TW202509060A - Glycosylation of immunoglobulin single variable domains - Google Patents

Abstract

The present invention relates to glycosylation of immunoglobulin single variable domains (ISVDs). The present invention particularly relates to specific positions within the amino acid sequence of an ISVD for use as glycosylation acceptor sites. The present invention also relates to ISVDs modified with specific glycans at the specified glycosylation acceptor sites and to conjugates thereof. The present invention also relates to polypeptides comprising at least one ISVD of the present technology, at least one ISVD with specific glycans or at least one conjugate thereof. The invention furthermore relates to a nucleotide sequence or nucleic acid encoding such ISVD and/or polypeptide, a method of making such ISVDs and/or polypeptides, a composition comprising such ISVDs, conjugates and/or polypeptides, and the use of the polypeptide, nucleic sequence or nucleic acid, conjugate or composition according to the present technology in a medicament.

Description

Glycosylation of immunoglobulin single variable domains

The present technology relates to the glycosylation domain of immunoglobulin single variable domain (ISVD). More specifically, the present technology provides specific positions within the amino acid sequence of ISVD to serve as glycosylation receptor sites. The present technology also relates to ISVDs modified with specific polysaccharides at specified glycosylation receptor sites and conjugates thereof. The present technology also relates to polypeptides comprising at least one ISVD of the present technology, at least one ISVD with specific polysaccharides or at least one conjugate thereof. The present invention also relates to a nucleotide sequence or nucleic acid encoding such an ISVD and/or polypeptide, a method for preparing such an ISVD and/or polypeptide, and a composition comprising such an ISVD, conjugate and/or polypeptide.

Over the past few years, antibody therapy has rapidly gained traction in the medical community. The ability to selectively target only tissue affected by disease while leaving healthy tissue intact is highly desirable for patients' quality of life.

Although antibody therapies are target-specific, problems are often encountered when using such complex biomolecules. For example, issues with stability or solubility. Therefore, there is a constant drive to improve existing antibody therapies.

Glycosylation is a post-translational modification commonly applied in antibody therapeutics, especially for monoclonal antibodies. Glycosylation is a process that may be required to obtain the desired therapeutic efficacy. In addition, glycosylation can facilitate the conjugation of one antibody to another antibody and/or to other moieties that can provide additional functions to the antibody.

Immunoglobulin single variable domains (ISVDs) present interesting therapeutic potential in the field of antibody therapeutics due to their small size. In addition, production is simpler, faster, and cheaper than that of monoclonal antibodies.

Glycosylation of ISVD at specified glycosylation receptor sites has been described in WO 2016/150845, WO 2018/206734 and WO 2021/116252.

However, glycosylation of ISVD has proven to be challenging. Depending on the host cell/organism used, glycosylation does not occur at all, or it occurs in a limited manner. Furthermore, the introduction of glycosylated receptor sites often results in interference with the folding of the ISVD protein, resulting in a loss of affinity and/or stability and, therefore, functionality.

However, since glycosylation promotes the conjugation of ISVD with other moieties and may have an impact on therapeutic efficacy, there is still a need for effective and stable glycosylated ISVD.

The present invention aims to solve the problems associated with the glycosylation of ISVD. Therefore, in a first aspect, the present invention relates to a polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at the following amino acid positions, the amino acid positions being selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 according to the Kabat numbering.

In another aspect, the present technology relates to a polypeptide comprising or consisting essentially of an 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 the Kabat numbering.

In another aspect, the present technology relates to a polypeptide comprising or consisting essentially of an 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 the Kabat numbering.

In another aspect, the present technology relates to a polypeptide comprising or consisting essentially 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 the Kabat numbering.

In yet another aspect, the present technology relates to a polypeptide comprising or consisting (essentially) 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.

In another aspect, the present technology relates to a polypeptide comprising or consisting essentially 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.

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

In another aspect, the present 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 present 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.

In yet another aspect, the present technology relates to a polypeptide comprising or consisting (essentially) of three ISVDs, wherein at least one of the three 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, 102, 105, 108, and 110 according to the Kabat numbering.

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

In another aspect, the present technology relates to a polypeptide comprising or consisting essentially of three ISVDs, wherein the C-terminal ISVD comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 1, 3, 15, 19, 26 and 105 according to Kabat numbering.

In yet another aspect, the present technology relates to a polypeptide comprising or consisting (essentially) of three ISVDs, wherein the ISVD at neither the C-terminus nor the N-terminus comprises a glycosylation acceptor site present 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 the Kabat numbering.

The present technology also relates to such polypeptides that are glycosylated at one or more of these glycosylation receptor sites.

In another aspect, the present technology relates to a nucleotide sequence or nucleic acid encoding a polypeptide according to the present technology.

In another aspect, the present technology relates to a method for producing a polypeptide according to the present technology, the method comprising the following steps: -          Expressing a nucleotide sequence or nucleic acid according to the present technology in a suitable host cell or host organism, wherein the host cell or host organism is capable of glycosylation of the expressed polypeptide.

In yet another aspect, the present technology relates to methods for conjugating the polypeptides to moieties.

The present invention also provides a conjugate comprising the glycosylated polypeptide and a conjugated portion, wherein the portion is conjugated to the polysaccharide on the polypeptide.

In another aspect, the present technology relates to a composition comprising a polypeptide, nucleic acid or conjugate according to the present technology.

1. Definition

Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art, which would be clear to one of ordinary skill in the art. For example, reference is made to the standard manuals, Sambrook et al. (Molecular Cloning: A Laboratory Manual (2nd edition) 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, N.Y., 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 edition) Mosby/Elsevier, Edinburgh, 2001), Roitt et al. (Roitt's Essential Immunology (10th edition) Blackwell Publishing, UK, 2001) and Janeway et al. (Immunobiology (6th edition) Garland Science Publishing/Churchill Livingstone, New York, 2005), and the general background art cited in this article.

Unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can be performed and are performed in a manner known per se, as will be clear to one having ordinary skill in the art. For example, reference is again made to the standard manuals and the general background art mentioned herein and to the other references cited therein; and to reviews such as 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), Gonzales et al. (Tumour Biol. 26(1): 31-43, 2005), which describe techniques for protein engineering (e.g., affinity maturation) and other techniques for improving the specificity and other desired properties of proteins (e.g., immunoglobulins).

The term "sequence" as used herein (e.g., in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood to include the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding the amino acid sequence, unless the context requires a more restricted interpretation.

A nucleic acid or an amino acid is considered to be "in (substantially) isolated (form)", e.g., when, compared to the reaction medium or medium from which it is obtained, it is separated from at least one other component with which it is normally associated in said source or medium, e.g., another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a nucleic acid or an amino acid is considered to be "(substantially) isolated" when it is purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more. Nucleic acids or amino acids "in (substantially) isolated form" are preferably substantially homogeneous, as determined using a suitable technique (e.g., a suitable analytic technique, such as polyacrylamide gel electrophoresis).

When a nucleotide sequence or an amino acid sequence is said to "comprise" another nucleotide sequence or amino acid sequence, respectively, or to "essentially consist of" another nucleotide sequence or amino acid sequence, respectively, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first-mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this means that the first-mentioned nucleotide sequence or amino acid sequence, respectively, contains within its sequence a stretch of nucleotide or amino acid residues, respectively, which has the same nucleotide sequence or amino acid sequence as the latter sequence, respectively, regardless of how the first-mentioned sequence is actually generated or obtained (for example, it may be generated or obtained by any suitable method described herein). By way of non-limiting example, when a polypeptide is said to comprise an immunoglobulin single variable domain, this may mean that the sequence of the immunoglobulin single variable domain has been incorporated into the sequence of the polypeptide, but more generally this means that the polypeptide contains the sequence of the immunoglobulin single variable domain within its sequence, regardless of how the polypeptide is produced or obtained. In addition, when a nucleic acid or nucleotide sequence is said to comprise another nucleotide sequence, the first mentioned nucleic acid or nucleotide sequence is preferably such that when it is 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 in the same reading frame as the first mentioned larger nucleic acid or nucleotide sequence).

"(essentially) consisting of" means that the following nucleic acid sequence or amino acid sequence is completely identical to or corresponds to a polypeptide (e.g., CDR region; ISVD) having a limited number of amino acid residues, such as 1-20 amino acid residues, such as 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, at the amino terminus, at the carboxyl terminus, or at both the amino terminus and the carboxyl terminus of an immunoglobulin single variable domain.

Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. See Table A-2 on page 48 of WO 08/020079.

2. Peptides with glycosylated receptor sites and ISVD

ISVD glycosylation presents a challenge. The inventors have now surprisingly found that glycosylation acceptor sites can be introduced at previously unknown positions and that these glycosylation acceptor sites provide ISVDs with good glycosylation properties, while at the same time the glycosylation does not interfere with the binding and folding of the ISVD, which in turn provides good conjugative properties, for example in the production of ISVD conjugates.

Glycosylation is a reaction in which a carbohydrate (or "glycan", i.e., glycosyl donor) is attached to the hydroxyl or other functional group of another molecule (glycosyl acceptor) 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. Proteins synthesized in the rough endoplasmic reticulum mostly undergo glycosylation. Glycosylation is also present in the cytoplasm and nucleus as O-GlcNAc modifications. Different classes of glycans are produced: N-linked glycans, which are attached to the nitrogen of the side chains of asparagine or arginine; O-linked glycans, which are attached to the hydroxyl oxygen of the side chains of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline, or to an oxygen on a lipid such as ceramide; phosphoglycans, which are linked through the phosphate group of phosphoserine; and C-linked glycans, a rare form of glycosylation in which a sugar is added to a carbon on the side chain of tryptophan.

As used herein, "glycans" generally refer to glycosidically linked monosaccharides, oligosaccharides, and polysaccharides. Thus, the carbohydrate portion of a glycoconjugate such as a glycoprotein, glycolipid, or proteoglycan is referred to herein as a "glycan." A glycan may be a homopolymer or heteropolymer of monosaccharide residues, and may be linear or branched. N-linked glycans may be composed of galactose, neuraminic acid, N-acetylglucosamine (GalNAc), fucose, mannose, and other monosaccharides, also as further exemplified herein. In eukaryotes, O-linked glycans are assembled 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, but O-linked glycosylation is favored by the position of the proline residue at -1 or +3 relative to the serine or threonine.

The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" to define an immunoglobulin molecule in which the antigen binding site is present on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementary determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will participate in the formation of the antigen binding site.

In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or a Fab fragment, F(ab')2 fragment, Fv fragment (such as a disulfide-linked Fv or scFv fragment) or a diabody (all known in the art) derived from such a conventional 4-chain antibody is usually not regarded as an immunoglobulin single variable domain, because in these cases, the binding to the corresponding epitope of the antigen is usually not carried out by one (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, which bind in combination to the epitope of the corresponding antigen.

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

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

The immunoglobulin single variable domain (ISVD) can be, for example, a heavy chain ISVD, such as VHH (including humanized VHH), VH (including camelized VH and human VH). In one embodiment, it is VHH, camelized VH or humanized VHH. The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

For example, the immunoglobulin single variable domain can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or ^NANOBODY® ISVD (as defined herein, and including but not limited to VHH); other single variable domains, or any suitable fragment of any of them. [Note: ^NANOBODY® and ^NANBODIES® are registered trademarks of Ablynx NV]

In particular, the immunoglobulin single variable domain may be ^NANOBODY® ISVD (such as VHH, including humanized VHH or camelized VH) or a suitable fragment thereof.

"VHH domains" (also referred to as VHH or VHH antibody fragments) were originally described as antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al. Nature 363: 446-448, 1993). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHH, see the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001).

The generation of immunoglobulin sequences such as VHHs has been extensively described in various publications including WO 94/04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74: 277-302, 2001). In these methods, camels are immunized with an antigen of interest to induce an immune response against the target antigen. The pool of VHHs obtained from the immunization is further screened for VHHs that bind to the target antigen. In these cases, the generation of antibodies requires purified antigens for immunization and/or screening. The antigens can be purified from a natural source or purified during recombinant production. Immunization and/or screening of immunoglobulin sequences can be carried out using peptide fragments of such antigens.

Immunoglobulin sequences of different origins can be used in the present invention, including mouse, rat, rabbit, donkey, human and camel immunoglobulin sequences. In addition, fully human sequences, humanized sequences or chimeric sequences can be used. For example, camel immunoglobulin sequences and humanized camel immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs described by Ward et al. (see, e.g., WO 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 multivalent and/or multispecific constructs (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10. 7346-7350), and e.g. WO 96/34103 and WO 99/23221).

However, it should be noted that the ISVD included in the present invention is not limited to the ISVD sequence of the origin (or the nucleotide sequence used to express it), nor is it limited to the way in which the ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Therefore, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a specific but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to a "humanized" (as defined herein) immunoglobulin sequence (such as a partially or fully humanized camel, mouse or rabbit immunoglobulin sequence, and in particular a partially or fully humanized VHH sequence), a "camelized" (as defined herein) immunoglobulin sequence (and in particular a camelized VH sequence), and an ISVD that has been obtained by techniques such as affinity maturation (e.g., starting from a synthetic, random or naturally occurring immunoglobulin sequence), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences that are well known to those of ordinary skill; or any suitable combination of any of the foregoing.

Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence and may, for example, be a sequence isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), a nucleotide sequence that has been isolated from a library (and in particular an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers or a nucleotide sequence that has been prepared using techniques known per se for DNA synthesis.

A "humanized VHH" comprises an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VHH domain, but said amino acid sequence has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequence) with one or more amino acid residues present at one or more corresponding positions in a VH domain of a conventional 4-chain antibody from humans. This can be done in a manner known per se, which is clear to a person of ordinary skill, for example based on the prior art (e.g. WO 2008/020079). Furthermore, it should be noted that such humanized VHH can be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VHH domain as starting material.

A "camelized VH" comprises an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VH domain, but said amino acid sequence has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with one or more amino acid residues present at one or more corresponding positions in a VHH domain of a (camelid) heavy chain antibody. This can be done in a manner known per se, which is clear to a person of ordinary skill, e.g. based on the description in the prior art (e.g. Davies and Riechman (1994 and 1996), supra). Such "camelization" substitutions are inserted at amino acid positions forming and/or present at the VH-VL interface, and/or at so-called camel marker residues, as defined herein (see, e.g., WO 94/04678, and Davies and Riechmann (1994 and 1996), supra). In one embodiment, the VH sequence used as a starting material or starting point for the generation or design of a camelized VH is a VH sequence from a mammal, such as a human VH sequence, such as a VH3 sequence. It should be noted, however, that such camelized VH can be obtained in any suitable manner known per se, and is therefore not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VH domain as a starting material.

The structure of an immunoglobulin single variable domain sequence can be considered to be composed of four framework regions ("FR"), which are referred to in the art and herein as "Framework Region 1" ("FR1"); "Framework Region 2" ("FR2"); "Framework Region 3" ("FR3"); and "Framework Region 4" ("FR4"); the framework regions are interrupted by three complementary determining regions ("CDR"), which are referred to in the art and herein as "Complementary Determining Region 1" ("CDR1"); "Complementary Determining Region 2" ("CDR2"); and "Complementary Determining Region 3" ("CDR3").

As further described in paragraph q) on pages 58 and 59 of WO 08/020079, the amino acid residues of the ISVD can be numbered according to the universal numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication number 91), as applied to VHH domains from camelids in the paper by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195, see e.g. Figure 2 of this publication ₎ . In the present application, unless otherwise indicated, the sequence of the ISVD is determined according to the Kabat numbering.

It should be noted that, as is well known in the art for VH domains and for VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. That is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering. This means that, in general, the numbering according to Kabat 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 domains and VHH domains is generally in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

The determination of CDR regions can also be carried out according to different methods. In the determination according to Kabat's CDR, the FR1 of ISVD comprises the amino acid residues at positions 1-30, the CDR1 of ISVD comprises the amino acid residues at positions 31-35, the FR2 of ISVD comprises the amino acid residues at positions 36-49, the CDR2 of ISVD comprises the amino acid residues at positions 50-65, the FR3 of ISVD comprises the amino acid residues at positions 66-94, the CDR3 of ISVD comprises the amino acid residues at positions 95-102, and the FR4 of ISVD comprises the amino acid residues at positions 103-113.

The framework sequence is an immunoglobulin framework sequence or a framework sequence (a suitable combination) that has been derived from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence can be a framework sequence derived from a light chain variable domain (e.g., a VL sequence) and/or from a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In a specific aspect, the framework sequence is a framework sequence that has been derived from a VHH sequence (wherein the framework sequence may, as the case may be, have been partially or fully humanized) or is a conventional VH sequence that has been camelized (as defined herein).

In particular, the framework sequences present in the ISVD sequences used in the methods described herein may contain one or more marker residues (as defined below) such that the ISVD sequence is a ^NANOBODY® ISVD, such as, for example, a VHH, including a humanized VHH or a camelized VH. Non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

Typically, NANOBODY ^® ISVD (in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences) may be characterized by the presence of one or more "marker residues" (as described herein) in one or more framework sequences (also as further described herein). Thus, typically, NANOBODY ^® ISVD may be defined as an immunoglobulin sequence having the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementary determining regions 1 to 3, respectively, and wherein one or more of the marker residues are as further defined herein.

In particular, ^NANOBODY® ISVD may be an immunoglobulin sequence having the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementary determining regions 1 to 3, respectively, and wherein the framework sequence is as further defined herein.

