Polypeptides modulating siglec dependent immune responses

Abstract

To provide glycosylated polypeptides based on mammalian proteins that exhibit a reduced immune response or increased immune tolerance due to modulated SIGLEC binding, to provide therapeutic methods using the glycosylated polypeptides, to provide protein complexes that exhibit a reduced immune response or increased immune tolerance due to modulated SIGLEC binding, and to provide therapeutic methods using the protein complexes.SOLUTION: Provided is a glycosylated polypeptide comprising an amino acid sequence being identical or homologous to at least a fragment of a mammalian, preferably a human protein, the glycosylated polypeptide containing one or more sialylated O-glycans and showing an increased binding affinity to one or more SIGLECs, selected from SIG-5, SIG-7, SIG-8, and SIG-9 compared to the mammalian protein or fragment thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to glycosylated polypeptides based on mammalian proteins that exhibit a reduced immune response or increased immune tolerance due to modulated SIGLEC binding, and to therapeutic methods using the glycosylated polypeptides. The present invention further relates to protein complexes that exhibit a reduced immune response or increased immune tolerance due to modulated SIGLEC binding, and to therapeutic methods using the protein complexes. [Background technology]

[0002] Hemophilia A is a group of genetic disorders that impair the body's ability to regulate blood clotting, or coagulation. In the most common form, hemophilia A, there is a deficiency of coagulation factor VIII (FVIII). Hemophilia A occurs in 1 in 5,000–10,000 live male births. The FVIII protein is an essential cofactor in blood clotting with multifunctional properties. FVIII deficiency can be treated with platelet-derived FVIII concentrates or recombinantly produced FVIII. Treatment with FVIII concentrates has led to a normalization of life for hemophilia patients. Historically, hemophilia A has been treated with FVIII derived from human plasma. In plasma, under normal conditions, FVIII molecules are constantly associated with their cofactor, von Willebrand factor (vWF), which stabilizes the FVIII molecule from various denatured forms.

[0003] Many methods for purifying factor VIII from plasma or recombinant factor VIII-producing cultures (rFVIII), with or without von Willebrand factor, have been described. In the 1990s, the first recombinant FVIII (rFVIII) products were commercially available. These products were divided into full-length rFVIII molecules, which mimic the predominant form of FVIII in plasma, and B-domain deleted rFVIII molecules, in which one inactive portion (the B domain) was removed (Eriksson et al., 2001). Both rFVIII products have high purity (all without vWF).

[0004] Patients with hemophilia A are treated with FVIII on demand or as prophylactic therapy administered several times a week. For prophylactic therapy, 15-25 IU / kg body weight of FVIII is administered three times a week, which is necessary due to the continuous need for FVIII and its short half-life in the blood system, which is only about 11 hours in humans (Ewenstein et al., 2004).

[0005] Frequently, continuous treatment with exogenously administered FVIII provokes a response of the patient's immune system (Saenko et al., Haemophilia 8:1-11 (2002)), which represents a serious limitation of the treatment.

[0006] Currently, the most common option for achieving immune tolerance in patients with hemophilia A (congenital FVIII deficiency) and inhibitors is immune tolerance induction (ITI), in which high doses of FVIII are administered over an extended period of time. However, this treatment can take up to two years, remains unsuccessful in approximately 30% of patients, is very costly, and cannot be used in a prophylactic manner to suppress the early development of inhibitory antibodies.

[0007] Therefore, approaches to attenuate the immune response are needed. One promising approach is the optimization of glycosylation of either FVIII or its binding partner vWF.

[0008] For example, WO 2014 / 176125A1 relates to an immunoconjugate for inducing antigen-specific immune tolerance to FVIII. The immunoconjugate is a FVIII protein conjugated to a specific glycan ligand that targets SIGLEC, i.e., SIG-1 or SIG-10 (or ortholog SIG-G), expressed on B cells. The glycan ligand is specifically coupled to a liposome into which FVIII is introduced.

[0009] Sialic Acid Binding Immunoglobulin Lectins (SIGLECs) comprise a family of 15 human and 9 murine cell surface receptors expressed on various leukocytes of the immune system, except for most T cells in mice and humans. SIGLECs are localized on different cell types and bind to different glycan structures (reviewed in Paulson et al. 2012). For example, binding of vWF and FVIII to SIG-5 has been demonstrated (Pegon 2012). However, the binding mechanism remains unclear.

[0010] A different approach is described in WO 2014 / 179184A1. The authors suggest the addition of a SIGLEC ligand to reduce unwanted antibody immune responses and induce immune tolerance to blood clotting factors, such as FVIII. The SIGLEC ligand is selected from 9-N-biphenylcarboxyl-NeuAca2-6Gal~l-4GlcNAc (6'-BPCNeuAc), NeuAca2-6Galwl-4GlcNAc, and NeuAca2-6Galwl-4(6-sulfo)GlcNAc. The SIGLEC ligand is conjugated to the clotting factor via a water-soluble polymer. Summary of the Invention [Means for solving the problem]

[0011] The present invention is based on the finding that naturally occurring glycan structures in plasma-derived proteins, particularly vWF, enable interaction with a group of SIGLECs, particularly SIG-5, SIG-7, SIG-8, and SIG-9. Furthermore, the inventors have found that modifying the glycan structure on a protein can increase interaction with SIGLECs, such as SIG-5, SIG-7, SIG-8, and SIG-9. This increase results in a reduced immune response and / or increased immune tolerance in patients to whom the protein is administered.

[0012] Thus, according to a first aspect, the present invention provides a glycosylated polypeptide comprising an amino acid sequence identical or homologous to at least a fragment of a mammalian, preferably human, protein, wherein the glycosylated polypeptide contains one or more sialylated O-glycans and has increased binding affinity for one or more SIGLECs selected from the group consisting of SIG-5, SIG-7, SIG-8, and SIG-9 compared to the mammalian protein or fragment thereof.

[0013] We specifically defined glycan structures that are required for interaction with SIGLEC but whose addition also results in increased binding to SIGLEC: sialylated core 2 type O-glycans and / or extended core 1 type O-glycans.

[0014] Thus, a glycosylated polypeptide according to the first aspect can also be defined as a glycosylated polypeptide comprising an amino acid sequence that is identical or homologous to at least a fragment of a mammalian, preferably human, protein, and that contains one or more sialylated O-glycans, wherein the combined number of sialylated core 2 type O-glycans and sialylated extended core 1 type O-glycans of the glycosylated polypeptide is greater than the combined number of sialylated core 2 type O-glycans and sialylated extended core 1 type O-glycans of the mammalian protein or fragment thereof.

[0015] Proteins having a glycan composition comprising sialylated core 2 type O-glycans and / or sialylated extended core 1 type O-glycans, particularly sialylated core 2 type O-glycans, can be used to modify a patient's immune response to therapeutic proteins by combined administration.

[0016] Thus, according to a second aspect, the present invention relates to the use of a glycosylated polypeptide containing one or more sialylated O-glycans and exhibiting binding to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8 and SIG-9 for reducing the immune response or increasing immune tolerance in a patient against a therapeutic protein.

[0017] By using modified glycosylated polypeptides that have binding affinity for SIGLEC, it is possible not only to directly modify the SIGLEC binding affinity of the modified polypeptide itself, but also to modify the SIGLEC binding affinity of a protein complex or composition of which the glycosylated polypeptide is a part, such as a complex of factor VIII (FVIII) and von Willebrand factor (vWF).

[0018] Thus, according to a third aspect, the present invention provides a protein composition comprising first and second polypeptides, wherein the first polypeptide is a glycosylated polypeptide containing one or more sialylated O-glycans, and the second polypeptide contains an amino acid sequence that is homologous or identical to a second mammalian, particularly human, protein, and has increased binding affinity for one or more SIGLECs selected from SIG-5, SIG-7, SIG-8, and SIG-9 compared to the second polypeptide. The first and second polypeptides of the composition according to the third aspect preferably form a protein complex.

[0019] According to a fourth aspect, the present invention provides an isolated polypeptide comprising a nucleic acid sequence encoding a glycosylated polypeptide according to the first aspect of the invention. In a fifth aspect, the present invention also relates to an expression vector comprising a polynucleotide according to the fourth aspect of the invention.

[0020] The glycosylated polypeptides according to the first aspect, in particular vWF or FVIII, and the compositions according to the third aspect, in particular complexes of FVIII and vWF, are particularly useful in medical therapy due to the reduced immune response.

[0021] Thus, according to a fourth aspect, the present invention provides a glycosylated polypeptide as defined in the first aspect or a composition as defined in the third aspect for use in the treatment or prevention of bleeding disorders. [Brief explanation of the drawings]

[0022] [Figure 1] The results of a vWF binding assay for different SIGLECs are shown. The absorbance at 492 nm is proportional to the vWF binding to the specified SIGLEC or control. SIG-2, SIG-5, SIG-7, SIG-F, SIG-9, and SIG-10 were immobilized on a microtiter plate at 500 ng / well via protein A. Biotinylated vWF was added at concentrations ranging from 0 to 0.8 μg / mL. After washing, binding was visualized with HRP-conjugated streptavidin, and the absorbance was measured at 492 nm. Anti-vWF and anti-chicken IgY were used as controls. [Figure 2] Schematic representation of the vWF domain structure including N- and O-glycosylation, V8 protease cleavage sites and the fragments obtained after V8 protease cleavage is shown. [Figure 3]Figure 1 shows the results of binding studies of N- and C-terminal fragments of vWF to SIGLECs SIG-5, SIG-7, SIG-F, and SIG-9. The absorbance at 492 nm is proportional to the vWF fragment binding to the specified SIGLEC or control, respectively. SIG-5, SIG-7, SIG-F, and SIG-9 were immobilized at 500 ng / well on a microtiter plate via protein A. Biotinylated N-terminal VWF fragments (dark gray bars) and C-terminal vWF fragments (light gray bars) were added at a concentration of 1 μg / mL. After washing, binding was visualized with HRP-conjugated streptavidin, and absorbance was measured at 492 nm. Anti-chicken IgY was used as a negative control. [Figure 4] The results of binding assays of desialylated, N-deglycosylated, and unprocessed vWF N-terminal fragments are shown. The absorbance at 492 nm is proportional to the vWF N-terminal fragment binding to the specified SIGLEC or control, respectively. Undigested N-terminal vWF fragments are represented by white bars, PNGase F-deglycosylated fragments by gray bars, and desialylated fragments by black bars. SIG-5, SIG-7, SIG-F, and SIG-9 were immobilized on microtiter plates at 500 ng / well via protein A. Biotinylated N-terminal vWF fragments (undigested, digested with PNGase F, or sialidase A) were added at a concentration of 8 μg / mL. After washing, binding was visualized with HRP-conjugated streptavidin, and absorbance was measured at 492 nm. Anti-chicken IgY was used as a control. [Figure 5]Figure 1 shows the results of binding studies of O-glycosylated cluster I and cluster II to SIGLEC. Absorbance at 492 nm is proportional to the cluster I fragment (light gray bars) or cluster II fragment (dark gray bars) binding to the specified SIGLEC or control, respectively. SIG-5, SIG-7, SIG-F, SIG-9, and SIG-10 were immobilized at 500 ng / well on a microtiter plate via protein A. Biotinylated cluster I and cluster II were added at a concentration of 4 μg / mL. After washing, binding was visualized with HRP-conjugated streptavidin, and absorbance was measured at 492 nm. Anti-chicken IgY was used as a negative control. [Figure 6] Figure 1 shows the results of binding assays of O-glycosylated cluster II to SIGLEC before and after treatment with sialidase A. Absorbance at 492 nm is proportional to the untreated cluster II fragment (light gray bars) or sialidase A-digested cluster II fragment (dark gray bars) binding to the specified SIGLEC or control, respectively. SIG-5, SIG-7, SIG-F, and SIG-9 were immobilized at 500 ng / well on a microtiter plate via protein A. Biotinylated cluster II before digestion (light gray bars) and after sialidase A digestion was added at a concentration of 2 μg / mL. After washing, binding was visualized with HRP-conjugated streptavidin, and absorbance was measured at 492 nm. Anti-chicken IgY was used as a negative control. [Figure 7] Schematic representation of recombinantly expressed vWF fragments Seq11 and Seq12 is shown. [Figure 8]Figure 1 shows MALDI MS spectra of an O-glycopeptide isolated from Seq11 after trypsin / chymotrypsin digestion, sialidase A digestion, and lectin enrichment. The identified peptide sequence is KVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHGSAW (SEQ ID NO: 6), with the last four amino acids (underlined) corresponding to a tag attached to the C-terminus of the sequence. The top spectrum shows a fully O-glycosylated glycopeptide, and the bottom spectrum shows the same glycopeptide after O-glycosidase digestion. [Figure 9] Figure 1 shows a MALDI MS spectrum of an O-glycopeptide isolated from Seq 12 after trypsin / chymotrypsin digestion, sialidase A digestion, and lectin enrichment. The identified peptide sequence is [KVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHGSAW (SEQ ID NO: 7)], where the last four amino acids (underlined) correspond to a tag attached to the C-terminus of the sequence. The top spectrum shows a fully O-glycosylated glycopeptide, and the bottom spectrum shows the same glycopeptide after O-glycosidase digestion. [Figure 10] Figure 1 shows the results of binding studies of recombinant polypeptides Seq11 and Seq12 to SIGLEC. Absorbance at 492 nm is proportional to Seq11 (dark gray bars) or Seq12 (light gray bars) binding to the specified SIGLEC or control, respectively. SIG-5, SIG-7, SIG-F, and SIG-9 were immobilized on microtiter plates at 500 ng / well via protein A. Strep-Tag-bearing sequences were applied to the plates at an equimolar concentration of 42 nM. After washing, binding was visualized with HRP-conjugated streptavidin, and absorbance was measured at 492 nm. Anti-chicken IgY was used as a negative control, and anti-vWF pAb was used as a positive control. [Figure 11]Figure 1 shows the results of binding assays of recombinant polypeptides Seq11 and Seq12 to SIGLEC after sialidase A treatment. Absorbance at 492 nm is proportional to Seq11 (dark gray bars) or Seq12 (light gray bars) binding to the specified SIGLEC or control, respectively. SIG-5, SIG-7, SIG-F, and SIG-9 were immobilized on microtiter plates at 500 ng / well via protein A. Strep-Tag-bearing sequences were enzymatically desialylated and applied to the plates at an equimolar concentration of 42 nM. After washing, binding was visualized with HRP-conjugated streptavidin, and absorbance was measured at 492 nm. Anti-chicken IgY was used as a negative control, and anti-vWF pAb was used as a positive control. [Figure 12] 1 shows the results of Scatchard analysis of the concentration-dependent binding and specific binding curves of recombinant polypeptide Seq11 to SIGLEC. [Figure 13] 1 shows the results of Scatchard analysis of the concentration-dependent binding and specific binding curves of recombinant polypeptide Seq12 to SIGLEC. [Figure 14] A summary of the KD values obtained from Scatchard analysis performed on the curves presented in Figures 12 and 13 is shown. [Figure 15] Figure 1 shows the calculated dissociation affinity constant (KD) values for the binding of Seq11, Seq12 and full-length plasma VWF to recombinant FVIII. Data were obtained by SPR. [Figure 16] Effect of N- and C-terminal VWF fragments on IL-12p70 and IFN-γ. moDCs were cultured with various concentrations of VWF fragments either without (left column) or with (right column) 0.1 μg / ml LPS. Extracellular levels of cytokines were simultaneously measured via cytometric bead array. IL-12p70 and IFN-γ levels in unstimulated cells were below the detection limit for most donors (b-d, 0.6 pg / ml for IL-12p70 and 1.8 pg / ml for IFN-γ). Data are presented as mean ± SEM, and each dot represents one donor. [Figure 17] Figure 1 shows the phosphorylation of SIGLEC and the adaptor molecules SHP-1 and SHP-2 involved in SIGLEC signaling after stimulation of moDCs with 500 nM of the N-terminal fragment of VWF for 10 minutes. Cells stimulated with the same volume of 100 mM NaCl served as a control. Analysis of immunoreceptor phosphorylation in cell lysates was performed using the Proteome Profiler Human Phospho-Immunoreceptor Array Kit. Results are shown as the mean pixel density ± SEM of two to four individual experiments. DETAILED DESCRIPTION OF THE INVENTION

[0023] In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given such terms, the following definitions are provided.

