WO2024068753A1 - Dégradation de protéine à médiation par glycane - Google Patents

Dégradation de protéine à médiation par glycane Download PDF

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WO2024068753A1
WO2024068753A1 PCT/EP2023/076745 EP2023076745W WO2024068753A1 WO 2024068753 A1 WO2024068753 A1 WO 2024068753A1 EP 2023076745 W EP2023076745 W EP 2023076745W WO 2024068753 A1 WO2024068753 A1 WO 2024068753A1
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glycan
certain embodiments
bifunctional
glycoengineered
degrader
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PCT/EP2023/076745
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English (en)
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Jonathan Albert BACK
Amirreza Faridmoayer
Tanmoy GANGULY
Ganesh Kaundinya
Manuela Mally
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Glycoera Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01041—Polypeptide N-acetylgalactosaminyltransferase (2.4.1.41)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/90—Protozoa ; Processes using protozoa

Definitions

  • the present application relates to glycoengineered bifunctional degraders, populations of glycoengineered bifunctional degraders, Leishmania host cells for producing glycoengineered bifunctional degraders, methods of engineering said Leishmania host cells, methods of culturing said Leishmania host cells, methods of making glycoengineered bifunctional degraders using Leishmania host cells, and methods of using glycoengineered bifunctional degraders.
  • the glycoengineered bifunctional degraders comprise a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2.
  • a glycoprotein is a glycoconjugate in which a protein carries one or more glycans covalently attached to a polypeptide backbone, usually via N- or O-linkages.
  • An N-glycan N-linked oligosaccharide, N-[Asn]-linked oligosaccharide
  • N-linked oligosaccharide is a sugar chain covalently linked to an asparagine residue of a polypeptide chain, commonly involving a GlcNAc residue in eukaryotes, and the consensus peptide sequence: Asn-X-Ser/Thr (Varki, Ajit (2009): Essentials of glycobiology. 2ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).
  • Protein glycosylation is a ubiquitous post-translational modification found in all domains of life. There is a significant complexity in animal systems and glycan structures have crucial biological and physiological roles, from contributions in protein folding and quality, control to involvement in a large number of biological events, like recognition, stability, action, and turnover of these molecules (Moremen et al. 2012).
  • Therapeutic Glycoproteins like monoclonal antibodies, enzymes, and hormones are the major products of the biotechnology industry (Lagasse, H A Daniel et al. 2017; Dimitrov 2012) and the impact of glycan heterogeneity has more and more been recognized as “critical quality attribute”.
  • glycosylation is regarded as even one of the most important ones: influencing the biological activity, serum half-life and immunogenicity of the protein.
  • Glycans are relevant for increased serum circulation times and many of the biologies approved or under development suffer from an insufficient half-life necessitating frequent applications in order to maintain a therapeutic concentration over an extended period of time.
  • Half-life extension strategies are key to allow the generation of long-lasting therapeutics with improved pharmacokinetics (Kontermann 2016).
  • Glycosylation also appears to improve protein solubility and stability, for example, through a reduced propensity for aggregation and leads to increased circulatory lifetimes due to the prevention of proteolytic degradation.
  • N-glycans with different terminating monosaccharides can be recognized by lectins leading to their degradation (Blasko et al., 2013; Varki, 2017). Consequently, monitoring and control of glycosylation is critical in biopharmaceutical manufacturing and a requirement of regulatory agencies (Costa et al. 2014; Eon-Duval et al. 2012; Reusch and Tejada 2015). For these reasons, glycoengineering of expression platforms is increasingly recognized as an important strategy to improve biopharmaceuticals in many aspects (Dicker and Strasser 2015).
  • Endocytic lectins are involved in receptor-mediated endocytosis by capturing glycosylated proteins via specific glycan structures to mediate degradation (Cummings et al., Cold Spring Harbor Laboratory Press, (2017). Endocytic lectins are ubiquitous in humans and can recognize various glycan structures.
  • Carbohydrate binding receptors are highly diverse and can be exploited by glycoengineering to develop novel therapeutics with unprecedented effectiveness for different diseases, including but not limited to: inflammatory, blood disorders, autoimmune and cancer. This allows development of novel therapeutics based on the concept of glycan- mediated protein degradation. Leveraging natural protein degradation through the glycosylation of monoclonal antibodies can lead to novel therapeutics.
  • the present invention shows a novel finding of Leishmania host cells are capable of producing polypeptides comprising a biantennary, GalNAc-terminated N-glycan, specifically A2GalNAc2, which mediates protein degradation.
  • compositions and methods provided herein address the unmet medical need of patients suffering from various difficult to treat diseases such as cancer, autoimmune and inflammatory diseases, and infectious diseases, treated with glycosylated proteins, such as monoclonal antibodies, and provide related advantages.
  • glycoengineered bifunctional degraders populations of glycoengineered bifunctional degraders, Leishmania host cells for producing glycoengineered bifunctional degraders, methods of engineering said Leishmania host cells, methods of culturing said Leishmania host cells, methods of making glycoengineered bifunctional degraders using Leishmania host cells, and methods of using glycoengineered bifunctional degraders.
  • the glycoengineered bifunctional degraders 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.
  • the bifunctional degrader simultaneously binds to the target protein and to an ASGPR receptor(s) present on liver cells, wherein the complex so formed is internalized and targeted for degradation via the lysosomal pathway.
  • the bifunctional degraders of the present invention are unexpectedly efficient for internalization and/or degradation of target proteins, as compared to similar molecules modified with other N-glycans, such as triantennary, GalNAc2 -terminated glycans, which have most often been described for use in so-called “Lysosome-Targeting Chimaerae” or “LYTACs.” See, Zhou et al. (2021) ACS Cent. Sci. 7:499-506; Ahn et al. Nat Chem Biol.
  • bifunctional degrader specifically binds to a target protein and (ii) comprises an N-glycan of the structure:
  • the N-glycan is linked to the bifunctional degrader at one or more N-glycosylation sites, wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N- acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the bifunctional degrader.
  • the N-glycan is linked to the bifunctional degrader at least one N- glycosylation site.
  • the N-glycan is linked to the bifunctional degrader at least two N-glycosylation sites.
  • the N-glycan is linked to the bifunctional degrader at one, two, three, or four N-glycosylation sites.
  • the N-glycan is linked to the bifunctional degrader at one N-glycosylation site.
  • the N-glycan is linked to the bifunctional degrader at two N- glycosylation sites.
  • the N-glycan is linked to the bifunctional degrader at three N-glycosylation sites.
  • the N-glycan is linked to the bifunctional degrader at four N-glycosylation sites.
  • the amino acid residue is Asn.
  • the N-glycosylation site comprises a consensus sequence of N-X-S/T or N-X-C, wherein X is any amino acid except proline.
  • the one or more N-glycosylation sites are distal to a target-specific binding location of the bifunctional degrader. In certain embodiments, at least one or at least two N-glycosylation sites are distal to the target-specific binding location. In certain embodiments, all of the N-glycosylation sites are distal to the target-specific binding location. In certain embodiments, the one or more N-glycosylation sites are not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader. In certain embodiments, the target-specific binding location is a variable region of an antibody or antigen-binding fragment (Fab), or an ectodomain of an Fc-fusion protein.
  • Fab antigen-binding fragment
  • one or more N-glycosylation sites present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader (“natural N- glycosylation sites”) have been deleted, mutated, or functionally inactivated.
  • at least one or at least two natural N-glycosylation sites have been deleted, mutated, or functionally inactivated.
  • all natural N-glycosylation sites have been deleted, mutated, or functionally inactivated.
  • the one or more natural N-glycosylation sites are located at or proximal to the target-specific binding location of the wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • the target-specific binding location is a variable region of an antibody or antigen-binding fragment (Fab), or an ectodomain of an Fc-fusion protein.
  • Fab antigen-binding fragment
  • one or more natural N-glycosylation sites have been deleted, mutated, or functionally inactivated and one or more glycoengineered N-glycosylation sites are present in the bifunctional degrader.
  • one or more glycoengineered N- glycosylation sites are located distal to a target-specific binding location of the bifunctional degrader.
  • the bifunctional degrader is an antibody or fragment thereof. In certain embodiments, the bifunctional degrader is a Fab fragment of an antibody. In certain embodiments, the glycoengineered bifunctional degrader 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 humanized, chimeric or fully human. In certain embodiments, the antibody has a glycan to protein ratio of 2 to 1, 4 to 1, 6 to 1, 8 to 1, or 10 to 1. In certain embodiments, the N-glycan is linked to an N-glycosylation site of the light chain of the antibody or fragment thereof.
  • the N-glycan is linked to an N-glycosylation site of the heavy chain of the antibody or fragment thereof.
  • one or more N-glycosylation sites are located in a constant domain of the antibody or fragment thereof.
  • one or more N-glycosylation sites are located in a variable domain of the antibody or fragment thereof.
  • one or more N-glycosylation sites are located on the Fab region of the antibody.
  • one or more N-glycosylation sites are located on the Fc region of the antibody.
  • one or more N- glycosylation sites are located in the hinge region of the antibody.
  • At least one of the N-glycosylation sites is not present in a wild-type form of the antibody.
  • at least two N-glycosylation sites of the Fab region of the antibody are glycosylated by the N-glycan.
  • one N-glycosylation site of the Fab region is located on each of the two heavy chain polypeptides of the antibody, and wherein each of said N-glycosylation sites is glycosylated by the N-glycan.
  • at least two N-glycosylation sites of the Fc region of the antibody are glycosylated by the N- glycan.
  • the Fab region contains more of the N-glycans than the Fc region. In certain embodiments, the Fab region contains two more of the N-glycans than the Fc region. In certain embodiments, the Fc region contains more of the N-glycans than the Fab region. In certain embodiments, the Fc region contains two or four more of the N- glycans than the Fab region. In certain embodiments, the Fc region and the Fab region contain the same number of the N-glycans. In certain embodiments, the glycoengineered bifunctional degrader binds to an autoantibody and comprises an autoantigen or immunogenic fragment thereof.
  • the glycoengineered bifunctional degrader comprises a moiety that specifically binds to the target protein, and wherein the target protein is associated with a disease.
  • the target protein is a cell surface molecule or a non-cell surface molecule.
  • the cell surface molecule is a receptor.
  • the non-cell surface molecule is an extracellular protein.
  • the extracellular protein is an autoantibody, a hormone, a cytokine, a chemokine, a blood protein, or a central nervous system (CNS) protein.
  • the target protein associated with a disease is upregulated in the disease compared to a non-disease state.
  • the target protein associated with a disease is expressed in the disease compared to a non-disease state. In certain embodiments, the target protein associated with a disease is involved in cancer progression. In certain embodiments, the target protein associated with disease comprises TNFa, HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, avp3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptors, EGF, c-MET, CCL2, CCR2, Frizzled receptors, Wnt, LRP5/6, CSF-1R, SIRPa, CD38, CD73, or TGF-p, Bombesin R, CAIX, CD 13, CD44, v6, Emmprin, Endoglin, EpCAM, EphA2, FAP-a, Folate R, GRP78, IGF-1R, Matriptase, Mesothel
  • the target protein associated with disease is involved in an autoimmune disease, and wherein the target protein is an antibody binding to TSHRa, MOG, AChR-al, noncollagen domain 1 of the a3 chain of type IV collagen (a3NCl), ADAMTS13, Desmoglein-1/3, or GPIb/IX, GPIIb/IIIa, GPIa/IIa, NMD A receptor, glutamic acid decarboxylase (GAD), amphiphysin, or gangliosides GM1, GD3 or GQ1B.
  • the disease comprises a cancer.
  • the disease comprises an autoimmune disease.
  • composition comprising a population of bifunctional degraders described herein, wherein the population of bifunctional degraders 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 the N-glycosylation site(s).
  • 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% homogenous at one of the N-glycosylation sites.
  • 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 site(s). In certain embodiments, the N-glycan profile is at least 60% homogeneous at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile is at least 70% homogenous at one or more of the N-glycosylation site(s).
  • the N-glycan profile is at least 80% homogeneous at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile is at least 90% homogeneous at one or more of the N-glycosylation site(s). In certain embodiments, the N- glycan profile is at least 95% homogenous at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile is at least 98% homogenous at one or more of the N-glycosylation site(s). In certain embodiments, the homogeneity of the N-glycan profile at one or more of the N-glycosylation sites is determined by N-glycan analysis, glycopeptide analysis or intact protein analysis.
  • 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
  • the N-glycan profile comprises about 90% to about 100% of the N-glycan at one N-glycosylation site.
  • 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 two N-glycosylation sites, respectively. In certain embodiments, the N-glycan profile comprises about 80% to about 90% of the N- glycan at two N-glycosylation sites, collectively. In certain embodiments, the N-glycan profile comprises about 90% to about 100% of the N-glycan at two N-glycosylation sites, collectively.
  • the N-glycan profile comprises about 90% to about 100% of the N-glycan at three or more of the N-glycosylation site(s), respectively. In certain embodiments, the N-glycan profile comprises about 70% to about 100% of the N-glycan at three or more of the N-glycosylation site(s), collectively. In certain embodiments, the relative amount of the N-glycan at one or more of the N-glycosylation sites in determined by N-glycan analysis or glycopeptide analysis.
  • the population of bifunctional degraders has an N-glycan profile comprising 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 an N-glycan of the structure: among all glycans in the N-glycan profile, wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the bifunctional degrader.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • the population of bifunctional degraders has an N-glycan profile comprising at least 30% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 40% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 50% of the N-glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising at least 60% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 70% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 80% of the N-glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising at least 90% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 95% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 98% of the N-glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising 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-glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising about 30% to about 40% of the N-glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising about 40% to about 50% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising about 50% to about 60% of the N-glycan among all glycans in the N- glycan profile. In certain embodiments, the population of bifunctional degraders has an N- glycan profile comprising about 60% to about 70% of the N-glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N- glycan profile comprising about 70% to about 80% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N- glycan profile comprising about 80% to about 90% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N- glycan profile comprising 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 determined by N-glycan analysis, glycopeptide analysis or intact protein analysis.
  • 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%. 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.
  • 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.
  • 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.
  • 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 determined by N-glycan analysis, glycopeptide analysis or intact protein analysis.
  • the bifunctional degrader in said population is expressed from one or more nucleic acid sequences in a Leishmania host cell.
  • glycan can refer to an N-glycan. Based on the specific structure, the skilled artisan would know if a specific glycan is an N-linked glycan.
  • a subject refers to an animal (e.g., birds, reptiles, and mammals).
  • a subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human).
  • a subject is a non-human animal.
  • a subject is a farm animal or pet (e.g., a dog, cat, horse, goat, sheep, pig, donkey, or chicken).
  • a subject is a human.
  • the terms “subject” and “patient” can be used herein interchangeably.
  • a[number]”, “a[number], [number]”, “Pfnumber]”, or “Pfnumber], [number]” refer to glycosidic bonds or glycosidic linkages which are covalent bonds that join a carbohydrate residue to another group.
  • An a-glycosidic bond is formed when both carbons have the same stereochemistry, whereas a P-glycosidic bond occurs when the two carbons have different stereochemistry.
  • glycoengineering means a process of glycosylating a target protein, or a target protein (e.g., bifunctional degrader) made by such process, wherein the process uses an in vivo host cell system that has one or more enzymes (e.g., pathways) that provides for glycosylation of the target protein.
  • a host cell system can be genetically engineered to introduce a glycosylation pathway to selectively glycosylate a target protein with a particular glycan structure.
  • a host cell used to generate a glycoengineered target protein can include, for example, a recombinant nucleic acid encoding a target protein; and a recombinant nucleic acid encoding a heterologous glycosyltransferase.
  • the host cell system used for glycoengineering (e.g., to generate a glycoengineered protein) can introduce N-linked glycosylation.
  • the host cell used for glycoengineering or to generate a glycoengineered target protein can be a mammalian cell, an insect cell, a yeast cell, a bacterial cell, a plant cell, a microalgae, or a protozoa.
  • the protozoa used for glycoengineering can be a species of Leishmania.
  • a glycoengineered binfunctional degrader also includes a bifunctional degrader that has been engineered to be selectively glycosylated at one or more specific sites when generated in the host cell system.
  • the term “glycosite” or “glycosylation site” refers to a site of glycosylation in a protein. Such a glycosite can be naturally present in the amino acid sequence of a protein or recombinantly engineered into the protein by addition or substitution or deletion of amino acids.
  • a glycosite is present in a so-called glycotag that is fused to a bifunctional degrader provided herein.
  • a glycotag is fused to a protein to create a bispecific binding protein.
  • a glycotag refers to a peptide containing consensus N-glycosylation site sequence fused to N- or a C- terminal or both termini of a protein or polypeptide.
  • the glycotag is fused to the C-terminus of the bifunctional degrader via a peptide linker.
  • the glycotag is fused to the N-terminus of the bifunctional degrader via a peptide linker.
  • the peptide linker is a consensus peptide sequence.
  • the consensus peptide sequence is 1, 2, 3, 4, 5, 6, 7 or more amino acid residues in length.
  • the bifunctional degrader provided herein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycotags.
  • a “glycoengineered bifunctional degrader” or “bifunctional degrader” is a polypeptide that mediates the degradation of a target protein by specifically binding to the target protein and engaging with endocytic receptors to activate degradation pathways.
  • a bifunctional degrader provided herein is “glycosylated” by an N-glycan if the N-glycan is linked to the bifunctional degrader at one or more site of the bifunctional degrader.
  • the N-glycan is linked to an N-glycosylation site of the bifunctional degrader.
  • an N-glycosylation site is “occupied” by an N-glycan if the N-glycan is linked to the N-glycosylation site.
  • N-glycosylation sites that are not present in a wild-type, natural, synthetic, and/or commercial precursor of the bifunctional degrader are referred to herein as “glycoengineered N-glycosylation sites”.
  • N-glycosylation sites that are not present in a wild-type precursor of the bifunctional degrader are referred to herein as “glycoengineered N-glycosylation sites”.
  • N-glycosylation sites that are not present in a natural precursor of the bifunctional degrader are referred to herein as “glycoengineered N- glycosylation sites”.
  • the term “natural” encompasses anything made in or derived from a biological system.
  • N-glycosylation sites that are not present in a synthetic precursor of the bifunctional degrader are referred to herein as “glycoengineered N-glycosylation sites”.
  • N-glycosylation sites that are not present in a commercial precursor of the bifunctional degrader are referred to herein as “glycoengineered N-glycosylation sites”.
  • the wild-type, natural, synthetic, and/or commercial precursor of the bifunctional degrader is selected from a wildtype, natural, synthetic, and/or commercial precursor of the degrader described in Section 7.1.
  • the wild-type, natural, synthetic, and/or commercial precursor of the bifunctional degrader is also referred to herein as an “unmuted form of the bifunctional degrader”.
  • the wild-type precursor of the bifunctional degrader is referred to herein as an “unmuted form of the bifunctional degrader”.
  • the natural precursor of the bifunctional degrader is referred to herein as an “unmuted form of the bifunctional degrader”.
  • the term “natural” encompasses anything made in or derived from a biological system.
  • the synthetic precursor of the bifunctional degrader is referred to herein as an “unmuted form of the bifunctional degrader”.
  • the commercial precursor of the bifunctional degrader is referred to herein as an “unmuted form of the bifunctional degrader”.
  • the wild-type, natural, synthetic, and/or commercial precursor of the bifunctional degrader is selected from a wild-type, natural, synthetic, and/or commercial precursor of the degrader described in Section 7.1.
  • one or more of the N-glycosylation sites that are present in a wild-type, natural, synthetic, and/or commercial precursor of the bifunctional degrader are referred to herein as “natural N-glycosylation sites”.
  • one or more of the N-glycosylation sites that are present in a wild-type precursor of the bifunctional degrader are referred to herein as “natural N-glycosylation sites”.
  • one or more of the N-glycosylation sites that are present in a natural precursor (i.e. anything made in or derived from a biological system) of the bifunctional degrader are referred to herein as “natural N-glycosylation sites”.
  • one or more of the N- glycosylation sites that are present in a synthetic precursor of the bifunctional degrader are referred to herein as “natural N-glycosylation sites”.
  • one or more of the N-glycosylation sites that are present in a commercial precursor of the bifunctional degrader are referred to herein as “natural N-glycosylation sites”.
  • the wild-type, natural, synthetic, and/or commercial precursor of the bifunctional degrader is selected from a wild-type, natural, synthetic, and/or commercial precursor of the degrader described in Section 7.1.
  • distal and proximal refer to the proximity in three- dimensional space of, for example, an N-glycan or N-glycosylation site to a specific region of the bifunctional degrader and, thus, relate to the quaternary structure of the bifunctional degrader as opposed to its primary amino acid sequence.
  • inflammatory disorder includes disorders, diseases or conditions characterized by inflammation.
  • inflammatory disorders include allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury and transplant rejection, among others.
  • blood disorder includes a disorders, diseases or conditions that affect blood.
  • blood disorders include anemia, bleeding disorders such as hemophilia, blood clots, and blood cancers such as leukemia, lymphoma, and myeloma, among others.
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed 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.
  • Mx number (x) of residues within the oligomannose series; Ax: number (x) of antennae; F: core fucose; Gx: number (x) of galactoses; S: number (x) of sialic acids.
  • Linkage information is given in () parentheses if applicable, e.g. A2G1S1(6) - a2-6 linked sialic acid.
  • Brackets [x] before G or GalNAc indicate which arm of the mannosyl core is galactosylated e.g. [3] G1 indicates that the galactose is on the antenna of the al-3 mannose.
  • FIG. 1 shows that an antibody displaying A2GalNAc2 glycan on Fab is highly internalized by HepG2 cells. HepG2 cells were incubated for 4 hours with pHrodo-labeled antibodies. The graph shows the average adjusted MFI of pHrodo of triplicate values ⁇ SEM. Graph shows data from one out of 3 representative experiments.
  • FIG. 2 shows that internalization of antibodies displaying A2GalNAc2 glycan on Fab by HepG2 cells is mediated by ASGPR.
  • HepG2 cells were incubated for 3 hours with pHrodo-labeled antibodies (3 pg/ml) and indicated inhibitors.
  • the graph shows the average and individual adjusted pHrodo MFI of 2 independent experiments.
  • Black circle No inhibitor.
  • Black triangle Fetuin.
  • Open square Asialofetuin.
  • Black diamond EGTA.
  • Open circle Chloroquine.
  • Open triangle Bafilomycin.
  • FIG. 3 shows that 50-fold higher concentration of Asialofetuin is required to block 50% of A-84.86-A2GalNAc2 uptake.
  • HepG2 cells were incubated with increasing concentrations (0.002- 4 pM) of asialofetuin for 30 min, before incubation with pHrodo- labelled antibodies.
  • the graph shows the data from 1 representative experiment out of 3.
  • FIGS. 4A-4C show that internalization of A-84.86-A2GalNAc2 is dependent on ASGPR1 and ASGPR2.
  • FIG. 4A shows flow cytometry staining for ASGPR1 and ASGPR2 on HepG2 treated with negative control siRNA or siRNA specific for ASGPR1.
  • FIG. 4B shows flow cytometry staining for ASGPR1 and ASGPR2 for HepG2 cells treated with negative control siRNA and ASGPR2 siRNA.
  • the graph shows the normalized expression to the negative control treated cells.
  • ASGPR2 siRNA leads to complete reduction of ASGPR2 surface level but not ASGPR1.
  • FIG. 4C shows that uptake of A-84.86-A2GalNAc2 antibody is completely abrogated by ASGPR1 and ASGPR2 siRNA while uptake of H-A2F (not an ASGPR engager) is unchanged.
  • the graph shows the average and individual data from 4 independent experiments (siRNA ASGPR1) or 1 experiment (siRNA ASGPR2). Ns: not statistically significant. ** statistically significant difference (p ⁇ 0.01) by two-way ANOVA followed by Tukey’s multiple comparisons test.
  • FIG. 5A shows the expression of ASGPR1, by flow cytometry normalized to isotype control staining in HepG2 (Sigma), HepG2 parental (HepG2-wt, Synthego) and HepG2-ASGPRlko (Synthego).
  • FIG. 5B shows the expression of ASGPR2 normalized to isotype control staining in same cells than in A.
  • FIG. 5C shows the DOL adjusted gMFI of the indicated pHRodo-labeled antibody incubated on same cells than in A & B.
  • FIG. 6A shows that internalized A-84.86-A2GalNAc2 antibody is colocalized with lysosomal vesicles in HepG2 cells.
  • the graph shows the number of pHrodo spots (DeepRed channel) colocalized with the lysosome ROI and normalized to cell number per well, plotted against time of incubation.
  • Lysosome ROI is defined by lysotracker green signal, as described in Example 5. Each data is an average of 3 wells and the errors bars show the standard deviation.
  • FIG. 6B shows the same readout than in 6A for a competitive inhibition experiment with unlabelled A-84.86-A2GalNAc2 titrated from lOOnM down to 0.160nM (some curves were removed for clarity) against InM of pHrodo A-84.86- A2GalNAc2.
  • FIG. 6C shows the same readout than in 6A for a competitive inhibition experiment of asialofetuin, titrated from lOOpM to 0.160pM, against InM of pHrodo A-
  • FIG. 7A shows Western blot images after HepG2 cells were incubated for indicated time with antibodies at 100 ug/ml. Western blot was done using anti-human IgG H+L antibody, detecting both heavy and light chain of adalimumab antibodies.
