84 and 85, or 83 and 84 of the IVD (according to the AHo numbering convention), and / or amino acids 28 to 40. , 29-40, 30-40, 31-40, 32-40, 33-40, 34-40, 35-40, 36-40, 37-40, 38-40, 39 and 40, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, 27-39, 28-39, 29-39, 30-39, 31-39, 32-39, 33-39, 34-39, 35-39, 36-39, 37-39, 38 and 39, 27-38, 28-38, 29-38, 30-38 , 31-38, 32-38, 33-38, 34-38, 35-38, 36-38, 37 and 38, 27-37, 28-37, 29-37, 30-37, 31-37, 32-37, 33-37, 34-37, 35-37, 36 and 37, 27-36, 28-36, 29-36, 30-36, 31-36, 32-36, 33-36, 34-36, 35-36, 27-35, 28-35, 29-35, 30-35, 31-35, 32-35, 33-35, 34 and 35 , 27-34, 28-34, 29-34, 30-34, 31-34, 32-34, 33 and 34, 27-33, 28-33, 29-33, 30-33, 31-33, 32 and 33, 27-32, 28-32, 29-32, 30-32, 31 and 32, 27-31, 28-31, 29-31, 30 and 31, 27-30, 28-30, 29 and 30, 27-29, 28 and 29, or 27 and 28.

[0096] In another alternative embodiment according to the invention, a nucleotide sequence is provided encoding a polypeptide comprising an IVD, wherein the IVD has a glycosylation acceptor site present at amino acid 86 and / or at amino acid 27 of the IVD (according to the AHo numbering convention). In another alternative embodiment according to the invention, a nucleotide sequence is provided encoding a polypeptide comprising an IVD, wherein the IVD has glycosylation acceptor sites present at amino acid 86 and amino acid 27 of the IVD (according to the AHo numbering convention).

[0097] In another alternative embodiment according to the invention, a nucleotide sequence is provided encoding a polypeptide comprising an IVD, wherein said IVD has a glycosylation acceptor site present at amino acid 86 of the IVD (according to the AHo numbering convention). In another alternative embodiment according to the invention, a nucleotide sequence is provided encoding a polypeptide comprising an IVD, wherein the IVD has a glycosylation acceptor site present at amino acid 27 of the IVD (according to the AHo numbering convention).

[0098] In a preferred embodiment, a nucleotide sequence is provided encoding a polypeptide comprising an IVD, wherein the IVD has a glycosylation acceptor site located at any of amino acids 83 to 88 of the IVD (according to the AHo numbering convention). More particularly, the glycosylation acceptor site of the IVD is located at amino acids 84 to 88, 83, 84, 85, 86, 87, 88, 85 to 88, 86 to 88, 87 and 88, 83 to 87, 84 to 87, 85 to 87, 86 and 87, 83 to 86, 84 to 86, 85 and 86, 83 to 85, 84 and 85, or 83 and 84 (according to the AHo numbering convention). Most particularly, the glycosylation acceptor site of the IVD is located at amino acid 86 of the IVD (according to the AHo numbering convention).

[0099] In another embodiment according to the invention, a nucleotide sequence is provided encoding a polypeptide comprising an IVD, wherein the IVD has a glycosylation acceptor site present at any of amino acids 27 to 40 of the IVD (according to the AHo numbering convention). More specifically, the glycosylation acceptor site of the IVD is present at any of amino acids 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 28-40, 29-40, 30-40, 31-40, 32-40, 33-40, 34-40, 35-40, 36-40, 37-40, 38-40, 39 and 40 of the IVD (according to the AHo numbering convention). , 27-39, 28-39, 29-39, 30-39, 31-39, 32-39, 33-39, 34-39, 35-39, 36-39, 37-39, 38 and 39, 27-38, 28-38, 29-38, 30-38, 31-38, 32-38, 33-38, 34-38, 35-38, 36-38, 37 and 38, 27-37, 28-37, 29-37, 30-37, 3 1-37, 32-37, 33-37, 34-37, 35-37, 36 and 37, 27-36, 28-36, 29-36, 30-36, 31-36, 32-36, 33-36, 34-36, 35-36, 27-35, 28-35, 29-35, 30-35, 31-35, 32-35, 33-35, 34 and 35, 27-34, 28-34, 29-34, 30-34, 31- 34, 32-34, 33 and 34, 27-33, 28-33, 29-33, 30-33, 31-33, 32 and 33, 27-32, 28-32, 29-32, 30-32, 31 and 32, 27-31, 28-31, 29-31, 30 and 31, 27-30, 28-30, 29 and 30, 27-29, 28 and 29, or 27 and 28.

[0100] Most specifically, the glycosylation acceptor site of the IVD is located at amino acid 27 of the IVD (according to the AHo numbering convention). It should be apparent to one of skill in the art based on the disclosure provided herein that additional positions in the IVD next to positions 83-88 and / or 27-40 may be selected that are prone to glycosylation.

[0101] Thus, in a specific embodiment according to the invention, a nucleotide sequence is provided encoding a polypeptide comprising an IVD, said IVD having a glycosylation acceptor site present at amino acids 83-88 and / or at amino acids 27-40 of the IVD (according to the AHo numbering convention) and an additional glycosylation acceptor site present in the IVD at a position such as position 14 and / or position 48. According to a specific embodiment, the IVD of the invention has an additional glycosylation acceptor site introduced into the IVD at any position in the IVD. According to another specific embodiment, the IVD of the invention has an additional glycosylation acceptor site present in the IVD at position 14 (according to the AHo numbering convention). According to another specific embodiment, the IVD of the invention has an additional glycosylation acceptor site present in the IVD at position 48 (according to the AHo numbering convention).

[0102] In another alternative embodiment, the IVD of the invention has additional glycosylation acceptor sites which, according to specific embodiments, are present at positions 16 and / or 49 and / or 139. In another alternative embodiment, the IVD of the invention has an extra terminal amino (N-)-terminus tag and / or an extra carboxy-terminus (C-), which tag comprises a glycosylation acceptor site, in particular an N-glycan acceptor site.

[0103] Thus, it is clear that the scope of the present invention encompasses the combined use of at least two or even more glycosylation acceptor sites within the IVD of the present invention. Based on this application, the skilled artisan will know how to select additional glycosylation acceptor sites within or next to a particular glycosylation acceptor site identified in the IVD of the present invention, and the identification and / or use of further positions and combinations thereof are also within the scope of the present invention as presented.

[0104] In a particular embodiment, the present invention provides a polypeptide encoded by an IVD nucleotide sequence as described hereinbefore.

[0105] According to another embodiment, the glycosylation acceptor site of the IVD is an asparagine residue which can be N-glycosylated. More specifically, the IVD contains a NXT, NXS, NXC or NXV motif, where X can be any amino acid except proline, such that the asparagine residue of the NXT / NXS / NXC / NXV motif can be present at any of amino acids 83 to 88 and / or at any of amino acids 27 to 40. Even more specifically, the IVD contains a NXT / NXS motif.

[0106] According to a particular embodiment, the present invention provides a nucleotide sequence encoding a polypeptide comprising an IVD as previously described, wherein said IVD is an immunoglobulin single variable domain (ISVD).

[0107] According to a specific embodiment, a nucleotide sequence is provided encoding a polypeptide comprising an ISVD as previously described, wherein said ISVD is a heavy chain variable domain sequence. According to a more specific embodiment, the ISVD is a heavy chain variable domain sequence derived from a heavy chain antibody, preferably a camelid heavy chain antibody.

[0108] In another specific embodiment, a nucleotide sequence is provided that encodes a polypeptide comprising an ISVD as described above, wherein said polypeptide consists of said ISVD. In another other embodiment, an expression vector is provided that comprises a nucleotide sequence that encodes a polypeptide comprising an IVD as described above.

[0109] In the present invention, the term "comprising a polypeptide comprising an ISVD" means that the ISVD can be fused (or coupled) to another polypeptide, such as a half-life extending polypeptide (e.g. a VHH directed to serum albumin), a second VHH (e.g. to create a bispecific or bivalent IgG), an enzyme, a therapeutic protein, an Fc domain, such as an IgA Fc domain or an IgG Fc domain.

[0110] In another aspect, the present invention provides a cell comprising an expression vector according to the present invention. In a specific aspect, the cell is a higher eukaryotic cell, such as a mammalian cell or a plant cell, a lower eukaryotic cell, such as a filamentous fungal cell or a yeast cell, or a prokaryotic cell.

[0111] The higher eukaryotic cells can be any higher eukaryote, but in a specific embodiment, mammalian cells are envisaged. The nature of the cells used will typically depend on the desired glycosylation characteristics and / or the ease and cost of producing the IVD or ISVD described herein. Mammalian cells may be used, illustratively, to avoid issues with immunogenicity. Higher eukaryotic cell lines for protein production, including cell lines with modified glycosylation pathways, are well known in the art.

