Glycan-mediated protein degradation

Abstract

The present application relates to glycoengineered bifunctional degradation derivatives, populations of glycoengineered bifunctional degradation derivatives, Leishmania host cells for producing the glycoengineered bifunctional degradation derivatives, methods for genetically engineering the Leishmania host cells, methods for culturing the Leishmania host cells, methods for making glycoengineered bifunctional degradation derivatives using the Leishmania host cells, and methods of using the glycoengineered bifunctional degradation derivatives. In particular, the glycoengineered bifunctional degradation derivatives comprise biantennary GalNAc-terminated N-glycans, specifically A2GalNAc2.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Patent Application No. 63 / 410,955, filed September 28, 2022, and U.S. Patent Application No. 63 / 410,936, filed September 28, 2022, each of which is incorporated by reference herein in its entirety.

[0002] 2. Sequence Listing This application contains a computer readable Sequence Listing, submitted herewith in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The Sequence Listing XML file submitted herewith is named "14688-005-228_SEQLISTING.xml", was created on September 22, 2023, and is 52,246 bytes in size.

[0003] 3. Preface The present application relates to glycoengineered bifunctional degradation derivatives, populations of glycoengineered bifunctional degradation derivatives, Leishmania host cells for producing the glycoengineered bifunctional degradation derivatives, methods for genetically engineering the Leishmania host cells, methods for culturing the Leishmania host cells, methods for making glycoengineered bifunctional degradation derivatives using the Leishmania host cells, and methods of using the glycoengineered bifunctional degradation derivatives. In particular, the glycoengineered bifunctional degradation derivatives comprise biantennary GalNAc-terminated N-glycans, specifically A2GalNAc2. [Background technology]

[0004] 4.Background technology Glycoproteins are glycoconjugates with one or more glycans covalently attached (usually via N- or O-linkages) to the polypeptide backbone. N-glycans (N-linked oligosaccharides, N-[Asn]-linked oligosaccharides) are sugar chains covalently attached to asparagine residues in polypeptide chains, and generally contain GlcNAc residues in eukaryotes, with the consensus peptide sequence Asn-X-Ser / Thr (Varki, Ajit (2009): Essentials of glycobiology. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

[0005] Protein glycosylation is a ubiquitous post-translational modification found in all domains of life. Given the considerable complexity of animal systems, glycan structures play crucial biological and physiological roles, ranging from their involvement in protein folding and quality control to their involvement in numerous biological events, such as the recognition, stability, action, and turnover of these molecules (Moremen et al. 2012). Therapeutic glycoproteins, such as monoclonal antibodies, enzymes, and hormones, are key products of the biotechnology industry (Lagasse, H.A., Daniel et al. 2017; Dimitrov 2012), and the impact of glycan heterogeneity is increasingly recognized as a "critical quality attribute." Among the many characteristics that determine product quality, glycosylation is considered one of the most important, affecting a protein's biological activity, serum half-life, and immunogenicity. Glycans are associated with increased serum circulation time, and many approved or developmental biologics have insufficient half-lives and require frequent application to maintain therapeutic concentrations over extended periods. Half-life extension strategies are key to enabling the generation of long-acting therapeutics with improved pharmacokinetics (Kontermann 2016). Glycosylation also appears to improve protein solubility and stability, for example, by reducing the tendency for aggregation, leading to prolonged circulatory life by preventing proteolysis. In addition, N-glycans with different terminal monosaccharides can be recognized by lectins, leading to their degradation (Blasko et al., 2013; Varki, 2017). Therefore, monitoring and controlling glycosylation is important in biopharmaceutical manufacturing and regulatory requirements (Costa et al., 2014; Eon-Duval et al., 2012; Reusch and Tejada, 2015). For these reasons, glycoengineering of expression systems is increasingly recognized as an important strategy for improving many aspects of biopharmaceuticals (Dicker and Strasser 2015).

[0006] Endocytic lectins are involved in receptor-mediated endocytosis by capturing glycosylated proteins via specific glycan structures and mediating their degradation (Cummings et al., Cold Spring Harbor Laboratory Press, (2017)). Endocytic lectins are ubiquitous in the human body and can recognize a variety of glycan structures.

[0007] Carbohydrate-binding receptors are highly diverse and can be exploited by glycoengineering to develop novel therapeutics with unprecedented efficacy against various diseases, including, but not limited to, inflammation, hematological disorders, autoimmunity, and cancer. This enables the development of novel therapeutics based on the concept of glycan-mediated proteolysis. Harnessing the natural proteolysis caused by glycosylation of monoclonal antibodies may lead to novel therapeutics. The present invention presents the novel finding that Leishmania host cells can produce polypeptides containing biantennary GalNAc-terminal N-glycans, specifically A2GalNAc2, that mediate proteolysis.

[0008] The compositions and methods provided herein address unmet medical needs and provide related benefits for patients suffering from a variety of intractable diseases, such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases, that are treated with glycosylated proteins, such as monoclonal antibodies. Summary of the Invention

[0009] 5. Summary of the Invention Provided herein are glycoengineered bifunctional degradation derivatives, populations of glycoengineered bifunctional degradation derivatives, Leishmania host cells for producing glycoengineered bifunctional degradation derivatives, methods of genetically engineering the Leishmania host cells, methods of culturing the Leishmania host cells, methods of making glycoengineered bifunctional degradation derivatives using the Leishmania host cells, and methods of using the glycoengineered bifunctional degradation derivatives.

[0010] Without being bound by theory, glycoengineered bifunctional degradative derivatives of the present invention, in which peptidyl molecules such as antibodies and fragments thereof are modified with one or more A2GalNAc2 glycans, are optimally suited for the bifunctional role of binding to a desired target protein and engaging an ASGPR receptor via the N-glycan moiety. Without being bound by theory, it is believed that the bifunctional degradative derivatives simultaneously bind to the target protein and the ASGPR receptor(s) present in hepatocytes, and the complex thus formed is internalized and targeted for degradation via the lysosomal pathway. The inventors have found that the bifunctional degradative derivatives of the present invention, modified with one or more A2GalNAc2 glycans, are unexpectedly more efficient at internalizing and / or degrading target proteins than similar molecules modified with other N-glycans, such as triantennary GalNAc2-terminal glycans (often previously described for use in so-called "Lysosome-Targeting Chimeras" or "LYTACs"). See Zhou et al. (2021) ACS Cent. Sci. 7:499-506; Ahn et al. Nat Chem Biol. 2021 Sep;17(9):937-946; Zhao et al. (2022) Signal Transduction and Targeted Therapy 7:113; and Zhong et al. (2022) Antibodies 11:5, the disclosures of each of which are incorporated by reference in their entireties.

[0011] Provided herein are glycoengineered bifunctional degradative derivatives, wherein the bifunctional degradative derivatives (i) specifically bind to a target protein, and (ii) comprise an N-glycan of the structure: [ka] The N-glycan is linked to the bifunctional degraded derivative at one or more N-glycosylation sites, where the black square represents an N-acetylgalactosamine (GalNAc), 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 bifunctional degraded derivative. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at at least one N-glycosylation site. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at at least two N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at one, two, three, or four N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at one N-glycosylation site. In certain embodiments, the N-glycan is linked to the bifunctional degradative derivative at two N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degradative derivative at three N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degradative derivative at four N-glycosylation sites. In certain embodiments, the amino acid residue is Asn. In certain embodiments, the N-glycosylation site comprises the consensus sequence NXS / T or NXC, where X is any amino acid other than proline.

[0012] In certain embodiments, one or more N-glycosylation sites are distal to the target-specific binding site of the bifunctional degraded derivative. In certain embodiments, at least one or at least two N-glycosylation sites are distal to the target-specific binding site. In certain embodiments, all of the N-glycosylation sites are distal to the target-specific binding site. In certain embodiments, one or more N-glycosylation sites are not present in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degraded derivative. In certain embodiments, the target-specific binding site is the variable region of an antibody or antigen-binding fragment (Fab), or the ectodomain of an Fc fusion protein.

[0013] In certain embodiments, one or more N-glycosylation sites ("native N-glycosylation sites") present in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degraded derivative are deleted, mutated, or functionally inactivated. In certain embodiments, at least one or at least two natural N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, all natural N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, the one or more natural N-glycosylation sites are located at or proximal to a target-specific binding site of the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degraded derivative. In certain embodiments, the target-specific binding site is the variable region of an antibody or antigen-binding fragment (Fab), or the ectodomain of an Fc-fusion protein. In certain embodiments, one or more native N-glycosylation sites are deleted, mutated, or functionally inactivated, and one or more glyco-engineered N-glycosylation sites are present in the bifunctional degradable derivative, hi certain embodiments, the one or more glyco-engineered N-glycosylation sites are located distal to the target-specific binding position of the bifunctional degradable derivative.

[0014] In certain embodiments, the bifunctional degraded derivative is an antibody or fragment thereof. In certain embodiments, the bifunctional degraded derivative is a Fab fragment of an antibody. In certain embodiments, the glycoengineered bifunctional degraded derivative is an antibody. In certain embodiments, the antibody is a monoclonal or polyclonal antibody. In certain embodiments, the antibody is a recombinant antibody. In certain embodiments, the antibody is a humanized antibody, a chimeric antibody, or a fully human antibody. In certain embodiments, the antibody has a glycan-to-protein ratio of 2:1, 4:1, 6:1, 8:1, or 10:1. In certain embodiments, the N-glycan is linked to an N-glycosylation site on the light chain of the antibody or fragment thereof. In certain embodiments, the N-glycan is linked to an N-glycosylation site on the heavy chain of the antibody or fragment thereof. In certain embodiments, the one or more N-glycosylation sites are located in the constant domain of the antibody or fragment thereof. In certain embodiments, the one or more N-glycosylation sites are located in the variable domain of the antibody or fragment thereof. In certain embodiments, one or more N-glycosylation sites are located in the Fab region of the antibody. In certain embodiments, one or more N-glycosylation sites are located in the Fc region of the antibody. In certain embodiments, one or more N-glycosylation sites are located in the hinge region of the antibody. In certain embodiments, at least one of the N-glycosylation sites is not present in the wild-type form of the antibody. In certain embodiments, at least two N-glycosylation sites in the Fab region of the antibody are glycosylated by an N-glycan. In certain embodiments, one N-glycosylation site in the Fab region is located in each of the two heavy chain polypeptides of the antibody, and each of the N-glycosylation sites is glycosylated by an N-glycan. In certain embodiments, at least two N-glycosylation sites in the Fc region of the antibody are glycosylated by an N-glycan. In certain embodiments, the Fab region contains more N-glycans than the Fc region. In certain embodiments, the Fab region contains two more N-glycans than the Fc region. In certain embodiments, the Fc region contains more N-glycans than the Fab region, hi certain embodiments, the Fc region contains two or four more N-glycans than the Fab region.In certain embodiments, the Fc region and the Fab region contain the same number of N-glycans. In certain embodiments, the glycoengineered bifunctional degraded derivative binds to an autoantibody and comprises an autoantigen or an immunogenic fragment thereof.

[0015] In certain embodiments, the glycoengineered bifunctional degradation derivative comprises a moiety that specifically binds to a target protein, wherein the target protein is associated with a disease. In certain embodiments, the target protein is a cell surface molecule or a non-cell surface molecule. In certain embodiments, the cell surface molecule is a receptor. In certain embodiments, the non-cell surface molecule is an extracellular protein. In certain embodiments, the extracellular protein is an autoantibody, hormone, cytokine, chemokine, blood protein, or central nervous system (CNS) protein. In certain embodiments, the disease-associated target protein is upregulated in disease compared to non-disease states. In certain embodiments, the disease-associated target protein is expressed in disease compared to non-disease states. In certain embodiments, the disease-associated target protein is involved in cancer progression. In certain embodiments, the target protein associated with the disease is TNFα, HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, CCR2, Frizzled receptor, Wnt, LRP5 / 6, CSF-1R, SIRPα, CD38, CD73, or TGF-β, bombesin R, CAIX, CD13, CD44, v6, EMMPRIN, Endoglin, EpCAM, EphA2, FAP-α, Folate R, GRP78, IGF- 1R, matriptase, mesothelin, sMET / HGFR, MT1-MMP, MT6-MMP, PSCA, PSMA, Tn antigen, and uPAR, TSHRα, MOG, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin and ganglioside GM1, GD3, GQ1B, LILRB1, LILRB2, VEGF-R, CXCR4, CXCL12, CSF-1, CD47, aggregated light chain, or aggregated transthyretin.In certain embodiments, the disease-related target protein is involved in autoimmune disease, and the target protein is TSHRα, MOG, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or an antibody that binds to GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin, or ganglioside GM1, GD3, or GQ1B.In certain embodiments, the disease includes cancer.In certain embodiments, the disease includes autoimmune disease.

[0016] Also provided herein is a composition comprising a population of bifunctional degraded derivatives described herein, wherein the population of bifunctional degraded derivatives has 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 sites. In certain embodiments, the N-glycan profile 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 at least 98% homogeneous at one of the N-glycosylation sites. In certain embodiments, the N-glycan profile 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 at least 98% homogeneous at two of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 50% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 60% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 70% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 80% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 90% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 95% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 98% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the homogeneity of the N-glycan profile at one or more of the N-glycosylation sites is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis.

[0017] In certain 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 following structure at one or more of the N-glycosylation sites: [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 a bifunctional degradation derivative. In certain embodiments, the N-glycan profile comprises about 90% to about 100% of the N-glycan at one N-glycosylation site. In certain embodiments, the N-glycan profile comprises about 95% to about 100% of the N-glycan at one N-glycosylation site. In certain embodiments, the N-glycan profile comprises about 90% to about 100% of the N-glycan at each of two N-glycosylation sites. In certain embodiments, the N-glycan profile comprises about 80% to about 90% of the N-glycans at two N-glycosylation sites collectively. In certain embodiments, the N-glycan profile collectively comprises about 90% to about 100% of the N-glycans at two N-glycosylation sites. In certain embodiments, the N-glycan profile collectively comprises about 90% to about 100% of the N-glycans at three or more N-glycosylation sites. In certain embodiments, the N-glycan profile collectively comprises about 70% to about 100% of the N-glycans at three or more N-glycosylation sites. In certain embodiments, the relative amounts of N-glycans at one or more N-glycosylation sites are measured by N-glycan analysis or glycopeptide analysis.

[0018] In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises 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 N-glycans of the following structure among all glycans in the N-glycan profile: [ka] wherein the black square represents an N-acetylgalactosamine (GalNAc), 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 bifunctional degradation derivative. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains at least 30% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains at least 40% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains at least 50% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains at least 60% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 70% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 80% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 90% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 95% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 98% of the N-glycan among all glycans in the N-glycan profile.

[0019] In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that contains the N-glycan at 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. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that contains the N-glycan at about 30% to about 40% of all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that contains the N-glycan at about 40% to about 50% of all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that contains the N-glycan at about 50% to about 60% of all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 60% to about 70% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 70% to about 80% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 80% to about 90% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 90% to about 100% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the relative amount of the N-glycan among all glycans in the N-glycan profile is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis.

[0020] In certain embodiments, the population has 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%, or at least 98% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 30% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 40% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 50% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 60% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 70% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 80% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 90% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 95% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 98% homogeneous.

[0021] In certain embodiments, the population has an N-glycan profile that is 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% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 30% to about 40% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 40% to about 50% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 50% to about 60% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 60% to about 70% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 70% to about 80% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 80% to about 90% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 90% to about 100% homogeneous. In certain embodiments, the homogeneity of the N-glycan profile is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis.

[0022] In certain embodiments, the bifunctional degradative derivatives of the population are expressed from one or more nucleic acid sequences in a Leishmania host cell.

[0023] 5.1 Definition As used herein, the term "glycan" may refer to an N-glycan. Based on the specific structure, one skilled in the art can know whether a particular glycan is an N-linked glycan.

[0024] The term "about," when used in conjunction with a numerical value, refers to any numerical value within ±1, ±5, or ±10% of the referenced numerical value.

[0025] As used herein, the term "patient" refers to animals (e.g., birds, reptiles, and mammals). In another embodiment, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a livestock or pet (e.g., a dog, cat, horse, goat, sheep, pig, donkey, or chicken). In certain embodiments, the subject is a human. The terms "subject" and "patient" may be used interchangeably herein.

[0026] The abbreviations "α[number]," "α[number], [number]," "β[number]," or "β[number], [number]" refer to a glycosidic bond or glycosidic linkage, which is a covalent bond that connects a carbohydrate residue to another group. An α-glycosidic bond is formed when both carbons have the same stereochemical configuration, while a β-glycosidic bond occurs when two carbons have different stereochemical configurations.

[0027] As used herein, the terms "glycoengineering," "glycoengineered," or their equivalents refer to a process for glycosylating a target protein, or a target protein (e.g., a bifunctional degradation derivative) produced by such a process, which uses an in vivo host cell system harboring one or more enzymes (e.g., pathways) that provide glycosylation of the target protein. Such host cell systems can be genetically engineered to incorporate glycosylation pathways that selectively glycosylate 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., production of glycoengineered proteins) can incorporate N-linked glycosylation. Host cells used for glycoengineering or production of 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 species of Leishmania. Glycoengineered bifunctional degradative derivatives also include bifunctional degradative derivatives that have been engineered to be selectively glycosylated at one or more specific sites when produced in a host cell system.

[0028] As used herein, the term "glycosylation site" or "glycosylation site" refers to a site of glycosylation in a protein. Such a glycosylation site 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 certain embodiments, the glycosylation site is present in a so-called glycotag fused to a bifunctional degradation derivative provided 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 the bifunctional degradation derivative via a peptide linker. In some embodiments, the glycotag is fused to the N-terminus of the bifunctional degradation derivative 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 degrading derivatives provided herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycotags.

[0029] As used herein, a "glycoengineered bifunctional degradative derivative" or "bifunctional degradative derivative" is a polypeptide that mediates the degradation of a target protein by specifically binding to the target protein and activating a degradative pathway in conjunction with an endocytic receptor.

[0030] As used herein, the bifunctional degraded derivatives provided herein are "glycosylated" by N-glycans when the N-glycans are linked to the bifunctional degraded derivative at one or more sites of the bifunctional degraded derivative. In certain embodiments, the N-glycans are linked to the N-glycosylation sites of the bifunctional degraded derivative. As used herein, the N-glycosylation site is "occupied" by an N-glycan when the N-glycan is linked to the N-glycosylation site.

[0031] In certain embodiments, an N-glycosylation site that is not present in the wild-type, natural, synthetic, and / or commercially available precursor of a bifunctional degradation derivative (i.e., that is incorporated into the amino acid sequence of the wild-type, natural, synthetic, and / or commercially available precursor of a bifunctional degradation derivative) is referred to herein as a "glyco-engineered N-glycosylation site." In certain embodiments, an N-glycosylation site that is not present in the wild-type precursor of a bifunctional degradation derivative is referred to herein as a "glyco-engineered N-glycosylation site." In certain embodiments, an N-glycosylation site that is not present in the natural precursor of a bifunctional degradation derivative is referred to herein as a "glyco-engineered N-glycosylation site." In this context, the term "natural" encompasses those that are produced in or derived from a living system. In certain embodiments, an N-glycosylation site that is not present in the synthetic precursor of a bifunctional degradation derivative is referred to herein as a "glyco-engineered N-glycosylation site." In certain embodiments, an N-glycosylation site that is not present in a commercially available precursor of a bifunctional degradative derivative is referred to herein as a "glyco-engineered N-glycosylation site." In certain embodiments, the wild-type, natural, synthetic, and / or commercially available precursor of a bifunctional degradative derivative is selected from the wild-type, natural, synthetic, and / or commercially available precursors of the degradative derivatives described in Section 7.1.

[0032] In certain embodiments, wild-type, naturally occurring, synthetic, and / or commercially available precursors of bifunctional degradation derivatives are also referred to herein as "unmutated forms of bifunctional degradation derivatives." In certain embodiments, wild-type precursors of bifunctional degradation derivatives are referred to herein as "unmutated forms of bifunctional degradation derivatives." In certain embodiments, natural precursors of bifunctional degradation derivatives are referred to herein as "unmutated forms of bifunctional degradation derivatives." In this context, the term "natural" encompasses those made in or derived from a biological system. In certain embodiments, synthetic precursors of bifunctional degradation derivatives are referred to herein as "unmutated forms of bifunctional degradation derivatives." In certain embodiments, commercially available precursors of bifunctional degradation derivatives are referred to herein as "unmutated forms of bifunctional degradation derivatives." In certain embodiments, wild-type, naturally occurring, synthetic, and / or commercially available precursors of bifunctional degradation derivatives are selected from wild-type, naturally occurring, synthetic, and / or commercially available precursors of degradation derivatives described in Section 7.1.

[0033] In certain embodiments, one or more N-glycosylation sites present in a wild-type, natural, synthetic, and / or commercially available precursor of a bifunctional degraded derivative are referred to herein as "native N-glycosylation sites." In certain embodiments, one or more N-glycosylation sites present in a wild-type precursor of a bifunctional degraded derivative are referred to herein as "native N-glycosylation sites." In certain embodiments, one or more N-glycosylation sites present in a natural precursor of a bifunctional degraded derivative (i.e., produced in or derived from a biological system) are referred to herein as "native N-glycosylation sites." In certain embodiments, one or more N-glycosylation sites present in a synthetic precursor of a bifunctional degraded derivative are referred to herein as "native N-glycosylation sites." In certain embodiments, one or more N-glycosylation sites present in a commercially available precursor of a bifunctional degraded derivative are referred to herein as "native N-glycosylation sites." In certain embodiments, the wild-type, natural, synthetic, and / or commercially available precursors of the bifunctional degradative derivatives are selected from the wild-type, natural, synthetic, and / or commercially available precursors of the degradative derivatives described in Section 7.1.

[0034] As used herein, the terms "distal" and "proximal" refer to the three-dimensional spatial proximity of, for example, an N-glycan or N-glycosylation site to a particular region of a bifunctional degradative, and thus relate to the quaternary structure of the bifunctional degradative, as opposed to its primary amino acid sequence.

[0035] As used herein, the term "inflammatory disorder" includes disorders, diseases, or conditions characterized by inflammation. Examples of inflammatory disorders include allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury, and transplant rejection, among others.

[0036] As used herein, the term "blood disorder" includes disorders, diseases, or conditions that affect the blood. Examples of blood disorders include, among others, anemia, bleeding disorders such as hemophilia, blood clots, and blood cancers such as leukemia, lymphoma, and myeloma.

[0037] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly in humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia.

[0038] The term "carrier" as used herein in the context of a pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. [Table 1] [Table 2] TIFF2025534987000007.tif79165 [Brief explanation of the drawings]

[0039] 6. Brief description of the drawings [Figure 1]This shows that antibodies displaying A2GalNAc2 glycans on the Fab are highly internalized by HepG2 cells. HepG2 cells were incubated with pHrodo-labeled antibodies for 4 hours. The graph shows the mean adjusted MFI (±SEM) of triplicate values ​​of pHrodo. The graph shows data from one representative experiment out of three.

[0040] [Figure 2] Figure 1 shows that internalization by HepG2 cells of antibodies displaying A2GalNAc2 glycans on the Fab is mediated by ASGPR. HepG2 cells were incubated with pHrodo-labeled antibodies (3 μg / ml) and the indicated inhibitors for 3 hours. Graph shows the average and individually adjusted pHrodo MFI of two independent experiments. Closed circles: no inhibitor. Closed triangles: fetuin. Open squares: asialofetuin. Closed diamonds: EGTA. Open circles: chloroquine. Open triangles: bafilomycin.

[0041] [Figure 3] A 50-fold higher concentration of asialofetuin was required to block 50% of A-84.86-A2 GalNAc2 incorporation. HepG2 cells were incubated with increasing concentrations of asialofetuin (0.002-4 μM) for 30 minutes, followed by incubation with pHrodo-labeled antibody. Graph shows data from one representative experiment out of three.

[0042] [Figure 4A] Figure 1 shows that internalization of A-84.86-A2GalNAc2 depends on ASGPR1 and ASGPR2. Flow cytometry staining for ASGPR1 and ASGPR2 on HepG2 cells treated with negative control siRNA or siRNA specific for ASGPR1 is shown. Graphs show expression normalized to negative control-treated cells. ASGPR1 siRNA results in a complete reduction of surface levels of ASGPR1 and also a strong reduction of ASGPR2. ASGPR2 homodimers fluctuate on cells. [Figure 4B] Figure 1 shows that internalization of A-84.86-A2GalNAc2 is dependent on ASGPR1 and ASGPR2. Flow cytometry staining for ASGPR1 and ASGPR2 on HepG2 cells treated with negative control siRNA and ASGPR2 siRNA is shown. Graph shows expression normalized to negative control-treated cells. ASGPR2 siRNA resulted in a complete reduction of ASGPR2 surface levels, but not ASGPR1. [Figure 4C] This shows that internalization of A-84.86-A2GalNAc2 is dependent on ASGPR1 and ASGPR2. The uptake of A-84.86-A2GalNAc2 antibody is completely inhibited by ASGPR1 and ASGPR2 siRNA, while the uptake of H-A2F (not an ASGPR-associated factor) remains unchanged. Graphs show average and individual data from four independent experiments (siRNA ASGPR1) or one experiment (siRNA ASGPR2). NS: Not statistically significant. **Statistically significant (p<0.01) by two-way ANOVA followed by Tukey's multiple comparison test.

[0043] [Figure 5A] Expression of ASGPR1 by flow cytometry normalized to isotype control staining in HepG2 (Sigma), HepG2 parent (HepG2-wt, Synthego), and HepG2-ASGPR1ko (Synthego) is shown. [Figure 5B] Expression of ASGPR2 normalized to isotype control staining in the same cells as in Figure 5A is shown. [Figure 5C] DOL-adjusted gMFI of the indicated pHRo-labeled antibodies incubated with the same cells as in panels A and B is shown.

[0044] [Figure 6A]This shows that internalized A-84.86-A2GalNAc2 antibody colocalizes with lysosomal vesicles in HepG2 cells. The graph shows the number of pHrodo spots (DeepRed channel) that colocalize with lysosomal ROIs, normalized to the number of cells per well, and plotted against incubation time. Lysosomal ROIs are revealed by LysoTracker green signals, as described in Example 5. Each data point is the average of three wells, and error bars indicate standard deviation. [Figure 6B] The same readouts as in Figure 6A are shown for a competitive inhibition experiment in which unlabeled A-84.86-A2GalNAc2 was titrated from 100 nM to 0.160 nM against 1 nM pHrodo A-84.86-A2GalNAc2 (some curves removed for clarity). [Figure 6C] The same readouts as in Figure 6A are shown for a competitive inhibition experiment in which asialofetuin was titrated from 100 μM to 0.160 μM against 1 nM pHrodo A-84.86-A2GalNAc2.

[0045] [Figure 7A] Western blot images are shown after incubating HepG2 cells with 100 μg / ml of antibody for the indicated times. Western blots were performed using anti-human IgG H+L antibody to detect both the heavy and light chains of the adalimumab antibody. [Figure 7B] Quantification of signals from Western blot images is shown. The graph shows the density of signals corresponding to internalized intact A-84.86-A2GalNAc2 (intact heavy chain + intact light chain indicated by arrows to the right of the 50 KDa and 25 KDa blots; black bars) or degradation fragments (indicated by arrows in the blot; white bars). Densities were normalized to the corresponding beta-actin signal.

[0046] [Figure 8]Figures A-B show that the antigen is well internalized when complexed with A-84.86-A2GalNAc2 but not when complexed with a control antibody. Figure A shows a Western blot image of an anti-lambda light chain blot (HCA202 detection) and beta-actin detection. Figure B shows quantification of the lambda light chain (HCA202) signal normalized to the corresponding beta-actin signal using the iBright system.

[0047] [Figure 9] These results demonstrate that the target HCA202 is rapidly internalized by hepatocytes after injection of GalNAc2-glycosylated antibodies, and that the internalized target is rapidly degraded after internalization. Mice (N = 3 / group) were intravenously injected with HCA202, followed immediately by injection of control mAb H-A2F, PBS, or A-84.86-A2GalNAc2. Livers were harvested at different time points, and HCA202 was quantified in liver protein extracts by Western blot. Panel A shows Western blot anti-lambda light chain (HCA202) from one representative animal per time point. Panel B shows the normalized lambda light chain band intensity of the Western blot. Each data point represents the mean ± SEM of n = 3 independent animals.

