Glycomodified polypeptides targeting anti-neutrophil autoantibodies and uses thereof

Abstract

Provided herein are glycomodified polypeptides comprising a first portion comprising one or more peptides that specifically bind to an anti-neutrophil autoantibody, and a second portion comprising one or more glycans conjugated to the first portion at one or more glycosylation sites. Also provided herein are nucleic acid sequences encoding the provided glycomodified polypeptides. Further provided herein are compositions comprising the glycomodified polypeptides and / or nucleic acids encoding same, as well as methods of making and using them.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 478,558, filed January 5, 2023, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Antineutrophil autoantibodies are commonly found in autoimmune diseases such as antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and are often involved in the pathogenesis of the disease. Summary of the Invention

[0003] The present disclosure identifies certain challenges with existing therapies used to treat diseases associated with antineutrophil antibodies (e.g., ANCA vasculitis). For example, the present disclosure identifies limitations of some of the most widely used treatment options. B cell-targeted therapy is one of the most common therapies for treating diseases associated with antineutrophil antibodies (e.g., ANCA vasculitis or AAV). However, these treatment options do not directly act on antineutrophil autoantibodies in the circulation, tissues, or organs; they do not reduce and / or deplete antineutrophil autoantibodies. Therefore, while currently available treatment options may provide patients with some relief, these treatment options do not treat the underlying cause of the disease.

[0004] The present disclosure also identifies limitations of certain nonspecific B cell-directed therapies (e.g., rituximab) used to treat ANCA vasculitis or AAV. Such nonspecific depletion of B cells is undesirable and can lead to undesirable side effects, such as organ damage or immunosuppression. Furthermore, nonspecific B cell-directed therapies are likely to require longer administration periods to achieve the desired results, e.g., a clinical response. Furthermore, nonspecific B cell-directed therapies are likely to fail to produce a rapid response due to their lack of specificity and targeting of disease-causing components, e.g., antineutrophil autoantibodies or immune complexes containing them. Such nonspecific therapies also fail to destroy and / or remove antineutrophil autoantibodies or pathogenic immune complexes containing them.

[0005] Without wishing to be bound by any particular theory, the technology provided herein can address certain limitations identified in existing therapies used to treat diseases associated with anti-neutrophil antibodies (e.g., ANCA vasculitis or AAV). The technology provided herein specifically targets autoantibodies or immune complexes containing the same, and when administered to a subject, produces a rapid, specific, and sustained response. In some embodiments, the technology provided herein is expected to have fewer undesirable side effects and / or less severe undesirable side effects than non-specific B cell-directed therapies due to its specificity in targeting immunogenic autoantibodies. In some embodiments, the technology provided herein can produce a response (e.g., a clinical response) in a shorter time period than non-specific B cell-directed therapies. In some embodiments, the technology provided herein does not require longer administration than, for example, non-specific B cell-directed therapies. In some embodiments, the technology provided herein can destroy and / or remove immune complexes containing the autoantibodies disclosed herein. In some embodiments, the technology provided herein can result in improved depletion and / or clearance of anti-neutrophil autoantibodies or immune complexes comprising same compared to non-specific B cell-directed therapies. The advantages associated with the technology disclosed herein can improve responses (e.g., clinical responses) in patients with or at risk of having a disease associated with anti-neutrophil antibodies (e.g., ANCA vasculitis or AAV).

[0006] Among other things, the present disclosure provides techniques for reducing and / or eliminating anti-neutrophil autoantibodies by providing glycoengineered polypeptides that can simultaneously bind to a target autoantibody (e.g., an anti-neutrophil autoantibody) and an endocytic receptor via one or more glycans. Without wishing to be bound by theory, the present disclosure proposes that binding of the glycoengineered polypeptides disclosed herein to the target autoantibody and the endocytic receptor induces internalization of the target autoantibody into the cell. In some embodiments, internalization of the target autoantibody results in degradation. The technology disclosed herein also relates to nucleic acid molecules encoding the glycoengineered polypeptides disclosed herein. Additionally, provided herein are compositions comprising the glycoengineered polypeptides disclosed herein or nucleic acid molecules encoding same, and methods for making the same.

[0007] The technology provided herein is particularly useful for reducing and / or eliminating anti-neutrophil autoantibodies in subjects having or at risk of having a disease associated with anti-neutrophil antibodies (e.g., ANCA vasculitis). Administration of a composition comprising a glycoengineered polypeptide disclosed herein, or a nucleic acid molecule encoding same, can reduce and / or deplete anti-neutrophil antibodies, thus treating the disease or ameliorating one or more symptoms of the disease.

[0008] Accordingly, the present disclosure provides glycomodified polypeptides comprising: (a) a first portion comprising one or more peptides that specifically bind to an anti-neutrophil autoantibody, or a fragment or complex thereof; and (b) a second portion comprising one or more glycans conjugated to the first portion at one or more glycosylation sites.

[0009] In some embodiments, the anti-neutrophil autoantibody is an anti-proteinase 3 (PR3) autoantibody, or a fragment or complex thereof. In some embodiments, the anti-PR3 autoantibody binds to PR3, or a variant or fragment thereof. In some embodiments, the anti-PR3 autoantibody binds to PR3 complexed with one or more proteins. In some embodiments, the one or more proteins complexed with PR3 include CD177.

[0010] In some embodiments, the anti-neutrophil autoantibody is an anti-myeloperoxidase (MPO) autoantibody, or a fragment or conjugate thereof. In some embodiments, the anti-MPO autoantibody binds to MPO, or a variant or fragment thereof.

[0011] In some embodiments, the glycoengineered polypeptide is capable of binding to (a) an anti-PR3 autoantibody, or a fragment or complex thereof, and (b) an anti-MPO autoantibody, or a fragment or complex thereof.

[0012] In some embodiments, the second portion specifically binds to one or more endocytic receptors.

[0013] In some embodiments, the endocytic receptor is or comprises an endocytic lectin. In some embodiments, the endocytic receptor is selected from asialoglycoprotein receptor (ASGPR); mannose-binding receptor, cluster of differentiation 206 (CD206) receptor, DC-SIGN (cluster of differentiation 209 or CD209) receptor; C-type lectin domain family 4 member G (LSECTin) receptor; macrophage-inducible Ca2+-dependent lectin receptor (Mincle); L-SIGN CD209L receptor; Dectin-1; Dectin-2, Langerin, macrophage mannose 2 receptor, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, CLEC10, DNGR1, CLEC12B, DEC-205, and mannose 6-phosphate receptor (M6PR), or a combination thereof.

[0014] In some embodiments, the endocytic receptor is ASGPR or a fragment or variant thereof, or a complex comprising ASGPR.

[0015] In some embodiments, the glycan structure comprises a biantennary structure. In some embodiments, the glycan structure comprises a biantennary GalNAc. In some embodiments, the biantennary GalNAc binds to an asialoglycoprotein receptor (ASGPR) or a fragment or variant thereof, or a complex comprising an ASGPR.

[0016] The present disclosure also provides compositions comprising the glycomodified polypeptides disclosed herein or compositions comprising polynucleotides encoding the glycomodified polypeptides disclosed herein.

[0017] In some embodiments, the composition comprises a glycoengineered polypeptide comprising a first portion that specifically binds to an anti-PR3 autoantibody or a fragment or complex thereof.

[0018] In some embodiments, the composition comprises a glycoengineered polypeptide comprising a first portion that specifically binds to an anti-MPO autoantibody or a fragment or complex thereof.

[0019] In some embodiments, a composition comprises (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to an anti-PR3 autoantibody, or a fragment or complex thereof, and (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to an anti-MPO autoantibody, or a fragment or complex thereof. In some embodiments, the ratio of the first glycoengineered polypeptide to the second glycoengineered polypeptide is about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2.5, about 1:2, about 1:1.5, about 1:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2.5:1, about 2:1, or about 1.5:1.

[0020] Also provided herein is a Leishmania host cell expressing a glycoengineered polypeptide disclosed herein.

[0021] The present disclosure further provides methods that include administering to a subject a pharmaceutical composition comprising a glycoengineered polypeptide disclosed herein or a polynucleotide encoding a glycoengineered polypeptide disclosed herein.

[0022] In some embodiments, the subject has or has been diagnosed with antineutrophil cytoplasmic antibody (ANCA) vasculitis.

[0023] In some embodiments, the ANCA vasculitis is granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA (formerly called Wegener's granulomatosis).

[0024] In some embodiments, the ANCA vasculitis is microscopic polyangiitis (MPA) / perinuclear ANCA.

[0025] In some embodiments, the glycomodified polypeptide can simultaneously bind to a target at a first portion and to an endocytic receptor-expressing cell at a second portion, thereby internalizing the target into the cell, which in some embodiments includes transport to lysosomes and / or degradation.

[0026] In some embodiments, administration of the pharmaceutical composition reduces the level of antineutrophil antibodies compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition, hi some embodiments, reducing the level of antineutrophil autoantibodies prevents neutrophil activation.

[0027] In some embodiments, administration of the pharmaceutical composition reduces one or more symptoms of ANCA vasculitis.

[0028] Further provided herein are methods comprising assessing the level of anti-neutrophil autoantibodies in a sample from a subject, and if the level of anti-neutrophil autoantibodies is higher than a comparison control, administering a pharmaceutical composition comprising a glycoengineered polypeptide disclosed herein or a polynucleotide encoding a glycoengineered polypeptide disclosed herein.

[0029] In some embodiments, a comparator comprises a predetermined reference sample, such as a sample obtained from an otherwise similar subject who does not have the disease or disorder or symptoms of the disease or disorder.

[0030] In some embodiments, the anti-neutrophil autoantibody comprises an anti-PR3 autoantibody, or a fragment or conjugate thereof; or an anti-MPO autoantibody, or a fragment or conjugate thereof, or a combination thereof.

[0031] In some embodiments, the subject has or is at risk of having ANCA vasculitis.

[0032] In some embodiments, the method is a therapeutic method.

[0033] In some embodiments, the method is a prophylactic method.

[0034] Additional features of the glycomodified polypeptides disclosed herein, nucleic acids encoding same, compositions comprising the glycomodified polypeptides or nucleic acids encoding same, and methods of making and using same are provided throughout this disclosure. [Brief explanation of the drawings]

[0035] [Figure 1A] The structure of PR3 is shown in the tetrameric form shown. The source structure RCSB 1FUJ was used to visualize the form shown. [Figure 1B]The structure of PR3 is shown in its monomeric form with the N-glycosylation site (N129; N174), the catalytic triad (H71, D118, S203), and membrane-binding residues (F180, F181, L228, F229) indicated by arrows. The source structure RCSB 1FUJ was used to visualize the described conformation.

[0036] [Figure 2A] Binding kinetics of an exemplary PR3 glycoengineered polypeptide with recombinant anti-PR3 antibody CLB12.8 are shown. SPR binding data were collected in single-cycle kinetic experiments using a Biacore 8K. [Figure 2B] Binding kinetics of an exemplary PR3 glycoengineered polypeptide with biotinylated recombinant human ASGPR1 (FIG. 2B) are shown. SPR binding data were collected in single-cycle kinetic experiments using a Biacore 8K.

[0037] [Figure 3A] Figure 1 shows the blocking potency of exemplary PR3 glycoengineered polypeptides against PR3+ ANCA patient autoantibodies from patient samples, compared to that of human neutrophil-derived PR3 (HN PR3). Serum samples from PR3+ ANCA patients were incubated with PR3 glycoengineered polypeptides or HN PR3 at the indicated concentrations (x-axis). Autoantibodies not blocked by PR3 glycoengineered polypeptides or HN PR3 were assessed by ELISA. Results are expressed as the percentage of residual autoantibodies remaining in the sample compared to untreated samples. For each concentration tested, data are shown as paired bars: the filled bar on the left indicates the percentage of residual binding after blocking with HN PR3, and the open bar on the right indicates the percentage of residual binding after blocking with PR3 glycoengineered polypeptide. [Figure 3B]Figure 1 shows the blocking potency of exemplary PR3 glycoengineered polypeptides against PR3+ ANCA patient autoantibodies from patient samples, compared to that of human neutrophil-derived PR3 (HN PR3). Serum samples from PR3+ ANCA patients were incubated with PR3 glycoengineered polypeptides or HN PR3 at the indicated concentrations (x-axis). Autoantibodies not blocked by PR3 glycoengineered polypeptides or HN PR3 were assessed by ELISA. Results are expressed as the percentage of residual autoantibodies remaining in the sample compared to untreated samples. For each concentration tested, data are shown as paired bars: the filled bar on the left indicates the percentage of residual binding after blocking with HN PR3, and the open bar on the right indicates the percentage of residual binding after blocking with PR3 glycoengineered polypeptide. [Figure 3C] Figure 1 shows the blocking potency of exemplary PR3 glycoengineered polypeptides against PR3+ ANCA patient autoantibodies from patient samples, compared to that of human neutrophil-derived PR3 (HN PR3). Serum samples from PR3+ ANCA patients were incubated with PR3 glycoengineered polypeptides or HN PR3 at the indicated concentrations (x-axis). Autoantibodies not blocked by PR3 glycoengineered polypeptides or HN PR3 were assessed by ELISA. Results are expressed as the percentage of residual autoantibodies remaining in the sample compared to untreated samples. For each concentration tested, data are shown as paired bars: the filled bar on the left indicates the percentage of residual binding after blocking with HN PR3, and the open bar on the right indicates the percentage of residual binding after blocking with PR3 glycoengineered polypeptide. [Figure 3D]Figure 1 shows the blocking potency of exemplary PR3 glycoengineered polypeptides against PR3+ ANCA patient autoantibodies from patient samples, compared to that of human neutrophil-derived PR3 (HN PR3). Serum samples from PR3+ ANCA patients were incubated with PR3 glycoengineered polypeptides or HN PR3 at the indicated concentrations (x-axis). Autoantibodies not blocked by PR3 glycoengineered polypeptides or HN PR3 were assessed by ELISA. Results are expressed as the percentage of residual autoantibodies remaining in the sample compared to untreated samples. For each concentration tested, data are shown as paired bars: the filled bar on the left indicates the percentage of residual binding after blocking with HN PR3, and the open bar on the right indicates the percentage of residual binding after blocking with PR3 glycoengineered polypeptide. [Figure 3E] Figure 1 shows the blocking potency of exemplary PR3 glycoengineered polypeptides against PR3+ ANCA patient autoantibodies from patient samples, compared to that of human neutrophil-derived PR3 (HN PR3). Serum samples from PR3+ ANCA patients were incubated with PR3 glycoengineered polypeptides or HN PR3 at the indicated concentrations (x-axis). Autoantibodies not blocked by PR3 glycoengineered polypeptides or HN PR3 were assessed by ELISA. Results are expressed as the percentage of residual autoantibodies remaining in the sample compared to untreated samples. For each concentration tested, data are shown as paired bars: the filled bar on the left indicates the percentage of residual binding after blocking with HN PR3, and the open bar on the right indicates the percentage of residual binding after blocking with PR3 glycoengineered polypeptide.

[0038] [Figure 4A]Figure 1 shows the autoantibody titer-independent blocking efficacy of an exemplary PR3 glycoengineered polypeptide against PR3+ ANCA patient autoantibodies. Fifteen serum samples from PR3+ ANCA patients with low autoantibody titers were incubated with the PR3 glycoengineered polypeptide at the concentrations indicated (x-axis). Autoantibodies not blocked by the PR3 glycoengineered polypeptide were then assessed by ELISA. The graph shows the residual autoantibodies remaining in the sample, expressed as a percentage, compared to untreated samples. The dotted line represents 50% blockage of autoantibodies by the PR3 glycoengineered polypeptide. [Figure 4B] Figure 1 shows the autoantibody titer-independent blocking efficacy of an exemplary PR3 glycoengineered polypeptide against PR3+ ANCA patient autoantibodies. Fifteen serum samples from PR3+ ANCA patients with moderate autoantibody titers were incubated with the PR3 glycoengineered polypeptide at the concentrations indicated (x-axis). Autoantibodies not blocked by the PR3 glycoengineered polypeptide were then assessed by ELISA. The graph shows the residual autoantibodies remaining in the sample, expressed as a percentage, compared to untreated samples. The dotted line represents 50% blockage of autoantibodies by the PR3 glycoengineered polypeptide. [Figure 4C] Figure 1 shows the autoantibody titer-independent blocking efficacy of an exemplary PR3 glycoengineered polypeptide against PR3+ ANCA patient autoantibodies. Fifteen serum samples from PR3+ ANCA patients with high autoantibody titers were incubated with the PR3 glycoengineered polypeptide at the concentrations indicated (x-axis). Autoantibodies not blocked by the PR3 glycoengineered polypeptide were then assessed by ELISA. The graph shows the residual autoantibodies remaining in the sample, expressed as a percentage, compared to untreated samples. The dotted line represents 50% blockage of autoantibodies by the PR3 glycoengineered polypeptide.

[0039] [Figure 5A]Figure 1 shows the depletion efficacy of exemplary PR3 glycoengineered polypeptides against anti-PR3 autoantibodies from ANCA patients. Serum samples from five PR3+ ANCA patients were incubated with magnetic beads coated with PR3 glycoengineered polypeptides, and the samples containing the magnetic beads were placed on a magnet. The flow-through was collected and analyzed for autoantibody levels by ELISA. Antibody levels, expressed as international units (IU) of anti-PR3 IgG / mL, in the indicated serum samples are shown after depletion with beads coated with a control protein (Ctrl) or PR3 glycoengineered polypeptides. [Figure 5B] Figure 5 shows the depletion efficacy of exemplary PR3 glycoengineered polypeptides against anti-PR3 autoantibodies from ANCA patients. Serum samples from five PR3+ ANCA patients were incubated with magnetic beads coated with PR3 glycoengineered polypeptides, and the samples containing the magnetic beads were placed on a magnet. The flow-through was collected and analyzed for autoantibody levels by ELISA. Data from Figure 5A are shown as the numerical percentage of anti-PR3 autoantibody depletion obtained with the PR3 glycoengineered polypeptides.

[0040] [Figure 6A] 1 shows the ability of a complex comprising an exemplary PR3 glycoengineered polypeptide and an anti-PR3 antibody to be internalized and degraded in hepatocytes. [Figure 6B] Figure 1 shows the ability of a complex comprising an exemplary PR3 glycoengineered polypeptide and an anti-PR3 antibody to be internalized and degraded in hepatocytes. Figure 2 shows a blot showing PR3 glycoengineered polypeptide protein levels using an anti-His antibody. The blot was reprobed with β-actin as a loading control. [Figure 6C] Figure 1 shows the ability of a complex comprising an exemplary PR3 glycoengineered polypeptide and an anti-PR3 antibody to be internalized and degraded in hepatocytes. Figure 2 shows a blot showing CLB12.8 (anti-PR3 mouse antibody) protein levels using an anti-mouse IgG antibody. The blot was reprobed with β-actin as a loading control. HC: heavy chain. LC: light chain.

[0041] [Figure 7] Figure 1 shows ASGPR-dependent internalization of a complex containing an exemplary PR3 glycoengineered polypeptide and an anti-PR3 antibody. The left side of the blot shows data from HepG2 wild-type cells (WT). The right side of the blot shows data from HepG2 ASGPR1 knockout cells (KO). The top panel of the entire blot shows CLB12.8 (anti-PR3 mouse antibody) protein levels using an anti-IgG antibody. The bottom panel shows β-actin levels as a loading control. HC: heavy chain. LC: light chain.

[0042] [Figure 8] Figure 1 shows the blocking efficacy of exemplary MPO glycoengineered polypeptides against autoantibodies in MPO+ANCA patients. Serum samples from one MPO+ANCA patient were incubated with the indicated concentrations of MPO glycoengineered polypeptides (x-axis). Autoantibodies that were not blocked by the MPO glycoengineered polypeptides were then assessed by ELISA. The graph shows autoantibody levels expressed as International Units (IU) of anti-MPO IgG / mL. The dotted line represents the lower limit of ELISA detection. DETAILED DESCRIPTION OF THE INVENTION

[0043] definition In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," (iii) the terms "comprising" and "including" may be understood to encompass itemized components or steps regardless of whether they are presented by themselves or with one or more additional components or steps, (iv) the terms "about" and "approximately" may be understood to allow for standard variations that would be understood by one of ordinary skill in the art, and (v) when ranges are specified, both endpoints are included.

[0044] MPO: The term "MPO" is used herein in reference to myeloperoxidase polypeptides as understood in the art. MPO is a heme-containing peroxidase. The MPO protein is encoded by the MPO gene. The amino acid sequence of full-length MPO and / or the nucleic acid encoding it can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human MPO (SEQ ID NO: 4, for which residues 1-45 represent the signal peptide) can be found under UniProt / Swiss-Prot accession number P05164; the nucleic acid sequence encoding human MPO (SEQ ID NO: 9) can be found under GenBank: J02694.1. Those skilled in the art will understand that the sequences provided in SEQ ID NOs: 4 and 9 are exemplary, and that certain variations (including, for example, conservative substitutions in SEQ ID NO: 4, codon-optimized variants of SEQ ID NO: 9, etc.) will also be understood to be human MPO or to encode human MPO; further, those skilled in the art will understand that homologs and orthologs of human MPO are known and / or may be known through the exercise of routine skill and may be useful in the present invention based, for example, on the degree of sequence identity, the presence of one or more characteristic sequence elements, and / or one or more shared activities.

[0045] PR3: The term "PR3" is used herein in reference to proteinase 3 (PR3) polypeptides as understood in the art. PR3 is a serine protease and is also known as myeloblastin, AGP7, neutrophil proteinase 4, P29, or Wegener's autoantigen. The PR3 protein is encoded by the myeloblastin gene. The amino acid sequence of full-length PR3 and / or the nucleic acid encoding it can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human PR3 (SEQ ID NO: 1, for which residues 1-25 represent the signal peptide) can be found under UniProt / Swiss-Prot accession number P24158; the nucleic acid sequence encoding human PR3 (SEQ ID NO: 7) is associated with GenBank: M75154.1. Those skilled in the art will understand that the sequences provided in SEQ ID NOs: 1 and 7 are exemplary, and that certain variations (including, for example, conservative substitutions in SEQ ID NO: 1, codon-optimized variants of SEQ ID NO: 7, etc.) will also be understood to be human PR3 or to encode human PR3; further, those skilled in the art will understand that homologs and orthologs of human PR3 are known and / or may be known through the exercise of routine skill and may be useful in the present invention based, for example, on the degree of sequence identity, the presence of one or more characteristic sequence elements, and / or one or more shared activities.

[0046] Glycan: As used herein, the term "glycan" refers to one or more sugars or sugar chains that can be attached to a protein or lipid to form a glycoconjugate. Glycans conjugated to proteins form glycoproteins. Glycans conjugated to the nitrogen atom of an amino acid residue are N-linked glycans, and glycans conjugated to the oxygen atom of an amino acid residue are O-linked glycans. As will be understood by those skilled in the art, the structure of a glycan indicates whether a particular glycan is an N-linked glycan.

[0047] Glycoengineering: As used herein, the term "glycoengineering" or its equivalents refers to a process of glycosylating a target protein (e.g., a glycoengineered polypeptide disclosed herein), or a target protein produced by such a process. In some embodiments, the process employs a host cell system harboring one or more enzymes (e.g., pathways) that provide glycosylation of the target protein; in other embodiments, the process is carried out by chemically attaching one or more glycans to the target protein, e.g., using click chemistry. Such host cell systems can be genetically engineered to incorporate glycosylation pathways for selectively glycosylating target proteins with specific glycan structures. Host cells used to produce glycoengineered target proteins can include, for example, a recombinant nucleic acid encoding the target protein; and a recombinant nucleic acid encoding a heterologous glycosyltransferase. Host cell systems used for glycoengineering (e.g., to produce glycoengineered proteins) can introduce, remove, or modify N-linked glycosylation. Host cell systems used for glycoengineering (e.g., to produce glycoengineered proteins) can introduce, remove, or modify O-linked glycosylation. The host cells used for glycoengineering or to produce the glycoengineered target protein can be mammalian cells, insect cells, yeast cells, bacterial cells, plant cells, microalgae, or protozoa. The protozoa used for glycoengineering can be a Leishmania species. Glycoengineered target proteins also include target proteins that have been engineered to be selectively glycosylated at one or more specific sites when produced in a host cell system.

[0048] Glycoengineered Polypeptide: As used herein, a "glycoengineered polypeptide" is a polypeptide that specifically binds to a target protein (e.g., an anti-neutrophil autoantibody) and mediates the internalization and / or degradation of the target protein by associating with one or more endocytic receptors. In some embodiments, binding (e.g., simultaneous binding) of a glycoengineered polypeptide to a target protein and an endocytic receptor results in the internalization of the target protein and / or activation of one or more degradative pathways.

[0049] Glycosylation site: As used herein, the term "glycosylation site" refers to a glycosylation site in a protein. Such glycosylation sites, also referred to herein as glycosites, can be naturally occurring in the amino acid sequence of a protein or can be recombinantly engineered into the protein by the addition, substitution, or deletion of amino acids. In some embodiments, the glycosylation site is present in a so-called glycotag fused to a glycoengineered polypeptide disclosed herein. In certain embodiments, a glycotag is fused to a protein to create a bispecific binding protein. As used herein, a glycotag refers to a peptide containing a consensus N-glycosylation site sequence fused to the N-terminus, C-terminus, or both termini of a protein or polypeptide. In some embodiments, the glycotag is fused to the C-terminus of a glycoengineered polypeptide disclosed herein via a peptide linker. In some embodiments, the glycotag is fused to the N-terminus of a glycoengineered polypeptide disclosed herein via a peptide linker. In some embodiments, the peptide linker is a consensus peptide sequence. In some embodiments, the consensus peptide sequence is 1, 2, 3, 4, 5, 6, 7, or more amino acid residues in length. In some embodiments, the bifunctional proteins provided herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycotags.

[0050] Endocytic receptor: As used herein, the term "endocytic receptor" refers to a receptor or fragment thereof that binds to a target and internalizes the target into a cell. In some embodiments, the endocytic receptor recognizes and binds to one or more glycans on the target. In some embodiments, the binding of the endocytic receptor to the target internalizes the target within the cell, for example, into a lysosome or phagosome. In some embodiments, the endocytic receptor is or includes an endocytic lectin. In some embodiments, the endocytic receptor is selected from asialoglycoprotein receptor (ASGPR); mannose-binding receptor, cluster of differentiation 206 (CD206) receptor, DC-SIGN (cluster of differentiation 209 or CD209) receptor; C-type lectin domain family 4 member G (LSECTin) receptor; macrophage-inducible Ca2+-dependent lectin receptor (Mincle); L-SIGN CD209L receptor; Dectin-1; Dectin-2, Langerin, macrophage mannose 2 receptor, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, CLEC10, DNGR1, CLEC12B, DEC-205, and mannose 6-phosphate receptor (M6PR), or a combination thereof.

[0051] Anti-neutrophil autoantibody. As used herein, the term "anti-neutrophil autoantibody" refers to an antibody that is produced in an organism (e.g., a mammal) and specifically binds to an epitope on an antigen endogenous to a neutrophil (e.g., a neutrophil autoantigen). Neutrophil autoantigens are expressed by neutrophils (e.g., present inside and / or on the surface of neutrophils) and are encoded by nucleic acid sequences naturally occurring in the neutrophil genome. In some embodiments, the neutrophil autoantigen comprises a PR3 polypeptide or a variant or fragment thereof, an MPO polypeptide or a variant or fragment thereof, or both. In some embodiments, the anti-neutrophil autoantibody is or comprises a complete antibody, an antigen-binding fragment of an antibody, or a complex thereof. In some embodiments, the anti-neutrophil autoantibody is or comprises an anti-PR3 autoantibody, or a fragment or complex thereof. In some embodiments, the anti-neutrophil autoantibody is or comprises an anti-MPO autoantibody, or a fragment or complex thereof.

[0052] Antineutrophil autoantibody-associated disease: As used herein, "antineutrophil autoantibody-associated disease" includes diseases, disorders, or conditions in which one or more antineutrophil autoantibodies are present and / or can be detected. In some embodiments, antineutrophil autoantibody-associated disease includes antineutrophil cytoplasmic antibody (ANCA) vasculitis, which is also referred to as ANCA-associated vasculitis (or AAV). In some embodiments, ANCA vasculitis includes granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA (formerly known as Wegener's granulomatosis). In some embodiments, ANCA vasculitis includes microscopic polyangiitis (MPA) / perinuclear ANCA. In some embodiments, ANCA vasculitis includes ANCA vasculitis, including renal-confined vasculitis. In some embodiments, ANCA vasculitis includes eosinophilic granulomatous polyangiitis (EGPA).

