Glycosylation of a single variable domain of immunoglobulin

Abstract

The present invention relates to the glycosylation of immunoglobulin single variable domains (ISVDs). In particular, the present invention relates to specific positions within the amino acid sequence of an ISVD for use as a glycosylation acceptor site. The present invention also relates to ISVDs modified with specific glycans at specific glycosylation acceptor sites and their conjugates. The present invention also relates to at least one ISVD of the present technology, at least one ISVD having a specific glycan, or at least one conjugate thereof. The present invention further relates to nucleotide sequences or nucleic acids encoding such ISVDs and / or polypeptides, methods for producing such ISVDs and / or polypeptides, compositions comprising such ISVDs, conjugates and / or polypeptides, and the use in pharmaceuticals of polypeptides, nucleic acid sequences or nucleic acids, conjugates, or compositions according to the present technology.

Description

[Technical Field]

[0001] This technology relates to the field of glycosylation of immunoglobulin monovariate domains (ISVDs). More specifically, this technology provides specific locations within the amino acid sequence of an ISVD for use as a glycosylation acceptor site. This technology also relates to ISVDs and their conjugates modified with specific glycans at specific glycosylation acceptor sites. This technology also relates to polypeptides comprising at least one ISVD of this technology, at least one ISVD having a specific glycan, or at least one conjugate thereof. The present invention further relates to nucleotide sequences or nucleic acids encoding such ISVDs and / or polypeptides, methods for producing such ISVDs and / or polypeptides, and compositions comprising such ISVDs, conjugates, and / or polypeptides. [Background technology]

[0002] In recent years, antibody therapy has rapidly gained attention in the medical community. Its ability to selectively target only disease-affected tissue while leaving healthy tissue intact is highly desirable for improving patients' quality of life.

[0003] Antibody therapy is target-specific, but it often encounters problems due to the handling of such complex biomolecules, such as issues with stability or solubility. As a result, there is a constant desire to improve existing antibody therapies.

[0004] Glycosylation is a post-translational modification commonly applied in antibody therapy, particularly in monoclonal antibodies. Glycosylation is a process that may be necessary to achieve the desired therapeutic efficacy. In addition, glycosylation can facilitate the conjugation of one antibody to another and / or other parts, potentially providing the antibody with additional functionality.

[0005] Immunoglobulin single variable domains (ISVDs) exhibit interesting therapeutic potential in the field of antibody therapy due to their small size. Furthermore, their production is far simpler, faster, and cheaper than the production of monoclonal antibodies.

[0006] The glycosylation of ISVD at specific glycosylation acceptor sites is described in International Publication No. 2016 / 150845, International Publication No. 2018 / 206734, and International Publication No. 2021 / 116252.

[0007] However, glycosylation of ISVD has proven difficult. Depending on the host cell / organism used, glycosylation may not occur at all or may occur in a limited manner. In addition, the introduction of glycosylation acceptor sites often leads to interference with the folding of the ISVD protein, resulting in loss of affinity and / or stability, and therefore loss of functionality.

[0008] However, glycosylation can promote conjugation to other sites of ISVD, potentially affecting therapeutic efficacy; therefore, effective and stable glycosylated ISVD remains necessary. [Overview of the Initiative] [Means for solving the problem]

[0009] This technology aims to solve problems related to the glycosylation of ISVD. Accordingly, in a first aspect, the technology relates to a polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariate domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0010] In another aspect, the technology relates to a polypeptide comprising or (essentially) consisting of one ISVD, wherein the ISVD comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0011] In another aspect, the technology relates to a polypeptide comprising or (essentially) consisting of one ISVD, wherein the ISVD comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 according to Kabat numbering.

[0012] In a further aspect, the technology relates to a polypeptide comprising or (essentially) consisting of two ISVDs, wherein at least one of the two ISVDs comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 105, 108, and 110 according to Kabat numbering.

[0013] In yet another aspect, the technology relates to a polypeptide comprising or (essentially) consisting of two ISVDs, wherein at least one of the two ISVDs comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 3, 19, 26, 55, 105 and 108 according to Kabat numbering.

[0014] In another aspect, the technology relates to a polypeptide comprising or (essentially) consisting of two ISVDs, wherein the N-terminal ISVD comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering.

[0015] In yet another embodiment, the technology relates to a polypeptide comprising or (essentially) two ISVDs, wherein the C-terminal ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 19, 26, 55, 105, and 108 according to Kabat numbering.

[0016] In a further embodiment, the technology relates to a polypeptide comprising at least three ISVDs, wherein at least one of the at least three ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0017] In yet another embodiment, the technology relates to a polypeptide comprising or (essentially) three ISVDs, wherein at least one of the three ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0018] In another embodiment, the technology relates to a polypeptide comprising or (essentially) three ISVDs, wherein the N-terminal ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 15, 19, 26, 55, 73, 75, 76, 105, 108, and 110 according to Kabat numbering.

[0019] In a further embodiment, the technology relates to a polypeptide comprising or (essentially) three ISVDs, wherein the C-terminal ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, and 105 according to Kabat numbering.

[0020] In yet another embodiment, the technology relates to a polypeptide comprising or (essentially) three ISVDs, wherein the ISVDs, which are neither C-terminal nor N-terminal, contain glycosylation acceptor sites located at amino acid positions selected from 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0021] This technology also relates to such polypeptides that are glycosylated at one or more of these glycosylated acceptor sites.

[0022] In another embodiment, the technology relates to a nucleotide sequence or nucleic acid encoding a polypeptide by the technology.

[0023] In a further embodiment, the technology is a method for producing polypeptides according to the technology, - To express nucleotide sequences or nucleic acids using this technology in suitable host cells or host organisms. The present invention relates to a method that includes a host cell or host organism capable of glycosylation of an expressed polypeptide.

[0024] In yet another embodiment, the technology relates to a method for partially conjugating polypeptides.

[0025] This technology also provides a conjugate comprising a glycosylated polypeptide and a conjugate site, wherein the portion is conjugated to a glycan on the polypeptide.

[0026] In another embodiment, the Technology relates to compositions comprising polypeptides, nucleic acids, or conjugates according to the Technology. [Brief explanation of the drawing]

[0027] [Figure 1]The glycan profile of glycosylated ISVD T043800005, measured by LC-QTOF, is shown. [Figure 2] The glycan profile of glycosylated ISVD T043800002, measured by LC-QTOF, is shown. [Figure 3] The MALDI-TOF measurement of the ISVD T043800005 conjugate using bis-mannose-6-phosphate as described in Example 12 is shown. [Figure 4] The MALDI-TOF measurement of the ISVD T043800002 conjugate using bis-mannose-6-phosphate as described in Example 12 is shown. [Figure 5A] Flow cytometry measurements are shown to evaluate TNF internalization in Jurkat cells (A) or K562 cells (B) after treatment with various concentrations of TNF complex and anti-TNF ISVD or bis-M6P-anti-TNF ISVD conjugate for 1 and 4 hours. [Figure 5B] Flow cytometry measurements are shown to evaluate TNF internalization in Jurkat cells (A) or K562 cells (B) after treatment with various concentrations of TNF complex and anti-TNF ISVD or bis-M6P-anti-TNF ISVD conjugate for 1 and 4 hours. [Figure 6A-6B] This shows the increased mean fluorescence intensity (MFI) in Jurkat cells (A) or K562 cells (B) that demonstrates TNF internalization by the bis-M6P-anti-TNF ISVD conjugate compared to anti-TNF ISVD alone. [Figures 7A-7B] Western blotting of TNF-α at various time points is shown in (A) cell lysates after internalization at 37°C for 2 hours, followed by treatment with or without bafilomycin for 4 hours and 24 hours, and in (B) supernatant from the same K562 cell lysates, using either anti-TNF ISVD or bis-M6P-anti-TNF ISVD. [Modes for carrying out the invention]

[0028] 1.Definition Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art and will be obvious to those skilled in the art. For example, Sambrook et al. (Molecular Cloning: A Laboratory Manual (2nd.Ed.) Vols. 1-3, Cold Spring Harbor Laboratory Press, 1989), F. Ausubel et al. (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York, 1987), Lewin (Genes II, John Wiley & Sons, New York, NY, 1985), Old et al. (Principles of Gene Manipulation: An Introduction to Genetic Engineering (2nd edition) University of California Press, Berkeley, CA, 1981); Roitt et al. (Immunology (6th Ed.) Mosby / Elsevier, Edinburgh, 2001), Roitt et al. (Roitt's Essential Immunology (10th Ed.) Blackwell Publishing, UK, 2001), and Janeway et al. al.(Immunobiology(6th See standard handbooks such as (ed.) Garland Science Publishing / Churchill Livingstone, New York, 2005), as well as the general background technologies cited herein.

[0029] Unless otherwise specified, all methods, processes, techniques, and operations not described in detail may be carried out or performed in a manner known to those skilled in the art. For example, in this case as well, please refer to the standard handbook and the general background techniques listed herein and the further references cited therein, as well as the following reviews which describe protein engineering techniques such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins: Presta (Adv. Drug Deliv. Rev. 58(5-6):640-56, 2006), Levin and Weiss (Mol. Biosyst. 2(1):49-57, 2006), Irving et al. (J. Immunol. Methods 248(1-2):31-45, 2001), Schmitz et al. (Placenta 21 Suppl. A:S106-12, 2000), and Gonzales et al. (Tumour Biol. 26(1):31-43, 2005).

[0030] As used herein, the term “sequence” (for example, “immunoglobulin sequence,” “antibody sequence,” “variable domain sequence,” “VHH sequence,” or “protein sequence”) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding it, unless a more restricted interpretation is required in the context.

[0031] Nucleic acids or amino acids are considered "(essentially) isolated" compared to, for example, reaction medium or culture medium if they have been separated from at least one other component normally associated with the source or medium, such as another nucleic acid, another protein / polypeptide, another biological component or macromolecule, or at least one contaminant, impurity or trace component. In particular, nucleic acids or amino acids are considered "(essentially) isolated" if they have been purified by at least 2 times, especially at least 10 times, even more especially at least 100 times, and up to 1000 times or more. Nucleic acids or amino acids in "(essentially) isolated form" are preferably essentially homogeneous when determined using a preferred technique, such as a preferred chromatographic technique such as polyacrylamide gel electrophoresis.

[0032] When a nucleotide sequence or amino acid sequence is said to "contain" or "essentially consist of" another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence is incorporated into the first-referenced nucleotide sequence or amino acid sequence, but more usually, it means that the first-referenced nucleotide sequence or amino acid sequence contains within its sequence stretches of nucleotide or amino acid residues having identical nucleotide or amino acid sequences to the latter sequence, regardless of how the first-referenced sequence was actually generated or obtained (e.g., by any preferred method described herein). Using a non-restrictive example, when a polypeptide is said to contain an immunoglobulin monovariate domain, this may mean that the immunoglobulin monovariate domain sequence is incorporated into the polypeptide sequence, but more usually, it means that the polypeptide contains the immunoglobulin monovariate domain sequence within its sequence, regardless of how the polypeptide was generated or obtained. Furthermore, when a nucleic acid or nucleotide sequence is said to contain another nucleotide sequence, the first-mentioned nucleic acid or nucleotide sequence is preferably such that, when expressed as an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of the expression product (in other words, the latter nucleotide sequence is within the same reading frame as the larger nucleic acid or nucleotide sequence first mentioned).

[0033] "Consists of (essentially)" means that either the latter nucleic acid sequence or amino acid sequence is identical to a polypeptide (e.g., CDR region; ISVD), or that it corresponds to a polypeptide (e.g., CDR region; ISVD) having a limited number of amino acid residues attached to the amino terminus, carboxyl terminus, or both the amino and carboxyl terminus of an immunoglobulin monovariable domain, for example, 1 to 20 amino acid residues, for example, 1 to 10 amino acid residues, preferably 1 to 6 amino acid residues, for example, 1, 2, 3, 4, 5, or 6 amino acid residues.

[0034] Amino acid residues are indicated according to standard three-letter or one-letter amino acid codes. See Table A-2 on page 48 of International Publication No. 08 / 020079.

[0035] 2. Polypeptides having glycosylated acceptor sites and ISVD The glycosylation of ISVD presents a challenging problem. We have now discovered that glycosylation acceptor sites can be introduced at previously unknown locations, and that these sites provide ISVDs with good glycosylation properties, while glycosylation does not hinder ISVD binding and folding, and consequently provides good conjugation properties, for example, in the production of ISVD conjugates.

[0036] Glycosylation is a reaction in which a carbohydrate (or "glycan"), i.e., a glycosylated donor, is bound to a hydroxyl or other functional group (glycosyl acceptor) of another molecule to form a glycoconjugate. In biology, glycosylation usually refers to an enzyme-catalyzed reaction. Glycosylation is a form of co-translational and post-translational modification. Most proteins synthesized in the rough endoplasmic reticulum undergo glycosylation. Glycosylation is also present in the cytoplasm and nucleus as O-GlcNAc modification. Different classes of glycans are produced: N-linked glycans, bonded to the nitrogen of the asparagine or arginine side chain; O-linked glycans, bonded to the hydroxyl oxygen of the serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chain, or to the oxygen on lipids such as ceramide; phosphoglycans, bonded through the phosphate of phosphoserine; and C-linked glycans, a rare form of glycosylation in which a sugar is added to the carbon on the tryptophan side chain.

[0037] As used herein, “glycan” generally refers to glycosidic monosaccharides, oligosaccharides, and polysaccharides. Therefore, the carbohydrate portion of glycoconjugates such as glycoproteins, glycolipids, or proteoglycans is referred to herein as “glycan.” Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. N-linked glycans may consist of galactose, neuraminic acid, N-acetylglucosamine (GalNAc), fucose, mannose, and other monosaccharides, as further illustrated herein. In eukaryotes, O-linked glycans assemble one sugar at a time on a serine or threonine residue of a peptide chain in the Golgi apparatus. Unlike N-linked glycans, there is no known consensus sequence, however, the position of either a -1 or +3 proline residue relative to serine or threonine is preferred for O-linked glycosylation.

[0038] The term "immunoglobulin single variable domain" (ISVD), used synonymously with "single variable domain," defines an immunoglobulin molecule in which the antigen-binding site resides on a single immunoglobulin domain and is formed by a single immunoglobulin domain. This term distinguishes an immunoglobulin single variable domain from "conventional" immunoglobulins (e.g., monoclonal antibodies) or their fragments (e.g., Fab, Fab', F(ab')2, scFv, di-scFv) in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL contribute to the antigen-binding site; that is, a total of six CDRs are involved in the formation of the antigen-binding site.

[0039] In light of the above definition, the antigen-binding domains of conventional four-chain antibodies (e.g., IgG, IgM, IgA, IgD, or IgE molecules known in the art), or of Fab fragments, F(ab')2 fragments, Fv fragments, such as disulfide-linked Fv or scFv fragments, or diabodies derived from such conventional four-chain antibodies (all known in the art) are not usually considered to be single immunoglobulin variable domains, because in these cases, the binding of the antigen to the corresponding epitope is usually not by a single immunoglobulin domain, but by a pair of (associated) immunoglobulin domains, such as light-chain and heavy-chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains that bind together to the epitope of the corresponding antigen.

[0040] In contrast, a single immunoglobulin variable domain can specifically bind to an antigen epitope without pairing with an additional immunoglobulin variable domain. The binding site of a single immunoglobulin variable domain is formed by a single VH, a single VHH, or a single VL domain.

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

[0042] The immunoglobulin single variable domain (ISVD) may be a heavy chain ISVD such as VHH containing humanized VHH, camelid VH, or VH containing human VH. In one embodiment, this is VHH, camelid VH, or humanized VHH. The heavy chain ISVD may be derived from a conventional quadruple-chain antibody or a heavy chain antibody.

[0043] For example, an immunoglobulin monovariate domain may be a monodomain antibody (or an amino acid sequence suitable for use as a monodomain antibody), "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or NANOBODY® ISVD (as defined herein, including but not limited to VHH); another monovariate domain, or any suitable fragment of any one of them. [Note: NANOBODY® and NANBODIES® are registered trademarks of Ablynx NV]

[0044] In particular, the immunoglobulin monovariate domain may be NANOBODY® ISVD (VHH including humanized VHH or camelid VH) or a suitable fragment thereof.

[0045] The "VHH domain," also known as VHH or VHH antibody fragment, was originally described as the antigen-binding immunoglobulin variable domain of "heavy chain antibodies" (i.e., "antibodies without a light chain"; Hamers-Casterman et al. Nature 363:446-448, 1993).

[0046] The term "VHH domain" is chosen to distinguish these variable domains from the heavy chain variable domains (referred to herein as "VH domains") present in conventional quadruple antibodies and from the light chain variable domains (referred to herein as "VL domains") present in conventional quadruple antibodies. For a further explanation of VHH, see the review by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0047] The generation of immunoglobulin sequences such as VHHs is widely described in various published literature, including International Publication No. 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001). In these methods, camelids are immunized with a target antigen to induce an immune response to the target antigen. The repertoire of VHHs obtained from this immunization is further screened for VHHs that bind to the target antigen. In these examples, antibody production requires purified antigens for immunization and / or screening. Antigens can be derived from natural sources or purified during recombinant production. Immunization and / or screening for immunoglobulin sequences can be carried out using peptide fragments of such antigens.

