Engineered and fully-functionally tailored glycoproteins

Abstract

Described herein are compositions and methods for producing glycosylated proteins in vitro and in vivo. The methods include using a host cell to produce a glycosylated protein. Glycosylated proteins produced using these methods and uses thereof are also described herein.

Description

[0001] Related applications

[0002] This application is a divisional application of Chinese Patent Application No. 201880056874.5, filed on June 28, 2018, entitled "Engineering and Fully-Functionally Customized Glycoprotein".

[0003] 1. Cross-references to related applications

[0004] This application claims priority to U.S. Provisional Patent Application No. 62 / 527,466, filed June 30, 2017, the entire contents of which are incorporated herein by reference.

[0005] 2. Sequence List

[0006] This application includes a sequence list submitted in ASCII format and whose entire contents are incorporated herein by reference. The ASCII copy created on June 28, 2018, is named 14197-005-228_ST25_FINAL.txt and has a size of 1,065,529 bytes. Technical Field

[0007] This document describes compositions and methods for producing glycosylated proteins in vitro and in vivo. The methods include using host cells to produce glycosylated proteins. This document also describes the glycosylated proteins produced using these methods and their uses. Background Technology

[0008] Glycosylation is a ubiquitous post-translational modification across all life systems. Animal systems exhibit immense complexity, and glycan structures play crucial biological and physiological roles, from their contributions to protein folding and quality control to their involvement in a wide range of biological events, such as the recognition, stability, function, and turnover of these molecules (Moremen et al., 2012). Therapeutic glycoproteins, such as monoclonal antibodies, enzymes, and hormones, are key products in the biotechnology industry (Lagassé, HA Daniel et al., 2017; Dimitrov 2012), and the impact of glycan heterogeneity is increasingly recognized as a “critical quality attribute.” Among the many properties determining product quality, glycosylation is considered among the most important: influencing protein bioactivity, serum half-life, and immunogenicity. Glycans are associated with increased serum circulation time, and many approved or developing biologics have insufficient half-lives, necessitating frequent administration to maintain therapeutic concentrations over extended periods. Half-life extension strategies are crucial for durable therapeutic agents that can produce pharmacokinetic improvements (Kontermann 2016). Glycosylation also appears to improve protein solubility and stability, for example, by reducing the tendency to aggregate and leading to increased cycle life due to protection against protein hydrolysis and degradation. Furthermore, N-glycans with different terminal monosaccharides can be identified by lectins that cause their degradation (Blasko et al., 2013; Varki, 2017). Therefore, the monitoring and control of glycosylation is important in biopharmaceutical manufacturing and is required by 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 for improving biopharmaceuticals in several ways (Dicker and Strasser 2015).

[0009] Most protein drugs are glycosylated by the attached glycan structures, thereby affecting the properties of the therapeutic protein. Generally, the number and composition of glycans play a crucial role in protein folding, solubility, and intracellular transport. Glycans can protect the protein backbone from immunogenic responses, and different cellular recognition events depend on the presence of specific glycan structures. Therefore, glycosylation has a significant impact on the biological activity of glycoproteins and requires careful control during production to achieve therapeutic efficacy. The glycosylation patterns of recombinant glycoproteins vary widely depending on the species, cell type, and physiological state of the producing host. Generally, the structures of glycans on proteins are very different and consist of a group of monosaccharides assembled by different bonds. The matured glycan at any glycosylation site can be as simple as a monosaccharide or as complex as a polymer with more than 200 sugars, and may be modified by phosphate esters, sulfate esters, acetate esters, or phosphorylcholine (Stanley 2011). It is estimated that approximately 700 proteins are required to produce all the diverse mammalian glycans (estimated to be ≥7,000 structures), which are assembled from only 10 monosaccharides: fucose, galactose, glucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), glucuronic acid (GlcA), iduronic acid (IdoA), mannose, sialic acid (Sia), and xylose (Xyl) (Moremen et al., 2012).

[0010] Antibody therapy

[0011] Antibodies (or immunoglobulins, Ig), and specifically IgG antibodies, are among the most successful therapeutic agents developed over the past few decades (e.g., bevacizumab, rituximab, infliximab, adalimumab, trastuzumab, or cetuximab, etc.). They are highly specific and have long serum half-lives, and they are generally expressed in mammalian cell cultures, which have been developed and improved over the past 30 years. The basic structure of an antibody molecule is assembled from two identical polypeptide heavy chains and two identical polypeptide light chains. These chains are linked by disulfide bonds, forming a “Y”-shaped structure. Due to their heavy chains, human immunoglobulins are classified into five classes: IgG, IgA, IgD, IgE, and IgM. IgG and IgA exist in four classes (IgG1-4) and two subclasses (IgA1-2), respectively. Specific antigen recognition is mediated by an antigen-binding fragment (Fab) comprising a variable region of both light and heavy chains and a constant domain. The binding of effector proteins, such as Fc receptors (FcRs), to crystalline fragment regions (Fc) corresponding to two additional domains (CH2 and CH3) of the heavy chain constant domain initiates effector function. Figure 1Therefore, the Fab fragment consists of variable and constant domains of both the light and heavy chains, while the Fc fragment consists entirely of the constant domain of the heavy chain. Due to its pH-dependent binding to the neonatal Fc receptor (FcRn), this Fc domain also prolongs the serum half-life of the antibody, protecting the protein from degradation in endosomes and allowing it to cycle back into the circulatory system (Jefferis 2009).

[0012] Antibody engineering has been used to further improve the clinical success of therapeutic antibodies, for example, by altering their binding properties to ligands or Fc receptors, or by further extending their half-life. Typical approaches include introducing mutations or altering antibody glycosylation. Introducing mutations into the Fc chain has an inherent disadvantage: it no longer works in conjunction with the native sequence. Glycosylation of therapeutic proteins is generally believed to prolong the circulating half-life; however, for antibodies, previous studies on the effect of glycosylation on the rate of Ig clearance from circulation have yielded confusing results, although recent observations suggest that differences in the glycan structure of the Fc moiety do indeed affect clearance (Millward et al., 2008; Liu 2015, 2017). During post-translational modifications of the antibody chain, enzymes in the endoplasmic reticulum and Golgi apparatus can link carbohydrate chains to the antibody's polypeptide backbone. A single N-linked glycan is present at the asparagine position 297 in the Fc moiety of all IgG subclasses. Approximately 20% of IgG antibodies contain glycans at other locations in the molecule. Most recombinant antibody drugs have been engineered or selected to contain only a single Fc glycosylation site.

[0013] When the antibody chain is correctly folded and bound, the oligosaccharide at position 297 is isolated within an internal space enclosed by the CH2 domain, and there are intense non-covalent interactions between the oligosaccharide and the antibody's amino acids, resulting in conformational effects. Oligosaccharides present at the conserved Asn-297 site typically belong to the fucosylated biantennary complex type. However, in antibody molecules, the carbohydrate structure (glycoform) is significantly heterogeneous due to varying branches, chain lengths, and / or the number of different carbohydrate moieties. Indeed, the structures of the linked N-linked oligosaccharides differ significantly based on the degree of processing, and these structures can include high-mannose and complex biantennary oligosaccharides with or without equal portions of GlcNAc and a core fucose residue (Wright and Morrison, 1997). Typically, the core oligosaccharide structure linked at a given glycosylation site exhibits heterogeneous processing, resulting in multiple glycoforms even for monoclonal antibodies. Importantly, major differences in antibody glycosylation occurred between antibody-producing cell lines, and differences were also observed for a given cell line grown under different culture conditions. Indeed, each step in mammalian N-glycan biosynthesis is <100% efficient and some enzymes compete for substrates, resulting in different glycoforms. This heterogeneous glycosylation has led to problems in the production of therapeutic proteins. For example, glycans can affect pharmacokinetics and biological activity (Ferrara et al., 2011; Elliott et al., 2003; Krapp et al., 2003). Since N-glycans are generally essential for protein folding, these difficulties cannot be easily overcome by completely removing N-glycosylation sites or interfering with glycosylation before or within the endoplasmic reticulum. However, antibodies do not require N-glycans for proper folding (Feige et al., 2010), but differences in glycoform lead to different or inconsistent effector functions, which can make the antibody difficult to use for therapeutic purposes or for regulatory purposes. Deglycosylation of the Fc moiety at N-297 can lead to the elimination of the function or reduction of the stability of effector molecules containing Fc. Importantly, glycoforms not synthesized in humans can be allergenic, immunogenic, and accelerate the cytoplasmic clearance of linked antibodies by antidrug antibodies that can be generated due to repeated therapeutic doses.

[0014] Improving antibody potency, reducing therapeutic doses, and enhancing overall clinical performance through changes in the Fc domain is the next challenge in engineered antibody development. Influences on the effector functions of the molecule, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADCP), and overall half-life, are key objectives for improving antibody therapeutic properties. Figure 1(Jennewein and Alter 2017). The overall market is expected to experience positive growth, with a healthy annual growth rate of nearly 40% between 2016 and 2026.

[0015] Based on the types of antibodies they recognize, Fc receptors (FcRs) are classified into different types: Fcγ receptors (FcγR; binds to IgG), Fcα receptors (FcαR; binds to IgA), Fcε receptors (FcεR; binds to IgE), and neonatal Fc receptors (FcRn). Among these, the "Fcγ" receptor is the most important. Fcγ receptors are responsible for activating the phagocytic activity of opsonized microorganisms. Due to their different molecular structures, Fcγ receptors are further divided into different types based on their affinity for antibodies. Some of these include FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRIII (CD16) is present on most natural killer (NK) cells, granulocytes, monocytes, macrophages, and T cell subtypes. The gene encoding this receptor is either FcγRIIIa or FcγRIIIb. FcγRIIIa is the Fc receptor responsible for NK cell-mediated ADCC. Fcα receptors comprise two extracellular Ig-like domains and have only one subgroup, FcαRI (CD89). These constitute part of both multi-chain immune recognition receptors (MIRRs) and the Ig superfamily. Fcε receptors have two types. One of these receptors is FcεRII (CD23), a low-affinity receptor, a C-type lectin, while the other is FcεRI, a high-affinity receptor, a class within the Ig superfamily. Neonatal receptors (FcRn) are known to play a role in preventing antibody lysosomal degradation, thereby increasing the half-life of therapeutic antibodies. FcRn in acidic endosomes binds to IgG internalized via transcytosis. IgG is recycled to the cell surface and released at blood pH, preventing lysosomal degradation. Although glycosylation has a significant impact on effector function, the interaction between IgG-Fc and FcRn is believed to be independent of Fc glycosylation.