[0112] Non-limiting examples of animal or mammalian host cells suitable for carrying, expressing and producing proteins followed by isolation and / or purification include CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; and Kolkekar et al., 1997, Biochemistry, 36:10901-10909), CHO-K1 Tet-On cell line (Clontech), ECACC 85050302 designated as CHO (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), ECACC 93061607 designated as CHO-K1 / SF (CAMR, Salisbury, Wiltshire, Chinese hamster ovary cells (CHO), such as ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), designated RR-CHOK1, dihydrofolate reductase negative CHO cells (CHO / -DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells subcloned to grow in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical cancer cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human liver cancer cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad. Sci., 383:44-68); MCR 5 cells; and FS4 cells. According to a specific embodiment, the cell is a mammalian cell selected from CHO cells, Hek293 cells, or COS cells. According to a further specific embodiment, the mammalian cell is selected from CHO cells and Hek293 cells.

[0113] According to another specific embodiment, the cell according to the invention is a plant cell. Exemplary plant cells include cells from tobacco, tomato, carrot, corn, algae, alfalfa, rice, soybean, Arabidopsis thaliana, Taxus cuspidata, Nicotiana benthamiana, and Catharanthus roseus. Further additional plant species that may be useful for producing IVD or ISVD polypeptides according to the invention are described in Weathers, PJ et al., Appl Microbiol Biotechnol. 85(5), 2010.

[0114] In more specific embodiments, the cell according to the invention is a lower eukaryotic cell, such as a filamentous fungal cell or a yeast cell. Specific examples of filamentous fungal and yeast cells have been reviewed earlier in this specification. In a more specific embodiment, the cell according to the invention is a prokaryotic cell, such as E. coli, a Lactococcus species or a Bacillus species.

[0115] In a more specific embodiment, the cell according to the invention as described above is a glycoengineered cell. The glycoengineered cell may be capable of removing unwanted N-glycosylation and / or O-glycosylation. The term glycoengineered cell has been reviewed hereinbefore. The glycoengineered cell may also be a non-mammalian cell that has been engineered to mimic the human glycosylation pathway as described above.

[0116] In a specific embodiment, a polypeptide is provided comprising an IVD encoded by a nucleotide sequence according to the invention as described above, wherein the polypeptide comprises a glycan (or more than one glycan), wherein the glycan is selected from the group consisting of terminal GlcNAc, GalNAc, galactose, sialic acid, glucose, glucosamine, galactosamine, bacillosamine (e.g., a rare amino sugar (2,4-diacetamido-2,4,6-trideoxyglucose) described in Bacillus subtilus and Campylobacter jejuni), The IVD polypeptides containing glycans with specific sugars can be made in vivo, having mannose or mannose-6-P sugars, or chemically modified monosaccharides such as GalNAz, azido-sialic acid (AzSia), or GlcNAz.

[0117] For example, higher eukaryotic cells will typically produce glycans with terminal sialic acid, yeast cells will typically produce glycans with terminal mannose or mannose-6P, some filamentous fungi will produce glycans with terminal galactose, some glycoengineered yeast cells produce terminal GlcNAc (e.g., as described in WO2010015722), and some glycoengineered higher eukaryotic cells produce a mixture of glycans with terminal GlcNAc, galactose, and sialic acid (e.g., as described in WO2010015722 and WO2015032899); Other glycoengineered higher eukaryotic cells produce glycans with terminal GlcNAc (see WO2017005925), eukaryotic cells containing certain mutant galactosyltransferases can enzymatically attach GalNAc to non-reducing GlcNAc sugars (see WO2004063344), and eukaryotic cells containing mutant galactosyltransferases supplied with UDP-GalNAz (a C2-substituted azidoacetamido-galactose UDP-derivative) will incorporate GalNAz at the terminal non-reducing GlcNAc of the glycan (see WO2007095506 and WO2008029281). Optionally, IVD polypeptides containing glycans with specific sugars can be made by a combination of in vivo trimming of the glycan followed by in vitro trimming until the desired terminal sugar is obtained. See, for example, WO2015057065 (Synaffix).

[0118] In another specific embodiment, the present invention provides a polypeptide comprising an IVD of the present invention, wherein the IVD comprises a glycan (or more than one glycan), and wherein the glycan consists of a glycan selected from the group consisting of GlcNAc, LacNAc, sialyl-LacNAc, Man5GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, hyper-mannosylated glycan, mannose-6-phosphate glycan, complex glycan, hybrid glycan, and chemically modified glycan (GlcNAz, GlcNAc-GalNAz, azido-sialic acid-LacNAc, etc.).

[0119] In another specific embodiment, the present invention provides a composition comprising a polypeptide comprising an IVD of the present invention, wherein the IVD comprises a glycan (or more than one glycan), and wherein the glycan consists of a glycan selected from the group consisting of GlcNAc, LacNAc, sialyl-LacNAc, Man5GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, hypermannosyl glycan, mannose-6-phosphate glycan, complex glycan, hybrid glycan, and chemically modified glycan (GlcNAz, GlcNAc-GalNAz, azido-sialic acid-LacNAc, etc.), and wherein the presence of one or more of these glycans at a specific position(s) in the polypeptide is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the same peptide in a sample.

[0120] It should be noted that while the various host cells described herein above may be particularly useful for producing the specific glycans present on the IVDs provided by the present invention, combined in vivo and in vitro approaches may also be used to obtain the desired glycan structures. Indeed, the IVDs or ISVDs of the present invention produced in eukaryotic hosts may be purified, the glycan structures may be trimmed by suitable endoglycosaminidases or exoglycosidases, and then rebuilt by the in vitro use of various glycosyltransferases (e.g., galactosyltransferases, sialyltransferases, polysialyltransferases, etc.).

[0121] IVD-conjugates In a specific embodiment, the present invention provides an IVD-conjugate. In a preferred embodiment, an IVD or ISVD polypeptide according to the present invention is coupled to a specific component (conjugated moiety as defined hereinbefore) via a glycan structure present on the IVD or ISVD polypeptide. Such specific coupling of a glycan to a specific component is referred to in the art as glycan-specific conjugation. A glycan structure with a specific terminal carbohydrate or a specific glycan structure, as described hereinbefore, present on the IVD or ISVD polypeptide is used as a starting point for coupling to a specific component.

[0122] Specific moieties that can be used for conjugation There is a plethora of conjugation moieties in the art that can be used for coupling to the glycan structures present in the IVD or ISVD of the present invention. Conjugation moieties include, for example, half-life extension moieties, therapeutic agents, detection units, targeting moieties, or even a second (same or different) IVD or ISVD polypeptide. One or more conjugation moieties, which may also be different from each other, can be linked to the IVD or ISVD of the present invention. Even one conjugation moiety can have more than one function, i.e., a half-life extension moiety can be useful as a targeting moiety at the same time.

[0123] i) Half-life extension ingredients Various half-life extension moieties are envisaged herein. Non-limiting and brief half-life extension strategies are described in Kontermann, RE, Expert Opin Biol Ther. 16(7), 2016 or van Witteloostuijn, SB, ChemMedChem. 11(22), 2016. In particular, various half-life extension techniques have been developed that rely on covalent chemical modification. These methods include PEGylation, fusion to unstructured polypeptide-based PEG mimics, employing polysialylation (e.g., enzymatic use of polysialyltransferase enzymes), biotin-coupling, polyoxazoline-coupling, conjugation with large polysaccharides, lipidation, fusion to albumin or IgG Fc domain or IgA Fc domain, and derivatization with bio-orthogonal moieties that direct self-assembly. Another alternative half-life extending component is an IVD (such as a VHH) directed against serum albumin.

[0124] ii) therapeutic ingredients In certain embodiments, the conjugated moiety comprises various therapeutic agents, including, namely, anti-inflammatory agents, anti-cancer agents, cytotoxic agents, anti-infective agents (e.g., anti-fungal agents, anti-bacterial agents, anti-parasitic agents, anti-viral agents, etc.), and anesthetic therapeutic agents. In certain embodiments, the conjugated moiety is an enzyme capable of converting a prodrug, which is converted into a toxic drug. Poisonous substances (e.g., toxins, cytotoxic drugs, radionuclides) may also be suitable for therapeutic purposes and are particularly useful in cancer treatment. Thus, a specific example of an IVD-conjugate is an antibody-drug-conjugate (ADC). In principle, any agent suitable for therapeutic purposes is contemplated herein. The therapeutic agents as described are typically small molecules or biologics, but the therapeutic agent may also be something else that would be obvious to the skilled artisan and the invention should not be limited thereto.

[0125] iii) Detection components In some embodiments, the conjugated moiety comprises a detection moiety. The term "detection moiety" or "detectable label" refers to any unit that possesses a property or function that can be used for detection purposes, i.e., selected from the group including chromophore units, fluorescent units, phosphorescent units, luminescent units, light absorbing units, radioactive units, and transition metal isotope mass tag units. Without limitation, the detection moiety can be a small molecule or a large molecule, as would be apparent to one skilled in the art.

[0126] Suitable fluorescent units are those known from the technical field of immunofluorescence techniques, e.g., flow cytometry or fluorescence microscopy. In these aspects of the invention, the conjugates containing the detection unit are detected by exciting the detection unit and detecting the resulting emission (photoluminescence). In this aspect, the detection unit is preferably a fluorescent unit.

[0127] Useful fluorescent units may be protein-based such as phycobiliproteins, polymers such as polyfluorenes, xanthenes such as fluorescein, or organic small molecule dyes such as rhodamines, cyanines, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyrromethenes, or organometallic complexes such as Ru, Eu, Pt complexes, etc. In addition to single molecular entities, clusters of fluorescent proteins or organic small molecule dyes, as well as nanoparticles such as quantum dots, upconverting nanoparticles, gold nanoparticles, dyed polymeric nanoparticles, etc. may also be used as fluorescent units.