[0048] Immunoglobulin sequences of different origins, including those from mouse, rat, rabbit, donkey, human, and camelid, can be used in this technology. Fully humanized, humanized, or chimeric sequences may also be used. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelid domain antibodies, such as camelid dAbs as described by Ward et al. (see, for example, International Publication No. 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot.Eng., 9:531-537, 1996), can be used herein. ISVDs can be fused to form polyvalent and / or multispecific constructs (see Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10. 7346-7350) for polyvalent and multispecific polypeptides containing one or more VHH domains and their preparations, as well as, for example, International Publication No. 96 / 34103 and International Publication No. 99 / 23221).

[0049] However, it should be noted that the ISVDs included in this technology are not limited in terms of the origin of the ISVD sequence (or the nucleotide sequence used to express it) or the way in which the ISVD sequence or nucleotide sequence is generated or obtained (or generated or obtained). Therefore, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In one specific but non-limiting embodiment, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, which includes, but is not limited to, “humanized” (as defined herein) immunoglobulin sequences (e.g., partially or fully humanized camelid, mouse, or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), “camelized” (as defined herein) immunoglobulin sequences (and in particular camelized VH sequences), and ISVDs obtained by techniques such as affinity maturation (e.g., starting from a synthetic, random, or naturally occurring immunoglobulin sequence), CDR grafting, veneering, conjugation of fragments derived from different immunoglobulin sequences, PCR assembly using overlap primers, and similar immunoglobulin sequence engineering techniques well known to those skilled in the art, or any suitable combination of any of the foregoing.

[0050] Similarly, a nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may also be, for example, a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from cells), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable method known to be known, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlap primers, or a nucleotide sequence prepared using a known method for DNA synthesis.

[0051] "Humanized VHH" includes an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain but is "humanized," that is, an amino acid sequence that is "humanized" by replacing one or more amino acid residues in the amino acid sequence of the said naturally occurring VHH sequence (and in particular in the framework sequence) with one or more amino acid residues that occur at the corresponding positions in the VH domain derived from a conventional human four-chain antibody. This can be carried out by methods known in itself, which will be apparent to those skilled in the art based, for example, on the prior art (e.g., International Publication No. 2008 / 020079). Again, it should be noted that such humanized VHH can be obtained by any suitable method known in itself and is therefore not strictly limited to polypeptides obtained using polypeptides containing a naturally occurring VHH domain as a starting material.

[0052] "Camelized VH" includes amino acid sequences that correspond to the amino acid sequence of a naturally occurring VH domain but are "camelized," that is, sequences in which one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain derived from a conventional four-chain antibody are "camelized" by substituting one or more amino acid residues that occur at the corresponding positions in the VHH domain of a (camelid) heavy-chain antibody. This can be carried out in a manner known to those skilled in the art, for example, based on the description in the prior art (e.g., Davies and Riechman (1994 and 1996), cited above). Such "camelized" substitutions are inserted into amino acid positions that form and / or exist at the VH-VL interface and / or so-called camelid hallmark residues, as defined herein (see, for example, International Publication No. 94 / 04678 and Davies and Riechmann (1994 and 1996), cited above). In one embodiment, the VH sequence used as a starting material or starting point for generating or designing camelid VH is a mammalian VH sequence, such as a human VH sequence, such as a VH3 sequence. However, it should be noted that such camelid VH can be obtained by any suitable method known in itself, and is therefore not strictly limited to polypeptides obtained using polypeptides containing naturally occurring VH domains as starting materials.

[0053] The structure of an immunoglobulin monovariable domain sequence can be considered to consist of four framework regions ("FRs"), which are referred to in the Art and Specified herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively. These framework regions are interrupted by three complementarity-determining regions ("CDRs"), which are referred to in the Art and Specified herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0054] As further described in paragraphs q) on pages 58 and 59 of the International Publication No. 08 / 020079, the amino acid residues of ISVD can be numbered according to the general numbering of VH domains assigned by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) (see, for example, Figure 2 of this document, V from Camelidae in the literature by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185~195). HH (As applied to the domain). In this application, unless otherwise specified, the sequence of ISVDs is determined according to Kabat numbering.

[0055] As is well known in the art with respect to VH and VHH domains, it should be noted that the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering. That is, one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number permitted by Kabat numbering. This generally means that Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in VH and VHH domains is usually in the range of 110 to 120, and often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0056] The determination of the CDR region can also be performed according to different methods. In CDR determination according to Kabat, FR1 of ISVD contains amino acid residues from positions 1 to 30, CDR1 of ISVD contains amino acid residues from positions 31 to 35, FR2 of ISVD contains amino acids from positions 36 to 49, CDR2 of ISVD contains amino acid residues from positions 50 to 65, FR3 of ISVD contains amino acid residues from positions 66 to 94, CDR3 of ISVD contains amino acid residues from positions 95 to 102, and FR4 of ISVD contains amino acid residues from positions 103 to 113.

[0057] The framework sequence is an immunoglobulin framework sequence or a preferred combination thereof derived from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence may be a framework sequence derived from a light chain variable domain (e.g., a VL sequence) and / or a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In a particular embodiment, the framework sequence is either a framework sequence derived from a VHH sequence (where the framework sequence is optionally partially or completely humanized) or a camelized conventional VH sequence (as defined herein).

[0058] In particular, the framework sequences present in the ISV sequences used in the methods described herein may include one or more Hallmark residues (as defined herein below), and the ISV sequences may include, for example, NANOBODY® ISVs such as humanized VHH or VHH containing camelid VH. Non-limiting examples of such framework sequences (or preferred combinations thereof) will become apparent from the disclosures herein.

[0059] Generally, NANOBODY® ISVD (in particular VHH sequences including (partially) humanized VHH sequences and camelized VH sequences) can be characterized by the presence of one or more "Hallmark residues" (again, as further described herein) of one or more framework sequences (as described herein). Therefore, generally, NANOBODY® ISVD can be defined as an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It may also be an immunoglobulin sequence having, Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more Hallmark residues are as further defined herein.

[0060] In particular, NANOBODY® ISVD can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It may also be an immunoglobulin sequence having, Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively, and the framework sequence is as further defined herein.

[0061] More specifically, NANOBODY® ISVD can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It may also be an immunoglobulin sequence having, Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. Here, one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108, according to Kabat numbering, are selected from the Hallmark residues mentioned in Table 1 below.

[0062] [Table 1]

[0063] In a first aspect, the technology relates to a polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0064] The term "glycosylation acceptor site" refers to a position within an ISVD that can be N-glycosylated or O-glycosylated. N-linked glycans are typically linked to asparagine (Asn), while O-linked glycans are generally linked to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chains. In some embodiments, the glycosylation acceptor site is the N-glycosylation site. In some embodiments, the glycosylation acceptor site for N-glycosylation is asparagine (Asn). In some embodiments, the glycosylation acceptor site for O-glycosylation is serine, threonine, tyrosine, hydroxylysine, or hydroxyproline.

[0065] Surprisingly, the inventors have discovered that, in addition to known glycosylation acceptor sites, it is possible to create novel glycosylation acceptor sites in the ISVD amino acid sequence at positions previously not known to be suitable for glycosylation.

[0066] For efficient glycan transport, in some embodiments, asparagine is located in a specific consensus sequence in the polypeptide's primary structure (NXS, NXT, or NXC). Therefore, the glycosylation acceptor site for N-linked glycosylation is typically contained within an NXT or NXS motif (where X can be any amino acid residue) that is glycosylated on an asparagine (N) residue. Thus, where it is stated in this art that a position can be glycosylated and that this position is intended to be N-glycosylated, the primary sequence of the ISVD according to this art generally contains an NXT or NXS motif (preferably introduced by mutation and / or substitution of the relevant amino acid position) such that it is located at the position where the asparagine residue of the motif is glycosylated, i.e., the glycosylation acceptor site.

[0067] Therefore, in a further embodiment, the glycosylation acceptor site is contained within the NXT or NXS motif (where X can be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 according to Kabat numbering.

[0068] In another embodiment, the glycosylation acceptor site is located at an amino acid position selected from amino acid positions 19, 26, and 105 according to Kabat numbering.

[0069] It was found that ISVDs having one of these three glycosylation acceptor positions 19, 26, and 105, when used in such constructs, exhibit a remarkably high degree of glycosylation in any format, monovalent or polyvalent, regardless of the ISVD's position within the construct.

[0070] In one embodiment, the polypeptide is a monovalent polypeptide containing or consisting of one ISVD. In this embodiment, the glycosylation acceptor site may be located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0071] In further embodiments, the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. In this embodiment, the glycosylation acceptor site may be located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 according to Kabat numbering.

[0072] The process of designing / selecting and / or preparing polypeptides starting from an immunoglobulin monovariate domain such as VHH, humanized VHH, camelid VH, domain antibody, or dAb is also referred to herein as “formatting” the immunoglobulin monovariate domain, and the immunoglobulin monovariate domain that becomes part of the polypeptide is said to be the “formatted” polypeptide or “in the format of” the polypeptide. Examples of methods by which an immunoglobulin monovariate domain can be formatted, and examples of such formats, will be apparent to those skilled in the art based on the disclosure herein, and such formatted immunoglobulin monovariate domains form further embodiments of the Art.

[0073] For example, but not limited to, one or more immunoglobulin monovariable domains may be used as “binding units,” “binding domains,” or “building blocks” for the preparation of polypeptides (these terms are used interchangeably), and the polypeptide may optionally contain one or more further immunoglobulin monovariable domains that can function as binding units.

[0074] This technology also provides polypeptides or constructs comprising, or essentially comprising, one or more immunoglobulin monovariate domains. A monovalent polypeptide comprises, or essentially comprises, only one binding unit (e.g., an immunoglobulin monovariate domain). A polypeptide comprising two or more binding units (e.g., an immunoglobulin monovariate domain) is also referred herein as a “polyvalent” polypeptide, and the binding unit / immunoglobulin monovariate domain present in such polypeptides is also referred herein as “polyvalent format.” For example, a "bivalent" polypeptide may contain two immunoglobulin monovariable domains linked via linker sequences in an optional manner, while a "trivalent" polypeptide may contain three immunoglobulin monovariable domains linked via two linker sequences in an optional manner; on the other hand, a "tetravalent" polypeptide may contain four immunoglobulin monovariable domains linked via three linker sequences in an optional manner; on the other hand, a "pentavalent" polypeptide may contain five immunoglobulin monovariable domains linked via four linker sequences in an optional manner; and on the other hand, a "hexavalent" polypeptide may contain six immunoglobulin monovariable domains linked via five linker sequences in an optional manner.

[0075] In another embodiment, the technology relates to a polypeptide comprising or (essentially) one ISVD, wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0076] In one embodiment, the technology relates to a polypeptide comprising or (essentially) one ISVD, wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 according to Kabat numbering.

[0077] In another embodiment, the technology relates to a monovalent polypeptide comprising or (essentially) one ISVD, wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0078] In further embodiments, the technology relates to a monovalent polypeptide comprising or (essentially) one ISVD, wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 according to Kabat numbering.

[0079] The inventors have surprisingly found that positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 are glycosylation acceptor sites in ISVD that can be glycosylated to have a functional and well-glycosylated ISVD for use, for example, as a monovalent polypeptide and / or in conjugation with further parts.

[0080] In one embodiment of the present technology, the polypeptide according to the present technology comprises at least two ISVDs. In this embodiment, at least one of the at least two ISVDs may contain a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0081] In one embodiment, at least one of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 55, 105, and 108 according to Kabat numbering.

[0082] The inventors have surprisingly found that glycosylation acceptor sites located at positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 can provide a good degree of glycosylation in polyvalent polypeptides, such as polypeptides containing at least two ISVDs. Interestingly, another position, namely position 3, was found to be suitable for use as a glycosylation acceptor site. This position provided an insufficient degree of glycosylation in the monovalent format, but a very high degree of glycosylation in polyvalent polypeptides. In particular, positions 3, 19, 26, 55, 105, and 108 yielded a high degree of glycosylation regardless of the position of the ISVDs in polyvalent polypeptides, such as polypeptides containing at least two ISVDs.

[0083] In a further embodiment, the polypeptide is a divalent polypeptide comprising or (essentially) two ISVDs. In another embodiment, at least one of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 105, 108, and 110 according to Kabat numbering.

[0084] In another embodiment, the polypeptide is a divalent polypeptide comprising or consisting of two ISVDs. In another embodiment, at least one of the at least two ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 55, 105, and 108 according to Kabat numbering.

[0085] Therefore, in another embodiment, the present technology relates to a polypeptide comprising or (essentially) two ISVDs, wherein at least one of the two ISVDs contains a glycosylation acceptor site at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0086] In one embodiment, at least one of the two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 55, 105, and 108 according to Kabat numbering.

[0087] The inventors have surprisingly found that the position of ISVD in the polypeptide affects the degree of glycosylation obtained due to the use of several positions as glycosylation acceptor sites. Specifically, in some embodiments, the degree of glycosylation depended on whether the ISVD containing the glycosylation acceptor site was a C-terminal ISVD or an N-terminal ISVD. When positions 3, 19, 26, 55, 105, and 108 were used as glycosylation acceptor sites, the position of the ISVD containing the glycosylation acceptor site in the polypeptide did not affect the degree of glycosylation obtained. However, when positions 53, 68, 75, and 110 were used as glycosylation acceptor sites, the inventors have surprisingly found that insufficient glycosylation was obtained when the ISVD containing the glycosylation acceptor site was a C-terminal ISVD. In addition, regarding position 1, insufficient glycosylation was obtained when the ISVD containing the glycosylation site was the N-terminal ISVD, but good glycosylation was obtained when the ISVD containing the glycosylation site was the C-terminal ISVD. For position 15, regardless of the ISVD's position, the degree of glycosylation in the divalent format was found to be appropriate but not high. Furthermore, for position 102, insufficient glycosylation was found in the divalent format, but good glycosylation was found in the trivalent format.

[0088] Therefore, in another embodiment, the N-terminal ISVD of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering.

[0089] In a further embodiment, the C-terminal ISVD of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 19, 26, 55, 73, 105, and 108 according to Kabat numbering.

[0090] Therefore, in a further embodiment, the technology relates to a polypeptide comprising or (essentially) two ISVDs, wherein the N-terminal ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering.

[0091] In a further embodiment, the C-terminal ISVD of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 19, 26, 55, 73, 105, and 108 according to Kabat numbering.

[0092] As previously stated, the inventors surprisingly found that the 3-position glycosylation acceptor site provides a good degree of glycosylation only in the polyvalent format containing the divalent polypeptide. In addition, the inventors found that the 102-position provides a good degree of glycosylation in both the monovalent and polyvalent formats, except for the divalent format. Other disclosed glycosylation acceptor sites provided a good degree of glycosylation in both the monovalent and polyvalent formats containing the divalent polypeptide.

[0093] In another embodiment, the polypeptide according to the present technology comprises at least three ISVDs. In one embodiment, at least one of the at least three ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0094] The inventors were surprised to find that the glycosylation acceptor site present in ISVD at position 15 resulted in a high degree of glycosylation only when ISVD was in (at least) a trivalent format. When ISVD was in a monovalent format, the glycosylation acceptor site at position 15 resulted in an insufficient degree of glycosylation, and in a divalent format, only an acceptable degree of glycosylation was observed. However, when such ISVD was formatted with a trivalent polypeptide, a surprisingly high degree of glycosylation was observed. In addition, the glycosylation acceptor sites present in ISVD at positions 1, 3, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110, which have already been discussed, also resulted in a high degree of glycosylation when ISVD was in (at least) a trivalent format.

[0095] In one embodiment, at least one of the three ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 15, 19, 26, and 105 according to Kabat numbering.

[0096] Surprisingly, the inventors found that when the glycosylation acceptor site is located at amino acid positions 3, 15, 19, 26, or 105 in the trivalent polypeptide, the position of ISVD within the peptide does not affect the degree of glycosylation.

[0097] In another embodiment, the polypeptide is a trivalent polypeptide comprising or (essentially) three ISVDs. As described above, the presence of a glycosylation acceptor site in the ISVD at position 15 surprisingly provided a high degree of glycosylation in the trivalent polypeptide. In addition, glycosylation acceptor sites present in the ISVD at positions 1, 3, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 provided a high degree of glycosylation in the polyvalent format containing the trivalent polypeptide.