[0016] The glycan determines the "open" of the horseshoe-shaped Fc fragment, and truncation of the Fc glycan results in a "closed" conformation. The "open" IgG-Fc structure is the most favorable conformation for interaction with FcγRIII, as observed with fully galactosylated but fucosylated IgG-Fc (Krapp et al., 2003). High-affinity binding between FcγRIII and antibodies lacking a core fucose requires a unique carbohydrate-carbohydrate interaction. However, core fucosylation prevents high-affinity FcγRIII binding due to "fucose clash." Fucose residues in the oligosaccharide moiety have been shown to sterically hinder the binding of therapeutic antibodies to FcγRIIIa (activating the Fc receptor). Figure 2A Defucosylation plays a crucial role in enhancing Fc receptor function and ADCC. Importantly, (Chung et al., 2012) showed a linear relationship between fucosylation % and ADCC. This results in the desirable properties of homogeneous glycans without core fucosylation residues. Antibodies against fucosylation were generated in GnT-III overexpressing cell lines by adding an equal amount of GlcNAc to the core β-Man, a structure that inhibits α1,6-core fucosylation and leads to reduced fucosylation. This reduction resulted in approximately 50% fucosylation-free structures, which were also observed through glycosylation profiles. Confirmed Figure 42 ).

[0017] To interpret previous data, new studies (Li et al., 2017; Chen et al., 2017) used a chemical enzymatic approach to produce homogeneous glycans and showed high retention of FcR affinity, even with the use of 2,6-extended biantennary sialic acid, which translates into improved effector function, such as ADCC. This data, based on structurally defined and homogeneous glycans with and without α1,6-linked core fucose, improves our understanding of FcR binding and related downstream effects (Li et al., 2017; Sondermann et al., 2013). Figure 2B Furthermore, the α2,3-terminal sialic acid linked to the N-glycan's two tentacles does not lead to increased FcγR affinity, and therefore it can be considered to act as an inhibitory glycan by activating the loss of Fc receptor function in the pathway. The sialic acid at the terminals explains the antibody's anti-inflammatory sialic acid-related effects and the increased circulating half-life. Obtaining antibodies and Fc-containing molecules with improved properties, such as longer circulating half-life and immune tolerance (e.g., antidrug antibodies against potentially immunogenic therapeutic mAbs), without the drawbacks such as heterogeneous glycosylation or reduced antigen binding, would be advantageous. Moreover, functionally customized N-glycans will enhance the function of downstream effector molecules targeting the molecule.

[0018] Glycosylation processes in the endoplasmic reticulum (ER) and Golgi apparatus compartments of eukaryotic cells produce heterogeneous glycan structures, which are the majority of recombinant glycoproteins. Due to its recognized importance in therapy, significant work has been done in recent years to overcome glycan heterogeneity and to establish in vivo and in vitro glycoengineering techniques for the efficient production of homogeneous therapeutic glycoproteins. Although progress has been made in altering glycosylation pathways to produce humanized glycans in several expression hosts, this can be accompanied by loss of health, yield, or even viability. Importantly, no in vivo system for producing well-defined, homogeneous N-glycans has yet been reported.

[0019] Therefore, expression technologies for producing such recombinant therapeutic proteins with such defined homogeneity and even functionally customized glycosylation represent a new class of safe and innovative next-generation drugs with enhanced biological activity and improved properties.

[0020] N-glycosylation pathway

[0021] The most prominent and best-characterized form of protein glycosylation is the bonding of glycans to the amide in the asparagine side chain of the newly synthesized protein (N-glycosylation). N-glycosylation of the protein is initiated in the lumen of the endoplasmic reticulum (ER) by transferring a conserved oligosaccharide precursor (Glc3Man9GlcNAc2) assembled on a lipid carrier to the exposed Asn-X-Ser / Thr sequence (where X is any amino acid except proline) on the nascent polypeptide chain. This initial glycan transfer reaction is catalyzed by a heterooligosaccharide transferase (OST) complex, and this reaction should occur before protein folding in the ER (Aebi 2013). Immediately after oligosaccharide transfer, the two terminal glucose residues are cleaved by glucosidases I and II, and the resulting polypeptide with a monoglucosylated glycan structure (Glc1Man9GlcNAc2) can interact with ER-resident membrane-bound lectin callinkin or its soluble homologous calreticulin. These lectins support protein folding in the glycan-dependent protein mass control cycle. Secretory glycoproteins that have acquired their native conformation are released from the cadherin / calreticulin cycle and the glycoproteins exit the ER into the Golgi apparatus. In the Golgi apparatus, further N-glycan elongation occurs on the ER-derived oligomannose N-glycans on the maturely folded glycoproteins, resulting in highly diverse and complex N-glycans (Stanley 2011).

[0022] Insects, yeast, and plants produce N-glycans that differ significantly from those produced by mammalian cells. The processing of the initial Glc2Man9GlcNAc2 oligosaccharide into Man8GlcNAc2 in the endoplasmic reticulum shows remarkable similarities across these species, from yeast to mammals; however, very different processing events occur in the Golgi apparatus. For example, yeast can add 50 or more Man residues to Man. 8-9 GlcNAc2, however, insect cells typically remove most or all of the Man residues to produce paucimannosidic Man. 3-1 GlcNAc2 N-glycan. Plant cells also remove Man residues to obtain Man. 4-5 GlcNAc2, occasionally with compound GlcNAc or Gal modifications, but usually with the addition of potentially immunogenic β1,2-linked xylose (Xyl) and, together with insect cells, the addition of core α1,3-linked fucose (Fuc) residues.

[0023] The N-glycans of animal glycoproteins typically include galactose, fucose, and terminal sialic acid. These sugars are not present in glycoproteins produced by yeast and filamentous fungi. In human and other non-human eukaryotic cells, all types of glyconucleotide precursors (e.g., UDP-N-acetylglucosamine, UDP-N-acetylglucosamine, CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.) are synthesized in the cytosol and transported to the Golgi apparatus, where they are linked to the core oligosaccharide via glycosyltransferases (“Gnt”).

[0024] Sugar Engineering Methods

[0025] Genetic and metabolic engineering efforts have been conducted to modify N-glycan processing pathways in insects, yeast, and plants, and sialylated complex-type N-glycans have been obtained in insect cells, yeast, and plants, demonstrating that cell lines can be engineered to produce mammalian-like glycoproteins with potential therapeutic value (Geisler et al., 2015a; Strasser 2016; Hamilton et al., 2006; Jacobs et al., 2009). Beneficial glycosylation has been tested in other heterologous hosts such as mosses, aquatic plants, algae, and silkworms, but these systems are far from optimal (Calow et al., 2016; Cox et al., 2006; Tada et al., 2015). To date, there is no reliable method to predict whether a specific heterologously expressed glycosyltransferase in lower eukaryotes is adequately translated, catalytically active, or located within the correct organelle in the secretory pathway. Furthermore, alterations in the glycosylation pathway can change cell viability and / or glycoprotein site occupancy, leading to reduced yield or product quality.

[0026] Sialic acid (Sia) is a group of N- or O-substituted N-acetylneuraminic acid (Neu5Ac) derivatives that are ubiquitous in deuterostome lineages ranging from starfish to humans. These compounds have also been identified in several other organisms, including certain bacteria, protozoa, and fungi. Sialic acid biosynthesis in pathogenic bacteria and mammalian cells is well understood. Although sialic acid on the surface of pathogenic organisms is primarily thought to be a means of evading the host immune system, these same sialic acid molecules are also involved in several processes in higher organisms, including protein targeting, cell-cell interactions, cell-substrate recognition, and adhesion (Varki 2017; Vimr et al., 2004; Schauer 2000). The presence of sialic acid influences the in vivo half-life of glycoproteins. For example, the importance of sialic acid has been demonstrated in studies of human erythropoietin (hEPO). Terminal sialic acid residues on the N-linked glycans of this glycoprotein prevent rapid clearance of hEPO from the blood and improve its in vivo activity. Asialo-hEPO with a galactose terminal residue significantly reduces erythropoietic activity in vivo. This reduction is due to increased clearance of asialo-hEPO via hepatic desialylate glycoprotein receptors (Fukuda et al., 1989). Similarly, the absence of terminal sialic acid on many therapeutic glycoproteins can reduce in vivo efficacy and thus require more frequent patient dosing regimens.

[0027] Generally speaking, the ability to generate mammalian and fully humanized N-glycans is expected to transform the biotechnology industry, as a new group of organisms will emerge to produce therapeutic agents valuable for human health. This invention describes the analysis of natural and novel N-glycosylation pathways in the zooplastic lizard *Leishmania tarentolae*, revealing relevant differences from conserved pathways and their potential applications in glycoengineering. Glycoengineering methods will be provided in several embodiments.

[0028] This kinetic plasmid glycoengineering expression platform, based on the simple engineered glycan construction of human oligomannose, has led to the production of defined and fully-customized N-glycans, and significantly advances the field of glycoengineering by enabling the production of therapeutic agents with highly homogeneous humanized glycosylation at a lower cost, with greater safety, and more consistent results. The specific and unexpectedly unique N-glycan biosynthesis is novel and has never been described before, distinguishing it from glycoengineering methods in any other organism, such as Pichia pastoris or other eukaryotes. Furthermore, the shorter development timeline and faster production of recombinant strains compared to mammalian cells significantly enhance the broad applicability of this simple yet fully functional and customizable expression platform for therapeutic proteins. Summary of the Invention

[0029] This article describes a single-celled kinematoplast eukaryotic host cell containing heteroglycosyltransferases, including N-acetylglucosamine transferase, galactosyltransferase, and sialyltransferase. The host cells described herein are capable of producing mammalian (e.g., human) therapeutic glycoproteins containing homogeneous and fully functional, customized N-glycans with high site occupancy. The host cells provided herein can be used to express full-length therapeutic antibodies (e.g., anti-CD20 (rituximab)) and other therapeutic proteins (e.g., erythropoietin).

[0030] This article also provides nucleic acid and combinatorial libraries that can be used to successfully target and express mammalian enzymatic activities (e.g., those involved in N-acetylglucosamine elongation, galactosylation, and sialylation) in intracellular compartments of kinetoplastic eukaryotic host cells. This method provides engineered host cells that can be used to express and target any desired gene involved in glycosylation. The design of a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins is also provided.

[0031] In a specific implementation, this document provides a Leishmania host cell containing (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a heteroglycosyltransferase.