[0053] About: The term "about," when used herein in reference to a value, refers to a value similar to a reference value in the context. Generally, a person skilled in the art familiar with the context will fully understand the relative degree of difference encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

[0054] Administration: As used herein, the term "administration" typically refers to administering a composition to a subject or system, e.g., to achieve delivery of an agent that is, is contained in, or is otherwise delivered by the composition. Those skilled in the art will recognize various routes that may be utilized for administration to a subject, e.g., an animal or a human, under appropriate circumstances. In some embodiments, the animal is a companion animal, e.g., a domestic animal such as a dog or cat; in some embodiments, the animal is an animal used for agriculture (e.g., farming [e.g., cattle, sheep, or horses]) or recreation. For example, in some embodiments, administration may be systemic or local. Those skilled in the art will recognize appropriate routes of administration for use with particular therapies described herein, including, for example, bronchial (e.g., by bronchial instillation), buccal, transdermal (which may be or include, e.g., one or more of topical, intradermal, interdermal, transdermal, etc., into the dermis), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), intravaginal, intravitreal, etc. In some embodiments, administration may be by injection (e.g., intramuscular, intravenous, or subcutaneous). In some embodiments, injection may comprise a bolus injection, infusion, perfusion, or infusion. In some embodiments, administration may comprise only a single dose. In some embodiments, administration may comprise the application of a number of doses. In some embodiments, administration may include administration that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) administration. In some embodiments, administration may include continuous administration (e.g., perfusion) for at least a selected period of time. In some embodiments, the antibody agent may be formulated for oral delivery.For example, one skilled in the art will understand that the antibody agents disclosed herein can be formulated for oral delivery using technology developed by Oramed (https: / / www.oramed.com / ) or Premas (https: / / www.premasbiotech.com / ).

[0055] Adult: As used herein, the term "adult" refers to a human over the age of 18. In some embodiments, a human adult has a weight within the range of about 90 pounds to about 250 pounds.

[0056] Affinity: As known in the art, "affinity" is a measure of the tightness with which two or more binding partners associate with one another. Those skilled in the art will be aware of various assays that can be used to assess affinity and will also be aware of appropriate controls for such assays. In some embodiments of the glycoengineered polypeptides disclosed herein, the first portion comprising one or more peptides that specifically bind to a target autoantibody (e.g., an anti-neutrophil autoantibody) has high affinity for the autoantigen (e.g., a neutrophil autoantigen). In some embodiments, high affinity is an affinity of about 100-1000 pM. In some embodiments, affinity is assessed in a quantitative assay. In some embodiments, affinity (e.g., of one binding partner at a time) is assessed across multiple concentrations. In some embodiments, affinity is assessed in the presence of one or more potential competitors (e.g., that may be present in a relevant, e.g., physiological, context). In some embodiments, affinity is assessed in comparison to a reference (e.g., with a known affinity above a certain threshold [see "positive control"] or with a known affinity below a certain threshold [see "negative control"]). In some embodiments, affinity may be assessed relative to a concurrent reference; in some embodiments, affinity may be assessed relative to a background reference. Typically, when affinity is assessed relative to a reference, it is assessed under comparable conditions.

[0057] Avidity: As known in the art, "binding activity" is a measure of the cumulative strength of multiple non-covalent interactions between two or more binding partners in a complex. Those skilled in the art will be aware of various assays that can be used to assess binding activity and will also be aware of appropriate controls for such assays. In some embodiments, binding activity can be determined by (1) the binding affinity of two or more binding partners in a complex; (2) the respective valencies of the binding partners in the complex; and / or (3) the structural configuration of two or more binding partners in the complex. In some embodiments, the avidity of binding between two or more binding partners is greater than the sum of the individual binding affinities between the two or more binding partners. In some embodiments, binding activity is also referred to as apparent affinity or functional affinity. In some embodiments of the glycoengineered polypeptides disclosed herein, a second moiety comprising one or more glycans capable of binding to a receptor (e.g., an endocytic receptor) contributes to the binding activity of the glycoengineered polypeptide. In some embodiments, the endocytic receptor is ASGPR or a fragment or variant thereof. In some embodiments, binding activity is assessed in a quantitative assay. In some embodiments, binding activity is assessed across multiple concentrations. In some embodiments, binding activity is assessed in the presence of one or more potential competitors (e.g., those that may be present in relevant physiological conditions). In some embodiments, binding activity may be assessed in comparison to a concurrent reference; in some embodiments, binding activity may be assessed in comparison to a background reference. Typically, when binding activity is assessed in comparison to a reference, it is assessed under comparable conditions.

[0058] Agent / Drug / Agent: As used herein, the term "agent / drug / agent" may refer to a physical entity or phenomenon. In some embodiments, an agent / drug / agent may be characterized by a particular property and / or effect. In some embodiments, an agent / drug / agent may be a compound, molecule, or entity of any chemical class, including, for example, a small molecule, polypeptide, nucleic acid, monosaccharide, lipid, metal, or combination or complex thereof. In some embodiments, the term "agent / drug / agent" may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymer moieties. In some embodiments, the term "agent / drug / agent" may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymer moiety. In some embodiments, the term may refer to a compound, molecule, or entity that is devoid of or substantially free of any polymer or polymer moiety.

[0059] Amino acid: As used herein in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a polypeptide, may contain structural modifications compared to the general structures above. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to one containing the same amino acid except for the unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to one containing the same amino acid except for the unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, the term may be used to refer to an amino acid residue of a polypeptide.

[0060] Animal: As used herein, refers to a member of the animal kingdom. In some embodiments, "animal" refers to a human; unless otherwise specified, in many embodiments, a human can be of either sex and / or at any stage of development. In some embodiments, "animal" refers to a non-human animal; unless otherwise specified, in many embodiments, a non-human animal can be of either sex and / or at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, the animal can be, for example, a mammal, bird, reptile, amphibian, fish, insect, worm, etc. In some embodiments, the animal can be a transgenic animal, a genetically modified animal, and / or a clone.

[0061] Antibody: As used herein, the term "antibody" refers to a polypeptide containing canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. In the case of autoimmune diseases, the antigen to which a pathogenic autoantibody binds is also referred to as an "autoantigen." As is known in the art, intact antibodies, as produced naturally, are approximately 150 kD tetrameric agents composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains, each about 110 amino acids long: an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 domain (located at the base of the Y stem). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in an intact antibody. Each light chain is composed of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch." An intact antibody tetramer is composed of two heavy-light chain dimers, in which the heavy and light chains are linked to each other by one disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to each other, thereby connecting the dimers to form a tetramer. Naturally produced antibodies are usually glycosylated in the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., a three-, four-, or five-stranded sheet) packed together in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4).When a natural antibody folds, the FR regions form beta sheets to provide the structural framework for the domain, and the CDR loop regions of both the heavy and light chains join in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to components of the complement system and also to receptors on effector cells, such as effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding properties of the Fc region for an Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present disclosure comprise a glycosylated Fc domain, including Fc domains with modified or altered glycosylation. In some embodiments, antibodies produced and / or utilized in accordance with the present disclosure comprise one or more modifications in the Fc domain, e.g., effector-null mutations, e.g., LALA, LAGA, FEGG, AAGG, or AAGA mutations. For the purposes of this disclosure, in certain embodiments, any polypeptide or polypeptide complex that contains a sufficient immunoglobulin domain sequence as found in a natural antibody may be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., generated by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of a canine, feline, murine, rabbit, primate, or human antibody. In some embodiments, the antibody sequence elements are human, humanized, primatized, chimeric, etc., as known in the art. Furthermore, as used herein, the term "antibody" can refer, in appropriate embodiments (unless otherwise stated or apparent from the context), to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations.For example, in some embodiments, antibodies utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., VHHs [e.g., camelid-derived VHH or NAR), alternative scaffolds or antibody mimetics (e.g., anticalins, FN3 monobodies, DARPins, affibodies, affilins, affimers, affitins, alphabodies, avimers, fynomers, Im7, VLR, VNAR, Trimbab, CrossMab, Trident); formats selected from nanobodies, binanobodies, F(ab')2, Fab', di-sdFv, trifunctional antibodies, diabodies, and minibodies. In some embodiments, suitable formats may be or include Adnectins®; Affibodies®; Affilins®; Anticalins®; Avimers®; BiTEs®; cameloid antibodies; Centyrins®; ankyrin repeat proteins or DARPINs®; dual affinity retargeting (DART) agents; Fynomers®; shark single domain antibodies, such as IgNAR; immune mobilizing monoclonal T cell receptors against cancer (ImmTACs); KALBITOR®; microproteins; Nanobodies® minibodies; masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAb®); TCR-like antibodies; Trans-bodies®; TrimerX®; VHHs. In some embodiments, the antibody may lack covalent modifications (eg, glycan attachment) that it would have if produced naturally.In some embodiments, the antibody format is or includes a VHH, e.g., a camelid-derived VHH. In some embodiments, the VHH is a multivalent VHH, e.g., a bivalent VHH. In some embodiments, the antibody includes a single-domain antibody, e.g., including one or more additional domains, e.g., an Fc, a half-Fc (e.g., including interchain cysteine ​​mutations), an albumin domain, or a combination thereof. In some embodiments, the antibody includes a single-chain Fv, e.g., including one or more additional domains, e.g., an Fc, a half-Fc (e.g., including interchain cysteine ​​mutations), an albumin domain, or a combination thereof. In some embodiments, the antibody includes a polypeptide-Fc fusion. In some embodiments, the antibody can contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.)).

[0062] Antibody fragment: As used herein, "antibody fragment" refers to a portion of an antibody or antibody drug described herein, and typically refers to an antigen-binding portion or portion comprising a variable region thereof. Antibody fragments can be produced by any means. For example, in some embodiments, antibody fragments can be produced enzymatically or chemically by fragmentation of an intact antibody or antibody drug. Alternatively, in some embodiments, antibody fragments can be produced recombinantly (i.e., by expression of an altered nucleic acid sequence). In some embodiments, antibody fragments can be fully or partially synthetically produced. In some embodiments, antibody fragments (particularly antigen-binding antibody fragments) are at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or more amino acids in length, and in some embodiments, are at least about 200 amino acids in length.

[0063] Antigen: As used herein, the term "antigen" refers to an agent that elicits an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including the production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., engagement of T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen binds to an antibody and may or may not induce a specific physiological response in an organism. Generally, an antigen may be or include any chemical entity, such as a small molecule, nucleic acid, polypeptide, carbohydrate, lipid, polymer (in some embodiments, other than a biological polymer [e.g., other than a nucleic acid or amino acid polymer]), etc. In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a glycan. Those skilled in the art will understand that, in general, antigens may be provided in isolated or pure form, or alternatively, may be provided in crude form (e.g., together with other materials, such as cell extracts or other relatively crude preparations of antigen-containing sources). In some embodiments, antigens utilized in accordance with the present invention are provided in crude form. In some embodiments, the antigen is a recombinant antigen.

[0064] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (above or below) of the stated reference value, unless otherwise specified or a different meaning is apparent from the context (except where such number would exceed 100% of the possible values).

[0065] Binding: Those skilled in the art will understand that the term "binding," as used herein, typically refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between the entities or moieties, while indirect binding involves a physical interaction due to physical contact through one or more intermediate entities. Typically, binding between two or more entities can be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied alone or in the context of a more complex system (e.g., while covalently bound or otherwise associated with a carrier entity and / or within a biological system or cell).

[0066] CDR: As used herein, refers to a complementarity-determining region within an antibody variable region. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. A "set of CDRs" or "CDR set" refers to a group of three or six CDRs present in either a single variable region capable of binding to an antigen or the CDRs of cognate heavy and light chain variable regions capable of binding to an antigen. Certain systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.); one of skill in the art will appreciate the differences between these systems and will be able to understand CDR boundaries to the extent necessary to understand and practice the claimed subject matter.

[0067] Composition: Those skilled in the art will understand that the term "composition" can be used to refer to a separate physical entity that includes one or more specified components. Generally, unless otherwise specified, a composition can be in any form, for example, a gas, a gel, a liquid, a solid, etc.

[0068] Comprising: Compositions or methods described herein as "comprising" one or more named elements or steps are open-ended, meaning that the named elements or steps are required, but that other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it should also be understood that any composition or method described as "comprising" (or "comprises") one or more named elements or steps also represents a corresponding, more limited composition or method that "consistes essentially of" (or "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps, and may include additional elements or steps that do not materially affect the basic and novel property(ies) of the composition or method. It should also be understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also represents a corresponding, more limited, closed-ended composition or method "consisting of" (or "consists of") the named elements or steps, excluding any other elements or steps not named. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0069] Conjugate: As used herein, the term "conjugate" refers to the linking of one moiety to another moiety by in vitro methods (e.g., chemical synthesis) or in vivo (e.g., within a cell). In some embodiments, a moiety comprising one or more glycans (e.g., a second moiety) is conjugated to a different moiety at one or more glycosylation sites in vivo, e.g., in a cell. In some embodiments, a moiety comprising one or more glycans (e.g., a second moiety) is conjugated to a different moiety at one or more glycosylation sites, e.g., by chemical conjugation.

[0070] Domain: As used herein, the term "domain" refers to a section or portion of an entity. In some embodiments, a "domain" relates to a particular structural and / or functional characteristic of an entity such that when the domain is physically separated from the remainder of its parent entity, it substantially or completely retains the particular structural and / or functional characteristic. Alternatively or additionally, a domain may be or comprise a portion of an entity that, when separated from its (parent) entity and associated with a different (recipient) entity, substantially retains and / or confers to the recipient entity one or more structural and / or functional characteristics that characterized the domain in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide, and in some such embodiments, a domain is characterized by particular structural elements (e.g., particular amino acid sequences or sequence motifs, alpha-helical properties, alpha-sheet properties, coiled-coil properties, random coil properties, etc.) and / or particular functional properties (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0071] Epitope: As used herein, includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional structure. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0072] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a characteristic property and / or activity.

[0073] Fragment: A "fragment" of a substance or entity as described herein comprises a distinct portion of the whole, but has a structure that lacks one or more portions found in the whole. In some embodiments, the fragment consists of such a distinct portion. In some embodiments, the fragment consists of or comprises a characteristic structural element or portion found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., residues) found throughout the polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomer units (e.g., residues) found in the whole polymer. The whole substance or entity may, in some embodiments, be referred to as the "parent" of the fragment.

[0074] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., polypeptide molecules. In some embodiments, polymer molecules, such as antibodies, are considered to be "homologous" to one another if their sequences are at least 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 80%, 85%, 90%, 95%, or 99% similar.

[0075] Human: In some embodiments, the human is an embryo, fetus, infant, child, teenager, adult, or elderly.

[0076] Humanized: As known in the art, the term "humanized" is generally used to refer to an antibody (or antibody component) whose amino acid sequence comprises VH and VL region sequences derived from a reference antibody produced in a non-human species (e.g., a mouse), but also includes modifications of these sequences relative to the reference antibody intended to make them more "human-like," i.e., more similar to human germline variable sequences. In some embodiments, a "humanized" antibody (or antibody component) is one that immunospecifically binds to an antigen of interest and has framework (FR) regions having amino acid sequences substantially those of a human antibody and complementarity-determining regions (CDRs) having amino acid sequences substantially those of a non-human antibody. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., a donor immunoglobulin) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In some embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region. In some embodiments, a humanized antibody contains at least the variable domains of both a light chain and a heavy chain. The antibody also comprises a C H 1. Hinge, C H 2. C H 3, and optionally, C of the heavy chain constant region H In some embodiments, the humanized antibody may comprise a humanized V L In some embodiments, the humanized antibody contains only a humanized V region. H In some particular embodiments, the humanized antibody contains only a humanized V region. H and V L Contains the region.

[0077] As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. For example, calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Nucleotides at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4:11-17), which is incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, comparison of nucleic acid sequences with the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix.

[0078] "Improve," "Increase," "Inhibit," or "Decrease": As used herein, the terms "improve," "increase," "inhibit," "decrease," or their grammatical equivalents refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in an equivalent system known or expected to respond in a particular way in the presence of the relevant agent or treatment.

[0079] Peptide: As used herein, the term "peptide" refers to a polypeptide that is typically relatively short, e.g., having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 40 amino acids, less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, or less than 10 amino acids.

[0080] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in an amount of a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in a particular form (e.g., a solid or liquid form) and / or may be specifically adapted for, for example, oral administration (e.g., specially formulated for buccal, sublingual, or systemic absorption, e.g., as a drench [aqueous or non-aqueous solution or suspension], tablet, capsule, bolus, powder, granules, paste, etc.); parenteral administration (e.g., subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation); topical application (e.g., as a cream, ointment, patch, or spray applied, e.g., to the skin, lungs, or buccal cavity); vaginal or rectal administration (e.g., as a pessary, suppository, cream, or foam); intraocular administration; nasal or pulmonary administration, etc.

[0081] Polypeptide: As used herein, refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-natural amino acid sequence. In some embodiments, a polypeptide has an altered amino acid sequence, in that it is artificially designed and / or manufactured. In some embodiments, a polypeptide can comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide can comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide can comprise only D-amino acids. In some embodiments, a polypeptide can comprise only L-amino acids. In some embodiments, a polypeptide can comprise one or more pendant groups or other modifications, e.g., modification of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications can be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., e.g., combinations thereof. In some embodiments, a polypeptide may be cyclic and / or include a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure; in such cases, it is used herein to refer to polypeptides that share a related activity or structure and therefore can be considered members of the same class or family of polypeptides.For each such class, the present specification provides, and / or one of skill in the art will recognize, exemplary polypeptides within the class whose amino acid sequence and / or function are known; in some embodiments, such exemplary polypeptides are the reference polypeptides of the class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity with the reference polypeptide of the class (in some embodiments, with all polypeptides within the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range). For example, in some embodiments, a member polypeptide exhibits an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contains at least one region (e.g., a conserved region that, in some embodiments, is or may contain a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically encompasses at least 3-4, and often up to 20 or more, amino acids, and in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids. In some embodiments, the related polypeptide may comprise or consist of a fragment of the parent polypeptide.In some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., fragments directly linked to the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), and thus the polypeptide of interest is a derivative of that parent polypeptide.

[0082] Reference: As used herein, refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular reference or control considered.

[0083] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in the environment in which the binding occurs. A binding agent that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to that interacting target. In some embodiments, specific binding is assessed by detecting or measuring the degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or measuring the ability of a binding agent to compete with an alternative interaction of its partner with another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a range of concentrations.

[0084] Specific: The term "specific," as used herein with respect to an active agent, is understood by those skilled in the art to mean that the agent distinguishes between potential target entities or aspects. For example, in some embodiments, an agent is said to bind "specifically" to a target if it preferentially binds to that target in the presence of one or more competing alternative targets. In many embodiments, the specific interaction depends on the presence of a particular structural feature (e.g., an epitope, cleft, binding site) of the target entity. It should be understood that specificity need not be absolute. In some embodiments, specificity can be assessed relative to the specificity of a binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to that of a reference specific binding agent. In some embodiments, specificity is assessed relative to that of a reference nonspecific binding agent. In some embodiments, an agent or entity does not directly bind to a competing alternative target under conditions in which it binds to its target entity. In some embodiments, a binding agent binds to its target entity with a higher on-rate, a lower off-rate, increased affinity, decreased dissociation, and / or increased stability when compared to competing surrogate target(s).

[0085] Specificity: As known in the art, "specificity" is the degree to which a particular ligand is able to distinguish its binding partner from other potential binding partners.

[0086] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting a complete or near-complete extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or perfection, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0087] Substantial identity: As used herein, refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences, BLASTP for amino acid sequences, gapped BLAST, and PSI-BLAST. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues, which in some embodiments is the entire sequence.In some embodiments, the relevant section is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues. In the context of CDRs, references to "substantial identity" typically refer to CDRs that have no more than a small number (e.g., 3, 2, or 1) of amino acid sequence changes compared to the amino acid sequence of the reference CDR. In some embodiments, a CDR that is substantially identical to a reference CDR differs from the reference CDR by one or more amino acid changes at the termini of the reference CDR; in some such embodiments, the relevant CDR is identical to the reference CDR except at one or both termini. As is known in the art, CDR elements typically have lengths ranging from a few amino acids (e.g., 3, 4, 5, 6, or 7) to about 20 or 30 amino acids (see, e.g., Collis et al. J. Mol. Biol. 325:337, 2003, incorporated herein by reference); thus, in some embodiments, a CDR may be considered substantially identical to a reference CDR if it shares at least about 80% (or less for shorter CDRs), at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100% identity with the reference CDR.

[0088] Substantial sequence homology: The phrase "substantial homology" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues with suitably similar structural and / or functional properties. For example, as is well known to those skilled in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another amino acid of the same type may often be considered a "homologous" substitution. Typical amino acid classifications are summarized below: [Table 4] [Table 5]

[0089] As is well known in the relevant art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences, BLASTP for amino acid sequences, gapped BLAST, and PSI-BLAST. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above programs typically provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of their corresponding residues are homologous over the relevant stretch of residues. In some embodiments, the relevant stretch is the entire sequence.In some embodiments, the relevant section is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more residues.

[0090] Treatment: As used herein, the terms "treat," "treatment," or "treating" are used to refer to one or more of the partial or complete alleviation, amelioration, palliation, inhibition, prevention, delay in onset, reduction in severity, and / or reduction in frequency (e.g., incidence) of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, treatment may be prophylactic; for example, it may be administered to a subject who does not show signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who shows early signs of a disease, disorder, and / or condition, for example, it may reduce the risk of developing pathology associated with the disease, disorder, and / or condition, and / or it may delay the onset of one or more characteristics of the disease, disorder, and / or condition, and / or it may reduce the rate of onset or worsening of one or more characteristics of the disease, disorder, and / or condition.

[0091] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that partially or completely alleviates, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of a subject who does not exhibit signs of the relevant disease, disorder, and / or condition and / or who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment of a subject who exhibits one or more symptoms of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who has been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who is known to have one or more susceptibility factors, e.g., susceptibility factors that statistically correlate with an increased risk of developing the relevant disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0092] Variant: As used herein, the term "variant" refers to a molecule or entity (e.g., a nucleic acid, protein, or small molecule, for example) that exhibits significant structural identity with a reference molecule or entity, but that is structurally different from the reference molecule or entity, e.g., in the presence or absence, or at the level, of one or more chemical moieties compared to the reference molecule or entity. In some embodiments, a variant also differs functionally from the reference molecule or entity. In many embodiments, whether a particular molecule or entity is properly considered a "variant" of a reference is based on the degree of structural identity with the reference molecule. As will be understood by those skilled in the art, biological or chemical reference molecules are typically characterized by certain characteristic structural elements. A variant, by definition, is a distinct molecule or entity that shares one or more such characteristic structural elements but differs from the reference molecule or entity in at least one aspect. To give a few examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function, and a nucleic acid may have characteristic sequence elements composed of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid exhibits an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of the reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to that of the reference, but with minor sequence modifications at specific positions. Typically, less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to the reference. Often, a variant polypeptide or nucleic acid contains very few (e.g., less than about 5, about 4, about 3, about 2, or about 1) substituted, inserted, or deleted functional residues (i.e., residues responsible for a particular biological activity) compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, the variant polypeptide or nucleic acid contains less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the reference polypeptide or nucleic acid is one found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0093] Detailed Description of Certain Embodiments Disclosed herein are glycoengineered polypeptides and compositions comprising the same that have the ability to degrade one or more anti-neutrophil autoantibodies by binding to an anti-neutrophil autoantibody at a first site and to an endocytic receptor at a second site comprising one or more glycans, thereby targeting the anti-neutrophil antibody for degradation. As exemplified herein, the glycoengineered polypeptide is modified by introducing glycosylation sites onto the glycoengineered polypeptide, resulting in an altered glycosylation profile that mediates degradation of the glycoengineered polypeptide and the target endocytic receptor to which it binds.

[0094] By customizing N-glycosylation, the polypeptides described herein: 1) have uniform glycosylation; 2) are capable of degrading large targets such as immune complexes; 3) have defined ligand-to-antibody ratios; 4) have defined glycosylation sites; 5) are capable of activating more diverse and potent degradation receptors; and / or 6) are capable of participating in proteolysis in a highly optimized manner. Glycoengineered polypeptides can be used as novel therapeutic agents for treating autoimmune diseases, such as diseases associated with anti-neutrophil autoantibodies, e.g., ANCA vasculitis.

[0095] Anti-neutrophil autoantibodies Neutrophils are the most common type of white blood cell. Anti-neutrophil autoantibodies are antibodies that specifically bind to antigens endogenous to neutrophils (e.g., neutrophil autoantigens). Neutrophil autoantigens are encoded by nucleic acid sequences that naturally occur in the genome of neutrophils. In some embodiments, neutrophil autoantigens are expressed in neutrophils, for example, in the cytoplasm, nucleus, perinuclear, or intracellular compartment. In some embodiments, neutrophil autoantigens are expressed on the cell surface of neutrophils.

[0096] In some embodiments, the anti-neutrophil autoantibody is an IgG antibody. In some embodiments, the anti-neutrophil autoantibody is an IgA antibody. In some embodiments, the anti-neutrophil autoantibody is an IgM antibody. In some embodiments, the anti-neutrophil autoantibody is an IgD antibody. In some embodiments, the anti-neutrophil autoantibody is an IgE antibody.

[0097] Antineutrophil autoantibodies are produced and released by B cells. Previous reports have shown that antigens, such as microbial components, can stimulate the expression of neutrophil autoantigens, such as PR3 and / or MPO, on the surface of neutrophils (Fijolet, J. et al. Clinical Rheumatology, 2019). When antineutrophil autoantibodies bind to neutrophil antigens, such as PR3 and / or MPO, the autoantibodies can activate neutrophils. Upon activation, neutrophils adhere to the vascular endothelium and release reactive oxygen species, inflammatory cytokines, toxic substances, and neutrophil extracellular traps (NETs), which can cause systemic vasculitis and damage to multiple organs. Neutrophil adhesion in target tissues, particularly the kidneys, is due to the release of harmful oxidants and enzymes.

[0098] Exemplary neutrophil autoantigens include, but are not limited to, proteinase 3 (PR3) or a variant or fragment thereof; myeloperoxidase (MPO) or a variant or fragment thereof; lysosomal membrane protein 2 (LAMP2) or a variant or fragment thereof; and pentraxin 3 (PTX3) or a variant or fragment thereof. In some embodiments, an anti-neutrophil autoantibody may specifically bind to a neutrophil autoantigen (e.g., a cytoplasmic granule protein), such as PR3, MPO, LAMP2, and / or PTX3. The peptide sequence of human PR3 is designated by UNIPROT protein number P24158. The peptide sequence of human MPO is designated by UNIPROT protein number P05164. The peptide sequence of human LAMP2 is designated by UNIPROT protein number P13473. The peptide sequence of human PTX3 is designated by UNIPROT protein number P26022. These peptide sequences, as well as other information available through the UNIPROT database, are incorporated herein by reference. See www.uniprot.org.

[0099] In some cases, antineutrophil cytoplasmic antibodies are associated with the administration of various drugs, most notably the antithyroid drug propylthiouracil (PTU). Gao and Zhao (2009) Nephrology 14(1):33-41.

[0100] Antineutrophil autoantibody-associated disorders Antineutrophil autoantibodies have been detected in many autoimmune disorders. In some embodiments, antineutrophil autoantibodies cause neutrophil activation, which can lead to endothelial damage in small and / or medium-sized blood vessels. In some embodiments, antineutrophil autoantibodies contribute to and / or result in vasculitis. In some embodiments, antineutrophil autoantibodies contribute to and / or result in antineutrophil cytoplasmic antibody (ANCA) vasculitis.

[0101] In some embodiments, diseases associated with antineutrophil autoantibodies are associated with increased and / or abnormal expression of one or more neutrophil autoantigens. Without wishing to be bound by any particular theory, it is proposed that antigens, such as microbial components, can stimulate the expression of neutrophil autoantigens on the cell surface of neutrophils and / or induce abnormal transcription of neutrophil autoantigens, for example, due to epigenetic dysregulation. (Fijolet, J. et al. Clinical Rheumatology, (2019); Jennette CJ et al., Curr Opin Nephrol Hypertens (2011) 20(3): 263-270).