[0128] Another group of photoluminescent detection units are phosphorescent units with time-delayed emission of light after excitation. Phosphorescent units include organometallic complexes such as Pd complexes, Pt complexes, Tb complexes, Eu complexes, or nanoparticles with incorporated phosphorescent dyes such as lanthanide-doped SrAl2O4.

[0129] In another embodiment of the invention, the conjugates containing the detection units are detected without prior excitation by irradiation. In this embodiment, the detection units can be radioactive labels. They can be non-radioactive isotopes or their radioactive counterparts (tritium, 32 P, 35 S or 14 C) or linked to tyrosine 125 I, in fluorodeoxyglucose 18 F, or organometallic complexes (i.e. 99 This may be in the form of radioisotope labeling by incorporating a covalently bound label, such as 1Tc-DTPA).

[0130] In another embodiment, the detection unit is capable of producing chemiluminescence, ie, a horseradish peroxidase label in the presence of luminol. In another embodiment of the invention, conjugates containing a detection unit are not detected by radiation emission but by absorption of UV, visible, or NIR radiation. Suitable light absorbing detection moieties are light absorbing dyes without fluorescent emission, such as N-arylrhodamines, azo dyes, and organic small molecule quencher dyes such as stilbenes.

[0131] In another embodiment, the light absorption detection unit can be illuminated by a pulsed light laser to generate a photoacoustic signal. In another aspect of the present invention, the conjugate containing the detection unit is detected by mass spectrometry detection of the transition metal isotope. The transition metal isotope mass tag label may be introduced as a component of a covalently bound organometallic complex or nanoparticle. Known in the art are isotope tags of lanthanides and adjacent late transition elements.

[0132] iv) targeting component In some embodiments, the conjugated moiety comprises a targeting moiety. As used herein, the term "targeting moiety" refers to a conjugated moiety that binds to a target molecule. Both small molecules or biologics can be employed as targeting moieties. Targeting moieties can include, without limitation, proteins, nucleotide sequences, lipids, other carbohydrates (e.g., certain glycans), and combinations thereof (e.g., glycoproteins, glycopeptides, and glycolipids). Any moiety that can bind to a target can be employed as a targeting moiety according to the present invention.

[0133] Linkers useful in IVD-conjugates In some embodiments, the IVD-conjugate comprises a linker between the glycan and the targeting moiety. Some linkers are more useful than others, and the use of a particular linker will depend on the application. For example, oximes and hydrazones, especially those derived from aliphatic aldehydes, show less stability over time in water or at lower pH. Aromatically stabilized structures may be more useful for stably linking glycans to conjugated moieties. Such stabilized linkers are also within the scope of the application, as they may limit adverse effects caused by premature release of the conjugated moiety, particularly when the conjugated moiety is a toxic substance intended to kill tumor cells.

[0134] Of particular interest are bicyclo[6.1.0]non-4-yne reagents (BICYCLO[6.1.0]NON-4-YNE REAGENTS) and aromatically stabilized triazole and sulfamide linkers. It is also within the scope of common technical knowledge that the increased stability of the conjugate reduces the tendency of any of the components contained within the conjugate to aggregate. For the production of IVD conjugates with increased stability, the reader is referred to, non-exclusively, WO2013036748, WO2014065661, WO2015057064 and WO2016053107, as well as other patent applications filed by Synaffix BV as expressly mentioned herein.

[0135] In general, various linkers known in the art can be used to link the IVD and the conjugated moiety according to the present invention. As should be clear, cleavable and non-cleavable linkers can be employed to obtain the desired release profile. In general, the optimal combination of linker and conjugation chemistry must be uniquely tailored to correlate each unique facet: IVD, conjugated moiety, and disease profile to be treated. For discussion of antibody-drug conjugates and linkers used herein, see, for example, Jessica R. McCombs and Shawn C. Owen, AAPS J. 17(2), 2015, and Lu, J. et al., Int J Mol Sci. 17(4), 2016, as well as a recent review by Pillow, TH, Pharm Pat Anal. 6(1), 2017, which describes novel quaternary ammonium salt linkers useful in conjugates for the treatment of cancer and infectious diseases.

[0136] Still other suitable spacers or linkers will be clear to the skilled artisan. These may generally be any linkers or spacers used in the art. In certain aspects, the linkers or spacers are suitable for use in applications intended for pharmaceutical use. For example, the linker between the glycan and the moiety in the ISVD-conjugate or IVD-conjugate may also be in certain aspects a suitable amino acid sequence, in particular an amino acid sequence of between 1 and 50, or more particularly between 1 and 30 amino acid residues. Some examples of such amino acid sequences are Gly-Ser (GS) linkers, such as (GS)n or (GGGSS)n or (GSS)n, as described in WO 99 / 42077, and (G4S)3, GS, as described in the applications by Ablynx mentioned herein (see, for example, WO 06 / 040153 and WO 06 / 122825). 30 , G.S. 15 , GS9 and GS7 linkers, and hinge-like regions such as the hinge region of a naturally occurring heavy chain antibody or similar sequences (such as those described in WO 94 / 04678).

[0137] Further suitable linkers generally include organic compounds or polymers, especially those suitable for use in polypeptides for pharmaceutical applications. By way of illustration, poly(ethylene glycol) moieties have been used to link antibody domains, see, for example, WO 04 / 081026. It is within the scope of the present invention that the length, flexibility and / or other properties of the linker may have a certain effect on the properties of the final IVD conjugate of the present invention, including but not limited to the affinity, specificity or avidity for a particular target. Based on the present disclosure herein, the skilled artisan will be able to determine, optionally after some limited routine experimentation, the optimal linker for use in a particular IVD or ISVD of the present invention. For example, in a multivalent ISVD of the present invention comprising building blocks directed against a first and a second target, the length and flexibility of the linker is preferably such that the linker allows each building block to bind to its cognate target.

[0138] Based on the disclosure herein, the skilled person will also be able to determine, optionally after some limited routine experimentation, the optimal linker for use in a particular IVD or ISVD of the invention. Finally, when more than one linker is used in an IVD or ISVD of the invention, these linkers may be the same or different. Based on the disclosure herein, the skilled person will also be able to determine, optionally after some limited routine experimentation, the optimal linker for use in a particular polypeptide of the invention. In certain embodiments, it is desirable to produce IVD conjugates with longer linkers (e.g., including carbohydrates) that may provide IVD conjugates with higher hydrophilicity and consequently improved water solubility. Thus, IVD conjugates containing linkers with more carbohydrates are also within the scope of the present application. Linkers modified with or consisting of PEG may also be useful to increase the hydrophilic properties of the IVD conjugate.

[0139] In another alternative embodiment, the present invention provides a method for producing a polypeptide comprising an IVD of the invention, said method comprising the steps of introducing into a suitable expression host an expression vector comprising a nucleotide sequence encoding an IVD of the invention, expressing said IVD of the invention and isolating it. Suitable conditions must be selected for expressing a polypeptide comprising an IVD according to the invention.

[0140] By the term "suitable cells" it is envisaged that higher eukaryotic cells, such as mammalian cells or plant cells, lower eukaryotic cells, such as filamentous fungal cells or yeast cells, optionally glycosylated, are envisaged as explained above.

[0141] Specifically contemplated herein is the production of a polypeptide comprising an IVD or IVSD according to the invention, wherein the polypeptide is glycosylated and comprises one or more glycans having terminal GlcNAc, GalNAc, galactose, sialic acid, glucose, glucosamine, galactosamine, bacillosamine, mannose or mannose-6-P sugars, or chemically modified monosaccharides such as GalNAz, AzSia, or GlcNAz.

[0142] For example, a polypeptide comprising an IVD of the invention, wherein the polypeptide is N-glycosylated and comprises a mixture of N-glycans with terminal GlcNAc, galactose or sialic acid, may typically be obtained by expression in a higher eukaryotic glycoengineered cell according to the invention, as described in WO2010015722 and WO2015032899. For example, a polypeptide comprising an IVD of the invention, wherein the polypeptide is N-glycosylated and comprises or essentially comprises an N-glycan with terminal GlcNAc, may be produced in a lower eukaryotic cell, as described in WO2010015722. For example, an N-glycan with terminal GlcNAc may be produced in a glycoengineered cell deficient in the expression and / or activity of endogenous UDP-galactose 4-epimerase (GalE), as described in WO2017005925.

[0143] Also specifically contemplated herein is the production of a polypeptide comprising an IVD according to the invention, wherein the glycosylation of the polypeptide consists of one or more glycans selected from the group consisting of GlcNAc, LacNAc, sialyl-LacNAc, Man5GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, complex glycans, hybrid glycans, and GlcNAc-GalNAz. Even more specifically contemplated herein is the production of a polypeptide comprising an IVD according to the invention, wherein the glycosylation of the polypeptide consists of one or more glycans selected from the group consisting of GlcNAc, LacNAc, sialyl-LacNAc, Man5GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, and complex glycans.