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

surface 1 : NANOBODY ^® ISVD The symbol of radical Location people VH3 Signs of cruelty 11 L, V; mainly L L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; preferably L 37 V, I, F; usually V F ⁽¹⁾ , Y, V, L, A, H, S, I, W, C, N, G, D, T, P, preferably F ⁽¹⁾ or Y 44 ⁽⁸⁾ G E ⁽³⁾ , Q ⁽³⁾ , G ⁽²⁾ , D, A, K, R, L, P, S, V, H, T, N, W, M, I; G ⁽²⁾ , E ⁽³⁾ or Q ⁽³⁾ is preferred; G ⁽²⁾ or Q ⁽³⁾ is most preferred. 45 ⁽⁸⁾ L L ⁽²⁾ , R ⁽³⁾ , P, H, F, G, Q, S, E, T, Y, C, I, D, V; preferably L ⁽²⁾ or R ⁽³⁾ 47 ⁽⁸⁾ W, Y F ⁽¹⁾ , L ⁽¹⁾ or W ⁽²⁾ , G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W ⁽²⁾ , L ⁽¹⁾ or F ⁽¹⁾ 83 R or K; usually R R, K ⁽⁵⁾ , T, E ⁽⁵⁾ , Q, N, S, I, V, G, M, L, A, D, Y, H; K or R preferred; K most preferred 84 A, T, D; mainly A P ⁽⁵⁾ , S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E; preferably P 103 W W ⁽⁴⁾ , R ⁽⁶⁾ , G, S, K, A, M, Y, L, F, T, N, V, Q, P ⁽⁶⁾ , E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L; preferably G 108 L, M or T; mainly L Q, L ⁽⁷⁾ , R, P, E, K, S, T, M, A, H; Q or L ⁽⁷⁾ is preferred Notes: (1) Particularly, but not exclusively, in combination with KERE or KQRE at positions 43-46. (2) Typically as GLEW at positions 44-47. (3) Typically as KERE or KQRE at positions 43-46, for example as KEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47. Alternatively, sequences such as the following are possible: TERE (e.g., TEREL), TQRE (e.g., TQREL), KECE (e.g., KECEL or KECER), KQCE (e.g., KQCEL), RERE (e.g., REREG), RQRE (e.g., RQREL, RQREF or RQREW), QERE (e.g., QEREG), QQRE (e.g., QQREW, QQREL or QQREF), KGRE (e.g., KGREG), KDRE (e.g., KDREV). Some other possible but less preferred sequences include, for example, DECKL and NVCEL. (4) Having both GLEW at positions 44-47 and KERE or KQRE at positions 43-46. (5) KP or EP at positions 83-84 as is usually the case with naturally occurring VHH domains. (6) Particularly, but not exclusively, in combination with GLEW at positions 44-47. (7) With the proviso that when positions 44-47 are GLEW, position 108 is always Q in a (non-humanized) _VHH sequence that also contains W at 103. (8) The GLEW group also contains GLEW-like sequences at positions 44-47, such as, for example, GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW.

In a first aspect, the present technology relates to a polypeptide comprising or consisting essentially of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD 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, 102, 105, 108 and 110 according to the Kabat numbering.

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 attached to asparagine (Asn), while O-linked glycans are usually attached to the hydroxyl oxygen of the side chain of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline. In one embodiment, the glycosylation acceptor site is an N-glycosylation site. In one embodiment, the glycosylation acceptor site for N-glycosylation is asparagine (Asn). In one embodiment, the glycosylation acceptor site for O-glycosylation is serine, threonine, tyrosine, hydroxylysine, or hydroxyproline.

Surprisingly, the inventors discovered that, in addition to known glycosylation receptor sites, new glycosylation receptor sites can be generated in the ISVD amino acid sequence at positions not previously found to be suitable for glycosylation.

For efficient transfer of glycans, in one embodiment, aspartate is located within a specific consensus sequence (NXS, NXT, or NXC) in the primary structure of the polypeptide. Thus, a glycosylation acceptor site for N-linked glycosylation will typically be contained within a NXT motif or a NXS motif (where X can be any amino acid residue) that is glycosylated on an aspartate (N) residue. Therefore, in the present invention, when it is stated that a position can be glycosylated, and the position is intended to be N-glycosylated, the primary sequence of the ISVD according to the present invention will generally contain a NXT or NXS motif (suitably introduced by mutation and/or substitution of one or more relevant amino acid positions), such that the asparagine residue of the motif is present at the position to be glycosylated, i.e., at the glycosylation receptor site.

Thus, in further embodiments, the glycosylation acceptor site is contained within a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is a glycosylation acceptor site and is present 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 the Kabat numbering.

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

ISVDs with one of the three glycosylation receptor positions 19, 26, and 105 were found to have significantly higher levels of glycosylation in any format (monovalent or multivalent) regardless of the position of the ISVD in the construct when used in the construct.

In one embodiment, the polypeptide is a monovalent polypeptide comprising or (essentially consisting of) one ISVD. In this embodiment, the glycosylation receptor site may be present at the following amino acid position, the amino acid position being selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 according to the Kabat numbering.

In another embodiment, the polypeptide is a monovalent polypeptide comprising or (essentially consisting of) one ISVD. In this embodiment, the glycosylation receptor site may be present at the following amino acid position, the amino acid position is selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108 and 110 according to the Kabat numbering.

The process of designing/selecting and/or preparing a polypeptide starting from an immunoglobulin single variable domain (such as a VHH, a humanized VHH, a camelized VH, a domain antibody or a dAb) is also referred to herein as "formatting" the immunoglobulin single variable domain; and the immunoglobulin single variable domain that constitutes part of the polypeptide is said to be "formatted" or "in the format of the polypeptide". Examples of ways in which an immunoglobulin single variable domain can be formatted and examples of such formats will be clear to a person of ordinary skill based on the disclosure herein; and such formatted immunoglobulin single variable domains form another aspect of the present technology.

For example and without limitation, one or more immunoglobulin single variable domains can be used as a "binding unit", "binding domain" or "building block" (these terms are used interchangeably) for preparation of a polypeptide, which can optionally contain one or more additional immunoglobulin single variable domains that can serve as binding units.

The present technology also provides polypeptides or constructs comprising or consisting essentially of one or more immunoglobulin single variable domains. A monovalent polypeptide comprises or consists essentially of only one binding unit (such as, for example, an immunoglobulin single variable domain). A polypeptide comprising two or more binding units (such as, for example, an immunoglobulin single variable domain) will also be referred to herein as a "multivalent" polypeptide, and the binding units/immunoglobulin single variable domains present in such polypeptides will also be referred to herein as a "multivalent format". For example, a "bivalent" polypeptide may comprise two immunoglobulin single variable domains, optionally linked via a linker sequence, and a "trivalent" polypeptide may comprise three immunoglobulin single variable domains, optionally linked via two linker sequences; and a "tetravalent" polypeptide may comprise four immunoglobulin single variable domains, optionally linked via three linker sequences; and a "pentavalent" polypeptide may comprise five immunoglobulin single variable domains, optionally linked via four linker sequences; and a "hexavalent" polypeptide may comprise six immunoglobulin single variable domains, optionally linked via five linker sequences, etc.

In another aspect, the present technology relates to a polypeptide comprising or consisting essentially of an 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 the Kabat numbering.

In one embodiment, the present technology relates to a polypeptide comprising or consisting essentially of an 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 the Kabat numbering.

In another embodiment, the present technology relates to a monovalent polypeptide comprising or consisting essentially 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 the Kabat numbering.

In further embodiments, the present technology relates to a monovalent polypeptide comprising or consisting essentially 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 the Kabat numbering.

The inventors unexpectedly discovered that positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108 and 110 are glycosylation acceptor sites that can be glycosylated to be functional ISVDs as well as fully glycosylated ISVDs (e.g., used as monovalent polypeptides and/or conjugated to other moieties).

In one aspect 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 comprise a glycosylation receptor site present at the following amino acid positions, the amino acid positions being selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 according to the Kabat numbering.

In one embodiment, at least one of the at least 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.

The inventors unexpectedly discovered that glycosylation acceptor sites present at positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 can also provide a good degree of glycosylation in a multivalent polypeptide (such as, for example, in a polypeptide comprising at least two ISVDs). Interestingly, another position (i.e., position 3) was found to be used as a glycosylation acceptor site that gave insufficient glycosylation in a monovalent format but gave a very high degree of glycosylation in a multivalent polypeptide. In particular, positions 3, 19, 26, 55, 105 and 108 gave a high degree of glycosylation regardless of the position of the ISVD within the multivalent polypeptide (such as, for example, a polypeptide comprising at least two ISVDs).

In a further embodiment, the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs. In another embodiment, at least one of the at least 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 the Kabat numbering.

In another embodiment, the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs. In another embodiment, at least one of the at least 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.

Thus, in another aspect, the present technology relates to a polypeptide comprising or consisting essentially 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, 102, 105, 108 and 110 according to the Kabat numbering.

In one embodiment, 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.

The inventors unexpectedly discovered that for the use of some positions as glycosylation receptor sites, the position of the ISVD in the polypeptide has an effect on the degree of glycosylation obtained. Specifically, in some embodiments, the degree of glycosylation depends on whether the ISVD with the glycosylation receptor site is a C-terminal ISVD or an N-terminal ISVD. For the use of positions 3, 19, 26, 55, 105 and 108 as glycosylation receptor sites, the position of the ISVD with the glycosylation receptor site in the polypeptide has no effect on the degree of glycosylation obtained. However, for the use of positions 53, 68, 75 and 110 as glycosylation receptor sites, the inventors unexpectedly found that when the ISVD containing the glycosylation receptor site was a C-terminal ISVD, poor glycosylation was obtained. In addition, for position 1, when the ISVD containing the glycosylation site was an N-terminal ISVD, poor glycosylation was obtained, while when the ISVD containing the glycosylation site was a C-terminal ISVD, good glycosylation was obtained. It was found that in the bivalent format, regardless of the position of the ISVD, position 15 had an acceptable but not high degree of glycosylation. In addition, it was found that position 102 had poor glycosylation when in the bivalent format, but had good glycosylation in the trivalent format.

Thus, in another embodiment, the N-terminal ISVD of the at least two ISVDs 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.

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

Thus, in another aspect, the present technology relates to a polypeptide comprising or consisting essentially 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 the Kabat numbering.

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

As mentioned above, the inventors unexpectedly discovered that position 3 as a glycosylation receptor site provided good glycosylation levels only in multivalent formats (including bivalent polypeptides). In addition, they discovered that position 102 provided good glycosylation levels in both monovalent and multivalent formats in addition to bivalent formats. Other disclosed glycosylation receptor sites provided good glycosylation levels in both monovalent and multivalent formats (including bivalent polypeptides).

In another aspect, 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 receptor site present at the following amino acid positions, the amino acid positions being 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.

The inventors surprisingly found that the glycosylation acceptor site present at position 15 in the ISVD results in hyperglycosylation of the ISVD only when the ISVD is in (at least) a trivalent format. When the ISVD is in a monovalent format, an insufficient degree of glycosylation is observed for the glycosylation acceptor site present at position 15 in the ISVD, and only a just acceptable degree of glycosylation is observed in a bivalent format. However, when such an ISVD is formatted in a trivalent polypeptide, extremely hyperglycosylation is observed. In addition, the glycosylation receptor sites present in the ISVD at positions 1, 3, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 that have been discussed previously also provide for high glycosylation when the ISVD is in (at least) a trivalent format.

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

The inventors surprisingly discovered that when the glycosylation receptor site is at amino acid position 3, 15, 19, 26 or 105 in an at least trivalent polypeptide, the position of the ISVD within the polypeptide has no effect on the degree of glycosylation.

In another embodiment, the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs. As mentioned above, the glycosylation acceptor site present at position 15 in the ISVD unexpectedly provides high glycosylation in the trivalent polypeptide. In addition, in a multivalent format (including in a trivalent polypeptide), the glycosylation acceptor sites present at positions 1, 3, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 in the ISVD provide high glycosylation.

The inventors unexpectedly discovered that for the use of some positions as glycosylation acceptor sites, the position of the ISVD in the formatted polypeptide has an effect on the degree of glycosylation obtained, also in the trivalent format. Specifically, in some cases, the degree of glycosylation depends on whether the ISVD with the glycosylation acceptor site is a C-terminal ISVD, an N-terminal ISVD, or an ISVD that is neither C-terminal nor N-terminal (i.e., an ISVD between the C-terminal ISVD and the N-terminal ISVD, such as an intermediate ISVD). For the use of positions 3, 15, 19, 26, 55, 73, 105, and 108 as glycosylation acceptor sites, the position of the ISVD with the glycosylation acceptor site in the polypeptide has no effect on the degree of glycosylation obtained. However, for the use of positions 53, 68, 75, 102 and 110 as glycosylation receptor sites, the inventors unexpectedly found that when the ISVD containing the glycosylation receptor site is a C-terminal ISVD, poor glycosylation is obtained. In addition, the inventors found that the glycosylation receptor site at position 1 can only be present in a C-terminal ISVD to obtain high glycosylation. For the use of positions 68, 75 and 110 as glycosylation receptor sites, any other position of the ISVD in the polypeptide except the C-terminus will result in high glycosylation.

Thus, in one embodiment, the N-terminal ISVD of the at least three ISVDs comprises a glycosylation acceptor site present 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.

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

In yet another embodiment, at least one of the at least three ISVDs that is neither at the C-terminus nor at the N-terminus comprises a glycosylation acceptor site present 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.

Specific terms for each specific glycosylation receptor site are provided below. The present invention should not be considered to be limited to these terms.

Location 1

Item 1.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 1 according to the Kabat numbering.

Item 1.2. The polypeptide according to Item 1.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 1.3. A polypeptide according to item 1.1 or 1.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site.

Item 1.4. The polypeptide according to any one of items 1.1 to 1.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 1.5. The polypeptide according to any one of items 1.1 to 1.3, wherein the polypeptide comprises or (essentially 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 comprises a glycosylation acceptor site at amino acid position 1 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation acceptor site at amino acid position 1.

Item 1.7. A polypeptide according to Item 1.6, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation acceptor site at amino acid position 1 according to Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation acceptor site at amino acid position 1.

Item 1.8. The polypeptide according to Item 1.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 1 according to the Kabat numbering.

Item 1.9. The polypeptide according to any one of items 1.5 to 1.8, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 1.10. The polypeptide according to any one of items 1.1 to 1.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 1.11. The polypeptide of Item 1.10, wherein at least one of the at least three ISVDs comprises a glycosylation acceptor site at amino acid position 1 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation acceptor site at amino acid position 1.

Item 1.12. A polypeptide according to Item 1.10, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 1 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 1.

Item 1.13. The polypeptide of Item 1.10, wherein at least two of the at least three ISVDs comprise a glycosylation acceptor site at amino acid position 1 according to Kabat numbering, and the remaining ISVDs do not comprise a glycosylation acceptor site at amino acid position 1.

Item 1.14. The polypeptide of Item 1.10, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 1 according to the Kabat numbering.

Item 1.15. The polypeptide according to items 1.10 to 1.14, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 3

Item 3.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 3 according to the Kabat numbering.

Item 3.2. The polypeptide according to Item 3.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 3.3. A polypeptide according to item 3.1 or 3.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 3 according to Kabat numbering.

Item 3.4. The polypeptide according to any one of items 3.1 to 3.3, wherein the polypeptide comprises 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 comprises a glycosylation acceptor site at amino acid position 3 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation acceptor site at amino acid position 3.

Item 3.6. The polypeptide according to Item 3.5, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation acceptor site at amino acid position 3 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation acceptor site at amino acid position 3.

Item 3.7. The polypeptide according to Item 3.5, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation acceptor site at amino acid position 3 according to Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation acceptor site at amino acid position 3.

Item 3.8. The polypeptide according to Item 3.4, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 3 according to the Kabat numbering.

Item 3.9. The polypeptide according to items 3.4 to 3.8, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 3.10. The polypeptide according to items 3.1 to 3.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 3.11. The polypeptide according to Item 3.10, wherein at least one of the at least three ISVDs comprises a glycosylation acceptor site at amino acid position 3 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation acceptor site at amino acid position 3.

Item 3.12. The polypeptide according to Item 3.11, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 3 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 3.

Item 3.13. The polypeptide according to Item 3.11, wherein only the ISVD that is neither at the C-terminus nor at the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 3 according to Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 3.

Item 3.14. The polypeptide according to Item 3.11, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 3 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 3.

Item 3.15. The polypeptide of Item 3.10, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 3 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 3.

Item 3.16. The polypeptide of Item 3.10, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 3 according to Kabat numbering.

Item 3.17. The polypeptide according to any of items 3.10-3.16, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 15

Item 15.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 15 according to the Kabat numbering.

Item 15.2. The polypeptide according to Item 15.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 15.3. A polypeptide according to item 15.1 or 15.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 15 according to Kabat numbering.

Item 15.4. The polypeptide according to any one of items 15.1 to 15.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 15.5. The polypeptide according to Item 15.4, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 15 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 15.

Item 15.6. The polypeptide according to Item 15.5, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 15 according to the Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 15.

Item 15.7. The polypeptide according to Item 15.5, wherein only the ISVD that is neither at the C-terminus nor at the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 15 according to the Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 15.

Item 15.8. The polypeptide according to Item 15.5, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 15 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 15.

Item 15.9. The polypeptide according to Item 15.4, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 15 according to the Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 15.

Item 15.10. The polypeptide according to Item 15.4, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 15 according to the Kabat numbering.

Item 15.11. The polypeptide according to any one of items 15.4 to 15.10, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 19

Item 19.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 19 according to the Kabat numbering.

Item 19.2. The polypeptide according to Item 19.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 19.3. A polypeptide according to item 19.1 or 19.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 19 according to Kabat numbering.