[0102] ANCA vasculitis ANCA vasculitis is a systemic disease that can involve the ears, nose and throat, lungs, kidneys, heart, digestive system, nervous system, eyes, skin, musculoskeletal tract, and rarely other organs (Hilhorst, M. et al. J Am Soc Neprhol, 2015). ANCA vasculitis can be triggered by autoimmunity (e.g., antineutrophil autoantibodies) against neutrophil granule proteins (e.g., neutrophil autoantigens), such as myeloperoxidase (MPO) or proteinase 3 (PR3), as described herein. Antineutrophil autoantibodies against PR3 or MPO, alone or in combination, can result in vasculitis (e.g., small- to medium-sized vessel vasculitis). In some embodiments, antineutrophil autoantibodies that specifically bind to PR3 and / or MPO cause ANCA vasculitis. Additional antineutrophil autoantibodies have been identified in subjects with ANCA vasculitis, which may be responsible for or contribute to the development or severity of neutrophil autoantibody-associated diseases, including LAMP-2 and PTX3.

[0103] Current treatments for ANCA vasculitis include cyclophosphamide, rituximab, methotrexate, mycophenolate mofetil, steroids, and plasma exchange. In some embodiments, one or more additional therapies (e.g., one or more of the current therapies listed above) may be administered before, substantially simultaneously with, or after one or more provided glycoengineered polypeptides.

[0104] Microscopic polyangiitis (MPA) / perinuclear ANCA Autoimmunity against MPO (e.g., production of anti-MPO autoantibodies) is strongly associated with microscopic polyangiitis (MPA) / perinuclear ANCA. MPA is often characterized by vasculitis limited to the kidney. Studies have shown that one in three patients with MPA progresses to dialysis or kidney transplantation (Hilhorst, M. et al. J Am Soc Neprhol, 2015).

[0105] In some embodiments, the ANCA vasculitis is microscopic polyangiitis (MPA) / perinuclear ANCA.

[0106] Current treatments for MPA vasculitis include immunosuppressive therapy using glucocorticoids (e.g., methylprednisone), cyclophosphamide, rituximab, methotrexate, mycophenolate mofetil, azathioprine, steroids, and plasma exchange. In some embodiments, one or more additional therapies (e.g., one or more of the current therapies listed above) may be administered before, substantially simultaneously with, or after one or more provided glycoengineered polypeptides.

[0107] Granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA Autoimmunity against PR3 (e.g., production of PR3 autoantibodies) is strongly associated with granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA (formerly known as Wegener's granulomatosis). GPA is often characterized by respiratory granulomatous inflammation, necrotizing small vessel vasculitis, and glomerulonephritis. Granulomatous inflammation is a hallmark of GPA. Granuloma formation is thought to be initiated by small aggregates of neutrophils (microabscesses) surrounding necrotic areas (Hilhorst, M. et al. J Am Soc Neprhol, 2015).

[0108] In some embodiments, the ANCA vasculitis is granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA.

[0109] Treatment options available for patients presenting with antineutrophil autoantibodies may include the administration of immunosuppressants such as methotrexate, cyclophosphamide, rituximab, and / or steroids (e.g., corticosteroids). Rituximab can be effective in managing GPA and / or MPA, but it has a delayed onset and a high risk of infection. Maintenance of remission can be achieved with methotrexate and, optionally, CTL4-immunoglobulin. Some patients may benefit from plasma exchange.

[0110] Glycomodified polypeptides Disclosed herein are glycomodified polypeptides comprising: (a) a first portion comprising one or more peptides that specifically bind to an anti-neutrophil autoantibody, or a fragment or complex thereof; and (b) a second portion comprising one or more glycans conjugated to the first portion at one or more glycosylation sites.

[0111] In some embodiments, the anti-neutrophil autoantibody is an anti-proteinase 3 (PR3) autoantibody, or a fragment or complex thereof. In some embodiments, the anti-PR3 autoantibody binds to PR3, or a variant or fragment thereof. In some embodiments, the anti-PR3 autoantibody binds to PR3 complexed with one or more proteins. In some embodiments, the one or more proteins complexed with PR3 include CD177.

[0112] In some embodiments, the anti-neutrophil autoantibody is an anti-myeloperoxidase (MPO) autoantibody, or a fragment or conjugate thereof. In some embodiments, the anti-MPO autoantibody binds to MPO, or a variant or fragment thereof.

[0113] In some embodiments, the glycoengineered polypeptide is capable of binding to an anti-PR3 autoantibody, or a fragment or complex thereof, and an anti-MPO autoantibody, or a fragment or complex thereof.

[0114] In some embodiments, the glycoengineered polypeptide is capable of binding to one or more anti-neutrophil autoantibodies in addition to anti-PR3 and / or anti-MPO autoantibodies.

[0115] In some embodiments, the glycomodified polypeptides disclosed herein comprise a first portion, a second portion, and one or more additional elements, hi some embodiments, the glycomodified polypeptide comprises an N-glycosylation site, a linker, a spacer, a signal peptide, a tag, a half-life extender, or a combination thereof.

[0116] In some embodiments, the glycomodified polypeptide comprises one or more N-glycosylation sites in the first portion. In some embodiments, the first portion comprises one or more naturally occurring N-glycosylation sites and / or one or more N-glycosylation sites that have been engineered into the first portion. In some embodiments, the engineered N-glycosylation site (also referred to herein as a glycosite or glycotag) is or comprises GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0117] In some embodiments, the glycomodified polypeptide comprises a linker. In some embodiments, the linker comprises a Gly-Ser linker or an EAAAK linker. In some embodiments, the linker comprises a (Gly-Gly-Gly-Gly-Ser)n linker, where n is an integer between 0 and 20.

[0118] In some embodiments, the glycomodified polypeptide comprises a spacer, hi some embodiments, the spacer comprises one or more nucleotides that separate a first nucleic acid sequence from a subsequent nucleic acid sequence.

[0119] In some embodiments, the spacer comprises a nucleic acid sequence encoding one or more peptides that separate a first encoded polypeptide sequence from a subsequent encoded polypeptide sequence.

[0120] In some embodiments, the glycomodified polypeptide comprises a signal peptide, e.g., as disclosed herein. In some embodiments, the signal peptide is a native signal peptide. In some embodiments, the signal peptide is not a native signal peptide.

[0121] In some embodiments, the signal peptide is derived from a Leishmania species. In certain embodiments, the signal peptide is derived from Leishmania tarentolae. In certain embodiments, the signal peptide is derived from Leishmania major.

[0122] In certain embodiments, the signal peptide is an invertase signal peptide from Leishmania tarentolae.

[0123] In certain embodiments, the signal peptide is the alkaline phosphatase signal peptide from Leishmania major.

[0124] In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 35, or a portion thereof. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 36, or a portion thereof. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 38, or a portion thereof. In certain embodiments, the signal peptide is processed and removed from the glycomodified polypeptide.

[0125] Exemplary signal peptide: SPinv, a modified signal peptide from Leishmania tarentolae invertase, SEQ ID NO: 35: MIASSVRHAVILLLVAVAMMAAVIA.

[0126] Exemplary signal peptide: SPinv, the native signal peptide from Leishmania tarentolae invertase, SEQ ID NO: 36: MIASSVRHAVILLLVAVAMMAAAVIA.

[0127] Exemplary signal peptide: The native signal peptide from Leishmania major alkaline phosphatase, SEQ ID NO: 38: MASRLVRVLAAAMLVAAAVS

[0128] In some embodiments, the glycoengineered polypeptide comprises a tag. In some embodiments, the tag is a moiety that can be used to purify and / or identify the glycoengineered polypeptides disclosed herein. In some embodiments, the tag is not a glycotag. In some embodiments, the tag comprises a His tag, a Myc tag, or a GST tag. In some embodiments, the tag comprises a cleavable tag.

[0129] In some embodiments, the tag is a His tag (HHHHHHHHHH; SEQ ID NO: 44).

[0130] In some embodiments, the half-life extender comprises albumin or a fragment or variant thereof.

[0131] In some embodiments, the half-life extender comprises an Fc domain, eg, with or without mutations in the Fc domain.

[0132] First part In some embodiments, the glycoengineered polypeptide comprises a first portion that comprises one or more peptides that specifically bind to an anti-neutrophil autoantibody or fragment thereof.

[0133] In some embodiments, the one or more peptides comprise an epitope from a neutrophil antigen recognized by an anti-neutrophil autoantibody or a fragment thereof (e.g., an antigen-binding fragment of an anti-neutrophil autoantibody). In some embodiments, the epitope comprises a fragment of a neutrophil antigen, e.g., an inactive fragment of a neutrophil antigen and / or a soluble fragment of a neutrophil antigen. In some embodiments, the epitope comprises an extracellular fragment of a neutrophil antigen.

[0134] In some embodiments, the first moiety comprising one or more peptides that specifically bind to an anti-neutrophil autoantibody, or a fragment or complex thereof, comprises one or more idiotopes specific for the anti-neutrophil autoantibody, or a fragment thereof. In some embodiments, the first moiety comprising one or more peptides that specifically bind to an anti-neutrophil autoantibody is an anti-idiotypic antibody, or a fragment thereof.

[0135] In some embodiments, the first portion comprises a peptide that is about 5 amino acids to about 500 amino acids in length, hi some embodiments, the first portion comprises a peptide that is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 amino acids in length.

[0136] In some embodiments, the first portion comprises a peptide that is at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, at least 65 amino acids, at least 70 amino acids, at least 75 amino acids, at least 80 amino acids, at least 85 amino acids, at least 90 amino acids, at least 95 amino acids, at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids in length.

[0137] In some embodiments, the first portion comprises one or more peptides that are a total of about 50 to about 5000 amino acids in length, or about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 amino acids in length.

[0138] In some embodiments, the first portion comprises one, two, three, four, five, or more peptides that specifically bind to anti-neutrophil autoantibodies.

[0139] In some embodiments, one or more peptides of the first portion that specifically binds to an anti-neutrophil autoantibody are the same, e.g., one or more peptides have the same sequence. In some embodiments, one or more peptides with the same sequence are separated by one or more intervening sequences (e.g., spacers and / or linkers). In some embodiments, one or more peptides with the same sequence are not separated by one or more intervening sequences.

[0140] In some embodiments, one or more peptides of the first portion that specifically binds to an anti-neutrophil autoantibody are different, e.g., one or more peptides do not have the same sequence. In some embodiments, one or more peptides with different sequences are separated by one or more intervening sequences (e.g., spacers and / or linkers). In some embodiments, one or more peptides with different sequences are not separated by one or more intervening sequences (e.g., spacers and / or linkers).

[0141] In some embodiments, the one or more peptides with different sequences include one or more peptides that specifically bind to the same autoantibody. In some embodiments, the one or more peptides bind to different epitopes of the autoantibody (e.g., different domains of the autoantibody). In some embodiments, the one or more peptides with different sequences include one or more peptides that specifically bind to an anti-PR3 autoantibody or a fragment thereof. In some embodiments, the one or more peptides with different sequences include one or more peptides that specifically bind to an anti-MPO autoantibody or a fragment thereof.

[0142] In some embodiments, the one or more peptides with different sequences include one or more peptides that specifically bind to a first autoantibody (e.g., an anti-PR3 autoantibody or fragment thereof) and one or more peptides that bind to a second autoantibody (e.g., an anti-MPO autoantibody or fragment thereof). In some embodiments, the one or more peptides with different sequences are separated by one or more intervening sequences (e.g., spacers and / or linkers). In some embodiments, the one or more peptides with different sequences are not separated by one or more intervening sequences (e.g., spacers and / or linkers).

[0143] In some embodiments, the linker separating the one or more peptides of the first portion comprises a Gly-Ser linker or an EAAAK linker. In some embodiments, the linker comprises a (Gly-Gly-Gly-Gly-Ser) linker, where n is an integer between 0 and 20.

[0144] In some embodiments, the spacer separating the one or more peptides of the first portion comprises 1 to 10 amino acid residues, or about 10 to 20 amino acid residues.

[0145] In some embodiments, each of the one or more peptides that specifically bind to an anti-neutrophil autoantibody is conjugated to a second moiety.

[0146] In some embodiments, each of the one or more peptides that specifically bind to an anti-neutrophil autoantibody is not conjugated to a second moiety.

[0147] In some embodiments, one or more peptides that specifically bind to anti-neutrophil autoantibodies are conjugated to each other, eg, located on a single polypeptide.

[0148] In some embodiments, one or more peptides that specifically bind to anti-neutrophil autoantibodies are separated by a protease cleavage site or IRES. In some embodiments, each of the one or more peptides is expressed as a separate peptide, for example, translated as a separate peptide by an IRES, or expressed as a separate peptide after cleavage of the protease cleavage site.

[0149] In some embodiments, the one or more peptides that specifically bind to anti-neutrophil autoantibodies are not separated by a protease cleavage site or IRES, eg, are expressed as a fusion protein.

[0150] In some embodiments, the one or more peptides of the first portion that specifically bind to an anti-neutrophil autoantibody comprise an epitope recognized by the anti-neutrophil autoantibody. In some embodiments, the epitope is a linear epitope. In some embodiments, the epitope is a conformational epitope.

[0151] In some embodiments, the epitope is or comprises a single contiguous epitope, hi some embodiments, the epitope comprises one or more additional amino acid residues, for example, at the 5' and / or 3' ends of the epitope.

[0152] In some embodiments, an epitope comprises one or more sequences separated by one or more intervening amino acid sequences, such that the one or more sequences form a single epitope, e.g., spatially, when expressed and folded into a polypeptide conformation. In some embodiments, the intervening amino acid sequences comprise a linker and / or spacer. For example, an epitope comprising one or more sequences separated by one or more intervening amino acid sequences has the following structure: Xn-[A1]-Xn-[A2]-Xn, where A1 is a first portion of the epitope, A2 is a second portion of the epitope, which together form a spatial epitope recognized by anti-neutrophil autoantibodies, X represents an intervening amino acid sequence, and n is an integer between 0 and 20. In some embodiments, the intervening amino acid sequence is a spacer or linker, e.g., as described herein.

[0153] In some embodiments, an epitope formed by one or more sequences can be broken down into three, four, five, or more fragments. For example, in such embodiments, a polypeptide may have the following structure: Xn-[A1]-Xn-[A2]-Xn-...-[An]-Xn, where A1 is the first portion of the epitope, A2 is the second portion of the epitope, and An is the nth portion of the epitope, which together form a spatial epitope recognized by anti-neutrophil autoantibodies, and X represents an intervening amino acid sequence, where n is an integer between 0 and 20. In some embodiments, the intervening amino acid sequence is a spacer or linker, e.g., as described herein.

[0154] In some embodiments, the first portion comprises multiple epitopes, for example, the same or different epitopes. In some embodiments, the first portion comprises multiple identical epitopes, for example, epitopes recognized by the same anti-neutrophil antibody. In some embodiments, the first portion comprises multiple different epitopes, for example, epitopes recognized by different anti-neutrophil antibodies. In some embodiments, the multiple epitopes are separated by a linker, an IRES, or a cleavage peptide.

[0155] In some embodiments, the one or more peptides that specifically bind to an anti-neutrophil antibody comprise an antibody agent. In some embodiments, the antibody agent comprises an antigen-binding fragment. In some embodiments, the antibody agent comprises a full antibody, a Fab fragment, an scFv, a nanobody, a duobody, or a single domain antibody (e.g., a VHH). In some embodiments, the antibody agent comprises a VHH, e.g., a camelid-derived VHH or a bivalent VHH.

[0156] a first portion comprising proteinase 3 (PR3) and a PR3 peptide; PR3 is a serine protease enzyme primarily expressed in neutrophils and monocytes. It is typically present in the azurophil granules of human polymorphonuclear leukocytes. PR-3 has broad proteolytic activity and degrades various extracellular matrix proteins, including fibronectin, type IV collagen, and laminin (Brockmann H et al., Arthritis Research & Therapy (2002) volume 4, Article number: 220). Typically, PR3 transcription is halted before neutrophils egress from the bone marrow; however, increased and / or abnormal PR3 expression has been reported in ANCA vasculitis. Furthermore, ANCA vasculitis has been associated with elevated levels of PR3 on the surface of circulating neutrophils (Jennette CJ et al., Curr Opin Nephrol Hypertens (2011) 20(3):263-270).

[0157] The human PR3 polypeptide sequence is provided herein as SEQ ID NO: 1, with the bold sequence indicating the signal peptide and propeptide (AE, bold italics) (corresponding to Uniprot accession number: P24158):

[0158] [ka]

[0159] Human PR3 signal peptide: MAHRPPSPALASVLLALLLSGAARAAE (SEQ ID NO: 6).

[0160] Human PR3 can be encoded by the following nucleic acid sequence (SEQ ID NO:7) derived from the myeloblastin gene: [ka]

[0161] In some embodiments, the PR3 polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the PR3 polypeptide is or comprises SEQ ID NO: 1 without the signal peptide of SEQ ID NO:6.

[0162] In some embodiments, the PR3 polypeptide comprises a sequence having at least 85% identity to SEQ ID NO:1 without the signal peptide of SEQ ID NO:6.

[0163] In some embodiments, a PR3 polypeptide comprising a sequence having at least 85% identity to SEQ ID NO: 1 without the signal peptide of SEQ ID NO: 6 further comprises a different signal peptide, e.g., as disclosed herein.

[0164] In some embodiments, the glycomodified polypeptide comprises a first portion comprising one or more peptides comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 1. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 1 without the signal peptide of SEQ ID NO:6.

[0165] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO:1 without the signal peptide of SEQ ID NO:6.

[0166] In some embodiments, the first portion comprising one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO:1 without the signal peptide of SEQ ID NO:6 further comprises a different signal peptide, e.g., as disclosed herein.

[0167] An exemplary PR3 polypeptide sequence is provided herein as SEQ ID NO:40, with the bolded sequence indicating the truncated signal peptide:

[0168] [ka]

[0169] Modified PR3 truncated signal peptide: MAHRPPSPALASVLLALLLSGAARA (SEQ ID NO: 39)

[0170] In some embodiments, the PR3 polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40. In some embodiments, the PR3 polypeptide is or comprises SEQ ID NO: 40 without the signal peptide of SEQ ID NO: 39.

[0171] In some embodiments, a PR3 polypeptide comprising a sequence having at least 85% identity to SEQ ID NO: 40 without the signal peptide of SEQ ID NO: 39 further comprises a different signal peptide, e.g., as disclosed herein.

[0172] In some embodiments, the glycomodified polypeptide comprises a first portion comprising one or more peptides comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 40 without the signal peptide of SEQ ID NO: 39.

[0173] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO:40 without the signal peptide of SEQ ID NO:39.

[0174] In some embodiments, the first portion comprising one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 40 without the signal peptide of SEQ ID NO: 39 further comprises a different signal peptide, e.g., as disclosed herein.

[0175] An exemplary PR3 polypeptide sequence without the signal peptide is provided herein as SEQ ID NO:45.

[0176] IVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIHPSFVLTAAHCLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLSSPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTFFCRPHNICTFVPRRKAGICFGDSGGPLICDGIIQGIDSFVIWGCATRLFP DFFTRVALYVDWIRSTLRRVEAKGRP

[0177] In some embodiments, the PR3 polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45. In some embodiments, the PR3 polypeptide is or comprises SEQ ID NO:45.

[0178] In some embodiments, the PR3 polypeptide comprising a sequence having at least 85% identity to SEQ ID NO:45 further comprises a signal peptide, e.g., as disclosed herein, in some embodiments, the signal peptide is selected from SEQ ID NO:39, SEQ ID NO:6, SEQ ID NO:38, SEQ ID NO:35, or SEQ ID NO:36.

[0179] In some embodiments, the glycomodified polypeptide comprises a first portion comprising one or more peptides comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO:45.

[0180] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 45 and a signal peptide, eg, as disclosed herein.

[0181] In some embodiments, the neutrophil autoantigen is a PR3 polypeptide or a variant or fragment thereof. In some embodiments, the PR3 polypeptide variant or fragment exhibits reduced or eliminated protease activity compared to the full-length wild-type PR3 polypeptide.

[0182] In some embodiments, the neutrophil autoantigen is a PR3 polypeptide comprising a mutation at one or more of amino acids 71 ​​(His), 118 (Asp), and 203 (Ser) of SEQ ID NO:1.

[0183] In some embodiments, the neutrophil autoantigen is a PR3 polypeptide having a mutation at one or more of amino acids 180 (Phe), 181 (Phe), 228 (Leu), or 229 (Phe) of SEQ ID NO:1.

[0184] In some embodiments, the anti-neutrophil autoantibody is an anti-PR3 autoantibody, or a fragment thereof. In some embodiments, the anti-PR3 autoantibody is characterized by binding to a PR3 polypeptide or a variant or fragment thereof.

[0185] In some embodiments, the first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides that specifically bind to an anti-PR3 autoantibody. In some embodiments, the one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a PR3 polypeptide, or a fragment or variant thereof.

[0186] In some embodiments, the one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a soluble fragment of a PR3 polypeptide.

[0187] In some embodiments, the one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, hi some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length PR3 polypeptide of SEQ ID NO: 1.

[0188] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, in some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length PR3 polypeptide of SEQ ID NO: 1 without the signal peptide.

[0189] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, hi some embodiments, the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length PR3 polypeptide of SEQ ID NO: 1.

[0190] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, in which the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length PR3 polypeptide of SEQ ID NO: 1 without the signal peptide.

[0191] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, in some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length PR3 polypeptide of SEQ ID NO: 40.

[0192] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, in some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length PR3 polypeptide of SEQ ID NO: 40 without the signal peptide.

[0193] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, in some embodiments, the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length PR3 polypeptide of SEQ ID NO: 40.

[0194] In some embodiments, one or more polypeptides that specifically bind to anti-PR3 autoantibodies comprise a fragment of a PR3 polypeptide, in which the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length PR3 polypeptide of SEQ ID NO: 40 without the signal peptide.

[0195] In some embodiments, the one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, in some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length PR3 polypeptide of SEQ ID NO: 45.

[0196] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a fragment of a PR3 polypeptide, in some embodiments, the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length PR3 polypeptide of SEQ ID NO: 45.

[0197] In some embodiments, the fragment comprises an epitope recognized by a PR3 autoantibody. In some embodiments, the epitope is a linear epitope. In some embodiments, the epitope is a conformational epitope. In some embodiments, the epitope is or comprises a single contiguous epitope. In some embodiments, the epitope comprises one or more additional amino acid residues, for example, at the 5' and / or 3' end of the epitope.

[0198] In some embodiments, an epitope comprises one or more sequences separated by one or more intervening amino acid sequences, such that the one or more sequences form a single epitope, e.g., spatially, when expressed and folded into a polypeptide conformation. In some embodiments, the intervening amino acid sequences comprise a linker and / or spacer. For example, an epitope comprising one or more sequences separated by one or more intervening amino acid sequences has the following structure: Xn-[A1]-Xn-[A2]-Xn, where A1 is a first portion of the epitope and A2 is a second portion of the epitope, which together form a spatial epitope recognized by anti-neutrophil autoantibodies, X represents an intervening amino acid sequence, and n is an integer between 0 and 20.

[0199] In some embodiments, an epitope formed by one or more sequences can be broken down into three, four, five, or more fragments. For example, in such embodiments, a polypeptide may have the following structure: Xn-[A1]-Xn-[A2]-Xn-[An]-Xn, where A1 is the first portion of the epitope, A2 is the second portion of the epitope, and An is the nth portion of the epitope, which together form a spatial epitope recognized by anti-neutrophil autoantibodies, X represents an intervening amino acid sequence, and n is an integer from 0 to 20.

[0200] In some embodiments, the first portion comprises multiple epitopes, for example, the same or different epitopes. In some embodiments, the first portion comprises multiple identical epitopes, for example, epitopes recognized by anti-PR3 autoantibodies. In some embodiments, the first portion comprises multiple different epitopes, for example, epitopes recognized by different anti-PR3 autoantibodies. In some embodiments, the multiple epitopes are separated by a linker, an IRES, or a cleavage peptide.

[0201] PR3 epitopes that can be recognized by anti-PR3 autoantibodies are disclosed in Van Der Geld YM et al., (2004) Clin Exp Immunol, Vol 137, pp. 451-459, the entire contents of which are incorporated herein by reference. In some embodiments, one or more epitopes of the first portion disclosed herein comprise the epitopes disclosed in Van Der Geld et al., 2004.

[0202] In some embodiments, the epitope recognized by PR3 autoantibodies comprises IVGGHEAQPHSRPYMASLQMR (SEQ ID NO: 2). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0203] In some embodiments, the epitope recognized by PR3 autoantibodies comprises IVGGHEAQPHSRPYMASLQM (SEQ ID NO: 3). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0204] In some embodiments, the epitope recognized by the PR3 autoantibody comprises GHEAQPHSRPY (SEQ ID NO: 11). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0205] In some embodiments, the epitope recognized by a PR3 autoantibody comprises TQEPTQQ (SEQ ID NO: 12). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0206] In some embodiments, the epitope recognized by a PR3 autoantibody comprises ATVQLPQQDQPVPHGTQ (SEQ ID NO: 13). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0207] In some embodiments, the epitope recognized by PR3 autoantibodies comprises RVGAHDPP (SEQ ID NO: 14). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0208] In some embodiments, the epitope recognized by PR3 autoantibodies comprises FCRPHNI (SEQ ID NO: 15). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0209] In some embodiments, the epitope recognized by a PR3 autoantibody comprises PRRKAGICFGDSGGP (SEQ ID NO: 16). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0210] In some embodiments, the epitope recognized by PR3 autoantibodies comprises IDSFVIW (SEQ ID NO: 17). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0211] In some embodiments, the epitope recognized by PR3 autoantibodies comprises PYMASL (SEQ ID NO: 18). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0212] In some embodiments, the epitope recognized by the PR3 autoantibody comprises AHCLRDIPQRLVNV (SEQ ID NO: 19). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0213] In some embodiments, the epitope recognized by a PR3 autoantibody comprises HGTQCLAMGWGRVGAH (SEQ ID NO: 20). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0214] In some embodiments, the epitope recognized by a PR3 autoantibody comprises NICTFVPRRKAGIC (SEQ ID NO: 21). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0215] In some embodiments, the epitope recognized by PR3 autoantibodies comprises CATRLFPDFFTRVAL (SEQ ID NO: 22). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0216] In some embodiments, the epitope recognized by PR3 autoantibodies comprises WIRSTRLLVEAKGRP (SEQ ID NO: 23). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0217] In some embodiments, the one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a variant of a PR3 polypeptide, hi some embodiments, the variant is an inactive variant compared to a wild-type PR3 polypeptide.

[0218] In some embodiments, the variant comprises a mutation at the valine residue at position 119, the alanine residue at position 135, the threonine residue at position 136, or a combination thereof.

[0219] In some embodiments, the variant comprises a mutation at one or more or all of amino acids 71 ​​(His), 118 (Asp), and 203 (Ser) of SEQ ID NO:1.

[0220] In some embodiments, the variant comprises a mutation at amino acid 71 (His) of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at amino acid 71 (His) further comprises one or more additional mutations relative to SEQ ID NO: 1, e.g., as described herein.

[0221] In some embodiments, the variant comprises a mutation at amino acid 118 (Asp) of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at amino acid 118 (Asp) further comprises one or more additional mutations relative to SEQ ID NO: 1, e.g., as described herein.

[0222] In some embodiments, the variant comprises a mutation at amino acid 203 (Ser) of SEQ ID NO: 1. In some embodiments, the variant comprises a serine to alanine mutation at position 203 of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at amino acid 203 (Ser) further comprises one or more additional mutations relative to SEQ ID NO: 1, e.g., as described herein.

[0223] In some embodiments, the variant comprises mutations at amino acids 71 ​​(His), 118 (Asp), and 203 (Ser) of SEQ ID NO: 1. In some embodiments, the variant comprising mutations at amino acids 71 ​​(His), 118 (Asp), and 203 (Ser) of SEQ ID NO: 1 further comprises one or more additional mutations relative to SEQ ID NO: 1, for example, as described herein.

[0224] In some embodiments, the variant comprises a mutation at one or more or all of amino acids 180 (Phe), 181 (Phe), 228 (Leu), or 229 (Phe) of SEQ ID NO:1.

[0225] In some embodiments, the variant comprises a mutation at amino acid 180 (Phe) of SEQ ID NO: 1. In some embodiments, the variant comprises a phenylalanine to alanine mutation at position 180 of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at amino acid 180 (Phe) further comprises one or more additional mutations relative to SEQ ID NO: 1, e.g., as described herein.

