Glyco-modified polypeptides targeting immunoglobulin A and complexes containing the same

Abstract

Provided herein are glycoengineered polypeptides comprising: a first portion comprising one or more peptides that specifically bind to a target antibody (e.g., IgA1 or an immune complex comprising same, gd-IgA1 or an immune complex comprising same, or an anti-gd-IgA1 autoantibody or an immune complex comprising same); 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 glycoengineered polypeptides. Further provided herein are compositions comprising the glycoengineered 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 / 478,578, filed January 5, 2023, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] IgA nephropathy (IgAN) is generally associated with the accumulation of IgA immune complexes in renal tissue. IgA immune complexes (also called renal deposits) may contain IgA antibodies and complement components 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 elevated and / or abnormal IgA (e.g., IgA1 nephropathy, or IgAN). For example, the present disclosure identifies limitations of some of the most widely used treatment options. Blood pressure medications and proteinuria management therapies are some of the most common therapies for treating diseases associated with elevated and / or abnormal IgA (e.g., IgAN). However, these treatment options do not directly affect and / or reduce (1) IgA1 levels or immune complexes containing IgA1, (2) galactose-deficient IgA1 (gd-IgA1) levels or immune complexes containing IgA1, or (3) anti-gd-IgA1 autoantibodies or immune complexes containing IgA1, (4) immune complexes containing IgA, or (5) antigenic variants of IgG. Thus, while currently available treatment options may provide some relief to patients, these treatment options do not treat the underlying cause of the disease.

[0004] The present disclosure also identifies limitations in certain nonspecific B cell-directed therapies proposed for the treatment of IgAN. For example, atacicept is a fusion protein that nonspecifically inhibits B cells by blocking their activation (see, e.g., Hans-Peter Hartung and Bernd C. Kieseier, Ther Adv Neurol Disord. 2010 Jul;3(4):205-216). Atacicept results in the nonspecific depletion of B cells, including antibody-producing plasma cells, reducing the levels of all antibodies in the subject. This nonspecific depletion of B cells is undesirable and may lead to undesirable side effects such as organ damage or immunosuppression. Furthermore, nonspecific therapies such as atacicept are likely to require longer-term administration to achieve the desired results, e.g., a clinical response. Furthermore, nonspecific therapies such as atacicept are unlikely to produce a rapid response due to their lack of specificity and targeting of disease-causing components, e.g., gd-IgA1 or immune complexes containing it, IgA1 or immune complexes containing it, or anti-gd-IgA1 or immune complexes containing it. Such nonspecific therapies also fail to disrupt and / or remove pathogenic immune complexes.

[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 increased and / or abnormal IgA, such as atacicept. The technology provided herein specifically targets autoantigens or immune complexes containing autoantibodies or immune complexes containing autoantibodies, and produces a rapid, specific, and sustained response when administered to a subject. In some embodiments, the technology provided herein is expected to produce 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 autoantigens and / or 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 periods than, for example, non-specific B cell-directed therapies. In some embodiments, the technology provided herein can destroy and / or remove immune complexes containing autoantigens and / or autoantibodies disclosed herein. In some embodiments, the technology provided herein can result in improved depletion and / or removal of specific autoantigens or autoantibodies, or immune complexes comprising them, e.g., gd-IgA1 and / or anti-gd-IgA1, compared to nonspecific B cell-directed therapies. Advantages associated with the technology disclosed herein can improve therapeutic responses (e.g., clinical responses) in patients having or at risk of having a disease associated with elevated and / or abnormal IgA (e.g., IgAN).

[0006] Among other things, the present disclosure provides techniques for degrading and / or removing immunogenic autoantigens by providing glycoengineered polypeptides that can simultaneously bind to a target antibody (e.g., IgA1 or an immune complex comprising it, gd-IgA1 or an immune complex comprising it, or an anti-gd-IgA1 autoantibody or an immune complex comprising it) and an endocytic receptor via one or more glycans, thereby depleting, reducing, and / or removing: (1) IgA1 levels or immune complexes comprising it, (2) galactose-deficient IgA1 (gd-IgA1) levels or immune complexes comprising it; or (3) anti-gd-IgA1 autoantibodies or immune complexes comprising it; (4) immune complexes comprising IgA; or (5) antigenic variants of IgG. Without wishing to be bound by theory, the present disclosure proposes that binding of the glycoengineered polypeptides disclosed herein to the target antibody and the endocytic receptor induces internalization of the target antibody into the cell. In some embodiments, internalization of the target antibody results in degradation. The technology disclosed herein also relates to nucleic acid molecules encoding the glycomodified polypeptides disclosed herein. Additionally, provided herein are compositions comprising the glycomodified polypeptides disclosed herein or nucleic acid molecules encoding same, and methods for making same.

[0007] The technology provided herein is particularly useful for reducing and / or eliminating target antibodies in subjects having or at risk of having a disease associated with elevated and / or abnormal IgA (e.g., IgAN). Administration of a composition comprising a glycoengineered polypeptide disclosed herein, or a nucleic acid molecule encoding same, can reduce and / or deplete target 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 a target antibody, 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 complex comprising the targeting antibody is or comprises an immune complex.

[0010] In some embodiments, the targeting antibody comprises galactose-deficient IgA1 (gd-IgA1), or a fragment or conjugate thereof.

[0011] In some embodiments, the target antibody comprises IgA1, or a fragment or complex thereof.

[0012] In some embodiments, the targeting antibody comprises an autoantibody that specifically binds to gd-IgA1 (an "anti-gd-IgA1 autoantibody"), or a fragment or complex thereof.

[0013] In some embodiments, the second moiety specifically binds to one or more endocytic receptors. 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 glycan structure comprises a biantennary GalNAc.

[0015] In some embodiments, the bisecting GalNac binds to the asialoglycoprotein receptor (ASGPR) or a fragment or variant thereof, or a complex comprising ASGPR.

[0016] The present disclosure also provides compositions comprising (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to IgA1, or a fragment or complex thereof, (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to gd-IgA1, or a fragment or complex thereof, and / or (iii) a third glycoengineered polypeptide comprising a first portion that specifically binds to an anti-gd-IgA1 autoantibody, or a fragment or complex thereof. In some embodiments, the compositions are pharmaceutical compositions.

[0017] Further provided herein are methods comprising administering to a subject a pharmaceutical composition comprising a glycomodified polypeptide disclosed herein or a nucleic acid encoding same.

[0018] Provided herein are methods comprising assessing the level of a target antibody in a sample from a subject, and if the level of the target antibody is higher than a comparison control, administering a pharmaceutical composition comprising a glycoengineered polypeptide disclosed herein or a nucleic acid encoding same.

[0019] 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.

[0020] In some embodiments, the subject has or is diagnosed with a disease associated with elevated and / or abnormal IgA. In some embodiments, the disease associated with elevated and / or abnormal IgA is IgA nephropathy. In some embodiments, the disease associated with elevated and / or abnormal IgA is dermatitis herpetiformis. In some embodiments, the disease associated with elevated and / or abnormal IgA is Henoch-Schönlein purpura.

[0021] In some embodiments, the method is a therapeutic method.

[0022] In some embodiments, the method is a prophylactic method.

[0023] 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]

[0024] [Figure 1]

[0023] Figures 1A-1C show exemplary configurations of glycoengineered polypeptide constructs described herein. For example, configurations can be used in glycoengineered polypeptides that specifically bind to IgA1, gd-IgA1, or anti-gd-IgA1. Exemplary configurations, shown in the 5' to 3' direction, are: (1) VHH-glycotag-cleavable HIS-tag; (2) HIS-tag-glycotag-VHH; (3) VHH-glycotag; (4) glycotag-VHH; (5) VHH-half-Fc-glycotag; (6) VHH-albumin domain-glycotag; (7) albumin domain-VHH-glycotag; (8) VHH-VHH-glycotag; (9) glycotag-VHH-VHH; (10) VHH-VHH-glycotag-HIS-tag; and (11) His-tag-glycotag-VHH-VHH.

[0025] [Figure 2]

[0033] Exemplary configurations of glycoengineered polypeptide constructs described herein are shown. For example, the configurations can be used for glycoengineered polypeptides comprising CD89 polypeptides or fragments thereof that specifically bind to IgA1. The glycoengineered polypeptides can contain native N-glycosylation sites, mutations in native N-glycosylation sites, engineered N-glycosylation sites (glycotags), or any combination thereof.

[0026] [Figure 3] Schematic diagram of the depletion assay protocol for the experiments in Example 1. Female Wistar rats were intravenously injected with human IgA at -0.5 h, followed by a subcutaneous injection of 2.0 mg of an exemplary soluble CD89 glycoengineered polypeptide (designated G-LyTAC in the figure; 4 rats) or control PBS solution (4 rats) at 0 h. Serum levels of total human IgA (free + bound) were quantified 1, 3, 6, 10, 24, and 48 h after treatment with sCD89 glycoengineered polypeptide or PBS.

[0027] [Figure 4] Depletion of IgA antibodies in rat serum using exemplary soluble CD89 glycoengineered polypeptides is shown in the format depicted in Figure 3. Results are expressed as the percentage of human IgA antibodies remaining in serum after treatment with sCD89 glycoengineered polypeptides (triangle markers) or PBS vehicle (circle markers) at each time point. DETAILED DESCRIPTION OF THE INVENTION

[0028] 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.

[0029] IgA1: The term "IgA1" is used herein to refer to an immunoglobulin A1 polypeptide as understood in the art. IgA1 belongs to the IgA family and is the major immunoglobulin produced in the mucosa. The IgA1 protein is encoded by the IGHA1 gene. The amino acid sequence of full-length IgA1 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 the human IgA1 constant region (SEQ ID NO: 1) can be found under UniProt / Swiss-Prot accession number P01876; nucleic acid sequences encoding human IgA1 can be readily found by those skilled in the art. Those skilled in the art will understand that the sequence presented in SEQ ID NO: 1 is exemplary and that certain variations (including, for example, conservative substitutions in SEQ ID NO: 1, codon-optimized variants of nucleic acid sequences encoding human IgA1, etc.) are also understood to be human IgA1 or to encode human IgA1; further, those skilled in the art will understand that homologs and orthologs of human IgA1 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.

[0030] gd-IgA1: As used herein, the term "gd-IgA1" or "galactose-deficient IgA1" refers to IgA1 having a glycan profile that differs from a reference glycan profile of IgA1. The reference glycan profile can be that of IgA1 from a healthy individual or a subject not at risk of having IgA nephropathy. In some embodiments, the reference glycan profile comprises 1, 2, 3, 4, 5, or 6 O-glycan chains. In some embodiments, gd-IgA1 has reduced glycosylation in the hinge region compared to the glycosylation of the hinge region of IgA1 in a healthy individual or a subject not at risk of having IgA nephropathy. In some embodiments, reduced glycosylation comprises reduced O-linked glycosylation. In some embodiments, reduced glycosylation comprises reduced galactosylation. In some embodiments, reduced galactosylation comprises altered glycosylation with fewer galactose moieties compared to the glycosylation of IgA1 in healthy individuals or subjects not at risk for IgA nephropathy. In some embodiments, gd-IgA1 has a glycan profile that includes terminal GalNAc. In some embodiments, gd-IgA1 has a glycan profile that includes terminal GalNAc with alpha 2,6-linked sialic acid. In some embodiments, sialylation of terminal GalNAc blocks effective galactosylation.

[0031] 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.

[0032] Glycoengineering: As used herein, the term "glycoengineering" or equivalents thereof 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, and / or modify N-linked glycosylation. Host cell systems used for glycoengineering (e.g., to produce glycoengineered proteins) can introduce, remove, and / or modify O-linked glycosylation. Host cells used for glycoengineering or to produce glycoengineered target proteins 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.

[0033] Glycoengineered Polypeptide: As used herein, a "glycoengineered polypeptide" is a polypeptide that specifically binds to a target protein (e.g., a targeting antibody) 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.

[0034] 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.

[0035] 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.

[0036] Targeted antibody: As used herein, the term "targeted antibody" refers to (1) IgA1, a fragment thereof, or an immune complex comprising same; (2) galactose-deficient IgA1 (gd-IgA1), a fragment thereof, or an immune complex comprising same; (3) an anti-gd-IgA1 autoantibody, a fragment thereof, or an immune complex comprising same; (4) an immune complex comprising IgA; or (5) an antigenic variant of IgG. In some embodiments, the immune complex comprises one or more antibodies, antigens recognized by one or more antibodies or autoantibodies, and / or one or more components of the complement system. In some embodiments, the complement components comprise C3, C5b, C6, C7, C8, and / or C9, or a fragment of any complement component, or a combination thereof. In some embodiments, the immune complex comprises C3 or a fragment of C3. In some embodiments, the C3 fragment comprises iC3b, C3c, C3dg, or a combination thereof. In some embodiments, the immune complex comprises one or more antibodies selected from IgG, IgA, IgM, IgD, IgE, or fragments or combinations thereof. In some embodiments, the target antibody comprises a gd-IgA1 antibody, a fragment thereof, or an immune complex comprising same. In some embodiments, the target antibody comprises an IgA1 antibody, a fragment thereof, or an immune complex comprising same. In some embodiments, the target antibody comprises an anti-gd-IgA1 autoantibody, a fragment thereof, or an immune complex comprising same. In some embodiments, the anti-gd-IgA1 autoantibody is IgG or IgM. In some embodiments, the target antibody comprises one or more antigenic variants of IgG, for example, as disclosed herein.

[0037] Diseases Associated with Increased and / or Abnormal IgA: As used herein, the term "diseases associated with increased and / or abnormal IgA" refers to diseases in which the following are present: (1) increased IgA expression; (2) abnormal glycosylation of IgA compared to wild-type IgA; (3) accumulation of IgA or immune complexes containing IgA; and / or (4) the presence of autoantibodies that specifically bind to gd-IgA1 or immune complexes containing IgA. In some embodiments, IgA comprises IgA1 or gd-IgA1. In some embodiments, increased or altered IgA expression comprises increased or abnormal expression of IgA1. In some embodiments, increased or altered IgA expression comprises increased or abnormal expression of gd-IgA1. In some embodiments, abnormally glycosylated IgA comprises IgA1 with reduced and / or altered glycosylation in the hinge region compared to the glycosylation of a reference IgA1 (e.g., wild-type IgA1). In some embodiments, increased or abnormal glycosylation comprises glycosylation of IgA1 with fewer galactose moieties compared to glycosylation of IgA1 in healthy individuals or subjects not at risk of having IgA nephropathy. In some embodiments, reduced glycosylation comprises reduced O-linked glycosylation. In some embodiments, the disease associated with increased and / or abnormal IgA is characterized by immune deposits comprising immune complexes (e.g., those described herein) observed in one or more tissues (e.g., renal tissue) or organs (e.g., kidney). In some embodiments, the immune deposits may accumulate in glomeruli. In some embodiments, the immune deposits activate mesangial cells. In some embodiments, the immune deposits induce renal injury. In some embodiments, the disease associated with increased and / or abnormal IgA comprises IgA nephropathy (e.g., primary IgA nephropathy or secondary IgA nephropathy). In some embodiments, the disease associated with increased and / or abnormal IgA comprises dermatitis herpetiformis. In some embodiments, the disease associated with increased and / or abnormal IgA comprises Henoch-Schönlein purpura.

[0038] About: When used herein in connection with a value, the term "about" refers to a value similar to the reference value in the context. Generally, a person skilled in the art who is 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.

[0039] 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 / ).

[0040] 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.

[0041] 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 the target autoantibody has high affinity for the 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 intact antibodies. 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 (e.g., camelid VHHs and / or bivalent 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.)).

[0047] 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.

[0048] 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, generally, 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.

[0049] 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).

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.).

[0056] 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).

[0057] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a characteristic property and / or activity.

[0058] Fragment: A "fragment" of a material or entity as described herein comprises a discrete 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 discrete 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.

[0059] 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.

[0060] Human: In some embodiments, the human is an embryo, fetus, infant, child, teenager, adult, or elderly.

[0061] Humanized: As known in the art, the term "humanized" refers to an antibody whose amino acid sequence is derived from a reference antibody raised in a non-human species (e.g., mouse). H and V L The term "humanized" is generally used to refer to antibodies (or antibody components) that contain variable domain sequences but also contain modifications of these sequences relative to a 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 the CDR regions of a non-human immunoglobulin (i.e., 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. 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.

[0062] Identity: 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 for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0) can be used to determine the percent identity between two nucleotide sequences. In some exemplary embodiments, comparison of nucleic acid sequences using 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.

[0063] "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.

[0064] 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.

[0065] 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.

[0066] 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. (including 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, may be or may include 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.

[0067] 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 simultaneously 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.

[0068] 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.

[0069] 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).

[0070] 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.

[0071] 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, proceed 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.

[0072] 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.

[0073] 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 with another amino acid of the same type may often be considered a "homologous" substitution. Typical amino acid classifications are summarized below: [Table 3-1] [Table 3-2] 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., "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.

[0074] 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.

[0075] 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.

[0076] 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. (Mode for Carrying Out the Invention)

[0077] Disclosed herein are glycoengineered polypeptides and compositions comprising the same that have the ability to degrade one or more target antibodies (e.g., IgA1 or an immune complex comprising same, gd-IgA1 or an immune complex comprising same, or anti-gd-IgA1 autoantibodies or an immune complex comprising same) by binding to the target antibody at a first site and binding to an endocytic receptor at a second site comprising one or more glycans, thereby targeting the target antibody for degradation. As exemplified herein, glycoengineered polypeptides are modified by introducing glycosylation sites onto the glycoengineered polypeptide, resulting in an altered glycosylation profile that mediates endocytic receptor degradation of the glycoengineered polypeptide and the target antibody to which it is bound.

[0078] 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 increased and / or abnormal IgA, e.g., IgA nephropathy. Diseases associated with increased and / or abnormal IgA

[0079] Immunoglobulin A (IgA) is the major immunoglobulin class found in human mucosal secretions. IgA is a polymeric antibody, typically containing two copies of IgA assembled in a single binding chain to form dimeric IgA. This dimeric IgA immunoglobulin protein reaches the gastrointestinal and respiratory tract fluids by binding to another polypeptide chain called a "polymeric immunoglobulin receptor" produced by mucosal epithelial cells. Once the dimeric IgA antibody binds to this receptor, it is transported to the apical surface of the epithelial cells via an endocytic pathway and released into the mucosal fluid cavity as secretory IgA (sIgA) (Selvskandan et al. Frontiers in Immunology, 2020 and Boyd et al. Kidney International, 2012).

[0080] IgA includes two isotypes, IgA1 and IgA2. The main difference between isotypes IgA1 and IgA2 is in the hinge region of the heavy chain polypeptide; the IgA2 hinge region is characterized by a deletion of 13 amino acids (Yamasaki K et al. Monoclon Antib Immunodiagn Immunother, 2018). IgA1 and IgA2 are also found in human plasma and serum.

