Glycosylated Polypeptides

Abstract

The present invention provides molecules comprising one or more copies of the following glycosylated amino acid sequence: X1ThrProX2X3, where X1, X2, and X3 are any amino acid, and the threonine (Thr) amino acid residue is O-glycosylated with a sialylated sugar. The sialylated sugar optionally comprises a chemical group that can be conjugated to a moiety. In some embodiments, the sialylated sugar is conjugated to a moiety. The present invention also provides uses and methods of using such molecules, particularly in generating conjugates of desired moieties through the use of glycosylation sequences.

Description

[Technical Field]

[0001] (Reference to sequence listing) This application has been submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file entitled "22760_WO_000 Sequence Listing ST26," created on February 12, 2024, and is 15 kilobytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to molecules comprising one or more copies of a glycosylated amino acid sequence for O-glycosylation, which provides a convenient method for conjugating the glycosylated amino acid sequence to a desired moiety. The present invention further relates to cell lines for producing such molecules. The present invention also relates to conjugation methods for producing the molecules of the present invention. The present invention also relates to various uses of the molecules and methods of using them, including therapeutics and diagnostics. [Background technology]

[0003] For further information regarding the headings herein, see MPEP 608.01(a) / 37 CFR 1.77. O-glycosylation is the covalent attachment of a sugar to the oxygen group of serine or threonine. Organisms utilize protein O-glycosylation for many important biological functions, including protection from protease degradation, regulation of serum half-life, functional regulation, intracellular trafficking, cell adhesion, and self-versus-foreign recognition during the immune response. The most abundant form of O-linked glycosylation in higher eukaryotes, termed "mucin-type," is characterized by N-acetylgalactosamine (GalNAc) attached to the hydroxyl group of a serine or threonine side chain (Hang et al. (2005) Bioorg Med Chem., 13(17):5021-5034). The primary amino acid consensus sequence (i.e., template) for mucin-type O-glycosylation is unknown.

[0004] Heterogeneity in protein glycosylation is a universal feature of life. There are two codified types of variability in glycosylation: macroheterogeneity and site occupancy. Macroheterogeneity is the observation that known glycosylation sites in proteins can have variable occurrence of glycans (i.e., site occupancy). Mucin-type O-linked glycans typically occur clustered together in "mucin domains" (Thanka et al. (2001) Biophys J., 80(2):952-960). Determining site occupancy is difficult due to the lack of recognized consensus sequences and the lack of universal enzymes for O-glycan removal (Jensen et al. (2010) FEBS J., 277(1):81-94). Contributing to this complexity is the potential importance of primary, secondary, tertiary, and quaternary protein structure on the efficiency of O-glycosylation of that protein (Wang et al. (1993) J Biol Chem., 268(31):22979-22983). Microheterogeneity is the observation of variation in glycan structure at the same site on a protein (Galleguillos et al. (2017) Comput Struct Biotechnol J., 15:212-221).

[0005] There is currently a need to provide methods for joining a given biological molecule to a desired moiety. The present invention uses glycosylation sequences to conjugate the two together in a convenient and highly versatile manner. The present invention also provides other embodiments based on the glycosylation sequences of the invention, which are discussed in more detail below. Summary of the Invention

[0006] The present invention is based on glycosylated amino acid sequences that allow O-glycosylation of threonine in the sequence. Preferably, after glycosylation of the amino acid sequence, the resulting O-glycan contains at least one sialic acid. The sialic acid may optionally further contain a chemical group for conjugation to a desired moiety, or a chemical group that is so conjugated to a desired moiety. The glycosylated amino acid sequences of the present invention are typically present in a polypeptide that is or forms part of a molecule of the present invention, providing a convenient and highly versatile method for joining a polypeptide to a desired moiety via conjugation. The present invention also provides molecules comprising one or more glycosylated amino acid sequences of the present invention that are not yet glycosylated. The present invention further provides molecules comprising one or more glycosylated amino acid sequences of the present invention that are O-glycosylated but do not contain a chemical group for conjugation. Thus, in a preferred embodiment, the present invention provides molecules comprising one or more glycosylated amino acid sequences of the present invention with terminal sialylation of the O-glycan, wherein the glycan does not contain a chemical group for conjugation. In a particularly preferred embodiment, the present invention thus provides a molecule comprising one or more glycosylated amino acid sequences of the present invention having terminal sialylation of the O-glycans, wherein the glycans comprise chemical groups that allow conjugation to a desired moiety. In a preferred embodiment, such conjugation has occurred, and thus the molecule comprises a moiety. In another embodiment, conjugation has not yet occurred, but the chemical group is capable of such conjugation.

[0007] Advantages of the present invention include that the glycosylation site, and therefore any conjugation site, is highly specific and can be selected. The approach of the present invention can also help avoid undesirable side reactions. The conjugation approach of the present invention also typically has the advantage of being bioorthogonal, i.e., conjugation can occur under physiological conditions and is not toxic to cells. The conjugation is typically biologically and chemically stable under physiological conditions and is not easily reversible. Conjugation also typically does not require the addition of a catalyst. The engineered O-glycan recognition sequences of the present invention typically induce complete or near-complete site occupancy in terms of the percentage of sites that are O-glycosylated. The high site occupancy and consistency in the specific O-glycan sugars added to amino acid sites means that the present invention provides an efficient method for generating homogeneous conjugates, making it a particularly good method for generating therapeutics when such consistency is important. The present invention can be used in a variety of different ways. Conjugation can be performed, for example, by the cell itself. Alternatively, the molecules of the invention can be purified from the cells that produce them and then conjugated to the desired moiety.

[0008] A particularly preferred approach for the conjugation of the present invention is to use click chemistry. The use of click chemistry allows for a robust, high-yield reaction that proceeds rapidly, selectively, and with few or no by-products under mild conditions. In such embodiments, the sialylated O-glycan and the desired conjugation partner moiety have complementary click chemistry groups that react with each other to produce a single product molecule. In one embodiment, the moiety is or includes a linker, which is conjugated to or already conjugated to an additional molecule or group. In a further preferred embodiment, the molecule comprising at least one glycosylated amino acid sequence of the present invention and the desired moiety are both polypeptides, and thus the present invention provides a convenient method for joining at least two polypeptides together.

[0009] The present invention also provides cells comprising polypeptides having glycosylation sequences of the present invention. The present invention also provides cells encoding molecules of the present invention. The cells can be any of the cell types discussed herein. In a particularly preferred embodiment, the cells are CHO cell lines. In another preferred embodiment, the cells are human cell lines. The present invention further provides cells suitable for producing molecules of the present invention. In a particularly preferred embodiment, the cells are used to produce glycosylated forms of molecules of the present invention in which one or more glycosylation sequences are O-glycosylated. In a preferred embodiment, the O-glycosyl sugar chain of the molecule is sialylated. In a preferred embodiment, sialylation terminates the O-glycan sugar chain. In one embodiment, the cell is a cell encoding a molecule comprising one or more glycosylation sequences of the present invention. In another embodiment, the cell further comprises a non-functional UDP-N-acetylglucosamine 2-epimerase gene. Unexpectedly, it has been discovered that both the epimerase and kinase functions of the endogenous UDP-N-acetylglucosamine 2-epimerase-N-acetylmannosamine kinase gene can be eliminated together, without the need to introduce a gene to compensate for the loss of endogenous N-acetylmannosamine kinase activity. Disruption of UDP-N-acetylglucosamine 2-epimerase activity means that the cell is deficient in sugar synthesis, providing a convenient method for promoting the incorporation of sialic acid into O-glycosyl sugar chains in glycosylation sequences of the invention by providing the cell with exogenous modified sugars.

[0010] Thus, the present invention provides a molecule comprising one or more copies of the following glycosylated amino acid sequence: X1Thr Pro X2X3 where: X1, X2, and X3 are any amino acids; Threonine (Thr) amino acid residues are O-glycosylated with sialylated sugars.

[0011] In a preferred embodiment, the present invention provides a molecule comprising one or more copies of the following glycosylated amino acid sequence: X1Thr Pro X2X3 where: X1, X2, and X3 are any amino acids; Threonine (Thr) amino acid residues are O-glycosylated with sialylated sugars that optionally contain chemical groups that can be or have been conjugated to a moiety.

[0012] In a particularly preferred embodiment, the sialylated sugar comprises such a chemical group. In a preferred embodiment, the chemical group is capable of being conjugated to a desired moiety, but is not actually yet conjugated to that moiety. In a further preferred embodiment, the chemical group is conjugated to a desired moiety.

[0013] The present invention further provides a pharmaceutical composition comprising a molecule of the invention and a pharmaceutically acceptable carrier.

[0014] The present invention further provides the molecules of the invention for use in therapy of the human or animal body.

[0015] The present invention further provides a molecule of the invention for use in treating a condition selected from cancer, cardiovascular disease, obesity, an autoimmune condition, an inflammatory condition, diabetes, or a CNS disorder.

[0016] The present invention also provides a method of treating a condition, which may be any of those described herein, comprising administering to a subject in need thereof an effective amount of a molecule of the invention.

[0017] The present invention also provides (a) an endogenous UDP-N-acetylglucosamine 2-epimerase-ManNAc kinase gene, wherein the gene has been mutated such that at least UDP-N-acetylglucosamine 2-epimerase function is reduced or eliminated; and (b) a sequence encoding a polypeptide comprising the amino acid sequence: X1Thr Pro X2X3 where: X1, X2, and X3 are any amino acids; The threonine (T) amino acid residue is O-glycosylated with a sialylated sugar.

[0018] The present invention further provides a method for producing a glycosylated polypeptide, comprising culturing a cell of the present invention in a medium supplemented with peracetylated ManNAz.

[0019] The present invention also provides a method for introducing a glycosylation site into a polypeptide, comprising modifying the sequence of the polypeptide to comprise the amino acid sequence: X1Thr Pro X2X3 where: X1, X2, and X3 are any amino acids; The threonine (T) amino acid residue is O-glycosylated with a sialylated sugar.

[0020] The present invention further provides a method of conjugating a molecule to a moiety, the method comprising: (a) providing a molecule of the invention, wherein the sialylated sugar comprises a chemical group that can be conjugated to a desired moiety having a compatible chemical group; (b) contacting the molecule of (a) with a desired moiety; (c) rendering the chemical group of the molecule susceptible to conjugation with a desired moiety via a compatible chemical group.

[0021] The present invention also provides a method of joining two molecules together, comprising: (a) providing a molecule of the invention, wherein the sialylated sugar comprises a chemical group that can be conjugated to a desired second molecule having a compatible chemical group that allows for conjugation; (b) contacting the molecule of (a) with a desired second molecule; (c) allowing conjugation of the first molecule to the second molecule via a compatible chemical group.

[0022] The present invention further provides a method for generating a combinatorial library, the method comprising: (a) providing a plurality of molecules of the invention, the molecules being different from one another, but wherein the sialylated sugar of each molecule contains the same chemical group that can be conjugated to a desired moiety; (b) providing a second plurality of molecules, the second plurality of molecules being different from each other and from the molecules of (a), but the second plurality of molecules containing compatible chemical groups to the chemical groups of the molecules of (a) to allow for conjugation; (c) contacting the molecules of (a) and (b) under conditions that allow for conjugation, thereby allowing for the generation of a combinatorial library.

[0023] The present invention also provides a method of conjugating an antibody to a desired moiety, comprising the steps of: providing a molecule of the invention, wherein the sialylated sugar of the molecule comprises a chemical group that can be conjugated to a compatible chemical group of a desired moiety, the molecule being either an antibody or a component part of an antibody; conjugating the desired moiety to the molecule via the chemical group; and if the molecule is a component part of an antibody rather than the antibody itself, the method further comprises assembling the whole antibody.

[0024] The present invention further provides a method of labeling a molecule, the method comprising: providing a molecule of the invention, wherein the sialylated sugar of the molecule comprises a chemical group that can be conjugated to a desired label that comprises a compatible chemical group that allows for conjugation; providing a label having a compatibility group that allows for conjugation of the molecule of (a) to the chemical group; contacting the molecule of (a) with the label of (b) under conditions suitable to result in a conjugate of the two.

[0025] The present invention also provides for the use of a molecule of the present invention as a capture agent for a desired moiety, wherein the sialylated sugar of the molecule comprises a chemical group that can be conjugated to the desired moiety, and the desired moiety comprises a compatible conjugation group for conjugation.

[0026] The present invention also provides a cell encoding a polypeptide comprising one or more copies of the following glycosylated amino acid sequence: X1Thr Pro X2X3 where: X1, X2, and X3 are any amino acids; The threonine (T) amino acid residue at the second position is O-glycosylated with a sialylated sugar.

[0027] The present invention also provides cells encoding the molecules of the present invention. [Brief explanation of the drawings]

[0028] [Figure 1] 1 shows the structures of potential O-glycosyl saccharides with potential sialylation and click chemistry groups. [Figure 2(a)] 1 shows the structures of potential O-glycosyl saccharides with potential sialylation and click chemistry groups. [Figure 2(b)] 1 shows the structures of potential O-glycosyl saccharides with potential sialylation and click chemistry groups. [Figure 2(c)] 1 shows the structures of potential O-glycosyl saccharides with potential sialylation and click chemistry groups. [Figure 3(a)]Exemplary protein mass spectrometry results for specific glycosylation sequences (without GNE knockout-3(a) and with GNE knockout-3(b)) are shown, with the peaks for antibody molecules bearing O-glycosyl saccharide chains with sialylation and click chemistry groups representing the highest peaks observed in each case. [Figure 3(b)] Exemplary protein mass spectrometry results for specific glycosylation sequences (without GNE knockout-3(a) and with GNE knockout-3(b)) are shown, with the peaks for antibody molecules bearing O-glycosyl saccharide chains with sialylation and click chemistry groups representing the highest peaks observed in each case. [Figure 4] The percentages of site occupancy, amounts of clickable glycans, unconjugated glycans, conjugation yield with DBCO, and final conjugate homogeneity are shown. [Figure 5(a)] The viable cell density (VCD) of cultures with an initial viable cell density >10 x 106 / mL is shown. [Figure 5(b)] In Example 4, the viable cell densities of cultures with initial viable cell densities >10 x 106 / mL are shown. [Figure 6] In Example 4, the productivity of molecule #1 is shown for cultures with initial viable cell densities >10 x 10 / mL. [Figure 7(a)] In Figure 7(a) growth of the culture is shown with an initial viable cell density of approximately 1 x 106 / mL. [Figure 7(b)] In Example 4, the viability of cultures with initial viable cell densities of approximately 1 x 106 / mL is shown. [Figure 8] In Example 4, the productivity of molecule #1 is shown for cultures with an initial viable cell density of approximately 1×10 6 / mL. [Figure 9] 1 shows the protein mass spectrometry results for the sugar titration performed in Example 4. [Figure 10]The product distribution obtained in the sugar titration performed in Example 4 is shown, along with the percentage of non-sialylated, sialylated, and sialylated molecules on the click sugar. [Figure 11] For molecule #2 of Example 5, cell growth of cultures with initial viable cell densities of approximately 10 x 106 / mL is shown. [Figure 12] For molecule #2 of Example 5, viability is shown for cultures with an initial viable cell density of approximately 10 x 106 / mL. [Figure 13] For molecule #2 of Example 5, productivity of cultures with initial viable cell densities of approximately 10 x 10 / mL is shown. [Figure 14] For molecule #2 of Example 5, product distribution for cultures with initial viable cell densities of approximately 10 x 10 / mL is shown. [Figure 15] 1 shows a CE-MS summary of a day 9 sample showing the incorporation of azidosialic acid into molecule #2 of Example 5. [Figure 16] We demonstrate the basic approach of how antibodies with provided glycosylation sequences can be conjugated to siRNA via the existing sialylated O-glycosyl sugar chains using click chemistry sugars, either with or without linkers. [Figure 17] To assess the effect of antibody concentration on conjugation, exemplary chromatograms are shown for the results of siRNA conjugation to azidosialic acid sugars with incorporated antibody, monitored by analytical anion exchange (aAEX) over time as a function of antibody concentration at 1 mg / mL, 5 mg / mL, and 10 mg / mL. Based on the % peak area from the aAEX chromatograms, the drug antibody ratio (DAR) was calculated. [Figure 18(a)] The results obtained for conjugation kinetics are shown (18(a) - 10 mg / mL, 18(b) - 5 mg / mL, and 18(c) - 1 mg / mL mAb). [Figure 18(b)]The results obtained for conjugation kinetics are shown (18(a) - 10 mg / mL, 18(b) - 5 mg / mL, and 18(c) - 1 mg / mL mAb). [Figure 18(c)] The results obtained for conjugation kinetics are shown (18(a) - 10 mg / mL, 18(b) - 5 mg / mL, and 18(c) - 1 mg / mL mAb). [Figure 19] 1 shows the conjugate profile obtained in Example 7 analyzed by analytical anion exchange. [Figure 20] 1 shows the stability of the siRNA conjugate produced in Example 7 in cynomolgus monkey plasma and mouse plasma. [Figure 21] 1 shows the target gene knockdown ability of mouse TfR-binding antibody-siRNA conjugates using either eCys or glyco-mAb conjugation chemistry (e.g., mTfR2-dsRNA No. 8 conjugate) in Example 7. [Figure 22] 1 shows the aAEX profile of the siRNA conjugate described in Example 7. [Figure 23(a)] 1 shows the effect of siRNA:antibody conjugates on gene expression studied in Example 7. [Figure 23(b)] 1 shows the effect of siRNA:antibody conjugates on gene expression studied in Example 7. [Figure 24(a)] 1 shows the effect of siRNA:antibody conjugates on gene expression studied in Example 7. [Figure 24(b)] 1 shows the effect of siRNA:antibody conjugates on gene expression studied in Example 7. [Figure 25(a)] 1 shows the effect of siRNA:antibody conjugates on gene expression studied in Example 7. [Figure 25(b)] 1 shows the effect of siRNA:antibody conjugates on gene expression studied in Example 7. [Figure 26]This study demonstrates the ability of the siRNA:Ab conjugates of the present invention to cross the blood-brain barrier (BBB) ​​via the antibody portion of the conjugate and then knock down target gene expression via siRNA. Results are also provided for an isotype control conjugate that does not target the TfR protein, which is required for antibody transport across the BBB. [Figure 27] 1 illustrates the use of the conjugation methods provided to generate multifunctional antibodies and combinatorial libraries. [Figure 28(a)] 1 illustrates the generation and use of preferred linkers of the present invention. [Figure 28(b)] 1 illustrates the generation and use of preferred linkers of the present invention. [Figure 28(c)] 1 illustrates the generation and use of preferred linkers of the present invention. [Figure 29] 1 illustrates the generation and use of preferred linkers of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0029] definition A number of definitions are provided in this section and elsewhere in this application. If a term is not defined, explained, or illustrated herein, it typically has the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, specific terms cited herein have the meanings ascribed to them herein.