[0226] In some embodiments, the variant comprises a mutation at amino acid 181 (Phe) of SEQ ID NO: 1. In some embodiments, the variant comprises a phenylalanine to alanine mutation at position 181 of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at amino acid 181 (Phe) further comprises one or more additional mutations relative to SEQ ID NO: 1, e.g., as described herein.

[0227] In some embodiments, the variant comprises a mutation at amino acid 228 (Leu) of SEQ ID NO: 1. In some embodiments, the variant comprises a leucine to alanine mutation at position 228 of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at amino acid 228 (Leu) further comprises one or more additional mutations relative to SEQ ID NO: 1, e.g., as described herein.

[0228] In some embodiments, the variant comprises a mutation at amino acid 229 (Phe) of SEQ ID NO: 1. In some embodiments, the variant comprises a phenylalanine to alanine mutation at position 229 of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at amino acid 229 (Phe) further comprises one or more additional mutations relative to SEQ ID NO: 1, e.g., as described herein.

[0229] In some embodiments, the variant comprises a mutation at amino acid 180(Phe), 181(Phe), 228(Leu), or 229(Phe) of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at amino acid 180(Phe), 181(Phe), 228(Leu), or 229(Phe), e.g., to alanine, further comprises one or more additional mutations relative to SEQ ID NO: 1, e.g., as described herein.

[0230] In some embodiments, the variant comprises a PR3 polypeptide or fragment thereof having one or more mutations at a glycosylation site. In some embodiments, the mutation is at an asparagine residue such that the glycosylation site is altered. In some embodiments, the mutation comprises a mutation at asparagine 129 and / or asparagine 174.

[0231] In some embodiments, the mutation is a valine to isoleucine mutation, in some embodiments, the mutation is an alanine to threonine mutation, in some embodiments, the mutation is a serine to threonine mutation.

[0232] In some embodiments, the glycoengineered polypeptide comprises a PR3 polypeptide or fragment thereof having one or more native glycosylation sites.

[0233] In some embodiments, the glycomodified polypeptide comprises a PR3 polypeptide or fragment thereof having two native glycosylation sites, hi some embodiments, the native glycosylation sites include N129 or N174.

[0234] In some embodiments, the glycomodified polypeptide comprises a PR3 polypeptide or fragment thereof having one or more engineered glycosylation sites, hi some embodiments, the engineered glycosylation site is or comprises GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0235] In some embodiments, the glycomodified polypeptide comprises a PR3 polypeptide or fragment thereof having two native glycosylation sites: N129 and N174, and one engineered glycosylation site having the sequence GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0236] In some embodiments, the glycomodified polypeptide further comprises one or more additional elements, in some embodiments, the one or more additional elements comprise: (a) a linker, (b) a spacer, (c) a cleavage peptide, e.g., an IRES or a protease cleavage site, (d) a signal peptide, (e) a tag, e.g., a cleavable tag, (f) a half-life extending domain, e.g., an Fc domain or albumin, or (g) any combination of (a)-(f).

[0237] In some embodiments, the glycoengineered polypeptide comprises a tag, eg, a His tag.

[0238] In some embodiments, the glycoengineered polypeptide comprises the following sequence (SEQ ID NO: 41):

[0239] MAHRPPSPALSVLLALLLSGAARAIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIHPSFVLTAAHCLRDIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLSSPANLSASVATVQLPQ QDQPVPHGTQCLAMGWGRVGAHDPPAQVLQELNVTVVTAACRPHNICTFVPRRKAGICFGDAGGPLICDGIIQGIDSFVIWGCATRAAPDFFTRVALYVDWIRSTLRRVEAKGRPGGGGANSTAPAPAPAHHHHHHHHHH

[0240] In some embodiments, the glycomodified polypeptide comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 41. In some embodiments, the glycomodified polypeptide comprises the sequence of SEQ ID NO: 41.

[0241] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a contiguous chain of amino acids comprising at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the amino acids of SEQ ID NO:1.

[0242] In some embodiments, one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a contiguous chain of amino acids comprising at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the amino acids of SEQ ID NO:41.

[0243] In some embodiments, one or more polypeptides that specifically bind to anti-PR3 autoantibodies comprise a contiguous stretch of amino acids comprising at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the amino acids of SEQ ID NO:45.

[0244] In some embodiments, the one or more polypeptides that specifically bind to anti-PR3 autoantibodies comprise the full-length PR3 protein set forth in SEQ ID NO: 1, for example, with or without the signal peptide.

[0245] In some embodiments, the one or more polypeptides that specifically bind to anti-PR3 autoantibodies include the PR3 protein set forth, for example, in SEQ ID NO:45.

[0246] In some embodiments, the first portion comprises one, two, three, four, five, or more peptides that specifically bind to an anti-PR3 autoantibody.

[0247] In some embodiments, one or more peptides of the first moiety that specifically binds to an anti-PR3 autoantibody are the same, e.g., one or more peptides have the same sequence of a PR3 polypeptide, or a fragment or variant thereof. In some embodiments, one or more peptides having the same sequence of a PR3 polypeptide, or a fragment or variant thereof, are separated by one or more intervening sequences (e.g., spacers and / or linkers). In some embodiments, one or more peptides having the same sequence of a PR3 polypeptide, or a fragment or variant thereof, are not separated by one or more intervening sequences (e.g., spacers and / or linkers).

[0248] In some embodiments, one or more peptides of the first portion that specifically binds to an anti-PR3 autoantibody are different, e.g., one or more peptides do not have the same sequence of a PR3 polypeptide, or a fragment or variant thereof. In some embodiments, one or more peptides having different sequences of a PR3 polypeptide, or a fragment or variant thereof, are separated by one or more intervening sequences (e.g., spacers and / or linkers). In some embodiments, one or more peptides having different sequences of a PR3 polypeptide, or a fragment or variant thereof, are not separated by one or more intervening sequences (e.g., spacers and / or linkers).

[0249] In some embodiments, each of the one or more peptides having a different sequence of a PR3 polypeptide, or a fragment or variant thereof, specifically binds to an anti-PR3 autoantibody or fragment thereof.

[0250] In some embodiments, the linker separating the one or more peptides of the first portion comprises a Gly-Ser linker or an EAAAK linker. In some embodiments, the linker comprises a (Gly-Gly-Gly-Gly-Ser) linker, where n is an integer between 0 and 20.

[0251] In some embodiments, each of the one or more peptides that specifically bind to an anti-PR3 autoantibody is conjugated to a second moiety.

[0252] In some embodiments, each of the one or more peptides that specifically bind to an anti-PR3 autoantibody is not conjugated to a second moiety.

[0253] In some embodiments, one or more peptides that specifically bind to anti-PR3 autoantibodies are conjugated to each other, eg, located on a single polypeptide.

[0254] In some embodiments, one or more peptides that specifically bind to an anti-PR3 autoantibody are separated by a protease cleavage site or IRES. In some embodiments, each of the one or more peptides is expressed as a separate peptide, for example, translated as a separate peptide by an IRES, or expressed as a separate peptide after cleavage of the protease cleavage site.

[0255] In some embodiments, the one or more peptides that specifically bind to an anti-PR3 autoantibody are not separated by a protease cleavage site or IRES, eg, are expressed as a fusion protein.

[0256] In some embodiments, the first portion comprises one or more peptides that specifically bind to an anti-PR3 antibody. In some embodiments, the one or more peptides that specifically bind to an anti-PR3 antibody comprise a PR3 polypeptide, or a variant or fragment thereof. In some embodiments, the first portion further comprises (a) a linker, (b) a spacer, (c) a cleavage peptide, e.g., an IRES or a protease cleavage site, (d) a signal peptide, (e) a tag, e.g., a cleavable tag, (f) a half-life extending domain, e.g., an Fc domain or albumin, or (g) any combination of (a)-(f).

[0257] In some embodiments, the one or more peptides that specifically bind to an anti-MPO antibody comprise an antibody agent. In some embodiments, the antibody agent comprises an antigen-binding fragment. In some embodiments, the antibody agent comprises a full antibody, a Fab fragment, an scFv, a nanobody, a duobody, or a single domain antibody (e.g., a VHH). In some embodiments, the antibody agent comprises a VHH, e.g., a camelid-derived VHH or a bivalent VHH.

[0258] A first portion comprising myeloperoxidase (MPO) and MPO peptides MPO is a heme-containing peroxidase expressed primarily in neutrophils and monocytes. MPO catalyzes the formation of reactive oxygen intermediates in the presence of hydrogen peroxide and halides (Aratani Y., Arch Biochem Biophys. 2018 Feb 15;640:47-52.). Typically, MPO transcription is halted before neutrophils egress from the bone marrow; however, increased and / or abnormal MPO expression has been reported in ANCA vasculitis. Furthermore, ANCA vasculitis has been shown to be associated with elevated levels of MPO on the surface of circulating neutrophils (Jennette CJ et al., Curr Opin Nephrol Hypertens (2011) 20(3):263-270).

[0259] The human MPO polypeptide sequence is provided herein as SEQ ID NO: 4, with the bold sequence indicating the signal peptide (corresponding to Uniprot accession number: P05164):

[0260] [ka]

[0261] Human MPO signal peptide: MGVPFFSSLRCMVDLGPCWAGGLTAEMKLLLALAGLLAILATPQPSEG (SEQ ID NO: 8)

[0262] Human MPO can be encoded by the following nucleic acid sequence (SEQ ID NO: 9) from the myeloperoxidase gene: [ka]

[0263] In some embodiments, the MPO polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the MPO polypeptide is or comprises SEQ ID NO: 4 without the signal peptide of SEQ ID NO: 8.

[0264] In some embodiments, an MPO polypeptide comprising a sequence having at least 85% identity to SEQ ID NO: 4 without the signal peptide of SEQ ID NO: 8 further comprises a different signal peptide, e.g., as disclosed herein.

[0265] In some embodiments, the glycomodified polypeptide comprises a first portion comprising one or more peptides comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 4. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 4 without the signal peptide of SEQ ID NO: 8.

[0266] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO:4 without the signal peptide of SEQ ID NO:8.

[0267] In some embodiments, the first portion comprising one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 4 without the signal peptide of SEQ ID NO: 8 further comprises a different signal peptide, e.g., as disclosed herein.

[0268] An exemplary modified MPO polypeptide sequence is provided herein as SEQ ID NO:42, with the bolded sequence indicating the signal peptide:

[0269] [ka]

[0270] Leishmania signal peptide: MASRLVRVLAAAMLVAAAVS (SEQ ID NO: 38)

[0271] In some embodiments, the MPO polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42. In some embodiments, the MPO polypeptide is or comprises SEQ ID NO: 42 without the signal peptide of SEQ ID NO:38.

[0272] In some embodiments, the MPO polypeptide comprises a sequence having at least 85% identity to SEQ ID NO:42 without the signal peptide of SEQ ID NO:38.

[0273] In some embodiments, an MPO polypeptide comprising a sequence having at least 85% identity to SEQ ID NO: 42 without the signal peptide of SEQ ID NO: 38 further comprises a different signal peptide, e.g., as disclosed herein.

[0274] In some embodiments, the glycomodified polypeptide comprises a first portion comprising one or more peptides comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 42. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 42 without the signal peptide of SEQ ID NO: 38.

[0275] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO:42 without the signal peptide of SEQ ID NO:38.

[0276] In some embodiments, the first portion comprising one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 42 without the signal peptide of SEQ ID NO: 38 further comprises a different signal peptide, e.g., as disclosed herein.

[0277] An exemplary MPO polypeptide without the signal peptide is provided herein as SEQ ID NO:46:

[0278] AAPAVLGEVDTSLVLSSMEEAKQLVDKAYKERRESIKQRLRSGSASPMELLSYFKQPVAATRTAVRAADYLHVALDLLERKLRSLWRRPFNVTDVLTPAQLNVLSKSSGCAYQDVGVTCPEQDKYRTITGMCNNRRSPTLGASNAFVRWLPAEYEDGFSLPYGWTPGVKRNGF PVALARAVSNEIVRFPTDQLTPDQERSLMFMQWGQLLDADLDFTPEPAARASFVTGVNCETSCVQQPPCFPLKIPPNDPRIKNQADCIPFFRSAPACPGSNITIRNQINALTSFVDASMVYGSEEPLARNLRNMSNQLGLLAVNQRFQDNGRALLPFDNLHDDPCLLTNRSARI PCFLAGDTRSSEMPELTSMHTLLLREHNRLATELKSLNPRWDGERLYQEARKIVGAMVQIITYRDYLPLVLGPTAMRKYLPTYRSYNDSVDPRIANVFTNAFRYGHTLIQPFMFRLDNRYQPMEPNPRVPLSRVFFASWRVVLEGGIDPILRGLMATPAKLNRQNQIAVDEIRE RLFEQVMRIGLDLPALNMQRSRDHGLPGYNAWRRFCGLPQPETVGQLGTVLRNLKLARKLMEQYGTPNNIDIWMGGVSEPLKRKGRVGPLLACIIGTQFRKLRDGDRFWWENEGVFSMQQRQALAQISLPRIICDNTGITTVSKNNIFMSNSYPRDFVNCSTLPALNLASWREAS

[0279] In some embodiments, the MPO polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46. In some embodiments, the MPO polypeptide is or comprises SEQ ID NO:46.

[0280] In some embodiments, the MPO polypeptide comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 46 further comprises a signal peptide disclosed herein. In some embodiments, the signal peptide is a Leishmania-derived signal peptide.

[0281] In some embodiments, the signal peptide is selected from SEQ ID NO:8, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:38.

[0282] In some embodiments, the glycomodified polypeptide comprises a first portion comprising one or more peptides comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO:46.

[0283] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 46 and a signal peptide, eg, as disclosed herein.

[0284] In some embodiments, the neutrophil autoantigen is an MPO polypeptide or a variant or fragment thereof. In some embodiments, the MPO polypeptide variant or fragment exhibits reduced or eliminated protease activity compared to full-length wild-type MPO polypeptide.

[0285] In some embodiments, the neutrophil autoantigen is an MPO polypeptide comprising a mutation at one or more of amino acids 261 (His), 316 (Cys), 405 (Arg), and 257 (Gln) of SEQ ID NO:4.

[0286] In some embodiments, the anti-neutrophil autoantibody is an MPO autoantibody, or a fragment thereof. In some embodiments, the anti-MPO autoantibody is characterized by binding to an MPO polypeptide or a variant or fragment thereof.

[0287] In some embodiments, the first portion of a glycomodified polypeptide disclosed herein comprises one or more peptides that specifically bind to an anti-MPO autoantibody, hi some embodiments, the one or more polypeptides that specifically bind to an anti-MPO autoantibody comprise an MPO polypeptide, or a fragment or variant thereof.

[0288] In some embodiments, the one or more polypeptides that specifically bind to an anti-MPO autoantibody comprise a soluble fragment of an MPO polypeptide.

[0289] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide. In some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length MPO polypeptide of SEQ ID NO: 4.

[0290] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide, hi some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length MPO polypeptide of SEQ ID NO: 4 without the signal peptide.

[0291] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide, hi some embodiments, the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length MPO polypeptide of SEQ ID NO: 4.

[0292] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide, hi some embodiments, the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length MPO polypeptide of SEQ ID NO: 4 without the signal peptide.

[0293] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide. In some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length MPO polypeptide of SEQ ID NO: 42.

[0294] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide, hi some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length MPO polypeptide of SEQ ID NO: 42 without the signal peptide.

[0295] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide, hi some embodiments, the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length MPO polypeptide of SEQ ID NO: 42.

[0296] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide, hi some embodiments, the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length MPO polypeptide of SEQ ID NO: 42 without the signal peptide.

[0297] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide, hi some embodiments, the fragment comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the full-length MPO polypeptide of SEQ ID NO: 46.

[0298] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide, hi some embodiments, the fragment comprises 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less of the full-length MPO polypeptide of SEQ ID NO: 46.

[0299] In some embodiments, the fragment comprises an epitope recognized by an MPO autoantibody. In some embodiments, the epitope is a linear epitope. In some embodiments, the epitope is a conformational epitope. In some embodiments, the epitope is or comprises a single contiguous epitope. In some embodiments, the epitope comprises one or more additional amino acid residues, for example, at the 5' and / or 3' end of the epitope.

[0300] In some embodiments, an epitope comprises one or more sequences separated by one or more intervening amino acid sequences, such that the one or more sequences form a single epitope, e.g., spatially, when expressed and folded into a polypeptide conformation. In some embodiments, the intervening amino acid sequences comprise a linker and / or spacer. For example, an epitope comprising one or more sequences separated by one or more intervening amino acid sequences has the following structure: Xn-[A1]-Xn-[A2]-Xn, where A1 is a first portion of the epitope and A2 is a second portion of the epitope, which together form a spatial epitope recognized by anti-neutrophil autoantibodies, X represents an intervening amino acid sequence, and n is an integer between 0 and 20.

[0301] In some embodiments, an epitope formed by one or more sequences can be broken down into three, four, five, or more fragments. For example, in such embodiments, a polypeptide may comprise the following structure: Xn-[A1]-Xn-[A2]-Xn-....-[An]-Xn, where A1 is the first portion of the epitope, A2 is the second portion of the epitope, and An is the nth portion of the epitope, which together form a spatial epitope recognized by anti-neutrophil autoantibodies, X represents an intervening amino acid sequence, and n is an integer from 0 to 20.

[0302] In some embodiments, the first portion comprises multiple epitopes, e.g., the same or different epitopes. In some embodiments, the first portion comprises multiple identical epitopes, e.g., epitopes recognized by anti-MPO autoantibodies. In some embodiments, the first portion comprises multiple different epitopes, e.g., epitopes recognized by different anti-MPO autoantibodies. In some embodiments, the multiple epitopes are separated by a linker, an IRES, or a cleavage peptide.

[0303] MPO epitopes that can be recognized by anti-MPO autoantibodies are disclosed in Bruner BF et al., (2011) Clin Exp Immunol., volume 164, pp. 330-336, the entire contents of which are incorporated herein by reference. In some embodiments, one or more epitopes of the first portion disclosed herein include an epitope disclosed in Bruner et al., 2011.

[0304] MPO epitopes that can be recognized by anti-MPO autoantibodies are disclosed in Van der Geld et al. (2004) Clin Exp Immunol 137:451-459, the entire contents of which are incorporated herein by reference. In some embodiments, one or more epitopes of the first portion disclosed herein include an epitope disclosed in Van Der Geld et al., 2004.

[0305] MPO epitopes that can be recognized by anti-MPO autoantibodies are disclosed in Free et al. (2020) J. Immunol. 106:102306; and 447-461, the entire contents of which are incorporated herein by reference. In some embodiments, one or more epitopes of the first portion disclosed herein include an epitope disclosed in Free et al., 2020.

[0306] In some embodiments, the epitope recognized by an MPO autoantibody comprises PRWDGERLYQEARKIVGAMV (SEQ ID NO: 5). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0307] In some embodiments, the epitope recognized by MPO autoantibodies comprises RLYQEARKIVG (SEQ ID NO: 10). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0308] In some embodiments, the epitope recognized by MPO autoantibodies comprises VNCETSCVQQPPCFPLKIPPNDPRIKNQADCIPFFRSCPACP GSNITIRNQI NALTSFVDASMVYGSEEPLARNLRNMSNQLGLLAVNQRFQDNGRALLPFD NLHDDPCLLTNRSARIPCFLAGDTRSSEMPELTSMHTLLLREHNRLATEL KSLNPRWDGERLYQEARKIVGAMVQIITYRDYLPLVLGPTAMRK (SEQ ID NO: 24). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0309] In some embodiments, the epitope recognized by MPO autoantibodies comprises GYNAWRRFCGLPQPETVGQLGTVLRNLKLARKLMEQYGTPNNIDIWM GGVSEPLKRKGRVGPLLACIIGTQFRKLRDGDRFWWENEGVFSMQQRQALAQIS LPRIICDNTGITTVSKNNIFMSNSYPRDFVNCSTLPALNLASWREAS (SEQ ID NO: 25). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' of any one or more of the epitope sequences. In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' of the epitope sequence.

[0310] In some embodiments, the epitope recognized by MPO autoantibodies comprises RKIVGAMVQIITY (SEQ ID NO: 26). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0311] In some embodiments, the epitope recognized by MPO autoantibodies comprises RKIVGAMVQIITYRD (SEQ ID NO: 27). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0312] In some embodiments, the epitope recognized by MPO autoantibodies comprises WTPGVKRNGF (amino acids 213-222) (SEQ ID NO: 28). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0313] In some embodiments, the epitope recognized by MPO autoantibodies comprises RLDNRYQPMEPN (AA 511-522) (SEQ ID NO: 29). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0314] In some embodiments, the epitope recognized by MPO autoantibodies comprises SARIPCFLAG (aa 393-402) (SEQ ID NO: 30). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence. In some embodiments, the epitope recognized by MPO autoantibodies comprises WDGERLYQEA (aa 437-446) (SEQ ID NO: 31). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0315] In some embodiments, the epitope recognized by MPO autoantibodies comprises YRSYNDSVDP (aa 479-488) (SEQ ID NO: 32). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0316] In some embodiments, the epitope recognized by MPO autoantibodies comprises FMSNSYPRD (aa 717-726) (SEQ ID NO: 33). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0317] In some embodiments, the epitope recognized by MPO autoantibodies comprises GSASPMELLS (aa 91-100) (SEQ ID NO: 34). In some embodiments, the epitope comprises one or more additional sequences 5' and / or 3' to the epitope sequence.

[0318] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a variant of an MPO polypeptide, hi some embodiments, the variant is an inactive variant compared to a wild-type MPO polypeptide.

[0319] In some embodiments, the variant comprises a mutation at one or more or all of amino acids 261 (His), 316 (Cys), 405 (Arg), and 257 (Gln) of SEQ ID NO:4.

[0320] In some embodiments, the variant comprises a mutation at 261(His) of SEQ ID NO: 4. In some embodiments, the variant comprises a histidine to alanine mutation at position 261 of SEQ ID NO: 4. In some embodiments, the variant comprising a mutation at 261(His) of SEQ ID NO: 4 further comprises one or more additional mutations in SEQ ID NO: 4, e.g., as described herein.

[0321] In some embodiments, the variant comprises a mutation at 405(Arg) of SEQ ID NO: 4. In some embodiments, the variant comprising a mutation at 405(Arg) of SEQ ID NO: 4 further comprises one or more additional mutations in SEQ ID NO: 4, e.g., as described herein.

[0322] In some embodiments, the variant comprises a mutation at 257(Gln) of SEQ ID NO: 4. In some embodiments, the variant comprising a mutation at 257(Gln) of SEQ ID NO: 4 further comprises one or more additional mutations in SEQ ID NO: 4, e.g., as described herein.

[0323] In some embodiments, the variant comprises a mutation at 316 (Cys) of SEQ ID NO: 4. In some embodiments, the variant comprises a cysteine ​​to alanine mutation at position 316 of SEQ ID NO: 1. In some embodiments, the variant comprising a mutation at 316 (Cys) of SEQ ID NO: 4 further comprises one or more additional mutations in SEQ ID NO: 4, e.g., as described herein.

[0324] In some embodiments, the variant comprises mutations at 261 (His), 405 (Arg), 316 (Cys), and 257 (Gln) of SEQ ID NO: 4. In some embodiments, the variant comprising mutations at 261 (His), 405 (Arg), 316 (Cys), and 257 (Gln) of SEQ ID NO: 4 further comprises one or more additional mutations in SEQ ID NO: 4, e.g., as described herein.

[0325] In some embodiments, the variant comprises an MPO polypeptide or fragment thereof having one or more mutations at a glycosylation site. In some embodiments, the mutation is at an asparagine residue such that the glycosylation site is altered. In some embodiments, the mutation comprises a mutation at asparagine 323, asparagine 355, asparagine 391, asparagine 483, asparagine 729, or a combination thereof.

[0326] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having one or more native glycosylation sites. In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having 1, 2, 3, 4, 5, or 6 native glycosylation sites. In some embodiments, the one or more native glycosylation sites comprise N139, N323, N355, N391, N483, and / or N729.

[0327] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having the native glycosylation site N139.

[0328] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having the native glycosylation site N323.

[0329] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having native glycosylation site N355.

[0330] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having the native glycosylation site N391.

[0331] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having the native glycosylation site N483.

[0332] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having the native glycosylation site N729.

[0333] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having an engineered glycosylation site comprising the sequence GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0334] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having one or more native glycosylation sites and one engineered glycosylation site, e.g., having the sequence GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0335] In some embodiments, the glycomodified polypeptide comprises an MPO polypeptide or fragment thereof having six native glycosylation sites: N139, N323, N355, N391, N483, and N729, and one engineered glycosylation site having the sequence GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0336] In some embodiments, the glycomodified polypeptide further comprises one or more additional elements, in some embodiments, the one or more additional elements comprise: (a) a linker, (b) a spacer, (c) a cleavage peptide, e.g., an IRES or a protease cleavage site, (d) a signal peptide, (e) a tag, e.g., a cleavable tag, (f) a half-life extending domain, e.g., an Fc domain or albumin, or (g) any combination of (a)-(f).

[0337] In some embodiments, the glycoengineered polypeptide comprises a tag, eg, a His tag.

[0338] In some embodiments, the glycoengineered polypeptide comprises the following sequence (SEQ ID NO: 43):

[0339] MASRLVRVLAAAMLVAAAVSAAPAVLGEVDTSLVLSSMEEAKQLVDKAYKERRESIKQRLRSGSASPMELLSYFKQPVAATRTAVRAADYLHVALDLLERKLRSLWRRPFNVTDVLTPAQLNVLSKSSGCAYQDVGVTCPEQDKYRTITGMCNNRRSPTLGASNRAFVRWLPAEYEDGFSL PYGWTPGVKRNGFPVALARAVSNEIVRFPTDQLTPDQERSLMFMQWGQLLDADLDFTPEPAARASFVTGVNCETSCVQQPPCFPLKIPPNDPRIKNQADCIPFFRSAPACPGSNITIRNQINALTSFVDASMVYGSEEPLARNLRNMSNQLGLLAVNQRFQDNGRALLPFDNLHDDPCLLTN RSARIPCFLAGDTRSSEMPELTSMHTLLLREHNRLATELKSLNPRWDGERLYQEARKIVGAMVQIITYRDYLPLVLGPTAMRKYLPTYRSYNDSVDPRIANVFTNAFRYGHTLIQPFMFRLDNRYQPMEPNPRVPLSRVFFASWRVVLEGGIDPILRGLMATPAKLNRQNQIAVDEIRERLF EQVMRIGLDLPALNMQRSRDHGLPGYNAWRRFCGLPQPETVGQLGTVLRNLKLARKLMEQYGTPNNIDIWMGGVSEPLKRKGRVGPLLACIIGTQFRKLRDGDRFWWENEGVFSMQQRQALAQISLPRIICDNTGITTVSKNNIFMSNSYPRDFVNCSTLPALNLASWREASHHHHHHHHHHH

[0340] In some embodiments, the glycomodified polypeptide comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 43. In some embodiments, the glycomodified polypeptide comprises the sequence of SEQ ID NO: 43.

[0341] In some embodiments, one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a contiguous stretch of amino acids that comprises at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the amino acids of SEQ ID NO:4.

[0342] In some embodiments, one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a contiguous stretch of amino acids comprising at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the amino acids of SEQ ID NO:43.

[0343] In some embodiments, one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a contiguous stretch of amino acids comprising at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the amino acids of SEQ ID NO:42.

[0344] In some embodiments, one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise a contiguous stretch of amino acids comprising at least 5%, at least 10%, 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the amino acids of SEQ ID NO:46.

[0345] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies comprise the full-length MPO protein set forth in SEQ ID NO:4, for example, with or without the signal peptide.

[0346] In some embodiments, the one or more polypeptides that specifically bind to anti-MPO autoantibodies include the MPO protein set forth in, for example, SEQ ID NO:46.

[0347] In some embodiments, the first portion comprises one, two, three, four, five, or more peptides that specifically bind to anti-MPO autoantibodies.

[0348] In some embodiments, the one or more peptides of the first moiety that specifically binds to an anti-MPO autoantibody are the same, e.g., the one or more peptides have the same sequence of an MPO polypeptide, or a fragment or variant thereof. In some embodiments, the one or more peptides having the same sequence of an MPO polypeptide, or a fragment or variant thereof, are separated by one or more intervening sequences (e.g., spacers and / or linkers). In some embodiments, the one or more peptides having the same sequence of an MPO polypeptide, or a fragment or variant thereof, are not separated by one or more intervening sequences (e.g., spacers and / or linkers).