[0081] Those skilled in the art will understand, upon reading this disclosure, that the glycoengineered polypeptides disclosed herein (and / or the nucleic acid(s) encoding them, and / or compositions comprising and / or delivering either) can be useful for treating a disease associated with elevated and / or abnormal IgA in a subject. In some embodiments, a disease associated with elevated and / or abnormal IgA is characterized by increased or elevated levels of IgA (e.g., circulating IgA1 and / or circulating gd-IgA1) and / or immune complexes comprising same, compared to healthy subjects who do not have or are not at risk of developing a disease associated with elevated and / or abnormal IgA. In some embodiments, a disease associated with elevated and / or abnormal IgA is characterized by having or having galactose-deficient gd-IgA1 and / or immune complexes comprising same, as described herein. In some embodiments, a disease associated with elevated and / or abnormal IgA is characterized by having or having anti-gd-IgA1 autoantibodies or immune complexes comprising same.

[0082] Deposition of immunoglobulin A1 (IgA1) in human tissues and organs is a hallmark of several human diseases, including IgA nephropathy (IgAN), dermatitis herpetiformis (DH), and Henoch-Schönlein purpura (HS) (Hall, RP & TJ Lawley, J. Immunol. (1985) 135(3): 1760-5, Clarindo MV et al., Bras Dermatol, 2014, and Xu L et al. Front Immunology, 2022). IgA1 deposition can be associated with various clinical symptoms, including renal failure, skin blisters, rash, arthritis, gastrointestinal bleeding, and abdominal pain.

[0083] In some embodiments, the disease associated with increased and / or abnormal IgA has or is characterized by IgA deposits (e.g., IgA1 deposits, gd-IgA1 deposits, and / or anti-gd-IgA1 deposits). Without wishing to be bound by any particular theory, it is suggested that IgA deposits may result from the accumulation of immune complexes as described herein.

[0084] Available treatment options for patients with abnormal IgA1 deposition include administration of corticosteroids, which have immunosuppressive and anti-inflammatory properties, dietary fish oil supplements to reduce renal inflammation, and angiotensin-converting enzyme inhibitors, which reduce the risk of progressive renal disease and failure. However, such treatments do not reduce levels of IgA1, gd-IgA1, and / or anti-gd-IgA1, or directly affect and / or remove IgA1, gd-IgA1, and / or anti-gd-IgA1 deposits in tissues or organs.

[0085] Diseases associated with increased and / or abnormal IgA include IgA nephropathy, dermatitis herpetiformis (DH), and Henoch-Schönlein purpura (HS). IgA nephropathy

[0086] IgAN is a kidney disease. It is considered an immune complex-mediated glomerulonephritis and is characterized by the deposition of IgA alone or in combination with other immunoglobulins (e.g., IgG and / or IgM) and / or complement components in the glomerular mesangium (Wyatt RJ and Julian BA, (2013) NEJM 368:25). Nephropathy results, which is defined by proliferative changes in glomerular mesangial cells. IgAN is one of the most common types of chronic glomerulonephritis and a frequent cause of end-stage renal disease.

[0087] Without wishing to be bound by any particular theory, it has been suggested that IgAN may develop through the following four steps: The first step is the appearance of elevated levels of abnormally O-galactosylated IgA1 (e.g., galactose-deficient IgA1 [gd-IgA1]) in the circulation. This IgA1 may also exhibit reduced O-linked sialylation and reduced N-acetylgalactosamine (GalNAc) residues in the hinge region of IgA1. The second step is the production of IgG and / or IgA autoantibodies against the abnormally O-galactosylated hinge region of gd-IgA1; and the third step is the formation of anti-gd-IgA1:gd-IgA1 immune complexes. The fourth step is the variable development of inflammatory and fibrotic processes in the kidney caused by the deposition of anti-gd-IgA1:gd-IgA1 immune complexes in the mesangium (Selvskandan et al. Frontiers in Immunology, 2020).

[0088] In some embodiments, the anti-gd-IgA1 is an IgG-type anti-gd-IgA1.

[0089] In some embodiments, the anti-gd-IgA1 is an IgM-type anti-gd-IgA1.

[0090] In some embodiments, the anti-gd-IgA1 is an IgE-type anti-gd-IgA1.

[0091] In some embodiments, the anti-gd-IgA1 is an IgD-type anti-gd-IgA1.

[0092] In some embodiments, IgA deposition occurs due to the accumulation of IgA1 immune complexes (e.g., gd-IgA1 immune complexes and / or IgA1 immune complexes with normal O-glycosylation). In some embodiments, IgA deposition occurs due to the accumulation of gd-IgA1 immune complexes. In some embodiments, IgA deposition occurs due to the accumulation of IgA1 immune complexes. In some embodiments, IgA deposition occurs due to the accumulation of anti-gd-IgA1 immune complexes, for example, together with gd-IgA1 antigens (Selvskandan, H. et al. Frontiers in Immunology, 2020). In some embodiments, immune complexes comprising IgA1, gd-IgA1, and / or anti-gd-IgA1 comprise an antigen recognized by an antibody, one or more components of the complement system, one or more additional immunoglobulins, or a combination thereof.

[0093] In some embodiments, the disease associated with increased and / or abnormal IgA is IgA nephropathy (IgAN). In some embodiments, the present disclosure provides methods for treating IgAN by delivering a glycomodified polypeptide or a composition comprising same, e.g., as disclosed herein, to a patient in need of such treatment.

[0094] Current therapies for IgAN include blood pressure medications, proteinuria management therapies, nephroprotective agents (e.g., SGLT2 inhibitors), and glucocorticoids. In some embodiments, one or more additional therapeutic agents (e.g., one or more of the listed current therapeutic agents) may be administered before, substantially simultaneously with, or after one or more provided glycoengineered polypeptides. Dermatitis herpetiformis (DH)

[0095] In some embodiments, the disease associated with increased and / or abnormal IgA is dermatitis herpetiformis (DH). In some embodiments, the present disclosure provides methods for treating dermatitis herpetiformis (DH) by delivering a glycoengineered polypeptide or a composition comprising the same, e.g., as disclosed herein, to a patient in need of such treatment.

[0096] Dermatitis herpetiformis is characterized by a chronic bullous skin disease with IgA1 deposits at the dermo-epidermal junction (Hall, RP & TJ Lawley, J. Immunol. (1985) 135(3):1760-5). Patients with DH have granular IgA1 deposits and often have associated gluten-sensitive enteropathy (GSE).

[0097] Current treatments for dermatitis herpetiformis include oral antibiotics such as dapsone. In some embodiments, one or more additional therapeutic agents (e.g., one or more of the current therapeutic agents listed) may be administered before, substantially simultaneously with, or after one or more provided glycoengineered polypeptides. Henoch-Schönlein purpura (HS)

[0098] In some embodiments, the disease associated with increased and / or abnormal IgA is Henoch-Schönlein purpura (HS). In some embodiments, the present disclosure provides methods for treating Henoch-Schönlein purpura (HS) by delivering a glycomodified polypeptide or a composition comprising the same, e.g., as disclosed herein, to a patient in need of such treatment.

[0099] Henoch-Schönlein purpura (HSP) is a disease of the skin, blood vessels, and kidneys. HSP is characterized by the deposition of IgA1-containing immune complexes in tissues. The disease is often diagnosed by observing evidence of IgA1 deposition in skin tissue or kidneys via immunofluorescence microscopy. Clinical symptoms typically include a rash, joint pain, abdominal pain, and renal disease.

[0100] Current treatments for Henoch-Schönlein purpura include corticosteroids. In some embodiments, one or more additional therapeutic agents (e.g., one or more of the listed current therapeutic agents) may be administered before, substantially simultaneously with, or after one or more provided glycomodified polypeptides. Target antibodies (IgA1, gd-IgA1, and anti-gd-IgA1)

[0101] IgAN, the most common primary glomerulonephritis, is typically diagnosed by pathological evaluation of a kidney biopsy showing IgA-containing immune deposits (Knoppova et al., Frontiers in Immunology (2016) volume 7, article 117). The immune deposits found in IgAN contain IgA1, including gd-IgA1, complement components, and / or other immunoglobulins such as anti-gd-IgA1.

[0102] Disclosed herein, inter alia, is the discovery that glycoengineered polypeptides comprising a first portion that specifically binds to a target antibody can be used to treat and / or prevent diseases associated with elevated and / or abnormal IgA or to ameliorate one or more symptoms associated with elevated and / or abnormal IgA.

[0103] In some embodiments, the targeting antibody disclosed herein comprises IgA1, or a fragment thereof, or an immune complex comprising same.

[0104] In some embodiments, the targeting antibody disclosed herein comprises gd-IgA1, or a fragment thereof, or an immune complex comprising same.

[0105] In some embodiments, the targeting antibody disclosed herein comprises an anti-gd-IgA1 autoantibody or a fragment thereof, or an immune complex comprising the same. In some embodiments, the anti-gd-IgA1 autoantibody is IgG. In some embodiments, the anti-gd-IgA1 autoantibody is IgM. In some embodiments, the anti-gd-IgA1 autoantibody is IgE. In some embodiments, the anti-gd-IgA1 autoantibody is IgD.

[0106] In some embodiments, the targeting antibodies disclosed herein comprise an immune complex comprising the targeting antibody. In some embodiments, the immune complexes disclosed herein comprise one or more antibodies, antigens recognized by the one or more antibodies, and / or one or more components of the complement system. In some embodiments, the complement components comprise C3, C5b, C6, C7, C8, and / or C9, or a fragment of any complement component, or a combination thereof. In some embodiments, the immune complex comprises C3 or a fragment of C3. In some embodiments, the C3 fragment comprises iC3b, C3c, C3dg, or a combination thereof. In some embodiments, the immune complex comprises one or more antibodies selected from IgG, IgA, IgM, IgD, IgE, or a fragment or combination thereof. Glycomodified polypeptides

[0107] Disclosed herein are glycoengineered polypeptides comprising: (a) a first portion comprising one or more peptides that specifically bind to a target antibody, 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.

[0108] In some embodiments, the target antibody is an IgA1 antibody, or a fragment or complex thereof. In some embodiments, the first portion of the glycoengineered polypeptide comprises one or more peptides that specifically bind to an IgA1 antibody, or a fragment or complex thereof. In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody, or a fragment or complex thereof, comprise a CD89 polypeptide, or a fragment or variant thereof. In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody, or a fragment or complex thereof, comprise soluble CD89.

[0109] In some embodiments, the targeting antibody is a gd-IgA1 antibody, or a fragment or complex thereof. In some embodiments, the first portion of the glycoengineered polypeptide comprises one or more peptides that specifically bind to a gd-IgA1 antibody, or a fragment or complex thereof.

[0110] In some embodiments, the target antibody is an anti-gd-IgA1 autoantibody, or a fragment or complex thereof. In some embodiments, the first portion of the glycoengineered polypeptide comprises one or more peptides that specifically bind to an anti-gd-IgA1 autoantibody, or a fragment or complex thereof. In some embodiments, the one or more peptides comprise an epitope recognized by the target antibody (e.g., an anti-gd-IgA1 autoantibody) or a fragment thereof (e.g., an antigen-binding fragment of an anti-gd-IgA1 autoantibody). In some embodiments, the epitope comprises a fragment of gd-IgA1, such as the hinge region described herein. In some embodiments, the one or more peptides specifically bind to one or more idiotopes of an anti-gd-IgA1 autoantibody. In some embodiments, the one or more peptides are anti-idiotypic antibodies that specifically bind to an anti-gd-IgA1 autoantibody.

[0111] In some embodiments, the glycoengineered polypeptide is capable of binding to an IgA1 antibody, or a fragment or complex thereof; a gd-IgA1 antibody, or a fragment or complex thereof; and / or an anti-gd-IgA1 autoantibody, or a fragment or complex thereof, or a combination thereof.

[0112] In some embodiments, the glycoengineered polypeptide is capable of binding to one or more target antibodies in addition to an IgA1 antibody, or fragment or complex thereof; a gd-IgA1 antibody, or fragment or complex thereof; and / or an anti-gd-IgA1 autoantibody, or fragment or complex thereof, or a combination thereof.

[0113] 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.

[0114] 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) is or comprises GGGGANSTAPAPAPA (SEQ ID NO: xx).

[0115] 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.

[0116] In some embodiments, the glycomodified polypeptide comprises a spacer. In some embodiments, the spacer comprises one or more nucleotides that separate a first nucleic acid sequence from a subsequent nucleic acid sequence. In some embodiments, the spacer comprises a nucleic acid sequence encoding one or more peptides that separate the first encoded polypeptide sequence from a subsequent encoded polypeptide sequence.

[0117] 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.

[0118] 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.

[0119] In certain embodiments, the signal peptide is an invertase signal peptide from Leishmania tarentolae.

[0120] In certain embodiments, the signal peptide is the alkaline phosphatase signal peptide from Leishmania major.

[0121] In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 21, or a portion thereof. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 22, or a portion thereof. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 42, or a portion thereof. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 43, or a portion thereof. In certain embodiments, the signal peptide is processed and removed from the glycomodified polypeptide.

[0122] Exemplary signal peptide: SPinv, a modified signal peptide from Leishmania tarentolae invertase, SEQ ID NO: 21: MIASSVRHAVILLLVAVAMMAAVIA.

[0123] Exemplary signal peptide: SPinv, the native signal peptide from Leishmania tarentolae invertase, SEQ ID NO: 22: MIASSVRHAVILLLVAVAMMAAAVIA.

[0124] Exemplary signal peptide: The native signal peptide from Leishmania tarentolae invertase Spinv4, SEQ ID NO: 42: MIASSVRHAVILLLVAVAMMGGVIA

[0125] Exemplary signal peptide: The native signal peptide from Leishmania major alkaline phosphatase ("LmSAP"), SEQ ID NO: 43: MASRLVRVLAAAMLVAAAVS

[0126] 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 comprises a His tag, a Myc tag, or a GST tag. In some embodiments, the tag comprises a cleavable tag. In some embodiments, the tag is not a glycotag.

[0127] In some embodiments, the tag is a His tag (HHHHHHHHHH; SEQ ID NO: 40).

[0128] In some embodiments, the half-life extender comprises albumin or a fragment or variant thereof. In some embodiments, the half-life extender comprises an Fc variant.

[0129] In some embodiments, the glycoengineered polypeptides disclosed herein have the configuration shown in FIG.

[0130] In some embodiments, the glycoengineered polypeptides disclosed herein have the configuration shown in FIG. First part

[0131] In some embodiments, the glycoengineered polypeptide comprises a first portion that comprises one or more peptides that specifically bind to a target antibody or fragment thereof or a complex comprising same.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] In some embodiments, the first portion comprises one, two, three, four, five, or more peptides that specifically bind to a target antibody.

[0136] In some embodiments, one or more peptides of the first portion that specifically binds to the target antibody 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.

[0137] In some embodiments, one or more peptides of the first portion that specifically binds to the target antibody 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, tags, 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, tags, and / or linkers).

[0138] In some embodiments, the one or more peptides having different sequences include one or more peptides that specifically bind to the same target antibody. In some embodiments, the one or more peptides bind to different epitopes of the target antibody (e.g., different domains of the target antibody). In some embodiments, the one or more peptides having different sequences include one or more peptides that specifically bind to IgA1 or a fragment thereof. In some embodiments, the one or more peptides having different sequences include one or more peptides that specifically bind to gd-IgA1 or a fragment thereof. In some embodiments, the one or more peptides having different sequences include one or more peptides that specifically bind to an anti-gdIgA1 autoantibody or a fragment thereof.

[0139] In some embodiments, the one or more peptides having different sequences include one or more peptides that specifically bind to a first target antibody (e.g., IgA1 or a fragment thereof), one or more peptides that bind to a second target antibody (e.g., gd-IgA1 or a fragment thereof), and one or more peptides that bind to a third target antibody (e.g., an anti-gd-IgA1 autoantibody or a fragment thereof). In some embodiments, the one or more peptides having different sequences are separated by one or more intervening sequences (e.g., spacers, tags, and / or linkers). In some embodiments, the one or more peptides having different sequences are not separated by one or more intervening sequences (e.g., spacers, tags, and / or linkers).

[0140] 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.

[0141] 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.

[0142] In some embodiments, each of the one or more peptides that specifically bind to a target antibody is conjugated to a second moiety.

[0143] In some embodiments, each of the one or more peptides that specifically bind to a target antibody is not conjugated to a second moiety.

[0144] In some embodiments, one or more peptides that specifically bind to a target antibody are conjugated to one another, eg, located on a single polypeptide.

[0145] In some embodiments, one or more peptides that specifically bind to a target antibody 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, e.g., translated as a separate peptide by an IRES, or expressed as a separate peptide after cleavage of the protease cleavage site.

[0146] In some embodiments, the one or more peptides that specifically bind to a target antibody are not separated by a protease cleavage site or IRES, eg, are expressed as a fusion protein.

[0147] In some embodiments, the one or more peptides of the first portion that specifically bind to the target antibody comprise an epitope recognized by the target antibody. In some embodiments, the epitope is a linear epitope. In some embodiments, the epitope is a conformational epitope.

[0148] 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.

[0149] 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 a target antibody, 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.

[0150] 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 a target antibody, 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.

[0151] 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 target antibody. In some embodiments, the first portion comprises multiple different epitopes, for example, epitopes recognized by different target antibodies. In some embodiments, the multiple epitopes are separated by a linker, an IRES, or a cleavage peptide. Immunoglobulin A1 (IgA1) and peptides that bind to IgA1

[0152] In humans, there are two types of IgA subclasses: IgA1 and IgA2. IgA1 has O-glycans attached to serine or threonine residues in the hinge region of the heavy chain (Knoppova 2017). The hinge region of IgA2 does not have serine or threonine residues, and IgA2 does not have O-glycans.

[0153] The human IgA1 constant region polypeptide sequence is provided herein as SEQ ID NO:2: ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0154] In some embodiments, the glycomodified polypeptide comprises a first portion comprising one or more peptides that specifically bind to IgA1, hi some embodiments, the one or more peptides that specifically bind to IgA1 recognize an epitope on IgA1.

[0155] 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.

[0156] 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 a target antibody, X represents an intervening amino acid sequence, and n is an integer between 0 and 20.

[0157] 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 a target antibody, X represents an intervening amino acid sequence, and n is an integer from 0 to 20.

[0158] 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 IgA1 antibodies. In some embodiments, the first portion comprises multiple different epitopes, e.g., epitopes recognized by different IgA1 antibodies. In some embodiments, the multiple epitopes are separated by a linker, an IRES, or a cleavage peptide.

[0159] In some embodiments, the one or more peptides that specifically bind to anti-IgA1 antibodies comprise an epitope that is not present or accessible in IgA1 from a healthy individual or an individual not at risk of developing IgAN.

[0160] In some embodiments, the first portion comprises 1, 2, 3, 4, 5, or more peptides that specifically bind to IgA1 antibodies.

[0161] In some embodiments, one or more peptides of the first portion that specifically binds to an IgA1 antibody 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 (e.g., spacers and / or linkers).

[0162] In some embodiments, one or more peptides of the first portion that specifically binds to an IgA1 antibody 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).

[0163] In some embodiments, each of the one or more peptides having different sequences specifically binds to an IgA1 antibody or fragment thereof.