Item 19.4. The polypeptide according to any one of items 19.1 to 19.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 19.5. The polypeptide according to any one of items 19.1 to 19.3, wherein the polypeptide comprises 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 comprises a glycosylation receptor site at amino acid position 19 according to the Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 19.

Item 19.7. The polypeptide according to Item 19.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 19 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 19.

Item 19.8. The polypeptide according to Item 19.6, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 19 according to Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 19.

Item 19.9. The polypeptide according to Item 19.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 19 according to the Kabat numbering.

Item 19.10. The polypeptide according to any one of items 19.5 to 19.9, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 19.11. The polypeptide according to any one of items 19.1 to 19.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 19.12. The polypeptide according to Item 19.11, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 19 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 19.

Item 19.13. The polypeptide according to Item 19.12, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 19 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 19.

Item 19.14. The polypeptide according to Item 19.12, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 19 according to the Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 19.

Item 19.15. The polypeptide according to Item 19.12, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 19 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 19.

Item 19.16. The polypeptide of Item 19.11, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 19 according to the Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 19.

Item 19.17. The polypeptide of Item 19.11, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 19 according to the 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 or (essentially) consisting of three ISVDs.

Location 26

Item 26.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 26 according to the Kabat numbering.

Item 26.2. The polypeptide according to Item 26.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 26.3. A polypeptide according to item 26.1 or 26.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present 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 or (essentially) consisting of one ISVD.

Item 26.5. The polypeptide according to any one of items 26.1 to 26.3, wherein the polypeptide comprises or (essentially 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 comprises a glycosylation receptor site at amino acid position 26 according to the Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 26.

Item 26.7. The polypeptide according to item 26.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 26 according to the Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 26.

Item 26.8. The polypeptide according to item 26.6, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 26 according to the Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 26.

Item 26.9. The polypeptide according to Item 26.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 26 according to the Kabat numbering.

Item 26.10. The polypeptide according to any one of items 26.5 to 26.9, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 26.11. The polypeptide according to any one of items 26.1 to 26.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 26.12. The polypeptide according to Item 26.11, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 26 according to the Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 26.

Item 26.13. The polypeptide according to item 26.12, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 26 according to the Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 26.

Item 26.14. The polypeptide according to item 26.12, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 26 according to the Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 26.

Item 26.15. The polypeptide according to item 26.12, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 26 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 26.

Item 26.16. The polypeptide of Item 26.11, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 26 according to the Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 26.

Item 26.17. The polypeptide according to Item 26.11, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 26 according to the Kabat numbering.

Item 26.18. A polypeptide according to items 26.11 to 26.17, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 53

Item 53.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 53 according to the Kabat numbering.

Item 53.2. The polypeptide according to Item 53.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 53.3. A polypeptide according to item 53.1 or 53.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present 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 or (essentially) consisting of one ISVD.

Item 53.5. The polypeptide according to any one of items 53.1 to 53.3, wherein the polypeptide comprises or (essentially 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 comprises a glycosylation receptor site at amino acid position 53 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 53.

Item 53.7. The polypeptide according to item 53.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 53 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 53.

Item 53.8. The polypeptide according to Item 53.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 53 according to the Kabat numbering.

Item 53.9. The polypeptide according to any one of items 53.5 to 53.8, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 53.10. The polypeptide according to any of items 53.1-53.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 53.11. The polypeptide according to item 53.10, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site present at amino acid position 53 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site present at amino acid position 53.

Item 53.12. The polypeptide according to item 53.11, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 53 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 53.

Item 53.13. The polypeptide according to item 53.11, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 53 according to Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 53.

Item 53.14. The polypeptide of Item 53.10, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 53 according to the Kabat numbering, and the remaining ISVDs do not comprise a glycosylation receptor site at amino acid position 53.

Item 53.15. The polypeptide according to Item 53.10, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 53 according to the Kabat numbering.

Item 53.16. A polypeptide according to any one of items 53.10-53.15, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 55

Item 55.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 55 according to the Kabat numbering.

Item 55.2. The polypeptide according to Item 55.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 55.3. A polypeptide according to item 55.1 or 55.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 55 according to Kabat numbering.

Item 55.4. A polypeptide according to any one of items 55.1 to 55.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 55.5. The polypeptide according to any one of items 55.1 to 55.3, wherein the polypeptide comprises or (essentially 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 comprises a glycosylation receptor site at amino acid position 55 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 55.

Item 55.7. The polypeptide according to item 55.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 55 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 55.

Item 55.8. The polypeptide according to item 55.6, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 55 according to Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 55.

Item 55.9. The polypeptide according to Item 55.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 55 according to the Kabat numbering.

Item 55.10. The polypeptide according to any one of items 55.5 to 55.9, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 55.11. A polypeptide according to any of items 55.1-55.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 55.12. The polypeptide according to item 55.11, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 55 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 55.

Item 55.13. The polypeptide according to item 55.12, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 55 according to the Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 55.

Item 55.14. A polypeptide according to item 55.12, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 55 according to the Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 55.

Item 55.15. The polypeptide according to item 55.12, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 55 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 55.

Item 55.16. The polypeptide of Item 55.11, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 55 according to the Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 55.

Item 55.17. The polypeptide according to Item 55.11, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 55 according to the Kabat numbering.

Item 55.18. A polypeptide according to items 55.11 to 55.17, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 68

Item 68.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 68 according to the Kabat numbering.

Item 68.2. The polypeptide according to Item 68.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 68.3. A polypeptide according to item 68.1 or 68.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 68 according to Kabat numbering.

Item 68.4. A polypeptide according to any one of items 68.1 to 68.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 68.5. The polypeptide according to any one of items 68.1 to 68.3, wherein the polypeptide comprises or (essentially 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 comprises a glycosylation receptor site at amino acid position 68 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 68.

Item 68.7. The polypeptide according to item 68.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 68 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 68.

Item 68.8. The polypeptide according to item 68.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 68 according to the Kabat numbering.

Item 68.9. The polypeptide according to any one of items 68.5 to 68.9, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 68.10. The polypeptide according to any one of items 68.1 to 68.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 68.11. The polypeptide according to item 68.10, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site present at amino acid position 68 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site present at amino acid position 68.

Item 68.12. A polypeptide according to item 68.11, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 68 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 68.

Item 68.13. A polypeptide according to item 68.11, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 68 according to Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 68.

Item 68.14. The polypeptide of Item 68.10, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 68 according to the Kabat numbering, and the remaining ISVDs do not comprise a glycosylation receptor site at amino acid position 68.

Item 68.15. The polypeptide according to item 68.10, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 68 according to the Kabat numbering.

Item 68.15. A polypeptide according to items 68.10 to 68.15, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 73

Item 73.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 73 according to the Kabat numbering.

Item 73.2. The polypeptide according to Item 73.1, wherein the glycosylation 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 a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 73 according to Kabat numbering.

Item 73.4. The polypeptide according to any one of items 73.1 to 73.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 73.5. The polypeptide according to any one of items 73.1 to 73.3, wherein the polypeptide comprises or (essentially 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 comprises a glycosylation receptor site at amino acid position 73 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 73.

Item 73.7. The polypeptide according to item 73.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 73 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 73.

Item 73.8. The polypeptide according to item 73.6, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 73 according to Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 73.

Item 73.9. The polypeptide according to item 73.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 73 according to the Kabat numbering.

Item 73.10. The polypeptide according to items 73.5 to 73.9, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 73.11. The polypeptide according to any of items 73.1-73.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 73.12. The polypeptide according to item 73.11, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 73 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 73.

Item 73.13. The polypeptide according to item 73.12, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 73 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 73.

Item 73.14. The polypeptide according to item 73.12, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site present at amino acid position 73 according to Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site present at amino acid position 73.

Item 73.15. A polypeptide according to item 73.12, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 73 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 73.

Item 73.16. The polypeptide of Item 73.11, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 73 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 73.

Item 73.17. The polypeptide according to Item 73.11, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 73 according to the Kabat numbering.

Item 73.18. A polypeptide according to items 73.11 to 73.17, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 75

Item 75.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 75 according to the Kabat numbering.

Item 75.2. The polypeptide according to Item 75.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 75.3. A polypeptide according to item 75.1 or 75.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 75 according to Kabat numbering.

Item 75.4. A polypeptide according to any one of items 75.1 to 75.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 75.5. The polypeptide according to any one of items 75.1 to 75.3, wherein the polypeptide comprises or (essentially consists of) at least two ISVDs.

Item 75.6. The polypeptide of Item 75.5, wherein at least one of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 75 according to the Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 75.

Item 75.7. The polypeptide according to item 75.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 75 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 75.

Item 75.8. The polypeptide according to item 75.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 75 according to the Kabat numbering.

Item 75.9. The polypeptide according to items 75.5 to 75.8, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 75.10. The polypeptide according to any one of items 75.1 to 75.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 75.11. The polypeptide of Item 75.10, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 75 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 75.

Item 75.12. The polypeptide according to item 75.11, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 75 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 75.

Item 75.13. A polypeptide according to item 75.11, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 75 according to the Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 75.

Item 75.14. The polypeptide of Item 75.10, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 75 according to the Kabat numbering, and the remaining ISVDs do not comprise a glycosylation receptor site at amino acid position 75.

Item 75.15. The polypeptide of Item 75.10, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 75 according to the Kabat numbering.

Item 75.16. A polypeptide according to any one of items 75.10 to 75.15, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 76

Item 76.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 76 according to the Kabat numbering.

Item 76.2. The polypeptide according to Item 76.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 76.3. A polypeptide according to item 76.1 or 76.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 76 according to Kabat numbering.

Item 76.4. A polypeptide according to any one of items 76.1 to 76.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 76.5. The polypeptide according to any one of items 76.1 to 76.3, wherein the polypeptide comprises or (essentially consists of) at least two ISVDs.

Item 76.6. The polypeptide of Item 76.5, wherein at least one of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 76 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 76.

Item 76.7. The polypeptide according to item 76.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 76 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 76.

Item 76.8. The polypeptide according to item 76.6, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 76 according to Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 76.

Item 76.9. The polypeptide according to item 76.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 76 according to the Kabat numbering.

Item 76.10. The polypeptide according to any one of items 76.5 to 76.9, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 76.11. The polypeptide according to any one of items 76.1 to 76.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 76.12. The polypeptide of Item 76.11, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 76 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 76.

Item 76.13. The polypeptide according to item 76.12, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 76 according to the Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 76.

Item 76.14. A polypeptide according to item 76.12, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 76 according to the Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 76.

Item 76.15. The polypeptide according to item 76.12, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 76 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 76.

Item 76.16. The polypeptide of Item 76.11, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 76 according to the Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 76.

Item 76.17. The polypeptide according to item 76.11, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 76 according to the Kabat numbering.

Item 76.18. A polypeptide according to any one of items 76.11 to 76.17, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 102

Item 102.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 102 according to the Kabat numbering.

Item 102.2. The polypeptide according to Item 1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 102.3. A polypeptide according to item 102.1 or 102.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 102 according to Kabat numbering.

Item 102.4. The polypeptide according to any one of items 102.1 to 102.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 102.5. The polypeptide according to any one of items 102.1 to 102.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 102.6. The polypeptide of Item 102.5, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 102 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 102.

Item 102.7. The polypeptide according to item 102.6, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site present at amino acid position 102 according to the Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site present at amino acid position 102.

Item 102.8. The polypeptide according to item 102.6, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 102 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 102.

Item 102.9. The polypeptide of Item 102.5, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site present at amino acid position 102 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site present at amino acid position 102.

Item 102.10. The polypeptide according to item 102.5, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 102 according to the Kabat numbering.

Item 102.11. A polypeptide according to any one of items 102.5 to 102.10, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 105

Item 105.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 105 according to the Kabat numbering.

Item 105.2. The polypeptide according to Item 105.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 105.3. A polypeptide according to item 105.1 or 105.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present 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 or (essentially) consisting of one ISVD.

Item 105.5. The polypeptide according to any one of items 105.1 to 105.3, wherein the polypeptide comprises or (essentially consists of) at least two ISVDs.

Item 105.6. The polypeptide of Item 105.5, wherein at least one of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 105 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 105.

Item 105.7. The polypeptide according to item 105.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 105 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 105.

Item 105.8. The polypeptide according to item 105.6, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 105 according to Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 105.

Item 105.9. The polypeptide according to item 105.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 105 according to the Kabat numbering.

Item 105.10. The polypeptide according to any one of items 105.5 to 105.9, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 105.11. The polypeptide according to any one of items 105.1 to 105.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 105.12. The polypeptide of Item 105.11, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 105 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 105.

Item 105.13. The polypeptide according to item 105.12, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 105 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 105.

Item 105.14. The polypeptide according to item 105.12, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 105 according to Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 105.

Item 105.15. The polypeptide according to item 105.12, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 105 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 105.

Item 105.16. The polypeptide of Item 105.11, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site at amino acid position 105 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 105.

Item 105.17. The polypeptide according to item 105.11, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 105 according to the Kabat numbering.

Item 105.18. A polypeptide according to any one of items 105.11 to 105.17, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 108

Item 108.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 108 according to the 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. A polypeptide according to item 108.1 or 108.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present at amino acid position 108 according to Kabat numbering.

Item 108.4. The polypeptide according to any one of items 108.1 to 108.3, wherein the polypeptide is a monovalent polypeptide comprising or (essentially) consisting of one ISVD.

Item 108.5. The polypeptide according to any one of items 108.1 to 108.3, wherein the polypeptide comprises or (essentially consists of) at least two ISVDs.

Item 108.6. The polypeptide of Item 108.5, wherein at least one of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 108 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 108.

Item 108.7. The polypeptide according to item 108.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 108 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 108.

Item 108.8. The polypeptide according to item 108.6, wherein only the C-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 108 according to Kabat numbering, and the remaining N-terminal ISVD does not comprise a glycosylation receptor site at amino acid position 108.

Item 108.9. The polypeptide according to item 108.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 108 according to Kabat numbering.

Item 108.10. The polypeptide according to any one of items 108.5 to 108.9, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 108.11. The polypeptide according to any one of items 108.1 to 108.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 108.12. The polypeptide of item 108.11, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 108 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site at amino acid position 108.

Item 108.13. The polypeptide according to item 108.12, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 108 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 108.

Item 108.14. The polypeptide according to item 108.12, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site present at amino acid position 108 according to Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site present at amino acid position 108.

Item 108.15. The polypeptide according to item 108.12, wherein only the C-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 108 according to Kabat numbering, and the remaining N-terminal ISVD and the middle ISVD do not comprise a glycosylation receptor site at amino acid position 108.

Item 108.16. The polypeptide of item 108.11, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site present at amino acid position 108 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site present at amino acid position 108.

Item 108.17. The polypeptide of Item 108.11, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 108 according to Kabat numbering.

Item 108.18. A polypeptide according to any one of items 108.11 to 108.17, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Location 110

Item 110.1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present at amino acid position 110 according to the Kabat numbering.

Item 110.2. The polypeptide according to Item 110.1, wherein the glycosylation acceptor site is an N-glycosylation site.

Item 110.3. A polypeptide according to item 110.1 or 110.2, wherein the glycosylation acceptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation acceptor site and is present 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 or (essentially) consisting of one ISVD.

Item 110.5. The polypeptide according to any one of items 110.1 to 110.3, wherein the polypeptide comprises or (essentially consists of) at least two ISVDs.

Item 110.6. The polypeptide of Item 110.5, wherein at least one of the at least two ISVDs comprises a glycosylation receptor site at amino acid position 110 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site at amino acid position 110.

Item 110.7. The polypeptide according to item 110.6, wherein only the N-terminal ISVD of the at least two ISVDs comprises a glycosylation receptor site present at amino acid position 110 according to Kabat numbering, and the remaining C-terminal ISVD does not comprise a glycosylation receptor site present at amino acid position 110.

Item 110.8. The polypeptide according to item 110.5, wherein both of the at least two ISVDs comprise a glycosylation acceptor site present at amino acid position 110 according to the Kabat numbering.

Item 110.9. The polypeptide according to any one of items 110.5 to 110.8, wherein the polypeptide is a bivalent polypeptide comprising or (essentially) consisting of two ISVDs.

Item 110.10. The polypeptide according to any one of items 110.1 to 110.3, wherein the polypeptide comprises or (essentially consists of) at least three ISVDs.

Item 110.11. The polypeptide according to item 110.10, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site present at amino acid position 110 according to Kabat numbering, and the remaining two ISVDs do not comprise a glycosylation receptor site present at amino acid position 110.

Item 110.12. The polypeptide according to item 110.11, wherein only the N-terminal ISVD of the at least three ISVDs comprises a glycosylation receptor site at amino acid position 110 according to Kabat numbering, and the remaining middle ISVD and C-terminal ISVD do not comprise a glycosylation receptor site at amino acid position 110.

Item 110.13. The polypeptide according to item 110.11, wherein only the ISVD at neither the C-terminus nor the N-terminus of the at least three ISVDs comprises a glycosylation receptor site present at amino acid position 110 according to Kabat numbering, and the remaining N-terminal ISVD and C-terminal ISVD do not comprise a glycosylation receptor site present at amino acid position 110.

Item 110.14. The polypeptide of item 110.10, wherein at least two of the at least three ISVDs comprise a glycosylation receptor site present at amino acid position 110 according to Kabat numbering, and the remaining ISVD does not comprise a glycosylation receptor site present at amino acid position 110.

Item 110.15. The polypeptide of Item 110.10, wherein all three of the at least three ISVDs comprise a glycosylation acceptor site present at amino acid position 110 according to the Kabat numbering.