  • FIG. 7B shows signal quantification from the Western blot images. The graph shows the density of signal corresponding to internalized intact A-84.86-A2GalNAc2 (intact Heavy chain + intact Light chain indicated by the arrows on the right side of the blot at 50 KDa and 25 KDa; black bars) or the degradation fragment (white bars, indicated by the arrows in the blot). Density was normalized to the Beta-actin corresponding signal.
  • FIGS. 8A-8B show that the antigen is well internalized when complexed with A-
  • FIG. 8A shows western blot images for anti-lambda light chain blot (HCA202 detection) and Beta- Actin detection.
  • FIG. 8B shows the quantification of the lambda light chain (HCA202) signal, normalized to beta-actin corresponding signal, using iBright system
  • FIG. 9 shows that the target HCA202 is rapidly internalized by liver cells after GalNAc2 glycosylated antibody injection and that the internalized target is rapidly degraded following internalization.
  • HCA202 mAb H-A2F, PBS or A-84.86-A2GalNAc2.
  • Liver of animals were harvested at different timepoint and HCA202 was quantified in liver protein extract by western blot.
  • Panel A shows the western blot anti-Lambda light (HCA202) for 1 representative animal per time point.
  • Panel B shows the normalized Lamdba light chain band intensity of the western blot.
  • FIG. 10 shows the amount of antibody depleted from the supernatant after HepG2 cells were plated in a 12-well plate and treated with 5ng/ml of the indicated antibodies for 24, 48 and 72 hours.
  • the graph shows the average ⁇ SD for the amount of antibody depleted from the supernatant, from 3 independent experiments.
  • FIG. 11 shows that an A2GalNAc2 glycan displayed by a glycotag on the Fc (C- terminus of HC; 1 lK2-gtl) drives efficient ASGPR-specific internalization of an antibody.
  • a glycotag located on the LC (LCLgtl) was less efficient than a glycotag on the Fc.
  • the internalization is ASGPR-specific as ASGPRko HepG2 cells did not internalize the variants.
  • a variant displaying glycotags on C-terminus of HC and C-terminus of LC (11K2- LCLgtl.gtl-A2GalNAc2) showed a slightly better internalization than 1 lK2-gtl- A2GalNac2.
  • the variant 1 lK2-84.gtl-A2GalNAc2 with a glycotag on HC plus a glycosite inserted at position 84 in the variable domain of HC showed a higher internalization than the gtl variant.
  • FIG. 12 shows internalization of 11K2 glycovariants quantified by western blot. HepG2 were incubated with antibodies at O.lmg/ml. Western blot using anti human IgG H+L & Beta-Actin was performed on cell protein extracts. The graph shows the intensity of total antibody signal (heavy chain + light chain signal + degradation fragment signal) for each antibody, normalized to corresponding Beta- Actin signal.
  • FIG. 13 shows that glycotag on C-terminus of light chain or heavy chain on a mAb drives efficient clearance in vivo and that AlGalNAcl glycan is not active.
  • Mice were injected with different glycoengineered variants of 11K2 mAb. The levels of the mAb were measured in serum of the animals by ELISA.
  • IgG4-A2F is the non glycoengineered mAb produced in CHO cells.
  • the graphs show average ⁇ SEM of mAb concentration. The graphs show the data of 2 independent experiments.
  • FIG. 14 shows that a Fab fragment displaying A2GalNAc2 glycan leads to efficient internalization of its target antigen by HepG2 cells.
  • HepG2-wt and HepG2- ASGPRlko cells were incubated with 3pg/ml of pHrodo-BSA-Fentanyl x Fab-Fent complexes for 4h.
  • the graph shows the average ⁇ SEM of BSA-fentanyl pHrodo geometric MFI (gMFI) in the indicated samples.
  • N 3 **** P ⁇ 0.0001, two-way ANOVA followed by Tukey’s multiple comparisons test.
  • Control Fab-Fent-LCLgtl-A2 displaying a non ASGPR engaging glycan on a glycotag did not lead to significant internalization of BSA-fentanyl antigen.
  • Fab-Fent-LCLgtl-A2GalNAc2 which displays on its glycotag a single A2GalNAc2 glycan, induced a clear internalization of the antigen. This internalization is ASGPR dependent as it is abrogated in HepG2 knock out for ASGPR.
  • the variant Fab-Fent-86.LCLgtl-A2GalNAc2 induced an even stronger ASGPR-dependent internalization of the antigen, as compared to the LCLgtl variant.
  • FIG. 15 shows that the BSA-Fentanyl antigen, internalized by A2GalNAc2 displaying Fab anti-fentanyl glycovariants is rapidly degraded.
  • HepG2 cells were treated as described in example 9.
  • the Figure shows the western blot image for detection with the anti- fentanyl antibody (upper blot) and for beta-actin (lower blot) at indicated time post wash.
  • FIG. 16 shows that the MOG-Fc-A2GalNAc2 construct was efficiently internalized by HepG2 cells, to a similar extent than the A-84.86-A2GalNAc2 antibody.
  • the internalization was ASGPR-specific as it was blocked by competition with asialofetuin.
  • the graph shows the average DOL-adjusted pHrodo gMFI and individual data point from 2 independent experiments.
  • FIG. 17 shows that position the A2GalNAc2 N-Glycan is critical for degrader compounds that are based on the autoantigen to capture autoantibodies.
  • Panel A shows that engagement of the 8-18C5 antibody prevents internalization of MOG-Fc degrader but not of variants containing a glycotag at C-term of the Fc.
  • HepG2 cells were incubated with MOG bifunctional degraders in complex with 8-18C5-pHrodo.
  • the graph shows the pHrodo signal detected in the cells after incubation.
  • Panel B shows the model of ASGPR engagement of endogenous glycan in presence of model autoantibody binding to MOG bifunctional degrader.
  • the model autoantibody is represented by the hashed structure.
  • N-glycans are indicated by the stars.
  • ASGPR cannot engage the endogenous glycan when the autoantibody is bound to bifunctional degrader.
  • Fc glycotag gtl
  • MOG bifunctional degrader is bound to the autoantibody.
  • FIG. 18 shows that MOG-Fc-N60Q-gtl-A2GalNAc2 bifunctional degrader efficiently depletes a model autoantibody injected in rat.
  • FIG. 19 shows that antibodies displaying A2GalNAc2 Glycan structure lead to potent elimination of a target antigen from blood circulation in rat. Rats were injected i.v.
  • HCA202 0.5 mg/kg
  • Antibodies i.v. 5 mg/kg Graph shows average ⁇ SD of HCA202 serum concentration in ng/ml of 3 or 4 animal /group.
  • Black circles show H-A2F (adalimumab, Humira) treated group.
  • Open squares show A-84-A2GalNAc2 treated group.
  • Black triangles show A-84.86-A2GalNAc2 treated group.
  • Black square show A-84.86- A2G2S2 treated group.
  • Open circles show PBS treated group (HCA202 only).
  • Open diamonds and dotted line show A-M3 treated group.
  • FIG. 20 shows that an antibody displaying A2GalNAc2 Glycan structure is distributed to the liver area with a fast kinetic as compared to control antibodies.
  • Mice were injected i.v. with CF750-labeled antibodies at 5 mg/kg and imaged using fluorescence tomography.
  • the graph shows the average fluorescence in pmol ⁇ SD of 3 animals / timepoint in the gated liver region of interest.
  • Open Squares and dotted line show Ptz-A2F treated group. Black Circles show H-A2F treated group. Open diamonds show A-84.86- A2G2S2 treated group. Black triangles show A-84.86-A2G2 treated group.
  • FIG. 21 shows that antibodies distributed to the liver are degraded.
  • Livers from mice injected intravenously (i.v.) with CF750-labelled antibodies at 5 mg/kg were harvested at indicated time points & protein extracts were obtained from -100 mg liver homogenized in RIPA buffer (+protease inhibitor). Fluorescence captured in the CF750 channel was quantified using iBright system. The extracts were also blotted for beta-Actin. Blot shows the CF750 fluorescence signal.
  • FIG. 22 shows the relative densitometric unit of the CF750 signal from the gel in FIG. 21 normalized to Beta-Actin control intensity.
  • FIG. 23 shows that injection by subcutaneous route (s.c.) prolongs the effect of antigen depletion from blood circulation as compared to intravenous (i.v.) route.
  • Rats were injected i.v with HCA202 (0.5 mg/kg) and 15 min later with PBS i.v. A-84.86-A2GalNAc2 i.v or s.c at 5 mg/kg.
  • Graph shows average ⁇ SD of total (free + bound) HCA202 serum concentration in ng/ml of 3 animal /group.
  • Rats were injected i.v. with HCA202 (0.5 mg/kg) and 15 min later with PBS s.c. or A-84.86-A2GalNAc2 s.c. at 10 mg/kg.
  • the graph shows average ⁇ SD total (free + bound) HCA202 serum levels.
  • the assay LLOQ is indicated by the dotted line (20 ng/ml).
  • FIG. 25 shows that a single injection of A-84.86-A2GalNAc2 by s.c route can lead to antigen depletion for up to 96 hours.
  • Rats were injected i.v. with HCA202 at timepoints indicated by the arrows.
  • Fifteen minutes after first HCA202 injection rats were injected with PBS s.c. or A-84.86-A2GalNAc2 s.c.
  • the graph shows average ⁇ SEM total (free + bound) HCA202 serum levels.
  • N 4 animal / group.
  • the assay LLOQ is indicated by the dotted line.
  • FIG. 26 shows that when HepG2 knock out for ASGPR1 (HepG2-ASGRlko) are treated with Ptz-gtl-A2GalNAc2 or Ptz-hgt-A2GalNAc2 variants, no HER2 reduction is observed, as compared to isotype control or Ptz-A2F treated cells.
  • the graph shows Her2 detection in HepG2 parental (-wt) or HepG2 knock out for ASGPR1 (-ASGRlko) treated with Ipg/ml of the indicated antibody for 4 hours.
  • FIG 27 shows that HER2-Fc/trastuzumab Immune complexes (ICs) form predominantly 600-1300 KDa structures.
  • Panel A shows the overlay of size-exclusion chromatogram of individual injections as well as pre-formed immune complexes of Trastuzumab and HER2-Fc in a molar ratio of 1 : 1. Sizes of formed ICs was estimated via protein MW standards as well as dynamic light scattering measurements.
  • Panel B shows the structure of the ICs formed and that Ptz-gtl antibody can bind to the formed ICs.
  • FIG. 28 shows that large immune complexes can be internalized and degraded by a mAb bifunctional degrader.
  • Panel A shows the anti-human IgG H+L western blot and antibeta actin western blot of HepG2 cell extracts. The position of heavy (H) and light chains (L) of the different components is indicated in the gel. Trtz is trastuzumab. Ptz-gtl heavy chain has a higher molecular weight than trastuzumab HC because of the glycotag addition.
  • FIG. 29 shows that HCA202 target depletion by A-gtl-A2GalNAc2 follows a dose response and that depletion requires low degrader (A-gtl) to target (HCA202) ratio.
  • Rats were injected i.v. with HCA202 followed by PBS or A-gtl -A2GalNAc2 i.v. at different doses.
  • the numbers above the graph indicate the molar degradertarget ratio.
  • FIG. 30 shows that target depletion by Fab-A-FLGT4 follows a dose response and that depletion requires low degrader (Fab-A-FLGT4) to target (HCA202) ratio.
  • the numbers above the graph indicate the molar degrader (target ratio.
  • the graph shows average ⁇ SEM of HCA202 depletion from theoretical Czero .
  • glycoengineered bifunctional degraders and populations comprising the same having improved functionalities as compared to a control antibody.
  • the glycoengineered bifunctional degrader is engineered by introduction of glycosylation sites on the glycoengineered bifunctional degrader, resulting in an engineered glycosylation profile that mediates endocytic receptor degradation of the glycoengineered bifunctional degrader and the target to which it binds.
  • the glycoengineered bifunctional degraders described herein 1) have homogeneous glycosylation; 2) can degrade large targets such as immune complexes; 3) have a defined ligand-to-antibody ratio; 4) have defined glycosylation sites; 6) can activate more diverse and powerful degradation receptors; and/or 6) can engage in protein degradation in a highly optimized manner.
  • the glycoengineered bifunctional degrader may be employed as novel therapeutics to treat diseases, which include but are not limited to inflammatory disorders, blood disorders, autoimmune disorders, infectious diseases, and cancer.
  • a glycoengineered bifunctional degrader wherein the bifunctional degrader (i) specifically binds to a target protein and (ii) comprises an N-glycan of the structure: linked to the bifunctional degrader at one or more N-glycosylation sites, wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N- acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the bifunctional degrader.
  • the black square represents an N-acetyl galactosamine (GalNAc)
  • the white square represents an N- acetylglucosamine (GlcNAc) residue
  • the black circle represents a mannose (Man) residue
  • X represents an amino acid residue of the bifunctional degrader.
  • the glycoengineered bifunctional degrader specifically binds to one or more target proteins, for example, but not limited to target proteins described in Section 7.7. In certain embodiments, the glycoengineered bifunctional degrader specifically binds to one target protein. In certain embodiments, the glycoengineered bifunctional degrader comprises a moiety that specifically binds to a target protein. In certain embodiments, the moiety comprises a heavy chain variable region and a light chain variable region of an antibody, or a functional fragment thereof. In certain embodiments, the moiety comprises a Fab region of a monoclonal antibody.
  • the glycoengineered bifunctional degrader comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties that each specifically bind to a target protein so that a single bifunctional degrader can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target protein molecules.
  • each of the moieties specifically binds to the same target protein.
  • two, three, four or more of the moieties bind to different target proteins.
  • the bifunctional degrader specifically binds to two different target proteins.
  • the bifunctional degrader comprises a first moiety that specifically binds to a first target protein and a second moiety that specifically binds to a second target protein.
  • the glycoengineered bifunctional degrader has: (i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more first moieties that specifically bind to a first target protein so that a single bifunctional degrader can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the 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 so that a single bifunctional degrader can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the second target protein molecules.
  • the bifunctional degrader specifically binds to three different target proteins.
  • the bifunctional degrader 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.
  • the glycoengineered bifunctional degrader has (i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more first moieties that specifically bind to a first target protein so that a single bifunctional degrader can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the 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 so that a single bifunctional degrader can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the 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 so that a single bifunctional degrader can bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the third target protein molecules.
  • the glycoengineered bifunctional degrader described herein activates natural degradation pathways.
  • the natural degradation pathways comprise receptor-mediated endocytosis.
  • receptor-mediated endocytosis comprises capture of glycosylated proteins via specific glycan structures (for example, by endocytic lectins) to mediate lysosomal degradation (Cummings et al., Cold Spring Harbor Laboratory Press, (2017). Endocytic lectins are ubiquitous in humans and can recognize various glycan structures.
  • Glycan engagement with endocytic carbohydrate-binding proteins and receptors enables essential biological pathways including, but not limited to, those involved in modulating immune responses, mediating protein clearance, protein turnover, and controlling trafficking of soluble glycoproteins, glycolipids and any natural molecule containing a glycan moiety.
  • activation of the natural degradation pathways by the glycoengineered bifunctional degrader reduces the concentration of a target protein in a subject.
  • activation of the natural degradation pathways by the glycoengineered bifunctional degrader reduces the concentration of a target protein in a 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 about 100% as compared to the concentration in the subject before administration of the glycoengineered bifunctional degrader.
  • the glycoengineered bifunctional degrader mediates endocytic receptor degradation of the glycoengineered bifunctional degrader and the target protein to which it binds.
  • the glycoengineered bifunctional degrader comprises an N-glycan of the structure:
  • the black square represents an N-acetyl galactosamine (GalNAc)
  • the white square represents an N- acetylglucosamine (GlcNAc) residue
  • the black circle represents a mannose (Man) residue
  • X represents an amino acid residue of the bifunctional degrader
  • the N- glycan specifically binds to one or more endocytic receptors that mediate lysosomal degradation.
  • the endocytic receptors are endocytic carbohydrate- binding proteins and/or lectin receptors.
  • the endocytic carbohydrate- binding protein is ASGPR.
  • the N-glycan specifically binds to ASGPR.
  • ASGPR-mediated degradation in the hepatocyte has many applications. ASPGR binding to the N-glycan structure disclosed herein can result in the selective degradation of one or more target protein described in Section 7.7.
  • ASGPR-mediated degradation can lead to removal of cytokines, chemokines and hormones.
  • ASGPR-mediated degradation can be used for the delivery of the target molecules to the hepatocyte endosome.
  • ASGPR-mediated degradation is applicable for various liver diseases, while limiting systemic toxicity.
  • the bifunctional degrader comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites (or glycosites; such as an N-glycosylation consensus sequence). In certain embodiments, the bifunctional degrader comprises 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 degrader comprises at least 2 N-glycosylation sites. In certain embodiments, the bifunctional degrader comprises at least 3 N-glycosylation sites. In certain embodiments, the bifunctional degrader comprises at least 4 N-glycosylation sites.
  • the bifunctional degrader comprises at least 5 N-glycosylation sites. In certain embodiments, the bifunctional degrader comprises at least 6 N-glycosylation sites. In certain embodiments, the bifunctional degrader comprises at least 7 N-glycosylation sites. In certain embodiments, the bifunctional degrader comprises at least 8 N-glycosylation sites. In certain embodiments, the bifunctional degrader comprises at least 9 N-glycosylation sites. In certain embodiments, the bifunctional degrader comprises at least 10 N- glycosylation sites. In certain embodiments, the bifunctional degrader comprises 2 N- glycosylation sites. In certain embodiments, the bifunctional degrader comprises 3 N- glycosylation sites.
  • the bifunctional degrader comprises 4 N- glycosylation sites. In certain embodiments, the bifunctional degrader comprises 5 N- glycosylation sites. In certain embodiments, the bifunctional degrader comprises 6 N- glycosylation sites. In certain embodiments, the bifunctional degrader comprises 7 N- glycosylation sites. In certain embodiments, the bifunctional degrader comprises 8 N- glycosylation sites. In certain embodiments, the bifunctional degrader comprises 9 N- glycosylation sites. In certain embodiments, the bifunctional degrader comprises 10 N- glycosylation sites.
  • one or more of the N-glycosylation sites are engineered into the amino acid sequence of the bifunctional degrader (i.e. one or more of the N- glycosylation sites are not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader; such sites are also referred to herein as “glycoengineered N- glycosylation sites”).
  • at least one of the N-glycosylation sites is engineered into the amino acid sequence of the bifunctional degrader.
  • at least two of the N-glycosylation sites are engineered into the amino acid sequence of the bifunctional degrader.
  • At least three of the N- glycosylation sites are engineered into the amino acid sequence of the bifunctional degrader. In certain embodiments, at least four of the N-glycosylation sites are engineered into the amino acid sequence of the bifunctional degrader. In certain embodiments, one or more of the N-glycosylation sites are engineered distal to a target-specific binding location of the bifunctional degrader. In certain embodiments, at least one or at least two N-glycosylation sites are engineered distal to the target-specific binding location. In certain embodiments, wherein all of the N-glycosylation sites are engineered distal to the target-specific binding location.
  • the target-specific binding location is a variable region of an antibody or antigen-binding fragment (Fab), or an ectodomain of an Fc-fusion protein.
  • one or more of the engineered N-glycosylation sites are glycotags fused to the N- and/or C-terminus of the the bifunctional degrader via a peptide linker.
  • a glycotag is fused to the N-terminus of the bifunctional degrader.
  • a glycotag is fused to the C-terminus of the bifunctional degrader.
  • a glycotag is fused to the N- and the C-terminus of the bifunctional degrader.
  • one or more of the N-glycosylation sites are natural N-glycosylation sites (i.e. one or more of the N-glycosylation sites are present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader).
  • at least one of the N-glycosylation sites is a natural N-glycosylation site.
  • at least two of the N-glycosylation sites are natural N-glycosylation sites.
  • one or more natural N-glycosylation sites have been deleted, mutated, or functionally inactivated.
  • at least one or at least two natural N- glycosylations sites have been deleted, mutated, or functionally inactivated.
  • all natural N-glycosylation sites have been deleted, mutated, or functionally inactivated.
  • the one or more natural N-glycosylation sites are located at or proximal to the target-specific binding location of the wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • one or more natural N-glycosylation sites have been deleted, mutated, or functionally inactivated and one or more glycoengineered N-glycosylation sites are present in the bifunctional degrader.
  • one or more glycoengineered N-glycosylation sites are located distal to a target-specific binding location of the bifunctional degrader.
  • the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites can be glycosylated by the N-glycan such that the resulting glycoengineered bifunctional degrader can engage with or bind to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endocytic receptor molecules.
  • the bifunctional degrader is glycosylated by the N- glycan 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.
  • the bifunctional degrader is glycosylated by the N-glycan at least 2 N-glycosylation sites.
  • the bifunctional degrader is glycosylated by the N-glycan at least 3 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at least 4 N- glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at least 5 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at least 6 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at least 7 N-glycosylation sites.
  • the bifunctional degrader is glycosylated by the N-glycan at least 8 N- glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at least 9 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at least 10 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at 2 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at 3 N-glycosylation sites.
  • the bifunctional degrader is glycosylated by the N-glycan at 4 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at 5 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at 6 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at 7 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at 8 N-glycosylation sites.
  • the bifunctional degrader is glycosylated by the N-glycan at 9 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated by the N-glycan at 10 N-glycosylation sites. In certain embodiments, the bifunctional degrader is glycosylated at an Asn amino acid residue of the bifunctional degrader. In certain embodiments, the N-glycosylation site is an N-glycosylation consensus sequence. In certain embodiments, the N-glycosylation site comprises a consensus sequence of N-X-S/T or N-X- C, wherein X is any amino acid except proline.
  • 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 an N-glycan.
  • 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 an N- glycan of the structure: linked to the bifunctional degrader at one or more N-glycosylation sites, wherein the black square represents an N-acetyl galactosamine (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 degrader.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • X represents an amino acid residue of the bifunctional degrader.
  • At least 10% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 20% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 30% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 40% of the N-glycosylation sites are occupied by the N- glycan. In certain embodiments, at least 50% of the N-glycosylation sites are occupied by the N-glycan. In certain embodiments, at least 60% of the N-glycosylation sites are occupied by the N-glycan.
  • 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.
  • the N-glycan is linked to the bifunctional degrader at least one N-glycosylation site. In certain embodiments, the N-glycan is linked to the bifunctional degrader at least two N-glycosylation sites. In certain embodiments, the N- glycan is linked to the bifunctional degrader at one, two, three, or four N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degrader at one N- glycosylation site. In certain embodiments, the N-glycan is linked to the bifunctional degrader at two N-glycosylation sites.
  • the N-glycan is linked to the bifunctional degrader at three N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degrader at four N-glycosylation sites. In certain embodiments, the N-glycan is linked to the bifunctional degrader at an Asn amino acid residue of the bifunctional degrader. In certain embodiments, the N-glycan is linked to the bifunctional degrader at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the bifunctional degrader at a consensus sequence of N-X-S/T or N-X-C, wherein X is any amino acid except proline.
  • the one or more N-glycosylation sites are distal to a target-specific binding location of the bifunctional degrader. In certain embodiments, at least one, at least two, at least three, or at least four N-glycosylation sites are distal to the targetspecific binding location. In certain embodiments, at least one N-glycosylation site is distal to the target-specific binding location. In certain embodiments, at least two N-glycosylation sites are distal to the target-specific binding location. In certain embodiments, at least three N-glycosylation sites are distal to the target-specific binding location. In certain embodiments, at least four N-glycosylation sites are distal to the target-specific binding location.
  • one N-glycosylation site is distal to the target-specific binding location. In certain embodiments, two N-glycosylation sites are distal to the target- specific binding location. In certain embodiments, three N-glycosylation sites are distal to the target-specific binding location. In certain embodiments, four N-glycosylation sites are distal to the target-specific binding location. In certain embodiments, all of the N- glycosylation sites are distal to the target-specific binding location. In certain embodiments, the one or more N-glycosylation sites are not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • At least one, at least two, at least three, or at least four of the N-glycosylation sites are not present in a wildtype, natural, synthetic, or commercial precursor of the bifunctional degrader. In certain embodiments, at least one of the N-glycosylation sites is not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader. In certain embodiments, at least two of the N-glycosylation sites are not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader. In certain embodiments, at least three of the N-glycosylation sites are not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • At least four of the N- glycosylation sites are not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • one of the N-glycosylation sites is not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • two of the N-glycosylation sites are not present in a wildtype, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • three of the N-glycosylation sites are not present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • the targetspecific binding location is a variable region of an antibody or antigen-binding fragment (Fab), or an ectodomain of an Fc-fusion protein.
  • 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.
  • the distal N-glycosylation site(s) and the target-specific binding location 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, abour 100-150, about 150-200, about 200-300, or about 300-400 amino acids.
  • the amino acid separation between the distal N-glycosylation site(s) and the target-specific binding location is the number of amino acids between the terminal amino acids of the N- glycosylation consensus sequence.
  • the bifunctional degrader folds in space and, thus, has a three-dimensional geometry in addition to its primary amino acid structure. Also without being bound by theory, this three-dimensional geometry, including the position of the N-glycan is not static but dynamic (see, for example, Re, S., et al Biophysical Reviews, 4, 179-187 (2012)).
  • the distance between the distal N-glycosylation site(s) and the target-specific binding location on a bifunctional degrader may be from an equilibrium geometry of the bifunctional degrader, as determined by any standard means known in the art, including for example computational modelling studies.