[0032] In some embodiments, the heteroglycosyltransferase is N-acetylglucosamine transferase; and / or heterogalactosyltransferase; and / or heterosialylate transferase. In some embodiments, host cells comprising two or more N-acetylglucosamine transferases are provided herein. In other embodiments, the host cell comprising the heterosialylate transferase also comprises a heterogeneous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0033] In another embodiment, this document provides a host cell in which one or more endogenous enzymes from the N-glycan biosynthetic pathway are missing, mutated, and / or functionally inactivated.

[0034] In other embodiments, the amino acid sequences of the N-acetylglucosamine transferase, galactosyltransferase, and / or sialic acid transferase are derived from the N-acetylglucosamine transferase, galactosyltransferase, or sialic acid transferase listed in Table 9, or any of their functional homologs, isoforms, or variants.

[0035] In other embodiments, the CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or any of its functional homologs, isoforms, or variants of the CMP-Sia biosynthetic pathway proteins listed in Table 11.

[0036] In another embodiment, the N-acetylglucosamine transferase is GnT-I. In another embodiment, the N-acetylglucosamine transferase is GnT-II. In another embodiment, the N-acetylglucosamine transferase is both GnT-I and GnT-II. In another embodiment, the galactosyltransferase is B4GALT1. In some embodiments, a host cell is provided herein, wherein the N-acetylglucosamine transferase is GnT-I and GnT-II, and the galactosyltransferase is B4GALT1. In another embodiment, the sialyltransferase is 2,6-SiaT or 2,3-SiaT. In some embodiments, a host cell is provided herein, wherein the N-acetylglucosamine transferase is GnT-I and GnT-II, the galactosyltransferase is B4GALT1, and the sialyltransferase is 2,6-SiaT or 2,3-SiaT. In a specific embodiment, a host cell is provided herein, wherein the N-acetylglucosamine transferase is GnT-I and GnT-II, the galactosyltransferase is B4GALT1, the sialyl transferase is 2,6-SiaT or 2,3-SiaT, and wherein the sialyl transferase further comprises a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0037] In some embodiments, the host cell is a Leishmania tarentolae cell. In other embodiments, the host cell is any strain listed in Table 13.

[0038] In some embodiments, the host cell comprises, for example, Figure 53 The CMP-Neu5Ac pathway with prokaryotic or eukaryotic enzymes is shown.

[0039] In other embodiments, a Leishmania signaling and / or retention sequence is added to N-acetylglucosamine transferase, galactosyltransferase, and / or sialic acid transferase, wherein the signaling sequence targets the N-acetylglucosamine transferase, galactosyltransferase, and / or sialic acid transferase to the endoplasmic reticulum of a Leishmania host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase, galactosyltransferase, and / or sialic acid transferase in the endoplasmic reticulum or Golgi apparatus. In another embodiment, the retention sequence retains the N-acetylglucosamine transferase and / or galactosyltransferase in the endoplasmic reticulum of the host cell. In another embodiment, the retention sequence retains the N-acetylglucosamine transferase and / or galactosyltransferase in the cis-faced membrane sac of the Golgi apparatus compartment of the host cell. In yet another embodiment, the retention sequence retains the N-acetylglucosamine transferase and / or galactosyltransferase in the intermediate membrane sac of the Golgi apparatus compartment of the host cell. In another embodiment, the retention sequence retains galactosyltransferase in the transmembrane sac of the Golgi compartment of the host cell. In another embodiment, the retention sequence retains sialyltransferase in the transmembrane sac of the Golgi compartment of the host cell. In yet another embodiment, the retention sequence retains both sialyltransferase and galactosyltransferase in the transmembrane sac of the Golgi compartment of the host cell.

[0040] In another embodiment, this document provides a host cell in which one or more endogenous enzymes from the N-glycan biosynthetic pathway are missing, mutated, and / or functionally inactivated.

[0041] In another embodiment, the signal sequence and / or preserved sequence is a signal sequence or preserved sequence derived from any species of Leishmania. In other embodiments, the signal sequence and / or preserved sequence is a signal sequence or preserved sequence derived from the lizard Leishmania tarentolae.

[0042] In another embodiment, the signal sequence is processed and removed.

[0043] In other embodiments, the preserved sequence is a cytoplasmic-transmembrane-stem (CTS) sequence derived from a Leishmania tarentolae protein. In another embodiment, the CTS sequence is derived from Leishmania tarentolae MAN1, NTPDase 1, or NTPDase 2. In yet another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or a functionally active fragment thereof. In another embodiment, the CTS sequence comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 24, or a functionally active fragment thereof; and identical to SEQ ID NO: 24. The sequence shown in NO:25 is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence or its functionally active fragment; or is identical to SEQ ID. The sequence shown in NO:26 is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the same sequence or its functionally active fragment.

[0044] In another embodiment, the CTS is derived from Leishmania tarentolae MAN1. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24 or a functionally active fragment thereof. In other embodiments, the preserved sequence comprises a GRIP sequence derived from Leishmania or a functionally active fragment thereof. In another embodiment, the GRIP sequence comprises the sequence of SEQ ID NO: 27 or a functionally active fragment thereof. In another embodiment, the GRIP sequence comprises a sequence or a functionally active fragment thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 27. In other embodiments, the retained sequence comprises a CTS sequence or a functionally active fragment thereof derived from a Leishmania protein, and a GRIP sequence or a functionally active fragment thereof derived from Leishmania.

[0045] In other embodiments, the target protein is heterologous to Leishmania host cells.

[0046] In another embodiment, the target protein has been engineered to include a signal sequence from Leishmania. In other embodiments, the signal sequence is a signal sequence from the lizard Leishmania tarentolae. In some embodiments, the signal sequence includes the sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29, or a functionally active fragment thereof. In a specific embodiment, the signal sequence includes the sequence shown in SEQ ID NO: 28, or a functionally active fragment thereof. In another embodiment, the signal sequence comprises a sequence or a functionally active fragment thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 28. In other embodiments, the signal sequence is processed and removed from the target protein.

[0047] In other embodiments, the target protein comprises the following amino acid sequence: human interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin-IL-2 (Denileukindiftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), tumor necrosis factor α (TNF-α), insulin, pramlintide, growth hormone (GH), insulin-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide-1 agonist (GLP-1), glucagon, growth hormone-releasing hormone (GHRH), secretin, and thyroid-stimulating hormone (TSH). H), human bone morphogenetic protein 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7), gonadotropin-releasing hormone (GnRH), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), fibroblast growth factor 7 (FGF7), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), neurotrophin-3, human fertility stimulating hormone (FSH), human chorionic gonadotropin (HCG), luteinizing hormone-α, erythropoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), extracellular domain of CTLA4 (e.g., FC-fusion protein), or extracellular domain of TNF receptor (e.g., FC-fusion protein).

[0048] In other embodiments, the target protein is a therapeutic protein. In other embodiments, the target protein is an Fc-fusion protein. In other embodiments, the target protein is an antibody.

[0049] In another embodiment, the target protein is an anti-human protein antibody. In other embodiments, the antibody has the following amino acid sequence: adalimumab (Humira); Remicade (Infliximab); ReoPro (Abciximab); Rituxan (Rituximab); Simulect (Basiliximab); Synagis (Palivizumab); Herceptin (Trastuzumab); Mylotarg (Gemtuzumab ozogamicin); Camppath (Alemtuzumab); Zevalin (Ibritumomab). tiuxetan); Xolair (Omalizumab); Bexxar (Tositumomab-I-131); Erbitux (Cetuximab); Avastin (Bevacizumab); Tysabri (Natalizumab); Actemra (Tocilizumab); Vectibix (Panitumumab); Lucentis (Ranibizumab); Soliris (Eculizumab); Cimzia (PEG-conjugated Certolizumab) pegol); Simponi (Golimumab); Ilaris (Canakinumab); Stelara (Ustekinumab); Arzerra (Ofatumumab); Prolia (Denosumab); Numax (Motavizumab); ABThrax (Raxibacumab); Benlysta (Belimumab); Yervoy (Ipilimumab); Adcetris (Brentuximab Vedotin); Perjeta (Pertuzumab).The amino acid sequences of Kadcyla (a new conjugate of trastuzumab and emtansine); or Gazyva (obinutuzumab).

[0050] In other embodiments, the antibody is a full-length antibody, Fab, F(ab')2, scFv, or sdAb. In other embodiments, the target protein comprises the amino acid sequence of an enzyme or its inhibitor. In another embodiment, the target protein comprises the following amino acid sequence: coagulation factor VII, coagulation factor VIII, coagulation factor IX, coagulation factor X, coagulation factor XIII, coagulation factor VIIa, antithrombin III (AT-III), protein C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, aglucosidase-α, laronidase (α-L-iduronidase), iduronidase (iduronate-2-sulfatase), thioesterase, galactosidase-β (human α-galactosidase A), botulinum toxin, collagenase, human deoxyribonuclease-I, hyaluronidase, papain, asparaginase, uricase (urate oxidase), glutamate carboxypeptidase (glutapeptidase), α1 protease inhibitor (α1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase.