[0098] To our surprise, we found that the position of ISVD in the formatted polypeptide affected the degree of glycosylation obtained, even in the trivalent format, when using several positions as glycosylation acceptor sites. Specifically, in some cases, it depended on whether the ISVD with glycosylation acceptor sites was a C-terminal ISVD, an N-terminal ISVD, or an ISVD that was neither C-terminal nor N-terminal (i.e., an ISVD between a C-terminal and an N-terminal ISVD, e.g., an intermediate ISVD). When positions 3, 15, 19, 26, 55, 73, 105, and 108 were used as glycosylation acceptor sites, the position of the ISVD with glycosylation acceptor sites in the polypeptide did not affect the degree of glycosylation obtained. However, when positions 53, 68, 75, 102, and 110 are used as glycosylation acceptor sites, the inventors surprisingly found that poor glycosylation was obtained when the ISVD containing the glycosylation acceptor site was a C-terminal ISVD. In addition, the inventors found that a high degree of glycosylation could be obtained when the glycosylation acceptor site at position 1 was present only in the C-terminal ISVD. When positions 68, 75, and 110 are used as glycosylation acceptor sites, any other position in the polypeptide other than the C-terminus of the ISVD would result in a high degree of glycosylation.

[0099] Therefore, in one embodiment, the N-terminal ISVD of the at least three ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 15, 19, 26, 55, 73, 75, 76, 105, 108, and 110 according to Kabat numbering.

[0100] In another embodiment, the C-terminal ISVD of the at least three ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, and 105 according to Kabat numbering.

[0101] In yet another embodiment, at least one of the three ISVDs that are neither C-terminal nor N-terminal includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

[0102] Here, the following specific items are provided for all specific glycosylation acceptor sites. The present invention should not be considered to be limited to these items.

[0103] 1st place Item 1.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 1 according to Kabat numbering. Item 1.2. The polypeptide according to Item 1.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 1.3. The polypeptide according to Item 1.1 or 1.2, wherein the glycosylated acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylated acceptor site. Item 1.4. A polypeptide according to any one of items 1.1 to 1.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 1.5. The polypeptide described in any one of items 1.1 to 1.3, wherein the polypeptide comprises or consists of at least two ISVDs. Item 1.6. The polypeptide according to Item 1.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 1 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 1. Item 1.7. The polypeptide described in Item 1.6, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 1 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 1. Item 1.8. The polypeptide according to Item 1.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 1 according to Kabat numbering. Item 1.9. The polypeptide described in any one of items 1.5 to 1.8, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 1.10. The polypeptide described in any one of items 1.1 to 1.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 1.11. The polypeptide according to Item 1.10, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 1 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 1. Item 1.12. The polypeptide according to Item 1.10, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 1 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylated acceptor site located at amino acid position 1. Item 1.13. The polypeptide according to Item 1.10, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 1 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 1. Item 1.14. A polypeptide as described in Item 1.10, wherein all three of at least three ISVDs contain glycosylation acceptor sites located at amino acid position 1 according to Kabat numbering. Item 1.15 The polypeptide according to any one of items 1.10 to 1.14, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) three ISVDs.

[0104] 3rd place Item 3.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 3 according to Kabat numbering. Item 3.2. The polypeptide according to Item 3.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 3.3. The polypeptide according to Item 3.1 or 3.2, wherein the glycosylated acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylated acceptor site, located at amino acid position 3 according to Kabat numbering. Item 3.4. The polypeptide described in any one of items 3.1 to 3.3, wherein the polypeptide contains or (essentially) consists of at least two ISVDs. Item 3.5. The polypeptide according to Item 3.4, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 3 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 3. Item 3.6. The polypeptide described in Item 3.5, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 3 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 3. Item 3.7. The polypeptide described in Item 3.5, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 3 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 3. Item 3.8. The polypeptide according to Item 3.4, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 3 according to Kabat numbering. Item 3.9. The polypeptide described in any one of items 3.4 to 3.8, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 3.10. The polypeptide described in any one of items 3.1 to 3.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 3.11. The polypeptide according to Item 3.10, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 3 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 3. Item 3.12. The polypeptide according to Item 3.11, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 3 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 3. Item 3.13. The polypeptide according to Item 3.11, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 3 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 3. Item 3.14. The polypeptide described in Item 3.11, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 3 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylated acceptor site located at amino acid position 3. Item 3.15. The polypeptide according to Item 3.10, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 3 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 3. Item 3.16. A polypeptide as described in Item 3.10, wherein all three of at least three ISVDs contain glycosylated acceptor sites located at amino acid position 3 according to Kabat numbering. Item 3.17. The polypeptide described in any one of items 3.10 to 3.16, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0105] 15th place Item 15.1. A polypeptide comprising or essentially comprising a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 15 according to Kabat numbering. Item 15.2. The polypeptide according to Item 15.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 15.3. The polypeptide according to item 15.1 or 15.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 15 according to Kabat numbering. Item 15.4. The polypeptide described in any one of items 15.1 to 15.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 15.5. The polypeptide according to Item 15.4, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 15 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 15. Item 15.6. The polypeptide according to Item 15.5, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 15 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 15. Item 15.7. The polypeptide according to Item 15.5, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 15 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 15. Item 15.8. The polypeptide according to Item 15.5, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 15 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylated acceptor site located at amino acid position 15. Item 15.9. The polypeptide according to Item 15.4, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 15 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 15. Item 15.10. A polypeptide as described in Item 15.4, wherein all three of at least three ISVDs contain glycosylation acceptor sites located at amino acid position 15 according to Kabat numbering. Item 15.11. The polypeptide described in any one of items 15.4 to 15.10, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0106] 19th place Item 19.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 19 according to Kabat numbering. Item 19.2. The polypeptide according to Item 19.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 19.3. The polypeptide according to item 19.1 or 19.2, wherein the glycosylated acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylated acceptor site, located at amino acid position 19 according to Kabat numbering. Item 19.4. The polypeptide described in any one of items 19.1 to 19.3, wherein the polypeptide is a monovalent polypeptide containing or consisting of one ISVD. Item 19.5. The polypeptide described in any one of items 19.1 to 19.3, wherein the polypeptide contains or (essentially) consists of at least two ISVDs. Item 19.6. The polypeptide according to Item 19.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 19 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 19. Item 19.7. The polypeptide according to Item 19.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 19 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 19. Item 19.8. The polypeptide described in Item 19.6, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 19 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 19. Item 19.9. The polypeptide according to Item 19.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 19 according to Kabat numbering. Item 19.10. The polypeptide described in any one of items 19.5 to 19.9, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 19.11. The polypeptide described in any one of items 19.1 to 19.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 19.12. The polypeptide according to Item 19.11, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 19 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 19. Item 19.13. The polypeptide according to Item 19.12, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 19 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 19. Item 19.14. The polypeptide according to Item 19.12, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 19 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 19. Item 19.15. The polypeptide according to Item 19.12, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 19 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylated acceptor site located at amino acid position 19. Item 19.16. The polypeptide according to Item 19.11, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 19 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 19. Item 19.17. A polypeptide as described in Item 19.11, wherein all three of at least three ISVDs contain glycosylation acceptor sites located at amino acid position 19 according to Kabat numbering. Item 19.18. A polypeptide according to any one of items 19.11 to 19.17, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0107] 26th place Item 26.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 26 according to Kabat numbering. Item 26.2. The polypeptide according to Item 26.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 26.3. The polypeptide according to item 26.1 or 26.2, wherein the glycosylated acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylated acceptor site, located at amino acid position 26 according to Kabat numbering. Item 26.4. The polypeptide according to any one of items 26.1 to 26.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 26.5. The polypeptide described in any one of items 26.1 to 26.3, wherein the polypeptide comprises or consists of at least two ISVDs. Item 26.6. The polypeptide according to Item 26.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 26 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 26. Item 26.7. The polypeptide according to Item 26.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 26 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 26. Item 26.8. The polypeptide according to Item 26.6, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 26 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 26. Item 26.9. The polypeptide according to Item 26.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 26 according to Kabat numbering. Item 26.10. The polypeptide according to any one of items 26.5 to 26.9, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 26.11. The polypeptide described in any one of items 26.1 to 26.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 26.12. The polypeptide according to Item 26.11, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 26 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 26. Item 26.13. The polypeptide according to Item 26.12, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 26 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 26. Item 26.14. The polypeptide according to Item 26.12, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain a glycosylated acceptor site located at amino acid position 26 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 26. Item 26.15. The polypeptide according to Item 26.12, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 26 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylated acceptor site located at amino acid position 26. Item 26.16. The polypeptide according to Item 26.11, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 26 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 26. Item 26.17. A polypeptide as described in Item 26.11, wherein all three of at least three ISVDs contain a glycosylation acceptor site located at amino acid position 26 according to Kabat numbering. Item 26.18. The polypeptide described in any one of items 26.11 to 26.17, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) three ISVDs.

[0108] 53rd place Item 53.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 53 according to Kabat numbering. Item 53.2. The polypeptide according to Item 53.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 53.3. The polypeptide according to item 53.1 or 53.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 53 according to Kabat numbering. Item 53.4. The polypeptide according to any one of items 53.1 to 53.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 53.5. The polypeptide described in any one of items 53.1 to 53.3, wherein the polypeptide contains or consists of at least two ISVDs. Item 53.6. The polypeptide according to Item 53.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 53 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 53. Item 53.7. The polypeptide according to Item 53.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 53 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 53. Item 53.8. The polypeptide according to Item 53.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 53 according to Kabat numbering. Item 53.9. The polypeptide according to any one of items 53.5 to 53.8, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 53.10. The polypeptide described in any one of items 53.1 to 53.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 53.11. The polypeptide according to Item 53.10, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 53 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 53. Item 53.12. The polypeptide according to item 53.11, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 53 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 53. Item 53.13. The polypeptide according to item 53.11, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 53 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 53. Item 53.14. The polypeptide according to Item 53.10, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 53 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 53. Item 53.15. A polypeptide as described in Item 53.10, wherein all three of at least three ISVDs contain a glycosylation acceptor site located at amino acid position 53 according to Kabat numbering. Item 53.16. The polypeptide described in any one of items 53.10 to 53.15, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0109] 55th place Item 55.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 55 according to Kabat numbering. Item 55.2. The polypeptide according to Item 55.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 55.3. The polypeptide according to item 55.1 or 55.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 55 according to Kabat numbering. Item 55.4. The polypeptide according to any one of items 55.1 to 55.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 55.5. The polypeptide described in any one of items 55.1 to 55.3, wherein the polypeptide comprises or consists of at least two ISVDs. Item 55.6. The polypeptide according to Item 55.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 55 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 55. Item 55.7. The polypeptide according to Item 55.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 55 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 55. Item 55.8. The polypeptide according to Item 55.6, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylation acceptor site located at amino acid position 55 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylation acceptor site located at amino acid position 55. Item 55.9. The polypeptide according to Item 55.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 55 according to Kabat numbering. Item 55.10. The polypeptide according to any one of items 55.5 to 55.9, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 55.11. The polypeptide described in any one of items 55.1 to 55.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 55.12. The polypeptide according to Item 55.11, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 55 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 55. Item 55.13. The polypeptide according to Item 55.12, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 55 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 55. Item 55.14. The polypeptide according to Item 55.12, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 55 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 55. Item 55.15. The polypeptide according to item 55.12, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 55 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylation acceptor site located at amino acid position 55. Item 55.16. The polypeptide according to Item 55.11, wherein at least two of the at least three ISVDs include a glycosylated acceptor site located at amino acid position 55 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 55. Item 55.17. A polypeptide as described in Item 55.11, wherein all three of at least three ISVDs contain glycosylation acceptor sites located at amino acid position 55 according to Kabat numbering. Item 55.18. The polypeptide described in any one of items 55.11 to 55.17, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0110] 68th place Item 68.1. A polypeptide comprising or essentially comprising a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 68 according to Kabat numbering. Item 68.2. The polypeptide according to Item 68.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 68.3. The polypeptide according to item 68.1 or 68.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 68 according to Kabat numbering. Item 68.4. The polypeptide according to any one of items 68.1 to 68.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 68.5. The polypeptide described in any one of items 68.1 to 68.3, wherein the polypeptide comprises or consists of at least two ISVDs. Item 68.6. The polypeptide according to Item 68.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 68 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 68. Item 68.7. The polypeptide according to Item 68.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 68 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 68. Item 68.8. The polypeptide according to Item 68.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 68 according to Kabat numbering. Item 68.9. The polypeptide according to any one of items 68.5 to 68.9, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 68.10. The polypeptide described in any one of items 68.1 to 68.3, wherein the polypeptide contains or consists of at least three ISVDs. Item 68.11. The polypeptide according to Item 68.10, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 68 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 68. Item 68.12. The polypeptide according to item 68.11, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 68 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 68. Item 68.13. The polypeptide according to item 68.11, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain a glycosylated acceptor site located at amino acid position 68 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 68. Item 68.14. The polypeptide according to Item 68.10, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 68 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 68. Item 68.15. A polypeptide as described in Item 68.10, wherein all three of at least three ISVDs contain a glycosylation acceptor site located at amino acid position 68 according to Kabat numbering. Item 68.15. The polypeptide described in any one of items 68.10 to 68.15, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0111] 73rd place Item 73.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 73 according to Kabat numbering. Item 73.2. The polypeptide according to Item 73.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 73.3. The polypeptide according to item 73.1 or 73.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 73 according to Kabat numbering. Item 73.4. The polypeptide described in any one of items 73.1 to 73.3, wherein the polypeptide is a monovalent polypeptide containing or consisting of one ISVD. Item 73.5. The polypeptide described in any one of items 73.1 to 73.3, wherein the polypeptide contains or consists of at least two ISVDs. Item 73.6. The polypeptide according to Item 73.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 73 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 73. Item 73.7. The polypeptide according to Item 73.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylation acceptor site located at amino acid position 73 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylation acceptor site located at amino acid position 73. Item 73.8. The polypeptide according to Item 73.6, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 73 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 73. Item 73.9. The polypeptide according to Item 73.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 73 according to Kabat numbering. Item 73.10. The polypeptide described in any one of items 73.5 to 73.9, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 73.11. The polypeptide described in any one of items 73.1 to 73.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 73.12. The polypeptide according to Item 73.11, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 73 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 73. Item 73.13. The polypeptide according to item 73.12, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 73 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 73. Item 73.14. The polypeptide according to item 73.12, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain a glycosylated acceptor site located at amino acid position 73 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 73. Item 73.15. The polypeptide according to item 73.12, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 73 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylated acceptor site located at amino acid position 73. Item 73.16. The polypeptide according to Item 73.11, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 73 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 73. Item 73.17. A polypeptide as described in Item 73.11, wherein all three of at least three ISVDs contain a glycosylation acceptor site located at amino acid position 73 according to Kabat numbering. Item 73.18. The polypeptide described in any one of items 73.11 to 73.17, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0112] 75th place Item 75.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 75 according to Kabat numbering. Item 75.2. The polypeptide according to Item 75.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 75.3. The polypeptide according to item 75.1 or 75.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 75 according to Kabat numbering. Item 75.4. The polypeptide according to any one of items 75.1 to 75.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 75.5. The polypeptide described in any one of items 75.1 to 75.3, wherein the polypeptide contains or consists of at least two ISVDs. Item 75.6. The polypeptide according to Item 75.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 75 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 75. Item 75.7. The polypeptide according to Item 75.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 75 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 75. Item 75.8. The polypeptide according to Item 75.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 75 according to Kabat numbering. Item 75.9. The polypeptide according to any one of items 75.5 to 75.8, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 75.10. The polypeptide described in any one of items 75.1 to 75.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 75.11. The polypeptide according to Item 75.10, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 75 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 75. Item 75.12. The polypeptide according to Item 75.11, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 75 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 75. Item 75.13. The polypeptide according to Item 75.11, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 75 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 75. Item 75.14. The polypeptide according to Item 75.10, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 75 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 75. Item 75.15. A polypeptide as described in Item 75.10, wherein all three of at least three ISVDs contain glycosylation acceptor sites located at amino acid position 75 according to Kabat numbering. Item 75.16. The polypeptide described in any one of items 75.10 to 75.15, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0113] 76th place Item 76.1. A polypeptide comprising or essentially comprising a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 76 according to Kabat numbering. Item 76.2. The polypeptide according to Item 76.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 76.3. The polypeptide according to item 76.1 or 76.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 76 according to Kabat numbering. Item 76.4. The polypeptide according to any one of items 76.1 to 76.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 76.5. The polypeptide described in any one of items 76.1 to 76.3, wherein the polypeptide comprises or consists of at least two ISVDs. Item 76.6. The polypeptide according to Item 76.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 76 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 76. Item 76.7. The polypeptide according to Item 76.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylation acceptor site located at amino acid position 76 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylation acceptor site located at amino acid position 76. Item 76.8. The polypeptide according to Item 76.6, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 76 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 76. Item 76.9. The polypeptide according to Item 76.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 76 according to Kabat numbering. Item 76.10. The polypeptide according to any one of items 76.5 to 76.9, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 76.11. The polypeptide described in any one of items 76.1 to 76.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 76.12. The polypeptide according to Item 76.11, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 76 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 76. Item 76.13. The polypeptide according to Item 76.12, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 76 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 76. Item 76.14. The polypeptide according to item 76.12, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 76 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 76. Item 76.15. The polypeptide according to item 76.12, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 76 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylation acceptor site located at amino acid position 76. Item 76.16. The polypeptide according to Item 76.11, wherein at least two of the at least three ISVDs include a glycosylated acceptor site located at amino acid position 76 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 76. Item 76.17. A polypeptide as described in Item 76.11, wherein all three of at least three ISVDs contain a glycosylation acceptor site located at amino acid position 76 according to Kabat numbering. Item 76.18. The polypeptide described in any one of items 76.11 to 76.17, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0114] 102nd place Item 102.1. A polypeptide comprising or essentially comprising a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 102 according to Kabat numbering. Item 102.2. The polypeptide according to Item 1, wherein the glycosylation acceptor site is an N-glycosylation site. Item 102.3. The polypeptide according to item 102.1 or 102.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 102 according to Kabat numbering. Item 102.4. A polypeptide according to any one of items 102.1 to 102.3, wherein the polypeptide is a monovalent polypeptide containing or consisting of one ISVD. Item 102.5 The polypeptide described in any one of items 102.1 to 102.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 102.6. The polypeptide according to Item 102.5, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 102 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 102. Item 102.7. The polypeptide according to Item 102.6, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain a glycosylated acceptor site located at amino acid position 102 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 102. Item 102.8. The polypeptide according to Item 102.6, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylated acceptor site located at amino acid position 102 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylated acceptor site located at amino acid position 102. Item 102.9. The polypeptide according to Item 102.5, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 102 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 102. Item 102.10. A polypeptide as described in Item 102.5, wherein all three of at least three ISVDs contain glycosylation acceptor sites located at amino acid position 102 according to Kabat numbering. Item 102.11. The polypeptide described in any one of items 102.5 to 102.10, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0115] 105th place Item 105.1. A polypeptide comprising or essentially comprising a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 105 according to Kabat numbering. Item 105.2. The polypeptide according to Item 105.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 105.3. The polypeptide according to item 105.1 or 105.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 105 according to Kabat numbering. Item 105.4. A polypeptide according to any one of items 105.1 to 105.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 105.5. The polypeptide described in any one of items 105.1 to 105.3, wherein the polypeptide contains or consists of at least two ISVDs. Item 105.6. The polypeptide according to Item 105.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 105 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 105. Item 105.7. The polypeptide according to Item 105.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylation acceptor site located at amino acid position 105 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylation acceptor site located at amino acid position 105. Item 105.8. The polypeptide described in Item 105.6, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 105 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 105. Item 105.9. The polypeptide according to Item 105.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 105 according to Kabat numbering. Item 105.10. The polypeptide described in any one of items 105.5 to 105.9, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 105.11. The polypeptide described in any one of items 105.1 to 105.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 105.12. The polypeptide according to Item 105.11, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 105 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 105. Item 105.13. The polypeptide according to Item 105.12, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 105 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 105. Item 105.14. The polypeptide according to Item 105.12, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 105 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 105. Item 105.15. The polypeptide according to Item 105.12, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 105 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylation acceptor site located at amino acid position 105. Item 105.16. The polypeptide according to Item 105.11, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 105 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 105. Item 105.17. A polypeptide as described in Item 105.11, wherein all three of at least three ISVDs contain glycosylation acceptor sites located at amino acid position 105 according to Kabat numbering. Item 105.18. The polypeptide described in any one of items 105.11 to 105.17, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0116] 108th place Item 108.1. A polypeptide comprising or essentially comprising a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 108 according to Kabat numbering. Item 108.2. The polypeptide according to Item 108.1, wherein the glycosylation acceptor site is an N-glycosylation site. Item 108.3. The polypeptide according to item 108.1 or 108.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 108 according to Kabat numbering. Item 108.4. A polypeptide according to any one of items 108.1 to 108.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 108.5. The polypeptide described in any one of items 108.1 to 108.3, wherein the polypeptide contains or consists of at least two ISVDs. Item 108.6. The polypeptide according to Item 108.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 108 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 108. Item 108.7. The polypeptide described in Item 108.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylation acceptor site located at amino acid position 108 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylation acceptor site located at amino acid position 108. Item 108.8. The polypeptide described in Item 108.6, wherein only the C-terminal ISVD of at least two ISVDs contains a glycosylation acceptor site located at amino acid position 108 according to Kabat numbering, and the remaining N-terminal ISVD does not contain a glycosylation acceptor site located at amino acid position 108. Item 108.9. The polypeptide according to Item 108.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 108 according to Kabat numbering. Item 108.10. The polypeptide described in any one of items 108.5 to 108.9, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 108.11. The polypeptide described in any one of items 108.1 to 108.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 108.12. The polypeptide according to Item 108.11, wherein at least one of the three ISVDs includes a glycosylation acceptor site located at amino acid position 108 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylation acceptor site located at amino acid position 108. Item 108.13. The polypeptide according to Item 108.12, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 108 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 108. Item 108.14. The polypeptide described in Item 108.12, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain a glycosylated acceptor site located at amino acid position 108 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain a glycosylated acceptor site located at amino acid position 108. Item 108.15. The polypeptide described in Item 108.12, wherein only the C-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 108 according to Kabat numbering, and the remaining N-terminal and intermediate ISVDs do not contain a glycosylation acceptor site located at amino acid position 108. Item 108.16. The polypeptide according to Item 108.11, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 108 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 108. Item 108.17. A polypeptide as described in Item 108.11, wherein all three of at least three ISVDs contain glycosylation acceptor sites located at amino acid position 108 according to Kabat numbering. Item 108.18. The polypeptide described in any one of items 108.11 to 108.17, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0117] 110th place Item 110.1. A polypeptide comprising or (essentially) a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 110 according to Kabat numbering. Item 110.2. The polypeptide according to Item 110.1, wherein the glycosylated acceptor site is an N-glycosylation site. Item 110.3. The polypeptide according to item 110.1 or 110.2, wherein the glycosylation acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site, located at amino acid position 110 according to Kabat numbering. Item 110.4. A polypeptide according to any one of items 110.1 to 110.3, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Item 110.5. The polypeptide described in any one of items 110.1 to 110.3, wherein the polypeptide contains or (essentially) consists of at least two ISVDs. Item 110.6. The polypeptide according to Item 110.5, wherein at least one of the at least two ISVDs includes a glycosylated acceptor site located at amino acid position 110 according to Kabat numbering, and the remaining ISVD does not include a glycosylated acceptor site located at amino acid position 110. Item 110.7. The polypeptide according to Item 110.6, wherein only the N-terminal ISVD of at least two ISVDs contains a glycosylated acceptor site located at amino acid position 110 according to Kabat numbering, and the remaining C-terminal ISVD does not contain a glycosylated acceptor site located at amino acid position 110. Item 110.8. The polypeptide according to Item 110.5, wherein both of the at least two ISVDs include a glycosylation acceptor site located at amino acid position 110 according to Kabat numbering. Item 110.9. The polypeptide described in any one of items 110.5 to 110.8, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Item 110.10. The polypeptide described in any one of items 110.1 to 110.3, wherein the polypeptide contains or (essentially) consists of at least three ISVDs. Item 110.11. The polypeptide according to Item 110.10, wherein at least one of the three ISVDs includes a glycosylated acceptor site located at amino acid position 110 according to Kabat numbering, and the remaining two ISVDs do not include a glycosylated acceptor site located at amino acid position 110. Item 110.12. The polypeptide according to Item 110.11, wherein only the N-terminal ISVD of at least three ISVDs contains a glycosylation acceptor site located at amino acid position 110 according to Kabat numbering, and the remaining intermediate and C-terminal ISVDs do not contain a glycosylation acceptor site located at amino acid position 110. Item 110.13. The polypeptide described in Item 110.11, wherein at least three ISVDs that are neither C-terminal nor N-terminal contain glycosylated acceptor sites located at amino acid position 110 according to Kabat numbering, and the remaining N-terminal and C-terminal ISVDs do not contain glycosylated acceptor sites located at amino acid position 110. Item 110.14. The polypeptide according to Item 110.10, wherein at least two of the three ISVDs include a glycosylated acceptor site located at amino acid position 110 according to Kabat numbering, and the remaining ISVDs do not include a glycosylated acceptor site located at amino acid position 110. Item 110.15. A polypeptide as described in Item 110.10, wherein all three of at least three ISVDs contain glycosylated acceptor sites located at amino acid position 110 according to Kabat numbering. Item 110.16. The polypeptide described in any one of items 110.10 to 110.15, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