  • the distal N-glycosylation site(s) and the targetspecific binding location 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.
  • 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 n
  • the distance between the distal N- glycosylation site(s) and the target-specific binding location is chosen to minimize steric hinderance, for example between the bifunctional degrader(s), the target protein(s), and/or the ASGPR receptor(s), when the target protein is bound to the bifunctional degrader.
  • the N-glycan is linked to the bifunctional degrader at an Asn amino acid residue of the bifunctional degrader.
  • the N-glycan is linked to the bifunctional degrader at an N-glycosylation consensus sequence.
  • the N-glycan is linked to the bifunctional degrader at a consensus sequence of N-X-S/T or N-X- C, wherein X is any amino acid except proline.
  • one or more N-glycosylation sites present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader (“natural N- glycosylation sites”) have been deleted, mutated, or functionally inactivated.
  • at least one, at least two, at least three, or at least four natural N-glycosylation sites have been deleted, mutated, or functionally inactivated.
  • at least one natural N-glycosylation site has been deleted, mutated, or functionally inactivated.
  • at least two natural N-glycosylation sites have been deleted, mutated, or functionally inactivated.
  • At least three natural N-glycosylation sites have been deleted, mutated, or functionally inactivated. In certain embodiments, at least four natural N-glycosylation sites have been deleted, mutated, or functionally inactivated. In certain embodiments, one natural N-glycosylation site has been deleted, mutated, or functionally inactivated. In certain embodiments, two natural N-glycosylation sites have been deleted, mutated, or functionally inactivated. In certain embodiments, three natural N- glycosylation sites have been deleted, mutated, or functionally inactivated. In certain embodiments, four natural N-glycosylation sites have been deleted, mutated, or functionally inactivated.
  • all natural N-glycosylation sites have been deleted, mutated, or functionally inactivated.
  • the one or more natural N- glycosylation sites are located at or proximal to the target-specific binding location of the wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • the target-specific binding location is a variable region of an antibody or antigen-binding fragment (Fab), or an ectodomain of an Fc-fusion protein.
  • the glycoengineered bifunctional degrader comprises two different N-glycans (i.e. a first and a second N-glycan), wherein each N-glycan is independently linked to the bifunctional degrader 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: wherein the black square represents an N-acetyl galactosamine (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 degrader.
  • N-glycans i.e. a first and a second N-glycan
  • the different N-glycans specifically bind to different endocytic receptors.
  • the first N-glycan specifically binds to ASGPR.
  • the other N-glycan i.e. the second N-glycan
  • the other N-glycan is an N-glycan depicted in Section 5.3.
  • the other N-glycan is an N-glycan described in PCT/EP2022/057556, which is incorporated herein by reference in its entirety.
  • 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.
  • the N- glycosylation sites predominantly or exclusively occupied by the larger N-glycan are more sterically accessible than the N-glycosylation sites predominantly or exclusively occupied by the smaller N-glycan.
  • the other N-glycan is A2.
  • the other N-glycan is AlGalNAcl or A2GalNAcl.
  • the N-glycans are linked to the bifunctional degrader at an Asn amino acid residue of the bifunctional degrader.
  • the N-glycans are linked to the bifunctional degrader at an N-glycosylation consensus sequence.
  • the N-glycans are linked to the bifunctional degrader at a consensus sequence of N-X-S/T or N-X-C, wherein X is any amino acid except proline.
  • the first N-glycan is linked to the bifunctional degrader at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites
  • the second N-glycan is linked to the bifunctional degrader at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites.
  • the bifunctional degrader further comprises a third N- glycan, wherein the third N-glycan is linked to the bifunctional degrader at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites.
  • the third N-glycan specifically binds to a different endocytic receptor than the first and/or second N-glycan.
  • the third N-glycan is an N-glycan depicted in Section 5.3.
  • the third N-glycan is an N-glycan described in PCT/EP2022/057556, which is incorporated herein by reference in its entirety.
  • the third N-glycan is A2.
  • the third N-glycan is AlGalNAcl or A2GalNAcl. In certain embodiments, the third N-glycan is linked to the bifunctional degrader at an Asn amino acid residue of the bifunctional degrader. In certain embodiments, the third N-glycan is linked to the bifunctional degrader at an N-glycosylation consensus sequence. In certain embodiments, the third N-glycan is linked to the bifunctional degrader at a consensus sequence of N-X-S/T or N-X-C, wherein X is any amino acid except proline.
  • the second and/or third N-glycan specifically bind to an endocytic lectin.
  • the endocytic lectin is a mannose binding receptor.
  • the endocytic lectin is a Cluster of Differentiation 206 (CD206) receptor.
  • the endocytic lectin is a DC-SIGN (Cluster of Differentiation 209 or CD209) receptor.
  • the endocytic lectin is a C- Type Lectin Domain Family 4 Member G (LSECTin) receptor.
  • the endocytic lectin is a macrophage inducible Ca 2+ -dependent lectin receptor (Mincle).
  • the endocytic receptor is L-SIGN CD209L.
  • the endocytic receptor is asialoglycoprotein (ASGPR).
  • the endocytic receptor is dectin-1.
  • the endocytic receptor is dectin-2.
  • the endocytic receptor is langerin.
  • the second and/or third N-glycan 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).
  • 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).
  • CD206 is a C-type lectin and phagocytic/endocytic recycling and signaling receptor. CD206 is expressed primarily by M2 anti-inflammatory macrophages, dendritic cells, and live sinusoidal endothelial cells.
  • DC-SIGN is a non-recycling, signaling receptor that targets both the ligand and receptor to the lysosome for degradation.
  • LSECTin is expressed on liver sinusoidal endothelial cells.
  • the glycoengineered bifunctional degrader is glycosylated at two or more N-glycosylation sites by an N-glycan of the structure: wherein the black square represents an N-acetyl galactosamine (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 degrader, and wherein 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.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • X represents an amino acid residue of the bifunctional degrader
  • the N-glycan is linked to the bifunctional degrader 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, abour 100-150, about 150-200, or about 200-300 amino acids.
  • 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.
  • the bifunctional degrader folds in space and, thus, has a three-dimensional geometry in addition to its primary amino acid structure.
  • this three-dimensional geometry, including the position of the N-glycan is not static but dynamic (see, for example, Re, S., et al Biophysical Reviews, 4, 179-187 (2012)).
  • the distance between N-glycosylation sites and/or N-glycans on a bifunctional degrader may be from an equilibrium geometry of the bifunctional degrader, as determined by any standard means known in the art, including for example computational modelling studies.
  • the N-glycan is linked to the bifunctional degrader at two N-glycosylation sites separated by a distance of at least 1.0 nm.
  • the N-glycan is linked to the bifunctional degrader at two N- glycosylation sites separated by a distance of about 1.0-5.0 nm. In certain embodiments, the N-glycan is linked to the bifunctional degrader at two N-glycosylation sites separated by a distance of about 1.5-3.0 nm. In certain embodiments, the N-glycan is linked to the bifunctional degrader at two N-glycosylation sites separated by a distance of about 1.5-2.5 nm. In certain embodiments, the N-glycan is linked to the bifunctional degrader at three N- glycosylation sites each separated by a distance of about 1.0-5.0 nm.
  • the N-glycan is linked to the bifunctional degrader at three N-glycosylation sites each separated by a distance of about 1.5-3.0 nm. In certain embodiments, the N-glycan is linked to the bifunctional degrader at three N-glycosylation sites each separated by a distance of about 1.5-2.5 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.
  • the distance between the N-glycosylation sites and/or N-glycans is chosen to minimize steric hinderance, for example between the bifunctional degrader(s), the target protein(s), and/or the ASGPR receptor(s). In certain embodiments, the distance between the N-glycosylation sites and/or N-glycans is chosen based on the separation of ASGPR receptors on a cell surface. In certain embodiments, the distance between the N-glycosylation sites and/or N-glycans is chosen to be similar (e.g. no more than twice, or no less than half) to the separation of ASGPR receptors on a cell surface.
  • the N-glycan is linked to the bifunctional degrader at an Asn amino acid residue of the bifunctional degrader. In certain embodiments, the N-glycan is linked to the bifunctional degrader at an N-glycosylation consensus sequence. In certain embodiments, the N-glycan is linked to the bifunctional degrader at a consensus sequence of N-X-S/T or N- X-C, wherein X is any amino acid except proline.
  • the bifunctional degrader 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.
  • the bifunctional degrader is a dimer comprising two polypeptide chains that have 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 degrader comprises four polypeptide chains. In certain embodiments, two of the four polypeptides 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 polypeptides chains are identical to each other.
  • the bifunctional degrader is an antibody or fragment thereof. In other embodiments, the antibody is a full length antibody, an Fab, an F(ab’)2, an Scfv, or a sdAb. In certain embodiments, the bifunctional degrader is a Fab, scFv, Fc, Fv fragment of an antibody. In certain embodiments, the bifunctional degrader 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 humanized, chimeric or fully human. In certain embodiments, the bifunctional degrader is an autoantigen. In certain embodiments, the bifunctional degrader is an autoantibody. In certain embodiments, the glycoengineered bifunctional degrader is a nanobody.
  • the glycoengineered bifunctional degrader is an antibody.
  • the antibody has the amino acid sequence of adalimumab (Humira® (Abb Vie 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 Biopharma Inc.) (Ibritumomab tiux
  • the bifunctional degrader is an antibody or fragment thereof, wherein the antibody comprises one or more N-glycosylation sites glycosylated by an N-glycan of structure: wherein the black square represents an N-acetyl galactosamine (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 antibody.
  • the N-glycan is linked to an N-glycosylation site of the light chain of the antibody or fragment thereof.
  • the N-glycan is linked to an N- glycosylation site of the heavy chain of the antibody or fragment thereof.
  • one or more of the N-glycosylation sites are located in a constant domain of the antibody or fragment thereof. In certain embodiments, one or more of the N- glycosylation sites are located in a 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 degrader at an Asn amino acid residue of the antibody.
  • 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 a consensus sequence of N-X-S/T or N-X-C, wherein X is any amino acid except proline. In certain embodiments, at least one of the N-glycosylation sites is not present in a wild-type form of the antibody. In certain embodiments, all but two of the N-glycosylation sites are not present in a wild-type form of the antibody.
  • 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, 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.
  • N- glycosylation sites in the Fab region of the antibody are glycosylated by an N-glycan of structure: wherein the black square represents an N-acetyl galactosamine (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 antibody.
  • at least two N-glycosylation sites of the Fab region of the antibody are glycosylated by the N-glycan.
  • two N-glycosylation sites of the Fab region of the antibody are glycosylated by the N-glycan.
  • one N- glycosylation site of the Fab region is located on each of the two heavy chain polypeptides of the antibody and each of said N-glycosylation sites is glycosylated by the N-glycan.
  • four N-glycosylation sites of the Fab region of the antibody are glycosylated by the N-glycan.
  • six N-glycosylation sites of the Fab region of the antibody are glycosylated by the N-glycan.
  • 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, 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.
  • N- glycosylation sites in the Fc region of the antibody are glycosylated by an N-glycan of structure: wherein the black square represents an N-acetyl galactosamine (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 antibody.
  • at least two N-glycosylation sites of the Fc region of the antibody are glycosylated by the N-glycan.
  • two N-glycosylation sites of the Fc region of the antibody are glycosylated by the N-glycan.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fc region of the antibody are glycosylated by an N-glycan depicted in Section 5.3.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fc region of the antibody are glycosylated by an N-glycan described in PCT/EP2022/057556, which is incorporated herein by reference in its entirety.
  • at least two N- glycosylation sites of the Fc region of the antibody are glycosylated by an N-glycan having the structure of A2.
  • two N-glycosylation sites of the Fc region of the antibody are glycosylated by an N-glycan having the structure of A2.
  • four N-glycosylation sites of the Fc region of the antibody are glycosylated by an N-glycan having the structure of A2.
  • 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 of:
  • the black square represents an N-acetyl galactosamine (GalNAc)
  • the white square represents an N-acetylglucosamine (GlcNAc) residue
  • the black circle represents a mannose (Man) residue
  • X represents an amino acid residue of the antibody.
  • at least two N-glycosylation sites of the Fc region of the antibody are glycosylated by the first N-glycan.
  • two N-glycosylation sites of the Fc region of the antibody are glycosylated by the first N-glycan.
  • at least two N-glycosylation sites of the Fc region of the antibody are glycosylated by the second N-glycan.
  • two N-glycosylation sites of the Fc region of the antibody are glycosylated by the second N-glycan.
  • the different N- glycans specifically bind to different endocytic receptors.
  • the first N-glycan specifically binds to ASGPR.
  • the second N-glycan is an N-glycan depicted in Section 5.3.
  • the second N-glycan is an N- glycan described in PCT/EP2022/057556, each of which is incorporated herein by reference in its entirety.
  • the first N-glycan is larger than the second N-glycan.
  • the first N-glycan is smaller than the second N-glycan.
  • the N-glycosylation sites predominantly or exclusively occupied by the larger N-glycan are more sterically accessible than the N-glycosylation sites predominantly or exclusively occupied by the smaller N-glycan.
  • the other N-glycan is A2.
  • the other N-glycan is AlGalNAcl or A2GalNAcl.
  • only the Fc region and/or the hinge region of the antibody has one or more N-glycosylation sites.
  • the Fab region and the Fc region of the antibody each independently have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N- glycosylation sites.
  • the Fab region and the Fc region of the antibody each independently have 2, 4, or 6 N-glycosylation sites.
  • the Fab region contains more N-glycosylation sites than the Fc region.
  • the Fab region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more N-glycosylation sites than the Fc region.
  • the Fab region contains 2 more N-glycosylation sites than the Fc region.
  • 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, or 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 the Fc region contain the same number of N-glycosylation sites.
  • the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fab region are glycosylated by an N-glycan having a structure of: wherein the black square represents an N-acetyl galactosamine (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 antibody.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • X represents an amino acid residue of the antibody.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fc region are glycosylated by an N-glycan having a structure of: wherein the black square represents an N-acetyl galactosamine (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 antibody.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fc region are glycosylated by an N-glycan depicted in Section 5.3.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fc region are glycosylated by an N-glycan described in PCT/EP2022/057556, each of which is incorporated herein by reference in its entirety.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fc region are glycosylated by an N-glycan having the structure of A2.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N-glycosylation sites of the Fc region are glycosylated by two different N-glycans.
  • the N-glycosylation sites of the Fab and/or Fc region that are distal to the hinge region of the antibody are glycosylated by the N-glycan having a structure of: wherein the black square represents an N-acetyl galactosamine (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 antibody.
  • the N-glycosylation sites of the Fab and/or Fc region that are proximal to the hinge region of the antibody are glycosylated by the N-glycan having the structure of A2.
  • 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.
  • the N-glycan structures in the Fab region and Fc region are identical (i.e., the same). In some embodiments, the N-glycan structures in the Fab region and Fc region are nonidentical (i.e., not the same).
  • 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.
  • the N-glycan structures in the Fab region and Fc region are identical (i.e., the same). In some embodiments, the N-glycan structures in the Fab region and Fc region are nonidentical (i.e., not the same).
  • the Fc region and/or the hinge region contain more A2GalNAc2 glycans than the Fab region.
  • the Fab region contains more A2GalNAc2 glycans than the Fc region and/or the hinge region.
  • the Fc region and/or the hinge region contain more A2GalNAc2 glycans than the Fab region.
  • the antibody has a N-glycan to protein ratio of 2 to 1, 4 to 1, 6 to 1, 8 to 1, or 10 to 1.
  • the antibody is glycosylated at a predetermined and specific residue. In other embodiments, the antibody is glycosylated at a random residue.
  • the glycoengineered bifunctional degrader binds to an autoantibody and comprises an autoantigen or immunogenic fragment thereof. In certain embodiments, the glycoengineered bifunctional degrader comprises a moiety that specifically binds to the target protein, and wherein the target protein is associated with a disease.
  • the glycoengineered bifunctional degrader a therapeutic polypeptide, i.e., a polypeptide used in the treatment of a disease or disorder.
  • the glycoengineered bifunctional degrader can be an enzyme, a cytokine, or an antibody.
  • the glycoengineered bifunctional degrader is selected from the group consisting of adalimumab, rituximab and erythropoietin (EPO).
  • EPO erythropoietin
  • the glycoengineered bifunctional degrader can be any polypeptide (or peptide/polypeptide corresponding to the polypeptide) known in the art and used in accordance with the methods described herein.
  • nucleic acid sequence of a known polypeptide can easily be deduced using methods known in the art, and thus it would be well within the capacity of one of skill in the art to introduce a nucleic acid that encodes any bifunctional degrader into a host cell provided herein (e.g., via an expression vector, e.g., a plasmid, e.g., a site specific integration by homologous recombination).
  • an expression vector e.g., a plasmid, e.g., a site specific integration by homologous recombination.
  • the glycoengineered bifunctional degrader comprises the amino acid sequence of human Interferon-a (INF-a), Interferon-P (INF-P), Interferon-y (INF- y), Interleukin-2 (IL2), Chimeric diphteria toxin-IL-2 (Denileukin diftitox), Interleukin- 1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), Tumor necrosis factor alpha (TNF-a), 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 morphogenic protein 2 (hBMP2), Human bone morphogenic protein 7 (
  • the glycoengineered bifunctional degrader is an enzyme or an inhibitor.
  • Exemplary enzymes and inhibitors that can be used as a glycoengineered bifunctional degrader include, without limitation, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor Vila, Antithrombin III (AT -III), Polypeptide C, Tissue plasminogen activator (tPA) and tPA variants, Urokinase, Hirudin, Streptokinase, Glucocerebrosidase, Alglucosidase-a, Laronidase (a-L-iduronidase), Idursulphase (Iduronate-2-sulphatase), Galsulphase, Agalsidase-P (human a-galactosidase A), Botulinum toxin, Collagenase, Human DNAse-I, Hyaluronidase, Papain, L-Asparaginase, Ur
  • the glycoengineered bifunctional degrader is a cytokine.
  • cytokines that can be used as a glycoengineered bifunctional degrader include, without limitation, Interferon-a (INF-a), Interferon-P (INF-P), Interferon-y (INF-y), Interleukin-2 (IL2), Chimeric diphteria toxin-IL-2 (Denileukin diftitox), Interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), and Tumor necrosis factor alpha (TNF-a).
  • the glycoengineered bifunctional degrader is a hormone or growth factor.
  • hormones and growth factors that can be used as a glycoengineered bifunctional degrader include, without limitation, 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 morphogenic protein 2 (hBMP2), Human bone morphogenic 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
  • the glycoengineered bifunctional degrader is a receptor.
  • exemplary receptors that can be used as a glycoengineered bifunctional degrader include, without limitation, the extracellular domain of human CTLA4 (e.g., fused to an Fc) and the soluble TNF receptor (e.g., fused to an Fc).
  • the glycoengineered bifunctional degrader is a therapeutic polypeptide.
  • the glycoengineered bifunctional degrader is an approved biologic drug.
  • the therapeutic polypeptide comprises the amino acid sequence of 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., Cered
  • 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.)
  • Dostarlimab e.g., Jemperli® (G
  • the glycoengineered bifunctional degrader comprises the amino acid sequence of an enzyme or an inhibitor thereof.
  • the glycoengineered bifunctional degrader comprises the amino acid sequence of Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor Vila, Antithrombin III (AT-III), Polypeptide C, Tissue plasminogen activator (tPA) and tPA variants, Urokinase, Hirudin, Streptokinase, Glucocerebrosidase, Alglucosidase-a, Laronidase (a-L-iduronidase), Idursulphase (Iduronate-2-sulphatase), Galsulphase, Agalsidase-P (human a-galactosidase A), Botulinum toxin, Collagenase, Human DNAse-I, Hyaluronidase, Papain, L-Asparaginase, Urokinase, Hirudin,
  • the glycoengineered bifunctional degrader is a receptor.
  • exemplary receptors that can be used as a glycoengineered bifunctional degrader include, without limitation, the extracellular domain of human CTLA4 (e.g., fused to an Fc) and the soluble TNF receptor (e.g., fused to an Fc).
  • the glycoengineered bifunctional degrader is secreted into the culture media. In certain embodiments, the glycoengineered bifunctional degrader is purified from the culture media. In another embodiment, the glycoengineered bifunctional degrader is purified from the culture media via affinity purification or ion exchange chromatography. In another embodiment, the glycoengineered bifunctional degrader contains an FC domain and is affinity purified from the culture media via polypeptide-A. In another embodiment, the glycoengineered bifunctional degrader contains an affinity tag and is affinity purified.
  • the glycoengineered bifunctional degrader can be a full length polypeptide, a truncation, a polypeptide domain, a region, a motif or a peptide thereof.
  • the glycoengineered bifunctional degrader is a soluble receptor.
  • the glycoengineered bifunctional degrader is an Fc-fusion polypeptide.
  • the glycoengineered bifunctional degrader is a biologic comprising an Fc domain of an IgG.
  • the glycoengineered bifunctional degrader is a ligand to a receptor.
  • the glycoengineered bifunctional degrader is localized in the secretory pathway.
  • localization within the secretory pathway includes, but is not limited to, localization to one or more of the following sub- cellular compartments: the endoplasmic reticulum, the Golgi apparatus, lysosomes, intracellular membrane proteins, cell surface anchored proteins, and membrane proteins.
  • localization in the secretory pathway comprises localization to one or more of said sub-cellular compartments.
  • the glycoengineered bifunctional degrader comprises a signal peptide localizing the glycoengineered bifunctional degrader in the secretory pathway.
  • the signal peptide is derived from the same source as the glycoengineered bifunctional degrader (i.e. the signal peptide is not added to the glycoengineered bifunctional degrader, but is one fused to the glycoengineered bifunctional degrader when naturally expressed in the source).
  • the glycoengineered bifunctional degrader is localized in the secretory pathway without adding a Leishmania signal peptide to the glycoengineered bifunctional degrader.
  • the signal peptide is added to the glycoengineered bifunctional degrader.
  • the signal peptide is derived from Leishmania species.
  • the signal peptide is derived from Leishmania tarentolae.
  • the signal peptide is an invertase signal peptide from derived from Leishmania tarentolae. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed and removed from the glycoengineered bifunctional degrader.
  • the glycoengineered bifunctional degrader has been engineered to comprise one or more tag(s).
  • the tag is processed and removed from the glycoengineered bifunctional degrader.
  • the glycoengineered bifunctional degrader is expressed from a Leishmania host cell described in Section 7.3.
  • the Leishmania host cells used to make the glycoengineered bifunctional degraders provided herein are genetically engineered using the methods described in Section 7.4.
  • the Leishmania host cells used to make the glycoengineered bifunctional degraders provided herein are cultured according to the methods described in Section 7.5.
  • compositions comprising the glycoengineered bifunctional degrader described herein.
  • the compositions described herein are useful in the treatment and/or prevention of diseases/disorders in subjects (e.g., human subjects) (see Section 7.8).
  • the pharmaceutical described herein comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed 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.
  • suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin.
  • the pharmaceutical compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the pharmaceutical compositions described herein may be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration.
  • the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary administration.
  • the pharmaceutical compositions described herein additionally comprise one or more buffers, e.g., phosphate buffer and sucrose phosphate glutamate buffer. In other embodiments, the pharmaceutical compositions described herein do not comprise buffers.
  • the pharmaceutical compositions described herein additionally comprise one or more salts, e.g., sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts).
  • the pharmaceutical compositions described herein do not comprise salts.
  • the pharmaceutical compositions described herein can be administered in a single dosage form, for example a single dosage form of a glycoengineered bifunctional degrader described here.
  • kits comprising the glycoengineered bifunctional degrader of the present disclosure is provided herein.
  • the kit further provides instructions for administering the bifunctional molecule or pharmaceutical composition to an individual in need thereof.
  • compositions described herein can be stored before use, e.g., the compositions can be stored frozen (e.g., at about -20 °C or at about -70 °C); stored in refrigerated conditions (e.g., at about 4 °C); or stored at room temperature.
  • composition comprising a population of bifunctional degraders described in Section 7.1, wherein the population of bifunctional degraders 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 the N-glycosylation site(s).
  • 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% homogenous at one of the N-glycosylation sites.
  • 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 site(s). In certain embodiments, the N-glycan profile is at least 60% homogeneous at one or more of the N- glycosylation site(s). In certain embodiments, the N-glycan profile is at least 70% homogenous at one or more of the N-glycosylation site(s).
  • the N- glycan profile is at least 80% homogeneous at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile is at least 90% homogeneous at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile is at least 95% homogenous at one or more of the N-glycosylation site(s). In certain embodiments, the N- glycan profile is at least 98% homogenous at one or more of the N-glycosylation site(s). In certain embodiments, the homogeneity of the N-glycan profile at one or more of the N- glycosylation sites is determined according to any standard means known in the art.
  • the homogeneity of the N-glycan profile at one or more of the N- glycosylation sites is determined by N-glycan analysis, glycopeptide analysis or intact protein analysis. In certain embodiments, the homogeneity of the N-glycan profile at one or more of the N-glycosylation sites is determined according to one or more assays described in Section 7.9.3.
  • composition comprising a population of bifunctional degraders described in Section 7.1, wherein the population of bifunctional degraders has an N-glycan profile comprising 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% of an N-glycan of the structure: at one or more of the N-glycosylation site(s), wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the bifunctional degrader.
