RU2776807C2 - Polypeptides modulating siglec-dependent immune responses - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: there are presented: a glycosylated polypeptide containing an amino acid sequence, having at least 90 % identity to at least a fragment of von Willebrand factor (vWF), as well as use thereof to reduce the immune response to a second polypeptide, in particular a therapeutic protein, composition for treating or preventing a bleeding disorder in a patient, comprising a first and a second polypeptide in an effective amount, an isolated polynucleotide encoding a glycosylated polypeptide, a vector for producing a glycosylated polypeptide and a host cell.

EFFECT: novel molecule comprising an amino acid sequence having at least 90 % identity to at least a human von Willebrand factor (vWF) fragment, allows using it for effective treatment and prevention of blood clotting disorders in patient.

24 cl, 17 dwg, 13 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to glycosylated mammalian protein-based polypeptides eliciting reduced immune response or increased immune tolerance due to modulated SIGLEC binding, and methods of treatment using the glycosylated polypeptide. In addition, the invention relates to protein complexes that cause a reduced immune response or increased immune tolerance due to the modulated binding of SIGLEC, and methods of treatment using these protein complexes.

BACKGROUND OF THE INVENTION

Hemophilia is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation. In the most common form, hemophilia A, clotting factor VIII (FVIII) is deficient, with hemophilia A occurring in about 1 in 5,000 to 10,000 male births. The FVIII protein is an essential clotting cofactor with multiple functions. FVIII deficiency can be replenished with plasma-derived FVIII concentrates or recombinantly produced FVIII. Treatment with FVIII concentrates has led to a normalization of the lives of patients with hemophilia. Historically, hemophilia has been treated with FVIII derived from human plasma. In blood plasma, under normal conditions, the FVIII molecule is always associated with its cofactor, von Willebrand factor (vWF), which ensures the stability of the FVIII molecule to various types of degradation.

Many methods have been described for purifying factor VIII from plasma or culture mass that recombinantly produces factor VIII (rFVIII), without and with von Willebrand factor. In the 1990s, the first commercially available recombinant FVIII (rFVIII) were divided into two types: full-length rFVIII molecules that mimic the main form of FVIII in blood plasma, and rFVIII molecules with a B-domain removed (Eriksson et al., 2001), in which one inactive region (B-domain) was removed, both types being of high purity (all without vWF).

Patients with hemophilia A are treated with FVIII as needed or as a prophylactic therapy several times a week. For prophylactic treatment, FVIII at 15-25 IU/kg body weight is administered three times a week, which is necessary because of the constant need for FVIII and its short circulatory half-life, which in humans is only about 11 hours. (Ewenstein et al., 2004).

Frequent chronic administration of exogenous FVIII elicits a response from the patient's immune system (Saenko et al., Haemophilia 8:1-11 (2002)), which is a serious limitation for therapy.

Currently, the most common option for achieving immune tolerance in patients with hemophilia A (congenital FVIII deficiency) and inhibitors is immune tolerance induction (ITI) when high doses of FVIII are administered over a long period of time. However, treatment can take up to two years, fails in approximately 30% of patients, is extremely expensive, and cannot be used prophylactically to suppress the initial occurrence of inhibitory antibodies.

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

For example, WO 2014/176125 A1 describes immune conjugates for inducing antigen-specific immune tolerance to FVIII. Immune conjugates are FVIII proteins conjugated to certain glycan ligands that target SIGLEC molecules expressed on B cells, namely SIG-1 or SIG-10 (or an orthologue of SIG-G). Glycan ligands, in particular, are associated with liposomes into which FVIII is introduced.

Sialic acid-binding immunoglobulin lectins (SIGLEC) are a family of 15 human and 9 murine cell surface receptors that are expressed on various white blood cells of the immune system, with the exception of most T cells in mice and humans. SIGLECs are localized to various cell types and bind to various glycan structures (see review 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 unknown.

Another approach is described in WO 2014/179184 A1. The authors suggest the reduction of unwanted humoral immune response and the induction of immune tolerance against blood clotting factors such as FVIII by the addition of SIGLEC ligands. SIGLEC ligands are selected from 9-N-biphenylcarboxy-NeuAca2-6Gal~l-4GlcNAc (6'-BPCNeuAc), NeuAca2-6Galwl-4GlcNAc and NeuAca2-6Galwl-4(6-sulfo)GlcNAc. The SIGLEC ligand is linked to the clotting factor via a water soluble polymer.

SUMMARY OF THE INVENTION

The present invention is based, inter alia, on the discovery that the glycan structure naturally occurring on plasma proteins, in particular vWF, allows interaction with a group of SIGLEC molecules, in particular SIG-5, SIG-7, SIG-8 and SIG- 9. In addition, the inventors found that by modifying the glycan structure on the protein, interaction with SIGLECs such as SIG-5, SIG-7, SIG-8, and SIG-9 can be increased. This increase results in a decrease in the immune response and/or an increase in immune tolerance in the patient receiving the protein.

Thus, according to a first aspect, the invention relates to a glycosylated polypeptide comprising an amino acid sequence identical or homologous to at least a fragment of a mammalian, preferably human protein, wherein said glycosylated polypeptide contains one or more sialylated O-glycans, and wherein the polypeptide has an 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 a mammalian protein or fragment thereof.

The authors of the present invention, in particular, have identified a glycan structure, which, on the one hand, is necessary for interaction with SIGLEC, but also the addition of which leads to increased binding to SIGLEC. Responsible for this are type 2 sialylated core O-glycans and/or extended type 1 core O-glycans.

Thus, a glycosylated polypeptide according to the first aspect can also be described as a glycosylated polypeptide comprising an amino acid sequence identical or homologous to at least a portion of a mammalian, preferably human, protein, wherein said glycosylated polypeptide contains one or more sialylated O-glycans, and where the combined amount of sialylated type 2 core O-glycans and type 1 sialylated core O-glycans of the glycosylated polypeptide exceeds the combined amount of type 2 sialylated core O-glycans and type 1 sialylated core O-glycans in the protein mammal or its fragment.

A protein with a glycan composition comprising type 2 sialylated core O-glycans and/or type 1 sialylated core O-glycans, in particular sialylated core 2 O-glycans, can be used to modify a patient's immune response to a therapeutic protein. when administered together.

Thus, in a second aspect, the invention relates to the use of a glycosylated polypeptide comprising one or more sialylated O-glycans and binding to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8, and SIG-9 to reduce immune response or increase the patient's immune tolerance to the therapeutic protein.

Using the modification of a glycosylated polypeptide that binds affinity to SIGLEC, one can not only directly modify the binding affinity for SIGLEC of the modified polypeptide itself, but also of the protein complex or composition of which the glycosylated polypeptide is a part, e.g. Willebrand (vWF).

Thus, according to a third aspect, the present invention relates to a protein composition comprising first and second polypeptides, where 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 the second mammalian protein, in particular, a human, where (compared to the second polypeptide) the composition has an increased binding affinity for one or more SIGLECs selected from SIG-5, SIG-7, SIG-8, and SIG-9. Preferably, the first and second polypeptide of the composition according to the third aspect form a protein complex.

According to a fourth aspect, the present invention relates to an isolated polynucleotide which contains a nucleic acid sequence encoding a glycosylated polypeptide according to the first aspect of the invention. In a fifth aspect, the invention also relates to an expression vector containing a polynucleotide according to the fourth aspect of the invention.

The glycosylated polypeptide according to the first aspect, in particular vWF or FVIII, and the composition according to the third aspect, in particular the complex of FVIII and vWF, due to the reduced immune response, are particularly useful in therapy.

Thus, according to a fourth aspect, the present invention relates to a glycosylated polypeptide according to the first aspect, or a composition according to the third aspect, for use in the treatment or prevention of bleeding disorders.

FIGURES

Figure 1 shows the results of the vWF binding test with various SIGLECs. The absorbance at 492 nm is proportional to the amount of vWF bound to the respectively indicated SIGLEC or controls. SIG-2, SIG-5, SIG-7, SIG-F, SIG-9 and SIG-10 were immobilized with protein A on a microtiter plate at a concentration of 500 ng/well. Biotinylated vWF was added at a concentration of 0 to 0.8 μg/ml, and after washing, binding was visualized using HRP-conjugated streptavidin and measuring absorbance at 492 nm. Anti-vWF and anti-chicken IgY antibodies were used as controls.

Figure 2 is a schematic representation of the vWF domain structure, including sites for N- and O-glycosylation, cleavage with V8 protease, and fragments obtained after cleavage with V8 protease.

Figure 3 shows the results of the binding test of the N-terminal and C-terminal vWF fragment with SIGLEC - SIG-5, SIG-7, SIG-F and SIG-9. The optical density at 492 nm is proportional to the amount of vWF fragment bound to the respective SIGLEC or control, respectively. SIG-5, SIG-7, SIG-F and SIG-9 were immobilized with protein A on a microtiter plate at a concentration of 500 ng/well. Biotinylated N-terminal VWF fragment (dark gray bars) and C-terminal VWF fragment (light gray bars)) were added at a concentration of 1 μg/ml, and after washing, binding was visualized using HRP-conjugated streptavidin and absorbance measured at 492 nm. Anti-chicken IgY antibodies were used as a negative control.

Figure 4 shows the results of the binding test of the N-terminal vWF fragment in desialylylated, de-N-glycosylated and raw form. The absorbance at 492 nm is proportional to the amount of N-terminal vWF fragment bound to the respective SIGLEC or control, respectively. The N-terminal fragment of VWF before cleavage is represented by white bars, the de-N-glycosylated PNGaseF fragment is shown by gray bars, and the desialylated fragment is shown by black bars. SIG-5, SIG-7, SIG-F and SIG-9 were immobilized with protein A on a microtiter plate at a concentration of 500 ng/well. Biotinylated N-terminal vWF fragments (before digestion, digested with PNGaseF or Sialidase A) were added at a concentration of 8 μg/ml, and after washing, binding was visualized using HRP-conjugated streptavidin and measuring absorbance at 492 nm. Anti-chicken IgY antibodies were used as a negative control.

In FIG. 5 shows the results of the Cluster I and Cluster II O-glycosylation binding assay with SIGLEC. Absorbance at 492 nm is proportional to the amount of cluster I fragment (light gray bars) or cluster II fragment (dark gray bars) bound to the respective SIGLEC or control, respectively. SIG-5, SIG-7, SIG-F, SIG-9 and SIG-10 were immobilized with protein A on a microtiter plate at a concentration of 500 ng/well. Biotinylated cluster I and cluster II were added at a concentration of 4 μg/ml, and after washing, binding was visualized using HRP-conjugated streptavidin and measuring absorbance at 492 nm. Anti-chicken IgY antibodies were used as a negative control.

Figure 6 shows the results of the Cluster II O-glycosylation binding test with SIGLEC before and after treatment with sialidase A. Absorbance at 492 nm is proportional to the amount of untreated fragment of cluster II (light gray bars) or fragment of cluster II cleaved with Sialidase A (dark gray bars) associated with the respective SIGLEC or control. SIG-5, SIG-7, SIG-F and SIG-9 were immobilized with protein A on a microtiter plate at a concentration of 500 ng/well. Biotinylated cassette II before digestion (light gray bars) and digested with Sialidase A was added at a concentration of 2 μg/ml, and after washing, binding was visualized using HRP-conjugated streptavidin and measuring absorbance at 492 nm. Anti-chicken IgY antibodies were used as a negative control.

Figure 7 shows a schematic representation of recombinantly expressed fragments of vWF fragments - Seq11 and Seq12.

8 shows MALDI mass spectra of O-glycopeptide isolated from Seq11 after trypsin/chymotrypsin digestion; cleavage with Sialidase A and enrichment on lectin. The peptide sequence identified is KVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLH GSAW (SEQ ID NO: 6) where the last four amino acids (underlined) correspond to the tag attached to the C-terminal sequence. Upper spectrum shows full length O-glycosylated glycopeptide, lower spectrum shows the same glycopeptide after cleavage with O-glycosidase.

9 shows MALDI mass spectra of O-glycopeptide isolated from Seq12 after trypsin/chymotrypsin digestion; sialidase A digestion and lectin enrichment. The identified peptide sequence is KVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLH GSAW (SEQ ID NO:7), where the last four amino acids (underlined) correspond to the tag attached to the C-terminal sequence. Upper spectrum shows full length O-glycosylated glycopeptide, lower spectrum shows the same glycopeptide after cleavage with O-glycosidase.

Figure 10 shows the results of the test for binding of recombinant polypeptides Seq11 and Seq12 with SIGLEC. Absorbance at 492 nm is proportional to the amount of Seq11 (dark gray bars) or Seq12 (light gray bars) bound to the respective SIGLEC or control, respectively. SIG-5, SIG-7, SIG-F and SIG-9 were immobilized with protein A on a microtiter plate at a concentration of 500 ng/well. Strep-Tag-tagged sequences were plated at equal molar concentrations of 42 nM, and after washing, binding was visualized using HRP-conjugated Strep-Tactin and measuring absorbance at 492 nm. Anti-chicken IgY was used as a negative control and anti-vWF pAb was used as a positive control.

Figure 11 shows the results of a binding assay of the recombinant Seq11 and Seq12 polypeptides after treatment with Sialidase A. Absorbance at 492 nm is proportional to the amount of Seq11 (dark gray bars)) or Seq12 (light gray bars) bound to respectively indicated SIGLEC or control. SIG-5, SIG-7, SIG-F and SIG-9 were immobilized with protein A on a microtiter plate at a concentration of 500 ng/well. Strep-Tag tagged sequences were enzymatically desiallylated and plated at equal molar concentrations of 42 nM, and after washing, binding was visualized using HRP-conjugated Strep-Tactin and measuring absorbance at 492 nm. Anti-chicken IgY was used as a negative control and anti-vWF pAb was used as a positive control.

Figure 12 shows the results of concentration-dependent binding of recombinant Seq11 polypeptide to SIGLEC, and Scatchard analysis of specific binding curves.

Figure 13 shows the results of concentration-dependent binding of the recombinant Seq12 polypeptide to SIGLEC, and Scatchard analysis of specific binding curves.

FIG. 14 shows the final K _D values obtained from the Scatchard analysis performed on the curves shown in FIGS. 12 and 13. FIG.

Figure 15 shows dissociation constants (K _D ) calculated for the binding of Seq11, Seq12 and full-length plasma VWF to recombinant FVIII. The data was obtained using SPR.

16 shows the 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 column) or supplemented (right column) with 0.1 μg/ml LPS. Extracellular levels of cytokines were determined simultaneously using cytometric analysis on beads. The levels of IL-12p70 and IFN-γ in unstimulated cells were below the detection limit for most donors (bd, 0.6 pg/ml for IL-12p70 and 1.8 μg/ml for IFN-γ). Data are presented as mean ± SEM, with each point representing data from a single donor.

Figure 17 shows the results of phosphorylation of the SIGLEC adapter molecules SHP-1 and SHP-2 involved in the SIGLEC signaling pathway after moDC stimulation for 10 minutes with a 500 nM N-terminal VWF fragment. Cells stimulated with the same volume of 100 mM NaCl served as controls. Analysis of immunoreceptor phosphorylation in cell lysates was performed using the Proteome Profiler Human Phosphosphoreceptor Array Kit. Results are shown as mean pixel density ± SEM for 2-4 separate experiments.

DETAILED DESCRIPTION OF THE INVENTION

For a clear and precise understanding of the description and claims, including the scope defined by such terms, definitions are provided below.