[0048] [Figure 10] HepG2 cells were plated in 12-well plates and treated with 5 ng / ml of the indicated antibodies for 24, 48, and 72 hours, and the amount of antibody depleted from the supernatant is shown. The graph shows the mean ± SD of the amount of antibody depleted from the supernatant of three independent experiments.

[0049] [Figure 11]We demonstrate that the A2GalNAc2 glycan displayed by a glycotag on the Fc (C-terminus of the HC; 11K2-gt1) drives efficient ASGPR-specific internalization of the antibody. The glycotag located on the LC (LCLgt1) was less efficient than the glycotag on the Fc. ASGPR-specific internalization is evident because ASGPR-specific HepG2 cells did not internalize the mutant. A mutant displaying glycotags at the C-terminus of the HC and the C-terminus of the LC (11K2-LCLgt1.gt1-A2GalNAc2) showed slightly better internalization than 11K2-gt1-A2GalNAc2. Similarly, the mutant 11K2-84.gt1-A2GalNAc2, which has a glycotag on the HC and a glycosylation site inserted at position 84 of the HC variable domain, showed higher internalization than the gt1 mutant. The graph shows the mean ± SEM of DOL-adjusted pHrodo geometric mean MFI (gMFI) from 4–6 independent experiments, excluding the 86-A1GalNAc2 variant (N=2). **Statistically significant difference (p<0.01) compared to the IgG4-PAA-A2F control antibody by Mann-Whitney test. # indicates that statistical testing was not performed because only two data points were available.

[0050] [Figure 12] Internalization of 11K2 glycovariants quantified by Western blot. HepG2 cells were incubated with 0.1 mg / ml of antibody. Western blots using anti-human IgG H+L and beta-actin were performed on cell protein extracts. The graph shows the intensity of the total antibody signal (heavy chain + light chain signal + degradation fragment signal) for each antibody, normalized to the corresponding beta-actin signal.

[0051] [Figure 13]This shows that glycotags at the C-terminus of the mAb light or heavy chain drive efficient clearance in vivo, and that the A1GalNAc1 glycan is inactive. Mice were injected with different glycoengineered variants of the 11K2 mAb. mAb levels were measured in the animals' serum by ELISA. IgG4-A2F is a non-glycoengineered mAb produced in CHO cells. The graph shows the mean ± SEM of mAb concentrations. The graph shows data from two independent experiments.

[0052] [Figure 14] This shows that Fab fragments displaying A2GalNAc2 glycans result in efficient internalization of target antigens by HepG2 cells. HepG2-wt and HepG2-ASGPR1ko cells were incubated with 3 μg / ml of pHrodo-BSA-fentanyl × Fab-Fent complex for 4 hours. Graphs show the mean ± SEM of the pHrodo geometric mean MFI (gMFI) of BSA-fentanyl in the indicated samples. N = 3. ********P < 0.0001, statistically significant difference by two-way ANOVA followed by Tukey's multiple comparison test. The control Fab-Fent-LCLgt1-A2, which displays a non-ASGPR-linked glycan on its glycotag, did not result in significant internalization of the BSA-fentanyl antigen. In contrast, Fab-Fent-LCLgt1-A2GalNAc2, which displays a single A2GalNAc2 glycan on its glycotag, induced clear internalization of the antigen. This internalization is ASGPR-dependent, as it is suppressed in HepG2 knockouts of ASGPR. Similarly, the mutant Fab-Fent-86.LCLgt1-A2GalNAc2 induced stronger ASGPR-dependent internalization of antigen compared with the LCLgt1 mutant.

[0053] [Figure 15]This shows that the internalized BSA-fentanyl antigen is rapidly degraded by the Fab anti-fentanyl glycovariant presenting A2GalNAc2. HepG2 cells were treated as described in Example 9. The figure shows Western blot images of anti-fentanyl antibody detection (top blot) and beta-actin (bottom blot) at the indicated time points after washing.

[0054] [Figure 16] The MOG-Fc-A2GalNAc2 construct was internalized by HepG2 cells as efficiently as the A-84.86-A2GalNAc2 antibody. Internalization was ASGPR-specific, as it was blocked by competition with asialofetuin. Graphs show the mean DOL-adjusted pHrodo gMFI and individual data points from two independent experiments.

[0055] [Figure 17] This figure shows that the location of the A2GalNAc2 N-glycan is important for autoantigen-based degradation compounds to capture autoantibodies. Panel A shows that engagement of the 8-18C5 antibody blocks internalization of MOG-Fc degradation derivatives but not of mutants containing a glycotag at the C-terminus of Fc. HepG2 cells were incubated with MOG bifunctional degradation derivatives complexed with 8-18C5-pHrodo. The graph shows the pHrodo signal detected in cells after incubation. Panel B shows a model of ASGPR engagement of endogenous glycans in the presence of a model autoantibody bound to a MOG bifunctional degradation derivative. The model autoantibody is represented by a cross-hatched structure. The N-glycan is indicated by an asterisk. ASGPR cannot engage endogenous glycans when the autoantibody is bound to the bifunctional degradation derivative. However, the Fc glycotag (gt1) remains accessible to the ASGPR when the MOG bifunctional degradation derivative is bound to the autoantibody.

[0056] [Figure 18]This shows that the MOG-Fc-N60Q-gt1-A2GalNAc2 bifunctional degradation derivative efficiently depletes model autoantibodies injected into rats. Rats (N=4 / group) were injected intraperitoneally with 8-18C5 antibody followed by subcutaneous injection of the MOG bifunctional degradation derivative. Total (free and bound) 8-18C5 levels were quantified in serum. Graphs show mean ± SEM (N=4 / group).

[0057] [Figure 19] This shows that antibodies displaying the A2GalNAc2 glycan structure result in potent clearance of target antigens from the circulation in rats. Rats were intravenously injected with HCA202 (0.5 mg / kg) and 5 mg / kg of antibody. The graph shows the mean ± SD of HCA202 serum concentrations (ng / ml) for 3-4 animals per group. Closed circles represent the H-A2F (adalimumab, Humira) treatment group. Open squares represent the A-84-A2GalNAc2 treatment group. Closed triangles represent the A-84.86-A2GalNAc2 treatment group. Closed squares represent the A-84.86-A2G2S2 treatment group. Open circles represent the PBS treatment group (HCA202 only). Open diamonds and dotted lines represent the A-M3 treatment group. For graphical representation, if HCA202 levels fell below the assay LLOQ (dotted LLOQ / MRD10 line = 20 ng / ml), they were taken as 19 ng / ml.

[0058] [Figure 20] This shows that antibodies displaying the A2GalNAc2 glycan structure distribute to liver regions at a rapid rate compared to control antibodies. Mice were intravenously injected with 5 mg / kg of CF750-labeled antibody and imaged using fluorescence tomography. Graphs show mean fluorescence in pmol ± SD for three animals / time points in the gated liver region of interest. Open squares and dotted lines indicate the Ptz-A2F treatment group. Closed circles indicate the H-A2F treatment group. Open diamonds indicate the A-84.86-A2G2S2 treatment group. Closed triangles indicate the A-84.86-A2G2 treatment group.

[0059] [Figure 21]This shows that the antibody distributed to the liver is degraded. Livers from mice intravenously (iv) injected with 5 mg / kg of CF750-labeled antibody were harvested at the indicated time points, and protein extracts were obtained from approximately 100 mg of liver homogenized in RIPA buffer (plus protease inhibitors). Fluorescence captured in the CF750 channel was quantified using the iBright system. Extracts were also blotted for beta-actin. The blot shows the CF750 fluorescent signal.

[0060] [Figure 22] Shown are relative densitometric units of the CF750 signal from the gel in Figure 21, normalized to beta-actin control intensity.

[0061] [Figure 23] This figure shows that subcutaneous (sc) injection prolongs antigen depletion from the circulation compared with intravenous (iv) injection. Rats were intravenously injected with HCA202 (0.5 mg / kg) and, 15 minutes later, received an intravenous injection of PBS, or an intravenous or subcutaneous injection of A-84.86-A2GalNAc2 at 5 mg / kg. The graph shows the mean ± SD of total (free + bound) HCA202 serum concentrations (ng / ml) for three animals / group. For graphical representation, if HCA202 levels fell below the assay LLOQ (dotted LLOQ / MRD10 line = 20 ng / ml), the value was set at 19 ng / ml.

[0062] [Figure 24] This shows that a single subcutaneous injection of the A-84.86-A2GalNAc2 pathway can result in antigen depletion for up to 48 hours. Rats were intravenously injected with HCA202 (0.5 mg / kg) and, 15 minutes later, received a subcutaneous injection of PBS or A-84.86-A2GalNAc2 at 10 mg / kg. The graph shows the mean ± SD of total (free + bound) HCA202 serum levels. The assay LLOQ is indicated by the dotted line (20 ng / ml).

[0063] [Figure 25]Figure 1 shows that a single injection of A-84.86-A2GalNAc2 via the subcutaneous route can result in antigen depletion for up to 96 hours. Rats were intravenously injected with HCA202 at the time indicated by the arrow. 15 minutes after the first HCA202 injection, rats were injected subcutaneously with PBS or A-84.86-A2GalNAc2. Graph shows mean ± SEM of total (free + bound) HCA202 serum levels. N=4 animals / group. Assay LLOQ is indicated by the dotted line.

[0064] [Figure 26] HepG2 knockout cells of ASGPR1 (HepG2-ASGR1ko) were treated with the Ptz-gt1-A2GalNAc2 or Ptz-hgt-A2GalNAc2 mutants, and no HER2 reduction was observed compared to isotype control or Ptz-A2F-treated cells. Graph shows Her2 detection in HepG2 parental (-wt) or HepG2 knockout cells of ASGPR1 (-ASGR1ko) treated with 1 μg / ml of the indicated antibody for 4 hours. Her2 detection is expressed as geometric mean fluorescence intensity (gMFI) normalized to isotype control treatment.

[0065] [Figure 27] These results demonstrate that HER2-Fc / trastuzumab immune complexes (ICs) primarily form 600-1300 kDa structures. Panel A shows an overlay of size-exclusion chromatograms of individual injections of trastuzumab and HER2-Fc at a 1:1 molar ratio and preformed immune complexes of trastuzumab and HER2-Fc. The size of the formed ICs was estimated using protein MW standards and dynamic light scattering measurements. Panel B demonstrates the structure of the formed ICs and that the Ptz-gt1 antibody can bind to the formed ICs.

[0066] [Figure 28]This demonstrates that large immune complexes can be internalized and degraded by mAb bifunctional degradation derivatives. Panel A shows anti-human IgG H+L Western blots and anti-beta-actin Western blots of HepG2 cell extracts. The positions of the heavy (H) and light (L) chains of different components are indicated within the gel. Trtz is trastuzumab. The Ptz-gt1 heavy chain has a higher molecular weight than trastuzumab HC due to glycotagging. Panel B shows a quantification graph of the total band intensity (all vertical lanes for each condition) of the anti-human IgG H+L Western blot using the IBright FL1500 analysis tool. The graph shows the adjusted total lane volume (= sum of pixel intensities including all bands within a lane) normalized to the corresponding beta-actin band intensity.

[0067] [Figure 29] These results demonstrate that HCA202 target depletion by A-gt1-A2GalNAc2 follows a dose-response and requires a low ratio of degradation derivative (A-gt1) to target (HCA202) for depletion. Rats were intravenously injected with HCA202 followed by various doses of PBS or A-gt1-A2GalNAc2. The numbers above the graph indicate the molar ratio of degradation derivative:target. The graph shows the mean ± SEM of HCA202 depletion from a theoretical C of zero for N=4 animals.

[0068] [Figure 30] These results demonstrate that target depletion by Fab-A-FLGT4 follows a dose-response and requires a low ratio of degradation derivative (Fab-A-FLGT4) to target (HCA202) for depletion. Rats (N=4 animals / group) were intravenously injected with HCA202 followed by various doses of PBS or Fab-A-FLGT4-A2GalNAc2. The numbers above the graph indicate the molar ratio of degradation derivative:target. The graph shows the mean ± SEM of HCA202 depletion from a theoretical C of zero. DETAILED DESCRIPTION OF THE INVENTION

[0069] 7. MODE FOR CARRYING OUT THE INVENTION Described herein are glycoengineered bifunctional degradation derivatives and populations comprising glycoengineered bifunctional degradation derivatives with improved functionality compared to control antibodies. As exemplified herein, glycoengineered bifunctional degradation derivatives can be engineered by introducing glycosylation sites into the glycoengineered bifunctional degradation derivative, resulting in an altered glycosylation profile that mediates endocytic receptor degradation of the glycoengineered bifunctional degradation derivative and its bound target. By customizing N-glycosylation, the glycoengineered bifunctional degradation derivatives described herein 1) have homogeneous 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 protein degradation in a highly optimized manner. Glycoengineered bifunctional degradation derivatives can be used as novel therapeutic agents to treat diseases, including, but not limited to, inflammatory disorders, hematological disorders, autoimmune disorders, infectious diseases, and cancer.

[0070] 7.1 Glycosylated bifunctional degradatives Provided herein are glycoengineered bifunctional degradative derivatives, wherein the bifunctional degradative derivatives (i) specifically bind to a target protein, and (ii) comprise an N-glycan of the structure: [ka] The N-glycans are linked to bifunctional degradatives at one or more N-glycosylation sites, where the black boxes represent N-acetylgalactosamine (GalNAc), the white boxes represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue of the bifunctional degradative.

[0071] In certain embodiments, the glycoengineered bifunctional degrading derivatives specifically bind to one or more target proteins, including, but not limited to, the target proteins described in Section 7.7. In certain embodiments, the glycoengineered bifunctional degrading derivatives specifically bind to one target protein. In certain embodiments, the glycoengineered bifunctional degrading derivatives comprise moieties that specifically bind to a target protein. In certain embodiments, the moieties comprise the heavy and light chain variable regions of an antibody, or functional fragments thereof. In certain embodiments, the moieties comprise the Fab region of a monoclonal antibody. In certain embodiments, the glycoengineered bifunctional degrading derivatives comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more moieties, each of which specifically binds to a target protein, such that a single bifunctional degrading derivative can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target protein molecules. In certain embodiments, each of the moieties specifically binds to the same target protein. In certain embodiments, 2, 3, 4, or more of the moieties bind to different target proteins.

[0072] In certain embodiments, the bifunctional degradative derivatives specifically bind to two different target proteins. In certain embodiments, the bifunctional degradative derivatives comprise a first moiety that specifically binds to a first target protein and a second moiety that specifically binds to a second target protein. In certain embodiments, the glycoengineered bifunctional degradative derivatives have (i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more first moieties that specifically bind to a first target protein, such that a single bifunctional degradative can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more first target protein molecules, and (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more second moieties that specifically bind to a second target protein, such that a single bifunctional degradative can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more second target protein molecules.

[0073] In certain embodiments, the bifunctional degraded derivative specifically binds to three different target proteins, hi certain embodiments, the bifunctional degraded derivative comprises a first moiety that specifically binds to a first target protein, a second moiety that specifically binds to a second target protein, and a third moiety that specifically binds to a third target protein. In certain embodiments, the glycoengineered bifunctional degradative derivatives have (i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more first moieties that specifically bind to a first target protein, such that a single bifunctional degradative derivative can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more first target protein molecules; (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more second moieties that specifically bind to a second target protein, such that a single bifunctional degradative derivative can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more second target protein molecules; and (iii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more third moieties that specifically bind to a third target protein, such that a single bifunctional degradative derivative can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more third target protein molecules.

[0074] In certain embodiments, the glycoengineered bifunctional degradation derivatives described herein activate natural degradation pathways. In certain embodiments, the natural degradation pathway comprises receptor-mediated endocytosis. Without being bound by theory, receptor-mediated endocytosis involves the capture of glycosylated proteins via specific glycan structures (e.g., by endocytic lectins) to mediate lysosomal degradation (Cummings et al., Cold Spring Harbor Laboratory Press, (2017)). Endocytic lectins are ubiquitous in the human body and can recognize a variety of glycan structures. Engagement of glycans with endocytic carbohydrate-binding proteins and receptors enables essential biological pathways, including, but not limited to, those involved in regulating immune responses, mediating protein clearance, protein turnover, and controlling the trafficking of soluble glycoproteins, glycolipids, and any natural molecule containing a glycan moiety.

[0075] In certain embodiments, activation of a natural degradation pathway by the glycoengineered bifunctional degradation derivative reduces the concentration of the target protein in a subject. In certain embodiments, activation of a natural degradation pathway by the glycoengineered bifunctional degradation derivative reduces the concentration of the target protein in the subject by 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%, at least 98%, or at least about 100% compared to the concentration in the subject prior to administration of the glycoengineered bifunctional degradation derivative. In certain embodiments, the glycoengineered bifunctional degradation derivative mediates endocytic receptor degradation of the glycoengineered bifunctional degradation derivative and the target protein to which it binds.

[0076] In certain embodiments, the glycoengineered bifunctional degrading derivative comprises an N-glycan of the structure: [ka] The N-glycans are linked to bifunctional degradation derivatives at one or more N-glycosylation sites, where 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 bifunctional degradation derivative, and the N-glycans specifically bind to one or more endocytic receptors that mediate lysosomal degradation. In certain embodiments, the endocytic receptors are endocytic carbohydrate-binding proteins and / or lectin receptors. In one embodiment, the endocytic carbohydrate-binding protein is ASGPR. In one embodiment, the N-glycans specifically bind to ASGPR.

[0077] ASGPR-mediated degradation in hepatocytes has many applications. Binding of ASGPR to the N-glycan structures disclosed herein can result in the selective degradation of one or more target proteins described in Section 7.7. For example, ASGPR-mediated degradation can result in the removal of cytokines, chemokines, and hormones. In addition, ASGPR-mediated degradation can be used to deliver target molecules to hepatocyte endosomes. Therefore, ASGPR-mediated degradation can be applied to various liver diseases while limiting systemic toxicity.

[0078] In certain embodiments, the bifunctional degradative derivatives comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites (or carbohydrate sites, such as N-glycosylation consensus sequences). In certain embodiments, the bifunctional degradative derivatives comprise 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 bifunctional degradative derivatives comprise at least 2 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivatives comprise at least 3 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivatives comprise at least 4 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivatives comprise at least 5 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivatives comprise at least 6 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivatives comprise at least 7 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises at least 8 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises at least 9 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises at least 10 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises 2 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises 3 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises 4 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises 5 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises 6 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises 7 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises 8 N-glycosylation sites. In certain embodiments, the bifunctional degradative derivative comprises 9 N-glycosylation sites. In a particular embodiment, the bifunctional degradative derivative comprises 10 N-glycosylation sites.

[0079] In certain embodiments, one or more N-glycosylation sites are incorporated into the amino acid sequence of the bifunctional degradative derivative (i.e., one or more N-glycosylation sites are not present in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradative derivative; such sites are also referred to herein as "glyco-engineered N-glycosylation sites"). In certain embodiments, at least one N-glycosylation site is incorporated into the amino acid sequence of the bifunctional degradative derivative. In certain embodiments, at least two N-glycosylation sites are incorporated into the amino acid sequence of the bifunctional degradative derivative. In certain embodiments, at least three N-glycosylation sites are incorporated into the amino acid sequence of the bifunctional degradative derivative. In certain embodiments, at least four N-glycosylation sites are incorporated into the amino acid sequence of the bifunctional degradative derivative. In certain embodiments, one or more N-glycosylation sites are engineered distal to the target-specific binding position of the bifunctional degradative derivative. In certain embodiments, at least one or at least two N-glycosylation sites are engineered distal to the target-specific binding site. In certain embodiments, all of the N-glycosylation sites are engineered distal to the target-specific binding site. In certain embodiments, the target-specific binding site is the variable region of an antibody or antigen-binding fragment (Fab), or the ectodomain of an Fc fusion protein. 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 bifunctional degradation derivative via a peptide linker. In certain embodiments, the glycotag is fused to the N-terminus of the bifunctional degradation derivative. In certain embodiments, the glycotag is fused to the C-terminus of the bifunctional degradation derivative. In certain embodiments, the glycotag is fused to both the N-terminus and the C-terminus of the bifunctional degradation derivative. In certain embodiments, one or more of the N-glycosylation sites are naturally occurring N-glycosylation sites (i.e., one or more of the N-glycosylation sites are present in a wild-type, natural, synthetic, or commercially available precursor of the bifunctional degraded derivative). In certain embodiments, at least one of the N-glycosylation sites is naturally occurring N-glycosylation site. In certain embodiments, at least two of the N-glycosylation sites are naturally occurring N-glycosylation sites.In certain embodiments, one or more native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, at least one or at least two native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, all native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, one or more native N-glycosylation sites are located at or proximal to the target-specific binding position of a wild-type, natural, synthetic, or commercially available precursor of the bifunctional degraded derivative. In certain embodiments, one or more native N-glycosylation sites are deleted, mutated, or functionally inactivated, and one or more glyco-engineered N-glycosylation sites are present in the bifunctional degraded derivative. In certain embodiments, one or more glyco-engineered N-glycosylation sites are located distal to the target-specific binding position of the bifunctional degraded derivative.

[0080] In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites can be glycosylated with N-glycans such that the resulting glycoengineered bifunctional degradable derivative is capable of engaging or binding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endocytic receptor molecules. In certain embodiments, the bifunctional degradable derivative 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 of the N-glycosylation sites. In certain embodiments, the bifunctional degradable derivative is glycosylated with N-glycans at at least two N-glycosylation sites. In certain embodiments, the bifunctional degradable derivative is glycosylated with N-glycans at at least three N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at at least four N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at at least five N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at at least six N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at at least seven N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at at least eight N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at at least nine N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at at least ten N-glycosylation sites. In certain embodiments, the bifunctional degraded derivatives are glycosylated with N-glycans at two N-glycosylation sites, hi certain embodiments, the bifunctional degraded derivatives are glycosylated with N-glycans at three N-glycosylation sites.In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at four N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at five N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at six N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at seven N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at eight N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at nine N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated with N-glycans at ten N-glycosylation sites. In certain embodiments, the bifunctional degraded derivative is glycosylated at the Asn amino acid residue of the bifunctional degraded derivative. 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 N-XC consensus sequence, where X is any amino acid other than proline.

[0081] 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 structure: [ka] The N-glycans are linked to the bifunctional degraded derivatives at one or more N-glycosylation sites, wherein a black box represents an N-acetylgalactosamine (GalNAc), an open box represents an N-acetylglucosamine (GlcNAc) residue, a black circle represents a mannose (Man) residue, and X represents an amino acid residue of the bifunctional degraded derivative. In certain embodiments, at least 10% of the N-glycosylation sites are occupied by the N-glycans. In certain embodiments, at least 20% of the N-glycosylation sites are occupied by the N-glycans. In certain embodiments, at least 30% of the N-glycosylation sites are occupied by the N-glycans. In certain embodiments, at least 40% of the N-glycosylation sites are occupied by the N-glycans. In certain embodiments, at least 50% of the N-glycosylation sites are occupied by the N-glycans. In certain embodiments, at least 60% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 70% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 80% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 90% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 95% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 98% of the N-glycosylation sites are occupied by the N-glycan.

[0082] In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at at least one N-glycosylation site. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at at least two N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at one, two, three, or four N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at one N-glycosylation site. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at two N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at three N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at four N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at an Asn amino acid residue of the bifunctional degraded derivative. In certain embodiments, the N-glycan is linked to the bifunctional degradative derivative at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the bifunctional degradative derivative at an NXS / T or NXC consensus sequence, where X is any amino acid other than proline.

[0083] In certain embodiments, one or more N-glycosylation sites are distal to the target-specific binding position of the bifunctional degraded derivative. In certain embodiments, at least one, at least two, at least three, or at least four N-glycosylation sites are distal to the target-specific binding position. In certain embodiments, at least one N-glycosylation site is distal to the target-specific binding position. In certain embodiments, at least two N-glycosylation sites are distal to the target-specific binding position. In certain embodiments, at least three N-glycosylation sites are distal to the target-specific binding position. In certain embodiments, at least four N-glycosylation sites are distal to the target-specific binding position. In certain embodiments, one N-glycosylation site is distal to the target-specific binding position. In certain embodiments, two N-glycosylation sites are distal to the target-specific binding position. In certain embodiments, three N-glycosylation sites are distal to the target-specific binding position. In certain embodiments, four N-glycosylation sites are distal to the target-specific binding site. In certain embodiments, all of the N-glycosylation sites are distal to the target-specific binding site. In certain embodiments, one or more N-glycosylation sites are absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradable derivative. In certain embodiments, at least one, at least two, at least three, or at least four N-glycosylation sites are absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradable derivative. In certain embodiments, at least one N-glycosylation site is absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradable derivative. In certain embodiments, at least two N-glycosylation sites are absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradable derivative. In certain embodiments, at least three N-glycosylation sites are absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradable derivative. In certain embodiments, at least four of the N-glycosylation sites are not present in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degraded derivative.In certain embodiments, one N-glycosylation site is absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradation derivative. In certain embodiments, two N-glycosylation sites are absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradation derivative. In certain embodiments, three N-glycosylation sites are absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradation derivative. In certain embodiments, four N-glycosylation sites are absent in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradation derivative. In certain embodiments, the target-specific binding site is the variable region of an antibody or antigen-binding fragment (Fab), or the ectodomain of an Fc-fusion protein.

[0084] In certain embodiments, the distal N-glycosylation site(s) and the target-specific binding location 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 distal N-glycosylation site(s) and the target-specific binding location are 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, about 200-300, or about 300-400 amino acids. In certain embodiments, the amino acid separation between the distal N-glycosylation site(s) and the target-specific binding position is the number of amino acids between the terminal amino acids of the N-glycosylation consensus sequence. Without being bound by theory, the bifunctional degradative derivative has a three-dimensional shape in addition to its primary amino acid structure because it folds in space. Also, without being bound by theory, this three-dimensional shape, including the location of the N-glycan, is not static but dynamic (see, for example, Re, S., et al. Biophysical Reviews, 4, 179-187 (2012)). Nevertheless, in certain embodiments, the distance between the distal N-glycosylation site(s) on the bifunctional degradative derivative and the target-specific binding position can be from the equilibrium shape of the bifunctional degradative derivative, as measured by any standard means known in the art, including, for example, computer modeling studies. In certain embodiments, the distal N-glycosylation site(s) and the target-specific binding position are separated by a distance of at least 4 nm, at least 5 nm, at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm, at least 10 nm, at least 11 nm, at least 12 nm, at least 13 nm, at least 14 nm, at least 15 nm, at least 16 nm, at least 17 nm, at least 18 nm, at least 19 nm, or at least 20 nm.In certain embodiments, the distal N-glycosylation site(s) and the target-specific binding location are separated by a distance of about 4 to 20 nm, about 5 to 20 nm, about 6 to 20 nm, about 7 to 20 nm, about 8 to 20 nm, about 9 to 20 nm, about 10 to 20 nm, about 11 to 20 nm, about 12 to 20 nm, about 13 to 20 nm, about 14 to 20 nm, about 15 to 20 nm, about 16 to 20 nm, about 17 to 20 nm, about 18 to 20 nm, about 4 to 6 nm, about 5 to 7 nm, about 7 to 9 nm, about 8 to 10 nm, about 9 to 11 nm, about 10 to 12 nm, about 11 to 13 nm, about 12 to 14 nm, about 13 to 15 nm, about 14 to 16 nm, about 15 to 17 nm, about 16 to 18 nm, or about 17 to 19 nm. In certain embodiments, the distance between the distal N-glycosylation site(s) and the target-specific binding site is selected to minimize steric hindrance between the bifunctional degradative derivative(s), the target protein(s), and / or the ASGPR receptor(s), for example, when the target protein is bound to the bifunctional degradative derivative. In certain embodiments, the N-glycan is linked to the bifunctional degradative derivative at an Asn amino acid residue of the bifunctional degradative derivative. In certain embodiments, the N-glycan is linked to the bifunctional degradative derivative at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the bifunctional degradative derivative at an NXS / T or N-XC consensus sequence, where X is any amino acid other than proline.