Item 110.16. A polypeptide according to any one of items 110.10 to 110.15, wherein the polypeptide is a trivalent polypeptide comprising or (essentially) consisting of three ISVDs.

Combination of glycosylation receptor sites

Item A.     A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 1 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item B. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 3 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item C. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 15 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item D.     A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 19 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 15, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item E. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 26 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 15, 19, 53, 55, 68, 73, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item F. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 53 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 15, 19, 26, 55, 68, 73, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item G.     A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 55 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any of positions 1, 3, 15, 19, 26, 53, 68, 73, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item H.     A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 68 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 15, 19, 26, 53, 55, 73, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item I. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 73 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 15, 19, 26, 53, 55, 68, 75, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item J. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 75 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 76, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item K.     A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 76 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108 and/or 110 according to the Kabat numbering.

Item L. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 102 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any one of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 105, 108 and/or 110 according to the Kabat numbering.

Item M.     A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 105 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 108 and/or 110 according to the Kabat numbering.

Item N.     A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 108 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site, wherein the at least one additional glycosylation receptor site is present at any of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105 and/or 110 according to the Kabat numbering.

Item O.     A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at amino acid position 110 according to the Kabat numbering, in combination with at least one additional glycosylation receptor site present at any one of positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105 and/or 108 according to the Kabat numbering.

3. ISVD Glycoprotein

The glycosylation receptor sites present in the polypeptide can (but need not be) modified with N- or O-linked glycans. The present technology also relates to polypeptides of the present technology that are glycosylated at one or more of the specified glycosylation receptor sites. After expressing the polypeptide having the glycosylation receptor site in a host or host cell capable of glycosylation of the polypeptide (as further defined herein), the resulting polypeptide is directly modified with one or more glycans in the host or host cell. The resulting glycosylated polypeptide, also referred to herein as ISVD glycoprotein, comprises one or more glycans. In one embodiment, the polypeptide may comprise one or more glycans having terminal N-acetylglucosamine (GlcNAc), (terminal) mannose, (terminal) sialic acid, (terminal) galactose, or a combination thereof. Thus, the present 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.

In some embodiments, the ISVD glycoproteins of the present technology show high affinity. In some embodiments, the ISVD glycoproteins of the present technology have the same affinity as a polypeptide that is not glycosylated at a given glycosylation receptor site.

Affinity is a measure of the strength of binding between a moiety and a binding site on a target molecule: the lower the dissociation constant ( _KD ) value, the stronger the binding strength between the target molecule and the targeting moiety. Typically, the binding units (e.g., ISVDs) used in the present technology will bind to their targets with a _KD of ^10-5 to ^10-12 mol/liter or less, ^10-7 to ^10-12 mol/liter or less, or ^10-8 to ^10-12 mol/liter (i.e., bind with an association constant ( _KA ) of ^10-5 to ^10-12 liter/mol or more, ^10-7 to ^10-12 liter/mol or more, or ^10-8 to ^10-12 liter/mol). Any _KD value greater than ^10-4 mol/liter (or any _KA value less than ¹⁰⁴ liter/mol) is generally considered to indicate nonspecific binding. Biological interactions considered specific (such as the binding of an immunoglobulin sequence to an antigen) typically have _KD values in the range of ^10-5 mol/liter (10,000 nM or 10 µM) to ^10-12 mol/liter (0.001 nM or 1 pM) or lower.

As will be clear to one having ordinary skill, the dissociation constant may be an actual or apparent dissociation constant. Methods for determining the dissociation constant will be clear to one having ordinary skill and include, for example, the techniques mentioned below. In this regard, it will also be clear that dissociation constants greater than ^10-4 mol/liter or ^10-3 mol/liter (e.g., ^10-2 mol/liter) may not be measured. Optionally, as will also be clear to one having ordinary skill, the (actual or apparent) dissociation constant may be calculated based on the (actual or apparent) association constant by means of the relationship [ _KD = 1/ _KA ].

The affinity of a molecular interaction between two molecules can be measured by various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, e.g., Ober et al., 2001, Intern. Immunology 13: 1551-1559). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentrations in a biosensor array, wherein one molecule is immobilized on a biosensor chip and the other molecule is passed under flow conditions past the immobilized molecule, thereby generating _kon , _koff measurements and, therefore, _KD (or _KA ) values. For example, this can be performed using the well-known ^BIAcore® system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, New Jersey). For further description, 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 Johnnson et al. 1991 (Anal. Biochem. 198: 268-277).

Another well-known biosensor technology used to determine 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 term "biolayer interferometry" or "BLI" refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on the biosensor tip (signal beam). Changes in the number of molecules bound to the biosensor tip result in 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 biosensor tip surface. Because the interactions can be measured in real time, association and dissociation rates as well as affinity can be determined. For example, BLI can be performed using the well-known ^Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

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

The GYROLAB ^® Immunoassay System provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

In some embodiments, the glycosylated polypeptides of the present technology specifically bind to their targets with a dissociation constant (K _D ) of 10 ^-5 to 10 ^-12 mol/liter or less, 10 ^-7 to 10 ^-12 mol/liter or less, or 10 ^-8 to 10 ^-12 mol/liter, as determined by surface plasmon resonance. In some embodiments, the glycosylated polypeptides of the present technology specifically bind to their targets with a dissociation constant (K _D ) of 10 ^-5 to 10 ^-12 mol/liter or less, 10 ^-7 to 10 ^-12 mol/liter or less, or 10 ^-8 to 10 ^-12 mol/liter, as determined by Meso Scale Discovery.

In some embodiments, the ISVD glycoprotein of the present technology has (substantially) the same or higher melting temperature (Tm) than a polypeptide that is not glycosylated at a given glycosylation receptor site. The denaturation midpoint of a protein is defined as the temperature (Tm) or denaturant concentration at which the two states, folded and unfolded, are equally distributed at equilibrium (assuming a two-state protein fold). Tm is typically 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 unfolded.

The components present in a polypeptide (e.g., an ISVD) can be linked to each other via one or more suitable linkers (e.g., 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 called "Gly-Ser" or "GS" linkers. These linkers are linkers that are essentially composed of glycine (G) and serine (S) residues and typically contain a peptide motif, such as one or more repeats of the GGGGS (SEQ ID NO: 150) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7 or greater). Some commonly used examples of such GS linkers are 9GS linker (GGGGSGGGS, SEQ ID NO: 151), 15GS linker (n = 3; SEQ ID NO: 152) and 35GS linker (n = 7; SEQ ID NO: 153). See, for example, 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 the present invention, the components of the polypeptide are linked to each other using a 9GS linker. In one embodiment of the polypeptide of the present invention, the components of the polypeptide are linked to each other using a 35GS linker.

The present technology also provides sequence-optimized ISVDs and polypeptides that show increased stability when stored during stability studies. Sequence-optimized ISVDs and polypeptides show reduced N-terminal pyroglutamate post-translational modification and therefore have increased product stability. Pyroglutamate modification leads to heterogeneity of the final product and needs to be avoided. By changing the N-terminal glutamate (E) to aspartic acid (D), the possibility of pGlu post-translational modification at the N-terminus is eliminated, resulting in improved product stability. Therefore, the present invention also relates to ISVDs and polypeptides as described above, wherein the glutamate at position 1 (the position is determined according to Kabat numbering) is changed to aspartic acid (E1D).

The present technology also provides "humanized" sequence-optimized ISVDs and polypeptides, i.e., one or more amino acid residues in the amino acid sequence (and in particular in the framework sequence) of the naturally occurring VHH sequence are replaced by one or more amino acid residues present at one or more corresponding positions in the VH domain of a conventional 4-chain antibody from humans (e.g., as indicated above). Therefore, the present invention also relates to humanized ISVDs and polypeptides as described above.

The present technology also provides sequence-optimized ISVDs and polypeptides that exhibit reduced binding to pre-existing antibodies present in human serum. To this end, in one embodiment, the polypeptide comprises valine (V) at amino acid position 11 (according to Kabat numbering) and leucine (L) at amino acid position 89 in at least one ISVD. In one embodiment, the polypeptide comprises valine (V) at amino acid position 11 (according to Kabat numbering) and leucine (L) at amino acid position 89 in each ISVD. Therefore, the present technology also relates to ISVDs and polypeptides as described above, which have been sequence-optimized with valine (V) at amino acid position 11 (according to Kabat numbering) and leucine (L) at amino acid position 89 in at least one ISVD (such as in all ISVDs).

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

In another embodiment, the polypeptide comprises lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVD comprises lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these embodiments, the C-terminus of the 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), such that, after addition of a single alanine, the C-terminus of the polypeptide comprises, 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). In one embodiment, the polypeptide comprises valine (V) at amino acid position 11 (according to Kabat numbering) and leucine (L) at amino acid position 89 in each ISVD, optionally lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and an extension of 1 to 5 (naturally occurring) amino acids (as defined above), such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD, such that the C-terminus of the polypeptide comprises, for example, the sequence VTVSSA (SEQ ID NO: 163), VKVSSA (SEQ ID NO: 164) or VQVSSA (SEQ ID NO: 165). For further information in this regard, see, for example, WO2012/175741 and WO2015/173325.

Some embodiments of ISVD glycoproteins are provided, which have a particularly high degree of glycosylation when the glycosylation receptor sites in the ISVD are as described herein.

In one aspect of the present technology, the ISVD glycoprotein is a monovalent polypeptide, wherein the sequence of the polypeptide is selected from SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73-78, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89-91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96 and SEQ ID NO: 177-179.

In another aspect of the present technology, the ISVD glycoprotein is a bivalent polypeptide, wherein the sequence of the polypeptide is selected from SEQ ID NO: 99-102, SEQ ID NO: 105-108, SEQ ID NO: 110, SEQ ID NO: 116-118, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 123-125, SEQ ID NO: 128-133, SEQ ID NO: 141-143, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 182, SEQ ID NO: 183 and SEQ ID NO: 187.

In another aspect of the present technology, the ISVD glycoprotein is a trivalent polypeptide, wherein the sequence of the polypeptide is selected from SEQ ID NO: 20, SEQ ID NO: 22-24, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44-52, SEQ ID NO: 55-57, SEQ ID NO: 59-61, SEQ ID NO: 63-65, SEQ ID NO: 186, SEQ ID NO: 190 and SEQ ID NO: 191.

4. Bonding

The present invention also relates to a conjugate comprising a polypeptide according to the present invention and a conjugation moiety that conjugates with a glycan. The polypeptide modified with a glycan at a designated glycosylation receptor site is an ideal starting point for glycan-based conjugation due to its high glycosylation.

The conjugation can be performed chemically (e.g., using periodate oxidation of the glycan components, followed by conjugation by methods known in the art such as oxime ligation, hydrazone ligation, or by reductive amination) or enzymatically (e.g., using galactose oxidase to oxidize galactose, followed by conjugation by oxime ligation, hydrazone ligation, or by reductive amination). Alternatively, labeled glycan residues can be incorporated to allow for subsequent conjugation reactions (e.g., using mutant galactosyltransferases to incorporate GalNAz into the glycan chain, followed by conjugation reactions by click chemistry).

In certain embodiments, the conjugate comprises a linker between the polysaccharide and the joining portion. The use of a specific linker will depend on the application and will be clear to one of ordinary skill. For example, oximes and hydrazones (particularly derived from aliphatic aldehydes) show lower stability over time in water or at lower pH. Aromatically stable structures may be more useful for stably connecting polysaccharides to the joining portion. Such stable linkers are also within the scope of the present application because they can limit adverse effects caused by premature release of the joining portion, particularly when the joining portion is a toxic substance (e.g., intended to kill tumor cells). Of particular interest are bicyclo[6.1.0]non-4-yne reagents as well as aromatic stabilized triazole linkers and sulfonamide linkers. In the art, the increased stability of the conjugate may also be caused by a reduced tendency to aggregate any moiety contained within the conjugate. For the generation of ISVD conjugates with increased stability, the reader is non-exclusively referred to WO 2013036748, WO 2014065661, WO 2015057064 and WO 2016053107 and other patent applications filed by Synaffix B.V.

A variety of linkers known in the art can be used to link the glycosylated polypeptide and the binding moiety. It should be clear that both cleavable and non-cleavable linkers can be employed to achieve the desired release profile. In general, the optimal combination of linker and conjugation chemistry must be tailored to relate to each unique aspect: glycosylated polypeptide, binding moiety, and profile of the disease to be treated. For a review of antibody-drug conjugates and linkers used herein, see, e.g., 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), as well as Pillow et al. 2017 (Pharm Pat Anal. 6(1)), which describes a novel quaternary ammonium salt linker that can be used in conjugates for the treatment of cancer and infectious diseases.

Other suitable linkers generally include organic compounds or polymers, particularly those suitable for use in polypeptides for pharmaceutical use. For example, poly(ethylene glycol) moieties have been used to link antibody domains, see, for example, WO 04/081026. It is contemplated within the scope of the present invention that the length, degree of flexibility and/or other properties of the linker may have some effect on the properties of the final conjugate, including but not limited to affinity, specificity or avidity for a particular target. Based on the disclosure herein, a person of ordinary skill will be able to determine the best linker for use in a particular conjugate, optionally after some limited routine experimentation.

In some embodiments, a conjugate comprising a polypeptide according to the present technology and a conjugation moiety has at least one additional function or property compared to an unconjugated polypeptide. For example, a conjugate comprising a polypeptide of the present technology and a cytotoxic drug as a conjugation moiety results in the formation of a conjugated 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, the conjugation of a second conjugated polypeptide to a polypeptide of the present technology can confer additional binding properties. As another example, the conjugation moiety can 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 a degrader function. As yet another example, the conjugation moiety can be a PEG molecule that provides half-life extension and/or stabilization/solubilization function. As another example, the conjugated moiety can be a proteolytic targeting chimera (PROTAC) that provides a degrader function (Sakamoto et al. 2001, PNAS 98: 8554-9). As another example, the conjugated moiety can be a therapeutic moiety that provides a therapeutic function (e.g., antifungal, antibacterial, antiviral, antiparasitic, cytotoxic, radionucleotide). Other moieties that can be conjugated to the glycans present at the glycosylated receptor site include half-life extension moieties (PEG or PEG analogs, large polysaccharides), detection units (chromophore units, fluorescent units, phosphorescent units, luminescent units, light absorbing units, radioactive units), targeting moieties (e.g., small molecules, antibodies).

5. Nucleic acid molecules

Another aspect of the present technology relates to a nucleotide sequence or nucleic acid encoding a polypeptide according to the present technology.

" Nucleic acid molecule " (interchangeably used with " nucleic acid ") is a chain of nucleotide monomers linked to each other via a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform/transfect host cells or host organisms, for example, for the expression and/or production of polypeptides. Suitable hosts or host cells for production purposes will be clear to those of ordinary skill, preferably host organisms capable of glycosylation of polypeptides, and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell strain or any suitable fungal, prokaryotic or eukaryotic organism. Hosts or host cells comprising nucleic acids encoding polypeptides of the present technology are also encompassed by the present technology.

The nucleic acid can be, for example, DNA, RNA, or a hybrid thereof, and can also contain (e.g., chemically) modified nucleotides, such as PNA. It can be single-stranded or double-stranded. In one embodiment, it is in the form of double-stranded DNA. For example, the nucleotide sequence of the present technology can be genomic DNA or cDNA.

The nucleic acids of the present invention can be prepared or obtained in a manner known per se and/or can be isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides can, for example, be subjected to site-directed mutagenesis in order to provide nucleic acid molecules encoding polypeptides with sequence variations. In addition, as will be clear to those with ordinary skill, in order to prepare nucleic acids, either several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety and, for example, a nucleic acid encoding one or more linkers can be linked together in a suitable manner.

Techniques for generating nucleic acids will be clear to those of ordinary skill, and may, for example, include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more portions thereof); introducing mutations that result in the expression of truncated expression products; introducing one or more restriction sites (e.g., to generate cassettes and/or regions that can be readily digested and/or ligated using appropriate restriction enzymes); and/or introducing mutations using one or more "mismatched" primers via PCR reactions.

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

In other 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 that is capable of glycosylation of a polypeptide encoded by the nucleotide sequence or nucleic acid. As used herein, a vector is a vehicle suitable for carrying genetic material into a cell. Vectors include naked nucleic acids (such as plasmids or mRNA) or nucleic acids embedded in larger structures (such as liposomes or viral vectors).

In some embodiments, the vector comprises at least one nucleic acid, which is 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 the encoded polypeptide or construct under appropriate conditions (e.g., when the vector is introduced into a (e.g., human) cell). DNA-based vectors include the presence of elements for transcription (e.g., promoters and polyadenylation signals) and translation (e.g., Kozak sequences).

In one embodiment, in the vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, if the promoter is able to initiate or otherwise control/regulate the transcription and/or expression of the coding sequence, the promoter is considered to be "operably linked" to the coding sequence (wherein the coding sequence is understood to be "under the control" of the promoter). Generally, when two nucleotide sequences are operably linked, they are in the same orientation and usually also in the same reading frame. They are also generally substantially contiguous, but this may not be required.

In one embodiment, any regulatory element of a vector enables it to provide its intended biological function in an intended host cell or host organism. For example, a promoter, enhancer or terminator should be "operable" in an intended host cell or host organism, which means, for example, that the promoter should be able to initiate or otherwise control/regulate the transcription and/or expression of a nucleotide sequence (e.g., a coding sequence) to which it is operably linked.

In another aspect, the present technology relates to a method for producing a polypeptide and/or ISVD glycoprotein according to the present technology, wherein the method comprises the following steps: -          expressing a nucleotide sequence or nucleic acid according to the present technology in a suitable (non-human) host cell or (non-human) host organism, wherein the host cell or host organism is capable of glycosylation of the expressed polypeptide; optionally subsequently -          isolating and/or purifying the obtained polypeptide and/or ISVD glycoprotein.