[0164] 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.

[0165] In some embodiments, each of the one or more peptides that specifically bind to an IgA1 antibody is conjugated to a second moiety.

[0166] In some embodiments, each of the one or more peptides that specifically bind to an IgA1 antibody is not conjugated to a second moiety.

[0167] In some embodiments, one or more peptides that specifically bind to IgA1 antibodies are conjugated to one another, eg, located on a single polypeptide.

[0168] In some embodiments, one or more peptides that specifically bind to an IgA1 antibody 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, e.g., translated as a separate peptide by an IRES, or expressed as a separate peptide after cleavage of the protease cleavage site.

[0169] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody are not separated by a protease cleavage site or IRES, eg, are expressed as a fusion protein.

[0170] In some embodiments, a glycoengineered polypeptide comprising a first portion comprising one or more peptides that specifically bind to an IgA1 antibody or fragment thereof has the configuration shown in FIG.

[0171] In some embodiments, the one or more peptides that specifically bind to an anti-IgA1 antibody comprise an antibody agent. In some embodiments, the antibody agent comprises an antigen-binding fragment. In some embodiments, the antibody agent comprises a whole 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. CD89 polypeptide as an IgA1 binding factor

[0172] CD89, also known as the immunoglobulin alpha Fc receptor (FcaR1), is a human myeloid IgA Fc receptor that binds both the IgA1 and IgA2 subclasses of IgA (Morton HC and Brandtzaeg P (2001) Arch Immunol Ther Exp(warsz) 49(3):217-29).

[0173] An exemplary human CD89 polypeptide sequence is provided herein as SEQ ID NO: 5, with bold indicating the signal sequence (corresponding to UniProt Accession No. P24071):

[0174] [ka]

[0175] Human CD89 signal peptide: MDPKQTTLLCLVLCLGQRIQA (SEQ ID NO: 44)

[0176] An exemplary human CD89 polypeptide sequence is provided herein as SEQ ID NO:9, with bold indicating the signal peptide of SEQ ID NO:44:

[0177] [ka]

[0178] The modified human CD89 polypeptide sequence is provided herein as SEQ ID NO:38, with bold indicating the signal peptide of SEQ ID NO:42:

[0179] [ka]

[0180] An exemplary modified CD89 polypeptide sequence without the signal peptide is provided herein as SEQ ID NO:39.

[0181] QEGDFPMPFISAKSSPVIPLDGSVKIQCQAIREAYLTQLMIIKNSTYREIGRRLKFWNETDPEFVIDHMDANKAGRYQCQYRIGHYRFRYSDTLELVVTGLYGKPFLSADRGLVLMPGEQISLTCSSAHIPFDRFSLAKEGELSLPQHQSGEHPAQFSLGPVDLQVSGIYRCYGWYQRSPYLWSFPSNALELVVT

[0182] In some embodiments, the one or more peptides, or fragments or conjugates thereof, in the first portion that specifically binds to IgA1 comprises a CD89 (FcaR1) polypeptide, or a variant thereof, or a fragment thereof.

[0183] In some embodiments, the one or more peptides comprise a soluble form of CD89, or a variant or fragment thereof. Soluble CD89 is described, for example, in van Zandbergen G et al., (1999) J Immunology 163, pp. 5806-5812; and van Der Boog PJM et al., (2002) J Immunology 168.3 pp. 1252-1258, the entire contents of each of which are expressly incorporated herein by reference.

[0184] In some embodiments, the glycoengineered polypeptide comprising a first portion comprising one or more peptides comprising a CD89 (FcaR1) polypeptide, or a variant or fragment thereof, has the configuration shown in FIG.

[0185] In some embodiments, the CD89 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: 5. In some embodiments, the CD89 polypeptide is or comprises SEQ ID NO: 5 or SEQ ID NO: 9 without the signal peptide of SEQ ID NO: 44.

[0186] In some embodiments, the CD89 polypeptide comprises a sequence having at least 85% identity to SEQ ID NO: 5 without the signal peptide of SEQ ID NO: 44. In some embodiments, the CD89 polypeptide comprises a sequence having at least 85% identity to SEQ ID NO: 9 without the signal peptide of SEQ ID NO: 44.

[0187] In some embodiments, a CD89 polypeptide comprising a sequence having at least 85% identity to SEQ ID NO: 5 without the signal peptide of SEQ ID NO: 44 further comprises a different signal peptide, e.g., as disclosed herein.

[0188] In some embodiments, a CD89 polypeptide comprising a sequence having at least 85% identity to SEQ ID NO: 9 without the signal peptide of SEQ ID NO: 44 further comprises a different signal peptide, e.g., as disclosed herein.

[0189] 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:5 or SEQ ID NO:9. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO:5 or SEQ ID NO:9. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO:5 without the signal peptide of SEQ ID NO:44. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO:9 without the signal peptide of SEQ ID NO:44.

[0190] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 5 without the signal peptide of SEQ ID NO: 44. In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 9 without the signal peptide of SEQ ID NO: 44.

[0191] In some embodiments, the first portion comprising one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 5 without the signal peptide of SEQ ID NO: 44 further comprises a different signal peptide, e.g., as disclosed herein.

[0192] In some embodiments, the first portion comprising one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 9 without the signal peptide of SEQ ID NO: 44 further comprises a different signal peptide, e.g., as disclosed herein.

[0193] In some embodiments, the CD89 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: 38. In some embodiments, the CD89 polypeptide is or comprises SEQ ID NO: 38 without the signal peptide of SEQ ID NO:42.

[0194] In some embodiments, the CD89 polypeptide comprises a sequence having at least 85% identity to SEQ ID NO:38 without the signal peptide of SEQ ID NO:42.

[0195] In some embodiments, a CD89 polypeptide comprising a sequence having at least 85% identity to SEQ ID NO: 38 without the signal peptide of SEQ ID NO: 42 further comprises a different signal peptide, e.g., as disclosed herein.

[0196] 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: 38. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 38. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 38 without the signal peptide of SEQ ID NO:42.

[0197] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO:38 without the signal peptide of SEQ ID NO:42.

[0198] In some embodiments, the first portion comprising one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 38 without the signal peptide of SEQ ID NO: 42 further comprises a different signal peptide, e.g., as disclosed herein.

[0199] In some embodiments, the CD89 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: 39. In some embodiments, the CD89 polypeptide is or comprises SEQ ID NO: 39.

[0200] In some embodiments, the CD89 polypeptide comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 39 comprises a signal peptide disclosed herein, e.g., a Leishmania-derived signal peptide or a CD89 signal peptide.

[0201] In some embodiments, the signal peptide is selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44.

[0202] 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: 39. In some embodiments, the first portion comprises one or more peptides comprising the sequence of SEQ ID NO: 39.

[0203] In some embodiments, the first portion comprises one or more peptides comprising a sequence having at least 85% identity to SEQ ID NO: 39 and a signal peptide, e.g., as disclosed herein. In some embodiments, the signal peptide is selected from SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44.

[0204] In some embodiments, the targeting antibody is an IgA1 antibody, or a fragment thereof. In some embodiments, the IgA1 antibody is characterized by binding to a CD89 polypeptide or a variant or fragment thereof.

[0205] In some embodiments, the first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides that specifically bind to an IgA1 antibody, hi some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a CD89 polypeptide, or a fragment or variant thereof.

[0206] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a soluble fragment of a CD89 polypeptide.

[0207] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 5 or SEQ ID NO: 9.

[0208] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 5 or SEQ ID NO: 9 without the signal peptide.

[0209] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 5 or SEQ ID NO: 9.

[0210] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 5 or SEQ ID NO: 9 without the signal peptide.

[0211] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 38.

[0212] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 38 without the signal peptide.

[0213] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 38.

[0214] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 38 without the signal peptide.

[0215] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 39.

[0216] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody comprise a fragment of a CD89 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 CD89 polypeptide of SEQ ID NO: 39.

[0217] In some embodiments, the fragment comprises a CD89 fragment that binds to IgA1. In some embodiments, the CD89 fragment that binds to IgA1 is a linear fragment. In some embodiments, the CD89 fragment that binds to IgA1 is a conformational fragment. In some embodiments, the fragment comprises one or more additional amino acid residues, for example, at the 5' and / or 3' end of the epitope.

[0218] In some embodiments, the IgA1-binding CD89 fragment comprises one or more sequences separated by one or more intervening amino acid sequences, which are configured to form a conformation to which IgA1 can bind. In some embodiments, the intervening amino acid sequences comprise a linker and / or spacer. For example, an IgA1-binding CD89 fragment 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 IgA1-binding CD89 fragment, A2 is a second portion of the IgA1-binding CD89 fragment, which together form a conformation to which IgA1 can bind, X represents an intervening amino acid sequence, and n is an integer of 0 to 20.

[0219] In some embodiments, the IgA1-binding CD89 fragment formed by one or more sequences can be broken down into three, four, five, or more fragments. For example, in such embodiments, the polypeptide may have the following structure: Xn-[A1]-Xn-[A2]-Xn-[An]-Xn, where A1 is a first portion of the IgA1-binding CD89 fragment, A2 is a second portion of the IgA1-binding CD89 fragment, and An is the nth portion of the IgA1-binding CD89 fragment, which together form a conformation to which IgA1 can bind, X represents an intervening amino acid sequence, and n is an integer between 0 and 20.

[0220] In some embodiments, the first portion comprises multiple CD89 fragments that bind to IgA1, e.g., the same or different CD89 fragments that bind to IgA1. In some embodiments, the first portion comprises multiple of the same CD89 fragments that bind to IgA1. In some embodiments, the first portion comprises multiple different CD89 fragments that bind to IgA1. In some embodiments, the multiple CD89 fragments that bind to IgA1 are separated by a linker, an IRES, or a truncation peptide.

[0221] In some embodiments, one or more peptides that specifically bind to IgA1 antibodies comprise a contiguous chain of amino acids that includes 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:5 or SEQ ID NO:9.

[0222] In some embodiments, one or more peptides that specifically bind to IgA1 antibodies comprise a continuous chain of amino acids that includes 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: 5 or SEQ ID NO: 9 without the signal peptide.

[0223] In some embodiments, one or more peptides that specifically bind to IgA1 antibodies 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: 38.

[0224] In some embodiments, one or more peptides that specifically bind to IgA1 antibodies 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: 39.

[0225] In some embodiments, one or more peptides that specifically bind to IgA1 antibodies 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.

[0226] In some embodiments, the one or more peptides that specifically bind to IgA1 antibodies comprise, for example, the full-length CD89 protein set forth in SEQ ID NO:5, with or without the signal peptide.

[0227] In some embodiments, the one or more polypeptides that specifically bind to an anti-IgA1 antibody comprise a variant of a CD89 polypeptide, hi some embodiments, the variant is an inactive variant compared to a wild-type CD89 polypeptide.

[0228] In some embodiments, the variant comprises a CD89 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 141, asparagine 177, asparagine 186, and / or asparagine 198.

[0229] In some embodiments, the glycomodified polypeptide comprises a CD89 polypeptide or fragment thereof having one or more native glycosylation sites. In some embodiments, the glycomodified polypeptide comprises a CD89 polypeptide or fragment thereof having two native glycosylation sites. In some embodiments, the native glycosylation sites comprise N65 and / or N79.

[0230] In some embodiments, the glycoengineered polypeptide comprises a CD89 polypeptide or a fragment thereof comprising the native glycosylation sites: N65 and N79.

[0231] In some embodiments, the glycomodified polypeptide comprises a CD89 polypeptide or fragment thereof having one or more modified glycosylation sites, hi some embodiments, the modified glycosylation site is or comprises GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0232] In some embodiments, the glycomodified polypeptide comprises a CD89 polypeptide or fragment thereof having two native glycosylation sites: N65 and N79, and one engineered glycosylation site having the sequence GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0233] 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).

[0234] In some embodiments, the glycoengineered polypeptide comprises a tag, eg, a His tag.

[0235] In some embodiments, the glycoengineered polypeptide comprises the following sequence (SEQ ID NO: 41):

[0236] MIASSVRHAVILLLVAVAMMGGVIAQEGDFPMPFISAKSSPVIPLDGSVKIQCQAIREAYLTQLMIIKNSTYREIGRRLKFWNETDPEFVIDHMDANKAGRYQCQYRIGHYRFRYSDTLELV VTGLYGKPFLSADRGLVLMPGEQISLTCSSAHIPFDRFSLAKEGELSLPQHQSGEHPAQFSLGPVDLQVSGIYRCYGWYQRSPYLWSFPSNALELVVTGGGGANSTAPAPAPAHHHHHHHHHH

[0237] 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.

[0238] In some embodiments, the first portion comprises 1, 2, 3, 4, 5, or more peptides that specifically bind to IgA1 antibodies.

[0239] In some embodiments, the one or more peptides of the first portion that specifically binds to an IgA1 antibody are the same, e.g., the one or more peptides have the same sequence of a CD89 polypeptide, or a fragment or variant thereof. In some embodiments, the one or more peptides having the same sequence of a CD89 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 a CD89 polypeptide, or a fragment or variant thereof, are not separated by one or more intervening sequences (e.g., spacers and / or linkers).

[0240] In some embodiments, one or more peptides of the first portion that specifically binds to an IgA1 antibody are different, e.g., one or more peptides do not have the same sequence of a CD89 polypeptide, or fragment or variant thereof. In some embodiments, one or more peptides having different sequences of a CD89 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 a CD89 polypeptide, or fragment or variant thereof, are not separated by one or more intervening sequences (e.g., spacers and / or linkers).

[0241] In some embodiments, each of the one or more peptides having a different sequence of a CD89 polypeptide, or a fragment or variant thereof, specifically binds to an IgA1 antibody or a fragment thereof.

[0242] 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.

[0243] In some embodiments, each of the one or more peptides that specifically bind to an IgA1 antibody is conjugated to a second moiety.

[0244] In some embodiments, each of the one or more peptides that specifically bind to an IgA1 antibody is not conjugated to a second moiety.

[0245] In some embodiments, one or more peptides that specifically bind to IgA1 antibodies are conjugated to one another, eg, located on a single polypeptide.

[0246] In some embodiments, one or more peptides that specifically bind to an IgA1 antibody 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, e.g., translated as a separate peptide by an IRES, or expressed as a separate peptide after cleavage of the protease cleavage site.

[0247] In some embodiments, the one or more peptides that specifically bind to an IgA1 antibody are not separated by a protease cleavage site or IRES, eg, are expressed as a fusion protein. Galactose-deficient IgA1 (gd-IgA1) and peptides that bind to gd-IgA1

[0248] As described herein, IgA1 has a hinge region containing serine and threonine residues that can be glycosylated. IgA1 has a hinge region containing nine serine and threonine amino acid residues, of which approximately three to six are typically attached to O-glycans (Knoppova 2017). In humans, IgA1 hinge region glycoforms with four and five glycans are most common.

[0249] As described by Knoppova (2017), each IgA1 heavy chain also contains two N-glycans: one in the CH2 domain (Asn263) and the second in the tailpiece (Asn459). Furthermore, normal human circulating IgA1 typically has a core 1 O-glycan consisting of N-acetylgalactosamine (GalNAc) with a β1,3-linked galactose. One or both sugars may be sialylated; the galactose is accompanied by an α2,3-linked sialic acid, and the GalNAc is accompanied by an α2,6-linked sialic acid.

[0250] The composition of O-glycans on normal serum IgA1 varies, with the most common form comprising GalNAc-galactose disaccharide and its monosialylated and disialylated forms. Typically, normal serum IgA1 has few or no galactose-deficient O-glycans, although it has been shown that terminal or sialylated GalNAc can be found in healthy individuals (Knoppova 2017).

[0251] IgA1 with abnormal O-glycosylation is commonly found in patients with IgA nephropathy. In particular, galactose-deficient IgA1 (gd-IgA1) has been shown to play a role in immune complex formation and glomerular deposition. Several reports suggest that IgA in mesangial deposits is primarily of the IgA1 subclass, with the Gd-IgA1 glycoform being enriched (Knoppova 2017).

[0252] A typical O-glycosylation profile of the IgA1 hinge region is described in detail in Knoppova et al., particularly Figure 3 therein. Knoppova et al., 2017, is incorporated herein by reference in its entirety.

[0253] As discussed in Knoppova 2017, normal serum IgA1 O-glycans consist primarily of the galactose-β1-3GalNAc disaccharide, also known as the T antigen, and its mono- and disialylated forms [NeuAcα2-3-galactose-β1-3GalNAc and NeuAcα2-3-galactose-β1-3(NeuAcα2-6)GalNAc, commonly referred to as sialic-T (ST) antigens] (see Figure 3, left panel in Knoppova 2017). After the initial addition of GalNAc to a Ser / Thr residue, galactose is added by UDP-galactose:GalNAc-α-Ser / Thr β1,3-galactosyltransferase (C1GalT1). The galactose-β1,3GalNAc structure is then modified by attachment of sialic acid from CMP-N-acetylneuraminic acid (CMP-NeuAc) to the galactose residue by the activity of galactose-β1,3GalNAcα2,3-sialyltransferase (ST3Gal) and / or to the GalNAc residue by the activity of α2,6-sialyltransferase (ST6GalNAc).

[0254] In some embodiments, gd-IgA1 comprises an aberrant glycosylation profile compared to a reference glycan profile. In some embodiments, the reference glycan profile disclosed herein comprises the O-glycosylation profile disclosed in Figure 3 of Knoppova et al. In some embodiments, the reference glycan profile disclosed herein comprises a glycan profile described herein. In some embodiments, gd-IgA1 comprises a glycosylation profile with a terminal GalNAc (also referred to as Tn antigen). In some embodiments, gd-IgA1 comprises a glycosylation profile with GalNAc with an alpha 2,6-linked sialic acid (also referred to as STn antigen). In some embodiments, sialylation of terminal GalNAc blocks effective galactosylation, resulting in gd-IgA1 with an aberrant glycosylation profile.

[0255] The IgA1 hinge region is provided as SEQ ID NO: 1: PVPSTPPTPSPSTPPTPSC.

[0256] In some embodiments, the IgA1 hinge region comprises a core sequence that is VPSTPPTPSPSTPPTPSPS (SEQ ID NO: 8).

[0257] In some embodiments, the hinge region comprises 9 O-glycosylation sites, hi some embodiments, at least 1, 2, 3, 4, 5, or 6 of the O-glycosylation sites are occupied.

[0258] In some embodiments, the first portion comprises one or more peptides that specifically bind to glycosylation-deficient IgA1 (gd-IgA1), or a fragment or complex thereof. In some embodiments, the one or more peptides bind to the hinge region of gd-IgA1. In some embodiments, the one or more peptides bind to SEQ ID NO: 1, or a portion or variant thereof. In some embodiments, the one or more peptides that bind to SEQ ID NO: 1 bind to one or more additional amino acid sequences other than SEQ ID NO: 1.