[0085] In certain embodiments, one or more N-glycosylation sites ("native N-glycosylation sites") present in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degraded derivative are deleted, mutated, or functionally inactivated. In certain embodiments, at least one, at least two, at least three, or at least four native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, at least one native N-glycosylation site is deleted, mutated, or functionally inactivated. In certain embodiments, at least two native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, at least three native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, at least four native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, one native N-glycosylation site is deleted, mutated, or functionally inactivated. In certain embodiments, two native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, three native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, four native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, all native N-glycosylation sites are deleted, mutated, or functionally inactivated. In certain embodiments, one or more native N-glycosylation sites are located at or proximal to a target-specific binding site of a wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradation derivative. In certain embodiments, the target-specific binding site is the variable region of an antibody or antigen-binding fragment (Fab), or the ectodomain of an Fc fusion protein.

[0086] In certain embodiments, the glycoengineered bifunctional degraded derivative comprises two different N-glycans (i.e., a first and a second N-glycan), wherein each N-glycan is independently linked to the bifunctional degraded derivative at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites, and wherein one of the N-glycans (i.e., the first N-glycan) has the 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 a bifunctional degradation derivative. In certain embodiments, different N-glycans specifically bind to different endocytic receptors. In certain embodiments, the first N-glycan specifically binds to ASGPR. In certain embodiments, the other N-glycan (i.e., the second N-glycan) is an N-glycan described in Section 5.3. In certain embodiments, the other N-glycan is an N-glycan described in PCT / EP2022 / 057556 (incorporated herein by reference in its entirety). 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, N-glycosylation sites that are predominantly or exclusively occupied by larger N-glycans are more sterically accessible than N-glycosylation sites that are predominantly or exclusively occupied by smaller N-glycans. 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 bifunctional degradation derivative at an Asn amino acid residue of the bifunctional degradation derivative. In certain embodiments, the N-glycan is linked to the bifunctional degradation derivative at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the bifunctional degradation derivative at an NXS / T or NXC consensus sequence, where X is any amino acid other than proline. In certain embodiments, the first N-glycan is linked to the bifunctional degradative derivative 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 bifunctional degradative derivative at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites.

[0087] In certain embodiments, the bifunctional degraded derivative further comprises a third N-glycan, wherein the third N-glycan is linked to the bifunctional degraded derivative 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 an endocytic receptor that is different from the first and / or second N-glycan. In certain embodiments, the third N-glycan is an N-glycan described in Section 5.3. In certain embodiments, the third N-glycan is an N-glycan described in PCT / EP2022 / 057556 (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 bifunctional degraded derivative at an Asn amino acid residue of the bifunctional degraded derivative. In certain embodiments, the third N-glycan is linked to the bifunctional degradative derivative at an N-glycosylation consensus sequence. In certain embodiments, the third N-glycan is linked to the bifunctional degradative derivative at an NXS / T or NXC consensus sequence, where X is any amino acid other than proline.

[0088] 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 R (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 tertiary N-glycans specifically bind to a receptor selected from the group consisting of macrophage mannose 2 receptor, BDCA-2, DCIR, MBL, MDL, MICL, CLEC2, DNGR1, CLEC12B, DEC-205, and mannose 6-phosphate receptor (M6PR).

[0089] CD206 is a C-type lectin and a phagocytosis / endocytosis recycling and signaling receptor. CD206 is primarily expressed by M2 anti-inflammatory macrophages, dendritic cells, and viable 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.

[0090] In certain embodiments, the glycoengineered bifunctional degradative derivative is glycosylated at two or more N-glycosylation sites with an N-glycan of the 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 a bifunctional degradation derivative, and two of the 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 bifunctional degradative derivative 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 separation 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, the bifunctional degradative derivative has a three-dimensional shape in addition to its primary amino acid structure as it folds in space. Also, without being bound by theory, this three-dimensional shape, including the location of the N-glycans, is not static but dynamic (see, e.g., Re, S., et al., Biophysical Reviews, 4, 179-187 (2012)). Nevertheless, in certain embodiments, the distance between the N-glycosylation sites and / or N-glycans on the bifunctional degraded derivative can be from the equilibrium shape of the bifunctional degraded derivative, as measured by any standard means known in the art, including, for example, computer modeling studies. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at two N-glycosylation sites separated by a distance of at least 1.0 nm. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at two 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 bifunctional degraded derivative at two 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 bifunctional degraded derivative 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 bifunctional degraded derivative 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 bifunctional degraded derivative 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 bifunctional degraded derivative 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 bifunctional degradation derivative(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 two-fold or more than half) the spacing of ASGPR receptors on the cell surface. In certain embodiments, the N-glycan is linked to the bifunctional degradation derivative at an Asn amino acid residue of the bifunctional degradation derivative. In certain embodiments, the N-glycan is linked to the bifunctional degradation derivative at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the bifunctional degradation derivative at an NXS / T or N-X consensus sequence, where X is any amino acid other than proline.

[0091] In certain embodiments, the bifunctional degradation derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptide chains. In certain embodiments, each chain can be produced in a different cell line. In certain embodiments, the bifunctional degradation derivative is a dimer comprising two polypeptide chains having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or about 100% sequence identity. In certain embodiments, the dimer comprises two identical polypeptide chains. In certain embodiments, the bifunctional degradation derivative comprises four polypeptide chains. In certain embodiments, two of the four polypeptide chains have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or about 100% sequence identity to each other. In certain embodiments, two of the four polypeptide chains are identical to each other.

[0092] In certain embodiments, the bifunctional degradation derivative is an antibody or a fragment thereof. In other embodiments, the antibody is a full-length antibody, Fab, F(ab')2, Scfv, or sdAb. In certain embodiments, the bifunctional degradation derivative is a Fab, scFv, Fc, or Fv fragment of an antibody. In certain embodiments, the bifunctional degradation derivative is an antibody. In certain embodiments, the antibody is isolated from a human subject. In certain embodiments, the antibody is a monoclonal or polyclonal antibody. In certain embodiments, the antibody is a recombinant antibody. In certain embodiments, the antibody is a humanized antibody, a chimeric antibody, or a fully human antibody. In certain embodiments, the bifunctional degradation derivative is an autoantigen. In certain embodiments, the bifunctional degradation derivative is an autoantibody. In certain embodiments, the glycoengineered bifunctional degradation derivative is a nanobody.

[0093] In certain embodiments, the glycoengineered bifunctional degrading derivative is an antibody. In certain embodiments, the antibody is selected from the group consisting of adalimumab (Humira® (AbbVie Inc.)), Remicade® (Janssen Biotech, Inc.) (infliximab), ReoPro® (Janssen Biotech, Inc.) (abciximab), Rituxan® (Genentech, Inc.) (rituximab), Simulect® (Novartis Pharmaceuticals Corporation) (basiliximab), Synagis® (Medimmune, LLC) (palivizumab), Herceptin® (Genentech, Inc.) (trastuzumab), Mylotarg® (Pfizer) (gemtuzumab ozogamicin), Campath® (Takeda Oncology Corporation) (alemtuzumab), Zevalin® (Acrotech Biopharmaceuticals Corporation) (anticoagulant), and anticoagulant. Inc.) (ibritumomab tiuxetan), Xolair® (Genentech, Inc.) (omalizumab), Bexxar® (GlaxoSmithKline) (tositumomab-I-131), Erbitux® (Lilly USA, Inc.) (cetuximab), Avastin® (Genentech, Inc.) (bevacizumab), Tysabri® (Biogen Idec Corporation) (natalizumab), Actemra® (Genentech) (tocilizumab), Vectibix® (Amgen, Inc.) (panitumumab), Lucentis® (Genentech, Inc.) (ranibizumab), Soliris® (Alexion) Pharmaceuticals Inc.) (eculizumab), Cimzia® (UCB Pharma Ltd.) (certolizumab pegol), Simponi® (Janssen Biotech, Inc.)) (golimumab), Ilaris® (Novartis Pharmaceuticals Corporation) (canakinumab), Stelara® (Janssen Biotech, Inc.) (ustekinumab), Arzerra® (GlaxoSmithKline) (ofatumumab), Prolia® and Xgeva® (Amgen, Inc.) (denosumab), Numax® (Medimmune, LLC) (motavizumab), AB Thrax® (GlaxoSmithKline) (raxibacumab), Benlysta® (GlaxoSmithKline) (belimumab), Yervoy® (Bristol-Myers Squibb) Squibb) (ipilimumab), Adcetris® (Seagen, Inc.) (brentuximab vedotin), Perjeta® (Genentech, Inc.) (pertuzumab), Kadcyla® (Genentech, Inc.) (Ado-trastuzumab emtansine), or Gazyva® (Genentech, Inc.) (obinutuzumab).

[0094] In certain embodiments, the bifunctional degradation derivative is an antibody or fragment thereof, wherein the antibody comprises one or more N-glycosylation sites that are glycosylated with an N-glycan of the 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, and X represents an amino acid residue of the antibody. In certain embodiments, the N-glycan is linked to an N-glycosylation site of the light chain of the antibody or fragment thereof. In certain embodiments, the N-glycan is linked to an N-glycosylation site of the heavy chain of the antibody or fragment thereof. In certain embodiments, one or more of the N-glycosylation sites are located in the constant domain of the antibody or fragment thereof. In certain embodiments, one or more of the N-glycosylation sites are located in the variable domain of the antibody or fragment thereof. In certain embodiments, one or more of the N-glycosylation sites are located in the Fab region of the antibody. In certain embodiments, one or more of the N-glycosylation sites are located in the Fc region of the antibody. In certain embodiments, one or more of the N-glycosylation sites are located in the hinge region of the antibody. In certain embodiments, the N-glycan is linked to the bifunctional degraded derivative at an Asn amino acid residue of the antibody. In certain embodiments, the N-glycan is linked to the antibody at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the antibody at an NXS / T or NXC consensus sequence, where X is any amino acid except proline. In certain embodiments, at least one of the N-glycosylation sites is not present in the wild-type form of the antibody. In certain embodiments, two of the N-glycosylation sites are not present in the wild-type form of the antibody.

[0095] In certain embodiments, the Fab region of the antibody has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites. In certain embodiments, the Fab region of the antibody has 2 N-glycosylation sites. In certain embodiments, the Fab region of the antibody has 4 N-glycosylation sites. In certain embodiments, the Fab region of the antibody has 6 N-glycosylation sites. In certain embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites in the Fab region of the antibody are glycosylated with an N-glycan of the structure: [ka] wherein the black box represents an N-acetylgalactosamine (GalNAc) residue, the white box represents an N-acetylglucosamine (GlcNAc) residue, the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the antibody. In certain embodiments, at least two N-glycosylation sites in the Fab region of the antibody are glycosylated by an N-glycan. In certain embodiments, two N-glycosylation sites in the Fab region of the antibody are glycosylated by an N-glycan. In certain embodiments, one N-glycosylation site in the Fab region is located in each of the two heavy chain polypeptides of the antibody, and each of the N-glycosylation sites is glycosylated by an N-glycan. In certain embodiments, four N-glycosylation sites in the Fab region of the antibody are glycosylated by N-glycans. In certain embodiments, six N-glycosylation sites in the Fab region of the antibody are glycosylated by N-glycans.

[0096] In certain embodiments, the Fc region of the antibody has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites. In certain embodiments, the Fc region of the antibody has 2 N-glycosylation sites. In certain embodiments, the Fc region of the antibody has 4 N-glycosylation sites. In certain embodiments, the Fc region of the antibody has 6 N-glycosylation sites. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites in the Fc region of the antibody are glycosylated with an N-glycan of the structure: [ka] wherein the black box represents an N-acetylgalactosamine (GalNAc) residue, the white box represents an N-acetylglucosamine (GlcNAc) residue, the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the antibody. In certain embodiments, at least two N-glycosylation sites in the Fc region of the antibody are glycosylated by an N-glycan. In certain embodiments, two N-glycosylation sites in the Fc region of the antibody are glycosylated by an N-glycan.

[0097] In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites in the Fc region of the antibody are glycosylated by N-glycans described in Section 5.3. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites in the Fc region of the antibody are glycosylated by N-glycans described in PCT / EP2022 / 057556 (incorporated herein by reference in its entirety). In certain embodiments, at least two N-glycosylation sites in the Fc region of the antibody are glycosylated by N-glycans having the A2 structure. In certain embodiments, two N-glycosylation sites in the Fc region of the antibody are glycosylated by N-glycans having the A2 structure. In certain embodiments, four N-glycosylation sites in the Fc region of the antibody are glycosylated by N-glycans having the A2 structure.

[0098] In certain embodiments, the Fc region comprises two different N-glycans (i.e., a first and a second N-glycan), wherein each N-glycan is independently linked to the Fc region at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites, and wherein one of the N-glycans (i.e., the first N-glycan) has the structure: [ka] wherein the black box represents an N-acetylgalactosamine (GalNAc) residue, the white box represents an N-acetylglucosamine (GlcNAc) residue, the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the antibody. In certain embodiments, at least two N-glycosylation sites in the Fc region of the antibody are glycosylated by a first N-glycan. In certain embodiments, at least two N-glycosylation sites in the Fc region of the antibody are glycosylated by a first N-glycan. In certain embodiments, at least two N-glycosylation sites in the Fc region of the antibody are glycosylated by a second N-glycan. In certain embodiments, two N-glycosylation sites in the Fc region of the antibody are glycosylated by a second N-glycan. In certain embodiments, different N-glycans specifically bind to different endocytic receptors. In certain embodiments, the first N-glycan specifically binds to ASGPR. In certain embodiments, the second N-glycan is an N-glycan described in Section 5.3. In certain embodiments, the second N-glycan is an N-glycan described in PCT / EP2022 / 057556 (each of which is incorporated herein by reference in its entirety). 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, N-glycosylation sites predominantly or exclusively occupied by larger N-glycans are more sterically accessible than N-glycosylation sites predominantly or exclusively occupied by smaller N-glycans. In certain embodiments, the other N-glycan is A2. In certain embodiments, the other N-glycan is A1GalNAc1 or A2GalNAc1.

[0099] In certain embodiments, only the Fc region and / or hinge region of an antibody have one or more N-glycosylation sites. In other embodiments, the Fab region and Fc region of an antibody each independently have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites. In certain embodiments, the Fab region and Fc region of an antibody each independently have 2, 4, or 6 N-glycosylation sites. In certain embodiments, the Fab region contains more N-glycosylation sites than the Fc region. In certain embodiments, the Fab region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites than the Fc region. In certain embodiments, the Fab region contains 2 more N-glycosylation sites than the Fc region. In other embodiments, the Fc region contains more N-glycosylation sites than the Fab region. In certain embodiments, the Fc region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 more N-glycosylation sites than the Fab region. In certain embodiments, the Fc region contains 2 or 4 more N-glycosylation sites than the Fab region. In still other embodiments, the Fab region and Fc region contain the same number of N-glycosylation sites. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fab region are glycosylated with N-glycans having the structure: [ka] wherein the black boxes represent N-acetylgalactosamine (GalNAc), the white boxes represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue of the antibody. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites in the Fc region are glycosylated with an N-glycan having the structure: [ka] wherein the black boxes represent N-acetylgalactosamine (GalNAc), the white boxes represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue in the antibody. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites in the Fc region are glycosylated with an N-glycan described in Section 5.3. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites in the Fc region are glycosylated with an N-glycan described in PCT / EP2022 / 057556 (each of which is incorporated herein by reference in its entirety). In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites in the Fc region are glycosylated with an N-glycan having the structure A2. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites in the Fc region are glycosylated with two different N-glycans. In certain embodiments, the N-glycosylation sites in the Fab and / or Fc region distal to the hinge region of the antibody are glycosylated with an N-glycan having the structure: [ka] wherein the black boxes represent N-acetylgalactosamine (GalNAc) residues, the white boxes represent N-acetylglucosamine (GlcNAc) residues, the black circles represent mannose (Man) residues, and X represents an amino acid residue in the antibody. In certain embodiments, the N-glycosylation site in the Fab and / or Fc region proximal to the hinge region of the antibody is glycosylated with an N-glycan having the structure A2.

[0100] In certain embodiments, the Fab region contains more N-glycans than the Fc region. In some embodiments, the Fab region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycans compared to the Fc region. In some embodiments, the Fab region contains 2 more N-glycans than the Fc region. In some embodiments, about 10% of the N-glycans are in the Fc region and about 90% of the N-glycans are in the Fab region. In some embodiments, about 20% of the N-glycans are in the Fc region and about 80% of the N-glycans are in the Fab region. In some embodiments, about 30% of the N-glycans are in the Fc region and about 70% of the N-glycans are in the Fab region. In some embodiments, about 40% of the N-glycans are in the Fc region and about 60% of the N-glycans are in the Fab region. In some embodiments, about 50% of the N-glycans are in the Fc region and about 50% of the N-glycans are in the Fab region. In some embodiments, the N-glycan structures in the Fab region and the Fc region are identical (i.e., the same). In some embodiments, the N-glycan structures in the Fab region and the Fc region are not identical (i.e., not the same).

[0101] In certain embodiments, the Fc region contains more N-glycans than the Fab region. In some embodiments, the Fc region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycans compared to the Fab region. In some embodiments, the Fc region contains 2 or 4 more N-glycans than the Fab region. In some embodiments, about 10% of the N-glycans are in the Fab region and about 90% of the N-glycans are in the Fc region. In some embodiments, about 20% of the N-glycans are in the Fab region and about 80% of the N-glycans are in the Fc region. In some embodiments, about 30% of the N-glycans are in the Fab region and about 70% of the N-glycans are in the Fc region. In some embodiments, about 40% of the N-glycans are in the Fab region and about 60% of the N-glycans are in the Fc region. In some embodiments, about 50% of the N-glycans are in the Fab region and about 50% of the N-glycans are in the Fc region. In some embodiments, the N-glycan structures in the Fab region and the Fc region are identical (i.e., the same). In some embodiments, the N-glycan structures in the Fab region and the Fc region are not identical (i.e., not the same).

[0102] In certain embodiments, the target protein is a membrane protein and the Fc region and / or hinge region contain more A2GalNAc2 glycans than the Fab region. In certain embodiments, the target protein is a soluble protein and the Fab region contains more A2GalNAc2 glycans than the Fc region and / or hinge region. In other embodiments, the target protein is a soluble protein and the Fc region and / or hinge region contain more A2GalNAc2 glycans than the Fab region.

[0103] In certain embodiments, the antibody has an N-glycan to protein ratio of 2:1, 4:1, 6:1, 8:1, or 10:1. In some embodiments, the antibody is glycosylated at predetermined and specific residues. In other embodiments, the antibody is glycosylated at random residues.

[0104] In certain embodiments, the glycoengineered bifunctional degradative derivatives bind to an autoantibody and comprise an autoantigen or an immunogenic fragment thereof, hi certain embodiments, the glycoengineered bifunctional degradative derivatives comprise a moiety that specifically binds to a target protein, wherein the target protein is associated with a disease.

[0105] In certain embodiments, the glycoengineered bifunctional degradative derivative is a therapeutic polypeptide, i.e., a polypeptide used in the treatment of a disease or disorder. For example, the glycoengineered bifunctional degradative derivative can be an enzyme, cytokine, or antibody. In certain embodiments, the glycoengineered bifunctional degradative derivative is selected from the group consisting of adalimumab, rituximab, and erythropoietin (EPO).

[0106] The glycoengineered bifunctional degradative derivatives can be any polypeptide (or peptide / polypeptide corresponding to that polypeptide) known in the art and used in accordance with the methods described herein. One of skill in the art will readily appreciate that the nucleic acid sequences of known polypeptides as well as newly identified polypeptides can be readily deduced using methods known in the art, and thus, it is well within the capabilities of one of skill in the art to introduce nucleic acids encoding any bifunctional degradative derivative into a host cell provided herein (e.g., via an expression vector, e.g., a plasmid, e.g., via site-specific integration by homologous recombination).

[0107] In certain embodiments, the glycoengineered bifunctional degraded derivatives are selected from the group consisting of human interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin-IL-2 (denileukin diftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), tumor necrosis factor alpha (TNF-α), insulin, pramlintide, growth hormone (GH), insulin-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide-1 agonist (GLP-1), glucagon, growth hormone-releasing hormone (GHRH), secretin, thyroid-stimulating hormone (TSH), human The glycoengineered bifunctional degradative derivatives include the amino acid sequence of bone morphogenetic protein 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7), gonadotropin-releasing hormone (GnRH), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), fibroblast growth factor 7 (FGF7), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), neurotrophin-3, human follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), lutropin-α, erythropoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), the extracellular domain of CTLA4 (e.g., Fc fusion), or the extracellular domain of a TNF receptor (e.g., Fc fusion). In certain embodiments, the glycoengineered bifunctional degradative derivatives are enzymes or inhibitors.Exemplary enzymes and inhibitors that can be used as glycoengineered bifunctional degradation derivatives include, but are not limited to, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor VIIa, antithrombin III (AT-III), polypeptide C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, alglucosidase-α, laronidase (α-L-iduronidase), idursulfur enzymes (iduronate-2-sulfatase), galsulfase, agalsidase-β (human α-galactosidase A), botulinum toxin, collagenase, human DNAse-I, hyaluronidase, papain, L-asparaginase, uricase (urate oxidase), glutamic acid carboxypeptidase (glucarpidase), α1 protease inhibitor (α1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase.

[0108] In certain embodiments, the glycoengineered bifunctional degradable derivatives are cytokines. Exemplary cytokines that can be used as glycoengineered bifunctional degradable derivatives include, but are not limited to, interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin-IL-2 (denileukin diftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), and tumor necrosis factor alpha (TNF-α).

[0109] In certain embodiments, the glycoengineered bifunctional degradation derivatives are hormones or growth factors. Exemplary hormones and growth factors that can be used as glycoengineered bifunctional degradation derivatives include, but are not limited to, insulin, pramlintide, growth hormone (GH), insulin-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide-1 agonist (GLP-1), glucagon, growth hormone-releasing hormone (GHRH), secretin, thyroid-stimulating hormone (TSH), human bone morphogenetic protein 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7), gonadotropin-releasing hormone (GnR), and the like. H), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), fibroblast growth factor 7 (FGF7), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), neurotrophin-3, human follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), lutropin-α, erythropoietin, granulocyte-colony stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0110] In certain embodiments, the glycoengineered bifunctional degradative derivatives are receptors. Exemplary receptors that can be used as glycoengineered bifunctional degradative derivatives include, but are not limited to, the extracellular domain of human CTLA4 (e.g., Fc fusions) and soluble TNF receptors (e.g., Fc fusions).