[0024] definition As used herein, a "peptide" can be composed of any type of any number of amino acids, preferably naturally occurring amino acids, linked by peptide bonds. In particular, a peptide comprises at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12, or at least 15 amino acids. Furthermore, there is no upper limit to the length of a peptide. However, preferably, a peptide according to the present invention does not exceed 500 amino acids in length, more preferably, it does not exceed 300 amino acids in length; even more preferably, it is not longer than 250 amino acids.

[0025] Thus, the term "peptide" includes "oligopeptide," which generally refers to a peptide having a length of 2 to 10 amino acids, and "polypeptide," which generally refers to a peptide having a length of more than 10 amino acids.

[0026] As used herein, the term "protein" refers to a peptide having at least 60, at least 80, and preferably at least 100 amino acids. The terms "polypeptide" and "protein" are used interchangeably. As used herein, polypeptides and proteins include chemically synthesized proteins and naturally synthesized proteins encoded by genes. Polypeptides or proteins can be obtained from natural sources, such as human blood, or produced as recombinant proteins in cell culture.

[0027] The term "mammalian protein" as used herein relates to a naturally occurring mammalian protein, i.e., a protein that is naturally expressed by a mammalian organism. Thus, a mammalian protein has a naturally occurring amino acid sequence and naturally occurring post-translational modifications, such as glycosylation. According to the present invention, the terms mammalian protein and naturally occurring mammalian protein can be used interchangeably.

[0028] The term "human protein" as used herein relates to a naturally occurring human protein, i.e., a protein that is naturally expressed by a human organism. Thus, a human protein has a naturally occurring amino acid sequence and naturally occurring post-translational modifications, such as glycosylation. According to the present invention, the terms human protein and naturally occurring human protein are used interchangeably.

[0029] As used herein, a "recombinant protein" or "recombinant polypeptide" is one that is encoded by a transgene that is introduced into a cell by molecular biology techniques. Proteins can be modified by chemical methods or enzymatically in post-translational processes.

[0030] The term "fusion protein" according to the present invention relates to a protein created through the joining of two or more genes, cDNAs or sequences that originally coded for separate proteins / peptides. The genes may be naturally occurring in the same organism or in different organisms, or may be synthetic polynucleotides.

[0031] The term "therapeutic protein" as used herein relates to a protein or polypeptide that has a therapeutic effect, i.e. a protein that is used as an active pharmaceutical ingredient.

[0032] The relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity." For the purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 3.0.0 or later. Optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output value of Needle labeled "longest identity" (obtained using the no brief option) is used as the percent identity and is calculated as follows: (Identical residues × 100) / (length of alignment − total number of gaps in the alignment)

[0033] The transitional term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended, not excluding additional, unrecited elements or method steps. The transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim, except for impurities normally associated therewith. When the phrase "consisting of" appears in a section of the body of a claim rather than immediately following the preamble, it limits only the elements set forth in that section; other elements are not excluded from the claim as a whole. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention. "'Consisting essentially of' in a claim occupies a neutral position between a closed claim written in a 'consisting of' format and a fully open claim drafted in a 'comprising' format."

[0034] As used herein, "homologous" means that the respective amino acid sequence and nucleotide sequence have a specified degree of identity with the reference amino acid sequence and the target nucleotide sequence. A homologous sequence is understood to include an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identical to the target sequence using the conventional sequence alignment tool Clustal V with default parameters. Typically, a homolog contains the same active site residues as the target amino acid sequence, but may contain any number of conservative amino acid substitutions. As used herein, "identical" refers to 100% amino acid or nucleotide sequence identity with the reference sequence.

[0035] The term "recombinant," when used with reference to a subject cell, nucleic acid, protein, or vector, indicates that the subject has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes at levels or under conditions that are different from those found in nature.

[0036] As used herein, the terms "transformed," "stable transformed," and "transgenic" when used with respect to cells mean that the cell contains a non-native (e.g., heterologous) nucleic acid sequence that is integrated into its genome or carried as an episome that is maintained through multiple generations.

[0037] As used herein, the term "fragment" refers to a polypeptide that has one or more amino acid amino- and / or carboxyl-terminal deletions compared to the native or wild-type protein, but the remaining amino acid sequence is identical to the corresponding portion in the amino acid sequence deduced from the full-length cDNA. Fragments are typically at least 50 amino acids in length.

[0038] As used herein, the term "glycosylation" refers to the attachment of glycans to a molecule, e.g., a protein. Glycosylation can be an enzymatic reaction. The attachment can be via a covalent bond. Thus, as used herein, a glycosylated polypeptide is a polypeptide to which glycans are attached. The phrase "hyperglycosylated" refers to a molecule, such as an enzyme, that is glycosylated at all or nearly all of the available glycosylation sites, e.g., O-linked or N-linked glycosylation sites.

[0039] As used herein, the term "glycan" refers to a polysaccharide or oligosaccharide, or the carbohydrate segment of a glycoprotein or glycosylated polypeptide. Glycans can be homo- or heteropolymers of monosaccharide residues. They can be linear or branched molecules. However, glycans typically contain at least three sugars and can be linear or branched. Glycans can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6' sulfo-N-acetylglucosamine, etc.).

[0040] As used herein, the term "O-glycan" refers to glycans commonly found covalently attached to serine and threonine residues of mammalian glycoproteins. O-glycans can be α-linked to the -OH of serine or threonine via an O-glycosidic bond via an N-acetylgalactosamine (GalNAc) moiety. Other linkages include α-linked O-fucose, β-linked O-xylose, α-linked O-mannose, β-linked O-GlcNAc (N-acetylglucosamine), α- or β-linked O-galactose, and α- or β-linked O-glucose glycans.

[0041] As used herein, the term "sialylated" refers to a molecule, particularly a glycan, that has been reacted with sialic acid or a derivative thereof.

[0042] As used herein, the term "binding affinity" or "affinity" refers to the strength of binding between two molecules, particularly a ligand and a protein target. Binding affinity is influenced by non-covalent intermolecular interactions between the two molecules, such as hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces.

[0043] As used herein, immune response refers to adaptive or innate immune response. Innate immune response refers to non-specific defense mechanisms that are activated immediately or within a few hours after the appearance of an antigen in the body. These mechanisms include immune system cells that attack physical barriers, such as skin, chemicals in the blood, and foreign cells in the body. Innate immune response is activated by the chemical properties of antigens. Adaptive immune response refers to antigen-specific immune response. For this reason, antigens must first be processed and recognized. Once an antigen is recognized, the adaptive immune system creates a large number of immune cells that are specifically designed to attack that antigen.

[0044] As used herein, "immune tolerance" (or simply "tolerance") is the process by which the immune system does not attack antigens. It occurs in three forms: central tolerance, peripheral tolerance, and acquired tolerance. Tolerance can be either "natural" or "self-tolerance," in which the body does not mount an immune response to self-antigens, or "induced tolerance," in which tolerance to antigens can be created by manipulation of the immune system.

[0045] Glycosylated Polypeptides According to a first aspect, the present invention provides a glycosylated polypeptide comprising an amino acid sequence identical or homologous to at least a fragment of a mammalian, preferably human, protein, wherein the glycosylated polypeptide contains one or more sialylated O-glycans and has increased binding affinity for one or more SIGLECs compared to the mammalian protein or fragment thereof.

[0046] The glycosylated polypeptides according to the present invention are based on mammalian proteins, i.e., contain amino acid sequences identical or homologous to those of mammalian proteins. Mammalian proteins are, in particular, human proteins. The human proteins to which the amino acid sequence of the glycosylated peptide is homologous or identical are preferably glycosylated proteins.

[0047] The human protein is more preferably a human blood protein, which may be a human blood coagulation factor, a transport protein, a protease inhibitor, an immunoglobulin, a cell-associated plasma protein, an apolipoprotein, a complement factor, a growth factor, an antiangionetic protein, a highly glycosylated protein, a blood factor, or another human blood protein.

[0048] The human blood coagulation factor is particularly selected from the group consisting of fibrinogen, fibrin monomer, prothrombin, thrombin, FV, FX, FIX, FVII, FVIII, FXI, FXII, and FXIII, von Willebrand factor, and ADAMTS13.

[0049] It is recognized that the coagulation factors FV, FX, FIX, FVII, FVIII, FXI, FXII, and FXIII exist in inactive and active forms. Thus, in the context of the present invention, references to FV, FX, FIX, FVII, FVIII, FXI, FXII, and FXIII include the activated forms FVa, FXa, FIXa, FVIIa, FVIIIa, FXIa, FXIIa, and FXIIIa, respectively, unless otherwise specified or the context indicates that the activated forms cannot logically be included. Thus, for example, in this context, FV, FX, FIX, FVII, FVIII, FXI, FXII, and FXIII can be read as FV / FVa, FX / FXa, FIX / FIXa, FVII / FVIIa, FVIII / FVIIIa, FXI / FXIa, FXII / FXIIa, and FXIII / FXIIIa.

[0050] The transport protein may be selected from albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobin, and hemopexin.

[0051] Possible protease inhibitors are, for example, β-antithrombin, α-antithrombin, oxidized antithrombin, 2-macroglobulin, Cl inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), protein C, protein S, and protein Z.

[0052] Examples of immunoglobulins are, for example, polyclonal antibodies (IgG), monoclonal antibodies, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, IgM, IgE, IgD, and Bence Jones proteins.

[0053] Cell-associated plasma proteins can be, for example, fibronectin, thromboglobulin, platelet factor 4. Examples of apolipoproteins are apo AI, apo A-II, and apo E.

[0054] Complement factors according to the present invention include, for example, factor B, factor D, factor H, factor I, C3b inactivating factor, properdin, C4 binding protein, and the like.

[0055] Examples of growth factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-α), fibroblast growth factor (FGF), and hepatocyte growth factor.

[0056] Anti-angiogenic proteins include latent antithrombin, pre-latent antithrombin, oxidized antithrombin and plasminogen.

[0057] Examples of hyperglycosylated proteins are alpha-1-acid glycoprotein, antichymotrypsin, inter-α-trypsin inhibitor, α-2-HS glycoprotein, C-reactive protein. Blood factors can be, for example, erythropoietin, interferon, tumor factor, tPA, gCSF, etc.

[0058] Other human blood proteins include histidine-rich glycoprotein, mannan-binding lectin, C4-binding protein, fibronectin, GC-globulin, plasminogen / plasmin, alpha-1 microglobulin, and C-reactive protein.

[0059] The human protein is in particular selected from vWF, FVIII, FVII, FIX, ADAMTS13.

[0060] Human factor VIII is encoded by the F8 gene, which contains 187,000 base pairs in six exons. The transcribed mRNA is 9,029 base pairs long and is translated into a protein with 2,351 amino acids, from which 19 amino acids are removed by post-translational modification. Human FVIII molecules are glycosylated on 31 amino acid side chains (25 N-glycosylations, 6 O-glycosylations).

[0061] After translation, the amino acid chain is cleaved by specific proteases at positions that result in the formation of a heavy chain with a molecular mass of approximately 200 kDa and a light chain with a molecular mass of approximately 80 kDa. The domain organization is typically characterized as A1-A2-B-A3-C1-C2. The light chain is composed of domains A3-C1-C2. The heavy chain is generally composed of domains A1-A2-B. The heavy chain found in plasma has a heterogeneous composition, with molecular masses ranging from 90 to 200 kDa. This is due to heterogeneity in its glycosylation, the presence of splice variants, and the presence of proteolytic products, such as the B-domain-depleted heavy chain A1A2. The amino acid sequence of full-length FVIII is identified by amino acids 20 to 2351 of P00451 in SwissProt, July 21, 1986.

[0062] The human protein is preferably full-length FVIII defined by amino acids 20 to 2351 of P00451 of SwissProt, July 21, 1986, B-domain deleted FVIII, or a FVIII protein in which part of the B-domain has been replaced by a linker.

[0063] vWF is a multimeric adhesive glycoprotein present in mammalian plasma that has multiple physiological functions. During primary hemostasis, vWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix, such as collagen. Additionally, vWF functions as a carrier and stabilizing protein for the procoagulant factor VIII. vWF is synthesized in endothelial cells and megakaryocytes as a 2,813-amino acid precursor molecule. The precursor polypeptide, prepro-vWF, consists of a 22-residue signal peptide, a 741-residue propeptide, and the 2,050-residue polypeptide found in mature plasma von Willebrand factor (Fischer et al., 1994). Full-length vWF is identified by Uniprot entry P04275.

[0064] Upon secretion into plasma, vWF circulates in the form of various species with different molecular sizes. These vWF molecules consist of oligomers and multimers of a mature subunit of 2,050 amino acid residues. vWF is normally found in plasma as multimers ranging in size from approximately 500 to 20,000 kDa (Furlan et al. 1996). vWF specifically has the amino acid sequence of any of the sequences in Uniprot entry P04275. More preferably, the vWF protein is identified by SEQ ID NO: 1.