[0259] In some embodiments, one or more peptides bind to and / or recognize a glycan profile on gd-IgA1. In some embodiments, one or more peptides that specifically bind to gd-IgA1 recognize a glycan profile on gd-IgA1. The gd-IgA1 comprises a glycan profile that differs from a reference glycan profile for IgA1. In some embodiments, the gd-IgA1 comprises a glycan profile that has at least one fewer glycan than the reference glycan profile for IgA1.

[0260] In some embodiments, the reference glycan profile comprises an O-glycosylation profile. In some embodiments, the reference O-glycosylation profile comprises 1, 2, 3, 4, 5, or 6 O-glycan chains. In some embodiments, the O-glycan chains of the reference O-glycosylation profile comprise N-acetylgalactosamine (GalNAc). In some embodiments, the O-glycan chains further comprise galactose (Gal), sialic acid, or a combination thereof.

[0261] In some embodiments, the gd-IgA1 glycan profile comprises at least one less galactosylation compared to the reference glycan profile.

[0262] In some embodiments, the gd-IgA1 glycan profile contains at least 5% less galactosylation compared to the reference glycan profile.

[0263] In some embodiments, the gd-IgA1 glycan profile comprises increased sialylation compared to a reference glycan profile.

[0264] In some embodiments, the gd-IgA1 glycan profile comprises terminal GalNAc (also referred to as the Tn antigen).

[0265] In some embodiments, the gd-IgA1 glycan profile comprises GalNAc with alpha-2,6-linked sialic acid (also referred to as STn antigen).

[0266] In some embodiments, sialylation of the terminal GalNac blocks effective galactosylation.

[0267] In some embodiments, one or more peptides that specifically bind to gd-IgA1 recognize an epitope in the hinge region of gd-IgA1. In some embodiments, the change in glycan profile causes a conformational change in gd-IgA1. In some embodiments, the conformational change exposes a neoepitope recognized by one or more peptides. In some embodiments, the neoepitope is not present in non-galactose-deficient IgA1, e.g., IgA1 with a reference glycan profile. In some embodiments, the neoepitope is a linear epitope. In some embodiments, the neoepitope is a conformational epitope.

[0268] In some embodiments, the one or more peptides that specifically bind to gd-IgA1 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. Anti-gd-IgA1 autoantibodies and peptides that bind to anti-gd-IgA1

[0269] In IgA nephropathy autoimmunity, the gd-IgA1 autoantigen produces anti-gd-IgA1 autoantibodies. In some embodiments, anti-gd-IgA1 autoantibodies or immune complexes containing them are deposited in one or more tissues or organs. In some embodiments, anti-gd-IgA1 autoantibodies or immune complexes containing them contribute to and / or result in IgA nephropathy.

[0270] In some embodiments, the anti-gd-IgA1 autoantibody is an IgG antibody. In some embodiments, the anti-gd-IgA1 autoantibody is an IgA antibody. In some embodiments, the anti-gd-IgA1 autoantibody is an IgM antibody. In some embodiments, the anti-gd-IgA1 autoantibody is an IgD antibody. In some embodiments, the anti-gd-IgA1 autoantibody is an IgE antibody.

[0271] In some embodiments, the gd-IgA1 autoantigen is a gd-IgA1 polypeptide or a variant or fragment thereof. In some embodiments, the gd-IgA1 autoantigen is a glycan profile found on gd-IgA1. In some embodiments, the anti-gd-IgA1 autoantibody, fragment thereof, or complex is characterized by binding to a gd-IgA1 polypeptide or a variant or fragment thereof. In some embodiments, the anti-gd-IgA1 autoantibody, fragment thereof, or complex is characterized by binding to one or more glycans on gd-IgA1.

[0272] In some embodiments, anti-gd-IgA1 antibody specifically binds to the glycan profile on gd-IgA1. In some embodiments, the glycan profile that anti-gd-IgA1 binds to is not present in reference IgA1, for example, IgA1 from a healthy individual or an individual who is not at risk of developing IgAN. In some embodiments, the glycan profile that anti-gd-IgA1 binds to is the gd-IgA1 glycan profile described herein.

[0273] In some embodiments, the first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides, or fragments thereof, that specifically bind to one or more idiotopes of an anti-gd-IgA1 autoantibody. In some embodiments, the one or more peptides comprise an anti-idiotypic antibody or fragment thereof (e.g., antigen-binding fragment).

[0274] In some embodiments, the anti-gd-IgA1 IgG comprises a mutation in the complementarity-determining region 3 (CDR3) of the Ig heavy chain (IgH) variable region. In some embodiments, the mutation comprises an alanine to serine mutation. In some embodiments, the alanine to serine mutation occurs in the YCAR or YCAK amino acid sequence of CDR3 of IgH. Exemplary mutations in anti-gd-IgA1 autoantibodies are disclosed in U.S. Patent No. 9,655,963, the entire contents of which are incorporated herein by reference.

[0275] In some embodiments, the first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides that specifically bind to the CDR3 of the IgH region of an anti-gd-IgA1 autoantibody, or a fragment thereof, in some embodiments, the CDR3 of the IgH region of the anti-gd-IgA1 autoantibody comprises, for example, a mutation described herein.

[0276] In some embodiments, the first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides that specifically bind to a mutation in the CDR3 of the IgH region of an anti-gd-IgA1 autoantibody. In some embodiments, the one or more peptides bind to the YCAR amino acid sequence in the CDR3 of the IgH where the alanine has been mutated to serine. In some embodiments, the one or more peptides bind to the YCAK amino acid sequence in the CDR3 of the IgH where the alanine has been mutated to serine.

[0277] In some embodiments, one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies bind to an IgG protein, or a fragment or variant thereof. In some embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IgG protein has a mutation in the CDR3 region.

[0278] In some embodiments, the wild-type IgG1 constant region polypeptide is provided as SEQ ID NO:3.

[0279] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0280] In some embodiments, the wild-type IgG4 constant region is provided as SEQ ID NO:4.

[0281] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0282] In some embodiments, a first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides, or fragments thereof, comprising an epitope recognized by an anti-gd-IgA1 autoantibody. In some embodiments, a first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides, or fragments or conjugates thereof, that compete with gd-IgA1 for binding to an anti-gd-IgA1 autoantibody. In some embodiments, a first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides, or fragments or conjugates thereof, that block binding of gd-IgA1 to an anti-gd-IgA1 autoantibody. In some embodiments, blocking binding of gd-IgA1 to an anti-gd-IgA1 autoantibody, or fragments or conjugates thereof, reduces and / or prevents the formation and / or deposition of immune complexes comprising anti-gd-IgA1 autoantibodies in a tissue or organ.

[0283] In some embodiments, the first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides, or fragments or conjugates thereof, that do not compete with gd-IgA1 for binding to anti-gd-IgA1 autoantibodies.

[0284] In some embodiments, the first portion of a glycoengineered polypeptide disclosed herein comprises one or more peptides that specifically bind to an anti-gd-IgA1 autoantibody. In some embodiments, the one or more peptides that specifically bind to an anti-gd-IgA1 autoantibody comprise a gd-IgA1 polypeptide, or a fragment or variant thereof.

[0285] In some embodiments, the one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies comprise gd-IgA1 polypeptide fragments. In some embodiments, the fragments comprise the sequence of SEQ ID NO: 1, or fragments or variants thereof. In some embodiments, the fragments comprise one or more additional amino acid residues at the 5' and / or 3' end of the sequence.

[0286] In some embodiments, the fragment comprises a gd-IgA1 polypeptide fragment having a glycan profile that differs from, for example, a reference glycan profile described herein. In some embodiments, the gd-IgA1 polypeptide fragment has a glycan profile with terminal GalNAc. In some embodiments, the gd-IgA1 has a glycan profile with terminal sialylated GalNAc. In some embodiments, the fragment comprises a gd-IgA1 amino acid sequence in which the GlcNAc or GalNAc is capped with sialic acid.

[0287] In some embodiments, the fragment comprises an epitope recognized by an anti-gd-IgA1 autoantibody. In some embodiments, the epitope comprises one or more glycans. In some embodiments, the epitope comprises a glycan profile, such as a gd-IgA1 glycan profile described herein.

[0288] 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-gd-IgA1 autoantibodies. In some embodiments, the first portion comprises multiple different epitopes, for example, epitopes recognized by different anti-gd-IgA1 autoantibodies. In some embodiments, the multiple epitopes are separated by a linker, an IRES, or a cleavage peptide.

[0289] In some embodiments, one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies comprise a fragment, e.g., an epitope, that is not present in or is inaccessible to IgA1 polypeptides from healthy individuals or individuals not at risk of developing IgAN.

[0290] In some embodiments, the first portion comprises one, two, three, four, five, or more peptides that specifically bind to anti-gd-IgA1 autoantibodies.

[0291] In some embodiments, one or more peptides of the first portion that specifically binds to an anti-gd-IgA1 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 (e.g., spacers and / or linkers).

[0292] In some embodiments, one or more peptides of the first portion that specifically binds to an anti-gd-IgA1 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).

[0293] 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.

[0294] In some embodiments, each of the one or more peptides that specifically bind to an anti-gd-IgA1 autoantibody is conjugated to a second moiety.

[0295] In some embodiments, each of the one or more peptides that specifically bind to an anti-gd-IgA1 autoantibody is not conjugated to a second moiety.

[0296] In some embodiments, one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies are conjugated to each other, eg, located on one polypeptide.

[0297] In some embodiments, one or more peptides that specifically bind to anti-gd-IgA1 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.

[0298] In some embodiments, the one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies are not separated by a protease cleavage site or IRES, eg, are expressed as a fusion protein.

[0299] In some embodiments, the one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies 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 one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies comprise a VHH, such as a camelid-derived VHH or a bivalent VHH. Second part

[0300] The glycoengineered polypeptides disclosed herein comprise a first portion that specifically binds to a target antibody, 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.

[0301] 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.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] In some embodiments, provided herein are glycomodified polypeptides comprising a first portion that specifically binds to a target antibody and a second portion comprising a glycan comprising a terminal GlcNAc.

[0306] In some embodiments, provided herein are glycomodified polypeptides comprising a first portion that specifically binds to a target antibody and a second portion comprising a glycan comprising a terminal GalNAc.

[0307] In some embodiments, provided herein are glycomodified polypeptides comprising a first portion that specifically binds to a target antibody and a second portion comprising a glycan comprising a terminal Gal.

[0308] 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 antibodies and (ii) binding specificity for one or more endocytic receptor(s).

[0309] In some embodiments, the glycoengineered polypeptide comprises one type of N-glycan that has binding specificity for one type of endocytic receptor.

[0310] 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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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).

[0315] 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).

[0316] 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.

[0317] 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.

[0318] In some embodiments, the glycosite is or comprises the sequence GGGGANSTAPAPAPA (SEQ ID NO: 37).

[0319] In some embodiments, the N-glycans are conjugated to the glycoengineered polypeptide at at least 1, 2, 3, or 4 N-glycosylation sites.

[0320] In some embodiments, the N-glycans are conjugated to the glycoengineered polypeptide at 1, 2, 3, or 4 N-glycosylation sites.

[0321] In some embodiments, the N-glycosylation site is naturally occurring.

[0322] In some embodiments, an N-glycosylation site is engineered into the amino acid sequence of the first portion.

[0323] 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.

[0324] In some embodiments, provided herein is a glycoengineered polypeptide that specifically binds to a targeting antibody associated with a disease disclosed herein, the glycoengineered polypeptide comprising a first portion and a second portion. In some embodiments, provided herein is a glycoengineered polypeptide comprising a first portion that specifically binds to a targeting antibody associated with a disease disclosed herein and a second portion that specifically binds to an endocytic receptor, wherein the second portion comprises a glycan structure.

[0325] In some embodiments, provided herein is a method for preparing a nucleotide sequence comprising administering to a subject a first moiety that specifically binds to a target protein and ... and a second portion comprising an N-glycan selected from the group consisting of GlcNAc2, GalNAc4GlcNAc4Man3GlcNAc2, Gal4GlcNAc4Man3GlcNAc2, or Man-6-PN-glycan.

[0326] 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.

[0327] 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.

[0328] In some embodiments, glycoengineered polypeptides disclosed herein comprise glycan structures with monoantennary structures.

[0329] In some embodiments, glycoengineered polypeptides disclosed herein comprise glycan structures having biantennary structures.

[0330] In some embodiments, glycoengineered polypeptides disclosed herein comprise a glycan structure having a triantennary structure.

[0331] In some embodiments, glycoengineered polypeptides disclosed herein comprise a glycan structure having a tetraantennary structure.

[0332] 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.

[0333] In some embodiments, the N-glycan has the following structure: [ka] 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.

[0334] 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.

[0335] 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.

[0336] 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 antibodies (e.g., those disclosed herein). 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.

[0337] 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.

[0338] 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.

[0339] 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] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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).

[0344] 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.

[0345] In certain embodiments, the glycoengineered polypeptide is glycosylated at two or more N-glycosylation sites with an N-glycan of the following structure: [ka] 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. Nucleic acid sequences encoding glycoengineered polypeptides

[0346] The present disclosure provides, inter alia, nucleic acids encoding the glycomodified polypeptides described herein.

[0347] In some embodiments, the nucleic acid 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).

[0348] 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.

[0349] 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. Vectors comprising any of the nucleic acids described herein, or fragments thereof, are further provided by the present disclosure. 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 constructing them are generally known to those of skill in the art.

[0350] 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.

[0351] 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). Compositions and pharmaceutical compositions

[0352] 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.

[0353] 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.

[0354] In some embodiments, a composition comprising multiple glycoengineered polypeptides comprises 1, 2, 3, 4, 5, or more glycoengineered polypeptides that comprise a first moiety that binds to a target antibody (e.g., first moieties that bind to the same target antibody).

[0355] In some embodiments, a composition comprising a plurality of glycoengineered polypeptides comprises glycoengineered polypeptides having a first portion that binds to gd-IgA1 or a 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 IgA1 or a fragment thereof, a second glycoengineered polypeptide comprises a different first portion that binds to IgA1 or a fragment thereof, etc.).

[0356] In some embodiments, a composition comprising a plurality of glycoengineered polypeptides comprises glycoengineered polypeptides having a first portion that binds to gd-IgA1 or a 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 gd-IgA1 or a fragment thereof, a second glycoengineered polypeptide comprises a different first portion that binds to gd-IgA1 or a fragment thereof, etc.).

[0357] In some embodiments, a composition comprising a plurality of glycoengineered polypeptides comprises glycoengineered polypeptides having a first portion that binds to an anti-gd-IgA1 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-gd-IgA1 autoantibody or fragment thereof, a second glycoengineered polypeptide comprises a different first portion that binds to an anti-gd-IgA1 autoantibody or fragment thereof, etc.).

[0358] 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 target antibody (e.g., IgA1 or a fragment thereof, or gd-IgA1 or a fragment thereof, or an anti-gd-IgA1 autoantibody or a 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 a target antibody (e.g., IgA1 or a fragment thereof), a second glycoengineered polypeptide comprising a first portion that binds to a different target antibody (e.g., gd-IgA1 or a fragment thereof), and a third glycoengineered polypeptide comprising a first portion that binds to a different target antibody (e.g., an anti-gd-IgA1 autoantibody or a fragment thereof).

[0359] In some embodiments, the glycoengineered polypeptides disclosed herein comprise: (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to IgA1, or a fragment or complex thereof; (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to gd-IgA1, or a fragment or complex thereof; and (iii) a third glycoengineered polypeptide comprising a first portion that specifically binds to an anti-gd-IgA1 autoantibody, or a fragment or complex thereof.

[0360] In some embodiments, the glycoengineered polypeptides disclosed herein comprise: (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to IgA1, or a fragment or complex thereof, and (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to a target antibody other than IgA1, or a fragment or complex thereof.

[0361] In some embodiments, the glycoengineered polypeptides disclosed herein comprise: (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to gd-IgA1, or a fragment or complex thereof, and (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to a target antibody other than gd-IgA1, or a fragment or complex thereof.

[0362] In some embodiments, the glycoengineered polypeptides disclosed herein comprise: (i) a first glycoengineered polypeptide comprising a first portion that specifically binds to an anti-gd-IgA1 autoantibody, or a fragment or complex thereof; and (ii) a second glycoengineered polypeptide comprising a first portion that specifically binds to a target antibody, or a fragment or complex thereof, other than an anti-gd-IgA1 autoantibody.

[0363] 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.

[0364] 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.

[0365] In some embodiments, the ratio of first glycomodified polypeptide to second glycomodified polypeptide is about 1:1.5 to about 1.5:1.

[0366] In some embodiments, the ratio of the first glycomodified polypeptide to the third 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.

[0367] In some embodiments, the ratio of first glycomodified polypeptide to third glycomodified polypeptide is from about 1:5 to about 5:1; from about 1:2.5 to about 2.5:1.

[0368] In some embodiments, the ratio of the first glycomodified polypeptide to the third glycomodified polypeptide is about 1:1.5 to about 1.5:1.

[0369] In some embodiments, the ratio of the second glycoengineered polypeptide to the third 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.

[0370] In some embodiments, the ratio of second glycomodified polypeptide to third glycomodified polypeptide is from about 1:5 to about 5:1; from about 1:2.5 to about 2.5:1.

[0371] In some embodiments, the ratio of the second glycomodified polypeptide to the third glycomodified polypeptide is about 1:1.5 to about 1.5:1.

[0372] In some embodiments, the first glycomodified polypeptide is present in an amount of about 10-90% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 90-10%.

[0373] In some embodiments, the first glycomodified polypeptide is present in an amount of about 20-80% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 80-20%.

[0374] In some embodiments, the first glycomodified polypeptide is present in an amount of about 30-70% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 70-30%.

[0375] In some embodiments, the first glycomodified polypeptide is present in an amount of about 40-60% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 60-40%.

[0376] In some embodiments, the first glycomodified polypeptide is present in an amount of about 10% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 90%.

[0377] In some embodiments, the first glycomodified polypeptide is present in an amount of about 20% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 80%.

[0378] In some embodiments, the first glycomodified polypeptide is present in an amount of about 30% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 70%.

[0379] In some embodiments, the first glycomodified polypeptide is present in an amount of about 40% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 60%.

[0380] In some embodiments, the first glycomodified polypeptide is present in an amount of about 50% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 50%.

[0381] In some embodiments, the first glycomodified polypeptide is present in an amount of about 60% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 40%.

[0382] In some embodiments, the first glycomodified polypeptide is present in an amount of about 70% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 30%.

[0383] In some embodiments, the first glycomodified polypeptide is present in an amount of about 80% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 20%.

[0384] In some embodiments, the first glycomodified polypeptide is present in an amount of about 90% and the additional (e.g., second or third) glycomodified polypeptide is present in an amount of about 10%.

[0385] In some embodiments, disclosed herein are compositions comprising a population of glycomodified polypeptides disclosed herein, 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).

[0386] 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.

[0387] 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.

[0388] 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.

[0389] In some embodiments, the glycomodified 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 increased and / or abnormal IgA), or for ameliorating symptoms associated with a disease, disorder, or condition described herein.

[0390] 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 increased and / or abnormal IgA. 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.