[0030] As used herein, the use of singular forms such as "a," "an," and "the" includes plural referents unless the context clearly dictates otherwise.

[0031] When terms such as "comprise," "comprising," and the like are used herein, the present invention also includes embodiments that "consist essentially of" and "consist of" what is recited. Thus, when embodiments are described using "comprising" or similar language, embodiments consisting of what is recited are also specifically disclosed.

[0032] The term "specific for," as used herein with respect to a binding domain, refers to the ability of a binding domain of a multispecific binding molecule to bind, associate with, and / or modulate a particular target molecule above background levels of non-target levels. In some embodiments, "specific for" may be demonstrated by an affinity (Kd) measure for the target over a non-target. When two antigen-binding sites have "different" specificities, they typically bind either two different antigens or two different epitopes of the same antigen.

[0033] As used herein, the terms "treatment," "treat," and "treating" include inhibiting, slowing, halting, controlling, delaying, or reversing the progression or severity of an existing condition or disorder, or ameliorating an existing condition or disorder, but do not necessarily indicate the complete elimination of an existing condition or disorder. Treatment includes the administration of a protein or nucleic acid or vector or composition for the treatment of a condition or disorder to a patient, particularly a human.

[0034] A "molecule" of the present invention comprises at least one glycosylated amino acid sequence of the present invention. The glycosylated amino acid sequence is O-glycosylated. The glycosylation may further comprise a chemical group for conjugation. If such a chemical group is present, it may already be conjugated to a desired moiety or may be capable of such conjugation. If such conjugation has already occurred, the molecule may also be said to comprise a conjugated desired moiety. A molecule may be or comprise a peptide, polypeptide, or protein comprising at least one glycosylated amino acid sequence of the present invention. Preferred molecules are polypeptides. However, a molecule is not limited to being a single polypeptide. For example, an IgG antibody comprises four polypeptide chains and represents an IgG molecule. A molecule may itself comprise smaller molecules as constituent parts. Thus, the term "molecule" in this application encompasses higher-order structures, e.g., polypeptides and / or other moieties joined together, e.g., via the present invention.

[0035] A "polypeptide" is a linear polymer of amino acids joined by amide bonds, particularly peptide bonds. Since the glycosylated amino acid sequences of the present invention are at least 5 amino acids in length, the polypeptides of the present invention are at least that length. The term "peptide" refers to short polypeptides, e.g., 5 to 50 amino acids, e.g., 10 to 50 amino acids. As used herein, the term "polypeptide" encompasses "peptide" sequences. However, it can refer to longer sequences, e.g., sequences at least 50 amino acids in length.

[0036] As used herein, "protein" includes one or more covalently or non-covalently associated polypeptides. Proteins typically have more defined tertiary and quaternary structures and may have homogeneous or heterogeneous post-translational modifications. Examples of proteins include, but are not limited to, antibodies, enzymes, and cytokines. A molecule of the present invention may be or include a protein comprising one or more glycosylated amino acid sequences of the present invention.

[0037] A "binding molecule" can be considered to be any molecule that binds, preferably specifically binds, to a target. Particularly preferred examples of binding molecules include antibodies, but this term is not limited to antibodies and includes any polypeptide, protein, or nucleic acid that specifically binds to a target. Binding molecules can be protein-binding molecules. They can also be nucleic acid-binding molecules. Examples of the latter include antisense nucleic acid molecules, siRNA molecules, and aptamers.

[0038] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0039] Embodiments of the invention, as used herein, also include antibody fragments or antigen-binding fragments comprising at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as a Fab, Fab', F(ab'), Fv fragment, scFv antibody fragment, scFab, disulfide-linked Fv (sdFv), Fd fragment, etc. It includes, for example, linear antibodies that may be fused to an Fc region or an IgG heavy chain constant region, e.g., scFv-CH3 minibody, scFv-Fc antibody, scFv-zipper antibody, Fab2 bispecific, bis-scFv, sdAb, tetrabody, triabody, diabody, or Fab3 triabody.

[0040] The term "antibody" includes single-chain antibody formats. It includes heavy chain-only antibodies such as VH and VHH domain antibodies. It also includes other types of antigen-binding molecules, such as antibody analogs such as DARPins (designed ankyrin repeat proteins). It also includes artificially constructed antibody formats, such as naturally occurring antibody format molecules, but with additional antigen-binding sites added, such as at the C-terminus of the light and / or heavy chain.

[0041] As used herein, the term "antigen-binding domain" refers to a portion of a binding molecule, antibody, antibody fragment, bispecific antibody, multispecific binding protein, etc. that binds to an antigen or an epitope of an antigen.

[0042] As used herein, the term "bispecific" refers to a molecule that contains two different antigen-binding domains. A bispecific binding molecule can bind to two different antigens or two different epitopes of the same antigen. Exemplary embodiments of bispecific molecules include the bispecific antibodies disclosed herein.

[0043] As used herein, the term "multispecific" refers to a molecule comprising two or more different antigen-binding domains. A multispecific binding molecule can bind to two or more different antigens or two or more different epitopes of the same antigen. Exemplary embodiments of multispecific binding molecules include bispecific, trispecific, or tetraspecific binding molecules known in the art, and single-chain multispecific binding molecules, such as diabodies, tandem scFvs, tandem VHHs, or tandem scFabs.

[0044] As used herein, the term "agonize" refers to the ability of an antibody, antibody fragment, or binding molecule to induce or increase one or more activities or functions associated with an antigen. In one embodiment, a molecule of the invention may be or include an agonist.

[0045] As used herein, the term "antagonize" refers to the ability of a molecule, antibody, antibody fragment, or binding molecule to reduce or eliminate one or more activities or functions associated with an antigen. In one embodiment, a molecule of the invention may be or include an antagonist.

[0046] As used herein, the term "neutralizing" refers to the ability of a molecule, antibody, antibody fragment, or binding molecule to oppose, inactivate, or neutralize at least one activity or function of an antigen or other target.

[0047] As used herein, the terms "bind" and "binds," unless otherwise specified, are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in proximity of the two proteins or molecules, as determined by common methods known in the art. The term "conjugation" involves the joining of a molecule to a desired moiety via a chemical bond.

[0048] As referred to herein, the term "epitope" refers to the amino acid residues of an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a hybrid epitope.

[0049] The term "structural epitope" can be used to describe the region of an antigen that is covered by an antibody (e.g., the footprint of the antibody when bound to the antigen). In some embodiments, a structural epitope can describe amino acid residues of an antigen that are within a particular proximity (e.g., within a particular number of angstroms) of amino acid residues of an antibody.

[0050] The term "functional epitope" can also be used to describe the amino acid residues of an antigen that interact with the amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody.

[0051] Epitopes can be determined according to different experimental techniques, also known as "epitope mapping techniques." It is understood that the determination of epitopes may vary based on the different epitope mapping techniques used, and may also vary depending on the different experimental conditions used, for example, due to the conformational changes or cleavage of the antigen induced by certain experimental conditions. Epitope mapping techniques are known in the art, including, but not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species swap mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd ed. 2018).

[0052] As used herein, the terms "compete for binding" or "compete with" refer to two antibodies that cross-compete (i.e., compete with each other) for binding to the same antigen. In some embodiments, two antibodies may compete for binding to the same antigen if they bind to spatially overlapping regions of the same antigen. In some embodiments, two antibodies may compete for binding to the same antigen, where the antibodies bind to non-overlapping regions of the antigen, but the binding of one antibody blocks binding by the other antibody, for example, due to steric hindrance or conformational changes in the antigen induced by the first antibody.

[0053] As used herein, the term "paratope" refers to the amino acid residues of an antigen that bind to the antigen. The amino acid residues of the paratope can be identified based on their proximity (e.g., within a certain number of angstroms) to the amino acid residues of the antigen, as can be determined, for example, by X-ray crystallography. The amino acid residues of the paratope can also be identified based on their contribution to the binding energy between the antigen and the antibody. For example, such amino acid residues of the paratope can be determined by examining protein binding in a functional binding assay of the antibody to the antigen when the antibody is mutated at different sites in the paratope.

[0054] "Specifically binds" refers to binding preferentially to a target over a non-target. The binding is to the target, but may not be to a significant degree to the non-target. The affinity of binding may be at least 5, 10, 50, 100, 1000, or more times greater for the target than for the non-target.

[0055] A "monoclonal antibody" is an antibody produced by a single clone of cells or cell line and consisting of identical antibody molecules.

[0056] A "chemical group for conjugation" refers to a chemical group that can be conjugated to a complementary chemical group, for example, but not limited to, by click chemistry.

[0057] As used herein, "glycosylated amino acid sequence" refers to the amino acid sequence X1ThrProX2X3, where X1, X1, and X1 can be any amino acid and the sequence can be glycosylated at a Thr residue. Such a sequence is referred to as a glycosylated amino acid sequence or glycosylation sequence of the present invention. The introduced O-glycan typically contains a sialic acid sugar, which may also contain a group that allows conjugation to a desired moiety. Particularly preferred is when the O-glycan contains a click chemistry group and the molecule desired to be conjugated also contains a compatible click chemistry group. The two click chemistry groups can react to conjugate the polypeptide and the desired moiety together.

[0058] As used herein, a "moiety" is any entity that can be conjugated to form part of a molecule of the invention. A moiety may itself be a molecule, but can be conjugated to form a larger molecule. Thus, for example, a molecule of the invention typically contains at least one glycosylation sequence that allows for conjugation to a moiety to form a larger, yet still representative, molecule of the invention. The moiety that is desired to be conjugated contains a complementary group for reaction with a conjugating sialic acid analog that contains a chemical group for conjugation. A "molecule" encompasses molecules that themselves contain component molecules joined or associated together; for example, the term molecule includes molecules such as IgG molecules, which typically contain four polypeptide chains.

[0059] The numbering of amino acid residues in the antibody sequences described herein is based on the EU index as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: US Department of Health and Human Services, Public Health Service, National Institutes of Health (1991)). The terms EU index numbering or EU numbering are used interchangeably herein. The assignment of amino acid residues to CDRs may be made according to any well-known scheme, including those described in Kabat, Chothia, North, or IMGT.

[0060] Molecular and glycosylation sequences The glycosylation sequence of the invention comprises the amino acid sequence X1ThrProX2X3. In particular, the invention provides molecules comprising a glycosylation sequence comprising the amino acid sequence: X1ThrProX2X3, where (i) X1, X2, and X3 are any amino acid, and (ii) the threonine (T) amino acid residue is O-glycosylated with a sialylated sugar. In particularly preferred embodiments, the sialylated sugar comprises a chemical group that can be conjugated to a desired moiety. In even more particularly preferred embodiments, the sialylated sugar comprises a chemical group that is actually conjugated to a desired moiety. As used herein, "glycosylation sequence" refers to such an amino acid sequence, unless otherwise specified.

[0061] The threonine in the amino acid sequence X1Thr Pro X2X3 in the glycosylation sequence of the present invention is glycosylated. In another aspect, the molecule may be glycosylated at other sites or in other ways, but minimally contains one or more glycosylation sequences of the present invention. In one embodiment, the only glycosylation of the molecule of the present invention is one that contains one or more glycosylation sequences of the present invention. In another embodiment, it is not the only glycosylation.

[0062] In one embodiment, in the glycosylation sequence of the present invention, (i) X1 is Pro, (ii) X3 is Pro, (iii) X1 and X3 are both Pro, (iv) X2 is Ala, (iv) one or both of X1 and X3 are Pro and X2 is Ala, or (v) X1 and X3 are both Pro and X2 is Ala. In one embodiment, the glycosylation sequence comprises the amino acid sequence X1Thr Pro X2X3, wherein at least one of X1 and X3 is Pro. In one embodiment, both X1 and X3 are Pro. In a preferred embodiment, X2 is Ala. In one embodiment, X1 is Pro and X2 is Ala. In another embodiment, X2 is Ala and X3 is Pro. In another preferred embodiment, X1 and X3 are Pro and X2 is Ala.

[0063] In particularly preferred embodiments, the glycosylation sequence comprises the amino acid sequence Pro Thr Pro Ala Pro (SEQ ID NO: 1). In any of the embodiments described herein, the glycosylation sequence may be, for example, any of the glycosylation sequences of the invention described herein. However, in particularly preferred embodiments, one or more of the glycosylation sequences in any of the embodiments described herein comprises the amino acid sequence Pro Thr Pro Ala Pro.

[0064] In a preferred embodiment, any of the above glycosylation sequences may be flanked on the N-terminus by an alanine (Ala) residue. In a preferred embodiment, any of the above glycosylation sequences may be flanked on the C-terminus by an Ala residue.

[0065] In a preferred embodiment, any of the above glycosylation sequences may be flanked on the N- and C-termini by Ala residues. In a preferred embodiment, the glycosylation sequence comprises or consists of Ala Pro Thr Pro Ala Pro Ala. In a preferred embodiment, the glycosylation sequence comprises or consists of Ala Ala Pro Thr Pro Ala Pro. In a preferred embodiment, the glycosylation sequence comprises or consists of Ala Ala Pro Thr Pro Ala Pro Ala.

[0066] In another preferred embodiment, the glycosylation sequence comprises or consists of Ala Ala Ala Thr Pro Ala Pro (SEQ ID NO: 2).

[0067] The glycosylated amino acid sequences of the present invention are typically present in one or more polypeptides that form part of the molecules of the present invention. In one embodiment, the molecules of the present invention comprise a polypeptide comprising at least one glycosylated amino acid sequence of the present invention. In one embodiment, the molecules of the present invention are such polypeptides. In one embodiment, the molecules of the present invention comprise or consist of a polypeptide that is glycosylated and comprises a glycosylated amino acid sequence that comprises a chemical group for conjugation.

[0068] Preferably, the glycosylation sequence of the present invention is O-glycosylated at the threonine residue of the glycosylation sequence. The present invention also provides molecules before glycosylation, and such intermediates form part of the present invention. In a preferred embodiment, the O-glycan comprises a sialic acid group. In a further preferred embodiment, the sialylated sugar comprises a chemical group that allows for conjugation to a desired moiety. As discussed further below, in a particularly preferred embodiment, the sialic acid group comprises a click chemistry group that allows for conjugation of the click chemistry group with a second, compatible click chemistry group on the moiety to which it is desired to conjugate.

[0069] One advantage of the present invention is that a glycosylation sequence can be easily introduced into the amino acid sequence of a given polypeptide at a desired position to which a moiety is to be conjugated. This means that the present invention can be applied to any desired molecule containing an amino acid sequence that can be modified to become a glycosylated amino acid sequence of the present invention. The amino acid sequence may be modified by altering an existing amino acid sequence to include a glycosylation sequence or by inserting a glycosylation sequence into the amino acid sequence. In another embodiment, a region of an amino acid sequence can be replaced with a glycosylation sequence of the present invention; for example, a region of the same length can be replaced with a glycosylation sequence of the present invention.

[0070] In one embodiment, a molecule of the invention comprises at least one glycosylation sequence of the invention. In one embodiment, a molecule of the invention comprises only one glycosylation sequence of the invention. In another embodiment, it comprises at least two such glycosylation sequences. In another embodiment, it comprises only two such glycosylation sequences. In one embodiment, a molecule of the invention comprises at least four glycosylation sequences. In one embodiment, it comprises only four such glycosylation sequences. In one embodiment, a molecule of the invention comprises 1, 2, 3, 4, 5, 6, 7, or more glycosylation sequences of the invention. In another embodiment, it comprises 1 to 7, e.g., 2 to 6, e.g., 2, such glycosylation sequences. In one embodiment, a molecule comprises an even number of such glycosylation sequences. In one embodiment, it comprises 2, 4, 6, 8, or 10 glycosylation sequences. In another, it comprises at least such number, or a range formed by such numbers, e.g., 2 to 10 such sequences. The presence of multiple glycosylation sequences can be used to generate molecules containing higher-order structures, such as dimers or multimers, by using O-glycosylation to join polypeptides. In one embodiment, two glycosylation amino acid sequences of the invention are present in a molecule of the invention, and the two are conjugated to each other. In another embodiment, a molecule of the invention comprises at least one such conjugated pair of glycosylation sequences. In one embodiment, it comprises 2, 3, 4, 5, or 6 such pairs of conjugated amino acid sequences of the invention.

[0071] In one embodiment, two or more glycosylation amino acid sequences may be present in a molecule of the present invention. The presence of O-glycans near the glycosylation site of the present invention can help promote O-glycosylation. Thus, in one embodiment, a molecule of the present invention may contain two or more glycosylation sequences of the present invention within 100 amino acids of each other, for example, within 75 amino acids of each other, preferably within 50 amino acids of each other, in the same polypeptide. In one embodiment, the glycosylation sequences of the present invention are present at regular intervals in the amino acid sequence present in a molecule of the present invention, for example, to enable conjugation to multiple moieties or to form multiple bridges between two molecules, thereby strengthening their binding to each other.

[0072] The present invention can be used to conjugate a desired moiety to a given protein to form a desired molecule of the present invention. In another embodiment, the present invention can be used to form a bridge between two polypeptides present in the molecule, e.g., to help stabilize the entire molecule. In one embodiment, the present invention is used to form a molecule that is or includes a "locked" polypeptide, in which the conjugated polypeptides are covalently linked via O-glycosyl bridges and therefore do not readily dissociate. In one embodiment, the present invention is used to form a molecule that is or includes a locked dimer or multimer, in which individual monomers are covalently linked via glycosyl bridges. The present invention can also be used to generate ligands that become covalently bound to a receptor upon binding to the receptor via the conjugation approach of the present invention, thereby forming a molecule of the present invention that is a complex of the covalently linked ligand and receptor.