[0051] In another embodiment, the therapeutic protein comprises the following amino acid sequence: abatacept (e.g., Orencia), aflibercept (e.g., Eylea), agalsidase beta (e.g., Fabrazyme), albiglutide (e.g., Eperzan), aldesleukin (e.g., Proleukin), alefacept (e.g., Amevive), alglucerase (e.g., Ceredase), and α-alglucosidase. Alfa (e.g., LUMIZYME), Aliskiren (e.g., Tektuma), α-1-protease inhibitor (e.g., Aralast), Alteplase (e.g., Activase), Anakinra (e.g., Kinet), Anistreplase (e.g., Eminase), Human anthrax immunoglobulin (e.g., ANTHRASIL), Antihemophilic factor (e.g., Advate), Anti-inhibitor coagulant complex (e.g., Feiba) Nf), antithrombin α, human antithrombin III, antithymocyte globulin (e.g., antithymocyte globulin), anti-thymocyte globulin (horse) (e.g., ATGAM), anti-thymocyte globulin (rabbit) (e.g., ATG-Fresenius), aprotinin (e.g., Trasylol), asfotase α, asparaginase (e.g., Elspar), Erwinia chrysanthemi asparaginase (e.g., Erwinaze), becaplermin (e.g., REGRANEX), belacept (e.g., Nulojix), beractant, bivalirudin (e.g., Angiomax), botulinum toxin type A (e.g., Botox). Botulinum toxin type B (e.g., Myobloc), Brentuximab vedotin (e.g., Adcetris), Buserelin (e.g., Suprecur), C1 esterase inhibitors (human), C1 esterase inhibitors (recombinant) (e.g., Ruconest), pegylated cerolizumab pegol (e.g., Cimzia), human chorionic gonadotropin (e.g., chorionic gonadotropin alpha), human chorionic gonadotropin (e.g., Ovidrel), human chorionic gonadotropin (recombinant) (e.g., Ovitrelle), coagulation factor ix (e.g., Alprolix), coagulation factor VIIa (e.g., NovoSeven), human coagulation factor X (e.g., Coagadex), coagulation factor XIII A-subunit (recombinant), collagenase (e.g., Cordase), Conestarα, adrenocorticotropic hormone (e.g., HPActhar), cosyntropin (e.g., Cortrosyn), darbepoetin α (e.g., Aranesp), defibrotide (e.g., Noravid), denileukin diftitox (e.g., Ontak), desirudin, digoxin, immune Fab (sheep) (e.g., DIGIBIND), and domase (a type of enzyme). alfa (e.g., Pulmozyme), Drotrecogin α (e.g., Xigris), Dulaglutide, Efmoroctocog α (e.g., ELOCTA), Elosulfase α, Enfuvirtide (e.g., FUZEON), Epoetin alfa (e.g., Binocrit), Epoetin zeta (e.g., Retacrit), Eptifibatide (e.g., INTEGRILIN), Etanercept (e.g., Enbrel), Exenatide (e.g., Byetta), Factor IX complex (human) (e.g., AlphaNine), Fibrinolysin, also known as cytoplasm (e.g., Elase), Filgrastim (e.g., ...NA), filgrastim-sndz, follicle-stimulating hormone (FSH) α (e.g., Gonal-F), follicle-stimulating hormone (FSH) β (e.g., Follistim AQ), galsulfase (e.g., Naglazyme), intragastric factor, gemtuzumab oxazolidin (e.g., Merota), glatiramer acetate (e.g., Copaxone), recombinant glucagon (e.g., Novogene), glutcarpidase (e.g., Voraxaze), gramicidin D (e.g., Neosporin), hepatitis B immunoglobulin, human calcitonin, human clostridium tetani toxoid immunoglobulin, human rabies virus immunoglobulin (e.g., Hyperab Rabies human immunoglobulin), human Rho(D) immunoglobulin (e.g., Hyp Rho D Inj 16.5%), human serum albumin (e.g., Albuminar), human varicella-zoster immunoglobulin (e.g., Varizig), hyaluronidase (e.g., HYLENEX), recombinant human hyaluronidase, irimotumab tiuxetan (e.g., Zevalin), idursulfase (e.g., Elaprase), imiglucerase (e.g., Cerezoyme), human immunoglobulin, insulin aspart (e.g., NovoLog), bovine insulin, insulin degludec (e.g., Tresiba), insulin detemir (e.g., LEVEMIR), insulin glargine (e.g., Lantus), insulin lissig (e.g., Insulin glulisine (e.g., APIDRA), insulin lispro (e.g., Humalog), porcine insulin (e.g., Iletin II), regular insulin (e.g., Humulin R), porcine insulin (e.g., vetsulin), insulin isopanaxylene (e.g., Novolin N), recombinant interferon alpha-2a (e.g., Roferon A), interferon alpha-2b (e.g., ...INTRON A), interferon αcon-1 (e.g., INFERGEN), interferon α-n1 (e.g., Wellferon), interferon α-n3 (e.g., Alferon), interferon β-1a (e.g., Avonex), interferon β-1b (e.g., Betaseron), interferon gamma-1b (e.g., Actimmune), intravenous immunoglobulins (e.g., Civacir), laronidase (e.g., Aldurazyme), lenograstim (e.g., Granocyte), lepiprazole Lepirudin (e.g., Refludan), Leuprolide (e.g., Eligard), Liraglutide (e.g., Saxenda), Lucinactant (e.g., Surfaxin), Lutropin α (e.g., Luveris), Mecasermin (e.g., NA), Menotropins (e.g., Menopur), Methoxy polyethylene glycol-betaeportin (e.g., Mircera), and more. Metreleptin (e.g., Myalept), natural alpha interferon or multiferon (e.g., Intron / Roferon-A), Nesiritide (e.g., NATRECOR), Ocriplasmin (e.g., Jetrea), Oprelvekin (e.g., Neumega), OspA lipoprotein (e.g., Lymerix), Oxytocin (e.g., Pitocin), Palifermin (e.g., Kepiv) Pancreatic lipase (e.g., Pancrecarb), bovine pegademase (e.g., Adagen), pegaspargase (e.g., Oncaspar), pegylated filgrastim (e.g., Neulasta), PEG-interferon α-2a (e.g., Pegasys), PEG-interferon α-2b (e.g., PEG-Intron), PEG-interferon β-1a (e.g., Plegridy), pegloticase (e.g., ...(Krystexxa)), Pegvisomant (e.g., SOMAVERT), Poractant α (e.g., Curosurf), Pramlintide (e.g., Symlin), Preotact (e.g., Protamine sulfate (e.g., protamine sulfate injection, USP), human protein S (e.g., human protein S), prothrombin (e.g., Feiba Nf), prothrombin complex (e.g., Cofact), prothrombin complex concentrate (e.g., Kcentra), rasburicase (e.g., Elitek), reteplase (e.g., Retavase), linonacept (e.g., Arcalyst), romiplostim (e.g., Nplate), sacrosidase (e.g., Sucraid), salmon calcitonin (e.g., Calcinar), sargramostim (e.g., Leucomax), and satumomab. Pendetide (e.g., OncoScint), lysosomal acid lipase α (e.g., Kanuma), secretin (e.g., SecretFlo), sermorelin (e.g., sermorelin acetate), serum albumin (e.g., Albunex), iodinated serum albumin (e.g., Megatope), simococog α (e.g., Nuwiq), sipuleucel-T (e.g., Provenge), recombinant growth hormone (e.g., NutropinAQ), recombinant growth hormone (e.g., BioTropin), streptokinase (e.g., Streptase), susoctocog α (e.g., Obizur), afatalizylase (Taliglucerase) alfa (e.g., Elelyso), teduglutide (e.g., Gattex), tenecteplase (e.g., TNKase), teriparatide (e.g., Forteo), tesamorelin (e.g., Egrifta), coagulation regulator alpha (e.g., Recomodulin), thymalfasin (e.g., Zadaxin), thyroglobulin, thyroid-stimulating hormone alpha (e.g., Thyrogen), tuberculin purified protein derivative (e.g., Aplisol), turoctocog alpha (e.g., Zonovate), follicle-stimulating hormone (e.g., BRAVELLE), urokinase (e.g., Kinlytic), vasopressin (e.g.,Pitressin, velaglucerase α (e.g., Vpriv), abciximab (e.g., ReoPro), adalimumab (e.g., Humira), alemtuzumab (e.g., CAMPATH), alirocumab (e.g., Praluent), arcitumomab (e.g., CEA-Scan), and artemizole (e.g., Atemizole) are all effective antiviral agents. Atezolizumab (e.g., Tecentriq), Basiliximab (e.g., Simulect), Belimumab (e.g., Benlysta), Bevacizumab (e.g., Avastin), Blinatumomab (e.g., Blincyto), Brodalumab (e.g., Siliq), Canazine (C anakinumab (e.g., ILARISE), canakinumab (e.g., Ilaris), capromab (e.g., ProstaScint), cetuximab (e.g., Erbitux), daclizumab (e.g., Zenapax), daratumumab (e.g., DARZALEX), denosumab (e.g., X) geva), Dinutuximab (e.g., unituxin), Eculizumab (e.g., Soliris), Efalizumab (e.g., RAPTIVA), Elotuzumab (e.g., EMPLICITI), Evolocumab (e.g., Repatha), Golimumab (e.g., Simponi Injection), Ibritumomab (e.g., Zevalin), Idarucizumab (e.g., Praxbind), Infliximab (e.g., Remicade), Ipilimumab (e.g., YERVOY), Ixekizumab (e.g., geva), Dinutuximab (e.g., unituxin), Eculizumab (e.g., Soliris), Efalizumab (e.g., RAPTIVA), Elotuzumab (e.g., EMPLICITI), Evolocumab (e.g., Repatha), Golimumab (e.g., Simponi Injection), Ibritumomab (e.g., Zevalin), Idarucizumab (e.g., Praxbind), Infliximab (e.g., Inmicade), Ipilimumab (e.g., YERVOY), Ixekizumab (e.g., geva), Dinutuximab (e.g., unituxin), Eculizumab (e.g., unituxin), Eculizumab (e.g., unituxin), Efal ...Taltz), mepolizumab (e.g., Nucala), and muromonab (e.g., ORTHOCLONE) OKT3), Natalizumab (e.g., Tysabri), Necitumumab (e.g., Portrazza), Nivolumab (e.g., Opdivo), Obiltoxaximab (e.g., Anthim), Obinutuzumab (e.g., Gazyva), Ofatumumab (e.g., Arzerra), Omalizumab (e.g., Xolair), Palivizumab (e.g., Synagis), Panitumumab (e.g., Vectibix), Pembrolizumab (e.g., Keytruda), Pertuzumab (e.g., Perjeta). Ramucirumab (e.g., Cyramza), ranibizumab (e.g., Lucentis), rexibacumab (e.g., RAXIBACUMAB), rituximab (e.g., Rituxan), secukinumab (e.g., Cosentyx), siltuximab (e.g., Sylvant), tocilizumab (e.g., ACTEMRA), tositumomab (e.g., Bexxar), trastuzumab (e.g., Herceptin), ustekinumab (e.g., Stelara), or vedolizumab (e.g., Entyvio).

[0052] In other embodiments, the host cell contains (a) Stt3 oligosaccharide transferase (OST) and (b) does not have endogenous N-glycan extension.

[0053] Another embodiment includes a method for preparing a glycosylated target protein, wherein the method includes culturing host cells and purifying the target protein from the culture.

[0054] Another embodiment includes a composition of glycosylated target proteins.

[0055] In another embodiment, the composition of the glycosylated target protein has at least about 90% to 100% of the N-linked glycosylated concordant sequence of the target protein in the composition having oligosaccharides comprising the following structures:

[0056]

[0057] The squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0058] In another embodiment, the composition of the glycosylated target protein has at least about 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein being a G0-Gn glycan, characterized by the following structure:

[0059]

[0060] The squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0061] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as a G0 glycan, characterized by the following structure:

[0062]

[0063] The squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0064] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as G1-Gn glycan, characterized by the following structure:

[0065]

[0066] Hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0067] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as a G2 glycan, characterized by the following structure:

[0068]

[0069] Hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0070] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as a G1 glycan, characterized by the following structure:

[0071]

[0072] Hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0073] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as a G1 glycan, characterized by the following structure:

[0074]

[0075] Hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0076] In another embodiment, the glycosylation on the target protein is further modified to optimize the pharmacokinetic properties of the target protein upon introduction into the target. In another embodiment, the glycosylation on the target protein is sialylated.