Definitions

Used in the present description, the term "peptide" may consist of any number of amino acids of any type, preferably natural amino acids, which are preferably connected by peptide bonds. In particular, the peptide contains at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12 or at least 15 amino acids. In addition, there is no upper limit to the length of the peptide. However, it is preferred that the peptide of the invention does not exceed 500 amino acids in length, more preferably does not exceed 300 amino acids in length, even more preferably does not exceed 250 amino acids in length.

Thus, the term "peptide" includes "oligopeptides", which generally refers to peptides between 2 and 10 amino acids in length, and "polypeptides", which generally refers to peptides greater than 10 amino acids in length.

As used herein, the term "protein" refers to a peptide of at least 60, at least 80, at least 80, preferably at least 100 amino acids. The terms "polypeptide" and "protein" are used interchangeably. Polypeptides and proteins in the context of the present invention include chemically synthesized proteins as well as naturally synthesized proteins that are encoded by genes. The polypeptides or proteins may be obtained from a natural source, such as human blood, or may be obtained in cell culture as recombinant proteins.

As used herein, the term "mammalian protein" refers to a naturally occurring mammalian protein, that is, a protein naturally expressed by a mammalian body. Therefore, a mammalian protein has a natural amino acid sequence and natural post-translational modifications such as glycosylation. In accordance with the present invention, the terms "mammalian protein" and "natural mammalian protein" can be used interchangeably.

As used herein, the term "human protein" refers to a naturally occurring human protein, that is, a protein naturally expressed by the human body. Thus, the human protein has a natural amino acid sequence and natural post-translational modifications such as glycosylation. In accordance with the present invention, the terms human protein and natural human protein are used interchangeably.

"Recombinant proteins" or "recombinant polypeptides" in the context of the present invention are those encoded by transgenes introduced into cells by molecular biology techniques. Proteins can be modified by chemical means or by enzymes in post-translational processes.

The term "fusion protein" according to the invention refers to proteins created by combining two or more genes, cDNA or sequences that originally code for separate proteins/peptides. In nature, genes may be found in the same organism or in different organisms, or may be synthetic polynucleotides.

The term "therapeutic protein" as used herein refers to proteins or polypeptides with a therapeutic effect, namely proteins used as an active pharmaceutical ingredient.

The relationship between two amino acid sequences or between 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 and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needle program in 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. The optional parameters used are the gap insertion penalty of 10, the gap lengthening penalty of 0.5, and the EBLOSUM62 substitution matrix (EMBOSS version of the BLOSUM62 matrix). The Needle output indicated as "longest identity" (obtained using the No brief option) is used as percent identity and is calculated as follows: (identical residuals × 100)/(alignment length - total number of gaps in alignment).

The transitional term "comprising" which is synonymous with "including", "including" or "characterized in that" is non-exclusive or open-ended and does not exclude additional, unspecified elements or method steps. The transitive phrase "consisting of" excludes any element, step, or ingredient not listed in the claims, except for the impurities normally associated with them. When the phrase "consists of" appears in a claim, and not immediately after the preamble, it limits only the element set forth in that claim; other elements are not excluded from the claims as a whole. The transitional phrase "consisting essentially of" limits the scope of the claim (claim) to the specified materials or steps "and those that do not significantly affect the essential and new properties (or property)" of the claimed invention. A claim "consisting essentially of" is intermediate between a closed object claim expressed in the format "consisting of" and a fully open claim expressed in the format "comprising".

The term "homologous" as used herein means that the corresponding nucleotide sequence of an amino acid sequence has a given degree of identity with a reference amino acid sequence and given nucleotide sequences. The homologous sequence is expected 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 given sequence using the normal Clustal V sequence alignment program with default parameters. Typically, homologues include the same residues at active sites with a given amino acid sequence, although they may include any number of conservative amino acid substitutions. The term "identical" as used herein refers to 100% amino acid or nucleotide sequence identity with a reference sequence.

The term "recombinant", when used with respect to the cell, nucleic acid, protein or vector in question, indicates that the subject has been modified by introducing a heterologous nucleic acid or protein, or altering a natural nucleic acid or protein, or that the cell is derived from cells modified in this way. For example, recombinant cells express genes that are not found in the natural (non-recombinant) form of the cell, or express natural genes at a different level or under different conditions than in nature.

As used herein, the terms "transformed", "stably transformed", and "transgenic" when used with respect to a cell means that the cell contains a non-natural (e.g., heterologous) nucleotide sequence incorporated into its genome or transferred as an episome that is maintained in a plurality of generations.

The term "fragment" as used in the present invention refers to a polypeptide that has at the amino and/or carboxyl terminus a deletion of one or more amino acids compared to the native or wild-type protein, but the remaining amino acid sequence is identical to the corresponding positions in the amino acid. sequence derived from the full length cDNA. Fragments are typically at least 50 amino acids in length.

The term "glycosylation" as used herein refers to the attachment of glycans to molecules, such as proteins. Glycosylation may be an enzymatic reaction. Attachment can occur through covalent bonds. Accordingly, a glycosylated polypeptide in the context of the present invention is a polypeptide to which glycans are attached. The term "highly glycosylated" refers to a molecule, such as an enzyme, that is glycosylated at all or nearly all of the available glycosylation sites, eg O- or N-linked glycosylation sites.

The term "glycan" as used herein refers to a polysaccharide or oligosaccharide, or a carbohydrate moiety of a glycoprotein or glycosylated polypeptide. Glycans may be homo- or heteropolymers of monosaccharide residues. They may be linear or branched molecules. Glycans typically contain at least three sugars and may be linear or branched. The glycan may include natural sugars (eg, glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and/or modified sugars (eg, 2'-fluororibose , 2'-deoxyribose, phosphomannose, 6'-sulfo-N-acetylglucosamine, etc.)

The term "O-glycans" as used herein refers to glycans that are typically covalently linked to serine and threonine residues in mammalian glycoproteins. O-glycans can be α-linked via an N-acetylgalactosamine (GalNAc) moiety to the OH group of serine or threonine via an O-glycosidic bond. Other glycans 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.

The term "sialylated" as used herein refers to molecules, in particular glycans, that have reacted with sialic acid or its derivatives.

The terms "binding affinity" or "affinity" as used herein indicate the strength of the bond between two molecules, in particular between a ligand and a target protein. Binding affinity is affected by non-covalent intermolecular interactions between two molecules, such as hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces.

Immune response in the context of the present description refers to an adaptive or innate immune response. The innate immune response refers to non-specific defense mechanisms that are activated immediately or within a few hours after a foreign antigen enters the body. These mechanisms include physical barriers such as the skin, chemicals in the blood, and immune system cells that attack foreign cells in the body. The innate immune response is activated by the chemical properties of the antigen. Adaptive immune response refers to an antigen-specific immune response. To do this, the antigen must be processed and recognized. Once an antigen is recognized, the adaptive immune system creates a large number of immune cells specifically designed to attack that antigen.

As used herein, the term "immune tolerance" (or simply "tolerance") is the process by which the immune system does not attack an antigen. This process exists in three forms: central tolerance, peripheral tolerance and acquired tolerance. Tolerance can be "natural" or "self-tolerance", when the body does not develop an immune response to self-antigens, or "induced tolerance", when tolerance to antigens can be developed by manipulating the immune system.

Glycosylated polypeptide

According to a first aspect, the invention relates to a glycosylated polypeptide comprising an amino acid sequence identical or homologous to at least a fragment of a mammalian, preferably human protein, wherein said glycosylated polypeptide contains one or more sialylated O-glycans, and wherein the polypeptide has an increased binding affinity to one or several SIGLECs compared to a mammalian protein or fragment thereof.

The glycosylated polypeptide of the invention is based on a mammalian protein, i. contains an amino acid sequence identical or homologous to a mammalian protein. The mammalian protein is in particular a human protein. The human protein with which the glycosylated peptide is homologous or identical in amino acid sequence is preferably a glycosylated protein.

More preferably, the human protein is a human blood protein. The human blood protein can be a human blood clotting factor, transport protein, protease inhibitor, immunoglobulin, plasma cellular protein, apolipoproteins, complement factor, growth factor, anti-angiogenic protein, highly glycosylated protein, blood factors, or other human blood protein.

The human blood clotting factor is specifically 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.

It is understood that the clotting factors FV, FX, FIX, FVII, FVIII, FXI, FXII and FXIII have an inactive and an activated form. Thus, in the context of the invention, reference to FV, FX, FIX, FVII, FVIII, FXI, FXII, and FXIII includes activated forms of FVa, FXa, FIXa, FVIIa, FVIIIa, FXIa, FXIIa, and FXIIIa, respectively, unless otherwise indicated, or from the context, the activated form may not be logically enabled. 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, FXIII/FXIIIa.

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

Potential protease inhibitors are e.g. , protein S and protein Z.

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

The cellular plasma protein may be, for example, fibronectin, thromboglobulin, platelet factor 4. Examples of apolipoproteins are apo A-I, apo A-II and apo E.

Complement factors according to the invention are, for example, factor B, factor D, factor H, factor I, C3b inactivator, properdin, C4 binding protein, etc.

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-β), fibroblast growth factor (FGF), and hepatocyte growth factor.

Antiangiogenic proteins include latent antithrombin, prelatent antithrombin, oxidized antithrombin, and plasminogen.

Examples of highly glycosylated 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 factors, tPA, gCSF.

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

The human protein is specifically selected from vWF, FVIII, FVII, FIX, ADAMTS13.

Factor VIII in humans is encoded by the F8 gene, which contains 187,000 base pairs in six exons. The transcribed mRNA is 9029 base pairs long and is translated into a 2351 amino acid protein, from which 19 amino acids are removed as a result of post-translational modification. The FVIII molecule in humans is glycosylated at 31 amino acid residues (25 nitrogen glycosylations and 6 O atom glycosylations).

After translation, the amino acid chain is cleaved by specific proteases at specific sites, resulting in a heavy chain of about 200 kDa and a light chain of about 80 kDa. The domain organization is usually characterized as A1-A2-B-A3-C1-C2. The light chain consists of A3-C1-C2 domains. The heavy chain mainly consists of A1-A2-B domains. The heavy chains found in plasma have a heterogeneous composition with molecular weights ranging from 90 to 200 kDa. The reason for this is the heterogeneity of glycosylation, the existence of splicing variants and proteolytic products such as the A ₁ A ₂ heavy chain without the B domain. The amino acid sequence of full length FVIII is represented by amino acids 20 to 2351 in the sequence P00451 (Swisssrot, July 21, 1986).

Preferably, the human protein is a full-length FVIII, represented by amino acids 20 to 2351 in the sequence P00451 (Swisssrot, July 21, 1986), an FVIII with a deletion of the B domain, or an FVIII protein in which a portion of the B domain has been replaced by a linker .

vWF is a multimeric adhesive glycoprotein present in mammalian plasma that has multiple physiological functions. During primary hemostasis, vWF acts as an intermediary between specific receptors on the platelet surface and extracellular matrix components such as collagen. In addition, vWF serves as a carrier and stabilizing protein for procoagulant factor VIII. vWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The polypeptide precursor, pre-pro-vWF, consists of a 22 amino acid signal peptide, a 741 amino acid propeptide, and a 2050 amino acid polypeptide in mature plasma von Willebrand factor (Fischer et al., 1994). The full size vWF is numbered P04275 in the Uniprot database.

After secretion into plasma, vWF circulates in the plasma as molecules of various molecular weights. These vWF molecules are composed of oligo- and multimers of the mature 2050 amino acid subunit. vWF is normally found in plasma as multimers ranging in size from 500 to 20,000 kDa (Furlan et al. 1996). In particular, the amino acid sequence of vWF corresponds to any of the sequences numbered P04275 in Uniprot. More preferably, the vWF protein has the sequence of SEQ ID NO: 1.

SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQR EGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSID INDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK (SEQ ID NO: 1)

The glycosylated polypeptide may, for example, contain a vWF fragment according to WO 2015/185758 A2. As shown in WO 2015/185758 A2, the complex of FVIII and vWF fragments according to the invention shows less binding to phospholipid membranes compared to FVIII alone, as well as reduced binding to collagen III and heparin compared to the FVIII complex and full length vWF.

The above vWF fragment is, in particular, the fragment starting at amino acid 1 in SEQ ID NO: 1. Amino acids 1-272 in SEQ ID NO: 1 contain a FVIII binding domain to vWF.

The vWF fragment, preferably starting at the 1st amino acid of SEQ ID NO 1, preferably ends with an amino acid of SEQ ID NO: 1 in the range of 1142 to 1390. More preferably, the fragment ends with an amino acid in the range of 1267 to 1390. More preferably, so that the vWF fragment ends with an amino acid from SEQ ID NO: 1 in the range from 1337 to 1390.

It should be understood that a glycosylated polypeptide has an increased binding affinity relative to a mammalian protein or fragment as defined by the amino acid sequence contained in the glycosylated peptide. Thus, if a glycosylated polypeptide contains the amino acid sequence of a full length mammalian protein, then the glycosylated polypeptide has a higher affinity for SIGLEC compared to the full length mammalian protein.

On the other hand, if the glycosylated polypeptide contains a fragment of a mammalian protein defined by a subsequence of the mammalian protein, then the glycosylated polypeptide has an increased binding affinity compared to an identical fragment from a natural protein. For example, if the amino acid sequence in a glycosylated polypeptide is identical or homologous to a vWF fragment, then the glycosylated polypeptide of the first aspect has increased binding affinity for one or more SIGLECs compared to the same fragment resulting from fragmentation of a plasma-derived vWF.

As shown in the examples, the vWF glycan structure was determined to bind specifically to at least SIGLEC SIG-5, SIG-7, SIG-8 and SIG-9 (see example 2). Thus, according to one embodiment of the first aspect, one or more SIGLECs are selected from the group SIG-5, SIG-7, SIG-8, and SIG-9.

The inventors unexpectedly 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 bind to these SIGLECs (see example 2). This is especially surprising because, up to now, N-glycans have been shown to interact with SIGLEC (Lai et al, 2015).

The inventors have also determined that not only must O-glycans be sialylated to bind to SIG-5, SIG-7, SIG-8, SIG-9, but a minimum percentage of type 2 core O-glycans must also be present (see example 4).

Therefore, SIGLEC binding and hence reduced immune response is based on an increased number or percentage of sialylated type 2 core O-glycans in the glycosylated protein compared to the number of sialylated type 2 core O-glycans in the mammalian protein or fragment thereof.

Because of the structural analogy, sialylated extended type 1 O-glycan cores are expected to have the same effect as type 2 sialylated core O-glycans. Thus, in order to increase binding affinity to the above SIGLECs, it is preferable that the combined amount or percentage of sialylated type 2 O-glycans and type 1 extended O-glycans is increased.

Thus, according to one embodiment of the invention, the number of sialylated type 2 core O-glycans and/or extended type 1 core O-glycans in the glycosylated polypeptide exceeds the number of sialylated type 2 core O-glycans and/or extended core O -glycans of the 1st type in a mammalian protein or its fragment. Accordingly, the percentage of sialylated type 2 core O-glycans and/or type 1 extended core O-glycans is also increased compared to the percentage of type 2 core O-glycans and/or extended 1-type core O-glycans. type in a mammalian protein.

This means that the combined amount of type 2 sialylated core O-glycans and type 1 sialylated core O-glycans of the glycosylated polypeptide exceeds the combined amount of type 2 sialylated core O-glycans and type 1 sialylated core O-glycans. type in a mammalian protein or fragment thereof.

Alternatively, only the amount of sialylated core O-glycans type 2 can be increased. Accordingly, the percentage of sialylated type 2 core O-glycans also increases compared to the percentage of sialylated type 2 core O-glycans in the mammalian protein.