[0073] The molecules of the present invention may be or comprise any suitable polypeptide. The molecules may be or comprise therapeutic proteins. In a preferred embodiment, the molecules of the present invention are or comprise cytokines. Accordingly, at least one polypeptide chain of the cytokine may comprise one or more glycosylation sequences of the present invention. In one embodiment, all polypeptide chains of the cytokine may do so. In one embodiment, the cytokine is an interleukin. In another embodiment, the cytokine is an interferon. Examples of cytokines to which the present invention can be applied include TNF-α, IL-1, IL-10, IL-12, INF-α, or INF-γ. In another embodiment, the cytokine is IL-2, IL-4, IL-5, TGF-β, or INF-λ. In a further embodiment, the cytokine is a colony-stimulating factor (CSF). In one embodiment, the cytokine is GM-CSF. Other preferred proteins include, for example, growth factors, hormones, blood clotting factors, tumor necrosis factors, interferons, and cytokines. The protein may be or include EPO (erythropoietin) or an EPO analogue. The protein may be an enzyme.

[0074] In one particularly preferred embodiment, the molecule of the invention may comprise or be an antibody, as discussed further below.

[0075] In preferred embodiments, the molecules of the invention may be or include vertebrate proteins. The proteins may be, for example, mammalian proteins. In particularly preferred embodiments, the proteins may be human proteins. The proteins may be animal proteins, such as mouse, rat, or monkey proteins. The proteins may be bovine, ovine, canine, or feline proteins.

[0076] In one embodiment, the molecules of the present invention can be secreted. In a preferred embodiment, the polypeptides of the present invention having one or more glycosylation sequences also contain a secretion signal. In another embodiment, the polypeptides contain a signal that allows them to be displayed on the cell surface.

[0077] In one embodiment, the present invention can be applied to dimers or multimers, for example, to form covalent crosslinks from one subunit of a dimer or multimer to another subunit. The present invention can be used to form covalent crosslinks that join at least two of the individual subunits of a multimer together. In one embodiment, the present invention can be used to form locked dimers or multimers, in which at least two subunits are joined together via a glycosylation sequence(s) of the present invention to form a crosslink. In one embodiment, cytokine dimers can be generated in which each cytokine subunit contains at least one glycosylation sequence of the present invention that allows for the addition of an O-glycan sugar chain and subsequent conjugation of one cytokine monomer to another. Such an approach can be used, for example, to form locked cytokine dimers. The generation of such cytokine dimers can be used, for example, to generate low-affinity cytokine dimers.

[0078] In an alternative embodiment of the invention, a molecule of the invention comprises one or more glycosylation sequences of the invention that are O-glycosylated with sialylation terminating the O-glycan sugar, but the O-glycan sugar does not comprise a group for conjugation. In a preferred embodiment, the presence of O-glycosylation is used to modify a property of the molecule, such as a physical property of the molecule. In another embodiment, O-glycosylation is used to promote immune tolerance. In another embodiment, O-glycosylation is used to mask another site in the polypeptide. In a further alternative embodiment, the invention further provides a molecule in which one or more glycosylation sequences of the invention are O-glycosylated but not sialylated.

[0079] In a particularly preferred embodiment, a molecule of the invention comprises a polypeptide comprising a glycosylation sequence, wherein the polypeptide is at least 10 amino acids in length. In another preferred embodiment, the polypeptide is at least 20 amino acids in length. In another embodiment, the polypeptide is at least 50 amino acids in length. In an even more preferred embodiment, the polypeptide is at least 100 amino acids in length.

[0080] In a further particularly preferred embodiment, the molecule of the present invention comprising a glycosylated amino acid sequence is present in a cell. In another embodiment, it is expressed in a cell. In a preferred embodiment, it is present in or expressed in a mammalian cell. Examples of preferred cells include human cells. They also include rodent cells, for example, CHO cells are particularly preferred.

[0081] Glycan Preferably, the threonine residue of the glycosylation sequence in the molecule of the present invention is O-glycosylated. Thus, a sugar chain is attached to the threonine. In a particularly preferred embodiment, the glycosylation sequence of the present invention is O-glycosylated with a sugar chain comprising an N-acetylhexosamine and a hexose. In a preferred embodiment, the O-glycan sugar chain is a glycan comprising an N-acetylhexosamine and a hexose. In a particularly preferred embodiment, the O-glycosylation comprises a sialic acid group. Typically, the sialylation terminates the O-glycan sugar chain with either a group that forms part of the sialic acid group or a group for conjugation attached thereto. In a preferred embodiment, the O-glycan sugar chain comprises or is a glycan having at least one sialic acid at its terminus. In one embodiment, the glycan may have one or two sialic acids.

[0082] In one particularly preferred embodiment, the O-glycosylation of threonine comprises a threonine-N-acetylhexosamine-hexose-sialic acid-conjugation group.

[0083] In a preferred embodiment, at least two glycosylation sequences are so glycosylated, where the conjugation groups are compatible with each other, and then conjugated together in a molecule of the invention via compatible conjugation groups. In another embodiment, one glycosylation sequence is so glycosylated and then conjugated to a moiety having a suitable compatible conjugation group, but no moiety requiring or having an O-glycosyl sugar chain, because the presence of a glycan on the moiety desired to be conjugated is not necessary for the conjugation reaction to occur in such an embodiment. In a preferred embodiment, both the polypeptide and the moiety to which the polypeptide is conjugated comprise at least one glycosylation sequence of the invention, such that both are O-glycosylated with a sialylated sugar chain having a compatible group for conjugation, allowing the polypeptide and the moiety to be conjugated to each other and thereby covalently bonded together. In one embodiment, the desired moiety for conjugation is or comprises a linker that can be conjugated to a chemical group of a sialylated sugar chain on a glycosylated amino acid sequence of the invention. In one embodiment, a molecule of the invention comprises a glycosylated amino acid sequence so conjugated to a linker. In another embodiment, a molecule of the invention comprises a glycosylated amino acid sequence conjugated to a desired moiety that comprises a linker that is itself conjugated to a second molecule that forms a further part of the desired moiety.

[0084] An advantage of the present invention may include that for molecules comprising glycosylation sequences of the present invention, a very high percentage of molecules having a glycosylation sequence are typically glycosylated. This is sometimes referred to as "site occupancy." In one embodiment, the percentage of glycosylation sites of the present invention that are glycosylated is at least 60%, at least 70%, preferably at least 80%, and more preferably at least 90%. In one embodiment, the percentage that is glycosylated is at least 95%. In another embodiment, this percentage is at least 99%. A further advantage of the present invention is that the glycan in each molecule for O-glycosylation is typically the same. Such consistency is advantageous for drug production. In one embodiment, at least 60%, at least 70%, preferably at least 80%, and more preferably at least 90% of the molecules in a sample of molecules of the present invention have the same glycan in the glycosylation sequence(s) of the present invention present in the molecule. In another embodiment, at least 95% of the glycans are the same. In another embodiment, at least 99% are the same.

[0085] In one embodiment, the present invention can be used to stabilize molecules. For example, in one embodiment, one or more pairs of glycosylated amino acid sequences of the present invention are conjugated to each other to stabilize molecules. In another embodiment, the present invention can be used, for example, to prevent dissociation between two polypeptides.

[0086] Conjugation Groups and Conjugation In a particularly preferred embodiment, the O-glycosyl sugar chain in the glycosylation sequence of the present invention comprises a chemical group that acts as a conjugation group. The first conjugation group can be conjugated to a compatible second conjugation group, allowing for a means of attaching a desired moiety to the polypeptide. The conjugation is typically a covalent bond. Typically, the first conjugation group and the second conjugation group are not identical, but they can be conjugated to each other. The conjugation chemistry used is typically bioorthogonal. Therefore, preferably, the conjugation can be performed under physiological conditions. In one embodiment, the conjugation can be performed without the need for an external catalyst, for example, without the need for the addition of exogenous copper. In one embodiment, the conjugation can be performed intracellularly without the need for the addition of any additional reagents to effect the conjugation.

[0087] A particularly preferred means for conjugation is click chemistry. As used herein, the term "click chemistry group" or "click chemistry handle" refers to a reactant or reactive group that can participate in a click chemistry reaction. A click reactive group can be a moiety that is rarely or never found in naturally occurring biomolecules and is chemically inert toward biomolecules. In one embodiment, the click chemistry group is an azide-reactive group or an alkyne-reactive group. Such groups can react efficiently under biologically relevant conditions, such as cell culture conditions, without the need for excessive heat or harsh reactants. In one embodiment, the click chemistry reaction is performed intracellularly. In one embodiment, it is performed in vitro. In another embodiment, it is performed ex vivo. In another embodiment, the conjugation is performed in vivo. In another embodiment, it can be performed ex vivo. In one embodiment, the invention includes a transgenic animal encoding and expressing a molecule of the invention. In one embodiment, the animal is any of the animals described herein. In one embodiment, the entities to be conjugated are simply mixed, e.g., in isolated form.

[0088] Generally, click chemistry reactions require at least two molecules containing click reaction partners capable of reacting with each other. Such mutually reactive click reaction partners are sometimes referred to as click chemistry handle pairs or click chemistry pairs. In some embodiments, the click reaction partners are a reactive alkene or alkyne and a suitable tetrazine. For example, trans-cyclooctene, norbornene, or bicyclononyne can be paired with a suitable tetrazine as a click reaction pair. In other embodiments, tetrazole can act as a potential source of nitrile imine, which can pair with an unactivated alkene in the presence of ultraviolet light to generate a click reaction pair referred to as a "photoclick" reaction pair. In some embodiments, the click reaction partners are an azide and an alkyne, particularly a strained alkyne, such as cyclooctyne, or any other alkyne.

[0089] Other suitable click chemistry handles are known to those skilled in the art (see, for example, Spicer et al., 2014, Nature Communications, 5:4740). In other embodiments, the click reaction partners are Staudinger ligation components such as a phosphine and an azide. In other embodiments, the click reaction partners are Diels-Alder reaction components such as a diene, e.g., a tetrazine, and an alkene, e.g., trans-cyclooctene (TCO) or norbornene. Exemplary click reaction partners are described, for example, in U.S. Patent Publication No. 2013 / 0266512 and International Publication No. WO 2015 / 073746 (both of which are incorporated by reference in their entireties), and are specifically described with respect to the associated descriptions of click reaction partners therein, both of which are incorporated by reference herein. According to a preferred embodiment, one of the first and second click reaction partners comprises an alkyne group, and the other click reaction partner comprises an azide. According to another preferred embodiment, one of the first and second Click reaction partners comprises an alkene group and the other Click reaction partner comprises a diene.

[0090] As used herein, the terms "alkyne," "alkyne group," or "alkyne moiety" refer to a functional group containing a carbon-carbon triple bond. Alkyne moieties include terminal alkynes and cyclic alkynes, preferably terminal alkynes and cyclic alkynes that are reactive with azide groups. Terminal alkynes have at least one hydrogen atom attached to a triple-bonded carbon atom. Cyclic alkynes are cycloalkyl rings containing one or more triple bonds. Examples of cyclic alkynes include, but are not limited to, cyclooctyne and cyclooctyne derivatives such as bicyclononyne (BCN), difluorinated cyclooctyne (DIFO), dibenzocyclooctyne (DIBO / DBCO), keto-DIBO, biarylazacyclooctynone (BARAC), dibenzoazacyclooctyne (DIBAC), dimethoxyazacyclooctyne (DIMAC), difluorobenzocyclooctyne (DIFBO), monobenzocyclooctyne (MOBO), and tetramethoxy DIBO (TMDIBO).

[0091] In a particularly preferred embodiment, one of the first and second click reaction partners comprises a cyclic alkyne, preferably DBCO. DBCO is a particularly preferred conjugation group. According to a preferred embodiment, the other click reaction partner comprises an azide. Thus, a particularly preferred "click pair" is DBCO with an azide.

[0092] As used herein, the term "diene" refers to a compound having two carbon-carbon double bonds, which are conjugated at the 1,3 position. The double bonds of a diene can be either cis or trans. Examples of dienes include, but are not limited to, tetrazine or tetrazole groups.

[0093] As used herein, the terms "alkene," "alkene group," or "alkene moiety" refer to an unsaturated hydrocarbon molecule containing a carbon-carbon double bond. In one embodiment, an alkene can contain 2 to 100 carbon atoms. Examples of alkenes include, but are not limited to, norbornene and trans-cyclooctene (TCO). According to another preferred embodiment, one of the first and second click reaction partners contains an alkene group, preferably norbornene or TCO. According to a preferred embodiment, the other click reaction partner contains a diene, preferably a tetrazine or tetrazole group.

[0094] As used herein, a "proteinogenic amino acid" is an amino acid that is biosynthetically incorporated into proteins during translation, preferably the 20 genetically encoded (proteinogenic) amino acids in the standard genetic code and the additional two (selenocysteine ​​and pyrrolysine) that can be incorporated by specialized translational machinery.

[0095] As used herein, the term "covalently linked" means that a molecule is linked to a first Click functional group via at least one covalent bond, and that a conjugation partner is linked to a second Click functional group via at least one covalent bond. The linkage can be direct, i.e., without a linker, or indirect, i.e., via a linker.

[0096] One advantage of the present invention is that it typically allows the conjugated entities to retain their activity or not suffer a significant reduction in activity. In one embodiment, the location of the glycosylation site(s) is selected to help avoid any loss, or at least any significant loss, of activity in the entities conjugated to each other. Thus, preferably, the moiety desired to be conjugated retains activity after conjugation. For example, if the molecule is or comprises an antibody and is conjugated to the desired moiety using the present invention, the antibody typically retains its antigen-binding activity.

[0097] Linker In one embodiment, the moiety may be or may include a linker, such that the O-glycosyl sugar chain is conjugated to the linker as a moiety or as part of a moiety. The linker may then be conjugated to an additional moiety or may already be conjugated. Thus, in any of the embodiments described herein, a linker may be present that bridges the O-glycosyl sugar and the additional molecule, with the linker and the additional molecule representing the desired moiety. Alternatively, in other embodiments, such a linker may not be present. A linker may be used as a bridge between the O-glycosylated amino acid sequence of the present invention and the additional molecule. Thus, for example, a linker may be attached to a sialylated sugar, and the other end of the linker may be attached to a molecule of choice. Thus, any of the embodiments described herein may use such a linker.

[0098] An example of a preferred linker is or comprises DBCO.

[0099] Any suitable linker capable of forming a bond with an O-glycosylated sugar can be used. In a preferred embodiment, a linker containing a polyarginine sequence can be used, for example, a linker having 2 to 15, preferably 3 to 10, and more preferably 4 to 8 consecutive arginine residues. In a particularly preferred embodiment, the linker contains six consecutive arginine residues. In a particularly preferred embodiment, the polyarginine sequence is flanked on each side by lysine residues. In a particularly preferred embodiment, the sequence is acetylated at the N-terminus and amidated at the C-terminus. In a preferred embodiment, the linker contains the amino acid sequence Lys-Arg-Arg-Arg-Arg-Arg-Arg-Lys with an N-terminal acetyl group and C-terminal amidation, in other words, Ac-Lys-(Arg)6-Lys-NH2. In a particularly preferred embodiment, the linker has the structure shown in Figure 28(c). Thus, in one embodiment, the linker comprises a Lys-(Arg)-Lys sequence in which both lysine side chains are modified with PEG-DBCO, thus Ac-Lys(PEG-DBCO)-(Arg)-Lys(PEG-DBCO)-NH. In a preferred embodiment, the linker comprises or is that shown in Figure 28(c).

[0100] In a particularly preferred embodiment, two molecules of the invention are joined via their O-glycosylation sites via a linker, in particular the linkers described above, to form a larger molecule of the invention.

[0101] portion In preferred embodiments, the molecules of the invention comprise a glycosylation sequence of the invention having a sialylated O-glycosyl sugar chain conjugated to a desired moiety, which in some embodiments may be or include a linker.

[0102] Thus, the present invention can be used to conjugate any desired entity using one or more glycosylation sequences of the present invention that are O-glycosylated with a sialylated sugar chain containing a suitable conjugation group. The present invention can be used, for example, to conjugate polypeptides that themselves represent molecules of the present invention to each other to form larger molecules of the present invention. In one embodiment, polypeptide refers to a segment that, when conjugated to a polypeptide containing one or more glycosylation sequences of the present invention, can ultimately form part of a molecule of the present invention. For example, the ability to conjugate two polypeptides together can be used to conjugate polypeptide chains within a larger molecule. As discussed further below, the ability to easily conjugate two molecules via the present invention can be used in multiplexing to generate permutations of different molecules that are conjugated together and then screen them for desired properties. Thus, the present invention is a highly versatile method for conjugating molecules and desired moieties together. While specific moieties are described below, reference thereto should not be considered limiting; the specific moieties are identified merely as illustrative examples.

[0103] In one embodiment, the moiety is selected from Fc, PEG, a fluorophore, a radiotracer, a fatty acid, a glycan, a peptide, a nucleic acid, an enzyme, and a steroid.

[0104] In one embodiment, the molecule of the present invention is conjugated to a nucleic acid or comprises a nucleic acid via conjugation. In one embodiment, the nucleic acid is single-stranded. In another embodiment, it is double-stranded. In one embodiment, the nucleic acid is DNA. In another embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is an antisense nucleic acid. In one embodiment, the nucleic acid is antisense RNA. In a particularly preferred embodiment, the nucleic acid is siRNA. In a particularly preferred embodiment, the conjugate of the present invention comprises an antibody conjugated to a nucleic acid molecule via the present invention. In a preferred embodiment, the nucleic acid can inhibit the expression of a target gene. In a preferred embodiment, the present invention provides a conjugate that is a conjugate of an antibody and siRNA using the conjugation approach of the present invention. Thus, in such an embodiment, the antibody comprises a glycosylation sequence of the present invention that allows conjugation to a selected nucleic acid.