[0144] Polypeptides comprising an IVD of the invention, where the polypeptide is glycosylated and where the glycosylation consists of GlcNAc, LacNAc and sialyl-LacNAc glycans, are typically obtained in mammalian glycoengineered cells according to the invention as described in WO2010015722 and WO2015032899, although such GlcNAc, LacNAc and sialyl-LacNAc glycans may also be engineered in lower eukaryotic cells (e.g. via introduction of a mammalian complex glycosylation pathway into yeast). Polypeptides comprising an IVD of the invention, where the polypeptide is glycosylated and where the glycosylation consists of GlcNAc, may be produced in glycoengineered cells according to the invention, which may lack endogenous UDP-galactose 4-epimerase (GalE) expression and / or activity as described in WO2017005925.

[0145] Polypeptides comprising an IVD of the invention, where the polypeptide is glycosylated and where the glycosylation consists of complex glycans, can be produced in higher eukaryotic cells according to the invention, which are optionally glycomodified. Polypeptides comprising an IVD of the invention, where the polypeptide is glycosylated and where the glycosylation consists of one or more glycans selected from the group consisting of Man5GlcNAc2 glycan, Man8GlcNAc2 glycan, Man9GlcNAc2 glycan, hypermannosylated glycan, mannose-6-phosphate modified glycan, and complex glycan, can be produced in glycomodified cells according to the invention, in particular yeast cells.

[0146] Coupling methods for linking specific components to the IVD In another alternative embodiment, the present invention provides a method for producing an IVD or ISVD conjugate of the invention. In general, such a method begins by introducing into a suitable cell of choice an expression vector comprising a nucleotide sequence encoding an IVD according to the invention, followed by expressing the IVD polypeptide for a period of time, purifying the IVD polypeptide, and coupling a specific conjugated moiety to the purified IVD polypeptide. The coupling method itself is generally carried out in vitro.

[0147] There are several possibilities in the art for linking a particular conjugated moiety to the IVD polypeptide of the invention, generally referred to as chemical, enzymatic and combined chemical-enzymatic conjugation strategies for carrying out the coupling reaction.

[0148] According to a particular embodiment, the method for producing an IVD-conjugate comprises: - oxidizing vicinal diols or diols present in the glycans of a polypeptide comprising an IVD of the invention; - reacting the resulting free aldehyde group with an aminooxy-containing molecule; This includes the steps:

[0149] For oxidation, sodium periodate or several other equivalent reagents known in the art can be used. In certain embodiments, the diol to be oxidized originates from a LacNAc disaccharaide or a sialyl-LacNAc trisaccharide present on the IVD of the invention. An oxime is subsequently formed by reaction of the resulting free aldehyde group with an aminooxy-containing molecule, generally described as LacNAc / sialyl-LacNAc oxidation-oxime ligation chemistry.

[0150] Well known in the art is the use of catalysts such as para-phenylenediamine, 2-aminophenol, or 2-(aminomethyl)benzimidazole. Oxime and hydrazine conjugation is a promising alternative to click chemistry, a more complex bioorthogonal modification strategy. Under reductive amination conditions, aldehydes (for example, galactose oxidation produces di-aldehydes and sialic acid oxidation produces mono-aldehydes) can react with amines, resulting in stable oxazepine derivatives.

[0151] According to another specific embodiment, the method for producing an IVD-conjugate optionally comprises: - oxidizing the C6 hydroxyl group of the Gal residue in the terminal LacNAc N-glycan present on the IVD of the invention - reacting the free aldehyde group with an aminooxy-containing molecule This includes the steps:

[0152] For oxidation, the enzyme galactose oxidase (GAO) can be used. Employing the steps mentioned above, typically an oxime bond is formed, optionally in the presence of catalase, all of which are well known in the art. To modulate, or specifically increase, the stability of the oximes and hydrazones, the use of linkers, as described above, is specifically contemplated herein.

[0153] In a more specific embodiment, the conjugated moiety is linked to a glycan present on the IVD, specifically a single GlcNAc or LacNAc, via a monosaccharide derivative. The monosaccharide derivative can be linked to a glycan present on the IVD in the presence of a glycosyltransferase, such as a β(1,4)-galactosyltransferase, a β(1,3)-galactosyltransferase, a β(1,4)-galactosyltransferase with a mutant catalytic domain, a β(1,3)-galactosyltransferase with a mutant catalytic domain, a sialyltransferase, or a GalNAc transferase. The monosaccharide can contain one, two, three, or four functional groups, which can be selected from an azide group, a keto group, an alkynyl group, or a thiol group, or precursors thereof. The functional group can also be selected from a halogen, a sulfonyloxy group, a halogenated acetamide group, a mercaptoacetamide group, or a sulfonylated hydroxyacetamide group. For detailed protocols, readers are referred to WO2015057064 and WO2015057065.

[0154] In yet another specific embodiment, the conjugated moiety is linked to the glycan via an oxime bond and / or via conjugation of an azide-modified form of UDP-GalNAc to an N-glycan with a terminal GlcNAc. In an even more specific embodiment, the conjugated moiety is linked to the glycan via conjugation of an azide-modified form of UDP-GalNAc to an N-glycan with a terminal GlcNAc, using a modified galactosyltransferase followed by attachment of the molecule of interest via a click chemistry-based reaction. Click chemistry-based reactions are well known to those active in this field and are also elucidated based on the examples provided herein. For reviews of current approaches in click chemistry, the reader is referred to Jain, N. et al. Pharm Res. 32(11), 2015; Qasba, PK et al., Biotechnol Prog. 24(3), 2008; or Nwe, K. and Brechbiel, MW, Cancer Biother Radiopharm. 24(3), 2009.

[0155] The use of linkers to modulate the stability of IVD conjugates as described above is specifically envisaged herein. Certain methods are identified in detail further below in the Examples section.

[0156] Applications of the IVD and IVD-conjugates of the invention In specific embodiments, polypeptide-conjugates comprising an IVD of the invention are used to modulate circulating half-life or to increase IVD stability, for selective targeting, to modulate the immunogenicity of the IVD-conjugate, or for detection purposes. In another alternative embodiment, the IVD-conjugates of the invention are used as medicines.

[0157] In another alternative embodiment, the IVD of the invention (not conjugated to any moiety) is used as a medicine. In another alternative embodiment, the IVD of the invention (not conjugated to any moiety) is used to prevent pre-binding of antibodies. In another alternative embodiment, the IVD of the invention (not conjugated to any moiety) is used to reduce immunogenicity.

[0158] With respect to the expression "to modulate the circulatory half-life", it is meant that the half-life of a polypeptide (e.g., an IVD-conjugate) can either be increased or decreased. For some applications, it may be useful for a polypeptide comprising an IVD of the invention or an IVD-conjugate of the invention to remain in the bloodstream for a shorter time than a polypeptide or conjugate lacking the specific properties of the polypeptide or IVD-conjugate as claimed. Often, a prolonged half-life is also aimed, as many therapeutic molecules are below the renal filtration threshold and are rapidly lost from the circulation, thereby limiting their therapeutic potential. As a non-limiting example, albumin or other half-life extending moieties as referenced above can be used in various ways known to the skilled practitioner to increase the circulatory half-life of such molecules.

[0159] By "selective targeting" it is meant that the polypeptides and IVD-conjugates of the invention may be useful to obtain an exclusive effect on the target of interest. An example of this is conventional chemotherapy, which often fails to selectively target cancer cells that do not interact with normal somatic cells. As a consequence thereof, severe side effects including organ damage are caused, leading to impaired treatment with lower doses and ultimately to low survival rates. The polypeptides and IVD-conjugates of the invention may be useful to overcome the disadvantages of conventional approaches, optionally including a targeting moiety, but not limited to cancer therapy.

[0160] Modulation of immunogenicity using the polypeptides and conjugates of the present invention can be achieved compared to polypeptides or IVD-conjugates lacking certain properties of the claimed polypeptides or IVD-conjugates. For example, for long-term treatment, it is preferable to reduce immunogenicity. Specifically and non-limitingly, glycans as described herein can be used as a tool to modify immunogenicity. Those skilled in the art can adapt immunogenicity based on general knowledge and the disclosure provided herein.

[0161] The polypeptides and conjugates as described herein can be used to prevent or reduce binding to existing antibodies.This effect has been described in the literature for glycans on ISVD (i.e. see WO2016150845).The use of the polypeptides and conjugates according to the present invention to prevent pre-binding of antibodies is within the scope of this disclosure and is also envisaged herein.

[0162] The polypeptides and conjugates of the invention are also provided for detection purposes, particularly when they comprise a detection unit as previously explained. In particular, the polypeptides and conjugates of the invention are more prone to detection purposes than polypeptides or conjugates lacking the specific properties of the claimed polypeptides or conjugates. Thus, in a specific embodiment, the IVD-conjugates of the invention can also be used for diagnostic purposes.

[0163] In another alternative embodiment, the present invention provides a kit comprising an IVD of the present invention. In another alternative embodiment, the present invention provides a kit comprising an IVD-conjugate of the present invention. In another aspect there is provided a pharmaceutical composition comprising a polypeptide comprising an IVD or an IVD-conjugate as described above.

[0164] Thus, the present invention encompasses pharmaceutical compositions comprised of a pharma- ceutically effective amount of the polypeptides, nucleotide sequences, and IVD-conjugates of the invention and a pharma- ceutically acceptable carrier, which is preferably relatively non-toxic and innocuous to a patient at concentrations consistent with the effective activity of the active ingredient, such that any side effects attributable to the carrier do not impair the beneficial effects of the active ingredient.