The host cell or host organism capable of glycosylation of 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 a cell from a unicellular organism. In other words, a higher eukaryotic cell is a cell from (or derived from, in the case of cell culture, from) a multicellular eukaryotic organism, such as a human cell strain or another mammalian cell strain (e.g., a CHO cell strain). Typically, a higher eukaryotic cell will not be a fungal cell. Specifically, the term generally refers to mammalian cells, human cell strains, and insect cell strains. More specifically, the term refers to a vertebrate cell, and even more specifically a mammalian cell or a human cell. A higher eukaryotic cell as described herein will typically be part of a cell culture (e.g., a cell line such as a HEK or CHO cell line), although this is not always strictly required (e.g., in the case of plant cells, the plant itself can be used to produce the recombinant protein).

In one embodiment, the host cell is a lower eukaryotic cell. "Lower eukaryotic cell" means a filamentous fungal cell or a yeast cell. The yeast cell may be from a species of 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 Ind Microbiol Biotechnol. 36 (11)), the Phaffia fusca was previously named Pichia pastoris and is better known by the old name, and the nomenclature will also be used further herein. According to a specific embodiment, the lower eukaryotic cell is a Pichia cell, and in the most specific embodiment is a Pichia pastoris cell.

Some non-limiting examples of suitable mammalian cell lines or yeast strains include Chinese hamster ovary (CHO) cells, human embryonic 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 strains that can be used for the expression/production/manufacturing of polypeptides and proteins intended for administration to human subjects and/or for therapeutic use.

The present invention also relates to a (non-human) host cell or (non-human) host organism comprising a polypeptide or ISVD glycoprotein of the present invention, a nucleic acid encoding a polypeptide or ISVD glycoprotein of the present invention, and/or a vector comprising the nucleic acid molecule.

6. Composition

In yet another aspect, the present invention relates to a composition comprising a polypeptide according to the present invention; a polypeptide or ISVD glycoprotein produced using the method of the present invention; a conjugate according to the present invention; a nucleic acid encoding a polypeptide of the present invention or a vector comprising such a nucleic acid molecule. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more additional pharmaceutically active polypeptides and/or compounds.

7. use

The polypeptides, ISVD glycoproteins or conjugates, nucleic acid molecules or vectors as described herein, or compositions comprising the polypeptides, ISVD glycoproteins, conjugates, nucleic acid molecules or vectors of the present technology can be used as medicaments. Therefore, the present technology provides polypeptides, ISVD glycoproteins or conjugates, nucleic acid molecules or vectors as described herein, or compositions comprising the polypeptides, ISVD glycoproteins, conjugates, nucleic acid molecules or vectors of the present technology for use as medicaments.

Thus, further provided are methods for diagnosing, preventing and/or treating at least one disease and/or disorder, the methods comprising administering to a subject in need thereof a pharmaceutically active amount of at least one polypeptide, ISVD glycoprotein or conjugate of the present technology, such as a nucleic acid molecule or vector described herein, or a composition comprising a polypeptide, ISVD glycoprotein, conjugate, nucleic acid molecule or vector of the present technology.

8. Embodiment

The present invention is further illustrated by but not limited to the following embodiments.

Embodiment 1. A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 according to the Kabat numbering.

Example 2. A polypeptide according to Example 1, wherein the glycosylation receptor site is an N-glycosylation site.

Example 3. A polypeptide according to Example 1 or 2, wherein the glycosylation receptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation receptor site.

Example 4. A polypeptide according to any one of Examples 1 to 3, wherein the glycosylation receptor site is present at the following amino acid positions, and the amino acid positions are selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 105, 108 and 110 according to Kabat numbering.

Example 5. A polypeptide according to any one of Examples 1 to 3, wherein the glycosylation receptor site is present at the following amino acid positions, and the amino acid positions are selected from amino acid positions 19, 26, 55, 73, 105 and 108 according to Kabat numbering.

Embodiment 6. A polypeptide according to any one of embodiments 1 to 3, wherein the glycosylation receptor site is present at the following amino acid positions, and the amino acid positions are selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108 and 110 according to Kabat numbering.

Example 7. A polypeptide according to Example 6, wherein the polypeptide is a monovalent polypeptide comprising or (substantially) consisting of an ISVD.

Embodiment 8. A polypeptide comprising or (essentially) consisting of an ISVD, wherein the ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 1, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 according to Kabat numbering.

Example 9. A polypeptide according to Example 8, wherein the glycosylation receptor site is present at the following amino acid positions, and the amino acid positions are 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.

Example 11. The polypeptide according to Example 10, wherein at least one of the at least two ISVDs comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are 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.

Example 12. The polypeptide according to Example 10, wherein at least one of the at least two ISVDs comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 1, 3, 19, 26, 53, 55, 68, 73, 75, 105, 108 and 110 according to Kabat numbering.

Example 13. The polypeptide according to Example 10, wherein at least one of the at least two ISVDs comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 19, 26, 55, 105 and 108 according to Kabat numbering.

Example 14. A polypeptide according to Example 10, wherein the polypeptide is a bivalent polypeptide comprising or (substantially) consisting of two ISVDs.

Example 15. The polypeptide according to Example 14, wherein the N-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108 and 110 according to Kabat numbering.

Example 16. The polypeptide according to Example 14, wherein the C-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 1, 3, 19, 26, 55, 105 and 108 according to Kabat numbering.

Example 17. The polypeptide according to Example 14, wherein at least one of the two ISVDs comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 19, 26, 55, 105 and 108 according to Kabat numbering.

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

Example 19. The polypeptide according to Example 18, wherein at least one of the two ISVDs comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 19, 26, 55, 105 and 108 according to Kabat numbering.

Example 20. The polypeptide according to Example 18, wherein the N-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108 and 110 according to Kabat numbering.

Example 21. The polypeptide according to Example 18, wherein the C-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 1, 3, 19, 26, 55, 105 and 108 according to Kabat numbering.

Example 22. A polypeptide comprising or (essentially) consisting of two ISVDs, wherein the N-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 19, 26, 53, 55, 68, 73, 75, 105, 108 and 110 according to Kabat numbering.

Example 23. A polypeptide comprising or (essentially) consisting of two ISVDs, wherein the C-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 1, 3, 19, 26, 55, 105 and 108 according to Kabat numbering.

Embodiment 24.       A polypeptide according to any one of embodiments 1 to 3, wherein the polypeptide comprises at least three ISVDs.

Example 25. The polypeptide according to Example 24, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are 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.

Example 26. The polypeptide according to Example 24, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 15, 19, 26 and 105 according to Kabat numbering.

Embodiment 27. A polypeptide according to any one of embodiments 24 to 26, wherein the polypeptide is a trivalent polypeptide comprising or (substantially) consisting of three ISVDs.

Example 28. The polypeptide according to Example 24, 25 or 27, wherein the N-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 15, 19, 26, 55, 73, 75, 76, 105, 108 and 110 according to Kabat numbering.

Example 29. The polypeptide according to Example 24, 25 or 27, wherein the C-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are 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 the at least three ISVDs that is neither at the C-terminus nor at the N-terminus comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are 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) consisting of at least three ISVDs, wherein at least one of the at least three ISVDs comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 1, 3, 15, 19, 26, 53, 55, 68, 73, 75, 76, 102, 105, 108 and 110 according to the Kabat numbering.

Example 32. The polypeptide according to Example 31, wherein the N-terminal ISVD comprises a glycosylation receptor site located at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 3, 15, 19, 26, 55, 73, 75, 76, 105, 108 and 110 according to Kabat numbering.

Example 33. The polypeptide according to Example 31, wherein the C-terminal ISVD comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are 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 the at least three ISVDs that is neither at the C-terminus nor at the N-terminus comprises a glycosylation receptor site present at the following amino acid positions, wherein the amino acid positions are 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. A polypeptide according to any one of embodiments 31 to 34, wherein the polypeptide is a trivalent polypeptide comprising or (substantially) consisting of three ISVDs.

Embodiment 36. According to any one of embodiments 1 to 35, the polypeptide is glycosylated by one or more polysaccharides at the glycosylation receptor site.

Example 37. The polypeptide according to Example 36, wherein the polysaccharide 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, wherein the sequence of the polypeptide is selected from SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73-78, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89-91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96 and SEQ ID NO: 177-179.

Embodiment 39. The polypeptide according to embodiment 1, wherein the ISVD glycoprotein is a bivalent polypeptide, wherein the sequence of the polypeptide is selected from SEQ ID NO: 99-102, SEQ ID NO: 105-108, SEQ ID NO: 110, SEQ ID NO: 116-118, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 123-125, SEQ ID NO: 128-133, SEQ ID NO: 141-143, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 182, SEQ ID NO: 183 and SEQ ID NO: 187.

Embodiment 40. The polypeptide according to embodiment 1, wherein the ISVD glycoprotein is a trivalent polypeptide, wherein the sequence of the polypeptide is selected from SEQ ID NO: 20, SEQ ID NO: 22-24, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44-52, SEQ ID NO: 55-57, SEQ ID NO: 59-61, SEQ ID NO: 63-65, SEQ ID NO: 186, SEQ ID NO: 190 and SEQ ID NO: 191.

Embodiment 41. A nucleotide sequence or nucleic acid encoding a polypeptide according to any one of Embodiments 1 to 40.

Example 42. According to the nucleotide sequence or nucleic acid described in Example 41, the nucleotide sequence or nucleic acid is optimized for expression in a host cell or host organism, and the host cell or host organism is capable of glycosylation of the polypeptide encoded by the nucleotide sequence or nucleic acid.

Embodiment 43. According to the nucleotide sequence or nucleic acid described in Embodiment 41 or 42, 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, and the host cell or host organism is capable of glycosylation of the polypeptide encoded by the nucleotide sequence or nucleic acid.

Example 44. A method for producing a polypeptide according to any one of Examples 1 to 40, wherein the method comprises the following steps: -          Expressing a nucleotide sequence or nucleic acid according to any one of Examples 41 to 43 in a suitable host cell or host organism, wherein the host cell or host organism is capable of glycosylation of the expressed polypeptide.

Example 45. A method for conjugating a moiety to a polypeptide according to any one of Examples 1 to 40, comprising the following steps: -           oxidation of one or more polysaccharides present on the polypeptide, optionally using periodate salt oxidation; and -           conjugating the oxidized polysaccharide to the moiety.

Example 46. The method according to Example 45, wherein the moiety is selected from (bis-)mannose-6-phosphate, PROTAC and PEG moieties.

Example 47. A conjugate comprising a polypeptide according to any one of Examples 1 to 40 and a conjugated portion, wherein the portion is conjugated to a polysaccharide.

Embodiment 48.       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 a polypeptide according to any one of Embodiments 1 to 40, a polypeptide produced using the method according to Embodiment 45 or 46, or a conjugate according to Embodiment 47 or 48.

Embodiment 50. The polypeptide according to any one of embodiments 1 to 40, the nucleotide sequence or nucleic acid according to any one of embodiments 41 to 43, the conjugate according to embodiment 47 or 48 or the composition according to embodiment 49 is used as a medicament.

Embodiment 51. Use of the polypeptide according to any one of embodiments 1 to 40, the nucleotide sequence or nucleic acid according to any one of embodiments 41 to 43, the conjugate according to embodiment 47 or 48, or the composition according to embodiment 49 for the manufacture of a medicament.

Preferred glycosylation receptor sites present in an ISVD according to the present technology, as well as one or more preferred formats of such an ISVD having the glycosylation receptor sites, are listed in Table 2A and Table 2B below.

surface 2A : ISVD Preferred location of glycosylation receptor site ( Kabat Number) Location Price 1 Unit price, multiple price 3 Multi-price 15 Multi-price 19 Unit price, multiple price 26 Unit price, multiple price 53 Unit price, multiple price 55 Unit price, multiple price 68 Unit price, multiple price 73 Unit price, multiple price 75 Unit price, multiple price 76 Multi-price 102 Unit price, multiple price 105 Unit price, multiple price 108 Unit price, multiple price 110 Unit price, multiple price

surface 2B : ISVD Other preferred locations for glycosylation receptor sites ( Kabat Number) Location Price 1 Monovalent; polyvalent, preferably polyvalent in the C-terminal ISVD 3 Multiple prices, preferably two prices, more preferably three prices 15 Multiple prices, preferably two prices, more preferably three prices 19 Unit price; multiple price 26 Unit price; multiple prices, preferably two or three prices 53 Monovalent; multivalent, preferably bivalent or trivalent in ISVDs other than the C-terminal ISVD 55 Unit price; multiple prices, preferably three prices, more preferably two prices 68 Monovalent; multivalent, preferably bivalent or trivalent in ISVDs other than the C-terminal ISVD 73 Unit price; multiple price 75 Monovalent; polyvalent, preferably in ISVDs other than the C-terminal ISVD 76 Multiple prices, preferably three prices 102 Monovalent; multivalent, preferably trivalent in ISVDs other than the C-terminal ISVD 105 Unit price; multiple prices, preferably two or three prices 108 Unit price; multiple prices, preferably three prices, more preferably two prices 110 Multivalent, preferably bivalent or trivalent in ISVDs other than the C-terminal ISVD

The present technology and its implementation examples will be further emphasized in the examples section below.

Examples

Examples 1 : ISVD Generation of expression constructs

DNA fragments encoding ISVDs obtained by PCR with specific combinations of forward FR1 and reverse FR4 primers, each carrying a unique restriction site, were digested with appropriate restriction enzymes and ligated into the matching selection cassette of the ISVD expression vector. The ligation mixture was then used to transform electrocompetent or chemically competent Escherichia coli TG1 (Lucigen, catalog number 60502 or custom, respectively) or TOP10 (ThermoFisher Scientific, catalog number C404052 or C4081201, respectively) cells, which were grown under appropriate antibiotic selection pressure (kanamycin or genomycin). Resistant clones were verified by Sanger sequencing of plastid DNA (LGC Genomics).

Used to express unit price in E. coli ISVD Preparation of constructs

The monovalent ISVD is expressed in E. coli TG1 cells from a plasmid expression vector containing the lac promoter, the kanamycin resistance gene, the E. coli replication origin, and the ISVD selection site following the coding sequence for the OmpA signal peptide, which directs the expressed ISVD to the periplasmic lumen of the bacterial host. In frame with the ISVD coding sequence, the vector encodes a C-terminal 3xFLAG and His6 tag.

Used in CHO Expression in cells ISVD Preparation of constructs

The mammalian expression vector for expression of ISVD protein contains the RSV-LTR promoter, the resistance gene for genomicin, and the signal peptide for the mouse light chain. DNA encoding the ISVD constructs and the GS linker are cloned in the expression vector by Golden Gate cloning (Engler C, Marillonnet S. Golden Gate cloning. Methods Mol. Biol. 2014;1116:119-31). The expression vector contains two BpiI restriction sites, which are used to clone the PCR-amplified monovalent ISVD DNA and the GS linker DNA in one or more vectors. All of these elements are flanked by BpiI sites. The use of unique nucleotide single-stranded overhangs at each position of the cloning cassette allows seamless ligation in a predetermined sequence. After confirmation by Sanger sequencing, plasmid DNA from E. coli TOP10 was transfected into CHOEBNALT85 cells.

Examples 2 : ISVD Expression in E. coli

E. coli cells containing the ISVD expression vector were grown at 37ºC for 2 hours and then grown at 30ºC for 29 hours (250 rpm) in baffled shake flasks containing "5052" autoinduction medium (50x stock: 25% glycerol, 2.5% glucose, 10% lactose). Cells were pelleted by centrifugation (20 minutes, 4500 rpm, 4ºC), the supernatant was discarded, and the pellet was frozen at -20ºC overnight. The frozen cell pellet was then dissolved in DPBS (Gibco, catalog number 14190-094) at 1/12.5 of the original culture volume and incubated at 4ºC for 1 hour with gentle rotation to disrupt the outer membrane of the cells. The cells were pelleted again (20 minutes, 8500 rpm, 4ºC), and the supernatant containing the ISVD protein was collected and filtered to immediately proceed to purification.

ISVD expressed in E. coli is considered a control because no glycosylation will occur upon expression by E. coli.

Examples 3 : ISVD Expression in mammalian cells

CHOEBNALT85-1E9 cells (QMCF technology licensed from Icosagen) were seeded at 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. Complexes were 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 1.5 hours of incubation at 37ºC, 10 mL of fresh CHO TF medium containing GlutaMAX™ supplement was added, and the cells were allowed to grow for 24 hours at 37ºC. Subsequently, 10 mL of fresh CHO TF medium containing GlutaMAX™ supplement and penicillin-streptomycin (Gibco, catalog number 15140-122) was added, and the cells were incubated for an additional 72 hours at 37ºC. The cell density was then determined, and when 3.5E06 cells/mL was reached and viability was > 90%, 1.8 mL of Basic Feed (Xell, catalog number 1092-0001) was added to the cells, and the incubation temperature was reduced to 30ºC. Basal feed was added again after 48 hours and again after another 48 hours. After the final incubation of 72 hours, cells were pelleted by centrifugation (10 minutes at 1000 rpm) and the supernatant was transferred to another tube. The supernatant was centrifuged again to remove remaining cell debris (30 minutes at 8500 rpm), and the medium containing ISVD was collected, filtered, and stored at -20ºC until purification. All incubations were performed in a humidified orbital shaker incubator at 200 rpm in the presence of 8% _CO2 .