[0077] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0078]

[0079] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0080] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0081]

[0082] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0083] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0084]

[0085] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0086] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0087]

[0088] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0089] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0090]

[0091] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0092] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0093]

[0094] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0095] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0096]

[0097] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0098] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0099]

[0100] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0101] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0102]

[0103] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0104] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0105]

[0106] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0107] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0108]

[0109] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0110] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0111]

[0112] In this context, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0113] In another embodiment, the N-linked glycosylation concordance sequence is Asn-X-Ser / Thr; where X is any amino acid other than proline.

[0114] In another embodiment, the glycosylation target protein is secreted into a culture medium, and wherein the glycosylation target protein is glycosylated. In some embodiments, the glycosylation target protein is purified from the culture medium. In another embodiment, the glycosylation target protein is purified from the culture medium by affinity purification or ion-exchange chromatography. In another embodiment, the glycosylation target protein contains an Fc domain and is affinity purified from the culture medium by protein-A. In another embodiment, the glycosylation target protein contains an affinity tag and is affinity purified.

[0115] In another embodiment, the glycosylated target protein population is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homogeneous. In other embodiments, 90% to 100% of the N-glycosylation sites on the target protein are occupied by glycosylation.

[0116] In a specific embodiment, this document provides a hybrid N-acetylglucosamine transferase, wherein the hybrid N-acetylglucosamine transferase comprises (a) a catalytic domain of an N-acetylglucosamine transferase not derived from Leishmania; and (b) an amino acid sequence responsible for localization and retention in the Golgi compartment of Leishmania.

[0117] In some embodiments, the hybridized N-acetylglucosamine transferase is derived from the lizard leishmania (Leishmania tarentolae).

[0118] In other embodiments, the hybridized N-acetylglucosamine transferase has been engineered to include a signal sequence and at least one retention sequence, wherein the signal sequence targets the N-acetylglucosamine transferase to the endoplasmic reticulum of a Leishmania tarentolae host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase in the endoplasmic reticulum or Golgi apparatus.

[0119] In another embodiment, the hybridized N-acetylglucosamine transferase retains the N-acetylglucosamine transferase in the endoplasmic reticulum. In another embodiment, the hybridized N-acetylglucosamine transferase retains the N-acetylglucosamine transferase in the cisfacial sac of the Golgi apparatus. In another embodiment, the hybridized N-acetylglucosamine transferase retains the N-acetylglucosamine transferase in the intermediate sac of the Golgi apparatus. In other embodiments, the hybridized N-acetylglucosamine transferase is a cytoplasmic transmembrane stem (CTS) sequence.

[0120] In some other embodiments, the N-acetylglucosamine transferase is derived from the N-acetylglucosamine transferases listed in Table 9, or their functional homologs, isoforms, or variants.

[0121] In a specific embodiment, this document provides a hybrid galactosyltransferase comprising (a) a catalytic domain of a galactosyltransferase not derived from Leishmania; and (b) an amino acid sequence responsible for localization and retention in the endoplasmic reticulum or Golgi apparatus compartment of Leishmania.

[0122] In another embodiment, the hybrid galactosyltransferase is derived from the lizard leishmania (Leishmania tarentolae).

[0123] In other embodiments, the hybridized galactosyltransferase has been engineered to include a signal sequence that targets the galactosyltransferase to the endoplasmic reticulum of a Leishmania tarentolae host cell, and the retention sequence retains the galactosyltransferase in the endoplasmic reticulum or Golgi apparatus.

[0124] In some embodiments, the hybridized galactosyltransferase retains the galactosyltransferase in the endoplasmic reticulum. In some embodiments, the hybridized galactosyltransferase retains the galactosyltransferase in the cis-facing sac of the Golgi apparatus. In another embodiment, the hybridized galactosyltransferase retains the galactosyltransferase in the intermediate sac of the Golgi apparatus. In yet another embodiment, the hybridized galactosyltransferase retains the galactosyltransferase in the trans-facing sac of the Golgi apparatus. In other embodiments, the hybridized galactosyltransferase is a cytoplasmic-transmembrane-stem (CTS) sequence. In other embodiments, the hybridized sialic acid transferase is a GRIP sequence. In some embodiments, the hybridized sialic acid transferase is a combination of a CTS sequence and a GRIP sequence.

[0125] In other embodiments, the galactosyltransferase is derived from the galactosyltransferases listed in Table 9, or their functional homologs, isoforms, or variants.

[0126] In a specific embodiment, this document provides a hybrid sialyltransferase comprising (a) a catalytic domain of a sialyltransferase not derived from Leishmania; and (b) an amino acid sequence responsible for localization and retention in the endoplasmic reticulum or Golgi apparatus compartment of Leishmania.

[0127] In another embodiment, the hybrid sialyltransferase is derived from the lizard leishmania (Leishmania tarentolae).

[0128] In some embodiments, the hybrid sialyltransferase has been engineered to include a signal sequence that targets the sialyltransferase to the endoplasmic reticulum of a Leishmania tarentolae host cell, and wherein the retention sequence retains the sialyltransferase in the endoplasmic reticulum or Golgi apparatus.

[0129] In some embodiments, the hybridized galactosyltransferase retains the galactosyltransferase in the endoplasmic reticulum. In another embodiment, the hybridized sialyltransferase retains the sialyltransferase in the Golgi apparatus transmembrane sac. In yet another embodiment, the hybridized sialyltransferase is a CTS sequence. In other embodiments, the hybridized sialyltransferase is a GRIP sequence. In still other embodiments, the hybridized sialyltransferase is a combination of CTS and GRIP sequences.

[0130] In some other embodiments, the hybrid sialyltransferase is derived from the sialyltransferases listed in Table 9, or their functional homologs, isoforms, or variants.

[0131] In other embodiments, this document provides a nucleic acid encoding the N-acetylglucosamine transferase of the hybridization. In other embodiments, this document provides a nucleic acid encoding the galactosyltransferase of the hybridization. In some embodiments, this document provides a nucleic acid encoding the sialylate transferase of the hybridization.

[0132] 5.1 Terms and Abbreviations

[0133] When used in conjunction with numerical values, the term “about” refers to any value within ±1, ±5, or ±10% of the reference value.

[0134] As used herein, the term "object" refers to an animal (e.g., birds, reptiles, and mammals). In another embodiment, the object is a mammal, including non-primates (e.g., camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In some embodiments, the object is a non-human animal. In some embodiments, the object is livestock or pets (e.g., dogs, cats, horses, goats, sheep, pigs, donkeys, or chickens). In a particular embodiment, the object is a human. The terms "object" and "patient" are used interchangeably herein.

[0135] The abbreviations “α[numerical]”, “α[numerical],[numerical]”, “β[numerical]”, or “β[numerical],[numerical]” represent glycosidic bonds or glycosidic linkages, which are covalent bonds that link carbohydrate residues to another group. An α-glycosidic bond is formed when the two carbons have the same stereochemistry, while a β-glycosidic bond is formed when the two carbons have different stereochemistry. Attached Figure Description

[0136] Figure 1 The diversity of antibody combinations obtained from modifications (Jennewein and Alter 2017) drives antibody effector function. Modification of the IgG Fc domain through two alterations—(i) glycosylation selection (36 options) and (ii) subclass selection (4 subclasses)—resulted in 144 theoretical combinations and associated functional states. Based on antibody-glycan combinations, various functional responses can be induced, including the induction of anti-inflammatory responses; functional responses such as antibody-dependent phagocytosis (ADCP) and antibody-dependent cytotoxicity (ADCC); or inflammatory responses including complement activation and cytokine secretion. Abbreviation: IL, interleukin.

[0137] Figure 2A , 2B 2C: Structural changes obtained by glycosylation of the IgG Fc fragment, based on modifications according to (Sondermann et al., 2013). Figure 2A Animation of structural changes within the proposed Fc fragment via sialylation. The non-galactosylated (G0F) Fc fragment maintains an open conformation that allows FcγR binding. Triangles represent fucose and low binding. Figure 2B In fully α2,6-sialylated Fc (G2FS2), the α1,3-arm binds to the protein core of the Cγ2 domain, resulting in a closed conformation. The closed conformation of the resulting Fc fragment with altered tertiary structure reveals the DC-SIGN binding site, however, the activation of FcγR is blocked. Figure 2CBased on (Chen et al., 2017; Li et al., 2017), the model can be modified to achieve an open conformation on fucosylated N-glycans, allowing for binding to FcγR with higher affinity. The conformational change can be both open and compressed when the fucose-free N-glycan is capped by sialic acid double tentacles. The Fc structure is stabilized by two glycan latches to maintain the correct Fc conformation, but this Fc structure is mediated by different glycan residues. Chen's results demonstrate the role of N-glycosylation in maintaining Fc structural integrity and show the possibility of different modes in glycan-glycan interactions regulated by terminal sialic acid.

[0138] Figure 3A and 3B Bioinformatic evaluation of endoplasmic reticulum (ER) N-glycan biosynthesis in Leishmania tarentolae and comparison with conserved pathways obtained based on (Varki 2009). Figure 3A The missing genes in the lizard leishmania tarentolae are indicated by crosses and strikethroughs. Figure 3B The enzymes catalyzing each step of biosynthesis were identified primarily from studies of mutants of the yeast *Saccharomyces cerevisiae*. The gene affected by each yeast mutation is designated as an ALG gene (used for alterations in glycosylation). The synthesis of polyterpene-PP-GlcNAc2Man9Glc3 is described. Polyterpene phosphate (Dol-P) located on the cytoplasmic side of the ER membrane accepts GlcNAc-1-P from UDP-GlcNAc in the cytoplasm to produce Dol-PP-GlcNAc. Before "flipping" across the ER membrane to the luminal side, Dol-PP-GlcNAc is extended to Dol-PP-GlcNAc2Man5 by the indicated ALG enzyme. On the luminal side of the ER membrane, four mannose residues are added from Dol-P-Man, and three glucose residues are added from Dol-P-Glc. Dol-P-Man and Dol-P-Glc are also prepared on the cytoplasmic side of the ER, and they are "flipped" to the luminal side. Homologs present in *L. tarentolae* are indicated by checkmarks, and missing homologs by crosses. The man3 structure is expected to flip and transfer directly to the polypeptide without further mannosylation due to the absence of ALG12, 6, 8, 9, and 10, and ALG3 is thought to contain a loss-of-function mutation. ALG5 (polyterpenoid-phosphoβ-glucosyltransferase) is absent. OST consists only of Stt3 (Table 4).