[0105] In one embodiment, the molecule may be or include a viral or microbial polypeptide. In one embodiment, a virus may be produced that contains one or more glycosylation sequences of the present invention in the viral capsid polypeptide. In another embodiment, the molecule of the present invention may be a molecule on the surface of a cell. In another embodiment, the molecule is on the surface of a pathogen. In one embodiment, the present invention may then be used to conjugate a desired molecule onto the surface of a virus, pathogen, or cell. The present invention may be used to combine different cells, where both cells have compatible conjugation groups, and at least one of the cells has such a group introduced through the use of a glycosylation sequence of the present invention. The present invention may also be used to help target a virus to a desired cell. The present invention may also be used to target cancer cells. In one embodiment, a molecule of the present invention may first be targeted to a desired cell, where the molecule has a compatible conjugation group, which then allows for conjugation to a target on the cell. In another embodiment, cells, viruses, liposomes, or other delivery vehicles can have sialylated O-glycosyl sugars of the present invention on their surface and then be used for conjugation to a desired targeting molecule. The targeting molecule can be selected for its specificity and can be exchanged for a different targeting molecule to change the specificity. In one embodiment, the targeting molecule is an antibody specific for the target cell. Thus, in a preferred embodiment, the molecule of the present invention can include a portion of a molecule that targets it to a specific target.

[0106] In another embodiment, the molecule may be or may comprise an antigen. In one embodiment, an antigen against which it is desired to elicit an immune response is modified to include one or more glycosylation sequences of the present invention, and the present invention is used to conjugate the antigen to a desired moiety. In one embodiment, the moiety is a carrier. In one embodiment, the present invention conjugates the antigen to a desired moiety such as diphtheria toxoid (DT), tetanus toxoid (TT), CRM, or other toxoids. 197, Haemophilus protein D (PD), or serogroup B meningococcus outer membrane protein complex (OMPC). The present invention also provides conjugate vaccines comprising an antigen and a protein carrier conjugated using the conjugation approach of the present invention. In one embodiment, a polypeptide comprising one or more glycosylation sequences of the present invention is the carrier, and the moiety conjugated to it using the present invention is a glycan. Thus, for example, the present invention provides a convenient method for conjugating a glycan to a polypeptide carrier. In another embodiment, the present invention can be used to conjugate alpha-gal to a polypeptide to help enhance the immune response to a polypeptide having one or more glycosylation sequences of the present invention. Thus, the present invention can be used to enhance vaccine efficacy.

[0107] In one embodiment, a molecule of the invention comprises one or more glycosylation sequences of the invention conjugated to a label. In one embodiment, the molecule comprises a fluorescent dye conjugated to a glycosylation sequence of the invention. In one embodiment, the label is a fluorescent protein. In another embodiment, the fluorescent protein is GFP. In one embodiment, the polypeptide sequence is conjugated to a dye using a glycosylation sequence of the invention. In another embodiment, the polypeptide is conjugated to biotin via O-glycosylation. In another embodiment, the polypeptide is conjugated to a steroid. In one embodiment, the steroid is a steroid hormone. In another embodiment, it is an androgen. In another embodiment, it is cortisone. In another embodiment, it is an anabolic steroid. In a further embodiment, the molecule is conjugated to a moiety that affects the stability and / or half-life of the molecule. For example, in one embodiment, the glycosylation amino acid sequence is conjugated to BSA, e.g., to alter the stability of the molecule in circulation. In one embodiment, the molecule is conjugated to PEG (polyethylene glycol) via a glycosylation sequence of the invention. The present invention can also be used to alter the isoelectric point (pi) of a molecule of the invention, for example, since the addition of sialic acid should result in a lower pi, which can be used, for example, to increase the half-life of a molecule of the invention.

[0108] In another preferred embodiment, the glycosylated amino acid sequence of the present invention can be conjugated to an anti-cancer drug. In one embodiment, the anti-cancer drug is monomethyl auristatin E (MMAE). In one embodiment, the glycosylated amino acid sequence is conjugated to a chemotherapeutic drug.Examples of chemotherapeutic agents that can be conjugated include: lenalidomide (REVLIMID®, Celgene), vorinostat (ZOLINZA®, Merck), panobinostat (FARYDAK®, Novartis), mocetinostat (MGCD0103), everolimus (ZORTRESS®, CERTICAN®, Novartis), bendamustine (TREAKISYM®, RIBOMUSTIN®, LEVACT®, TREANDA®, Mundipharma International), erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS 0103), rifacillin (RIBA), rifampicin (RIBA ... No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diaminedichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb), Oncology, Princeton, NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS number 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1 / 2, HPPD, and rapamycin.In another embodiment, the present invention may be used to form molecules of the invention that include radionucleotides, for example for use in cell killing or alternatively in labeling or imaging.

[0109] In one embodiment, the molecules of the present invention may include a label, for example, the label may be conjugated to the molecule via a glycosylated amino acid sequence of the present invention. Such labels may be useful, for example, in diagnosis and / or imaging. In one embodiment, the label is a fluorescent dye. In another embodiment, it is a radioactive label.

[0110] In one embodiment, the moiety is a polypeptide or peptide. The present invention can be used to join any suitable polypeptides. In one embodiment, conjugation serves to stabilize molecules comprising at least two polypeptides. In another embodiment, conjugation is used to help stabilize dimeric or multimeric proteins through conjugation between glycosylation sequences at the positions of the present invention in different polypeptides. In another embodiment, conjugation joins subunits of a protein together to make it more stable. In one embodiment, it is used to join a ligand to a receptor, for example, after the ligand has bound to its receptor.

[0111] In one embodiment, the present invention is used to provide a method for recovering a molecule of the present invention by its ability to conjugate to a compatible conjugation group. In one embodiment, the compatible conjugation group is provided by a support such as a bead or plate. In another embodiment, the present invention is used to localize the molecule to the site of a cell that produces the molecule; for example, the molecule is labeled, and conjugation to the support provides a localized region of the labeled molecule, or simply a localized region of the molecule, that can be detected using a secondary label. In another embodiment, the present invention can be used in techniques such as ELISA to immobilize a molecule of interest to a support prior to detection.

[0112] Nucleic Acid Sequences and Vectors Also provided are nucleic acid sequence(s) encoding at least the molecules of the invention or polypeptide portions thereof. Thus, the present invention provides nucleic acid molecules encoding polypeptides comprising one or more glycosylation sequences of the invention. In a preferred embodiment, the present invention provides nucleic acid molecule(s) encoding the molecules of the invention that are or comprise antibodies of the invention. The nucleic acid typically further comprises regulatory elements for expression of the polypeptides or proteins of the invention, such as, for example, a promoter and a polyadenylation signal.

[0113] The present invention also provides a vector comprising such nucleic acid(s) of the present invention.The vector may be, for example, an expression vector.The vector may be a cloning vector.The present invention also provides a sequence for introducing the nucleic acid sequence of the present invention into the genome of a cell via CRISPR.

[0114] Binding molecules and antibodies The present invention is highly versatile and can be used to conjugate any desired molecules, provided that at least one of them contains one or more glycosylation sequences of the present invention. The glycosylation sequence can either be inserted into an amino acid sequence forming part of a molecule of the present invention, such as a polypeptide, or used to replace part of the original sequence. In a particularly preferred embodiment, the present invention is applied to binding molecules, particularly antibodies. The present invention provides binding molecules comprising one or more glycosylation sequences of the present invention. In particular, the present invention provides such molecules in which one or more glycosylation sequences are O-glycosylated at a threonine amino acid residue, and the O-glycosylation comprises a sialylated sugar bearing a group that allows conjugation to a further moiety. Particularly preferred binding molecules of the present invention are or comprise antibodies.

[0115] Particularly preferred molecules of the present invention are antibody conjugates. Thus, the present invention provides antibodies in which at least one polypeptide of the antibody comprises one or more glycosylation sequences of the present invention. In particular, the present invention provides such antibodies comprising at least one glycosylation sequence in which a threonine residue is O-glycosylated. In a preferred embodiment, the O-glycosylated sugar comprises a sialylated sugar group. In a preferred embodiment, the O-glycan comprises a sialylated sugar bearing a chemical group functional for conjugation. In a preferred embodiment, the antibody comprises at least one glycosylation sequence of the present invention in which a threonine residue is O-glycosylated and the O-glycosyl sugar group is conjugated to another molecule. For example, such an antibody can be represented by the formula: Ab-O-DM (where Ab represents the antibody, O represents the O-glycosyl sugar chain, and DM represents the desired moiety conjugated to the antibody).

[0116] In one embodiment, a linker (L) can be used to join the O-glycan to the selected molecule (the linker itself can be considered part of the desired moiety). In one embodiment, the molecule can have the format Ab-O-DM, where DM comprises a linker and a second molecule. In one embodiment, the molecule can have more than one glycosylation sequence of the invention. For example, it can have two such sequences and take the format DM-O-Ab-O-DM. Again, DM can include or not include a linker, or can be a linker. In one embodiment, the binding molecule can be symmetrical, e.g., it can have two glycosylation sites at the same site on the antibody heavy chain and / or at the same position on the antibody light chain.

[0117] The present invention can be applied to any antibody format that can contain the glycosylation sequences of the present invention. Thus, the molecules of the present invention can be or comprise antibodies. In a preferred embodiment, the antibody is an antibody comprising two light chains and two heavy chains. In one embodiment, at least the light chain variable regions of the two light chains are identical to each other. In one embodiment, at least two heavy chain variable regions of the two heavy chains are identical to each other. In one embodiment, the light chain variable regions of the two light chains are identical to each other, and the heavy chain variable regions of the two heavy chains are identical to each other. However, the present invention can also be used to combine antigen-binding sites of different specificities, such as a light and heavy chain pair for a first specificity and a different heavy and light chain pair for a second specificity. In some embodiments, the light chains of both pairs are the same, but the heavy chains are different, resulting in different specificities. This is discussed further below.

[0118] In one embodiment, the antibody comprises at least two glycosylation sequences of the invention. In one embodiment, the antibody comprises at least four such sequences. In one embodiment, the antibody comprises two or four glycosylation sequences of the invention. In one embodiment, the antibody comprises at least two glycosylation sequences of the invention, one of the sequences being present in each heavy chain. In one embodiment, the antibody comprises at least two glycosylation sequences of the invention, one such amino acid sequence being present in each light chain. In one embodiment, the antibody comprises glycosylation sequences of the invention in each of its two light chains and two heavy chains. In one embodiment, the glycosylation sequence(s) of the invention are present in the antibody constant region. In one embodiment, the glycosylation sequences of the invention are present only in the antibody constant region. In another embodiment, the glycosylation sequences of the invention are present in the light chain variable region or the heavy chain variable region of the antibody, but not in the framework regions of the antibody. In one embodiment, the glycosylation sequences present are in the C-terminal half of the light chain. In another embodiment, the glycosylation sequences present are in the C-terminal half of the heavy chain. In another embodiment, the glycosylation sequence present is in the N-terminal half of the heavy chain. In another embodiment, the glycosylation sequence present is in the N-terminal half of the light chain. In one embodiment, the glycosylation sequence present is at the C-terminus of the light chain. In one embodiment, the glycosylation sequence present is at the C-terminus of the heavy chain. In one embodiment, the glycosylation sequence present is at the C-terminus of the light and heavy chains of the antibody. In a particularly preferred embodiment, the glycosylation sequence(s) of the invention are present in the hinge region of the heavy chain of the antibody.

[0119] An exemplary antibody is an immunoglobulin G (IgG) antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of approximately 100 to 125 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region refers to the region of an antibody that contains the Fc region and CH1 domain of the antibody heavy chain. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). The numbering of amino acid residues in the constant region is based on the EU index as in Kabat. Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: US Department of Health and Human Services, Public Health Service, National Institutes of Health (1991). The terms EU index numbering or EU numbering are used interchangeably herein.

[0120] The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs is based on the Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987)), Al-Lazikani et al., "Standard conformations for the canonical structures of 5 immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international This can be done according to well-known schemes, including those described in the ImMunoGeneTics database, available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212. The North CDR definitions are used in the exemplary anti-human CD33 antibodies described herein.

[0121] The present invention may also be applied to other antibody formats, for example, the antibody may be a heavy chain-only antibody. The antibody may be a VHH format antibody. The antibody may be a camelid antibody. The antibody may be derived from a cartilaginous fish, for example, an IgNAR format antibody. The antibody may be a heavy chain-only antibody comprising a heavy chain lacking a CH3 region. The antibody may be in tandem with another VHH of the same or different identity, for example, a VHH comprising a half-life extender that binds to human serum albumin (HSA). The present invention may be applied to antibody-like molecules, for example, DARPins and affibodies.

[0122] Considering that the present invention provides a method for joining two molecules, each containing a glycosylation sequence of the present invention, the present invention can also be used to join separate antigen-binding sites. In one embodiment, the present invention is used to join two antibodies together to produce an antibody with higher valency (i.e., a greater number of antigen-binding sites). The joining can be direct, or two molecules, each containing an antigen-binding site, can be combined with a molecule containing another scaffold sequence and antigen-binding site. The "valency" of a linked molecule typically refers to the number of binding sites present in the linked molecule. For example, in the case of an antibody, the "valency" of the antibody indicates the number of antigen-binding sites the antibody possesses. Reference to "antibody" herein includes structures comprising individual antibodies joined together with "O" glycosyl groups that act as "bridges" joining the antibodies. For example, the present invention provides antibodies in which an additional antibody is conjugated to each of the two light chains. The present invention also provides antibodies in which an additional antibody is conjugated to each of the two heavy chains. In one embodiment, the further antibodies are conjugated to or near the C-terminus of the polypeptide chain to which they are attached. In one embodiment, the further antibodies conjugated to the antibody molecule are scFv antibodies or heavy chain-only antibodies. Thus, for example, the invention provides Ig molecules in which the valency of the antibody is increased from 2 to 4 by conjugating an antibody to each of two light chains or two heavy chains. In one embodiment, the molecule of the invention is a multispecific antibody. In a preferred embodiment, it is a bispecific antibody. The invention also provides the use of the molecule of the invention in generating multispecific antibodies, in particular bispecific antibodies.

[0123] In an alternative embodiment, rather than conjugating an additional antibody to the first antibody, the present invention is used to conjugate a ligand to the antibody, such that the ligand effectively represents the moiety. In one embodiment, the ligand is specific for the same target as the antigen-binding site of the antibody. The present invention also provides a method of joining two antigen-binding sites together by forming a bridge between the two antigen-binding sites using the glycosylation sequences of the present invention.

[0124] The present invention also provides a method for modifying a known antibody by introducing one or more glycosylation sequences of the present invention into the primary sequence of the antibody. The glycosylation sequences of the present invention can be introduced at any of the positions described above. For example, known antibodies such as Humira®, Herceptin®, Avastin®, Keytruda®, Rituximab®, Remicade®, Stelara®, Enbrel®, Imbruvica®, Opdivo®, Cosentrx®, Ocrevus®, and other known antibodies can be modified to introduce one or more glycosylation sequences of the present invention, thereby providing a convenient method for conjugating desired moieties to these antibody drugs.

[0125] The present invention also provides a convenient method for producing antibody dimers or multimers by providing a method for conjugating antibodies via their respective glycosylation sequences. In one embodiment, the present invention provides an antibody dimer in which two antibodies each have one or more glycosylation sequences of the present invention that allow conjugation of the two individual antibodies. In another embodiment, the present invention is used to form antibody trimers. In another embodiment, the present invention is used to form antibody hexamers. All of the individual antibodies within a dimer or multimer may be identical except for the conjugation group. For example, two batches of the same antibody can be produced that differ only in the conjugation group in the O-glycosyl sugar chain of the glycosylation sequence of the present invention, with the two batches having different but compatible conjugation groups. The antibodies from the two batches can then be conjugated together to form a dimer. In another embodiment, antibodies with different specificities are conjugated together into dimers or higher-order structures using the present invention.

[0126] In a particularly preferred embodiment, the antibody binds to a target that allows transport across the blood-brain barrier (BBB). In one embodiment, such an antibody conjugate can be used to deliver a moiety to the CNS, particularly the brain. In one embodiment, the moiety may be for inhibiting expression of a target gene. In a particularly preferred embodiment, the moiety may be for inhibiting expression of a target gene in the brain. In a particularly preferred embodiment, an antibody specific for a target that allows transport across the blood-brain barrier (BBB) ​​can be conjugated to a moiety that is an siRNA using the present invention. The antibody can also be conjugated to a chemotherapeutic agent or other anti-cancer agent that would otherwise not be able to easily cross the BBB. Such a conjugate can be used, for example, to treat cancer.

[0127] Bispecific and multispecific binding molecules A binding molecule, particularly an antibody, of the present invention can have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more valencies. It can have a range of valencies. It can have at least these values. It can have valencies up to or including these values. In a preferred embodiment, an antibody can have a valency of two. In one embodiment, all antigen-binding sites of a binding molecule, particularly an antibody, can have the same specificity. In an alternative embodiment, an antibody can contain at least two different antigen-binding sites with different specificities. The antigen-binding sites can have different specificities, meaning that they bind to different antigens. In another embodiment, they can have different specificities, meaning that they bind to two different epitopes on the same antigen.

[0128] In one embodiment, the present invention provides a bispecific antibody comprising one or more glycosylation sequences of the present invention. In a preferred embodiment, the bispecific antibody comprises at least two glycosylation sequences of the present invention that are O-glycosylated at threonine, and the sugar chains are conjugated via sialic acid groups on the O-glycans to form a bridge between two different antigen-binding sites. In one embodiment, the bispecific antibody comprises an Ig antibody comprising two heavy chains and two light chains, each of whose light chain constant regions comprises a glycosylation sequence of the present invention conjugated to a Fab comprising an antigen-binding site with a specificity different from that of the antigen-binding site of the Ig antibody. In such an embodiment, typically, each of the Fab fragments conjugated to the Ig is identical and has the same antigen specificity, but the specificity is different from that of the antigen-binding site of the Ig. In another embodiment, the Fab fragment is conjugated to a heavy chain.

[0129] In a particularly preferred embodiment, a bispecific antibody is provided comprising two heavy chains and two light chains, wherein each heavy chain comprises an O-glycosylated glycosylation sequence of the present invention, the two sugar chains are conjugated to each other, and the antigen-binding site formed by the first light chain and the first heavy chain has a different specificity from the antigen-binding site formed by the second light chain and the second heavy chain. In a preferred embodiment, the glycosylation sequence of the present invention is substituted for a cysteine ​​present in the heavy chain hinge.