[0391] In some embodiments, the pharmaceutical composition is or comprises a composition according to the present disclosure.

[0392] 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.).

[0393] 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).

[0394] 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).

[0395] 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. Methods of Treatment and / or Prevention

[0396] The present disclosure provides, inter alia, methods for treating and / or preventing a disease associated with increased and / or abnormal IgA (e.g., IgAN) in a subject, comprising administering a composition described herein, thereby improving at least one sign or symptom of a disease associated with increased and / or abnormal IgA (e.g., IgAN) in the subject after administration.

[0397] In some embodiments, provided herein are methods for treating and / or preventing diseases associated with increased and / or abnormal IgA (e.g., IgAN), comprising administering a composition described herein.

[0398] Among other things, disclosed herein is the identification of glycoengineered polypeptides that specifically bind to one or more target antibodies. In some embodiments, the glycoengineered polypeptides disclosed herein have therapeutic value, for example, in the treatment of diseases associated with increased and / or abnormal IgA (e.g., IgAN).

[0399] Glycoengineered polypeptides that specifically bind to one or more target antibodies of the present disclosure can be used to treat, prevent, and / or ameliorate, among other diseases associated with increased and / or abnormal IgA (e.g., IgAN).

[0400] Additionally or alternatively, glycoengineered polypeptides that specifically bind to one or more target antibodies of the present disclosure can be used to treat, prevent, and / or ameliorate a number of diseases, among others, in which target antibody levels are abnormally high and / or in which reduction of target antibody levels is desired.

[0401] 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 increased and / or abnormal IgA (e.g., IgAN).

[0402] Those skilled in the art reading this disclosure will understand that the provided compositions may be useful for treating diseases associated with elevated and / or abnormal IgA (e.g., IgAN). In some embodiments, a subject having a disease associated with elevated and / or abnormal IgA (e.g., IgAN) has or is characterized by elevated levels of a target antibody and / or immune complexes comprising the same, e.g., compared to a healthy subject or a subject not at risk for developing a disease associated with elevated and / or abnormal IgA (e.g., IgAN). In some embodiments, a subject having a disease associated with elevated and / or abnormal IgA (e.g., IgAN) has or is characterized by having an abnormal target antibody and / or immune complexes comprising the same, e.g., compared to a healthy subject or a subject not at risk for developing a disease associated with elevated and / or abnormal IgA (e.g., IgAN).

[0403] In some embodiments, the targeting antibody disclosed herein comprises IgA1, or a fragment thereof, or an immune complex comprising same.

[0404] In some embodiments, the targeting antibody disclosed herein comprises gd-IgA1, or a fragment thereof, or an immune complex comprising same.

[0405] In some embodiments, the targeting antibody disclosed herein comprises an anti-gd-IgA1 autoantibody, or a fragment thereof, or an immune complex comprising the same. In some embodiments, the anti-gd-IgA1 autoantibody is IgG. In some embodiments, the anti-gd-IgA1 autoantibody is IgM. In some embodiments, the anti-gd-IgA1 autoantibody is IgE. In some embodiments, the anti-gd-IgA1 autoantibody is IgD.

[0406] In some embodiments, the subject has increased levels of IgA1 or immune complexes containing IgA1 compared to subjects without a disease associated with increased and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the composition reduces the level of IgA1 or immune complexes containing IgA1 compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition. In some embodiments, the reduced level of IgA1 or immune complexes containing IgA1 comprises degradation of IgA1 or immune complexes containing IgA1. In some embodiments, the reduced level of IgA1 or immune complexes containing IgA1 is the result of internalization into cells. In some embodiments, internalization comprises transport to lysosomes and / or degradation. In some embodiments, administration of the pharmaceutical composition prevents IgA1 from binding to an antigen or binding partner. In some embodiments, the binding partner of IgA1 comprises CD89, e.g., soluble CD89.

[0407] In some embodiments, the subject has increased levels of IgA1 or immune complexes containing IgA1 compared to subjects without a disease associated with increased and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the pharmaceutical composition prevents and / or reduces the formation of immune complexes containing IgA1. In some embodiments, the immune complexes disclosed herein comprise IgA1, an antigen recognized by IgA1, one or more components of the complement system, and / or one or more additional immunoglobulins. In some embodiments, the complement components comprise C3, C5b, C6, C7, C8, and / or C9, or fragments of any complement component or combinations thereof. In some embodiments, the immune complex comprises C3 or a fragment of C3. In some embodiments, the C3 fragment comprises iC3b, C3c, C3dg, or a combination thereof. In some embodiments, the immune complex comprises one or more antibodies selected from IgG, IgA, IgM, IgD, IgE, or fragments or combinations thereof.

[0408] In some embodiments, the subject has increased levels of IgA1 or immune complexes containing IgA1 compared to a subject who does not have a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the pharmaceutical composition reduces activation of the complement system compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition. In some embodiments, the complement system comprises the lectin pathway, the alternative pathway, or the classical pathway, or a combination thereof.

[0409] In some embodiments, the subject has increased levels of IgA1 or immune complexes containing IgA1 compared to a subject without IgAN, hi some embodiments, administration of the pharmaceutical composition prevents and / or reduces IgA deposition in the kidneys of the subject compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition.

[0410] In some embodiments, a subject has detectable levels of gd-IgA1 or immune complexes comprising it compared to subjects without a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the composition reduces the level of gd-IgA1 or immune complexes comprising it compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition. In some embodiments, the reduction in the level of gd-IgA1 or immune complexes comprising it comprises degradation of gd-IgA1 or immune complexes comprising it. In some embodiments, the reduction in the level of gd-IgA1 or immune complexes comprising it is the result of internalization into cells. In some embodiments, internalization comprises transport to lysosomes and / or degradation.

[0411] In some embodiments, the subject has increased levels of gd-IgA1 or immune complexes containing gd-IgA1 compared to subjects without a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the pharmaceutical composition prevents and / or reduces the formation of immune complexes containing gd-IgA1. In some embodiments, the immune complexes disclosed herein comprise gd-IgA1, an antigen recognized by gd-IgA1, one or more components of the complement system, and / or one or more additional immunoglobulins. In some embodiments, the complement components comprise C3, C5b, C6, C7, C8, and / or C9, or fragments of any complement component or combinations thereof. In some embodiments, the immune complexes comprise C3 or a fragment of C3. In some embodiments, the C3 fragments comprise iC3b, C3c, C3dg, or combinations thereof. In some embodiments, the immune complex comprises one or more antibodies selected from IgG, IgA, IgM, IgD, IgE, or fragments or combinations thereof.

[0412] In some embodiments, the subject has increased levels of gd-IgA1 or immune complexes containing same compared to a subject without a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the pharmaceutical composition reduces activation of the complement system compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition. In some embodiments, the complement system comprises the lectin pathway, the alternative pathway, or the classical pathway, or a combination thereof.

[0413] In some embodiments, the subject has increased levels of gd-IgA1 or immune complexes containing same, compared to subjects who do not have a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the pharmaceutical composition prevents and / or reduces gd-IgA1 deposition in the subject's kidneys, compared to a subject who has not been administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition.

[0414] In some embodiments, a subject has detectable levels of anti-gd-IgA1 or immune complexes comprising same compared to subjects without a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the composition reduces the levels of anti-gd-IgA1 or immune complexes comprising same compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition. In some embodiments, the reduction in the levels of anti-gd-IgA1 or immune complexes comprising same comprises degradation of gd-IgA1 or immune complexes comprising same. In some embodiments, the reduction in the levels of anti-gd-IgA1 or immune complexes comprising same is the result of internalization into cells. In some embodiments, internalization comprises transport to lysosomes and / or degradation.

[0415] In some embodiments, the subject has increased levels of anti-gd-IgA1 or immune complexes containing the same compared to subjects without a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of a pharmaceutical composition prevents and / or reduces the formation of immune complexes containing anti-gd-IgA1. In some embodiments, the immune complexes disclosed herein comprise anti-gd-IgA1, an antigen recognized by anti-gd-IgA1, one or more components of the complement system, and / or one or more additional immunoglobulins. In some embodiments, the complement components comprise C3, C5b, C6, C7, C8, and / or C9, or fragments of any complement component, or combinations thereof. In some embodiments, the immune complexes comprise C3 or a fragment of C3. In some embodiments, the C3 fragments comprise iC3b, C3c, C3dg, or combinations thereof. In some embodiments, the immune complex comprises one or more antibodies selected from IgG, IgA, IgM, IgD, IgE, or fragments or combinations thereof.

[0416] In some embodiments, the subject has increased levels of anti-gd-IgA1 or immune complexes containing same compared to a subject without a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the pharmaceutical composition reduces activation of the complement system compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition. In some embodiments, the complement system comprises the lectin pathway, the alternative pathway, or the classical pathway, or a combination thereof.

[0417] In some embodiments, the subject has increased levels of anti-gd-IgA1 or immune complexes containing same, compared to subjects who do not have a disease associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, administration of the pharmaceutical composition prevents and / or reduces anti-gd-IgA1 deposition in the kidneys of the subject, compared to a subject who has not been administered the pharmaceutical composition, or compared to the same subject before administration of the pharmaceutical composition.

[0418] Additionally or alternatively, glycoengineered polypeptides (or compositions comprising same) that specifically bind to one or more targeting antibodies of the present disclosure can be used to inhibit elevated production of gd-IgA1. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject reduces the production of gd-IgA1. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject modulates enzyme expression in IgA1-producing cells, e.g., such that the expressed IgA1 is not gd-IgA1. In some embodiments, the expressed IgA1 has a glycan profile similar to a reference glycan profile. In some embodiments, the reference glycan profile is an IgA1 glycan profile produced in a healthy individual.

[0419] In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a subject reduces the number of cells that secrete gd-IgA1.

[0420] Additionally or alternatively, glycoengineered polypeptides (or compositions comprising same) that specifically bind to one or more targeting antibodies of the present disclosure can be used to modulate the production of anti-gd-IgA1 autoantibodies. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject reduces the production of anti-gd-IgA1 autoantibodies. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject depletes cells that produce anti-gd-IgA1 autoantibodies. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject manipulates the affinity maturation of anti-gd-IgA1 autoantibodies, reducing their affinity for the autoantigen (gd-IgA1). In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject removes anti-gd-IgA1 autoantibodies from the circulation.

[0421] Additionally or alternatively, glycoengineered polypeptides (or compositions comprising same) that specifically bind to one or more targeting antibodies of the present disclosure can be used to inhibit the formation of pathogenic IgA1-containing immune complexes. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject prevents and / or inhibits immune complex formation, and / or promotes the clearance of immune complexes from the circulation, and / or promotes the catabolism of immune complexes. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject blocks an epitope on an autoantigen (Gd-IgA1) with a non-crosslinking antibody. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject inhibits binding of anti-gd-IgA1 autoantibodies to antigens bearing epitope-bearing glycopeptides or glycomimetics. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject prevents and / or inhibits complement activation.

[0422] Additionally or alternatively, glycoengineered polypeptides (or compositions comprising same) that specifically bind to one or more targeting antibodies of the present disclosure can be used to prevent glomerular deposition and / or damage. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject inhibits mesangial cell activation. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject reduces complement activation in situ. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject prevents and / or inhibits binding of IgA1-containing immune complexes to mesangial cells. In some embodiments, administration of a glycoengineered polypeptide disclosed herein to a cell, tissue, or subject prevents and / or inhibits mesangial cell signaling induced by IgA1-containing immune complexes. Administration

[0423] 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.

[0424] 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).

[0425] 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.

[0426] In some embodiments, the administering step comprises intravenous, intraperitoneal, subcutaneous, intradermal, or intramuscular injection.

[0427] In particular, disclosed herein is the identification of glycoengineered polypeptides and compositions comprising same that have therapeutic value, for example, in the treatment of diseases associated with increased and / or abnormal IgA (e.g., IgAN).

[0428] In some embodiments, glycoengineered polypeptides and compositions comprising same are delivered to a subject suffering from or susceptible to a disorder associated with increased and / or abnormal IgA as described herein (e.g., by administering a pharmaceutical composition described herein that contains or delivers such an agent, or a nucleic acid encoding same).

[0429] In some embodiments, the subject is a human.

[0430] In some embodiments, the subject has detectable levels of one or more target antibodies, eg, IgA1, gd-IgA1, and / or anti-gd-IgA1.

[0431] In some embodiments, compositions according to the present disclosure are delivered to a subject suffering from or susceptible to a disease associated with increased and / or abnormal IgA (e.g., IgAN).

[0432] In some embodiments, the subject has or has been diagnosed with a disease associated with increased and / or abnormal IgA (e.g., IgAN). In some embodiments, the subject is human.

[0433] In some embodiments, administration of a composition according to the present disclosure reduces one or more symptoms of a disease associated with elevated and / or abnormal IgA (e.g., IgAN).

[0434] In some embodiments, administration of a glycoengineered polypeptide that binds to IgA1 or a fragment or complex thereof treats and / or prevents diseases associated with increased and / or abnormal IgA (eg, IgAN).

[0435] In some embodiments, administration of a glycoengineered polypeptide that binds to gd-IgA1 or a fragment or complex thereof treats and / or prevents diseases associated with increased and / or abnormal IgA (e.g., IgAN).

[0436] In some embodiments, administration of a glycoengineered polypeptide that binds to anti-gd-IgA1 or a fragment or conjugate thereof treats and / or prevents diseases associated with increased and / or abnormal IgA (e.g., IgAN).

[0437] In some embodiments, the methods disclosed herein are therapeutic methods.

[0438] In some embodiments, the methods disclosed herein are prophylactic methods. Methods for assessing target antibody levels

[0439] Also provided herein are methods of assessing the level of a target antibody in a sample from a subject, and administering a pharmaceutical composition comprising a glycoengineered polypeptide disclosed herein if the level of the target antibody is higher than a comparator. In some embodiments, the comparator comprises a predetermined reference sample, such as a sample obtained from an otherwise similar subject who is free of the disease or disorder or symptoms of the disease or disorder.

[0440] In some embodiments, the disease or disorder is IgA nephropathy.

[0441] In some embodiments, the disease or disorder is dermatitis herpetiformis.

[0442] In some embodiments, the disease or disorder is Henoch-Schönlein purpura.

[0443] In some embodiments, the level of target antibodies is assessed using an assay that detects the level and / or activity of IgA1, gd-IgA1, or anti-gd-IgA1, or a combination thereof.

[0444] In some embodiments, the assay is a dot blot assay.

[0445] In some embodiments, the assay is a capture ELISA.

[0446] An exemplary assay that can be used to detect levels of anti-gd-IgA1 or immune complexes containing it is disclosed in U.S. Pat. No. 9,655,963, the entire contents of which are incorporated herein by reference.

[0447] Among other things, the present disclosure provides the discovery that assays, such as those disclosed in U.S. Patent No. 9,655,963, for detecting anti-gd-IgA1 or immune complexes comprising same can be used to identify subjects having or at risk of developing a disease associated with elevated and / or abnormal IgA (e.g., IgAN). In some embodiments, subjects identified as having or at risk of developing a disease associated with elevated and / or abnormal IgA, e.g., IgAN, have increased levels of anti-gd-IgA1 or immune complexes comprising same compared to healthy subjects. In some embodiments, subjects identified as having or at risk of developing a disease associated with elevated and / or abnormal IgA, e.g., IgAN, can benefit from the administration of a glycoengineered polypeptide as disclosed herein.

[0448] Additional assays that can be used to detect levels of anti-gd-IgA1 or immune complexes containing it are disclosed in Suzuki H. et al., (2009) J Clin Investigation 119:6, the entire contents of which are incorporated herein by reference. Suzuki teaches a dot blot assay for detecting glycan-specific IgG antibodies in IgAN patients. The assay disclosed in Suzuki can distinguish IgAN patients from healthy controls with high specificity and sensitivity.

[0449] Among other things, the present disclosure provides the discovery that assays, such as those disclosed in Suzuki et al., 2009, for detecting anti-gd-IgA1 or immune complexes comprising same can be used to identify subjects having or at risk of developing a disease associated with elevated and / or abnormal IgA (e.g., IgAN). In some embodiments, subjects identified as having or at risk of developing a disease associated with elevated and / or abnormal IgA, e.g., IgAN, have increased levels of anti-gd-IgA1 or immune complexes comprising same compared to healthy subjects. In some embodiments, subjects identified as having or at risk of developing a disease associated with elevated and / or abnormal IgA, e.g., IgAN, can benefit from the administration of a glycoengineered polypeptide as disclosed herein. Dosing regimen

[0450] In some embodiments, the method comprises administering the composition once. In some embodiments, the method comprises administering the composition repeatedly.

[0451] In some embodiments, administration of the composition continues to maintain remission (e.g., to maintain low and / or undetectable levels of the target antibody and / or immune complexes comprising same) and / or to avoid relapse.

[0452] 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.

[0453] In some embodiments, the glycoengineered polypeptide according to the present disclosure is delivered in an amount effective to reduce the levels of one or more target antibodies or fragments or immunoconjugates comprising same.

[0454] In some embodiments, the glycoengineered polypeptide according to the present disclosure is delivered in an amount effective to reduce levels of IgA1 antibodies, or fragments or immune complexes comprising same.

[0455] In some embodiments, the glycoengineered polypeptide according to the present disclosure is delivered in an amount effective to reduce levels of gd-IgA1 antibodies, or fragments or immune complexes comprising same.

[0456] In some embodiments, the glycoengineered polypeptide according to the present disclosure is delivered in an amount effective to reduce levels of anti-gd-IgA1 autoantibodies or fragments or immune complexes comprising same. Combination therapy

[0457] According to the present disclosure, glycoengineered polypeptides may be administered in combination with one or more therapies, such as standard therapies typically used to treat and / or manage disorders associated with increased and / or abnormal IgA, e.g., IgAN.

[0458] 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 disorders associated with elevated and / or abnormal IgA, e.g., IgAN (e.g., agents that ameliorate the symptoms of disorders associated with elevated and / or abnormal IgA, e.g., IgAN), 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 agents prescribed by a clinician for the treatment of disorders associated with elevated and / or abnormal IgA (e.g., IgAN).

[0459] 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 a blood pressure medication, a proteinuria management therapy, a renal protective agent (e.g., an SGLT2 inhibitor), a glucocorticoid, or a combination thereof. In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with one or more therapies and / or agents prescribed by a clinician for the treatment of a disorder associated with increased and / or abnormal IgA (e.g., IgAN).

[0460] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with a blood pressure medication, such as, for example, typically used to manage high blood pressure.

[0461] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with a therapy used to manage proteinuria.

[0462] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with a nephroprotective agent, such as those used in non-diabetic kidney disease, hi some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with an SGLT2 inhibitor.

[0463] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with a glucocorticoid.