[0139] Figure 4A ,4B 4C: Bioinformatics evaluation of endoplasmic reticulum (ER) N-glycan biosynthesis, pruning, and translocation to protein receptors. Figure 4A A cross indicates the absence of ALG homologs in biosynthesis, and a strikethrough indicates a reduced Man3 precursor. Although it is assumed that any Man4 or Man5 precursors are missing (due to the absence of ALG3, 9, 11, 12) and glucosylation precursors (due to the absence of ALG6, 8, 10, and ALG5), pruning enzymes (GCS1, GNAB, MAN1) are present (enzymes missing are indicated by strikethrough), such as... Figure 4B As shown. Figure 4C It is expected that only the final glycan Man3 will be transferred to the N-common site of the protein receptor via Stt3. The folding intermediate Glc1Man9GlcNAc2 was not expected to be used as a ligand for the lectin it is responsible for in quality control.

[0140] Figure 5Based on (Moremen et al., 2012), a bioinformatics evaluation of protein N-glycosylation and protein folding quality control. a) During glycoprotein biosynthesis, after translation of nascent peptides, they translocate through SEC61 pores, and glycans are simultaneously transferred from lipid-linked intermediates to peptide receptor sequences via oligosaccharide transferases (OSTs). A cleft in the STT3 subunit of the OST searches for the Asn-X-Ser / Thr receptor sequence, while adjacent clefts bind glycan donors. b) Immediately after transfer, glycan pruning occurs via Glc removal through α-glucosidase I (GIsI) and α-glucosidase II α-β heterodimers (GIsIIα / β). The folding intermediate containing the Glc1Man9GlcNAc2 structure is a ligand for either lectin-calcified protein (absent in *L. tarentolae*) or calreticulin (absent in *L. tarentolae*), which functions in complex with ERp57. Further Glc cleavage follows dissociation from the lectin. Other chaperone proteins are facilitated by the ATP-driven chaperone protein BiP (also known as GRP78). The properly folded glycoprotein package is used for transport to the Golgi apparatus. c) Incompletely folded glycoproteins are recognized by the fold-sensing molecule UDP-Glc:glycoprotein glucosyltransferase (UGGT1, absent in *L. tarentolae*). They are then reglycosylated and reintegrated into the calcified protein cycle by adding Glc residues back into the glycan structure. d) Endoplasmic reticulum (ER) treatment of terminally misfolded glycoproteins via Man pruning (through the activity of ER α-mannosidase I (ERManI, =MAN1, present in Leishmania tarentolae) or GolgiManIA, GolgiManIB, and GolgiManIC (not shown), followed by ER degradation-enhanced α-mannosidase-like 1 (EDEM1), EDEM2, and EDEM3 (which are homologous to Htm1, also known as Mnl1 in yeast, all absent in Leishmania tarentolae). The pruned glycans bind to ER lectins OS9 or XTP3B (not shown) and utilize the driving force of cytosolic ubiquitin-binding proteins and the ATPase function of valocin-containing proteins (VCP; also known as TERATPase; known as Cdc48, Ufd1, or Npl4 in yeast) via derlin The complex of DER1, DER2, and DER3 (SEL1L complex; Hrd3 in yeast) translocates into the cytosol. The peptides are deglycosylated by cytosol PNG enzymes (absent in Leishmania tarentolae) and degraded by proteasome 149.A cross indicates the absence of a homolog in *L. tarentolae*, and the homologs of the present invention are shown in boxes. See also Table 8 for detailed comparison information.

[0141] Figure 6 The mammalian N-glycosylation pathway, modified from (Kellokumpu et al., 2016) compared to the wild-type lizard Leishmania tarentolae, is illustrated in the schematic animation showing the sequential processing of N-glycans via glycosyltransferases in the endoplasmic reticulum (ER) and Golgi apparatus. It is generally assumed that the involved enzymes (glycosyltransferases and glycosidases) act sequentially and individually, over a specified time, by adding or removing sugar residues through a reciprocating motion of the growing oligosaccharide chain, as shown on the left (“mammals”). Figure 6 The right side represents the wild-type lizard leishmania (Leishmania tarentolae), which, compared to the mammalian system, has a reduced precursor and different mature N-glycans.

[0142] Figure 7 The mammalian N-glycosylation pathway was modified from (Kellokumpu et al., 2017) compared to the wild-type lizard leishmania (Leishmania tarentolae). A schematic animation of the mammalian pathway is provided by illustrating the glycoengineering steps in glycoengineered Leishmania to produce the indicated glycans. Figure 6 .

[0143] Figure 8 The proposed N-glycosylation biosynthetic pathway in the lizard leishmania (Leishmania tarentolae). The N-glycan precursor assembles as Man3 on the cytoplasmic side and then flips into the ER lumen. This Man3 glycan is then directly transferred via Stt3 (the only part of the normally present OST complex). The final glycan at the N-glycosyl site of the receptor protein is Man3, which is transferred to the Golgi cis-faced sac. In the Golgi cis-faced sac, a heterologous enzyme catalyzes a glycosyltransferase reaction to construct the human biantennae N-glycan. These are Gnt-I and Gnt-II derived from mammals or insects, with the addition of the first two GlcNAc residues. The corresponding glycoforms are described by their abbreviations in the following boxes. In the Golgi apparatus transmembrane sac, mammalian-derived GalT and SiaT enzymes expressed in Leishmania catalyze the reaction to complete N-glycans, which then become homogeneous, fucose-free, but biantennae-sialyzed humanized glycans based on α2,3 or α2,6-linked sialic acid (Neu5Ac), mediating different intended functions.

[0144] Figure 9 In three different wt strains, UPLC separation and determination by MS m / z of PNGase F or PNGase A released from total Leishmania tarentolae cell precipitates and RF-labeled N-glycans or blank-treated N-glycans were performed. (Man)3(GlcNAc)2 = Man3 was detected by MS, with a calculated m / z = 1222.7166; [M+Na] + 1244.6985; [M+K] + 1260.6724. Man3 was observed in all precipitated sample particles, with RTs of 9.16, 9.27, and 9.34 when released via PNGase F or PNGase A, but not in the blank-treated samples. The blank-treated samples showed contamination in the strain (middle and bottom plots), for example, contamination from cell wall glycolipids (characterized by hexoses). Another peak was observed in the FLR trace at RT 15.76, but the compound could not be identified despite obtaining excellent MS spectra. Generally, there was little background observed in the FLR trace, but there was a lot of background in the MS trace at earlier RTs (<8 min RT, not shown).

[0145] Figure 10A and 10B Following the in vitro activity assays of SfGnT-I, TbGnT-I, or TbGnT-II, the 2-AB-labeled N-glycan reaction products were separated by HPLC. Immunoenrichment of recombinant leishmaniae (L. tarentolae) expressing SfGnT-I, TbGnT-I, or TbGnT-II was used in the in vitro assays with free paucimannose (Man3) as a substrate. Figure 10A or dissolved portion ( Figure 10B The clean and 2AB-labeled reaction products were separated by HPLC and compared with standards, Man3-2AB, G0-Gn(NGA3-N), and G0(NGA2). The peak at RT 76.7' represents the activity of the heterologous GnT-I enzyme, which is marked with an asterisk.

[0146] Figure 11A and 11B Following the in vitro activity assay of MGAT1, the reaction product of 2-AB-labeled N-glycan was separated by HPLC. Immunoenriched MGAT1 from SN or precipitated particles, dissolved fractions, and lysis buffers from recombinant MGAT1-expressing leishmania (L. tarentolae) were used in the in vitro assays of the following substances: Figure 11AFor 2AB-labeled high-mannose (Man5), the commonly used biosynthetic receptor for MGAT1 in the known N-glycosylation pathway, or ( Figure 11B For the substrate 2AB-oligomannose (Man3), it mimics the Man3 receptor used for extension in the Golgi apparatus of the Leishmania N-glycan pathway. As a negative control, lysates from *L. tarentolae* cells were used in the reaction. Clean 2AB-labeled reaction products were separated by HPLC and compared with standards. Figure 11A Man5 or Figure 11B Comparison of Man3 and G0-Gn (NGA2-N, green). Peaks marked with asterisks indicate the activity of heterologous GnT-I enzymes.

[0147] Figure 12A and 12B Following the in vitro activity assay of MGAT2, the reaction product of 2-AB-labeled N-glycan was separated by HPLC. Immunoenriched MGAT2 from SN and precipitate particles from recombinant leishmania (L. tarentolae) were used in the in vitro assays of the following substances: for 2AB-labeled G0-Gn (NGA2-N), the commonly used biosynthetic receptor for MGAT2 in the known N-glycosylation pathway (…). Figure 12A ), or for 2AB-oligomannose (Man3) as a substrate, which mimics the Man3 receptor used for elongation in the Golgi apparatus of the Leishmania N-glycan pathway ( Figure 12B The reaction product, labeled with 2AB, was not extended via MGAT2. Clean 2AB-labeled products were separated by HPLC and compared with standards Man3, G0-Gn(NGA2-N), and G0(NGA2). Peaks marked with asterisks indicate the activity of the heterologous GnT-II enzyme.

[0148] Figure 13A , 13B 13C: HPLC separation of the 2-AB-labeled N-glycan reaction products following the in vitro activity assay of SfGnT-II. Crude lysates from recombinant Leishmania tarentolae expressing SfGnT-II were used in the in vitro assays for the following substances: 2AB-oligomannose (Man3) as a substrate, which mimics the Man3 receptor used for extension in the Golgi apparatus of the Leishmania N-glycan pathway (…). Figure 13A For 2AB-labeled G0-Gn (NGA2-N), the commonly used biosynthetic receptor for Gnt-II in the known N-glycosylation pathway is ( Figure 13BOr for Man5 labeled 2AB, non-GnT-II substrates ( Figure 13C The clean 2AB-labeled reaction product was separated by HPLC and compared with standards Man3, G0-Gn(NGA2-N), and Man5. Peaks marked with an asterisk represent the activity of the heterologous GnT-II enzyme.