[0111] In another embodiment, the glycoengineered bifunctional degradable derivative is a therapeutic polypeptide. In another embodiment, the glycoengineered bifunctional degradable derivative is an approved biologic. In another embodiment, the therapeutic polypeptide is abatacept (e.g., Orencia® (Bristol-Myers Squibb)), aducanumab-avwa (e.g., Aduhelm® (Biogen Corporation)), aflibercept (e.g., Eylea® (Regeneron Corp.)), agalsidase beta (e.g., Fabrazyme® (Genzyme Corp.)), albiglutide (e.g., Eperzan® (GlaxoSmithKline Corp.)), aldesleukin (e.g., Proleukin® (Clinigen, Inc.)), alefacept (e.g., Amevive® (Astellas Pharma, Inc.)), alglucerase (e.g., Ceredase® (Genzyme Corp.)), alglucosidase alfa (e.g., Lumizyme® (Genzyme Corp.)), or rifaceptin (e.g., Amevive® (Astellas Pharma, Inc.)). Corp.), aliskiren (e.g., Tekturna® (Noden Pharma)), alpha-1-polypeptidase inhibitors (e.g., Aralast® (Takeda Pharmaceuticals, Inc.)), alteplase (e.g., Activase® (Genentech)), anakinra (e.g., Kineret® (Sobi, Inc.)), anistreplase (e.g., Eminase® (SmithKlineBeecham)), anthrax human immune globulin (e.g., Anthrasil® (Cangene Corp.)), antihemophilic factor (e.g., Advate® (Baxter Healthcare Corp.)), antihemophilic factor Fc-VWF-XTEN fusion protein (e.g., Altuviiio® (Bioverativ Therapeutics, Inc.)), anti-inhibitor blood coagulation complex (e.g., Feiba Nf® (Takeda Pharmaceuticals, Inc.)), Pharmaceuticals, Inc.)), antithrombin-alpha, antithrombin III human, antithymocyte globulin (e.g., antithymocyte globulin), antithymocyte globulin (horse) (e.g., ATGAM® (Pfizer)), antithymocyte globulin (rabbit) (e.g., ATG-Fresenius), aprotinin (e.g., Trasylol® (Bayer AG)), asfotase alfa (e.g., Strensiq® (AstraZeneca)), asparaginase (e.g., Elspar® (Merck & Co., Inc.)), asparaginase blackleg (e.g., Erwinaze® (EUSA Pharma, Inc.)), becaplermin (e.g., Regranex® (Smith & Nephew, Inc.)), belatacept (e.g., Nulojix® (Bristol-Myers Squibb) Squibb), beractant, bivalirudin (e.g., Angiomax® (The Medicines Co.)), botulinum toxin type A (e.g., Botox® (Allergan, Inc.)), botulinum toxin type B (e.g., Myobloc® (Supernus Pharmaceuticals)), brentuximab vedotin (e.g., Adcetris® (Seagen Inc.)), buserelin (e.g., Suprecur® (Sanofi-Aventis)), C1 esterase inhibitor (human) (e.g., Cinryze® (Takeda Corporation)), C1 esterase inhibitor (recombinant) (e.g., Ruconest® (Salix Pharmaceuticals, Inc.)), cerliponase alfa (e.g., Brineura® Biomarin Pharmaceutical, Inc.)), certolizumab pegol (e.g., Cimzia® (UCB Pharma Ltd.)), chorionic gonadotropin alpha (e.g., Choriogonadotropin alfa), chorionic gonadotropin (human) (e.g., Ovidrel® (EMD Serono)), chorionic gonadotropin (recombinant) (e.g., Ovitrelle® (Merck Serono)), coagulation factor IX (e.g., Alprolix® (Bioverativ Therapeutics, Inc.)), coagulation factor VIIa (e.g., NovoSeven® (Novo Nordisk A / S)), coagulation factor X human (e.g., Coagadex® (Bio Products Laboratory, Ltd.)), coagulation factor XIII A-subunit (recombinant), collagenase (e.g., Cordase® (Headway Pharma PVT Ltd.)), Conestat alfa, corticotropin (e.g., HPActhar® (Mallinckrodt Pharmaceuticals), corticotropin (e.g., Cortrosyn® (Amphastar Pharmaceuticals, Inc.)), darbepoetin alfa (e.g., Aranesp® (Amgen Inc.)), defibrotide (e.g., Noravid® (Gentium SpA)), denileukin diftitox (e.g., Ontak® (Eisai Medical Research)), desirudin, digoxin immune Fab (ovine) (e.g., Digibind® (GlaxoSmithKline LLC)), dornase alfa (e.g., Pulmozyme® (Genentech Inc.)), drotrecogin alfa (e.g., Xigris® (Eli Lilly & Co.)), dulaglutide (e.g., Trulicity® (Eli Lilly and Co.)), efgartigimod Alpha (e.g., Vyvgart® Hytrulo (Argenx, US, Inc.)), ecallantide (e.g., Kalbitor® (Dyax Corp.)), elapeguademase (e.g., Revcovi® (Leadiant Biosciences, Inc.)), efmoroctocog alfa (e.g., Elocta® (Swedish Orphan Biovitrum AB)), elosulfase alfa (e.g., Vimizim® (Biomarin Pharmaceutical, Inc.)), enfuvirtide (e.g., Fuzeon® (Genentech)), eptinezumab (e.g., Vyepti® (Lundbeck Seattle Biopharmaceuticals, Inc.)), epoetin alfa (e.g., Binocrit® (Sandoz GmbH)), epoetin zeta (e.g., Retacrit® (Pfizer)), eptifibatide (e.g., Integrilin® (COR Therapeutics, Inc.), etanercept (e.g., Enbrel® (Amgen Inc.)), exenatide (e.g., Byetta® (AstraZeneca)), factor IX complex (human) (e.g., AlphaNine® (Grifols Biologicals LLC)), fibrinolysin, also known as plasmin (e.g., Elase® (Parke-Davis)), filgrastim (e.g., NA), filgrastim-sndz, follitropin alfa (e.g., Gonal-F® (EMD Serono)), follitropin beta (e.g., Follistim AQ® (Organon & Co.)), galsulfase (e.g., Naglazyme® (BioMarin Pharmaceuticals)), Inc.), gastric intrinsic factor, gemtuzumab ozogamicin (e.g., Mylotarg® (Pfizer)), glatiramer acetate (e.g., Copaxone® (Teva Neuroscience)), glucagon recombinant (e.g., GlucaGen® (Novo Nordisk, Inc.))), glucarpidase (e.g., Voraxaze® (BTG Pharmaceuticals)), gramicidin D (e.g., Neosporin® (Johnson & Johnson Consumer, Inc.)), hepatitis B immune globulin, human calcitonin, human tetanus toxoid immune globulin, human rabies virus immune globulin (e.g., Hyperab Rabies Immune Globulin Human), human Rho(D) immune globulin (e.g., Hyp Rho D Inj 16.5%), human serum albumin (e.g., Albuminar® (CSL Behring LLC)), human varicella-zoster immune globulin (e.g., Varizig® (Cangene Corporation)), hyaluronidase (e.g., Hylenex® (Henry Schein, Inc.), hyaluronidase (human recombinant), ibritumomab tiuxetan (e.g., Zevalin® (Acrotech Biopharma Inc.)), idursulfase (e.g., Elaprase® (Takeda Pharmaceuticals, Inc.)), imiglucerase (e.g., Cerezyme® (Genzyme Corporation)), immunoglobulin human, insulin aspart (e.g., NovoLog® (Novo Nordisk A / S)), insulin steroid, insulin degludec (e.g., Tresiba® (Novo Nordisk A / S)), insulin detemir (e.g., Levemir® (Novo Nordisk A / S)), insulin glargine (e.g., Lantus® (Sanofi-Aventis US LLC)), insulin glulisine (e.g., Apidra® (Sanofi-Aventis US LLC)), insulin lispro (e.g., Humalog® (Eli Lilly Corp.)), insulin pork (e.g., Iletin II® (Eli Lilly Corporation)), insulin regular (e.g., Humulin R® (Eli Lilly Corp.)), insulin, porcine (e.g., Vetsulin® (Merck & Co.)), insulin, isophane (e.g., Novolin N® (Novo Nordisk A / S), interferon alfa-2a, recombinant (e.g., Roferon A® (Hoffman-LaRoche, Inc.)), interferon alfa-2b (e.g., Intron A® (Merck & Co., Inc.)), interferon alfacon-1 (e.g., Infergen® (Three Rivers Pharmaceuticals, LLC)), interferon alfa-n1 (e.g., Wellferon® (GlaxoSmithKline)), interferon alfa-n3 (e.g., Alfe. ron® (AIM Immunotech Inc.)), interferon beta-1a (e.g., Avonex® (Biogen-Idec Corporation)), interferon beta-1b (e.g., Betaseron® (Bayer Healthcare Pharmaceuticals)), interferon gamma-1b (e.g., Actimmune® (Horizon Pharma USA, Inc.)), intravenous immunoglobulin (e.g., Xembify® (Grifols Therapeutics LLC)), laronidase (e.g., Aldurazyme® (Genzyme Corporation)), lenograstim (e.g., Granocyte® (Chugai Pharmaceuticals, Inc.)), lepirudin (e.g., Refludan® (Behring GmbH)), leuprolide (e.g., Eligard® (Sanofi-Aventis) US, LLC)), liraglutide (e.g., Saxenda® (Novo-Nordisk, Inc.)), lucinactant (e.g., Surfaxin), lutropin alfa (e.g., Luveris® (EMD Serono)), mecasermin (e.g., NA), menotropins (e.g., Menopur® (Ferring Pharmaceuticals)), methoxypolyethylene glycol-epoetin beta (e.g., Mircera® (Vifor Pharma)), metreleptin (e.g., Myalept® (AstraZeneca)), natural alpha interferon or multiferon (e.g., Intron / Roferon-A® (Merck & Co., Inc.)), Co. / Hoffman-LaRoche, Inc.), nesiritide (e.g., Natrecor® (Scios, Inc.)), ocriplasmin (e.g., Jetrea® (ThromboGenics, Inc.)), oprelvekin (e.g., Neumega® (Wyeth Pharmaceuticals Inc.)),)), OspA lipopolysaccharide peptide (e.g., Lymerix® (GlaxoSmithKline)), oxytocin (e.g., Pitocin® (Pfizer)), palifermin (e.g., Kepivance® (Amgen, Inc.)), pancrelipase (e.g., Creon® (AbbVie Inc.)), pegademase (e.g., Adagen® (Enzon Pharmaceuticals, Inc.)), pegaspargase (e.g., Oncaspar® (Sigma-Tau Pharmaceuticals, Inc.)), pegfilgrastim (e.g., Neulasta® (Amgen, Inc.)), pegylated interferon alpha-2a (e.g., Pegasys® (Genentech USA, Inc.)), pegylated interferon alpha-2b (e.g., PEG-Intron® (Merck & Co., Inc.)), Co.), pegylated interferon beta-1a (e.g., Plegridy® (Biogen Corporation)), pegloticase (e.g., Krystexxa® (Horizon Therapeutics)), pegvisomant (e.g., Somavert® (Pfizer)), boractant alpha (e.g., Curosurf® (Chiesi USA, Inc. / Cornerstone Therapeutics, Inc.)), pramlintide (e.g., Symlin® (AstraZeneca Pharmaceuticals)), preotact (e.g., Preotact® (Nycomed A / S)), protamine sulfate (e.g., Protamine Sulfate Injection, USP), polypeptide S human (e.g., Polypeptide S human), prothrombin (e.g., Feiba Nf® (Takeda Pharmaceuticals, Inc.)), prothrombin complex (e.g., Cofact® (Sanquin Plasma Products BV)), prothrombin complex concentrate (e.g., Kcentra® (CSL Behring LLC)), rasburicase (e.g., Elitek® (Sanofi-Aventis US, LLC)), reteplase (e.g., Retavase® (Chiesi USA, Inc.)), rilonacept (e.g., Arcalyst® (Kiniksa Pharmaceuticals, Ltd.)), romiplostim (e.g., Nplate® (Amgen, Inc.)), sacrosidase (e.g., Sucraid® (QOL Medical, LLC)), salmon calcitonin (e.g., Calcimar® (Sandoz GmbH)), sargramostim (e.g., Leucomax® (Novartis)), satumomab pendetide (e.g., OncoScint® (Cytogen Corporation), sebelipase alfa (e.g., Kanuma® (Alexion Pharmaceuticals, Inc.)), secretin (e.g., SecreFlo® (Repligen Corp.)), sermorelin (e.g., sermorelin acetate), serum albumin (e.g., Albunex® (Mallinckrodt Medical, Inc.)), iodinated serum albumin (e.g., Megatope® (Iso-Tex Diagnostics, Inc.)), simoctocog alfa (e.g., Nuwiq® (Octapharma USA, Inc.)), sipuleucel-T (e.g., Provenge® (Dendreon Corporation)), somatotropin recombinant (e.g., Nutropin AQ® (Genentech)), somatropin recombinant (e.g., BioTropin® (Bio-Technology General), streptokinase (e.g., Streptase® (CSL Behring LLC)), susoctocog alfa (e.g., Obizur® (Baxalta US, Inc.)), taliglucerase alfa (e.g., Elelyso® (Pfizer, Inc.)),)), teduglutide (e.g., Gattex® (NPS Pharmaceuticals, Inc.)), tenecteplase (e.g., TNKase® (Genentech, Inc.)), teriparatide (e.g., Forteo® (Lilly US, LLC)), tesamorelin (e.g., Egrifta® (Theratechnologies, Inc.)), thrombomodulin alpha (e.g., Recomodulin® (Asahi Kasei Pharma)), thymalfasin (e.g., Zadaxin® (SciClone Pharmaceuticals, Int'l)), thyroglobulin, thyrotropin alpha (e.g., Thyrogen® (Genzyme Corporation)), purified tuberculin polypeptide derivative (e.g., Aplisol® (Par Pharmaceuticals)), turoctocog alfa (e.g., Zonovate® (Novo Pharmaceuticals)), Nordisk), urofollitropin (e.g., Bravelle® (Ferring Pharmaceuticals, Inc.)), urokinase (e.g., Kinlytic® (ImaRx Therapeutics, Inc.)), vasopressin (e.g., Pitressin® (JHP Pharmaceuticals, LLC)), velaglucerase alfa (e.g., Vpriv® (Takeda Pharmaceuticals)), abciximab (e.g., ReoPro® (Janssen Biotech, Inc.)), adalimumab (e.g., Humira® (AbbVie, Inc.)), alemtuzumab (e.g., Campath® (Takeda Oncology, Inc.)), alirocumab (e.g., Praluent® (Regeneron / Sanofi)), arcitumomab (e.g., CEA-Scan® (Immunomedics, Inc.)),)), atezolizumab (e.g., Tecentriq® (Genentech)), basiliximab (e.g., Simulect® (Novartis Pharmaceuticals Corporation)), belimumab (e.g., Benlysta® (GlaxoSmithKline Inc.)), benralizumab (e.g., Fasenra® (AstraZeneca)), bevacizumab (e.g., Avastin® (Genentech, Inc.)), bezlotoxumab (e.g., Zinplava® (Merck & Co., Inc.)), blinatumomab (e.g., Blincyto® (Amgen, Inc.)), brodalumab (e.g., Siliq® (Valeant Pharmaceuticals)), brolucizumab (e.g., Beovu® (Novartis Pharmaceuticals)), Corporation)), burosumab (e.g., Crysvita® (Ultragenyx, Inc.)), calaspargase pegol (e.g., Asparlas® (Servier Pharmaceuticals, LLC)), canakinumab (e.g., Ilaris® (Novartis Pharmaceuticals Corporation)), caplacizumab (e.g., Cablivi® (Ablynx, NV)), capromab (e.g., ProstaScint® (EUSA Pharma (USA) Inc.)), cemiplimab (e.g., Libtayo® (Regeneron Pharmaceuticals, Inc.)), cetuximab (e.g., Erbitux® (Lilly USA, LLC)), crizanlizumab (e.g., Adakveo® (Novartis Pharmaceuticals Corporation), daclizumab (e.g., Zenapax® (Hoffmann-LaRoche, Inc.)), daratumumab (e.g., Darzalex® (Janssen Biotech, Inc.)), denosumab (e.g., Prolia® (Amgen, Inc.)),, Xgeva® (Amgen, Inc.)), dinutuximab (e.g., Unituxin® (United Therapeutics Corp.)), dostallimab (e.g., Jemperli® (GlaxoSmithKline, LLC)), durvalumab (e.g., Imfinzi® (AstraZeneca)), dupilumab (e.g., Dupixent® (Regeneron Pharmaceuticals, Inc.)), eculizumab (e.g., Soliris® (Alexion Pharmaceuticals, Inc.)), efalizumab (e.g., Rapti. va® (Genentech, Inc.)), elotuzumab (e.g., Empliciti® (Bristol-Myers Squibb)), erlanatamab (e.g., Elrexfio® (Pfizer, Inc.)), emapalumab (e.g., Gamifant® (Sobi, Inc.)), emicizumab (e.g., Hemlibra® (Genentech, Inc.)), erenumab (e.g., Aimovig® (Amgen, Inc.)), evinacumab (e.g., Evkeeza® (Regeneron Pharmaceuticals, Inc.)), evolocumab (e.g., Repatha® (Amgen, Inc.)), fam-trastuzumab deruxtecan-nxki (e.g., Enhertu® (Daiichi Sankyo, Inc.), fremanezumab (e.g., Ajovy® (Teva Pharmaceuticals, Inc.)), galcanezumab (e.g., Emgality® (Eli Lilly and Company)), golimumab (e.g., Simponi® (Janssen Biotech, Inc.)), guselkumab (e.g., Tremfya® (Janssen Biotech, Inc.)), ibalizumab (e.g., Trogarzo® (Theratechnologies, Inc.)), ibritumomab (e.g., Zevalin® (Acrotech Biopharma Inc.)), idarucizumab (e.g., Praxbind® (Boehringer Ingelheim Pharmaceuticals, Inc.)), infliximab (e.g., Remicade® (Janssen Biotech, Inc.), ipilimumab (e.g., Yervoy® (Bristol-Myers Squibb)), isatuximab (e.g., Sarclisa® (Sanofi-Aventis, US, LLC)), ixekizumab (e.g., Taltz® (Eli Lilly & Co.)), lanadelumab (e.g., Takhzyro® (Takeda Pharmaceuticals, USA, Inc.)), magrolimab (Gilead Sciences, Inc.), margetuximab (e.g., Margenza® (Macrogenics, Inc.)), mepolizumab (e.g., Nucala® (GlaxoSmithKline)), muromonab (e.g., Orthoclone OKT3® (Centocor Ortho Biotech Products, LP)), natalizumab (e.g., Tysabri® (Biogen Idec Corporation)), necitumumab (e.g., Portrazza® (Eli Lilly and Company)), nivolumab (e.g., Opdivo® (Bristol-Myers Squibb)), obiltoxaximab (e.g., Anthim® (Elysys Therapeutics, Inc.), obinutuzumab (e.g., Gazyva® (Genentech, Inc.)), ofatumumab (e.g., Arzerra® (GlaxoSmithKline)), omalizumab (e.g., Xolair® (Genentech, Inc.)), palivizumab (e.g., Synagis® (Medimmune, LLC)), panitumumab (e.g., Vectibix® (Amgen, Inc.)), pembrolizumab (e.g., Keytruda® (Merck & Co.)), pertuzumab (e.g., Perjeta® (Genentech, Inc.)), polatuzumab (e.g., Polivy® (Genentech, Inc.)), pozelimuab (e.g., Veopoz (Regeneron Pharmaceuticals, Inc.), ramucirumab (e.g., Cyramza® (Eli Lilly and Company)), ranibizumab (e.g., Lucentis® (Genentech, Inc.))), ravulizumab-cwvz (e.g., Ultomoris® (AstraZeneca)), raxibacumab (GlaxoSmithKline), risanizumab (e.g., Risanizumab-rzaa, Skyrizi® (AbbVie Inc.)), rituximab (e.g., Rituxan® (Genentech, Inc.)), rozanolixizumab (e.g., Rystiggo® (UCB, Inc.)), sarilumab (e.g., Kevzara® (Sanofi-Aventis, US, LLC)), satralizumab® (e.g., Enspryng (Genentech, Inc.)), secukinumab (e.g., Cosentyx® (Novartis Pharmaceuticals Corporation)), siltuximab (e.g., Sylvant® (Janssen The amino acid sequence of the antibody or antibody fragments includes the amino acid sequence of thrombin time-dependent ...

[0112] In another embodiment, the glycoengineered bifunctional degradative derivative comprises the amino acid sequence of an enzyme or its inhibitor. In another embodiment, the glycoengineered bifunctional degradative derivative comprises the amino acid sequence of an enzyme or its inhibitor, such as Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor VIIa, antithrombin III (AT-III), polypeptide C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, alglucosidase-α, laronidase (α-L-iduronidase), idursulfase (iduronic acid-2-sulfuronidase), or a combination thereof. These include the amino acid sequences of: agalactosidase (human α-galactosidase A), galsulfase, agalsidase-β (human α-galactosidase A), botulinum toxin, collagenase, human DNAse-I, hyaluronidase, papain, L-asparaginase, uricase (urate oxidase), glutamic acid carboxypeptidase (glucarpidase), α1 protease inhibitor (α1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase.

[0113] In certain embodiments, the glycoengineered bifunctional degradative derivatives are receptors. Exemplary receptors that can be used as glycoengineered bifunctional degradative derivatives include, but are not limited to, the extracellular domain of human CTLA4 (e.g., Fc fusions) and soluble TNF receptors (e.g., Fc fusions).

[0114] In another embodiment, the glycoengineered bifunctional degradative derivative is secreted into the culture medium. In certain embodiments, the glycoengineered bifunctional degradative derivative is purified from the culture medium. In another embodiment, the glycoengineered bifunctional degradative derivative is purified from the culture medium via affinity purification or ion exchange chromatography. In another embodiment, the glycoengineered bifunctional degradative derivative contains an FC domain and is affinity purified from the culture medium via polypeptide A. In another embodiment, the glycoengineered bifunctional degradative derivative comprises an affinity tag and is affinity purified.

[0115] In certain embodiments, the glycoengineered bifunctional degraded derivatives are full-length polypeptides, truncations, polypeptide domains, regions, motifs, or peptides thereof.

[0116] In certain embodiments, the glycoengineered bifunctional degraded derivative is a soluble receptor. In certain embodiments, the glycoengineered bifunctional degraded derivative is an Fc fusion polypeptide.

[0117] In certain embodiments, the glycoengineered bifunctional degrading derivative is a biologic comprising the Fc domain of IgG.

[0118] In certain embodiments, the glycoengineered bifunctional degrading derivative is a ligand for a receptor.

[0119] In certain embodiments, the glycoengineered bifunctional degradative derivatives are localized within the secretory pathway. Without being bound by theory, localization within the secretory pathway includes, but is not limited to, localization within one or more of the following intracellular compartments: the endoplasmic reticulum, the Golgi apparatus, lysosomes, intracellular membrane proteins, cell surface-anchored proteins, and membrane proteins. In certain embodiments, localization within the secretory pathway includes localization within one or more of the above intracellular compartments.

[0120] In certain embodiments, the glycoengineered bifunctional degradation derivative comprises a signal peptide that localizes the glycoengineered bifunctional degradation derivative within the secretory pathway. In certain embodiments, the signal peptide is derived from the same source as the glycoengineered bifunctional degradation derivative (i.e., the signal peptide is not appended to the glycoengineered bifunctional degradation derivative, but rather is fused to the glycoengineered bifunctional degradation derivative upon natural expression in the source). In certain embodiments, the glycoengineered bifunctional degradation derivative is localized within the secretory pathway without appending a Leishmania signal peptide to the glycoengineered bifunctional degradation derivative. In other embodiments, a signal peptide is appended to the glycoengineered bifunctional degradation derivative. In certain 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 an invertase signal peptide from Leishmania tarentolae. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed away from the glycoengineered bifunctional degradation derivatives.

[0121] In another embodiment, the glycoengineered bifunctional degradative derivatives are engineered to contain one or more tag(s), hi other embodiments, the tags are processed away from the glycoengineered bifunctional degradative derivatives.

[0122] In certain embodiments, the glycoengineered bifunctional degradative derivatives are expressed from Leishmania host cells described in Section 7.3. In certain embodiments, the Leishmania host cells used to make the glycoengineered bifunctional degradative derivatives provided herein are genetically engineered using the methods described in Section 7.4. In certain embodiments, the Leishmania host cells used to make the glycoengineered bifunctional degradative derivatives provided herein are cultured according to the methods described in Section 7.5.

[0123] 7.1.1 Pharmaceutical Formulations Containing Glycoengineered Bifunctional Degrading Derivatives In another aspect, provided herein are pharmaceutical compositions comprising the glycoengineered bifunctional degrading derivatives described herein. The compositions described herein are useful for treating and / or preventing diseases / disorders in a subject (e.g., a human subject) (see Section 7.8).

[0124] In certain embodiments, in addition to comprising the glycoengineered bifunctional degrading derivatives described herein, the pharmaceutical compositions described herein comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more specifically, in humans. The term "carrier," as used herein in the context of a pharmaceutically acceptable carrier, refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0125] In certain embodiments, the pharmaceutical compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the pharmaceutical compositions described herein may be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration. In certain embodiments, the pharmaceutical compositions may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal, or pulmonary administration.

[0126] In certain embodiments, the pharmaceutical compositions described herein further comprise one or more buffers, such as phosphate buffer and sucrose phosphate glutamate buffer, hi other embodiments, the pharmaceutical compositions described herein do not comprise a buffer.

[0127] In certain embodiments, the pharmaceutical compositions described herein further comprise one or more salts, such as sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or mixtures of such aluminum salts). In other embodiments, the pharmaceutical compositions described herein do not comprise a salt.

[0128] In some embodiments, the pharmaceutical compositions described herein can be administered in a single dosage form, for example, a single dosage form of a glycoengineered bifunctional degrading derivative described herein.

[0129] The pharmaceutical compositions described herein can be included in a kit, container, pack, or dispenser along with instructions for administration. In some embodiments, kits are provided herein that include the glycoengineered bifunctional degrading derivatives of the present disclosure. In some embodiments, the kits further provide instructions for administering the bifunctional molecule or pharmaceutical composition to an individual in need thereof.

[0130] The pharmaceutical compositions described herein can be stored prior to use, for example, the compositions can be stored frozen (e.g., at about -20°C or about -70°C), refrigerated (e.g., at about 4°C), or at room temperature.

[0131] 7.2 A collection of glycosylated bifunctional degradatives Also provided herein are compositions comprising a population of bifunctional degraded derivatives described in Section 7.1, wherein the population of bifunctional degraded derivatives has 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 sites. In certain embodiments, the N-glycan profile 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 of the N-glycosylation sites. In certain embodiments, the N-glycan profile 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 two of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 50% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 60% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 70% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 80% homogeneous at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile is at least 90% homogeneous at one or more of the N-glycosylation sites (several). In certain embodiments, the N-glycan profile is at least 95% homogeneous at one or more of the N-glycosylation sites (several). In certain embodiments, the N-glycan profile is at least 98% homogeneous at one or more of the N-glycosylation sites (several). In certain embodiments, the homogeneity of the N-glycan profile at one or more of the N-glycosylation sites is measured according to any standard method known in the art.In certain embodiments, the homogeneity of the N-glycan profile at one or more of the N-glycosylation sites is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis. In certain embodiments, the homogeneity of the N-glycan profile at one or more of the N-glycosylation sites is measured according to one or more of the assays described in Section 7.9.3.

[0132] Also provided herein are compositions comprising a population of bifunctional degraded derivatives as described in Section 7.1, wherein the population of bifunctional degraded derivatives has an N-glycan profile that 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 structure: [ka] where 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 a bifunctional degradation derivative. In certain embodiments, the N-glycan profile contains 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 the N-glycans at one N-glycosylation site. In certain embodiments, the N-glycan profile 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 the N-glycans at each of two N-glycosylation sites. In certain embodiments, the N-glycan profile comprises about 30% to about 40% of the structural N-glycans at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile comprises about 40% to about 50% of the structural N-glycans at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile comprises about 50% to about 60% of the structural N-glycans at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile comprises about 60% to about 70% of the N-glycans at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile comprises about 70% to about 80% of the N-glycans at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile comprises about 80% to about 90% of the N-glycans at one or more of the N-glycosylation sites. In certain embodiments, the N-glycan profile comprises about 90% to about 100% of the N-glycans at one or more of the N-glycosylation sites. In certain embodiments, the relative amount of N-glycans at one or more of the N-glycosylation sites is measured according to any standard means known in the art.In certain embodiments, the relative amount of N-glycans at one or more of the N-glycosylation sites is measured by N-glycan analysis or glycopeptide analysis. In certain embodiments, the relative amount of N-glycans at one or more of the N-glycosylation sites is measured according to one or more of the assays described in Section 7.9.3.

[0133] Without being bound by theory, N-glycan structures that are not fully capped with GalNAc (e.g., A1GalNAc1 or A2GalNAc1 compared to A2GalNAc2) do not engage the ASGPR. In certain preferred embodiments, the N-glycan profile comprises about 90% to about 100% N-glycans of the following structure at one or more of the N-glycosylation sites: [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 a bifunctional degradation derivative. In one embodiment, the N-glycan profile contains about 90% to about 100% of the N-glycan at one N-glycosylation site. In one embodiment, the N-glycan profile contains about 95% to about 100% of the N-glycan at one N-glycosylation site. In one embodiment, the N-glycan profile contains about 90% to about 100% of the N-glycan at each of two N-glycosylation sites. In one embodiment, the N-glycan profile contains about 95% to about 100% of the N-glycan at each of two N-glycosylation sites. In one embodiment, the N-glycan profile collectively comprises about 80% to about 90% of the N-glycans at two N-glycosylation sites. In one embodiment, the N-glycan profile collectively comprises about 90% to about 100% of the N-glycans at two N-glycosylation sites. In certain embodiments, the relative amount of N-glycans at one or more N-glycosylation sites is measured according to any standard means known in the art. In certain embodiments, the relative amount of N-glycans at one or more N-glycosylation sites is measured by N-glycan analysis or glycopeptide analysis. In certain embodiments, the relative amount of N-glycans at one or more N-glycosylation sites is measured according to one or more assays described in Section 7.9.3.

[0134] Also provided herein are compositions comprising a population of bifunctional degraded derivatives as described in Section 7.1, wherein the population of bifunctional degraded derivatives has an N-glycan profile that comprises 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% of N-glycans of the following structure among all glycans in the N-glycan profile: [ka] wherein the black square represents an N-acetylgalactosamine (GalNAc), 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 bifunctional degradation derivative. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains at least 30% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains at least 40% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains at least 50% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains at least 60% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 70% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 80% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 90% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 95% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraded derivatives has an N-glycan profile that comprises at least 98% of the N-glycan among all glycans in the N-glycan profile.

[0135] Also provided herein are compositions comprising a population of bifunctional degraded derivatives as described in Section 7.1, wherein the population of bifunctional degraded derivatives 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 N-glycans of the following structure among all glycans in the N-glycan profile: [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 bifunctional degradation derivative. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains about 30% to about 40% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains about 40% to about 50% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that contains about 50% to about 60% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 60% to about 70% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 70% to about 80% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 80% to about 90% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 90% to about 100% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the relative amount of the N-glycan among all glycans in the N-glycan profile is measured according to any standard means known in the art. In certain embodiments, the relative amount of the N-glycan among all glycans in the N-glycan profile is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis. In certain embodiments, the relative amount of the N-glycan among all glycans in the N-glycan profile is measured according to one or more of the assays described in Section 7.9.3.

[0136] Also provided herein are compositions comprising a population of bifunctional degraded derivatives described in Section 7.1, wherein the population of bifunctional degraded derivatives has an N-glycan profile that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or about 100% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 60% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 70% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 80% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 90% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 95% homogeneous. In certain embodiments, the population has an N-glycan profile that is at least 98% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 100% homogeneous. In certain embodiments, the homogeneity of N-glycan profile is measured according to any standard method known in the art.In certain embodiments, the homogeneity of N-glycan profile is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis.In certain embodiments, the homogeneity of N-glycan profile is measured according to one or more assays described in section 7.9.3.

[0137] Also provided herein are compositions comprising a population of bifunctional degraded derivatives described in Section 7.1, wherein the population of bifunctional degraded derivatives has an N-glycan profile that is about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 30% to about 40% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 40% to about 50% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 50% to about 60% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 60% to about 70% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 70% to about 80% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 80% to about 90% homogeneous. In certain embodiments, the population has an N-glycan profile that is about 90% to about 100% homogeneous. In certain embodiments, the homogeneity of the N-glycan profile is measured according to any standard means known in the art. In certain embodiments, the homogeneity of the N-glycan profile is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis. In certain embodiments, the homogeneity of the N-glycan profile is measured according to one or more assays described in Section 7.9.3.

[0138] In certain embodiments, the populations of bifunctional degradative derivatives described in this section are produced by Leishmania host cells described in Section 7.3. In certain embodiments, the Leishmania host cells used to produce the populations of glycoengineered bifunctional degradative derivatives described in this section are genetically engineered using the methods described in Section 7.4. In certain embodiments, the Leishmania host cells used to produce the populations of glycoengineered bifunctional degradative derivatives described in this section are cultured according to the methods described in Section 7.5.