SIGLEC molecules that have been shown to bind are involved in the immune response in humans and mice. SIGLECs have in common an N-terminal immunoglobulin V domain that binds sialic acid containing ligands and a variable number of immunoglobulin C2 domains that remove the ligand binding site from the membrane surface.

In addition, many SIGLECs have cytoplasmic tyrosine motifs, including the immunoreceptor tyrosine inhibitory motif (ITIM) and ITIM-like motifs commonly found in co-receptors involved in the regulation of cell signaling. Other SIGLECs do not contain tyrosine motifs but contain a positively charged transmembrane region that allows binding to adapter proteins. SIGLECs do not recognize danger-associated molecular patterns (DAMPS), but rather recognize the determinants of "self-identification".

SIGLECs bind to sialylated self-identification ligands (self-ligands) on the same cell in a "cis" manner and on adjacent cells in a "trans" manner. Human SIGLECs are commonly referred to as SIG-1 through SIG-14. Mouse SIGLECs SIG-E, SIG-F and SIG-G, respectively, are orthologues of human SIG-9, SIG-8 and SIG-10.

The SIG-1 to SIG-4 molecules are also referred to as Sialoatezin, CD22, CD33 and MAG, respectively. CD22 and SIG-10 are present in B cells, SIG-5 in neutrophils and monocytes, SIG-7 in NK cells, SIG-8 in eosinophils, SIG-9 in monocytes, neutrophils and dendritic cells (Paulsen et al 2012).

SIGLECs bind to various glycan structures. SIG-2, SIG-5, SIG-7, SIG-8, SIG-9 and SIG-10 each preferentially bind to different glycans (Paulson et al., 2012). SIGLEC play a role in innate and adaptive immunity. Specifically, SIG-2 and SIG-10, which are present on human and mouse B cells.

According to Paulsen et al., SIG-2 and SIG-10 appear to have a synergistic effect on peripheral B cell tolerance. In addition, SIGLECs act as inhibitory co-receptors for Toll-like receptors (TRLs). In this regard, it has been shown that cross-linking SIG-7 or SIG-9 with activating receptors leads to inhibition of the cytolytic activity of NK cells against tumor cells and, accordingly, to the release of chemical mediators from mast cells.

Moreover, cross-linking of SIG-E (SIG-9) and SIG-11 with immobilized antibody results in inhibition of cytokine production in macrophages in response to LPS.

It should be noted that expression of SIG-5 and SIG-9 in a macrophage cell line inhibits TNF-alpha production and increases IL-10 production in response to peptide glycan, ATLR2 ligand, LPS, and CpG. In addition, LPS-induced expression of SIG-E (SIG-9) in macrophages has been shown to affect signal transduction from TRL. In addition, sialylated pathogens attenuate the immune response through SIGLEC. For example, group B streptococcus expresses the Neu-Acα-1 Galβ-1 4GlcNAc residue on envelope polysaccharides and recruits SIGLEC-9 on neutrophils, resulting in suppression of the microbicidal function of neutrophils.

Accordingly, without being bound by any particular theory, it is believed that binding to SIGLEC on antigen-presenting cells (eg, dendritic cells) results in decreased expression of pro-inflammatory receptors and increased expression of immunosuppressive receptors on the cell surface. In addition, binding leads to increased production of anti-inflammatory cytokines, reduces the production of pro-inflammatory cytokines and, as a result, leads to inhibition of T cell proliferation and antibody production. Thus, binding of SIG-5, SIG-7, SIG-8, and SIG-9 results in reduced immune response or increased immune tolerance when a glycosylated polypeptide is administered to a patient.

Thus, a glycosylated polypeptide according to the first aspect can also be described as a glycosylated polypeptide comprising an amino acid sequence identical or homologous to at least a portion of a mammalian, preferably human protein, wherein said glycosylated polypeptide contains one or more sialylated O-glycans, and where compared to a mammalian protein or fragment:

- the human immune response to the glycosylated polypeptide is reduced; and/or

- the human immune tolerance to the glycosylated polypeptide is increased.

On the other hand, a more structural definition of the first aspect of the invention is as follows: a glycosylated polypeptide comprising an amino acid sequence identical or homologous to at least a portion of a mammalian, preferably human protein, wherein said glycosylated polypeptide contains one or more sialylated O-glycans, and where the combined amount of sialylated type 2 core O-glycans and type 1 sialylated core O-glycans of the glycosylated polypeptide exceeds the combined amount of type 2 sialylated core O-glycans and type 1 sialylated core O-glycans in a mammalian protein or a fragment of it.

Preferably, the glycosylated polypeptide comprising an amino acid sequence is identical or homologous to at least a fragment of a mammalian, preferably human protein, wherein said glycosylated polypeptide contains one or more sialylated O-glycans, and where the number of type 2 sialylated core O-glycans is glycosylated polypeptide will exceed the combined amount of sialylated core O-glycans type 2 in the mammalian protein or its fragment.

To link O-glycans to the amino acid sequence of a glycosylated polypeptide, it contains one or more O-glycosylation sites. The O-glycosylation sites in the glycosylated polypeptide may be the standard O-glycosylation sites serine (Ser) and threonine (Thr). However, the binding of O-glycans to tyrosine (Tyr), hydroxylysine (Hydroxy-Lys) or hydroxyproline (Hydroxy-Pro) has also been described in the context of the invention. Thus, one or more O-glycosylation sites in a glycosylated polypeptide can be selected from Ser, Thr, Tyr, Hydroxy-Lys and Hydroxy-Pro at any other possible O-glycosylation site. Preferably, the O-glycosylation sites are selected from Ser and Thr.

The standard Ser and Thr glycosylation sites are, as a rule, occupied to the greatest extent by O-glycans. Thus, in one embodiment, one or more glycosylation sites are selected from Ser and Thr.

In the context of the invention, for practical reasons, the term "glycosylated polypeptide" is used in one form. Typically, the glycosylated polypeptide will be in the form of a composition of one type of polypeptide. In this respect, glycosylated polypeptides in their original form are a composition of glycosylated polypeptides having the same amino acid sequence, but with differences in glycosylation. For example, not all individual molecules of the composition may be 100 percent glycosylated. In addition, there may be differences in the glycans associated with a particular O-glycosylation site. Accordingly, the present invention also relates to a composition comprising at least first type glycosylated polypeptide molecules, wherein the amino acid sequence of the first type protein molecules is identical or homologous to at least a fragment of a mammalian, preferably human protein, and the protein molecules contain one or more glycosylation sites .

Preferably, the polypeptide contains one or more clusters of glycosylation sites. Although a single glycosylation site may be sufficient to bind SIGLEC, it is expected that the formation of a cluster of O-glycosylation sites results in improved binding to SIGLEC. Glycosylation site clustering is frequently observed in mammalian proteins, examples being human IgA containing clustered O-glycans in the hinge region (see Franc et al. 2013) and human mucin (see Guzman-Aranguez and Argüeso 2010).

In this context, already two closely spaced O-glycosylation sites are considered as an O-glycosylation cluster. Thus, one or more O-glycosylation site clusters contain at least two O-glycosylation sites. Clusters of O-glycosylation sites can have varying numbers of O-glycosylation sites. For example, a glycosylated polypeptide may contain one cassette with two and a second cassette with three glycosylation sites. There may also be N-glycosylation sites between the O-glycosylation sites in the cluster. Preferably, there are no N-glycosylation sites in the O-glycosylation cassette.

In addition, one cluster may contain three O-glycosylation sites and another four O-glycosylation sites. A series of three O-glycosylation sites yields three adjacent O-glycans that can interact with SIGLEC and therefore increase the effect. In one embodiment, one or more clusters of O-glycosylation sites preferably contain at least three O-glycosylation sites.

vWF contains two clusters with four O-glycosylation sites each. Thus, it is preferred that the polypeptide contains one or more clusters with at least four O-glycosylation sites. It is currently believed that the higher the number of O-glycosylation sites, the higher the binding affinity.

A cluster of O-glycosylation sites can be characterized by the presence of two or more O-glycosylation sites in close proximity to each other in an amino acid sequence. These clusters are also referred to as "sequential clusters". However, due to the three-dimensional structure of the glycosylated polypeptide, the O-glycosylation cluster may also include O-glycosylation sites that are far apart in the amino acid sequence but are in close proximity after folding. This type of clusters is also called "structural cluster".

In vWF, the 2nd O-glycosylation cluster contains 4 O-glycosylation sites within 20 amino acids, which are arranged in a beta turn. Accordingly, the distance between O-glycans or O-glycosylation sites is 27.2 to 34.0 Å, giving an average distance of 6.8 to 8.5 Å. Thus, the average distance between two O-glycosylation sites in a cluster can range from 4.0 to 15.0 Å. At a distance of less than 4.0 Å, steric hindrance between O-glycans may occur, in particular, glycosylation of both O-glycosylation sites may be impossible. With an average distance between two amino acids of more than 15.0 Å, there is probably no combined effect of O-glycans. The combined effect is, for example, interaction with SIGLEC on the same cell. Preferably, the average distance between two O-glycosylation sites in a cluster is in the range of 5.0 to 12.0 Å. More preferably, the average distance between two O-glycosylation sites in a cluster is in the range of 6.0 to 9.0 Å.

Structural clusters may span amino acid sequences of more than 100 amino acids. However, it is preferable that the spatial arrangement of the cluster does not exceed 80 Å. If the O-glycosylation sites are separated from each other by more than 80 Å, it is assumed that the O-glycans do not exhibit a combined effect. The combined effect of O-glycans in a cluster is strongest if O-glycans are located in a region with a diameter of 50 Å. Thus, it is more preferable that the spatial arrangement of the cluster does not exceed 50 Å.

In one embodiment, one or more clusters, such as consecutive clusters, contain at least one O-glycosylation site in ten amino acids. If the O-glycosylation sites are located at a greater distance, it is believed that the O-glycans associated with the O-glycosylation site may not interact with the same cell containing SIGLEC.

Preferably, one or more clusters contain at least one O-glycosylation site in four amino acids. With an average distance between O-glycosylation sites of four amino acids, there is a high probability that the O-glycosylation sites are also closely spaced after folding. Spatial proximity allows co-interaction of glycans in a cluster with SIGLEC on the same cell.

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

As shown in the examples, already one of these O-glycosylation clusters is sufficient for a strong interaction with SIGLEC. Moreover, as also shown by comparing two vWF polypeptides, more O-glycosylation site clusters result in increased peptide binding affinity for SIGLECs, specifically SIG-5, SIG-7, SIG-8 and SIG-9. Thus, the glycosylated polypeptide preferably contains at least two glycosylation cassettes, more preferably at least three glycosylation cassettes.

Without wishing to be bound by any particular theory, the binding affinity of a glycosylated polypeptide to SIGLEC is higher the closer the clusters are. In this context, if two clusters are present, they are preferably separated by less than 100 amino acids. A distance of less than 100 amino acids allows the cooperative action of glycan clusters upon 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.

According to one embodiment of the invention, the distance between any two adjacent clusters is less than 100 amino acids, preferably, the distance between any two clusters in the glycosylated polypeptide is less than 50 amino acids. More preferably, the distance between any two adjacent clusters is less than 30 amino acids.

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

As described above, like all glycosylated polypeptides, the glycosylated polypeptide of the invention is a composition of glycosylated polypeptide molecules. These molecules have a certain degree of heterogeneity in glycosylation, in particular not all glycosylation sites are necessarily occupied by O-glycans. O-glycan occupancy depends, in part, on the host cell in which the recombinant glycosylated polypeptide is produced. Preferably, the host cell, ie the expression system, is chosen such that the percentage of O-glycosylation sites occupied is above 70%. Below 70% occupancy, insufficient O-glycans may be present to bind SIGLEC. 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. In a preferred embodiment, more than 95% of the O-glycosylation sites should be occupied by O-glycans.

The composition of the glycans attached to the glycosylated polypeptide depends on the method of preparation. O-glycans can be natural or synthetic glycans. Natural O-glycans are, for example, glycans with the following core structure:

Type 1 core O-glycan : Galβ1→3GalNAcα1→Ser/Thr

Extended core O-glycan type 1 : Galβ1→4GlcNAcβ1→3Galβ1→3GalNAcα1→Ser/Thr

Type 2 core O-glycan : Galβ1→3(Galβ1→3GlcNAcβ1→6)GalNAc α1→Ser/Thr

SIGLECs are known to bind to sialic acid. Accordingly, the examples show that desialylation abolishes binding to SIGLEC (see example 3). Thus, it is confirmed that sialylation of O-glycans is essential for binding. Accordingly, a high percentage of O-glycan sialylation in a glycosylated polypeptide is preferred.

Accordingly, the O-glycans of the glycosylated polypeptide are preferably sialylated, that is, they contain at least one sialic acid in the glycan molecule.

Preferably, the sialylated O-glycans contain at least two sialic acid molecules in an alpha 2-3 glycosidic bond. Alternatively, sialylated O-glycans may contain two sialic acid molecules in an alpha-2-8 glycosidic bond. Siallylated O-glycans can also include alpha-2-3 and alpha-2-8 glycosidic bonds. In one embodiment, the sialylated O-glycans contain at least three sialic acid molecules in 2-3 and/or 2-8 glycosidic bonds. The sialylated O-glycans in the glycosylated polypeptide are, in particular, core 1 or core 2 O-glycans. Below are the general structures of O-glycans: core type 1, extended core type 1, and core type 2:

Type 1 sialylated core O-glycan : NeuNAcα2→3Galβ1→3GalNAcα1→Ser/Thr

Type 1 sialylated core O-glycan: NeuNAcα2→3Galβ1→4GlcNAcβ1→3Galβ1→3GalNAcα1→Ser/Thr

Type 2 sialylated core O-glycan: NeuNAcα2→3Galβ1→4GlcNAcβ1→6(NeuNAcα2→3Galβ1→3)GalNAcα1→Ser/Thr

It is possible that O-glycans of both core type 1 and core type 2 and/or extended core type 1 should be present on the glycosylated polypeptide. In one embodiment, the glycosylated polypeptide comprises type 1 sialylated core O-glycans as well as type 1 sialylated core O-glycans and/or extended type 1 core O-glycans. As shown in example 4, the percentage of type 2 core glycans, which is 2.5% of the total number of O-glycans, is not enough to interact with SIGLEC. Thus, according to one embodiment of the glycosylated polypeptide, the percentage of core type 2 O-glycans relative to the number of O-glycans is at least 5%. In the same example, Cluster 2 with a Type 2 core O-glycan content of 10.78% of O-glycans was shown to have a strong interaction with SIGLEC. Thus, according to a preferred embodiment of the invention, the percentage of core type 2 O-glycans relative to the number of O-glycans in the glycosylated polypeptide is at least 8%. More preferably, the percentage of core type 2 O-glycans relative to the number of O-glycans is at least 10%.

In Example 7, about 80% of the glycopeptide molecules of the recombinantly produced vWF peptides were determined to contain either a type 2 core O-glycan or an extended type 1 core O-glycan. Accordingly, the percentage of sialylated type 2 core O-glycans and/or extended type 1 core O-glycans of the total O-glycans is at least 20%. Thus, in one embodiment, the concentration of type 2 core O-glycans and/or type 1 extended core O-glycans of the total O-glycans in the glycosylated polypeptide is at least 15%, more preferably at least 18% and most preferably at least 20%.