[0065] The glycosylated polypeptide may, for example, contain a fragment of vWF as defined in WO 2015 / 185758 A2. As shown in WO 2015 / 185758 A2, a complex of FVIII and a vWF fragment as defined therein exhibits reduced binding to phospholipid membranes compared to FVIII alone and reduced binding to collagen III and heparin compared to a complex of FVIII and full-length vWF.

[0066] In this regard, a fragment of vWF is in particular a fragment starting from amino acid 1 of SEQ ID NO: 1. Amino acids 1 to 272 of SEQ ID NO: 1 comprise the FVIII binding domain of vWF.

[0067] The fragment of vWF preferably comprises the amino acid sequence of SEQ ID NO: 1. 1 Starting from, preferably 1142~1390 The fragment more preferably ends with an amino acid of SEQ ID NO: 1 in the range of: 1267~1390 More preferably, the vWF fragment ends with an amino acid in the range of 1337~1390 and ending with an amino acid of SEQ ID NO: 1 in the range of

[0068] It should be understood that glycosylated polypeptides have increased binding affinity compared to mammalian proteins or fragments defined by the amino acid sequence contained in the glycosylated peptide. Thus, when a glycosylated polypeptide comprises the amino acid sequence of a full-length mammalian protein, the glycosylated polypeptide has a higher affinity for SIGLEC compared to the full-length mammalian protein.

[0069] On the other hand, if the glycosylated polypeptide comprises a fragment of a mammalian protein defined by a subsequence of the mammalian protein, the glycosylated polypeptide will have an increased binding affinity compared to the same fragment derived from a naturally occurring protein, for example, if the amino acid sequence in the glycosylated polypeptide is identical or homologous to a fragment of vWF, the glycosylated polypeptide according to the first aspect will have an increased binding affinity for one or more SIGLECs compared to the same fragment obtained from fragmentation of plasma-derived vWF.

[0070] As shown in the Examples, the glycan structures of vWF that specifically bind to at least SIGLECs SIG-5, SIG-7, SIG-8 and SIG-9 have been determined (see Example 1). Thus, according to one embodiment of the first aspect, the one or more SIGLECs are selected from the group of SIG-5, SIG-7, SIG-8 and SIG-9.

[0071] We surprisingly found that in human vWF, O-glycans are responsible for binding to SIG-5, SIG-7, SIG-8, and SIG-9. In contrast, N-glycans do not exhibit any binding to their SIGLEC counterparts (see Example 2). This is particularly surprising, since so far, it has been N-glycans that have been shown to interact with SIGLEC (Lai et al., 2015).

[0072] We further determined that not only must O-glycans be sialylated for binding to SIG-5, SIG-7, SIG-8, and SIG-9, but a minimum proportion of core 2 glycans must also be present (see Example 4).

[0073] Thus, SIGLEC binding and the resulting reduced immune response are based on an increased number or proportion of sialylated core type 2 O-glycans in the glycosylated protein compared to the number of sialylated core type 2 O-glycans on the mammalian protein or fragment thereof.

[0074] Due to structural similarity, it is assumed that sialylated extended core 1 type O-glycans have the same effect as sialylated core 2 glycans. Therefore, to increase binding affinity to SIGLEC as defined above, it is preferable to increase the combined number or proportion of sialylated core 2 type and extended core 1 type O-glycans.

[0075] Thus, according to one embodiment, the number of sialylated core type 2 and / or extended core type 1 O-glycans of the glycosylated polypeptide is greater than the number of sialylated core type 2 and / or extended core type 1 O-glycans of the mammalian protein or fragment thereof, and in this regard, the proportion of sialylated core type 2 and / or extended core type 1 O-glycans is also increased compared to the proportion of core type 2 and / or extended core type 1 O-glycans of the mammalian protein.

[0076] This means that the combined number of sialylated core 2 type O-glycans and sialylated extended core 1 type O-glycans of a glycosylated polypeptide is greater than the combined number of sialylated core 2 type O-glycans and sialylated extended core 1 type O-glycans of a mammalian protein or fragment thereof.

[0077] Alternatively, only the number of sialylated core 2 type O-glycans can be increased, and in this regard, the proportion of sialylated core 2 type O-glycans is also increased relative to the proportion of core 2 type O-glycans of mammalian proteins.

[0078] The SIGLECs shown to bind are involved in human and mouse immune responses. SIGLECs share a single N-terminal V-set Ig domain that binds sialic acid-containing ligands and a variable number of C2-set Ig domains that extend the ligand-binding side distal to the membrane surface.

[0079] Furthermore, many SIGLECs possess cytoplasmic tyrosine motifs commonly found in coreceptors involved in regulating cell signaling, such as immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and ITIM-like motifs. Other SIGLECs do not contain tyrosine motifs but contain positively charged transmembrane regions that allow association with adaptor proteins. SIGLECs do not recognize danger-associated molecular patterns (DAMPs), but instead recognize determinants of "self."

[0080] SIGLECs bind to such sialylated autoligands in cis on the same cell and in trans on neighboring cells. Human SIGLECs are commonly referred to as SIG-1 through SIG-14. Mouse SIGLECs SIG-E, SIG-F, and SIG-G are orthologues of human SIGLECs SIG-9, SIG-8, and SIG-10, respectively.

[0081] SIG-1 to SIG-4 are also known as sialoatesin, CD22, CD33, and MAG, respectively. CD22 and SIG-10 are localized on B cells, SIG-5 on neutrophils and monocytes, SIG-7 on NK cells, SIG-8 on eosinophils, and SIG-9 on monocytes, neutrophils, and dendritic cells (Paulsen et al., 2012).

[0082] SIGLECs bind to a variety of different glycan structures. SIGLECs SIG-2, SIG-5, SIG-7, SIG-8, SIG-9, and SIG-10 each have different glycan preferences (Paulson et al., 2012). SIGLECs play a role in innate and adaptive immunity. In particular, SIG-2 and SIG-10 are localized on human and mouse B cells.

[0083] According to Paulsen et al., SIG-2 and SIG-10 are thought to synergistically contribute to peripheral B cell tolerance. Furthermore, SIGLEC is thought to act as an inhibitory coreceptor for Toll-like receptors (TRLs). In this regard, cross-linking of SIG-7 or SIG-9 to activating receptors has been shown to inhibit the cytolytic activity of NK cells against tumor cells and the release of chemical mediators from mast cells, respectively.

[0084] Furthermore, cross-linking of SIG-E (SIG-9) and SIG-11 with immobilized antibodies results in inhibition of cytokine production in response to LPS in macrophages.

[0085] Notably, local expression of SIG-5 and SIG-9 in macrophage cell lines has been shown to inhibit TNF-alpha production and enhance IL-10 production in response to peptidoglycan, ATLR2 ligands, LPS, and CpG. Furthermore, LPS-induced SIG-E (SIG-9) expression in macrophages is thought to mediate TRL signaling. Furthermore, sialylated pathogens attenuate immune responses via SIGLEC. For example, group B streptococcus expresses Neu-Acα-1Galβ-14GlcNAc residues on its capsular polysaccharide, which recruits SIGLEC-9 on neutrophils, resulting in the suppression of neutrophil bactericidal function.

[0086] Therefore, without being bound by theory, it is believed that binding to SIGLEC on antigen-presenting cells (e.g., dendritic cells) results in downregulation of pro-inflammatory cytokines and upregulation of immunosuppressive receptor expression on the cell surface. Furthermore, binding results in increased production of anti-inflammatory cytokines and reduced production of pro-inflammatory cytokines, resulting in inhibition of T cell proliferation and antibody production. Thus, binding of SIGLEC to SIG-5, SIG-7, SIG-8, and SIG-9 results in a reduced immune response or increased immune tolerance when glycosylated polypeptides are administered to patients.

[0087] Thus, a glycosylated polypeptide according to the first aspect is a glycosylated polypeptide comprising an amino acid sequence that is identical or homologous to at least a fragment of a mammalian, preferably human, protein, and that contains one or more sialylated O-glycans, and that, compared to the mammalian protein or fragment thereof, - the human immune response to glycosylated polypeptides is reduced; and / or - Increased human immune tolerance to glycosylated polypeptides It may also be defined as a glycosylated polypeptide.

[0088] On the other hand, a more structural definition of the first aspect of the invention is a glycosylated polypeptide comprising an amino acid sequence that is identical or homologous to at least a fragment of a mammalian, preferably human, protein, and that contains one or more sialylated O-glycans, wherein the combined number of sialylated core 2 type O-glycans and sialylated extended core 1 type O-glycans of the glycosylated polypeptide is greater than the combined number of sialylated core 2 type O-glycans and sialylated extended core 1 type O-glycans of the mammalian protein or fragment thereof.

[0089] Preferably, a glycosylated polypeptide comprising an amino acid sequence that is identical or homologous to at least a fragment of a mammalian, preferably human, protein, and that contains one or more sialylated O-glycans, wherein the number of sialylated core 2 type O-glycans of the glycosylated polypeptide is greater than the combined number of sialylated core 2 type O-glycans of the mammalian protein or fragment thereof.

[0090] To couple O-glycans to the amino acid sequence of the glycosylated polypeptide, it contains one or more O-glycosylation sites. The O-glycosylation site of the glycosylated polypeptide can be the standard O-glycosylation site serine (Ser) and threonine (Thr). However, further attachment of O-glycans to tyrosine (Tyr), hydroxylysine (hydroxy-Lys), or hydroxyproline (hydroxy-Pro) has been described and is considered in the context of the present invention. Thus, one or more O-glycosylation sites in the glycosylated polypeptide can be selected from Ser, Thr, Tyr, hydroxy-Lys, and hydroxy-Pro among any further possible O-glycosylation sites. Preferably, the O-glycosylation site is selected from Ser and Thr.

[0091] The canonical glycosylation sites Ser and Thr generally show the greatest occupancy by O-glycans. Thus, according to one embodiment, the one or more glycosylation sites are selected from Ser and Thr.

[0092] For practical reasons, the term "glycosylated polypeptide" is used in the singular form in the context of the present invention. Generally, glycosylated polypeptides occur in the form of a composition of polypeptides of the same type. In this regard, the early form of glycosylated polypeptide is a composition of glycosylated polypeptides having the same amino acid sequence but with a variance in glycosylation. For example, not all of the individual molecules of the composition may be 100% glycosylated. Furthermore, differences may occur in the glycans attached to a given O-glycosylation site. Thus, the present invention also relates to a composition comprising at least a first type of glycosylated polypeptide molecule, wherein the amino acid sequence of the first type of protein molecule is identical or homologous to at least a fragment of a mammalian, preferably human, protein, and the protein molecule contains one or more glycosylation sites.

[0093] Preferably, the polypeptide contains one or more clusters of glycosylation sites. Although a single glycosylation site may be sufficient for SIGLEC binding, it is assumed that the formation of clusters of O-glycosylation sites results in improved binding to SIGLEC. Clusters of glycosylation sites are often observed in mammalian proteins, such as human IgA, which contains clustered O-glycans in the hinge region (see Franc et al. 2013) and human mucins (see Guzman-Aranguez and Arguesso 2010).

[0094] In this regard, two adjacent O-glycosylation sites are considered in an O-glycosylation cluster. Thus, one or more clusters of O-glycosylation sites contain at least two O-glycosylation sites. Clusters of O-glycosylation sites can have different numbers of O-glycosylation sites. For example, a glycosylated polypeptide can contain one cluster with two glycosylation sites and a second cluster with three glycosylation sites. N-glycosylation sites can also be present between the O-glycosylation sites of a cluster. Preferably, there are no N-glycosylation sites in an O-glycosylation cluster.

[0095] Furthermore, one cluster may contain three O-glycosylation sites, while another cluster may contain four. A large number of three O-glycosylation sites results in three adjacent O-glycans that can all interact with SIGLEC, thus resulting in increased efficacy. According to one embodiment, one or more clusters of O-glycosylation sites preferably contain at least three O-glycosylation sites.

[0096] In vWF, there are two clusters, each with four O-glycosylation sites. Therefore, preferably, a polypeptide contains one or more clusters with at least four O-glycosylation sites. It is currently believed that the greater the number of O-glycosylation sites, the greater the binding affinity.

[0097] A cluster of O-glycosylation sites can be defined by two or more O-glycosylation sites within a short distance in the amino acid sequence. Such clusters are also referred to as "sequence clusters." However, due to the three-dimensional assembly of glycosylated polypeptides, an O-glycosylation cluster can also include O-glycosylation sites that are located at long distances in the amino acid sequence but are closely located after folding. This latter cluster is also referred to as a "folding cluster."

[0098] vWF O-glycosylation cluster 2 contains four O-glycosylation sites within 20 amino acids, arranged as a beta turn. Therefore, the distance between O-glycans or O-glycosylation sites is 27.2 Å to 34.0 Å, resulting in an average distance of 6.8 Å to 8.5 Å. Therefore, the average distance between two O-glycosylation sites in a cluster can range from 4.0 Å to 15.0 Å. If the distance is less than 4.0 Å, steric hindrance of the O-glycans may exist, particularly in cases where it is not possible to glycosylate both O-glycosylation sites. If the average distance between two amino acids exceeds 15.0 Å, there is likely no cooperative effect of the O-glycans. A cooperative effect could be, for example, interaction with SIGLEC on the same cell. Preferably, the average distance between two O-glycosylation sites in a cluster ranges from 5.0 Å to 12.0 Å. More preferably, the average distance between two O-glycosylation sites in a cluster is in the range of 6.0 Å to 9.0 Å.

[0099] Folding clusters can span an amino acid sequence of more than 100 amino acids.However, it is preferred that the spatial arrangement of clusters does not exceed 80 Å.If O-glycosylation sites are separated by more than 80 Å, it is assumed that O-glycans do not exhibit combinatorial effect.The combinatorial effect of O-glycans in clusters is strongest when O-glycans are located within a region with a diameter of 50 Å.Therefore, it is more preferred that the spatial arrangement of clusters does not exceed 50 Å.

[0100] According to one embodiment, one or more clusters, i.e., sequence clusters, contain at least one O-glycosylation site within 10 amino acids. If O-glycosylation were more diffuse, it is conceivable that the O-glycans attached to the O-glycosylation sites would likely not act together on the same cell containing SIGLEC.

[0101] Preferably, one or more clusters contain at least one O-glycosylation site within four amino acids. The average distance between O-glycosylation sites within four amino acids is such that the O-glycosylation sites are likely to be close together after folding. This spatial proximity allows for cooperative interaction of glycans within a cluster with SIGLEC on the same cell.