  • the black square represents an N-acetyl galactosamine (GalNAc)
  • the white square represents an N-acetylglucosamine (GlcNAc) residue
  • the black circle represents a mannose (Man) residue
  • X
  • 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-glycan 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-glycan at two N-glycosylation sites, respectively.
  • the N-glycan profile comprises about 30% to about 40% of the N- glycan of the structure at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile comprises about 40% to about 50% of the N-glycan of the structure at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile comprises about 50% to about 60% of the N-glycan of the structure at one or more of the N- glycosylation site(s). In certain embodiments, the N-glycan profile comprises about 60% to about 70% of the N-glycan at one or more of the N-glycosylation site(s).
  • the N-glycan profile comprises about 70% to about 80% of the N-glycan at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile comprises about 80% to about 90% of the N-glycan at one or more of the N-glycosylation site(s). In certain embodiments, the N-glycan profile comprises about 90% to about 100% of the N-glycan at one or more of the N-glycosylation site(s). In certain embodiments, the relative amount of the N-glycan at one or more of the N-glycosylation sites is determined according to any standard means known in the art.
  • the relative amount of the N-glycan at one or more of the N-glycosylation sites is determined by N- glycan analysis or glycopeptide analysis. In certain embodiments, the relative amount of the N-glycan at one or more of the N-glycosylation sites is determined according to one or more assays described in Section 7.9.3.
  • N-glycan structures that are not fully capped with GalNAc (for example, AlGalNAcl or A2GalNAcl as compared to A2GalNAc2) do not engage ASGPR.
  • the N-glycan profile comprises about 90% to about 100% of the N-glycan of structure: at one or more of the N-glycosylation site(s), wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the bifunctional degrader.
  • the N-glycan profile comprises about 90% to about 100% of the N-glycan at one N-glycosylation site. In one embodiment, the N-glycan profile comprises about 95% to about 100% of the N-glycan at one N-glycosylation site. In one embodiment, the N-glycan profile comprises about 90% to about 100% of the N-glycan at two N-glycosylation sites, respectively. In one embodiment, the N-glycan profile comprises about 95% to about 100% of the N-glycan at two N- glycosylation sites, respectively. In one embodiment, the N-glycan profile comprises about 80% to about 90% of the N-glycan at two N-glycosylation sites, collectively.
  • the N-glycan profile comprises about 90% to about 100% of the N-glycan at two N-glycosylation sites, collectively.
  • the relative amount of the N-glycan at one or more of the N-glycosylation sites is determined according to any standard means known in the art. In certain embodiments, the relative amount of the N-glycan at one or more of the N-glycosylation sites is determined by N-glycan analysis or glycopeptide analysis. In certain embodiments, the relative amount of the N-glycan at one or more of the N-glycosylation sites is determined according to one or more assays described in Section 7.9.3.
  • composition comprising a population of bifunctional degraders described in Section 7.1, wherein the population of bifunctional degraders has an N-glycan profile comprising 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 an N-glycan of the structure: among all glycans in the N-glycan profile, wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the bifunctional degrader.
  • the black square represents an N-acetyl galactosamine (GalNAc)
  • the white square represents an N-acetylglucosamine (GlcNAc) residue
  • the black circle represents a mannose (Man) residue
  • X represents
  • the population of bifunctional degraders has an N-glycan profile comprising at least 30% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 40% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 50% of the N-glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising at least 60% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 70% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 80% of the N-glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising at least 90% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 95% of the N-glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising at least 98% of the N-glycan among all glycans in the N-glycan profile.
  • composition comprising a population of bifunctional degraders described in Section 7.1, wherein the population of bifunctional degraders has an N-glycan profile comprising 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 an N-glycan of the structure: among all glycans in the N-glycan profile, wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the bifunctional degrader.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • the population of bifunctional degraders has an N-glycan profile comprising about 30% to about 40% of the N- glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising about 40% to about 50% of the N- glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising about 50% to about 60% of the N- glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising about 60% to about 70% of the N- glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising about 70% to about 80% of the N- glycan among all glycans in the N-glycan profile. In certain embodiments, the population of bifunctional degraders has an N-glycan profile comprising about 80% to about 90% of the N- glycan among all glycans in the N-glycan profile.
  • the population of bifunctional degraders has an N-glycan profile comprising about 90% to about 100% of the N-glycan among all glycans in the N-glycan profile.
  • the relative amount of the N-glycan among all glycans in the N-glycan profile is determined 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 determined 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 determined according to one or more assays described in Section 7.9.3.
  • composition comprising a population of bifunctional degraders described in Section 7.1, wherein the population of bifunctional degraders 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.
  • the population has an N- glycan profile that is at least 60% homogeneous.
  • the population has an N-glycan profile that is at least 70% homogeneous.
  • the population has an N-glycan profile that is at least 80% homogeneous.
  • the population has an N-glycan profile that is at least 90% homogeneous.
  • 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 the N-glycan profile is determined according to any standard means known in the art. In certain embodiments, the homogeneity of the N-glycan profile is determined by N-glycan analysis, glycopeptide analysis or intact protein analysis. In certain embodiments, the homogeneity of the N-glycan profile is determined according to one or more assays described in Section 7.9.3.
  • composition comprising a population of bifunctional degraders described in Section 7.1, wherein the population of bifunctional degraders 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.
  • the population has an N-glycan profile that is about 30% to about 40% homogeneous.
  • the population has an N-glycan profile that is about 40% to about 50% homogeneous.
  • the population has an N-glycan profile that is about 50% to about 60% homogeneous.
  • the population has an N-glycan profile that is about 60% to about 70% homogeneous.
  • 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 determined according to any standard means known in the art. In certain embodiments, the homogeneity of the N-glycan profile is determined by N-glycan analysis, glycopeptide analysis or intact protein analysis. In certain embodiments, the homogeneity of the N-glycan profile is determined according to one or more assays described in Section 7.9.3.
  • the population of bifunctional degraders described in this Section is produced by Leishmania host cells described in Section 7.3.
  • the Leishmania host cells used to produce the population of glycoengineered bifunctional degraders described in this Section aree genetically engineered using the methods described in Section 7.4.
  • the Leishmania host cell used to produce the population of glycoengineered bifunctional degraders described in this Section are cultured according to the methods described in Section 7.5.
  • compositions Comprising a Population of Bifunctional Degraders
  • compositions comprising a population of glycoengineered bifunctional degraders described herein.
  • the compositions described herein are useful in the treatment and/or prevention of diseases/disorders in subjects (e.g., human subjects) (see Section 7.8).
  • the pharmaceutical described herein comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed 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.
  • suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin.
  • the pharmaceutical compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the pharmaceutical compositions described herein may be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration.
  • the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary administration.
  • the pharmaceutical compositions described herein additionally comprise one or more buffers, e.g., phosphate buffer and sucrose phosphate glutamate buffer. In other embodiments, the pharmaceutical compositions described herein do not comprise buffers.
  • the pharmaceutical compositions described herein additionally comprise one or more salts, e.g., sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts).
  • the pharmaceutical compositions described herein do not comprise salts.
  • 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 degraders described here.
  • kits comprising the population of glycoengineered bifunctional degraders of the present disclosure is provided herein.
  • the kit further provides instructions for administering the population of glycoengineered bifunctional degraders or pharmaceutical composition to an individual in need thereof.
  • compositions described herein can be stored before use, e.g., the compositions can be stored frozen (e.g., at about -20 °C or at about -70 °C); stored in refrigerated conditions (e.g., at about 4 °C); or stored at room temperature.
  • Leishmania host cells for the production of glycoengineered bifunctional degraders described in Section 7.1 or a population of glycoengineered bifunctional degraders described in Section 7.2, wherein the Leishmania host cells comprise: (a) a recombinant nucleic acid encoding a glycoengineered bifunctional degrader; and (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine
  • the Leishmania host cells provided herein are capable of producing glycoengineered bifunctional degraders comprising a biantennary, GalNAc-terminated N-glycan.
  • the Leishmania host cells provided herein are capable of producing glycoengineered bifunctional degraders comprising an N-glycan of the following structure: wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the glycoengineered bifunctional degrader.
  • the 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.
  • the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases described in Section 7.3.2.
  • one or more endogenous enzymes described in Section 7.3.3 from the glycan biosynthesis pathway of the the Leishmania host cells provided herein have been deleted, mutated and/or functionally inactivated.
  • the Leishmania host cells provided herein further comprise a recombinant nucleic acid encoding heterologous UDP- GalNAc biosynthetic pathway proteins as described in Section 7.3.4 capable of generating UDP-GalNAc.
  • the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.3.5 capable of transporting UDP-GalNAc to the secretory pathway.
  • the strain of the Leishmania host cells provided herein is described in Section 7.3.6.
  • the Leishmania host cells provided herein below are genetically engineered using the methods described in Section 7.4. In certain embodiments, the Leishmania host cells provided herein below are cultured according to the methods described in Section 7.5.
  • Suitable host cells comprise liver cells, myeloid cells, immune cells, endothelial cells, parenchymal cells or epithelial cells.
  • the immune cell is a dendritic cell, a macrophage, a monocyte, a microglia cell, a granulocyte or a B lymphocyte.
  • the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases.
  • the GalNAc transferase or a functionally active variant thereof, is capable of catalyzing the addition of a GalNAc to a N-acetyl glucosamine-terminated glycan.
  • the GalNAc transferase is heterologous to the Leishmania host cell.
  • the GalNAc transferase is derived from Homo sapiens, Caenorhabditis elegans, Parasteatoda lepidariorum, Salmo Irulla, or Hucho hucho.
  • the GalNAc transferase is derived from a mammalian source.
  • the mammalian source is Homo sapiens.
  • the GalNAc transferase is selected from the group consisting of [34-GalNAcT3, [34-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or functionally active variants thereof.
  • the GalNAc transferase is selected from the group consisting of P4-GalNAcT3, P4-GalNAcT4, CeP4GalNAcT, Ptp4GalNAcT, and Stp4GalNAcT, or variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.
  • the GalNAc transferase comprises P4-GalNAcT3, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises P4-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 P4-GalNAcT3. In certain embodiments, the GalNAc transferase comprises an N-terminally truncated variant of [34-GalNAcT3.
  • the [34-GalNAcT3 comprises [34-GalNAcT3 of Homo sapiens, or a functionally active variant thereof.
  • the GalNAc transferase comprises [34-GalNAcT3 of Homo sapiens.
  • the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 1.
  • the GalNAc transferase comprises one that is homologous to [34-GalNAcT3 of Homo sapiens.
  • 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 [34-GalNAcT3 of Homo sapiens.
  • the GalNAc transferase comprises an N-terminally truncated variant of [34- GalNAcT3 of Homo sapiens comprising an amino acid sequence of SEQ ID NO: 2.
  • the GalNAc transferase comprises [34-GalNAcT4, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase comprises [34-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 [34-GalNAcT4. In certain embodiments, the GalNAc transferase comprises an N-terminally truncated variant of [34-GalNAcT4.
  • the [34-GalNAcT4 comprises [34-GalNAcT4 of Homo sapiens, or a functionally active variant thereof.
  • the GalNAc transferase comprises [34-GalNAcT4 of Homo sapiens.
  • the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 3.
  • the GalNAc transferase comprises one that is homologous to [34-GalNAcT4 of Homo sapiens.
  • 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 [34-GalNAcT4 of Homo sapiens.
  • the GalNAc transferase comprises an N-terminally truncated variant of [34- GalNAcT4 of Homo sapiens comprising an amino acid sequence of SEQ ID NO: 4.
  • the GalNAc transferases comprise [34-GalNAcT3 and [3- GalNAcT4, or functionally active variants thereof.
  • the GalNAc transferases comprise [34-GalNAcT3 and [34-GalNAcT4.
  • the GalNAc transferases comprise variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to [34-GalNAcT3 and [3-GalNAcT4, respectively.
  • the GalNAc transferases comprise N-terminally truncated variants of [34-GalNAcT3 and/or [34-GalNAcT4.
  • the GalNAc transferases comprise P4-GalNAcT3 and P4-GalNAcT4 of Homo sapiens, or functionally active variants thereof.
  • the GalNAc transferases comprise [34-GalNAcT3 and [34-GalNAcT4 of Homo sapiens.
  • the GalNAc transferases comprise amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 3.
  • the GalNAc transferases comprise ones that are homologous to [34- GalNAcT3 and [34-GalNAcT4 of Homo sapiens.
  • the GalNAc transferases comprise variants that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous to [34-GalNAcT3 and [34-GalNAcT4 of Homo sapiens, respectively.
  • the GalNAc transferases comprise N- terminally truncated variants of [34-GalNAcT3 and/or [34-GalNAcT4 of Homo sapiens comprising amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
  • the GalNAc transferase is Ce
  • the GalNAc transferase is Ce
  • the GalNAc transferase is a
  • the GalNAc transferase is Pt
  • the Ptp4GalNAcT is a P4GalNAcT of Parasteatoda tepidariorum, or a functionally active variant thereof.
  • the GalNAc transferase is a Ptp4GalNAcT of Parasteatoda tepidariorum.
  • the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 7.
  • the GalNAc transferase is one that is homologous to a Ptp4GalNAcT of Parasteatoda tepidariorum.
  • 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 a Ptp4GalNAcT of Parasteatoda tepidariorum.
  • the GalNAc transferase comprises an N-terminally truncated variant of Ptp4GalNAcT of Parasteatoda tepidariorum comprising an amino acid sequence of SEQ ID NO: 8.
  • the GalNAc transferase is Stp4GalNAcT, or a functionally active variant thereof.
  • the GalNAc transferase is Stp4GalNAcT. 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 Stp4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminally truncated variant of Stp4GalNAcT. In certain embodiments, the Stp4GalNAcT is a P4GalNAcT of Salmo IriiUa, or a functionally active variant thereof.
  • the GalNAc transferase is a p4GalNAcT of Salmo trutta. In certain embodiments, the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 9. In certain embodiments, the GalNAc transferase is one that is homologous to a P4GalNAcT of Salmo trutta. 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 a p4GalNAcT of Salmo trutta.
  • the GalNAc transferase is Hhp4GalNAcT, or a functionally active variant thereof. In certain embodiments, the GalNAc transferase is Hhp4GalNAcT. 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 Hhp4GalNAcT. In certain embodiments, the GalNAc transferase is an N-terminally truncated variant of Hhp4GalNAcT.
  • the Hhp4GalNAcT is a P4GalNAcT of Hucho hucho, or a functionally active variant thereof.
  • the GalNAc transferase is a P4GalNAcT of Hucho hucho.
  • the GalNAc transferase comprises an amino acid sequence of SEQ ID NO: 10.
  • the GalNAc transferase is one that is homologous to a p4GalNAcT of Hucho hucho.
  • 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 a P4GalNAcT of Hucho hucho.
  • the GalNAc transferase is localized in the secretory pathway.
  • localization within the secretory pathway includes, but is not limited to, localization to one or more of the following sub-cellular compartments: the endoplasmic reticulum, the Golgi apparatus, lysosomes, intracellular membrane proteins, cell surface anchored proteins, and membrane proteins.
  • localization in the secretory pathway comprises localization to one or more of said sub-cellular compartments.
  • the GalNAc transferase comprises a signal peptide localizing the GalNAc transferase in the secretory pathway.
  • 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 one contained in the GalNAc transferase when naturally expressed in the source).
  • the GalNAc transferase is localized in the secretory pathway without adding Leishmania signal peptide to the GalNAc transferase. In other embodiments, the signal peptide is added to the GalNAc transferase.
  • 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 peptides is fused to one or more amino acids within the polypeptide of the GalNAc transferase. In certain embodiments, the signal peptide is fused to the N-terminus of an N-terminally truncated variant of the GalNAc transferase. In certain embodiments, the signal peptide is fused to one or more amino acids within the polypeptide of an N-terminally truncated variant of the GalNAc transferase.
  • the signal peptide is derived from 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 derived from Leishmania tarentolae. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the signal peptide is processed and removed from the GalNAc transferase.
  • the GalNAc transferase and the additional recombinant glycosyltransferase described in Section 7.3.2 are co-localized in the secretory pathway.
  • the GalNAc transferase and the glycoengineered bifunctional degrader described in Section 7.1 are co-localized in the secretory pathway. 7.3.2 Additional Recombinant Glycosyltransferases
  • the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding one or more additional recombinant glycosyltransferases.
  • the additional recombinant glycosyltransferase, or a functionally active variant thereof is capable of catalyzing the addition of a first glycan to a second glycan.
  • the additional recombinant glycosyltransferase is a N-acetyl glucosamine transferase, or a functionally active variant thereof, capable of catalyzing the addition of a N-acetyl glucosamine (GlcNAc) to a mannose-terminated glycan, for example, a Man3GlcNAc2 glycan (Man3, see Section 5.3).
  • GlcNAc N-acetyl glucosamine transferase
  • Man3GlcNAc2 glycan Man3, see Section 5.3
  • the additional recombinant glycosyltransferase comprises one or more N-acetyl glucosamine transferases.
  • the N-acetyl glucosamine transferase is heterologous to the host cell.
  • the N- acetyl glucosamine transferase is derived from Homo sapiens, Spodoptera frugiperda, Trypanosoma brucei.
  • the additional recombinant glycosyltransferase is derived from a mammalian source.
  • the mammalian source is Homo sapiens.
  • the N-acetyl glucosamine transferase is selected from the group consisting of MGAT1 (alpha-1, 3-mannosyl-glycoprotein 2-beta-N- acetylglucosaminyltransf erase) and MGAT2 (alpha- 1,6-mannosylgly coprotein 2-beta-N- acetylglucosaminyltransferase), or functionally active variants thereof.
  • the additional recombinant glycosyltransferases comprise MGAT1 and MGAT2.
  • the N-acetyl glucosamine transferase comprises MGAT1, or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1. In certain embodiments, the N-acetyl glucosamine 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 MGAT1. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Homo sapiens (accession number P26572), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferase comprises an amino acid sequence of SEQ ID NO: 13. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MG ATI of Homo sapiens. In certain embodiments, the N-acetyl glucosamine 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 MGAT1 of Homo sapiens.
  • the N-acetyl glucosamine transferase comprises MGAT2, or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2. In certain embodiments, the N-acetyl glucosamine 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 MGAT2. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT2 of Homo sapiens (accession number: Q10469.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT2 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferase comprises an amino acid sequence of SEQ ID NO: 14. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT2 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine 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 MGAT2 of Homo sapiens.
  • the N-acetyl glucosamine transferases comprise MGAT1 and MGAT2, or functionally active variants thereof. In certain embodiments, the N-acetyl glucosamine transferases comprise MGAT1 and MGAT2. In certain embodiments, the N- acetyl glucosamine transferases are 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-acetyl glucosamine transferases are MGAT1 and MGAT2 of Homo sapiens, or functionally active variants thereof.
  • the N-acetyl glucosamine transferases are MGAT1 and MGAT2 of Homo sapiens. In certain embodiments, the N-acetyl glucosamine transferases comprise amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In certain embodiments, the N-acetyl glucosamine transferases are ones that are homologous to MGAT1 and MGAT2 of Homo sapiens.
  • the N-acetyl glucosamine transferases are 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 of Homo sapiens, respectively.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Spodoptera frugiperda (SfGnT-I, accession number: AEX00082), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MG ATI of Spodoptera frugiperda.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Spodoptera frugiperda.
  • the N-acetyl glucosamine 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 MG ATI of Spodoptera frugiperda.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Trypanosoma brucei (TbGnT-I, accession number: XP 844156), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MG ATI of Trypanosoma brucei.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Trypanosoma brucei.
  • the N-acetyl glucosamine 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 MGAT1 of Trypanosoma brucei.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Pan troglodytes (PtMGATl, accession number: XP 001155433.2), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MG ATI of Pan troglodytes .
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Pan troglodytes.
  • the N-acetyl glucosamine 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 MGAT1 of Pan troglodytes.
  • the N-acetyl glucosamine transferase comprises MGAT1 Macaco mulatto (MaMGATl, accession number: NP_001244759), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT1 mulatto.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Macaco mulatto.
  • the N-acetyl glucosamine 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 MGAT1 of Macaco mulatto.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Mus musculus (MuMGATl, accession number: NP 001103620.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT1 oiMus musculus.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 oiMus musculus.
  • the N-acetyl glucosamine 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 MGAT1 of Mus musculus.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Rattus norvegicus (RnMGATl, accession number: NP_110488.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MG ATI of Rattus norvegicus.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Rattus norvegicus.
  • the N-acetyl glucosamine 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 MGAT1 of Rattus norvegicus.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Danio rerio A (DrMGATla, accession number: NP 956970.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MG ATI of Danio rerio A.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Danio rerio A.
  • the N-acetyl glucosamine 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 MGAT1 of Danio rerio A.
  • the N-acetyl glucosamine transferase comprises an MGAT1 of Caenorhabditis elegans (Cel4MGATl, accession number: NP 497719.1 or Cel3MGATl, accession number: NP 509566.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Caenorhabditis elegans.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Caenorhabditis elegans.
  • the N-acetyl glucosamine 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 MGAT1 of Caenorhabditis elegans.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Arabidopsis thaliana (AtMGATl, accession number: NP 195537.2), or a functionally active variant thereof. In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Arabidopsis thaliana. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 of Arabidopsis thaliana.
  • the N-acetyl glucosamine 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 MGAT1 of Arabidopsis thaliana.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Oryza sativa Japonica (OsJMGATl, accession number: XP 015624616.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Oryza sativa Japonica.
  • the N- acetyl glucosamine transferase comprises one that is homologous to MGAT1 Oryza sativa Japonica.
  • the N-acetyl glucosamine 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 MGAT1 of Oryza sativa Japonica.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Xenopus tropicalis (XtMGATl, accession number: NP 001011350.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Xenopus tropicalis.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Xenopus tropicalis.
  • the N-acetyl glucosamine 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 MGAT1 of Xenopus tropicalis.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Canis lupus (C1MGAT1, accession number: XP 855658.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Canis lupus.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Canis lupus.
  • the N-acetyl glucosamine 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 MGAT1 of Canis lupus.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Bos taurus (BtMGATl, accession number: NP 001015653.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MG ATI of Bos taurus.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Bos taurus.
  • the N- acetyl glucosamine 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 MGAT1 of Bos taurus.
  • the N-acetyl glucosamine transferase comprises MGAT1 oi Danio rerio B (DrMGATlb, accession number: NP 001073440.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MG ATI of Danio rerio B.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Danio rerio B. In certain embodiments, the N- acetyl glucosamine 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 MGAT1 of Danio rerio B. [00184] In certain embodiments, the N-acetyl glucosamine transferase comprises MGAT1 of Gekko japonicus (GjMGATl, accession number: XP 015280466.1), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT1 of Gekko japonicus. In certain embodiments, the N-acetyl glucosamine transferase comprises one that is homologous to MGAT1 Gekko japonicus. In certain embodiments, the N-acetyl glucosamine 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 MGAT1 of Gekko japonicus.
  • the N-acetyl glucosamine transferase comprises MGAT2 of Rattus norvegicus (rMGAT2, accession number: NP 446056), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT2 of Rattus norvegicus.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT2 of Rattus norvegicus.
  • the N-acetyl glucosamine 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 MGAT2 of Rattus norvegicus.
  • the N-acetyl glucosamine transferase comprises MGAT2 of Spodoptera frugiperda (SfGnT-II, accession number: AEX00083), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT2 of Spodoptera frugiperda.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT2 of Spodoptera frugiperda.
  • the N-acetyl glucosamine 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 MGAT2 of Spodoptera frugiperda.
  • the N-acetyl glucosamine transferase comprises MGAT2 of Trypanosoma brucei (TbGnT-II, accession number: XP 845654), or a functionally active variant thereof.
  • the N-acetyl glucosamine transferase comprises MGAT2 of Trypanosoma brucei.
  • the N-acetyl glucosamine transferase comprises one that is homologous to MGAT2 of Trypanosoma brucei.
  • the N-acetyl glucosamine 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 MGAT2 of Trypanosoma brucei.
  • the additional recombinant glycosyltransferase is localized in the secretory pathway.
  • localization within the secretory pathway includes, but is not limited to, localization to one or more of the following sub-cellular compartments: the endoplasmic reticulum, the Golgi apparatus, lysosomes, intracellular membrane proteins, cell surface anchored proteins, and membrane proteins.
  • localization in the secretory pathway comprises localization to one or more of said sub-cellular compartments.
  • the additional recombinant glycosyltransferase comprises a signal peptide localizing the additional recombinant glycosyltransferase in the secretory pathway.
  • the signal peptide is derived from the same source as the additional recombinant glycosyltransferase (i.e. the signal peptide is not added to additional recombinant glycosyltransferase, but is one fused to the additional recombinant glycosyltransferase when naturally expressed in the source).
  • the additional recombinant glycosyltransferase is localized in the secretory pathway without adding a Leishmania signal peptide to the additional recombinant glycosyltransferase.
  • the signal peptide is added to the additional recombinant glycosyltransferase.
  • the signal peptide is fused to the C-terminus of the additional recombinant glycosyltransferase.
  • the signal peptide is fused to the N-terminus of the additional recombinant glycosyltransferase.
  • the signal peptides is fused to one or more amino acids within the polypeptide of the additional recombinant glycosyltransferase.
  • the signal peptide is derived from Leishmania species.
  • the signal peptide is derived from Leishmania tarentolae.
  • the signal peptide is an invertase signal peptide from derived from Leishmania tarentolae.