[0118] Combinations of glycosylated acceptor sites Item A. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 1 in combination with at least one further glycosylation acceptor site located at any of positions 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item B. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariate domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 3 in combination with at least one further glycosylation acceptor site located at any of positions 1, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item C. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 15 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item D. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 19 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item E. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 26 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 53, 55, 68, 73, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item F. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariate domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 53 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 55, 68, 73, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item G. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariate domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 55 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 68, 73, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item H. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariate domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 68 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 55, 73, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item I. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 73 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 75, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item J. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 75 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 76, 102, 105, 108 and / or 110 according to Kabat numbering. Item K. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 76 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108 and / or 110 according to Kabat numbering. Item L. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 102 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 105, 108 and / or 110 according to Kabat numbering. Item M. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariate domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 105 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 108 and / or 110 according to Kabat numbering. Item N. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariate domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 108 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105 and / or 110 according to Kabat numbering. Item O. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at amino acid position 110 in combination with at least one further glycosylation acceptor site located at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, and / or 108 according to Kabat numbering.

[0119] 3. ISVD glycoprotein The glycosylation acceptor sites present in a polypeptide can be (but not necessarily) modified with N-linked or O-linked glycans. The technology also relates to a polypeptide of the technology that is glycosylated at one or more of the specified glycosylation acceptor sites. When a polypeptide having such a glycosylation acceptor site is expressed in a host or host cell capable of glycosylating the polypeptide (as further defined herein), the produced polypeptide is directly modified with one or more glycans in the host or host cell. The resulting glycosylated polypeptide (also referred to herein as an ISVD glycoprotein) contains one or more glycans. In one embodiment, the polypeptide can contain one or more glycans having terminal N-acetylglucosamine (GlcNAc), (terminal) mannose, (terminal) sialic acid, (terminal) galactose, or combinations thereof. Thus, the technology provides a polypeptide comprising an ISVD as described herein, wherein the polypeptide is glycosylated with one or more glycans selected from N-acetylglucosamine (GlcNAc), mannose, galactose, fucose, and sialic acid.

[0120] In some embodiments, the ISVD glycoproteins of the technology exhibit high affinity. In some embodiments, the ISVD glycoproteins of the technology have the same affinity as a polypeptide that does not undergo glycosylation at the specified glycosylation acceptor site.

[0121] Affinity is a measure of the binding strength between a moiety and a binding site on a target molecule, and the smaller the value of the dissociation constant (K D ), the stronger the binding strength between the target molecule and the targeting moiety. Typically, the binding units used in the technology, such as ISVD, are at 10 -5 ~10 -12 mol / liter or less, 10 -7 ~10 -12 mol / liter or less, or 10 -8 ~10 -12 mol / liter (i.e., 10 5~10 12 liters / mol or more, 10 7 ~10 12 liters / mol or more, or 10 8 ~10 12 The association constant (K) of liters / moles A ) and) bind to that target. 10 -4 Any K greater than moles / liter D value (or 10) 4 Any K less than liter / mol A The K value is generally considered to indicate nonspecific binding. K values ​​are considered to indicate specific biological interactions, such as the binding of immunoglobulin sequences to antigens. D Typically, 10 -5 moles / liter (10000 nM or 10 μM) ~ 10 -12 The concentration is in the range of moles / liter (0.001 nM or 1 pM) or less.

[0122] The dissociation constant may be the actual dissociation constant or the apparent dissociation constant, as will be obvious to those skilled in the art. Methods for determining the dissociation constant are obvious to those skilled in the art, and include, for example, the techniques mentioned below. In this regard, 10 -4 moles / liter or 10 -3 moles / liter greater than (for example, 10 -2 It will also be apparent that it may be impossible to measure the dissociation constant (moles / liter). Optionally, and as will be apparent to those skilled in the art, the (actual or apparent) dissociation constant can be expressed by the relation [K] based on the (actual or apparent) association constant. D = 1 / K A It can be calculated by [ ].

[0123] The affinity of molecular interactions between two molecules can be measured by various techniques known on their own, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, e.g., Ober et al. 2001, Intern. Immunology 13:1551-1559). The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flow conditions, k on , k off Measured value, therefore K D (or K A This yields a value. This can be done, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson et al. 1993 (Ann. Biol. Clin. 51:19-26), Jonsson et al. 1991 (Biotechniques 11:620-627), Johnsson et al. 1995 (J. Mol. Recognit. 8:125-131), and Johnsson et al. 1991 (Anal. Biochem. 198:268-277).

[0124] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). As used herein, the terms “biolayer interferometry” or “BLI” refer to a label-free optical technique that analyzes the interference patterns of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on a biosensor chip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the tip surface of the biosensor. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0125] Alternatively, affinity can be measured using a kinetic exclusion assay (KinExA) (see, e.g., Drake et al. 2004, Anal. Biochem., 328:35-43) with the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of an unmodified molecule. An equilibrium solution of the antibody / antigen complex is passed through a column containing beads pre-coated with the antigen (or antibody) to bind the free antibody (or antigen) to the coated molecule. Detection of the thus captured antibody (or antigen) is achieved using a fluorescently labeled protein that binds to the antibody (or antigen).

[0126] The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5:1765-74).

[0127] In some embodiments, the glycosylated polypeptide of the technology is determined by surface plasmon resonance, so as to be 10 -5 ~10 -12 moles / liter or less, 10 -7 ~10 -12 moles / liter or less, or 10 -8 ~10 -12 The dissociation constant (K) in moles / liter D ) specifically binds to those targets. In some embodiments, the glycosylated polypeptide of the technology is determined by Meso Scale Discovery, as 10 -5 ~10 -12 moles / liter or less, 10 -7 ~10 -12 moles / liter or less, or 10 -8 ~10 -12 The dissociation constant (K) in moles / liter D ) then specifically binds to those targets.

[0128] In some embodiments, the ISVD glycoproteins of this technology have (essentially) the same or higher melting temperature (Tm) compared to polypeptides that do not undergo glycosylation at a designated glycosylation acceptor site. The denaturation midpoint of a protein is defined as the temperature (Tm) or concentration of the denaturant at which both folded and unfolded states are equally positioned in equilibrium (assuming two-state protein folding). Tm is often determined using a thermal shift assay, such as the thermal shift assay described in the Examples section of this application. The melting temperature is defined as the temperature of the protein at which 50% of the protein is in an unfolded state.

[0129] Components present in a polypeptide, such as ISVD, may be linked to one another by one or more suitable linkers, such as peptide linkers. The use of linkers to link two or more (poly)peptides is well known in the art. One commonly used class of peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues and typically contain one or more repeats of a peptide motif, such as the GGGGS (SEQ ID NO: 150) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser)n, where n may be 1, 2, 3, 4, 5, 6, 7 or greater). Some commonly used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 151), the 15GS linker (n=3; SEQ ID NO: 152), and the 35GS linker (n=7; SEQ ID NO: 153). For example, see Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10):1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sel. 27(10):325-330). In one embodiment of the polypeptide of this technology, a 9GS linker is used to link the polypeptide components together. In one embodiment of the polypeptide of this technology, a 35GS linker is used to link the polypeptide components together.

[0130] This technology also provides sequence-optimized ISVDs and polypeptides that exhibit improved stability when stored during stability testing. The sequence-optimized ISVDs and polypeptides exhibit reduced N-terminal post-translational modification of pyroglutamate, thus improving product stability. Pyroglutamate modification leads to heterogeneity in the final product and should be avoided. By changing the N-terminal glutamate (E) to aspartate (D), the possibility of N-terminal pGlu post-translational modification is eliminated, resulting in improved product stability. Therefore, the present invention also relates to the above-mentioned ISVDs and polypeptides in which the glutamate at position 1 (the position determined according to Kabat numbering) is changed to aspartate (E1D).

[0131] The technology also provides sequence-optimized ISVDs and polypeptides that are "humanized," that is, "humanized" by replacing one or more amino acid residues in the amino acid sequence (and particularly in the framework sequence) of the naturally occurring VHH sequence with one or more amino acid residues (e.g., those shown above) that occur at the corresponding positions in the VH domain derived from conventional human four-chain antibodies. Accordingly, the present invention also relates to the humanized ISVDs and polypeptides described above.

[0132] This technology also provides sequence-optimized ISVDs and polypeptides that exhibit reduced binding by existing antibodies present in human serum. For this purpose, in one embodiment, the polypeptide contains valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 in at least one ISVD (according to Kabat numbering). In one embodiment, the polypeptide contains valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 in each ISVD (according to Kabat numbering). Thus, this technology also relates to the above-described ISVDs and polypeptides sequence-optimized with valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD, e.g., all ISVDs.

[0133] In one embodiment, the ISVD or polypeptide has the C-terminus of the sequence VTVSS(X)n (SEQ ID NO: 154), where n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5, and each X is an independently selected amino acid residue. In one embodiment, the polypeptide contains such an ISVD at its C-terminus. In one embodiment, n is 1 or 2, for example 1. In one embodiment, X is a naturally occurring amino acid. In one embodiment, X is selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I).