[0102] More preferably, one or more clusters contain at least one O-glycosylation site within three amino acids. As shown in the examples, the test vWF peptide contains a cluster with one glycosylation site within two amino acids. Thus, according to a preferred embodiment, one or more clusters contain at least one glycosylation site within two amino acids.

[0103] As shown in the Examples, even one such O-glycosylation cluster is sufficient for strong interaction with SIGLEC. Furthermore, comparison of two vWF polypeptides also shows that a larger number of clusters of O-glycosylation sites results in increased binding affinity of the peptide to SIGLEC, particularly SIG-5, SIG-7, SIG-8, and SIG-9. Therefore, a glycosylated polypeptide preferably contains at least two glycosylation clusters, more preferably at least three glycosylation clusters.

[0104] Without being bound by theory, the closer the clusters are located, the greater the binding affinity of the glycosylated polypeptide to SIGLEC. In this regard, when two clusters are present, they are preferably separated by less than 100 amino acids. A distance of less than 100 amino acids allows for the cooperative effect of the glycan clusters in SIGLEC binding. More preferably, the two clusters are separated by less than 50 amino acids. Most preferably, the two clusters are separated by less than 30 amino acids.

[0105] According to one embodiment, the distance between any two adjacent clusters is less than 100 amino acids, preferably the distance between any two clusters in a glycosylated polypeptide is less than 50 amino acids, more preferably the distance between any two adjacent clusters is less than 30 amino acids.

[0106] The glycosylated polypeptide preferably contains at least one additional cluster of O-glycosylation sites compared to the human protein to which the sequence is homologous or identical.

[0107] As defined above, like all glycosylated polypeptides, the glycosylated polypeptides of the present invention represent a composition of glycosylated polypeptide molecules. These molecules exhibit a certain degree of heterogeneity in the glycosylation pattern, in particular, not all glycosylation sites are necessarily occupied by O-glycans. O-glycan occupation is particularly dependent on the host cell in which the recommended glycosylated polypeptide is produced. Preferably, the host cell, i.e., the expression system, is selected so that the percentage of O-glycosylation sites is greater than 70%. Below 70% occupancy, there may be insufficient O-glycans for SIGLEC binding. Preferably, more than 80% of the O-glycosylation sites are occupied by O-glycans. More preferably, more than 90% of the O-glycosylation sites are occupied by O-glycans. According to a preferred embodiment, more than 95% of the O-glycosylation sites are occupied by O-glycans.

[0108] The composition of the glycans attached to glycosylated polypeptides depends on the method of production. O-glycans can be natural or synthetic glycans. Natural O-glycans are, for example, glycans with the following core structure: Core 1 O-glycan: Galβ1→3GalNAcα1→Ser / Thr Elongated core 1: O-glycan: Galβ1→4GlcNAcβ1→3Galβ1→3GalNAcα1→Ser / Thr Core 2 O-glycan: Galβ1→3(Galβ1→3GlcNAcβ1→6)GalNAc α1→Ser / Thr

[0109] SIGLEC is known to bind to sialic acid. Consistent with this, the Examples show that desialylation abolished binding to SIGLEC (see Example 3). This confirms that sialylation of O-glycans is a prerequisite for binding. Therefore, sialylation of a high proportion of O-glycans in glycosylated polypeptides is preferred.

[0110] Thus, the O-glycans of the glycosylated polypeptide are preferably sialylated, ie, contain at least one sialic acid as part of the glycan molecule.

[0111] Preferably, the sialylated O-glycan contains at least two sialic acids in alpha 2-3 glycosidic linkages. Alternatively, the sialylated O-glycan may contain two sialic acids in alpha 2-8 glycosidic linkages. The sialylated O-glycan may also contain alpha 2-3 and alpha 2-8 glycosidic linkages. According to one embodiment, the sialylated O-glycan contains at least three sialic acids in 2-3 and / or 2-8 glycosidic linkages. The sialylated O-glycan in the glycosylated polypeptide is, in particular, a core 1 or core 2 O-glycan. The structures of core 1, extended core 1 and core 2 O-glycans are summarized below: Sialylated core 1: O-glycan: NeuNAcα2→3Galβ1→3GalNAcα1→Ser / Thr Sialylated extended core type 1 O-glycans: NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→3GalNAcα1→Ser / Thr Sialylated core type 2 O-glycan: NeuNAcα2→3Galβ1→4GlcNAcβ1→6(NeuNAcα2→3Galβ1→3)GalNAcα1→Ser / Thr

[0112] It is believed that both core 1 and core 2 and / or extended core 1 type O-glycans must be present on the glycosylated polypeptide. According to one embodiment, the glycosylated polypeptide contains sialylated core 1 type O-glycans and sialylated core 2 type and / or extended core 1 type O-glycans. As shown in Example 4, a core 2 glycan ratio of 2.5% based on the total number of O-glycans is not sufficient for SIGLEC interaction. Therefore, according to one embodiment of the glycosylated polypeptide, the core 2 type O-glycan ratio based on the number of O-glycans is at least 5%. In the same example, it is shown that cluster 2, which has a core 2 type O-glycan ratio of 10.78% based on the number of O-glycans, provides a strong interaction with SIGLEC. Therefore, according to a preferred embodiment, the core 2 type O-glycan ratio based on the number of O-glycans in the glycosylated polypeptide is at least 8%. More preferably, the core 2 type O-glycan ratio based on the number of O-glycans is at least 10%.

[0113] In Example 7, it was determined that approximately 80% of the glycopeptide molecules of a recombinantly produced vWF peptide contain either core 2 type O-glycans or extended core 1 glycans. Thus, the percentage of sialylated core 2 type and / or extended core 1 type O-glycans based on the total number of O-glycans is at least 20%. Thus, according to one embodiment, the concentration of core 2 type and / or extended core 1 type O-glycans based on the number of O-glycans in the glycosylated polypeptide is at least 15%, more preferably at least 18%, and most preferably at least 20%.

[0114] The number or proportion of core 2 O-glycans in a glycosylated polypeptide, in particular the number or proportion of individual molecules of a glycosylated polypeptide bearing core 2 O-glycans, can be increased by any of the following strategies:

[0115] One strategy is to use the enzyme β1,6-N-acetylglucosaminyltransferase, which is involved in the formation of core 2 O-glycans. Thus, an increase in the number of core 2 O-glycans per core 2-bearing molecule and / or polypeptide molecule can be achieved by expressing glycosylated polypeptides in cell lines that overexpress the enzyme β1,6-N-acetylglucosaminyltransferase.

[0116] Another option is the expression of glycosylated polypeptides in expression cell lines derived from cancer cell lines, which are often shown to produce glycosylated proteins with higher amounts of core 2 O-glycans.

[0117] One strategy for increasing the proportion of extended core 1 is to use the enzyme β1,3-N-acetylglucosaminyltransferase. This enzyme is involved in the formation of extended core 1 type O-glycans. Therefore, an increase in the number of extended core 1 type O-glycans per extended core 1-bearing molecule and / or polypeptide molecule can be obtained by expressing glycosylated polypeptides in cell lines overexpressing the enzyme β1,6-N-acetylglucosaminyltransferase.

[0118] The concentration of core 2 and / or extended core 1 sialylated O-glycans can be increased by chemical synthesis of the glycans.

[0119] Therefore, based on the teachings of the present invention, one skilled in the art can tailor the glycans to determine synthetic glycans with high binding affinity. According to a preferred embodiment, the O-glycans are naturally occurring glycans.

[0120] According to one embodiment, at least a portion of the sialylated core 2-O-glycans in the glycosylated polypeptide contain sulfate groups attached to galactose (Gal) or N-acetylglucosaminidase (GlcNAc) or fucose attached to GlcNAc in core 2 type O-glycans, which represent high affinity binding ligands for SIGLEC7, 8, and 8 (Paulson et al. 2012).

[0121] There are various techniques known to those skilled in the art for generating additional O-glycosylation sites. Glycosylated polypeptides can have an increased number of sialylated core type 2 O-glycans due to the increased number of O-glycosylation sites compared to the human protein or fragment thereof.

[0122] In this regard, the glycosylated polypeptide may be a fusion protein in which a second amino acid sequence containing one or more O-glycosylation sites is covalently linked to an amino acid sequence (first amino acid sequence) that is identical or homologous to a human protein or fragment thereof. The second amino acid sequence may be located N-terminally with respect to the first amino acid sequence. Alternatively, the second amino acid sequence may be located C-terminally with respect to the first amino acid sequence. The glycosylated polypeptide may contain additional amino acid sequences both N- and C-terminally with respect to the first amino acid sequence.

[0123] Thus, in such fusion proteins, there may be present a second and optionally further amino acid sequence containing primarily O-glycosylation sites, particularly O-glycosylation clusters, which may be based on the amino acid sequences of known mammalian, particularly human, protein glycosylated proteins.

[0124] The second amino acid sequence may include one or more of the following O-glycosylation clusters: VVPPTXAPVXPTTXYVXXXSXPP (SEQ ID NO: 8), VVPPTDAPVSPTTLYVEDISEPP (SEQ ID NO: 9), PPPTXPPXXAXVTVXPXXXXVSTXXP (SEQ ID NO: 10), PPPTLPPDMAQVTVGPGLLGVSTLGP (SEQ ID NO: 11), VSSTSXXXXSTXPSXXXAAXTXXTSSXXPPSXPVXXXSXXXTTXXXX (SEQ ID NO: 12), VSSTSNNLISTIPSDNLAAGTDDTSSLGPPSMPVHYDSQLDTTLFGK (SEQ ID NO: 13), XXXATTXPXXXXXXTXPXXX (SEQ ID NO: 14), QFNATTIPENDIEKTDPWFA (SEQ ID NO: 15), XXTTAATXXX (SEQ ID NO: 16), LGTTAATELK (SEQ ID NO: 17), XXPTPXXXSXSXXXEAX (SEQ ID NO: 18), QSPTPHGLSLSDLQEAK (SEQ ID NO: 19); VXXXXXXXXXTXTSXXSPXXXXXVXXSXXXXTXXAXX (SEQ ID NO: 20), and VHIYQKDLFFTETSDGSPGHLDLVEGSLLQGTEGAIK (SEQ ID NO: 21).

[0125] In the sequences SEQ ID NO: 8, 10, 12, 14, 16, 18 and 20, X means any of the naturally occurring amino acids.

[0126] SEQ ID NOs: 9 and 11 are found in vWF, and SEQ ID NOs: 13, 15, 17, 19 and 21 are derived from the B domain of FVIII.

[0127] The second amino acid sequence may comprise one or more of the sequences selected from SEQ ID NOs: 8, 10, 12, 14, 16, 18, and 20. The second amino acid sequence may contain a combination of sequences. The second amino acid sequence preferably comprises multiple copies of one of the sequences of SEQ ID NOs: 8, 10, 12, 14, 16, 18, and 20. The second amino acid sequence may further comprise a combination of multiple copies of SEQ ID NOs: 8, 10, 12, 14, 16, 18, and 20.

[0128] The second amino acid sequence may comprise one or more sequences selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, and 21. The second amino acid sequence may contain a combination of sequences. The second amino acid sequence preferably comprises multiple copies of one of the sequences of SEQ ID NOs: 9, 11, 13, 15, 17, 19, and 21. The second amino acid sequence may further comprise a combination of multiple copies of SEQ ID NOs: 9, 11, 13, 15, 17, 19, and 21.

[0129] According to a preferred embodiment, the second amino acid sequence contains one or more copies of SEQ ID NO:8.

[0130] Furthermore, the second amino acid sequence may have a certain percentage of identity with the sequence of a naturally occurring glycosylated protein. The level of identity with the naturally occurring protein is preferably 80%, more preferably at least 90%.

[0131] Alternatively, the amino acid sequence of the O-glycosylation site in the second amino acid sequence can be completely synthetic. As used herein, a completely synthetic amino acid sequence is a sequence that is not based on a known protein, particularly a mammalian protein.

[0132] According to one embodiment, the covalent linker connecting the second amino acid sequence in the glycosylated polypeptide to the amino acid sequence identical or homologous to the human protein or fragment thereof is selected from a peptide bond, a chemical linker, or a glycosidic bond. Suitable chemical linkers in this regard are: amine-amine linkers, such as bismaleimidoethane, 1,8-bismaleimido-diethylene glycol, - amine-sulfhydryl linkers, such as succinimidyl iodoacetate, N-α-maleimidoaceto-oxysuccinimide ester, - carboxyl-amine linkers dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and - sulfhydryl-carbohydrate linkers, e.g., N-β-maleimidopropionic acid hydrazide, N-ε-maleimidocaproic acid hydrazide is.

[0133] The linker of the fusion protein of the present invention can be formed by a spacer peptide sequence that separates the first and second amino acid sequences that define the fusion protein. The spacer peptide sequence can facilitate correct folding of the individual protein or peptide moieties, thereby making them more likely to retain their individual functional properties. The spacer peptide sequence can be inserted into the fusion protein DNA sequence during the in-frame assembly of the individual DNA fragments that make up the complete fusion protein DNA sequence, i.e., during overlapping PCR or DNA ligation.

[0134] Peptide bonds have the advantage that the entire glycosylated polypeptide can be expressed at once as a fusion protein.

[0135] The second amino acid sequence can be attached to the first amino acid sequence by a chemical linker and can therefore be added after expression of the protein.

[0136] As shown in the examples, therefore, in particular vWF and fragments containing O-glycosylation clusters 1 and / or 2 bind to SIGLEC.

[0137] Thus, according to one embodiment, the human protein is FVIII or a fragment thereof. Thus, the sequence identity of the glycosylated polypeptide with FVIII is at least 90%, more preferably at least 95%, and most preferably at least 98%. According to a preferred embodiment, the first amino acid sequence is the amino acid sequence of SEQ ID NO: 1. 1~505 be identical or homologous to

[0138] The length of the second amino acid sequence is preferably in the range of 5 to 100 amino acids, more preferably 10 to 80 amino acids, and most preferably 20 to 70 amino acids.