[0165] A pharma- ceutically effective amount of the polypeptides, nucleotide sequences, and conjugates of the invention and pharma- ceutically acceptable carriers is preferably an amount that results in or has an effect on the specific condition being treated. The polypeptides, nucleotide sequences, and conjugates of the invention and pharma- ceutically acceptable carriers may be administered using any effective conventional dosage form, including immediate release, sustained release, and timed release preparations, with pharma- ceutically acceptable carriers that are well known in the art, and by any suitable route, including any route generally known to those of skill in the art.

[0166] For treatment, the pharmaceutical composition of the present invention can be administered to any patient according to standard techniques. Administration can be by any suitable mode, including orally, parenterally, topically, nasally, ophthalmically, intrathecally, intracerebroventricularly, sublingually, rectally, vaginally, etc. Nanotechnology formulations and other techniques of aerosols and inhalants are also within the scope of the present invention. The dosage and frequency of administration will depend on the age, sex, and condition of the patient, coadministration with other drugs, counter-indications, and other parameters to be considered by the clinician.

[0167] The pharmaceutical compositions of the present invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use.

[0168] When prepared as a lyophilizate or liquid, physiologically acceptable carriers, excipients, and stabilizers are required to be added to the pharmaceutical composition of the present invention (Remington's Pharmaceutical Sciences 22th edition, Ed. Allen, Loyd V, Jr. (2012)). The dosage and concentration of the carriers, excipients, and stabilizers should be safe for subjects (humans, mice, and other mammals), and they include buffers such as phosphate, citrate, and other organic acids; antioxidants such as vitamin C; small polypeptides, such as serum albumin, gelatin, or immunoglobulins, proteins; hydrophilic polymers such as PVP; amino acids such as aminoacetic acid, glutamate, asparagine, arginine, and lysine; glycoses, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol, sorbitol; Na + and / or surfactants such as TWEEN™, PLURONICS™, or PEG.

[0169] Preparations containing the pharmaceutical compositions of the invention must be sterilized prior to injection, which can be accomplished using sterile filtration membranes, prior to or following lyophilization and reconstitution. Pharmaceutical compositions are often packaged in a container with a sterile access port, such as a corked IV solution bottle, which can be pierced by a hypodermic injection needle.

[0170] Although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein for the nucleotide sequences, cells, polypeptides, conjugates, and methods according to the present invention, it should be understood that various changes or modifications may be made in form and detail without departing from the scope and spirit of the present invention. The following examples are provided to better illustrate specific embodiments and should not be considered as limiting the present application. The present application is limited only by the claims.

[0171] Example 1: Using crystallographic data of ISVD structures as a rational design approach to introduce N-glycans In this example, we started from the available crystallographic structure of a representative immunoglobulin single variable domain polypeptide (chain B from entry 3K74 isolated from the RCSB Protein Data Bank or in brief: PDB database; 3K74 contains two chains, namely A chain (dihydrofolate reductase) and B chain (nanobody that binds dihydrofolate reductase); further herein we use 3K74 to identify only the nanobody (B chain)) to identify regions in the structure suitable for the introduction of artificial N-glycosylation sites. This 3K74 ISVD polypeptide is a nanobody originally described by Oyen D. et al (2011) J.Mol.Biol. 407: 138-148 and its protein secondary structure is depicted diagrammatically in FIG. 1.

[0172] In our rational design approach, we reasoned that potential regions in the secondary structure for the introduction of N-glycans should not interfere with (or hinder) the antigen recognition site of the antibody and, importantly, should not block the formation of beta-sheets during folding. Where the CDR regions of nanobodies are important for antigen recognition and the beta-sheet structure is important for correct folding, we hypothesized that the protein regions between the CDR regions and the beta-strands would likely be less sensitive to minor modifications such as the attachment of N-glycans.

[0173] Example 2: Selection of nine virtual regions in ISVD for the introduction of N-glycosylation signatures Based on our rational design approach and specific criteria outlined in Example 1, we selected a total of nine regions present in the representative nanobody 3K74 (see FIG. 1, right panel, the nine regions selected for the introduction of artificial N-glycan acceptor sites are depicted in black). In addition to the regions present in the beta-sheet structural elements, we planned the addition of N- and C-terminal glycosylation tags to the actual nanobody amino acid sequence represented by SEQ ID NO:1.

[0174] The amino acid sequence of nanobody 3K74, without any N- and C-terminal tags, is represented in SEQ ID NO: 1. In SEQ ID NO: 1, the CDR1, CDR2 and CDR3 regions are underlined. SEQ ID NO:2 represents CDR1, SEQ ID NO:3 represents CDR2, SEQ ID NO:4 represents CDR3, SEQ ID NO:5 represents FR1, SEQ ID NO:6 represents FR2, SEQ ID NO:7 represents FR3, and SEQ ID NO:8 represents FR4.

[0175] SEQ ID NO:1 (115 amino acids): QLQESGGGLVQPGGSLRLSCAAS GFTFNNYW MYWVRRAPGKGLEWVSM INPGIITK YAESVKGRFTISRDNAKNTLYLQMNSLTSEDTAVYYC AKDWATGLA KKGQGTQVTVSS

[0176] SEQ ID NO:2 (CDR1): GFTFNNYW SEQ ID NO:3 (CDR2): INPGGIITK SEQ ID NO:4 (CDR3):AKDWATGLA SEQ ID NO:5 (FR1): QLQESGGGLVQPGGSLRLSCAAS SEQ ID NO:6 (FR2): MYWVRRAPGKGLEWVSM SEQ ID NO:7 (FR3): YAESVKGRFTISRDNAKNTLYLQMNSLTSEDTAVYYC SEQ ID NO:8 (FR4):KKGQGTQVTVSS

[0177] Also based on the criteria outlined in Example 1, we selected specific amino acid sequences within nine selected protein regions of nanobody 3K74 (see Figure 2 which depicts the ten proposed N-glycan acceptor sites to be introduced into the nine different regions of the nanobody structure).

[0178] Example 3: Proof-of-concept using GFP-binding nanobodies The GFP-binding nanobody (abbreviated GBP and published by Kubala, MH et al (2010) Protein Sci. 19(12)) was selected as the benchmark ISVD for the incorporation of the 10 rationally designed proposed N-glycosylation acceptor sites identified in Example 2. We included an artificial N-glycosylated tagged variant (QADDANATVQLVESGGA, instead of QVQLVESGGA in wild-type GBP). We also included a C-glyco-HIS tagged variant (VSSLQAAAAAANATVAAASGDVWDIHHHHHH, instead of VSSHHHHHH in wild-type (HIS-tagged) GBP). All of the GBP variants were equipped with a C-terminal histidine tag (6xHIS) to facilitate purification and / or detection.

[0179] The amino acid sequence of Nanobody GBP is represented in SEQ ID NO: 9. In SEQ ID NO: 9, the CDR1, CDR2 and CDR3 regions are underlined. SEQ ID NO:10 represents CDR1, SEQ ID NO:11 represents CDR2, SEQ ID NO:12 represents CDR3, SEQ ID NO:13 represents FR1, SEQ ID NO:14 represents FR2, SEQ ID NO:15 represents FR3, and SEQ ID NO:16 represents FR4.

[0180] SEQ ID NO:9: QVQLVESGGALVQPGGSLRLSCAAS GFPVNRYS MRWYRQAPGKEREWVAG MSSAGDRSS YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYC NVNVGFE YWGQGTQVTVSSHHHHH (121 amino acids)

[0181] SEQ ID NO: 10 (CDR1): GFPVNRYS SEQ ID NO: 11 (CDR2): MSSAGDRSS SEQ ID NO: 12 (CDR3): NVNVGFE SEQ ID NO: 13 (FR1): QVQLVESGGALVQPGGSLRLSCAAS SEQ ID NO: 14 (FR2): MRWYRQAPGKEREWVAG SEQ ID NO: 15 (FR3): YEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYC SEQ ID NO: 16 (FR4): YWGQGTQVTVSS

[0182] Figure 3 depicts an amino acid alignment of the GBP nanobody with the reference nanobody 3K74 from Example 1. N-linked glycosylation signatures were introduced into the GBP protein at hypothetical locations identified through rational analysis of the 3K74 reference nanobody crystal structure. The specific mutations introduced to obtain N-glycan acceptor sites in nanobody GBP are depicted in Figure 4.

[0183] Several chimeric genes were constructed: the coding sequences of wild-type GBP nanobody and various mutants with N-glycosylation acceptor sites introduced at specific positions were operably linked to the AOX1 promoter of Pichia pastoris (a methanol-inducible promoter) as depicted in Figure 4. The resulting expression vectors were introduced into three different strains of Pichia pastoris: wild-type (WT), GlycoSwitch M5 (GSM5) and GlycoDelete (GD). The GlycoSwitch M5 strain modifies its glycoproteins primarily with Man5GlcNAc2 structures (Jacobs, PP et al., (2009) Nat Protoc. 4(1)), whereas proteins expressed in the GlycoDelete strain are uniformly modified with a single GlcNAc residue (see, e.g., Claes, K. et al. (2016) ACS Synth Biol. 5(10), and the general GlycoDelete technology discussed in application WO2010015722).