  • the signal peptide comprises an amino acid sequence of SEQ ID NO: 11.
  • the signal peptide comprises an amino acid sequence of SEQ ID NO: 12.
  • the signal peptide is processed and removed from the additional recombinant glycosyltransferase.
  • the GalNAc transferase described in Section 7.3.1 and the additional recombinant glycosyltransferase are co-localized in the secretory pathway. 7.3.3 Deletion, Mutation and/or Functionally Inactivation of Endogenous Enzymes from the Glycan Biosynthesis Pathway
  • the Leishmania host cells provided herein are characterized in that one or more endogenous enzymes from the glycan biosynthesis pathway have been deleted, mutated and/or functionally inactivated.
  • the Leishmania host cell does not have endogenous N-glycan elongation.
  • the Leishmania host cells do not have endogenous N-glycan elongation as described in WO 2019/002512, which is incorporated herein by reference in its entirety.
  • the Leishmania host cell has been genetically engineered such that the formation of an O-linked GlcNAc on a polypeptide in the host cell is reduced or eliminated.
  • 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.
  • the gene encoding the at least one GlcNAc-transferase in the Leishmania host cell is functionally inactivated, downregulated, deleted, and/or mutated.
  • the enzyme that catalyzes the formation of O-linked GlcNAc is an N-acetylglucosamine (GlcNAc)-transferase.
  • the GlcNAc-transferase is selected from the group consisting of OGNT1, OGNT2, OGNTL, and homologous GlcNAc-transf erases thereof.
  • OGNT1, OGNT2, OGNTL were identified based on homology to Trypanosoma enzymes and not mammalian (e.g. human) enzymes (Heise, N., et al Glycobiology, 19(8), 918-933 (2009) and Chiribao, M.L.
  • the GlcNAc-transferase is OGNTL In other embodiments, the GlcNAc-transferase is OGNT2. In yet other embodiments, the GlcNAc-transferase is OGNTL. In certain embodiments, the GlcNAc-transferase is a GlcNAc-transferase that is homologous to OGNTL In certain embodiments, the GlcNAc- transferase is a GlcNAc-transferase that is homologous to OGNT2.
  • 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. Nonlimiting examples of GlcNAc-transferases in Trypanosomatida are listed in Table 1, in which one representative genome per species is listed.
  • Table 1 Exemplary GlcNAc-transferases in Trypanosomatida.
  • GlcNAc is derived from species other than Trypanosomatida species.
  • the enzyme is a human O-GlcNAc transferase (OGT, Uniprot: 015294) and homologous enzymes thereof.
  • the O-GlcNAc transferase (OGT; uridine diphospho-N-acetylglucosamine:polypeptide P-N-acetylglucosaminyltransferase; EC 2.4.1.255) can catalyze the transfer of a single N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue in cytoplasmic and nuclear proteins resulting in their modification with a beta-linked N-acetylglucosamine (O-GlcNAc).
  • the enzyme that catalyzes the formation of O-linked GlcNAc may be different isoforms of OGT.
  • Exemplary isoforms of OGT include but are not limited to: (1) the 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 that is targeted to the mitochondria (mOGT; which may be 90 kDa).
  • OGT may appear to form multimers in the nucleus and cytoplasm, consisting of one or more 110-kDa subunits and 78-kDa subunits (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)).
  • the enzyme that catalyzes the formation of O-linked GlcNAc is human EOGT (Uniprot: Q5NDL2).
  • the enzyme catalyzes the transfer of a single N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue in extracellular proteins resulting in their modification with a beta-linked N-acetylglucosamine (O-GlcNAc).
  • the enzyme catalyzes Specific glycosylation of the Thr residue located between the fifth and sixth conserved cysteines of folded EGF-like domains.
  • the enzyme that catalyzes the formation of O-linked GlcNAc may transfer in alpha-linkage. In other embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc may transfer in beta-linkage.
  • 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, for example one, two, three, four, five, six, seven, eight, nine or ten enzymes as described in this Section.
  • 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 Trypanosomatida species, for example Leishmania tarentolae.
  • the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc-transferase, for example one, two, three, four, five, six, seven, eight, nine or ten GlcNAc-transferases, one or more of which is derived from Trypanosomatida species.
  • the number of the at least one GlcNAc- transferase is one, two or three.
  • the at least one GlcNAc-transferase is selected from the group consisting of 0GNT1, 0GNT2, OGNTL and homologous GlcNAc-transferases thereof.
  • at least one GlcNAc-transferase is a GlcNAc-transferase that is homologous to 0GNT1, 0GNT2 and/or OGNTL.
  • 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 species that is other than Trypanosomatida species, for example human.
  • the formation of O-linked GlcNAc in the Leishmania host cell prior to genetic engineering is catalyzed by at least one GlcNAc-transferase, for example one, two, three, four, five, six, seven, eight, nine or ten GlcNAc-transferases, one or more of which is derived from human.
  • the number of the at least one GlcNAc-transferase is one, two or three.
  • the at least one GlcNAc-transferase is selected from the group consisting of human O-GlcNAc transferase and human EOGT and homologous enzymes thereof.
  • at least one GlcNAc-transferase is an enzyme that is homologous to human O-GlcNAc transferase and/or human EOGT.
  • the enzyme catalyzes the formation of O-linked GlcNAc prior to the genetic engineering of the Leishmania host cell. In certain embodiments, the enzyme still catalyzes the formation of O-linked GlcNAc after the genetic engineering of the Leishmania host cell. In certain embodiments, the enzyme does not catalyze the formation of O-linked GlcNAc after the genetic engineering of the Leishmania host cell.
  • the gene encoding the at least one GlcNAc-transferase in the Leishmania host cell is functionally inactivated. In certain embodiments, the gene encoding the at least one GlcNAc-transferase in the Leishmania host cell is downregulated. In certain embodiments, the gene encoding the at least one GlcNAc-transferase in the Leishmania host cell is overexpressed.
  • the Leishmania host cell provided herein comprises at least one gene deletion.
  • the gene encoding the at least one GlcNAc- transferase is deleted.
  • the gene encoding the at least one GlcNAc- transferase is mutated.
  • the gene encoding the at least one GlcNAc- transferase is overexpressed.
  • additional modifications may be introduced (e.g., using recombinant techniques) into the Leishmania host cell described herein.
  • the 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 catalyzes the formation of O- linked GlcNAc may be functionally inactivated. In certain embodiments, the 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 catalyzes the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding three enzymes that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.
  • the genes and/or genetic loci that may be functionally inactivated include but are not limited to OGNT1, OGNT2, and OGNTL.
  • the 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-transferase that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.
  • the genes encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 GlcNAc- transferase that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.
  • the genes encoding three GlcNAc-transferases that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.
  • the genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all enzymes that each catalyzes the formation of O- linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all enzymes that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.
  • the genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all GlcNAc-transferases that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated. In certain embodiments, the genes encoding 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all GlcNAc-transferases that each catalyzes the formation of O-linked GlcNAc may be functionally inactivated.
  • the at least one GlcNAc-transferase is selected from the group consisting of OGNT1, OGNT2 and OGNTL, and homologous GlcNAc-transferases thereof.
  • the Leishmania host cell is a OGNT1, OGNT2 and OGNTL triple knockout.
  • the formation of the 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 the O-linked GlcNAc in a reference Leishmania cell.
  • the formation of the 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 the O-linked GlcNAc in a reference Leishmania cell.
  • the reference Leishmania cell is wild-type.
  • the reference Leishmania cell is genetically engineered differently as the genetically engineered Leishmania cells described herein.
  • some of the engineering of the reference Leishmania cell may be the same of the engineering of the genetically engineered Leishmania cells described herein, for example the deletion of one or more enzymes that catalyze the formation of O-linked GlcNAc.
  • the reference Leishmania cell may comprise a recombinant nucleic acid encoding a heterologous glycosyltransferase, for example the Leishmania cells described in International Publication No. W02019/002512 A2, incorporated by reference in its entirety herein.
  • the formation of the 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 the O-linked GlcNAc in a wild-type Leishmania cell.
  • the formation of the 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 the O-linked GlcNAc in a wild-type Leishmania cell.
  • the formation of the 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 the O- linked GlcNAc in a Leishmania cell that comprises a recombinant nucleic acid encoding a heterologous glycosyltransferase.
  • the formation of the 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 the O-linked GlcNAc in a Leishmania cell that comprises a recombinant nucleic acid encoding a heterologous glycosyl transferase.
  • the growth rate of the 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 the 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 wildtype. In certain embodiments, the reference Leishmania cell is genetically engineered differently as the genetically engineered Leishmania cells described herein.
  • some of the engineering of the reference Leishmania cell may be the same of the engineering of the genetically engineered Leishmania cells described herein, for example the deletion of one or more enzymes that catalyze the formation of O-linked GlcNAc.
  • the growth rate of the Leishmania cell is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of a wild-type Leishmania cell.
  • the growth rate of the Leishmania cell is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of a wild-type Leishmania cell.
  • the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding UDP-GalNAc biosynthetic pathway proteins capable of generating UDP-GalNAc.
  • the recombinant UDP-GalNAc biosynthetic pathway proteins are heterologous to the Leishmania host cell.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting GalNAc to UDP-GalNAc.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc are derived from a mammalian source.
  • the mammalian source is Homo sapiens.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting N- Acetyl galactosamine 1 -phosphate (GalNAc- 1-P) to UDP-GalNAc.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting GalNAc- 1-P and UTP to UDP-GalNAc.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1), or a functionally active variant thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) of Homo sapiens, or a functionally active variant thereof.
  • the heterologous UDP- GalNAc biosynthetic pathway proteins comprise UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) of Homo sapiens, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP- N-acetyl hexosamine pyrophosphorylase (UAP1) of Homo sapiens.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise a variant of UDP-N- acetyl hexosamine pyrophosphorylase (UAP1) of Homo sapiens that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.
  • the AGX1 isoform of UDP-N-acetyl hexosamine pyrophosphorylase is about two to three times more active towards GalNAc-l-P than GlcNAc-1-P, whereas the AGX2 isoform is about eight times more active towards GlcNAc-1-P than GalNAc-l-P.
  • the UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) is the AGX1 isoform of UAP1.
  • the UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) is the AGX2 isoform of UAP1.
  • the UDP-N-acetyl hexosamine pyrophosphorylase (UAP1) comprises an amino acid sequence of SEQ ID NO: 15.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting GalNAc to GalNAc-l-P.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2), or a functionally active variant thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2) of Homo sapiens, or a functionally active variant thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N- acetyl galactosamine kinase (GALK2) of Homo sapiens, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise N-acetyl galactosamine kinase (GALK2) of Homo sapiens.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise a variant of N-acetyl galactosamine kinase (GALK2) of Homo sapiens that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.
  • the N-acetyl galactosamine kinase (GALK2) comprises an amino acid sequence of SEQ ID NO: 15.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins are capable of converting UDP-GlcNAc to UDP-GalNAc.
  • the heterologous UDP-GalNAc biosynthetic pathway protein capable of converting UDP- GlcNAc to UDP-GalNAc comprises a NAD-dependent epimerase that converts UDP- GlcNAc to UDP-GalNAc.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting UDP-GlcNAc to UDP-GalNAc is derived from a mammalian source.
  • the mammalian source is Homo sapiens.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP -galactose 4-epimerase (GalE), or a functionally active variant thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise UDP -galactose 4-epimerase (GalE) of Homo sapiens (hGalE), or a functionally active variant thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway protein comprise hGalE, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise hGalE.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise 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 thereof.
  • the hGalE comprises an amino acid sequence of SEQ ID NO: 17.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins capable of converting UDP- GlcNAc to UDP-GalNAc are derived from a bacterial source.
  • the bacterial source is Campylobacter jejuni.
  • the heterologous UDP- GalNAc biosynthetic pathway proteins comprise UDP-GlcNAc/Glc 4-epimerase of Campylobacter jejuni (CjGne), or a functionally active variant thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise CjGne.
  • the heterologous UDP-GalNAc biosynthetic pathway proteins comprise comprise 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 thereof.
  • the CjGne comprises an amino acid sequence of SEQ ID NO: 18.
  • the Leishmania host cell comprises (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to UDP-GalNAc as described in this Section; and (b) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway as described in Section 7.3.5.
  • the recombinant nucleic acid in (a) encodes UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc- 1-P to UDP-GalNAc and UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to GalNAc- 1-P, as described in this Section.
  • 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 transporter protein capable of transporting UDP-GalNAc to the secretory pathway as described in Section 7.3.5.
  • the Leishmania host cell comprises (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins 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.
  • the recombinant nucleic acid in (a) encodes UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc- 1-P to UDP-GalNAc and UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to GalNAc- 1-P, as described in this Section.
  • the Leishmania host cell comprises (a) a recombinant nucleic acid encoding recombinant UDP-GalNAc biosynthetic pathway proteins 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; and (c) a recombinant nucleic acid encoding a recombinant UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway as described in Section 7.3.5.
  • the recombinant nucleic acid in (a) encodes UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc- 1-P to UDP-GalNAc and UDP-GalNAc biosynthetic pathway proteins capable of converting GalNAc to GalNAc- 1-P, as described in this Section.
  • the Leishmania host cells provided herein comprise a recombinant nucleic acid encoding a UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway.
  • the UDP- GalNAc transporter protein is heterologous to the Leishmania host cell.
  • the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is derived from a nematode source.
  • the nematode source is C. elegans.
  • the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises UDP-GalNAc transporter of C. elegans (CeC03H5.2), or a functionally active variant thereof.
  • the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises CeC03H5.2, or a variant that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homologous thereof.
  • the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway comprises CeC03H5.2.
  • the UDP-GalNAc transporter protein capable of transporting UDP-GalNAc to the secretory pathway is CeC03H5.2. In certain embodiments, the UDP-GalNAc transporter protein capable of transporting UDP- GalNAc to 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 thereof.
  • the UDP-GalNAc transporter protein capable of transporting UDP- GalNAc to 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 thereof.
  • the CeC03H5.2 has an amino acid sequence of SEQ ID NO: 19.
  • 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.
  • 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.
  • 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.
  • the method may be used to accomplish the introduction of one or more genes encoding a GalNAc transferase as described in Section 7.3.1. In certain embodiments, the method may be used to accomplish the introduction of one or more genes encoding an additional recombinant glycosyltransferase as described in Section 7.3.2. In certain embodiments, the method may be used to accomplish the functional inactivation of one or more genes encoding an enzyme that catalyzes the formation of O-linked GlcNAc as described in Section 7.3.3.
  • the method may be used to accomplish the introduction of one or more genes encoding a heterologous UDP-GalNAc biosynthetic pathway proteins as described in Section 7.3.4. In certain embodiments, the method may be used to accomplish the introduction of one or more genes encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.3.5. In certain embodiments, the strain of the engineered Leishmania host cell is described in Section 7.3.6.
  • nucleic acids are introduced into the host cells described herein using a plasmid, e.g., the heterologous nucleic acids are expressed in the host cells by a plasmid (e.g., an expression vector), and the plasmid is introduced into the modified host cells by transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation.
  • a plasmid e.g., an expression vector
  • said plasmid is introduced into the modified host cells by stable transfection.
  • linearized nucleic acids are introduced into the host cells described herein using transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation.
  • heterologous nucleic acids are integrated site-specifically into the host cell genome by homologous recombination.
  • the method of engineering the Leishmania host cell comprises introducing one or more genes encoding a GalNAc transferase as described in Section 7.3.1.
  • the method of engineering the Leishmania host cell comprises introducing one or more genes encoding an additional recombinant glycosyltransferase as described in Section 7.3.2.
  • the method of engineering the 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.
  • the method comprises downregulating the gene encoding the at least one GlcNAc-transferase.
  • the method comprises deleting the gene encoding the at least one GlcNAc-transferase.
  • the method comprises mutating the gene encoding the at least one GlcNAc-transferase.
  • the method comprises overexpressing the gene encoding the at least one GlcNAc-transferase.
  • the method comprises functionally inactivating the gene encoding an enzyme that catalyzes the formation of O-linked GlcNAc using the methods described in the Assay or Example Sections (Sections 7.7 and 8. , respectively). In certain embodiments, the method comprises functionally inactivating the gene encoding an enzyme that catalyzes the formation of O-linked GlcNAc using any method known in the art, for example methods described in International Publication No. W02019/002512 A2, incorporated by reference in its entirety herein.
  • Non-limiting exemplary mutagenesis approaches include site directed mutagenesis using targeted gene editing techniques such as TALENs , ZFNs, CRISPR/Cas9; in combination with a repair scaffold 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), 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), pp.
  • targeted gene editing techniques such as TALENs , ZFNs, CRISPR/Cas9
  • a repair scaffold for directed, homologous recombination mediated repair Zhang, W et al. (2017) mSphere 2 (1); Gupta, R. and Mus
  • RNA interference (Lye, L. et al. (2010) PLoS Pathog 6 (10), elOOl 161), conditional knock-down using Cre/LoxP or FRT/FLP (Duncan, S. (2017) Molecular and Biochemical Parasitology 216: 30-38).
  • Overexpression may be accomplished by the following non-limiting exemplary approaches, such as gene copy number increase by introduction of additional copies into separate loci (Beverley, S. (1991): Gene amplification in Leishmania. In d/w//. Rev. Microbiol. 45, pp. 417-444), high expression 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.
  • the method of engineering the Leishmania host cell comprises introducing one or more genes encoding a heterologous UDP-GalNAc biosynthetic pathway proteins as described in Section 7.3.4.
  • the method of engineering the Leishmania host cell comprises introducing one or more genes encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.3.5.
  • the method of engineering the Leishmania host cell comprises (i) functionally inactivating one or more genes encoding an enzyme that catalyzes the formation of O-linked GlcNAc as described in Section 7.3.3; (ii) introducing one or more genes encoding a heterologous UDP-GalNAc biosynthetic pathway proteins as described in Section 7.3.4; (iii) introducing one or more genes encoding a heterologous UDP-GalNAc transporter protein as described in Section 7.3.5; (iv) introducing one or more genes encoding a GalNAc transferase as described in Section 7.3.1; and (v) introducing one or more genes encoding an additional recombinant glycosyltransferase as described in Section 7.3.2.
  • the method comprises conducting steps (i)-(v) in sequential order. In other embodiments, steps (i)-(v) are conducted in a different order. For example, in certain embodiments, steps (ii) and (iii) are conducted before step (i). In other embodiments, step (iv) and/or (v) are conducted before step (i). In certain embodiments, step (v) is conducted before step (iv). In certain embodiments, step (v) is conducted first.
  • steps (i)-(v) may be conducted simultaneously, for example by introducing the genes in a single module.
  • steps (ii) and (iii) are conducted simultaneously.
  • step (v) is conducted before step (iv).
  • step (v) is conducted first.
  • step (i) is conducted, followed by steps (ii) and (iii), which are conducted simultaneously, and then step (iv) separately.
  • steps (ii) and (iii) are conducted simultaneously and before step (iv), and step (iv) is conducted before step (i).
  • step (i) is conducted, and steps (ii), (iii), and (iv) are conducted simultaneously after step (i).
  • step (v) is conducted before step (iv).
  • step (v) is conducted first.
  • the Leishmania host cells may be engineered using the methods described in the Assay and Examples Sections (Sections 7.7 and 8. , respectively).
  • the Leishmania host cells are cultured using any of the standard culturing techniques known in the art. For example, cells are routinely grown in rich media like Brain Heart Infusion, Trypticase Soy Broth or Yeast Extract, all containing 5 pg /ml Hemin. Additionally, incubation is done at 26°C in the dark as static or shaking cultures for 2-3 days. In some embodiments, cultures of recombinant cell lines contain the appropriate selective agents. Non-limiting exemplary selective agents are provided in Table 2.
  • the Leishmania host cells are cultured in a growth medium comprising GalNAc.
  • the growth medium comprises at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, at least 12 mM, at least 13 mM, at least 14 mM, at least 15 mM, at least 16 mM, at least 17 mM, at least 18 mM, at least 19 mM, or at least 20 mM GalNAc.
  • the growth medium comprises about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 15 mM, or 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 about 10 mM GalNAc.
  • the Leishmania host cells are cultured in a growth medium comprising GlcNAc.
  • the growth medium comprises at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, at least 12 mM, at least 13 mM, at least 14 mM, at least 15 mM, at least 16 mM, at least 17 mM, at least 18 mM, at least 19 mM, or at least 20 mM GlcNAc.
  • the growth medium comprises about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 15 mM, or 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.
  • the Leishmania host cells may be cultured using the methods described in the Assay and Examples Sections (Sections 7.7 and 8. , respectively).
  • Leishmania host cell described in Section 7.3 may be used as an expression system for making a glycoengineered bifunctional degrader described in Section 7.1 or a population of glycoengineered bifunctional degraders described in Section 7.2.
  • the glycoengineered bifunctional degrader may be a heterologous, non-Leishmania protein, such as a therapeutic protein (e.g., an antibody).
  • the Leishmania host cells may be engineered as described in Sections 7.4 and cultured as described in Section 7.5.
  • Leishmania host cells for use as expression systems are known and may also be used, for example, see WO 2019/002512, WO 2021/140144 and WO 2021/140143, each of which are incorporated herein by reference in their entirety.
  • Use of Leishmania host cells to make monoclonal antibodies are also known. Exemplary methods are described in WO 2022/053673, which is incorporated herein by reference in its entirety.
  • the Leishmania host cells may be used as an expression system for producing a glycoengineered bifunctional degrader according to the methods described in the Assay and Examples Sections (Sections 7.7 and 8. , respectively).
  • compositions comprising the Leishmania host cells described in Section 7.3.
  • Such compositions can be used in methods for generating a glycoengineered bifunctional degrader as described in Section 7.1 or a population of glycoengineered bifunctional degraders as described in Section 7.2.
  • the compositions comprising Leishmania host cells can be cultured under conditions suitable for the production of glycoengineered bifunctional degraders. Subsequently, the glycoengineered bifunctional degrader can be isolated from said compositions comprising Leishmania host cells using methods known in the art.
  • compositions comprising the Leishmania host cells can comprise additional components suitable for maintenance and survival of the Leishmania host cells, and can additionally comprise additional components required or beneficial to the production of glycoengineered bifunctional degraders by the Leishmania host cells, e.g., inducers for inducible promoters, such as arabinose, IPTG.
  • inducers for inducible promoters such as arabinose, IPTG.
  • provided herein are methods for making a glycoengineered bifunctional degrader, for example, one described in Section 7.1.
  • a method of producing a glycoengineered bifunctional degrader in vivo using a. Leishmania host cell described in Section 7.3.
  • a method for producing a glycoengineered bifunctional degrader comprising (i) culturing a Leishmania host cell described in Section 7.3 under conditions suitable for polypeptide production and (ii) isolating said glycoengineered bifunctional degrader.
  • the Leishmania host cell comprises: (a) a recombinant nucleic acid encoding a glycoengineered bifunctional degrader; and (b) a recombinant nucleic acid encoding one or more recombinant N-acetylgalactosamine (GalNAc) transferases.
  • the Leishmania host cell is capable of producing glycoengineered bifunctional degraders comprising a biantennary, GalNAc-terminated N-glycan.
  • the Leishmania host cells provided herein is capable of producing glycoengineered bifunctional degraders comprising an N-glycan of the following structure: wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the glycoengineered bifunctional degrader.
  • glycoengineered bifunctional degraders described herein are made according to methods described in ‘GLYCOENGINEERING USING LEISHMANIA CELLS’, filed September 27, 2023 as an international application with the European Receiving Office, which claims priority to U.S. Provisional Application Nos. 63/410,936 and 63/410,955, and which is incorporated herein in its entirety.
  • the glycoengineered bifunctional degrader produced by the Leishmania host cell is a therapeutic polypeptide, /. ⁇ ., a polypeptide used in the treatment of a disease or disorder.
  • the glycoengineered bifunctional degrader produced by the Leishmania host cell can be an enzyme, a cytokine, or an antibody.
  • a list of nonlimiting exemplary polypeptides of interest is provided in Section 7.1.
  • nucleic acid sequence of a known protein e.g., a monoclonal antibody
  • a newly identified protein e.g., a monoclonal antibody
  • nucleic acid that encodes any glycoengineered bifunctional degrader into a host cell provided herein (e.g., via an expression vector, e.g., a plasmid, e.g., a site specific integration by homologous recombination).
  • the target protein is a cell surface molecule or a non-cell surface molecule.
  • the cell surface molecule is a receptor.
  • the non-cell surface receptor is an extracellular protein.
  • 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).
  • the target protein associated with a disease is upregulated in the disease compared to a non-disease state. In some embodiments, the target protein associated with a disease is expressed in the disease compared to a non-disease state. In some embodiments, the target protein associated with a disease 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 cancer progression. In some embodiments the disease is an autoimmune disease. In some embodiments, the disease is neurodegenerative disease.
  • the disease is Graves’ disease.
  • Graves’ disease is the most common cause of hyperthyroidism. Prevalence in the US is 1.2% (1), with lifetime risk in women as high as 3%. Production of agonistic anti-TSH Receptor (TSHR) antibodies (TRAb) leading to over production of thyroxine hormone (> 90% of patients are TRAb+) (2). Current treatments have not advanced in the 50 years and are limited by high risk of recurrence or severe side effects such as hypothyroidism.
  • the target protein associated with Graves’ disease is an autoantibody binding TSHR. In other embodiments, the target protein associated with Graves’ disease is TSHR.