[0139] According to one embodiment, the second amino acid is the amino acid of SEQ ID NO: 1 475~505 Preferably, the second amino acid sequence is at least 98% homologous to the amino acid sequence of SEQ ID NO: 1. 475~505 According to a more preferred embodiment, the second amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 1. 475~505 Preferably, the second amino acid sequence is at least 98% homologous to two consecutive copies of the amino acid sequence of SEQ ID NO: 1. 475~505 is identical to two successive copies of

[0140] An exemplary fusion protein according to the present invention is Seq12, which has the following amino acid sequence (SEQ ID NO:2): JPEG2025121974000001.jpg58170

[0141] The following sequence (SEQ ID NO: 3) contains additional 22 Figure 1 shows Seq 12 with an amino acid signal peptide (bold and underlined). Expression of this peptide provides a monomeric form of Seq 12. The signal peptide is enzymatically cleaved. JPEG2025121974000002.jpg62170

[0142] A further exemplary fusion protein according to the invention is Pro-Seq12, which comprises a propeptide (bold) with Seq12 and a signal peptide (bold and underlined). Pro-Seq12 is identified by SEQ ID NO: 4: JPEG2025121974000003.jpg124170

[0143] Expression of Pro-Seq12 leads to the formation of a dimer, which remains intact after cleavage of the propeptide.

[0144] According to one embodiment, the glycosylated polypeptide comprises the amino acid sequence of SEQ ID NO: 1 1~505 The second amino acid sequence comprises a first amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1. 475~505 It is at least 98% homologous to two consecutive copies of

[0145] According to one embodiment, the glycosylated polypeptide is produced by expression in a human cell line. Generally, any human cell line is suitable for expressing glycosylated polypeptides. Preferred glycosylated polypeptides are obtained using the HEK cell line in particular.

[0146] Examples of HEK cell lines for the production of glycosylated polypeptides are HEK293F, Flp-In™-293 (Invitrogen, R75007), 293 (ATCC® CRL-1573), 293EBNA, 293H (ThermoScientific 11631017), 293S, 293T (ATCC® CRL-3216™), 293T / 17 (ATCC® CRL11268™), 293T / 17SF (ATCC® ACS4500™), HEK293STF (ATCC® CRL3249™), HEK-293.2sus (ATCC® CRL-1573™). The preferred cell line for the production of polypeptides is HEK293F as a cell line.

[0147] Other cell lines suitable as expression host cells include cell lines derived from human myeloid leukemia cells. Specific examples of host cells include K562, NM-F9, NM-D4, NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X, GT-5s, and cells derived from any one of the above host cells. K562 is a human myeloid leukemia cell line present in the American Type Culture Collection (ATCC CCL-243). The remaining cell lines are derived from K562 cells and selected for their defined glycosylation characteristics.

[0148] According to alternative embodiments, one or more glycosylation sites are located within an amino acid sequence that is homologous or identical to a mammalian protein or fragment thereof. It should be understood that an O-glycosylation site that is not present in the amino acid sequence of the mammalian protein or fragment is found within a homologous or identical amino acid sequence within the glycosylated polypeptide.

[0149] One or more O-glycosylation sites within an amino acid sequence homologous or identical to a mammalian protein or a fragment thereof can be inserted into the sequence. Alternatively, one or more O-glycosylation sites can be substituted for amino acids in the mammalian protein. Amino acid substitution is preferred because it does not change the size of the polypeptide chain and is therefore unlikely to affect the three-dimensional structure of the protein.

[0150] The one or more O-glycosylation sites in the amino acid sequence are preferably located in a portion of the sequence that does not form the binding site or active center of the protein. Furthermore, the one or more O-glycosylation sites are preferably added in amino acid positions that are exposed on the surface of the folded protein. To achieve minimal impact on the activity or integrity of the protein, the one or more O-glycosylation sites can be added to a flexible loop of the protein.

[0151] In the case of the FVIII protein, one or more O-glycosylation sites are preferably added in or in place of the B domain.

[0152] In one embodiment, glycosylated polypeptides are modified by attachment of one or more biocompatible polymers, for example, to improve half-life or stability. Suitable biocompatible polymers include polyalkylene oxides, such as, but not limited to, polyethylene glycol (PEG), dextran, colominic acid or other polymer-based carbohydrates, polymers of amino acids, biotin derivatives, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyethylene-co-maleic anhydride, polystyrene-co-malic anhydride, polyoxazolines, polyacryloylmorpholines, heparin, albumin, cellulose, chitosan hydrolysates, starches, such as hydroxyethyl-starch and hydroxypropyl-starch, glycogen, agarose and its derivatives, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, other biopolymers, and any equivalents thereof. In one embodiment, the polymer is polyethylene glycol (PEG). In another embodiment, the polymer is methoxypolyethylene glycol (mPEG). Other useful polyalkylene glycol compounds are polypropylene glycol (PPG), polybutylene glycol (PBG), PEG-glycidyl ether (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethylene glycol, linear polyethylene glycol, forked polyethylene glycol, and multiarmed or "hyperbranched" polyethylene glycol (star-PEG). The biocompatible polymer is preferably linked to the polypeptide by one of the following residues: -SH, OH, -COOH.

[0153] According to one embodiment, glycosylated polypeptides can form dimers or multimers. The formation of dimers, particularly multimers, increases the number of adjacent O-glycans or O-glycan clusters. Therefore, more O-glycans can interact with SIGLEC on a single cell. Furthermore, O-glycans can interact with several SIGLEC-expressing cells that are closely located together, thereby increasing immune tolerance.

[0154] Multimerization can be the result of a multimerization domain in the amino acid sequence of the mammalian protein on which the glycosylated polypeptide is based. Alternatively, multimers of glycosylated polypeptides can be formed by introducing a multimerization domain into the amino acid sequence of the glycosylated polypeptide.

[0155] An example of a fusion protein according to the invention that forms a multimer is Pro-Seq12-Mult, which contains a propeptide (bold) with Seq12 and a signal peptide (bold and underlined) and the multimerization sequence "cysteine knot domain" of vWF (underlined). Pro-Seq12-Mult is identified by SEQ ID NO: 5: JPEG2025121974000004.jpg131170

[0156] Alternatively, multimerization of glycosylated polypeptides can be obtained by conjugation to polymers or liposomes.

[0157] Uses of Glycosylated Polypeptides As defined above, the present inventors have found that proteins having a glycan composition comprising sialylated core type 2 O-glycans and / or sialylated extended core type 1 O-glycans interact with defined SIGLECs and thus affect cells of the mammalian immune system. In particular, glycosylated polypeptides exhibit reduced immune responses. Therefore, when administered with a second protein, such glycosylated polypeptides can affect a patient's immune response to the second protein. Therefore, glycosylated polypeptides having sialylated core type 2 O-glycans and / or sialylated extended core type 1 O-glycans can be used to modify, and in particular reduce, a patient's immune response to proteins, particularly therapeutic proteins in combination administration.

[0158] Thus, according to a second aspect, the present invention relates to the use of a glycosylated polypeptide containing one or more sialylated O-glycans and exhibiting binding to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8 and SIG-9 for reducing the immune response of a therapeutic protein.

[0159] Preferably, the glycosylated polypeptide used for reducing the immune response comprises sialylated core type 2 O-glycans and / or sialylated extended core type 1 O-glycans. More preferably, the glycosylated polypeptide is defined as a glycosylated polypeptide according to the first aspect.

[0160] The use may also be described as a method of treating a patient with a therapeutic protein, comprising administering a glycosylated polypeptide containing one or more sialylated O-glycans and exhibiting binding to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8, and SIG-9 to reduce an immune response to the therapeutic protein.

[0161] Compositions and Protein Complexes Thus, the concept according to the present invention, i.e., reducing the immune response of a human protein by, for example, adding one or more sialylated core 2-O-glycans, can be achieved not only by preparing a fusion protein or by inserting or replacing an amino acid with an O-glycosylation site, but also by adding an additional polypeptide as described in the use according to the second aspect, which results in the formation of a composition of a glycosylated polypeptide and a second polypeptide that should reduce the immune response.

[0162] Thus, in a third aspect, the present invention also relates to a composition comprising a first and a second polypeptide, wherein the first polypeptide is a glycosylated polypeptide containing one or more sialylated O-glycans, and the second polypeptide contains an amino acid sequence that is homologous or identical to a second mammalian, particularly a human, protein, and has increased binding affinity to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8, and SIG-9 compared to the second polypeptide.

[0163] It is also possible to provide a binding partner to a polypeptide having increased binding affinity for SIGLEC, such that a complex of the two polypeptides has increased binding affinity for SIGLEC compared to the polypeptide.

[0164] Thus, according to a third aspect, the present invention provides a composition comprising a first and a second polypeptide, wherein the first polypeptide is a glycosylated polypeptide containing one or more sialylated O-glycans, and the second polypeptide contains an amino acid sequence that is homologous or identical to a second mammalian, in particular human, protein, and wherein, compared to the second polypeptide: - has an increased binding affinity to a SIGLEC selected from: - the human immune response to the complex is reduced; and / or - Human immune tolerance to the complex is increasing A composition is provided.

[0165] The second human protein is preferably a human blood protein, which may be a human blood coagulation factor, a transport protein, a protease inhibitor, an immunoglobulin, a cell-associated plasma protein, an apolipoprotein, a complement factor, a growth factor, an anti-angiogenic protein, a highly glycosylated protein, a blood factor, or another human blood protein.

[0166] The human blood coagulation factor is particularly selected from the group consisting of fibrinogen, fibrin monomer, prothrombin, thrombin, FV / FVa, FX / FXa, FIX / FIXa, FVII / FVIIa, FVIII / FVIIIa, FXI / FXIa, FXII / FXIIa, FXIII / FXIIIa, von Willebrand factor, and ADAMTS13.

[0167] The transport protein may be selected from albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobin, and hemopexin.

[0168] Possible protease inhibitors are, for example, β-antithrombin, α-antithrombin, oxidized antithrombin, 2-macroglobulin, Cl inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), protein C, protein S, and protein Z.

[0169] Examples of immunoglobulins are, for example, polyclonal antibodies (IgG), monoclonal antibodies, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, IgM, IgE, IgD, and Bence Jones proteins.

[0170] Cell-associated plasma proteins can be, for example, fibronectin, thromboglobulin, platelet factor 4. Examples of apolipoproteins are apo AI, apo A-II, and apo E.

[0171] Complement factors according to the present invention include, for example, factor B, factor D, factor H, factor I, C3b inactivating factor, properdin, C4 binding protein, and the like.

[0172] Examples of growth factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-α), fibroblast growth factor (FGF), and hepatocyte growth factor.

[0173] Anti-angiogenic proteins include latent antithrombin, pre-latent antithrombin, oxidized antithrombin and plasminogen.

[0174] Examples of hyperglycosylated proteins are alpha-1-acid glycoprotein, antichymotrypsin, inter-α-trypsin inhibitor, α-2-HS glycoprotein, C-reactive protein. Blood factors can be, for example, erythropoietin, interferon, tumor factor, tPA, gCSF, etc.

[0175] Other human blood proteins include histidine-rich glycoprotein, mannan-binding lectin, C4-binding protein, fibronectin, GC-globulin, plasminogen / plasmin, alpha-1 microglobulin, and C-reactive protein.

[0176] The second human protein is in particular selected from vWF, FVIII, FVII / FVIIa, FIX, ADAMTS13.

[0177] The composition according to the third aspect is in particular a protein complex of the first and second polypeptides.

[0178] The first polypeptide is preferably glycosylated and contains one or more sialylated O-glycans. The second polypeptide contains an amino acid sequence identical to a mammalian, particularly a human, protein. The first polypeptide forms a complex with the second polypeptide to reduce a human immune response to the second polypeptide.

[0179] For protein complex formation, the first polypeptide comprises, in particular, a binding domain that allows binding to the second polypeptide and a glycosylation domain, the glycosylation domain comprising, in particular, one or more O-glycosylation sites, preferably an O-glycosylation cluster.

[0180] According to one embodiment, the second polypeptide is a FVIII protein and the first polypeptide comprises a FVIII binding domain of vWF and one or more O-glycosylation sites to which sialylated core type 2 O-glycans are attached.

[0181] A preferred example of a composition is a FVIII protein having an amino acid sequence that is 95% identical to the sequence defined by amino acids 20 to 2351 of P00451 and the amino acid sequence of SEQ ID NO: 1. 1~172 and a first polypeptide as a binding partner comprising an amino acid sequence that is at least 95% identical to

[0182] According to one embodiment of the protein complex of the third aspect, the first polypeptide is a polypeptide according to the first aspect.

[0183] According to further embodiments, the second polypeptide may be selected from, for example, FVIII, FVII, FIX and ADAMTS13.

[0184] In one embodiment of the third aspect, the first polypeptide comprises at least a fragment of human vWF and the second polypeptide is a FVIII protein, in particular a full-length FVIII protein, a B-domain deleted FVIII protein or a FVIII protein in which part of the B-domain is replaced by a linker. According to one embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 1. 1~505 and it is produced in HEK cells, in particular HEK293F cells.

[0185] According to a further embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 1. 1~505 and the amino acid sequence of SEQ ID NO: 1 475~505 Additionally, the first polypeptide can be defined by an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0186] As shown in the examples, proteins with improved binding affinity to SIGLEC, particularly SIG-5, SIG-7, SIG-8 and / or SIG-9, can be produced using the cell line HEK293F. Thus, according to a further embodiment of the protein complex, the first and second polypeptides are produced by recommended expression in a human cell line, preferably a HEK cell line. Examples of HEK cell lines for producing glycosylated polypeptides are HEK293F, Flp-In™-293, 293, 293EBNA, 293H, 293S, 293T, 293T / 17, 293T / 17SF, HEK293STF, and HEK-293.2sus. The preferred cell line for producing polypeptides is HEK293F as a cell line.

[0187] The first and second polypeptides can be produced by separate recombinant expression and then combined. Alternatively, the first and second polypeptides can be recombinantly expressed in the same cell. Thus, the first and second polypeptides can be encoded on the same vector or on two different vectors.

[0188] Polynucleotides According to a fourth aspect, the present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a glycosylated polypeptide according to the first aspect of the invention.

[0189] The isolated polynucleotide may be a DNA molecule or an RNA molecule. The isolated polynucleotide is preferably a DNA molecule, particularly a cDNA molecule. Techniques used to isolate or clone a polynucleotide encoding a peptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. Cloning of such polynucleotides from genomic DNA can be performed, for example, by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with common structural features (see, for example, Innis et al., 1990, "PCR: A Guide to Methods and Applications," Academic Press, New York). Other nucleic acid amplification procedures, such as ligase chain reaction (LCR), ligation-activated transcription (LAT), and polynucleotide-based amplification (NASBA), can be used.

[0190] The isolated polynucleotide can be a DNA molecule that encodes a glycosylated polypeptide having an amino acid sequence similar or identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5.