[0349] In some embodiments, one or more peptides of the first moiety that specifically binds to an anti-MPO autoantibody are different, e.g., one or more peptides do not have the same sequence of an MPO polypeptide, or fragment or variant thereof. In some embodiments, one or more peptides having different sequences of an MPO polypeptide, or fragment or variant thereof, are separated by one or more intervening sequences (e.g., spacers and / or linkers). In some embodiments, one or more peptides having different sequences of an MPO polypeptide, or fragment or variant thereof, are not separated by one or more intervening sequences (e.g., spacers and / or linkers).

[0350] In some embodiments, each of the one or more peptides having a different sequence of an MPO polypeptide, or a fragment or variant thereof, specifically binds to an anti-MPO autoantibody or fragment thereof.

[0351] In some embodiments, the linker separating the one or more peptides of the first portion comprises a Gly-Ser linker or an EAAAK linker. In some embodiments, the linker comprises a (Gly-Gly-Gly-Gly-Ser) linker, where n is an integer between 0 and 20.

[0352] In some embodiments, each of the one or more peptides that specifically bind to an anti-MPO autoantibody is conjugated to a second moiety.

[0353] In some embodiments, each of the one or more peptides that specifically bind to an anti-MPO autoantibody is not conjugated to a second moiety.

[0354] In some embodiments, one or more peptides that specifically bind to anti-MPO autoantibodies are conjugated to each other, eg, located on a single polypeptide.

[0355] In some embodiments, one or more peptides that specifically bind to an anti-MPO autoantibody are separated by a protease cleavage site or IRES, hi some embodiments, each of the one or more peptides is expressed as a separate peptide, e.g., translated as a separate peptide by an IRES, or expressed as a separate peptide after cleavage of the protease cleavage site.

[0356] In some embodiments, the one or more peptides that specifically bind to an anti-MPO autoantibody are not separated by a protease cleavage site or IRES, eg, are expressed as a fusion protein.

[0357] In some embodiments, the first portion comprises one or more peptides that specifically bind to an anti-MPO antibody. In some embodiments, the one or more peptides that specifically bind to an anti-MPO antibody comprise an MPO polypeptide, or a variant or fragment thereof. In some embodiments, the first portion further comprises (a) a linker, (b) a spacer, (c) a cleavage peptide, e.g., an IRES or a protease cleavage site, (d) a signal peptide, (e) a tag, e.g., a cleavable tag, (f) a half-life extending domain, e.g., an Fc domain or albumin, or (g) any combination of (a)-(f).

[0358] In some embodiments, the one or more peptides that specifically bind to an anti-MPO antibody comprise an antibody agent. In some embodiments, the antibody agent comprises an antigen-binding fragment. In some embodiments, the antibody agent comprises a full antibody, a Fab fragment, an scFv, a nanobody, a duobody, or a single domain antibody (e.g., a VHH). In some embodiments, the antibody agent comprises a VHH, e.g., a camelid-derived VHH or a bivalent VHH.

[0359] Combination of PR3 and MPO peptides In some embodiments of the glycoengineered polypeptides disclosed herein, when the first portion comprises one or more anti-PR3 autoantibody binding polypeptides, the first portion further comprises one or more anti-MPO autoantibody binding polypeptides.

[0360] In some embodiments of the glycoengineered polypeptides disclosed herein, when the first portion comprises one or more anti-MPO autoantibody binding polypeptides, the first portion further comprises one or more anti-PR3 autoantibody binding polypeptides.

[0361] In some embodiments, the glycomodified polypeptides disclosed herein comprise a first portion comprising (i) one or more anti-PR3 autoantibody-binding polypeptides and (ii) one or more anti-MPO autoantibody-binding polypeptides.

[0362] In some embodiments, the one or more anti-PR3 autoantibody-binding polypeptides and the one or more anti-MPO autoantibody-binding polypeptides are located on the same polypeptide.

[0363] In some embodiments, the one or more anti-PR3 autoantibody-binding polypeptides and the one or more anti-MPO autoantibody-binding polypeptides are located on different polypeptides.

[0364] In some embodiments, one or more anti-PR3 autoantibody binding polypeptides are located on a first glycoengineered polypeptide and one or more anti-MPO autoantibody binding polypeptides are located on a second glycoengineered polypeptide.

[0365] In some embodiments, each of the anti-PR3 autoantibody-binding polypeptides and each of the anti-MPO autoantibody-binding polypeptides is conjugated to a second moiety.

[0366] Second part The glycoengineered polypeptides disclosed herein comprise a first portion that specifically binds to an anti-neutrophil autoantibody, or a fragment or complex thereof; and a second portion that comprises one or more glycans conjugated to the first portion at one or more glycosylation sites.

[0367] Without being bound by any particular theory, glycan association with endocytic carbohydrate-binding proteins and receptors enables distinct biological pathways. These essential biological pathways are involved in regulating immune responses, mediating protein clearance, protein turnover, and controlling the trafficking of soluble glycoproteins, glycolipids, and any natural molecules containing glycan moieties. Glycan-receptor interactions are determined by glycan structure. Glycan-binding receptors are highly diverse and can be exploited through glycomodification to develop novel therapeutics based on the concept of glycan-mediated protein degradation, thereby treating a variety of diseases, including but not limited to the autoimmune diseases disclosed herein.

[0368] Furthermore, without being bound by any particular theory, it is predicted that the polypeptides described herein that include a second portion having one or more glycans activate natural degradation pathways.

[0369] In some embodiments, the second portion of the glycomodified polypeptide disclosed herein comprises one or more glycans and specifically binds to one or more endocytic receptors. The endocytic receptors described herein capture glycoproteins via specific glycan structures and mediate degradation, e.g., lysosomal degradation. Endocytic receptors are ubiquitous in humans and can be found on a variety of cells.

[0370] In some embodiments, the endocytic receptor is or comprises an endocytic lectin. In some embodiments, the endocytic receptor is selected from asialoglycoprotein receptor (ASGPR); mannose-binding receptor, cluster of differentiation 206 (CD206) receptor, DC-SIGN (cluster of differentiation 209 or CD209) receptor; C-type lectin domain family 4 member G (LSECTin) receptor; macrophage-inducible Ca2+-dependent lectin receptor (Mincle); L-SIGN CD209L receptor; Dectin-1; Dectin-2, Langerin, macrophage mannose 2 receptor, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, DNGR1, CLEC12B, DEC-205, CLEC10, and mannose 6-phosphate receptor (M6PR), or a combination thereof.

[0371] In some embodiments, provided herein are glycoengineered polypeptides comprising a first portion that specifically binds to a target protein (e.g., an anti-neutrophil autoantibody) and a second portion comprising a glycan comprising a terminal GlcNAc.

[0372] In some embodiments, provided herein are glycomodified polypeptides comprising a first portion that specifically binds to a target protein (e.g., an anti-neutrophil autoantibody) and a second portion comprising a glycan comprising a terminal GalNAc.

[0373] In some embodiments, provided herein are glycomodified polypeptides comprising a first portion that specifically binds to a target protein (e.g., an anti-neutrophil autoantibody) and a second portion comprising a glycan comprising a terminal Gal.

[0374] In some embodiments, the polypeptides provided herein may comprise one or more N-glycan(s) that have (i) binding specificity for one or more target protein(s) (e.g., one or more anti-neutrophil autoantibodies) and (ii) binding specificity for one or more endocytic receptor(s).

[0375] In some embodiments, the glycoengineered polypeptide comprises one type of N-glycan that has binding specificity for one type of endocytic receptor.

[0376] In some embodiments, the glycomodified polypeptide comprises one or more N-glycosylation sites in the first portion. In some embodiments, the one or more N-glycosylation sites in the first portion are native N-glycosylation sites. In some embodiments, the one or more N-glycosylation sites in the first portion are engineered N-glycosylation sites. In some embodiments, the glycomodified polypeptide comprises one or more native N-glycosylation sites and one or more engineered N-glycosylation sites.

[0377] In some embodiments, a second moiety comprising one or more glycans is conjugated, eg, linked, to the first moiety at one or more N-glycosylation sites.

[0378] In some embodiments, the glycoengineered polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites (or glycosites; e.g., N-glycosylation consensus sequences). These N-glycosylation sites can be glycosylated with N-glycans, and the resulting glycoengineered bifunctional binding protein can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more endocytic receptor molecules.

[0379] In some embodiments, the glycomodified polypeptide comprises two types of N-glycans with binding specificities for two different endocytic receptors. In certain embodiments, the glycomodified polypeptides provided herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptide chains. Each chain can be produced in a different cell line. In certain embodiments, the glycomodified polypeptide can be an antibody, wherein one type of N-glycan is on the Fc domain and another type of N-glycan is on the Fab domain (e.g., variable region) of the antibody.

[0380] In some embodiments, the glycomodified polypeptide comprises (i) a first type of N-glycan that has binding specificity for a first endocytic receptor, the first type of N-glycan being present on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosites, thereby associating or binding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more molecules of the first endocytic carbohydrate-binding protein or receptor; and (ii) a second type of N-glycan that has binding specificity for a second endocytic receptor, the second type of N-glycan being present on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosites, such that a single bifunctional binding protein can bind or associate to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more molecules of the second endocytic receptor(s).

[0381] In some embodiments, the glycoengineered polypeptide comprises: (i) a first type of N-glycan that has binding specificity for a first endocytic receptor, the first type of N-glycan being present on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosites, thereby associating with or binding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more molecules of the first endocytic receptor; and (ii) a second type of N-glycan that has binding specificity for a second endocytic receptor, the second type of N-glycan being present on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosites, thereby forming a single bifunctional bond. (iii) a second type of N-glycan that the binding protein can bind to or associate with one, two, three, four, five, six, seven, eight, nine, ten, or more molecules of the second endocytic receptor(s); and (iv) a third type of N-glycan that has binding specificity for a third endocytic receptor and is present on one, two, three, four, five, six, seven, eight, nine, ten, or more glycosites, such that a single bifunctional binding protein can bind to or associate with one, two, three, four, five, six, seven, eight, nine, ten, or more molecules of the third endocytic receptor(s).

[0382] In some embodiments, glycoengineered polypeptides provided herein have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosites. In some embodiments, in a population of glycoengineered polypeptides, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the glycosites at a particular position in the population are glycosylated. In certain embodiments, in a population of glycoengineered polypeptides, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the glycosites in the population are glycosylated. N-glycans that can be present on the glycosites of the glycoengineered polypeptides described herein are described herein.

[0383] In some embodiments, the glycosite is an N-glycosylation consensus sequence, which is NXS / T or NXC, where X is any amino acid except proline.

[0384] In some embodiments, the glycosite is or comprises the sequence GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0385] In some embodiments, the N-glycans are conjugated to the glycoengineered polypeptide at at least 1, 2, 3, or 4 N-glycosylation sites.

[0386] In some embodiments, the N-glycans are conjugated to the glycoengineered polypeptide at 1, 2, 3, or 4 N-glycosylation sites.

[0387] In some embodiments, the N-glycosylation site is naturally occurring.

[0388] In some embodiments, an N-glycosylation site is engineered into the amino acid sequence of the first portion.

[0389] In certain embodiments, one or more of the N-glycosylation sites are engineered into the amino acid sequence of the first portion of the glycomodified polypeptide (i.e., the one or more N-glycosylation sites are not present in the wild-type, i.e., naturally occurring, form of the first portion). In certain embodiments, at least one of the N-glycosylation sites is engineered into the amino acid sequence of the first portion of the glycomodified polypeptide. In certain embodiments, at least two of the N-glycosylation sites are engineered into the amino acid sequence of the first portion of the glycomodified polypeptide. In certain embodiments, at least three of the N-glycosylation sites are engineered into the amino acid sequence of the first portion of the glycomodified polypeptide. In certain embodiments, at least four of the N-glycosylation sites are engineered into the amino acid sequence of the first portion of the glycomodified polypeptide. In certain embodiments, one or more of the engineered N-glycosylation sites is a glycotag fused to the N-terminus and / or C-terminus of the amino acid sequence of the first portion of the glycomodified polypeptide via a peptide linker. In certain embodiments, the glycotag is fused to the N-terminus of the first portion of the glycomodified polypeptide. In certain embodiments, the glycotag is fused to the C-terminus of the first portion of the glycomodified polypeptide. In certain embodiments, the glycotag is fused to the N-terminus and C-terminus of the first portion of the glycomodified polypeptide. In certain embodiments, one or more of the N-glycosylation sites is a native N-glycosylation site (i.e., one or more N-glycosylation sites are present in the wild-type, i.e., naturally occurring, form of the first portion). In certain embodiments, at least one of the N-glycosylation sites is a native N-glycosylation site. In certain embodiments, at least two of the N-glycosylation sites are native N-glycosylation sites.

[0390] In some embodiments, provided herein are glycoengineered polypeptides that specifically bind to a target protein associated with a disease disclosed herein (e.g., an anti-neutrophil autoantibody), the glycoengineered polypeptide comprising a first portion and a second portion. In some embodiments, provided herein are glycoengineered polypeptides comprising a first portion that specifically binds to a target protein associated with a disease disclosed herein (e.g., an anti-neutrophil autoantibody) and a second portion that specifically binds to an endocytic receptor, wherein the second portion comprises a glycan structure.

[0391] In some embodiments, provided herein is a method for preparing a nucleotide sequence comprising a first moiety that specifically binds to a target protein and a nucleotide sequence comprising GlcNAc2Man3GlcNAc2, GalNAc2GlcNAc2Man3 GlcNAc2, Gal2GlcNAc2Man3GlcNAc2, Man3 GlcNAc, GlcNAc1Man3 GlcNAc2, Gal2GlcNAc2Man3 GlcNAc2, Gal1 GlcNAc2Man3 GlcNAc2, GalNAc1 GlcNAc2Man3 GlcNAc2, GlcNAc3Man3 GlcNAc2, GlcNAc4Man3 GlcNAc2, Gal3GlcNAc3Man3 GlcNAc2, GalNAc3 GlcNAc3Man3 GlcNAc2, GalNAc4GlcNAc4Man3GlcNAc2, Gal4GlcNAc4Man3GlcNAc2, or Man-6-P and a second portion comprising an N-glycan selected from the group consisting of: -N-glycans.

[0392] In some embodiments, an increased number of glycan structures on a glycoengineered polypeptide increases the rate of lysosomal degradation compared to an otherwise similar glycoengineered polypeptide having a lower number of glycan structures.

[0393] In some embodiments, the number of glycan structures on a glycomodified polypeptide disclosed herein is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more glycan structures.

[0394] In some embodiments, glycoengineered polypeptides disclosed herein comprise glycan structures with monoantennary structures.

[0395] In some embodiments, glycoengineered polypeptides disclosed herein comprise glycan structures having biantennary structures.

[0396] In some embodiments, glycoengineered polypeptides disclosed herein comprise a glycan structure having a triantennary structure.

[0397] In some embodiments, glycoengineered polypeptides disclosed herein comprise a glycan structure having a tetraantennary structure.

[0398] In some embodiments, the glycan structure comprises a biantennary structure. In some embodiments, the glycan structure comprises a biantennary GalNAc. In some embodiments, the biantennary GalNAc binds to an asialoglycoprotein receptor (ASGPR) or a fragment or variant thereof, or a complex comprising an ASGPR.

[0399] In some embodiments, the N-glycan has the following structure: [ka]

[0400] Here, the black squares represent N-acetylgalactosamine (GalNAc), the white squares represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue in the first moiety.

[0401] In some embodiments, the N-glycans specifically bind to one or more endocytic receptors that mediate, for example, lysosomal degradation. In some embodiments, the N-glycans specifically bind to ASGPR.

[0402] In some embodiments, the endocytic receptor is or comprises ASGPR or a fragment or variant thereof, or a complex comprising ASGPR. In some embodiments, when the endocytic receptor is ASGPR, the glycan structure of the second moiety comprises a terminal GalNac.

[0403] ASGPR-mediated degradation in hepatocytes has many applications. Binding of ASPGR to the N-glycan structures disclosed herein can result in the selective degradation of one or more target proteins (e.g., anti-neutrophil autoantibodies). For example, ASGPR-mediated degradation can lead to the removal of cytokines, chemokines, and hormones. Furthermore, ASGPR-mediated degradation can be used to deliver target molecules to hepatocyte endosomes. Therefore, ASGPR-mediated degradation can be applied to various diseases while limiting systemic toxicity.

[0404] In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites can be glycosylated with N-glycans, and the resulting glycomodified polypeptide can associate with or bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more endocytic receptor molecules. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least 2 N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least 3 N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least four N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least five N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least six N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least seven N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least eight N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least nine N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at at least ten N-glycosylation sites.

[0405] In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at two N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at three N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at four N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at five N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at six N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at seven N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at eight N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at 9 N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated with N-glycans at 10 N-glycosylation sites. In certain embodiments, the glycomodified polypeptide is glycosylated at an Asn amino acid residue of the glycomodified polypeptide. In certain embodiments, the N-glycosylation site is an N-glycosylation consensus sequence. In certain embodiments, the N-glycosylation site comprises an NXS / T or NXC consensus sequence, where X is any amino acid except proline.

[0406] In certain embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the N-glycosylation sites are occupied by N-glycans, In certain embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of the N-glycosylation sites are occupied by N-glycans of the following structure, which are linked to the glycomodified polypeptide at one or more N-glycosylation sites: [ka]

[0407] wherein the black squares represent N-acetylgalactosamine (GalNAc) residues, the white squares represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue of the glycomodified polypeptide. In certain embodiments, at least 10% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 20% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 30% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 40% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 50% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 60% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 70% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 80% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 90% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 95% of the N-glycosylation sites are occupied by N-glycans. In certain embodiments, at least 98% of the N-glycosylation sites are occupied by N-glycans.

[0408] In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at at least one N-glycosylation site. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at at least two N-glycosylation sites. In certain embodiments, the N-glycan is attached to the glycomodified polypeptide at one, two, three, or four N-glycosylation sites. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at one N-glycosylation site. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at two N-glycosylation sites. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at three N-glycosylation sites. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at four N-glycosylation sites. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at an Asn amino acid residue of the glycomodified polypeptide. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at an N-glycosylation consensus sequence, hi certain embodiments, the N-glycan is linked to the glycomodified polypeptide at an NXS / T or NXC consensus sequence, where X is any amino acid except proline.

[0409] In certain embodiments, the glycomodified polypeptide comprises two different N-glycans (i.e., a first and a second N-glycan), each N-glycan independently linked to the glycomodified polypeptide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites, and one of the N-glycans (i.e., the first N-glycan) has the following structure: [ka] wherein the black squares represent N-acetylgalactosamine (GalNAc), the white squares represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue of the glycomodified polypeptide. In certain embodiments, different N-glycans specifically bind to different endocytic receptors. In certain embodiments, a first N-glycan specifically binds to ASGPR. In certain embodiments, the other N-glycan is an N-glycan described in PCT / EP2022 / 057556, the entire contents of which are incorporated herein by reference. In certain embodiments, the first N-glycan is larger than the second N-glycan. In other embodiments, the first N-glycan is smaller than the second N-glycan. In certain embodiments, the N-glycosylation site occupied predominantly or exclusively by the larger N-glycan is sterically more accessible than the N-glycosylation site occupied predominantly or exclusively by the smaller N-glycan. In certain embodiments, the other N-glycan is A2. In certain embodiments, the other N-glycan is A1GalNAc1 or A2GalNAc1. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at an Asn amino acid residue of the glycomodified polypeptide. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at an NXS / T or NXC consensus sequence, where X is any amino acid except proline. In certain embodiments, the first N-glycan is linked to the glycomodified polypeptide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites and the second N-glycan is linked to the glycomodified polypeptide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites.

[0410] In certain embodiments, the glycomodified polypeptide further comprises a third N-glycan, wherein the third N-glycan is linked to the glycomodified polypeptide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites. In certain embodiments, the third N-glycan specifically binds to a different endocytic receptor than the first and / or second N-glycan. In certain embodiments, the third N-glycan is an N-glycan described in PCT / EP2022 / 057556, which is incorporated herein by reference in its entirety. In certain embodiments, the third N-glycan is A2. In certain embodiments, the third N-glycan is A1GalNAc1 or A2GalNAc1. In certain embodiments, the third N-glycan is linked to the glycomodified polypeptide at an Asn amino acid residue in the glycomodified polypeptide. In certain embodiments, the third N-glycan is linked to the glycomodified polypeptide at an N-glycosylation consensus sequence, hi certain embodiments, the third N-glycan is linked to the glycomodified polypeptide at an NXS / T or NXC consensus sequence, where X is any amino acid except proline.

[0411] In certain embodiments, the second and / or third N-glycans specifically bind to an endocytic lectin. In some embodiments, the endocytic lectin is a mannose-binding receptor. In some embodiments, the endocytic lectin is a cluster of differentiation 206 (CD206) receptor. In some embodiments, the endocytic lectin is a DC-SIGN (cluster of differentiation 209 or CD209) receptor. In some embodiments, the endocytic lectin is a C-type lectin domain family 4 member G (LSECTin) receptor. In some embodiments, the endocytic lectin is a macrophage-induced Ca 2+In some embodiments, the endocytosis receptor is a phospholipase C-dependent lectin receptor (Mincle). In some embodiments, the endocytosis receptor is L-SIGN CD209L. In some embodiments, the endocytosis receptor is asialoglycoprotein A (ASGPR). In some embodiments, the endocytosis receptor is dectin-1. In some embodiments, the endocytosis receptor is dectin-2. In some embodiments, the endocytosis receptor is langerin. In some embodiments, the second and / or third N-glycan specifically binds to a receptor selected from the group consisting of macrophage mannose 2 receptor, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, CLEC10, DNGR1, CLEC12B, DEC-205, and mannose 6-phosphate receptor (M6PR).

[0412] CD206 is a C-type lectin and a phagocytic / endocytic recycling and signaling receptor. CD206 is primarily expressed by M2 anti-inflammatory macrophages, dendritic cells, and liver sinusoidal endothelial cells. DC-SIGN is a non-recycling signaling receptor that targets both the ligand and receptor to lysosomes for degradation. LSECTin is expressed on liver sinusoidal endothelial cells.

[0413] In certain embodiments, the glycoengineered polypeptide is glycosylated at two or more N-glycosylation sites with an N-glycan of the following structure: [ka]

[0414] wherein the black box represents an N-acetylgalactosamine (GalNAc), the white box represents an N-acetylglucosamine (GlcNAc) residue, the black circle represents a mannose (Man) residue, X represents an amino acid residue of the glycomodified polypeptide, and the two N-glycosylation sites are separated by at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at two N-glycosylation sites separated by a distance of about 5-10, about 10-20, about 20-30, about 30-40, about 40-50, about 50-60, about 60-70, about 70-80, about 80-90, about 90-100, about 100-150, about 150-200, or about 200-300 amino acids. In certain embodiments, the amino acid spacing between the N-glycosylation sites is the number of amino acids between the terminal amino acids of the N-glycosylation consensus sequence. Without being bound by theory, glycomodified polypeptides are spatially folded and therefore have a three-dimensional shape in addition to their primary amino acid structure. Also, without being bound by theory, this three-dimensional shape, including the location of the N-glycans, is dynamic rather than static (see, e.g., Re, S., et al. Biophysical Reviews, 4, 179-187 (2012)). Nevertheless, in certain embodiments, the distance between N-glycosylation sites and / or N-glycans on a glycomodified polypeptide may be from the equilibrium shape of the glycomodified polypeptide, as determined by any standard means known in the art, including, for example, computational modeling studies. In certain embodiments, an N-glycan is linked to the glycomodified polypeptide at two N-glycosylation sites separated by a distance of at least 1.0 nm. In certain embodiments, an N-glycan is linked to the glycomodified polypeptide at two N-glycosylation sites separated by a distance of about 1.0-5.0 nm. In certain embodiments, an N-glycan is linked to the glycomodified polypeptide at two N-glycosylation sites separated by a distance of about 1.5-3.0 nm.In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at two N-glycosylation sites separated by a distance of about 1.5 to 2.5 nm. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at three N-glycosylation sites separated by a distance of about 1.0 to 5.0 nm. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at three N-glycosylation sites separated by a distance of about 1.5 to 3.0 nm. In certain embodiments, the N-glycan is linked to the glycomodified polypeptide at three N-glycosylation sites separated by a distance of about 1.5 to 2.5 nm. In certain embodiments, the N-glycans are separated by a distance of at least 1.0 nm. In certain embodiments, the N-glycans are separated by a distance of about 1.0 to about 5.0 nm. In certain embodiments, the N-glycans are separated by a distance of about 1.5 to about 2.5 nm. In certain embodiments, the distance between N-glycosylation sites and / or N-glycans is selected, for example, to minimize steric hindrance between the glycoengineered polypeptide(s), the target protein(s), and / or the ASGPR receptor(s). In certain embodiments, the distance between N-glycosylation sites and / or N-glycans is selected based on the spacing of ASGPR receptors on the cell surface. In certain embodiments, the distance between N-glycosylation sites and / or N-glycans is selected to be similar to (e.g., less than or equal to two-fold, or more than or equal to) the spacing of ASGPR receptors on the cell surface. In certain embodiments, the N-glycan is linked to the glycoengineered polypeptide at an Asn amino acid residue of the glycoengineered polypeptide. In certain embodiments, the N-glycan is linked to the glycoengineered polypeptide at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the glycoengineered polypeptide at an NXS / T or NXC consensus sequence, where X is any amino acid except proline.

[0415] Nucleic acid sequences encoding glycoengineered polypeptides The present disclosure provides, inter alia, nucleic acid sequences encoding the glycomodified polypeptides described herein.

[0416] In some embodiments, the nucleic acid sequence is or comprises single-stranded DNA (e.g., as in the case of certain viral vectors). In some embodiments, the nucleic acid is or comprises double-stranded DNA (e.g., as in the case of certain viral vectors and / or certain plasmids). In some embodiments, the nucleic acid is or comprises RNA (e.g., as in the case of certain viral vectors and / or mRNA therapeutics).

[0417] Nucleic acids encoding glycomodified polypeptides may be modified to contain codons optimized for expression in a particular cell type (e.g., Leishmania cells) or organism. A codon-optimized sequence is a synthetic sequence that preferably encodes the same polypeptide (or a biologically active fragment of the full-length polypeptide having substantially the same activity as the full-length polypeptide) as encoded by a non-codon-optimized parent polynucleotide. In some embodiments, all or a portion of the coding region of a nucleic acid encoding a glycomodified polypeptide described herein may contain a sequence modified to optimize codon usage for a particular cell type (e.g., a eukaryotic or prokaryotic cell). For example, the coding sequence of an antibody agent (e.g., an antigen-binding fragment) described herein may be optimized for expression in bacterial cells. Alternatively, the coding sequence may be optimized for expression in mammalian cells (e.g., CHO cells). Such sequences may be described as codon-optimized sequences.

[0418] The nucleic acid constructs of the present disclosure may be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid may be operably linked to an expression control sequence. Further provided by the present disclosure are vectors comprising any of the nucleic acids described herein, or fragments thereof. Any of the nucleic acids described herein, or fragments thereof, can be cloned into any suitable vector and used to transform or transfect any suitable host (e.g., a Leishmania host cell). The selection of vectors and methods for their construction are generally known to those of skill in the art.

[0419] In some embodiments, the nucleic acids and vectors of the present disclosure are isolated and / or purified. The present disclosure also provides compositions comprising isolated or purified nucleic acids, optionally in the form of vectors. Isolated nucleic acids and vectors may be prepared using standard techniques known in the art, including, for example, alkali / SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis, and / or other techniques known in the art. The compositions may include other components as further described herein.

[0420] Any method known to those of skill in the art for inserting nucleic acids into a vector may be used to construct expression vectors encoding glycomodified polypeptides described herein under the control of transcriptional and / or translational regulatory signals. These methods may include in vitro recombinant DNA and synthetic techniques, as well as in vivo recombination (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994), the contents of each of which are incorporated herein by reference in their entireties).

[0421] Compositions and pharmaceutical compositions The compositions disclosed herein may comprise and / or deliver one or more glycomodified polypeptides disclosed herein or nucleic acids encoding one or more glycomodified polypeptides disclosed herein.

[0422] In some embodiments, a composition disclosed herein comprises a glycomodified polypeptide comprising a first portion and a second portion, hi some embodiments, a composition disclosed herein comprises a plurality of glycomodified polypeptides comprising a first portion and a second portion.

[0423] In some embodiments, a composition comprising multiple glycoengineered polypeptides comprises one, two, three, four, five, or more glycoengineered polypeptides that comprise a first moiety that binds to an anti-neutrophil autoantibody (e.g., a first moiety that binds to the same anti-neutrophil autoantibody).