[0149] Figure 14A , 14B 14C: HPLC separation of the 2-AB-labeled N-glycan reaction products after in vitro activity assay of B4GALT1. Figure 14A In the in vitro assay of 2AB-G0-Gn(NGA2-N), B4GALT1 immunoenriched from recombinant leishmania (L. tarentolae) was used. Clean 2AB-labeled reaction products were separated by HPLC and compared with standards, G0-Gn(NGA2-N), G1(NA2G1), and NA2(G2). Peaks marked with asterisks indicate the activity of the heterologous GalT enzyme. Figure 14B The schematic representation of the composition of the commercially available NGA2-N(G0-Gn) standard and the expected reaction outcome after in vitro determination with B4GALT1 are shown, which show that G1-Gn is formed on two different branches. Figure 14C This indicates the expected reaction product when G0(NGA2) is used as the substrate for GalT.

[0150] Figure 15 MALDI-TOF MS spectra of wt% of fully methylated PNGase F and N-glycans from glycoengineered Leishmania tarentolae in the m / z range of 1000–2200. Glycan shapes are annotated above the m / z peaks.

[0151] Figure 16 MALDI-TOF / TOF MS fragmentation profiles of the identified sugar forms. Fragment ions present in the profile at m / z 1171.6 (=Man3) (top) from wt St10569, at m / z 1416.7 (=G0-Gn) (middle) from the profile of the recombinant strain St11707 expressing SfGnT-I, and at m / z 1661.8 (=G0) (bottom) from the profile of the recombinant strain St12320 co-expressing SfGnT-I and SfGnT-II confirm the presence of fully methylated N-glycans in Man3 (top), G0-Gn (middle), and G0 (bottom).

[0152] Figure 17A , 17B And 17C: Figure 17AThe image shows UPLC separation and m / z determination by MS of PNGase F-released and RF-labeled N-glycans or blank-treated N-glycans from recombinant glycoengineered Leishmania tarentolae cell precipitates. The dashed lines represent background peaks visible in the blank sample. RF-labeled N-glycans were derived from: 16.260 - St11707 (SfGnT-I) using PNGase F; 16.261 - St11707 (SfGnT-I) - blank; 16.263 - St12525 (SfGnT-I and SfGnT-II) - PNGase F and 16.264 - St12767 (SfGnT-I and MGAT2) - PNGase F superimposed. Bold arrows indicate m / z-confirmed glycan peaks. Figure 17B The N-glycans (MGAT1 and MGAT2) from St 13065 are shown, and the peaks of St 13066 (SfGnT-I and B4GalT1) are confirmed in the peaks schematically annotated above with [M+H] m / z = 1222 Man3, m / z = 1425 G0-Gn, m / z = 1628 (G0) and m / z = 1587 (G1-Gn). Figure 17C Unlabeled peaks were also present in the blank hydrolysate and contained putative cell wall glycolipid fragments (dashed arrow, "background").

[0153] Figure 18 N-glycan extension of recombinant hEPO expressed in glycoengineered Leishmania. Recombinant expression of the GnT-I candidate SfGnT-I to extend the native N-glycan to G0-Gn. Recombinant target protein human erythropoietin (rhEPO) expressed alone (St11521) or co-expressed with SfGnT-I (St11895). rhEPO secreted via the Leishmania secretory pathway and purified from culture supernatant. SDSPAGE of two purified EPO samples stained with Coomassie Brilliant Blue. Molecular weight change upon extension to higher molecular weight with the addition of GlcNAc.

[0154] Figure 19: RhEPO sites occupied by peptide mapping. Light gray: EPO peptides in the flow portion during the glycopeptide enrichment step. Gray: Peptides present in the flow portion and the enriched glycopeptide portion. Dark gray: EPO peptides covering N-glycosylation sites N24 and N38 in their deacylated form in the enriched glycopeptide portion after deglycosylation with PNGase A+F. This peptide was not detected in its non-glycosylated form. In this study, peptides with N-glycosylation site N83 were not clearly detected (neither in their non-glycosylated form nor their deglycosylated form). Bold indicates N-glycosylation sites. SEQ ID NO: 108 is shown.

[0155] Figure 20 : A glycopeptide of rhEPO containing the identified N-glycan. N-glycosylation sites N24 and N38 are shown. SEQ ID NO: 109 is shown.

[0156] Figure 21A and 21B Identification of naturally secreted glycoproteins. Figure 21A The workflow is summarized in the boxes, and the Coomassie Brilliant Blue stained SDS-PAGE shows the elution profiles (E1 to E5) after ConA enrichment. Major proteins are in the 60-80 kDa range. Elution was used for N-glycan release, and peptide mapping was employed to identify 2AB markers and proteins using proteomics methods. Figure 21B This shows the number of proteins identified and the number of peptides identified by representing a sequence coverage probability >96%.

[0157] Figure 22A and 22B Identification of the N-terminus of naturally secreted glycoprotein invertases. Figure 22A The workflow is summarized in the box. The supernatant of St10881 was precipitated with (NH4)2SO4 and purified using ConA. ConA elution fractions E1 to E8 are shown in Coomassie Brilliant Blue stained SDS-PAGE. Following size exclusion chromatography, the most prominent secreted proteins, excluding rhEPO, were collected and subjected to EDMAN N-terminal sequencing. Figure 22B The sequences of the convertases identified by peptide mapping are shown, with light gray markings indicating the identified peptides. Circles indicate processed N-terminal DGVPYEx (SEQ ID NO: 157) derived from EDMAN N-terminal sequencing. SEQ ID NO: 110 is also shown.

[0158] Figure 23When the gene is fused to the invertase secretion sequence derived from the invertase, rhEPO is secreted. EPO is expressed using two different signal peptides for secretion translocation to secrete into the supernatant. The supernatant from the TCA precipitate was loaded onto an SDS-PAGE corresponding to 15 OD cells and subjected to Western blotting (left panel) or Coomassie Brilliant Blue staining (right panel). In the SN assay, two rhEPO variants, one with an LMSAP secretion signal (St11376) or derived from the invertase (St11377), were identified at their molecular weights representing their N-glycosylated forms, tested using five different monoclonal samples.

[0159] Figure 24A and 24B Domain prediction of proteins natively located in the Golgi apparatus. Most Golgi-localized glycosyltransferases are type II membrane proteins with a short N-terminal cytoplasmic domain, an α-helix™ domain of approximately 20 amino acids, a stem domain, and a C-terminal globular catalytic domain located in the lumen of the secretory pathway. (Kellokumpu et al., 2016) Figure 24A Representatives of Gnt (Gnt-x) or SfGnT-I with secretion signal and CTS located at the N-terminus followed by a globular / catalytic domain are shown. Figure 24B The natural and putative Golgi proteins MAN1, LtaNTPDase1, and 2 from the lizard *L. tarentolae*, identified by bioinformatics comparison, are shown, with the predicted amino acid sequences of the CTS domain listed below the schematic bar chart. The GRIP domain was identified in LTAR_110005600.1 (previous naming scheme: LtaP11.0070), and its C-terminal amino acid sequence is shown below the bar chart. SEQ ID NOs: 24-27 are shown.

[0160] Figure 25A , 25B 25C: Gnts and domain predictions for hybridization designs for improved Golgi targeting, retention, and homo- or heterodimerization. Gnt-X is represented by a white bar chart, which is Sf-Gnt-I shown as an example. In the example MGAT1 shown, Gnt-Y is in dark gray. The numbers below indicate the fusion point used for hybridization design after the CTS of Gnt-y / MGAT1. Figure 25BThis shows 90, 110, 130, or 150 amino acids starting from the N-terminus of Gnt-Y / MGAT1, which is a nucleotide sequence fused to the N-terminus of Gnt-X / SfGnT-I. Full-length (FL) Gnt-Y (MGAT1) fused to the top of Gnt-X / SfGnT-I (FL) or N-terminal CTS-truncated Gnt-X / SfGnT-I (bottom) are also shown. Figure 25C As shown.

[0161] Figure 26A and 26B Growth behavior of rhEPO or SfGnT-I recombinant cells and the expression and localization of rhEPO or SfGnT-I over time. Growth of cells expressing rhEPO (St11357) or SfGnT-I (St11521) was monitored in static culture versus shaking culture - growth (OD) and morphology over time are shown in the figure, as shown in the figure. Figure 26A As shown. In the supernatant of TCA precipitation (SN) and intact cell extract (WCE), the expression rates and localization of rhEPO or SfGnT-I over time were determined by Western blotting using anti-Strep antibody. Figure 26B ).

[0162] Figure 27A and 27B The localization of SfGnT-I, TbGnT-I, and TbGnT-II was determined. Sf-GnT-I, a Strep-tagged cell, was detected by Western blotting from cultured SNs via TCA precipitation at a load corresponding to 15 OD, or by affinity enrichment from cell lysates dissolved in Triton X using streptavidin. Figure 27A TbGnT-I and TbGnT-II derived from Trypanosoma and HA-tagged were detected by immunoblotting in the crude fraction and the Triton-X lysate fraction. Figure 27B ).

[0163] Figure 28A and 28B Localization of hybrid Gnt variants of SfGnT-I. Expression of natural SfGnT-I or hybrids of SfGnT-I with Man1-CTS or LTaNTPDase1 or 2CTS, and localization of the hybrids from cultured SN( Figure 28A ), or precipitate from crude lysis buffer using triton (TrX+) or without triton (TrX-), or homogenize from the insoluble fraction (Ins). Figure 28B ).

[0164] Figure 29MGAT1 localization and affinity-enrichment. HA-tagged MGAT1 was detected by immunoblotting in the purified fraction (Purif.) of the affinity-enriched portion from recombinant cell culture SN or precipitate particles. Elu was the elution fraction used for in vitro assays. For localization, MGAT1 was detected in lysis buffer with or without triton (TrX+), or in the insoluble fraction (Insol.fr.), or in the lysis buffer homogenized from fragments.

[0165] Figure 30 Localization and affinity-enrichment of SfGnT-II. HA-tagged SfGnT-II was detected by Western blotting in the purified fraction (Purif.) of the affinity-enriched portion from recombinant cell culture SN or precipitate particles. Elu was the elution fraction used for in vitro assays. For localization, SfGnT-II was detected in lysis buffer with or without triton (TrX+), or in the insoluble fraction (Insol.fr.), or in the lysis buffer homogenized from fragments.

[0166] Figure 31A and 31B MGAT2 localization and affinity-enrichment. HA-tagged MGAT2 was detected by immunoblotting in purified fractions (W, wash; Elu, elution) from affinity-enriched fractions (Pell) of recombinant cell culture SN or precipitate particles (Pell). Cells were grown for 96 h or 72 h. Elu was the elution fraction used for in vitro assays. For localization, SfGnT-II was detected in lysis buffer with or without triton (TrX+) or in lysis buffer from insoluble fractions (Insol.fr.) or from fragment homogenized lysis buffer. Figure 31A The localization and expression levels of MGAT2 in SNs from different passages of precipitated cells were shown by anti-HA immunoblotting. Figure 31B ).