[0191] In particular, the isolated polynucleotide may be a DNA molecule encoding a glycosylated polypeptide having an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 2. Furthermore, the isolated polynucleotide may be a DNA molecule encoding a glycosylated polypeptide having an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 3. According to one embodiment, the isolated polynucleotide is a DNA molecule encoding a glycosylated polypeptide having an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 4. According to one embodiment, the isolated polynucleotide is a DNA molecule encoding a glycosylated polypeptide having an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% identical to SEQ ID NO: 5.

[0192] Expression vector In a fifth aspect, the present invention also relates to an expression vector comprising a polynucleotide according to the fourth aspect of the invention.

[0193] The expression vector preferably further comprises control elements, such as a promoter, and transcription and translation termination signals.The polynucleotide according to the fourth aspect and the control elements can be linked together to produce a recombinant expression vector, which may contain one or more restriction sites to allow the insertion or substitution of the polynucleotide encoding the polypeptide at the restriction site.The polynucleotide can be inserted into an appropriate expression vector for expression.In creating an expression vector, the coding sequence is located in the expression vector so that the coding sequence is operably linked to the appropriate control sequence for expression.

[0194] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can result in expression of the polynucleotide of the fourth aspect of the present invention. The choice of expression vector typically depends on the compatibility of the expression vector with the host cell into which the expression vector is to be introduced. The expression vector may be a linear or closed circular plasmid.

[0195] The expression vector is preferably adapted for expression in mammalian cells. The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. For autonomous replication, the vector may further comprise a replication origin that allows the vector to replicate autonomously in the host cell. The replication origin may be any plasmid replication factor that mediates autonomous replication and functions in cells. The term "replication origin" or "plasmid replication factor" refers to a polynucleotide that allows a plasmid or vector to replicate in vivo.

[0196] The vector is preferably one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated. For integration into the host cell genome, the expression vector may rely on any other element of the expression vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration into the host cell genome at a precise location in the chromosome by homologous recombination.

[0197] Vectors of the present invention preferably contain one or more (e.g., several) selectable markers which allow easy selection of transformed, transfected, transduced cells, etc. A selectable marker is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0198] The procedures used to ligate the above elements to construct the recombinant expression vectors of the present invention are well known to those of skill in the art (see, eg, Sambrook et al., 1989, supra).

[0199] According to one embodiment, the vector backbone of the vector according to the fifth aspect is selected from pCDNA3, pCDNA3.1, pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1, pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES, and pSCAS.

[0200] The vector according to the fifth aspect can be transiently or non-transiently transformed into a host cell. The host cell can be any of the cells listed above. Preferably, the host cell is HEK293F.

[0201] Medical Uses and Treatment Methods As mentioned above, the glycosylated polypeptides and compositions, in particular protein complexes, according to the present invention have the advantage of a reduced immune response in patients, in particular human patients, and are therefore particularly useful as active ingredients for medical treatment.

[0202] According to a sixth aspect, the present invention provides a glycosylated polypeptide as defined in the first aspect for use in medical therapy. Alternatively, according to the sixth aspect, the present invention provides a composition as defined in the third aspect for use in medical therapy, preferably for the treatment or prevention of bleeding disorders.

[0203] Thus, a sixth aspect of the invention also relates to a method for treating or preventing a bleeding disorder in a patient, comprising administering to said patient a glycosylated polypeptide according to the first aspect or a composition, in particular a protein complex, according to the third aspect.

[0204] As used herein, the term "bleeding disorder" refers to a disease or condition that impairs normal hemostasis. Examples of bleeding disorders include hemophilia A, hemophilia B, factor VIII deficiency, factor XI deficiency, von Willebrand disease, Glanzmann thrombasthenia, Bernard-Soulier syndrome, idiopathic thrombocytopenic purpura, intracerebral hemorrhage, trauma, and traumatic brain injury.

[0205] As used herein, "hemophilia" refers to a group of bleeding disorders involving increased blood clot formation times compared to those in healthy individuals without hemophilia. "Hemophilia" refers to both hemophilia A, a disorder resulting in a deficiency of factor VIII, and hemophilia B, a disorder resulting in a deficiency of factor IX.

[0206] The bleeding disorder is preferably hemophilia. The treatment can be, for example, hemophilia treatment for PUPS (treatment-naive patients) or immune tolerance induction (ITI) treatment.

[0207] According to an alternative embodiment of the third aspect, the present invention provides a protein complex as defined according to the second aspect for use in the treatment or prevention of bleeding disorders.

[0208] Treatment preferably involves administering to the patient an effective amount of a glycosylated polypeptide or composition, particularly a protein complex.

[0209] The glycosylated polypeptides or compositions described herein, particularly protein conjugates, can be administered alone or in the form of pharmaceutical compositions. Pharmaceutical compositions according to the present invention can comprise an effective amount of the conjugate in combination with at least one pharmaceutically acceptable carrier. Pharmaceutical compositions of the present invention can be prepared and administered to a subject by any method known in the pharmaceutical arts. See, for example, Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, Hardman et al., eds., McGraw-Hill Professional (10th ed., 2001); *Remington: The Science and Practice of Pharmacy*, Gennaro, ed., Lippincott Williams & Wilkins (20th ed., 2003); and *Pharmaceutical Dosage Forms and Drug Delivery Systems*, Ansel et al. (eds.), Lippincott Williams & Wilkins (7th ed., 1999). Furthermore, pharmaceutical compositions of the present invention can be formulated to contain other medically useful drugs or biological agents. Pharmaceutical compositions typically contain a therapeutically effective amount of a glycosylated polypeptide or protein complex in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are any carriers known or established in the art. Exemplary pharmaceutically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free physiological saline solution. Other forms of pharmaceutically acceptable carriers that can be used in this embodiment include binders, disintegrants, surfactants, absorption enhancers, moisture-retaining agents, absorbents, lubricants, fillers, extenders, moisturizing agents, preservatives, stabilizers, emulsifiers, solubilizers, salts for regulating osmotic pressure, diluents (e.g., buffers, and excipients), which are commonly used depending on the form of use of the formulation. These may be selected and used depending on the unit dosage of the resulting formulation.

[0210] For in vivo use, glycosylated polypeptides, protein complexes or pharmaceutical compositions can be administered to patients by any conventional route of administration, for example, orally, parenterally, or by inhalation. Parenteral administration includes intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, and intraperitoneal injection, liquid medicine, suspension, emulsion, and drop. For parenteral administration, glycosylated polypeptides, protein complexes or pharmaceutical compositions should be injectable, for example, liquid medicine or suspension.

[0211] In other embodiments, glycosylated polypeptides, protein complexes, or pharmaceutical compositions are orally administered to patients. In these embodiments, the drug forms include solid formulations, such as tablets, coated tablets, powders, granules, capsules, and pills; liquid formulations, such as liquids (e.g., eye drops, nasal drops), suspensions, emulsions, and syrups; inhalants, such as aerosols, atomizers, and nebulizers; and liposome-encapsulated formulations. In yet some other embodiments, glycosylated polypeptides, protein complexes, or pharmaceutical compositions are administered by inhalation into the patient's respiratory tract to target the trachea and / or lungs of the subject. In these embodiments, they are commercially available.

[0212] According to one embodiment of the sixth aspect, the glycosylated polypeptide or composition for use, in particular the protein complex, comprises intravenous or non-intravenous injection, wherein the non-intravenous injection is preferably subcutaneous injection.

[0213] The present invention is further described by the following non-limiting examples. [Example]

[0214] Example 1 Binding of full-length Willebrand factor (vWF) to SIGLEC: Experimental Procedure Recombinant SIG-2, SIG-5, SIG-7, SIG-F (the mouse equivalent of human SIG-8), SIG-9, and SIG-10 were obtained as Fc-fusion proteins from R&D Systems. Protein A (SERVA Feinbiochemica GmbH & Co.) was first coated onto plates at a concentration of 0.5 μg / well overnight at 4°C. After a blocking and washing step with washing buffer (20 mM HEPES, 125 mM NaCl, 1 mM EDTA, 1% BSA), Fc-fused SIGLEC or control antibodies were bound to Protein A at a concentration of 5 μg / ml by incubation at 37°C for 1 hour. Anti-vWF-pAb (Dako, #A0082) was used as a positive control, and anti-chicken IgY (Sigma Aldrich, #C2288) was used as a negative control. Antibodies were immobilized via their Fc moieties.

[0215] Plasma-derived VWF (pdVWF) was biotinylated using the EZ-Link™ Sulfo-NHS-Biotin Biotinylation Kit (Thermo Fisher Scientific). A concentration series of biotinylated vWF was applied to the wells at concentrations ranging from 0 to 0.8 μg / mL. After five washing steps with wash buffer, HRP-coupled streptavidin (Thermo Fisher Scientific, #31001) was added to the wells and incubated at 37°C for 1 hour, followed by five washing steps.

[0216] To visualize the bound biotinylated pdvWF, the wells were incubated with o-phenylenediamine dihydrochloride substrate (SIGMAFAST™ OPD, #P9187, Sigma Aldrich), followed by measuring the absorbance at 492 nm.

[0217] 1.2 Results As shown in Figure 1, the absorbance at 492 nm increases with the starting concentration of vWF in binding experiments using SIG-5, SIG-7, SIG-F, and SIG-9. The values are slightly higher (SIG-F and SIG-9) or lower (SIG-5 and SIG-7) than the positive control (anti-vWF). In contrast, the absorbance in binding experiments using SIG-2, SIG-10, and the negative control anti-chicken IgY was approximately 0, regardless of concentration.

[0218] Thus, vWF binds to SIG-5, SIG-7, SIG-F, and SIG-9 in a concentration-dependent manner, whereas vWF does not bind to SIG-2 and SIG-10.

[0219] Example 2 Binding of vWF fragments to SIGLEC 2.1 Experimental procedure C- and N-terminal fragments of vWF were prepared by digestion with V8 protease (Thermo Fisher Scientific, #201959) performed at 37°C and 300 rpm for 3 hours using a 1:100 enzyme-to-protein w / w ratio in 50 mM Tris-HCl, 150 mM NaCl, pH 7.8 buffer. Purified by anion exchange chromatography on a MonoQ5 / 50GL column (GE Healthcare #17-5166-01). The running buffer was 20 mM Tris-HCl, pH 7.4, and the elution buffer was 20 mM Tris-HCl, 500 mM NaCl, pH 7.4. The fragments were further purified and desalted by size exclusion chromatography on a Superose6 10 / 300GL column (GE Healthcare #17-5172-01) using 100 mM NaCl as the running buffer.

[0220] The resulting fragments (C- and N-terminal) are shown schematically in Figure 2, with domains and glycosylation sites identified.

[0221] The purified vWF C-terminal fragment and vWF N-terminal fragment were biotinylated using the EZ-Link™ Sulfo-NHS-Biotin Biotinylation Kit (Thermo Fisher Scientific), and binding of the vWF C-terminal fragment and vWF N-terminal fragment to SIGLEC was measured at a concentration of 1 μg / mL as described in Example 1.

[0222] 2.2 Results Based on the absorbance values shown in Figure 3, the vWF N-terminal fragment, which contains most of the O-glycosylation sites and the two O-glycan clusters (cluster 1 and cluster 2), binds to SIG-5, SIG-7, SIG-F, and SIG-9. In contrast, little or no absorbance was measured for the vWF C-terminal fragment. Therefore, the latter fragment does not bind to SIGLEC.

[0223] Example 3 Binding of the N-terminal portion of vWF to SIGLEC: Experimental Procedure A portion of the N-terminal vWF fragment obtained in Example 2 was enzymatically desialylated using sialidase A. Incubation was carried out for 3 hours at 37°C in 50 mM sodium phosphate, pH 6.0, using 2 µl of enzyme (Sialidase A™ #GK80040 obtained from Prozyme) per 100 µg of VWF fragment.

[0224] The second portion of the N-terminal vWF fragment was de-N-glycosylated by incubation overnight at 37°C in 50 mM sodium phosphate, pH 7.5 buffer, using 1 μl of enzyme (PNGase F #P0704 from New England Biolabs) per 20 μg of VWF fragment.

[0225] Samples of desialylated, N-deglycosylated, and unprocessed N-terminal vWF fragments were tested for binding to SIG-5, SIG-7, SIG-8, and SIG-9. Binding experiments were performed as described in Example 1 at a concentration of 8 μg / mL of N-terminal vWF fragments.

[0226] 3.2 Results: As shown in Figure 4, the absorbance values measured for the N-deglycosylated and unprocessed vWF N-terminal fragments are only slightly different, thus indicating that N-deglycosylation does not affect binding, which is mediated via O-glycans.

[0227] Desialylation of O-glycans strongly reduces or abolishes the binding of vWF N-terminal fragments to SIGLEC, as shown in Figure 4. Thus, binding of vWF N-terminal fragments is mediated by sialic acid attached to the O-glycan chains.

[0228] Example 4 SIGLEC binding of peptides containing O-glycan clusters 1 and 2 4.1 Experimental procedure vWF contains two clusters of fully occupied O-glycosylation sites (see Solecka et al. 2016), which are shown schematically in Figure 2. Both clusters differ in the relative amount of core 2 structures. Only 4.9% of glycopeptide molecules contain core 2 structures in cluster 1. Therefore, the percentage of sialylated core 2 O-glycans based on the total number of O-glycans in cluster 1 is 1.25%.

[0229] On the other hand, 34.86% of glycopeptide molecules contain core 2 structures in cluster 2 (see Solecka et al., 2016). Therefore, the proportion of sialylated core 2 O-glycans based on the total number of O-glycans in cluster 2 is 10.78%.

[0230] To measure the binding of the two clusters independently, the vWF N-terminal fragment was treated with trypsin and the following fragment: AA of VWF of SEQ ID NO: 1 449~511 Cluster 1 fragment encompassing AA 674~728 / 729A cluster 2 fragment encompassing the fragments was generated. The fragments were purified by reverse-phase HPLC. Briefly, pdVWF was reduced, and free cysteines were blocked with maleimide-PEG2-biotin according to the manufacturer's instructions (EZ-Link™ Maleimide-PEG2-Biotin, #21901BID, obtained from Thermo Fisher Scientific). After overnight digestion with trypsin at 37°C, high molecular weight peptides were concentrated using a 10 kDa cutoff centrifugal filter device (Millipore). Peptides were then separated on a Jupiter 5μ 300Å C18 column (Phenomenex). The mobile phases were A - 0.1% trifluoroacetic acid (TFA) in H2O; B - 0.085% TFA in acetonitrile, with a flow rate of 0.3 mL / min. Eluted peptides / glycopeptides were detected by ultraviolet absorption at 215 nm wavelength. The fractions of interest were collected, lyophilized, and then reconstituted in 10 μL of H O. Because both clusters contain cysteine, both were supplied with biotin.