  • the target protein comprises a protein selected from the group consisting of TNFa, HER2, EGFR, HER3, VEGFR, CD20, CD19, CD22, avp3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptors, EGF, c-MET, CCL2, CCR2 Frizzled receptors, Wnt, LRP5/6 , CSF-1R, SIRPa, CD38, CD73, TGF-p, TSHRa, AChR-al, noncollagen domain 1 of the a3 chain of type IV collagen (a3NCl), ADAMTS13, Desmoglein-1/3, GPIb/IX, GPIIb/IIIa, GPIa/IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin and gangliosides GM
  • the target protein comprises an antibody that binds to TSHRa, MOG, AChR-al, noncollagen domain 1 of the a3 chain of type IV collagen (a3NCl), ADAMTS13, Desmoglein-1/3, GPIb/IX, GPIIb/IIIa, GPIa/IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin and gangliosides GM1, GD3, and GQ1B.
  • the target protein is a protein that is upregulated in cancer. In some embodiments, the target protein is a protein that is involved in cancer progression.
  • target proteins that are upregulated in cancer or involved in cancer progression that can be bound by a glycoengineered bifunctional degrader provided herein include, but are not limited to TNFa, HER2, EGFR, HER3, VEGFR, CD20, CD 19, CD22, avP3 integrin, CEA, CXCR4, MUC1, LCAM1, EphA2, PD-1, PD-L1, TIGIT, TIM3, CTLA4, VISTA, Notch receptors, EGF, c-MET, CCL2, CCR2 Frizzled receptors, Wnt, LRP5/6 , CSF-1R, SIRPa, CD38, CD73, TGF-p, Bombesin R, CAIX, CD13, CD44v6, Emmprin, Endoglin, EpCAM, EphA2, FAP-a, Folate R,
  • the target protein is an autoantibody, such as those associated with an autoimmune disease.
  • an autoantibody that can be bound by a glycoengineered bifunctional degrader include, but are not limited to, autoantibodies directed against TSHRa, MOG, AChR-al, noncollagen domain 1 of the a3 chain of type IV collagen (a3NCl), ADAMTS13, Desmoglein-1/3, or GPIb/IX, GPIIb/IIIa, GPIa/IIa, NMDA receptor, glutamic acid decarboxylase (GAD), amphiphysin and gangliosides GM1, GD3, GQ1B.
  • the target protein comprises a protein that is upregulated or expressed in tumor associated macrophages (TAMs).
  • TAMs tumor associated macrophages
  • the target protein is upregulated or expressed in pro-tumor TAMs.
  • target proteins that are upregulated or expressed in TAMs comprise SIRPa, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, or CXCR4 (9*).
  • the target proteins comprise CCL2, CXCL12, CSF-1 or CD47 (9*). These targets play a role in promoting pro-tumor TAMs particularly by promoting TAM recruitment and programming.
  • the target protein is a protein that is upregulated or expressed in a neurodegenerative disease.
  • target proteins that are upregulated or expressed in neurodegenerative diseases comprise alpha-synuclein, amyloid beta or complement cascade component.
  • the target protein is a protein that is upregulated or expressed in systemic amyloidosis or localized amyloidosis. In some embodiments, the target protein that is upregulated or expressed in systemic amyloidosis is transthyretin.
  • provided herein are methods of preventing or treating a disease or disorder in a subject comprising administering to the subject a glycoengineered bifunctional degrader described in Section 7.1 (including pharmaceutical compositions thereof) or a population of glycoengineered bifunctional degraders described in Section 7.2 (including pharmaceutical compositions thereof). Further provided herein are methods of preventing a disease or disorder in a subject comprising administering to the subject a glycoengineered bifunctional degrader or a population thereof.
  • provided herein are methods of treating a disease or disorder in a subject comprising administering to the subject a glycoengineered bifunctional degrader described herein or a population thereof.
  • methods of preventing a disease or disorder in a subject comprising administering to the subject a glycoengineered bifunctional degrader described herein or a population thereof.
  • a method for treating or preventing a disease or disorder in a subject comprising administering to the subject a glycoengineered bifunctional degrader produced according to the methods described herein, wherein the glycoengineered bifunctional degrader is glycosylated with an N-glycan of the following structure: wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and wherein X represents an amino acid residue of the glycoengineered bifunctional degrader.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • the disease or disorder may be caused by the presence of a defective version of a glycoengineered bifunctional degrader in a subject, the absence of a glycoengineered bifunctional degrader in a subject, diminished expression of a glycoengineered bifunctional degrader in a subject can be treated or prevented using the glycoengineered bifunctional degrader produced using the methods described herein.
  • the diseases or disorder may be mediated by a receptor that is bound by a glycoengineered bifunctional degrader produced using the methods described herein, or mediated by a ligand that is bound by a glycoengineered bifunctional degrader produced using the methods described herein (e.g., where the glycoengineered bifunctional degrader is a receptor for the ligand).
  • the methods of preventing or treating a disease or disorder in a subject comprise administering to the subject an effective amount of a glycoengineered bifunctional degrader described herein or a population thereof.
  • the effective amount is the amount of a therapy which has a prophylactic and/or therapeutic effect(s).
  • an “effective amount” refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a disease/disorder or symptom associated therewith; (ii) reduce the duration of a disease/disorder or symptom associated therewith; (iii) prevent the progression of a disease/disorder or symptom associated therewith; (iv) cause regression of a disease/disorder or symptom associated therewith; (v) prevent the development or onset of a disease/disorder, or symptom associated therewith; (vi) prevent the recurrence of a disease/disorder or symptom associated therewith; (vii) reduce organ failure associated with a disease/disorder; (viii) reduce hospitalization of a subject having a disease/disorder; (ix) reduce hospitalization length of a subject having a disease/disorder; (x) increase the survival of a subject with a disease/disorder; (i) reduce or
  • a method of treating or preventing a disease in a patient comprising administering to the patient a glycoengineered bifunctional degrader described herein or a population described herein.
  • the disease is an autoimmune disease, a cancer or tumor, a liver disease, an inflammatory disorder, or a blood disorder.
  • the autoimmune disease is selected from Graves’ Disease, Myasthenia Gravis, Anti-GBM Disease, Immune Thrombotic Thrombocytopenic Purpura, Acquired Pemphigus Vulgaris, Immune Thrombocytopenia, Guillain-Barre Syndrome, and Membranous Nephropathy.
  • 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.
  • a method of treating or preventing a disease provided herein includes an administration step that comprises intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection, or intramuscular injection of a glycoengineered bifunctional degrader described herein or a population described herein.
  • a method of treating or preventing a disease provided herein requires a lower dose and/or lower administration frequency to achieve the same effect as compared to the same antibody having a different glycosylation profile; and/or can be administered for an extended 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 does not trigger an immune response against the glycoengineered bifunctional degrader in the patient.
  • a suitable dose of a glycoengineered bifunctional degrader described herein is the amount corresponding to the lowest dose effective to produce a therapeutic effect.
  • an effective amount of an anti-TSH receptor antibody can be an amount that inhibits TSH activity in a subject suffering from a Graves’ disease.
  • the amount of glycoengineered bifunctional degrader described herein administered to a patient is less than the amount listed in the label of a drug product of the same glycoengineered bifunctional degrader having a different glycosylation profile from that of the glycoengineered bifunctional degrader described herein.
  • the accumulated amount of a glycoengineered bifunctional degrader described herein administered to a patient over a period of time is less than the accumulated amount indicated in the label of a drug product of the same glycoengineered bifunctional degrader having a different glycosylation profile from that of the glycoengineered bifunctional degrader described herein.
  • the reduced accumulated amount could be administered in reduced doses on a reduced frequency.
  • the reduced accumulated amount could be administered in one or more doses that are the same or higher than the dose in the label on a reduced frequency.
  • the reduced accumulated amount could be administered in one or more reduced doses on a frequency that is the same or higher than the frequency in the label.
  • the reduced accumulated amount could be administered over a shorter period of time than the period of time for the drug product to achieve the same level of effect in treatment or prevention.
  • the amount of the glycoengineered bifunctional degrader described herein in a single dose administered to a patient can be from 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.
  • the amount of glycoengineered bifunctional degrader described herein in a single dose administered to a patient can be from about 5 to about 80 mg.
  • the amount of glycoengineered bifunctional degrader described herein in a single dose administered to a patient can be from about 25 to about 50 mg. In some embodiments, the amount of a glycoengineered bifunctional degrader described herein in a single dose administered to a patient can from about 15 mg to about 35 mg. [00270] In some embodiments, the amount of a glycoengineered bifunctional degrader described herein in a single dose administered to a patient can be no more than 40 mg, for example 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.
  • the amount of a glycoengineered bifunctional degrader described herein in a single dose administered to a patient can be no more than 80 mg, for example 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.
  • the amount of a glycoengineered bifunctional degrader described herein in a single dose administered to a patient can be no more than 160 mg, for example 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.
  • the amount of a glycoengineered bifunctional degrader described herein in a single dose administered to a patient can be equal to or more than 160 mg, for example 170 mg, 180 mg, 200 mg, 250 mg, and 300 mg.
  • a glycoengineered bifunctional degrader of the disclosure can be administered on a frequency that is every other week, namely every 14 days. In some embodiments, a glycoengineered bifunctional degrader of the disclosure can be administered on a frequency that is lower than every 14 days, for example, every half a month, every 21 days, monthly, every 8 weeks, bimonthly, every 12 weeks, every 3 months, every 4 months, every 5 months, or every 6 months. In some embodiments, a glycoengineered bifunctional degrader of the disclosure can be administered on a frequency that is the same or higher than every 14 days, for example, every 14 days, every 10 days, every 7 days, every 5 days, every other day, or daily.
  • the administration of a glycoengineered bifunctional degrader of the disclosure can comprise an induction dose that is higher than the following doses, for example the following maintenance doses.
  • the administration of a glycoengineered bifunctional degrader of the disclosure can comprise a second dose that is lower than the induction dose and higher than the following maintenance doses.
  • the administration of a glycoengineered bifunctional degrader of the disclosure can comprise the same amount of the glycoengineered bifunctional degrader in all the doses throughout the treatment period.
  • a method of treating an acute condition associated with increased levels of a target protein comprises administering to a patient in need thereof a glycoengineered bifunctional degrader described herein, wherein the method results in a half-life that is at least 50%, 60%, 70%, 80%, 90% or 99% of the bifunctional degrader without any glycosylation.
  • the halflife of the target protein in the presence of a bifunctional degrader provided herein in a patient is 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours.
  • a method of treating a chronic condition associated with increased levels of a target protein comprises administering to a patient in need thereof a bifunctional degrader described herein, wherein the method results in a half-life that is at least 50%, 60%, 70%, 80%, 90% or 99% of the bifunctional degrader without any glycosylation in the patient.
  • 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.
  • a method of treating a chronic condition associated with increased levels of a target protein comprising administering to a patient in need thereof a bifunctional degrader described herein, wherein the bifunctional degrader (i) specifically binds to the target protein and (ii) comprises an N- glycan of the following structure: wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the glycoengineered bifunctional degrader, and wherein the N-glycan is linked to the bifunctional degrader 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 the patient in the absence of the bifunctional degrader
  • the chronic condition is an autoimmune disease, a cancer or tumor, a liver disease, an inflammatory disorder, or a blood coagulation disorder.
  • the autoimmune disease is selected from Graves’ Disease, Myasthenia Gravis, Anti-GBM Disease, Immune Thrombotic Thrombocytopenic Purpura, Acquired Pemphigus Vulgaris, Immune Thrombocytopenia, autoimmune encephalitis, Guillain-Barre Syndrome, and Membranous Nephropathy.
  • the cancer comprises a solid tumor.
  • the cancer comprises a blood-borne cancer or tumor.
  • the cancer may be a carcinoma or a sarcoma.
  • 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, or others), bone cancer (e.g., osteosarcoma), cervical cancer, cholangiocarcinoma, digestive tract cancer (e.g., oral, esophageal, stomach, colon or rectal cancer), head and neck cancer, leiomyosarcoma, liposarcoma, liver cancer (e.g., hepatocellular carcinoma), mesothelioma, nasopharyngeal cancer, neuroendocrine cancer, ovarian
  • lung cancer small cell or non-small cell
  • breast cancer gastric cancer
  • the cancer can be relapsed following a previous therapy, or refractory to conventional therapy. In certain embodiments, the cancer can be disseminated or metastatic.
  • the blood-borne cancer or tumor is selected from leukemia, myeloma (e.g., multiple myeloma) lymphoma (e.g., Hodgkin’s lymphoma or nonHodgkin’s lymphoma).
  • the leukemia is chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, acute myelogenous leukemia and acute myeloblastic leukemia.
  • treatment comprises reprogramming tumor associated macrophages (TAMs) by administering the bifunctional degrader under conditions to mediate endocytosis of a target protein.
  • TAMs tumor associated macrophages
  • the target protein is upregulated or expressed in TAMs.
  • the target protein upregulated or expressed in TAMs comprises SIRPa, CCR2, CSF-1R, LILRB1, LILRB2, VEGF-R, CXCR4, CCL2, CXCL12, CSF-1 or CD47.
  • the administration step comprises intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection, or intramuscular injection.
  • a method of delivering a target protein to a hepatocyte endosome comprises contacting the target protein with any of the glycoengineered bifunctional degraders disclosed herein under conditions to mediate endocytosis of any of the target proteins disclosed herein.
  • the method of delivering the target protein to a hepatocyte endosome occurs in vivo.
  • the mode of delivering a target protein to a hepatocyte endosome in vivo comprises intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal injection or intramuscular injection.
  • the method of delivering the target protein to a hepatocyte endosome occurs ex vivo.
  • the rate of delivery can be increased based on the number of N-glycan structures present on the glycoengineered bifunctional degrader. In some embodiments, increasing the number of N-glycan structures on the glycoengineered bifunctional degrader increases the rate of delivery.
  • the glycoengineered bifunctional degrader can 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 structure: wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the glycoengineered bifunctional degrader.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • X represents an amino acid residue of the glycoengineered bifunctional degrader.
  • a method of degrading a target protein comprises contacting the target protein with any of the glycoengineered bifunctional degraders disclosed herein under conditions to mediate degradation of any of the target proteins disclosed herein by a host cell.
  • degradation is lysosomal degradation.
  • degradation is mediated by endocytosis or phagocytosis.
  • the method comprises degrading a structure comprising at least one target protein bound to at least one glycoengineered bifunctional degrader.
  • 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.
  • 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 higher than degradation mediated by a glycoengineered bifunctional degrader not comprising an at least one or at least two N-glycans of the following structure: wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the glycoengineered bifunctional degrader.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • the glycoengineered bifunctional degrader enhances degradation of any of the disclosed target proteins relative to degradation of the target protein in the presence of a glycoengineered bifunctional degrader not comprising at least one or at least two of the N-glycans.
  • increasing the rate and/or efficiency of internalization of the glycoengineered bifunctional degrader increases the rate and/or activity of lysosomal degradation.
  • the rate and/or efficiency of internalization of the glycoengineered bifunctional degrader is proportional to the rate and/or activity of lysosomal degradation.
  • relative changes in the rate and/or efficiency of internalization of the glycoengineered bifunctional degrader are determined from relative changes in the rate and/or activity of lysosomal degradation.
  • the rate and/or efficiency of internalization can be regulated through glycoengineering. In some embodiments, the rate and/or efficiency of internalization can be regulated based on the number of N-glycan structures present on the glycoengineered bifunctional degrader. 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 degrader. In some embodiments, increasing the number of N- glycan structures on the glycoengineered bifunctional degrader increases the rate and/or efficiency of internalization.
  • increasing the number of N-glycan structures on the glycoengineered bifunctional degrader by one N-glycan increases the rate and/or efficiency of internalization as compared to a glycoengineered bifunctional degrader comprising one fewer N-glycan.
  • the increase in rate and/or efficiency of internalization resulting from increasing the number of N-glycan structures on the glycoengineered bifunctional degrader by one N-glycan is more than additive.
  • the increase in rate and/or efficiency of internalization resulting from increasing the number of N-glycan structures on the glycoengineered bifunctional degrader by one N-glycan is about 10% or more, about 20% or more, about 30% or more, 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 than the rate and/or efficiency of internalization per N-glycan for a glycoengineered bifunctional degrader comprising one less, two less, three less, or four less N-glycans.
  • the glycoengineered bifunctional degrader can 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 structure: wherein the black square represents an N-acetyl galactosamine (GalNAc), the white square represents an N-acetylglucosamine (GlcNAc) residue and the black circle represents a mannose (Man) residue, and X represents an amino acid residue of the glycoengineered bifunctional degrader.
  • GalNAc N-acetyl galactosamine
  • Man mannose
  • X represents an amino acid residue of the glycoengineered bifunctional degrader.
  • the presence of two or more of the N-glycans on the glycoengineered bifunctional degrader can increase the rate and/or efficiency of internalization relative to a glycoengineered bifunctional degrader comprising only one of the N-glycan.
  • the rate and/or efficiency of internalization can be finetuned. 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. For the treatment of an acute condition, rapid internalization of the complex between a bifunctional degrader provided herein bound to its target protein(s) would be desired.
  • N-glycosylation sites can be introduced and linked to the N-glycan, which in turn results in a rapid internalization and low half lifes of the target protein of less than 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, two hours, three hours, or less than four hours.
  • the method comprises administering to a patient in need thereof a bifunctional degrader, wherein the bifunctional degrader (i) specifically binds to the target protein and (ii) comprises the N-glycan, wherein the N-glycan is linked to the bifunctional degrader 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 degrader or in the absence of any treatment.
  • the rate and/or efficiency of internalization of the bifunctional degrader can be regulated based on location of the N-glycan(s) on the bifunctional degrader. In some embodiments, the rate and/or efficiency of internalization can be increased based on the location of the N-glycans(s) on the bifunctional degrader. In certain embodiments, the rate and/or efficiency of internalization of the bifunctional degrader is enhanced by engineering the bifunctional degrader so as to comprise one or more N- glycosylation sites distal to a target-specific binding location of the bifunctional degrader.
  • the rate and/or efficiency of internalization of the bifunctional degrader is enhanced by engineering the bifunctional degrader so as to comprise at least one or at least two N-glycosylation sites distal to the target-specific binding location. In certain embodiments, the rate and/or efficiency of internalization of the bifunctional degrader is enhanced by engineering the bifunctional degrader so as to comprise all N-glycosylation sites distal to the target-specific binding location.
  • the efficiency of target engagement and internalization by a bifunctional degrader is enhanced relative to an unmutated form of the bifunctional degrader by engineering the bifunctional degrader so as to delete, mutate, or functionally inactivate N-glycosylation sites present in a wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader (“natural N-glycosylation sites”).
  • the efficiency of target engagement and internalization by the bifunctional degrader is enhanced relative to an unmutated form of the bifunctional degrader by engineering the bifunctional degrader so as to delete, mutate, or functionally inactivate at least one or at least two natural N-glycosylation sites.
  • the efficiency of target engagement and internalization by the bifunctional degrader is enhanced relative to an unmutated form of the bifunctional degrader by engineering the bifunctional degrader so as to delete, mutate, or functionally inactivate all natural N- glycosylation sites.
  • efficiency of target engagement and internalization by the bifunctional degrader is enhanced relative to an unmutated form of the bifunctional degrader by engineering the bifunctional degrader so as to delete, mutate, or functionally inactivate one or more natural N-glycosylation sites are located at or proximal to the target-specific binding location of the wild-type, natural, synthetic, or commercial precursor of the bifunctional degrader.
  • the target-specific binding location is a variable region of an antibody or antigen-binding fragment (Fab), or an ectodomain of an Fc-fusion protein.
  • the efficiency of target degradation by a mutated form of the bifunctional degrader is enhanced relative to the unmutated form of the bifunctional degrader by engineering the bifunctional degrader so as to delete, mutate, or functionally inactivate one or more N-glycosylation sites present in a wildtype, natural, synthetic, or commercial precursor of the bifunctional degrader (“natural N- glycosylation sites”).
  • the efficiency of target degradation is enhanced by eliminating or decreasing the number of N-glycosylation sites located at or proximal to the target-specific binding location.
  • eliminating or decreasing the number of A2GalNAc2 glycans located at or proximal to the target-specific binding location can reduce the rate of internalization and clearance of the bifunctional degrader before target binding (i.e. of an unbound form of the degrader) as compared to after target binding (i.e. to a bound form of the degrader).
  • eliminating or decreasing the number of A2GalNAc2 glycans located at or proximal to the target-specific binding location can increase the probability that the bifunctional degrader binds to its target before being internalized via ASGPR.
  • the number of N-glycosylation sites located at or proximal to the target-specific binding location is eliminated or reduced by deleting, mutating, or functionally inactivating one or more natural N-glycosylation sites.
  • all glycoengineered N-glycosylation sites are distal to the target-specific binding location of the bifunctional degrader.
  • the rate and/or efficiency of internalization of the bifunctional degrader is enhanced by engineering the bifunctional degrader so as to comprise two N-glycosylation sites in the Fab region of the antibody, wherein one N-glycosylation site is located on each of the two heavy chain polypeptides of the antibody and each of said heavy chain N-glycosylation sites is glycosylated by the N- glycan, as compared to engineering the bifunctional degrader so as to comprise two N- glycosylation sites in the Fab region of the antibody, wherein one N-glycosylation site is located on each of the two light chain polypeptides of the antibody and each of said light chain N-glycosylation sites is glycosylated by the N-glycan.
  • rate and/or efficiency of internalization of the bifunctional degrader is enhanced by engineering the bifunctional degrader so as to comprise at least two N-glycosylation sites distal (in terms of the quaternary structure of the antibody) to the hinge region of the antibody, and wherein at least two of the distal N- glycosylation sites are glycosylated by the N-glycan, as compared to a bifunctional degrader comprising N-glycosylation sites proximal (in terms of the quaternary structure of the antibody) to the hinge region of the antibody, and wherein only the proximal N-glycosylation sites are glycosylated by the N-glycan.
  • the bifunctional degrader has been engineered as described in Section 7.1.
  • a method of degrading a target protein comprises GalNAc mediated degradation.
  • GalNAc degradation is optimal due to engagement of endocytic receptors.
  • the method of degrading a target protein via GalNAc mediated degradation is selective.
  • GalNAc degradation removes inflammatory cytokines from circulation, removes unwanted blood factors, removes autoantibodies, removes pathogenic antibodies, removes cell surface receptors, removes protein aggregates and removes extracellular soluble proteins.
  • Host cells are cultured using any of the standard culturing techniques known in the art. For example, cells are routinely grown in rich media like Brain Heart Infusion, Trypticase Soy Broth or Yeast Extract, all containing 5 pg/ml Hemin. In some embodiments, incubation is done at 26°C in the dark as static or shaking cultures for 2-3 days. In some embodiments, cultures of recombinant cell lines contain the appropriate selective agents. In some embodiments, cultures contain Biopterin at a final concentration of 10 pM to support growth.
  • Table 2 Selective agents used during transfection (50% concentration for preselection and 100% concentration for main selection) and standard culturing of L. tarentolae. Double amounts of the selective agents could be used if higher selection pressure was intended.
  • Table 3 Summary of exemplary strains. Some of the strains were produced by several rounds of transfection building on top of each other.
  • Plasmids were derived from a pUC57 vector backbone for E. coli propagation and contained an ampicillin or kanamycin section marker.
  • the expression cassettes are flanked by restriction sites suitable for excision. The composition of the cassettes depends on the intended use and is described in the respective methods and examples.
  • the genes of interest are included as ORFs that were codon usage optimized for L. tarentolae. Optimized sequences were manually curated for avoidance of restriction sites and deletion of repeats or homopolymer stretches.
  • the plasmids were generated and sequenced by a gene synthesis provider. Plasmids and descriptions are found in the sequence listings.
  • codon usage optimization protein sequences were back-translated to nucleotide sequences using a custom Python3 script that stochastically selects codons based on the /.. tarentolae codon usage frequency while excluding rare codons (frequency ⁇ 10%).
  • the codon usage has been calculated using cusp (Rice, et al. (2000) Trends in genetics: TIG 16 (6), pp. 276-277) on all annotated L. tarentolae nucleotide coding sequences.
  • Restriction digest (12 pg DNA in total volume of 240 pL) was performed using standard restriction enzymes (ThermoFisher, preferably FastDigest) according to the manufacturer’s instructions. The restriction digest was performed until completion or o/n at 30°C and DNA was purified by EtOH precipitation (2 volume 100% ice cold EtOH was added to 1 volume digested DNA, incubated 30min on ice, centrifuged for 30 min 17'500 x g at 4°C. Pellet was washed with 70% EtOH and subsequently dried for maximum 15 min and resuspended in ddH20.
  • EtOH precipitation 2 volume 100% ice cold EtOH was added to 1 volume digested DNA, incubated 30min on ice, centrifuged for 30 min 17'500 x g at 4°C. Pellet was washed with 70% EtOH and subsequently dried for maximum 15 min and resuspended in ddH20.
  • the cell pellet was resuspended in the DNA mix and transfected using a 16-well electroporation strip with pulse FI-158 (in some examples alternative pulses FP167, CM150, EO115, DN100, FP 158, FBI 58 were used).
  • pulse FI-158 in some examples alternative pulses FP167, CM150, EO115, DN100, FP 158, FBI 58 were used.
  • a ribonucleoprotein complex formed of the endonuclease SpCas9 and bipartite guideRNAs (gRNA) are transfected into L.