[0464] In some embodiments, a pharmaceutical composition according to the present disclosure is administered in combination with the administration of an intravenous immune globulin composition (IVIG). Characterization of glycoengineered polypeptides

[0465] The present disclosure provides, among other things, glycoengineered polypeptides that specifically bind to a target antibody, thereby causing degradation of the target antibody and / or immune complexes comprising it. In some embodiments, degradation of the target antibody comprises internalization within the cell for degradation (e.g., by transporting the target antibody to lysosomes). In some embodiments, glycoengineered polypeptides that specifically bind to a target antibody according to the present disclosure are characterized by inhibiting one or more pathological functions of the target antibody, including its ability to form immune complexes and / or activate the complement pathway.

[0466] In some embodiments, glycoengineered polypeptides according to the present disclosure are used to degrade and / or reduce levels of target antibodies and / or immune complexes comprising same. In some embodiments, glycoengineered polypeptides are used to reduce levels of target antibodies, e.g., to reduce elevated plasma target antibody levels in subjects with disorders associated with elevated and / or abnormal IgA, e.g., IgAN. In some embodiments, glycoengineered polypeptides are used to reduce and / or eliminate immune complexes comprising one or more target antibodies in subjects with disorders associated with elevated and / or abnormal IgA, e.g., IgAN.

[0467] In some embodiments, the present disclosure provides glycoengineered polypeptides characterized in that, when administered to a cell, tissue, or subject, the glycoengineered polypeptide, bound to a target antibody via a first moiety and to an endocytic receptor via a second moiety, results in degradation of the target antibody.

[0468] 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.

[0469] In some embodiments, the targeting antibody comprises an immune complex comprising it. In some embodiments, the immune complexes disclosed herein comprise one or more antibodies, antigens recognized by the one or more antibodies, and / or one or more components of the complement system. In some embodiments, the complement components comprise C3, C5b, C6, C7, C8, and / or C9, or a fragment of any complement component, or a combination thereof. In some embodiments, the immune complex comprises C3 or a fragment of C3. In some embodiments, the C3 fragment comprises iC3b, C3c, C3dg, or a combination thereof. In some embodiments, the immune complex comprises one or more antibodies selected from IgG, IgA, IgM, IgD, IgE, or a fragment or combination thereof.

[0470] In some embodiments, the target antibody is an IgA1 antibody, or a fragment thereof, or an immune complex comprising same.

[0471] In some embodiments, the target is a gd-IgA1 antibody, or a fragment thereof, or an immune complex comprising same.

[0472] In some embodiments, the target is a gd-IgA1 autoantibody, or a fragment thereof, or an immune complex comprising same.

[0473] In some embodiments, the present disclosure provides glycoengineered polypeptides, wherein when administered to a cell, tissue, or subject, the glycoengineered polypeptide attached to a targeting antibody via a first moiety prevents activation of a complement pathway or a component thereof, in some embodiments, the complement pathway comprises the classical pathway, the alternative pathway, or the lectin pathway.

[0474] In some embodiments, the present disclosure provides glycoengineered polypeptides, wherein the glycoengineered polypeptide attached to a targeting antibody via a first moiety when administered to a cell, tissue, or subject prevents proteinuria, reduced albumin levels, and / or edema.

[0475] In some embodiments, the present disclosure provides glycoengineered polypeptides, wherein the glycoengineered polypeptide, when administered to a cell, tissue, or subject, is attached to a targeting antibody via a first moiety, prevents and / or reduces the formation of immune complexes comprising the targeting antibody.

[0476] In some embodiments, the disclosure provides glycoengineered polypeptides, wherein, when administered to a cell, tissue, or subject, the glycoengineered polypeptide attached to a targeting antibody via a first moiety prevents and / or reduces IgA deposition in one or more tissues or organs. In some embodiments, the tissue is renal tissue. In some embodiments, the organ is a kidney. Method for producing glycosylated polypeptides

[0477] 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., a targeting antibody or fragment or complex thereof) and a second portion comprising one or more glycans conjugated to the first portion.

[0478] 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, and 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.

[0479] 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.

[0480] 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.

[0481] 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.

[0482] 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.

[0483] 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.

[0484] 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.

[0485] 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] 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.

[0486] 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. Leishmania host cells

[0487] 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] 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.

[0488] 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 to the secretory pathway.

[0489] 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.

[0490] 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.

[0491] 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. Methods for culturing Leishmania host cells

[0492] 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 1.

[0493] [Table 1]

[0494] 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.

[0495] 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.

[0496] 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.

[0497] 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. Yeast or filamentous fungal host cells

[0498] 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.

[0499] 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.

[0500] 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.

[0501] 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.

[0502] In some embodiments, the yeast or filamentous fungal host cell is genetically modified to produce glycoproteins with predominant N-glycan glycoforms.

[0503] 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] 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]

[0504] Example 1: Construction of glycoengineered polypeptides of CD89 This example describes the structure and properties of exemplary glycoengineered polypeptides of CD89 for use in binding to IgA antibodies and complexes containing same.

[0505] The immunoglobulin alpha Fc receptor FcαRI, or CD89, is a type I transmembrane glycoprotein expressed on the surface of myeloid cells and is a member of the Ig superfamily. CD89 binds to both IgA1 and IgA2 with similar affinity (Ka approximately 106 M-1). The interaction site between CD89 and IgA was identified in the first extracellular domain of CD89 and the Cα2 / Cα3 junction of IgA. (van der Boog et al; J Immunol 1 February 2002;168(3):1252-1258, herein "van der Boog 2002"). Different glycosylation of soluble CD89 (sCD89) has been shown to affect the binding affinity of CD89 for IgA (Goritzer, K. et al. J. Biol. Chem. 294, 13995-14008 (2019), hereafter "Goritzer 2009"). Goritzer 2009 demonstrated that shorter glycans confer better affinity, with GlcNAc providing the best affinity for IgA. This data also predicts a 1:1 binding model rather than the 2:1 listed in most literature and crystal structures.

[0506] Methods: Construction of a soluble CD89 glycoengineered polypeptide (sCD89) consisted of three major steps. First, to express sCD89 in Leishmania tarentolae, the native CD89 signal peptide (amino acids 1-21) was replaced with a Leishmania-derived signal sequence (MIASSVRHAVILLLVAVAMMGGVIA; SEQ ID NO: 42) in the CD89 sequence, spanning amino acids 22 to 216 (numbering according to Uniprot P24071). While the Leishmania signal peptide was used in this example, other suitable signal peptides readily conceivable by those skilled in the art could also be used. Second, while retaining the native N-glycosylation sites in the D1 domain (N65, N79), the glycosylation sites in the D2 domain were mutated to glutamine (N141Q, N177Q, N186Q, N198Q). Third, the glycoengineered polypeptide was modified at its C-terminus by adding a peptide sequence containing an N-glycosylation consensus site ("glycotag"; SEQ ID NO: 37) and a His-tag for purification (FLGT13; SEQ ID NO: 40). These construction steps resulted in a sCD89 glycoengineered polypeptide with three N-glycosylation sites (two native sites and a third engineered site within the C-terminal glycotag sequence; SEQ ID NO: 41).

[0507] The corresponding nucleotide sequence was transfected as an expression cassette into the glycoengineered Leishmania tarentolae host cell line StCGP4564. The resulting cell line, StCGP5907, was grown in a bioreactor for expression and secretion of sCD89. sCD89 containing A2GalNAc2 glycans was purified from cell supernatants by IMAC purification and analyzed (Table 2). Size exclusion chromatography (SEC) was used to measure protein aggregation and degradation.

[0508] Results: The glycoengineering process yielded the sCD89 glycoengineering polypeptide. Analysis of the construct (Table 2) showed that the sCD89 glycoengineering polypeptide had a high percentage of A2GalNAc2 (82.8%) and high occupancy at all three glycosites (all >95% occupancy).

[0509] [Table 2] Example 2: Glycoengineered sCD89 polypeptides deplete IgA antibodies

[0510] This example demonstrates the depletion of human IgA antibodies in a rat model using exemplary glycoengineered polypeptides, including sCD89 polypeptides.

[0511] Methods: Standard animal clinical monitoring was used in the experiment. First, eight female Wistar rats were intravenously administered 1 mg of purified total human IgA at -0.5 h on day 1 (D1) (30 min before administration of the glycoengineered polypeptide; see below) to ensure that theoretical circulating IgA levels immediately after administration approached 0.1 mg / mL (theoretical C0). For reference, note that typical circulating IgA levels in humans are approximately 2.6 mg / mL. Next, at 0 h (30 min after IgA administration), the rats were subcutaneously injected with 2.0 mg of an exemplary sCD89 glycoengineered polypeptide (4 animals) or a control PBS solution (4 animals). Blood samples were obtained 1, 3, 6, 10, 24, and 48 h after injection of the exemplary glycoengineered polypeptide or PBS control. All blood samples were processed to serum, which was aliquoted into four vials and stored at -80°C until analysis. Serum human IgA levels were assessed using a human IgA quantification ELISA kit (catalog number E88-102, BioMethyl Laboratories) according to the manufacturer's instructions. Because IgA binding by the glycoengineered sCD89 polypeptide did not interfere with the detection of human IgA by the ELISA kit (data not shown, validated during assay development), the ELISA method quantifies total human IgA (free + bound). Results are expressed as the percentage of target (IgA) remaining in the serum sample.

[0512] Results: The percentage of human IgA targets remaining in rat serum was rapidly depleted after administration of the exemplary glycoengineered polypeptides. At 6 hours post-administration, less than 5% of the theoretical C0 IgA remained in the serum of samples treated with sCD89 glycoengineered polypeptides, whereas the majority of the IgA remained in the serum from PBS-treated samples (Figure 4). The maximum effect of IgA depletion in the sCD89 glycoengineered polypeptide-treated group was achieved at 6 hours compared to the PBS control group.

[0513] This data demonstrates in vivo depletion of human IgA in rats administered subcutaneously with sCD89 glycoengineered polypeptides. This data supports the development of glycoengineered polypeptides for use as therapeutic agents for the treatment and / or prevention of IgAN (e.g., in subjects with one or more IgA antibodies). This data further supports the use of glycoengineered polypeptides to reduce IgA antibody levels in a subject. Example 3: Identification of glycoengineered polypeptides that bind to IgA

[0514] This example describes a process that can be used to identify polypeptides capable of preferentially binding to IgA antibodies with a particular glycosylation profile. For example, the particular glycosylation profile can be a glycan profile that is rare in wild-type IgA and increased in galactose-deficient IgA1. In some embodiments, the glycan profile of galactose-deficient IgA1 is characterized by terminal GalNac. Such glycan profiles are also known in the art as "Tn antigens." See, e.g., Knoppova et al., Frontiers in Immunology (2016) volume 7, article 117, the entire contents of which are incorporated herein by reference.

[0515] Method: To identify exemplary polypeptides capable of binding to IgA1 with a specific glycosylation profile (e.g., Tn-IgA), humanized or hyperimmunized mouse strains are first immunized with peptides mimicking IgA1 hinge TnO-glycosylation. This immunization generates a diverse library of fully human binding factors capable of recognizing and binding to the antigen (Tn-IgA). Once the library of binding factors is obtained, the specificity of the binding factors is determined by comparing their binding to Tn-IgA1 with that to wild-type IgA1 (which rarely has TnO-glycosylation). Binding affinity is quantified using standard methods, such as Biacore assays. Tn-IgA1 is obtained by treating wt-IgA1 with neuraminidase (Sigma reference number 10269611001) and β-galactosidase (Sigma reference number G4142-.2UN) enzymes according to standard protocols. This treatment converts the normal O-glycosylation profile of wild-type IgA1 to one enriched for Tn antigens.

[0516] Results: Polypeptides with preferential binding specificity for galactose-deficient IgA1 (Tn-IgA1) compared to wt-IgA1 are identified. In some embodiments, polypeptides that preferentially bind to a favorable glycosylation profile on IgA1 (e.g., Tn-IgA) show increased binding to Tn-IgA1 compared to normal IgA1.

[0517] Polypeptides identified using this method can be used to construct glycoengineered polypeptides capable of specifically binding to galactose-deficient IgA1, for example, using the methods described herein in Example 1. In some embodiments, glycoengineered polypeptides comprising one or more polypeptides identified using the methods disclosed in this Example exhibit preferential binding to antibodies of IgAN patients and may be useful as therapeutic agents in the treatment and / or prevention of IgAN (e.g., in subjects with one or more IgA autoantibodies). Enumerated Embodiments

[0518] Embodiment 1. A glycoengineered polypeptide comprising: (a) a first portion comprising one or more peptides that specifically bind to a target antibody, 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.

[0519] Embodiment 2. The glycoengineered polypeptide of embodiment 1, wherein the complex comprising the targeting antibody is or comprises an immunoconjugate.

[0520] Embodiment 3. The glycoengineered polypeptide of embodiment 1 or 2, wherein said targeting antibody comprises galactose-deficient IgA1 (gd-IgA1), or a fragment or conjugate thereof.

[0521] Embodiment 4. The glycoengineered polypeptide of embodiment 1 or 2, wherein said targeting antibody comprises IgA1, or a fragment or complex thereof.

[0522] Embodiment 5. The glycoengineered polypeptide of embodiment 1 or 2, wherein said targeting antibody comprises an autoantibody that specifically binds to gd-IgA1 (an "anti-gd-IgA1 autoantibody"), or a fragment or conjugate thereof.

[0523] Embodiment 6. The glycoengineered polypeptide of any one of the preceding embodiments, wherein said second portion specifically binds to one or more endocytic receptors.

[0524] Embodiment 7. The glycoengineered polypeptide of embodiment 6, wherein said endocytic receptor is or comprises an endocytic lectin.

[0525] Embodiment 8. The glycomodified polypeptide of embodiment 6 or 7, 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.

[0526] Embodiment 9. The glycomodified polypeptide of any one of the preceding embodiments, wherein the glycan comprises a terminal GlcNac.

[0527] Embodiment 10. The glycomodified polypeptide of any one of embodiments 1 to 8, wherein the glycan comprises a terminal GalNac.

[0528] Embodiment 11. The glycomodified polypeptide of any one of embodiments 1 to 8, wherein the glycan comprises a terminal Gal.

[0529] Embodiment 12. The glycomodified polypeptide of any one of the preceding embodiments, wherein the glycan is an N-glycan.

[0530] Embodiment 13. The glycomodified polypeptide of embodiment 12, wherein the N-glycan is linked to the glycomodified polypeptide at 1, 2, 3, 4, or 5 N-glycosylation sites.

[0531] Embodiment 14. 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.

[0532] Embodiment 15. 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.

[0533] Embodiment 16. 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.

[0534] Embodiment 17. 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.

[0535] Embodiment 18. The glycomodified polypeptide of any one of the preceding embodiments, wherein the glycan structure comprises a biantennary structure.

[0536] Embodiment 19. The glycomodified polypeptide of embodiment 18, wherein the glycan structure comprises a bisecting GalNAc.

[0537] Embodiment 20. The glycoengineered polypeptide of embodiment 18 or 19, wherein said bisecting GalNac binds to the asialoglycoprotein receptor (ASGPR) or a fragment or variant thereof, or a complex comprising ASGPR.

[0538] Embodiment 21. 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.

[0539] Embodiment 22. The glycomodified polypeptide of any one of Embodiments 12 to 21, wherein the N-glycan is conjugated to the glycomodified polypeptide at at least 1, 2, 3, or 4 N-glycosylation sites.

[0540] Embodiment 23. The glycomodified polypeptide of any one of Embodiments 12 to 21, wherein the N-glycan is conjugated to the glycomodified polypeptide at one, two, three, or four N-glycosylation sites.

[0541] Embodiment 24. 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.

[0542] Embodiment 25. The glycoengineered polypeptide of any one of the preceding embodiments, wherein the N-glycosylation site is naturally occurring.

[0543] Embodiment 26. The polypeptide of embodiment 25, wherein the N-glycosylation site is engineered into the amino acid sequence of the first portion, and optionally, the engineered N-glycosylation site comprises the sequence of SEQ ID NO: 37.

[0544] Embodiment 27. The glycomodified polypeptide of any one of Embodiments 6 to 26, wherein the endocytic receptor is or comprises ASGPR or a fragment or variant thereof, or a complex containing ASGPR.

[0545] Embodiment 28. The glycomodified polypeptide of embodiment 27, wherein the glycan structure of the second portion comprises a terminal GalNac when the endocytic receptor is ASGPR.

[0546] Embodiment 29. The glycoengineered polypeptide of any one of the preceding embodiments, wherein the first portion comprises one or more peptides that specifically bind to glycosylation-deficient IgA1 (gd-IgA1), or a fragment or complex thereof.

[0547] Embodiment 30. The polypeptide of embodiment 29, wherein the gd-IgA1 comprises a glycosylated hinge region.

[0548] Embodiment 31. The polypeptide of embodiment 30, wherein the hinge region comprises the sequence PVPSTPPTPSPSTPPTPSPSC (SEQ ID NO: 1) or a variant or fragment thereof.

[0549] Embodiment 32. The glycomodified polypeptide of embodiment 30 or 31, wherein the hinge region comprises nine O-glycosylation sites.

[0550] Embodiment 33. The glycoengineered polypeptide of embodiment 32, wherein at least 1, 2, 3, 4, 5, or 6 of the O-glycosylation sites are occupied.

[0551] Embodiment 34. A glycomodified polypeptide according to any one of embodiments 29 to 33, wherein said one or more peptides that specifically bind to gd-IgA1 recognize a glycan profile on gd-IgA1.

[0552] Embodiment 35. The glycoengineered polypeptide of embodiment 34, wherein said gd-IgA1 comprises a glycan profile that differs from a reference glycan profile of IgA1.

[0553] Embodiment 36. The glycomodified polypeptide of any one of embodiments 34 to 35, wherein the gd-IgA1 comprises a glycan profile having at least one less glycan compared to a reference glycan profile of IgA1.

[0554] Embodiment 37. The glycomodified polypeptide of any one of embodiments 34 to 36, wherein the reference glycan profile comprises an O-glycosylation profile.

[0555] Embodiment 38. The glycomodified polypeptide of embodiment 37, wherein said reference O-glycosylation profile comprises 1, 2, 3, 4, 5, or 6 O-glycan chains.

[0556] Embodiment 39. The glycomodified polypeptide of embodiment 37 or 38, wherein the O-glycan chain of the reference O-glycosylation profile comprises N-acetylgalactosamine (GalNAc).

[0557] Embodiment 40. The glycomodified polypeptide of any one of embodiments 37 to 39, wherein the O-glycan chain further comprises galactose (Gal), sialic acid, or a combination thereof.

[0558] Embodiment 41. The glycomodified polypeptide of any one of embodiments 34 to 40, wherein the gd-IgA1 glycan profile comprises at least one less galactosylation compared to the reference glycan profile.

[0559] Embodiment 42. The glycomodified polypeptide of any one of embodiments 34 to 41, wherein the gd-IgA1 glycan profile comprises at least 5% less galactosylation compared to the reference glycan profile.

[0560] Embodiment 43. The glycomodified polypeptide of any one of embodiments 34 to 42, wherein the gd-IgA1 glycan profile comprises increased sialylation compared to the reference glycan profile.

[0561] Embodiment 44. The glycomodified polypeptide of any one of embodiments 34 to 43, wherein the gd-IgA1 glycan profile comprises a terminal GalNAc (also referred to as the Tn antigen).