[0231] Binding experiments were performed as described in Example 1 using SIGLEC SIG-5, SIG-7, SIG-F, SIG-9 and SIG-10 at a concentration of 4 μg / mL of cluster 1 and cluster 2 fragments.

[0232] Additionally, samples of cluster 1 and cluster 2 fragments were treated by desialation and then tested in binding experiments as described in Example 1 at a concentration of 2 μg / mL of desialylated cluster 1 and cluster 2 fragments.

[0233] 4.2 Results: Based on the absorbance values shown in Figure 5, the cluster 2 fragment bound to SIGLEC SIG-5, SIG-7, SIG-F, and SIG-9. No or little absorbance was detected for SIG-10, confirming the results from other experiments.

[0234] Consequently, a high proportion of core 2 structures on the O-glycan cluster is a requirement for binding to SIG-5, SIG-7, SIG-F, and SIG-9.

[0235] Example 5 Sialic acid dependence of cluster 2 SIGLEC binding 5.1 Experimental procedure Samples of the cluster 2 fragments obtained were desialylated as described in Example 4. The desialylated cluster 2 fragments and untreated cluster 2 fragments were then tested in binding experiments with SIGLEC SIG-5, SIG-7, SIG-F, and SIG-9 at a concentration of 2 μg / mL of desialylated cluster 1 and cluster 2 fragments as described in Example 1.

[0236] 5.2 Results The absorbance values detected for the untreated cluster 2 fragments confirmed the results found in Example 4 (see Figure 6). Desialylated cluster 2 fragments showed no or only weak binding. Thus, sialylation of the core 2 structure on the O-glycan cluster is a requirement for binding to SIG-5, SIG-7, SIG-F, and SIG-9.

[0237] Example 6 Recombinant expression of VWF fragments with or without O-linked glycan repeats containing the FVIII binding site: Two recombinant vWF fragments were expressed in the HEK cell line 293F. The first fragment, Seq11, contains the AA 1~505 and an O-glycosylation site 485、492、493、500 ) contains cluster 1 with

[0238] The second fragment, Seq12, is AA of SEQ ID NO:1. 1~505 and A.A. 475~505 Two additional iterations of (AA 1~505 +2× 475~505 ) and therefore encompasses two additional copies of the cluster 1 O-glycan cluster repeat.

[0239] Seq11 and Seq12 were transiently expressed in HEK293 cells with a C-terminal Strep-Tag and purified by Strep-tactin affinity chromatography (IBA GmbH). Therefore, the genes encoding Seq11 and 12 were synthesized by GeneArt (Thermo Fisher Scientific) and cloned into the pDSG-expression vector (IBA GmbH) containing a Twin-Strep-Tag. TOP10 E. coli (IBA GmbH) was transformed with the constructs, and single clones were selected after overnight incubation at 37°C on ampicillin-containing LB agar plates. Plasmid DNA preparation was performed using the QIAamp DNA-Mini or Maxi kit (Qiagen) according to the manufacturer's recommendations. Correct orientation and integration of the cloned constructs were confirmed by sequencing. For eukaryotic expression of both vWF fragments, MEXi-293 cells (IBA GmbH) grown in MEXi transfection medium (IBA GmbH) were transfected with 1.5 mg / ml of the construct using 4.5 mg / ml of 25 kDa linear polyethyleneimine. After 2-4 hours of incubation at 37°C, 5% CO2, and 100-150 rpm, the culture was diluted 1:2 with MEXi transfection medium and continued to grow until cell viability reached 75%. The supernatant was then separated from the cells by centrifugation at 4°C and 300 × g. To minimize the inhibitory effect of biotin in the cell culture medium and to adjust the pH, 0.1 volume of buffer (1 M Tris-HCl, 1.5 mM NaCl, 10 mM EDTA, pH 8.0) and 0.09% (v / v) BioLock solution (IBA GmbH) were added to the supernatant and incubated for 20 min at 4° C. After centrifugation, the supernatant was applied to a Strep-Tactin XT column (IBA GmbH), washed five times with wash buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0), and the bound Strep-tag-containing proteins were eluted with elution buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10 mM desthiobiotin, pH 8.0).

[0240] Both fragments, Seq11 and Seq12, are shown schematically in FIG.

[0241] Example 7 Analysis of O-glycosylation of vWF fragments Seq11 and Seq12 7.1 Experimental Procedure The O-glycosylation of fragments Seq11 and Seq12 produced according to Example 6 was analyzed by mass spectrometry.

[0242] For this purpose, Seq11 and Seq12 were first reduced and alkylated by incubation with 50 mM dithiothreitol at 60°C, followed by 100 mM iodoacetamide for 20 minutes. After trypsin and chymotrypsin digestion, the resulting peptides were rebuffered in sialidase A digestion buffer and desialylated overnight using the conditions described in Example 3. O-glycopeptides were specifically enriched by jacalin (Artocarpus integrifolia lectin) affinity chromatography using agarose-immobilized lectin (Vector Laboratories). Jacalin-agarose was packed into a gravity-driven column, and chromatography was performed according to the manufacturer's instructions. The eluted O-glycopeptides were purified for MALDI MS measurement using C4 Ziptip pipette tips (Millipore) and subjected to measurement in linear positive ion mode using 25 mg / ml SuperDHB matrix dissolved in 50% acetonitrile / 0.1% trifluoroacetic acid.

[0243] Aliquots of the concentrated glycopeptides were further treated with O-glycosidase (endo-α-N-acetylgalactosaminidase, #P0733, New England Biolabs). Briefly, peptides were incubated with the enzyme for 2 hours at 37°C, using 1 μl of enzyme per 10 μl of glycoprotein. Because O-glycosidase is specific for only core 1 O-glycans (Galβ1→3GalNAcα1→Ser / Thr disaccharides), it leaves core 2 and / or extended core 1 O-glycans attached to the peptide backbone.

[0244] 7.2 Results The results are summarized in Figures 8 and 9.

[0245] The O-glycopeptides were identified by post-source decay (PSD) MALDI. The peptide sequence of the identified Seq11 fragment was: KVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLH GSAW (SEQ ID NO: 6). The last four amino acids (underlined) correspond to the C-terminal Strep-Tag. This peptide contains four O-glycosylation sites.

[0246] The top spectrum in Figure 8 shows a fully O-glycosylated glycopeptide, and the bottom spectrum in Figure 8 shows the same glycopeptide after O-glycosidase digestion. The mass shift of 1460.3 Da (labeled by arrows) after O-glycosidase digestion corresponds to four core 1 O-glycans, each with a mass of 365 Da. The 365 Da mass distance observed in the top spectrum corresponds to different glycoforms of the same peptide, while each additional 365 Da mass addition corresponds to a Galβ1→4GlcNAc disaccharide-forming core 2 structure. After O-glycosidase treatment, a fully deglycosylated form of the peptide (8358.6 Da) and glycoforms containing the core 2 structure (Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAc, 730 Da) and / or the extended core 1 structure (Galβ1→4GlcNAcβ1→3Galβ1→3GalNAc, 730 Da) are observed.

[0247] The identified peptide sequence of the Seq12 fragment is KVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLH GSAW (SEQ ID NO: 7). The last four amino acids (underlined) correspond to the C-terminal Strep-Tag.

[0248] The upper spectrum in Figure 9 shows a fully O-glycosylated glycopeptide, and the lower spectrum in Figure 9 shows the same glycopeptide after O-glycosidase digestion. The mass shift of 4380.3 Da after O-glycosidase digestion corresponds to 12 core 1 O-glycans, each with a mass of 365 Da, confirming that all 12 O-glycosylation sites in Seq12 are occupied by O-glycans. Similar to that observed for Seq11, the distances of 365 Da and 730 Da observed in the spectrum correspond to Galβ1→4GlcNAc disaccharides or core 2 and / or extended core 1 structures, respectively.

[0249] Quantitation of core 1 and core 2 O-glycans is based on the relative amounts of glycopeptides to which each O-glycan is attached. Quantitation of different glycoforms of a given peptide is performed by evaluation of signal intensity in the MALDI spectrum.

[0250] For glycopeptide Seq11, quantification is based on the MALDI signal intensity of different glycoforms of the same peptide. The total peak intensity of all glycoforms of this peptide (8358 Da, 8724 Da, 9089 Da, 9454 Da, 9819 Da, 10181 Da, 10543 Da, 10910 Da, 11277 Da) is equal to 41275 a.u., which represents 100%.

[0251] A glycoform containing only core 1 type O-glycans with a mass of 8358 Da exhibits an intensity of 8410 a.u., which corresponds to 20% of the total. Thus, all other glycoforms (80%) contain at least one core 2 and / or extended core 1 glycan type glycan attached. In this measurement, core 2 and extended core 1 are indistinguishable. Therefore, the proportion of core 2 and / or extended core 1 type O-glycans based on the total number of O-glycans is at least 20%.

[0252] Thus, four O-glycosylation sites in Seq11 and all 12 O-glycosylation sites in Seq12, recombinantly produced in HEK cell lines, are occupied by core 1 O-glycans and to a large extent by core 2 O-glycans and / or extended core 1 O-glycans. Given the abundance of core 2 O-glycans, both sequences may be good ligands for SIGLEC binding. Addition of two additional sequence repeats containing four O-glycosylation sites successfully resulted in proteins with 12 clustered and fully occupied O-glycans.

[0253] Example 8 Analysis of SIGLEC binding of Seq11 and Seq12 8.1 Experimental Procedure The Strep-Tag-bearing recombinant proteins Seq11 and Seq12 were tested in SIGLEC binding ELISA as described in Example 1, except for the following detection strategy: instead of streptavidin-HRP, Strep-Tactin-HRP (#2-1502-001, IBA GmbH) conjugate was used to detect the Strep-tagged proteins. The applied concentration of Strep-Tactin-HRP was 0.25 μg / ml. Both proteins Seq11 and Seq12 were tested at an equimolar concentration of 42 nM.

[0254] 8.2 Results As shown in Figure 10, both polypeptides Seq11 and Seq12 exhibited binding to SIG-5, SIG-7, SIG-F, and SIG-9. The absorbance measured for both polypeptides Seq11 and Seq12 against all four SIGLECs was in the same range as the binding of Seq11 and Seq12 to anti-vWF.

[0255] In contrast, the absorbance of Seq11 and Seq12 in the experiments using SIG-2 and SIG10 was in the same range as the negative control experiment using an anti-chicken antibody, indicating that neither Seq11 nor Seq12 bound to SIG-2 or SIG-10 (see Figure 10).

[0256] Example 9 Sialic acid dependence of SIGLEC binding of Seq11 and Seq12 9.1 Experimental Procedure The experimental protocol according to Example 8 was repeated with the addition of sialidase A digestion of the strep-tagged polypeptides Seq11 and Seq12. Desialylation was carried out as described in Example 3.

[0257] 9.2 Results The results of binding experiments using desialylated Seq11 and Seq12 are shown in Figure 11. According to the measured absorbance, binding of desialylated Seq11 and Seq12 to SIG-5 is strongly reduced compared to the untreated polypeptides. Binding to SIG-7, SIG-F, and SIG-9 is completely abolished; i.e., the absorbance is at the same level as that measured for binding to SIG-2 and SIG10. Thus, binding of both polypeptides, Seq11 and Seq12, to SIG-5, SIG-7, SIG-F, and SIG-9 is sialic acid dependent.

[0258] Example 10 Comparison of SIGLEC binding in Seq11 and Seq12 10.1 Experimental Procedure To measure and compare the apparent binding affinities of Seq11 and Seq12, SIGLEC ELISA with Scatchard analysis of binding curves was applied. ELISA was performed as described in Examples 8 and 9. Scatchard analysis was performed using Graph Pad Prism software.

[0259] 10.2 Results The binding curves and corresponding Scatchard plots for sequences 11 and 12 are shown in Figures 12 and 13. The apparent binding affinities (K D) are summarized in Figure 14. Increasing the O-glycan repeats in Seq12 had a significant effect on SIGLEC binding affinity. The affinity for SIGLEC5 increased from 0.494 μM for Seq11 to 0.14 μM for Seq12. The affinity for SIGLEC7 increased from 0.371 μM for Seq11 to 0.005 μM for Seq12. The affinity for SIGLEC8 increased from 1.027 μM for Seq11 to 0.015 μM for Seq12. Finally, the affinity for SIGLEC9 increased from 0.591 μM for Seq11 to 0.041 μM for Seq12. Note in the text: "From this ELISA experiment, dissociation affinity constants are calculated for all Seq11 and 12-SIGLEC interactions."

[0260] Example 11 Measurement of FVIII binding affinity of Seq11 and Seq12 11.1 Experimental Procedure FVIII binding of both sequences was assessed by surface plasmon resonance (SPR). Analysis was performed using a Biacore3000 (GE Healthcare) instrument. Sequences 11 and 12 polypeptides were immobilized on a CM5 chip using an amine coupling kit (GE Healthcare). Full-length plasma VWF (Wilate, Octapharma) was immobilized as a positive control. Subsequently, a concentration series of FVIII (Nuwiq, Octapharma) (0.2 nM, 0.6 nM, 1.7 nM, 5.0 nM, 15 nM, 45 nM) was injected across the sensor chip surface. The running buffer was 150 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 0.05%.

[0261] 11.2 Results SPR measurements revealed that the FVIII binding affinity (KD) of both sequences was equal to 1.4 nM, therefore the additional O-glycan repeats had no effect on the binding affinity to FVIII.

[0262] Example 12 N-terminal VWF fragments, but not C-terminal VWF fragments, reduce extracellular levels of IL-12p70 and IFN-γ 12.1 Background SIGLECs are expressed on various cells of the immune system, such as monocytes and dendritic cells, and exhibit roles in modulating cell adhesion, endocytosis, and adaptive and innate immune signaling pathways (Macauley et al. 2014). Most SIGLECs contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or ITIM-like motifs in their cytoplasmic domains, which have been shown to function in attenuating inflammatory responses by inhibiting cell proliferation and activation (Vitale et al. 1999; Ikehara et al. 2004), inducing apoptosis (Nutku et al. 2003), and suppressing cytokine production (Erdmann et al. 2009; Chen et al. 2013).

[0263] To determine whether the levels of inflammatory cytokines produced by moDCs were altered in the presence of SIGLEC-engaging vWF fragments, the amounts of IL-12p70 and IFN-γ in the supernatants of stimulated immature monocyte-derived dendritic cells (moDCs) were simultaneously analyzed by flow cytometry.