[0184] Various cultures of recombinant Pichia pastoris were then first grown for 48 h at 28° C. in medium containing glycerol as the sole carbon source, and recombinant protein expression was then induced by replacing glycerol with methanol. After another 48 h at 28° C., growth medium (supernatant) was harvested from each recombinant culture. Culture supernatants were then either treated with endoglycosidase PNGaseF (to remove N-glycans) or mock treated, and assayed via Coomassie Blue stained SDS-PAGE and His-tag specific Western Blot. The results of this analysis are shown in Figures 5, 6 and 7.

[0185] Our data show that glycosylation of nanobody GBP was obtained for almost all their glycan variants (except for the S73N-K75T variant (aHo numbering)), although the efficiency of N-glycosylation varied. See Table 1 for an overview specifying the glycosylation efficiency. Note that this is also shown at the top of FIG. 4 (denoted as +++, +, and - for glycosylation efficiency). Surprisingly, four specific positions leading to extremely efficient glycosylation were identified from the nanobody protein structure: position 14, position 27, position 48, and position 86. Also surprising, two of these positions (27 and 86) have never been described for nanobodies regarding the introduction of N-glycan sites. Positions 14 and 48 are coincidentally cited in WO2016150845. Positions 14, 27, 48, and 86 follow the Aho numbering.

[0186] The GBP-R86N variant (aHo numbering) produced in the Pichia pastoris GlycoDelete background - modified in the loop between the D and E strands - was particularly highly efficiently glycosylated. 2+ It was extensively characterized after purification using affinity chromatography and gel filtration, followed by analysis using an ESI-QTOF mass spectrometer. The results are depicted in FIG.

[0187] Nanobody GBP-R86N occurs predominantly in its glycosylated form, and we show that this glycovariant allows highly efficient glycosylation.

[0188] To verify whether the functionality of the nanobody was retained, we analyzed the thermostability and GFP-binding affinity of both the unmodified GBP nanobody and four selected glycosylation variants at positions 14, 27, 48 and 86. The selected glycan variants were recombinantly produced in Pichia pastoris Kai3 (see Vervecken, N. et al (2007) Modification of the N-glycosylation pathway to produce homogeneous, human-like glycans using GlycoSwitch plasmids, in: JM Cregg (Ed.), Pichia Protocols, Humana Press, New York, pp. 119-138). The Pichia strain produces Man5GlcNAc2-type N-glycans on recombinant proteins. Melting curves of GBP-WT and its glycosylation variants were obtained from thermal shift assays using SYPRO Orange dye in a qPCR machine (Huynh K & Partch CL in Current Protocols in Protein Sciences 79, 2015).

[0189] If Man5GlcNAc2-type N-glycans are introduced at positions 14, 27 or 48, the shape of the melting curve changes: it shows only one denaturation peak instead of the two obtained for GBP-WT and the glycan variant R86N (see FIG. 12). However, the temperature at which thermal denaturation begins does not shift. Surprisingly, we observed that the nanobody function (antigen binding) is not impaired by the presence of N-glycans in the four specific regions: the GFP binding affinity is in the sub-nanomolar range (see FIG. 13). Only when the Man5GlcNAc2-type N-glycan occurs at position 48, the antigen affinity is moderately reduced (dissociation constant K D increases), which is mostly due to a decreased association rate (k on) (see Figure 13).

[0190] Example 4: Generalizing the approach To assess whether we could extrapolate these findings to other nanobodies, we introduced N-linked glycosylation signatures into several other nanobodies. In nanobody Nb41, designated NbCA4141 in Claes, K. et al (2016) ACS Synth Biol. 5(10), we introduced an N-glycosylation sequon at the site corresponding to K86N in the reference nanobody 3K74 and R86N in the GBP nanobody. The sequence of Nb41 is represented by SEQ ID NO: 17. SEQ ID NO: 18 represents CDR1, SEQ ID NO: 19 represents CDR2, SEQ ID NO: 20 represents CDR3, SEQ ID NO: 21 represents FR1, SEQ ID NO: 22 represents FR2, SEQ ID NO: 23 represents FR3, and SEQ ID NO: 24 represents FR4.

[0191] In nanobodies F-VHH-4 and F-VHH-L66 (Rossey, I. et al. (2017) Nat. Commun. 8, 14158), we introduced N-glycosylation sequons at the sites corresponding to G27N and / or R86N in the GBP, thus resulting in nanobodies carrying either one or two N-linked glycosylation signatures. The sequences of F-VHH-4 and F-VHH-L66 are represented in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

[0192] SEQ ID NO:17: QVQLQESGGGLVQPGGSLRLSCVAS GSIFSINA MGWYRQAPGKQRELVAA ISSGGRTN YADSVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYC NVGSWGFRSHSYLSGS SWGQGTQVTVSSHHHHHH (129 amino acids) The three CDR regions are underlined.

[0193] SEQ ID NO: 18 (CDR1): GSIFSINA SEQ ID NO: 19 (CDR2): ISSGGRTN SEQ ID NO: 20 (CDR3): NVGSWGFRSHSYLSGS SEQ ID NO: 21 (FR1): QVQLQESGGGLVQPGGSLRLSCVAS SEQ ID NO: 22 (FR2): MGWYRQAPGKQRELVAA SEQ ID NO: 23 (FR3): YADSVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYC SEQ ID NO: 24 (FR4): SWGQGTQVTVSS

[0194] SEQ ID NO:25 (F-VHH-4): QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGWFRQAPGKEREAVSCISGSSGSTYYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATIRSSSWGGCVHYGMDYWGKGTQVTVSSGSHHHHHHHH (135 amino acids) SEQ ID NO:26 (F-VHH-L66): QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGWFRQAPGKEREGVSCISSSHGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVAVAHFRGCGVDGMDYWGKGTQVTVSSGSHHHHHHHH (135 amino acids)

[0195] FIG. 9 depicts the sequence alignment of nanobodies Nb41, F-VHH-4, and F-VHH-L66 with nanobodies GBP and 3K74.

[0196] Wild-type Nb41 (without any N-glycosylation signature) was recombinantly produced in Pichia WT as outlined in Example 3. In Nb41, the K86N mutation is equivalent to the GBP-R86N mutation. Nb41-K86N was recombinantly produced in Pichia WT, Pichia GSM5, and Pichia GlycoDelete as outlined in Example 3. After induction of expression, supernatants of all recombinant products were harvested, PNGaseF-treated or mock-treated, and assayed via Coomassie Blue-stained SDS-PAGE and / or His-tag-specific Western blot analysis. A compilation of the data is shown in FIG. 10.

[0197] For Nb41, we show that glycosylation was absent in the WT nanobody (as expected), whereas introduction of a glycosylation signature at position 86 (AHo numbering) allowed efficient N-glycan modification.

[0198] Wild-type F-VHH-4 and F-VHH-L66 (without any N-glycosylation signature) and their variants carrying G27N and / or K86N mutations (corresponding to G27N and R86N in GBP, respectively; AHo numbering) were recombinantly produced in Pichia WT, Pichia GSM5 (same name as Pichia Kai3 strain) and Pichia GlycoDelete as outlined in Example 3. After expression induction, supernatants of all recombinant productions were harvested and assayed via Coomassie blue stained SDS-PAGE analysis, His-tag specific Western blot analysis and mass spectrometry analysis. Coomassie blue and Western blot data are shown in Figure 23.

[0199] For F-VHH-4 and F-VHH-L66, we found that glycosylation was absent in the WT nanobody (as expected), whereas introduction of glycosylation signatures at positions 27 or 86 (AHo numbering) allowed efficient N-glycan modification. Simultaneous introduction of N-linked glycosylation signatures at positions 27 and 86 in the same backbone led to efficient N-glycan modification at both sites.

[0200] To objectively evaluate the glycosylation efficiency at positions 27 and 86 (AHo numbering), we performed densitometry analysis on His-tag-specific Western blots of VHH glycovariants produced in Pichia pastoris GlycoSwitchM5. The site occupancy of F-VHH-4-G27N was determined to be 96% (Figure 23), that of F-VHH-4-R86N was 93% (Figure 23), that of GBP-G27N-P30T was 86% (Figure 7), and that of GBP-R86N was 93% (Figure 5). For all other nanobody variants with N-glycosylation sites at positions 27 and 86, the lower bands (corresponding to non-glycosylated nanobodies) could be detected, even if the fluorescence increment was set low enough to prevent overexposure of the upper bands (corresponding to N-glycosylated nanobodies). This suggests at least 80% N-glycosylation.

[0201] A compilation of glycosylation efficiencies obtained with GBP, Nb41, F-VHH-4 and F-VHH-L66 nanobodies in different recombinant Pichia pastoris backgrounds is summarized in Table 1 (also see Figure 11 specifically for GBP mutants). [Table 2-1] [Table 2-2]

[0202] Table 1 Overview of N-glycosylated nanobody variants. Pichia strains: WT = wild type, GSM5 = GlycoSwitchM5 (alternative name for Pichia Kai3 strain), GD = GlycoDelete. N-glycosylation types: HighMan = high-mannose N-glycosylation, Man5 = Man5GlcNAc2, and GlcNAc = single GlcNAc residue. * For the nanobody GBP-R86N produced in Pichia GlycoDelete, N-glycosylation was verified and quantified by ESI-QTOF mass spectrometry. CB = Coomassie Brilliant Blue stained SDS-PAGE analysis, WB = His-tag specific Western blot analysis, MS = Mass spectrometric analysis.