  • tarentolae to introduce double-strand breaks in the 5’ and 3’ regions of the open reading frames encoding OGNT1, OGNT2 and OGNTL.
  • the gRNAs are formed by a scaffold RNA (tracrRNA) and one of the six sequence specific targeting RNAs (crRNA), used in this method.
  • crRNA sequence specific targeting RNAs
  • a selection marker expression construct consisting of two linear DNA fragments is transfected into the cells.
  • the linear DNA pieces are integrated at the former OGNT expression sites by homologous recombination with each other and the 5’ and 3’ untranslated regions of the OGNT gene.
  • the selection marker expression construct does not introduce additional flanking untranslated regions and thus results in transcription of the marker by endogenous PolII.
  • gRNA for CRISPR/Cas9 mediated genome editing was assembled from equimolar amounts of tracrRNA and crRNA (Microsynth) as above by denaturation for 5 min at 95°C and subsequent slow cool down at 0. l°C/s in a thermo cycler. This was done separately for every crRNA used before the different gRNAs were subsequently mixed in equimolar amounts.
  • 122 pmol recombinantly expressed Cas9 protein i.e. Alt-R® S.p. HiFi Cas9 Nuclease V3 (IDT, #1081061) were added to 360 pmol of the gRNA mix and incubated for 15 min at RT to allow RNP formation.
  • the final volume used for a transfection by Nucleofector should not exceed 6 pl.
  • the RNP mix was added to the repair DNA containing transfection solution described below along with 1 pl of Alt-R® Cas9 Electroporation Enhancer (IDT, #1081072).
  • the linear DNA fragments for integration are mixed for transfection in the needed combinations at 1 pg per fragment and the gRNA was prepared as described above and mixed with the integration fragments.
  • the volume of the mix was reduced to maximum 2 pl per transfection in a vacuum concentrator at 30°C.
  • the cell pellet was resuspended in 20 pl of the DNA (or DNA/RNA or DNA/RNP) mix and transfected using a 16-well electroporation strip with pulse FI-158 (in some examples alternative pulses FP167, CM150, EO115, DN100, FP158, FBI 58 were used).
  • pulse FI-158 in some examples alternative pulses FP167, CM150, EO115, DN100, FP158, FBI 58 were used.
  • FI-158 in some examples alternative pulses FP167, CM150, EO115, DN100, FP158, FBI 58 were used.
  • PCR confirmation of OGNT knock-outs was performed by either amplification of the complete locus (OGNT1, OGNT2, OGNTL or OGNT1+L, where OGNT1+L comprises OGNT1 and OGNTL in tandem on the chromosome) or by amplification of the shorter fragments covering the integration sites.
  • amplification of shorter regions with primer binding within the OGNT coding sequence was preferred to test for presence of remaining wt genes (see Table 6).
  • the DreamTaq DNA polymerase (Thermo Fisher Scientific) was used.
  • the correct integration of the selection marker gene into the respective OGNT locus could be tested by combinations of a primer binding in the genome with one primer binding to the selection marker CDS or the intergenic regions of the integrated construct and the other one targeting the genome.
  • Table 5 PCRs for analysis of OGNT deletions. PCR primers used for confirmation OGNT knock-outs by absence of the respective OGNT wt gene and the expected amplicon sizes are summarized. * KO amplicon length for whole locus PCRs depends on the combination of selection marker and intergenic regions used.
  • HRP horse reddish peroxidase
  • secondary antibodies anti-mouse polyvalent-HRP (A0412, Sigma) 1 :2000 diluted or anti-rabbit-HRP conjugate (Jackson ImmunoResearch #111-035-008) 1 :2000 diluted
  • TMB 3,3’,5,5’-tetramethylbenzidine
  • Host cells were routinely grown in 50 ml culture in BHIH or YEH for 48 h at 26°C shaking at 140 rpm. Cultures were harvested and centrifuged for 10 min at 1800*g at RT. Media SN was filtered through 0.22 pm filter (Steriflip, SCGP00525) and EDTA(0.5 M pH8) was added to each load in a 1 : 100 dilution. Media SNs of each strain were subjected to 4h incubation with 100 pl of proteinA resin (ProteinA-Sepharose 4B Fast Flow, Sigma Aldrich, P9424) per Falcon tube in batch while rotating at RT.
  • proteinA resin ProteinA resin
  • Elution fractions were pooled and immediately neutralized by adding 100 mM Tris-HCl (1 M pH8). Afterwards, the pooled elutions were buffer exchanged to PBS pH 6 using 2 ml 7K ZebaSpin desalting columns and optionally concentrated using Amicon 0.5 ml 30 K concentrators.
  • Elution fractions were pooled and immediately neutralized by adding 100 mM Tris-HCl (1 M pH8). Afterwards, the pooled elutions were buffer exchanged to PBS pH 6 using 2 ml 7K ZebaSpin desalting columns and optionally concentrated using Amicon 0.5 ml 30 K concentrators.
  • SDS PAGE was performed under reduced or non-reducing conditions using 10 pg for Coomassie, 2.5 pg for WB, separated on 4-12% Gel with MOPS buffer for 55 minutes. Determination of Protein purity was done by Coomassie Stained SDS-PAGE with 10 pg protein sample and compared to a BSA standard curve. Impurities were quantified by ImageQuant. Capillary Gel Electrophoresis (CGE) was performed using an Agilent Protein 230 Kit (5067-1518), according to protocol.
  • CGE Capillary Gel Electrophoresis
  • MAbPac SEC-1 (4x300 mm) is a size exclusion chromatography (SEC) column specifically designed for separation and characterization of monoclonal antibodies (mAbs) and was used according to manufacturer’s recommendation (Temperature: 30 °C; Eluent: PBS 50mM NaPO4, 300 mM NaCl pH 6.8; Elution: isocratic, 30 minutes; Flow: 0.2 mL/minute; Detection: 215 nm; Injection V: 5 pL corresponding to 5 pg protein).
  • SEC size exclusion chromatography
  • Intact monoclonal antibodies were analyzed by mass spectrometry employing the state-of-the-art instrumentation (Orbitrap FTMS), data processing and data analysis (bioinformatics) tools by SpectroSwiss. In addition to the intact measurement the antibody was reduced with TCEP or enzymatically cleaved (IdeS) to generate Fd, LC and Fc/2 subunits, which were analyzed using the same instrumentation.
  • Orbitrap FTMS state-of-the-art instrumentation
  • bioinformatics bioinformatics
  • Enzymatic release of N-glycans from cell surfaces was performed using PNGase F (New England Biolabs). Cells (grown for 48 or 72 h at 26°C shaking at 140 rpm) were harvested and washed with PBS by centrifugation for 10 min at 1800*g at RT.50 mg of cell pellet were re-suspended in Glyco Buffer 2 and incubated with 1 pl PNGase F for 1 h at 37 °C and 650 rpm. Cells were again pelleted by centrifugation and 75 pl of the supernatant was dried down in a SpeedVac concentrator. Glycans were resuspended in 10 pl of water.
  • PNGase F New England Biolabs
  • glycans were directly labeled with procainamide as described previously (Behrens, et al. (2016) Glycobiology 28 (11), pp. 825-831). Briefly, released glycans were mixed with 1 pl acetic acid, 8 pl of a procainamide stock solution (550 mg/ml in DMSO) and 12 pl of a sodium cyanoborohydride stock solution (200 mg/ml in H2O). Samples were incubated for 60 min at 65°C and cleaned up using LC-PROC-96 clean up plates (Ludger Ltd) according to the manufacturer’s instructions.
  • Enzymatic release of N-glycans from purified proteins was performed using Rapid PNGase F (New England Biolabs) as recommended by the supplier. 8 pl of sample (15 pg of protein) were mixed with 2 pl Rapid Buffer and 1 pl of Rapid PNGase F. The mixture was incubated at 50°C for 10 min followed by 1 min at 90°C.
  • Rapid PNGase F New England Biolabs
  • IgGl mAb was either cleaved with IdeZ to F(ab’)2 and Fc/2, or heavy and light chains were reduced before separation on SDS PAGE. Bands were excised and enzymatic release of N-glycans from the monoclonal antibody was performed using PNGase F. Following release, glycans were directly labeled with procainamide (PC).
  • PC procainamide
  • Procainamide-labeled N-glycans were analyzed by hydrophilic interaction chromatography-ultra performance liquid chromatography-mass spectrometry (HILIC- UPLC-MS) using am Acquity UPLC System (Waters) with fluorescence detection coupled to a Synapt G2-Si mass spectrometer (Waters). Glycans were separated using an Acquity BEH Amide column (130 A, 1.7 pm, 2.1 mM x 150 mM; Waters) with 50 mM ammonium formate, pH 4.4 as solvent A and acetonitrile as solvent B. The separation was performed using a linear gradient of 72-55 % solvent B at 0.5 ml/min for 40 min.
  • MeOH/Chloroform extraction procedure for L. tarentolae cell pellets was performed on 2 OD of each sample, which were harvested by centrifugation and washed 2x with IxPBS (2200 g, 10 min, RT) and frozen.
  • pellets were thawed, resuspended in 480 pl MeOH , supplemented with 20 pl water and sonicated in a water bath at RT for 15 min.
  • the samples were spun in a table-top centrifuge at 18000 g and 4°C for 10 min.
  • the SN was transferred into a glass vial, supplemented with 268 pl chloroform and vortexed.
  • HPAEC-PAD High performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) using CarboPac PAI, 4x250 mm (Thermo), based on Tomiya et al. 2001 : Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Celles and Mammalian Cells. Analytical Biochemistry 293, p. 129-137, with an adjusted gradient.
  • Antibodies were prepared as described in the Section 7.9.1, in particular Section 7.9.1(v)(A).
  • Table 7 shows the protein structure and the main N-glycoform displayed at engineered glycosites for indicated antibodies.
  • the glycan nomenclature is described in Section 5.3.
  • HC Heavy chain.
  • LC light chain.
  • Table 8 shows the formats and N-Glycan structure on the engineered glycosites of antibody fragments or non-antibody molecules.
  • Table 8 Structure of Antibody fragments or non-antibodies and glycoengineering.
  • Table 8 shows the protein structure and the main N-glycoform displayed at engineered glycosites for indicated antibody fragment or non antibody scaffold.
  • the glycan nomenclature is described in Section 5.3.
  • HC Heavy chain.
  • LC light chain.
  • MOG myelin oligodendrocyte protein.
  • EDC Extracellular domain.
  • Fc Fragment cristalizable.
  • Table 9 shows the quality profile of the main antibodies and fragments described herein.
  • N-Glycan analysis was performed using the method described in Section 7.9.3. Aggregation and degradation data were generated using size-exclusion HPLC as further described in section 5.9.3. Unoccupancy means the relative amount of glycosylation sites not carrying a N-glycan as determined using the in-house IP- MS method (Section 5.9.3).
  • the target binding (HCA202 binding for adalimumab variants and BSA-fentanyl for Fab-Fent variants) was considered conserved if the binding was within 50-200% of the reference molecule determined by ELISA EC50 values.
  • the reference molecule was H-A2F for adalimumab glycovariants and was fab-Fent-LCLgtl-A2 for the anti-fentanyl Fab compounds. ND: Not done.
  • Multi-cycle kinetics were measured using a Biacore 8k+ with 10 mM HEPES pH 7.4, 150 mM NaCl, 40 mM CaC12 and at a temperature of 25°C. Steady state and 1 : 1 binding analysis was performed using Biacore Insights Software. [00337] Briefly, Cytiva Series S CM5 sensor chips (Cat # 29104988) were prepared for amine-based capture using the Cytiva Amine Coupling kit (Cat # BRI 00050).
  • the surface was activated by 0.2M l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.05M N-hydroxysuccinimide (NHS) prior to injection of anti-human Fc from the Cytiva Human Antibody Capture Kit, type 2 (Cat # 29234600).
  • Antibody was immobilized at a final concentration of 10 pg/ml with final resonance units (RU) reaching approximately 2000. Unbound activated groups were blocked by IM ethanolamine.
  • ASGPRl-Fc fusion and ASGPR2-Fc fusion were immobilized on 4 channels each at a final concentration of 1.175 pM (500-1000 RU) in 10 mM HEPES pH 7.4, 150 mM NaCl.
  • Analytes were prepared by diluting stocks of A84.86-A2GalNAc2 to 2 pm. A 5- fold dilution series was performed for a total of 8 points. Binding to either ASGPR1 or ASGPR2 was measured for each analyte. Association and dissociation time was measured for 180 seconds with a flow rate of 20 pl/min. Channels were regenerated between each injection with 20 mM EDTA. Blanks were included before, during, and after cycle
  • a multicycle experiment with a 12-point titration with a 2-fold dilution was run.
  • the contact time was 300 s and the dissociation time was 900 s at 30 pL/min.
  • the regeneration solution was HBS-N+, 50mM EDTA, TW20 0.05% with a contact time of 60s at a flow rate of 30 pL/min.
  • the data are shown in Table 10.B. Detectable binding and kinetic parameters were obtained only with A-84.86-A2GalNAc2.
  • HepG2 hepatocarcinoma cells express ASGPR and were maintained in a low glucose DMEM medium (Sigma, Ref. D5546) supplemented with 10% FBS.
  • Adalimumab antibody glycovariants were labeled with pHrodo dye (pHrodo iFL Red STP Ester [amine-reactive], ThermoFisher, ref. P36011) according to manufacturer instructions.
  • pHrodo dye pHrodo iFL Red STP Ester [amine-reactive], ThermoFisher, ref. P36011
  • the fluorescence of pHrodo is activated at low pH and therefore will allow for the visualization of protein internalization and targeting to the lysosomal pathway.
  • the pHrodo Degree of Labeling (DOL) for each antibody was determined as follows. Antibodies were diluted 1 :2 in denaturing buffer and analyzed with Nanodrop at 280 nm and 560 nm wavelength (A280 and A560). Protein concentration and pHrodo DOL
  • MW is the molecular weight of the antibody used: 144000 g/Mol.
  • Zmax is the absorbance measured at 560 nm.
  • edye is the Extinction coefficient: 65000 M' 1 cm' 1 .
  • Dilution factor is 2.
  • HepG2 cell monolayers were incubated for 3 to 4 hours or 24 hours with pHrodo- antibodies (3 pg/ml) + IVIg (1 mg/ml) (Hizentra, obtained from pharmacy), at 37°C.
  • cells were also treated with the following reagents: fetuin (Sigma, Ref. F3385) at 2 pM; asialofetuin (Sigma, Ref. A4781) at 2 pM; Chloroquine (Sigma, Ref. C6628) at 50 pM; Bafilomycin (Millipore, Ref. 19148) at 10 nM; cytochalasin D (Sigma, Ref.
  • FIG. 1 shows the data obtained comparing H-A2F (Adalimumab; Humira, obtained from pharmacy), A-84.86-A2, A-84.86-A2G2, A-84.86-A2GalNAc2 and A-84.86- M3 antibodies. After 4 hours of incubation, only GalNAc2 displaying antibodies were internalized in HepG2 cells, indicating that GalNAc2 is a potent glycan for recognition and internalization by hepatocyte cells.
  • FIG. 2 shows the data obtained in this inhibition experiment.
  • the internalization of A-84.86-A2GalNAc2 was inhibited by EGTA, a calcium ion chelator, indicating that uptake of the antibody is likely mediated by a calcium-dependent C-type lectin receptor.
  • internalization was selectively inhibited by asialofetuin (ligand for ASGPR) but not by fetuin (not a ligand for ASGPR), indicating that the recognition and internalization of A2GalNAc2-antibodies is mediated by ASGPR (Braun et al.
  • FIG. 3 shows that a 50-fold molar excess of asialofetuin is required to reach 50% blocking of A-84.86-A2GalNAc2 internalization response.
  • HepG2 cells (Sigma, 85011430) were treated with siRNA targeting ASGPR1 or ASGPR2 mRNA to knock-down their expression.
  • ASGPR1 Silencer Select Pre-designed (Ambion, 4392420-sl663)
  • ASGPR2 Silencer Select Pre-designed (Ambion, 4392420-sl665)
  • RNAiMAX Lipofectamine RNAiMAX
  • OptiMEM I Reduced Serum Medium Thermo Fisher Scientific, 31985062
  • HepG2 cells were plated in a 24-well plate at a concentration of 50000 cells/ well in 500pl of antibiotic free DMEM low glucose medium (Sigma, D5546) supplemented with 10% heat inactivated FBS (Pan-Biotech, P30-5500), 2mM L-Glutamine (Sigma, G7513) and 100 pl of the siRNA- lipofectamine complexes.
  • HepG2 cells were washed once, and the medium changed with complete HepG2 cells medium containing 10 Units/ml penicillin and lOOpg/ml Streptomycin. HepG2 cells were used for pHRodo internalization assays 48-72 hours post transfection.
  • FIGS. 4A-C show that ASGPR1 siRNA treated HepG2 cells do not internalize A-
  • ASGPR1 siRNA induced a complete reduction of ASGPR1 and ASGPR2 protein on cell surface, as probed by specific antibody staining by flow cytometry.
  • ASGPR2 siRNA induced only reduction of ASGPR2 protein from cell surface.
  • HepG2 cells knock-out for ASGR1 were constructed using Crispr-Cas9 technique.
  • Specific guide RNA targeting exon 4 of ASGR1 were designed and complexed together with the sp Cas9 to form a ribonucleoprotein (RNP). RNPs were then delivered to HepG2 cells via electroporation.
  • the edited site was PCR-amplified, and the amplicons were Sanger sequenced.
  • HepG2 cells were submitted to mock transfection in absence of RNP.
  • Cells were expanded and maintained in DMEM low glucose medium (Sigma, D5546) supplemented with 10% heat inactivated FBS (Pan-Biotech, P30- 5500), 2mM L-Glutamine (Sigma, G7513), 10 Units/ml penicillin and lOOpg/ml Streptomycin
  • FIGS. 5A-5C show the data obtained on ASGPR1 knock-out HepG2. These data confirm the siRNA data and show that internalization of a A2GalNAc2 displaying antibody is abrogated in the absence of ASGPR1. The ASGPR knockout and siRNA experiment therefore demonstrate that the internalization of an antibody displaying A2GalNAc2 structure by hepatocyte cells is totally dependent on ASGPR1 and ASGPR2 expression.
  • pre-prepared spin columns (3500 MWCO, Component D) were spun at 1000g for 2min to remove storage buffer, then 0.5mL of sterile PBS was added to the column and spun again for 1000g for 2min to equilibrate the column. Finally, the reaction mixture was added to the column and spun for a final time and the flow through was collected.
  • the labeled compound was analyzed with a Nanodrop at A280 and A647 under acidic conditions.
  • the concentration of the A-84.86- A2GalNAc2 was analyzed with the provided correction factors outlined in ThermoFisher’s protocol as well as the degree of labeling.
  • the labeled conjugates were stored at 4°C until use.
  • HepG2 cells were detached from their flask with Accutase (ThermoFisher, Cat # 00-4555-56) according to the manufacturer’s instructions. The cells were spun and counted and plated in a 384-well PhenoPlate (Perkin Elmer, Cat # 6057300) at 2000 cells/well in complete media with 1% Pen/Strep (ThermoFisher, Cat # 15140122). The plate was returned to the incubator at 37°C and 5% CO2to recover overnight.
  • A-84.86-A2GalNAc2-pHrodo starting concentration was 200nM
  • A-84.86-A2GalNAc2-pHrodo inhibition by unlabeled A- 84.86-A2GalNAc2 was also 200nM while the A-84.86-A2GalNAc2-pHrodo across the concentration series was held at InM
  • the top concentration asialofetuin was 200 pM while A-84.86-A2GalNAc2-pHrodo across the concentration series was held at InM.
  • the HepG2 cells are counter stained. First the media is decanted from the 384-well PhenoPlate. The cells are washed once with warmed OptiMEM, then the staining media is added. The staining media is OptiMEM with CellMask Orange (1 :20000) (ThermoFisher, Cat # C10045) and Hoechst (1 :20000) (ThermoFisher, Cat # H3570). The cells are then placed back in the incubator for 5 min. After incubation the media is decanted and the cells are washed once with warmed OptiMEM.
  • A-84.86-A2GalNAc2-pHrodo spots were searched for in the lysosome ROI.
  • the statistics of each of these three object types namely, object count, intensity and size were evaluated for each well. These data were exported to PRISM to generate graphs and statistical analysis.
  • pHrodo labeled A-84.86-A2GalNAc2 shows a dose and time dependent increase in internalization and lysosomal localization (FIGS. 6A-6C). This process is biphasic with a rapid initial step observed in the first 4 hours following which equilibrium is reached between 4-8 hours. There was no saturation in the concentration range tested (O-lOOnM) suggesting additional capacity of this in-vitro experimental system.
  • the internalization and colocalization of A-84.86-A2GalNAc2 was competitively inhibited by increasing concentrations of either unlabeled A-84.86-A2GalNAc2 or asialofetuin indicating specificity and ASGPR dependency of this process respectively.
  • HepG2 were plated at 0.8 million cell / well in 1 ml complete medium (DMEM low glucose supplemented with 10% FBS, 2 mM L-Glutamine and Penicillin-Streptomycin) in a 12-well plate and incubated overnight at 37°C in a cell culture incubator.
  • Cell culture medium was aspirated and 0.3 ml of complete medium with Fc block at 1 :25 dilution (Thermofisher #14-9161-73) was added and cells were incubated 15 minutes at 37°C.
  • Adalimumab antibodies were added to achieve a final concentration of 0.1 mg/ml. Cells were incubated for various time (1.5 to 24 hours) at 37°C in a cell culture incubator.
  • the cells were then washed with PBSlx and detached with accutase, collected in a 1.5 ml tube and pelleted by centrifugation (3 min 400g). The supernatant was aspirated and the cells were washed with 1 ml PBS and pelleted by centrifugation. The PBS supernatant was aspirated and the cell pellet was resuspended in 0.2 ml of RIP A lysis buffer complemented with protease inhibitor cocktail (Complete Protease Inhibitor Cocktail tablets, Roche). To lyse the cells, the ependorf was kept on ice and vortexed vigorously (least 30 seconds) 3-4 times during this period.
  • protease inhibitor cocktail Complete Protease Inhibitor Cocktail tablets, Roche
  • the cells were then sonicated in an ice cold bath at 35 kHz.
  • the lysate was frozen at -80°C during at least 15 hours.
  • the lysates were thawed and centrifuged 20 min at > 15’000 g to pellet the cell debris.
  • the supernatant was transferred in a new 1.5 ml Ependorf tube.
  • the protein content was quantified using BCA protein assay kit (Thermofisher # #23225), according to the supplier instructions.
  • the cell protein extracts were adjusted to similar concentration and stored at -80°C until analysis.
  • the protein extracts (approximately 20pg/lane) were run on a SDS-PAGE using a 4-12% Bis-Tris pre-casted gel (Thermofisher).
  • the proteins were transferred to nitrocellulose membrane using dry blotting system (iBlot 2, Thermofisher).
  • the membrane was blotted using anti-human IgG H+L (Jackson Immuno Research #709-035- 149), used at dilution 1 :2500.
  • the membrane was reblotted for anti-Beta Actin (Thermofisher #MA1-14O) used at dilution 1 :4000, detected with Goat antiMouse IgG H+L (HRP) (Thermofisher #31430), used at dilution 1 :20’000.
  • anti-Beta Actin Thermofisher #MA1-14O
  • HRP Goat antiMouse IgG H+L
  • FIGS. 7A-7B show that A-84.86-A2GalNAc2 is internalized specifically in HepG2 cells, while the control H-A2F is not detectably internalized. A degradation fragment is visible and is increasing in intensity overtime while intact heavy chain and intact light chain signal are decreasing overtime, indicating that the internalized A-84.86-A2GalNAc2 antibody is degraded overtime.
  • the HepG2 cells were incubated with the immune complexes added in the cell culture media at a final concentration corresponding to O.lmg/ml of adalimumab, for 3 hours at 37°C in a cell culture incubator. The cells were then washed to remove immune complexes and the cells were then further incubated with new cell culture media for 1, 3 or 24 hours, at 37°C in a cell culture incubator. The cells were washed and processed for protein extract as described above. The protein extracts were submitted to SDS-PAGE and western blot for anti-human lambda light chain with same method as described above.
  • the anti-lambda light chain detects only the HCA202 antigen as adalimumab is a kappa light chain antibody.
  • FIGS. 8A-8B show that the antigen is well internalized when complexed with A-84.86-A2GalNAc2 and not with an isotype control.
  • HCA202 was complexed to H-A2F same minimal internalization of HCA202 than with the isotype control was observed (not shown).
  • the signal corresponding to the antigen is decreasing overtime during the washout period. At 3 hours following the washout, the signal has decreased by 75% and is reduced to background level at 24hours.
  • HepG2 cells were plated in a 12-well plate in 600pl of DMEM low glucose medium (Sigma, D5546) supplemented with 10% heat inactivated FBS (Pan-Biotech, P30-5500), 2mM L-Glutamine (Sigma, G7513), 10 Units/ml penicillin and lOOpg/ml Streptomycin.
  • a 9-point standard curve ranging from 1000 ng/ml to 0.15 ng/ml in 1 :3 dilutions was prepared in PBST. After washing the plates 3 times with wash buffer, standards and undiluted samples were added in duplicate and incubated for 1 hour at room temperature. Plates were washed 3 times with wash buffer and the detection antibody (Goat anti -human IgG, y-specific, HRP, Sigma, A6029) diluted lOOOOx in PBST was added and incubated for 1 hour at room temperature. ELISA plates were then washed 3 times with wash buffer and revealed by addition of TMB substrate followed by addition of stop solution (H2SO4).