[0134] In another embodiment, the polypeptide contains lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVD contains lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these embodiments, the C-terminus of ISVD is VKVSS (SEQ ID NO: 155), VQVSS (SEQ ID NO: 156), VTVKS (SEQ ID NO: 157), VTVQS (SEQ ID NO: 158), VKVKS (SEQ ID NO: 159), VKVQS (SEQ ID NO: 160), VQVKS (SEQ ID NO: 161), or VQVQS (SEQ ID NO: 162), and therefore, after the addition of a single alanine, the C-terminus of the polypeptide includes, for example, the sequence VTVSSA (SEQ ID NO: 163), VKVSSA (SEQ ID NO: 164), VQVSSA (SEQ ID NO: 165), VTVKSA (SEQ ID NO: 166), VTVQSA (SEQ ID NO: 167), VKVKSA (SEQ ID NO: 168), VKVQSA (SEQ ID NO: 169), VQVKSA (SEQ ID NO: 170), or VQVQSA (SEQ ID NO: 171). In one embodiment, the polypeptide contains valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD, optionally containing lysine (K) or glutamine (Q) (according to Kabat numbering) at position 110 in at least one ISVD, and containing 1 to 5 (naturally occurring) amino acid elongations, such as a single alanine (A) elongation, at the C-terminus of the C-terminal ISVD, so that the C-terminus of the polypeptide contains, for example, the sequence VTVSSA (SEQ ID NO: 163), VKVSSA (SEQ ID NO: 164), or VQVSSA (SEQ ID NO: 165). For further information relating thereto, see, for example, International Publication No. 2012 / 175741 and International Publication No. 2015 / 173325.

[0135] When the glycosylation acceptor site in ISVD is as described herein, several embodiments are provided for ISVD glycoproteins having a particularly high degree of glycosylation.

[0136] In one embodiment of this technology, the ISVD glycoprotein is a monovalent polypeptide, and the polypeptide sequence is selected from SEQ ID NOs: 11, 14, 69, 70, 73-78, 81, 83, 85, 87, 89-91, 93, 94, 96, and 177-179.

[0137] In another aspect of this technology, the ISVD glycoprotein is a bivalent polypeptide, and the polypeptide sequence is selected from SEQ ID NOs: 99-102, SEQ ID NOs: 105-108, SEQ ID NOs: 110, SEQ ID NOs: 116-118, SEQ ID NOs: 120, SEQ ID NOs: 121, SEQ ID NOs: 123-125, SEQ ID NOs: 128-133, SEQ ID NOs: 141-143, SEQ ID NOs: 145, SEQ ID NOs: 146, SEQ ID NOs: 148, SEQ ID NOs: 149, SEQ ID NOs: 182, SEQ ID NOs: 183, and SEQ ID NOs: 187.

[0138] In a further embodiment of this technology, the ISVD glycoprotein is a trivalent polypeptide, and the polypeptide sequence is selected from SEQ ID NOs: 20, 22-24, 37, 38, 40, 44-52, 55-57, 59-61, 63-65, 186, 190, and 191.

[0139] 4. Conjugate This technology also relates to a conjugate comprising a polypeptide produced by this technology and a conjugate moiety conjugated to a glycan. A polypeptide modified with glycan at a designated glycosylation acceptor site is an ideal starting point for glycan-based conjugations due to its high degree of glycosylation.

[0140] Conjugation can be carried out either chemically (e.g., via methods known in the art, such as periodic acid oxidation and oxime ligation of glycan components, hydrazone ligation, or subsequent conjugation via reductive amination) or enzymatically (e.g., via galactose oxidase and oxime ligation, hydrazone ligation, or subsequent conjugation via reductive amination). Alternatively, tagged glycan residues can be incorporated to enable subsequent conjugation reactions (e.g., incorporation of GalNAz into the glycan chain using mutant galactosyltransferase, and subsequent conjugation reactions via click chemistry).

[0141] In certain embodiments, the conjugate includes a linker between the glycan and the conjugate portion. The use of a particular linker will depend on the application, which will be apparent to those skilled in the art. For example, oximes and hydrazones, particularly those derived from aliphatic aldehydes, exhibit poor stability over time in water or at lower pH. Aromatically stabilizing structures may be more useful for stably linking the glycan to the conjugate portion. Such stabilizing linkers are also within the scope of this application, as they can limit adverse effects resulting from the premature release of the conjugate portion, especially when the conjugate portion is a toxic substance intended, for example, to kill tumor cells. Of particular interest are the BICYCLO[6.1.0]NON-4-YNE reagent, as well as aromatically stabilizing triazole and sulfamide linkers. It is within the scope of the technical knowledge that improved conjugate stability may also result from a reduced tendency of any of the portions contained within the conjugate to aggregate. For the manufacture of improved stability ISVD conjugates, the Leaders non-exclusively refer to International Publication No. 2013036748, International Publication No. 2014065661, International Publication No. 2015057064, and International Publication No. 2016053107, as well as other patent applications filed by Synaffix BV.

[0142] Various linkers known in the art can be used to link glycosylated polypeptides and conjugate moieties. As should be made clear, cleavable and non-cleavable linkers can be used to achieve the desired release profile. In general, the optimal combination of linker and conjugation chemistry needs to be uniquely tailored to correlate with the profile of each intrinsic facet: glycosylated polypeptide, conjugate moiety, and the disease being treated. For a review of the antibody-drug conjugates and linkers used herein, see, for example, McCombs and Owen 2015 (AAPS J.17(2), 2015) and Lu et al. 2016 (Int.J.Mol.Sci.17(4:561); doi:10.3390 / ijms17040561) and Pillow et al. 2017 (Pharm Pat Anal.6(1)) describing novel quaternary ammonium salt linkers useful for conjugates for the treatment of cancer and infectious diseases.

[0143] Other suitable linkers generally include those suitable for use with organic compounds or polymers, particularly polypeptides for pharmaceutical applications. For example, poly(ethylene glycol) moieties have been used to link antibody domains; see, for example, International Publication No. 04 / 081026. The length, degree of flexibility, and / or other properties of the linker may have some influence on the properties of the final conjugate, including, but not limited to, affinity, specificity, or avidity to a particular target, and this is included within the scope of the invention. Based on the disclosure herein, those skilled in the art will be able to determine the optimal linker for use with a particular conjugate after several limited, standardized experiments of their choice.

[0144] In some embodiments, a conjugate comprising a polypeptide and a conjugate moiety according to the present technology has at least one additional function or property compared to a non-conjugate polypeptide. For example, a conjugate comprising a polypeptide according to the present technology and a cytotoxic drug that is the conjugate moiety results in the formation of a binding polypeptide having drug cytotoxicity as a second function (i.e., in addition to antigen binding conferred by the ISVD contained in the polypeptide). As another example, a conjugate of a second binding polypeptide to the polypeptide according to the present technology may confer additional binding properties. As yet another example, the conjugate moiety may be another sugar moiety such as glucose that provides glucose transport function (Ancey et al. 2018, FEBS J.285:2926) or bis-mannose-6-phosphate that provides degradation function. As yet another example, the conjugate moiety may be a PEG molecule that provides half-life extension and / or stabilization / solubilization function. As yet another example, the conjugate moiety may be a chimeric targeting proteolytic (PROTAC) that provides degradation function (Sakamoto et al. 2001, PNAS 98:8554-9). As a further example, the conjugated portion may be a therapeutic portion that provides therapeutic function (e.g., antifungal, antibacterial, antiviral, antiparasitic, cytotoxic, radioactive nucleotide). Other portions that can be conjugated to glycans present at the glycosylated acceptor site include half-life extension portions (PEG or PEG mimetics, large polysaccharides), detection units (chromophore units, fluorescent units, phosphorescent units, luminescent units, light-absorbing units, radioactive units), and targeting portions (e.g., small molecules, antibodies).

[0145] 5. Nucleic acid molecules Another aspect of this technology relates to a nucleotide sequence or nucleic acid encoding a polypeptide by this technology.

[0146] A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked together via a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform / transfect host cells or host organisms for, for example, polypeptide expression and / or production. Suitable hosts or host cells for production purposes will be obvious to those skilled in the art, and are preferably host organisms capable of glycosylation of polypeptides, such as any suitable fungus, prokaryotic, or eukaryotic cell or cell lineage, or any suitable fungus, prokaryotic, or eukaryote. Hosts or host cells containing nucleic acids encoding the polypeptides of the Art are also included in the Art.

[0147] Nucleic acids can be, for example, DNA, RNA, or hybrids thereof, and may also include (e.g., chemically) modified nucleotides such as PNA. They can be single-stranded or double-stranded. In one embodiment, this is in the form of double-stranded DNA. For example, the nucleotide sequence of this technology may be genomic DNA or cDNA.

[0148] The nucleic acids of this technology can be prepared or obtained by known methods and / or isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides can be subjected to site-directed mutagenesis, for example, to provide nucleic acid molecules encoding polypeptides with sequence mutations. As will be apparent to those skilled in the art, nucleic acids, or several nucleotide sequences, can be prepared by, for example, linking and integrating at least one nucleotide sequence encoding a target moiety and nucleic acids encoding one or more linkers in a suitable manner.

[0149] Nucleic acid generation techniques are apparent to those skilled in the art and include, but are not limited to, automated DNA synthesis, site-directed mutagenesis, combination of two or more naturally occurring and / or synthetic sequences (or two or more parts thereof), introduction of mutations resulting in the expression of truncated expression products, introduction of one or more restriction sites (e.g., creation of cassettes and / or regions that can be readily digested and / or ligated using a suitable restriction enzyme), and / or introduction of mutations using PCR reactions with one or more "mismatch" primers.

[0150] In one embodiment, the nucleotide sequence or nucleic acid is optimized for expression in a host cell or host organism capable of glycosylation of the polypeptide encoded by the nucleotide sequence or nucleic acid.

[0151] In further embodiments, the nucleotide sequence or nucleic acid is in the form of a construct or (expression) vector that can be expressed in a host cell or host organism capable of glycosylation of the polypeptide encoded by the nucleotide sequence or nucleic acid. When used herein, a vector is a suitable vehicle for delivering genetic material to a cell. A vector may include a naked nucleic acid such as a plasmid or mRNA, or a nucleic acid embedded in a larger structure such as a liposome or viral vector.

[0152] In some embodiments, the vector comprises at least one nucleic acid optionally linked to one or more regulatory elements, such as one or more suitable promoters, enhancers, terminators, etc. In one embodiment, the vector is an expression vector, i.e., a vector suitable for expressing a coding polypeptide or construct under suitable conditions when the vector is introduced into (e.g., human) cells. DNA-based vectors include the presence of elements for transcription (e.g., promoters and polyA signals) and translation (e.g., Kozak sequences).

[0153] In one embodiment, within a vector, the at least one nucleic acid and the regulatory element are “operably linked” to each other, which generally means they are functionally related to each other. For example, a promoter is considered “operably linked” to a coding sequence if the promoter can initiate or otherwise control / regulate the transcription and / or expression of the coding sequence (where the coding sequence should be understood as being “under the control” of the promoter). Generally, when two nucleotide sequences are operably linked, they are oriented in the same direction and usually within the same reading frame. They are usually contiguous in nature, but this is also not always necessary.

[0154] In one embodiment, any regulatory element of the vector is capable of providing its intended biological function in the intended host cell or host organism. For example, a promoter, enhancer, or terminator must be "operable" in the intended host cell or host organism, meaning, for example, that the promoter must be capable of initiating or otherwise controlling / regulating the transcription and / or expression of the nucleotide sequence, such as a coding sequence, to which it is operably linked.

[0155] In a further embodiment, the technology is a method for producing polypeptides and / or ISVD glycoproteins by the technology, - A step of expressing a nucleotide sequence or nucleic acid in a suitable (non-human) host cell or (non-human) host organism according to the Technology, wherein the host cell or host organism is capable of glycosylation of the expressed polypeptide; optionally, a step of subsequently The present invention relates to a method comprising the steps of: isolating and / or purifying the obtained polypeptide and / or ISVD glycoprotein.

[0156] The host cell or host organism capable of glycosylating the expressed polypeptide is typically a eukaryotic cell or organism. In one embodiment, the host cell is a higher eukaryotic cell. As used herein, “higher eukaryotic cell” refers to a eukaryotic cell that is not derived from a single-celled organism. In other words, a higher eukaryotic cell is a cell from a multicellular eukaryote such as a human cell line or another mammalian cell line (e.g., a CHO cell line) (or, in the case of a cell culture, derived from it). Typically, a higher eukaryotic cell is not a fungal cell. In particular, the term generally refers to mammalian cells, human cell lines, and insect cell lines. More specifically, the term refers to vertebrate cells, and more particularly mammalian cells or human cells. Higher eukaryotic cells as described herein are typically part of a cell culture (e.g., a cell line such as a HEK or CHO cell line), but this is not strictly required (e.g., in the case of plant cells, the plant itself can be used to produce recombinant proteins).

[0157] In one embodiment, the host cell is a lower eukaryotic cell. "Lower eukaryotic cell" means a filamentous fungal cell or a yeast cell. Yeast cells include those of the genera 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), Aspergillus niger, or Komagataella phaffii (Kurtzman, CP (2009) J innd Microbiol). They may be from the species Biotechnol.36(11) (which was previously named and is more commonly known under the old nomenclature as Pichia pastoris, and this name will also be used further herein). According to certain embodiments, the inferior eukaryotic cells are Pichia cells, and in the most specific embodiments, Pichia pastoris cells.

[0158] Some non-limiting examples of suitable mammalian cell lines or yeast lines include Chinese hamster ovary (CHO) cells, human fetal kidney (HEK) cells, SP2 / 0 or Ns0 mouse myeloma cells and baby hamster kidney (BHK) cells, as well as other mammalian cell lines and yeast lines that can be used for the expression / production / manufacturing of polypeptides and proteins intended for administration and / or therapeutic use in human subjects.

[0159] The technology also relates to (non-human) host cells or (non-human) host organisms containing the polypeptide or ISVD glycoprotein of the technology, nucleic acids encoding the polypeptide or ICVD glycoprotein of the technology, and / or vectors containing said nucleic acid molecules.

[0160] 6. Composition In further embodiments, the Technology relates to compositions comprising polypeptides according to the Technology; polypeptides or ISVD glycoproteins produced using the Method of the Technology; conjugates according to the Technology; nucleic acids encoding polypeptides according to the Technology, or vectors comprising such nucleic acid molecules. The compositions may be pharmaceutical compositions. The compositions may further comprise at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds.

[0161] 7.Applications The polypeptides, ISVD glycoproteins or conjugates of this technology, nucleic acid molecules or vectors described herein, or compositions comprising the polypeptides, ISVD glycoproteins, conjugates, nucleic acid molecules or vectors of this technology are useful as pharmaceuticals. Therefore, this technology provides compositions comprising the polypeptides, ISVD glycoproteins or conjugates of this technology, nucleic acid molecules or vectors described herein, or the polypeptides, ISVD glycoproteins, conjugates, nucleic acid molecules or vectors of this technology for use as pharmaceuticals.

[0162] Therefore, a method for the diagnosis, prevention and / or treatment of at least one disease and / or disorder is further provided, comprising administering to a subject in need a pharmaceutically active amount of at least one polypeptide, ISVD glycoprotein or conjugate, nucleic acid molecule or vector described herein, or a composition comprising the polypeptide, ISVD glycoprotein, conjugate, nucleic acid molecule or vector described herein.