Examples 4 : Use egg white A Affinity chromatography purification ISVD

ISVD constructs were purified on protein A followed by a desalting step and, if necessary, preparative SEC in D-PBS. Concentrations were determined by OD280/OD340 measurement. Quality control was performed by SDS-PAGE and mass spectrometry.

The complete list of generated ISVDs can be found in Table 3 shown in Example 5 below.

Examples 5 ：Glycosylation analysis

ISVD glycoproteins obtained from mammalian cells were analyzed by SDS page for glycosylation at different glycosylation receptor sites. N-linked oligosaccharides were removed by PNGase to confirm that the changes in molecular weight (MW) in SDS-PAGE were in fact due to glycosylation. More specifically, if the changes in MW did not disappear after PNGase treatment compared to wild-type non-glycosylated controls, it would mean that something other than the added sugar caused the increase in MW.

PNG Enzymes F Steps

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

plan SDS-PAGE analyze

2 µg of sample was analyzed on pre-made 4%-12% BT NuPAGE SDS PAGE gels (Invitrogen, #NP0321BOX or NP0323BOX) under both reducing (R), heated and non-reducing (NR), non-heated conditions. The loading buffer was NuPAGE, 4x LDS sample buffer (Invitrogen, #NR0008). Heating was performed at 98ºC for 3 min. 5 µl of marker (Sharp pre-stained protein standard, Novex, #LC5800) was loaded and the gel was then run at 180 V in 1x MES running buffer during 40 min. Finally, the gel was stained with Instant Blue and destained with tap water for 8 h.

Mass spectrometry analysis

Intact mass LC-MS analysis was performed 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 Hermo Fisher Scientific). After online desalting using a MassPREP desalting cartridge (Waters), the molecular mass of one or more major products was determined after charge state deconvolution of the raw MS data.

result

Only ISVDs with a glycosylation degree of more than 50% were considered fully glycosylated. A complete list of the ISVDs tested can be found in Table 3 below.

surface 3 : Generated ISVD Glycosylation analysis ISVD ID SEQ ID NO Glycosylation receptor site ( Multiple ) Price / Position mutation Glycosylation degree ALB00606 1 15N Unit price N/A ALB00607 2 19N Unit price N/A ALB00608 3 26N Unit price N/A ALB00609 4 55N Unit price N/A ALB00610 5 105N Unit price N/A ALB00611 6 108N Unit price N/A ALB00612 7 75T* Unit price N/A ALB00613 8 15N Unit price 20% ALB00614 9 19N Unit price 50% ALB00615 10 26N Unit price 60% ALB00616 11 55N Unit price 100% ALB00617 12 105N Unit price 40% ALB00618 13 108N Unit price 20% ALB00619 14 75T* Unit price 100% ALB00620 15 3N, 5T** Unit price 5% ALB00621 16 3N, 5T** Unit price N/A ALB00622 17 without Unit price N/A ALB00623 18 without Unit price 0% T043800001 19 without Three prices 0% T043800002 20 3N, 5T** Trivalent / C-terminal 100% T043800003 twenty one 5N Trivalent / C-terminal 10% T043800004 twenty two 15N Trivalent / C-terminal 80% T043800005 twenty three 19N Trivalent / C-terminal 95% T043800006 twenty four 26N Trivalent / C-terminal 99% T043800007 25 53N Trivalent / C-terminal 10% T043800008 26 55N Trivalent / C-terminal 50% T043800009 27 68N Trivalent / C-terminal 0% T043800010 28 A74N, T76** Trivalent / C-terminal 10% T043800011 29 102N Trivalent / C-terminal 5% T043800012 30 105N Trivalent / C-terminal 70% T043800013 31 108N Trivalent / C-terminal 50% T043800014 32 110N Trivalent / C-terminal 0% T043800015 33 75N Trivalent / C-terminal 10% T043800016 34 75T* Trivalent / C-terminal 50% T043800055 35 without Three prices 0% T043800056 36 15N Trivalent / N-terminal 60% T043800057 37 19N Trivalent / N-terminal 100% T043800058 38 26N Trivalent / N-terminal 100% T043800059 39 53N Trivalent / N-terminal nd T043800060 40 55N Trivalent / N-terminal 100% T043800061 41 68N Trivalent / N-terminal nd T043800062 42 74N Trivalent / N-terminal 60% T043800063 43 102N Trivalent / N-terminal nd T043800064 44 105N Trivalent / N-terminal 100% T043800065 45 75N Trivalent / N-terminal 100% T043800066 46 76N Trivalent / N-terminal 100% T043800067 47 15N Triple / Middle 90% T043800068 48 19N Triple / Middle 100% T043800069 49 26N Triple / Middle 100% T043800070 50 53N Triple / Middle 90% T043800071 51 55N Triple / Middle 100% T043800072 52 68N Triple / Middle 100% T043800073 53 74N Triple / Middle 50% T043800074 54 102N Triple / Middle 90% T043800075 55 105N Triple / Middle 100% T043800076 56 75N Triple / Middle 100% T043800089 57 3N, 5T** Trivalent / N-terminal 90% T043800090 58 5N Trivalent / N-terminal nd T043800091 59 3N, 5T** Triple / Middle 100% T043800092 60 108N Trivalent / N-terminal 100% T043800093 61 110N Trivalent / N-terminal 100% T043800094 62 5N Triple / Middle 0% T043800095 63 108N Triple / Middle 100% T043800096 64 110N Triple / Middle 100% T043800097 65 76N Triple / Middle 100% T043800098 66 without Three prices 0% T043800118 67 3N, 5T** Unit price 20% T043800119 68 110N Unit price 50% T043800120 69 75N Unit price 100% T043800121 70 75T* Unit price 100% T043800122 71 5N Unit price 0% T043800123 72 15N Unit price 10% T043800124 73 19N Unit price 100% T043800125 74 26N Unit price 90% T043800126 75 55N Unit price 100% T043800127 76 68N Unit price 90% T043800128 77 105N Unit price 90% T043800129 78 108N Unit price 90% T043800130 79 without Unit price 0% T043800131 80 5N Unit price 5% T043800133 81 55N Unit price 90% T043800134 82 68N Unit price 40% T043800135 83 108N Unit price 90% T043800136 84 110N Unit price 30% T043800137 85 75T* Unit price 95% T043800138 86 without Unit price 0% T043800139 87 110N Unit price 80% T043800143 88 15N Unit price 20% T043800144 89 19N Unit price 80% T043800145 90 26N Unit price 90% T043800146 91 55N Unit price 80% T043800147 92 68N Unit price 30% T043800148 93 105N Unit price 90% T043800149 94 108N Unit price 90% T043800150 95 without Unit price 0% T043800159 96 1N, 3S** Unit price 100% T043800161 97 3N, 5T** Unit price 10% T043800178 98 without Bivalent/N-terminal 0% T043800179 99 3N, 5T** Bivalent/N-terminal 90% T043800180 100 110N Bivalent/N-terminal 99% T043800181 101 75N Bivalent/N-terminal 99% T043800182 102 75T* Bivalent/N-terminal 99% T043800183 103 5N Bivalent/N-terminal 0% T043800184 104 15N Bivalent/N-terminal 40% T043800185 105 19N Bivalent/N-terminal 100% T043800186 106 26N Bivalent/N-terminal 99% T043800187 107 55N Bivalent/N-terminal 99% T043800188 108 108N Bivalent/N-terminal 100% T043800189 109 without Bivalent/C-terminal 0% T043800190 110 3N, 5T** Bivalent/C-terminal 99% T043800191 111 110N Bivalent/C-terminal 0% T043800192 112 75N Bivalent/C-terminal 30% T043800193 113 75T* Bivalent/C-terminal 50% T043800194 114 5N Bivalent/C-terminal 0% T043800195 115 15N Bivalent/C-terminal 30% T043800196 116 19N Bivalent/C-terminal 100% T043800197 117 26N Bivalent/C-terminal 99% T043800198 118 55N Bivalent/C-terminal 90% T043800199 119 68N Bivalent/C-terminal 10% T043800200 120 105N Bivalent/C-terminal 90% T043800201 121 108N Bivalent/C-terminal 90% T043800202 122 without Bivalent/N-terminal 0% T043800203 123 110N Bivalent/N-terminal 99% T043800204 124 75N Bivalent/N-terminal 100% T043800205 125 73N Bivalent/N-terminal 100% T043800206 126 5N Bivalent/N-terminal 0% T043800207 127 15N Bivalent/N-terminal 50% T043800208 128 19N Bivalent/N-terminal 99% T043800209 129 26N Bivalent/N-terminal 100% T043800210 130 55N Bivalent/N-terminal 100% T043800211 131 68N Bivalent/N-terminal 100% T043800212 132 105N Bivalent/N-terminal 100% T043800213 133 108N Bivalent/N-terminal 100% T043800214 134 3N, 5T** Bivalent/N-terminal 60% T043800215 135 without Bivalent/C-terminal 0% T043800216 136 110N Bivalent/C-terminal 0% T043800217 137 75N Bivalent/C-terminal 40% T043800218 138 73N Bivalent/C-terminal 60% T043800219 139 5N Bivalent/C-terminal 0% T043800220 140 15N Bivalent/C-terminal 50% T043800221 141 19N Bivalent/C-terminal 90% T043800222 142 26N Bivalent/C-terminal 100% T043800223 143 55N Bivalent/C-terminal 80% T043800224 144 68N Bivalent/C-terminal 30% T043800225 145 105N Bivalent/C-terminal 90% T043800226 146 108N Bivalent/C-terminal 80% T043800227 147 3N, 5T** Bivalent/C-terminal 50% T043800228 148 68N Bivalent/N-terminal 95% T043800229 149 105N Bivalent/N-terminal 100% T043800899 177 53N Unit price 100% T043800901 178 53N Unit price 95% T043800902 179 102N Unit price 100% T043800969 180 1N Bivalent/N-terminal 10% T043800970 181 1N Bivalent/N-terminal 10% T043800971 182 1N Bivalent/C-terminal 95% T043800972 183 1N Bivalent/C-terminal 90% T043800973 184 1N Trivalent/N-terminal 10% T043800974 185 1N Tri-Price/Middle 0% T043800975 186 1N Trivalent/C-terminal 100% T043800976 187 53N Bivalent/N-terminal 95% T043800977 188 53N Bivalent/C-terminal 5% T043800978 189 53N Trivalent/N-terminal nd T043800979 190 73N Trivalent/N-terminal 90% T043800980 191 73N Tri-Price/Middle 100% T043800981 192 102N Bivalent/N-terminal 0% T043800982 193 102N Bivalent/C-terminal 0% T043800983 194 102N Trivalent/N-terminal nd T043800984 195 102N Tri-Price/Middle nd T043800985 196 102N Trivalent/C-terminal 0% Notes: N/A stands for “not applicable” nd stands for “not determined” * = actual glycosylation receptor site is at position 73, but mutation is at position 75 ** = complete NXS / NXT motif is mutated; actual glycosylation receptor site is Asn.

Surprisingly, the inventors discovered that some glycosylation receptor sites are glycosylated only in some ISVD formats and/or in certain specific configurations of the ISVD, while other glycosylation receptor sites have different requirements for becoming highly glycosylated.

Based on the glycosylation analysis performed, it was concluded that the following positions (Kabat numbering) used as glycosylation acceptor sites in the ISVD had a good degree of glycosylation when the ISVD was in a monovalent format as well as in a multivalent format: 1, 19, 26, 53, 55, 68, 73, 75, 102, 105, 108 and 110. However, surprisingly, the use of a glycosylation acceptor site at one of positions 53, 68, 75, 102 and 110 present in the C-terminal ISVD in a multivalent format resulted in a low degree of glycosylation, while the use of a glycosylation acceptor site at these positions not present in the C-terminal ISVD in a multivalent format resulted in a high degree of glycosylation. This was particularly true for position 102 in trivalent or higher formats.

In the opposite manner, for position 1, it was observed that in the multivalent format, high glycosylation was only observed when the glycosylation acceptor site was present in the C-terminal ISVD.

For the glycosylation receptor sites at positions 3 and 15 in ISVD, it was observed that high glycosylation could be obtained only when the ISVD was in a multivalent format. Specifically, for the glycosylation receptor site at position 15 in ISVD, it was observed that high glycosylation could be obtained only when the ISVD was in a trivalent format or higher. Position 76 was only tested in the trivalent format and resulted in high glycosylation in that format.

Examples 6 ：Thermal shift measurement

In this assay, it was determined whether glycosylation of the ISVD had an effect on 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), ALB00622 (SEQ ID NO: 17).

Among them, ALB00606, ALB00607, ALB00608, ALB00609, ALB00610, ALB00611, ALB00612, ALB00621, and ALB00622 were produced in E. coli (in which no glycosylation occurred) and used as controls.

ALB00616 is produced in CHO cells and therefore will be glycosylated and is used as an exemplary glycosylated ISVD. Since the sequence of ALB00616 is identical to that of ALB00609, the only difference between these differently produced ISVDs is whether they are glycosylated.

Thermal shift assays (TSA) were performed in 96-well plates on a qPCR machine (LightCycler 480II, Roche). Per row, one ISVD was analyzed in the following pH ranges: 4, 5, 6, 7, 8, and 9. In some cases, six different formulation buffers were also included, namely 20 mM acetate (pH 5), 20 mM histidine (pH 6), or 20 mM phosphate (pH 7), with or without 8% sucrose. Per well, 5 µL of ISVD sample (0.8 mg/mL in D-PBS) was added to 5 µL of Sypro Orange (40× in MilliQ water; Invitrogen, catalog number S6551) and 10 µL of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate, and 115 mM NaCl, pH range from 4 to 9). A temperature gradient was applied (37ºC to 99ºC at a rate of 0.03ºC/s), which induces the unfolding of ISVD and thus exposes the hydrophobic patches. The binding of Sypro Orange to those hydrophobic patches causes an increase in fluorescence intensity, which is measured (Ex/Em = 465/580 nm). The inflection point of the first derivative of the fluorescence intensity curve at pH 7 was used as a measure of the melting temperature (Tm).

result

The results of the thermal measurements are shown in Table 4 below.

surface 4 ：When E. coli or CHO When produced in cells, the measured ISVD Melting temperature ISVD ID SEQ ID NO Melting temperature ( ºC ） ALB00606 1 71 ALB00607 2 72 ALB00608 3 71 ALB00609 4 74 ALB00610 5 74 ALB00611 6 72 ALB00612 7 74 ALB00616 11 74 ALB00621 16 72 ALB00622 17 74

As can be seen in Table 4, the melting temperatures of the non-glycosylated ISVD and the glycosylated ISVD are the same. Specifically, ISVDs having the same sequence ALB00609 (control ISVD produced in E. coli) and ALB00616 (exemplary ISVD produced in CHO cells) showed no difference in melting temperature, whether or not they were glycosylated.

Therefore, it can be concluded that the melting temperature of glycosylated ISVD is not affected by the glycosylation of the ISVD.

Examples 7 ：Using surface plasmon resonance ISVD/ Affinity analysis of human serum albumin interactions

The affinity of ISVDs for 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.

HSA (Sigma-Aldrich, catalog number A8763000) was directly immobilized on flow cell 2 (FC) of a C1 Biacore chip on a Biacore 8K(+) instrument: the chip was primed by a 7 min injection of EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, 200 mM, Sigma Aldrich, catalog number 39391)/NHS (N-hydroxysuccinimide, 50 mM, Sigma Aldrich, catalog number 130672), HSA was diluted to 4 µg/ml in 10 mM acetate buffer (pH 4.5) and flowed through channels 1 to 8 of FC1 for immobilization, followed by a deactivation step with ethanolamine HCl (1 M, Cytiva) for 7 min. The flow rate during priming, fixation and inactivation was set to 10 µL/min. Affinity determinations were set up with a 6-point dilution series of ISVD ranging between 1000 nM and 1 nM. Various concentrations were tested in multi-cycle kinetic mode in 1x HBS-EP+ buffer at a flow rate of 30 µL/min, an association time of 120 s and a dissociation time of 600 s. Regeneration conditions after each interaction analysis were performed by flowing 10 mM glycine pH 1.5 buffer over the chip surface at a flow rate of 30 µL/min for 60 s. Data analysis was performed using Biacore insight evaluation software.

result

The affinity measurement results of the tested ISVDs can be found in Table 5 below.

surface 5 : ISVD right HSA Affinity determination ( SPR ） ISVD ID SEQ ID NO ISVD Price / mutation K _D （ M ） T04380001 (wild type) 19 Trivalent / C-terminal 8,40E-09 T043800002 20 Trivalent / C-terminal 1,50E-08 T043800004 twenty two Trivalent / C-terminal 1,10E-08 T043800005 twenty three Trivalent / C-terminal 1,30E-08 T043800008 26 Trivalent / C-terminal 1,30E-08 T043800012 30 Trivalent / C-terminal 1,50E-08

As can be seen from Table 5, the affinity of the glycosylated ISVD is comparable to that of the wild-type reference ISVD. This indicates that the glycosylated ISVD maintains its high affinity to its target.

Examples 8 ：Using surface plasmon resonance ISVD/EGFR Affinity analysis of interactions

The affinity of ISVDs for EGFR was determined by surface plasmon resonance (SPR). ISVDs 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 ISVDs T043800178 (SEQ ID NO: 98) and T043800189 (SEQ ID NO: 109) were used as references.