[0139] 7.2.1 Pharmaceutical Compositions Comprising a Population of Bifunctional Degradation Derivatives In another aspect, provided herein are pharmaceutical compositions comprising a population of glycoengineered bifunctional degrading derivatives described herein. The compositions described herein are useful for treating and / or preventing a disease / disorder in a subject (e.g., a human subject) (see Section 7.8).

[0140] In certain embodiments, in addition to comprising the population of glycoengineered bifunctional degradation derivatives described herein, the pharmaceutical compositions described herein comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more specifically, in humans. As used herein in the context of a pharmaceutically acceptable carrier, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0141] In certain embodiments, the pharmaceutical compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the pharmaceutical compositions described herein may be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration. In certain embodiments, the pharmaceutical compositions may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal, or pulmonary administration.

[0142] In certain embodiments, the pharmaceutical compositions described herein further comprise one or more buffers, such as phosphate buffer and sucrose phosphate glutamate buffer, hi other embodiments, the pharmaceutical compositions described herein do not comprise a buffer.

[0143] In certain embodiments, the pharmaceutical compositions described herein further comprise one or more salts, such as sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or mixtures of such aluminum salts). In other embodiments, the pharmaceutical compositions described herein do not comprise a salt.

[0144] In some embodiments, the pharmaceutical compositions described herein can be administered in a single dosage form, for example, a single dosage form of a population of glycoengineered bifunctional degrading derivatives described herein.

[0145] The pharmaceutical compositions described herein can be included in a kit, container, pack, or dispenser together with instructions for administration. In some embodiments, kits comprising a population of glycoengineered bifunctional degrading derivatives of the present disclosure are provided herein. In some embodiments, the kits further provide instructions for administering the population of glycoengineered bifunctional degrading derivatives or pharmaceutical composition to an individual in need thereof.

[0146] The pharmaceutical compositions described herein can be stored prior to use, for example, the compositions can be stored frozen (e.g., at about -20°C or about -70°C), refrigerated (e.g., at about 4°C), or at room temperature.

[0147] 7.3 Leishmania host cells

[0013] Provided herein are Leishmania host cells for the production of a glycoengineered bifunctional degradation derivative described in Section 7.1 or a population of glycoengineered bifunctional degradation derivatives described in Section 7.2, wherein the Leishmania host cells comprise (a) a recombinant nucleic acid encoding the glycoengineered bifunctional degradation derivative, 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 glycoengineered bifunctional degradation derivatives comprising biantennary GalNAc-terminal N-glycans. In particular, the Leishmania host cells provided herein are capable of producing glycoengineered bifunctional degradation derivatives comprising an N-glycan of the 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 of the glycoengineered bifunctional degradative derivative.

[0148] In certain embodiments, Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases described in Section 7.3.1. In certain embodiments, Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases described in Section 7.3.2. In certain embodiments, one or more endogenous enzymes described in Section 7.3.3 from the glycan biosynthetic pathway of the Leishmania host cells provided herein are deleted, mutated, and / or functionally inactivated. In certain embodiments, Leishmania host cells provided herein further comprise a recombinant nucleic acid encoding a heterologous UDP-GalNAc biosynthetic pathway protein, such as described in Section 7.3.4, capable of producing UDP-GalNAc. In certain embodiments, Leishmania host cells provided herein comprise a recombinant nucleic acid encoding a heterologous UDP-GalNAc transport protein, such as described in Section 7.3.5, capable of transporting UDP-GalNAc into the secretory pathway. In certain embodiments, the strains of Leishmania host cells provided herein are described in Section 7.3.6.

[0149] In certain embodiments, the Leishmania host cells provided hereinbelow are genetically engineered using the methods described in Section 7.4. In certain embodiments, the Leishmania host cells provided hereinbelow are cultured according to the methods described in Section 7.5.

[0150] Notwithstanding anything in this section, 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.

[0151] 7.3.1 N-acetylgalactosamine (GalNAc) transferase The Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases. In certain embodiments, the GalNAc transferases, or functionally active variants thereof, are capable of catalyzing the addition of GalNAc to N-acetylglucosamine-terminal glycans.

[0152] In certain embodiments, the GalNAc transferase is heterologous to the Leishmania host cell. In certain embodiments, the GalNAc transferase is derived from Homo sapiens, Caenorhabditis elegans, Parasteatoda tepidariorum, Salmo trutta, or Hucho hucho. In certain embodiments, the GalNAc transferase is derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens.

[0153] In certain embodiments, the GalNAc transferase is selected from the group consisting of β4-GalNAcT3, β4-GalNAcT4, Ceβ4GalNAcT, Ptβ4GalNAcT, and Stβ4GalNAcT, or functionally active variants thereof. In certain embodiments, the GalNAc transferase is selected from the group consisting of β4-GalNAcT3, β4-GalNAcT4, Ceβ4GalNAcT, Ptβ4GalNAcT, and Stβ4GalNAcT, or variants thereof that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto.

[0154] In certain embodiments, the GalNAc transferase comprises β4-GalNAcT3 or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises β4-GalNAcT3. In certain embodiments, the GalNAc transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to β4-GalNAcT3. In certain embodiments, the GalNAc transferase comprises an N-terminal truncated variant of β4-GalNAcT3. In certain embodiments, the β4-GalNAcT3 comprises Homo sapiens β4-GalNAcT3 or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises Homo sapiens β4-GalNAcT3. In certain embodiments, the GalNAc transferase comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the GalNAc transferase includes those homologous to Homo sapiens β4-GalNAcT3. In certain embodiments, the GalNAc transferase includes a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Homo sapiens β4-GalNAcT3. In certain embodiments, the GalNAc transferase includes an N-terminal truncated variant of Homo sapiens β4-GalNAcT3 comprising the amino acid sequence of SEQ ID NO:2.

[0155] In certain embodiments, the GalNAc transferase comprises β4-GalNAcT4 or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises β4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to β4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises an N-terminal truncated variant of β4-GalNAcT4. In certain embodiments, the β4-GalNAcT4 comprises Homo sapiens β4-GalNAcT4 or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises Homo sapiens β4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, GalNAc transferases include those homologous to Homo sapiens β4-GalNAcT4. In certain embodiments, GalNAc transferases include variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Homo sapiens β4-GalNAcT4. In certain embodiments, GalNAc transferases include N-terminal truncated variants of Homo sapiens β4-GalNAcT4 comprising the amino acid sequence of SEQ ID NO:4.

[0156] In certain embodiments, the GalNAc transferase comprises β4-GalNAcT3 and β-GalNAcT4, or functionally active variants thereof. In certain embodiments, the GalNAc transferase comprises β4-GalNAcT3 and β4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to β4-GalNAcT3 and β-GalNAcT4, respectively. In certain embodiments, the GalNAc transferase comprises an N-terminal truncation mutant of β4-GalNAcT3 and / or β4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises Homo sapiens β4-GalNAcT3 and β4-GalNAcT4, or functionally active variants thereof. In certain embodiments, the GalNAc transferase comprises Homo sapiens β4-GalNAcT3 and β4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 3. In certain embodiments, the GalNAc transferase comprises homologous sequences to Homo sapiens β4-GalNAcT3 and β4-GalNAcT4. In certain embodiments, the GalNAc transferase comprises variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Homo sapiens β4-GalNAcT3 and β4-GalNAcT4, respectively. In certain embodiments, the GalNAc transferase comprises N-terminal truncated variants of Homo sapiens β4-GalNAcT3 and / or β4-GalNAcT4, comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0157] In certain embodiments, the GalNAc transferase is Ceβ4GalNAcT or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Ceβ4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Ceβ4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminal truncated variant of Ceβ4GalNAcT. In certain embodiments, the Ceβ4GalNAcT is Caenorhabditis elegans β4GalNAcT or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Caenorhabditis elegans β4GalNAcT. In certain embodiments, the GalNAc transferase comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the GalNAc transferase is homologous to Caenorhabditis elegans Ceβ4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Caenorhabditis elegans Ceβ4GalNAcT. In certain embodiments, the GalNAc transferase comprises an N-terminal truncated variant of Caenorhabditis elegans Ceβ4GalNAcT comprising the amino acid sequence of SEQ ID NO:6.

[0158] In certain embodiments, the GalNAc transferase is Ptβ4GalNAcT or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Ptβ4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Ptβ4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminal truncated variant of Ptβ4GalNAcT. In certain embodiments, the Ptβ4GalNAcT is Parasteatoda tepidariorum β4GalNAcT or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Parasteatoda tepidariorum Ptβ4GalNAcT. In certain embodiments, the GalNAc transferase comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the GalNAc transferase is homologous to Ptβ4GalNAcT of Parasteatoda tepidariorum. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Ptβ4GalNAcT of Parasteatoda tepidariorum. In certain embodiments, the GalNAc transferase comprises an N-terminal truncated variant of Ptβ4GalNAcT of Parasteatoda tepidariorum comprising the amino acid sequence of SEQ ID NO:8.

[0159] In certain embodiments, the GalNAc transferase is Stβ4GalNAcT or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Stβ4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Stβ4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminal truncated variant of Stβ4GalNAcT. In certain embodiments, the Stβ4GalNAcT is Salmo trutta β4GalNAcT or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Salmo trutta β4GalNAcT. In certain embodiments, the GalNAc transferase comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the GalNAc transferase is homologous to Salmo trutta β4GalNAcT, hi certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Salmo trutta β4GalNAcT.

[0160] In certain embodiments, the GalNAc transferase is Hhβ4GalNAcT or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Hhβ4GalNAcT. In certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Hhβ4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminal truncated mutant of Hhβ4GalNAcT. In certain embodiments, the Hhβ4GalNAcT is Hucho hucho β4GalNAcT or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Hucho hucho β4GalNAcT. In certain embodiments, the GalNAc transferase comprises the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the GalNAc transferase is homologous to the β4GalNAcT of Hucho hucho, hi certain embodiments, the GalNAc transferase is a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to the β4GalNAcT of Hucho hucho.

[0161] In certain embodiments, the GalNAc transferase is localized within the secretory pathway. Without being bound by theory, localization within the secretory pathway includes, but is not limited to, localization within one or more of the following intracellular compartments: the endoplasmic reticulum, the Golgi apparatus, lysosomes, intracellular membrane proteins, cell surface-anchored proteins, and membrane proteins. In certain embodiments, localization within the secretory pathway includes localization within one or more of the above intracellular compartments.

[0162] In certain embodiments, the GalNAc transferase comprises a signal peptide that localizes the GalNAc transferase within the secretory pathway. In certain embodiments, the signal peptide is derived from the same source as the GalNAc transferase (i.e., the signal peptide is not added to the GalNAc transferase, but is included in the GalNAc transferase when naturally expressed in the source). In certain embodiments, the GalNAc transferase is localized within the secretory pathway without the addition of a Leishmania signal peptide to the GalNAc transferase. In other embodiments, the signal peptide is added to the GalNAc transferase. In certain embodiments, the signal peptide is fused to the C-terminus of the GalNAc transferase. In certain embodiments, the signal peptide is fused to the N-terminus of the GalNAc transferase. In certain embodiments, the signal peptide is fused to one or more amino acids within the GalNAc transferase polypeptide. In certain embodiments, the signal peptide is fused to the N-terminus of an N-terminal truncated mutant of the GalNAc transferase. In certain embodiments, a signal peptide is fused to one or more amino acids within the polypeptide of the N-terminal truncated mutant of GalNAc transferase. In certain 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 an invertase signal peptide from Leishmania tarentolae. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed and removed from the GalNAc transferase.

[0163] In certain embodiments, the GalNAc transferase and the additional recombinant glycosyltransferase described in Section 7.3.2 are co-localized within the secretory pathway. In certain embodiments, the GalNAc transferase and the glycoengineered bifunctional degrading derivative described in Section 7.1 are co-localized within the secretory pathway.

[0164] 7.3.2 Additional recombinant glycosyltransferases In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases. In certain embodiments, the additional recombinant glycosyltransferase, or a functionally active variant thereof, is capable of catalyzing the addition of a first glycan to a second glycan. In certain embodiments, the additional recombinant glycosyltransferase is an N-acetylglucosaminyltransferase, or a functionally active variant thereof, capable of catalyzing the addition of N-acetylglucosamine (GlcNAc) to a mannose-terminal glycan, e.g., a Man3GlcNAc2 glycan (Man3, see Section 5.3).

[0165] In certain embodiments, the additional recombinant glycosyltransferase comprises one or more N-acetylglucosaminyltransferases. In certain embodiments, the N-acetylglucosaminyltransferase is heterologous to the host cell. In certain embodiments, the N-acetylglucosaminyltransferase is derived from Homo sapiens, Spodoptera frugiperda, Trypanosoma brucei, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Danio rerio A, Drosophila melanogaster, Anopheles gambiae, Caenorhabditis elegans, Arabidopsis thaliana, Oryza sativa japonica, Xenopus tropicalis, Canis lupus, Bos taurus, Danio rerio B, or Gekko japonicus. In certain embodiments, the additional recombinant glycosyltransferase is derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens.

[0166] In certain embodiments, the N-acetylglucosaminyltransferase is selected from the group consisting of MGAT1 (alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase) and MGAT2 (alpha-1,6-mannosylglycoprotein 2-beta-N-acetylglucosaminyltransferase), or a functionally active variant thereof. In certain embodiments, the additional recombinant glycosyltransferase comprises MGAT1 and MGAT2.

[0167] In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT1 or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises Homo sapiens MGAT1 (accession number P26572) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Homo sapiens MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Homo sapiens MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Homo sapiens.

[0168] In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT2 or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises Homo sapiens MGAT2 (accession number Q10469.1) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Homo sapiens MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Homo sapiens MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT2 of Homo sapiens.

[0169] In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT1 and MGAT2, or functionally active variants thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT1 and MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 and MGAT2, respectively. In certain embodiments, the N-acetylglucosaminyltransferase is Homo sapiens MGAT1 and MGAT2, or functionally active variants thereof. In certain embodiments, the N-acetylglucosaminyltransferase is Homo sapiens MGAT1 and MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In certain embodiments, the N-acetylglucosaminyltransferase is homologous to Homo sapiens MGAT1 and MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase includes variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Homo sapiens MGAT1 and MGAT2, respectively.

[0170] In certain embodiments, the N-acetylglucosaminyltransferase comprises Spodoptera frugiperda MGAT1 (SfGnT-I, accession number: AEX00082) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Spodoptera frugiperda MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Spodoptera frugiperda MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Spodoptera frugiperda MGAT1.

[0171] In certain embodiments, the N-acetylglucosaminyltransferase comprises Trypanosoma brucei MGAT1 (TbGnT-I, Accession Number: XP_844156), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Trypanosoma brucei MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Trypanosoma brucei MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Trypanosoma brucei MGAT1.

[0172] In certain embodiments, the N-acetylglucosaminyltransferase comprises Pan troglodytes MGAT1 (PtMGAT1, accession number: XP_001155433.2) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Pan troglodytes MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Pan troglodytes MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Pan troglodytes MGAT1.

[0173] In certain embodiments, the N-acetylglucosaminyltransferase comprises Macaca mulatta MGAT1 (MaMGAT1, accession number: NP_001244759), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Macaca mulatta MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Macaca mulatta MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Macaca mulatta MGAT1.

[0174] In certain embodiments, the N-acetylglucosaminyltransferase comprises Mus musculus MGAT1 (MuMGAT1, accession number: NP_001103620.1), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Mus musculus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Mus musculus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Mus musculus MGAT1.

[0175] In certain embodiments, the N-acetylglucosaminyltransferase comprises Rattus norvegicus MGAT1 (RnMGAT1, accession number: NP_110488.1), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Rattus norvegicus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Rattus norvegicus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Rattus norvegicus MGAT1.

[0176] In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT1 of Danio rerio A (DrMGAT1a, accession number: NP_956970.1), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT1 of Danio rerio A. In certain embodiments, the N-acetylglucosaminyltransferase comprises a homologue of MGAT1 of Danio rerio A. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Danio rerio A.

[0177] In certain embodiments, the N-acetylglucosaminyltransferase comprises Caenorhabditis elegans MGAT1 (Ce14MGAT1, Accession Number: NP_497719.1 or Ce13MGAT1, Accession Number: NP_509566.1), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Caenorhabditis elegans MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Caenorhabditis elegans MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Caenorhabditis elegans MGAT1.

[0178] In certain embodiments, the N-acetylglucosaminyltransferase comprises Arabidopsis thaliana MGAT1 (AtMGAT1, accession number: NP_195537.2), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Arabidopsis thaliana MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Arabidopsis thaliana MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Arabidopsis thaliana MGAT1.

[0179] In certain embodiments, the N-acetylglucosaminyltransferase comprises Oryza sativa Japonica MGAT1 (OsJMGAT1, accession number: XP_015624616.1) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Oryza sativa Japonica MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Oryza sativa Japonica MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Oryza sativa Japonica MGAT1.

[0180] In certain embodiments, the N-acetylglucosaminyltransferase comprises Xenopus tropicalis MGAT1 (XtMGAT1, accession number: NP_001011350.1) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Xenopus tropicalis MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a homologue of Xenopus tropicalis MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Xenopus tropicalis MGAT1.

[0181] In certain embodiments, the N-acetylglucosaminyltransferase comprises Canis lupus MGAT1 (ClMGAT1, accession number: XP_855658.1) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Canis lupus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a homologue of Canis lupus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Canis lupus MGAT1.

[0182] In certain embodiments, the N-acetylglucosaminyltransferase comprises Bos taurus MGAT1 (BtMGAT1, accession number: NP_001015653.1) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Bos taurus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Bos taurus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Bos taurus MGAT1.

[0183] In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT1 of Danio rerio B (DrMGAT1b, accession number: NP_001073440.1), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises MGAT1 of Danio rerio B. In certain embodiments, the N-acetylglucosaminyltransferase comprises a homologue of MGAT1 of Danio rerio B. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to MGAT1 of Danio rerio B.

[0184] In certain embodiments, the N-acetylglucosaminyltransferase comprises Gekko japonicus MGAT1 (GjMGAT1, accession number: XP_015280466.1) or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Gekko japonicus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Gekko japonicus MGAT1. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Gekko japonicus MGAT1.

[0185] In certain embodiments, the N-acetylglucosaminyltransferase comprises Rattus norvegicus MGAT2 (rMGAT2, Accession Number: NP_446056), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Rattus norvegicus MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises a homologue to Rattus norvegicus MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Rattus norvegicus MGAT2.

[0186] In certain embodiments, the N-acetylglucosaminyltransferase comprises Spodoptera frugiperda MGAT2 (SfGnT-II, accession number: AEX00083), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Spodoptera frugiperda MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Spodoptera frugiperda MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Spodoptera frugiperda MGAT2.

[0187] In certain embodiments, the N-acetylglucosaminyltransferase comprises Trypanosoma brucei MGAT2 (TbGnT-II, Accession Number: XP_845654), or a functionally active variant thereof. In certain embodiments, the N-acetylglucosaminyltransferase comprises Trypanosoma brucei MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises one that is homologous to Trypanosoma brucei MGAT2. In certain embodiments, the N-acetylglucosaminyltransferase comprises a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to Trypanosoma brucei MGAT2.

[0188] In certain embodiments, the additional recombinant glycosyltransferase is localized within the secretory pathway. Without being bound by theory, localization within the secretory pathway includes, but is not limited to, localization within one or more of the following intracellular compartments: the endoplasmic reticulum, the Golgi apparatus, a lysosome, an intracellular membrane protein, a cell surface-anchored protein, and a membrane protein. In certain embodiments, localization within the secretory pathway includes localization within one or more of the above intracellular compartments.

[0189] In certain embodiments, the additional recombinant glycosyltransferase comprises a signal peptide that localizes the additional recombinant glycosyltransferase within the secretory pathway. In certain embodiments, the signal peptide is derived from the same source as the additional recombinant glycosyltransferase (i.e., the signal peptide is not added to the additional recombinant glycosyltransferase, but is fused to the additional recombinant glycosyltransferase upon natural expression in the source). In certain embodiments, the additional recombinant glycosyltransferase is localized within the secretory pathway without adding a Leishmania signal peptide to the additional recombinant glycosyltransferase. In other embodiments, the signal peptide is added to the additional recombinant glycosyltransferase. In certain embodiments, the signal peptide is fused to the C-terminus of the additional recombinant glycosyltransferase. In certain embodiments, the signal peptide is fused to the N-terminus of the additional recombinant glycosyltransferase. In certain embodiments, the signal peptide is fused to one or more amino acids within the polypeptide of the additional recombinant glycosyltransferase. In certain 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 an invertase signal peptide from Leishmania tarentolae. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed to remove from the additional recombinant glycosyltransferase.

[0190] In certain embodiments, the GalNAc transferase described in Section 7.3.1 and the additional recombinant glycosyltransferase are co-localized within the secretory pathway.

[0191] 7.3.3 Deletion, mutation, and / or functional inactivation of endogenous enzymes from the glycan biosynthetic pathway In certain embodiments, the Leishmania host cells provided herein are characterized in that one or more endogenous enzymes from the glycan biosynthetic pathway have been deleted, mutated, and / or functionally inactivated. In certain embodiments, the Leishmania host cell does not have an endogenous N-glycan elongation. In certain embodiments, the Leishmania host cell does not have an endogenous N-glycan elongation as described in WO2019 / 002512 (incorporated herein by reference in its entirety). In certain embodiments, the Leishmania host cell has been genetically engineered to reduce or eliminate the formation of O-linked GlcNAc on polypeptides in the host cell. In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one N-acetylglucosamine (GlcNAc) transferase. In certain embodiments, the gene encoding at least one GlcNAc transferase in the Leishmania host cell is functionally inactivated, downregulated, deleted, and / or mutated.

[0192] In certain embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc is an N-acetylglucosamine (GlcNAc) transferase. In certain embodiments, the GlcNAc transferase is selected from the group consisting of OGNT1, OGNT2, OGNTL, and their homologous GlcNAc transferases. Without being bound by theory, OGNT1, OGNT2, and OGNTL were identified based on their homology to Trypanosoma enzymes rather than mammalian (e.g., human) enzymes (Heise, N., et al. Glycobiology, 19(8), 918-933 (2009) and Chiribao, M Let al. Gene, 498(2), 147-154 (2012), each of which is incorporated herein by reference in its entirety). In certain embodiments, the GlcNAc transferase is OGNT1. In other embodiments, the GlcNAc transferase is OGNT2. In still other embodiments, the GlcNAc transferase is OGNTL. In certain embodiments, the GlcNAc transferase is a GlcNAc transferase that is homologous to OGNT1. In certain embodiments, the GlcNAc transferase is a GlcNAc transferase that is homologous to OGNT2. In certain embodiments, the GlcNAc transferase is a GlcNAc transferase that is homologous to OGNTL. In certain embodiments, the GlcNAc transferase is derived from Leishmania tarentolae. In certain embodiments, the GlcNAc transferase is derived from other Trypanosomatida species. Non-limiting examples of Trypanosomatida GlcNAc transferases are listed in Table 1, where one representative genome is listed for each species. [Table 3] TIFF2025534987000026.tif214165TIFF2025534987000027.tif237165TIFF2025534987000028.tif237165TIFF2025534987000029.tif90165

[0193] In certain embodiments, the enzyme catalyzing the formation of O-linked GlcNAc is derived from a species other than Trypanosomatida. In certain embodiments, the enzyme is human O-GlcNAc transferase (OGT, Uniprot: O15294) and its homologs. In certain embodiments, O-GlcNAc transferase (OGT; uridine diphospho N-acetylglucosamine:polypeptide β-N-acetylglucosaminyltransferase; EC 2.4.1.255) can catalyze the transfer of a single N-acetylglucosamine from UDP-GlcNAc to serine or threonine residues in cytoplasmic and nuclear proteins, resulting in their modification with beta-linked N-acetylglucosamine (O-GlcNAc). In certain embodiments, the enzyme catalyzing the formation of O-linked GlcNAc can be a different isoform of OGT. Exemplary isoforms of OGT include, but are not limited to, (1) a nucleocytoplasmic or full-length variant (ncOGT) (which may be 110 kDa), (2) a short isoform of OGT (sOGT) (which may be 78 kDa), and (3) a variant of OGT targeted to mitochondria (mOGT, which may be 90 kDa). In certain embodiments, OGT may be found in the nucleus and cytoplasm to form multimers composed of one or more 110 kDa and 78 kDa subunits (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor, NY). In certain embodiments, the enzyme catalyzing the formation of O-linked GlcNAc is human EOGT (Uniprot:Q5NDL2). In certain embodiments, the enzyme catalyzes the transfer of a single N-acetylglucosamine from UDP-GlcNAc to serine or threonine residues in extracellular proteins, resulting in their modification with beta-linked N-acetylglucosamine (O-GlcNAc). In certain embodiments, the enzyme catalyzes the specific glycosylation of the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domain.

[0194] In certain embodiments, the enzyme catalyzing the formation of O-linked GlcNAc can transfer via an alpha linkage, while in other embodiments, the enzyme catalyzing the formation of O-linked GlcNAc can transfer via a beta linkage.

[0195] In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one enzyme as described in this section, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 enzymes as described in this section.

[0196] In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc transferase derived from a Trypanosomatida species, e.g., Leishmania tarentolae. In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc transferase, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GlcNAc transferases, one or more of which are derived from a Trypanosomatida species. In certain embodiments, the number of at least one GlcNAc transferase is 1, 2, or 3. In certain embodiments, the at least one GlcNAc transferase is selected from the group consisting of OGNT1, OGNT2, OGNTL, and their homologous GlcNAc transferases. In certain embodiments, the at least one GlcNAc transferase is a GlcNAc transferase that is homologous to OGNT1, OGNT2, and / or OGNTL.

[0197] In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc transferase derived from a species other than Trypanosomatida, e.g., human. In certain embodiments, the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc transferase, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GlcNAc transferases, one or more of which are derived from human. In certain embodiments, the number of at least one GlcNAc transferase is 1, 2, or 3. In certain embodiments, the at least one GlcNAc transferase is selected from the group consisting of human O-GlcNAc transferase and human EOGT and homologous enzymes thereof. In certain embodiments, the at least one GlcNAc transferase is an enzyme homologous to human O-GlcNAc transferase and / or human EOGT.

[0198] In certain embodiments, the enzyme catalyzes the formation of O-linked GlcNAc before genetic engineering of the Leishmania host cell. In certain embodiments, the enzyme also catalyzes the formation of O-linked GlcNAc after genetic engineering of the Leishmania host cell. In certain embodiments, the enzyme does not catalyze the formation of O-linked GlcNAc after genetic engineering of the Leishmania host cell.

[0199] In certain embodiments, a gene encoding at least one GlcNAc transferase in a Leishmania host cell is functionally inactivated. In certain embodiments, a gene encoding at least one GlcNAc transferase in a Leishmania host cell is downregulated. In certain embodiments, a gene encoding at least one GlcNAc transferase in a Leishmania host cell is overexpressed.

[0200] In certain embodiments, the Leishmania host cells provided herein comprise at least one gene deletion. In certain embodiments, the gene encoding at least one GlcNAc transferase is deleted. In certain embodiments, the gene encoding at least one GlcNAc transferase is mutated. In certain embodiments, the gene encoding at least one GlcNAc transferase is overexpressed. In certain embodiments, additional modifications may be introduced (e.g., using recombinant techniques) into the Leishmania host cells described herein.

[0201] In certain embodiments, genes encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 enzymes that each catalyze the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, genes encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 enzymes that each catalyze the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, genes encoding three enzymes that each catalyze the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, genes and / or genetic loci that may be functionally inactivated include, but are not limited to, OGNT1, OGNT2, and OGNTL.

[0202] In certain embodiments, genes encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 GlcNAc transferases, each of which catalyzes the formation of O-linked GlcNAc, may be functionally inactivated. In certain embodiments, genes encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 GlcNAc transferases, each of which catalyzes the formation of O-linked GlcNAc, may be functionally inactivated. In certain embodiments, genes encoding three GlcNAc transferases, each of which catalyzes the formation of O-linked GlcNAc, may be functionally inactivated.