[0130] In a preferred embodiment, bispecific antibodies of the invention are generated via antibody arm exchange. A well-recognized problem with bispecific antibodies is that when a bispecific antibody is formed from two different light chains and two different heavy chains, expressing all four antibody chains in the same cell results not only in bispecific antibodies, but also in a much larger proportion of undesired monospecific antibodies and other undesired antibody species. In one embodiment, the present invention provides a solution for facilitating antibody arm exchange between two "parent" monospecific antibody species to generate bispecific antibodies.

[0131] Thus, the present invention provides a method for the production of a bispecific antibody, the method comprising: (a) contacting a first antibody having a first specificity with a second antibody having a second specificity, wherein the first antibody and the second antibody each comprise one or more glycosylation sequences of the invention that are O-glycosylated with a sialylated sugar chain bearing a conjugation group, and the first antibody and the second antibody comprise compatible conjugation groups that allow them to be conjugated to each other; (b) subjecting the first and second antibodies to antibody arm exchange and conjugation between complementary functionalized sialylated sugar chains to produce a bispecific antibody, wherein the resulting bispecific antibody comprises an antigen-binding site derived from the first antibody and an antigen-binding site derived from the second antibody, and thus two antigen-binding sites with two different specificities.

[0132] In a preferred embodiment, the first antibody and the second antibody comprise one or more glycosylation sequences of the present invention in the heavy chain constant region of the antibody. The glycosylation sequences of the present invention are typically located in a position in the heavy chain that allows antibody arm exchange. In a preferred embodiment, the glycosylation sequence of the present invention is present in place of a section of the sequence containing a cysteine ​​normally present in the heavy chain hinge. In a different embodiment, the cysteine ​​normally present in the heavy chain hinge is replaced with a serine residue, and one or more glycosylation sequences of the present invention are present elsewhere, for example, in the C-terminal portion of the heavy chain.

[0133] In one embodiment, the method may further comprise purifying the bispecific antibody. For example, in one embodiment, the method comprises: (c) the further step of binding the antibody to a support containing an antigen or epitope for one of the specificities of the bispecific antibody, removing unbound antibody, and then recovering the antibody bound to the antigen or epitope present on the support; (d) the further step of binding the antibody to a support containing an antigen or epitope directed against the other of the specificities of the bispecific antibody, removing unbound antibody, and then recovering antibody bound to the antigen or epitope present on the support.

[0134] In one embodiment, the first and second antibodies differ not only in their heavy chain variable regions but also in their heavy chain constant regions. For example, the heavy chain constant regions of the first and second antibodies may contain amino acid sequence differences that either promote the formation of bispecific antibodies (heterodimers) over monospecific antibodies (monomers) or aid in the purification of bispecific antibodies from monospecific / undesired antibody species. In a preferred embodiment, the heavy chains have differences in their heavy chain constant regions that promote the formation of bispecific antibodies over monospecific antibodies. For example, the heavy chain constant regions may have charge and / or shape modifications that promote bispecific formation over monospecific antibodies. An example of a heavy chain modification that promotes the formation of bispecific antibodies over monospecific antibodies is a "knob-and-hole" amino acid modification. Thus, in one embodiment, the first or second antibody has a heavy chain constant region "knob" modification, and the other of the first and second antibody has a "hole" modification.

[0135] Another example of a modification that can be used is a modification that alters the affinity for the purification agent. For example, one parent monospecific may have a heavy chain constant region modification that alters the affinity of the antibody for the purification agent Protein A, for example, eliminating binding to Protein A. The other parent monospecific may lack that modification and therefore have a heavy chain that normally binds to Protein A. Thus, a bispecific antibody may comprise one heavy chain that binds to Protein A and the other heavy chain that either does not bind to Protein A or has reduced affinity for Protein A. Overall, this means that the bispecific antibody has a moderate affinity for Protein A and can be separated from the parent monospecific antibody on this basis.

[0136] Cells and cell lines In a further preferred embodiment, the present invention provides a cell line in which the cell's ability to produce sialic acid has been disrupted, such that the cell's natural ability to produce sialic acid is reduced or absent. Such cell lines can also be used in the present invention. Such cell lines can then be supplemented with synthetic compounds containing functional groups for conjugation that are preferentially incorporated when polypeptides containing one or more glycosylation sequences of the present invention are O-glycosylated, particularly peracetylated ManNAz compounds that are incorporated, deacetylated, and converted to sialic acid containing a click chemistry group. This provides a convenient method for ensuring that the click chemistry group is incorporated as part of the O-glycosylation of one or more glycosylation sequences of the present invention.

[0137] Thus, the present invention provides cell lines that cannot produce sialic acid by themselves unless supplemented, particularly with peracetylated ManNAz, which can be taken up, deacetylated, and converted to sialic acid. It is possible to disrupt endogenous synthesis of sialic acid in eukaryotic cells by mutating the gene encoding UDP-GlcNAc-2-epimerase / ManAc kinase (GNE) (SEQ ID NO: 6, NCBI Accession No. NM_001246709). Unexpectedly, it has been found that the epimerase function of the gene can be reduced without either retaining kinase function or introducing a kinase-encoding sequence to compensate for its loss. In one embodiment, the sequence encoding the UDP-N-acetylglucosamine 2-epimerase portion of GNE is mutated such that UDP-N-acetylglucosamine 2-epimerase function is either reduced or eliminated. In one embodiment, N-acetylmannosamine kinase function is also reduced or eliminated. The knowledge that N-acetylmannosamine kinase function does not need to be retained or reintroduced means that cell lines are easier to produce. In one embodiment, UDP-N-acetylglucosamine 2-epimerase is knocked out, while the sequence encoding N-acetylmannosamine kinase is not modified, so that the kinase remains active. The cells typically contain a polynucleotide encoding a polypeptide comprising one or more glycosylation sequences of the present invention. The present invention also provides a method for producing such cells, comprising disrupting the gene encoding endogenous UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase (GNE) and, either before or after, introducing a sequence encoding a polypeptide having one or more glycosylation sequences of the present invention.

[0138] Disruption of the gene encoding UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase (GNE) may be performed by any suitable means, and in one embodiment is performed by CRISPR.

[0139] In one embodiment, the present invention also provides a cell culture comprising the cell line of the present application, wherein the cell culture medium is supplemented to allow sialic acid production, particularly with peracetylated ManNAz, which is incorporated, deacetylated, and converted to sialic acid. In a preferred embodiment, the peracetylated ManNAz comprises a conjugation group. In one embodiment, the synthetic sialic acid precursor comprises a click chemistry group. In another embodiment, the synthetic precursor is mannosamine with an azide group. In a preferred embodiment, the synthetic compound is N-azidoacetylmannosamine (ManNAz).

[0140] Any suitable cell type capable of O-glycosylating and sialylating the glycosylation sequences of the present invention may be used. Typically, the cell type used is a eukaryote. In a preferred embodiment, the cell line is mammalian. A particularly preferred cell line is CHO. Further examples of mammalian cell lines include HeLa cells, HEK293 cells, WI-38 cells, MRC-5 cells, and HepG2 cells. In one embodiment, the cell line is an HEK cell line. Examples of rodent cells that may be used include 3T3 cells, L929 cells, and BHK-21 cells. In one embodiment, the cell line is a stem cell line. In one embodiment, the cell line is an embryonic stem (ES) cell line.

[0141] The present invention also provides transgenic animals expressing polypeptides having one or more glycosylation sequences of the present invention. In one embodiment, the transgenic animals also include sialic acid biosynthesis engineering. In one embodiment, the animals can be fed artificial sugars to effect sialylation of the glycans with the glycosylation sequences of the present invention. In one embodiment, the animals can be fed food or water supplemented with peracetylated ManNAz, which is taken up, deacetylated, and converted to sialic acid. In one embodiment, the animals are used to produce the molecules of the present invention. In another embodiment, the animals are used as models. In one embodiment, the animals are fed a desired moiety with a compatible conjugation group to, for example, target the moiety to a specific location or cell type. Such animals are typically non-human.

[0142] Combination with other conjugation methods In a preferred embodiment, the use of glycosylation sequences of the present invention to effect conjugation to a desired moiety can be combined with a different conjugation approach, such that both are used. In a particularly preferred embodiment, glycosylation sequences of the present invention can be used to conjugate a first moiety to a molecule, and a second conjugation approach can be used to conjugate a second, different moiety to the molecule. The use of such a combined approach can be used, for example, to introduce two different functionalities into a molecule of the present invention via the conjugation of a first moiety and a second moiety thereto.

[0143] In a particularly preferred embodiment, the second conjugation approach used can be the use of a cysteine ​​amino acid present in the molecule to provide a means for conjugating to a second moiety. In one embodiment, cysteine ​​is engineered into the primary amino acid sequence of the polypeptide in the molecule, for example, into the same polypeptide that contains the glycosylation sequence of the present invention (this approach can be referred to as eCys). The approach of introducing cysteine ​​as a means for conjugation is described in WO 2018 / 232088, both of which are incorporated by reference in their entirety, and are specifically incorporated with respect to conjugation via cysteine ​​residues.

[0144] In one embodiment, when a combination of a glycosylation sequence of the invention and an introduced cysteine ​​residue is used, the molecule is an antibody. In one embodiment, the antibody of the invention comprises an IgG heavy chain constant region and a light chain constant region, at least one polypeptide of the antibody comprises at least one glycosylation sequence of the formula X1Thr Pro X2X3, wherein the threonine (T) amino acid residue in the second position is O-glycosylated with a sialylated sugar comprising a chemical group that can be or is conjugated to a moiety Y; ● The antibody contains a cysteine ​​at at least one of the following residues: residue 124 in the CH1 domain, residue 157 in the CH1 domain, residue 162 in the CH1 domain, residue 262 in the CH2 domain, residue 378 in the CH3 domain, residue 397 in the CH3 domain, residue 415 in the CH3 domain, residue 156 in the C kappa domain, residue 171 in the C kappa domain, residue 191 in the C kappa domain, residue 193 in the C kappa domain, residue 202 in the C kappa domain, or residue 208 in the C kappa domain.

[0145] In one embodiment, the antibody comprises a cysteine ​​at residue 124 in the CH1 domain, and further comprises cysteines at residues 157 and 162 in the CH1 domain, residue 262 in the C2 domain, and one but not all of residues 378 and 415 in the CH3 domain. In one embodiment, the antibody comprises a cysteine ​​at residue 157 in the CH1 domain. In one embodiment, the antibody comprises a cysteine ​​at residue 378 in the CH3 domain. In one embodiment, the antibody comprises a cysteine ​​at residue 415 in the CH3 domain.

[0146] In one embodiment, the IgG heavy chain constant region is a human, mouse, rat, or rabbit IgG constant region. In one embodiment, the IgG heavy chain constant region of the antibody is of the human IgG1, human IgG4, or human IgG2 isotype. In one embodiment, the IgG heavy chain constant region is a human IgG1 constant region.

[0147] In a preferred embodiment, the IgG1 heavy chain constant region of the antibody further comprises an isoleucine substituted at residue 247, a glutamine substituted at residue 339, and optionally a glutamic acid substituted at residue 332. In another embodiment, the IgG heavy chain constant region is a human IgG4 constant region. In a further preferred embodiment, the IgG4 heavy chain constant region of the antibody further comprises a proline substituted at residue 228, an alanine substituted at residue 234, and an alanine substituted at residue 235, and a glutamine substituted at residue 339.

[0148] In one embodiment, the antibody is an antibody comprising two heavy chains and two light chains, and each heavy chain comprises an IgG heavy chain constant region comprising a cysteine ​​at one of the following residues: residue 124 in the CH1 domain, residue 378 in the CH3 domain, and residue 397 in the CH3 domain.

[0149] In one embodiment, the antibody comprises a cysteine ​​at residue 124 in the CH1 domain of each heavy chain, and further comprises cysteines at residues 378 and 397 in the CH3 domain and at one but not all of residue 157 in the CH1 domain. In one embodiment, the antibody comprises a cysteine ​​at residue 378 in the CH3 domain of each heavy chain. In a further embodiment, the antibody comprises a cysteine ​​at residue 397 in the CH3 domain.

[0150] In one embodiment, the introduced cysteine ​​replaces a native serine, valine, alanine, glutamine, asparagine, threonine, or glycine. In one embodiment, the total number of engineered cysteines is between 2 and 6.

[0151] Any of the embodiments outlined herein regarding cysteine ​​residues as a means for conjugation can be combined with any of the embodiments outlined herein using the glycosylation sequence(s) of the invention.

[0152] method In one embodiment, the invention provides a method for producing a molecule of the invention, comprising: (a) culturing a cell line expressing a polypeptide comprising one or more glycosylation sequences of the invention under conditions that allow O-glycosylation of the glycosylation sequence(s) of the invention with an O-glycan comprising a sialic acid group and a group for conjugation, and (b) harvesting the O-glycosylated polypeptide. The O-glycosylated polypeptide may be harvested by any suitable means; for example, the polypeptide may also comprise a sequence for binding to Protein A.

[0153] In one embodiment, step (a) is performed using cells cultured in a medium containing an artificial sugar, for example, to promote the incorporation of modified sialic acid. In a preferred embodiment, the artificial sugar is N-azidoacetylmannosamine (ManNAz). In a preferred embodiment, the sugar is peracetylated ManNAz. In one embodiment, the cell line used is a cell line that cannot synthesize sialic acid de novo by itself. In a preferred embodiment, the cell line is a cell line of the present invention that lacks UDP-N-acetylglucosamine 2-epimerase activity. In one embodiment, the cells lack both UDP-N-acetylglucosamine 2-epimerase activity and N-acetylmannosamine kinase activity. In one embodiment, the cell line is a cell line in which the endogenous GNE gene has been disrupted. In a particularly preferred embodiment, step (a) comprises using both such a cell line and a medium containing such an artificial sugar.

[0154] In one embodiment, the method further comprises a step (c) in which the purified polypeptide is then conjugated to a desired moiety. Thus, in one preferred embodiment, the present invention provides a method for producing a polypeptide comprising: (a) culturing a cell line expressing a polypeptide comprising one or more glycosylation sequences of the present invention under conditions that allow O-glycosylation of the glycosylation sequence with an O-glycan comprising a sialic acid group and a group for conjugation, wherein the cell line has at least the UDP-N-acetylglucosamine 2-epimerase activity of the disrupted GNE gene and is cultured in a medium containing artificial sugars that promote the incorporation of modified sialic acids, preferably peracetylated ManNAz, which sugars are incorporated, deacetylated, and converted to sialic acids; (b) obtaining an O-glycosylated polypeptide having an O-glycan comprising a sialic acid and a group for conjugation; (c) conjugating the harvested polypeptide with a desired moiety having a group compatible for conjugation to the conjugation group of the polypeptide.

[0155] The present invention also provides a method that does not include the previous steps and that includes only step (c). (a) a molecule comprising one or more glycosylated amino acid sequences of the present invention, which are O-glycosylated with a sugar chain comprising sialic acid and a group for conjugation; (b) contacting a desired moiety comprising a conjugation group that is compatible with the conjugation group of the molecule; and causing conjugation.

[0156] Screening and Multiplexing Methods One advantage of the present invention is that it allows for combinatorial screening to identify combinations with desired properties. For example, the present invention provides: (a) providing a pool A comprising a plurality of molecules of the invention, each comprising one or more glycosylation sequences of the invention, each O-glycosylated with an O-glycosyl sugar chain comprising a sialic acid sugar and a chemical group for conjugation; (b) providing a pool B containing a plurality of moieties, each containing a chemical group for conjugation that is compatible with the conjugation group of a member of pool A; (c) testing different pairwise combinations of molecules from pool A conjugated with moieties from pool B for a desired property.

[0157] In one embodiment, the molecules of pool B are also glycosylated molecules of the invention that contain chemical groups that match the conjugation groups of the molecules of pool A.

[0158] In one embodiment, each pool has at least 5, 10, 20, 100, 500 or more different molecules of the invention. In one embodiment, the members of a pool are not physically present together, but are grouped into a pool because they all have the same conjugation groups that are compatible with the conjugation groups used in other pools.

[0159] Such methods can be used to screen any desired combination of molecules, provided that one of the conjugation partners contains one or more glycosylation sequences of the present invention that are terminally sialylated and O-glycosylated with a group that allows conjugation to a compatible conjugation group. While evaluating pairwise combinations of antibodies represents a particularly preferred embodiment, the present invention is not limited thereto. Thus, any combination can be screened, for example, screening possible combinations of antibodies with different drugs or different permutations of cytokines. In one embodiment, one or more molecules screened may be variants of the molecule, and screening is performed to identify which variant forms function best or have particular desired properties.

[0160] In a preferred embodiment, the method is used to evaluate different combinations of antigen-binding sites. In one embodiment, the molecules in pool A and pool B are both antibodies. In one embodiment, the antibodies in pool A target one antigen and the antibodies in pool B target a different antigen. In one embodiment, the molecules in pool A and pool B are monospecific antibodies. In one embodiment, the antibodies in the pools are antibodies that can undergo arm exchange via conjugation using glycosylation sequences of the present invention. In one embodiment, the property being screened is the ability to bind to both specificities, i.e., the ability to act as a bispecific antibody. In one embodiment, rather than requiring arm exchange, both pool A and pool B contain antibodies having one or more glycosylation sequences of the present invention that are O-glycosylated with a sugar chain bearing sialic acid and a conjugation group, where the conjugation group in pool A is compatible with the conjugation group in pool B, and the antibodies in pool A and B have different specificities, for example, for different antigens or different epitopes of the same antigen.

[0161] The screening method of the present invention can include screening for any desired property. For example, in one embodiment, the method can evaluate the ability of different combinations to kill cancer cells. In another embodiment, the method can evaluate binding ability. In another embodiment, the method can screen for the ability of combinations to bind to and optionally activate receptors. In another embodiment, the method can evaluate the ability of different combinations to stimulate cells to release molecules.

[0162] The present invention also provides libraries comprising the molecules of the invention. The present invention further provides combinatorial libraries comprising the molecules of the invention, which are divided into at least two portions having compatible conjugation groups, allowing for pairwise combination screening.