The number or percentage of type 2 core O-glycans in a glycosylated polypeptide, in particular the number or percentage of individual glycosylated polypeptide molecules bearing a type 2 core O-glycan, can be increased by any of the following strategies:

One strategy is to use the enzyme β-1,6-N-acetylglucosaminyltransferase. This enzyme is involved in the formation of type 2 core O-glycans. Thus, an increase in the number of type 2 core O-glycan molecules and/or the number of type 2 core O-glycans per polypeptide molecule can be obtained by expression of a glycosylated polypeptide in a cell line that overexpresses the enzyme β-1,6-N -acetylglucosaminyltransferase.

Another option is to express the glycosylated polypeptide in an expression cell line derived from a cancer cell line. It has been shown that often cancer cell lines produce glycosylated proteins with a large number of type 2 core O-glycans.

One strategy to increase the percentage of extended type 1 O-glycans is to use the enzyme β-1,3-N-acetylglucosaminyltransferase. This enzyme is involved in the formation of extended type 1 O-glycans. Thus, an increase in the number of molecules bearing type 1 core extended O-glycans and/or the number of type 1 extended core O-glycans per polypeptide molecule can be obtained by expression of a glycosylated polypeptide in a cell line that overexpresses the β-1 enzyme. ,6-N-acetylglucosaminyltransferase.

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

Accordingly, based on the teachings of the invention, one skilled in the art can tailor glycans to create synthetic glycans with high binding affinity. According to a preferred embodiment, the O-glycans are natural glycans.

In one embodiment, at least a portion of the type 2 sialylated core O-glycans in the glycosylated polypeptide contains a sulfate group attached to galactose (Gal) or N-acetylglucosamine (GlcNAc), or fucose attached to GlcNAc in the core O-glycan. type 2, which is a high affinity binding ligand for SIGLEC 7, 8 and 8 (Paulson et al., 2012).

One of skill in the art is aware of various methods for creating additional O-glycosylation sites. The glycosylated polypeptide may have an increased number of sialylated core type 2 O-glycans due to an increase in the number of O-glycosylation sites compared to a human protein or fragment thereof.

In this case, the glycosylated polypeptide may be 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 a human protein or protein fragment (first amino acid sequence). The second amino acid sequence may be located N-terminally relative to the first amino acid sequence. Alternatively, the second amino acid sequence may be located C-terminally relative to the first amino acid sequence. The glycosylated polypeptide may contain additional amino acid sequences both at the N-terminus and at the C-terminus relative to the first amino acid sequence.

Accordingly, a second amino acid sequence and optionally additional amino acid sequences may be present in such a fusion protein, which primarily contain O-glycosylation sites, in particular O-glycosylation cassettes. The cluster of O-glycosylation sites may be based on an amino acid sequence from a known mammal, in particular a human glycosylated protein.

The second amino acid sequence may contain 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),

QSPTPPHGLSLSDLQEAK (SEQ ID NO: 19);

VXXXXXXXXXTXTSXXSPXXXXXVXXSXXXXTXXAXX (SEQ ID NO: 20) and

VHIYQKDLFFTETSDGSPGHLDLVEGSLLQGTEGAIK (SEQ ID NO: 21).

In the sequences of SEQ ID NOs: 8,10,12, 14, 16, 18 and 20, X is any of the naturally occurring amino acids.

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.

The second amino acid sequence may contain one or more sequences selected from SEQ ID NOs: 8, 10, 12, 14, 16, 18 and 20. The second amino acid sequence may contain combinations of sequences. The second amino acid sequence preferably contains 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 contain combinations of multiple copies of SEQ ID NOs: 8, 10, 12, 14 , 16, 18 and 20

The second amino acid sequence may contain one or more sequences selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19 and 21. The second amino acid sequence may contain combinations of sequences. The second amino acid sequence preferably contains 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 combinations of multiple copies of SEQ ID NOs: 9, 11, 13, 15 , 17, 19 and 21.

In a preferred embodiment, the second amino acid sequence contains one or more copies of SEQ ID NO: 8.

In addition, the second amino acid sequence may have some percent identity with the natural glycosylated protein sequence. The level of identity with natural protein is preferably 80%, more preferably at least 90%.

Alternatively, the amino acid sequence of the O-glycosylation sites in the second amino acid sequence may be entirely synthetic. The fully synthetic amino acid sequence used in the present invention is one that is not based on a known protein, in particular a mammalian protein.

According to one embodiment of the present invention, the covalent linker connecting the second amino acid sequence to an amino acid sequence identical or homologous to a human protein or fragment thereof in a glycosylated polypeptide is selected from a peptide bond, a chemical linker, or a glycosidic bond. Suitable chemical linkers for this are:

- Amine with amine linkers such as bismaleimidoethane, 1,8-bismaleimido-diethylene glycol,

- Amine with sulfhydryl linkers such as succinimidyl iodine acetate, N-α-maleimidoacetamido-oxysuccinimide ester,

- Carboxyl with amine linkers dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and

- Sulfhydryl with carbohydrate linkers such as N-β-maleimidopropionic acid hydrazide, N-ε-maleimidocaproic acid hydrazide.

The fusion protein linker of the invention may be formed by a spacer peptide sequence separating the first and second amino acid sequences that form the fusion protein. A spacer peptide sequence may assist in the proper folding of individual portions of a protein or peptide and may provide a greater likelihood of maintaining individual functional properties for individual portions of a protein or peptide. Spacer peptide sequences can be inserted into the DNA sequences of the fusion protein during assembly while maintaining the reading frame of the individual DNA fragments that make up the complete DNA sequence of the fusion protein, such as during overlapping PCR or DNA ligation.

The advantage of a peptide bond is that the complete glycosylated polypeptide can be immediately expressed as a fusion protein.

The second amino acid sequence can be added to the first amino acid sequence via a chemical linker and hence after protein expression.

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

Thus, in one embodiment, the human protein is FVIII or a fragment thereof. Accordingly, the sequence identity with FVIII in the glycosylated polypeptide is at least 90%, more preferably at least 95%, and most preferably at least 98%. In a preferred embodiment, the first amino acid sequence is identical or homologous to amino acids 1 to 505 of SEQ ID No: 1.

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, most preferably 20 to 70 amino acids.

In one embodiment, the second amino acid sequence has at least 98% homology to amino acids 475-505 of SEQ ID NO: 1. Preferably, the second amino acid sequence is identical to amino acids 475-505 of SEQ ID NO: 1. More preferably, The second amino acid sequence is at least 98% homologous to two consecutive copies of amino acids 475-505 of SEQ ID NO: 1. Preferably, the second amino acid sequence is identical to two consecutive copies of amino acids 475-505 of SEQ ID NO: 1.

An example of a chimeric protein according to the present invention is Seq12. Seq12 has the following amino acid sequence (SEQ ID NO: 2):

SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCЕVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLH

The following sequence (SEQ ID NO: 3) represents Seq12 with an additional 22 amino acid signal peptide (bold and underlined). Expression of this peptide provides the monomeric form of Seq12. The signal peptide is cleaved off enzymatically.

MIPARFAGVLLALALILPGTLC SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCЕVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLH (SEQ ID NO: 3)

An additional example of a fusion protein of the invention is Pro-Seq12, comprising Seq12 and a propeptide (in bold) with a signal peptide (in bold and underlined). Pro-Seq12 is listed as SEQ ID NO: 4:

MIPARFAGVLLALALILPGTLC AEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLH (SEQ ID NO: 4)

Expression of Pro-Seq12 leads to the formation of dimers. Peptide dimers also remain after propeptide cleavage.

In one embodiment, the glycosylated polypeptides comprise a first amino acid sequence that is at least 98% identical to amino acids 1-505 of SEQ ID NO: 1. A second amino acid sequence is at least 98% homologous to two consecutive copies of amino acids 475-505 of SEQ ID NO: 1.

According to one embodiment of the invention, the glycosylated polypeptide is obtained by expression in a human cell line. In general, any human cell line is suitable for expression of the glycosylated polypeptide. The peptide with the desired glycosylation, in particular, is obtained using HEK cell lines.

Examples of HEK cell lines for glycosylated polypeptide production are HEK 293 F, Flp-In™-293 (Invitrogen, R75007), 293 (ATCC® CRL-1573), 293 EBNA, 293 H (ThermoScientific 11631017), 293S, 293T (ATCC® CRL-3216™), 293T/17 (ATCC® CRL11268™), 293T/17 SF (ATCC® ACS4500™), HEK 293 STF (ATCC® CRL 3249™), HEK-293.2sus (ATCC® CRL-1573™) . The preferred cell line for producing the polypeptide is HEK 293 F.

Other cell lines suitable as host cells for expression include cell lines derived from human myeloid leukemia cells. Specific examples of host cells are K562, NM-F9, NM-D4, NM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12, GT-2X, GT-5s, and cells derived from any of these host cells. K562 is a human myeloid leukemia cell line from the American Type Culture Collection (ATCC CCL-243). The remaining cell lines were derived from K562 cells and were selected for specific glycosylation features.

In an alternative embodiment of the invention, the one or more glycosylation sites are within an amino acid sequence that is homologous or identical to a mammalian protein or fragment thereof. It should be understood that O-glycosylation sites that are not present in the amino acid sequence of a mammalian protein or fragment thereof are in the homologous or identical amino acid sequence in the glycosylated polypeptide.

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

One or more O-glycosylation sites in an amino acid sequence are preferably located in portions of the sequence that do not form protein binding sites or active sites. In addition, one or more O-glycosylation sites are preferably added at an amino acid position that will be exposed on the surface of the folded protein. To achieve the least effect on the activity or integrity of the protein, one or more O-glycosylation sites can be added to the flexible loop of the protein.

In the case of FVIII proteins, one or more O-glycosylation sites are preferably added to or replacing the B domain.

In one embodiment, the glycosylated polypeptide is modified by adding one or more biocompatible polymers to increase, for example, half-life or stability. Suitable biocompatible polymers include polyalkylene oxides such as, but not limited to, polyethylene glycol (PEG), dextrans, colominic acids or other carbohydrate based polymers, amino acid polymers, biotin derivatives, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyethylene/maleic anhydride copolymer , polystyrene/malic anhydride copolymer, polyoxazoline, polyacryloylmorpholine, heparin, albumin, celluloses, chitosan hydrolysates, starches such as hydroxyethyl starches and hydroxypropyl starches, glycogen, agaroses and their 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 glycols (PPG), polybutylene glycols (PBG), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethylene glycols, linear polyethylene glycols, bifurcated polyethylene glycols, and multi-branched or "super-branched" polyethylene glycols ( star PEGs). The biocompatible polymer is preferably linked to the polypeptide via one of the following residues: -SH, OH, -COOH.

In one embodiment, the glycosylated polypeptide is capable of forming dimers or multimers. The formation of dimers and, in particular, multimers increases the number of closely spaced O-glycans or clusters of O-glycans. Thus, more O-glycans can interact with SIGLEC on a single cell. Moreover, O-glycans can interact with multiple SIGLEC-expressing cells that are located close to each other, thereby increasing immune tolerance.

Multimerization may result from the presence of a multimerization domain in the amino acid sequence of the mammalian protein on which the glycosylated polypeptide is based. Alternatively, multimers of a glycosylated polypeptide can be formed by introducing multimerization domains into the amino acid sequence of the glycosylated polypeptide.

An example of a fusion protein of the invention that forms multimers is Pro-Seq12-Mult, comprising Seq12 and a propeptide (in bold) with a signal peptide (in bold and underlined) and a multimerizing sequence (cysteine knot domain) from vWF (underlined). Pro-Seq12-Mult is listed as SEQ ID NO: 5:

MIPARFAGVLLALALILPGTLC AEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHQEPGGLVVPPTDAPVPTTLYVEDISEPPLHEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK

Alternatively, multimerization of the glycosylated polypeptide can be achieved by binding to a polymer or liposome.

Use of glycosylated polypeptide

As described above, the inventors found that a protein with a glycan composition, including type 2 sialylated core O-glycans and/or type 1 sialylated core O-glycans, interacts with certain SIGLECs and therefore affects cells of the mammalian immune system. In particular, the glycosylated polypeptide elicits a reduced immune response. Therefore, such a glycosylated polypeptide, when administered with a second protein, can influence the patient's immune response to the second protein. Therefore, a glycosylated polypeptide with type 2 sialylated core O-glycans and/or type 1 sialylated core O-glycans can be used for modification, in particular to reduce the patient's immune response to a protein, in particular a therapeutic protein in joint introduction.

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

Preferably, the glycosylated polypeptide used to reduce the immune response contains type 2 sialylated core O-glycans and/or type 1 sialylated core O-glycans. More preferably, the glycosylated polypeptide is the glycosylated polypeptide of the first aspect.

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

Composition and protein complex

Accordingly, the concept of the invention, namely, a reduced immune response to a human protein, for example, by adding one or more type 2 sialylated core O-glycans, can be achieved not only by obtaining a fusion protein or inserting or replacing amino acids with O-glycosylation sites, but also by adding an additional polypeptide as described in the application of the second aspect. This results in a composition of the glycosylated polypeptide and the second polypeptide to which the immune response is to be reduced.

Accordingly, in a third aspect, the present invention also relates to a 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 comprises an amino acid sequence homologous or identical to a second mammalian protein, in in particular a human, wherein, compared to the second polypeptide, the composition has an increased binding affinity for a SIGLEC selected from one or more SIGLECs selected from SIG-5, SIG-7, SIG-8 and SIG-9.

It is also possible to design a polypeptide binding partner that has an increased binding affinity for SIGLEC such that a complex of two polypeptides will have an increased binding affinity for SIGLEC relative to the polypeptide.

Thus, according to a third aspect, the invention relates to a 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 comprises an amino acid sequence homologous or identical to a second mammalian protein, in particular, a human, where compared to the second polypeptide:

- the composition has an increased binding affinity for SIGLEC selected from; and/or

- the human immune response to the complex is reduced; and/or

- human immune tolerance to the complex is increased.

The second human protein is preferably a human blood protein. The human blood protein can be a human blood clotting factor, a transport protein, a protease inhibitor, an immunoglobulin, a cellular plasma protein, apolipoproteins, a complement factor, a growth factor, an anti-angiogenic protein, a highly glycosylated protein, blood factors, or another human blood protein.

The human blood clotting factor is selected in particular 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.

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

Potential protease inhibitors are e.g. , protein S and protein Z.

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

The cellular plasma protein may be, for example, fibronectin, thromboglobulin, platelet factor 4. Examples of apolipoproteins are apo A-I, apo A-II and apo E.

Complement factors according to the invention are, for example, factor B, factor D, factor H, factor I, C3b inactivator, properdin, C4 binding protein, etc.

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-β), fibroblast growth factor (FGF), and hepatocyte growth factor.

Antiangiogenic proteins include latent antithrombin, prelatent antithrombin, oxidized antithrombin, and plasminogen.

Examples of highly glycosylated 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 factors, tPA, gCSF.

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

The second human protein is specifically selected from vWF, FVIII, FVII/FVIIa, FIX, ADAMTS13.

The composition according to the third aspect is, in particular, a protein complex of the first and second polypeptide.

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 protein of a mammal, in particular a human. To reduce the human immune response to the second polypeptide, the first polypeptide forms a complex with the second polypeptide.

For the formation of a protein complex, the first polypeptide, in particular, contains a binding domain that provides binding to the second polypeptide, and a glycosylation domain. The glycosylation domain in particular contains one or more O-glycosylation sites, preferably an O-glycosylation cluster.

In one embodiment, the second polypeptide is a FVIII protein and the first polypeptide comprises a FVIII-vWF binding domain and one or more O-glycosylation sites to which type 2 sialylated core O-glycans are associated.