[0424] In some embodiments, a composition comprising a plurality of glycoengineered polypeptides comprises glycoengineered polypeptides having a first portion that binds to an anti-PR3 autoantibody or fragment thereof. In some embodiments, each of the plurality of glycoengineered polypeptides comprises the same first portion. In some embodiments, each of the plurality of glycoengineered polypeptides comprises a different first portion (e.g., a first glycoengineered polypeptide comprises a first portion that binds to an anti-PR3 autoantibody or fragment thereof, a second glycoengineered polypeptide comprises a different first portion that binds to an anti-PR3 autoantibody or fragment thereof, etc.).

[0425] In some embodiments, a composition comprising a plurality of glycoengineered polypeptides comprises glycoengineered polypeptides having a first portion that binds to an anti-MPO autoantibody or fragment thereof. In some embodiments, each of the plurality of glycoengineered polypeptides comprises the same first portion. In some embodiments, each of the plurality of glycoengineered polypeptides comprises a different first portion (e.g., a first glycoengineered polypeptide comprises a first portion that binds to an anti-MPO autoantibody or fragment thereof, a second glycoengineered polypeptide comprises a different first portion that binds to an anti-MPO autoantibody or fragment thereof, etc.).

[0426] In some embodiments, a composition comprising a plurality of glycoengineered polypeptides comprises one, two, three, four, five, or more glycoengineered polypeptides, each comprising a first portion that binds to a different anti-neutrophil autoantibody (e.g., an anti-PR3 autoantibody or fragment thereof, or an anti-MPO autoantibody or fragment thereof). In some embodiments, a composition comprising a plurality of glycoengineered polypeptides comprises a first glycoengineered polypeptide comprising a first portion that binds to an anti-neutrophil autoantibody (e.g., an anti-PR3 autoantibody or fragment thereof) and a second glycoengineered polypeptide comprising a first portion that binds to a different anti-neutrophil autoantibody (e.g., an anti-MPO autoantibody or fragment thereof).

[0427] In some embodiments, the glycoengineered polypeptides disclosed herein comprise: (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to an anti-PR3 autoantibody, or a fragment or complex thereof; and (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to an anti-MPO autoantibody, or a fragment or complex thereof.

[0428] In some embodiments, the glycoengineered polypeptides disclosed herein comprise: (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to an anti-PR3 autoantibody, or a fragment or complex thereof, and (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to an anti-neutrophil antibody, or a fragment or complex thereof, other than an anti-PR3 autoantibody.

[0429] In some embodiments, the glycoengineered polypeptides disclosed herein comprise: (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to an anti-MPO autoantibody, or a fragment or complex thereof, and (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to an anti-neutrophil antibody, or a fragment or complex thereof, other than an anti-MPO autoantibody.

[0430] In some embodiments, the ratio of the first glycomodified polypeptide to the second glycomodified polypeptide is about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2.5, about 1:2, about 1:1.5, about 1:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2.5:1, about 2:1, or about 1.5:1.

[0431] In some embodiments, the ratio of first glycomodified polypeptide to second glycomodified polypeptide is from about 1:5 to about 5:1; from about 1:2.5 to about 2.5:1.

[0432] In some embodiments, the ratio of first glycomodified polypeptide to second glycomodified polypeptide is about 1:1.5 to about 1.5:1.

[0433] In some embodiments, the first glycomodified polypeptide is present in an amount of about 10-90% and the second glycomodified polypeptide is present in an amount of about 90-10%.

[0434] In some embodiments, the first glycomodified polypeptide is present in an amount of about 20-80% and the second glycomodified polypeptide is present in an amount of about 80-20%.

[0435] In some embodiments, the first glycomodified polypeptide is present in an amount of about 30-70% and the second glycomodified polypeptide is present in an amount of about 70-30%.

[0436] In some embodiments, the first glycomodified polypeptide is present in an amount of about 40-60% and the second glycomodified polypeptide is present in an amount of about 60-40%.

[0437] In some embodiments, the first glycoengineered polypeptide is present in an amount of about 10% and the second glycoengineered polypeptide is present in an amount of about 90%.

[0438] In some embodiments, the first glycomodified polypeptide is present in an amount of about 20% and the second glycomodified polypeptide is present in an amount of about 80%.

[0439] In some embodiments, the first glycoengineered polypeptide is present in an amount of about 30% and the second glycoengineered polypeptide is present in an amount of about 70%.

[0440] In some embodiments, the first glycoengineered polypeptide is present in an amount of about 40% and the second glycoengineered polypeptide is present in an amount of about 60%.

[0441] In some embodiments, the first glycoengineered polypeptide is present in an amount of about 50% and the second glycoengineered polypeptide is present in an amount of about 50%.

[0442] In some embodiments, the first glycomodified polypeptide is present in an amount of about 60% and the second glycomodified polypeptide is present in an amount of about 40%.

[0443] In some embodiments, the first glycomodified polypeptide is present in an amount of about 70% and the second glycomodified polypeptide is present in an amount of about 30%.

[0444] In some embodiments, the first glycomodified polypeptide is present in an amount of about 80% and the second glycomodified polypeptide is present in an amount of about 20%.

[0445] In some embodiments, the first glycoengineered polypeptide is present in an amount of about 90% and the second glycoengineered polypeptide is present in an amount of about 10%.

[0446] In some embodiments, disclosed herein are compositions comprising a population of glycomodified polypeptides, wherein the population of glycomodified polypeptides have an N-glycan profile that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or about 100% homogeneous at one or more N-glycosylation site(s).

[0447] In some embodiments, the homogeneity of the N-glycan profile at one or more of the N-glycosylation sites is determined by N-glycan analysis, glycopeptide analysis, or intact protein analysis.

[0448] In some embodiments, the N-glycan profile comprises about 30% to 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% N-glycans of the structures provided herein.

[0449] In some embodiments, a population of glycomodified polypeptides has an N-glycan profile that comprises about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% of all glycans in the N-glycan profile that are N-glycans having the structures provided herein.

[0450] In some embodiments, the glycoengineered polypeptides disclosed herein, or compositions comprising same, may be useful for treating and / or preventing a disease described herein (e.g., a disease associated with anti-neutrophil autoantibodies) or for ameliorating symptoms associated with a disease, disorder, or condition described herein.

[0451] The present disclosure also provides pharmaceutical compositions that, when administered to a subject (e.g., a human subject), deliver a glycoengineered polypeptide described herein to such a subject, e.g., when administered to a subject suffering from a disease associated with anti-neutrophil autoantibodies. Thus, in some embodiments, the present disclosure provides pharmaceutical compositions that comprise or deliver one or more glycoengineered polypeptides described herein, or one or more polynucleotides encoding them.

[0452] In some embodiments, the pharmaceutical composition is or comprises a composition according to the present disclosure.

[0453] Typically, pharmaceutical compositions comprise a glycomodified polypeptide described herein, or a nucleic acid encoding same, in combination with one or more pharmaceutically acceptable carriers or excipients, such as one or more buffers, diluents, fillers, salts, solubilizers, stabilizers, and / or other substances known in the art. One of skill in the art will recognize various carrier components that are appropriate for a particular active form (e.g., polypeptide vs. nucleic acid, viral vector vs. plasmid vs. RNA, etc.) and / or route of administration (e.g., parenteral, enteral, etc.).

[0454] In some embodiments, a pharmaceutical composition can comprise or deliver two or more different glycoengineered polypeptides, such that such agents can be administered in combination (e.g., substantially simultaneously or sequentially) to a subject(s).

[0455] In some embodiments, the pharmaceutical composition may contain one or more agents that can, for example, improve the stability of the composition and / or its active agent(s) (e.g., for particular storage conditions and / or time period(s)), facilitate delivery of the composition and / or its active agent(s), and / or otherwise enhance the effectiveness of the active agent(s) or composition after administration (and / or reduce one or more undesirable side effects).

[0456] Alternatively or additionally, in some embodiments, the provided pharmaceutical compositions can comprise or deliver another active agent in addition to the glycoengineered polypeptides described herein.

[0457] Methods of Treatment and / or Prevention The present disclosure provides, inter alia, methods for treating and / or preventing an anti-neutrophil autoantibody-associated disease in a subject, comprising administering a composition described herein, thereby improving at least one sign or symptom of the anti-neutrophil autoantibody-associated disease in the subject after administration.

[0458] In some embodiments, provided herein are methods for treating and / or preventing diseases associated with anti-neutrophil autoantibodies, comprising administering a composition described herein.

[0459] In some embodiments, the disease associated with anti-neutrophil autoantibodies is an autoimmune disease and / or an inflammatory disorder, hi some embodiments, the autoimmune disease is ANCA vasculitis.

[0460] Among other things, disclosed herein is the identification of glycoengineered polypeptides that specifically bind to anti-neutrophil autoantibodies, or fragments or complexes thereof (e.g., anti-PR3 autoantibodies and / or anti-MPO autoantibodies). In some embodiments, the glycoengineered polypeptides disclosed herein have therapeutic value, for example, in the treatment of diseases associated with anti-neutrophil autoantibodies, or fragments or complexes thereof (e.g., ANCA).

[0461] Glycoengineered polypeptides that specifically bind to anti-neutrophil autoantibodies (e.g., anti-PR3 autoantibodies and / or anti-MPO autoantibodies) of the present disclosure can be used to treat, prevent, and / or ameliorate, inter alia, anti-neutrophil autoantibody-associated diseases, including, but not limited to, any number of diseases in which anti-neutrophil autoantibody levels are abnormally high and / or in which a reduction in anti-neutrophil autoantibody levels is desired.

[0462] A subject treated with the methods described herein can be, for example, a patient having, at risk of having, or diagnosed as having a disease associated with anti-neutrophil autoantibodies (e.g., ANCA vasculitis).

[0463] Those skilled in the art who read this disclosure will understand that the provided compositions may be useful for treating diseases associated with anti-neutrophil autoantibody-associated diseases. In some embodiments, the anti-neutrophil autoantibody-associated disease is characterized by elevated or increased levels of anti-neutrophil autoantibodies. In some embodiments, the anti-neutrophil autoantibody-associated disease is characterized by having abnormal anti-neutrophil autoantibodies or having abnormal anti-neutrophil autoantibodies.

[0464] In some embodiments, a method for treating and / or preventing granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA (formerly known as Wegener's granulomatosis) in a subject comprises administering to the subject a composition according to the present disclosure. In some embodiments, the subject has anti-PR3 autoantibodies. In some embodiments, administration of the composition reduces the level of PR3 autoantibodies compared to a subject not administered the composition or compared to the same subject before administration of the composition. In some embodiments, the reduction in the level of anti-PR3 autoantibodies prevents neutrophil activation.

[0465] In some embodiments, a method for treating and / or preventing microscopic polyangiitis (MPA) / perinuclear ANCA in a subject comprises administering to the subject a composition according to the present disclosure. In some embodiments, the subject has anti-MPO autoantibodies. In some embodiments, administration of the composition reduces the level of MPO autoantibodies compared to a subject not administered the composition or compared to the same subject before administration of the composition. In some embodiments, the reduction in the level of MPO autoantibodies prevents neutrophil activation.

[0466] In some embodiments, compositions according to the invention are used to reduce and / or resolve anti-neutrophil autoantibodies (e.g., anti-PR3 autoantibodies and / or anti-MPO autoantibodies).

[0467] In some embodiments, compositions according to the invention are used to reduce the risk of diseases associated with anti-neutrophil autoantibodies.

[0468] In some embodiments, compositions according to the invention are used to ameliorate one or more symptoms of a disease associated with anti-neutrophil autoantibodies.

[0469] Also disclosed herein are methods that include assessing the level of anti-neutrophil autoantibodies in a sample from a subject, and administering a pharmaceutical composition disclosed herein if the level of anti-neutrophil autoantibodies is higher than a comparison control. In some embodiments, the comparison control comprises a predetermined reference sample, such as a sample obtained from an otherwise similar subject who does not have the disease or disorder or symptoms of the disease or disorder.

[0470] In some embodiments, the anti-neutrophil autoantibody comprises an anti-PR3 autoantibody, or a fragment or conjugate thereof; or an anti-MPO autoantibody, or a fragment or conjugate thereof, or a combination thereof.

[0471] Administration In some embodiments of the present disclosure, a method is provided comprising administering to a subject a composition according to the present disclosure, hi some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising a glycomodified polypeptide according to the present disclosure.

[0472] Delivery of glycoengineered polypeptides can be achieved, for example, by administering a pharmaceutical composition described herein, e.g., a pharmaceutical composition comprising a glycoengineered polypeptide or a nucleic acid encoding same, e.g., by oral ingestion, inhalation, topical application, or parenteral administration (e.g., cutaneous, subcutaneous, intraperitoneal, intramuscular, or intravenous injection). In some embodiments, administration is by intravenous or intramuscular injection. In some embodiments, local administration can be or include topical administration (e.g., to the skin) or parenteral administration (e.g., by injection into a site of deposition such as the kidney).

[0473] In some embodiments, delivery of the glycoengineered polypeptide can be achieved, for example, by administration of a pharmaceutical composition described herein, e.g., a pharmaceutical composition comprising the glycoengineered polypeptide or a nucleic acid encoding same, and can be oral, rectal, ocular (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal, or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural). Those of skill in the art will be aware of typical guidelines for formulating pharmaceutical compositions for administration by such routes. For example, such techniques can include the step of bringing into association the glycoengineered polypeptide or a nucleic acid encoding same with the pharmaceutical carrier(s) or excipient(s). In some embodiments, compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0474] In some embodiments, the administering step comprises intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal injection, or intramuscular injection.

[0475] In some embodiments, compositions according to the present disclosure are delivered to a subject suffering from or susceptible to a disease associated with anti-neutrophil antibodies described herein.

[0476] In some embodiments, the subject has or has been diagnosed with ANCA vasculitis. In some embodiments, the individual is a human.

[0477] In some embodiments, the subject has or has been diagnosed with anti-PR3 and / or anti-MPO autoantibodies. In some embodiments, the subject with anti-PR3 autoantibodies is treated with a glycoengineered polypeptide that specifically binds to the anti-PR3 autoantibody or a nucleic acid encoding same. In some embodiments, the subject with anti-MPO autoantibodies is treated with a glycoengineered polypeptide that specifically binds to the anti-MPO autoantibody or a nucleic acid encoding same.

[0478] In some embodiments, administration of a composition according to the present disclosure reduces one or more symptoms of ANCA vasculitis.

[0479] In some embodiments, granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA is treated and / or prevented by administration of a glycoengineered polypeptide that binds to an anti-PR3 autoantibody.

[0480] In some embodiments, microscopic polyangiitis (MPA) / perinuclear ANCA is treated and / or prevented by administration of glycoengineered polypeptides that bind to anti-MPO autoantibodies. Dosing regimen

[0481] In some embodiments, the methods involve administering the composition once. In some embodiments, the methods involve administering the composition repeatedly (e.g., two or more times over a period of time).

[0482] In some embodiments, administration of the composition continues to maintain remission (e.g., to keep anti-neutrophil autoantibodies low and / or undetectable) and / or to avoid relapse.

[0483] The amount of glycomodified polypeptide administered in a single dose may depend on the nature and / or severity of the condition being treated and / or the nature of previous treatments the patient has undergone. In some embodiments, the attending physician determines the amount of glycomodified polypeptide to use to treat an individual patient. In some embodiments, the attending physician initially administers a low dose of the glycomodified polypeptide of the invention and observes the patient's response. In some embodiments, higher doses are administered until an optimal therapeutic effect is achieved for the patient, after which the dosage is not further increased.

[0484] In some embodiments, the glycoengineered polypeptide according to the present disclosure is delivered in an amount effective to reduce levels of anti-neutrophil autoantibodies or fragments thereof or immune complexes.

[0485] In some embodiments, the glycoengineered polypeptide according to the present disclosure is delivered in an amount effective to reduce levels of anti-PR3 autoantibodies or fragments thereof or immune complexes.

[0486] In some embodiments, the glycomodified polypeptide according to the present disclosure is delivered in an amount effective to reduce levels of anti-MPO autoantibodies or fragments thereof or immune complexes.

[0487] Combination therapy According to the present disclosure, glycoengineered polypeptides may be administered in combination with one or more therapies. In some embodiments, glycoengineered polypeptides may be administered in combination with one or more pharmaceutical agents. For example, glycoengineered polypeptides may be administered in combination with one or more therapeutic agents for antineutrophil cytoplasmic antibody-associated disorders (e.g., agents that ameliorate the symptoms of antineutrophil cytoplasmic antibody-associated disorders) and / or in combination with one or more other pharmaceutical agents. In some embodiments, glycoengineered polypeptides may be administered in combination with one or more therapies and / or pharmaceutical agents prescribed by a clinician for the treatment of antineutrophil cytoplasmic antibody-associated disorders.

[0488] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with one or more additional therapies. In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with an anti-CD20 antibody, a C5a receptor antagonist, an anti-CTLA4 antibody, an anti-CD52 antibody, a BAFF antagonist, a glucocorticoid, or a combination thereof. Exemplary therapies that can be used to treat ANCA vasculitis are disclosed in Hillhorst J. et al., (2015) J Am Soc Nephrol 26: 2314-2327, the entire contents of which are incorporated herein by reference. In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with one or more therapies and / or drugs prescribed by a clinician for the treatment of antineutrophil cytoplasmic antibody-associated disorders.

[0489] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with an anti-CD20 antibody, hi some embodiments, the anti-CD20 antibody is rituximab.

[0490] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with a C5a receptor antagonist, hi some embodiments, the C5a receptor antagonist is CCX168.

[0491] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with an anti-CTLA4 antibody.

[0492] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with an anti-CD52 antibody.

[0493] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with a BAFF antagonist.

[0494] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with a glucocorticoid.

[0495] Characterization of glycoengineered polypeptides The present disclosure provides, among other things, glycoengineered polypeptides that specifically bind to anti-neutrophil autoantibodies (e.g., anti-PR3 autoantibodies and / or anti-MPO autoantibodies), thereby causing degradation of the anti-neutrophil autoantibody. In some embodiments, degradation of the anti-neutrophil autoantibody comprises internalization within the cell for degradation (e.g., by transporting the anti-neutrophil autoantibody to lysosomes). In some embodiments, glycoengineered polypeptides that specifically bind to anti-neutrophil autoantibodies according to the present disclosure are characterized in that they inhibit the biological function of the anti-neutrophil autoantibody (e.g., binding to a neutrophil antigen), including its ability to activate neutrophils.

[0496] In some embodiments, glycoengineered polypeptides according to the present disclosure are used to degrade and / or reduce levels of anti-neutrophil autoantibodies, hi some embodiments, glycoengineered polypeptides are used to reduce anti-neutrophil autoantibody levels, e.g., to reduce elevated plasma anti-neutrophil autoantibody levels in subjects with diseases associated with anti-neutrophil autoantibodies described herein.

[0497] In some embodiments, the present disclosure provides glycoengineered polypeptides characterized in that, when administered to a cell, tissue, or subject, the glycoengineered polypeptide binds to a target via a first moiety and to an endocytic receptor via a second moiety, resulting in degradation of the target.

[0498] In some embodiments, the degradation comprises internalization into a cell. In some embodiments, the degradation comprises lysosomal degradation. In some embodiments, the degradation occurs in hepatocytes.

[0499] In some embodiments, the present disclosure provides glycoengineered polypeptides, wherein, when administered to a cell, tissue, or subject, the glycoengineered polypeptide, which is bound to a target via a first moiety, prevents binding of the target to an immune cell. In some embodiments, the immune cell is a neutrophil. In some embodiments, administration of a glycoengineered polypeptide described herein prevents neutrophil activation.

[0500] In some embodiments, the target is an anti-PR3 autoantibody or fragment thereof, and / or an anti-MPO autoantibody or fragment thereof.

[0501] In some embodiments, the target comprises an anti-MPO autoantibody or a fragment thereof.

[0502] In some embodiments, targets include anti-PR3 autoantibodies or fragments thereof and anti-MPO autoantibodies or fragments thereof.

[0503] In some embodiments, the glycoengineered polypeptides disclosed herein are characterized in that, when administered to a cell, tissue, or subject, the glycoengineered polypeptide or a composition comprising same reduces the level and / or activity of an autoantibody or fragment thereof (e.g., an anti-MPO autoantibody or fragment thereof, and / or an anti-PR3 autoantibody or fragment thereof). In some embodiments, this reduction is compared to a comparable cell, tissue, or subject administered a different therapeutic agent or no therapeutic agent. In some embodiments, this reduction is compared to the same cell, tissue, or subject prior to administration of the glycoengineered polypeptide or a composition comprising same.

[0504] In some embodiments, the level and / or activity of the autoantibody or fragment thereof is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0505] In some embodiments, the level and / or activity of the autoantibody or fragment thereof is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0506] Method for producing glycosylated polypeptides The present disclosure provides, inter alia, methods for making glycomodified polypeptides comprising a first portion comprising one or more peptides that specifically bind to a target (e.g., an anti-neutrophil autoantibody or a fragment or complex thereof) and a second portion comprising one or more glycans conjugated to the first portion.

[0507] The glycoengineered polypeptides disclosed herein can be made using the methods disclosed in U.S. Provisional Patent Application No. 63 / 410,955, filed September 28, 2022, U.S. Provisional Patent Application No. 63 / 410,936, filed September 28, 2022, and International Patent Application No. PCT / EP2023 / 076767, filed September 27, 2023, the entire contents of each of which are incorporated herein by reference.

[0508] For example, Section 5.3 of US 63 / 410,955 discloses Leishmania host cells; Section 5.4 discloses exemplary methods for genetically modifying Leishmania cells to express glycomodified polypeptides; Section 5.5 discloses exemplary methods for culturing Leishmania host cells; and Section 5.6 discloses exemplary uses of Leishmania host cells as expression systems.

[0509] As another example, International Patent Application PCT / EP2023 / 076767 discloses Leishmania host cells in section 7.1, including modifications that can be made to the Leishmania host cells to produce glycomodified polypeptides, discloses methods for genetically modifying Leishmania host cells to produce glycomodified polypeptides, and discloses methods for culturing Leishmania host cells in section 7.3. An exemplary method for producing glycomodified polypeptides using Leishmania host cells is provided in Example 1 herein.

[0510] In particular, exemplary Leishmania strains that can be used to make the glycoengineered polypeptides disclosed herein include StCGP3558, StCGP4564, StCGP5359, or StCGP5942, as disclosed in PCT / EP2023 / 076767.

[0511] As will be appreciated by those of skill in the art, such methods and host cells can also be used to produce the glycoengineered polypeptides disclosed herein.

[0512] In some embodiments, one or more glycans of the second moiety are conjugated to the first moiety at one or more glycosylation sites by in vivo glycosylation, e.g., within a cell. In some embodiments, the cell is a Leishmania host cell. In some embodiments, the cell is a glycoengineered yeast host cell, e.g., a glycoengineered Pichia pastoris host cell.

[0513] In some embodiments, one or more glycans of the second moiety are conjugated to the first moiety at one or more glycosylation sites by chemical conjugation, for example, using click chemistry.

[0514] Also disclosed herein are methods for making glycomodified polypeptides. In one embodiment, provided herein is a method for producing a glycomodified polypeptide in vivo using a Leishmania host cell described herein. In some embodiments, provided herein is a method for producing a glycomodified polypeptide, the method comprising: (i) culturing a Leishmania host cell under conditions suitable for polypeptide production, and (ii) isolating the glycomodified polypeptide. In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding a glycomodified polypeptide; and (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases. In certain embodiments, the Leishmania host cell is capable of producing a polypeptide comprising a biantennary GalNAc-terminal N-glycan. In particular, the Leishmania host cell provided herein is capable of producing a glycomodified polypeptide comprising an N-glycan of the following structure: [ka]

[0515] Here, the black square represents an N-acetylgalactosamine (GalNAc) residue, the white square represents an N-acetylglucosamine (GlcNAc) residue, the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the glycomodified polypeptide.

[0516] In certain embodiments, the glycoengineered polypeptide produced by the Leishmania host cell is a therapeutic polypeptide, i.e., a polypeptide used to treat a disease or disorder, For example, the glycoengineered polypeptide produced by the Leishmania host cell can be a peptide or an antibody.

[0517] Leishmania host cells Provided herein are Leishmania host cells for producing a glycomodified polypeptide or a population of glycomodified polypeptides disclosed herein, the Leishmania host cell comprising: (a) a recombinant nucleic acid encoding a glycomodified polypeptide disclosed herein; and (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases. In certain embodiments, the Leishmania host cells provided herein are capable of producing glycomodified polypeptides comprising biantennary GalNAc-terminal N-glycans. In particular, the Leishmania host cells provided herein are capable of producing glycomodified polypeptides comprising an N-glycan of the following structure: [ka]

[0518] Here, the black square represents an N-acetylgalactosamine (GalNAc) residue, the white square represents an N-acetylglucosamine (GlcNAc) residue, the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the glycomodified polypeptide.

[0519] In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases disclosed herein. In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases disclosed herein. In certain embodiments, one or more endogenous enzymes disclosed herein from the glycan biosynthetic pathway of the Leishmania host cells provided herein are deleted, mutated, and / or functionally inactivated. In certain embodiments, the Leishmania host cells provided herein further comprise a recombinant nucleic acid encoding a heterologous UDP-GalNAc biosynthetic pathway protein capable of producing UDP-GalNAc. In certain embodiments, the Leishmania host cells provided herein further comprise a recombinant nucleic acid encoding a heterologous UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway.

[0520] In certain embodiments, the Leishmania host cells provided herein have been genetically modified to reduce or eliminate the formation of O-linked GlcNAc on polypeptides produced in the Leishmania host cells. Leishmania host cells genetically modified to reduce or eliminate the formation of O-linked GlcNAc are described, for example, in WO2021 / 140143, which is incorporated herein by reference in its entirety.

[0521] In certain embodiments, the Leishmania host cells provided herein below have been genetically modified using the methods described herein. In certain embodiments, the Leishmania host cells provided herein below are cultured according to the methods described herein.

[0522] Other suitable host cells include hepatocytes, bone marrow cells, immune cells, endothelial cells, parenchymal cells, or epithelial cells. In some embodiments, the immune cells are dendritic cells, macrophages, monocytes, microglial cells, granulocytes, or B lymphocytes.

[0523] Methods for culturing Leishmania host cells Provided herein are methods for culturing Leishmania host cells. In one embodiment, Leishmania host cells are cultured using any of the standard culture techniques known in the art. For example, cells are routinely grown in a rich medium such as brain heart infusion, trypticase soy broth, or yeast extract, all containing 5 μg / ml hemin. Further incubation is performed in the dark at 26°C for 2-3 days, either as static or shaking cultures. In some embodiments, cultures of recombinant cell lines contain an appropriate selection agent. Non-limiting exemplary selection agents are listed in Table 2.

[0524] [Table 1]

[0525] In certain embodiments, Leishmania host cells are cultured in a growth medium containing GalNAc. In certain embodiments, the growth medium comprises at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, at least 12 mM, at least 13 mM, at least 14 mM, at least 15 mM, at least 16 mM, at least 17 mM, at least 18 mM, at least 19 mM, or at least 20 mM GalNAc. In certain embodiments, the growth medium comprises from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, from about 10 mM to about 15 mM, or from about 15 mM to about 20 mM GalNAc. In certain embodiments, the growth medium comprises about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM GalNAc. In certain embodiments, the growth medium comprises about 10 mM GalNAc.

[0526] In certain embodiments, Leishmania host cells are cultured in a growth medium containing GlcNAc. In certain embodiments, the growth medium comprises at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, at least 12 mM, at least 13 mM, at least 14 mM, at least 15 mM, at least 16 mM, at least 17 mM, at least 18 mM, at least 19 mM, or at least 20 mM GlcNAc. In certain embodiments, the growth medium comprises from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, from about 10 mM to about 15 mM, or from about 15 mM to about 20 mM GlcNAc. In certain embodiments, the growth medium comprises about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM GlcNAc.

[0527] In certain embodiments, Leishmania host cells may be used as expression systems to produce the glycoengineered polypeptides or populations of polypeptides disclosed herein. In certain embodiments, the glycoengineered polypeptide degrader may be a heterologous non-Leishmania protein, such as a therapeutic protein (e.g., an antibody). Other methods for generating Leishmania host cells for use as expression systems are also known and can be used; see, e.g., WO2019 / 002512, WO2021 / 140144, and WO2021 / 140143, each of which is incorporated herein by reference in its entirety. The use of Leishmania host cells to produce monoclonal antibodies is also known. An exemplary method is described in WO2022 / 053673, which is incorporated herein by reference in its entirety.