[0203] RSV-specific VHHs produced in different Pichia backgrounds, F-VHH-L66 and F-VHH-4 (WT, GSM5 (shown in FIG. 14 as GS Man5), and GlycoDelete), were assayed for their neutralizing capacity in a RSV neutralization assay. Briefly: Vero cells were seeded at a density of 15.000 cells / well in 96-well plates and cultured at 37° C. The next day, 4-fold serial dilutions of purified VHHs (starting at 400 ng / ml) in Optimem were mixed with an equal volume of RSV A2 (1.34 PFU / μl) and incubated for 30 min at 37° C.

[0204] Vero cells were then washed once with Optimem and the Optimem was replaced with 50 μl of the VHH-virus mixture. The cells were incubated at 37° C. for 3 h. After 3 h, 50 μl of DMEM+1.2% Avicel+1% FCS was added to the cells and the cells were further incubated for 3 days at 37° C. Finally, the cells were fixed with 2% paraformaldehyde, stained with a polyclonal goat anti-RSV antibody and the plaques were counted. The results for VHH F-VHH-L66 are shown in FIG. 14.

[0205] Our results show that the introduction of glycosylation sites in these VHHs does not block VHH binding to RSV and neutralization of RSV. Nevertheless, the effect of the (type of) glycan(s) present can be discerned. The size of the glycan appears to be inversely correlated with the neutralization capacity of the VHH, suggesting that glycans with a large hydrodynamic radius interfere more with neutralization than smaller glycans (WT glycan > Man5 glycan > GlycoDelete glycan). Moreover, for these VHHs, the glycan at site 27 (AHo numbering) appears to interfere slightly more with neutralization than the glycan at site 86 (AHo numbering); the presence of glycans at both sites has an additive effect.

[0206] We therefore conclude, surprisingly, that the introduction of small GlycoDelete glycans (GlcNAc, LacNAc, and sialyl-LacNAc) at various positions in a VHH does not lead to a reduction in the efficiency of the VHH, as shown here for a VHH directed against the RSV virus.

[0207] We conclude that positions within the ISVD that allow efficient N-glycosylation (as applied here to nanobodies) can be rationally selected based on structural criteria, that these positions are conserved within the ISVD, and that our concept can be applied to a variety of nanobodies. Specifically preferred positions within the ISVD sequence that lead to efficient N-glycan production without compromising ISVD function are the two regions 83-88 and 27-40 (AHo numbering).

[0208] Example 5: Generalization of positions identified for efficient N-glycan introduction in a genus of ISVDs The sequences of 222 VHH coding strands (PDB search was performed with the query "VHH" or nanobody and one strand was selected per resulting PDB ID containing either the macromolecule name "nanobody", "nb", "nab", "vhh" or "cab"; or the source species is Camelus dromedarius, Lama glama, or Vicugna pacos; or the conserved amino acid sequence "VQL" is in the first 40 amino acid residues) were extracted from the RCSB Protein Data Bank (in short PDB) and aligned using PyMOL (PyMOL Molecular Graphics System, Version 1.3 Schroedinger, LLC.) In each of these VHHs, the region between amino acids 83 and 88 (AHo) and the region between amino acids 27 and 40 (AHo) constitute the amino acid strands connecting the beta strands of the secondary structure.

[0209] We subsequently confirmed the presence of these conserved linker regions in almost 100% (1667 out of 1668) of the VHH coding sequences aligned in the Antibody Variable Domain Database (ABVDDB). Without being limited to a specific hypothesis, we hypothesize that in these selected regions, the glycan substituents project away from the antigen-binding region so as to minimize interference with antigen recognition. We conclude that glycosylation acceptor sites introduced at positions located within these two conserved regions can be used for the introduction of artificial N-glycan sites that are predicted to be efficiently N-glycosylated in all nanobodies.

[0210] In a next step, we generated a range of expression plasmids encoding GBP nanobody variants by introducing an NxT sequon at any single position between amino acids 83 and 88 (AHo) and between amino acids 27 and 40 (AHo), such that in each of the variants, an N (Asn) is present in the sequon but at various positions (e.g., 82, 83, 84, 85, the artificially inserted sequence between 85 and 86, 86, 87, 88, 26, 27, 29, 30, 31, 32, 33, the artificially inserted sequence between 33 and 39, 39, or 40) (see FIG. 15 ).

[0211] Specifically, note that for the GBP nanobody amino acid sequence (AHo-numbering), positions 28 and 34-38 are absent. All these nanobody variants were produced in recombinant Pichia pastoris Kai3 strain as described above - variants with amino acids mutated to arginine at positions 29 and 85 (F29N-P30A-V31T and A85N) did not transform efficiently in Pichia pastoris and were not evaluated. Supernatants of all recombinant variants were subsequently harvested, PNGaseF-treated or mock-treated, and assayed for the presence of N-glycosylation via Coomassie Brilliant Blue-stained SDS-PAGE and / or His-tag specific Western blot analysis (see FIG. 16).

[0212] The presence of N-glycans could be confirmed in each of the expressed nanobody variants; however, the efficiency of N-glycan addition (site occupancy) potentially depends on the exact amino acid position chosen for the introduction of the N-glycosylation sequon (see Table 2). To confirm that the glycans present are indeed (GlcNAc)2Man5, supernatant samples of wild-type GBP, glycovariants in each selected region (GBP-P30A-V31N-R33T in region 27-40, and GBP-D84N-A85T in region 83-88), and glycovariants containing an artificial insert in the first region (GBP-33-39-longinsert) were analyzed by intact protein mass spectrometry (Figure 16, bottom right). For each glycovariant, a peak was observed indicating the presence of a 1216 Da (GlcNAc)2Man5 modification.

[0213] The above results show that nanobodies can be effectively glycosylated in rationally selected and conserved regions where the presence of N-glycans does not affect antigen recognition and protein folding. These data pave the way for glycan-mediated targeting and site-specific glycan-based conjugation strategies. [Table 3]

[0214] Table 2 : Overview of N-glycosylated GBP variants within selected regions. Pichia strains GSM5=GlycoSwitchM5. Man5=Man5GlcNAc2. * For nanobody GBP-R86N produced in Pichia GlycoDelete, N-glycosylation was quantified by SDS-PAGE and anti-HIS Western blot (WB) detection with Coomassie Brilliant Blue (CB). The origin of the pKai61 vector is described in Schoonooghe S et al (2009) BMC Biotechnol. 9, 70.

[0215] Example 6: Development of glycan-specific conjugation methods The data from the previous examples convincingly show that ISVDs (exemplified by nanobodies) can be effectively glycosylated in rationally selected and conserved regions where the presence of glycans does not affect antigen recognition and protein folding. These data pave the way for glycan-mediated targeting of ISVDs and site-specific glycan-based conjugation strategies. In the following examples, we use nanobodies with simple and uniform N-linked glycans at artificially engineered N-glycosylation sites (as outlined in Examples 3, 4 and 5) for the application of glycan-specific conjugation methods.

[0216] Indeed, N-glycans containing only GlcNAc and / or only LacNAc and / or only Sialyl-LacNAc can be obtained by in vitro approaches or in vivo via the GlycoDelete technology as described in WO2010015722 or via the GlycoDoubleDelete technology as described in WO2017005925. Simple N-glycans provide a bioorthogonal stalk on the protein that can be used for coupling to a wide variety of desired components - e.g. PEG chains, chelators, toxic drugs, etc. Different glycan-based conjugation chemistries are evaluated / optimized using commercially available biotinylated PEG. Essentially, there are two broad methods for conjugation: a traditional chemical method as exemplified in Example 7 and a combined chemical and enzymatic conjugation method as exemplified in Example 8.

[0217] Example 7: Chemical conjugation strategies In this example, we show how nanobodies with an artificially introduced N-glycan at position 86 (Aho numbering) can be specifically modified with PEG-biotin on the glycan. Nanobodies are first recombinantly expressed in HEK293 GlycoDelete cells (see WO2010015722), HEK293 GlycoDoubledelete cells (see WO2017005925), Pichia-GlycoDelete cells, or Pichia-GlycoDelete cells overexpressing galactosyltransferase. Depending on the GlycoDelete method used, nanobodies modified with glycans selected from the group consisting of GlcNAc, LacNAc, and sialyl-LacNAc are obtained.

[0218] According to the first conjugation strategy, vicinal diol(s) in the glycan are oxidized by sodium periodate (NaIO4). Early versions of this chemistry have been used for decades, for example to generate fluorescently labeled antibodies. Glycoengineered nanobodies obtained via the GlycoDelete technology carry glycans where periodate oxidation produces pure products (in contrast to the situation for wild-type glycans). LacNAc-type glycans (GlcNAc-Gal) contain a single vicinal "cis" diol in a galactose residue (at the C3 and C4 ring positions) that can be oxidized.