[0163] 8. Embodiments The present invention is further illustrated by, but is not limited to, the following embodiments. Embodiment 1. A polypeptide comprising or (essentially) a heavy chain immunoglobulin monovariate domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 2. The polypeptide according to Embodiment 1, wherein the glycosylated acceptor site is an N-glycosylation site. Embodiment 3. The polypeptide according to Embodiment 1 or 2, wherein the glycosylated acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylated acceptor site. Embodiment 4. The polypeptide according to any one of Embodiments 1 to 3, wherein the glycosylated acceptor site is located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering. Embodiment 5. The polypeptide according to any one of Embodiments 1 to 3, wherein the glycosylated acceptor site is located at an amino acid position selected from amino acid positions 19, 26, 55, 73, 105, and 108 according to Kabat numbering. Embodiment 6. The polypeptide according to any one of Embodiments 1 to 3, wherein the glycosylated acceptor site is located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 7. The polypeptide according to Embodiment 6, wherein the polypeptide is a monovalent polypeptide comprising (essentially) one ISVD. Embodiment 8. A polypeptide comprising or (essentially) one ISVD, wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 9. The polypeptide according to Embodiment 8, wherein the glycosylation acceptor site is located at an amino acid position selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 10. The polypeptide according to any one of Embodiments 1 to 3, wherein the polypeptide comprises at least two ISVDs. Embodiment 11. The polypeptide according to Embodiment 10, wherein at least one of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 12. The polypeptide according to Embodiment 10, wherein at least one of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 19, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering. Embodiment 13. The polypeptide according to Embodiment 10, wherein at least one of the at least two ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 55, 105, and 108 according to Kabat numbering. Embodiment 14. The polypeptide according to Embodiment 10, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs. Embodiment 15. The polypeptide according to Embodiment 14, wherein the N-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering. Embodiment 16. The polypeptide according to Embodiment 14, wherein the C-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 19, 26, 55, 105, and 108 according to Kabat numbering. Embodiment 17. The polypeptide according to Embodiment 14, wherein at least one of the two ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 55, 105, and 108 according to Kabat numbering. Embodiment 18. A polypeptide comprising or (essentially) two ISVDs, wherein at least one of the two ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 105, 108, and 110 according to Kabat numbering. Embodiment 19. The polypeptide according to Embodiment 18, wherein at least one of the two ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 55, 105, and 108 according to Kabat numbering. Embodiment 20. The polypeptide according to Embodiment 18, wherein the N-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering. Embodiment 21. The polypeptide according to Embodiment 18, wherein the C-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 19, 26, 55, 105, and 108 according to Kabat numbering. Embodiment 22. A polypeptide comprising or (essentially) two ISVDs, wherein the N-terminal ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering. Embodiment 23. A polypeptide comprising or (essentially) two ISVDs, wherein the C-terminal ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 19, 26, 55, 105, and 108 according to Kabat numbering. Embodiment 24. The polypeptide according to any one of Embodiments 1 to 3, wherein the polypeptide comprises at least three ISVDs. Embodiment 25. The polypeptide according to Embodiment 24, wherein at least one of the at least three ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 26. The polypeptide according to Embodiment 24, wherein at least one of the at least three ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 15, 19, 26, and 105 according to Kabat numbering. Embodiment 27. The polypeptide according to any one of Embodiments 24 to 26, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) three ISVDs. Embodiment 28. The polypeptide according to Embodiment 24, 25, or 27, wherein the N-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 15, 19, 26, 55, 73, 75, 76, 105, 108, and 110 according to Kabat numbering. Embodiment 29. The polypeptide according to Embodiment 24, 25, or 27, wherein the N-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, and 105 according to Kabat numbering. Embodiment 30. The polypeptide according to Embodiment 24, 25, or 27, wherein at least one of at least three ISVDs that are neither C-terminus nor N-terminus comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 31. A polypeptide comprising or (essentially) three ISVDs, wherein at least one of the three ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 32. The polypeptide according to Embodiment 31, wherein the N-terminal ISVD includes a glycosylation acceptor site at an amino acid position selected from amino acid positions 3, 15, 19, 26, 55, 73, 75, 76, 105, 108, and 110 according to Kabat numbering. Embodiment 33. The polypeptide according to Embodiment 31, wherein the C-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26, and 105 according to Kabat numbering. Embodiment 34. The polypeptide according to Embodiment 31, wherein at least one of at least three ISVDs that are neither C-terminus nor N-terminus comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering. Embodiment 35. The polypeptide according to any one of Embodiments 31 to 34, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) three ISVDs. Embodiment 36. A polypeptide according to any one of Embodiments 1 to 35, wherein the glycosylated acceptor site is glycosylated with one or more glycans. Embodiment 37. The polypeptide according to Embodiment 36, wherein the glycan is selected from terminal N-acetylglucosamine (GlcNAc), (terminal) mannose, (terminal) sialic acid, (terminal) galactose, or a combination thereof. Embodiment 38. The polypeptide according to Embodiment 1, wherein the ISVD glycoprotein is a monovalent polypeptide, and the polypeptide sequence is selected from SEQ ID NOs: 11, 14, 69, 70, 73-78, 81, 83, 85, 87, 89-91, 93, 94, 96, and 177-179. Embodiment 39. The polypeptide according to Embodiment 1, wherein the ISVD glycoprotein is a bivalent polypeptide, and the polypeptide sequence is selected from SEQ ID NOs: 99-102, 105-108, 110, 116-118, 120, 121, 123-125, 128-133, 141-143, 145, 146, 148, 149, 182, 183, and 187. Embodiment 40. The polypeptide according to Embodiment 1, wherein the ISVD glycoprotein is a trivalent polypeptide, and the polypeptide sequence is selected from SEQ ID NOs: 20, 22-24, 37, 38, 40, 44-52, 55-57, 59-61, 63-65, 186, 190, and 191. Embodiment 41. A nucleotide sequence or nucleic acid encoding a polypeptide described in any one of Embodiments 1 to 40. The nucleotide sequence or nucleic acid according to embodiment 41, optimized for expression in a host cell or host organism capable of glycosylating the polypeptide encoded by the nucleotide sequence or nucleic acid. The nucleotide sequence or nucleic acid according to embodiment 41 or 42, in the form of a construct or (expression) vector that can be expressed in a host cell or host organism capable of glycosylating the polypeptide encoded by the nucleotide sequence or nucleic acid. Embodiment 44. A method for producing the polypeptide according to any one of embodiments 1 to 40, - a step of expressing the nucleotide sequence or nucleic acid according to any one of embodiments 41 to 43 in a suitable host cell or host organism comprising a method in which the host cell or host organism can glycosylate the expressed polypeptide. Embodiment 45. A method for conjugation of a moiety to the polypeptide according to any one of embodiments 1 to 40, - optionally, a step of oxidizing one or more of the glycans present on the polypeptide using periodate oxidation; - a step of conjugating the oxidized glycan to the moiety; comprising The method according to embodiment 45, wherein the moiety is selected from (bis-)mannose-6-phosphate, PROTAC and PEG moieties. Embodiment 47. A conjugate comprising the polypeptide according to any one of embodiments 1 to 40 and a conjugate moiety, wherein the moiety is conjugated to a glycan. The conjugate according to embodiment 47, wherein the moiety is selected from (bis-)mannose-6-phosphate, PROTAC and PEG moieties. Embodiment 49. A composition comprising the polypeptide according to any one of embodiments 1 to 40, the polypeptide produced using the method according to embodiment 45 or 46, or the conjugate according to embodiment 47 or 48. Embodiment 50. A polypeptide according to any one of Embodiments 1 to 40, a nucleotide sequence or nucleic acid according to any one of Embodiments 41 to 43, a conjugate according to Embodiment 47 or 48, or a composition according to Embodiment 49 for use as a medicament. Embodiment 51. Use of a polypeptide according to any one of Embodiments 1 to 40, a nucleotide sequence or nucleic acid according to any one of Embodiments 41 to 43, a conjugate according to Embodiment 47 or 48, or a composition according to Embodiment 49 for the manufacture of a medicament.

[0164] The following Tables 2A and 2B are lists of preferred glycosylation acceptor sites present in ISVD according to the present technology and preferred formats of such ISVDs having glycosylation acceptor sites.

[0165] [Table 2]

[0166] [Table 3]

[0167] The present technology and its embodiments are further highlighted in the following Examples section. [Examples]

[0168] Example 1: Generation of an ISVD Expression Construct ISVD-coding DNA fragments obtained by PCR using specific combinations of forward FR1 primers and reverse FR4 primers, each possessing unique restriction sites, were digested with appropriate restriction enzymes and ligated to the corresponding cloning cassettes of ISVD expression vectors. These ligated mixtures were then used to transform electrocompetent or chemically competent Escherichia coli TG1 (Lucigen, catalog numbers 60502 or custom-made, respectively) or TOP10 (ThermoFisher Scientific, catalog numbers C404052 or C4081201, respectively) cells, which were then grown under selective antibiotic pressure (kanamycin or zeosin). Resistance clones were identified by Sanger sequencing of the plasmid DNA (LGC Genomics).

[0169] Preparation of constructs for monovalent ISVD expression in Escherichia coli (E. coli) Monovalent ISVD is expressed in *E. coli* TG1 cells from an ISVD cloning site preceding a plasmid expression vector containing a lac promoter, a kanamycin resistance gene, an *E. coli* replication start site, and the coding sequence for the OmpA signal peptide. This directs the expressed ISVD to the periplasmic compartment of the bacterial host. In the frame containing the ISVD coding sequence, the vector encodes the C-terminal 3xFLAG and His6 tag.

[0170] Preparation of constructs for ISVD expression in CHO cells The mammalian expression vector used for ISVD protein expression contained the RSV-LTR promoter, a zeosin resistance gene, and a mouse light chain signal peptide. The DNA encoding the ISVD building block and the GS linker were cloned into the expression vector by Golden Gate cloning (Engler C, Marillonnet S. Golden Gate cloning. Methods Mol. Biol. 2014;1116:119-31). The expression vector contained two BpiI restriction sites for cloning PCR-amplified monovalent ISVD DNA, along with one or more GS linkers in the vector. All of these elements were adjacent to the BpiI sites. Seamless ligation in a predetermined order was possible by using nucleotide overhangs specific to each position in the cloning cassette. After Sanger sequence confirmation, plasmid DNA derived from E. coli TOP10 was transfected into CHOEBNALT85 cells.

[0171] Example 2: Expression of ISVD in Escherichia coli (E. coli) E. coli cells containing an ISVD expression vector were grown in a baffled shaker flask containing "5052" auto-induction medium (50x stock: 25% glycerol, 2.5% glucose, 10% lactose) at 37°C for 2 hours, followed by 29 hours at 30°C (250 rpm). The cells were pelletized by centrifugation (20 minutes, 4500 rpm, 4°C), the supernatant was discarded, and the pellet was frozen overnight at -20°C. The frozen cell pellet was then lysed in DPBS (Gibco, catalog no. 14190-094) to 1 / 12.5 of the original culture volume and incubated at 4°C for 1 hour with gentle rotation to disrupt the cell outer membrane. The cells were pelletized again (20 minutes, 8500 rpm, 4°C), the supernatant containing ISVD was collected, filtered, and immediately purified.

[0172] ISVD expressed in Escherichia coli (E. coli) was considered a control because glycosylation does not occur during E. coli-mediated expression.

[0173] Example 3: Expression of ISVD in mammalian cells CHOEBNALT85-1E9 cells (QMCF Technology licensed from Icosagen) were seeded at a density of 1.5E06 cells / mL in 10 mL of CHO TF medium (Xell, catalog no. 8860001) containing GlutaMAX® supplement (Gibco, catalog no. 35050-038) and transfected with DNA / transfection reagent 007 complex. The complex was formed by mixing 10 μg of plasmid DNA in 300 μL of water with 50 μg of transfection reagent 007 (Icosagen, catalog no. R007P001) in 200 μL of water and incubating at room temperature for 5 minutes. After incubation at 37°C for 1.5 hours, 10 mL of fresh CHO TF medium containing GlutaMAX® supplement was added, and the cells were grown at 37°C for 24 hours. Next, 10 mL of fresh CHO TF medium containing GlutaMAX® supplement and penicillin-streptomycin (Gibco, catalog no. 15140-122) was added, and the cells were incubated at 37°C for a further 72 hours. Then, the cell density was determined, and when it reached 3.5E06 cells / mL with a viability of >90%, 1.8 mL of Basic Feed (Xell, catalog no. 1092-0001) was added to the cells, and the incubation temperature was lowered to 30°C. Basic Feed was added again after 48 hours, and again after another 48 hours. After the final 72-hour incubation, the cells were pelleted by centrifugation (1000 rpm for 10 minutes), and the supernatant was transferred to another tube. The supernatant was centrifuged again (8500 rpm for 30 minutes) to remove any remaining cell debris, and the medium containing ISVD was collected, filtered, and stored at -20°C until purified. All incubation was performed in a humidified orbital shaker incubator at 200 rpm in the presence of 8% CO2.

[0174] Example 4: Purification of ISVD using Protein A affinity chromatography The ISVD construct was purified with Protein A, followed by desalting and, if necessary, preparative SEC in D-PBS. The concentration was determined by OD280 / OD340 measurement. Quality control was performed by SDS-PAGE and mass spectrometry.

[0175] A complete list of the generated ISVDs can be found in Table 3, shown in Example 5 below.

[0176] Example 5: Glycosylation Analysis ISVD glycoproteins obtained from mammalian cells were analyzed by SDS-PAGE for their glycosylation at different glycosylation acceptor sites. N-linked oligosaccharides were removed by PNGase, and the shift in molecular weight (MW) in SDS-PAGE was confirmed to be actually due to glycosylation. More specifically, if the MW shift did not disappear after PNGase treatment compared to the wild-type non-glycosylated control, it meant that something other than the added sugar caused the increase in MW.

[0177] PNGase F process N-linked oligosaccharides were removed from ISVD glycoprotein by PNGase F (N-glycosidase F). The method was carried out according to the manufacturer's instructions (NEB, catalog number P0704S): 5-10 μg of glycosylated ISVD, 1 μL of 10× glycoprotein denaturation buffer, and H2O were added to a total volume of 10 μL. The mixture was heated at 100°C for 10 minutes, then cooled on ice and briefly centrifuged for 10 seconds. Subsequently, 2 μL of 10× glycobuffer, 2 μL of 10% NP-40, and 6 μL of H2O were added, followed by 1 μL of PNGase F. The reaction products were gently mixed and incubated at 37°C for 1 hour. 2 μg of the mixture was finally analyzed using SDS-PAGE analysis according to the protocol described below.

[0178] Protocol SDS-PAGE Analysis 2 μg of the sample was analyzed on a precast 4-12% Bis-Tris NuPAGE SDS PAGE gel (Invitrogen, #NP0321BOX or NP0323BOX) under both reducing (R), heated and non-reducing (NR), non-heated conditions. The loading buffer was NuPAGE, 4×LDS Sample Buffer (Invitrogen, #NR0008). Heating was carried out at 98 °C for 3 minutes. After loading 5 μl of the marker (Sharp Pre-stained Protein Standard, Novex, #LC5800), the gel was electrophoresed at 180 V for 40 minutes in 1×MES running buffer. Finally, the gel was stained with Instant Blue and decolorized with tap water for 8 hours.

[0179] Mass Spectrometry Intact mass LC-MS analysis was performed either on an Agilent 1290 series UHPLC coupled to an Agilent Q-TOF 6530 mass spectrometer (both from Agilent Technologies) or on a Vanquish Flex UHPLC coupled to a Q-Exactive Plus mass spectrometer (both from Thermo Fisher Scientific). After online desalting using a MassPREP microdesalting column (Waters), the molecular weight of the major product was determined after charge state deconvolution of the raw MS data.

[0180] Results Only ISVDs with a degree of glycosylation exceeding 50% were considered to be sufficiently glycosylated. The complete list of ISVDs tested can be found in Table 3 below.

[0181] [Table 4]

[0182] [Table 5]

[0183] [Table 6]

[0184] [Table 7]

[0185] [Table 8]

[0186] Surprisingly, the inventors found that while some glycosylation acceptor sites are glycosylated only in certain ISVD formats and / or specific configurations of ISVD, other glycosylation acceptor sites have different requirements for achieving a high degree of glycosylation.

[0187] Based on the glycosylation analysis performed, it was concluded that the following positions in the ISVD (Kabat numbering) for use as glycosylation acceptor sites exhibited a good degree of glycosylation when the ISVD was in monovalent and polyvalent formats: 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110. However, surprisingly, the use of glycosylation acceptor sites located at one of positions 53, 68, 75, 102, and 110 in the C-terminal ISVD of the polyvalent format resulted in a low degree of glycosylation, while the use of glycosylation acceptor sites at these positions, when absent in the C-terminal ISVD of the polyvalent format, resulted in a high degree of glycosylation. Specifically, for position 102, this was in trivalent or higher formats.

[0188] Conversely, for the 1st position, a high degree of glycosylation was observed in the polyvalent format only when the glycosylation acceptor site was present in the C-terminal ISVD.

[0189] It was observed that high glycosylation levels could only be achieved at the glycosylation acceptor sites at positions 3 and 15 of ISVD when the ISVD was in a polyvalent format. Similarly, high glycosylation levels could only be achieved at the glycosylation acceptor site at position 15 of ISVD when the ISVD was in a trivalent or higher format. Position 76 was tested only in a trivalent format, and this format yielded a high degree of glycosylation.

[0190] Example 6: Thermal Shift Assay This assay determined whether glycosylation of ISVDs affects the melting temperature. The following ISVDs were used: ALB00606 (SEQ ID NO: 1), ALB00607 (SEQ ID NO: 2), ALB00608 (SEQ ID NO: 3), ALB00609 (SEQ ID NO: 4), ALB00610 (SEQ ID NO: 5), ALB00611 (SEQ ID NO: 6), ALB00612 (SEQ ID NO: 7), ALB00616 (SEQ ID NO: 11), ALB00621 (SEQ ID NO: 16), and ALB00622 (SEQ ID NO: 17).

[0191] Of these, ALB00606, ALB00607, ALB00608, ALB00609, ALB00610, ALB00611, ALB00612, ALB00621, and ALB00622 were produced in Escherichia coli (E. coli) (without glycosylation) and served as controls.

[0192] Since ALB00616 was produced in CHO cells, glycosylation occurred, and it functioned as an exemplary glycosylated ISVD. Because the sequence of ALB00616 is identical to that of ALB00609, the only difference between these differently produced ISVDs is whether or not they are glycosylated.