[0528] The composition comprising the Leishmania host cell may contain additional components suitable for the maintenance and survival of the Leishmania host cell, and may further contain additional components necessary or beneficial for the production of the glycosylated bifunctional degrading agent by the Leishmania host cell, for example, an inducer for an inducible promoter such as arabinose, IPTG, etc.

[0529] Yeast or filamentous fungal host cells The present invention provides yeast or filamentous fungal host cells for producing a glycoengineered polypeptide or a population of glycoengineered polypeptides disclosed herein. In some embodiments, the yeast or filamentous fungal host cell is a K. lactis host cell. In some embodiments, the yeast or filamentous fungal host cell is a Pichia pastoris host cell. In some embodiments, the yeast or filamentous fungal host cell is a Pichia methanolica host cell. In some embodiments, the yeast or filamentous fungal host cell is a Hansenula host cell.

[0530] Exemplary yeast or filamentous fungal host cells that can be used to produce the glycoengineered polypeptides disclosed herein are disclosed in U.S. Patent 8,206,949, the entire contents of which are incorporated herein by reference.

[0531] Exemplary yeast or filamentous fungal host cells that can be used to produce the glycoengineered polypeptides disclosed herein are disclosed in U.S. Patent 7,981,660, the entire contents of which are incorporated herein by reference.

[0532] Exemplary yeast or filamentous fungal host cells that can be used to produce the glycoengineered polypeptides disclosed herein are disclosed in U.S. Patent 8,883,483, the entire contents of which are incorporated herein by reference.

[0533] In some embodiments, the yeast or filamentous fungal host cell is genetically modified to produce glycoproteins with predominant N-glycan glycoforms.

[0534] In certain embodiments, the yeast or filamentous fungal host cells provided herein are capable of producing glycoengineered polypeptides comprising biantennary GalNAc-terminal N-glycans. In particular, the yeast or filamentous fungal host cells provided herein are capable of producing glycoengineered polypeptides comprising an N-glycan of the following structure: [ka]

[0535] Here, the black square represents an N-acetylgalactosamine (GalNAc) residue, the white square represents an N-acetylglucosamine (GlcNAc) residue, the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the glycomodified polypeptide. [Example]

[0536] Example 1: Construction of glycoengineered polypeptides of PR3 This example describes the structure and properties of exemplary PR3 glycoengineered polypeptides for use in binding to anti-PR3 autoantibodies.

[0537] The serine protease enzyme PR3 (Figure 1A) is an antigen of one subset of anti-neutrophil cytoplasmic receptor (ANCA)-associated vasculitis, granulomatosis with polyangiitis (Wegener's granulomatosis). PR3 is expressed by neutrophil granulocytes, normally stored in the cytoplasm, and externalized upon neutrophil apoptosis. The function of PR3 is unknown, but it has been implicated in the proteolytic generation of antimicrobial peptides during bacterial infection. PR3 can interact with membranes via hydrophobic, charged clusters, and these interactions are understood to be important for its proinflammatory function.

[0538] Methods: The construction of a glycoengineered polypeptide of PR3 consists of four major steps. First, we catalytically inactivated the glycoengineered PR3 due to its protease function. The putative catalytic triad of PR3 is H71, D118, and S203 (Figure 1B; numbering based on Uniprot sequence P24158). Catalytic inactivation was achieved by mutating the S203 residue to alanine. Previous studies have shown that inactivating the S203A catalytic site does not affect ANCA binding (Pang, Y.-P. et al. Remote Activation of a Latent Epitope in an Autoantigen Decoded With Simulated B-Factors. Front Immunol 10, 2467 (2019)). Second, we incorporated a minimal truncation of the native signal peptide by deleting two amino acids (AE) from the N-terminal propeptide. Because the PR3 ANCA epitope is heterogeneous and distributed across multiple sites on the PR3 protein, a truncated native signal peptide was used at the N-terminus of glycoengineered PR3. Third, the glycoengineered polypeptide was modified by adding a peptide sequence containing an N-glycosylation consensus site ("glycotag") and a C-terminal His-tag (FLGT13) for purification. Fourth, due to hydrophobic regions that may potentially result in membrane association, additional sites F180, F181, L228, and F229 were mutated to A to allow soluble expression of recombinant PR3. These construction steps resulted in the PR3 glycoengineered polypeptide protein shown in Figure 1B (provided herein as SEQ ID NO: 41) with three N-glycosylation sites (two native sites N129 and N174 and a third engineered site within the terminal glycotag sequence).

[0539] The corresponding nucleotide sequence was transfected as an expression cassette into a glycoengineered Leishmania tarentolae host cell line, as disclosed herein. The resulting cell line, stCGP05880 (based on the parent strain StCGP4564 disclosed herein), was grown in a bioreactor for PR3 expression and secretion. PR3 glycoengineered polypeptides containing A2GalNAc2 glycans were then purified from cell supernatants by IMAC purification and analyzed. Protein aggregation and degradation were measured using size exclusion chromatography (SEC).

[0540] Results: The glycoengineering process yielded a PR3 glycoengineering polypeptide with 81.5% A2GalNAc2 (Figure 1B and Table 2). Purification analysis further demonstrated that 80–90% of the N-glycosylation sites were fully occupied. [Table 2]

[0541] Example 2: Glycoengineered PR3 polypeptides bind to anti-PR3 antibodies and human ASGPR1 This example demonstrates the binding of exemplary glycoengineered polypeptides, including PR3 polypeptides, to anti-PR3 antibodies and human ASGPR1.

[0542] Methods: Surface plasmon resonance (SPR) was used to measure the binding kinetics (i.e., ka, kd, KD, equilibrium) of PR3 glycoengineered polypeptide analytes with recombinant CLB12.8 ligand (anti-PR3 antibody) or biotinylated recombinant human ASGPR1 ligand (Acro Biosystems). Data were collected using a Biocore 8K. Either recombinant CLB12.8 or ASGPR1 was immobilized on the surface of a sensor chip, and sequential injections of increasing concentrations of PR3 glycoengineered polypeptide were performed in single-cycle kinetic experiments. For the ASGPR1 assay, 2 mM CaCl2 was included in the running buffer. Data were fitted using a 1:1 binding model for CLB12.8 and a heterologous binding model for ASGPR1 using fitting software provided by Cytiva.

[0543] Results: Using SPR, we measured the affinity of the PR3 glycoengineered polypeptide for ASGPR1 and CLB12.8 using a single-cycle kinetics approach. The PR3 glycoengineered polypeptide exhibited an association with CLB12.8 with an on-rate (kA) of 3.05 × 10 5 1 / Ms, dissociation from CLB12.8, off-rate (kd1) 7.54 × 10 -2 1 / s, a pre-steady-state KD of 24.7 nM, and a steady-state KD of 32.4 nM (Fig. 2A). The PR3 glycosylated polypeptide exhibited an association with ASGPR1 with an on-rate (kA) of 1.61 × 10 8 1 / Ms, dissociation from ASGPR1, off-rate (kd1) was 7.54 1 / s, pre-steady-state KD1 was 46.9 nM, pre-steady-state KD2 was 70.6 nM, and steady-state KD was 364 nM (Figure 2B).

[0544] This data demonstrates that PR3 glycoengineered polypeptides bind to target anti-PR3 antibodies and human ASGPR1, and therefore supports the development of glycoengineered polypeptides comprising a PR3 polypeptide and a glycan moiety for binding to endocytic receptors, such as ASGPR1, for binding to anti-PR3 autoantibodies and for targeting anti-PR3 autoantibodies for degradation.

[0545] Example 3: Glycoengineered PR3 polypeptides bind to and block ANCA autoantibodies This example demonstrates that exemplary glycoengineered polypeptides, including PR3 polypeptides, block autoantibodies in the serum of PR3+ ANCA patients.

[0546] Methods: Serum from PR3+ ANCA patients was obtained and diluted 1:100. The diluted serum was either left untreated or incubated with various concentrations of PR3 glycoengineered polypeptides or human neutrophil-derived PR3 (HN PR3) for 30 minutes at room temperature. The concentrations of PR3 glycoengineered polypeptides or HN PR3 ranged from 0 to 100 μg / ml in the first experiment and from 0 to 600 μg / ml in the second experiment. The levels of anti-PR3 autoantibodies in the samples were quantified using a c-ANCA ELISA kit (catalog number 30112111, Tecan / IBL) according to the manufacturer's instructions. ELISA data were quantitatively evaluated using a standard curve with a four-parameter function. Results are expressed as a residual binding index, calculated as the percentage of residual autoantibodies remaining in samples treated at a given concentration compared to untreated samples.

[0547] Results: In the first experiment, PR3 glycoengineered polypeptides reduced anti-PR3 autoantibody levels to approximately 50% or less of untreated levels in four of five PR3-positive patient serum samples at one or more concentrations tested (Figures 3A-3E; corresponding to patient samples 1, 7, 24, and 58). Furthermore, patient samples 1, 7, 24, and 58 showed similar residual binding percentages for HN PR3 and PR3 glycoengineered polypeptides at most of the concentrations tested (Figures 3A-3E, filled bars vs. open bars). In these patient samples, the residual binding percentages were dose-dependent (residual binding increased with decreasing concentration of PR3 glycoengineered polypeptide), as observed with HN PR3. These data demonstrate that PR3 glycoengineered polypeptides block autoantibodies in the serum of PR3+ ANCA patients.

[0548] Additionally, PR3 glycoengineered polypeptides significantly reduced anti-PR3 autoantibody levels in 14 of 15 PR3+ ANCA patient sera. Residual binding percentages of 50% or less were achieved at one or more of the concentrations tested for all ANCA patient samples, except for the sample from patient 100 (Figure 4A–C). Residual binding decreased in a concentration-dependent manner in all patient samples. Furthermore, residual binding did not correlate with patient grouping based on initial antibody titer, as residual binding decreased with increasing concentrations of PR3 glycoengineered polypeptides in samples with low (Figure 4A), intermediate (Figure 4B), and high (Figure 4C) autoantibody titers. These data are consistent with those from the first experiment and further demonstrate that autoantibody blockade in ANCA patient samples is independent of the patient's serum antibody titer.

[0549] This data further supports the development of glycoengineered polypeptides comprising a PR3 polypeptide for use as therapeutic agents in the treatment and / or prevention of ANCA (e.g., in subjects with one or more anti-PR3 autoantibodies). This data also supports the use of glycoengineered polypeptides comprising a PR3 polypeptide to reduce the level of anti-PR3 autoantibodies in a subject.

[0550] Example 4: Depletion of ANCA autoantibodies by glycoengineered PR3 polypeptides This example demonstrates the depletion of anti-PR3 autoantibodies in the serum of PR3+ ANCA patients using exemplary glycoengineered polypeptides, including a PR3 polypeptide.

[0551] Methods: PR3 glycoengineered polypeptides or control proteins were conjugated to Dynabeads (catalog no. 10103D, Invitrogen) according to the manufacturer's instructions. Following the manufacturer's protocol, serum was diluted 50-fold with 500 μl of binding / wash buffer, and 1 mg of PR3 glycoengineered polypeptide or control protein-conjugated beads was added to the sample and incubated at room temperature for 10 minutes. The tubes were then placed on a magnet, and the supernatant was collected (depleted serum). Autoantibody levels were assessed using a c-ANCA ELISA kit (catalog no. 30112111, Tecan / IBL) according to the manufacturer's instructions. The ELISA data were then quantitatively evaluated using a standard curve with a four-parameter function. Results were expressed as international units (IU) of anti-PR3 IgG / ml remaining in serum (Fig. 5A) or as percentage of depletion by PR3 glycoengineered polypeptides (Fig. 5B) and calculated as 100 − (autoantibody level in treated sample / autoantibody level in control sample × 100).

[0552] Results: Sera from all five patients showed depletion of anti-PR3 autoantibodies after blocking with the PR3 glycoengineered polypeptide compared with treatment with the control protein (Figure 5A). In all patients tested, anti-PR3 autoantibodies were depleted by the PR3 glycoengineered polypeptide at levels greater than 50% compared with the control protein (Figure 5B). This data indicates that the PR3 glycoengineered polypeptide, but not the control protein, depletes anti-PR3 autoantibodies from the serum of PR3+ ANCA patients. The percentage of depletion was independent of the starting autoantibody titer (Figure 5B). These data suggest that the PR3 glycoengineered polypeptide can bind to the majority of autoantibodies in the serum of ANCA patients in solution.

[0553] This data supports the development of glycoengineered polypeptides for use as therapeutic agents for the treatment and / or prevention of ANCA (e.g., in subjects with one or more anti-PR3 antibodies). This data further supports the use of glycoengineered polypeptides to reduce anti-PR3 autoantibody levels in a subject.

[0554] Example 5: Internalization and degradation of glycoengineered PR3 polypeptide complexed with anti-PR3 antibody in hepatocytes. This example demonstrates that a complex comprising an exemplary glycoengineered polypeptide and an anti-PR3 antibody can be internalized and degraded in hepatocytes.

[0555] Methods: The experimental setup is shown in Figure 6A. PR3 glycoengineered polypeptides were preincubated with anti-PR3 antibody (clone CLB12.8) to form complexes. The complexes were added to HepG2 cells for 4 hours to allow for internalization. Cells were then washed and harvested at 0, 1, 2, and 4 hours after washing. Cell extracts were prepared using RIPA lysis and extraction buffer (catalog no. 89900, ThermoFisher Scientific) according to the manufacturer's instructions. For internalization analysis, cell extracts were analyzed by immunoblotting using anti-His or anti-mouse IgG antibodies. In both analyses, blots were reprobed using β-actin as a loading control.

[0556] Results: As shown in Figure 6B, Western blot analysis demonstrated that PR3 glycoengineered polypeptides were present (i.e., internalized) in hepatocytes both when added alone and after complexation with the anti-PR3 antibody clone CLB12.8. No change in internalization was observed when PR3 glycoengineered polypeptides were added in complex with the surrogate autoantibody, anti-PR3 clone CLB12.8. After the complex was washed out of the medium, the internalized PR3 glycoengineered polypeptides were rapidly degraded, and almost no PR3 glycoengineered polypeptides were detected in HepG2 cells 4 hours after washing (Figure 6B). These results indicate that the PR3 glycoengineered polypeptide and CLB12.8 complexes were internalized and subsequently degraded in hepatocytes.

[0557] Furthermore, the surrogate autoantibody, anti-PR3 clone CLB12.8, was internalized only when it formed a complex with the PR3 glycoengineered polypeptide. As shown in Figure 6C, the presence of anti-PR3 antibody clone CLB12.8 was detected by blotting (i.e., internalized) only when it was able to form a complex with the PR3 glycoengineered polypeptide (Figure 6C, compare left and right panels). After the complex was washed out of the medium, anti-PR3 clone CLB12.8 was also rapidly degraded, with almost no remaining antibody detected 4 hours after washing. These results indicate that the anti-PR3 antibody was internalized only when it formed a complex with the PR3 glycoengineered polypeptide, further indicating that the internalized complex was degraded within hepatocytes.

[0558] Taken together, the results of this example suggest that exemplary PR3 glycoengineered polypeptides can promote the internalization and degradation of anti-PR3 autoantibodies in hepatocytes. This data further supports the development of glycoengineered polypeptides for use as therapeutic agents for the treatment and / or prevention of ANCA (e.g., in subjects with one or more anti-PR3 antibodies). This data also supports the development of glycoengineered polypeptides for reducing anti-PR3 autoantibody levels in a subject. Example 6: Internalization of glycoengineered PR3 polypeptides complexed with anti-PR3 antibodies is ASGPR-dependent.

[0559] This example demonstrates that internalization of exemplary glycoengineered polypeptides, including PR3 polypeptides complexed with anti-PR3 antibodies, is ASGPR dependent.

[0560] Methods: PR3 glycoengineered polypeptides or control materials produced in Chinese hamster ovary (CHO) cells were preincubated with anti-PR3 antibody (clone CLB12.8) to allow complex formation. The complexes were added to wild-type (WT) or ASGPR1 knockout (KO) HepG2 cells for 4 hours to allow internalization. Cells were then washed, and cell extracts were prepared using RIPA lysis and extraction buffer (catalog no. 89900, ThermoFisher Scientific) according to the manufacturer's instructions. For internalization analysis, cell extracts were analyzed by immunoblotting using an anti-mouse IgG antibody. Blots were reprobed using β-actin as a loading control.

[0561] Results: The data indicate that only the complex formed with the PR3 glycoengineered polypeptide, but not the control protein produced in CHO cells, is internalized in HepG2 cells. Western blot analysis (Figure 7) detected the presence of CLB12.8 in HepG2 cells only when it was allowed to form a complex with the PR3 glycoengineered polypeptide, but not when it was incubated with the control protein produced in CHO cells (Figure 7, "WT," left side of blot, sixth column). No internalization was observed in ASGPR knockout HepG2 cells, confirming that internalization of the complex is ASGPR-dependent. As shown in Figure 7, when the CLB12.8-PR3 glycoengineered polypeptide complex was added to ASGPR knockout HepG2 cells, CLB12.8 was not detected (Figure 7, "KO," right side, last column).

[0562] These data indicate that the complex formed with the PR3 glycoengineered polypeptide and anti-PR3 antibody is preferentially internalized in HepG2 cells compared with the complex of the same antibody and a control protein, and further demonstrate that internalization of the complex is dependent on ASGPR1.

[0563] Example 7: Construction of glycosylated polypeptides of MPO This example describes the structure and properties of exemplary MPO glycoengineered polypeptides for use in conjugating anti-MPO autoantibodies.

[0564] MPO is a lysosomal heme enzyme present in the azurophilic granules of human neutrophils and monocytes and has bactericidal activity against a wide range of organisms. MPO catalyzes the production of hypohalous acids, primarily hypochlorous acid under physiological conditions, as well as other toxic intermediates that enhance PMN bactericidal activity. MPO undergoes processing and activation to generate disulfide-linked homodimers, where each monomer consists of a heavy chain and a light chain.

[0565] Methods: The construction of glycoengineered MPO polypeptides consisted of four major steps. First, a full-length MPO apo-pro variant was used to maximize coverage of potential epitopes. Second, the putative cysteine ​​sulfenic acid PTM at C316 (numbering according to Uniprot P05164) was mutated to alanine. Third, a signal peptide from Leishmania major was used at the N-terminus of MPO instead of the native signal peptide, and a His tag was added to the C-terminus. The native N-glycosylation sites N139, N323, N355, N391, N483, and N729 were maintained. Fourth, catalytic inactivation was achieved by mutating the H261 proton acceptor active site to alanine. These construction steps resulted in the MPO glycoengineered polypeptide (SEQ ID NO: 43).

[0566] The corresponding nucleotide sequences were transfected as expression cassettes into a glycoengineered Leishmania tarentolae host cell line, as disclosed herein. The resulting cell line, StCGP 4940 (based on the parent strain StCGP4564 disclosed herein), was grown in a bioreactor for MPO expression and secretion. MPO glycoengineered polypeptides containing A2GalNAc2 glycans were then purified from cell supernatants by IMAC purification and analyzed.

[0567] Results: The glycosylation process yielded an MPO glycosylated polypeptide with 69.1% A2GalNAc2 (Table 3). Purification analysis further demonstrated occupancy of >90% of the N-glycosylation sites, with no unoccupied sites observed above the detection limit. [Table 3]

[0568] Example 8: Glycoengineered MPO polypeptides block anti-MPO autoantibodies in ANCA patients This example demonstrates that exemplary glycoengineered polypeptides, including MPO polypeptides, can block autoantibodies in MPO+ANCA patient sera.

[0569] Methods: Serum samples from one patient with MPO-positive x-ANCA (atypical ANCA) were diluted 1:100 and incubated for 30 minutes with MPO glycosylated polypeptides at concentrations ranging from 0.0001 µg / ml to 10 µg / ml. Anti-MPO autoantibody levels were measured using an MPO ELISA kit (catalog no. 75601, Tecan / IBL) according to the manufacturer's instructions. ELISA data were quantitatively evaluated using a standard curve with a four-parameter function and expressed in international units of anti-MPO IgG / ml.

[0570] Results: The data show that MPO glycoengineered polypeptides block autoantibodies present in the serum of MPO+ANCA patients. The levels of anti-MPO autoantibodies in the serum of MPO+ANCA patients were reduced compared to untreated samples at all concentrations of MPO glycoengineered polypeptides tested (Figure 8). At both 1 μg / ml and 10 μg / ml concentrations, MPO glycoengineered polypeptides reduced the levels of anti-MPO autoantibodies below the lower limit of ELISA detection (Figure 8, second and third bars from the left). These data demonstrate that MPO glycoengineered polypeptides bind to and block the activity of anti-MPO autoantibodies in the sera of MPO+ANCA patients.

[0571] This data supports the development of glycoengineered polypeptides, including MPO polypeptides, for use as therapeutic agents in the treatment and / or prevention of ANCA (e.g., in subjects with one or more anti-MPO antibodies). This data further supports the development of glycoengineered polypeptides for reducing anti-MPO autoantibody levels in a subject.

[0572] Enumerated Embodiments Embodiment 1. A glycoengineered polypeptide comprising: (a) a first portion comprising one or more peptides that specifically bind to an anti-neutrophil autoantibody, or a fragment or complex thereof; and (b) a second portion comprising one or more glycans conjugated to said first portion at one or more glycosylation sites.

[0573] Embodiment 2. The glycoengineered polypeptide of embodiment 1, wherein said anti-neutrophil autoantibody is an anti-proteinase 3 (PR3) autoantibody, or a fragment or complex thereof.

[0574] Embodiment 3. The glycoengineered polypeptide of embodiment 2, wherein said anti-PR3 autoantibody binds to PR3, or a variant or fragment thereof.

[0575] Embodiment 4. The glycoengineered polypeptide of embodiment 2 or 3, wherein said anti-PR3 autoantibody binds to PR3 complexed with one or more proteins.

[0576] Embodiment 5. The glycoengineered polypeptide of embodiment 4, wherein said one or more proteins that form a complex with PR3 comprises CD177.

[0577] Embodiment 6. The glycoengineered polypeptide of embodiment 1, wherein said anti-neutrophil autoantibody is an anti-myeloperoxidase (MPO) autoantibody, or a fragment or conjugate thereof.

[0578] Embodiment 7. The glycoengineered polypeptide of embodiment 6, wherein said anti-MPO autoantibody binds to MPO, or a variant or fragment thereof.

[0579] Embodiment 8. The glycoengineered polypeptide of embodiment 1, comprising: (a) an anti-PR3 autoantibody or a fragment or complex thereof, and (b) a glycomodified polypeptide capable of binding to an anti-MPO autoantibody, or a fragment or complex thereof.

[0580] Embodiment 9. The glycoengineered polypeptide of any one of the preceding embodiments, wherein said second portion specifically binds to one or more endocytic receptors.

[0581] Embodiment 10. The glycoengineered polypeptide of embodiment 9, wherein said endocytic receptor is or comprises an endocytic lectin.

[0582] Embodiment 11. The glycomodified polypeptide of embodiment 9 or 10, wherein the endocytic receptor is selected from asialoglycoprotein receptor (ASGPR); mannose-binding receptor, cluster of differentiation 206 (CD206) receptor, DC-SIGN (cluster of differentiation 209 or CD209) receptor; C-type lectin domain family 4 member G (LSECTin) receptor; macrophage-inducible Ca2+-dependent lectin receptor (Mincle); L-SIGN CD209L receptor; Dectin-1; Dectin-2, Langerin, macrophage mannose 2 receptor, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, CLEC10, DNGR1, CLEC12B, DEC-205, and mannose-6-phosphate receptor (M6PR), or a combination thereof.

[0583] Embodiment 12. The glycoengineered polypeptide of embodiment 9 or 10, wherein the endocytic receptor is ASGPR or a fragment or variant thereof, or a complex comprising ASGPR.

[0584] Embodiment 13. The glycomodified polypeptide of any one of the preceding embodiments, wherein the glycan comprises a terminal GlcNac.

[0585] Embodiment 14. The glycomodified polypeptide of any one of embodiments 1 to 12, wherein the glycan comprises a terminal GalNac.

[0586] Embodiment 15. The glycomodified polypeptide of any one of embodiments 1 to 12, wherein the glycan comprises a terminal Gal.

[0587] Embodiment 16. The glycomodified polypeptide of any one of the preceding embodiments, wherein the glycan is an N-glycan.

[0588] Embodiment 17. The glycomodified polypeptide of embodiment 16, wherein the N-glycan is linked to the glycomodified polypeptide at 1, 2, 3, 4, or 5 N-glycosylation sites.

[0589] Embodiment 18. The glycan structure is GlcNAc2-Man3-GlcNAc2, GalNAc2-GlcNAc2-Man3-GlcNAc2, Gal2-GlcNAc2-Man3-GlcNAc2, GlcNAc1-Man3-GlcNAc2, Gal2-GlcNAc2-Man3-GlcNAc2, Gal1-GlcNAc2-Man3-GlcNAc2, GalNAc1-GlcNAc2-Man3-GlcNAc2, Glc 10. The glycomodified polypeptide of any one of the preceding embodiments, comprising cNAc3-Man3-GlcNAc2, GlcNAc4-Man3-GlcNAc2, Gal3-GlcNAc3-Man3-GlcNAc2, GalNAc3-GlcNAc3-Man3-GlcNAc2, GalNAc4-GlcNAc4-Man3-GlcNAc2, Gal4-GlcNAc4-Man3-GlcNAc2, or Man-6-PN-glycan.

[0590] Embodiment 19. A glycomodified polypeptide according to any one of the preceding embodiments, wherein an increased number of glycan structures on said glycomodified polypeptide increases the rate of lysosomal degradation compared to an otherwise similar glycomodified polypeptide having a lower number of glycan structures.

[0591] Embodiment 20. The glycomodified polypeptide of any one of the preceding embodiments, wherein the number of glycan structures comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more.

[0592] Embodiment 21. The glycomodified polypeptide of any one of the preceding embodiments, wherein the glycan structure comprises a monoantennary structure, a biantennary structure, a triantennary structure, or a tetraantennary structure.

[0593] Embodiment 22. The glycomodified polypeptide of any one of the preceding embodiments, wherein the glycan structure comprises a biantennary structure.

[0594] Embodiment 23. The glycomodified polypeptide of embodiment 22, wherein the glycan structure comprises a bisecting GalNAc.

[0595] Embodiment 24. The glycomodified polypeptide of embodiment 22 or 23, wherein said biantennacy GalNac binds to the asialoglycoprotein receptor (ASGPR) or a fragment or variant thereof, or a complex comprising ASGPR.

[0596] Embodiment 25. The N-glycan has the following structure: [ka] wherein the black squares represent N-acetylgalactosamine (GalNAc) residues, the white squares represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue in the first portion.

[0597] Embodiment 26. The glycomodified polypeptide of any one of Embodiments 16 to 25, wherein the N-glycan is conjugated to the glycomodified polypeptide at at least 1, 2, 3, or 4 N-glycosylation sites.

[0598] Embodiment 27. The glycomodified polypeptide of any one of embodiments 16 to 25, wherein the N-glycan is conjugated to the glycomodified polypeptide at one, two, three, or four N-glycosylation sites.

[0599] Embodiment 28. The glycoengineered polypeptide of any one of the preceding embodiments, wherein said N-glycosylation site comprises a consensus sequence of NXS / T or NXC, where X is any amino acid except proline.

[0600] Embodiment 29. The glycomodified polypeptide of any one of the preceding embodiments, wherein said N-glycosylation site is naturally occurring.

[0601] Embodiment 30. The glycomodified polypeptide of any one of embodiments 1 to 28, wherein the N-glycosylation site has been engineered into the amino acid sequence of the first portion, and optionally, the engineered N-glycosylation site comprises the sequence GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0602] Embodiment 31 The glycoengineered polypeptide of any one of the preceding embodiments, wherein said endocytic receptor is or comprises ASGPR or a fragment or variant thereof.

[0603] Embodiment 32. The glycomodified polypeptide of embodiment 31, wherein the glycan structure of the second portion comprises a terminal GalNac when the endocytic receptor is ASGPR.

[0604] Embodiment 33. A glycoengineered polypeptide according to any one of the preceding embodiments, comprising a first portion comprising one or more peptides that specifically bind to an anti-PR3 autoantibody or fragment thereof.

[0605] Embodiment 34. The glycoengineered polypeptide of embodiment 33, wherein the first portion comprises one, two, three, four, five, or more peptides that specifically bind to an anti-PR3 autoantibody.