  • detection antibody Goat anti -human IgG, y-specific, HRP, Sigma, A6029
  • ELISA plates were read at 450 and 650 nm on BioTek Synergy Hl instrument using Gen5 Software.
  • H-A2F and A-84.86-A2GalNAc2 concentration in the supernatant was calculated using the standard curve fitting with 4PL and blank reduction.
  • FIG. 10 shows the amount of H-A2F or A-84.86-A2GalNAc2 removed from the supernatants by HepG2 cells at 24, 48 and 72 hours. HepG2 cells depleted up to 3000 pg/ml of A-84.86-A2GalNAc2 after 72 hours of incubation. No significant depletion of H-A2F was observed at any of the timepoint tested. 8.8 EXAMPLE 8 - Glycotag-A2GalNAc2 antibody is internalized by HepG2 cells
  • FIG. 11 shows that 1 lK2-gtl-A2GalNAc2, with a glycotag added on C-terminus of HC was efficiently internalized, as compared to the non-ASGPR engaging antibody 1 lK2-IgG4-PAA-A2F.
  • the internalization is ASGPR-specific as ASGPRko HepG2 cells did not internalize the variant.
  • a variant displaying a glycotag on C-terminus of LC (1 lK2-LCLgtl-A2GalNAc2) showed some internalization, albeit lower than the HC glycotag variant.
  • a variant displaying with glycotags on C-terminus of HC and C-terminus of LC (1 lK2-LCLgtl.gtl-A2GalNAc2) showed a slightly better internalization than 1 lK2-gtl-A2GalNAc2.
  • the variant 1 lK2-84.gtl-A2GalNAc2 with a glycotag on HC plus a glycosite inserted at position 84 in the variable domain showed a higher internalization than the gtl variant.
  • AlGalNAcl glycan is not a ligand for ASGPR in vitro as neither the 1 lK2-84.86-AlGalNAcl nor 84. gtl -AlGalNAcl variants were internalized.
  • the ASGPR engagement activity of these 11K2 antibody variants was tested in vivo in mouse. It was reasoned that antibody PK profile and particularly the clearance rate of the antibody is directly correlated to ASGPR engagement potency. Mice were injected with the antibodies i.v at 2.5 mg/kg and the PK profile of the antibodies was measured in serum using an ELISA method. The data in FIG.
  • Fab-Fent variants Glycovariants of a Fab fragment of a fentanyl specific antibody were produced (Fab-Fent variants) (See Tables 7-9).
  • BSA-fentanyl biorbyt # orb738533
  • BSA-fentanyl was pHrodo labeled using method described in Example 3.
  • HepG2-wt and HepG2-ASGPRlko cells were incubated with 3pg/ml of pHrodo-BSA-Fentanyl x Fab- Fent complexes for 4h.
  • the pHrodo fluorescence intensity was analyzed as described in example 3.
  • MOG-Fc fusion protein was constructing, by fusing the extracellular domain of human MOG to the CH2 and CH3 domain of human IgG.
  • the MOG extracellular domain displays a natural glycosite at position N60 (considering the entire sequence including the signal peptide).
  • a MOG-Fc- A2GalNAc2 was produced. The construct was verified to be a homodimer.
  • the MOG-Fc- A2GalNAc2 was labeled with pHrodo using method described in example 3.
  • FIG. 16 shows that the MOG-Fc- A2GalNAc2 construct was efficiently internalized by HepG2 cells, to a similar extent than the A-84.86- A2GalNAc2 antibody.
  • the internalization was ASGPR specific as it was block by competition with asialofetuin.
  • MOG extracellular domain contains an endogenous N-glycosite at position N60, which displays A2GalNAc2 when produced in accordance to the methods described herein and drives efficient ASGPR-mediated internalization (FIG. 16).
  • Target internalization by glycoengineered MOG constructs was tested, using 8-18C5 mAb as a model anti-MOG autoantibody (Sun et al. (2021) Mol. Ther. 29: 1312-1323). Two additional MOG constructs were generated.
  • the MOG-Fc-gtl construct has an added glycotag at the C-terminus of the Fc.
  • the MOG-N60Q-gtl construct has the endogenous MOG ECD glycosite (N60) mutated and a Fc glycotag added at the C-terminus.
  • the 8-18C5 mAb was labeled with pHrodo (same method than in Example 3) and incubated with each of the 3 different MOG constructs on HepG2 cells, similar to the procedures described above. The binding of pHrodo-8-18C5 was verified to be equivalent on the 3 constructs, by ELISA (data not shown).
  • FIG. 17 shows the internationalization data. Interestingly the MOG-Fc-A2GalNAc2 construct was unable to drive internalization in the presence of the 8-18C5 mAb anti-MOG autoantibody.
  • each of the MOG constructs with a glycotag at the C-terminus of the Fc were able to drive efficient target internalization.
  • binding of the anti-MOG autoantibody to the MOG-Fc construct may prevent recognition of the A2GalNAc2 at the endogenous MOG glycosite by ASGPR.
  • the presence of a glycotag distal from the target binding site e.g. at the C-terminus of the Fc
  • the data suggest that placement of the A2GalNAc2 glycan distal to the target engagement epitope (e.g. an ECD or antigenbinding region) may be important to maintain efficient ASGPR mediated internalization in the presence of the target (e.g. an autoantibody or antigen, respectively).
  • A2GalNAc2 displayed by a glycotag can replace A2GalNAc2 displayed by an endogenous glycosite (such as the N60 position in MOG ECD).
  • an endogenous glycosite such as the N60 position in MOG ECD.
  • the endogenous glycosite of Fc, located at amino acid 297 is generally considered to be inaccessible, and therefore largely inactive.
  • A2GalNAc2 display may be gained by addition of one or more glycotags situated at other positions and/or the deletion of endogenous glycosites, allowing more precise control of the total A2GalNAc2 load displayed by the degrader.
  • the data presented here indicate that a single A2GalNAc2 glycan per molecule of degrader is sufficient to drive efficient ASGPR- mediated internalization. Without being bound by theory, avoiding too high an A2GalNAc2 load per degrader molecule may be beneficial to prevent too strong or unphysiological interaction with ASGPR and/or fine-tune the relative internalization rates of the degrader in the presence and absence of a target molecule.
  • the glycan-modified antibodies and the glycoengineered host cells that produce them are elements of the inventors' "Custom Glycan Platform", which is also referred to as “CGP”) and displaying GalNAc terminated glycans (e.g., an A2GalNAc2 structure) to deplete a circulating antigen
  • CGP Customer Glycan Platform
  • GalNAc terminated glycans e.g., an A2GalNAc2 structure
  • Rats were injected with an antigen (i.e. a target) and with antibodies displaying A2GalNAc2 glycan structure on their Fab or control antibodies, specific for the antigen.
  • the level of the circulating antigen in the serum of treated animals was quantified along time to measure the extend of depletion of the antigen from peripheral blood compartment. All antibodies showed aggregate levels below 5% as assessed by size exclusion HPLC method and higher than 94% purity as assessed by reducing polyacrylamide gel electrophoresis analysis and had an endotoxin level lower than 1 EU/mg (LAL assay). All glycoengineered antibodies (Table 9) conserved high binding to HCA202.
  • HCA202 Wistar female rats (Janvier Labs, St Berthevin, France, ref. RjHamWI) 180-220 g at start of experiment were injected i.v. bolus with anti-adalimumab Fab fragment HCA202 (Biorad, ref. HCA202) (the antigen or target) at 0.5 mg/kg dose, 0.5 ml/rat.
  • HCA202 compound was submitted prior to injection to an endotoxin removal step using PierceTM High Capacity Endotoxin Removal Spin Columns (Thermofisher, ref. 88274).
  • HCA202 levels were measured by ELISA method.
  • Anti-Penta-His antibody (Qiagen, Ref. 34660) was coated on 96-well ELISA plates at 5 pg/ml in coating buffer (PBS pH 7.4, final composition: 8 mM Na-Phosphate; 8 mM K- Phosphate, 0.15 M NaCl, 10 mM KC1) overnight at 4°C.
  • Blocking buffer 2% (w/v) Bovine serum albumin (BSA) in PBST
  • BSA Bovine serum albumin
  • the spiked serum was diluted 10-fold (MRD10) by adding diluent B (2% (w/v) Bovine serum albumin (BSA) in PBST).
  • BSA Bovine serum albumin
  • a serial 1 :3 dilution of the immune complex standard curve was performed using diluent B.
  • Study samples were processed similarly. Study serum samples were diluted 10-fold in dilution plates (to achieve MRD10 samples) using diluent B. MRD10 samples were further diluted if needed in diluent A (1/10 wistar rat pooled serum diluted in 2% BSA + 0.05% PBST) to achieve a signal within the linear range of the calibration standard curve.
  • ELISA plates ware washed 3 times with wash buffer.
  • ELISA plates were washed 3 times with wash buffer and a Humira solution at 1000 ng/ml was added to each sample. ELISA plates were incubated for Ih at room temperature. Plates were washed 3 times with wash buffer.
  • a detection antibody solution was prepared by diluting goat anti-human kappa LC-HRP (Thermofisher, ref. A18853) 1 :5000 in diluent B. The detection antibody solution was added to the ELISA plates and incubated for Ih at room temperature, protected from light.
  • ELISA plates were then washed 3 times with wash buffer and revealed by addition of TMB substrate followed by quenching with H2SO4. ELISA plates were read at 450 and 650 nM on a plate reader such as BioTek Synergy HL Data analysis was made using standard software such as Gen5 (Biotek).
  • Antibody levels in serum sample can be quantified by ELISA method.
  • the assay consists of a coating step with human TNFa to capture adalimumab and adalimumab variants present in the sample. Detection can be performed via an anti-human gamma HC specific HRP -tagged detection antibody. The assay therefore quantifies only free antibodies (having at least one Fab arm not bound to HCA202). Briefly, recombinant Human TNF-a (Peprotech, ref. AF-300-01 A) is coated on 96-well ELISA plates, typically atl pg/ml in PBS pH 7.4 at 4 °C overnight.
  • Blocking buffer, dilution buffer A and B and wash buffer are the same than used for the HCA202 ELISA. Plates are washed 3 times and blocked with blocking buffer as described in the HCA202 ELISA. Typically, a 7-point calibration curve, for example from 333.3 ng/ml to 0.5 ng/ml in 1 :3 dilutions is prepared by spiking pooled wistar rat serum diluted 10-fold (minimal required 10-fold dilution, MRD10) in dilution buffer B with 1 pg/ml adalimumab. Study serum samples are also diluted in dilution buffer B (MRD10 samples minimum).
  • Diluted study samples and standard calibration curve samples are then transferred to the ELISA plate, after blocking step and incubated Ih at room temperature. Plates are then washed 3 times and solution of detection antibody is added.
  • Solution of detection antibody can be prepared by diluting for example a Goat Anti-Human IgG (y-chain specific)-HRP (Sigma, ref. A6029) antibody (typical dilution 1 : 10’000) in dilution buffer B.
  • ELISA plates are incubated with detection antibody typically Ih at room temperature, protected from light. Plates are then washed 3 times and revealed by adding TMB substrate as described in the HCA202 ELISA.
  • FIG. 19 shows the data obtained for HCA202 levels.
  • Table 11 shows the HCA202 depletion numbers.
  • HCA202 decays slowly over period of 48h, as expected for a Fab fragment.
  • H-A2F (non-engineered adalimumab) treatment led to increase levels of HCA202 at 24 hours and 48 hours time point.
  • A-M3 and A-84.86-A2G2S2 also led to increased HCA levels as compared to PBS treatment (72% depletion from Czero with PBS vs 52-63% depletion with H-A2F, A-M3 and A-84.86-A2G2S2 at 6h), showing that these antibodies have no depleting potency.
  • Czero is the theoretical concentration (of HCA) in serum that would have been achieved immediately post injection, considering immediate homogeneous whole blood distribution.
  • injection of A-84-A2GalNAc2 led to a significant depletion of HCA202 as compared to non-depleting antibodies and PBS at 1 hour (74% depletion) and 6 hour (93% depletion).
  • A-84.86-A2GalNAc2 treatment led to a more extensive and faster HCA202 depletion (97% depletion at Ih, 100% at 6h).
  • Table 11 shows the % of HCA202 depletion from C zero .
  • the collected fluorescence data were reconstructed by FMT 2500 system software (TrueQuant V2.0, PerkinElmer) for the quantification of the three-dimensional fluorescence signal in whole animal body.
  • FMT 2500 system software TrueQuant V2.0, PerkinElmer
  • Three-dimensional regions of interest (RO I) were drawn encompassing the relevant biology on the thorax, abdomen and liver areas. The quantity and percentage of dose of labeled antibodies in each ROI was determined for each time point.
  • thyroids with trachea
  • lungs heart, liver, spleen, kidneys were harvested and submitted to FMT imaging.
  • Table 12 shows the antibody characteristics that were included in the study.
  • Table 12 Antibody characteristics.
  • Main N-glycan structure on Fc is on the canonical N-297 position.
  • Main N-glycan structure on Fab means on the engineered glycosite by point mutations (Eu numbering).
  • LC is light chain, HC is heavy chain.
  • Black striped circle represents mannose (Man), white square is N-acetyl glucosamine (GlcNAc), white circle is galactose (Gal), black square is N-acetyl galactosamine (GalNAc), white diamond is sialic acid, N- acetyl neuraminic acid (Neu5Ac), and white triangle is fucose (Fuc).
  • FIG. 20 shows the FMT imaging data for thorax and liver ROI along time for each antibody.
  • Table 13 shows the FMT imaging data obtained on harvested organs at 6 hours.
  • H-A2F adalimumab
  • Ptz-A2F pertuzumab
  • A-84.86-A2G2S2 and A-M3 showed a distribution profile similar to H-A2F and Ptz-A2F control antibodies, with broad organ distribution and relatively low liver distribution.
  • 20% of the injected dose for A-84.86-A2G2S2 and A-M3 was present in the liver (Table 13).
  • A-84.86-A2GalNAc2 antibody showed a rapid and essentially exclusive distribution to the liver area, followed by a decline after 6 hours (FIG. 20). The decline observed after 6 hours is likely due to degradation of the antibody into peptides by the lysosomal machinery and excretion of the product of degradation (including the fluorophore- couple peptides) from the liver cells.
  • A-84.86-A2GalNAc2 was present in the liver (Table 13).
  • A-84.86-A2GalNAc2 antibody was absent from Thyroid, lungs, heart and spleen and detectable at low level in the kidneys. This pattern of distribution is characteristics of an antibody that is essentially exclusively distributed to the liver and therefore not present in the blood and other organs.
  • Table 13 Organ distribution data at 6 hour time point.
  • Extracts were then sonicated for 20 minutes in an ice bath and spun to remove debris. Protein content in each sample was quantify using the BCA Assay Kit (Pierce). The protein extracts were loaded on polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane using the iBlot dry transfer system and CF750 fluorescence was read using an iBrightTM system (Invitrogen). Fluorescence densitometric signal was quantified using the iBright software. The CF750 fluorescence signals directly indicate the quantity of labeled antibody (intact or fragments of) present in the liver extract. Relative densitometric units were determined by normalizing the fluorescent signal to the signal density of the loading control P-actin [00391] FIG.
  • FIG. 21 shows the gel fluorescence blot.
  • FIG. 22 shows the quantification of antibody signal from the blot. Livers from A-84.86-A2GalNAc2 injected animals, collected at 6h after injection contained significantly higher amount of antibody (227% more) as compared to livers from H-A2F treated animals confirming that A-84.86-A2GalNAc2 is more directed to the liver as compared to the non-ASGPR engaging H-A2F control, as shown by the biodistribution data.
  • the blot shows clearly bands corresponding to fragmentation of the heavy chain of the H-A2F and A-84.86-A2GalNAc2 antibodies, providing strong evidence that the liver targeted A2GalNAc2 antibody is submitted to lysosomal degradation.
  • the level of A-84.86-A2GalNAc2 antibody in liver cells has dramatically decreased to 25% compared to the 6 hour timepoint, further highlighting that A-84.86-A2GalNAC2 is degraded.
  • the rest of the liver was returned to the -80°C.
  • the liver tissue was transferred to a 2mL microcentrifuge tube with round bottom and rinsed with 1 ml ice-cold PBS. Then ice-cold RIPA (with 50X PI, 100X PMSF, and 100X Na3VO4) at 500 pL/30mg of tissue was added to the livers. A single 5 mm grinding ball (OPS Diagnostics) was added to each sample. The samples were then placed in the TissueLyserll (Qiagen) at 4°C. The livers were processed for 5 min at 20Hz and then allowed to rest for an additional 15 min. The samples were then spun at Max Speed in a pre-cooled microcentrifuge for 15 min at 4°C.
  • the supernatant was collected, and a BCA was performed on a 1 :5 dilution of the stock liver lysates.
  • the liver extracts were processed using NuPAGETM 12%, Bis-Tris, 1.0 mm, 17 wells Mini Protein Gels (NP0349BOX).
  • the gel was analyzed by western blot using Goat AntiHuman Lambda LC IgG (Milipore Sigma # AP506P) at 1 :2000 in 5% Milk-TBST.
  • HCA202 is a human Lambda Fab antibody while H-A2F and A-84.86 are Kappa light chain antibodies. Therefore anti-Lambda LC western blot detects specifically HCA202.
  • Total protein was imaged in AF680 fluorescent channel right after protein transfer.
  • the signal density of the antibody band and total protein quantitation was performed using the iBright FL 1500 analysis tool. Ratio of antibody band to corresponding total protein was normalized to HCA202 + A- 84.86-A2GalNAC2 at 0.5 h timepoint.
  • FIG. 23 shows the data obtained.
  • HCA202 decays slowly over period of 48h, as expected for a Fab fragment.
  • Injection of A-84.86-A2GalNAc2 i.v. led to a fast and strong decrease of HCA202 levels.
  • Injection of A-84.86-A2GalNAc2 s.c. led to a delayed HC202 depletion reaching complete depletion at 24 hours.
  • A-84.86-A2GalNAC2 is able to prolong depletion of HCA202 antigen up to 48 hours.
  • A-84.86- A2GalNAc2 administration of A-84.86- A2GalNAc2 at 10 mg/kg was able to deplete 4 consecutive HCA202 doses (FIG. 25), indicating that s.c dosing can maintain depletion efficiency for up to 96 hours.
  • the calculated molar degrader (A-84.86-A2GalNAC2) to target (HCA202) ratio in this experiment is 1.67 further highlighting that low degradertarget ratio are sufficient to drive efficient target depletion by a mAb displaying A2GalNAc2.
  • A2GalNAC2 structure To assess whether an antibody specific for a target receptor, and displaying GalNAc2 terminated glycan (A2GalNAC2 structure) can lead to degradation of the target surface receptor on surface of cells expressing ASGPR, an experiment using CGP-produced glycovariants of the pertuzumab (Ptz) anti-HER2 antibody was performed. HepG2 cells coexpress HER2 and ASGPR. The hypothesis tested was that A2GalNAc2 displaying Ptz CGP- produced variant is able to co-engage HER2 and ASGPR on surface of HepG2 cells and trigger internalization and degradation of the formed complex, leading to reduction of HER2 levels on HepG2 cells.
  • Ptz pertuzumab
  • Pertuzumab antibodies (Ptz-A2, Ptz-86-A2GalNAc2, Ptz-gtl- A2GalNAc2, Ptz-hgt-A2GalNAc2) were purified from cell culture supernatant with Protein A HiTrap Mabselect PrismA, (Cytiva) and formulated in PBS buffer pH 7, by using Amicon Concentrator, (4ml, 30K MWCO). Due to lower levels of A2GalNAc2 ( ⁇ 70%) on the purified antibodies, the material was further polished by in-vitro glycoengineering to increase abundance of A2GalNAc2 glycan on Ptz.
  • the GalNAc addition was performed using in vitro glycosylation (IVGE) in a reaction using 10 mM UDP-GalNAc, 2% (w/w) GalTl(Y285L), lOOmM MnC12 in 25mM Tris, pH 8 at 30 °C under mild rotation.
  • the glycosylated mAb was purified from the reaction mixture with ProteinA sepharose (HiTrap MabSelect PrismA column GE Healthcare) according to manufacturer’s recommendation using FPLC (Bio-Rad NGC, Germany). Thereafter, a desalting procedure using PD-10 (Sephadex 25, Sigma, Switzerland) was carried out for a buffer exchange to PBS pH 7.
  • Ptz-gtl antibody comprises a glycotag at the C-terminal part of the heavy chain (ANSTMMS addition with C- terminal lysine replaced by Alanine of the glycotag sequence).
  • Ptz-hgt antibody comprises an inserted glycosite in the upper hinge region (LNLSS insertion after T223 position).
  • HepG2 cells were harvested using Accutase (Sigma/Merck, SCR005) and plated in flat-bottom 24-well plates at 0.1 million cells/well. Cells were left to recover for 72 hours at 37°C in a cell culture incubator. Cells were then incubated for 24 hours with nonengineered pertuzumab (Ptz-A2F; Perjeta, obtained from pharmacy) or CGP -produced pertuzumab glycovariant antibodies at 1 pg/ml + IVIg (Hizentra, obtained from pharmacy) at 1 mg/ml, in cell culture medium, at 37°C in cell culture incubator.
  • Ptz-A2F Perjeta, obtained from pharmacy
  • IVIg Hizentra, obtained from pharmacy
  • Antibody MAB1129 was shown to be non-competitive with pertuzumab for binding to HER2, ensuring that MAB1129 can bind to HER2 even if pertuzumab is bound.
  • MFI geometric mean fluorescence intensity
  • Table 14 presents the adjusted and normalized HER2 MFI.
  • Treatment with the control antibody Ptz-A2, which has no engineered glycosite and displays an A2 structure on the N297 Fc site did not reduce HER2 levels as compared to Ptz-A2F (104% of normalized HER2 MFI after treatment).
  • treatment with Ptz-gtl-A2GalNAc2 reduced normalized HER2 MFI to 55% and treatment with Ptz-hgt-A2GalNAc2 reduced normalized HER2 MFI to 68%.
  • A2GalNAc2 antibodies containing the equivalent engineered glycosite at position 86 in the Fab fragment did show efficient uptake by HepG2 cells (Example 3, Example 5, Example 8), and high depletion potency of a circulating antigen, when injected in animals indicating efficient ASGPR engagement (Example 11), indicating that position 86 displays accessible, active glycans. This indicates that the position of glycan displayed on the antibody is important to enable efficient engagement of ASGPR when the antibody is bound on HER2.
  • FIG. 26 shows that when HepG2 knock out for ASGPR1 (HepG2-ASGRlko) are treated with Ptz-gtl-A2GalNAc2 or Ptz-hgt-A2GalNAc2 variants, no HER2 reduction is observed, as compared to isotype control or Ptz-A2F treated cells. Therefore, the HER2 reduction observed is mediated by an ASGPR-dependent mechanism.
  • A2GalNAc2 displaying antibodies can be used to remove a target molecule from a cell surface, by leveraging the ASGPR endocytic and lysosomal degradation pathway.
  • ICs immune complexes
  • Pertuzumab does not compete with trastuzumab for binding to HER2 and therefore can bind to HER2-Fc engaged in immune complexes with trastuzumab (FIG. 27, Panel B).
  • ICs and large size protein aggregates can cause severe pathologies such as IgA nephropathy; systemic lupus erythematosus; amyloidosis; CO VID; iTTP; cutaneous necrotizing vasculitis. Therefore, these data indicate that biologies compounds displaying A2GalNAc2 and able to bind a component of large size immune complexes or protein aggregates can be used to remove these pathogenic units from circulating and cause their degradation in hepatocytes via the lysosomal pathway.
  • Rats were injected i.v with HCA202 target, followed by PBS or different doses of A-gtl-A2GalNAC2 or Fab-A-FLGT4-A2GalNAc2.
  • the levels of HCA202 target were quantified in serum and expressed as % of HCA202 depletion from theoretical Czero concentration.
  • FIG. 29 shows the data with A-gtl mAb
  • FIG. 30 shows the data with Fab-A-FLGT4 Fab compound.
  • the depletion of HCA202 by A-gtl -A2GalNAc2 followed a dose response. When the degrader :target ratio fell below 1, the depletion of HCA202 at early timepoints was incomplete.
  • viruses, nucleic acids, methods, host cells, and compositions disclosed herein are not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the viruses, nucleic acids, methods, host cells, and compositions in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

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Abstract

La présente demande concerne des agents de dégradation bifonctionnels glycomodifiés, des populations d'agents de dégradation bifonctionnels glycomodifiés, des cellules hôtes de Leishmania pour produire des agents de dégradation bifonctionnels glycomodifiés, des méthodes d'ingénierie desdites cellules hôtes Leishmania, des méthodes de culture desdites cellules hôtes Leishmania, des méthodes de fabrication d'agents de dégradation bifonctionnels glycomodifiés à l'aide de cellules hôtes Leishmania, et des méthodes d'utilisation d'agents de dégradation bifonctionnels glycomodifiés. En particulier, les agents de dégradation bifonctionnels glycomodifiés comprennent une N-glycane à terminaison GalNAc, biantennaire, spécifiquement A2GalNAc2.
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