[0203] In certain embodiments, the genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all enzymes that each catalyze the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all enzymes that each catalyze the formation of O-linked GlcNAc may be functionally inactivated.

[0204] In certain embodiments, genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all GlcNAc transferases that each catalyze the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all GlcNAc transferases that each catalyze the formation of O-linked GlcNAc may be functionally inactivated.

[0205] In certain embodiments, the at least one GlcNAc transferase is selected from the group consisting of OGNT1, OGNT2, and OGNTL, and their homologous GlcNAc transferases, hi certain embodiments, the Leishmania host cell is a triple knockout of OGNT1, OGNT2, and OGNTL.

[0206] In certain embodiments, the formation of O-linked GlcNAc in a Leishmania host cell is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of O-linked GlcNAc in a reference Leishmania cell. In certain embodiments, the formation of O-linked GlcNAc in a Leishmania host cell is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of O-linked GlcNAc in a reference Leishmania cell. In certain embodiments, the reference Leishmania cell is wild-type. In certain embodiments, the reference Leishmania cell is genetically engineered differently from the genetically engineered Leishmania cells described herein. In certain embodiments, some of the engineering of the reference Leishmania cell can be the same as the engineering of the genetically engineered Leishmania cells described herein (e.g., deletion of one or more enzymes that catalyze the formation of O-linked GlcNAc). In certain embodiments, the reference Leishmania cell may comprise a recombinant nucleic acid encoding a heterologous glycosyltransferase, e.g., a Leishmania cell described in International Publication No. WO2019 / 002512 A2, which is incorporated by reference in its entirety. In certain embodiments, the formation of O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of O-linked GlcNAc in a wild-type Leishmania cell.In certain embodiments, the formation of O-linked GlcNAc is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of O-linked GlcNAc in wild-type Leishmania cells. In certain embodiments, the formation of O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of O-linked GlcNAc in a Leishmania cell comprising a recombinant nucleic acid encoding a heterologous glycosyltransferase. In certain embodiments, the formation of O-linked GlcNAc is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% from the formation of O-linked GlcNAc in a Leishmania cell comprising a recombinant nucleic acid encoding a heterologous glycosyltransferase.

[0207] In certain embodiments, the growth rate of a Leishmania host cell described herein is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of a reference Leishmania cell. In certain embodiments, the growth rate of a Leishmania host cell is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of a reference Leishmania cell. In certain embodiments, the reference Leishmania cell is wild-type. In certain embodiments, the reference Leishmania cell is genetically engineered differently from the genetically engineered Leishmania cells described herein. In certain embodiments, some of the engineering of the reference Leishmania cell can be the same as the engineering of the genetically engineered Leishmania cells described herein (e.g., deletion of one or more enzymes that catalyze the formation of O-linked GlcNAc). In certain embodiments, the growth rate of the Leishmania cells is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of wild-type Leishmania cells. In certain embodiments, the growth rate of the Leishmania cells is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of wild-type Leishmania cells.

[0208] 7.3.4 Heterologous UDP-GalNAc Biosynthetic Pathway Proteins In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding a UDP-GalNAc biosynthetic pathway protein capable of producing UDP-GalNAc. In certain embodiments, the recombinant UDP-GalNAc biosynthetic pathway protein is heterologous to the Leishmania host cell.

[0209] In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein is capable of converting GalNAc to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc to UDP-GalNAc is derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens.

[0210] In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein is capable of converting N-acetylgalactosamine 1-phosphate (GalNAc-1-P) to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein is capable of converting GalNAc-1-P and UTP to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises UDP-N-acetylhexosamine pyrophosphorylase (UAP1), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises Homo sapiens UDP-N-acetylhexosamine pyrophosphorylase (UAP1), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises Homo sapiens UDP-N-acetylhexosamine pyrophosphorylase (UAP1) or a variant thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises Homo sapiens UDP-N-acetylhexosamine pyrophosphorylase (UAP1). In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises a variant of Homo sapiens UDP-N-acetylhexosamine pyrophosphorylase (UAP1) that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. Without being bound by theory, the AGX1 isoform of UDP-N-acetylhexosamine pyrophosphorylase is approximately 2-3 times more active with GalNAc-1-P than with GlcNAc-1-P, while the AGX2 isoform is approximately 8 times more active with GlcNAc-1-P than with GalNAc-1-P. In certain embodiments, the UDP-N-acetylhexosamine pyrophosphorylase (UAP1) is the AGX1 isoform of UAP1. In other embodiments, the UDP-N-acetylhexosamine pyrophosphorylase (UAP1) is the AGX2 isoform of UAP1.In certain embodiments, the UDP-N-acetylhexosamine pyrophosphorylase (UAP1) comprises the amino acid sequence of SEQ ID NO:15.

[0211] In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein is capable of converting GalNAc to GalNAc-1-P. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises N-acetylgalactosamine kinase (GALK2), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises Homo sapiens N-acetylgalactosamine kinase (GALK2), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises Homo sapiens N-acetylgalactosamine kinase (GALK2), or a variant at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises Homo sapiens N-acetylgalactosamine kinase (GALK2). In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises a variant of Homo sapiens N-acetylgalactosamine kinase (GALK2) that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the N-acetylgalactosamine kinase (GALK2) comprises the amino acid sequence of SEQ ID NO: 15.

[0212] In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein is capable of converting UDP-GlcNAc to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc comprises an NAD-dependent epimerase that converts UDP-GlcNAc to UDP-GalNAc. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc is derived from a mammalian source. In certain embodiments, the mammalian source is Homo sapiens. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises UDP-galactose 4-epimerase (GalE), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises Homo sapiens UDP-galactose 4-epimerase (GalE) (hGalE), or a functionally active variant thereof. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises hGalE, or a variant thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises hGalE. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises a variant of hGalE that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the hGalE comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc is derived from a bacterial source. In certain embodiments, the bacterial source is Campylobacter jejuni. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises Campylobacter jejuni UDP-GlcNAc / Glc 4-epimerase (CjGne), or a functionally active variant thereof.In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises CjGne or a variant thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises CjGne. In certain embodiments, the heterologous UDP-GalNAc biosynthetic pathway protein comprises a variant of CjGne that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, CjGne comprises the amino acid sequence of SEQ ID NO: 18.

[0213] In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc to UDP-GalNAc, as described in this section, and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transport protein capable of transporting UDP-GalNAc to the secretory pathway, as described in Section 7.3.5. In certain embodiments, the recombinant nucleic acid encodes (a) a UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc-1-P to UDP-GalNAc, and a UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc to GalNAc-1-P, as described in this section.

[0214] In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc, as described in this section, and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway, as described in Section 7.3.5.

[0215] In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc to UDP-GalNAc, as described in this section, and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc, as described in this section. In certain embodiments, the recombinant nucleic acid encodes (a) a UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc-1-P to UDP-GalNAc, and a UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc to GalNAc-1-P, as described in this section.

[0216] In certain embodiments, the Leishmania host cell comprises (a) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc to UDP-GalNAc, as described in this section, (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc biosynthetic pathway protein capable of converting UDP-GlcNAc to UDP-GalNAc, as described in this section, and (c) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transport protein capable of transporting UDP-GalNAc to the secretory pathway, as described in Section 7.3.5. In certain embodiments, the recombinant nucleic acid encodes (a) a UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc-1-P to UDP-GalNAc, and a UDP-GalNAc biosynthetic pathway protein capable of converting GalNAc to GalNAc-1-P, as described in this section.

[0217] 7.3.5 Heterologous UDP-GalNAc Transport Proteins In certain embodiments, the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding a UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway. In certain embodiments, the UDP-GalNAc transport protein is heterologous to the Leishmania host cell.

[0218] In certain embodiments, the UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway is derived from a nematode source, hi certain embodiments, the nematode source is C. elegans.

[0219] In certain embodiments, the UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway comprises a C. elegans UDP-GalNAc transporter (CeC03H5.2) or a functionally active variant thereof. In certain embodiments, the UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway comprises CeC03H5.2 or a variant thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway comprises CeC03H5.2. In certain embodiments, the UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway is CeC03H5.2. In certain embodiments, the UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway comprises a variant of CeC03H5.2 that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, the UDP-GalNAc transport protein capable of transporting UDP-GalNAc into the secretory pathway is a variant of CeC03H5.2 that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereto. In certain embodiments, CeC03H5.2 has the amino acid sequence of SEQ ID NO: 19.

[0220] 7.3.6 Leishmania host cell strains In certain embodiments, the Leishmania host cell is a Leishmania tarentolae cell. In certain embodiments, the Leishmania host cell is a Leishmania aethiopica cell. In certain embodiments, the Leishmania host cell is part of the Leishmania aethiopica species complex. In certain embodiments, the Leishmania host cell is a Leishmania aristidesi cell. In certain embodiments, the Leishmania host cell is a Leishmania deanei cell. In certain embodiments, the Leishmania host cell is part of the Leishmania donovani species complex. In certain embodiments, the Leishmania host cell is a Leishmania donovani cell. In certain embodiments, the Leishmania host cell is a Leishmania chagasi cell. In certain embodiments, the Leishmania host cell is a Leishmania infantum cell. In certain embodiments, the Leishmania host cell is a Leishmania hertigi cell. In certain embodiments, the Leishmania host cell is part of the Leishmania major species complex. In certain embodiments, the Leishmania host cell is a Leishmania major cell. In certain embodiments, the Leishmania host cell is a Leishmania martiniquensis cell. In certain embodiments, the Leishmania host cell is part of the Leishmania mexicana species complex. In certain embodiments, the Leishmania host cell is a Leishmania mexicana cell. In certain embodiments, the Leishmania host cell is a Leishmania pifanoi cell. In certain embodiments, the Leishmania host cell is part of the Leishmania tropica species complex. In certain embodiments, the Leishmania host cell is a Leishmania tropica cell.

[0221] 7.4 Methods for Genetic Manipulation of Leishmania Cells Also provided herein are methods for genetically engineering Leishmania host cells, such as those described in Section 7.3. In certain embodiments, the methods can be used to achieve the introduction of one or more genes encoding GalNAc transferases, such as those described in Section 7.3.1. In certain embodiments, the methods can be used to achieve the introduction of one or more genes encoding additional recombinant glycosyltransferases, such as those described in Section 7.3.2. In certain embodiments, the methods can be used to achieve the functional inactivation of one or more genes encoding enzymes catalyzing the formation of O-linked GlcNAc, such as those described in Section 7.3.3. In certain embodiments, the methods can be used to achieve the introduction of one or more genes encoding heterologous UDP-GalNAc biosynthetic pathway proteins, such as those described in Section 7.3.4. In certain embodiments, the methods can be used to achieve the introduction of one or more genes encoding heterologous UDP-GalNAc transport proteins, such as those described in Section 7.3.5. In certain embodiments, engineered Leishmania host cell strains are described in Section 7.3.6.

[0222] Any method known in the art can be used to engineer Leishmania host cells, such as Leishmania tarentolae. In certain embodiments, nucleic acids are introduced into the host cells described herein using a plasmid, for example, heterologous nucleic acids are expressed in the host cell by a plasmid (e.g., an expression vector), and the plasmid is introduced into the modified host cell by transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or complexation. In certain embodiments, the plasmid is introduced into the modified host cell by stable transfection.

[0223] In certain embodiments, the linearized nucleic acid is introduced into the host cell described herein using transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or complexation. In further embodiments, the heterologous nucleic acid is site-specifically integrated into the host cell genome by homologous recombination.

[0224] In certain embodiments, the method of engineering a Leishmania host cell comprises introducing one or more genes encoding a GalNAc transferase, as described in Section 7.3.1.

[0225] In certain embodiments, the method of engineering a Leishmania host cell comprises introducing one or more genes encoding additional recombinant glycosyltransferases as described in Section 7.3.2.

[0226] In certain embodiments, the method of engineering a Leishmania host cell comprises functionally inactivating one or more genes encoding an enzyme that catalyzes the formation of O-linked GlcNAc, as described in Section 7.3.3. In certain embodiments, the method comprises downregulating a gene encoding at least one GlcNAc transferase. In certain embodiments, the method comprises deleting a gene encoding at least one GlcNAc transferase. In certain embodiments, the method comprises mutating a gene encoding at least one GlcNAc transferase. In certain embodiments, the method comprises overexpressing a gene encoding at least one GlcNAc transferase. In certain embodiments, the method comprises functionally inactivating a gene encoding an enzyme that catalyzes the formation of O-linked GlcNAc using the methods described in the Assays or Examples sections (Sections 7.7 and 8, respectively). In certain embodiments, the method comprises functionally inactivating a gene encoding an enzyme that catalyzes the formation of O-linked GlcNAc using any method known in the art, for example, the method described in International Publication No. WO2019 / 002512 A2, the entire contents of which are incorporated herein by reference.

[0227] Non-limiting exemplary mutagenesis approaches include site-directed mutagenesis using targeted gene editing technologies such as TALEN, ZFN, and CRISPR / Cas9; transposon mutagenesis (Damasceno, J. et al. (2015) Christopher Peacock (Ed.): Parasite Genomics Protocols, vol. 1201. New York, NY: Springer New York (Methods in Molecular Biology)) combined with repair scaffolds for directed homologous recombination-mediated repair (Zhang, W et al. (2017) mSphere 2(1); Gupta, R. and Musunuru, K. (2014) The Journal of clinical investigation 124(10):4154-4161); Biology), pp. 235-245); in situ replacement of the endogenous copy by integration via homologous recombination, possibly in combination with a selectable marker and a mutant gene version (Roberts, S. (2011) Bioeng Bugs 2(6):320-326); RNA interference (RNAi) (Lye, L. et al. (2010) PLoS Pathog 6(10), e1001161), and conditional knockdown using Cre / LoxP or FRT / FLP (Duncan, S. (2017) Molecular and Biochemical Parasitology 216:30-38).

[0228] Overexpression can be achieved by the following non-limiting exemplary approaches, for example, increasing gene copy number by introducing additional copies into separate loci (Beverley, S. (1991): Gene amplification in Leishmania. In Annu. Rev. Microbiol. 45, pp. 417-444), highly expressed loci (ribosomal DNA loci) or episomal constructs (Lodes, M. et al. (1995) Mol Cell Biol 15(12), pp. 6845-6853. DOI: 10.1128 / mcb.15.12.6845; Boucher, N. (2004) Nucleic Acids Res 32(9): 2925-2936), modifying native UTRs flanking the coding sequence; introducing additional promoter regions, such as the endogenous PolI promoter or T7 promoter, in combination with the expression of bacterial T7 polymerase to increase expression levels (Boucher, N. et al. al. (2002) Molecular and Biochemical Parasitology 119(1):153-158; Gu, P. et al. (2015) Scientific reports 5, p.9684), the use of transposable elements or recombinase systems, such as FRT-FLP or Cre / LoxP, to introduce multiple copies of expression constructs (Duncan, S. et al. (2017) Molecular and Biochemical Parasitology 216, pp.30-38), minichromosome integration (Zomerdijk, J. et al. (1992) Nucleic acids research 20(11):2725-2734), and forced chromosomal translocations using CRISPR (Zhang, W. et al. (2017) mSphere 2(1). DOI:10.1128 / mSphere.00340-16).

[0229] In certain embodiments, methods of engineering Leishmania host cells include introducing one or more genes encoding heterologous UDP-GalNAc biosynthetic pathway proteins, as described in Section 7.3.4.

[0230] In certain embodiments, methods of engineering Leishmania host cells include introducing one or more genes encoding heterologous UDP-GalNAc transport proteins, such as those described in Section 7.3.5.

[0231] In certain embodiments, methods of engineering Leishmania host cells include (i) functionally inactivating one or more genes encoding enzymes that catalyze the formation of O-linked GlcNAc, as described in Section 7.3.3; (ii) introducing one or more genes encoding heterologous UDP-GalNAc biosynthetic pathway proteins, as described in Section 7.3.4; (iii) introducing one or more genes encoding heterologous UDP-GalNAc transport proteins, as described in Section 7.3.5; (iv) introducing one or more genes encoding GalNAc transferases, as described in Section 7.3.1; and (v) introducing one or more genes encoding additional recombinant glycosyltransferases, as described in Section 7.3.2.

[0232] In certain embodiments, the method includes performing steps (i) through (v) in order. In other embodiments, steps (i) through (v) are performed in a different order. For example, in certain embodiments, steps (ii) and (iii) are performed before step (i). In other embodiments, steps (iv) and / or (v) are performed before step (i). In certain embodiments, step (v) is performed before step (iv). In certain embodiments, step (v) is performed first.

[0233] In some embodiments, one or more of steps (i)-(v) may be performed simultaneously, e.g., by introducing genes into a single module. For example, in certain embodiments, steps (ii) and (iii) are performed simultaneously. In certain embodiments, step (v) is performed before step (iv). In certain embodiments, step (v) is performed first.

[0234] In certain embodiments, step (i) is performed, followed by steps (ii) and (iii) being performed simultaneously, and then step (iv) being performed separately. In another particular embodiment, steps (ii) and (iii) are performed simultaneously and before step (iv), and step (iv) is performed before step (i). In yet another particular embodiment, step (i) is performed, and then steps (ii), (iii), and (iv) are performed simultaneously after step (i). In certain embodiments, step (v) is performed before step (iv). In certain embodiments, step (v) is performed first.

[0235] In certain embodiments, Leishmania host cells may be engineered using the methods described in the Assays and Examples sections (sections 7.7 and 8, respectively).

[0236] 7.5 Methods for culturing Leishmania host cells Methods for culturing the Leishmania host cells described in Section 7.3 are provided herein.

[0237] 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 rich media such as brain heart infusion, trypticase soy broth, or yeast extract (all of which contain 5 μg / ml hemin). In addition, incubation is performed in the dark at 26°C as static or shaking cultures for 2-3 days. In some embodiments, cultures of recombinant cell lines contain an appropriate selective agent. Non-limiting exemplary selective agents are listed in Table 2.

[0238] In certain embodiments, Leishmania host cells are cultured in a growth medium comprising 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, 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.

[0239] In certain embodiments, Leishmania host cells are cultured in a growth medium comprising 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, 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.

[0240] In certain embodiments, Leishmania host cells may be cultured using the methods described in the Assays and Examples sections (sections 7.7 and 8, respectively).

[0241] 7.6 Use of Leishmania host cells as expression systems In certain embodiments, Leishmania host cells described in Section 7.3 may be used as an expression system to generate a population of glycoengineered bifunctional degradative derivatives described in Section 7.1 or glycoengineered bifunctional degradative derivatives described in Section 7.2. In certain embodiments, the glycoengineered bifunctional degradative derivatives may be a heterologous non-Leishmania protein, such as a therapeutic protein (e.g., an antibody). Leishmania host cells are engineered as described in Section 7.4 and cultured as described in Section 7.5. Other methods of generating Leishmania host cells for use as expression systems are also known and may be used. See, e.g., WO 2019 / 002512, WO 2021 / 140144, and WO 2021 / 140143 (each of which is incorporated by reference in its entirety). The use of Leishmania host cells to generate monoclonal antibodies is also well known. Exemplary methods are described in WO 2022 / 053673, which is incorporated herein by reference in its entirety.

[0242] In certain embodiments, Leishmania host cells may be used as an expression system to produce glycoengineered bifunctional degrading derivatives according to the methods described in the Assays and Examples sections (Sections 7.7 and 8, respectively).

[0243] 7.6.1 Compositions Comprising Host Cells In one aspect, provided herein are compositions comprising Leishmania host cells as described in Section 7.3. Such compositions can be used in methods for generating glycoengineered bifunctional degradative derivatives as described in Section 7.1 or populations of glycoengineered bifunctional degradative derivatives as described in Section 7.2. In certain embodiments, compositions comprising Leishmania host cells can be cultured under conditions suitable for producing glycoengineered bifunctional degradative derivatives. The glycoengineered bifunctional degradative derivatives can then be isolated from the compositions comprising Leishmania host cells using methods known in the art.

[0244] Compositions comprising Leishmania host cells may contain additional components suitable for the maintenance and survival of the Leishmania host cells, and may additionally contain additional components necessary or beneficial for the production of glycoengineered bifunctional degradation derivatives by the Leishmania host cells, for example, inducers of inducible promoters such as arabinose, IPTG, etc.

[0245] 7.6.2 Methods for producing glycosylated bifunctional degradable derivatives In one aspect, provided herein are methods for making glycoengineered bifunctional degradation derivatives, such as those described in Section 7.1. In one embodiment, provided herein are methods for producing glycoengineered bifunctional degradation derivatives in vivo using Leishmania host cells described in Section 7.3. In certain embodiments, provided herein are methods for producing glycoengineered bifunctional degradation derivatives, the methods comprising (i) culturing Leishmania host cells described in Section 7.3 under conditions suitable for polypeptide production, and (ii) isolating the glycoengineered bifunctional degradation derivatives. In certain embodiments, the Leishmania host cells comprise (a) a recombinant nucleic acid encoding a glycoengineered bifunctional degradation derivative, and (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases. In certain embodiments, the Leishmania host cells are capable of producing glycoengineered bifunctional degradation derivatives comprising biantennary GalNAc-terminal N-glycans. In particular, the Leishmania host cells provided herein are capable of producing glycoengineered bifunctional degradative derivatives comprising N-glycans of the 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 of the glycoengineered bifunctional degradative derivative.

[0246] In certain embodiments, the glycoengineered bifunctional degrading derivatives described herein are made according to the methods described in "GLYCOENGINEERING USING LEISHMANIA CELLS," an international application filed with the European Receiving Office on September 27, 2023, which claims priority to U.S. Provisional Applications Nos. 63 / 410,936 and 63 / 410,955, the entire contents of which are incorporated herein by reference.

[0247] In certain embodiments, the glycoengineered bifunctional degradative derivatives produced by the Leishmania host cells are therapeutic polypeptides, i.e., polypeptides used in the treatment of a disease or disorder. For example, the glycoengineered bifunctional degradatives produced by the Leishmania host cells can be enzymes, cytokines, or antibodies. A non-limiting list of exemplary polypeptides of interest is provided in Section 7.1.

[0248] Those skilled in the art will readily appreciate that the nucleic acid sequences of known proteins (e.g., monoclonal antibodies) as well as newly identified proteins (e.g., monoclonal antibodies) can be readily deduced using methods known in the art, and thus, it is well within the capabilities of one of skill in the art to introduce a nucleic acid encoding any glycoengineered bifunctional degrading derivative into a host cell provided herein (e.g., via an expression vector, e.g., a plasmid, e.g., via site-specific integration by homologous recombination).

[0249] Other methods for producing the glycoengineered bifunctional degradative derivatives provided herein can also be used, for example, chemical conjugation or chemoenzymatic modification can be used to produce the bifunctional degradative derivatives provided herein.

[0250] 7.7 Target Protein In some embodiments, the target protein is a cell surface molecule or a non-cell surface molecule. In some embodiments, the cell surface molecule is a receptor. In some embodiments, the non-cell surface receptor is an extracellular protein. In some embodiments, the extracellular protein is an autoantibody, a hormone, a cytokine, a chemokine, a blood protein, or a protein expressed in the central nervous system (CNS).

[0251] In some embodiments, the disease-associated target protein is upregulated in the disease compared to a non-disease state. In some embodiments, the disease-associated target protein is expressed in the disease compared to a non-disease state. In some embodiments, the disease-associated target protein is involved in the progression of the disease. In some embodiments, the disease is a cancer or tumor. In some embodiments, the target protein is involved in the progression of cancer. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is a neurodegenerative disease.

[0252] In some embodiments, the disease is Graves' disease. Graves' disease is the most common cause of hyperthyroidism. Its prevalence in the United States is 1.2% (1), with a lifetime risk of as high as 3% in women. The production of agonist anti-TSH receptor (TSHR) antibodies (TRAb) leads to overproduction of the hormone thyroxine (more than 90% of patients are TRAb+) (2). Current treatments have not progressed in 50 years and are limited by a high risk of relapse and severe side effects, including hypothyroidism. In some embodiments, the target protein associated with Graves' disease is autoantibody-bound TSHR. In other embodiments, the target protein associated with Graves' disease is TSHR.

[0253] In some embodiments, the target protein is TNFα, HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, CCR2 The protein includes a protein selected from the group consisting of Frizzled receptor, Wnt, LRP5 / 6, CSF-1R, SIRPα, CD38, CD73, TGF-β, TSHRα, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin, ganglioside GM1, GD3, and GQ1B. In other embodiments, target proteins include antibodies that bind to TSHRα, MOG, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin, and gangliosides GM1, GD3, and GQ1B.

[0254] In some embodiments, the target protein is a protein that is upregulated in cancer. In some embodiments, the target protein is a protein involved in cancer progression. Examples of target proteins that are upregulated in cancer or involved in cancer progression that can be bound by the glycoengineered bifunctional degradation derivatives provided herein include TNFα, HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CCL2, and CCR2. These include, but are not limited to, Frizzled receptors, Wnt, LRP5 / 6, CSF-1R, SIRPα, CD38, CD73, TGF-β, bombesin R, CAIX, CD13, CD44v6, emmprin, endoglin, EpCAM, EphA2, FAP-α, folate R, GRP78, IGF-1R, matriptase, mesothelin, sMET / HGFR, MT1-MMP, MT6-MMP, PSCA, PSMA, Tn antigen, and uPAR.

[0255] In some embodiments, the target protein is an autoantibody, such as one associated with an autoimmune disease. Examples of autoantibodies that can be bound by the glycoengineered bifunctional degradation derivatives provided herein include, but are not limited to, autoantibodies directed against TSHRα, MOG, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin, and gangliosides GM1, GD3, and GQ1B.

[0256] In some embodiments, the target protein comprises a protein upregulated or expressed in tumor-associated macrophages (TAMs). In some embodiments, the target protein is upregulated or expressed in tumor-promoting TAMs. Examples of target proteins upregulated or expressed in TAMs include SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, or CXCR4(9*). In other embodiments, the target protein comprises CCL2, CXCL12, CSF-1, or CD47(9*). These targets play a role in promoting tumor-promoting TAMs, particularly by promoting TAM recruitment and programming.

[0257] In certain embodiments, the target protein is a protein that is upregulated or expressed in neurodegenerative diseases. Examples of target proteins that are upregulated or expressed in neurodegenerative diseases include alpha-synuclein, amyloid beta, or complement cascade components.

[0258] In certain embodiments, the target protein is a protein that is upregulated or expressed in systemic amyloidosis or localized amyloidosis, hi some embodiments, the target protein that is upregulated or expressed in systemic amyloidosis is transthyretin.

[0259] 7.8 Methods of Use of Glycoengineered Bifunctional Degradatives In one aspect, provided herein are methods of preventing or treating a disease or disorder in a subject, the methods comprising administering to the subject a glycoengineered bifunctional degrading derivative (including pharmaceutical compositions thereof) described in Section 7.1, or a population of glycoengineered bifunctional degrading derivatives (including pharmaceutical compositions thereof) described in Section 7.2. Further provided herein are methods of preventing a disease or disorder in a subject, the methods comprising administering to the subject a glycoengineered bifunctional degrading derivative or population thereof.

[0260] In one aspect, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject a glycoengineered bifunctional degradation derivative or population thereof described herein. In another aspect, provided herein is a method of preventing a disease or disorder in a subject, the method comprising administering to the subject a glycoengineered bifunctional degradation derivative or population thereof described herein. In certain embodiments, provided herein is a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a glycoengineered bifunctional degradation derivative produced according to the methods described herein, wherein the glycoengineered bifunctional degradation derivative is glycosylated with an N-glycan of the 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 of the glycoengineered bifunctional degradative derivative.

[0261] In certain embodiments, a disease or disorder that may be caused by the presence of a defective form of a glycoengineered bifunctional degradation derivative in a subject, the absence of a glycoengineered bifunctional degradation derivative in a subject, or reduced expression of a glycoengineered bifunctional degradation derivative in a subject can be treated or prevented using a glycoengineered bifunctional degradation derivative produced using the methods described herein. In certain embodiments, the disease or disorder may be mediated by a receptor bound by a glycoengineered bifunctional degradation derivative produced using the methods described herein, or by a ligand bound by a glycoengineered bifunctional degradation derivative produced using the methods described herein (e.g., where the glycoengineered bifunctional degradation derivative is a receptor for the ligand).