A preferred example of a composition is a complex of a FVIII protein with an amino acid sequence 95% identical to the sequence defined by amino acids 20 to 2351 of p00451 and a first polypeptide as a binding partner containing an amino acid sequence at least 95% identical to amino acids 1- 172 of SEQ ID NO: 1.

According to one variant of the protein complex according to the third aspect, the first polypeptide is a polypeptide according to the first aspect.

In yet another embodiment, the second polypeptide may be selected from, for example, FVIII, FVII, FIX, and ADAMTS13

In one embodiment of the third aspect, the first polypeptide comprises at least a human vWF fragment and the second polypeptide is a FVIII protein, in particular a full-length FVIII protein, a FVIII protein with a B domain deleted, or a FVIII protein in which a portion of the B domain has been replaced linker. In one embodiment, the first polypeptide is defined by amino acids 1-505 of SEQ ID NO: 1, which is produced in HEK cells, in particular HEK 293F cells.

According to another embodiment of the invention, the first polypeptide is defined by amino acids 475-505 of SEQ ID NO: 1, and one copy of amino acids 475-505 of SEQ ID NO: 1. In addition, the first polypeptide may 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.

As shown in the examples, a protein with increased binding affinity for SIGLEC, in particular SIG-5, SIG-7, SIG-8 and/or SIG-9, can be obtained in the HEK 293F cell line. Thus, according to a further embodiment of the protein complex, the first and second polypeptides are produced by recombinant expression in a human cell line, preferably a HEK cell line. Examples of HEK cell lines for the production of a glycosylated polypeptide are HEK 293 F, Flp-In™-293, 293, 293 EBNA, 293 H, 293S, 293T, 293T/17, 293T/17 SF, HEK 293 STF and HEK-293.2sus. The preferred cell line for producing the polypeptide is HEK 293 F.

The first and second polypeptides can be obtained by separate recombinant expression and then connected. Alternatively, the first and second polypeptides are recombinantly expressed in the same cell. To do this, the first and second polypeptides can be encoded in the same vector or in two different vectors.

Polynucleotide

According to a fourth aspect, the present invention relates to an isolated polynucleotide which contains a nucleic acid sequence encoding a glycosylated polypeptide according to the first aspect of the invention.

An isolated polynucleotide may be a DNA molecule or an RNA molecule. The isolated polynucleotide is preferably a DNA molecule, in particular a cDNA molecule. Methods 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 polynucleotides from such genomic DNA can be accomplished, for example, using the well-known polymerase chain reaction (PCR) or screening of antibody 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.

An isolated polynucleotide may be a DNA molecule encoding a glycosylated polypeptide with 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.

In particular, the isolated polynucleotide may be a DNA molecule encoding a glycosylated polypeptide having an amino acid sequence identity of at least 90%, preferably at least 95%, more preferably at least 98%, most preferably 100% with SEQ ID NO: 2. In addition, the isolated polynucleotide may be a DNA molecule encoding a glycosylated polypeptide having an amino acid sequence identity of at least 90%, preferably at least 95%, more preferably at least at least 98%, most preferably 100% with SEQ ID NO: 3. According to one embodiment of the invention, the isolated polynucleotide is a DNA molecule encoding a glycosylated polypeptide having an amino acid sequence with at least 90% identity, preferably at least 95%, more preferably at least 98%, most more preferably 100% with SEQ ID NO: 4. According to one embodiment of the invention, the isolated polynucleotide is a DNA molecule encoding a glycosylated polypeptide having an amino acid sequence identity of at least 90%, preferably at least 95% , more preferably at least 98%, most preferably 100% with SEQ ID NO: 5.

Expression vector

In a fifth aspect, the invention also relates to an expression vector containing a polynucleotide according to the fourth aspect of the invention.

Preferably, the expression vector further contains regulatory elements such as a promoter and transcriptional and translational stop signals. The polynucleotide according to the fourth aspect and the regulatory elements may be joined together to form a recombinant expression vector, which may include one or more restriction sites to allow the introduction or replacement of a polynucleotide encoding a polypeptide at such sites. The polynucleotide can be inserted into an appropriate expression vector for expression. When creating an expression vector, the coding sequence is located in the expression vector in such a way that the coding sequence is operably linked to the appropriate regulatory sequences for expression.

The recombinant expression vector can be any vector (eg, plasmid or virus) that is suitable for recombinant DNA techniques and can express the polynucleotide of the fourth aspect of the invention. The choice of expression vector generally depends on the compatibility of the expression vector with the host cell into which the expression vector is to be introduced. Expression vectors can be linear or closed circular plasmid.

Preferably, the expression vector is adapted for expression in mammalian cells. The expression vector may be an autonomously replicating vector, ie a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. For autonomous replication, the vector may further comprise an origin of replication allowing the vector to replicate autonomously in an appropriate host cell. The point of origin of replication can be any plasmid replicon that mediates autonomous replication that functions in the cell. The terms "point of origin of replication" or "plasmid replicon" means a polynucleotide that allows a plasmid or vector to replicate in vivo .

The vector is preferably one which, when introduced into a host cell, is integrated into the genome and replicated along with the chromosome(s) into which it is inserted. For insertion into the genome of the host cell in the expression vector, any other element of the expression vector can be used to insert into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for targeted insertion by homologous recombination into the genome of the host cell at a precise location on the chromosome.

The vectors of the present invention preferably contain one or more (eg, several) selectable markers that allow for easy selection of transformed, transfected, transduced, or similarly modified cells. A selectable marker is a gene whose product provides biocidal or viral resistance, heavy metal resistance, auxotrophic prototrophy, and the like.

The techniques used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, supra).

According to one embodiment of the present invention, the vector framework 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 .

The vector according to the fifth aspect can be temporarily or permanently transformed into a host cell. The host cell may be any of the cells listed above. Preferably, the host cell is HEK 293F.

Medical use and method of treatment

As described above, the glycosylated polypeptide and the composition, in particular the protein complex of the invention, have the advantage of a reduced immune response in patients, in particular in humans. Thus, the glycosylated polypeptide and the protein complex are particularly useful as active ingredients for treatment.

According to a sixth aspect, the invention relates to glycosylated polypeptides according to the first aspect for use in therapy. Alternatively, according to a sixth aspect, the invention relates to a composition according to the third aspect for use in therapy. Preferably for the treatment or prevention of a bleeding disorder.

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

As used herein, the term "blood clotting disorder" refers to a disease or condition that interferes with normal hemostasis. The coagulation disorder can be, for example, hemophilia A, hemophilia B, factor VIII deficiency, factor XI deficiency, von Willebrand disease, Glanzmann's thrombasthenia, Bernard Soulier's syndrome, idiopathic thrombocytopenic purpura, intracerebral hemorrhage, trauma, traumatic brain injury, etc. .

As used herein, the term "hemophilia" refers to a group of bleeding disorders associated with an increase in the time to form a blood clot compared to the time to form a blood clot in healthy individuals without hemophilia. "Hemophilia" refers to both hemophilia A, a disorder resulting in the production of defective factor VIII, and hemophilia B, which is a disease resulting in the production of defective factor IX.

The bleeding disorder is preferably hemophilia. Treatment can be, for example, treatment of hemophilia in previously untreated patients (PUP) or induction of immunotolerance (ITI).

According to an alternative embodiment of the third aspect, the invention relates to a protein complex according to the second aspect for use in the treatment or prevention of bleeding disorders.

Treatment preferably comprises administering to the patient an effective amount of the glycosylated polypeptide or composition, in particular the protein complex.

The glycosylated polypeptide or composition, in particular the protein complex described herein, may be administered alone or in the form of pharmaceutical compositions. The pharmaceutical compositions of the invention may contain an effective amount of the conjugates, in combination with at least one pharmaceutically acceptable carrier. Pharmaceutical compositions according to embodiments may be prepared and administered to a subject by any means well known in the pharmaceutical art. 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). In addition, the pharmaceutical compositions of the present invention may also be formulated to include other therapeutically useful drugs or biological agents. The pharmaceutical composition typically contains a therapeutically effective amount of the glycosylated polypeptide or protein complex in association with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is any carrier known or known in the art. Examples of pharmaceutically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free saline. Other forms of pharmaceutically acceptable carriers that may be used for the present embodiments include binders, disintegrators, surfactants, absorption accelerators, moisture retaining agents, absorbents, lubricants, fillers, bulking agents, humectants, preservatives. , stabilizers, emulsifiers, solubilizing agents, salts for adjusting the osmotic pressure and diluting agents such as buffers and excipients commonly used depending on the form of application of the composition. They are optionally selected and used depending on the dosage form of the resulting composition.

For in vivo use, the glycosylated polypeptide, protein complex, or pharmaceutical composition may be administered to a patient 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 agents, suspensions, emulsions, and drip agents. For parenteral administration, the glycosylated polypeptide, protein complex, or pharmaceutical composition must be an injectable agent, such as a liquid agent or suspension.

In other embodiments, the glycosylated polypeptide, protein complex, or pharmaceutical composition is orally administered to a patient. In these embodiments, the drug form includes solid formulations such as tablets, coated tablets, powdered agents, granules, capsules, and pills, liquid formulations such as liquid agents (e.g., eye drops, nasal drops, and drops), suspensions, emulsions and syrups, inhalation agents such as aerosol agents, nebulizers and nebulizers, and agents incorporated into liposomes. In some other embodiments, the glycosylated polypeptide, protein complex, or pharmaceutical composition is administered by inhalation into the respiratory tract of a patient for targeted administration to the trachea and/or lung of the subject.

According to one embodiment of the sixth aspect, the glycosylated polypeptide or composition, in particular the protein complex, is for use, the use comprising intravenous or non-intravenous injection. Non-intravenous injection is preferably subcutaneous injection.

The invention will now be described by way of the following non-limiting examples.

EXAMPLES

Example 1 - Associating the von Willebrand factor (vWF) with SIGLEC:

1.1. Experimental technique

Recombinant SIG-2, SIG-5, SIG-7, SIG-F (mouse equivalent of human SIG-8), SIG-9 and SIG-10 were obtained as Fc fusion proteins from R&D Systems. First, the plate was coated with protein A (SERVA Feinbiochemica GmbH & Co) at a concentration of 0.5 μg/well at 4°C overnight (O/N). After blocking and washing with wash buffer (20 mM HEPES, 125 mM NaCl, 1 mM EDTA, 1% BSA), Fc-fusion SIGLEC or control antibodies were bound to protein A by 1-hour incubation at 37°C at a concentration of 5 µg/ ml. An anti-vWF pAb (Dako, #A0082) was immobilized as a positive control, and an anti-chicken IgY antibody (Sigma Aldrich, #C2288) was immobilized through the Fc portion of the antibody as a negative control.

Plasma vWF (pdVWF)) was biotinylated using the EZ-Link™ Sulfo-NHS-Biotin biotinylation kit (Thermo Fisher Scientific). Serial dilutions of biotinylated vWF were added to wells at concentrations ranging from 0 to 0.8 µg/mL. After washing five times with wash buffer, HRP-linked streptavidin (Thermo Fisher Scientific, #31001) was added to the wells and incubated for 1 hour at 37° C. followed by washing five times.

To visualize bound biotinylated pdvWF, wells were incubated with o-phenylenediamine dihydrochloride substrate (SIGMA FAST ™ OPD, #P9187, Sigma Aldrich). The absorbance was then measured at 492 nm.

1.2 Results

As shown in Figure 1, absorbance at 492 nm increases with initial vWF concentration in binding experiments with SIG-5, SIG-7, SIG-F and SIG-9. Values are slightly above (SIG-F and SIG-9) or below (SIG-5 and SIG-7) the positive control (anti-vWF) values. In contrast, absorbance in binding experiments with SIG-2, SIG-10, and anti-chicken IgY negative controls was about 0 regardless of concentration.

Accordingly, vWF binds to SIG-5, SIG-7, SIG-F, and SIG-9 in a concentration dependent manner. On the other hand, vWF does not bind to SIG-2 and SIG-10.

Example 2 Linking vWF fragments to SIGLEC

2.1 Experimental technique

C- and N-terminal vWF fragments were generated by cleavage with V8 protease (Thermo Fisher Scitific, #201959) carried out for 3 hours at 37°C, shaking at 300 rpm using a 1:100 weight ratio of enzyme to protein in 50 mM Tris-HCl, 150 mM NaCl, pH 7.8, and purified by anion exchange chromatography on a MonoQ 5/50 GL column (GE Healthcare # 17-5166-01). The working 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 Superose 6 10/300 GL column (GE Healthcare # 17-5172-01) using 100 mM NaCl as running buffer.

The resulting fragments (C-terminus and N-terminus) are shown schematically in FIG. 2 indicating domains and glycosylation sites.

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 to SIGLEC was measured as described in Example 1, with concentrations of C-terminal vWF fragment and N-terminal vWF fragment were 1 μg/ml.

2.2 Results

Based on the absorption values shown in FIG. 3, the N-terminal vWF fragment, which contains most of the O-glycosylation sites and 2 O-glycan clusters (cluster 1 and cluster 2), binds to SIG-5, SIG-7, SIG-F, and SIG-9. On the contrary, in the case of the C-terminal vWF fragment, little or no absorption was observed. Accordingly, the last fragment is not associated with SIGLEC.

Example 3 - Linking the N-terminal part of the vWF to SIGLEC:

3.1 Experimental procedure

One aliquot of the N-terminal fragment of vWF obtained in example 2 was subjected to enzymatic desialylation using Sialidase A. Incubation was carried out at 37°C for 3 hours in 50 mm sodium phosphate, pH 6.0, using 2 μl of enzyme per 100 μg fragment of VWF (Sialidase A™, #GK80040, Prozyme).

A second aliquot of the N-terminal vWF fragment was de-N-glycosylated. Incubation was carried out overnight at 37° C. in 50 mM sodium phosphate, pH 7.5, using 1 μl of enzyme per 20 μg of VWF fragment (PNGaseF, #P0704, New England Biolabs).

Samples of the desialylated, de-N-glycosylated, and unprocessed N-terminal vWF fragment were tested for binding to SIG-5, SIG-7, SIG-8, and SIG-9. The binding experiment was carried out as described in Example 1 with a concentration of N-terminal vWF fragments of 8 μg/ml.

3.2 Results:

As shown in Figure 4, the absorbance values determined for the de-N-glycosylated vWF N-terminal fragment and the untreated vWF N-terminal fragment differ only slightly. Thus, de-N-glycosylation does not affect binding, demonstrating that binding is mediated by O-glycans.

Desialylation of O-glycans greatly reduces or abolishes binding of the N-terminal vWF fragment to SIGLEC, as shown in FIG. 4. Binding of the N-terminal vWF fragment is mediated by sialic acid attached to O-glycan chains.

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), shown schematically in Fig.2. Both clusters differ in the relative number of type 2 crustal structures. Only 4.9% of glycopeptide molecules contain type 2 core structures in cluster 1. Accordingly, the percentage of sialylated core type 2 O-glycans of the total number of O-glycans in cluster 1 is 1.25%.

On the contrary, 34.86% of glycopeptide molecules contain type 2 core structures in cluster 2 (see Solecka et al., 2016). In accordance with this, the percentage of sialylated core type 2 O-glycans of the total number of O-glycans in cluster 2 is 10.78%.