[0163] Pharmaceutical Compositions and Treatment Methods Also provided are pharmaceutical compositions comprising the molecules of the invention and a pharmaceutically acceptable carrier or excipient. The invention also provides pharmaceutical compositions comprising the nucleic acids or vectors of the invention and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers in therapeutic compositions can include or be liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, or pH buffering substances, can be present in such compositions. Pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; gelatin; talc; wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as ethylene glycol and propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. Pharmaceutically acceptable carriers may also contain manufacturing aids (e.g., lubricants, talc, magnesium, calcium stearate or zinc stearate, or stearic acid), solvents, or encapsulating materials. If desired, certain sweeteners and / or flavorings and / or coloring agents may be added. Such carriers enable the pharmaceutical composition to be formulated for ingestion by the patient in formats such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. The pharmaceutical composition can be formulated taking into account the properties of the active agent.Compositions containing pharmaceutically acceptable carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000, which are incorporated by reference in their entireties).

[0164] The pharmaceutical composition of the present invention can be administered to a subject by any suitable route. Administration can be, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, or rectal. Administration can also be, for example, parenteral. Administration can be, for example, by injection or infusion. Examples of such administration include bolus injection or continuous injection. The pharmaceutical composition can be provided, for example, in a form suitable for such an administration route. For example, a preferred administration route is by injection, and therefore the pharmaceutical composition can be provided in a format suitable for injection, for example, a format suitable for intravenous injection.

[0165] The pharmaceutical composition provides a therapeutically effective dose of the active agent. For example, the pharmaceutical composition may provide a dosage of about 0.01 mg / kg to about 50 mg / kg of the active agent, e.g., 0.05 mg / kg to 50 mg / kg, e.g., about 0.10 mg / kg to about 5 mg / kg of the active agent, or about 0.10 mg / kg to about 0.50 mg / kg. The dosage may be adjusted, for example, depending on the molecular weight of the molecule; for example, in some embodiments, a lower mg / kg dosage may be given if the molecule has a small molecular weight, such as a peptide. The dosage may be selected by a physician as an appropriate dose for a given condition. The present invention also provides unit dosage forms of the pharmaceutical composition. Furthermore, various formats are provided to facilitate administration to a subject. For example, an autoinjector or pen delivery device filled with the pharmaceutical composition of the present invention is also provided. Also provided is an infusion bag filled with the pharmaceutical composition of the present invention. Also provided is a lyophilized form of the pharmaceutical composition that can be reconstituted and administered to a subject.

[0166] The pharmaceutical compositions of the present invention can be administered to any suitable subject. As used herein, the term "subject" refers to mammals, including but not limited to humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. In a preferred embodiment, the subject is a mammalian subject. In a preferred embodiment, the subject is a human.

[0167] The pharmaceutical compositions of the present invention can be used to treat a subject. The present invention provides a method for treating a condition in a subject, comprising administering a therapeutically effective amount of the composition to a subject in need thereof. Also provided is a pharmaceutical composition of the present invention for use in a method of treating the human or animal body. The present invention also provides a pharmaceutical composition of the present invention for use in a method of treating a condition. The pharmaceutical composition may further comprise another therapeutic agent in addition to the conjugate of the present invention, allowing both to be administered to the subject simultaneously. However, the two may also be administered to the subject separately. For example, when administered separately, the two may be administered to the subject simultaneously, sequentially, or separately.

[0168] The present invention also provides any of the active molecules of the invention for use in the manufacture of a medicament, for example for treating any of the conditions described herein.

[0169] The versatility of the present invention means that it can be used to treat any suitable condition. In one embodiment, it is used to treat a condition selected from cancer, heart disease, infectious diseases, autoimmune disorders, respiratory diseases, diabetes, dementia, pain, neurodegeneration, and liver disease. In one embodiment, the cancer is selected from bladder cancer, breast cancer, colon or rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, skin cancer (e.g., melanoma), non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer. In one embodiment, the present invention can be used to induce tolerance to treat autoimmune disorders. For example, tolerance can be induced via SIGLEC engagement with sialic acids on engineered O-glycans without any conjugation. Alternatively, click chemistry can be used to conjugate glycans known to induce immune tolerance. The present invention can be used to treat autoimmune diseases and allergies, for example, allergies to severe allergens such as peanut proteins. Therapeutic proteins with immunogenic regions can be engineered with nearby glycosylation sequences of the present invention to promote immune tolerance.

[0170] In one embodiment, the present invention is used to help facilitate targeting. For example, a moiety conjugated to a polypeptide having a glycosylation sequence of the present invention can target the molecule of the present invention to a specific location. In one embodiment, the conjugated moiety is specific to a particular organ or cell type. In one embodiment, targeting is to the liver. In one embodiment, the moiety is or includes a lysosomal targeting chimera (LYTAC). LYTACs typically bind to the ASGPR receptor, thus providing targeted delivery to the liver. Similarly, molecules that can target the heart, lungs, brain, kidney, or other organs can be used. In one embodiment, targeting is to a specific cell type. In a further embodiment, conjugation of the moiety to the molecule results in targeting to a specific compartment within the cell.

[0171] In a preferred embodiment, the targeting is to the CNS, particularly the brain. In a preferred embodiment, the binding molecule of the present invention binds to a target that is intended to be transported across the blood-brain barrier (BBB). In one embodiment, it binds to the transferrin receptor.

[0172] The present invention can also be used for diagnosis. For example, the conjugated moiety can be a label that allows the location of the molecule of the present invention to be identified. For example, the moiety can be a radioactive label that allows the location of the molecule to be identified. Such labeled molecules can be used, for example, in ADME (absorption, distribution, metabolism, and excretion) studies. Such methods can be in vivo; for example, the molecule of the present invention can be administered to a subject, and its location can then be identified via the conjugated label. The present invention can be used in imaging techniques such as MRI and PET imaging.

[0173] Further Use In one embodiment, the desired moiety conjugated to the molecule is one that results in targeting of the molecule to a particular cell or cell receptor, hi one embodiment, the invention is used for modulating protection from protease degradation, modulating serum half-life, functional modulation, intracellular trafficking, cell adhesion, and self versus foreign recognition during an immune response.

[0174] The present invention can be used for a wide variety of purposes. In one embodiment, the present invention is used to increase the stability of a molecule. The presence of glycosylation in the glycosylated amino acid sequence of the present invention can prevent, reduce, or slow down the degradation of the molecule. In one embodiment, a glycosylation sequence is introduced at one or more sites such that the glycosylation reduces the ability of a second entity to bind to the molecule. This may be the case for glycosylation prior to any conjugation, or the conjugated moiety may provide protection for the molecule. In one embodiment, one or more glycosylation sequences are introduced near the cleavage site of a protease.

[0175] In one embodiment, the present invention reduces the ability of an enzyme to bind to a molecule. In another embodiment, it reduces the ability of a molecule to bind to a receptor. In another embodiment, the present invention can change the serum half-life of a molecule. Thus, for example, a molecule can have a longer serum half-life when conjugated to a desired moiety. In one embodiment, the desired moiety is PEG, and conjugation results in an increase in serum half-life. In another embodiment, the moiety is serum albumin.

[0176] In one embodiment, the conjugated moiety facilitates the purification of the molecule of the present invention. For example, the desired moiety can be conjugated to biotin to enable rapid purification. The present invention can also be used to immobilize a desired molecule on a surface through conjugation. Thus, the present invention can be used to produce immobilized proteins for techniques such as ELISA. The present invention can also be used to immobilize a specific protein on a bead through conjugation.

[0177] The present invention can also be used to replace one or more native glycosylation sites in a polypeptide with one or more glycosylation sequences of the present invention.

[0178] In another embodiment, the present invention can be used to conjugate a moiety having a desired activity to a protein of interest, for example, an enzyme such as hyaluronidase (e.g., for tumor penetration), a reporter (e.g., luciferase, dyes), an affinity tag (e.g., biotin, FLAG, HA), a small molecule (e.g., solubilized steroid), an albumin-binding lipid, or a membrane-bound lipid.

[0179] The present invention can also be used to change the solubility of molecules. In one embodiment, this can be achieved by conjugating a desired moiety with a specific solubility. Thus, for example, water-soluble groups can be conjugated to organic groups to enhance their solubility. For example, in one embodiment, steroid-loaded cyclodextrin or HPMA can be conjugated to an antibody for targeted delivery.

[0180] kit The present invention also provides kits comprising the molecules of the present invention. In one embodiment, the kit comprises a library of the present invention. In another embodiment, the kit may further comprise a cell line of the present invention. In one embodiment, the kit may comprise instructions for use.

[0181] All patents, published patent applications and publications cited herein are incorporated by reference as if fully set forth herein. [Example]

[0182] Example 1: Optimization of O-glycosylation sequences preface Completely uniform O-linked glycosylation of a protein is unprecedented and highly desirable. Therefore, a suitable test system was used to evaluate the ability of test sequences to act as motifs for O-glycosylation in cells. Immunoglobulins such as human IgA1 or IgG3 are known to be O-glycosylated to some extent at the hinge, and therefore the hinge region was used as a model system to explore possible O-glycosylation sequences.

[0183] To mimic the location of O-glycosylation in wild-type human IgG3 and IgA1, given its prevalence in antibody-based drugs, a putative O-glycosylation motif was inserted into the heavy chain of human IgG1.

[0184] The following motifs were inserted into the hinge: ● PTPSP (O-glycosylation motif in IgA1) ● DTPPP (O-glycosylation motif in IgG3) ● ATPAP (variant of the above sequence) ● PTPAP (variant of the above sequence)

[0185] The protein was expressed in HEK293 cells, isolated with protein A, and characterized by mass spectrometry to study the level of O-glycosylation.

[0186] Materials and Methods Transient protein expression in HEK293 cells Human embryonic kidney cells (also known as HEK (Human Embryonic Kidney) 293) were maintained in logarithmic growth phase in DMEM / F-12 medium as serum-free suspension cultures in shake flasks. Cultures were grown at 37°C, 7% CO2, humidified, 160 RPM, 50 mm orbit. On the day of transfection, HEK293 cells were grown at 2 x 10 per ml in DMEM-F-12. 6The DNA-carrier complex was then prepared by mixing 10 μg of DNA expression vector into 1 ml of growth medium. 20 μL of polyethyleneimine (25k MW free base, 1 mg / mL in water) was added to 1 mL of DNA solution and mixed gently by inversion. The mixture was incubated at room temperature for approximately 20 minutes to allow complex formation. 10% volume / volume of the DNA-carrier complex was added to the prepared HEK culture. The transfected culture was incubated at 37°C, 7% CO2, humidified, 160 RPM, 50 mm orbital for 5 days.

[0187] Protein isolation Antibodies were isolated using a protein A affinity resin as described below.

[0188] Running Buffer: ● Buffer A-1x PBS pH 7.2. ● Buffer B-20mM acetic acid + 5mM citric acid pH 2.7. ● Neutralization buffer - 1M Tris pH 8.0. ● Resin: mAb Select SuRe (GE Healthcare catalog: 17543801).

[0189] Resin preparation: The following protocol was used. • 50 mL of mAb Select SuRe resin slurry was added to a Falcon tube. • The tubes were centrifuged at 3500 rpm for 10 minutes. ● The supernatant was discarded. • The resin was washed with 50% v:v 1x PBS pH 7.2 and the centrifugation repeated a total of 6 times. The resin was equilibrated with buffer A-1x PBS pH 7.2 to prepare a 50% slurry.

[0190] Batch Procedure: To harvest a 100 mL culture (1 g / L titer), 4 mL of resin was added for binding (assuming 25 mg / mL), and the culture was mixed with shaking for approximately 1 hour. The supernatant / resin was combined into a 10 mL PolyPrep column (BioRad catalog #: 7311550), and the flow-through was collected. The resin was then washed with 10x resin bed volume of Buffer A (1x PBS + pH 7.4), and the wash was again collected. 10% of the final elution volume of Neutralization Buffer - 1 M Tris pH 8.0 was pipetted into a new collection tube. The column was transferred to a collection tube, and the protein was eluted with 5x resin bed volume of Buffer B - 20 mM acetic acid + 5 mM citric acid.

[0191] Protein Mass Spectrometry After reduction with dithioerythritol, the product was characterized by mass spectrometry. An Agilent G7100A CE system was used for the analysis. The CE capillary was a neutral-coated, 60 cm long, 360 μm OD x 50 μm ID capillary (Agilent, PVA-coated) with a custom tapered end. The running buffer was 1% formic acid. The sheath fluid was 12% methanol, 0.1% formic acid. The spray tip was pulled in-house from an uncoated glass rod to a tip opening of approximately 10 μm. Samples were injected by pressure (50 mbar for 20 seconds). Separation was performed at 30,000 V for 15 minutes.

[0192] An Agilent 6550 Q-TOF mass analyzer was used for the analysis, with a custom nanosheath flow CEMS interface with 3000 V for nanosheath spray. Mass spectra were collected in the Q-TOF at 1 scan / s from m / z 600 to 3200. All spectra were collected in profile and centroid mode. Data were analyzed using Agilent Qualitative Analysis Software vB08.

[0193] result Figures 1 and 2 show potential O-glycosylated glycan structures depending on whether the glycan is sialylated and whether click sugars are incorporated. Figure 3 provides an example of the protein mass spectrometry results obtained, showing the different peaks for the products. The overall results obtained are summarized in Table 1 below.

[0194] [Table 1]

[0195] Example 2: GNE knockout CHO cells were generated in which the UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase (GNE) gene was modified to disrupt both epimerase and kinase activity. Using CRISPR technology, the genetic modification was introduced into CHO cells, resulting in a translational frameshift in all GNE gene alleles in the CHO cell line. Lentivirus was used to deliver the CRISPR gene into CHO cells, and stable CHO pools were generated after puromycin selection (10 μg / ml). Constitutive expression of the CRISPR gene ensured that GNE activity was abrogated. The constructs utilized in preparing the cell lines were hamster Gne-1-pLentiCRISPRv2 (T15257), Gne-2-pLentiCRISPRv2 (T15258), Gne-3-pLentiCRISPRv2 (T15259), Gne-4-pLentiCRISPRv2 (T15260), and Gne-5-pLentiCRISPRv2 (T15261), the sequences of which are provided as SEQ ID NOs: 4-8, respectively, with the actual CRISPR sequences provided as SEQ ID NOs: 9-13.

[0196] Anti-polysialic acid antibody binding followed by a secondary fluorescent antibody in flow cytometry was used to determine the level of sialylation in the modified CHO cells compared to parental cells (positive control) and neuraminidase-treated parental cells (negative control). The reduction in cell surface fluorescence confirmed the lack of sialic acid production in all five CRISPR-modified cell lines, and the results are shown in Table 2 below.

[0197] [Table 2]

[0198] Example 3: Transient Expression: mAb with O-Linkage at the Light Chain C-Terminus preface A therapeutic antibody (molecule #1) was engineered to contain an optimal O-linked glycan recognition site (AAAPTPAPAAA) of the present invention at the C-terminus of the light chain. DNA vectors expressing this glycan-engineered LC and unmodified HC were transiently transfected into CHO cells deficient in de novo sialic acid synthesis, as described below. Glycosylation and sialylation levels were then analyzed.

[0199] Materials and Methods Cell culture and transient expression of secreted proteins CHO cells were grown as a suspension in a perfusion bioreactor at a cell density of 5 × 10 7 Cells were grown at 37°C in fully defined medium allowing up to 2 x 10 cells / ml. Cells were centrifuged and then diluted to 2 x 10 in fresh medium in disposable shake flasks. 7 The cells were resuspended at 14.5 mg / mL. Expression vector DNA was added to the cells at 14.5 mg / L, followed immediately by the addition of 27 mg / L polyethyleneamine (PEI, 25k MW, linear). The transfected cultures were then temperature-shifted to a 32°C incubator, 6% CO, 160 RPM, 50 mm orbit, for increased protein production. Nutrient feeding allowed for 7 days of production with an average antibody titer of approximately 1 g / L.

[0200] Stable cell generation and expression of secreted proteins Recombinant stable CHO pools were generated by transfection of CHO cells with one or more DNA vectors containing transposon elements. DNA vectors expressing the appropriate transposase enzymes were co-transfected to induce recombination of the transposable DNA vectors with chromosomal DNA. CHO stable pools carrying one or more recombinant genes were collected at 5 x 10 6 The cultures were grown in suspension at 37°C until they reached a cell density of 2 x 10. The cultures were centrifuged at 500 x g and diluted to 2 x 10. 7 The cultures were immediately temperature-shifted to a 32°C incubator with 6% CO, 160 RPM, and a 50 mm orbit to allow for increased protein production. Nutrient feeding allowed for 14 days of production with an average antibody titer of approximately 5 g / L.

[0201] Click chemistry sugar supply Artificial sugars, such as peracetylated ManNAz, were formulated in DMSO, typically at approximately 500 mM for a stock solution stored at -20°C. Acetylated sugars were used to allow robust passive diffusion into cells. On the day of transfection, the click sugar solution was warmed to room temperature and added to the culture at a final concentration of 800 μM. Each day after culture, click sugars were added to a concentration of 400 μM. By adding click sugars to 800 μM the day before skipping a feed, it was possible to skip feeding the culture for one day. Click chemistry sugar concentrations could be adjusted accordingly to the cell density to avoid toxicity.

[0202] After 5 days of production using the peracetylated ManNAz feed, the antibody was purified using Protein A affinity chromatography. A280 spectrophotometry showed a volumetric expression level of 0.8 g / L.

[0203] Post-expression processing (purification, click chemistry) Due to the bioorthogonal nature of click chemistry, a simple one-step purification of the engineered glycoprotein was sufficient to prevent conjugation with host cell proteins in the raw supernatant. Click chemistry was typically performed under physiological conditions by adding the complementary click conjugate. A copper catalyst was included for reactions requiring copper ions (e.g., azide-bearing alkynes).

[0204] result Figures 1 and 2 show various O-glycosyl chains resulting from O-glycosylation, sialylation of O-glycosyl sugar chains, and incorporation of click chemistry groups. Mass spectrometry results indicate the percentage of O-glycosylation sites that are O-glycosylated ("occupancy") and the percentage of O-glycosyl chains sialylated with azidosialic acid. The resulting mass spectrometry results indicated a glycan occupancy rate of >99% with >95% azidosialic acid. Without the GNE knockout, cells incorporated native sialic acid, and the azidosialic acid content was reduced to <90%. Figure 3 shows exemplary protein mass spectrometry results for specific glycosylation sequences (without GNE knockout—3(a) and with GNE knockout—3(b)), showing the highest peaks observed in each case for antibody molecules bearing sialylated O-glycosyl sugar chains with click chemistry groups. The results obtained also demonstrated the utility of the generated GNE knockout and the advantage of the glycosylation sequence provided to achieve high site occupancy.