[0262] In certain embodiments, a method for preventing or treating a disease or disorder in a subject comprises administering to the subject an effective amount of a glycoengineered bifunctional degrading derivative or population thereof described herein, in certain embodiments, an effective amount is the amount of therapeutic agent that has prophylactic and / or therapeutic effect(s). In certain embodiments, an "effective amount" refers to the amount of a therapeutic agent sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or alleviating the severity of a disease / disorder or its associated symptoms; (ii) reducing the duration of a disease / disorder or its associated symptoms; (iii) preventing the progression of a disease / disorder or its associated symptoms; (iv) causing regression of a disease / disorder or its associated symptoms; (v) preventing the onset or onset of a disease / disorder or its associated symptoms; (vi) preventing the recurrence of a disease / disorder or its associated symptoms; (vii) reducing organ failure associated with a disease / disorder; (viii) reducing hospitalization of a subject with a disease / disorder; (ix) reducing the length of hospitalization of a subject with a disease / disorder; (x) increasing the survival rate of a subject with a disease / disorder; (xi) eliminating the disease / disorder in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another therapeutic agent.

[0263] In some embodiments, provided herein are methods of treating or preventing a disease in a patient, the methods comprising administering to the patient a glycoengineered bifunctional degrading derivative described herein or a population described herein. In some embodiments, the disease is an autoimmune disease, a cancer or tumor, a liver disease, an inflammatory disorder, or a hematological disorder. In some embodiments, the autoimmune disease is selected from Graves' disease, myasthenia gravis, anti-GBM disease, immune thrombotic thrombocytopenic purpura, pemphigus vulgaris acquisita, immune thrombocytopenia, Guillain-Barré syndrome, and membranous nephropathy. In some embodiments, the cancer or tumor is selected from breast cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, hepatocellular carcinoma, and hematological T-cell and B-cell malignancies.

[0264] In some embodiments, the methods of treating or preventing a disease provided herein comprise an administration step comprising intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular injection of a glycoengineered bifunctional degrading derivative described herein or a population described herein.

[0265] In some embodiments, the methods of treating or preventing a disease provided herein require lower doses and / or less frequent administration to achieve the same effect compared to the same antibody with a different glycosylation profile, and / or can be administered over a longer period of time (at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or at least 12 months, at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 years), and / or do not elicit an immune response in the patient against the glycoengineered bifunctional degraded derivative.

[0266] In some embodiments, a suitable dose of a glycoengineered bifunctional degrading derivative described herein corresponds to the minimum effective dose to produce a therapeutic effect. For example, an effective amount of an anti-TSH receptor antibody can be an amount that inhibits TSH activity in a subject suffering from Graves' disease.

[0267] In some embodiments, the amount of a glycoengineered bifunctional degradable derivative described herein administered to a patient is less than the amount described on a pharmaceutical label of the same glycoengineered bifunctional degradable derivative having a different glycosylation profile from the glycoengineered bifunctional degradable derivative described herein.

[0268] In some embodiments, the cumulative amount of a glycoengineered bifunctional degrading derivative described herein administered to a patient over a period of time is less than the cumulative amount indicated on the label of a pharmaceutical product of the same glycoengineered bifunctional degrading derivative having a different glycosylation profile from the glycoengineered bifunctional degrading derivative described herein. In some embodiments, the reduced cumulative amount may be administered at a reduced frequency and in a reduced dose. In some embodiments, the reduced cumulative amount may be administered at a reduced frequency and at one or more doses that are the same or higher than the labeled dose. In some embodiments, the reduced cumulative amount may be administered at one or more reduced doses that are the same or higher than the labeled dose. In some embodiments, the reduced cumulative amount may be administered for a shorter period of time than the period over which the pharmaceutical product achieves the same level of therapeutic or prophylactic efficacy.

[0269] In some embodiments, the amount of a glycosylated bifunctional degradable derivative described herein in a single dose administered to a patient can be about 1 to 150 mg, about 5 to 145 mg, about 10 to 140 mg, about 15 to 135 mg, about 20 to 130 mg, about 25 to 125 mg, about 30 to 120 mg, about 35 to 115 mg, about 40 to 110 mg, about 45 to 105 mg, about 50 to 100 mg, about 55 to 95 mg, about 60 to 90 mg, about 65 to 5 mg, about 70 to 80 mg, or about 75 mg. In some embodiments, the amount of a glycosylated bifunctional degradable derivative described herein in a single dose administered to a patient can be about 5 to about 80 mg. In some embodiments, the amount of a glycosylated bifunctional degradable derivative described herein in a single dose administered to a patient can be about 25 to about 50 mg. In some embodiments, the amount of glyco-engineered bifunctional degrading derivatives described herein in a single dose administered to a patient may be from about 15 mg to about 35 mg.

[0270] In some embodiments, the amount of glycoengineered bifunctional degrading derivatives described herein in a single dose administered to a patient can be 40 mg or less, e.g., 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 7 mg, 5 mg, and 2 mg. In some embodiments, the amount of glycoengineered bifunctional degrading derivatives described herein in a single dose administered to a patient can be 80 mg or less, e.g., 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 20 mg, 15 mg, 10 mg, 5 mg, and 2 mg. In some embodiments, the amount of glycoengineered bifunctional degrading derivatives described herein in a single dose administered to a patient can be 160 mg or less, e.g., 150 mg, 140 mg, 130 mg, 120 mg, 110 mg, 100 mg, 90 mg, 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 20 mg, 15 mg, 10 mg, 5 mg, and 2 mg. In some embodiments, the amount of glycoengineered bifunctional degrading derivatives described herein in a single dose administered to a patient can be 160 mg or more, e.g., 170 mg, 180 mg, 200 mg, 250 mg, and 300 mg.

[0271] In some embodiments, glycoengineered bifunctional degradable derivatives of the present disclosure can be administered every other week, i.e., every 14 days. In some embodiments, glycoengineered bifunctional degradable derivatives of the present disclosure can be administered less frequently than every 14 days, for example, every half month, every 21 days, monthly, every 8 weeks, every other month, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months. In some embodiments, glycoengineered bifunctional degradable derivatives of the present disclosure can be administered the same or more frequently than every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or every day.

[0272] In some embodiments, administration of a glycoengineered bifunctional degrading derivative of the present disclosure can include a following dose, for example, an induction dose that is higher than the following maintenance dose. In some embodiments, administration of a glycoengineered bifunctional degrading derivative of the present disclosure can include a second dose that is lower than the induction dose and higher than the following maintenance dose. In some embodiments, administration of a glycoengineered bifunctional degrading derivative of the present disclosure can include the same amount of glycoengineered bifunctional degrading derivative in all doses throughout the entire treatment period.

[0273] In some embodiments, provided herein are methods of treating acute conditions associated with increased levels of a target protein, the methods comprising administering a glycoengineered bifunctional degradation derivative described herein to a patient in need thereof, wherein the method results in a half-life that is at least 50%, 60%, 70%, 80%, 90%, or 99% of the half-life of the bifunctional degradation derivative without any glycosylation. In some embodiments, the half-life of the target protein in the patient in the presence of the bifunctional degradation derivative provided herein is 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours.

[0274] In some embodiments, provided herein are methods of treating a chronic condition associated with increased levels of a target protein, the method comprising administering a bifunctional degradation derivative described herein to a patient in need thereof, the method resulting in a half-life in the patient that is at least 50%, 60%, 70%, 80%, 90%, or 99% of the half-life of the bifunctional degradation derivative without any glycosylation. In some embodiments, the half-life of the target protein is at least 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0275] In some embodiments, provided herein are methods of treating a chronic condition associated with increased levels of a target protein, the method comprising administering to a patient in need thereof a bifunctional degrading derivative as described herein, wherein the bifunctional degrading derivative (i) specifically binds to the target protein, and (ii) comprises an N-glycan of the 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 a glyco-engineered bifunctional degradative derivative, and the N-glycan is linked to the bifunctional degradative derivative at one, two or more N-glycosylation sites such that the half-life is at most 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the half-life of the target protein in a patient in the absence of the bifunctional degradative derivative or in the absence of any therapeutic agent.

[0276] In some embodiments, the chronic condition is an autoimmune disease, a cancer or tumor, a liver disease, an inflammatory disorder, or a blood clotting disorder.

[0277] In some embodiments, the autoimmune disease is selected from Graves' disease, myasthenia gravis, anti-GBM disease, immune thrombotic thrombocytopenic purpura, pemphigus vulgaris acquisita, immune thrombocytopenia, autoimmune encephalitis, Guillain-Barré syndrome, and membranous nephropathy.

[0278] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a blood-borne cancer or tumor. In some embodiments, the cancer may be a carcinoma or sarcoma. In some embodiments, the cancer is selected from lung cancer (small cell or non-small cell), breast cancer, gastric cancer, colorectal cancer, bladder cancer, malignant melanoma, brain cancer (e.g., astrocytoma, glioma, meningioma, neuroblastoma, etc.), bone cancer (e.g., osteosarcoma), cervical cancer, bile duct cancer, gastrointestinal cancer (e.g., oral cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, etc.), head and neck cancer, leiomyosarcoma, liposarcoma, liver cancer (e.g., hepatocellular carcinoma), mesothelioma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer, spindle cell carcinoma, testicular cancer, thyroid cancer, or uterine cancer (e.g., endometrial cancer). In certain embodiments, the cancer may have recurred after previous treatment or may be resistant to conventional treatment. In certain embodiments, the cancer may be disseminated or metastatic. In some embodiments, the blood-borne cancer or tumor is selected from leukemia, myeloma (e.g., multiple myeloma), lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma). In certain embodiments, the leukemia is chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, and acute myeloblastic leukemia.

[0279] In some embodiments, the treatment comprises reprogramming tumor-associated macrophages (TAMs) by administering a bifunctional degradation derivative under conditions that mediate the endocytosis of a target protein. In some embodiments, the target protein is upregulated or expressed in TAMs. In some embodiments, the target protein upregulated or expressed in TAMs comprises SIRPα, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1, or CD47.

[0280] In some embodiments, the administering step comprises intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal injection, or intramuscular injection.

[0281] In some embodiments, provided herein are methods for delivering a target protein to hepatocyte endosomes. In some embodiments, the method for delivering a target protein to hepatocytes comprises contacting the target protein with any of the glycoengineered bifunctional degradation derivatives disclosed herein under conditions that mediate endocytosis of any of the target proteins disclosed herein. In some embodiments, the method for delivering a target protein to hepatocyte endosomes is performed in vivo. In some embodiments, modes of delivering a target protein to hepatocyte endosomes in vivo include intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection, or intramuscular injection. In some embodiments, the method for delivering a target protein to hepatocyte endosomes is performed ex vivo.

[0282] In some embodiments, the delivery rate can be increased based on the number of N-glycan structures present on the glycoengineered bifunctional degradable derivative. In some embodiments, increasing the number of N-glycan structures on the glycoengineered bifunctional degradable derivative increases the delivery rate. In some embodiments, the glycoengineered bifunctional degradable derivative may comprise 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 N-glycans of the following structures: [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 of the glycoengineered bifunctional degradative derivative.

[0283] In some embodiments, provided herein are methods for degrading a target protein. In some embodiments, the method for degrading a target protein comprises contacting the target protein with any of the glycoengineered bifunctional degradation derivatives disclosed herein under conditions that mediate degradation of any of the target proteins disclosed herein by a host cell. In some embodiments, the degradation is lysosomal degradation. In some embodiments, the degradation is mediated by endocytosis or phagocytosis. In certain embodiments, the method comprises degrading a structure comprising at least one target protein bound to at least one glycoengineered bifunctional degradation derivative. In certain embodiments, the structure has a size of about 50 kDa or more, about 75 kDa or more, about 100 kDa or more, about 150 kDa or more, about 200 kDa or more, about 250 kDa or more, about 300 kDa or more, about 400 kDa or more, about 500 kDa or more, about 600 kDa or more, about 700 kDa or more, about 800 kDa or more, about 900 kDa or more, about 1000 kDa or more, about 1100 kDa or more, about 1200 kDa or more, or about 1300 kDa or more. In some embodiments, the degradation is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 18-fold, 20-fold, 25-fold, or 30-fold greater than degradation mediated by a glycoengineered bifunctional degradative derivative that does not contain at least one or at least two N-glycans of 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 a glycoengineered bifunctional degradative derivative. In other embodiments, the glycoengineered bifunctional degradative derivative enhances degradation of any of the disclosed target proteins compared to degradation of the target protein in the presence of a glycoengineered bifunctional degradative derivative that does not contain at least one or at least two of the N-glycans.

[0284] Without being bound by theory, increasing the rate and / or efficiency of internalization (e.g., ASGPR-mediated) of a glycoengineered bifunctional degradation derivative increases the rate and / or activity of lysosomal degradation. In certain embodiments, the rate and / or efficiency of internalization (e.g., ASGPR-mediated) of a glycoengineered bifunctional degradation derivative is proportional to the rate and / or activity of lysosomal degradation. In certain embodiments, the relative change in the rate and / or efficiency of internalization (e.g., ASGPR-mediated) of a glycoengineered bifunctional degradation derivative is determined from the relative change in the rate and / or activity of lysosomal degradation. In some embodiments, the rate and / or efficiency of internalization can be modulated through glycoengineering techniques. In some embodiments, the rate and / or efficiency of internalization can be modulated based on the number of N-glycan structures present on the glycoengineered bifunctional degradation derivative. In some embodiments, the rate and / or efficiency of internalization can be increased based on the number of N-glycan structures present on the glycoengineered bifunctional degradation derivative. In some embodiments, increasing the number of N-glycan structures on a glycoengineered bifunctional degradable derivative increases the rate and / or efficiency of internalization. In some embodiments, increasing the number of N-glycan structures on a glycoengineered bifunctional degradable derivative by one N-glycan increases the rate and / or efficiency of internalization compared to a glycoengineered bifunctional degradable derivative comprising one fewer N-glycan. In some embodiments, the increase in rate and / or efficiency of internalization resulting from increasing the number of N-glycan structures on a glycoengineered bifunctional degradable derivative by one N-glycan is more than an additive effect. In certain embodiments, the increase in the rate and / or efficiency of internalization resulting from increasing the number of N-glycan structures on a glycoengineered bifunctional degrading derivative by one N-glycan is about 10% or more, about 20% or more, about 30%, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the rate and / or efficiency of internalization per N-glycan for a glycoengineered bifunctional degrading derivative comprising one, two, three, or four fewer N-glycans.In some embodiments, the glycoengineered bifunctional degrading derivative may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more N-glycans of the following structure: [ka] where 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, and X represents an amino acid residue of the glycoengineered bifunctional degradative derivative. In some embodiments, the presence of two or more N-glycans on the glycoengineered bifunctional degradative derivative can increase the rate and / or efficiency of internalization compared to a glycoengineered bifunctional degradative derivative containing only one N-glycan. In some embodiments, the rate and / or efficiency of internalization can be fine-tuned. That is, the rate and / or efficiency of internalization can be increased by increasing the number of N-glycan structures present. Different internalization rates are desired depending on the condition being treated. For the treatment of acute conditions, rapid internalization of the complex resulting from binding of the bifunctional degradative derivatives provided herein to their target protein(s) is desired. To achieve this, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 N-glycosylation sites can be introduced and linked to N-glycans, resulting in rapid internalization and a short half-life of the target protein of less than 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or less than 4 hours. For the treatment of acute conditions associated with increased target protein levels, the method comprises administering to a patient in need thereof a bifunctional degrading derivative, wherein the bifunctional degrading derivative (i) specifically binds to the target protein and (ii) comprises an N-glycan that is linked to the bifunctional degrading derivative at one, two or more N-glycosylation sites such that the half-life of the target protein is at most 0.1%, 0.5%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the half-life of the target protein in the patient in the absence of the bifunctional degrading derivative or in the absence of any therapeutic agent.

[0285] In some embodiments, the rate and / or efficiency of internalization of a bifunctional degradation derivative can be adjusted based on the location of the N-glycan(s) on the bifunctional degradation derivative. In some embodiments, the rate and / or efficiency of internalization can be increased based on the location of the N-glycan(s) on the bifunctional degradation derivative. In certain embodiments, the rate and / or efficiency of internalization of a bifunctional degradation derivative is enhanced by engineering the bifunctional degradation derivative to contain one or more N-glycosylation sites distal to the target-specific binding position of the bifunctional degradation derivative. In certain embodiments, the rate and / or efficiency of internalization of a bifunctional degradation derivative is enhanced by engineering the bifunctional degradation derivative to contain at least one or at least two N-glycosylation sites distal to the target-specific binding position. In certain embodiments, the rate and / or efficiency of internalization of a bifunctional degradation derivative is enhanced by engineering the bifunctional degradation derivative to contain all N-glycosylation sites distal to the target-specific binding position. In certain embodiments, the efficiency of target engagement and internalization by a bifunctional degradation derivative is enhanced compared to the unmutated form of the bifunctional degradation derivative by engineering the bifunctional degradation derivative to delete, mutate, or functionally inactivate N-glycosylation sites present in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradation derivative ("native N-glycosylation sites"). In certain embodiments, the efficiency of target engagement and internalization by a bifunctional degradation derivative is enhanced compared to the unmutated form of the bifunctional degradation derivative by engineering the bifunctional degradation derivative to delete, mutate, or functionally inactivate at least one or at least two native N-glycosylation sites. In certain embodiments, the efficiency of target engagement and internalization by a bifunctional degradation derivative is enhanced compared to the unmutated form of the bifunctional degradation derivative by engineering the bifunctional degradation derivative to delete, mutate, or functionally inactivate all native N-glycosylation sites.In certain embodiments, the efficiency of target engagement and internalization by a bifunctional degradation derivative is enhanced, compared to an unmutated form of the bifunctional degradation derivative, by engineering the bifunctional degradation derivative to delete, mutate, or functionally inactivate one or more native N-glycosylation sites located at or proximal to the target-specific binding position of a wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradation derivative. In certain embodiments, the target-specific binding position is the variable region of an antibody or antigen-binding fragment (Fab), or the ectodomain of an Fc-fusion protein. In certain embodiments, the efficiency of target degradation by a mutant form of a bifunctional degradation derivative is enhanced, compared to an unmutated form of the bifunctional degradation derivative, by engineering the bifunctional degradation derivative to delete, mutate, or functionally inactivate one or more N-glycosylation sites ("native N-glycosylation sites") present in the wild-type, natural, synthetic, or commercially available precursor of the bifunctional degradation derivative. In certain embodiments, the efficiency of target degradation is enhanced by eliminating or reducing the number of N-glycosylation sites located at or proximal to the target-specific binding position. Without being bound by theory, eliminating or reducing the number of A2GalNAc2 glycans located at or proximal to the target-specific binding site can reduce the rate of internalization and clearance of the bifunctional degradation derivative before target binding (i.e., the unbound form of the degradation derivative) compared to after target binding (i.e., the bound form of the degradation derivative). Thus, without being bound by theory, eliminating or reducing the number of A2GalNAc2 glycans located at or proximal to the target-specific binding site can increase the probability that the bifunctional degradation derivative will bind to its target before being internalized via ASGPR. In certain embodiments, the number of N-glycosylation sites located at or proximal to the target-specific binding site is eliminated or reduced by deleting, mutating, or functionally inactivating one or more native N-glycosylation sites. In certain embodiments, all of the glyco-engineered N-glycosylation sites are distal to the target-specific binding site of the bifunctional degradation derivative.In certain embodiments, the rate and / or efficiency of internalization of a bifunctional degradation derivative, wherein the bifunctional degradation derivative is an antibody, is enhanced by engineering the bifunctional degradation derivative to contain two N-glycosylation sites in the Fab region of the antibody, where one N-glycosylation site is located on each of the two heavy chain polypeptides of the antibody, and where each of the heavy chain N-glycosylation sites is glycosylated by an N-glycan, as compared to engineering a bifunctional degradation derivative to contain two N-glycosylation sites in the Fab region of the antibody, where one N-glycosylation site is located on each of the two light chain polypeptides of the antibody, and where each of the light chain N-glycosylation sites is glycosylated by an N-glycan. In certain embodiments, the rate and / or efficiency of internalization of a bifunctional degradative derivative (wherein the bifunctional degradative derivative is an antibody) is enhanced by engineering the bifunctional degradative derivative to contain at least two N-glycosylation sites distal to the hinge region of the antibody (in terms of the antibody's quaternary structure), wherein at least two of the distal N-glycosylation sites are glycosylated with an N-glycan, compared to a bifunctional degradative that contains N-glycosylation sites proximal to the hinge region of the antibody (in terms of the antibody's quaternary structure), and wherein only the proximal N-glycosylation sites are glycosylated with an N-glycan. In certain embodiments, the bifunctional degradative derivative is engineered as described in Section 7.1.

[0286] In some embodiments, the method for degrading a target protein comprises GalNAc-mediated degradation. In some embodiments, GalNAc degradation is optimal due to the involvement of endocytic receptors. In some embodiments, the method for degrading a target protein via GalNAc-mediated degradation is selective. In some embodiments, GalNAc degradation removes inflammatory cytokines from the circulation, removes undesirable blood factors, removes autoantibodies, removes pathogenic antibodies, removes cell surface receptors, removes protein aggregates, and removes extracellular soluble proteins.

[0287] 7.9 Assay 7.9.1 Strains, propagation, and genetic methods Methods are provided herein for culturing the Leishmania host cells described in Section 7.3 for the production of bifunctional degradative derivatives.

[0288] Host cells are cultured using any of the standard culture techniques known in the art. For example, cells are routinely grown in rich media such as brain heart infusion, trypticase soy broth, or yeast extract (all of which contain 5 μg / ml hemin). In some embodiments, incubation is performed in the dark at 26°C as a static or shaking culture for 2-3 days. In some embodiments, cultures of recombinant cell lines contain an appropriate selective agent. In some embodiments, cultures contain biopterin at a final concentration of 10 μM to support growth.

[0289] A non-limiting list of selective agents is provided in Table 2. [Table 4] [Table 5] TIFF2025534987000038.tif239165TIFF2025534987000039.tif239165

[0290] (i) Plasmid Plasmids were derived from a pUC57 vector backbone for propagation in E. coli and contained ampicillin or kanamycin selection markers. The expression cassette was flanked by restriction enzyme sites suitable for removal. The cassette composition was determined by the intended use and is described in the corresponding methods and examples. The gene of interest was included as an ORF with codon usage optimized for L. tarentolae. The optimized sequence was manually curated to avoid restriction enzyme sites and remove repetitive or homopolymeric stretches. Plasmids were generated and sequenced by a gene synthesis provider. Plasmids and further details can be found in the sequence listing.

[0291] For codon usage optimization, protein sequences were reverse-translated into nucleotide sequences using a custom Python script that probabilistically selects codons based on their codon usage in L. tarentolae, while excluding rare codons (frequency <10%). Codon usage was previously calculated using cusp (Rice, et al. (2000) Trends in Genetics: TIG 16(6), pp. 276-277) based on the entire annotated nucleotide coding sequence of L. tarentolae.

[0292] (ii) Transfection Method (A) DNA preparation Restriction enzyme digestion (12 μg DNA in a total volume of 240 μL) was performed using standard restriction enzymes (ThermoFisher, preferably FastDigest) according to the manufacturer's instructions. Restriction enzyme digestion was carried out at 30°C until completion or overnight, and the DNA was purified by EtOH precipitation (2 volumes of 100% ice-cold EtOH was added to 1 volume of digested DNA, incubated on ice for 30 minutes, and centrifuged at 17,500 × g for 30 minutes at 4°C). The pellet was washed with 70% EtOH, then dried for up to 15 minutes and resuspended in ddH2O. For optimal removal of circularized plasmid, one or two restriction enzymes with recognition sites in the vector backbone were selected, digested for 1 hour at 37°C, and purified with EtOH as described above. Digestion was analyzed by agarose gel electrophoresis in 0.7-2% agarose gels (TAE buffer). Optionally, undigested plasmid was removed from the preparation by gel extraction using the NucleoSpin® Gel and PCR Clean-up kit (Macherey & Nagel) according to the manufacturer's instructions.

[0293] (B) Transfection using Nucleofector One day before transfection, a densely grown culture of the parent strain was diluted 1:10 into fresh medium containing all antibiotics (brain heart infusion and hemin "BHIH"; or yeast extract and hemin "YEH") that had been previously integrated with the selectable marker and grown overnight at 26°C.

[0294] For transfection, linear DNA fragments for integration were mixed in the combination required for homologous recombination with multiple DNA fragments, with 1 μg of each fragment. The volume of the mixture was reduced to approximately 2 μl per transfection in a vacuum concentrator at 30°C. For episomal transfection of plasmids, 0.1–1 μg of plasmid DNA was used directly for transfection.

[0295] Transfection was performed using 4D-Nucleofector™ Core X with the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza). For this, the DNA prepared as described above was mixed with 16.4 μl of P3 Primary Cell solution and 3.6 μl of Supplement Solution. 10 of an equivalent culture volume was used. 7 Cells (OD should be approximately 0.3-1.0 / ml, and cell shape should be round to droplet-like) were pelleted by centrifugation at 1800 g for 5 minutes, and the supernatant was removed. The cell pellet was resuspended in the DNA mixture and transfected with pulsed FI-158 using a 16-well electroporation strip (in some cases, pulsed FP167, CM150, EO115, DN100, FP158, or FB158 was used instead). As a negative control, an additional culture was transfected with ddH2O only.

[0296] 80 μl of fresh medium (BHIH or YEH and parental selection marker) was added to each well, and 2 × 45 μl of the mixture was transferred to individual wells of a 96-well culture plate pre-filled with 200 μl of fresh medium. After 24 hours of incubation in the dark at 26°C (recovery), a new selection marker was added at a 50% concentration (pre-selection; see Table 2). After 1–2 days of further incubation, the selection marker was replenished to 100% (main selection; see Table 2), and several dilutions from 1:2 to 1:10 were performed in a 96-well format (final volume of 250 μl). The cultures were further incubated in the dark at 26°C for up to 7 days. If no growth was observed, the medium was replaced (centrifugation at 1800 g for 10 minutes at room temperature), and the cultures were again incubated for up to 7 days. This process was repeated as necessary. Prior to analysis, growing cultures were expanded to higher culture volumes by dilutions ranging from 1:5 to 1:20.

[0297] For clonal selection, cells were streaked onto BHIH or YEH plates (containing 1.4% agar and 100% of the appropriate selective agent) as soon as the liquid culture became turbid. The plates were sealed with parafilm, inverted, and incubated in the dark at 26°C for 7–10 days. Single colonies (1–2 mm in size) were transferred to 24-well plates containing 1 ml of BHIH or YEH, sealed with parafilm, and incubated in the dark at 26°C for approximately 7–10 days. Next, 1 ml of culture from the 24-well plate was transferred to 10 ml of BHI or YEH in a flask and further grown statically as usual.

[0298] (iii) Methods for engineering Leishmania tarentolae cells that are deficient in O-glycosylation Without being bound by theory, it has been shown that one of the most effective methods for controlling O-linked GlcNAc modification in L. tarentolae is CRISPR / Cas9-mediated RNP transfection, which replaces all three OGNT genes with a single selectable marker. See WO2021 / 140143. However, other methods have been reported and may also be utilized. See WO2021 / 140143, which is incorporated herein by reference in its entirety. In one embodiment, a ribonucleoprotein complex formed from the endonuclease SpCas9 and a bipartite guide RNA (gRNA) is transfected into L. tarentolae to introduce double-stranded breaks within the 5' and 3' regions of the open reading frames encoding OGNT1, OGNT2, and OGNTL. The gRNA is formed from a scaffold RNA (tracrRNA) and one of six sequence-specific targeting RNAs (crRNAs) used in this method. A selectable marker expression construct consisting of two linear DNA fragments is transfected into cells along with the RNP complex. During double-strand break repair in L. tarentolae, the linear DNA pieces are integrated into the previous OGNT expression site by homologous recombination with each other and the 5' and 3' untranslated regions of the OGNT gene. In the setup described herein, the selectable marker expression construct does not contain additional flanking untranslated regions, allowing transcription of the marker by endogenous Pol II.