[0193] A thermal shift assay (TSA) was performed on a 96-well plate on a qPCR machine (LightCycler 480II, Roche). One ISVD per row was analyzed within the following pH ranges: 4, 5, 6, 7, 8, and 9. In some cases, six different formulation buffers were also included, either with or without 8% sucrose, i.e., 20 mM acetic acid (pH 5), 20 mM histidine (pH 6), or 20 mM phosphate (pH 7). Per well, 5 μL of ISVD sample (0.8 mg / mL in D-PBS) was added to 5 μL of Sypro Orange (MilliQ 40× in water; Invitrogen, catalog no. S6551) and 10 μL of buffer (100 mM phosphate, 100 mM boric acid, 100 mM citrate, and 115 mM NaCl, pH range 4–9). A temperature gradient (37–99°C at a rate of 0.03°C / sec) was applied, which induced the unfolding of ISVD and thus the exposure of hydrophobic patches. Binding of Sypro Orange to these hydrophobic patches resulted in an increase in fluorescence intensity, which was measured (Ex / Em = 465 / 580 nm). The inflection point of the first derivative in the fluorescence intensity curve at pH 7 served as a measure of the melting point (Tm).

[0194] result The results of the thermal assay are shown in Table 4 below.

[0195] [Table 9]

[0196] As can be seen from Table 4, the melting temperatures were the same for non-glycosylated ISVD and glycosylated ISVD. In particular, ISVDs with identical sequences, ALB00609 (control ISVD produced in Escherichia coli (E. coli)) and ALB00616 (exemplary ISVD produced in CHO cells) showed no difference in melting temperature regardless of whether they were glycosylated or not.

[0197] As a result, it can be concluded that the melting temperature of glycosylated ISVD is not affected by the glycosylation of the ISVD.

[0198] Example 7: Affinity analysis of ISVD / human serum albumin interaction using surface plasmon resonance The affinity of ISVD to human serum albumin (HSA) was determined by surface plasmon resonance (SPR). T043800002 (SEQ ID NO: 20), T043800003 (SEQ ID NO: 21), T043800004 (SEQ ID NO: 22), T043800005 (SEQ ID NO: 23), T043800008 (SEQ ID NO: 26), and T043800012 (SEQ ID NO: 30) were tested. Wild-type ISVD T04380001 (SEQ ID NO: 19) was used as a reference.

[0199] HSA (Sigma-Aldrich, catalog number A8763000) was directly immobilized on flow cell 2 (FC) of a C1 Biacore tip on a Biacore 8K(+) instrument. Tip activation was performed by injecting EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, 200 mM, Sigma Aldrich catalog number 39391) / NHS (N-hydroxysuccinimide, 50 mM, Sigma Aldrich catalog number 130672) for 7 minutes. HSA was diluted to 4 μg / ml in 10 mM acetate buffer, pH 4.5, and flowed over channels 1-8 of FC1 for immobilization. Subsequently, a de-inactivation step was performed for 7 minutes using ethanolamine HCl (1 M, Cytiva). The flow rate during activation, immobilization, and deactivation was set to 10 μL / min. Affinity determination was set up in a 6-step dilution series ranging from 1000 to 1 nM ISVD. Various concentrations were tested in multi-cycle reaction rate mode with a flow rate of 30 μL / min, an association time of 120 seconds in 1×HBS-EP+ buffer, and a dissociation time of 600 seconds. Regeneration conditions after each interaction analysis were performed by flowing 10 mM glycine (pH 1.5) buffer onto the tip surface at a flow rate of 30 μL / min for 60 seconds. Data analysis was performed using Biacore insight evaluation software.

[0200] result The results of the affinity measurements of the tested ISVD can be found in Table 5 below.

[0201] [Table 10]

[0202] As can be seen from Table 5, the affinity of glycosylated ISVD to wild-type reference ISVD is equivalent. This indicates that glycosylated ISVD maintains a high affinity for those targets.

[0203] Example 8: Affinity analysis of ISVD / EGFR interaction using surface plasmon resonance The affinity of ISVD to EGFR was determined by surface plasmon resonance (SPR). ISVD T043800179 (SEQ ID NO: 99), T043800180 (SEQ ID NO: 100), T043800181 (SEQ ID NO: 101), T043800182 (SEQ ID NO: 102), T043800185 (SEQ ID NO: 105), T043800187 (SEQ ID NO: 107), T043800190 (SEQ ID NO: 110), T043800196 (SEQ ID NO: 116), T043800198 (SEQ ID NO: 118), and T043800200 (SEQ ID NO: 120) were tested. Wild-type ISVD T043800178 (SEQ ID NO: 98) and T043800189 (SEQ ID NO: 109) were used as reference.

[0204] EGFR (Sino Biological, catalog no. LC14JA1103) was directly immobilized on flow cell 2 (FC) of a CM5 Biacore tip on a Biacore 8K(+) instrument. Tip activation was performed by injecting EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, 200 mM, Sigma Aldrich catalog no. 39391) / NHS (N-hydroxysuccinimide, 50 mM, Sigma Aldrich catalog no. 130672) for 7 minutes. EGFR was diluted to 4 μg / ml in 10 mM acetate buffer, pH 4.5, and flowed over channels 1-8 of FC1 for immobilization. Subsequently, a de-inactivation step was performed for 7 minutes using ethanolamine HCl (1 M, Cytiva). The flow rate during activation, immobilization, and deactivation was set to 10 μL / min. Affinity determination was set up in a 6-step dilution series ranging from 2500 to 0.06 nM ISVD. Various concentrations were tested in multi-cycle reaction rate mode with a flow rate of 30 μL / min, an association time of 120 seconds, and a dissociation time of 600 seconds in 1×HBS-EP+ buffer. Regeneration conditions after each interaction analysis were performed by flowing 10 mM glycine (pH 2.5) buffer onto the tip surface at a flow rate of 45 μL / min for 30 seconds twice. Data analysis was performed using Biacore insight evaluation software.

[0205] result The results of the affinity analysis can be found in Table 6 below.

[0206] [Table 11]

[0207] As can be seen from Table 6, the affinity of glycosylated ISVDs to wild-type reference ISVDs decreased by up to four times, while all ISVDs maintained high affinity. This indicates that glycosylated ISVDs maintain high affinity for their targets.

[0208] Example 9: Affinity analysis of ISVD / human serum albumin interaction using Meso Scale Discovery The affinity of ISVD to HSA was further determined by Meso Scale Discovery (MSD). ISVD T043800056 (SEQ ID NO: 36), T043800057 (SEQ ID NO: 37), T043800060 (SEQ ID NO: 40), T043800064 (SEQ ID NO: 44), T043800065 (SEQ ID NO: 45), T043800066 (SEQ ID NO: 46), T043800067 (SEQ ID NO: 47), T043800068 (SEQ ID NO: 48), T043800070 (Distribution) The following samples were tested: (sequence number 50), T043800071 (sequence number 51), T043800072 (sequence number 52), T043800074 (sequence number 54), T043800075 (sequence number 55), T043800076 (sequence number 56), T043800089 (sequence number 57), T043800093 (sequence number 61), and T043800096 (sequence number 64). Wild-type ISVD T043800055 (sequence number 35) and T043800098 (sequence number 66) were used as references.

[0209] Human serum albumin (HSA, Sigma-Aldrich, catalog number A8763000) was biotinylated using NHS-LC-biotin (ThermoFisher, catalog number 21336) according to the manufacturer's instructions, with an average labeling ratio of 1. The biotinylated HSA was captured at a concentration of 1 μg / mL on an MSD GOLD 96-well Small Spot streptavidin SECTOR plate (MSD, catalog number L45SA-1). Subsequently, 25 μL of pre-equilibrium mixture (at room temperature for 2 hours) containing a fixed concentration of 100 pM of the ISVD compound of interest, each with HSA ranging from 1.13 pM to 10 μM (23 dilutions, 1 / 3 dilution ratio), was added to the plate. After incubating the plate for 10 minutes to capture the free ISVD compound, it was washed with 3 × 150 μL of PBS + 0.05% Tween-20. During the final detection step, 25 μL of sulfo-tagged anti-ISVD antibody was added at a concentration of 2 μg / ml and incubated for 1 hour, followed by a final wash of 150 μL with 1×PBS + 0.05% Tween-20. After adding 150 μL of MSD read buffer (Meso Scale Diagnostics, catalog no. R92TG-1), the plate was read using an MSD QuickPlex SQ120 reader. Data were analyzed using a 4-parameter logistic (4PL) scale fitted to a GraphPad Prism 8.

[0210] result The results of the affinity analysis can be found in Table 7 below.

[0211] [Table 12]

[0212] As can be seen from Table 7, the affinity of glycosylated ISVD to wild-type reference ISVD is equivalent. This indicates that glycosylated ISVD maintains a high affinity for those targets.

[0213] Example 10: Affinity analysis of ISVD / TNFα interactions using Meso Scale Discovery The affinity of ISVD to TNFα was determined by Meso Scale Discovery (MSD). T043800002 (SEQ ID NO: 20), T043800003 (SEQ ID NO: 21), T043800004 (SEQ ID NO: 22), T043800005 (SEQ ID NO: 23), T043800008 (SEQ ID NO: 26), T043800012 (SEQ ID NO: 30), and T043800016 (SEQ ID NO: 34) were tested. Wild-type ISVD T04380001 (SEQ ID NO: 19) was used as a reference.

[0214] Human TNFα (Bio-techne, catalog no. 210-TA) was biotinylated using NHS-LC-biotin (ThermoFisher, catalog no. 21336) according to the manufacturer's instructions, with an average labeling ratio of 1. The biotinylated TNFα was captured at a concentration of 0.5 μg / mL on MSD GOLD 96-well Small Spot streptavidin SECTOR plates (MSD, catalog no. L45SA-1). Subsequently, 25 μL of pre-equilibrium mixtures (at room temperature for 24 hours) containing a fixed concentration of 12.8 pM of the ISVD compound under test, each with TNFα ranging from 78 fM to 10 μM (23 dilutions, 1 / 10 dilution ratios from 1 to 0.01 μM; 1 / 1.8 dilution ratios from 10 nM to 78 fM), were added to the plates. After incubating the plate for 10 minutes to capture free ISVD compounds, it was washed with 3 × 150 μL of PBS + 0.05% Tween-20. During the final detection step, 25 μL of sulfo-tagged anti-ISVD antibody at a concentration of 2 ug / ml was added and incubated for 1 hour, followed by a final wash of 150 μL with 1 × PBS + 0.05% Tween-20. After adding 150 μL of MSD read buffer, the plate was read with an MSD QuickPlex SQ120 reader. Data were analyzed using a 4-parameter logistic (4PL) scale fitted to a GraphPad Prism 8.

[0215] result The results of the affinity analysis can be found in Table 8 below.

[0216] [Table 13]

[0217] As can be seen from Table 8, the affinity of glycosylated ISVD to wild-type reference ISVD is equivalent. This indicates that glycosylated ISVD maintains a high affinity for those targets.

[0218] Example 11: Glycan profiling of ISVD glycoprotein The glycan patterns on ISVD glycoproteins T043800002 (SEQ ID NO: 20), T043800004 (SEQ ID NO: 22), T043800005 (SEQ ID NO: 23), T043800006 (SEQ ID NO: 24), T043800008 (SEQ ID NO: 26), T043800012 (SEQ ID NO: 30), and T043800016 (SEQ ID NO: 34) were further analyzed by LC-MS.

[0219] Samples for glycan analysis were prepared according to the RapiFluor-MS (Waters) protocol. Prior to analysis, the samples were diluted 9:31 using 2.1:1 acetonitrile:dimethylformamide (Waters). The samples were analyzed using a 6545XT LC-QTOF and concurrently with the RapiFluor-MS performance standard (Waters) at a rate of 0.4 mL / min over 35 minutes using a column (glycan BEH amide, Waters) maintained at 60°C, utilizing a linear gradient from 25% 50 mM ammonium formate in water (Fisher), pH 4.4 (Waters) to 56% acetonitrile (Thermo Scientific). Data were analyzed using a Genedata Expressionist.

[0220] result Examples of glycan patterns of ISVD glycoproteins on LC-QTOF are shown in Figures 1 and 2. All ISVD glycoproteins showed similar patterns of glycan species; typically, they contained a high proportion of sialic acids containing glycans such as G1FS1, G2FS1, and G2FS2, in addition to neutral glycans such as G0F, G1F, and G2F. The amount of glycan species varied slightly among ISVD glycoproteins. Sialized species with one or two sialic acids were the most abundant.

[0221] Example 12: Bis-mannose 6-phosphate conjugation ISVD glycoproteins T043800005 (SEQ ID NO: 23), T043800002 (SEQ ID NO: 20), T043800124 (SEQ ID NO: 73), T043800125 (SEQ ID NO: 74), T043800126 (SEQ ID NO: 75), T043800127 (SEQ ID NO: 76), T043800128 (SEQ ID NO: 77), and T043800129 (SEQ ID NO: 78) were used in conjugation with bis-mannose 6-phosphate (bis-M6P).

[0222] The conjugation of bis-mannose 6-phosphate (manufactured in-house) containing glycans to glycosylated ISVD was first completed by oxidizing ISVD with sodium periodate (Sigma) at concentrations of 2.5 mg / mL and 20 mM, respectively, in 100 mM sodium acetate (Sigma), pH 5.6, for 30 minutes at 4°C with gentle shaking. The reaction was then quenched with 3% glycerol (Sigma) at 4°C for 15 minutes. The ISVD was purified in 100 mM sodium acetate, pH 5.6 by centrifugation through a molecular weight cutoff filter (Millipore) for at least 5 exchanges.

[0223] After determining the protein concentration via Stunner (Unchained Labs), oxidized ISVD was conjugated with bis-M6P glycan in 100 mM sodium acetate, pH 5.6. The reaction was allowed to proceed for at least 16 hours with gentle shaking at room temperature. The ISVD was then purified into PBS by centrifugation through a molecular weight cutoff filter for at least 5 exchanges. The resulting ISVD concentration and polydispersity were then determined via Stunner. The conjugates were then analyzed by completing intact mass LC-QTOF and MALDI-TOF (protocols described below).

[0224] Protocol-based intact mass spectrometry (LC-QTOF) For intact mass spectrometry, the sample was diluted to 1 mg / mL, then 20 mM DTT (final concentration, Sigma) was added and incubated at 37°C for 30 minutes. The sample was then analyzed using a 6545XT LC-QTOF (Agilent) at a linear gradient from 75% water + 0.1% formic acid (Fisher) to 60% acetonitrile + 0.1% formic acid (Fisher) over 7.5 minutes at a rate of 0.5 mL / min using a column (PLRP-S, Agilent) maintained at 60°C. The data were analyzed using Expressionist (Genedata).

[0225] Protocol MALDI-TOF analysis For MALDI-TOF, the sample was first diluted to 1 mg / mL using a sample diluent and 0.1% formic acid (Sigma) in water, then mixed 1:1 with the matrix solution, 50% acetonitrile in water, and 1% formic acid. The matrix-diluted samples were spotted three times onto each MALDI target plate (Bruker) and dried at room temperature. Repeat samples were then analyzed using a MALDI-TOF instrument (Bruker), and the data were analyzed using FlexAnalysis software (Bruker).

[0226] result The MALDI-TOF analysis of the conjugate with T043800005 can be seen in Figure 3. Conjugates with one and two bis-M6P per ISVD are visible. An average labeling degree (DoL) of 0.9 bis-M6P per ISVD was obtained.

[0227] The MALDI-TOF analysis of the conjugate with T043800002 can be seen in Figure 4. Conjugates with 1, 2, and 3 bis-M6P per ISVD are visible. An average labeling degree (DoL) of 2.1 bis-M6P per ISVD was obtained.

[0228] Example 13: PROTAC Conjugate ISVD glycoprotein T043800005 (SEQ ID NO: 23) was conjugated to an alkoxyamine-DBCO linker, followed by PROTAC. Conjugation of PROTAC BRD4 Degrader-5-CO-PEG3-N3 (PROTAC, MedChemExpress) to glycosylated ISVD T04380005 was first completed by oxidizing ISVD with sodium periodate at concentrations of 2.5 mg / mL and 20 mM, respectively, in 100 mM sodium acetate, pH 5.6. After allowing oxidation to proceed with gentle shaking at 4°C for 30 minutes, the mixture was quenched with 3% glycerol at 4°C for 15 minutes. Subsequently, ISVD was purified in 100 mM sodium acetate, pH 5.6 by centrifugation through a molecular weight cutoff filter for at least 5 exchanges.

[0229] After determining the protein concentration via Stunner, ISVD was conjugated to aminooxy-PEG2-bis-PEG3-DBCO (linker, Conju-Probe) at a final concentration of 2.5 mg / mL for both materials in 100 mM sodium acetate, pH 5.6. The reaction was allowed to proceed for at least 16 hours with gentle shaking at room temperature. ISVD was then purified into PBS by centrifugation through a molecular weight cutoff filter for at least 5 exchanges.