[0205] Example 4: Stable cell generation and protein expression of an IgG1 mAb with an O-linkage at the light chain C-terminus preface As described above, a therapeutic IgG1 monoclonal antibody (molecule #1) was engineered to have a preferred O-linked glycan recognition site (AAAPTPAPAAA) at the C-terminus of the light chain. A stable cell line expressing molecule #1 was generated, and the ability of the cell line to produce molecule #1 was further studied.

[0206] Materials and Methods DNA vectors expressing this glycan-engineered light chain (LC) and unmodified heavy chain (HC) with transposon elements flanking the antibody operon were transfected into CHO cells deficient in de novo sialic acid synthesis (see Stable cell generation and expression of secreted proteins), and a second vector expressing the appropriate transposase was co-transfected with the transposable DNA vector (containing no transposable elements to allow only transient expression of the transposase).

[0207] After generating a stable pool, a 7-day production run was carried out using ManNAz feed. 6 / mL or approximately 1 × 10 6 100 mL fed-batch shake flask cultures with a starting viable cell density (VCD) of either 10 x 10 / mL were grown in a chemically defined, proprietary medium. A concentrated, chemically defined, proprietary feed was added periodically to each flask during the process. 6 Cultures with an initial VCD of 1 x 10 / mL were incubated at 32°C, 6% CO2, and shaking at 150 rpm at a ¾" slow speed for 10 days. Approximately 1 x 10 6 The incubation conditions for the cultures with an initial VCD of 1 / mL were the same except that the incubation temperature was started at 36°C and then shifted to 32°C on day 6. The process duration for these cultures was 14 days.

[0208] For all shake flasks, peracetylated N-azidoacetylmannosamine (Ac4ManNAz) was added from a 500 mM stock dissolved in dimethyl sulfoxide (DMSO) starting on day 0. Ac4ManNAz was added daily at a rate of 20 μM per unit of integrated cell area (ICA) until a predetermined limit was reached. Ac4ManNAz was then added daily at that limit until the final day of the process. 6For cultures with an initial VCD of 1 × 10 / mL, the Ac4ManNAz addition limits were 200 μM, 400 μM, 600 μM, and 800 μM. 6 For cultures initiated with 1 mL VCD, the only limit tested was 400 μM. Control cultures were run in which DMSO alone was added in a volume equal to the maximum volume of DMSO added to the Ac4ManNAz test cultures. Additional controls were run as normal fed-batch processes. All conditions were run in duplicate.

[0209] Cultures were monitored daily for cell growth using a Vicell XR Viability Analyzer (Beckman Coulter, Indianapolis, IN). At the end of the cell culture process, cell-free media (CFM) samples from each flask were assayed by CE-MS analysis to determine azidosialic acid incorporation. Additional CFM samples from earlier days in the process were assayed for productivity using a Cedex BioHT Analyzer (Roche Diagnostics, Indianapolis, IN).

[0210] Samples from all sets of shake flasks were then analyzed by CE-MS as described in the previous examples.

[0211] result The results obtained are shown in FIGS. 5 to 9 as follows. ● Figure 5(a): Initial viable cell density >10 × 10 6 Growth of the culture is / mL. ● Figure 5(b): Initial viable cell density >10 × 10 6 Viability of the culture in / mL. ● Figure 6: Initial viable cell density >10 x 10 6 Productivity of molecule #1 for the culture in mL / mL. ● Figure 7(a): The initial viable cell density was approximately 1 × 10 6 Growth of the culture is / mL. ● Figure 7(b): The initial viable cell density was approximately 1 × 106 Viability of the culture in / mL. ● Figure 8: Initial viable cell density was approximately 1 × 10 6 The productivity of molecule #1 of the culture in / mL. Figure 9 shows the results of the sugar titration.

[0212] Initial VCD (viable cell density) >10 x 10 6 For cultures with 100 μM / mL, the 800 μM and 600 μM Ac4ManNAz limits became toxic on days 8 / 9 and 9 / 10, respectively. For lower Ac4ManNAz limits, growth was similar to control levels. There was some detrimental effect of Ac4ManNAz on productivity, except for the 200 μM limit, which was at or near control levels (Figure 2). All data are the average of duplicate shake flasks.

[0213] Approximately 1×10 6 For cultures with an initial VCD of 1 / mL, the 400 μM Ac4ManNAz limit became toxic for one of the two cultures on day 13 / 14 and also had some detrimental effect on productivity.

[0214] When samples from all sets of shake flasks were analyzed by CE-MS, all samples had nearly 100% incorporation of azidosialic acid, regardless of productivity and viability at the end of the process, and the results obtained are shown in Figures 9 and 10.

[0215] Example 5: Stable cell production and protein expression of an IgG4 mAb with an O-linkage at the light chain C-terminus preface Next, a study similar to that carried out in Example 4 was carried out using an IgG4 monoclonal antibody, molecule #2.

[0216] Materials and Methods The cell line for molecule #2 was generated in the same manner as described in Example 4 for molecule #1. Molecule #2 was then produced in bulk culture. Approximately 10 x 10 6A 100 mL fed-batch shake flask culture with a starting viable cell density (VCD) of >10 x 10 for molecule #1 was cultured in a 100 mL shake flask with a starting viable cell density (VCD) of >10 x 10 for molecule #1. 6 The cultures were grown under the same conditions as the / mL cultures.

[0217] As before, Ac4ManNAz was added from a 500 mM stock dissolved in DMSO at a daily rate of 20 μM per ICA unit starting on day 0 up to a predetermined limit, and then added daily at that limit until the final day of the process. Based on the toxicity, productivity, and incorporation results from molecule #1, the Ac4ManNAz addition limit was varied to 50 μM, 100 μM, 200 μM, and 400 μM. Control cultures were run in which DMSO alone was added at a volume equal to the maximum volume of DMSO added to the Ac4ManNAz test cultures. Additional controls were run as in the normal fed-batch process. All conditions were run in duplicate and monitored daily for growth. For the extended environment, all processes were terminated on day 9. Productivity was determined by BioHT on days 7 and 9. Azidosialic acid incorporation was determined by CE-MS from the day 9 sample.

[0218] result The results obtained are shown below. Figure 11: Initial viable cell density for molecule #2 was approximately 10 x 10 6 Growth of the culture is / mL. Figure 12: Initial viable cell density for molecule #2 was approximately 10 x 10 6 Viability of the culture in / mL. ● Figure 13: For molecule #2, the initial viable cell density was approximately 10 x 10 6 The productivity of the culture is / mL. Figure 14: Initial viable cell density for molecule #2 was approximately 10 x 10 6 Product distribution of the culture in mL / mL. ● Figure 15: CE-MS summary of the day 9 sample showing the incorporation of azidosialic acid into molecule #2.

[0219] As the Ac4ManNAz limit was reduced to lower levels for molecule #2, no toxicity to growth was observed (Figures 11 and 12). As with molecule #1, there was a detrimental effect on productivity at the 400 μM limit, but not at the lower limit (Figure 13). When samples were analyzed by CE-MS, there was a dose response of azidosialic acid incorporation relative to the loading limit (Figures 14 and 15). The 400 μM level was again at or near 100%, and 200 μM was approximately 98% incorporation.

[0220] Example 6: Transient mAb expression using mAbs with O-linkages at the light chain C-terminus and heavy chain C-terminus preface A therapeutic antibody, molecule #3, was engineered to have optimal O-linked glycan recognition sites (AAAPTPAPAAA) at the C-terminus of the light chain (LC) and the C-terminus of the heavy chain (HC). Transient expression of the antibody was studied.

[0221] Materials and Methods The DNA vector, molecule #3, expressing the glycan-engineered light chain (LC) and glycan-engineered heavy chain (HC) was transiently transfected into GNE knockout CHO cells, which are deficient in de novo sialic acid synthesis (as described in the previous example). After 5 days of production on Ac4ManNAz feed using the same approach as in the previous example, the antibody was purified using Protein A affinity chromatography. Expression levels, glycan occupancy, and azidosialic acid incorporation, as determined by A280 spectrophotometry, were all recorded.

[0222] result Expression levels as determined by A280 spectrophotometry, glycan occupancy, and azidosialic acid incorporation as determined by mass spectrometry are shown in Table 3 below.

[0223] [Table 3]

[0224] Example 7: Conjugation of O-glycosylated antibodies to siRNA preface Having demonstrated successful generation of sialylated O-glycosylated sugars with high occupancy and incorporating click sugars, antibodies were then used to demonstrate the ease with which desired moieties can be conjugated to antibodies using click chemistry groups. The moiety chosen for conjugation to antibodies was siRNA, and the molecules listed in Table 5 were generated and evaluated.

[0225] Materials and Methods Conjugation Conjugation was performed in two different formats. In the first format, siRNA was functionalized with a clickable DBCO group. DBCO enabled direct conjugation of siRNA duplexes to antibodies containing azidosialic acid sugars on either the heavy or light chain. In the second format, a DBCO-methyltetrazine bifunctional linker was used, in which the linker was first conjugated to the antibody azidosialic acid site. Excess linker was removed by desalting the antibody into 1x PBS pH 7.2, followed by the addition of TCO-functionalized siRNA duplexes clicked onto the methyltetrazine of the linker. In both conjugation formats, 4 to 1 molar equivalents of siRNA relative to antibody were used.

[0226] Figure 16 shows the basic approach of how antibodies with provided glycosylation sequences can be conjugated to siRNA via the existing sialylated O-glycosyl sugar chains using click chemistry sugars, either with or without linkers.

[0227] Conjugation was monitored using analytical anion exchange chromatography using a ProPac™ SAX-10 HPLC column (10 um particles, 4 mm diameter, 250 mm length) with the following procedure: flow rate 1 mL / min, Buffer A: 20 mM TRIS pH 7.0, Buffer B: 20 ​​mM TRIS pH 7.0 + 1.5 M NaCl, 30°C.

[0228] [Table 4]

[0229] Drug-to-antibody ratios (DAR) were calculated based on peak area % from analytical anion exchange (aAEX) chromatograms. An illustrative example of a chromatogram is shown in Figure 17.

[0230] Conjugation kinetics Conjugation of siRNA to antibodies incorporating azidosialic acid sugars was monitored by aAEX over time as a function of antibody concentration at 1 mg / mL, 5 mg / mL, and 10 mg / mL to assess the effect of antibody concentration on conjugation, and the results are shown in Figure 18 (18(a) - 10 mg / mL, 18(b) - 5 mg / mL, and 18(c) - 1 mg / mL mAb).

[0231] Purification of antibody-siRNA conjugates: After siRNA conjugation to the antibody, excess siRNA and unconjugated antibody were removed by further purification. Either preparative size exclusion chromatography (SEC) or preparative analytical anion exchange chromatography was used to purify the final conjugate. Preparative SEC was performed using a Cytiva Superdex 200 column in 1x PBS. Alternatively, Cytiva Q-FF was used with a starting buffer of 20 mM TRIS pH 7.0 and eluted with a 20-column volume gradient using a buffer containing 20 mM TRIS pH 7.0 and 1 M NaCl. These resulted in purified antibody-siRNA conjugates devoid of excess siRNA and unconjugated antibody. The resulting conjugate profiles were analyzed by analytical anion exchange; Figure 19 and Table 5 show the results. After AEX purification, DARs of 1.86 and 1.72 were obtained for ARC-180 and ARC-181, respectively.

[0232] [Table 5-1]

[0233] Ex vivo plasma stability The antibody-siRNA conjugates were subjected to ex vivo plasma stability evaluation to observe the conjugate stability and any dissociation of siRNA from the antibody. The antibody-siRNA conjugates were incubated in mouse and / or cynomolgus monkey plasma at 37°C for 0, 24, and 48 hours, respectively, with rotation at 5 rpm. The antibodies were immunoprecipitated from the plasma samples using biotinylated goat anti-human IgG. The solution was then incubated with streptavidin beads for 30 minutes with rotation at room temperature. After multiple washing steps with 1x PBS, the samples were eluted with 20% acetonitrile elution buffer containing 1% formic acid by mixing at 2000 rpm for 15 seconds, followed by a 5-minute static benchtop hold. The eluted samples were then injected into an LC-MS for analysis.

[0234] LCMS method: ● Equipment: Sciex I. ● Column: Agilent, PLRP-S 1000A 5μM 50×1.0MM, PN: PL1312-1502. ● Injection volume: 20μL. ● Column temperature: 80°C. ● Autosampler temperature: 5℃. ● Mobile phase A: Water containing 0.05% TFA. ● Mobile phase B: acetonitrile with 0.05% TFA. ● Gradient:

[0235] [Table 5-2] ● MS: m / z 2000-5000

[0236] The results obtained are shown in FIG.

[0237] In vitro target gene knockdown efficacy assessment in mouse cortical neurons Primary mouse cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18. Cells were plated at a density of 40k cells / well in poly-D-lysine-coated 96-well plates and cultured for 7 days at 37°C in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antimycotic (Corning) in a tissue culture incubator in a humidified chamber with 5% CO2. On day 7, half of the medium was removed from each well, and a 2x concentration of one of the following (i)–(iii) was added to the wells in culture medium containing 2% FBS for treatment as CRCs and incubated with the cells for another 7 days: (i) siRNA (targeting the desired therapeutic target in the CNS), (ii) control siRNA (a non-targeting therapeutic target in the CNS), and (iii) a human IgG4 isotype antibody with siRNA linked to the C-terminus of the light chain via azido-sialic acid of either a control non-targeting antibody or a CNS-targeting transferrin receptor (TfR) antibody. At the end of siRNA treatment, RT-qPCR was performed to quantify target mRNA levels using the TaqMan Fast Advanced Cell-to-CT Kit. Specifically, cells were lysed, cDNA was generated using a Mastercycler X50a (Eppendorf), and qPCR was performed using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Gene expression levels of therapeutic targets were normalized to β-actin (ThermoFisher Mm02619580_g1) using their respective probes.

[0238] The results are provided in Figure 26 and Table 6. The results provided in Table 6 demonstrate that exemplary mouse TfR binding protein-siRNAs generated via either eCys or glyco-mAb conjugation chemistries (e.g., mTfR2-dsRNA No. 8 conjugates) successfully targeted mouse genes, providing an order of magnitude greater knockdown than isotype Ab-siRNAs.

[0239] [Table 6]

[0240] Multiplexed conjugation using sugar chemistry and alternative conjugation chemistries To generate multimodal antibody conjugates, sugar-based conjugation was combined with orthogonal chemistries, such as lysine- or cysteine-based conjugation. Antibodies containing azidosialic acid sugars were engineered to have cysteine ​​sites for site-selective conjugation, or structural cysteines or lysines were utilized for secondary conjugation. For engineered cysteine ​​conjugation, the antibody was first reduced with 20 molar equivalents of a reducing agent and subsequently reoxidized with 10 molar equivalents of dehydroascorbic acid (DHAA). DBCO-functionalized siRNA1 and thiol-reactive siRNA2 were then added.

[0241] Conjugation of both siRNAs was monitored by analytical AEX, and the resulting conjugates were further purified using the methods described above. An example of a multiplex siRNA conjugate aAEX profile is provided in Figure 21.

[0242] In vitro target gene knockdown efficacy assessment of multiple targets in human cells Antibody-siRNA conjugates containing siRNAs against two targets (target 1 and target 2) were evaluated for functional target knockdown activity in cultured cells. The EFO-21 cell line, derived from ovarian cystadenocarcinoma, was used as an exemplary cell line for generating knockdown data. EFO-21 cells were seeded at 10,000 cells per well in 96-well plates and treated with siRNA to generate concentration response curves (CRCs). After 72 hours of incubation, cell lysates were prepared for Cell to CT qPCR to examine target gene expression, evaluate the efficacy of siRNAs for target gene knockdown, and compare CRC potency between lipid-conjugated and antibody-conjugated siRNAs. mRNA expression data were generated using a Cell to CT kit. Examples of target knockdown results using antibody-siRNA conjugates are provided in Figures 23(a and b), 24(a and b), and 25(a and b).

[0243] Example 8: Use of O-glycosylated antibodies conjugated to siRNA for in vivo gene knockdown To further demonstrate the efficacy of the conjugates of the present invention, we investigated the ability of the antibody-siRNA conjugates of the present invention to inhibit target gene expression in vivo. An antibody specific for mouse transferrin receptor (TfR) was modified to include the O-glycosylation sequence of the present invention at the C-terminus of the antibody's light chain. The O-glycosylation of the sequence allowed for the introduction of a DBCO (dibenzocyclooctyne) linker, which was used to generate conjugates between the antibody and siRNA specific for selected target genes expressed in the brain. The O-glycosylation included an azide group on the terminal sialic acid, allowing for the conjugation of functionalized siRNA to the DBCO group. Thus, the generated antibody-siRNA conjugate (ARC-181) contained an antibody specific for TfR, allowing the conjugate to cross the blood-brain barrier (BBB), and then allowing the siRNA to reduce target gene expression in the CNS. A control conjugate, ARC-180, was also produced in the same manner, except that the antibody used in the conjugate was an isotype control that recognizes a different target to the TfR, meaning that the control should not be transported across the BBB via the TfR. Table 5 provides further details of the ARC-180 and ARC-181 conjugate preparations subsequently used in studies in mice.

[0244] To demonstrate that mouse TfR binding protein (mTBP)-siRNA conjugates (ARC) cross the blood-brain barrier (BBB) ​​and deliver siRNA cargo to the CNS to reduce target mRNA gene expression, a proof-of-concept study was conducted to evaluate the pharmacodynamic efficacy of the constructs using peripheral delivery in mice. PBS control, isotype Ab-siRNA conjugates, or mTBP2-siRNA were administered intravenously as a single dose at an effective siRNA concentration of 10 mg / kg to 8-week-old FVB mice, which were sacrificed after 14, 28, and 70 days, respectively (see Figure 26). Additionally, a mouse anti-CD4 antibody (GK1.5) was administered at 10 mg / kg 2–3 days before the study to deplete CD4+ T cells and mitigate undesirable pharmacokinetic consequences resulting from spurious anti-drug antibody responses to the injected compound.