[0562] Embodiment 45. The glycomodified polypeptide of any one of embodiments 34 to 43, wherein the gd-IgA1 glycan profile comprises GalNAc with alpha 2,6-linked sialic acid (also referred to as STn antigen).

[0563] Embodiment 46. A glycomodified polypeptide according to any one of embodiments 34 to 45, wherein sialylation of the terminal GalNac blocks effective galactosylation.

[0564] Embodiment 47. A glycomodified polypeptide according to any one of embodiments 29 to 46, wherein the one or more peptides that specifically bind to gd-IgA1 recognize an epitope in the hinge region of gd-IgA1.

[0565] Embodiment 48. The glycomodified polypeptide of embodiment 47, wherein the altered glycan profile results in a conformational change in gd-IgA1.

[0566] Embodiment 49. The glycoengineered polypeptide of embodiment 48, wherein said conformational change exposes a neoepitope recognized by said one or more peptides.

[0567] Embodiment 50. The glycoengineered polypeptide of embodiment 49, wherein said neoepitope is not present in a non-galactose-deficient IgA1, such as an IgA1 comprising a reference glycan profile.

[0568] Embodiment 51. The glycoengineered polypeptide of embodiment 49 or 50, wherein the neoepitope is a linear epitope.

[0569] Embodiment 52. The glycoengineered polypeptide of embodiment 49 or 50, wherein the neoepitope is a conformational epitope.

[0570] Embodiment 53. The glycoengineered polypeptide of any one of embodiments 29 to 52, wherein the first portion comprises one, two, three, four, five, or more peptides that specifically bind to gd-IgA1 or a fragment thereof.

[0571] Embodiment 54. A glycomodified polypeptide according to any one of embodiments 29 to 53, wherein said one or more peptides that specifically bind to gd-IgA1 or a fragment thereof are the same.

[0572] Embodiment 55. The glycoengineered polypeptide of embodiment 54, wherein said one or more peptides that specifically bind to gd-IgA1 or a fragment thereof are separated by an intervening sequence.

[0573] Embodiment 56. The glycomodified polypeptide of embodiment 55, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0574] Embodiment 57. A glycomodified polypeptide according to any one of embodiments 29 to 53, wherein said one or more peptides that specifically bind to gd-IgA1 or a fragment thereof are different.

[0575] Embodiment 58. The glycomodified polypeptide of embodiment 57, wherein said one or more peptides that specifically bind to gd-IgA1 or a fragment thereof are separated by an intervening sequence.

[0576] Embodiment 59. The glycomodified polypeptide of embodiment 58, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0577] Embodiment 60. The glycomodified polypeptide of any one of Embodiments 57 to 59, wherein the different peptides form spatial epitopes.

[0578] Embodiment 61. A glycomodified polypeptide according to any one of embodiments 29 to 60, wherein said one or more peptides that specifically bind to gd-IgA1 or a fragment thereof are each conjugated to a second moiety.

[0579] Embodiment 62. A glycomodified polypeptide according to any one of embodiments 29 to 60, wherein each of the one or more peptides that specifically bind to gd-IgA1 or a fragment thereof is not conjugated to the second moiety.

[0580] Embodiment 63. A glycomodified polypeptide according to any one of embodiments 29 to 62, wherein said one or more peptides that specifically bind to gd-IgA1 or a fragment thereof are conjugated to each other.

[0581] Embodiment 64. The glycomodified polypeptide of embodiment 63, wherein said one or more peptides are located on a single polypeptide.

[0582] Embodiment 65. The glycomodified polypeptide of embodiment 63 or 64, wherein said one or more peptides are separated by an intervening amino acid sequence.

[0583] Embodiment 66. The glycomodified polypeptide of embodiment 65, wherein said intervening amino acid sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0584] Embodiment 67. The glycomodified polypeptide of any one of embodiments 29 to 66, wherein said one or more peptides that specifically bind to gd-IgA1 comprise an antibody agent.

[0585] Embodiment 68. The glycoengineered polypeptide of embodiment 67, wherein the antibody agent comprises an antigen-binding fragment.

[0586] Embodiment 69. The glycoengineered polypeptide of embodiment 68, 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).

[0587] Embodiment 70. The glycoengineered polypeptide of embodiment 68, wherein the antibody agent comprises a VHH, e.g., a camelid-derived VHH or a bivalent VHH.

[0588] Embodiment 71. The glycoengineered polypeptide of any one of embodiments 1 to 28, wherein the first portion comprises one or more peptides that specifically bind to IgA1, or a fragment or complex thereof.

[0589] Embodiment 72. The glycomodified polypeptide of embodiment 71, wherein said first portion comprises 1, 2, 3, 4, 5, or more peptides that specifically bind to IgA1 or a fragment thereof.

[0590] Embodiment 73. A glycoengineered polypeptide according to embodiment 71 or 72, wherein said one or more peptides that specifically bind to IgA1 or a fragment thereof are the same.

[0591] Embodiment 74. The glycomodified polypeptide of embodiment 73, wherein said one or more peptides that specifically bind to IgA1 or a fragment thereof are separated by an intervening sequence.

[0592] Embodiment 75. The glycomodified polypeptide of embodiment 74, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0593] Embodiment 76. A glycoengineered polypeptide according to embodiment 71 or 72, wherein said one or more peptides that specifically bind to IgA1 or a fragment thereof are different.

[0594] Embodiment 77. The glycomodified polypeptide of embodiment 76, wherein said one or more peptides that specifically bind to IgA1 or a fragment thereof are separated by an intervening sequence.

[0595] Embodiment 78. The glycomodified polypeptide of embodiment 77, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0596] Embodiment 79. The glycomodified polypeptide of any one of embodiments 76 to 78, wherein the different peptides form spatial epitopes.

[0597] Embodiment 80. The glycoengineered polypeptide of any one of embodiments 71 to 79, wherein said one or more peptides that specifically bind to IgA1 or a fragment thereof are each conjugated to a second moiety.

[0598] Embodiment 81. The glycomodified polypeptide of any one of embodiments 71 to 79, wherein each of said one or more peptides that specifically bind to IgA1 or a fragment thereof is not conjugated to said second moiety.

[0599] Embodiment 82. A glycomodified polypeptide according to any one of embodiments 71 to 81, wherein said one or more peptides that specifically bind to IgA1 or a fragment thereof are conjugated to each other.

[0600] Embodiment 83. The glycomodified polypeptide of embodiment 82, wherein said one or more peptides are located on a single polypeptide.

[0601] Embodiment 84. The glycomodified polypeptide of embodiment 82 or 83, wherein said one or more peptides are separated by an intervening amino acid sequence.

[0602] Embodiment 85. The glycomodified polypeptide of embodiment 84, wherein said intervening amino acid sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0603] Embodiment 86. A glycomodified polypeptide according to any one of embodiments 71 to 85, comprising a first portion comprising a peptide that specifically binds to IgA1 or a fragment thereof.

[0604] Embodiment 87. The glycoengineered polypeptide of any one of embodiments 71 to 86, wherein the IgA1 amino acid sequence is provided as SEQ ID NO:2.

[0605] Embodiment 88. A glycomodified polypeptide according to any one of embodiments 71 to 87, wherein said one or more peptides that specifically bind to IgA1 recognize an epitope on IgA1.

[0606] Embodiment 89. The glycomodified polypeptide of embodiment 88, wherein the epitope is a conformational epitope.

[0607] Embodiment 90. The glycomodified polypeptide of embodiment 88, wherein the epitope is a linear epitope.

[0608] Embodiment 91. The glycomodified polypeptide of any one of embodiments 71 to 90, wherein said first portion comprising one or more peptides, or fragments or conjugates thereof, that specifically bind to IgA1 comprises a CD89 (FcaR1) polypeptide, or a variant thereof, or a fragment thereof.

[0609] Embodiment 92. The glycoengineered polypeptide of embodiment 91, wherein said CD89 comprises soluble CD89 or a fragment or variant thereof.

[0610] Embodiment 93. The glycomodified polypeptide of embodiment 91 or 92, wherein the CD89 polypeptide is provided as SEQ ID NO:5, with or without a signal peptide, SEQ ID NO:9, with or without a signal peptide, SEQ ID NO:38, with or without a signal peptide, or SEQ ID NO:39.

[0611] Embodiment 94. The glycoengineered glycopolypeptide of any one of embodiments 91-93, wherein said one or more peptides, or fragments or conjugates thereof, that specifically bind to IgA1 comprise a fragment of a CD89 polypeptide.

[0612] Embodiment 95. The glycomodified polypeptide of embodiment 94, wherein said fragment comprises at least 5% of a full-length CD89 polypeptide, with or without the signal peptide.

[0613] Embodiment 96. The glycomodified polypeptide of embodiment 94, wherein the fragment comprises 99% or less of a full-length CD89 polypeptide, with or without the signal peptide.

[0614] Embodiment 97. The glycoengineered polypeptide of any one of Embodiments 91 to 96, wherein the fragment comprises a CD89 amino acid sequence that binds to IgA1.

[0615] Embodiment 98. The glycomodified polypeptide of any one of embodiments 91 to 97, wherein the fragment comprises one or more additional amino acid sequences 5' and / or 3' to the fragment sequence.

[0616] Embodiment 99. The glycomodified polypeptide of any one of embodiments 91 to 98, wherein said one or more peptides, or fragments or complexes thereof, that specifically bind to IgA1 comprise a variant of a CD89 polypeptide.

[0617] Embodiment 100. A glycomodified polypeptide according to any one of embodiments 91 to 99, wherein the one or more peptides that specifically bind to an IgA1 antibody comprise a contiguous amino acid stretch comprising at least 5% of the amino acids of SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:38, or SEQ ID NO:39.

[0618] Embodiment 100.1. The one or more peptides, or fragments or conjugates thereof, that specifically bind to IgA1 are: (i) SEQ ID NO: 5, or (ii) The glycomodified polypeptide of any one of embodiments 91-100, comprising a sequence having at least 85% identity to SEQ ID NO: 5 without the signal peptide, optionally further comprising a different signal peptide, e.g., as disclosed herein.

[0619] Embodiment 100.2. The one or more peptides, or fragments or conjugates thereof, that specifically bind to IgA1 are: (i) SEQ ID NO: 9, or (ii) The glycomodified polypeptide of any one of embodiments 91-100, comprising a sequence having at least 85% identity to SEQ ID NO: 9 without the signal peptide, optionally further comprising a different signal peptide, e.g., as disclosed herein.

[0620] Embodiment 100.3. The one or more peptides, or fragments or conjugates thereof, that specifically bind to IgA1 are: (i) SEQ ID NO: 38, or (ii) The glycomodified polypeptide of any one of embodiments 91-100, comprising a sequence having at least 85% identity to SEQ ID NO: 38 without the signal peptide, optionally further comprising a different signal peptide, e.g., as disclosed herein.

[0621] Embodiment 100.4. The glycoengineered polypeptide of any one of embodiments 91-100, wherein said one or more peptides, or fragments or conjugates thereof, that specifically bind to IgA1 comprise a sequence having SEQ ID NO: 39, optionally further comprising a signal peptide, e.g., as disclosed herein. Embodiment 100.5. The glycoengineered polypeptide of any one of embodiments 91-100, wherein said one or more peptides that specifically bind to IgA1 comprise a sequence having at least 85% identity to SEQ ID NO: 41.

[0622] Embodiment 101. The glycoengineered polypeptide of any one of embodiments 71 to 100.2, wherein said one or more peptides that specifically bind to IgA1 comprise an antibody agent.

[0623] Embodiment 102. The glycoengineered polypeptide of embodiment 101, wherein the antibody agent comprises an antigen-binding fragment.

[0624] Embodiment 103. The glycoengineered polypeptide of embodiment 102, 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).

[0625] Embodiment 104. The glycoengineered polypeptide of embodiment 102, wherein the antibody agent comprises a VHH, such as a camelid-derived VHH or a bivalent VHH.

[0626] Embodiment 105. The glycoengineered polypeptide of any one of embodiments 1 to 27, wherein the first portion comprises one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies.

[0627] Embodiment 106. The glycoengineered polypeptide of embodiment 105, wherein said first portion comprises one, two, three, four, five, or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof.

[0628] Embodiment 107. The glycoengineered polypeptide of embodiment 105 or 106, wherein said one or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof are the same.

[0629] Embodiment 108. The glycomodified polypeptide of embodiment 107, wherein said one or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof are separated by an intervening sequence.

[0630] Embodiment 109. The glycomodified polypeptide of embodiment 107, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0631] Embodiment 110. The glycoengineered polypeptide of embodiment 108 or 109, wherein said one or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof are different.

[0632] Embodiment 111. The glycomodified polypeptide of embodiment 110, wherein said one or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof are separated by an intervening sequence.

[0633] Embodiment 112. The glycomodified polypeptide of embodiment 111, wherein said intervening sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0634] Embodiment 113. The glycomodified polypeptide of any one of Embodiments 105 to 112, wherein said different peptides form spatial epitopes.

[0635] Embodiment 114. A glycomodified polypeptide according to any one of embodiments 105 to 112, wherein said one or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof are each conjugated to a second moiety.

[0636] Embodiment 115. The glycomodified polypeptide of any one of embodiments 105 to 114, wherein each of the one or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof is not conjugated to the second moiety.

[0637] Embodiment 116. A glycomodified polypeptide according to any one of embodiments 105 to 115, wherein said one or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof are conjugated to each other.

[0638] Embodiment 117. The glycomodified polypeptide of embodiment 116, wherein said one or more peptides are located on a single polypeptide.

[0639] Embodiment 118. The glycomodified polypeptide of embodiment 116 or 117, wherein said one or more peptides are separated by an intervening amino acid sequence.

[0640] Embodiment 119. The glycomodified polypeptide of embodiment 118, wherein said intervening amino acid sequence is an IRES, a protease cleavage site, a linker, or a spacer, or a combination thereof.

[0641] Embodiment 120. A glycomodified polypeptide according to any one of embodiments 105 to 119, comprising a first portion comprising a peptide that specifically binds to anti-gd-IgA1 or a fragment thereof.

[0642] Embodiment 121. The glycomodified polypeptide of any one of Embodiments 105 to 120, wherein the anti-gd-IgA1 autoantibody is IgG, IgM, IgE, IgD, or IgM.

[0643] Embodiment 122. The glycomodified polypeptide of any one of Embodiments 105 to 121, wherein the anti-gd-IgA1 autoantibody is an IgG.

[0644] Embodiment 123. The glycoengineered polypeptide of embodiment 122, wherein said IgG comprises a mutation in the complementarity-determining region 3 (CDR3) of the Ig heavy chain (IgH) variable region.

[0645] Embodiment 124. The glycomodified polypeptide of embodiment 123, wherein the mutation comprises an alanine to serine mutation.

[0646] Embodiment 125. The glycomodified polypeptide of embodiment 124, wherein the alanine to serine mutation occurs in the YCAR or YCAK amino acid sequence of IgH.

[0647] Embodiment 126. The glycomodified polypeptide of any one of embodiments 105 to 125, wherein the first portion comprises one or more peptides that specifically bind to a mutation in the CDR3 of the IgH region of an anti-gd-IgA1 autoantibody.

[0648] Embodiment 127. The glycomodified polypeptide of any one of embodiments 105 to 126, wherein the first portion comprises one or more peptides that specifically bind to a YCAR amino acid sequence in the CDR3 of IgH with an alanine to serine mutation.

[0649] Embodiment 128. The glycomodified polypeptide of any one of embodiments 105 to 126, wherein the first portion comprises one or more peptides that specifically bind to a YCAK amino acid sequence in the CDR3 of IgH with an alanine to serine mutation.

[0650] Embodiment 129. The glycoengineered polypeptide of any one of embodiments 105 to 128, wherein said one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies bind to an IgG protein, or a fragment or variant thereof.

[0651] Embodiment 130. The glycomodified polypeptide of embodiment 129, wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

[0652] Embodiment 131. The glycomodified polypeptide of embodiment 129 or 130, wherein the wild-type IgG1 protein is provided as SEQ ID NO: 3.

[0653] Embodiment 132. The glycomodified polypeptide of embodiment 129 or 130, wherein the wild-type IgG4 protein is provided as SEQ ID NO: 4.

[0654] Embodiment 133. The glycomodified polypeptide of any one of embodiments 105 to 132, wherein said one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies bind to a variant of an IgG protein.

[0655] Embodiment 134. The glycoengineered polypeptide of any one of embodiments 105 to 132, wherein the first portion comprising one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies comprises a gd-IgA1 polypeptide, or a variant or fragment thereof.

[0656] Embodiment 135. The glycoengineered polypeptide of embodiment 134, wherein said gd-IgA1 comprises a glycosylated hinge region.

[0657] Embodiment 136. The glycomodified polypeptide of embodiment 135, wherein the hinge region comprises the sequence PVPSTPPTPSPSTPPTPSPSC (SEQ ID NO: 1) or a fragment thereof.

[0658] Embodiment 137. The glycomodified polypeptide of embodiment 135 or 136, wherein the hinge region comprises nine O-glycosylation sites.

[0659] Embodiment 138. The glycomodified polypeptide of embodiment 137, wherein at least 1, 2, 3, 4, 5, or 6 of the O-glycosylation sites are occupied.

[0660] Embodiment 139. The glycomodified polypeptide of any one of embodiments 134 to 138, wherein the first portion comprising one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies comprises a gd-IgA1 polypeptide fragment.

[0661] Embodiment 140. The glycomodified polypeptide of embodiment 139, wherein the fragment comprises the sequence of SEQ ID NO: 1, or a fragment or variant thereof.

[0662] Embodiment 141. The glycomodified polypeptide of embodiment 140, wherein the fragment comprises one or more additional amino acid residues at the 5' and / or 3' end of the sequence.

[0663] Embodiment 142. The glycomodified polypeptide of any one of embodiments 134 to 142, wherein the fragment has a glycan profile that differs from a reference glycan profile.

[0664] Embodiment 143. The glycomodified polypeptide of embodiment 142, wherein the fragment has a glycan profile with terminal GalNAc.

[0665] Embodiment 144. The glycomodified polypeptide of embodiment 142, wherein the fragment has a glycan profile with terminal sialylated GalNAc.

[0666] Embodiment 145. The glycomodified polypeptide of any one of embodiments 134 to 144, wherein the fragment comprises a gd-IgA1 sequence that provides for capping of GlcNac or GalNac with sialic acid.

[0667] Embodiment 146. The glycoengineered polypeptide of any one of embodiments 105 to 145, wherein said one or more peptides that specifically bind to anti-gd-IgA1 comprise an antibody agent.

[0668] Embodiment 147. The glycoengineered polypeptide of embodiment 146, wherein the antibody agent comprises an antigen-binding fragment.

[0669] Embodiment 148. The glycoengineered polypeptide of embodiment 147, wherein the antibody agent comprises a complete antibody, a Fab fragment, an scFv, a nanobody, a duobody, or a single domain antibody (e.g., a VHH).