To measure the independent binding of the two clusters, the N-terminal vWF fragment was trypsinized to give the following fragments: Cluster 1 fragment spanning amino acids 449-511 and Cluster 2 fragment spanning amino acids 674-728/729 from the VWF sequence (SEQ ID NO: 1) . Fragments were purified by reverse phase HPLC. Briefly, pdVWF was reduced and free cysteines blocked with maleimide-PEG2-Biotin according to the manufacturer's instructions (EZ-Link™ Maleimide-PEG2-Biotin, #21901BID, Thermo Fisher Scientific). After digestion with trypsin at 37° C. overnight, high molecular weight peptides were concentrated using a 10 kDa centrifuge filter (Millipore). The peptides were then separated on a Jupiter 5 micron 300Å C18 column (Phenomenex). Mobile phase: A - 0.1% trifluoroacetic acid (TFA) in H ₂ O; B - 0.085% TFA in acetonitrile and the flow rate was 0.3 ml/min. Eluted peptides/glycopeptides were detected by absorbance in the ultraviolet region at a wavelength of 215 nm. Fractions of interest were collected, lyophilized and then resuspended in 10 μl H ₂ O. Since both clusters contain cysteines, biotin was added to both.

The binding experiment was carried out as described in Example 1 using SIGLEC SIG-5, SIG-7, SIG-F, SIG-9 and SIG-10, and the concentrations of cluster 1 and cluster 2 fragments were 4 μg/ml.

In addition, samples of cluster 1 and cluster 2 fragments were desiallylated and then tested in a binding experiment as described in Example 1 with a concentration of desiallylated cassette 1 and 2 fragments of 2 μg/ml.

4.2 Results:

Based on the absorption values given in FIG. 5, cluster fragment 2 binds to SIG-5, SIG-7, SIG-F, SIG-9,. No uptake or little uptake was detected using SIG-10, confirming the results from other examples.

Therefore, a high percentage of type 2 core structures in O-glycan clusters is required for binding to SIG-5, SIG-7, SIG-F, SIG-9.

Example 5 - Dependency of SIGLEC Cluster 2 Binding on Sialic Acid

5.1 Experimental procedure

Cluster 2 sample, prepared as described in Example 4, was desialylated. The desiallylated cassette 2 fragment and the untreated cassette 2 fragments were then tested in a binding experiment as described in Example 1 using SIG-5, SIG-7, SIG-F and SIG-9 and a concentration of desiallylated cassette 1 and 2 fragments of 2 mcg/ml.

5.2 Results

The absorbance values detected for the untreated fragment of cluster 2 confirmed the results obtained in example 4 (see Fig.6). The desialylated cluster 2 fragment shows no binding or only slight binding. Thus, sialylation of the core structures of type 2 glycans is necessary for binding to SIG-5, SIG-7, SIG-F, SIG-9.

Example 6 - Recombinant expression of VWF fragments with O-linked glycan repeats containing the FVIII binding site:

Two recombinant vWF fragments were expressed in the HEK 293 F cell line. The first fragment, Seq11, includes amino acids 1-505 of SEQ ID NO: 1 and contains cluster 1 with O-glycosylation sites at positions 485, 492, 493, 500.

The second fragment, Seq 12, includes amino acids 1-505 of SEQ ID NO: 1 and 2 additional repeats of amino acids 475-505 (AA1-505+2×475-505) and thus two additional copies of the O-glycan clustered repeat from cluster 1.

Seq 11 and Seq 12 were transiently expressed in the C-terminal Strep-Tag HEK293 cell line and purified by affinity chromatography on StrepTactin Sepharose (IBA GmbH). For this, the genes encoding Seq 11 and 12 were synthesized at GeneArt (Thermo Fisher Scientific) and cloned into a pDSG expression vector (IBA GmbH) containing a Twin-Strep-tag. E. coli TOP10 (IBA gmbH) cells were transmuted with constructs and individual clones were selected after overnight incubation at 37° C. on ampicillin-containing LB-agar plates. Isolation of plasmid DNA was performed using the QIAamp DNA-Mini or Maxi kit (Qiagen) according to the manufacturer's recommendations. The correct orientation and integrity of the cloned constructs was verified 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/l constructs using 4.5 mg/ml 25 kDa linear polyethyleneimine. After a 2-4 hour incubation at 37° C., 5% CO ₂ and shaking 100-150 rpm, the culture was diluted 1:2 with MEXi transfection medium and culture continued until the cells reached 75% confluence. Then the supernatant was 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 regulate pH, 0.1 volume of buffer (1 M Tris-HCl, 1.5 mM NaCl, 10 mM EDTA, pH 8.0) and 0.09 vol. BioLock solution (IBA GmbH) 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 bound Strep-tag proteins were eluted with elution buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10 mM dethiobiotin, pH 8.0).

Both fragments, Seq11 and Seq12, are schematically depicted in Fig.7

Example 7 O-Glycosylation Analysis of vWF, Seq11 and Seq12 Fragments

7.1 Experimental procedure

The O-glycosylation of the Seq11 and Seq12 fragments obtained according to Example 6 was analyzed by mass spectrometry.

For this, Seq11 and Seq12 were first reduced and alkylated by incubation overnight with 50 mM dithiothreitol at 60° C. and then for 20 minutes with 100 mM iodoacetamide. After digestion with trypsin and chymotrypsin, the resulting peptides were resuspended in Sialidase A digestion buffer and desialylated overnight using the conditions described in Example 3. O-glycopeptides were specifically enriched by affinity chromatography on Yacalin (a lectin from Artocarpus integrifolia ) using lectin immobilized on agarose (Vector Laboratories). Yacalin-agarose was packed into a gravity column and chromatography was performed according to the manufacturer's instructions. Eluted O-glycopeptides were purified for MALDI-MS analysis using C4 Ziptip pipette tips (Millipore) and analyzed in linear positive ionization mode using 25 mg/ml Super-DHB matrix dissolved in 50% acetonitrile/0.1% th trifluoroacetic acid.

An aliquot of enriched glycopeptides was further treated with O-glycosidase (endo-α-N-acetylgalactosaminidase, #P0733, New England Biolabs). Briefly, the peptides were incubated with the enzyme for 2 hours at 37° C. using 1 μM enzyme per 10 μM glycoprotein. Since O-glycosidase is specific for type 1 core O-glycans (Galβ1→3GalNAcα1→Ser/Thr disaccharide), it leaves type 2 core O-glycans and/or extended type 1 core O-glycans, attached to the peptide chain.

7.2 Results

The results are shown in Fig.8 and 9.

O-glycopeptides were identified using MALDI in PSD mode (post-source degradation). The peptide sequence of the identified Seq11 fragment is 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.

In the upper spectrum in Fig. 8 shows a fully O-glycosylated glycopeptide, the lower spectrum in FIG. 8 shows the same glycopeptide after cleavage with O-glycosidase. The 1460.3 Da mass shift after O-glycosidase cleavage (marked by an arrow) corresponds to four type 1 core O-glycans, each of which has a mass of 365 Da. The shifts at 365 Da observed in the upper spectrum correspond to different glycoforms of the same peptide, since each additional addition of mass at 365 Da corresponds to the disaccharide Galβ1→4GlcNAc, which forms the structure of the type 2 core O-glycan. After treatment with O-glycosidase, a completely deglycosylated form of the peptide (8358.6 Da) and glycoforms containing type 2 core structures (Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAc, 730 Da) and/or elongated 1- type (Galβ1→4GlcNAcβ1→3Galββ1→3GalNAc, 730 Da).

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.

In the upper spectrum in Fig. 9 shows a fully O-glycosylated glycopeptide, the lower spectrum in FIG. 9 shows the same glycopeptide after cleavage with O-glycosidase. The 4380.3 Da mass shift after O-glycosidase cleavage corresponds to twelve type 1 core O-glycans, each of which has a mass of 365 Da, confirming that all twelve 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 spectra correspond to the disaccharide Galβ1→4GlcNAc or a type 2 core and/or an extended type 1 core, respectively.

The quantification of type 1 and type 2 core O-glycans is based on the relative amount of glycopeptides with O-glycans attached to them. Quantification of various glycoforms of a given peptide is carried out by assessing the signal intensity in the MALDI spectrum.

For the Seq11 glycopeptide, the quantification is based on the intensity of the MALDI signal from various glycoforms of the same peptide. The total intensity of the peaks 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 is 100%.

The glycoform containing only type 1 core glycans with a mass of 8358 Da has an intensity of 8410 a.u., which corresponds to 20% of the total intensity. Thus, all other glycoforms (80%) contain at least one attached type 2 core glycan and/or extended type 1 core glycan. In this assay, type 2 core glycan and type 1 extended core glycan are indistinguishable. Accordingly, the percentage of type 2 core O-glycans and/or type 1 extended core O-glycans of the total O-glycans is at least 20%.

Accordingly, four O-glycosylation sites in Seq11 and all twelve O-glycosylation sites in Seq12 recombinantly produced in the HEK cell line are occupied by type 1 core O-glycans and also to a high degree by type 2 core O-glycans and/or or extended core O-glycans of the 1st type. Given the large number of type 2 O-glycan cores, both sequences may be good ligands for SIGLEC binding. The addition of two additional repeats of sequences containing four O-glycosylation sites made it possible to successfully obtain a protein with twelve O-glycosylation sites clustered and completely occupied by O-glycans.

Example 8 SIGLEC Binding Analysis of Seq11 and Seq12 Sequences

8.1 Experimental procedure

The Strep-Tag-bearing recombinant proteins, Seq11 and Seq12, were tested in ELISA for SIGLEC binding as described in Example 1, except for the detection strategy; Strep-Tactin-HRP conjugate (#2-1502-001, IBA GmbH) was used instead of Streptavidin-HRP to detect Strep-Tag-tagged proteins. The concentration of applied Strep-Tactin-HRP was 0.25 μg/ml. Both proteins, Seq11 and Seq12, were tested at the same molar concentration of 42 nM.

8.2 Results

As shown in Figure 10, both Seq11 and Seq12 polypeptides bind to SIG-5, SIG-7, SIG-F and SIG-9. The measured uptake for both Seq11 and Seq12 polypeptides with all four SIGLECs was the same as the binding of Seq11 and Seq12 to anti-vWF.

In contrast, the uptake for Seq11 and Seq12 in the SIG-2 and SIG-10 experiment was in the same range as the negative control (anti-chicken IgY) experiment. Thus, neither Seq11 nor Seq12 bind to SIG-2 or SIG-10 (see FIG. 10).

Example 9 Dependence of SIGLEC binding by Seq11 and Seq12 on sialic acid

9.1 Experimental procedure

The experimental protocol of Example 8 was repeated with the addition of Sialidase digestion of Strep-labeled Seq11 and Seq12 polypeptides. Desialylation was carried out as described in example 3.

9.2 Results

The results of binding experiments with desialylated Seq11 and Seq12 are shown in FIG. Binding to SIG-7, SIG-F and SIG-9 is completely blocked, ie. absorption is at the same level as when binding to SIG-2 and SIG-10. Thus, the binding of both Seq11 and Seq12 polypeptides to SIG-5, SIG-7, SIG-F and SIG-9 is sialic acid dependent.

Example 10 - Binding comparison of Seq11 and Seq12 with SIGLEC

10.1 Experimental procedure

In order to measure and compare the apparent binding affinity of Seq11 and Seq12, SIGLEC binding ELISA was performed with Scatchard analysis of binding curves. ELISA was performed as described in examples 8 and 9.

10.2 Results

The binding curves for Seq11 and Seq12 and the corresponding Scatchard plots are shown in FIG. 12 and 13. The apparent binding affinity (K _D ) derived from the Scatchard plots is shown in FIG. 14. Increasing the number of O-glycan repeats in Seq12 has a significant effect on the binding affinity of SIGLEC. The affinity for SIGLEC 5 can be increased from 0.494 μM for Seq 11 to 0.14 μM for Seq12. The affinity for SILEC 7 can be increased from 0.371 μM for Seq11 to 0.005 μM for Seq12. The affinity for SIGLEC 8 can be increased from 1.027 μM for Seq11 to 0.015 μM for Seq12. Finally, the affinity for SIGLEC 9 can be increased from 0.591 μM for Seq11 to 0.041 μM for Seq12. Statement in the text: "From this ELISA experiment, the dissociation constants for all interactions of Seq11 and 12 with SIGLEC will be calculated."

Example 11 - Determination of binding affinity of Seq11 and Seq12 to FVIII

11.1 Experimental procedure

FVIII binding to both sequences was assessed using surface plasma resonance (SPR). The analysis was performed using a Biacore 3000 instrument (GE Healthcare). The Seq11 and Seq12 polypeptides were immobilized on the CM5 chip using an amino attachment kit (GE Healthcare). Full length plasma VWF (Wislat, Ocpharma) was immobilized as a positive control. Thereafter, a series of concentrations of FVIII (Nuwiq, Octapharma) (0.2 nM, 0.6 nM, 1.7 nM, 5.0 nM, 15 nM, 45 nM) were passed over the sensor chip surface. The working buffer was 150 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ , 0.05%.

11.2 Results

The results of SPR measurements showed that the FVIII binding affinity (K _D ) of both sequences was 1.4 nM, thus additional O-glycan repeats did not affect the binding affinity for FVIII.

Example 12 N-terminal but not C-terminal VWF fragment reduces extracellular levels of IL-12p70 and IFN-γ

12.1 Theoretical background

SIGLECs are expressed on various cells of the immune system, including monocytes and dendritic cells, and play a role in cell adhesion, endocytosis, and modulation of adaptive and innate immune signaling pathways (Macauley et al. 2014). Most SIGLECs contain an immunoreceptor tyrosine inhibitory motif (ITIM) or an ITIM-like motif in their cytoplasmic domain, which has been shown to act to attenuate the inflammatory response by inhibiting cell proliferation and activation (Vitale et al. 1999; Ikehara et al. 2004) , inducing apoptosis (Nutku et al. 2003) and downregulating cytokine production (Erdmann et al. 2009; Chen et al. 2013).

In order to determine whether the levels of inflammatory cytokines produced by moDC are altered in the presence of a SIGLEC-binding vWF fragment, a simultaneous analysis of the amount of IL-12p70 and IFN-γ in the supernatant of stimulated immature monocyte-derived dendritic cells (moDC) was performed by flow cytometry.

12.2 Design of the experiment

Monocytes from healthy donors were enriched in a ficol gradient and then CD14+ monocytes were isolated by magnetic sorting. To obtain moDC, 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. The profile of cytokines secreted by moDCs was analyzed 24 hours after stimulation with the respective vWF fragments using CBA Flex Bead Cytometry (BD) for IL-12p70 and IFN-γ, according to the manufacturer's recommendations. Cells treated with the same volume of 100 mM NaCl served as controls. Samples were analyzed on a FACSVerse flow cytometer. The final analysis and calculation of cytokine concentration was performed using the FCAP Array (BD) software.

12.3 Results

Figure 16 shows the cytokine concentration after cell incubation with two fragments of vWF with and without LPS stimulation. Consistent with these results, the N-terminus, but not the C-terminus, of vWF reduces the production of pro-inflammatory cytokines synthesized in response to LPS stimulation. Without LPS stimulation, no effect of vWF fragments on the secretion of pro-inflammatory cytokines was found.

Example 13 Phosphorylation Assay of SIGLECs and Their Adapter Molecules

13.1 Theory

Upon binding of a sialic acid-containing ligand, ITIM and ITIM-like motifs in SIGLEC are phosphorylated by SRC-family tyrosine kinases, resulting in the recruitment of SH2-domain-containing (2nd homologous SRC domain) tyrosine phosphatases SHP-1 and SHP-2. Once activated, these phosphatases can dephosphorylate cellular substrates, thus controlling the activation of various signaling pathways (Crocker et al. 2007). While SHP-1 has been thought to be involved in inhibitory signaling pathways, SHP-2 enhances signaling in most signaling pathways, but has also been reported in the literature to be involved in the negative regulation of intracellular signaling (An et al., 2006; Avril et al. , 2004; Boyd et al., 2009; Qu, 2000; Salmond and Alexander, 2006).