EGFR (Sino Biological, catalog number LC14JA1103) was directly immobilized on flow cell 2 (FC) of a CM5 Biacore chip on a Biacore 8K(+) instrument: the chip was primed by a 7 min injection of EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, 200 mM, Sigma Aldrich, catalog number 39391)/NHS (N-hydroxysuccinimide, 50 mM, Sigma Aldrich, catalog number 130672), EGFR was diluted to 4 µg/ml in 10 mM acetate buffer (pH 4.5) and flowed through channels 1 to 8 of FC1 for immobilization, followed by a deactivation step with ethanolamine HCl (1 M, Cytiva) for 7 min. The flow rate during priming, fixation and inactivation was set at 10 µL/min. Affinity determinations were set up with a 6-point dilution series of ISVD ranging between 2500 nM and 0.06 nM. Various concentrations were tested in multi-cycle kinetic mode at a flow rate of 30 µL/min, an association time of 120 s and a dissociation time of 600 s in 1x HBS-EP+ buffer. Regeneration conditions after each interaction analysis were performed by flowing 10 mM glycine pH 2.5 buffer over the chip surface twice for 30 s at a flow rate of 45 µL/min. Data analysis was performed using Biacore insight evaluation software.

result

The results of the affinity analysis can be found in Table 6 below.

surface 6 : ISVD right EGFR Affinity determination ( SPR ） ISVD ID SEQ ID NO ISVD Price / mutation K _D （ M ） T043800178 (wild type) 98 Bivalent/N-terminal 2,30E-06 T043800179 99 Bivalent/N-terminal 3,40E-06 T043800180 100 Bivalent/N-terminal 2,20E-06 T043800181 101 Bivalent/N-terminal 3,20E-06 T043800182 102 Bivalent/N-terminal 6,00E-06 T043800185 105 Bivalent/N-terminal 2,50E-06 T043800187 107 Bivalent/N-terminal 3,60E-06 T043800189 (wild type) 109 Bivalent/C-terminal 7,50E-06 T043800190 110 Bivalent/C-terminal 6,40E-05 T043800196 116 Bivalent/C-terminal 9,30E-06 T043800198 118 Bivalent/C-terminal 9,00E-06 T043800200 120 Bivalent/C-terminal 3,20E-05

As can be seen from Table 6, the affinity of the glycosylated ISVDs decreased by up to 4-fold relative to the wild-type reference ISVD, and all ISVDs maintained high affinity. This indicates that the glycosylated ISVDs maintain their high affinity for their targets.

Examples 9 :use Meso Scale Discovery right ISVD/ Affinity analysis of human serum albumin interactions

The affinity of ISVD for HSA was further determined by Meso Scale Discovery (MSD). For 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 (SEQ ID NO: 50), T043800071 (SEQ ID NO: 51), T043800072 (SEQ ID NO: 52), T043800074 (SEQ ID NO: 54), T043800075 (SEQ ID The wild-type ISVDs T043800055 (SEQ ID NO: 35) and T043800098 (SEQ ID NO: 66) were used as reference.

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 density of 1. Biotinylated HSA was captured at a concentration of 1 µg/mL on MSD GOLD 96-well small plaque streptavidin SECTOR plates (MSD, catalog number L45SA-1). Subsequently, 25 µL of a pre-equilibrated mixture (2 hours at room temperature) containing a fixed concentration of 100 pM of the ISVD compound to be tested and HSA ranging from 1.13 pM to 10 µM (23 dilutions, 1/3 dilution factor) was added to the plate. The plates were allowed to incubate for 10 minutes to capture free ISVD compounds and then washed with 3 x 150 µL PBS + 0.05% Tween-20. During the final detection step, 25 µL of Sulfo-tag-labeled anti-ISVD antibody was added at a concentration of 2 µg/ml and allowed to incubate for 1 hour, followed by a final wash with 150 μL of 1x PBS + 0.05% Tween-20. The plates were read on an MSD QuickPlex SQ120 reader after adding 150 µL of MSD reading buffer (Meso Scale Diagnostics, Catalog No. R92TG-1). Data were analyzed using a four-parameter logic (4PL) fit in GraphPad Prism 8.

result

The results of the affinity analysis can be found in Table 7 below.

surface 7 : ISVD right HSA Affinity determination ( MSD ） ISVD ID SEQ ID NO ISVD Price / mutation K _D （ M ） T043800055 (wild type) 35 Trivalent / N-terminal 3,10E-09 T043800056 36 Trivalent / N-terminal 1,60E-09 T043800057 37 Trivalent / N-terminal 2,00E-09 T043800060 40 Trivalent / N-terminal 2,30E-09 T043800064 44 Trivalent / N-terminal 4,30E-10 T043800065 45 Trivalent / N-terminal 5,90E-10 T043800066 46 Trivalent / N-terminal 1,60E-09 T043800067 47 Triple / Middle 5,7E-09 T043800068 48 Triple / Middle 4,2E-09 T043800070 50 Triple / Middle 4,7E-09 T043800071 51 Triple / Middle 4,9E-09 T043800072 52 Triple / Middle 4,2E-09 T043800074 54 Triple / Middle 5,8E-09 T043800075 55 Triple / Middle 5,4E-09 T043800076 56 Triple / Middle 2,9E-09 T043800089 57 Trivalent / N-terminal 8,8E-10 T043800093 61 Trivalent / N-terminal 4,5E-10 T043800096 64 Triple / Middle 5,10E-09 T043800098 (wild type) 66 Triple / Middle 2,1E-09

As can be seen from Table 7, the affinity of the glycosylated ISVD is comparable to that of the wild-type reference ISVD. This indicates that the glycosylated ISVD maintains its high affinity to its target.

Examples 10 :use Meso Scale Discovery right ISVD/TNFα Affinity analysis of interactions

The affinity of ISVDs for 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.

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 density of 1. Biotinylated TNFα was captured on MSD GOLD 96-well small plaque streptavidin SECTOR plates (MSD, Catalog No. L45SA-1) at a concentration of 0.5 µg/mL. Subsequently, 25 µL of a pre-equilibrated mixture (24 hours at room temperature) containing a fixed concentration of 12.8 pM of the ISVD compound to be tested and TNFα ranging from 78 fM to 10 µM (23 dilutions, 1/10 dilution factor from 1 µM to 0,01 µM; 1/1.8 dilution factor from 10 nM to 78 fM) were added to the plate. The plate was allowed to incubate for 10 minutes to capture free ISVD compounds and then washed with 3 x 150 µL PBS + 0.05% Tween-20. During the final detection step, 25 µL of Sulfo-tag anti-ISVD antibody was added at a concentration of 2 ug/ml and allowed to incubate for 1 hour, followed by a final wash with 150 µL of 1x PBS + 0.05% Tween-20. 150 µL of MSD reading buffer was added and the plate was read on an MSD QuickPlex SQ120 reader. Data were analyzed using a Four Parameter Logistic (4PL) fit in GraphPad Prism 8.

result

The results of the affinity analysis can be found in Table 8 below.

surface 8 : ISVD right TNF Affinity determination ( MSD ） ISVD ID SEQ ID NO ISVD Price / mutation K _D （ M ） T04380001 (wild type) 19 Trivalent / C-terminal 1,6E-11 T043800002 20 Trivalent / C-terminal 1,3E-11 T043800004 twenty two Trivalent / C-terminal 3,4E-11 T043800005 twenty three Trivalent / C-terminal 1,2E-11 T043800008 26 Trivalent / C-terminal 1,5E-11 T043800012 30 Trivalent / C-terminal 1,6E-11 T043800016 34 Trivalent / C-terminal 3,4E-12

As can be seen from Table 8, the affinity of the glycosylated ISVD is comparable to that of the wild-type reference ISVD. This indicates that the glycosylated ISVD maintains its high affinity to its target.

Examples 11 : ISVD Glycoprotein Glycogram Analysis

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.

Samples were prepared for glycan analysis according to the RapiFluor-MS (Waters) protocol. Prior to analysis, samples were diluted 9:31 using 2.1:1 acetonitrile: dimethylformamide (Waters). Samples were analyzed on a 6545XT LC-QTOF along with the RapiFluor-MS Performance Test Standard (Waters) using a linear gradient from 25% 50 mM ammonium formate pH 4.4 (Waters) in water (Fisher) to 56% acetonitrile (Thermo Scientific) at 0.4 mL/min over 35 minutes with the column (Glycan BEH Amide, Waters) maintained at 60ºC. Data were analyzed using Genedata Expressionist.

result

Examples of glycan patterns of ISVD glycoproteins on LC-QTOF are shown in Figures 1 and 2. All ISVD glycoproteins show similar patterns of glycan species; typically containing a high proportion of sialic acid-containing glycans (such as G1FS1, G2FS1, and G2FS2) in addition to neutral glycans (such as G0F, G1F, and G2F). The amounts of glycan species vary slightly between ISVD glycoproteins. Sialyl species with one or two sialic acids are present in the most abundant amounts.

Examples 12 :pair - 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 for conjugation to bis-mannose 6-phosphate (bis-M6P).

Conjugation of glycan-containing di-mannose 6-phosphate (produced in-house) to glycosylated ISVD was accomplished by first oxidizing ISVD with sodium periodate (Sigma) at 2.5 mg/mL and 20 mM in 100 mM sodium acetate (Sigma), pH 5.6, at 4°C with gentle shaking for 30 min. The reaction was then quenched with 3% glycerol (Sigma) at 4°C for 15 min. ISVD was purified by centrifugation through a molecular weight cutoff filter (Millipore) into 100 mM sodium acetate, pH 5.6, for a minimum of five exchanges.

After protein concentration was determined by Stunner (Unchained Labs), oxidized ISVD was conjugated to bis-M6P glycan in 100 mM sodium acetate (pH 5.6). The reaction was allowed to proceed for at least 16 hours at room temperature with gentle shaking. The ISVD was then purified into PBS via a molecular weight cutoff filter by centrifugation for a minimum of five exchanges. The concentration and polydispersity of the resulting ISVD was then determined by Stunner. Intact mass LC-QTOF and MALDI-TOF were then completed to analyze the conjugate (protocol described below).

Complete quality analysis of the solution LC-QTOF

Samples for intact mass analysis were diluted to 1 mg/mL, then 20 mM DTT (final concentration, Sigma) was added and incubated at 37°C for 30 minutes. Samples were then analyzed on a 6545XT LC-QTOF (Agilent) using a linear gradient from 75% water + 0.1% formic acid (Fisher) to 60% acetonitrile + 0.1% formic acid (Fisher) at 0.5 mL/min over 7.5 minutes, with the column (PLRP-S, Agilent) maintained at 60°C. Data were analyzed using Expressionist (Genedata).

plan MALDI-TOF analyze

Samples for MALDI-TOF were first diluted to 1 mg/mL with sample diluent (0.1% formic acid in water (Sigma)) and then mixed 1:1 with matrix solution (50% acetonitrile and 1% formic acid in water). Matrix diluted samples were each spotted 3 times on MALDI target plates (Bruker) and allowed to dry at room temperature. Sample replicates were then analyzed on a MALDI-TOF instrument (Bruker), and the data were analyzed via FlexAnalysis software (Bruker).

result

MALDI-TOF analysis of conjugates with T043800005 can be seen in Figure 3. Conjugates with one and two bis-M6Ps per ISVD are visible. An average degree of labeling (DoL) of 0.9 bis-M6Ps per ISVD was obtained.

MALDI-TOF analysis of conjugates with T043800002 can be seen in Figure 4. Conjugates with one, two and three bis-M6Ps per ISVD are visible. An average degree of labeling (DoL) of 2.1 bis-M6Ps per ISVD was obtained.

Examples 13 : PROTAC Bonding

ISVD glycoprotein T043800005 (SEQ ID NO: 23) was further conjugated with an alkoxyamine-DBCO linker and subsequently conjugated with PROTAC. Conjugation of PROTAC BRD4 degrader-5-CO-PEG3-N3 (PROTAC, MedChemExpress) to glycosylated ISVD T04380005 was accomplished by first oxidizing ISVD with sodium periodate at 2.5 mg/mL and 20 mM in 100 mM sodium acetate (pH 5.6). Oxidation was allowed to proceed for 30 minutes at 4°C with gentle shaking and then quenched with 3% glycerol at 4°C for 15 minutes. ISVD was then purified by centrifugation at least five times through a molecular weight cutoff filter into 100 mM sodium acetate (pH 5.6).

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

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

plan SDS-PAGE

Samples for SDS-PAGE were first diluted to 1 mg/mL in phosphate buffered saline (PBS, Gibco). Samples were then incubated at 70ºC for 10 minutes. After incubation, samples were mixed with 4X NuPAGE LDS sample buffer (Invitrogen) and water and then loaded onto 4%-12% bis-tris NuPAGE gels (Invitrogen) along with PageRuler ladder (Thermo Scientific). Gels were run in an Xcell Surelock cell with a Powerease power supply (Invitrogen) and MES running buffer (Invitrogen) using the NuPAGE gel setting. After the program was completed, the gel was extracted from the cartridge and placed in InstantBlue stain (Abcam) for 15 minutes. The gels were then visualized in a ChemiDoc imager and Image Lab software (Bio-Rad).

result

Based on SDS-PAGE, binding to PROTAC was observed at a DoL (degree of labeling) of 0.42.

Examples 14 ：PEGylation

ISVD glycoprotein T043800005 (SEQ ID NO: 23) was further conjugated with alkoxyamine functionalized PEG. Conjugation of 2 kDa aminooxy-PEG (PEG, BroadPharm) to glycosylated ISVD T043800005 was accomplished by first oxidizing ISVD with sodium periodate at 2.5 mg/mL and 20 mM in 100 mM sodium acetate (pH 5.6) at 4ºC with gentle shaking for 30 minutes. The reaction was then quenched with 3% glycerol at 4ºC for 15 minutes. ISVD was then purified through a molecular weight cutoff filter into 100 mM sodium acetate (pH 5.6) by centrifugation for a minimum of five exchanges.

After determining the protein concentration by 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 for at least 16 hours at room temperature with gentle shaking. The ISVD was then purified into PBS by centrifugation through a molecular weight cutoff filter at least five times. The concentration and polydispersity of the resulting ISVD were then determined by Stunner. SDS-PAGE (protocol described in Example 13) and MALDI-TOF (protocol described in Example 12) were then completed to analyze the conjugate.

result

According to SDS-PAGE, conjugation with PEG was observed at a DoL of 0.55.

Examples 15 :Depend on ISVD Glycovariate-mediated TNF-α Internalization

Materials and methods

Material

Biotinylated TNF was purchased from R&D systems, and Alexa Fluor™ 647-labeled streptavidin was obtained from Thermo Fisher Scientific. All other chemicals were purchased from Millipore Sigma, except for the in-house produced Bis-M6P glycan, unless otherwise stated.

ISVD Expression and purification of glycoforms

The trivalent ISVD glycovariant T043800005 (SEQ ID NO: 23) was engineered by introducing an N-glycosylation site with R19N, and mutants were generated using site-directed mutagenesis. The ISVD construct was produced by Chinese hamster ovary (CHO) cells. The protocol is as described in Examples 1 and 4.

With double M6P Site specificity of glycans ISVD Bonding

The conjugation was performed based on techniques known in the art. Briefly, anti-TNF ISVD was oxidized with 20 mM sodium periodate in phosphate buffer (pH 7.2) on ice for 30 minutes. The oxidation reaction was protected from light and quenched with glycerol (3% v/v) for 15 minutes. The oxidized ISVD buffer was exchanged to 100 mM sodium acetate (pH 5.6) by 5 rounds of ultrafiltration through an ^Amicon® ultracentrifuge filter. The desalted ISVD was then reacted with a 30-fold molar excess of bi-M6P polysaccharide at room temperature overnight. Unconjugated free polysaccharides were removed by ultrafiltration using the same protocol as for the oxidized ISVD.

ISVD Characterization

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

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

The molecular weight of ISVDs and ISVD bis-M6P conjugates was measured using MALDI-TOF MS to determine the copy number of bis-M6P glycans for each ISVD. The analysis was run on a Bruker Autoflex III. Using linear positive mode, intact protein mass was determined on target plates spotted with ISVD or conjugate samples mixed with sinapic acid matrix. Data for each sample were obtained 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 dividing the difference by the molecular weight of the glycan.

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

Characterize the target protein by flow cytometry ( POI ) Internalization

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-hydroxyethanol, and 10 mM HEPES (all from Gibco). They were plated at 1 x 10 ⁵ cells/well in U-bottom 96-well plates.

Biotinylated recombinant human TNF-α and streptavidin labeled with Alexa Fluor ^™ 647 were added sequentially at final concentrations of 50 nM and 100 nM, respectively. ISVD T043800005 or double M6P-conjugated ISVD were subsequently introduced at various concentrations between 0.78-50 nM.

After incubation at 37ºC for 1 or 4 hours, cells were washed twice with cold phosphate-buffered saline (PBS) (pH 7.2) and subjected to flow cytometry to assess the fluorescence of TNF complexes containing Alexa Fluor ^™ .

Characterization by Western blot POI degradation

K562 cells or Jurkat cells were plated at 1 x 10 ⁵ cells/well in U-bottom 96-well plates. Recombinant human TNF-α (50 nM, Peprotech) and anti-TNF ISVD (T043800005) or double M6P-conjugated ISVD were added sequentially at a final concentration of 25 nM.

After incubation at 37°C for 2 hours, the cells were washed twice with medium and lysed with RIPA buffer (Boston BioProducts). Some cells were kept in culture for another 4 or 24 hours in the presence of DMSO or 100 nM bafilomycin A1 (InvivoGen).

At each time point, cells were washed twice with cold PBS and lysed with RIPA buffer. Lysates were prepared for western blotting using Jess (ProteinSimple) to detect TNF-α or β-actin using anti-TNF (strain D5G9, Cell Signaling Technology) and subsequent anti-rabbit detection module (ProteinSimple) or anti-β-actin (strain 8H10D10, Cell Signaling Technology) and subsequent anti-mouse detection module (ProteinSimple).