[0245] At the indicated time points, mice were fully anesthetized and then cardiac perfused with cold PBS (6 ml / min for 5 min) until blood was completely removed. The brains and spinal cords were then collected, and target mRNA levels were assessed by RT-qPCR. For RT-qPCR, RNA was isolated using the RNeasy Plus Universal Mini Kit (Qiagen 73404). Briefly, half-brain tissue homogenates were prepared with FastPrep-24 Lysing Matrix D beads, and the tissue was homogenized in an MP Fastprep24 (MP Biomedical) at 6 m / s for 40 s at 4°C. The vials were then centrifuged to collect the supernatant. RNA was then recovered. After determining the RNA quantity by the A260 / A280 ratio using a spectrophotometer, cDNA was generated using a Mastercycler X50a (Eppendorf) and qPCR was performed using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Gene expression levels of target genes were normalized by β-actin using their respective probes (ThermoFisher).

[0246] As shown in Figure 26, a single IV administration of the mTfR-siRNA conjugate (ARC-181) in mice resulted in robust and selective reduction of the target in the brain (61% mRNA reduction) starting 14 days after administration compared to PBS-administered controls. Importantly, the isotype Ab-siRNA conjugate (ARC-180) did not induce a significant reduction of target mRNA, demonstrating that active TfR-mediated transport is required to deliver siRNA cargo to the CNS. This further provided evidence of the successful use of the present invention to enable crossing the blood-brain barrier and delivery of cargo to the brain.

[0247] Example 9: Use of the conjugation methods provided to generate multifunctional antibodies and combinatorial libraries Figure 27 illustrates how the provided conjugation methods can be used to generate multifunctional binding molecules, such as multifunctional antibodies, as well as combinatorial libraries. The conjugation groups introduced as part of the O-glycosyl sugar chain are highly versatile and can be used to join any two desired molecules. This approach can also be used to generate different permutations of a panel of molecules, so that the combinations can be evaluated for desired properties (e.g., synergy or the ability to bind more than one desired epitope).

[0248] Example 10: Further conjugates preface Additional linkers were prepared and used in producing conjugates with the glycosylation sequences of the invention.

[0249] Materials and Methods We used the following: - peptide acetyl-KRRRRRRK-NH2 (5 mg Genscript lot#: U047MGA130-1 / PE1083) (shown in Figure 28(a)), • DBCO-PEG5-NHS (2 vials of 2 mg) Click Chemistry Tools Catalog #A102P-2 Lot #:3955 (shown in Figure 28(b)); ● N-methylpyrrolidone (NMP), and ● N-N'-Diisopropylethylamine (DIEA) Sigma Catalog #: 496219 Lot #: 33496KJ.

[0250] Preparation of the linker A 2 mg vial of DBCO-PEG5-NHS was dissolved in 100 μL of NMP and then transferred to a new vial of DBCO-PEG5-NHS and mixed to dissolve. Once dissolved, the contents of the vial were transferred to the peptide vial and 1 μL of DIEA was added. The contents of the vial were mixed and the vial was incubated at room temperature.

[0251] 0.5 μL of the vial contents was diluted 1:500. 20 μL of the dilution was placed in an Agilent microvial and CE / MS analysis was performed to confirm the completeness of the reaction. The resulting conjugation is shown in Figure 28(c).

[0252] After the reaction was judged complete by CE / MS, 1 mL of water and 5 μL of 5% acetic acid were added to adjust the pH to approximately pH 5.5, as measured by using pH strips. No precipitation was observed, and the preparation was stored at 4° C.

[0253] Linker purification and lyophilization Peptides were purified by preparative HPLC on a Shimadzu LC system (system controller CBM-20A, pump model LC-20AP, oven model CTO-20A, detector SPD-20A, fraction collector FRC-10A): ● Buffer A: 0.05% TFA in water. ● Buffer B: Acetonitrile. ● Gradient: 18% B to 30% B over 39 minutes ● Column: Waters SymmetryPrep C18 7 um, 19x300mm, Part No WAT066245, S / N01553030811204. ● Column oven: 50℃ ● Flow rate: 20mL / min

[0254] The purity of the linker fractions was assessed using analytical RP-HPLC (Agilent 1290 Infinity II LC system), and fractions >95% pure were pooled. Subsequent lyophilization of the final major product pool yielded the lyophilized linker TFA salt (3.3 mg, >95% pure). The molecular weight was determined by LC / MS and was consistent with the structure shown in Figure 28c (observed: [M + 3H] = 804.1, calculated [M + 3H] = 804.3, observed MW (average) = 2409.3, calculated MW (average): 2409.88).

[0255] Linker conjugation to Fab antibody fragments via O-glycosylated glycosylation sequences 0.5 μmol of Fab was placed in a 50 mL tube and 1.37 mmol of DBCO linker peptide was added. The tube was then mixed by inverting several times and placed at 4°C over the weekend. The extent of the reaction was assessed using CE / MS, which indicated approximately 75% conversion. The material was concentrated using a Millipore Ultracell-15 30 kDa MWCO filter, 50 mL tube style, and then washed three times with PBS buffer. A final recovery of 12.6 mg was expected, resulting in approximately 80% conjugation.

[0256] All patents, published patent applications and publications cited herein are incorporated by reference as if fully set forth herein.

[0257] array SEQ ID NO: 1 provides the sequence of a preferred glycosylated amino acid sequence of the present invention.

[0258] PTPAP.

[0259] SEQ ID NO: 2 provides the sequence of a further preferred glycosylated amino acid sequence of the invention.

[0260] AAAPTPAPAAA.

[0261] SEQ ID NO: 3 provides the sequence of the glycosylated test amino acid sequence used in the examples of this application.

[0262] AAATPAP.

[0263] SEQ ID NO: 4 provides the sequence of a further glycosylation test amino acid sequence used in the examples of the present application.

[0264] PTPSP.

[0265] SEQ ID NO: 5 provides the sequence of a further glycosylation test amino acid sequence used in the examples of the present application. DTPPP.

[0266] SEQ ID NO: 6 provides the sequence of the gene encoding bifunctional UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase (UDP-GlcNAc-2-epimerase / ManAc kinase, also known as GNE).

[0267] SEQ ID NO: 7 provides the sequence of hamster Gne-1-pLentiCRISPRv2(T15257). ATGGGAATAACCGAAAGCTT.

[0268] SEQ ID NO: 8 provides the sequence of hamster Gne-2-pLentiCRISPRv2(T15258). CCGTGCAGATTACTCCAAAT.

[0269] SEQ ID NO: 9 provides the sequence of hamster Gne-3-pLentiCRISPRv2(T15259). CCAATTTGGAGTAATCTGCA.

[0270] SEQ ID NO: 10 provides the sequence of hamster Gne-4-pLentiCRISPRv2(T15260). CTTAATGCCGAACATGATCG.

[0271] SEQ ID NO: 11 provides the sequence of hamster Gne-5-pLentiCRISPRv2(T15261). ACATCCAGCTCAAAGAAGGC.

[0272] SEQ ID NO: 12 provides the amino acid sequence of the linker. Ac-K(PEG5-DBCO)-RRRRRR-K(PEG5-DBCO)-NH2.

Claims

1. A molecule comprising one or more copies of the following glycosylated amino acid sequence: X １ Thr Pro X ２ X ３ where: X １ , X ２ , and X ３ is any amino acid, A molecule in which the threonine (Thr) amino acid residue is O-glycosylated with a sialylated sugar.

2. X １ But it is Pro, X ３ But it is Pro, X １ and X ３ are both Pro, or X ２ The molecule of claim 1 , wherein is Ala.

3. X １ and X ３ and are both Pro.

4. The amino acid sequence X １ Thr Pro X ２ X ３ The molecule of any one of claims 1 to 3, wherein is the amino acid sequence ProThrProAlaPro.

5. 5. The molecule of any one of claims 1 to 4, wherein the sialylated sugar is a sialylated N-acetylhexosamine linked to a hexose.

6. The molecule of any one of claims 1 to 5, wherein the molecule comprises a binding molecule, an interleukin, a cytokine, a chemokine, a hormone, or an enzyme.

7. The molecule of claim 6 , wherein the binding molecule is an antibody.

8. The molecule of claim 7 , wherein the antibody is a bispecific antibody.

9. The molecule of any one of claims 1 to 8, wherein the molecule comprises a nucleic acid molecule.

10. The molecule of claim 9 , wherein the nucleic acid molecule is an siRNA molecule.

11. The sialylated sugar is (a) a chemical group that can be conjugated to a moiety; or The molecule of any one of claims 1 to 10, comprising a chemical group conjugated to the (b) moiety.

12. The molecule of claim 11 , wherein the chemical group comprises a click chemistry group.

13. The molecule of claim 12 , wherein the click chemistry group is an azide group or an alkyne group.

14. 14. The molecule of any one of claims 11 to 13, wherein the sialylated sugar comprises a chemical group conjugated to a moiety.

15. The molecule of claim 14 , wherein the moiety comprises a linker.

16. The molecule of claim 15 , wherein the linker comprises a polyarginine amino acid sequence.

17. The moiety itself is １ Thr Pro X ２ X ３ The molecule of any one of claims 14 to 16, which also comprises at least one copy of a glycosylated amino acid sequence.

18. X present in the moiety １ Thr Pro X ２ X ３ The glycosylated amino acid sequence may be such that the moiety is not an X elsewhere in the molecule. １ ThrProX ２ X ３ 18. The molecule of claim 17, which is used to conjugate to a glycosylated amino acid sequence.

19. the molecule is a bispecific antibody; (a) the X in the molecule １ Thr Pro X ２ X ３ a copy of a glycosylated amino acid sequence is present in an antibody heavy chain and is conjugated to a moiety comprising an antibody light chain, such that the antibody heavy chain and the antibody light chain form an antigen-binding site having a first specificity; (b) the X in the molecule １ Thr Pro X ２ X ３ 19. The molecule of any one of claims 14 to 18, wherein a second copy of the glycosylated amino acid sequence is present in a second antibody heavy chain and is conjugated to a moiety comprising a second antibody light chain, such that the second antibody heavy chain and the second antibody light chain form an antigen-binding site with a second specificity, and wherein the first specificity and the second specificity are not the same.

20. The molecule has two X １ Thr Pro X ２ X ３ a glycosylated amino acid sequence, wherein the Thr of each of said sequences is selected from the two X １ ThrProX ２ X ３ 20. The molecule of any one of claims 1 to 19, which is O-glycosylated with a sugar chain comprising a sialylated sugar containing a chemical group that allows conjugation between glycosylated amino acid sequences.

21. 21. The molecule of claim 20, wherein the conjugation acts as a bridge joining a first binding molecule to a second binding molecule to form a whole molecule, or joining scaffolds each comprising an antigen binding site to form a whole molecule.

22. 22. The molecule of any one of claims 1-21, wherein the sialylated sugar comprises a chemical group that allows for conjugation to a moiety, and the molecule further comprises different means for conjugation at distinct site(s) in the molecule.

23. 23. The molecule of claim 22, wherein the different means for conjugation are one or more cysteine ​​residues.

24. The molecule is (a) X conjugated to a first moiety １ Thr Pro X ２ X ３ a glycosylated amino acid sequence; (b) a cysteine ​​residue conjugated to a second moiety different from the first moiety.

25. The X １ Thr Pro X ２ X ３ 25. The molecule of claim 24, wherein the glycosylated amino acid sequence and the cysteine ​​residue are present together in the same polypeptide in the molecule.

26. The molecule has 2 to 10 of the X １ Thr Pro X ２ X ３ 26. The molecule of any one of claims 1 to 25, comprising a glycosylated amino acid sequence.

27. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 26 and a pharmaceutically acceptable carrier.

28. A molecule according to any one of claims 1 to 26 for use in therapy of the human or animal body.

29. 27. A molecule according to any one of claims 1 to 26 for use in treating a condition selected from cancer, cardiovascular disease, obesity, an autoimmune condition, an inflammatory condition, diabetes, or a CNS disorder.

30. 30. The molecule for use according to claim 29, wherein the molecule is administered to or targeted to the CNS.

31. (a) the molecule comprises an antibody specific for a protein that enables delivery across the blood-brain barrier; and / or (b) the molecule is １ Thr Pro X ２ X ３ 31. The molecule for use according to claim 30, comprising a moiety conjugated to a glycosylated amino acid sequence, said moiety comprising a nucleic acid molecule capable of inhibiting expression of a target gene.

32. 32. The molecule for use according to any one of claims 28 to 31, wherein said molecule comprises an antibody specific for the transferrin receptor (TfR) that enables transport across the blood-brain barrier.

33. 27. A method of treating a condition, comprising administering to a subject in need thereof an effective amount of a molecule according to any one of claims 1 to 26.

34. 34. The method of claim 33, wherein the condition is selected from cancer, cardiovascular disease, obesity, an autoimmune condition, an inflammatory condition, diabetes, or a CNS disorder.

35. (a) the molecule comprises an antibody specific for a protein that enables delivery of the molecule across the blood-brain barrier; and / or 35. The method of claim 34, wherein (b) the molecule comprises a portion that is a nucleic acid molecule capable of inhibiting expression of a target gene.

36. 36. The method of claim 35, wherein the molecule comprises an antibody specific for the transferrin receptor (TfR) that enables transport across the blood-brain barrier.

37. (a) an endogenous UDP-N-acetylglucosamine 2-epimerase-ManNAc kinase gene, wherein the gene has been mutated such that at least UDP-N-acetylglucosamine 2-epimerase function is reduced or eliminated; and (b) a sequence encoding a polypeptide comprising the following amino acid sequence: X １ Thr Pro X ２ X ３ where: X １ , X ２ , and X ３ is any amino acid, A cell in which a threonine (T) amino acid residue is O-glycosylated with a sialylated sugar.

38. the sialylated sugar comprises a chemical group, the chemical group comprising: (a) can be conjugated to a moiety, or 38. The cell of claim 37, conjugated to the (b) moiety.

39. 39. The cell of claim 37 or 38, wherein the UDP-N-acetylglucosamine 2-epimerase-ManNAc kinase gene is mutated such that both the epimerase function and kinase function are reduced or eliminated.

40. 40. The cell of claim 39, wherein both the epimerase function and the kinase function are knocked out.

41. A method for producing a glycosylated polypeptide, comprising culturing the cell according to any one of claims 37 to 40 in a medium supplemented with peracetylated ManNAz.

42. 1. A method for introducing a glycosylation site into a polypeptide, comprising modifying the sequence of said polypeptide to comprise the following amino acid sequence: X １ Thr Pro X ２ X ３ where: X １ , X ２ , and X ３ is any amino acid, The method wherein the threonine (T) amino acid residue is O-glycosylated with a sialylated sugar.

43. the sialylated sugar comprises a chemical group, the chemical group comprising: (a) can be conjugated to a moiety, or 43. The method of claim 42, wherein (b) is conjugated to the moiety.

44. 1. A method of conjugating a molecule to a moiety, said method comprising: (a) providing a molecule according to any one of claims 1 to 26, wherein the sialylated sugar comprises a chemical group that can be conjugated to a desired moiety having a compatible chemical group; (b) contacting the molecule of (a) with the desired moiety; (c) allowing said chemical group of said molecule to undergo conjugation with said desired moiety via said compatible chemical group.

45. 45. The method of claim 44, wherein the conjugation of the molecule and the moiety is via click chemistry.

46. 46. ​​The method of claim 44 or 45, wherein the contacting is in vitro, in vivo, or ex vivo.

47. 1. A method for joining two molecules together, comprising: (a) providing a molecule according to any one of claims 1 to 26, wherein the sialylated sugar comprises a chemical group that can be conjugated to a desired second molecule having a compatible chemical group that allows for said conjugation; (b) contacting the molecule of (a) with the desired second molecule; (c) allowing conjugation of the first molecule with the second molecule via the compatible chemical group.

48. 1. A method for generating a combinatorial library, said method comprising: (a) providing a plurality of molecules according to any one of claims 1 to 26, wherein the molecules are different from one another but the sialylated sugar of each molecule comprises the same chemical group that can be conjugated to a desired moiety; (b) providing a second plurality of molecules, the second plurality of molecules being different from each other and from the molecules of (a), but the second plurality of molecules comprising compatible chemical groups to the chemical groups of the molecules of (a) to allow for conjugation; (c) contacting said molecules of (a) and (b) under conditions that allow for conjugation, thereby allowing for generation of said combinatorial library.

49. 1. A method of conjugating an antibody to a desired moiety, comprising: (a) providing a molecule according to any one of claims 1 to 26, wherein the sialylated sugar of said molecule comprises a chemical group that can be conjugated to a compatible chemical group of said desired moiety, said molecule being either said antibody or a component part of said antibody; (b) conjugating the desired moiety to the molecule via the chemical group, wherein if the molecule is a component part of the antibody rather than the antibody itself, the method further comprises assembling the whole antibody.

50. 1. A method of labeling a molecule, said method comprising: (a) providing a molecule according to any one of claims 1 to 26, wherein the sialylated sugar of said molecule comprises a chemical group that can be conjugated to a desired label that comprises a compatible chemical group that allows for conjugation; (b) providing the label with the compatibility group that allows conjugation of the molecule of (a) to the chemical group; (c) contacting said molecule of (a) with said label of (b) under conditions suitable to result in a conjugate of the two.

51. 27. Use of the molecule of any one of claims 1 to 26 as a capture agent for a desired moiety, wherein the sialylated sugar of the molecule comprises a chemical group that can be conjugated to a desired moiety, said desired moiety comprising a compatible conjugation group for said conjugation.

52. 1. A cell encoding a polypeptide comprising one or more copies of the following glycosylated amino acid sequence: X １ Thr Pro X ２ X ３ where: X １ , X ２ , and X ３ is any amino acid, A cell wherein the threonine (T) amino acid residue at the second position is O-glycosylated with a sialylated sugar.

53. the molecule comprises a chemical group, (a) the chemical group may be conjugated to a moiety, or 53. The cell of claim 52, wherein (b) the chemical group is conjugated to a moiety.

54. A cell encoding the molecule of any one of claims 1 to 26.

Images