13.2 Experimental procedure

moDCs were prepared as described in Example 12. To determine the effect of the N-terminal vWF fragment on tyrosine phosphorylation of SIGLEC and their adapter molecules SHP-1 and SHP-2, 6×10 ⁶ moDCs were incubated for 10 minutes with 500 nM N- end fragment vWF. Cells stimulated with the same volume of 100 mM NaCl served as controls. Immunoreceptor phosphorylation analysis was performed using the Proteome Profiler Human Phospho-Immunoreceptor Array Kit (R&D Systems) according to the manufacturer's recommendations using 500 µg of cell lysate.

13.3 Results

The results obtained in Phospho-Immunoreceptor Array are shown in Fig.17. According to the measured pixel density, the vWF N-terminal fragment specifically changes the phosphorylation of SHP-1, SHP-2, SIG-5 and SIG-7 compared to the control. Phosphorylation of SIG-2 and SIG-10 is not observed, which directly correlates with the absence of N-terminal binding of the vWF fragment of these SIGLECs.

Taking advantage of the inventive concept presented in the above description and the corresponding drawings, many modifications and other embodiments of the invention will be apparent to those skilled in the art to which the invention set forth in the present description pertains. Thus, it is to be understood that the invention is not limited to the specific embodiments described, and modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are used in the present specification, they are used in a general and descriptive sense only and not for purposes of limitation.

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Franc V, Řehulka P, Raus M, Stulík J, Novak J, Renfrow MB, Šebela 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, Argüeso 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. Dec 2012; 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. April 2012; 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.

--->

SEQUENCE LIST

<110> Octpharma AG

<120> POLYPEPTIDES MODULATING SIGLEC-DEPENDENT IMMUNE RESPONSES

<130> 4830/P/1095-WO

<160> 21

<170>PatentIn version 3.5

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<213> Homo sapiens

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Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp

1 5 10 15

Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr

20 25 30

Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro

35 40 45

Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys

50 55 60

Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys

65 70 75 80

Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr

85 90 95

Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr

100 105 110

Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr

115 120 125

Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile

130 135 140

Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys

145 150 155 160

Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly

165 170 175

Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val

180 185 190

Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser

195 200 205

Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr

210 215 220

Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln

225 230 235 240

Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val

245 250 255

Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg

260 265 270

Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met

275 280 285

Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val

290 295 300

Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

305 310 315 320

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys

325 330 335

Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly

340 345 350

Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu

355 360 365

Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn

370 375 380

Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu

385 390 395 400

Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro

405 410 415

Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp

420 425 430

Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys

435 440 445

Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys

450 455 460

Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu

465 470 475 480

Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val

485 490 495

Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu

500 505 510

Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala

515 520 525

Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu

530 535 540

Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp

545 550 555 560

Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu

565 570 575

Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala

580 585 590

Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys

595 600 605

Ile Asp Arg Pro Glu Ala Ser Arg Ile Thr Leu Leu Leu Met Ala Ser

610 615 620

Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly

625 630 635 640

Leu Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His

645 650 655

Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn

660 665 670

Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp

675 680 685

Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro

690 695 700

Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly Pro Gly Leu Leu

705 710 715 720

Gly Val Ser Thr Leu Gly Pro Lys Arg Asn Ser Met Val Leu Asp Val

725 730 735

Ala Phe Val Leu Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn

740 745 750

Arg Ser Lys Glu Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly

755 760 765

Gln Asp Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr

770 775 780

Val Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln

785 790 795 800

Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr Gly

805 810 815

Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val Ser Gln Gly

820 825 830

Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val Thr Gly Asn Pro

835 840 845

Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile Gln Val Val Pro

850 855 860

Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu Leu Glu Arg Ile Gly

865 870 875 880

Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp Phe Glu Thr Leu Pro Arg

885 890 895

Glu Ala Pro Asp Leu Val Leu Gln Arg Cys Cys Ser Gly Glu Gly Leu

900 905 910

Gln Ile Pro Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro Leu Asp

915 920 925

Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala Ser Tyr Phe

930 935 940

Asp Glu Met Lys Ser Phe Ala Lys Ala Phe Ile Ser Lys Ala Asn Ile

945 950 955 960

Gly Pro Arg Leu Thr Gln Val Ser Val Leu Gln Tyr Gly Ser Ile Thr

965 970 975

Thr Ile Asp Val Pro Trp Asn Val Val Pro Glu Lys Ala His Leu Leu

980 985 990

Ser Leu Val Asp Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly

995 1000 1005

Asp Ala Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His

1010 1015 1020

Gly Ala Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr

1025 1030 1035

Asp Val Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg

1040 1045 1050

Ser Asn Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp Arg Tyr

1055 1060 1065

Asp Ala Ala Gln Leu Arg Ile Leu Ala Gly Pro Ala Gly Asp Ser

1070 1075 1080

Asn Val Val Lys Leu Gln Arg Ile Glu Asp Leu Pro Thr Met Val

1085 1090 1095

Thr Leu Gly Asn Ser Phe Leu His Lys Leu Cys Ser Gly Phe Val

1100 1105 1110

Arg Ile Cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp

1115 1120 1125

Val Trp Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro

1130 1135 1140

Asp Gly Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg

1145 1150 1155

Gly Leu Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys Val

1160 1165 1170

Glu Glu Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys Val Cys Thr

1175 1180 1185

Gly Ser Ser Thr Arg His Ile Val Thr Phe Asp Gly Gln Asn Phe

1190 1195 1200

Lys Leu Thr Gly Ser Cys Ser Tyr Val Leu Phe Gln Asn Lys Glu

1205 1210 1215

Gln Asp Leu Glu Val Ile Leu His Asn Gly Ala Cys Ser Pro Gly

1220 1225 1230

Ala Arg Gln Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala

1235 1240 1245

Leu Ser Val Glu Leu His Ser Asp Met Glu Val Thr Val Asn Gly

1250 1255 1260

Arg Leu Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn

1265 1270 1275

Val Tyr Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly

1280 1285 1290

His Ile Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe Gln Leu Gln

1295 1300 1305

Leu Ser Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly

1310 1315 1320

Ile Cys Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly

1325 1330 1335

Thr Val Thr Thr Asp Trp Lys Thr Leu Val Gln Glu Trp Thr Val

1340 1345 1350

Gln Arg Pro Gly Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys

1355 1360 1365

Leu Val Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu

1370 1375 1380

Phe Ala Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala

1385 1390 1395

Ile Cys Gln Gln Asp Ser Cys His Gln Glu Gln Val Cys Glu Val

1400 1405 1410

Ile Ala Ser Tyr Ala His Leu Cys Arg Thr Asn Gly Val Cys Val

1415 1420 1425

Asp Trp Arg Thr Pro Asp Phe Cys Ala Met Ser Cys Pro Pro Ser

1430 1435 1440

Leu Val Tyr Asn His Cys Glu His Gly Cys Pro Arg His Cys Asp

1445 1450 1455

Gly Asn Val Ser Ser Cys Gly Asp His Pro Ser Glu Gly Cys Phe

1460 1465 1470

Cys Pro Pro Asp Lys Val Met Leu Glu Gly Ser Cys Val Pro Glu

1475 1480 1485

Glu Ala Cys Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln

1490 1495 1500

Phe Leu Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln Ile Cys

1505 1510 1515

Thr Cys Leu Ser Gly Arg Lys Val Asn Cys Thr Thr Gln Pro Cys

1520 1525 1530

Pro Thr Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu Val Ala Arg

1535 1540 1545

Leu Arg Gln Asn Ala Asp Gln Cys Cys Pro Glu Tyr Glu Cys Val

1550 1555 1560

Cys Asp Pro Val Ser Cys Asp Leu Pro Pro Val Pro His Cys Glu

1565 1570 1575

Arg Gly Leu Gln Pro Thr Leu Thr Asn Pro Gly Glu Cys Arg Pro

1580 1585 1590

Asn Phe Thr Cys Ala Cys Arg Lys Glu Glu Cys Lys Arg Val Ser

1595 1600 1605

Pro Pro Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr

1610 1615 1620

Gln Cys Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val Asn Ser

1625 1630 1635

Thr Val Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn

1640 1645 1650

Asp Cys Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp Lys Val Cys

1655 1660 1665

Val His Arg Ser Thr Ile Tyr Pro Val Gly Gln Phe Trp Glu Glu

1670 1675 1680

Gly Cys Asp Val Cys Thr Cys Thr Asp Met Glu Asp Ala Val Met

1685 1690 1695

Gly Leu Arg Val Ala Gln Cys Ser Gln Lys Pro Cys Glu Asp Ser

1700 1705 1710

Cys Arg Ser Gly Phe Thr Tyr Val Leu His Glu Gly Glu Cys Cys

1715 1720 1725

Gly Arg Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro

1730 1735 1740

Arg Gly Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser Gln Trp

1745 1750 1755

Ala Ser Pro Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg Val

1760 1765 1770

Lys Glu Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys Pro Gln

1775 1780 1785

Leu Glu Val Pro Val Cys Pro Ser Gly Phe Gln Leu Ser Cys Lys

1790 1795 1800

Thr Ser Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu Ala

1805 1810 1815

Cys Met Leu Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val Met

1820 1825 1830

Ile Asp Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly Val

1835 1840 1845

Ile Ser Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro

1850 1855 1860

Cys Pro Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys

1865 1870 1875

Gly Arg Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly Gly

1880 1885 1890

Gln Ile Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp Gly Cys

1895 1900 1905

Asp Thr His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe Trp

1910 1915 1920

Glu Lys Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His Lys Cys

1925 1930 1935

Leu Ala Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys Cys

1940 1945 1950

Asp Thr Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu

1955 1960 1965

Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp

1970 1975 1980

Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser

1985 1990 1995

Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser Pro

2000 2005 2010

Thr Arg Thr Glu Pro Met Gln Val Ala Leu His Cys Thr Asn Gly

2015 2020 2025

Ser Val Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys

2030 2035 2040

Ser Pro Arg Lys Cys Ser Lys

2045 2050

<210> 2

<211> 567

<212> PROTEIN

<213> Homo sapiens

<400> 2

Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp

1 5 10 15

Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr

20 25 30

Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro

35 40 45

Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys

50 55 60

Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys

65 70 75 80

Ile Gly Cys Asn Thr Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr

85 90 95

Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr

100 105 110

Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr

115 120 125

Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile

130 135 140

Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys

145 150 155 160

Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly

165 170 175

Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val

180 185 190

Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser

195 200 205

Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr

210 215 220

Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln

225 230 235 240

Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val

245 250 255

Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg

260 265 270

Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met

275 280 285

Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val

290 295 300

Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

305 310 315 320

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys

325 330 335

Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly

340 345 350

Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu

355 360 365

Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn

370 375 380

Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu

385 390 395 400

Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro

405 410 415

Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp

420 425 430

Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys

435 440 445

Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys

450 455 460

Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu

465 470 475 480

Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val

485 490 495

Glu Asp Ile Ser Glu Pro Pro Leu His Gln Glu Pro Gly Gly Leu Val

500 505 510

Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu

515 520 525

Asp Ile Ser Glu Pro Pro Leu His Gln Glu Pro Gly Gly Leu Val Val

530 535 540

Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp

545 550 555 560

Ile Ser Glu Pro Pro Leu His

565

<210> 3

<211> 589

<212> PROTEIN

<213> Homo sapiens

<400> 3

Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile

1 5 10 15

Leu Pro Gly Thr Leu Cys Ser Leu Ser Cys Arg Pro Pro Met Val Lys

20 25 30

Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr

35 40 45

Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met Ser Met Gly Cys Val

50 55 60

Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg His Glu Asn Arg Cys

65 70 75 80

Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln Gly Lys Glu Tyr Ala

85 90 95

Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr Cys Val Cys Gln Asp

100 105 110

Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp Ala Thr Cys Ser Thr

115 120 125

Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe

130 135 140

Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp Tyr Cys Gly Ser Asn

145 150 155 160

Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys Gly Cys Ser His Pro

165 170 175

Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu Val Glu Gly Gly Glu

180 185 190

Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys Arg Pro Met Lys Asp

195 200 205

Glu Thr His Phe Glu Val Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu

210 215 220

Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg His Leu Ser Ile Ser

225 230 235 240

Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly

245 250 255

Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln

260 265 270

Val Glu Glu Asp Pro Val Asp Phe Gly Asn Ser Trp Lys Val Ser Ser

275 280 285

Gln Cys Ala Asp Thr Arg Lys Val Pro Leu Asp Ser Ser Pro Ala Thr

290 295 300

Cys His Asn Asn Ile Met Lys Gln Thr Met Val Asp Ser Ser Cys Arg

305 310 315 320

Ile Leu Thr Ser Asp Val Phe Gln Asp Cys Asn Lys Leu Val Asp Pro

325 330 335

Glu Pro Tyr Leu Asp Val Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser

340 345 350

Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His

355 360 365

Val Cys Ala Gln His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu

370 375 380

Cys Pro Gln Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu

385 390 395 400

Cys Glu Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys

405 410 415

Gln His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys

420 425 430

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr

435 440 445

Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly Arg Arg

450 455 460

Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp Pro Glu His

465 470 475 480

Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr Cys Glu Ala Cys

485 490 495

Gln Glu Pro Gly Gly Leu Val Val Pro Thr Asp Ala Pro Val Ser

500 505 510

Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro Pro Leu His Gln

515 520 525

Glu Pro Gly Gly Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro

530 535 540

Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro Pro Leu His Gln Glu

545 550 555 560

Pro Gly Gly Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr

565 570 575

Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro Pro Leu His

580 585

<210> 4

<211> 1330

<212> PROTEIN

<213> Homo sapiens

<400> 4

Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile

1 5 10 15

Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr

20 25 30

Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly

35 40 45

Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly

50 55 60

Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys

65 70 75 80

Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu

85 90 95

Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro

100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys

115 120 125

Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly

130 135 140

Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly

145 150 155 160

Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln

165 170 175

Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala

180 185 190

Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser

195 200 205

Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln

210 215 220

Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu

225 230 235 240

Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu

245 250 255

Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala

260 265 270

Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His

275 280 285

Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys

290 295 300

Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met

305 310 315 320

Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu

325 330 335

Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His

340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn

355 360 365

Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys

370 375 380

Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp

385 390 395 400

Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg

405 410 415

Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys

420 425 430

Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu

435 440 445

Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val

450 455 460

Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu Lys Gly Asp Leu

465 470 475 480

Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu

485 490 495

Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu

500 505 510

Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn

515 520 525

Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro

530 535 540

Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln

545 550 555 560

Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met

565 570 575

Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe

580 585 590

Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys

595 600 605

Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly

610 615 620

Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val

625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln

645 650 655

Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu

660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe

675 680 685

Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys

690 695 700

Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp

705 710 715 720

Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met

725 730 735

His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val

740 745 750

Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg

755 760 765

Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu

770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met

785 790 795 800

Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg

805 810 815

His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln

820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr

835 840 845

Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp

850 855 860

Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly

865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp

885 890 895

Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys

900 905 910

Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu

915 920 925

Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys

930 935 940

Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg

945 950 955 960

Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg

965 970 975

His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val

980 985 990

Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr

995 1000 1005

Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn

1010 1015 1020

Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro

1025 1030 1035

Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln

1040 1045 1050

Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe

1055 1060 1065

Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

1070 1075 1080

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala

1085 1090 1095

Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln

1100 1105 1110

His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln

1115 1120 1125

Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu

1130 1135 1140

Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln

1145 1150 1155

His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys

1160 1165 1170

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln

1175 1180 1185

Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly

1190 1195 1200

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp

1205 1210 1215

Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr

1220 1225 1230

Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr

1235 1240 1245

Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser

1250 1255 1260

Glu Pro Pro Leu His Gln Glu Pro Gly Gly Leu Val Val Pro Pro

1265 1270 1275

Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile

1280 1285 1290

Ser Glu Pro Pro Leu His Gln Glu Pro Gly Gly Leu Val Val Pro

1295 1300 1305

Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp

1310 1315 1320

Ile Ser Glu Pro Pro Leu His

1325 1330

<210> 5

<211> 1423

<212> PROTEIN

<213> Homo sapiens

<400> 5

Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile

1 5 10 15

Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr

20 25 30

Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly

35 40 45

Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly

50 55 60

Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys

65 70 75 80

Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu

85 90 95

Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro

100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys

115 120 125

Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly

130 135 140

Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly

145 150 155 160

Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln

165 170 175

Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala

180 185 190

Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser

195 200 205

Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln

210 215 220

Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu

225 230 235 240

Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu

245 250 255

Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala

260 265 270

Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His

275 280 285

Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys

290 295 300

Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met

305 310 315 320

Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu

325 330 335

Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His

340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn

355 360 365

Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys

370 375 380

Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp

385 390 395 400

Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg

405 410 415

Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys

420 425 430

Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu

435 440 445

Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val

450 455 460

Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu Lys Gly Asp Leu

465 470 475 480

Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu

485 490 495

Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu

500 505 510

Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn

515 520 525

Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro

530 535 540

Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln

545 550 555 560

Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met

565 570 575

Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe

580 585 590

Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys

595 600 605

Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly

610 615 620

Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val

625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln

645 650 655

Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu

660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe

675 680 685

Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys

690 695 700

Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp

705 710 715 720

Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met

725 730 735

His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val

740 745 750

Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg

755 760 765

Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu

770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met

785 790 795 800

Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg

805 810 815

His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln

820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr

835 840 845

Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp

850 855 860

Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly

865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp

885 890 895

Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys

900 905 910

Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu

915 920 925

Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys

930 935 940

Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg

945 950 955 960

Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg

965 970 975

His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val

980 985 990

Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr

995 1000 1005

Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn

1010 1015 1020

Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro

1025 1030 1035

Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln

1040 1045 1050

Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe

1055 1060 1065

Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val

1070 1075 1080

Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala

1085 1090 1095

Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln

1100 1105 1110

His Gly Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln

1115 1120 1125

Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu

1130 1135 1140

Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln

1145 1150 1155

His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys Val Glu Gly Cys

1160 1165 1170

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gln

1175 1180 1185

Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val Ala Gly

1190 1195 1200

Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp

1205 1210 1215

Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr

1220 1225 1230

Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr

1235 1240 1245

Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser

1250 1255 1260

Glu Pro Pro Leu His Gln Glu Pro Gly Gly Leu Val Val Pro Pro

1265 1270 1275

Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile

1280 1285 1290

Ser Glu Pro Pro Leu His Gln Glu Pro Gly Gly Leu Val Val Pro

1295 1300 1305

Pro Thr Asp Ala Pro Val Pro Thr Thr Leu Tyr Val Glu Asp Ile

1310 1315 1320

Ser Glu Pro Pro Leu His Glu Glu Pro Glu Cys Asn Asp Ile Thr

1325 1330 1335

Ala Arg Leu Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu Val

1340 1345 1350

Glu Val Asp Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala

1355 1360 1365

Met Tyr Ser Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser Cys

1370 1375 1380

Cys Ser Pro Thr Arg Thr Glu Pro Met Gln Val Ala Leu His Cys

1385 1390 1395

Thr Asn Gly Ser Val Val Tyr His Glu Val Leu Asn Ala Met Glu

1400 1405 1410

Cys Lys Cys Ser Pro Arg Lys Cys Ser Lys

1415 1420

<210> 6

<211> 62

<212> PROTEIN

<213> Homo sapiens

<400> 6

Lys Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His

1 5 10 15

Cys Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly

20 25 30

Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr

35 40 45

Val Glu Asp Ile Ser Glu Pro Pro Leu His Gly Ser Ala Trp

50 55 60

<210> 7

<211> 124

<212> PROTEIN

<213> Homo sapiens

<400> 7

Lys Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His

1 5 10 15

Cys Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly

20 25 30

Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr

35 40 45

Val Glu Asp Ile Ser Glu Pro Pro Leu His Gln Glu Pro Gly Gly Leu

50 55 60

Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val

65 70 75 80

Glu Asp Ile Ser Glu Pro Pro Leu His Gln Glu Pro Gly Gly Leu Val

85 90 95

Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu

100 105 110

Asp Ile Ser Glu Pro Pro Leu His Gly Ser Ala Trp

115 120

<210> 8

<211> 23

<212> PROTEIN

<213> Homo sapiens

<220>

<221> Other indications

<222>(6)..(6)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (10)..(10)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (14)..(14)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (17)..(19)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (21)..(21)

<223> Xaa can be any naturally occurring amino acid

<400> 8

Val Val Pro Pro Thr Xaa Ala Pro Val Xaa Pro Thr Thr Xaa Tyr Val

1 5 10 15

Xaa Xaa Xaa Ser Xaa Pro Pro

twenty

<210> 9

<211> 23

<212> PROTEIN

<213> Homo sapiens

<400> 9

Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val

1 5 10 15

Glu Asp Ile Ser Glu Pro Pro

twenty

<210> 10

<211> 26

<212> PROTEIN

<213> Homo sapiens

<220>

<221> Other indications

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222>(8)..(9)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (11)..(11)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (15)..(15)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (17)..(20)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (24)..(25)

<223> Xaa can be any naturally occurring amino acid

<400> 10

Pro Pro Pro Thr Xaa Pro Pro Xaa Xaa Ala Xaa Val Thr Val Xaa Pro

1 5 10 15

Xaa Xaa Xaa Xaa Val Ser Thr Xaa Xaa Pro

20 25

<210> 11

<211> 26

<212> PROTEIN

<213> Homo sapiens

<400> 11

Pro Pro Pro Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly Pro

1 5 10 15

Gly Leu Leu Gly Val Ser Thr Leu Gly Pro

20 25

<210> 12

<211> 47

<212> PROTEIN

<213> Homo sapiens

<220>

<221> Other indications

<222>(6)..(9)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (15)..(17)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (20)..(20)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (22)..(23)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (27)..(28)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (32)..(32)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (35)..(37)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (39)..(41)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (44)..(47)

<223> Xaa can be any naturally occurring amino acid

<400> 12

Val Ser Ser Thr Ser Xaa Xaa Xaa Xaa Ser Thr Xaa Pro Ser Xaa Xaa

1 5 10 15

Xaa Ala Ala Xaa Thr Xaa Xaa Thr Ser Ser Xaa Xaa Pro Pro Ser Xaa

20 25 30

Pro Val Xaa Xaa Xaa Ser Xaa Xaa Xaa Thr Thr Xaa Xaa Xaa Xaa

35 40 45

<210> 13

<211> 47

<212> PROTEIN

<213> Homo sapiens

<400> 13

Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn

1 5 10 15

Leu Ala Ala Gly Thr Asp Asp Thr Ser Ser Leu Gly Pro Pro Ser Met

20 25 30

Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys

35 40 45

<210> 14

<211> 20

<212> PROTEIN

<213> Homo sapiens

<220>

<221> Other indications

<222> (1)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222>(7)..(7)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (9)..(14)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (16)..(16)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (18)..(20)

<223> Xaa can be any naturally occurring amino acid

<400> 14

Xaa Xaa Xaa Ala Thr Thr Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa

1 5 10 15

Pro Xaa Xaa Xaa

twenty

<210> 15

<211> 20

<212> PROTEIN

<213> Homo sapiens

<400> 15

Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys Thr Asp

1 5 10 15

Pro Trp Phe Ala

twenty

<210> 16

<211> 10

<212> PROTEIN

<213> Homo sapiens

<220>

<221> Other indications

<222> (1)..(2)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (8)..(10)

<223> Xaa can be any naturally occurring amino acid

<400> 16

Xaa Xaa Thr Thr Ala Ala Thr Xaa Xaa Xaa

1 5 10

<210> 17

<211> 10

<212> PROTEIN

<213> Homo sapiens

<400> 17

Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys

1 5 10

<210> 18

<211> 17

<212> PROTEIN

<213> Homo sapiens

<220>

<221> Other indications

<222> (1)..(2)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222>(6)..(8)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (10)..(10)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (12)..(14)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (17)..(17)

<223> Xaa can be any naturally occurring amino acid

<400> 18

Xaa Xaa Pro Thr Pro Xaa Xaa Xaa Ser Xaa Ser Xaa Xaa Xaa Glu Ala

1 5 10 15

xaa

<210> 19

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 19

Gln Ser Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala

1 5 10 15

Lys

<210> 20

<211> 37

<212> PROTEIN

<213> Homo sapiens

<220>

<221> Other indications

<222> (2)..(10)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (12)..(12)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (15)..(16)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (19)..(23)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (25)..(26)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (28)..(31)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (33)..(34)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> Other indications

<222> (36)..(37)

<223> Xaa can be any naturally occurring amino acid

<400> 20

Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Thr Ser Xaa Xaa

1 5 10 15

Ser Pro Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Ser Xaa Xaa Xaa Xaa Thr

20 25 30

Xaa Xaa Ala Xaa Xaa

35

<210> 21

<211> 37

<212> PROTEIN

<213> Homo sapiens

<400> 21

Val His Ile Tyr Gln Lys Asp Leu Phe Phe Thr Glu Thr Ser Asp Gly

1 5 10 15

Ser Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr

20 25 30

Glu Gly Ala Ile Lys

35

<---

Claims (24)

1. Glycosylated polypeptide for the treatment or prevention of bleeding disorders in a patient, containing an amino acid sequence having at least 90% identity to at least a fragment of human von Willebrand factor (vWF) having the amino acid sequence of SEQ ID NO: 1, where the specified glycosylated polypeptide contains one or more clusters of O-glycosylation sites, where these clusters contain at least two O-glycosylation sites and one or more sialylated O-glycans, where the combined number of type 2 sialylated core O-glycans and sialylated extended core O-glycans The type 1 glycosylated polypeptide exceeds the number of sialylated type 2 core O-glycans and sialylated extended type 1 core O-glycans of human vWF or a fragment thereof, and wherein the glycosylated polypeptide has an increased binding affinity for one or more SIGLECs selected from SIG-5, SIG-7, S IG-8 and SIG-9, compared to native human vWF. 2. The glycosylated polypeptide of claim 1, wherein the glycosylated polypeptide has increased binding to SIGLEC SIG-5, SIG-7, SIG-8 and SIG-9. 3. A glycosylated polypeptide according to any one of the preceding claims, wherein the polypeptide contains at least three O-glycosylation sites and most preferably at least four O-glycosylation sites. 4. The glycosylated polypeptide of claim 1, wherein one or more clusters contain at least one O-glycosylation site in ten amino acids, preferably at least one O-glycosylation site in four amino acids, more preferably at least one O-glycosylation site in three amino acids, and most preferably one O-glycosylation site in two amino acids. 5. Glycosylated polypeptide according to any one of paragraphs. 1-4 containing at least two clusters, more preferably at least three clusters. 6. Glycosylated polypeptide according to any one of paragraphs. 1-5, where the clusters are separated by less than 100 amino acids, preferably less than 50 amino acids, more preferably less than 30 amino acids. 7. A glycosylated polypeptide according to any one of the preceding claims, wherein the sialylated O-glycans contain at least two sialic acids in an α2-3 and/or α2-8 glycosidic bond, more preferably at least three sialic acids. 8. The glycosylated polypeptide of claim 7, wherein the percentage of type 2 core O-glycans relative to the amount of sialylated O-glycans is at least 5%, more preferably at least 10%, more preferably at least 35%, most preferably at least 35%. at least 50%. 9. A glycosylated protein according to any one of the preceding claims, comprising a multimerization domain. 10. Glycosylated polypeptide according to any one of paragraphs. 1-9, wherein one or more O-glycosylation sites are located in the amino acid sequence of human VWF, specifically replacing the amino acids of human VWF. 11. Glycosylated polypeptide according to any one of paragraphs. 1-10, wherein the glycosylated polypeptide is a fusion protein, wherein a second amino acid sequence containing one or more O-glycosylation sites is covalently linked to a human VWF amino acid sequence. 12. The glycosylated polypeptide of claim 11, wherein the covalent linker is selected from a peptide bond, a chemical linker, or a glycosidic bond. 13. A glycosylated polypeptide according to claim 11 or 12, wherein the second amino acid sequence is at least 95% homologous and preferably identical to amino acids 475-505 of SEQ ID NO: 1 or two consecutive copies of amino acids 475-505 of SEQ ID NO: 1. 14. Glycosylated polypeptide according to any one of paragraphs. 1-13, preferably produced by expression in a human cell line. 15. The use of a glycosylated polypeptide according to any one of paragraphs. 1-14 containing one or more sialylated O-glycans and binding to one or more SIGLECs selected from SIG-5, SIG-7, SIG-8 and SIG-9 to reduce the immune response to a second polypeptide, in particular a therapeutic protein . 16. Composition for the treatment or prevention of bleeding disorders in a patient, containing the first and second polypeptides in an effective amount, where the first polypeptide is a glycosylated polypeptide according to any one of paragraphs. 1-14, and the second polypeptide contains an amino acid sequence homologous or identical to the second protein of a mammal, in particular a human, where the first and second polypeptides form a protein complex, where, compared to the second polypeptide, the composition has an increased binding affinity for one or more SIGLECs selected from SIG-5, SIG-7, SIG-8 and SIG-9. 17. The composition of claim 16, wherein the second polypeptide is selected from FVIII, FVII, FIX, ADAMTS13 protein, in particular the full-length FVIII protein, the FVIII protein with the B domain deleted, or the FVIII protein in which a portion of the B domain has been replaced by a linker. 18. The composition according to claim 16 or 17, wherein the first polypeptide comprises at least a human vWF fragment, in particular a vWF fragment at least 95% identical to the sequence limited to amino acids 1-505 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, and the second polypeptide is a FVIII protein. 19. An isolated polynucleotide encoding a glycosylated polypeptide according to any one of paragraphs. 1-14. 20. The isolated polynucleotide of claim 19, wherein the glycosylated polypeptide has an amino acid sequence with at least 90%, preferably at least 95%, more preferably at least 98%, most preferably 100% identity with the sequence selected of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5. 21. A vector for obtaining a glycosylated polypeptide according to paragraphs. 1-14, where the vector contains a vector framework and a polynucleotide according to claim 19 or 20, where the vector framework is preferably selected from pCDNA3, pCDNA3.1, pCDNA4, pCDNA5, pCDNA6, pCEP4, pCEP-puro, pCET1019, pCMV, pEF1, pEF4, pEF5, pEF6, pExchange, pEXPR, pIRES and pSCAS. 22. The host cell to obtain a glycosylated polypeptide according to paragraphs. 1-14, where the host cell contains a polynucleotide according to claim 19 or 20 or a vector according to claim 21, where the host is preferably a previously established mammalian cell line, preferably a human cell line, more preferably a human kidney cell line, most preferably a cell line human embryonic kidneys, in particular the HEK293 cell line such as HEK293F. 23. Glycosylated polypeptide according to any one of paragraphs. 1-14, or a composition according to any one of paragraphs. 16-18 for use in the treatment or prevention of a bleeding disorder, preferably selected from the treatment of previously untreated patients (PUP) or the induction of immunotolerance (ITI). 24. The glycosylated polypeptide or composition for use according to claim 23, wherein the use comprises intravenous or non-intravenous injection, preferably subcutaneous injection.

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