[0299] (A) Preparation of ribonucleoprotein (RNP) complexes for transfection gRNAs for CRISPR / Cas9-mediated genome editing were assembled from equimolar amounts of tracrRNA and crRNA (Microsynth) as described above by denaturing at 95°C for 5 minutes, followed by slow cooling at 0.1°C / second in a thermocycler. This was done separately for each crRNA used, and then the different gRNAs were mixed in equimolar amounts. Next, 122 pmol of recombinantly expressed Cas9 protein (i.e., Alt-R® SpHiFi Cas9 Nuclease V3 (IDT, No. 1081061)) was added to the 360 ​​pmol gRNA mixture and incubated at room temperature for 15 minutes to allow RNP formation. The final volume used for Nucleofector transfection (as described in this section) should not exceed 6 μl. Finally, the RNP mixture was added to the transfection solution containing the repair DNA described below, along with 1 μl of Alt-R® Cas9 Electroporation Enhancer (IDT, No. 1081072).

[0300] (B) Preparation of DNA for transfection The linear DNA fragments for integration were mixed in the required combination for transfection, with 1 μg of each fragment. gRNA was prepared as described above and mixed with the integration fragments. The volume of the mixture was reduced to a maximum of 2 μl per transfection in a vacuum concentrator at 30°C.

[0301] (C) Transfection using Nucleofector One day before transfection, a densely grown culture of the parent strain was diluted 1:10 into fresh medium (BHIH) containing all antibiotics that had been previously integrated with the selection marker and grown overnight at 26°C.

[0302] Transfection was performed using 4D-Nucleofector™ Core X with the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza). For this, DNA (or DNA / RNA or DNA / RNP) prepared as described above was mixed with 16.4 μl of P3 Primary Cell solution and 3.6 μl of Supplement Solution. 10 of an equivalent culture volume was used. 7 Cells (OD should be approximately 0.3-1.0 / ml, and cell shape should be round to droplet-like) were pelleted by centrifugation at 1800 g for 5 minutes, and the supernatant was removed. The cell pellet was resuspended in 20 μl of DNA (or DNA / RNA or DNA / RNP) mixture and transfected with pulsed FI-158 using 16-well electroporation strips (in some cases, pulsed FP167, CM150, EO115, DN100, FP158, or FB158 was used instead). As a negative control, additional cultures were transfected with ddH2O only.

[0303] 80 μl of fresh medium (containing the selection marker of the parent cell line) was added to each well, and 2 × 45 μl of the mixture was transferred to individual wells of a 96-well culture plate pre-filled with 200 μl of fresh medium. After 24 hours of incubation in the dark at 26°C (recovery), a new selection marker was added at a 50% concentration (pre-selection; see Table 2). After 1–2 days of further incubation, the selection marker was replenished to 100% (main selection), and several dilutions from 1:2 to 1:10 were made in a 96-well format (final volume of 250 μl). The cultures were further incubated in the dark at 26°C for up to 7 days. If no growth was observed, the medium was replaced (centrifugation at 1800 g for 10 minutes at room temperature), and the cultures were again incubated for up to 7 days. This process was repeated as necessary. Prior to analysis, growing cultures were expanded to higher culture volumes by dilutions ranging from 1:5 to 1:20.

[0304] (D) crRNA design crRNAs were designed by EuPaGDT (http: / / grna.ctegd.uga.edu / ) for use with SpCas9 (PAM=NGG) based on the target region (usually the coding sequence of the OGNT gene) and validated for on- and off-target effects by blasting against the entire L. tarentolae genome. Next, crRNAs were selected to ideally target the ends of the coding sequence to be replaced. [Table 6]

[0305] (iv) PCR and sequence analysis of deletion strains (A) gDNA preparation - Genomic DNA isolation using a Tissue Kit Two ml of densely grown L. tarentolae culture was pelleted at 1800 g, and the supernatant was discarded. This pellet was used for the preparation of genomic DNA using the NucleoSpin® Tissue Kit (Macherey-Nagel). For this, the pellet was resuspended in 200 μl of buffer T1 and further processed according to the manufacturer's instructions until elution. For efficient elution, 50 μl of preheated (50°C) buffer BE was added to the column and incubated at room temperature for 3 minutes. The eluate was collected by centrifugation at 11000 g for 1 minute. Repeating this process, as well as reloading the eluate, can be used to increase the yield.

[0306] (B) Preparation of crude cell extracts for PCR analysis 50 μl of the culture was washed in 1 ml of PBS and pelleted at 1800 g for 5 min. The supernatant was removed, and the pellet was resuspended in 50 μl of PBS and boiled at 95°C for 5 min with intermittent vortexing. 1 μl was used in place of template DNA in the PCR reaction.

[0307] (C) PCR analysis of OGNT KO PCR confirmation of OGNT knockout was performed by either amplifying the entire locus (OGNT1, OGNT2, OGNTL, or OGNT1+L, where OGNT1+L includes OGNT1 and OGNTL in tandem on the chromosome) or by amplifying a shorter fragment covering the integration site.

[0308] Typically, successful replacement of the wild-type OGNT sequences (OGNT1 = 3.4 kbp, OGNT2 = 1.9 kbp, and OGNTL = 3.4 kbp) with selectable marker coding sequences (0.4–1.0 kbp) with or without the additional intergenic region (approximately 0.9 kbp total) can be easily confirmed by the size of the amplicon obtained from PCR targeting the entire native locus, since the amplicon is much shorter when the wild-type gene is correctly replaced. These PCRs used LA Taq DNA polymerase (TaKaRa) in combination with a buffer for amplification of GC-rich sequences, which allowed for the amplification of long wild-type regions (see Table 5). In some cases, amplification of shorter regions was performed using primers within the OGNT coding sequence to test for remaining wild-type genes (see Table 6). For these PCRs, DreamTaq DNA polymerase (Thermo Fisher Scientific) was used. Alternatively, whether the selectable marker gene has been correctly integrated into the corresponding OGNT locus can be tested by combining a primer that binds within the genome with one primer that binds to the selectable marker CDS or intergenic region in the integrated construct (but with the other primer targeting the genome). [Table 7] [Table 8]

[0309] (v) Expression analysis (A) Sample preparation from Leishmania tarentolae Cells were grown statically at 26°C for 2–3 days (e.g., 3 ml in a 6-well plate). Whole-cell extract (WCE) cell-free culture supernatants were analyzed by Western blot. For supernatant analysis, grown cultures were centrifuged at 1800 g for 5 minutes at room temperature, and the cell-free supernatant was transferred to a new tube and mixed with Laemmli dye under reducing or non-reducing conditions. The WCE cell pellet was washed with 1x PBS, centrifuged again at 1800 g for 5 minutes at room temperature, and frozen at -80°C for a minimum of 30 minutes. After thawing again at room temperature, the pellet was then dissolved in Laemmli (reducing) buffer and boiled again at 95°C for 10 minutes with vigorous vortexing.

[0310] (B) Expression analysis by Western blot Samples were run on a 4-12% Bis-Tris SDS PAGE gel at 200V for 60 minutes using MOPS running buffer. The gel was blotted onto a PVDF membrane using an Iblot device for 7 minutes. The membrane was blocked in 10% milk at room temperature for at least 30 minutes. Primary antibodies (i.e., goat anti-human IgG-HRP (A6029, Sigma) at a 1:2000 dilution and mouse anti-human kappa light chain (K4377, Sigma) at a 1:5000 dilution) were used in 1% milk, 1x PBST and incubated overnight at 4°C. Blots were then washed three times for 5 min each in 1x PBST and then detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse polyvalent HRP (A0412, Sigma) 1:2000 dilution or anti-rabbit HRP conjugate (Jackson ImmunoResearch, no. 111-035-008) 1:2000 dilution) rotated in 1% milk, 1x PBST for 3 h at 30°C, followed by three 5-min washes in 1x PBST and colorimetric detection with one-component 3,3',5,5'-tetramethylbenzidine (TMB) substrate stain (TMBM-1000-01, Surmodics).

[0311] 7.9.2 Small-Scale Expression and Purification of Monoclonal Antibodies Host cells were routinely grown in 50 ml cultures in BHIH or YEH at 26°C for 48 hours with shaking at 140 rpm. Cultures were harvested and centrifuged at 1800 x g for 10 minutes at room temperature. Medium SN was filtered through a 0.22 μm filter (Steriflip, SCGP00525), and EDTA (0.5 M, pH 8) was added to each load at a 1:100 dilution. Medium SN of each strain was incubated in batches with 100 μl of Protein A resin (Protein A-Sepharose 4B Fast Flow, Sigma Aldrich, P9424) per Falcon tube for 4 hours with rotation at room temperature. After treatment with Protein A resin, samples were centrifuged at 500 x g for 5 minutes, the FT was discarded, and the resin was transferred to a spin column. Washing was performed with Buffer A (pH 7.2, 20 mM NaHPO, 150 mM NaCl, pH adjusted to 7.20 with HCl) for 3 x 5 CVs using 500 μl per 100 μl of resin, centrifuged at 1000 × g for 1 min at room temperature between each step. Elution was performed with 100 μl of Buffer B (0.1 M acetic acid, 100 mM NaCl, pH adjusted to 3.20 with 1 M NaOH) for several CVs per 100 μl of resin, centrifuged at 1000 × g for 1 min at room temperature between each step (e.g., 3 x 1 CV and 1 x 0.5 CV). Elution fractions were pooled and immediately neutralized by adding 100 mM Tris-HCl (1 M, pH 8). The pooled eluate was then buffer exchanged into PBS pH 6 using a 2 ml 7K ZebaSpin desalting column and optionally concentrated using an Amicon 0.5 ml 30K concentrator.

[0312] The elution fractions were pooled and immediately neutralized by adding 100 mM Tris-HCl (1 M, pH 8). The pooled eluate was then buffer exchanged into PBS pH 6 using a 2 ml 7K ZebaSpin desalting column and optionally concentrated using an Amicon 0.5 ml 30K concentrator.

[0313] The samples were then subjected to analysis such as HILIC-UPLC-MS as described below.

[0314] 7.9.3 Analysis (i) SDS PAGE and capillary gel electrophoresis SDS-PAGE was performed under reducing or non-reducing conditions, using 10 μg of Coomassie gel or 2.5 μg of WB gel, separated on a 4-12% gel, in MOPS buffer for 55 minutes. Protein purity was measured by Coomassie-stained SDS-PAGE using 10 μg of protein sample and compared to a BSA standard curve. Impurities were quantified using ImageQuant. Capillary gel electrophoresis (CGE) was performed using the Agilent Protein 230 Kit (5067-1518) according to the protocol.

[0315] (ii) Analytical SEC The MAbPac SEC-1 (4 × 300 mm) is a size-exclusion chromatography (SEC) column specifically designed for the separation and characterization of monoclonal antibodies (mAbs) and was used according to the manufacturer's recommendations (temperature: 30 °C; eluent: PBS 50 mM NaPO, 300 mM NaCl pH 6.8; elution: isocratic, 30 min; flow rate: 0.2 mL / min; detection: 215 nm; injection volume: 5 μL equivalent to 5 μg of protein).

[0316] (iii) Identification of O-HexNAc Intact monoclonal antibodies were analyzed by mass spectrometry using a state-of-the-art instrument (Orbitrap FTMS) and data processing and analysis (bioinformatics) tools from SpectroSwiss. In additi...

Claims

1. A glyco-engineered bifunctional degradative derivative, the bifunctional degradative derivative (i) specifically binds to a target protein, and (ii) comprises an N-glycan of the structure: 【Chemical 1】 The glycosylated bifunctional degradative derivative, wherein the N-glycan is linked to the bifunctional degradative at one or more N-glycosylation sites, wherein a black box represents an N-acetylgalactosamine (GalNAc), an open box represents an N-acetylglucosamine (GlcNAc) residue, a black circle represents a mannose (Man) residue, and X represents an amino acid residue of the bifunctional degradative.

2. 2. The glyco-modified bifunctional degrading derivative of claim 1, wherein the N-glycan is linked to the bifunctional degrading derivative at at least one N-glycosylation site.

3. The glyco-modified bifunctional degrading derivative of claim 2, wherein the N-glycan is linked to the bifunctional degrading derivative at at least two N-glycosylation sites.

4. The glycosylated bifunctional degrading derivative of any one of claims 1 to 3, wherein the N-glycans are linked to the bifunctional degrading derivative at one, two, three, or four N-glycosylation sites.

5. The glycosylated bifunctional degrading derivative of claim 4, wherein the N-glycan is linked to the bifunctional degrading derivative at one N-glycosylation site.

6. The glyco-modified bifunctional degrading derivative of claim 4, wherein the N-glycan is linked to the bifunctional degrading derivative at two N-glycosylation sites.

7. The glyco-modified bifunctional degrading derivative of claim 4, wherein the N-glycan is linked to the bifunctional degrading derivative at three N-glycosylation sites.

8. The glyco-modified bifunctional degrading derivative of claim 4, wherein the N-glycans are linked to the bifunctional degrading derivative at four N-glycosylation sites.

9. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 8, wherein the one or more N-glycosylation sites are distal to the target-specific binding position of the bifunctional degradable derivative.

10. The glyco-engineered bifunctional degrading derivative of claim 9, wherein at least one or at least two N-glycosylation sites are distal to said target-specific binding site.

11. The glycoengineered bifunctional degradable derivative of claim 9, wherein all of the N-glycosylation sites are distal to the target-specific binding site.

12. The glycoengineered bifunctional degradable derivative according to any one of claims 9 to 11, wherein the target-specific binding site is the variable region of an antibody or antigen-binding fragment (Fab), or the ectodomain of an Fc fusion protein.

13. The glyco-engineered bifunctional degrading derivative of any one of claims 1 to 12, wherein said one or more N-glycosylation sites are not present in a wild-type, natural, synthetic, or commercially available precursor of said bifunctional degrading derivative.

14. 14. The glycosylated bifunctional degrading derivative of any one of claims 1 to 13, wherein one or more N-glycosylation sites present in the wild-type, natural, synthetic or commercially available precursor of said bifunctional degrading derivative ("native N-glycosylation site") are deleted, mutated or functionally inactivated.

15. 15. The glyco-engineered bifunctional degrading derivative of claim 14, wherein at least one or at least two of the native N-glycosylation sites are deleted, mutated or functionally inactivated.

16. 16. The glycoengineered bifunctional degrading derivative of claim 15, wherein all native N-glycosylation sites are deleted, mutated, or functionally inactivated.

17. 17. The glycoengineered bifunctional degrading derivative of any one of claims 14 to 16, wherein the one or more native N-glycosylation sites are located at or proximal to the target-specific binding position of a wild-type, natural, synthetic, or commercially available precursor of the bifunctional degrading derivative.

18. The glycosylated bifunctional degrading derivative according to any one of claims 1 to 17, wherein the amino acid residue is Asn.

19. The glycosylated bifunctional degradable derivative according to any one of claims 4 to 18, wherein the N-glycosylation site comprises a consensus sequence of N-X-S / T or N-X-C, where X is any amino acid except proline.

20. The glycosylated bifunctional decomposition derivative according to any one of claims 1 to 19, wherein the bifunctional decomposition derivative is an antibody or a fragment thereof.

21. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 20, wherein the bifunctional degradable derivative is a Fab fragment of an antibody.

22. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 21, wherein the glycosylated bifunctional degradable derivative is an antibody.

23. The glycoengineered bifunctional degrading derivative of claim 22 , wherein the antibody is a monoclonal or polyclonal antibody.

24. The glycoengineered bifunctional degrading derivative of claim 22 , wherein the antibody is a recombinant antibody.

25. The glycoengineered bifunctional degrading derivative of claim 22 , wherein the antibody is a humanized antibody, a chimeric antibody, or a fully human antibody.

26. 23. The glycoengineered bifunctional degrading derivative of claim 22, wherein the antibody has a glycan to protein ratio of 2:1, 4:1, 6:1, 8:1, or 10:

1.

27. The glycoengineered bifunctional degrading derivative of any one of claims 20 to 26, wherein said N-glycan is linked to an N-glycosylation site of the light chain of said antibody or fragment thereof.

28. The glycoengineered bifunctional degrading derivative of any one of claims 20 to 27, wherein said N-glycan is linked to an N-glycosylation site of the heavy chain of said antibody or fragment thereof.

29. The glycoengineered bifunctional degrading derivative of any one of claims 20 to 28, wherein one or more N-glycosylation sites are located in the constant domain of said antibody or fragment thereof.

30. The glycoengineered bifunctional degrading derivative of any one of claims 20 to 29, wherein one or more N-glycosylation sites are located in the variable domain of said antibody or fragment thereof.

31. The glycoengineered bifunctional degrading derivative of any one of claims 20 to 30, wherein one or more N-glycosylation sites are located in the Fab region of said antibody.

32. The glycoengineered bifunctional degrading derivative of any one of claims 20 to 31, wherein one or more N-glycosylation sites are located in the Fc region of said antibody.

33. The glycoengineered bifunctional degrading derivative of any one of claims 20 to 32, wherein one or more N-glycosylation sites are located in the hinge region of said antibody.

34. 34. The glycoengineered bifunctional degrading derivative of any one of claims 31 to 33, wherein at least one of said N-glycosylation sites is not present in the wild-type form of said antibody.

35. The glycoengineered bifunctional degrading derivative of any one of claims 1 to 34, wherein at least two N-glycosylation sites in the Fab region of said antibody are glycosylated by said N-glycan.

36. The glycosylated bifunctional degrading derivative according to any one of claims 1 to 35, wherein one N-glycosylation site of the Fab region is located in each of the two heavy chain polypeptides of the antibody, and each of the N-glycosylation sites is glycosylated by the N-glycan.

37. The glycoengineered bifunctional degrading derivative of any one of claims 1 to 36, wherein at least two N-glycosylation sites in the Fc region of said antibody are glycosylated by said N-glycan.

38. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 37, wherein the Fab region contains more of the N-glycans than the Fc region.

39. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 38, wherein the Fab region contains two more N-glycans than the Fc region.

40. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 37, wherein the Fc region contains more of the N-glycans than the Fab region.

41. The glycosylated bifunctional degradable derivative of any one of claims 1 to 37 or 40, wherein the Fc region contains two or four more N-glycans than the Fab region.

42. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 37, wherein the Fc region and the Fab region contain the same number of N-glycans.

43. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 42, wherein the glycosylated bifunctional degradable derivative binds to an autoantibody and comprises an autoantigen or an immunogenic fragment thereof.

44. The glycosylated bifunctional degradable derivative according to any one of claims 1 to 43, wherein the glycosylated bifunctional degradable derivative comprises a moiety that specifically binds to the target protein, and the target protein is associated with a disease.

45. The glycosylated bifunctional degrading derivative according to any one of claims 1 to 44, wherein the target protein is a cell surface molecule or a non-cell surface molecule.

46. The glycoengineered bifunctional degrading derivative of claim 45, wherein the cell surface molecule is a receptor.

47. 46. ​​The glycoengineered bifunctional degrading derivative of claim 45, wherein the non-cell surface molecule is an extracellular protein.

48. 48. The glycoengineered bifunctional degrading derivative of claim 47, wherein the extracellular protein is an autoantibody, a hormone, a cytokine, a chemokine, a blood protein, or a central nervous system (CNS) protein.

49. The glycosylated bifunctional degrading derivative according to any one of claims 44 to 48, wherein the target protein associated with a disease is upregulated in the disease compared to a non-disease state.

50. The glycosylated bifunctional degrading derivative according to any one of claims 44 to 49, wherein the target protein associated with a disease is expressed in the disease compared to a non-disease state.

51. The glycosylated bifunctional degrading derivative according to any one of claims 44 to 50, wherein the disease-related target protein is involved in the progression of cancer.

52. The target proteins associated with the disease include TNFα, HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, αvβ3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptor, EGF, c-MET, CC L2, CCR2, Frizzled receptor, Wnt, LRP5 / 6, CSF-1R, SIRPα, CD38, CD73, or TGF-β, bombesin R, CAIX, CD13, CD44, v6, EMMPRIN, endoglin, EpCAM, EphA2, FAP-α, folate R, GRP78, IGF-1R, matriptase, mesothelin, sME The glycosylated bifunctional degradable derivative according to any one of claims 44 to 51, comprising T / HGFR, MT1-MMP, MT6-MMP, PSCA, PSMA, Tn antigen, and uPAR, TSHRα, MOG, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin, and gangliosides GM1, GD3, GQ1B, LILRB1, LILRB2, VEGF-R, CXCR4, CXCL12, CSF-1, CD47, aggregated light chain, or aggregated transthyretin.

53. The glycosylated bifunctional degradable derivative according to any one of claims 44 to 52, wherein the disease-associated target protein is involved in an autoimmune disease, and the target protein is TSHRα, MOG, AChR-α1, non-collagenous domain 1 of the α3 chain of type IV collagen (α3NC1), ADAMTS13, desmoglein-1 / 3, or an antibody that binds to GPIb / IX, GPIIb / IIIa, GPIa / IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin, or ganglioside GM1, GD3, or GQ1B.

54. The glycosylated bifunctional degrading derivative according to any one of claims 44 to 53, wherein the disease includes cancer.

55. The glycosylated bifunctional degrading derivative according to any one of claims 44 to 53, wherein the disease includes an autoimmune disease.

56. 57. A composition comprising a population of glycoengineered bifunctional degraded derivatives according to any one of claims 1 to 56, wherein said population of bifunctional degraded derivatives has 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 of said N-glycosylation site(s).

57. 57. The composition of claim 56, wherein the N-glycan profile 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 at least 98% homogeneous at one of the N-glycosylation sites.

58. 58. The composition of claim 56 or 57, wherein the N-glycan profile 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 at least 98% homogeneous across two of the N-glycosylation sites.

59. 59. The composition of any one of claims 56 to 58, wherein the N-glycan profile is at least 50% homogeneous at one or more of the N-glycosylation site(s).

60. 59. The composition of any one of claims 56 to 58, wherein the N-glycan profile is at least 60% homogeneous at one or more of the N-glycosylation site(s).

61. 59. The composition of any one of claims 56 to 58, wherein the N-glycan profile is at least 70% homogeneous at one or more of the N-glycosylation site(s).

62. 59. The composition of any one of claims 56 to 58, wherein the N-glycan profile is at least 80% homogeneous at one or more of the N-glycosylation site(s).

63. 59. The composition of any one of claims 56 to 58, wherein the N-glycan profile is at least 90% homogeneous at one or more of the N-glycosylation site(s).

64. 59. The composition of any one of claims 56 to 58, wherein the N-glycan profile is at least 95% homogeneous at one or more of the N-glycosylation site(s).

65. 59. The composition of any one of claims 56 to 58, wherein the N-glycan profile is at least 98% homogeneous at one or more of the N-glycosylation site(s).

66. 66. The composition of any one of claims 56 to 65, wherein the homogeneity of the N-glycan profile at one or more of the N-glycosylation sites is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis.

67. 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 following structure at one or more of the N-glycosylation site(s): 【Chemistry 2】 67. The composition of any one of claims 56 to 66, 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 said bifunctional degradation derivative.

68. 68. The composition of claim 67, wherein the N-glycan profile comprises about 90% to about 100% of the N-glycans at one N-glycosylation site.

69. 69. The composition of claim 67 or 68, wherein the N-glycan profile comprises about 95% to about 100% of the N-glycans at one N-glycosylation site.

70. 70. The composition of any one of claims 67 to 69, wherein the N-glycan profile comprises about 90% to about 100% of the N-glycans at each of two N-glycosylation sites.

71. 70. The composition of any one of claims 67 to 69, wherein the N-glycan profile collectively comprises about 80% to about 90% of the N-glycans at two N-glycosylation sites.

72. 70. The composition of any one of claims 67 to 69, wherein the N-glycan profile collectively comprises about 90% to about 100% of the N-glycans at two N-glycosylation sites.

73. 70. The composition of any one of claims 67 to 69, wherein the N-glycan profile comprises about 90% to about 100% of the N-glycans at each of three or more of the N-glycosylation site(s).

74. 70. The composition of any one of claims 67 to 69, wherein the N-glycan profile collectively comprises about 70% to about 100% of the N-glycans at three or more of the N-glycosylation site(s).

75. 75. The composition of any one of claims 67 to 74, wherein the relative amount of N-glycans at one or more of the N-glycosylation sites is measured by N-glycan analysis or glycopeptide analysis.

76. The population of bifunctional degradation derivatives has an N-glycan profile that comprises 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 N-glycans of the following structure among all glycans in the N-glycan profile: 【Chemistry 3】 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 said bifunctional degradation derivative.

77. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 30% of said N-glycan among all glycans in said N-glycan profile.

78. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 40% of said N-glycan among all glycans in said N-glycan profile.

79. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 50% of said N-glycan among all glycans in said N-glycan profile.

80. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 60% of said N-glycan among all glycans in said N-glycan profile.

81. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 70% of said N-glycan among all glycans in said N-glycan profile.

82. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 80% of said N-glycan among all glycans in said N-glycan profile.

83. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 90% of said N-glycan among all glycans in said N-glycan profile.

84. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 95% of said N-glycan among all glycans in said N-glycan profile.

85. 77. The composition of any one of claims 56 to 76, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises at least 98% of said N-glycan among all glycans in said N-glycan profile.

86. 86. The composition of any one of claims 56 to 85, wherein the population of bifunctional degraded derivatives 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 the N-glycans among all glycans in the N-glycan profile.

87. The composition of any one of claims 56 to 86, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 30% to about 40% of the N-glycan among all glycans in the N-glycan profile.

88. The composition of any one of claims 56 to 86, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 40% to about 50% of the N-glycan among all glycans in the N-glycan profile.

89. The composition of any one of claims 56 to 86, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 50% to about 60% of the N-glycan among all glycans in the N-glycan profile.

90. The composition of any one of claims 56 to 86, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 60% to about 70% of the N-glycan among all glycans in the N-glycan profile.

91. The composition of any one of claims 56 to 86, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 70% to about 80% of the N-glycan among all glycans in the N-glycan profile.

92. The composition of any one of claims 56 to 86, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 80% to about 90% of the N-glycan among all glycans in the N-glycan profile.

93. The composition of any one of claims 56 to 86, wherein the population of bifunctional degradation derivatives has an N-glycan profile that comprises about 90% to about 100% of the N-glycan among all glycans in the N-glycan profile.

94. 94. The composition of any one of claims 76 to 93, wherein the relative amount of the N-glycan among all glycans of the N-glycan profile is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis.

95. 95. The composition of any one of claims 56 to 94, wherein the population has an N-glycan profile that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% homogeneous.

96. 96. The composition of claim 95, wherein the population has an N-glycan profile that is at least 60% homogeneous.

97. 96. The composition of claim 95, wherein the population has an N-glycan profile that is at least 70% homogeneous.

98. 96. The composition of claim 95, wherein the population has an N-glycan profile that is at least 80% homogeneous.

99. 96. The composition of claim 95, wherein the population has an N-glycan profile that is at least 90% homogeneous.

100. 96. The composition of claim 95, wherein the population has an N-glycan profile that is at least 95% homogeneous.

101. 96. The composition of claim 95, wherein the population has an N-glycan profile that is at least 98% homogeneous.

102. 102. The composition of any one of claims 56 to 101, wherein the population has an N-glycan profile that is about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% homogeneous.

103. 103. The composition of claim 102, wherein the population has an N-glycan profile that is about 60% to about 70% homogeneous.

104. 103. The composition of claim 102, wherein the population has an N-glycan profile that is about 70% to about 80% homogeneous.

105. 103. The composition of claim 102, wherein the population has an N-glycan profile that is about 80% to about 90% homogeneous.

106. 103. The composition of claim 102, wherein the population has an N-glycan profile that is about 90% to about 100% homogeneous.

107. The composition of claims 95 to 106, wherein the homogeneity of the N-glycan profile is measured by N-glycan analysis, glycopeptide analysis, or intact protein analysis.

108. 108. The composition of any one of claims 56 to 107, wherein the bifunctional degradative derivatives of the population are expressed from one or more nucleic acid sequences in a Leishmania host cell.

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