[0670] Embodiment 149. The glycoengineered polypeptide of embodiment 147, wherein the antibody agent comprises a VHH, such as a camelid-derived VHH or a bivalent VHH.

[0671] Embodiment 150. The glycomodified polypeptide of any one of the preceding embodiments, wherein said first portion is: (i) one or more peptides that specifically bind to gd-IgA1 or a fragment or complex thereof; (ii) one or more peptides that specifically bind to IgA1 or a fragment or complex thereof; (iii) one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies or fragments or complexes thereof; (iv) The glycosylated polypeptide as described above, comprising any one or all of (i) to (iii).

[0672] Embodiment 151. The glycomodified polypeptide of embodiment 150, wherein (i), (ii) and / or (iii) are located on the same polypeptide.

[0673] Embodiment 152. The glycomodified polypeptide of embodiment 150, wherein (i), (ii) and / or (iii) are located on different polypeptides.

[0674] Embodiment 153. The glycomodified polypeptide of embodiment 152, wherein (i) is located on a first glycomodified polypeptide, (ii) is located on a second glycomodified polypeptide, and / or (iii) is located on a third glycomodified polypeptide.

[0675] Embodiment 154. The glycomodified polypeptide of embodiment 150, wherein each of (i), (ii), and (iii) is conjugated to a second moiety.

[0676] Embodiment 155. The targeting antibody bound by the first moiety comprises an immune complex, and optionally, the immune complex comprises one of the following: 10. The glycoengineered polypeptide of any one of the preceding embodiments, comprising the targeting antibody or fragment thereof, an antigen recognized by the targeting antibody, one or more components of the complement system, one or more additional immunoglobulins, or a combination thereof.

[0677] Embodiment 156. A glycomodified polypeptide according to embodiment 155, comprising: (i) the one or more immunoglobulins comprise IgG, IgA, IgM, IgD, IgE, or a fragment or combination thereof; and / or (ii) The glycomodified polypeptide, wherein the immune complex comprises a gd-IgA1 antibody, an IgA1 antibody, an anti-gd-IgA1 autoantibody, or a combination thereof.

[0678] Embodiment 157. The glycoengineered polypeptide of embodiment 156, wherein the anti-gd-IgA1 autoantibody is IgG or IgM.

[0679] Embodiment 158. A glycomodified polypeptide according to any one of the preceding embodiments, wherein said polypeptide comprises one or more additional elements.

[0680] Embodiment 159. The glycomodified polypeptide of embodiment 158, wherein said additional element comprises a linker, a signal peptide (e.g., SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or a portion of any of the foregoing), a tag, a half-life extender, or a combination thereof.

[0681] Embodiment 160. The glycomodified polypeptide of any one of the preceding embodiments, wherein the second moiety is conjugated to the first moiety at at least 1, 2, 3, or 4 N-glycosylation sites.

[0682] Embodiment 161. The glycomodified polypeptide of any one of the preceding embodiments, wherein the second moiety is conjugated to the first moiety at one, two, three, or four N-glycosylation sites.

[0683] Embodiment 162. The glycomodified 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.

[0684] Embodiment 163. The glycomodified polypeptide of any one of the preceding embodiments, wherein said second moiety is conjugated to said first moiety in vivo.

[0685] Embodiment 164. The glycomodified polypeptide of embodiment 163, wherein the conjugation occurs intracellularly.

[0686] Embodiment 165. The glycoengineered polypeptide of embodiment 164, wherein the cell is a Leishmania cell.

[0687] Embodiment 166. The glycomodified polypeptide of any one of embodiments 1 to 162, wherein said second moiety is conjugated to said first moiety by chemical conjugation.

[0688] Embodiment 167. The glycomodified polypeptide of embodiment 166, wherein the chemical conjugation comprises click chemistry.

[0689] Embodiment 168. The glycoengineered polypeptide of any one of the preceding embodiments, wherein the first moiety is an antibody, and wherein the antibody is a monoclonal or polyclonal antibody.

[0690] Embodiment 169. The glycomodified polypeptide of embodiment 168, wherein the antibody is a recombinant antibody.

[0691] Embodiment 170. The glycoengineered polypeptide of embodiment 169, wherein the antibody is humanized, chimeric, or fully human.

[0692] Embodiment 171. The glycomodified polypeptide of any one of embodiments 168 to 170, wherein the antibody has a glycan-to-protein ratio of 2:1, 4:1...

Claims

1. 1. A glycomodified polypeptide comprising: (a) a first portion comprising one or more peptides that specifically bind to a target antibody, 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; The targeting antibody may be: (i) galactose-deficient IgA1 (gd-IgA1), or a fragment or complex thereof; (ii) IgA1, or a fragment or complex thereof; or (iii) the glycoengineered polypeptide, comprising an autoantibody that specifically binds to gd-IgA1 (an "anti-gd-IgA1 autoantibody"), or a fragment or complex thereof.

2. 2. The glycomodified polypeptide of claim 1, wherein the second portion specifically binds to one or more endocytic receptors.

3. 3. The glycomodified polypeptide of claim 2, 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, CLEC10, DNGR1, CLEC12B, DEC-205, and mannose-6-phosphate receptor (M6PR), or a combination thereof.

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

5. 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.

6. 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.

7. 7. The glycomodified polypeptide of claim 6, wherein the glycan structure comprises a monoantennary structure, a biantennary structure, a triantennary structure, or a tetraantennary structure.

8. 8. The glycomodified polypeptide of claim 6 or 7, wherein said glycan structure comprises a biantennary structure, and optionally said glycan structure comprises a biantennary GalNAc.

9. 9. The glycomodified polypeptide of claim 8, wherein the bisecting GalNac binds to the asialoglycoprotein receptor (ASGPR) or a fragment or variant thereof, or a complex comprising an ASGPR.

10. The N-glycan has the following structure: 【Chemistry 12】 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.

11. 11. The glycomodified polypeptide of any one of claims 5 to 10, wherein the N-glycan is conjugated to the glycomodified polypeptide at at least 1, 2, 3, or 4 N-glycosylation sites.

12. 12. The glycomodified polypeptide of claim 5, 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.

13. 13. The glycomodified polypeptide of any one of claims 5 to 12, wherein the N-glycosylation site is naturally occurring.

14. 13. The glycomodified polypeptide of any one of claims 5 to 12, wherein the N-glycosylation site is engineered into the amino acid sequence of the first portion, and optionally the engineered N-glycosylation is SEQ ID NO:

37.

15. 15. The glycomodified polypeptide of any one of claims 2 to 14, wherein the endocytosis receptor is or comprises ASGPR or a fragment or variant thereof, or a complex comprising ASGPR, and optionally, when the endocytosis receptor is ASGPR, the glycan structure of the second portion comprises a terminal GalNac.

16. 10. The glycoengineered polypeptide of any one of the preceding claims, wherein said first portion comprises one or more peptides that specifically bind to glycosylation-deficient IgA1 (gd-IgA1), or a fragment or complex thereof, and optionally said gd-IgA1 comprises a glycosylated hinge region.

17. 17. The glycoengineered polypeptide of claim 16, wherein the one or more peptides that specifically bind to gd-IgA1 recognize a glycan profile on gd-IgA1 that is different from a reference glycan profile of IgA1.

18. 18. The glycomodified polypeptide of claim 17, wherein said reference glycan profile comprises an O-glycosylation profile, and optionally said reference O-glycosylation profile comprises N-acetylgalactosamine (GalNac).

19. 19. The glycoengineered polypeptide of claim 17 or 18, wherein the gd-IgA1 glycan profile comprises terminal GalNAc (also referred to as Tn antigen).

20. 19. The glycomodified polypeptide of claim 17 or 18, wherein the gd-IgA1 glycan profile comprises GalNAc with alpha 2,6-linked sialic acid (also referred to as STn antigen).

21. 21. The glycomodified polypeptide of any one of claims 16 to 20, wherein the one or more peptides that specifically bind to gd-IgA1 comprise an antibody agent comprising an antigen-binding fragment.

22. 22. The glycoengineered polypeptide of claim 21 , wherein the antibody agent comprises a whole antibody, a Fab fragment, a scFv, a nanobody, a duobody, a single domain antibody (e.g., a VHH).

23. 16. The glycomodified polypeptide of any one of claims 1 to 15, wherein the first portion comprises one or more peptides that specifically bind to IgA1, or a fragment or complex thereof.

24. 24. The glycoengineered polypeptide of claim 23, wherein said one or more peptides that specifically bind to IgA1 or a fragment thereof are each conjugated to a second moiety.

25. 25. The glycomodified polypeptide of claim 23 or 24, wherein the IgA1 amino acid sequence is provided as SEQ ID NO:

2.

26. The glycomodified polypeptide according to any one of claims 23 to 25, wherein the one or more peptides that specifically bind to IgA1 recognize an epitope on IgA1.

27. 27. The glycomodified polypeptide of any one of claims 23 to 26, wherein the first portion comprising one or more peptides, or fragments or complexes thereof, that specifically bind to IgA1 comprises a CD89 (FcaR1) polypeptide, or a variant thereof, or a fragment thereof.

28. 28. The glycomodified polypeptide of claim 27, wherein said CD89 comprises a soluble CD89 or a fragment or variant thereof.

29. 29. The glycomodified polypeptide of claim 27 or 28, wherein the CD89 polypeptide is provided as SEQ ID NO: 5 (e.g., with or without a signal peptide), SEQ ID NO: 9 (e.g., with or without a signal peptide), SEQ ID NO: 38 (e.g., with or without a signal peptide), or SEQ ID NO:

39.

30. 30. The glycomodified polypeptide of any one of claims 27 to 29, wherein the one or more peptides, or fragments or complexes thereof, that specifically bind to IgA1 comprise a fragment of a CD89 polypeptide.

31. 31. The glycomodified polypeptide of claim 30, wherein the one or more peptides comprise a full-length CD89 polypeptide or at least 5% of the CD89 polypeptide sequence provided in SEQ ID NO: 5 (e.g., with or without the signal peptide), SEQ ID NO: 9 (e.g., with or without the signal peptide), SEQ ID NO: 38 (e.g., with or without the signal peptide), or SEQ ID NO:

39.

32. The glycomodified polypeptide of any one of claims 27 to 31, wherein the fragment comprises one or more additional amino acid sequences on the 5' and / or 3' side of the fragment sequence.

33. 33. The glycomodified polypeptide of any one of claims 27 to 32, wherein the one or more peptides, or fragments or complexes thereof, that specifically bind to IgA1 comprise a variant of a CD89 polypeptide.

34. The one or more peptides, or fragments or conjugates thereof, that specifically bind to IgA1 are selected from the group consisting of: (i) SEQ ID NO: 5, with or without the signal peptide of SEQ ID NO: 44; (ii) SEQ ID NO: 9, with or without the signal peptide of SEQ ID NO: 44; (iii) SEQ ID NO: 38, with or without the signal peptide of SEQ ID NO: 42; or (iv) The glycomodified polypeptide of any one of claims 27 to 33, comprising a sequence having at least 85% identity to the CD89 polypeptide sequence provided in SEQ ID NO:

39.

35. 35. The glycomodified polypeptide of any one of claims 23 to 34, wherein the one or more peptides that specifically bind to IgA1 comprise a sequence having at least 85% identity to SEQ ID NO:

41.

36. 36. The glycomodified polypeptide of any one of claims 23 to 35, wherein the one or more peptides that specifically bind to IgA1 comprise an antibody agent comprising an antigen-binding fragment.

37. 37. The glycoengineered polypeptide of claim 36, wherein the antibody agent comprises a whole antibody, a Fab fragment, a scFv, a nanobody, a duobody, a single domain antibody (e.g., a VHH).

38. 16. The glycomodified polypeptide of any one of claims 1 to 15, wherein the first portion comprises one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies.

39. 39. The glycoengineered polypeptide of claim 38, wherein said one or more peptides that specifically bind to anti-gd-IgA1 or a fragment thereof are each conjugated to a second moiety.

40. The glycomodified polypeptide of claim 38 or 39, wherein the anti-gd-IgA1 autoantibody is IgG, IgM, IgE, IgD, or IgM.

41. The glycosylated polypeptide according to any one of claims 38 to 40, wherein the anti-gd-IgA1 autoantibody is an IgG containing a mutation in complementarity-determining region 3 (CDR3) of the Ig heavy chain (IgH) variable region.

42. 42. The glycomodified polypeptide of any one of claims 38 to 41, wherein the first portion comprises one or more peptides that specifically bind to mutations in CDR3 of the IgH region of an anti-gd-IgA1 autoantibody.

43. 43. The glycomodified polypeptide of any one of claims 38 to 42, wherein the one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies bind to an IgG protein, or a fragment or variant thereof.

44. 44. The glycoengineered polypeptide of any one of claims 38 to 43, wherein the first portion comprising one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies comprises a gd-IgA1 polypeptide, or a variant or fragment thereof.

45. 45. The glycoengineered polypeptide of claim 44, wherein the gd-IgA1 comprises a glycosylated hinge region.

46. 46. ​​The glycoengineered polypeptide of claim 45, wherein the first portion comprising one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies comprises a gd-IgA1 polypeptide fragment, optionally wherein the fragment comprises the sequence of SEQ ID NO: 1, or a fragment or variant thereof.

47. 47. The glycomodified polypeptide of claim 46, wherein the fragment comprises one or more additional amino acid residues at the 5' and / or 3' end of the sequence.

48. 48. The glycomodified polypeptide of any one of claims 38 to 47, wherein the one or more peptides that specifically bind to anti-gd-IgA1 comprise an antibody agent comprising an antigen-binding fragment.

49. 49. The glycoengineered polypeptide of claim 48, wherein the antibody agent comprises a whole antibody, a Fab fragment, a scFv, a nanobody, a duobody, a single domain antibody (e.g., a VHH).

50. 10. The glycomodified polypeptide of any one of the preceding claims, wherein said first portion is: (i) one or more peptides that specifically bind to gd-IgA1 or a fragment or complex thereof; (ii) one or more peptides that specifically bind to IgA1 or a fragment or complex thereof; (iii) one or more peptides that specifically bind to anti-gd-IgA1 autoantibodies or fragments or complexes thereof; or (iv) The glycomodified polypeptide, comprising any one or all of (i) to (iii).

51. 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) signal peptide (e) a tag, e.g., a cleavable tag; (f) a half-life prolonging domain, e.g., an Fc domain or albumin, or (g) The glycomodified polypeptide, comprising one or more additional elements selected from any combination of (a) to (f).

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

53. 53. The glycomodified polypeptide of claim 52, wherein said conjugation occurs intracellularly, and optionally said cell is a Leishmania cell.

54. 53. The glycomodified polypeptide of any one of claims 1 to 52, wherein the second moiety is conjugated to the first moiety by chemical conjugation, optionally wherein the chemical conjugation comprises click chemistry.

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

56. A composition comprising a glycomodified polypeptide according to any one of claims 1 to 54.

57. 55. A composition comprising a population of glycomodified polypeptides according to any one of claims 1 to 54, 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).

58. 58. The composition of claim 57, wherein the N-glycan profile comprises about 30% of the N-glycans of the structure provided in claim 10.

59. The composition of any one of claims 56 to 58, wherein the composition is a pharmaceutical composition.

60. 55. A Leishmania host cell expressing a glycomodified polypeptide according to any one of claims 1 to 54, said cell comprising a polynucleotide sequence encoding the glycomodified polypeptide.

61. 60. A method comprising administering to a subject the pharmaceutical composition of claim 59.

62. 62. The method of claim 61, wherein the subject has or is diagnosed as having a disease associated with increased and / or abnormal IgA.

63. 63. The method of claim 62, wherein the disease associated with increased and / or abnormal IgA is IgA nephropathy, dermatitis herpetiformis, or Henoch-Schonlein purpura.

64. 64. The method of any one of claims 61 to 63, wherein the method is a therapeutic or prophylactic method.

65. 60. A method for treating and / or preventing IgA nephropathy (IgAN) in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 59.

66. 66. The method of any one of claims 61 to 65, wherein the glycomodified polypeptide is capable of simultaneously binding to the targeting antibody at the first portion and to an endocytic receptor-expressing cell at the second portion, thereby internalizing the targeting antibody or an immune complex comprising it into the cell.

67. 67. The method of claim 66, wherein internalization comprises transport to lysosomes and / or degradation.

68. 68. The method of claim 66 or 67, wherein the endocytic receptor is ASGPR or a variant or fragment thereof.

69. The method of any one of claims 61 to 68, wherein the subject has increased levels of IgA1 or immune complexes containing IgA1 compared to a subject who does not have IgAN.

70. 70. The method of claim 69, wherein administration of the pharmaceutical composition reduces the level of IgA1 or an immune complex containing IgA1 compared to a subject not administered the pharmaceutical composition or compared to the same subject before administration of the pharmaceutical composition, and optionally, the reduction in the level of IgA1 or an immune complex containing IgA1 includes degradation of IgA1 or an immune complex containing IgA1.

71. 71. The method of any one of claims 61 to 70, wherein administering the pharmaceutical composition comprises: (i) preventing IgA1 from binding to an antigen or binding partner, optionally wherein the IgA1 binding partner is CD89 or a fragment thereof; or (ii) preventing and / or reducing the formation of immune complexes containing IgA1.

72. 72. The method of any one of claims 61 to 71, wherein administering the pharmaceutical composition comprises: (i) preventing gd-IgA1 from binding to an antigen or binding partner; or (ii) preventing and / or reducing the formation of immune complexes containing gd-IgA1.

73. 73. The method of any one of claims 61 to 72, wherein administering the pharmaceutical composition comprises: (i) prevent anti-gd-IgA1 from binding to an antigen or binding partner; or (ii) preventing and / or reducing the formation of immune complexes containing anti-gd-IgA1.

74. 74. The method of any one of claims 61-73, wherein administration of the pharmaceutical composition reduces activation of the complement system compared to a subject not administered the pharmaceutical composition or compared to the same subject prior to administration of the pharmaceutical composition, and optionally the complement system comprises the lectin pathway, the alternative pathway, or the classical pathway, or a combination thereof.

75. 75. The method of claim 74, wherein the complement system comprises C3, C5b, C6, C7, C8, and / or C9, or a fragment of any complement component or a combination thereof.

76. The method according to any one of claims 61 to 75, wherein the disease is treated and / or prevented by administering the pharmaceutical composition.

77. 77. The method of any one of claims 61 to 76, wherein administering the pharmaceutical composition reduces one or more symptoms of the disease.

78. assessing the level of the target antibody in a sample from the subject; and 60. A method comprising administering the pharmaceutical composition of claim 59 if the level of the target antibody is higher than a control.

79. 79. The method of any one of claims 61-78, comprising administering the pharmaceutical composition in combination with one or more additional therapies, optionally wherein the one or more additional therapies comprise an agent that inhibits activation of the complement system, an agent that treats proteinuria, an agent that treats blood pressure, a nephroprotectant, or a combination thereof.

80. 80. The method of any one of claims 61-79, wherein the administering step comprises intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular injection.

81. 81. The method of any one of claims 61 to 80, wherein the subject is a mammal.

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