[0230] After purification, the protein concentration was determined via Stunner, and then ISVD was conjugated with PROTAC at final concentrations of 2.5 and 1.8 mg / mL, respectively, in PBS. The reaction was allowed to proceed for at least 16 hours with gentle shaking at room temperature. The ISVD was then purified into PBS by centrifugation through a molecular weight cutoff filter for at least 5 exchanges. The resulting ISVD concentration and polydispersity were then determined via Stunner. The conjugation was then analyzed by completing the SDS-PAGE protocol described below and the MALDI-TOF protocol described in Example 12.

[0231] Protocol SDS-PAGE The sample for SDS-PAGE was first diluted to 1 mg / mL in phosphate-buffered saline (PBS, Gibco). The sample was incubated at 70°C for 10 minutes. After incubation, the sample was mixed with 4X NuPAGELDS sample buffer (Invitrogen) and water, and then loaded onto a 4-12% Bis-Tris NuPAGE gel (Invitrogen) with a PageRuler standard ladder (Thermo Scientific). The gel was electrophoresed in an Xcell Surelock cell equipped with a Powerease power supply (Invitrogen) and MES running buffer (Invitrogen) using the NuPAGE gel setting. After program completion, the gel was removed from the cassette and placed in InstantBlue staining (Abcam) for 15 minutes. The gel was then visualized using ChemiDoc Imager and Image Lab software (Bio-Rad).

[0232] result Conjugation with PROTAC was observed by SDS-PAGE with a DoL (degree of labeling) of 0.42.

[0233] Example 14: PEGylation ISVD glycoprotein T043800005 (SEQ ID NO: 23) was further conjugated to alkoxyamine-functionalized PEG. Conjugation of 2 kDa aminooxy-PEG (PEG, BroadPharm) to glycosylated ISVD T043800005 was first completed by oxidizing ISVD with sodium periodate at concentrations of 2.5 mg / mL and 20 mM, respectively, in 100 mM sodium acetate, pH 5.6, with gentle stirring at 4°C for 30 minutes. The reaction product was then quenched with 3% glycerol at 4°C for 15 minutes. ISVD was then purified in 100 mM sodium acetate, pH 5.6 by centrifugation through a molecular weight cutoff filter for at least 5 exchanges.

[0234] After determining the protein concentration via Stunner, ISVD was PEGylated in 100 mM sodium acetate, pH 5.6, at concentrations of 2.5 mg / mL and 7.5 mg / mL, respectively. The reaction was allowed to proceed at room temperature with gentle shaking for at least 16 hours. The ISVD was then purified in PBS by centrifugation at least five times through a molecular weight cutoff filter. The resulting ISVD concentration and polydispersity were then determined via Stunner. The conjugation was then analyzed by completing SDS-PAGE, the protocol described in Example 13, and MALDI-TOF, the protocol described in Example 12.

[0235] result SDS-PAGE revealed conjugation with PEG at a DoL of 0.55.

[0236] Example 15: TNF-α internalization mediated by ISVD glycovariant Materials and methods material Biotinylated TNF was purchased from R&D Systems, while Alexa Fluor® 647-labeled streptavidin was obtained from Thermo Fisher Scientific. Except for the in-house manufactured bis-M6P glycan, all other chemical samples were purchased from Millipore Sigma unless otherwise specified.

[0237] Expression and purification of ISVD glycoside variants The trivalent ISVD sugar variant, T043800005 (SEQ ID NO: 23), was manipulated by introducing an N-glycosylation site with R19N, and mutants were generated using site-directed mutagenesis. The ISVD constructs were produced from Chinese hamster ovary (CHO) cells. The protocol is as described in Examples 1 and 4.

[0238] Site-specific ISVD conjugation with bis-M6P glycan Conjugation was carried out based on methods known in the art. Briefly, anti-TNF ISVD was oxidized with 20 mM sodium periodate in sodium phosphate buffer (pH 7.2) on ice for 30 minutes. The oxidized reaction product was protected from light and quenched with glycerol (3% (v / v)) for 15 minutes. The oxidized ISVD was buffer-changed to 100 mM sodium acetate (pH 5.6) by ultrafiltration through an Amicon® ultracentrifuge filter for 5 rounds. The desalted ISVD was then reacted with bis-M6P glycan in a 30-fold molar excess overnight at room temperature. Unconjugated free glycan was removed by ultrafiltration using the same protocol as for the oxidized ISVD.

[0239] Characterization of ISVD ISVD and ISVD conjugate were characterized using LC-MS intact protein analysis. The assay was performed by reducing 0.1 mg / mL of ISVD or ISVD conjugate with 20 mM dithiothreitol at 37°C for 30 minutes. After quenching the reaction with 0.1% trifluoroacetic acid, the reduced samples were analyzed on an Agilent 1290 / 6545XT Q-ToF UPLC / MS system.

[0240] UPLC-MS was performed using an Agilent PLRP column (2.1 mm × 50 mm, 5 μm) with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at 55°C in the m / z range of 100 to 9000 Da. The acquired data were processed using Expressionist 16.5 software. The strongest charge state in each spectrum was used for deconvolution using the MaxEnt algorithm (resolution: 1.0 Da, mass range 20 to 200 kDa).

[0241] The molecular weights of ISVD and ISVD bis-M6P conjugates were measured using MALDI-TOF MS, and the copy number of bis-M6P glycans per ISVD was determined. The analysis was performed using a Bruker Autoflex III. The intact protein mass was determined on target plates spotted with ISVD or conjugate samples mixed with a synapic acid matrix using linear positive mode. Data for each sample were acquired in triplicate. The number of bis-M6P glycans conjugated per ISVD was calculated by subtracting the molecular weight of the ISVD from the molecular weight of the conjugate, and then dividing the difference by the molecular weight of the glycan.

[0242] Size exclusion ultrahigh performance liquid chromatography (SEC-UPLC) was performed using a Water ACQUITY UPLC H-class PLUS Bio System. ISVD or bis-M6P conjugated ISVD (approximately 5 μg) was separated at room temperature using a Superdex 200 increase 10 / 300 gl column under isocratic conditions with a flow rate of 0.3 mL / min, using PBS (pH 7.2) as the mobile phase.

[0243] Characterization of internalization of target protein (POI) by flow cytometry Jurkat cells or K562 cells (ATCC) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.25 μg / mL amphotericin B, 55 μM 2-mercaptoethanol, and 10 mM HEPES (all from Gibco). These cells were seeded at 1 × 10⁵ cells / well in U-bottom 96-well plates.

[0244] Biotinylated recombinant human TNF-α and streptavidin labeled with Alexa Fluor® 647 were sequentially added at final concentrations of 50 nM and 100 nM, respectively. Subsequently, ISVD T043800005 or bis-M6P conjugate ISVD were sequentially added at various concentrations from 0.78 to 50 nM.

[0245] After culturing the cells at 37°C for 1 or 4 hours, they were washed twice with cold phosphate-buffered saline (PBS) (pH 7.2), and then flow cytometry was performed to evaluate the fluorescence of the TNF complex containing Alexa Fluor®.

[0246] Characterization of POI decomposition by Western blotting K562 cells or Jurkat cells were placed in a U-bottom 96-well plate, 1 × 10⁶ cells per well. 5Cells were seeded in wells. Recombinant human TNF-α (50 nM, Peprotech) and anti-TNF ISVD (T043800005) or bis-M6P conjugate ISVD were sequentially added to a final concentration of 25 nM.

[0247] After culturing the cells at 37°C for 2 hours, they were washed twice with culture medium and lysed with RIPA buffer (Boston BioProducts). Some cells were further cultured for 4 or 24 hours in the presence of DMSO or 100 nM bafilomycin A1 (InvivoGen).

[0248] At each time point, cells were washed twice with cold PBS and lysed with RIPA buffer. Western blotting to detect TNF-α or β-actin was performed using lysates prepared with anti-TNF (clone D5G9, Cell Signaling Technology), followed by an anti-rabbit detection module (ProteinSimple), or anti-β-actin (clone 8H10D10, Cell Signaling Technology), followed by an anti-mouse detection module (ProteinSimple) using Jess (ProteinSimple).

[0249] To measure TNF-α in the supernatant of the culture medium after internalization, K562 cells (1 × 10⁶) 5 Cells (per well) were treated with 50 nM TNF-α and 25 nM ISVD or bis-M6P conjugate ISVD at 37°C.

[0250] During the incubation period, the culture medium supernatant was collected at different time points (24, 48, and 72 hours) and Western blotting was performed using ISVD or conjugate as described above.

[0251] result Glycosylated ISVD contained one copy of bis M6P per ISVD, as determined by MALDI-TOF MS.

[0252] ISVD conjugates containing approximately one bis-M6P bound to a single introduced N-glycosylation site efficiently induced TNF-α internalization in Jurkat cells or K562 cells (Figure 5).

[0253] Bis-M6P conjugated ISVD constructs showed dose-dependent TNF-α internalization after 1-hour and 4-hour incubation, but a reduction in internalization was observed at high ISVD concentrations of 50 nM, likely due to the Hooke effect (Figure 6).

[0254] This was further confirmed by Western blotting analysis of cell lysates (Figure 7A) and supernatants (Figure 7B). The amount of internalized TNF-α was already quite low at 4 hours and was undetectable at 22 hours post-incubation. The addition of bafilomycin A1 significantly delayed TNF-α degradation at 4 and 22 hours, suggesting that TNF-α degradation by bis-M6P conjugate ISVD occurs in lysosomes. The amount of TNF-α remaining in the cell culture medium was also determined. There was little change in TNF-α levels in the culture supernatant derived from human K562 cells treated with control anti-TNF ISVD alone, but the amount of TNF-α was significantly reduced during cell incubation with bis-M6P conjugate ISVD from 24 to 72 hours.

[0255] This demonstrates that TNF-α internalization mediated by an ISVD sugar variant containing one copy of bis-M6P is effective, suggesting that a single bis-M6P on ISVD is sufficient to induce internalization and degradation of soluble POIs in lysosomes.

[0256] The following table shows the sequences disclosed herein.

[0257] [Table 14]

[0258] [Table 15]

[0259] Table 16

[0260] Table 17

[0261] Table 18

[0262] Table 19

[0263] Table 20

[0264] Table 21

[0265] Table 22

[0266] Table 23

[0267] Table 24

[0268] Table 25

[0269] Table 26

[0270] Table 27

[0271] Table 28

[0272] Table 29

[0273] Table 30

[0274] Table 31

[0275] Table 32

[0276] Table 33

[0277] Table 34

Claims

1. A polypeptide comprising or essentially comprising a heavy-chain immunoglobulin monovariable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 15, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

2. The polypeptide according to claim 1, wherein the glycosylated acceptor site is an N-glycosylation site.

3. The polypeptide according to claim 1 or 2, wherein the glycosylated acceptor site is contained in an NXT or NXS motif (where X may be any amino acid), and the asparagine residue of the NXT or NXS motif is the glycosylated acceptor site.

4. The polypeptide according to any one of claims 1 to 3, wherein the glycosylation acceptor site is located at an amino acid position selected from amino acid positions 19, 1, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering.

5. The polypeptide according to any one of claims 1 to 3, wherein the glycosylated acceptor site is located at an amino acid position selected from amino acid positions 19, 26, 55, 73, 105, and 108 according to Kabat numbering.

6. The polypeptide according to any one of claims 1 to 3, wherein the glycosylation acceptor site is located at an amino acid position selected from amino acid positions 19, 1, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 according to Kabat numbering.

7. The polypeptide according to claim 6, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) one ISVD.

8. A polypeptide comprising or (essentially) one ISVD, wherein the ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

9. The polypeptide according to claim 8, wherein the glycosylation acceptor site is located at an amino acid position selected from amino acid positions 19, 1, 26, 53, 55, 68, 73, 75, 102, 105, 108, and 110 according to Kabat numbering.

10. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises at least two ISVDs.

11. The polypeptide according to claim 10, wherein at least one of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 15, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

12. The polypeptide according to claim 10, wherein at least one of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering.

13. The polypeptide according to claim 10, wherein at least one of the at least two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 26, 55, 105, and 108 according to Kabat numbering.

14. The polypeptide according to claim 10, wherein the polypeptide is a divalent polypeptide comprising (essentially) two ISVDs.

15. The polypeptide according to claim 14, wherein the N-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering.

16. The polypeptide according to claim 14, wherein the C-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 26, 55, 105, and 108 according to Kabat numbering.

17. The polypeptide according to claim 14, wherein at least one of the two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 26, 55, 105, and 108 according to Kabat numbering.

18. A polypeptide comprising or (essentially) two ISVDs, wherein at least one of the two ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 15, 26, 53, 55, 68, 73, 75, 76, 105, 108, and 110 according to Kabat numbering.

19. The polypeptide according to claim 18, wherein at least one of the two ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 26, 55, 105, and 108 according to Kabat numbering.

20. The polypeptide according to claim 18, wherein the N-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering.

21. The polypeptide according to claim 18, wherein the C-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 26, 55, 105, and 108 according to Kabat numbering.

22. A polypeptide comprising or (essentially) two ISVDs, wherein the N-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 26, 53, 55, 68, 73, 75, 105, 108, and 110 according to Kabat numbering.

23. A polypeptide comprising or (essentially) two ISVDs, wherein the C-terminal ISVD comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 26, 55, 105, and 108 according to Kabat numbering.

24. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises at least three ISVDs.

25. The polypeptide according to claim 24, wherein at least one of the at least three ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 15, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

26. The polypeptide according to claim 24, wherein at least one of the at least three ISVDs includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 15, 26, and 105 according to Kabat numbering.

27. The polypeptide according to any one of claims 24 to 26, wherein the polypeptide is a trivalent polypeptide comprising (essentially) three ISVDs.

28. The polypeptide according to claim 24, 25, or 27, wherein the N-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 15, 26, 55, 73, 75, 76, 105, 108, and 110 according to Kabat numbering.

29. The polypeptide according to claim 24, 25, or 27, wherein the C-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 15, 26, and 105 according to Kabat numbering.

30. The polypeptide according to claim 24, 25, or 27, wherein at least one of the at least three ISVDs, which are not located at the C-terminus or the N-terminus, comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 15, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

31. A polypeptide comprising or (essentially) three ISVDs, wherein at least one of the three ISVDs comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 15, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

32. The polypeptide according to claim 31, wherein the N-terminal ISVD includes a glycosylation acceptor site at an amino acid position selected from amino acid positions 19, 3, 15, 26, 55, 73, 75, 76, 105, 108, and 110 according to Kabat numbering.

33. The polypeptide according to claim 31, wherein the C-terminal ISVD includes a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 1, 3, 15, 26, and 105 according to Kabat numbering.

34. The polypeptide according to claim 31, wherein at least one of the at least three ISVDs, which are not located at the C-terminus or the N-terminus, comprises a glycosylation acceptor site located at an amino acid position selected from amino acid positions 19, 3, 15, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108, and 110 according to Kabat numbering.

35. The polypeptide according to any one of claims 31 to 34, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) three ISVDs.

36. The polypeptide according to any one of claims 1 to 35, wherein the glycosylated acceptor site is glycosylated with one or more glycans.

37. The polypeptide according to claim 36, wherein the glycan is selected from terminal N-acetylglucosamine (GlcNAc), (terminal) mannose, (terminal) sialic acid, (terminal) galactose, or a combination thereof.

38. A nucleotide sequence or nucleic acid encoding the polypeptide according to any one of claims 1 to 37.

39. The nucleotide sequence or nucleic acid according to claim 38, which is optimized for expression in a host cell or host organism capable of glycosylation of the polypeptide encoded by the nucleotide sequence or nucleic acid.

40. The nucleotide sequence or nucleic acid according to claim 38 or 39, in the form of a construct or (expression) vector that can be expressed in a host cell or host organism capable of glycosylation of the polypeptide encoded by the nucleotide sequence or nucleic acid.

41. A method for producing a polypeptide according to any one of claims 1 to 37, - A step of expressing a nucleotide sequence or nucleic acid according to any one of claims 38 to 40 in a suitable host cell or host organism. Includes, A method by which the host cell or host organism can glycosylate the expressed polypeptide.

42. A method for partial conjugation of a polypeptide according to any one of claims 1 to 37, - Optionally, the process involves oxidizing one or more glycans present on the polypeptide using periodic acid oxidation. - The process of conjugating the oxidized glycan to the aforementioned portion, Methods that include...

43. The method according to claim 42, wherein the portion is selected from (bis-)mannose-6-phosphate, PROTAC, and PEG portions.

44. A conjugate comprising a polypeptide according to any one of claims 1 to 37 and a conjugate portion, wherein the portion is conjugated to a glycan.

45. The conjugate according to claim 44, wherein the aforementioned portion is selected from (bis-)mannose-6-phosphate, PROTAC, and PEG portions.

46. A composition comprising a polypeptide according to any one of claims 1 to 37, a polypeptide produced using the method described in claim 42 or 43, or a conjugate according to claim 44 or 45.

47. A polypeptide according to any one of claims 1 to 37, a nucleotide sequence or nucleic acid according to any one of claims 38 to 40, a conjugate according to claim 44 or 45, or a composition according to claim 46, for use as a pharmaceutical.

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