[0264] 12.2 Experimental Design Monocytes from healthy donors were enriched via a Ficoll gradient, followed by purification of CD14+ monocytes by magnetic cell sorting. To obtain moDCs, CD14+ monocytes were cultured for 5–6 days in RPMI medium supplemented with 10% fetal bovine serum, 1000 U / ml interleukin-4, and 1000 U / ml granulocyte-macrophage colony-stimulating factor. Cytokine profiles secreted by moDCs were analyzed 24 h after stimulation with the respective vWF fragments using a cytometric bead array (CBA Flex, BD) detecting IL-12p70 and IFN-γ according to the manufacturer's recommendations. Cells treated with an identical volume of 100 mM NaCl served as a control. Samples were analyzed using a FACS Verse flow cytometer. Final analysis and calculation of cytokine concentrations were performed using FCAP Array software (BD).

[0265] 12.3 Results Figure 16 shows cytokine concentrations after incubation of moDCs with two vWF fragments with and without LPS stimulation. These results indicate that the N-terminal portion of vWF, but not the C-terminal portion, reduces the production of pro-inflammatory cytokines synthesized in response to LPS stimulation. Without LPS stimulation, no effect of the vWF fragments on the secretion of pro-inflammatory cytokines could be detected.

[0266] Example 13 Analysis of phosphorylation of SIGLECs and their adaptor molecules 13.1 Background Upon binding of sialic acid-containing ligands, the ITIM and ITIM-like motifs of SIGLECs are phosphorylated by SRC family tyrosine kinases, leading to the recruitment of SRC homology 2 (SH2) domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2. These phosphatases, upon activation, can dephosphorylate cellular substrates, thereby regulating the activation of various signaling pathways (Crocker et al., 2007). While SHP-1 plays a role in inhibitory signaling, SHP-2 has been reported to enhance signal transduction in most signaling pathways, but also to negatively regulate intracellular signaling processes (An et al., 2006; Avril et al., 2004; Boyd et al., 2009; Qu, 2000; Salmond and Alexander, 2006).

[0267] 13.2 Experimental Procedure moDCs were prepared as described in Example 12. To measure the effect of N-terminal vWF fragments on the tyrosine phosphorylation of SIGLECs and their adaptor molecules SHP-1 and SHP-2, 6 * 10^6 moDCs were incubated with 500 nM N-terminal vWF fragment for 10 min. Cells stimulated with the same volume of 100 mM NaCl served as a control. Analysis of immunoreceptor phosphorylation was performed using 500 μg of cell lysate using the Proteome Profiler Human Phospho-Immunoreceptor Array Kit (R&D Systems) according to the manufacturer's recommendations.

[0268] 13.3 Results The results of the phospho-immunoreceptor array are shown in Figure 17. According to the measured pixel density, the N-terminal vWF fragment specifically alters the phosphorylation of SHP-1, SHP-2, SIG-5, and SIG-7 compared to the control. No phosphorylation of SIG-2 and SIG-10 could be observed, which correlates closely with the lack of binding of the vWF N-terminal fragment to these SIGLECs.

[0269] Many modifications and other embodiments of the invention described herein will come to mind to those skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing detailed description and the associated drawings. It is to be understood, therefore, that the invention is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although certain terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0270] References An, H., Zhao, W., Hou, J., Zhang, Y., Xie, Y., Zheng, Y., Xu, H., Qian, C., Zhou, J., Yu, Y., Liu, S., Feng, G., and Cao, X. (2006). SHP-2 phosphatase negatively regulates the TRIF adapter protein-dependent type I interferon and proinflammatory cytokine production.Immunity.25,919-928. Avril, T., Floyd, H., Lopez, F., Vivier, E., and Crocker, PR (2004).The membrane-proximal immunoreceptor tyrosine-based inhibitory motif is critical for the inhibitory signaling mediated by Siglecs-7 and-9,CD33-related Siglecs expressed on human monocytes and NK. cells.J.Immunol.173,6841-6849. Boyd,C.R.,Orr,S.J.,Spence,S.,Burrows,J.F.,Elliott,J.,Carroll,H.P.,Brennan,K.,Ni,G.J.,Coulter,W.A.,Jones,C.,Crocker,P.R.,Johnston,J.A.,and Jefferies,C.A.(2009).Siglec-E is up-regulated and phosphorylated following lipopolysaccharide stimulation in order to limit TLR-driven cytokine production.J.Immunol.183,7703-7709. Chen,Weilin;Han,Chaofeng;Xie,Bin;Hu,Xiang;Yu,Qian;Shi,Liyun et al.(2013):Induction of Siglec-G by RNA viruses inhibits the innate immune response by promoting RIG-I degradation.In:Cell 152(3),S.467-478.DOI:10.1016 / j.cell.2013.01.011. Crocker,P.R.,Paulson,J.C.,and Varki,A.(2007).Siglecs and their roles in the immune system.Nat.Rev.Immunol.7,255-266. Ewenstein BM,Collins P,Tarantino MD,Negrier C,Blanchette V,Shapiro AD,Baker D,Spotts G,Sensel M,Yi SE,Gomperts ED.Hemophilia therapy innovation development of an advanced category recombinant factor VIII by a plasma / albumin-free method Proceedings of a Special Symposium at the XIXth Congress of the International Society on Thrombosis and Haemostasis;2004,vol.41,pg.1-16. Erdmann,Hanna;Steeg,Christiane;Koch-Nolte,Friedrich;Fleischer,Bernhard;Jacobs,Thomas(2009):Sialylated ligands on pathogenic Trypanosoma cruzi interact with Siglec-E(sialic acid-binding Ig-like lectin-E).In:Cellular microbiology 11(11),S.1600-1611.DOI:10.1111 / j.1462-5822.2009.01350.x. Franc V,Rehulka P,Raus M,Stulik J,Novak J, Renfrow MB,Sebela M.Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF / TOF mass spectrometry:role of cysteine alkylation during sample processing.Journal of Proteomics,2013,Oct 30;vol.92,pg.299-312 Guzman-Aranguez A,Argueeso P.Structure and Biological Roles of Mucin-type O-glycans at the Ocular Surface.The ocular surface.2010;vol.8(1)pg-8-17. Ikehara,Yuzuru;Ikehara,Sanae Kabata;Paulson,James C.(2004):Negative regulation of T cell receptor signaling by Siglec-7(p70 / AIRM)and Siglec-9.In:The Journal of biological chemistry 279(41),S.43117-43125.DOI:10.1074 / jbc.M403538200. Lai JD Georgescu MT,Hough C,Lillicrap D.To clear or to fear:An innate perspective on factor VIII immunity,Cellular Immunology,2016 Mar;vol.301,pg.82-89. Nutku,Esra;Aizawa,Hideyuki;Hudson,Sherry A.;Bochner,Bruce S.(2003):Ligation of Siglec-8:a selective mechanism for induction of human eosinophil apoptosis.In:Blood 101(12),S.5014-5020.DOI:10.1182 / blood-2002-10-3058. Pegon JN,Kurdi M,Casari C,Odouard S,Denis CV,Christophe OD,Lenting PJ.Factor VIII and von Willebrand factor are ligands for the carbohydrate-receptor Siglec-5,Haematologica.2012 Dec;97(12):1855-63. Paulson JC,Macauley MS,and Kawasaki N.Siglecs as sensors of self in innate and adaptive immune responses.Ann N Y Acad Sci.2012 April;1253(1):37-48. Qu,C.K.(2000).The SHP-2 tyrosine phosphatase:signaling mechanisms and biological functions.Cell Res.10,279-288. Salmond,R.J.,and Alexander,D.R.(2006).SHP2 forecast for the immune system:fog gradually clearing.Trends Immunol.27,154-160. Solecka BA,Weise C,Laffan MA,Kannicht C,Site-specific analysis of von Willebrand factor O-glycosylation,J Thromb Haemost.2016 Jan 19. Vitale,C.;Romagnani,C.;Falco,M.;Ponte,M.;Vitale,M.;Moretta,A.et al.(1999):Engagement of p75 / AIRM1 or CD33 inhibits the proliferation of normal or leukemic myeloid cells.In:Proceedings of the National Academy of Sciences of the United States of America 96(26),S.15091-15096.

Claims

1. A glycosylated polypeptide comprising an amino acid sequence identical or homologous to at least a fragment of a mammalian, preferably human, protein, wherein the glycosylated polypeptide contains one or more sialylated O-glycans and exhibits increased binding affinity to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8, and SIG-9 compared to said mammalian protein or fragment thereof.

2. 2. The glycosylated polypeptide of claim 1, which exhibits increased binding to SIGLECs SIG-5, SIG-7, SIG-8, and SIG-9.

3. 3. The glycosylated polypeptide of claim 1, wherein the combined number of sialylated core type 2 and extended core type 1 O-glycans of the glycosylated polypeptide is greater than the number of sialylated core type 2 and extended core type 1 O-glycans of the mammalian protein or fragment thereof.

4. 3. The glycosylated polypeptide of claim 1, wherein the number of sialylated core type 2 O-glycans of the glycosylated polypeptide is greater than the number of sialylated core type 2 O-glycans of the mammalian protein or fragment thereof.

5. 5. The glycosylated polypeptide of any one of claims 1 to 4, which contains one or more clusters of O-glycosylation sites, wherein the cluster contains at least two O-glycosylation sites, more preferably at least three O-glycosylation sites, and most preferably at least four O-glycosylation sites.

6. 6. The glycosylated polypeptide of claim 5, wherein the one or more clusters contain at least one O-glycosylation site among 10 amino acids, preferably at least one O-glycosylation site among 4 amino acids, more preferably at least one O-glycosylation site among 3 amino acids, and most preferably one O-glycosylation site among 2 amino acids.

7. 7. A glycosylated polypeptide according to claim 5 or 6, containing at least two clusters, more preferably at least three clusters.

8. 8. The glycosylated polypeptide of claim 5, wherein the clusters are separated by less than 100 amino acids, preferably less than 50, more preferably less than 30 amino acids.

9. 9. The glycosylated polypeptide according to any one of claims 1 to 8, wherein the sialylated O-glycan contains at least two sialic acids, more preferably at least three sialic acids, in α2-3 and / or α2-8 glycosidic linkages.

10. 10. The glycosylated polypeptide of claim 9, wherein the percentage of core 2 type O-glycans based on the number of sialylated O-glycans is at least 5%, more preferably at least 10%, more preferably at least 35%, and most preferably at least 50%.

11. A glycosylated protein according to any one of claims 1 to 10, further comprising a multimerization domain.

12. The glycosylated polypeptide according to any one of claims 1 to 11, wherein the human protein is selected from vWF, FVIII, FVII, FIX, ADAMTS13.

13. The glycosylated polypeptide of claim 12, wherein the human protein or fragment thereof is a FVIII protein, in particular a full-length FVIII, a B-domain deleted FVIII, or a FVIII protein in which part of the B-domain has been replaced by a spacer peptide.

14. 14. The glycosylated polypeptide according to any one of claims 3 to 13, wherein one or more O-glycosylation sites are located within an amino acid sequence that is homologous or identical to said mammalian protein or a fragment thereof, in particular in place of an amino acid of said mammalian protein or a fragment thereof.

15. 14. The glycosylated polypeptide of any one of claims 3 to 13, which is a fusion protein, wherein a second amino acid sequence containing one or more O-glycosylation sites is covalently linked to an amino acid sequence identical or homologous to said human protein or a fragment thereof.

16. 16. The glycosylated polypeptide of claim 15, wherein the covalent linker is selected from a peptide bond, a chemical linker, or a glycosidic bond.

17. 17. The glycosylated polypeptide of claim 15 or 16, wherein the second amino acid sequence is at least 98% homologous, preferably identical, to amino acids 1238 to 1268 of SEQ ID NO:1 or to two consecutive copies of amino acids 1238 to 1268 of SEQ ID NO:

1.

18. Glycosylated polypeptide according to any one of claims 1 to 17, preferably produced by expression in a human cell line.

19. The use of a glycosylated polypeptide containing one or more sialylated O-glycans and exhibiting binding to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8, and SIG-9 to reduce the immune response of a second polypeptide, particularly a therapeutic protein.

20. A composition comprising a first and a second polypeptide, wherein the first polypeptide is a glycosylated polypeptide containing one or more sialylated O-glycans, and the second polypeptide contains an amino acid sequence that is homologous or identical to a second mammalian, particularly a human, protein, and has increased binding affinity to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8, and SIG-9 compared to the second polypeptide.

21. 20. The composition of claim 19, wherein the first and second polypeptides form a protein complex.

22. The composition of claim 21, wherein the first polypeptide is a polypeptide according to any one of claims 1 to 18.

23. The composition according to any one of claims 20 to 22, wherein the second polypeptide is selected from a FVIII protein, FVII, FIX, ADAMTS13, in particular a full-length FVIII protein, a B-domain deleted FVIII protein, or a FVIII protein in which part of the B-domain has been replaced by a linker.

24. The composition according to any one of claims 20 to 23, wherein the first polypeptide comprises at least a fragment of human vWF, in particular the vWF fragment is at least 95% identical to the sequence defined by 764 to 1268 of SEQ ID NO: 1, at least 95% identical to SEQ ID NO: 2, at least 95% identical to SEQ ID NO: 3, at least 95% identical to SEQ ID NO: 4, or at least 95% identical to SEQ ID NO: 5, or and the second polypeptide is a FVIII protein.

25. An isolated polynucleotide encoding a glycosylated polypeptide according to any one of claims 1 to 18.

26. An isolated polynucleotide encoding the glycosylated polypeptide having an amino acid sequence that has at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% identity to a sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:

5.

27. 27. A vector containing a vector backbone and a polynucleotide according to claim 25 or 26, wherein the vector backbone is preferably selected from pCDNA3, pCDNA3.1, pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1, pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES and pSCAS.

28. 28. A host cell containing a polynucleotide according to claim 25 or 26 or a vector according to claim 27, wherein the host cell is preferably a mammalian cell, preferably a human cell, more preferably a human kidney cell, most preferably a human embryonic kidney cell line, in particular a HEK293 cell line, such as HEK293F.

29. A glycosylated polypeptide according to any one of claims 1 to 18 or a composition according to any one of claims 20 to 24 for use in the treatment or prevention of bleeding disorders, preferably selected from the treatment of PUP and ITI treatment.

30. 30. The glycosylated polypeptide or protein complex for use according to claim 29, wherein said use comprises intravenous or non-intravenous injection, preferably subcutaneous injection.

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