To measure TNF-α in culture supernatants after internalization, K562 cells (1 x ¹⁰⁵ cells/well) were treated with 50 nM TNF-α and 25 nM ISVD or double M6P-conjugated ISVD at 37ºC.

During the incubation period, culture supernatants were collected at different time points (24, 48 and 72 h) and subjected to Western blotting with ISVD or conjugates as mentioned above.

result

Glycosylated ISVDs contained one copy of two M6P per ISVD as determined by MALDI-TOF MS.

ISVD conjugates containing approximately one diM6P attached to a single introduced N-glycosylation site efficiently induced TNF-α internalization in Jurkat cells or K562 cells ( FIG. 5 ).

The double M6P-conjugated ISVD construct showed dose-dependent TNF-α internalization after 1 and 4 h of incubation, while decreased internalization was observed at a high ISVD concentration of 50 nM, which may be due to the hook effect (Figure 6).

This was further confirmed by Western blot analysis of cell lysates (Fig. 7A) and supernatants (Fig. 7B). The amount of internalized TNF-α was already quite low 4 hours after incubation and was undetectable 22 hours after incubation. The addition of bafilomycin A1 significantly delayed TNF-α degradation at 4 and 22 hours, indicating that TNF-α degradation by the double M6P-conjugated ISVD occurs in lysosomes. The amount of TNF-α remaining in the cell culture medium was also determined. Although the TNF-α level in the culture supernatant derived from human K562 cells treated with only the control anti-TNF ISVD hardly changed, the amount of TNF-α decreased significantly during the incubation of cells with the double M6P-conjugated ISVD for 24 to 72 hours.

This shows that TNF-α internalization mediated by ISVD glycovariants with one copy of di-M6P is efficient, indicating that a single di-M6P on an ISVD is sufficient to induce internalization and degradation of soluble POI in lysosomes.

The following table shows the sequence disclosed in this article: ISVD ID SEQ ID NO sequence ALB00606 1 EVQLVESGGGVVQPNGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00607 2 EVQLVESGGGVVQPGGSLNLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00608 3 EVQLVESGGGVVQPGGSLRLSCAASNFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00609 4 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGNDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00610 5 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSNGTLVTVSS ALB00611 6 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTNVTVSS ALB00612 7 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSTNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00613 8 EVQLVESGGGVVQPNGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00614 9 EVQLVESGGGVVQPGGSLNLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00615 10 EVQLVESGGGVVQPGGSLRLSCAASNFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00616 11 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGNDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00617 12 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSNGTLVTVSS ALB00618 13 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTNVTVSS ALB00619 14 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSTNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00620 15 EVNLTESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS ALB00621 16 EVNLTESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS ALB00622 17 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS ALB00623 18 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS T043800001 19 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800002 20 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVNLTESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800003 twenty one EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLNESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800004 twenty two EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPNGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800005 twenty three EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLNLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800006 twenty four EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASNFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800007 25 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGNGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800008 26 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGNDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800009 27 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFNISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800010 28 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNNKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800011 29 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSNSSQGTLVTVSS T043800012 30 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSNGTLVTVSS T043800013 31 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTNVTVSS T043800014 32 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVNVSS T043800015 33 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSNNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800016 34 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSTNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800055 35 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800056 36 EVQLVESGGGVVQPNGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800057 37 EVQLVESGGGVVQPGGSLNLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800058 38 EVQLVESGGGVVQPGGSLRLSCAASNFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800059 39 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGNGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800060 40 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGNDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800061 41 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFNISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800062 42 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNNKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800063 43 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSNSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800064 44 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSNGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800065 45 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSNNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800066 46 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSTNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800067 47 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPNGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800068 48 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLNLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800069 49 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASNFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800070 50 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGNGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800071 51 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGND TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800072 52 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFNISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800073 53 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNNNKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800074 54 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSNSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800075 55 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSNGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800076 56 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSNNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800089 57 EVNLTESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800090 58 EVQLNESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800091 59 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVNLTESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800092 60 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTNVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800093 61 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVNVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800094 62 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLNESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800095 63 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTNVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800096 64 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVNVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800097 65 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSTNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800098 66 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800118 67 AVNLTESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800119 68 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVNVSS T043800120 69 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDANNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800121 70 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNATNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800122 71 AVQLNESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800123 72 AVQLVESGGGSVQANGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800124 73 AVQLVESGGGSVQAGGSLNLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800125 74 AVQLVESGGGSVQAGGSLRLTCAASNRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800126 75 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGNSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800127 76 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFNISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800128 77 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGNGTLVTVSS T043800129 78 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTNVTVSS T043800130 79 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800131 80 EVQLNESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800133 81 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGNITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800134 82 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFNISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800135 83 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTNVTVSS T043800136 84 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVNVSS T043800137 85 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNATNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800138 86 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800139 87 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVNVSS T043800143 88 EVQLVESGGGVVQPNGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800144 89 EVQLVESGGGVVQPGGSLNLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800145 90 EVQLVESGGGVVQPGGSLRLSCTASNFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800146 91 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDNTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800147 92 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFNISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800148 93 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGNGTLVTVSS T043800149 94 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTNVTVSS T043800150 95 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800159 96 NVSLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800161 97 DVNLTESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800178 98 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800179 99 AVNLTESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800180 100 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVNVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800181 101 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDANNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800182 102 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNATNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800183 103 AVQLNESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800184 104 AVQLVESGGGSVQANGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800185 105 AVQLVESGGGSVQAGGSLNLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800186 106 AVQLVESGGGSVQAGGSLRLTCAASNRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800187 107 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGNSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800188 108 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTNVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800189 109 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800190 110 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVNLTESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800191 111 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVNVSS T043800192 112 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDANNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800193 113 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNATNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800194 114 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLNESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800195 115 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQANGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800196 116 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLNLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800197 117 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASNRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800198 118 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGNSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800199 119 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFNISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800200 120 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGNGTLVTVSS T043800201 121 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTNVTVSS T043800202 122 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800203 123 EVQLVESGGGVVQPGGSLRLSCTASGFTFTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVNVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800204 124 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSNNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800205 125 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSTNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800206 126 EVQLNESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800207 127 EVQLVESGGGVVQPNGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800208 128 EVQLVESGGGVVQPGGSLNLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800209 129 EVQLVESGGGVVQPGGSLRLSCTASNFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800210 130 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDNTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800211 131 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFNISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800212 132 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGNGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800213 133 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTNVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800214 134 DVNLTESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800215 135 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800216 136 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVNVSS T043800217 137 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSNNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800218 138 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSTNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800219 139 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLNESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800220 140 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPNGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800221 141 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLNLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800222 142 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASNFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800223 143 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDNTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800224 144 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFNISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800225 145 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGNGTLVTVSS T043800226 146 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSEVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTNVTVSS T043800227 147 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSDVNLTESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800228 148 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFNISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800229 149 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGNGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS 5GS connector 150 GGGGS 9GS connector 151 GGGGSGGGS 15GS connector 152 GGGGSGGGGSGGGGS 35GS connector 153 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS VTVSS(X) ₁₀ 154 VTVSSXXXXXXXXXX C-terminal sequence 155 VKVSS C-terminal sequence 156 VQ C-terminal sequence 157 VTVKS C-terminal sequence 158 VTVQS C-terminal sequence 159 VKVKS C-terminal sequence 160 VxDV C-terminal sequence 161 VQ C-terminal sequence 162 VQVQ Ala extension 163 VTVSSA Ala extension 164 VKVSSA Ala extension 165 VQ Ala extension 166 VTVKSA Ala extension 167 VTVQSA Ala extension 168 VKVKSA Ala extension 169 VxDV Ala extension 170 VxV Ala extension 171 QVQ VTVSS(X) ₁ 172 VTVSSX VTVSS(X) ₂ 173 VTVSSXX VTVSS(X) ₃ 174 VTVSSXXX VTVSS(X) ₄ 175 VTVSSXXXX VTVSS(X) ₅ 176 VTVSSXXXXX T043800899 177 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGSITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800901 178 NVSLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800902 179 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGNDSTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800969 180 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDNWSQGTLVTVSS T043800970 181 NVSLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800971 182 NVSLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800972 183 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS NVSLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800973 184 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGG GSNVSLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSS T043800974 185 NVSLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800975 186 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSNVSLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800976 187 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSNVSLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800977 188 EVQLVESGGGVVQPGGSLRLSCTASGFFTFSTADMGWFRQAPGKGREFVARISGNDSTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSG GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS T043800978 189 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWNGSSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS T043800979 190 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGNGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800980 191 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSTNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800981 192 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSTNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800982 193 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGS AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDNWSQGTLVTVSS T043800983 194 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSNSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTI SRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800984 195 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSNSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSS T043800985 196 EVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKG LEWVSEINTNGLITKYPDSVKGRFTISSRDNAKNTLYLQMNSLRPEDTALYYCARSPSGFNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSNSSQGTLVTVSS

without

FIG1 shows the glycan spectrum of glycosylated ISVD T043800005 measured by LC - QTOF.

FIG. 2 shows the glycan spectrum of glycosylated ISVD T043800002 measured by LC-QTOF.

FIG. 3 shows the MALDI-TOF measurement results of the phosphorylation of the conjugate of ISVD T043800005 and di-mannose-6- as described in Example 12.

FIG. 4 shows the MALDI-TOF measurement results of the conjugate of ISVD T043800002 and bis-mannose-6-phosphate as described in Example 12.

FIG. 5 shows flow cytometry measurements for evaluating TNF internalization in Jurkat cells ( A ) or K562 cells ( B ) after treatment with TNF complex and anti-TNF ISVD or dual M6P-anti-TNF ISVD conjugate at different concentrations for 1 hour and 4 hours.

FIG6 shows the fold increase in mean fluorescence intensity (MFI) in Jurkat cells ( A ) or K562 cells ( B ), indicating that the dual M6P-anti-TNF ISVD conjugate exceeded TNF internalization by anti-TNF ISVD alone.

Figure 7 shows Western blots of TNF-α after treatment with anti-TNF ISVD or dual M6P-anti-TNF ISVD at different time points under the following conditions: ( A ) in cell lysates, after internalization in K562 cells or Jurkat cells at 37ºC for 2 h, followed by treatment with or without bafilomycin for 4 h and 24 h, and ( B ) in supernatants from the same K562 cell cultures.

TW202509060A_113117040_SEQL.xml

Claims (47)

A polypeptide comprising or (essentially) consisting of a heavy chain immunoglobulin single variable domain (ISVD), wherein the ISVD comprises a glycosylation acceptor site present 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 the Kabat numbering. The polypeptide of claim 1, wherein the glycosylation receptor site is an N-glycosylation site. The polypeptide of claim 1 or 2, wherein the glycosylation receptor site is contained in a NXT or NXS motif, wherein X can be any amino acid, and wherein the asparagine residue of the NXT or NXS motif is the glycosylation receptor site. The polypeptide of any one of claims 1 to 3, wherein the glycosylation acceptor site is present 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. The polypeptide of any one of claims 1 to 3, wherein the glycosylation acceptor site is present at an amino acid position selected from amino acid positions 19, 26, 55, 73, 105 and 108 according to Kabat numbering. The polypeptide of any one of claims 1 to 3, wherein the glycosylation acceptor site is present 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. A polypeptide as described in claim 6, wherein the polypeptide is a monovalent polypeptide, and the monovalent polypeptide comprises or (essentially consists of) an ISVD. A polypeptide comprising or consisting essentially of an ISVD, wherein the ISVD comprises a glycosylation acceptor site present 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 the Kabat numbering. The polypeptide of claim 8, wherein the glycosylation acceptor site is present 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. The polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises at least two ISVDs. The polypeptide of claim 10, wherein at least one of the at least two ISVDs comprises a glycosylation acceptor site present 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. The polypeptide of claim 10, wherein at least one of the at least two ISVDs comprises a glycosylation acceptor site present 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. The polypeptide of claim 10, wherein at least one of the at least two ISVDs comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 19, 3, 26, 55, 105, and 108 according to Kabat numbering. The polypeptide of claim 10, wherein the polypeptide is a bivalent polypeptide comprising or (essentially consisting of) two ISVDs. The polypeptide of claim 14, wherein the N-terminal ISVD comprises a glycosylation acceptor site present 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. The polypeptide of claim 14, wherein the C-terminal ISVD comprises a glycosylation acceptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 19, 1, 3, 26, 55, 105 and 108 according to Kabat numbering. The polypeptide of claim 14, wherein at least one of the two ISVDs comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 19, 3, 26, 55, 105, and 108 according to Kabat numbering. 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 19, 1, 3, 15, 26, 53, 55, 68, 73, 75, 76, 105, 108 and 110 according to the Kabat numbering. The polypeptide of claim 18, wherein at least one of the two ISVDs comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 19, 3, 26, 55, 105, and 108 according to Kabat numbering. The polypeptide of claim 18, wherein the N-terminal ISVD comprises a glycosylation acceptor site present 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. The polypeptide of claim 18, wherein the C-terminal ISVD comprises a glycosylation acceptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 19, 1, 3, 26, 55, 105 and 108 according to Kabat numbering. A polypeptide comprising or consisting essentially of two ISVDs, wherein the N-terminal ISVD comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 19, 3, 26, 53, 55, 68, 73, 75, 105, 108 and 110 according to the Kabat numbering. A polypeptide comprising or (essentially) consisting of two ISVDs, wherein the C-terminal ISVD comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 19, 1, 3, 26, 55, 105 and 108 according to the Kabat numbering. The polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises at least three ISVDs. The polypeptide of claim 24, wherein at least one of the at least three ISVDs comprises a glycosylation acceptor site present 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. The polypeptide of claim 24, wherein at least one of the at least three ISVDs comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 19, 3, 15, 26, and 105 according to Kabat numbering. A polypeptide as described in any one of claims 24 to 26, wherein the polypeptide is a trivalent polypeptide comprising or (essentially consisting of) three ISVDs. The polypeptide of claim 24, 25 or 27, wherein the N-terminal ISVD comprises a glycosylation acceptor site present 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. The polypeptide of claim 24, 25 or 27, wherein the C-terminal ISVD comprises a glycosylation acceptor site present at an amino acid position selected from amino acid positions 19, 1, 3, 15, 26 and 105 according to Kabat numbering. The polypeptide of claim 24, 25 or 27, wherein at least one of the at least three ISVDs that is neither at the C-terminus nor at the N-terminus comprises a glycosylation acceptor site present 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. A polypeptide comprising or (essentially) consisting of at least three ISVDs, wherein at least one of the at least three ISVDs comprises a glycosylation acceptor site present 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 the Kabat numbering. The polypeptide of claim 31, wherein the N-terminal ISVD comprises 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. The polypeptide of claim 31, wherein the C-terminal ISVD comprises a glycosylation acceptor site present at the following amino acid positions, wherein the amino acid positions are selected from amino acid positions 19, 1, 3, 15, 26 and 105 according to Kabat numbering. The polypeptide of claim 31, wherein at least one of the at least three ISVDs that is neither at the C-terminus nor at the N-terminus comprises a glycosylation acceptor site present 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. A polypeptide as described in any one of claims 31 to 34, wherein the polypeptide is a trivalent polypeptide comprising or (essentially consisting of) three ISVDs. A polypeptide as described in any one of claims 1 to 35, which is glycosylated with one or more polysaccharides at the glycosylation receptor site. The polypeptide of claim 36, wherein the polysaccharide is selected from terminal N-acetyl glucosamine (GlcNAc), (terminal) mannose, (terminal) sialic acid, (terminal) galactose or a combination thereof. A nucleotide sequence or nucleic acid encoding a polypeptide as described in any one of claims 1 to 37. The nucleotide sequence or nucleic acid of claim 38, wherein the nucleotide sequence or nucleic acid is optimized for expression in a host cell or host organism, wherein the host cell or host organism is capable of glycosylation of a polypeptide encoded by the nucleotide sequence or nucleic acid. A nucleotide sequence or nucleic acid as described in claim 38 or 39, which is in the form of a construct or (expression) vector that can be expressed in a host cell or host organism, and the host cell or host organism is capable of glycosylation of a polypeptide encoded by the nucleotide sequence or nucleic acid. A method for producing a polypeptide as described in any one of claims 1 to 37, wherein the method comprises the following steps: - expressing a nucleotide sequence or nucleic acid as described in any one of claims 38 to 40 in a suitable host cell or host organism, wherein the host cell or host organism is capable of glycosylation of the expressed polypeptide. A method for conjugating a moiety to a polypeptide as claimed in any one of claims 1 to 37, the method comprising the steps of: - oxidation of one or more polysaccharides present on the polypeptide, optionally using periodate salt oxidation; and - conjugating the oxidized polysaccharide to the moiety. The method of claim 42, wherein the moiety is selected from (bis-)mannose-6-phosphate, PROTAC and PEG moieties. A conjugate comprising the polypeptide of any one of claims 1 to 37 and a conjugated portion, wherein the portion is conjugated to the polysaccharide. The conjugate of claim 44, wherein the moiety is selected from (bis-)mannose-6-phosphate, PROTAC and PEG moieties. A composition comprising a polypeptide as described in any one of claims 1 to 37, a polypeptide produced using the method as described in claim 42 or 43, or a conjugate as described in claim 44 or 45. A polypeptide as described in any one of claims 1 to 37, a nucleotide sequence or a nucleic acid as described in any one of claims 38 to 40, a conjugate as described in claim 44 or 45, or a composition as described in claim 46, for use as a medicament.