[0606] Embodiment 35. The glycoengineered polypeptide of embodiment 33 or 34, wherein said one or more peptides that specifically bind to anti-PR3 autoantibodies are the same.

[0607] Embodiment 36. The glycoengineered polypeptide of embodiment 35, wherein said one or more peptides that specifically bind to an anti-PR3 autoantibody are separated by an intervening sequence.

[0608] Embodiment 37. The glycomodified polypeptide of embodiment 36, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0609] Embodiment 38. The glycoengineered polypeptide of embodiment 33 or 34, wherein said one or more polypeptides that specifically bind to anti-PR3 autoantibodies are different.

[0610] Embodiment 39. The glycoengineered polypeptide of embodiment 38, wherein said one or more peptides that specifically bind to an anti-PR3 autoantibody are separated by an intervening sequence.

[0611] Embodiment 40. The glycomodified polypeptide of embodiment 39, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0612] Embodiment 41. The glycomodified polypeptide of any one of Embodiments 38 to 40, wherein the different peptides form spatial epitopes.

[0613] Embodiment 42. The glycoengineered polypeptide of any one of embodiments 33 to 41, wherein said one or more peptides that specifically bind to an anti-PR3 autoantibody are each conjugated to a second moiety.

[0614] Embodiment 43. The glycomodified polypeptide of any one of Embodiments 33 to 41, wherein each of the one or more peptides that specifically bind to an anti-PR3 autoantibody is not conjugated to the second moiety.

[0615] Embodiment 44. The glycomodified polypeptide of any one of embodiments 33 to 43, wherein said one or more peptides that specifically bind to anti-PR3 autoantibodies are conjugated to each other.

[0616] Embodiment 45. The glycomodified polypeptide of embodiment 44, wherein said one or more peptides are located on a single polypeptide.

[0617] Embodiment 46. The glycomodified polypeptide of embodiment 44 or 45, wherein said one or more peptides are separated by an intervening amino acid sequence.

[0618] Embodiment 47. The glycomodified polypeptide of embodiment 46, wherein the intervening amino acid sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0619] Embodiment 48. A glycomodified polypeptide according to any one of Embodiments 33 to 47, comprising a first portion comprising one or more peptides that specifically bind to an anti-PR3 autoantibody.

[0620] Embodiment 49. The glycomodified polypeptide of any one of embodiments 33 to 48, wherein said one or more peptides that specifically bind to anti-PR3 autoantibodies are soluble polypeptides.

[0621] Embodiment 50. The glycoengineered polypeptide of any one of embodiments 33-49, wherein said one or more peptides that specifically bind to anti-PR3 autoantibodies comprise a PR3 protein, or a fragment or variant thereof.

[0622] Embodiment 51. The glycomodified polypeptide of embodiment 50, wherein said PR3 protein is provided as SEQ ID NO: 1, with or without a signal peptide, SEQ ID NO: 40, with or without a signal peptide, or SEQ ID NO: 45.

[0623] Embodiment 52. The glycoengineered polypeptide of embodiment 50 or 51, wherein said one or more polypeptides that specifically bind to anti-PR3 autoantibodies comprise a fragment of a PR3 protein.

[0624] Embodiment 53. The glycomodified polypeptide of embodiment 52, wherein the fragment comprises at least 5% of the full-length PR3 protein, with or without the signal peptide.

[0625] Embodiment 54. The glycomodified polypeptide of embodiment 52, wherein the fragment comprises 99% or less of the full-length PR3 protein, with or without the signal peptide.

[0626] Embodiment 55. The glycoengineered polypeptide of any one of Embodiments 52 to 54, wherein the fragment comprises an epitope recognized by a PR3 autoantibody.

[0627] Embodiment 56. The glycoengineered polypeptide of embodiment 55, wherein the epitope is a linear epitope.

[0628] Embodiment 57. The glycoengineered polypeptide of embodiment 55, wherein the epitope is a conformational epitope.

[0629] Embodiment 58. The glycomodified polypeptide of embodiment 57, wherein said conformational epitope comprises one or more peptides that spatially form said epitope.

[0630] Embodiment 59. The glycomodified polypeptide of any one of Embodiments 55 to 58, wherein the epitope comprises SEQ ID NO:2 or SEQ ID NO:3, or a portion of SEQ ID NO:2 or SEQ ID NO:3.

[0631] Embodiment 60. The glycomodified polypeptide of any one of embodiments 55 to 58, wherein the epitope comprises any one of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or a portion of any of the foregoing SEQ ID NOs.

[0632] Embodiment 61. The glycomodified polypeptide of any one of embodiments 55 to 60, wherein the epitope comprises one or more additional amino acid residues at the 5' and / or 3' end of the sequence.

[0633] Embodiment 62. The glycoengineered polypeptide of any one of embodiments 33 to 60, wherein said one or more polypeptides that specifically bind to anti-PR3 autoantibodies are variants of the PR3 protein.

[0634] Embodiment 63. The glycomodified polypeptide of embodiment 62, wherein the variant is an inactive variant compared to a wild-type PR3 polypeptide.

[0635] Embodiment 64. The glycomodified polypeptide of embodiment 62 or 63, wherein said variant comprises a mutation at a valine residue at position 119, an alanine residue at position 135, a threonine residue at position 136, or a combination thereof.

[0636] Embodiment 65. The mutation is: (a) valine to isoleucine mutation; (b) an alanine to threonine mutation; and / or (c) is a serine to threonine mutation;

[0637] Embodiment 66. The glycomodified polypeptide of any one of embodiments 62 to 65, wherein the variant comprises a mutation at one or more or all of amino acids 71 ​​(His), 118 (Asp), and 203 (Ser) of SEQ ID NO: 1.

[0638] Embodiment 67. The glycomodified polypeptide of any one of embodiments 62 to 66, wherein the variant comprises a mutation at one or more or all of amino acids 180 (Phe), 181 (Phe), 228 (Leu), or 229 (Phe) of SEQ ID NO: 1.

[0639] Embodiment 68. The glycoengineered polypeptide of any one of embodiments 33 to 67, wherein the one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise a contiguous amino acid stretch comprising at least 5% of the amino acids of SEQ ID NO: 1, SEQ ID NO: 40, or SEQ ID NO: 45.

[0640] Embodiment 68.1 wherein said one or more peptides that specifically bind to anti-PL3 autoantibodies are selected from the following: (i) SEQ ID NO: 1; or (ii) The glycomodified polypeptide of any one of the preceding embodiments, comprising a sequence having at least 85% identity to SEQ ID NO: 1 without the signal peptide, optionally further comprising a different signal peptide, e.g., as disclosed herein.

[0641] Embodiment 68.2. The one or more peptides that specifically bind to anti-PL3 autoantibodies are selected from the following: (i) SEQ ID NO: 40; or (ii) The glycomodified polypeptide of any one of embodiments 1 to 68, comprising a sequence having at least 85% identity to SEQ ID NO: 40 without the signal peptide, optionally further comprising a different signal peptide, e.g., as disclosed herein.

[0642] Embodiment 68.3 The glycomodified polypeptide of any one of embodiments 1 to 68, wherein the one or more peptides that specifically bind to an anti-PL3 autoantibody comprise a sequence having at least 85% identity to SEQ ID NO: 45, and optionally, the sequence further comprises a signal peptide, e.g., as disclosed herein.

[0643] Embodiment 68.4 The glycomodified polypeptide of any one of embodiments 1 to 68, wherein the one or more peptides that specifically bind to anti-PL3 autoantibodies comprise a sequence having at least 85% identity to SEQ ID NO: 41.

[0644] Embodiment 69. The glycoengineered polypeptide of any one of embodiments 33 to 68, wherein said one or more polypeptides that specifically bind to anti-PR3 autoantibodies comprise a full-length PR3 protein.

[0645] Embodiment 70. The glycoengineered polypeptide of any one of embodiments 33 to 69, wherein said one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise an antibody agent.

[0646] Embodiment 71. The glycoengineered polypeptide of embodiment 70, wherein the antibody agent comprises an antigen-binding fragment.

[0647] Embodiment 72. The glycoengineered polypeptide of embodiment 71, wherein the antibody agent comprises a full antibody, a Fab fragment, an scFv, a nanobody, a duobody, or a single domain antibody (e.g., a VHH).

[0648] Embodiment 73. A glycomodified polypeptide according to any one of Embodiments 1 to 32, comprising a first portion comprising one or more peptides that specifically bind to an anti-MPO autoantibody or fragment thereof.

[0649] Embodiment 74. The glycomodified polypeptide of embodiment 73, wherein the first portion comprises one, two, three, four, five, or more peptides that specifically bind to an anti-MPO autoantibody.

[0650] Embodiment 75. The glycoengineered polypeptide of embodiment 72 or 73, wherein said one or more peptides that specifically bind to anti-MPO autoantibodies are the same.

[0651] Embodiment 76. The glycoengineered polypeptide of embodiment 75, wherein said one or more peptides that specifically bind to anti-MPO autoantibodies are separated by an intervening sequence.

[0652] Embodiment 77. The glycomodified polypeptide of embodiment 76, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0653] Embodiment 78. The glycoengineered polypeptide of embodiment 72 or 73, wherein said one or more peptides that specifically bind to anti-MPO autoantibodies are different.

[0654] Embodiment 79. The glycoengineered polypeptide of embodiment 78, wherein said one or more peptides that specifically bind to anti-MPO autoantibodies are separated by an intervening sequence.

[0655] Embodiment 80. The glycomodified polypeptide of embodiment 79, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0656] Embodiment 81. The glycomodified polypeptide of any one of Embodiments 78 to 80, wherein the different peptides form spatial epitopes.

[0657] Embodiment 82. The glycoengineered polypeptide of any one of embodiments 73 to 81, wherein said one or more peptides that specifically bind to an anti-MPO autoantibody are each conjugated to a second moiety.

[0658] Embodiment 83. The glycoengineered polypeptide of any one of embodiments 73 to 81, wherein each of the one or more peptides that specifically bind to an anti-MPO autoantibody is not conjugated to the second moiety.

[0659] Embodiment 84. The glycoengineered polypeptide of embodiment 83, wherein said one or more peptides that specifically bind to anti-MPO autoantibodies are conjugated to each other.

[0660] Embodiment 85. The glycomodified polypeptide of embodiment 83 or 84, wherein said one or more peptides are separated by an intervening amino acid sequence.

[0661] Embodiment 86. The glycomodified polypeptide of embodiment 85, wherein said intervening amino acid sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0662] Embodiment 87. A glycomodified polypeptide according to any one of embodiments 73 to 86, comprising a first portion comprising a peptide that specifically binds to an anti-MPO autoantibody or a fragment thereof.

[0663] Embodiment 88. The glycomodified polypeptide of any one of embodiments 73 to 87, wherein said one or more peptides that specifically bind to anti-MPO autoantibodies are soluble polypeptides.

[0664] Embodiment 89. The glycomodified polypeptide of any one of embodiments 73 to 88, wherein the one or more peptides that specifically bind to anti-MPO autoantibodies comprise an MPO polypeptide, or a fragment or variant thereof.

[0665] Embodiment 90. The glycomodified polypeptide of embodiment 89, wherein the MPO polypeptide is provided as SEQ ID NO: 4, with or without a signal peptide, SEQ ID NO: 42, with or without a signal peptide, or SEQ ID NO: 46.

[0666] Embodiment 91. The glycomodified polypeptide of embodiment 88 or 89, wherein said one or more peptides that specifically bind to anti-MPO autoantibodies comprise a fragment of an MPO polypeptide.

[0667] Embodiment 92. The glycomodified polypeptide of embodiment 91, wherein the fragment comprises at least 5% of a full-length MPO polypeptide, with or without the signal peptide.

[0668] Embodiment 93. The glycomodified polypeptide of embodiment 91, wherein the fragment comprises 99% or less of a full-length MPO polypeptide, with or without the signal peptide.

[0669] Embodiment 94. The glycoengineered polypeptide of any one of embodiments 91 to 93, wherein the fragment comprises an epitope recognized by an MPO autoantibody.

[0670] Embodiment 95. The glycoengineered polypeptide of embodiment 94, wherein the epitope is a linear epitope.

[0671] Embodiment 96. The glycoengineered polypeptide of embodiment 94, wherein the epitope is a conformational epitope.

[0672] Embodiment 97. The glycomodified polypeptide of embodiment 95, wherein said conformational epitope comprises one or more peptides that spatially form said epitope.

[0673] Embodiment 98. The glycomodified polypeptide of any one of Embodiments 94 to 97, wherein the epitope comprises SEQ ID NO:5 or SEQ ID NO:10, or a portion of SEQ ID NO:5 or SEQ ID NO:10.

[0674] Embodiment 99. The glycomodified polypeptide of any one of embodiments 94 to 97, wherein the epitope comprises any one of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or a portion of any of the foregoing SEQ ID NOs.

[0675] Embodiment 100. The glycomodified polypeptide of any one of embodiments 94 to 99, wherein the epitope comprises one or more additional amino acid residues at the 5' and / or 3' end of the sequence.

[0676] Embodiment 101. The glycomodified polypeptide of any one of embodiments 73 to 100, wherein the one or more peptides that specifically bind to anti-MPO autoantibodies are variants of an MPO polypeptide, and optionally, the variants are inactive compared to a wild-type MPO protein....

Claims

1. 1. A glycomodified polypeptide comprising: (a) a first portion comprising one or more peptides that specifically bind to an anti-neutrophil autoantibody, or a fragment or complex thereof; and (b) a second portion comprising one or more glycans conjugated to the first portion at one or more glycosylation sites, wherein the anti-neutrophil autoantibody is (a) an anti-proteinase 3 (PR3) autoantibody, or a fragment or complex thereof; or (b) The glycomodified polypeptide, which is an anti-myeloperoxidase (MPO) autoantibody or a fragment or complex thereof.

2. 2. The glycoengineered polypeptide of claim 1, wherein the anti-PR3 autoantibody binds to PR3, or a variant or fragment thereof, and optionally the anti-PR3 autoantibody binds to PR3 in a complex with one or more proteins.

3. 2. The glycoengineered polypeptide of claim 1, wherein said anti-MPO autoantibody binds to MPO, or a variant or fragment thereof.

4. 10. A glycomodified polypeptide according to any one of the preceding claims, comprising: (a) an anti-PR3 autoantibody or a fragment or complex thereof; and (b) the glycoengineered polypeptide capable of binding to an anti-MPO autoantibody, or a fragment or complex thereof.

5. 10. The glycomodified polypeptide of claim 1, wherein said second portion specifically binds to one or more endocytic receptors.

6. 6. The glycomodified polypeptide of claim 5, wherein the endocytic receptor is selected from the group consisting of asialoglycoprotein receptor (ASGPR); mannose-binding receptor, cluster of differentiation 206 (CD206) receptor, DC-SIGN (cluster of differentiation 209 or CD209) receptor; C-type lectin domain family 4 member G (LSECTin) receptor; macrophage-inducible Ca2+-dependent lectin receptor (Mincle); L-SIGN CD209L receptor; Dectin-1; Dectin-2, Langerin, macrophage mannose 2 receptor, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, DNGR1, CLEC12B, DEC-205, CLEC10, mannose-6-phosphate receptor (M6PR), or any combination thereof.

7. 10. The glycomodified polypeptide of claim 1, wherein the one or more glycans comprise a terminal GlcNAc, a terminal GalNAc, or a terminal Gal.

8. 10. The glycomodified polypeptide of any one of the preceding claims, wherein said one or more glycans are N-glycans, and optionally said N-glycans are linked to said first portion of said glycomodified polypeptide at 1, 2, 3, 4, or 5 N-glycosylation sites.

9. The one or more glycans are selected from the group consisting of GlcNAc2-Man3-GlcNAc2, GalNAc2-GlcNAc2-Man3-GlcNAc2, Gal2-GlcNAc2-Man3-GlcNAc2, GlcNAc1-Man3-GlcNAc2, Gal2-GlcNAc2-Man3-GlcNAc2, Gal1-GlcNAc2-Man3-GlcNAc2, GalNAc1-GlcNAc2-Man3-GlcNAc2, GlcNAc3-M 10. The glycomodified polypeptide of any one of the preceding claims, comprising a glycan structure comprising an-an3-GlcNAc2, GlcNAc4-Man3-GlcNAc2, Gal3-GlcNAc3-Man3-GlcNAc2, GalNAc3-GlcNAc3-Man3-GlcNAc2, GalNAc4-GlcNAc4-Man3-GlcNAc2, Gal4-GlcNAc4-Man3-GlcNAc2, or Man-6-P-N-glycan.

10. 10. The glycomodified polypeptide of claim 9, wherein the glycan structure comprises a monoantennary structure, a biantennary structure, a triantennary structure, or a tetraantennary structure.

11. 11. The glycomodified polypeptide of claim 9 or 10, wherein said glycan structure comprises a biantennary structure, and optionally said glycan structure comprises a biantennary GalNAc.

12. 12. The glycoengineered polypeptide of claim 11, wherein the bisecting GalNAc binds to the asialoglycoprotein receptor (ASGPR) or a fragment or variant thereof, or a complex comprising an ASGPR.

13. The N-glycan has the following structure: 【Chemistry 15】 wherein the black squares represent N-acetylgalactosamine (GalNAc) residues, the white squares represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue in the first portion.

14. 14. The glycomodified polypeptide of any one of claims 8 to 13, wherein the N-glycan is conjugated to the first portion of the glycomodified polypeptide at at least 1, 2, 3, or 4 N-glycosylation sites.

15. 15. The glycomodified polypeptide of any one of claims 8 to 14, wherein the N-glycosylation site comprises the consensus sequence N-X-S / T or N-X-C, where X is any amino acid except proline.

16. 16. The glycomodified polypeptide of any one of claims 8 to 15, wherein the N-glycosylation site is naturally occurring.

17. The glycomodified polypeptide of any one of claims 8 to 16, wherein the N-glycosylation site is engineered into the amino acid sequence of the first portion.

18. 18. The glycomodified polypeptide of any one of claims 5 to 17, wherein said endocytic receptor is or comprises ASGPR or a fragment or variant thereof, and optionally said glycan structure of said second portion comprises a terminal GalNAc.

19. 10. A glycoengineered polypeptide according to any one of the preceding claims, comprising a first portion comprising one or more peptides that specifically bind to an anti-PR3 autoantibody or fragment thereof.

20. 20. The glycoengineered polypeptide of claim 19, wherein the one or more peptides that specifically bind to an anti-PR3 autoantibody are each conjugated to a second moiety.

21. 21. The glycomodified polypeptide of claim 19 or 20, wherein the one or more peptides that specifically bind to anti-PR3 autoantibodies are soluble polypeptides.

22. 22. The glycomodified polypeptide of any one of claims 19 to 21, wherein the one or more peptides that specifically bind to anti-PR3 autoantibodies comprise a PR3 protein, or a fragment or variant thereof.

23. 23. The glycomodified polypeptide of claim 22, wherein the PR3 protein is provided as SEQ ID NO: 1 (e.g., with or without a signal peptide), SEQ ID NO: 40 (e.g., with or without a signal peptide), or SEQ ID NO:

45.

24. 24. The glycomodified polypeptide of claim 22 or 23, wherein the one or more peptides comprise at least 5% of a full-length PR3 protein, or a PR3 polypeptide as provided in SEQ ID NO: 1 (e.g., with or without the signal peptide), or SEQ ID NO: 40 (e.g., with or without the signal peptide), or SEQ ID NO:

45.

25. The glycomodified polypeptide of any one of claims 19 to 24, wherein the fragment comprises an epitope recognized by a PR3 autoantibody.

26. The glycomodified polypeptide of any one of claims 19 to 25, wherein the one or more polypeptides that specifically bind to an anti-PR3 autoantibody are variants of the PR3 protein.

27. 27. The glycomodified polypeptide of claim 26, wherein the variant comprises a mutation at a valine residue at position 119, an alanine residue at position 135, a threonine residue at position 136, or a combination thereof.

28. The mutation is (a) a valine to isoleucine mutation; (b) an alanine to threonine mutation; and / or 28. The glycomodified polypeptide of claim 27, wherein (c) is a serine to threonine mutation.

29. The variant may be: (i) 71 (His), 118 (Asp), and 203 (Ser) of SEQ ID NO: 1; and / or (ii) The glycomodified polypeptide of any one of claims 26 to 28, comprising mutations at one or more or all of amino acids 180 (Phe), 181 (Phe), 228 (Leu), or 229 (Phe) of SEQ ID NO:

1.

30. The one or more peptides are selected from the group consisting of: (i) SEQ ID NO: 1, with or without the signal peptide of SEQ ID NO: 6; (ii) SEQ ID NO: 40, with or without the signal peptide of SEQ ID NO: 39; or (iii) SEQ ID NO:

45. The glycomodified polypeptide of any one of claims 19 to 29, comprising a sequence having at least 85% identity to the PR3 polypeptide sequence provided in SEQ ID NO:

45.

31. 31. The glycomodified polypeptide of any one of claims 19 to 30, wherein the one or more polypeptides that specifically bind to an anti-PR3 autoantibody comprise an antibody agent comprising an antigen-binding fragment.

32. 32. The glycoengineered polypeptide of claim 31 , wherein the antibody agent comprises a whole antibody, a Fab fragment, a scFv, a nanobody, a duobody, or a single domain antibody (e.g., a VHH).

33. 19. A glycomodified polypeptide according to any one of claims 1 to 18, comprising a first portion comprising one or more peptides that specifically bind to an anti-MPO autoantibody or fragment thereof.

34. 34. The glycoengineered polypeptide of Claim 33, wherein said one or more peptides that specifically bind to an anti-MPO autoantibody are each conjugated to a second moiety.

35. 35. The glycoengineered polypeptide of claim 33 or 34, wherein said one or more peptides that specifically bind to anti-MPO autoantibodies are soluble polypeptides.

36. 36. The glycomodified polypeptide of any one of claims 33-35, wherein the one or more peptides that specifically bind to anti-MPO autoantibodies comprise an MPO polypeptide, or a fragment or variant thereof.

37. 37. The glycomodified polypeptide of claim 36, wherein the MPO polypeptide is provided as SEQ ID NO: 4 (e.g., with or without a signal peptide), SEQ ID NO: 42 (e.g., with or without a signal peptide), or SEQ ID NO:

46.

38. 38. The glycomodified polypeptide of claim 36 or 37, wherein the fragment comprises at least 5% of a full-length MPO polypeptide or an MPO polypeptide provided in SEQ ID NO:4 (e.g., with or without the signal peptide), SEQ ID NO:42 (e.g., with or without the signal peptide), or SEQ ID NO:

46.

39. 39. The glycomodified polypeptide of any one of claims 36 to 38, wherein said fragment comprises an epitope recognized by an MPO autoantibody.

40. 40. The glycomodified polypeptide of any one of claims 32-39, wherein the one or more peptides that specifically bind to anti-MPO autoantibodies are variants of an MPO polypeptide, optionally wherein the variants are inactive compared to a wild-type MPO protein.

41. 41. The glycomodified polypeptide of claim 40, wherein the variant comprises a mutation at one or more or all of amino acids 261 (His), 316 (Cys), 405 (Arg) and 257 (Gln) of SEQ ID NO:

4.

42. The one or more peptides that specifically bind to anti-MPO autoantibodies are selected from the group consisting of: (i) SEQ ID NO: 4, with or without the signal peptide of SEQ ID NO: 8; (iii) SEQ ID NO: 42, with or without the signal peptide of SEQ ID NO: 38, or (iii) the glycomodified polypeptide of any one of claims 32-41, comprising a sequence having at least 85% identity to the MPO polypeptide sequence provided in SEQ ID NO:

46.

43. 43. The glycomodified polypeptide of any one of claims 32-42, wherein the one or more peptides that specifically bind to anti-MPO autoantibodies comprise an antibody agent comprising an antigen-binding fragment.

44. 44. The glycoengineered polypeptide of claim 43, wherein the antibody agent comprises a full antibody, a Fab fragment, a scFv, a nanobody, a duobody, or a single domain antibody (e.g., a VHH).

45. 10. The glycoengineered polypeptide of any one of the preceding claims, wherein said first portion comprises (i) one or more anti-PR3 autoantibody binding polypeptides and (ii) one or more anti-MPO autoantibody binding polypeptides.

46. 10. A glycomodified polypeptide according to any one of the preceding claims, wherein said polypeptide comprises: (a) a linker, (b) a spacer, (c) a cleavage peptide, e.g., an IRES or protease cleavage site; (d) a signal peptide, (e) a tag, e.g., a cleavable tag; (f) a half-life prolonging domain, e.g., an Fc domain or albumin; (g) The glycomodified polypeptide, comprising one or more additional elements selected from any combination of (a) to (f).

47. 10. The glycoengineered polypeptide of claim 1, wherein said second moiety is conjugated to said first moiety in vivo.

48. 48. The glycomodified polypeptide of claim 47, wherein said conjugation occurs intracellularly, and optionally said cell is a Leishmania cell.

49. 47. The glycomodified polypeptide of any one of claims 1 to 46, wherein the second moiety is conjugated to the first moiety by chemical conjugation, optionally wherein the chemical conjugation comprises click chemistry.

50. A polynucleotide encoding a glycomodified polypeptide according to any one of the preceding claims.

51. A composition comprising a glycomodified polypeptide according to any one of claims 1 to 50.

52. 51. A composition comprising a population of glycomodified polypeptides according to any one of claims 1 to 50, wherein said population of glycomodified polypeptides has an N-glycan profile that is at least 30% homogeneous at one or more of said N-glycosylation site(s).

53. 53. The composition of claim 52, wherein the N-glycan profile comprises about 30% of the N-glycans of the structure provided in claim 13.

54. 54. The composition of claim 52 or 53, which is a pharmaceutical composition.

55. 51. A Leishmania host cell expressing a glycomodified polypeptide according to any one of claims 1 to 50, said cell comprising a polynucleotide sequence encoding the glycomodified polypeptide.

56. 55. A method comprising administering to a subject the pharmaceutical composition of claim 54.

57. 57. The method of claim 56, wherein the subject has or has been diagnosed with antineutrophil cytoplasmic antibody (ANCA) vasculitis.

58. 58. The method of claim 56 or 57, which is a therapeutic or prophylactic method.

59. 58. The method of claim 56 or 57, wherein the ANCA vasculitis is granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA (formerly called Wegener's granulomatosis).

60. 60. The method of any one of claims 56 to 59, wherein the glycomodified polypeptide, when administered to a subject, is capable of simultaneously binding to the target at the first portion and to an endocytic receptor-expressing cell at the second portion, thereby internalizing the target into the cell.

61. 61. The method of claim 60, wherein internalization comprises transport to lysosomes and / or degradation.

62. 62. The method of claim 60 or 61, wherein the endocytic receptor is ASGPR or a variant or fragment thereof.

63. 63. The method of any one of claims 56-62, wherein administration of the pharmaceutical composition reduces the level of anti-neutrophil antibodies compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition.

64. 64. The method of claim 63, wherein the reduction in the level of anti-neutrophil autoantibodies prevents neutrophil activation.

65. 65. The method of any one of claims 56-64, wherein administration of the pharmaceutical composition reduces one or more symptoms of ANCA vasculitis.

66. 55. A method for treating and / or preventing granulomatosis with polyangiitis (GPA) / cytoplasmic ANCA (formerly known as Wegener's granulomatosis) in a subject, comprising administering to the subject the pharmaceutical composition of claim 54, wherein the subject has anti-PR3 autoantibodies.

67. 67. The method of claim 66, wherein administration of the pharmaceutical composition reduces the level of PR3 autoantibodies compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition.

68. 55. A method for treating and / or preventing microscopic polyangiitis (MPA) / perinuclear ANCA in a subject, comprising administering to the subject the pharmaceutical composition of claim 54, wherein the subject has MPO autoantibodies.

69. 69. The method of claim 68, wherein administration of said pharmaceutical composition reduces the level of MPO autoantibodies compared to a subject not administered said pharmaceutical composition or compared to the same subject prior to administration of said pharmaceutical composition.

70. assessing the level of anti-neutrophil autoantibodies in a sample from the subject; and 55. A method comprising administering the pharmaceutical composition of claim 54 if the level of the anti-neutrophil autoantibody is higher than in a comparison subject.

71. 71. The method of any one of claims 56-70, wherein the administering step comprises intravenous, intraperitoneal, subcutaneous, intradermal, or intramuscular injection.

72. 72. The method of any one of claims 56 to 71, wherein the subject is a mammal.

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