[0219] Sialyl-LacNAc type glycans contain vicinal diols in the glycerol side chains of terminal sialic acid residues, in addition to vicinal cis diols in galactose residues, which are susceptible to periodate oxidation. The vicinal diols in sialic acid are much more readily oxidized by periodate than galactose, yet allow for the use of milder oxidation conditions that favor sialic acid oxidation while still retaining product homogeneity. Periodate oxidation of vicinal diols present in glycans creates free aldehyde groups that can readily react with aminooxy-containing molecules to form oximes, which are readily stable in water.

[0220] Alternatively, the free aldehyde can be reacted with a hydrazine-containing molecule to form a stable hydrazone linkage or can be linked to an amine-containing molecule via reductive amination. LacNAc and sialyl-LacNAc glycans conjugated in this manner retain an intact GlcNAc residue directly linked to an asparagine of the protein, which is preferred in terms of conjugate degradability in the lysosome. Schematic diagrams of LacNAc- and sialyl-LacNAc-based periodate oxidation followed by oxime ligation are illustrated in Figure 17 (LacNAc) and Figure 18 (sialyl-LacNAc).

[0221] Briefly, GBP carrying the R86N mutation was recombinantly produced in HEK293 GlycoDelete cells and purified to produce a mixture of non-glycosylated protein and protein carrying either a single LacNAc chain or a sialyl-LacNAc chain. The purified protein was then subjected to mild periodate oxidation, followed by oxime ligation to a short biotinylated and aminooxy-modified PEG chain. Mass spec analysis showed that the PEG chain was selectively ligated to the sialyl-LacNAc-bearing GBP.

[0222] Example 8: Chemoenzymatic conjugation strategies In this example, an alternative conjugation strategy is applied. Instead of periodate oxidation of glycans, we use the enzyme galactose oxidase (GAO) to oxidize the C6 hydroxyl group of Gal residues in LacNAc glycans artificially introduced on the nanobody of interest (as obtained in Example 7). This enzymatic oxidation also creates free aldehyde groups, which can be linked to the molecule of interest via oxime ligation, hydrazone ligation, or reductive amination as described in Example 7 (Park, A. et al., Endocrinology 154, 2013).

[0223] A schematic diagram of GAO-based LacNAc oxidation followed by oxime ligation is illustrated in Figure 19. In a similar approach, a mutant form of galactose oxidase (GAO-F2; see Rannes, JB et al. (2011), J. Am. Chem. Soc., 133, 8436-8439) can be used to oxidize the C6 hydroxyl group of GlcNAc; a cargo of interest can then be directly linked to the GlcNAc residue. A schematic diagram of GAO-F2-based GlcNAc oxidation followed by oxime ligation is illustrated in Figure 20.

[0224] Briefly, GBP carrying the R86N mutation was recombinantly produced in Pichia GlycoDelete cells or in Pichia GlycoDelete cells co-expressing galactosyltransferase. Proteins were purified and mixtures of non-glycosylated protein and protein carrying a single GlcNAc residue (Pichia-GlycoDelete) or mixtures of non-glycosylated protein and protein carrying GlcNAc or LacNAc (Pichia-GlycoDelete co-expressing galactosyltransferase) were produced.

[0225] The purified protein from Pichia-GlycoDelete co-expressing galactosyltransferase was then oxidized with GAO and linked to a short PEG chain that was biotinylated and aminooxy modified in a one-pot reaction. Mass spectrometry showed that the PEG chain was selectively linked to the LacNAc-bearing GBP. The purified protein from Pichia-GlycoDelete was then oxidized with GAO-F2 and linked to a short PEG chain that was biotinylated and aminooxy modified in a one-pot reaction. Mass spectrometry showed that the PEG chain was selectively linked to the GlcNAc-bearing GBP.

[0226] In an alternative chemical-enzymatic strategy, we can use the enzyme hST6Gal1 (Wu, ZL, Carbohydrate Research 412, 2015) to conjugate an azide-modified form of Sia (AzSia) to LacNAc or a mutant form of human beta-1,4-galactosyl / GalNAc transferase (van Geel, R., Bioconjug. Chem. 26, 2015) to conjugate an azide-modified form of GalNAc (GalNAz) to the N-glycan of a single GlcNAc present on our nanobody of interest. GalNAz / AzSia introduced on the nanobody via the azide functionality can be homogeneously and site-specifically functionalized with PEG chains or another molecule of interest employing click chemistry (e.g., copper-free azide-alkyne cycloaddition reaction). Schematics are illustrated in FIG. 21 (GalNAz) and FIG. 22 (AzSia).

[0227] Azide-modified GlcNAc, LacNAc, and sialyl-LacNAc glycans can also be obtained by feeding azide-modified monosaccharide precursors (GlcNAz, GalNAz, AzSia) to GlycoDelete cells producing the protein of interest; this allows for subsequent site-specific functionalization via click chemistry.

[0228] Briefly, GBP carrying the R86N mutation was recombinantly produced in Pichia GlycoDelete cells or in Pichia GlycoDelete cells co-expressing galactosyltransferase. Proteins were purified and mixtures of non-glycosylated protein and protein carrying a single GlcNAc residue (Pichia-GlycoDelete) or mixtures of non-glycosylated protein and protein carrying GlcNAc or LacNAc (Pichia-GlycoDelete co-expressing galactosyltransferase) were produced.

[0229] The purified protein from Pichia-GlycoDelete co-expressing galactosyltransferase was then incubated with CMP-azido-sialic acid and recombinant hST6Gal1 enzyme, which adds AzSia residues to LacNAc chains, followed by a click reaction with a short PEG chain that was biotinylated and DBCO-modified. Mass spectrometry showed that the PEG chain was selectively linked to the LacNAc-bearing GBP. The purified protein from Pichia-GlycoDelete was incubated with UDP-GalNAz and a mutant form of human beta-1,4-galactosyl / GalNAc transferase enzyme, which adds GalNAz residues to single GlcNAc glycans, followed by a click reaction with a short PEG chain that was biotinylated and DBCO-modified. Mass spectrometry showed that the PEG chain was selectively linked to the GlcNAc-bearing GBP.

Claims

1. 1. A polynucleotide encoding a polypeptide comprising an immunoglobulin single variable domain (ISVD) derived from a camelid heavy chain antibody, wherein the ISVD comprises an amino acid sequence comprising four framework regions (FRs) and three complementarity determining regions (CDRs) according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(1), wherein the ISVD has an N-glycosylation acceptor site present at at least an asparagine residue at amino acid 86 of the ISVD (according to the AHo numbering convention).

2. 2. A polynucleotide encoding a polypeptide comprising the ISVD of claim 1, wherein the ISVD contains an NXT or NXS motif, where X can be any amino acid, such that an asparagine residue in the NXT / NXS motif is present at an amino acid selected from the amino acid range 83-88 of the ISVD (according to the AHo numbering convention).

3. An expression vector comprising the polynucleotide of claim 1 or 2.

4. A cell comprising the expression vector of claim 3.

5. 5. The cell of claim 4, wherein the cell is a higher eukaryotic cell, such as a mammalian cell or a plant cell, a lower eukaryotic cell, such as a filamentous fungal cell or a yeast cell, or a prokaryotic cell.

6. The cell of claim 4 or 5, wherein the cell is a glycomodified cell.

7. A polypeptide comprising an ISVD encoded by the polynucleotide of claim 1 or 2.

8. 8. A polypeptide comprising the ISVD of claim 7 which is glycosylated and comprises one or more glycans, wherein the glycans have terminal GlcNAc, GalNAc, galactose, sialic acid, glucose, glucosamine, galactosamine, bacillosamine, mannose or mannose-6-P sugars, or chemically modified monosaccharides such as GalNAz, GlcNAz and azido-sialic acid.

9. 9. A polypeptide comprising the ISVD of claim 8, wherein the glycosylation of the polypeptide is comprised of one or more glycans selected from the group consisting of GlcNAc, LacNAc, sialyl-LacNAc, Man5GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, hyper-mannosylated glycans, mannose-6-phosphate glycans, complex glycans, hybrid glycans, and chemically modified glycans such as GlcNAz, GlcNAc-GalNAz, and azido-sialic acid-LacNAc.

10. Use of a polypeptide according to any one of claims 7 to 9 for glycan-specific conjugation.

11. An ISVD conjugate comprising a polypeptide according to any one of claims 7 to 9 and a conjugated component.

12. The ISVD conjugate of claim 11, wherein the conjugated moiety is attached to an N-linked glycan.

13. The ISVD conjugate of claim 11 or 12, wherein the conjugated moiety comprises a half-life extending moiety, a therapeutic agent, a detection unit or a targeting moiety.

14. 10. A method for producing a polypeptide according to any one of claims 7 to 9, the method comprising introducing an expression vector according to claim 3 into a suitable cell, expressing the polypeptide and isolating it.

15. A method for producing an ISVD conjugate according to any one of claims 11 to 13, the method comprising introducing an expression vector according to claim 3 into a suitable cell, expressing a polypeptide according to any one of claims 7 to 9, and linking a conjugated component to the polypeptide.

16. Use of a polypeptide comprising an ISVD according to any one of claims 7 to 9 or an ISVD conjugate according to any one of claims 11 to 13 for modulating circulating half-life, for increasing glycoprotein stability, for selective targeting, for modulating immunogenicity, for preventing antibody pre-binding or for detection purposes.

17. A pharmaceutical composition comprising a polypeptide according to any one of claims 7 to 9 or an ISVD conjugate according to any one of claims 11 to 13.

Images