[0167] Figure 32Customized Fc glycans mediate desired downstream effector functions. Immuno-activating glycans mediate effects such as ADCC, ADCP, and CDC. The glycans indicated above were customized using a glycoengineered Leishmania platform for Fc molecule or antibody expression to mediate immune activation, immunosuppression, and / or PK optimization. If the biantennary glycan is capped with α2,6-linked Neu5Ac, the function will be activating, but if it is capped with α2,3-linked Neu5Ac, the function will be immunosuppressive. Supporting evidence is discussed in (Chen et al., 2017; Li et al., 2017). If the biantennary N-glycan is capped with sialic acid, regardless of their specific bonds, PK will be improved by increasing the circulating half-life due to the presence of sialic acid. Furthermore, the expected immune tolerance benefit of α2,6-linked Neu5Ac will avoid the generation of antidrug antibodies (ADAs) against potential immunogenic therapeutic agents. The immunosuppressive glycan at the bottom is the core α1,6-fucosylated glycan, a typical glycan derivative from the CHO cell platform (without terminal sialic acid).

[0168] Figure 33 The amino acid sequence of recombinant rituximab (anti-CD20 monoclonal antibody) is shown. The protein sequences of the light chain (LC) and heavy chain (HC) with modified convertase secretion sequences are shown in bold, and the Fc N-glycosylation sites are shown in bold and underlined. SEQ ID NOs: 111 and 112 are shown.

[0169] Figure 34 : An expression cassette containing recombinant rituximab (anti-CD20 monoclonal antibody) for stable integration into Leishmania tarentolae. The diagram illustrates the relevant regions: 5'ssu is a 5' homology site for site-specific integration into the ssu-locus encoding 18S rRNA via homologous recombination; 5'UTR is a 5' untranslated terminal repeat containing a splicing leader receptor sequence; a light chain sequence optimized for Leishmania codons containing the invertase secretion signal; IR1, a first intergenic region containing a 3'UTR for polyadenylation and a 5'UTR for downstream genes; a heavy chain sequence optimized for Leishmania codons containing the invertase secretion signal; IR2, a second intergenic region containing a 3'UTR for polyadenylation and a 5'UTR for downstream resistance markers (sat), followed by its 3'UTR and a 3' homology (3'ssu) region for site-specific recombination into the genome. KpnI is a restriction endonuclease used to obtain the expression cassette excised from the donor *E. coli* maintenance plasmid for transfection with linearized DNA.

[0170] Figure 35 Expression and purification of full-length monoclonal antibody from recombinant Leishmania tarentolae secreting rituximab_LMTB. The workflow is described at the top as a box. The harvested supernatant and SDS-PAGE of 5× concentrated SN stained with Coomassie Brilliant Blue are shown (left panel). Protein A elution (right panel) shows two main forms of Coomassie Brilliant Blue staining, one migrating as FL rituximab on SDS-PAGE under non-reducing conditions (“non”), and the other migrating as a degradation product at approximately 100 kDa. Under reducing conditions (“red”), the degradation product is separated by HC and LC. Hydrophobic interaction chromatography (HIC) elution shows the separation of these two forms when separated under non-reducing or reducing conditions. Coomassie Brilliant Blue staining of the SDS-PAGE gel.

[0171] Figure 36A and 36B Rituximab LMTB, purified from recombinant Leishmania, is a commercial product containing the same protein expressed in CHO cells. A comparison. Figure 36A Coomassie brilliant blue stained SDSPAGE shows FL antibodies that release N-linked glycans under non-reducing conditions, with or without (+) PNGase F treatment. Figure 36B A schematic diagram of the antibody molecule, domains, and disulfide bridging bonds is shown. N-glycosyl sites are represented by carbohydrates. On the left side of the animated molecule, the homogeneous Man3 N-glycan of the Leishmania-derived antibody is shown; however, on the right side, the expected slight heterogeneity of the N-glycan of the antibody derived from CHO expression is shown.

[0172] Figure 37 Rituximab LMTB, purified from recombinant Leishmania, is a commercial product containing the same protein expressed in CHO cells. Capillary gel electrophoresis and comparison. CGE shows the release of N-linked glycans by FL antibody under non-reducing conditions, without (-) or with (+) PNGase F treatment.

[0173] Figure 38A and 38B Rituximab LMTB, purified from recombinant Leishmania, was obtained. Figure 38B (and commercial products that express the same protein in CHO cells) ( Figure 38A Size exclusion chromatography and comparison.

[0174] Figure 39A and 39B Rituximab LMTB, purified from recombinant Leishmania, was obtained. Figure 39B (and commercial products that express the same protein in CHO cells) ( Figure 39A N-glycan spectra and comparisons using RF. N-glycans were released via PNGase F and labeled with RF. UPLC separation is shown, and m / z values ​​were confirmed for the N-glycans annotated above the peaks.

[0175] Figure 40 Rituximab LMTB, purified from recombinant Leishmania, is a commercial product containing the same protein expressed in CHO cells. N-glycan spectra and quantitative comparisons were performed using fully methylated N-glycans. N-glycans were released via PNGase F and fully methylated. The relative intensity (%) of N-glycans was calculated to establish the N-glycan spectrum for each spectrum. For this purpose, the sum of the peak intensities of deisotopized N-glycans was determined and set to 100%. The relative intensity (%) of each glycan was then determined relative to this value. Black column: Rituximab _LMTB, Gray column: Roche confirmed the m / z values ​​for the N-glycans annotated above the peaks, including confirmation through their fragment spectra.

[0176] Figure 41A and 41B Rituximab LMTB, purified from recombinant Leishmania, was obtained. Figure 41B (and commercial products that express the same protein in CHO cells) ( Figure 41A The N-glycan spectra of fully methylated N-glycans were used for comparison. Data from three different sampling times were collected. ( Figure 41A ) and from rituximab_LMTB during 2 independent collections ( Figure 41B MALDI-TOF MS spectra of fully methylated N-glycans released by PNGase F.

[0177] Figure 42 Rituximab LMTB, purified from recombinant Leishmania, and a commercial product expressing the same protein in CHO cells. And sugar-engineered third-generation anti-CD-20 Comparison of micro-heterogeneities and quantification of N-glycans by Roche. N-glycans were released via PNGase F and fully methylated. The relative intensities (%) of N-glycans were calculated to establish the N-glycan spectrum for each spectrum. For this purpose, the sum of the peak intensities of deisotopized N-glycans was determined and set to 100%. The relative intensity (%) of each glycan was then determined relative to this value. Gray bars: Rituximab _LMTB, Dark gray bars: Roche, light gray column: Roche confirmed the m / z values ​​for the N-glycans annotated above the peaks, including confirmation via their fragment spectra.

[0178] Figure 43A and 43B Functional assay of antigen binding of rituximab_LMTB, purified from recombinant Leishmania. Figure 43A Showing Or, a FACS bar chart of rituximab_LMTB relative to control IgG1κ. Raji cells expressing CD20 antigen were used to test the functionality of the anti-CD20 antibody. Staining sequences included specified concentrations of FCR blocking, IgG1 blocking, and primary antibody. Rituximab LMTB or negative control IgG1κ, followed by the secondary antibody anti-IgG1 APC. After gating, each sample represents the analysis of 10,000–13,000 cells. Arrows indicate average fluorescence migration, which indicates the binding of anti-CD20 antibody to the CD20 antigen on the surface of Raji cells. Figure 43B The results show the recombinant expression of the GST fusion protein on the membrane in *E. coli* (E. coli) at gradually increasing concentrations. Different concentrations of... Alternatively, rituximab-LMTB, or a control antibody (anti-maltose-binding protein, anti-GroEL heat shock protein from E. coli, or anti-His IgG antibody), may be used to culture the membrane strips, or no primary antibody may be used. After washing, the secondary antibody is anti-human IgG-HRP.

[0179] Figure 44 Rituximab LMTB, purified from recombinant Leishmania, is a commercial product containing the same protein expressed in CHO cells. A schematic diagram and comparative summary. For rituximab_LMTB (right side) and for... (Left side) Showing the N-terminal sequences, C-terminal amino acids, and glycan profiles of HC and LC. All indicated disulfide bridging bonds were identified in both samples.

[0180] Figure 45N-glycan spectra and quantitative comparisons using fully methylated N-glycans from different kinematosomes. N-glycans from *Crithidia fasciculata*, *Crithidia deanei* (Angomonas), and *Phytomonas davidi* were released and fully methylated after defatting with PNGase F. The relative intensities (%) of the N-glycans were calculated to establish the N-glycan spectrum for each spectrum. For this purpose, the sum of the peak intensities of the deisotopized N-glycans was determined and set to 100%. The relative intensity (%) of each glycan was then determined relative to this value. m / z values ​​were confirmed for all N-glycans annotated above the peaks, including confirmation via their fragment spectra.

[0181] Figure 46A and 46B Location and expression of the SfGnT-I hybrid Gnt variant. Figure 46B Schematic representation of hybrids of natural SfGnT-I or SfGnT-I catalytic domains, precipitated from cultured SN or from crude lysis buffer (WCE) using Triton X, as shown. Figure 46A As shown.

[0182] Figure 47A and 47B : Expressing different Gnt-I ( Figure 47A ) and different Sf-Gnt-I hybrids ( Figure 47B Quantification and comparison of N-glycans in Leishmania using RF. Total N-glycans from cell-precipitated particles were released via PNGase F and labeled with RF. Relative quantification of UPLC peaks is shown as G0-Gn conversion %, representing the in vivo addition of the first GlcNAc for glycoengineering. Error bars represent the standard deviation of several independent experiments. m / z values ​​were confirmed for UPLC peaks containing G0-Gn and Man3, while no other peak differences were found between the blank and PNGase F-released samples.

[0183] Figure 48A and 48B This shows the localization of different sialyl transferases expressed in Leishmania. Figure 48A ) and relative activity ( Figure 48B ) HA-tagged sialyltransferases were detected from the affinity concentrate (elu) of cell lysates from triton-lysed cells by anti-HA immunoblotting, compared with cell culture SN. Elu is the elution fraction used for in vitro assays ( Figure 48A ).

[0184] Figure 49A , 49B49C and 49D: In vitro activity of 2-AB-labeled G2 standards of HA affinity-enriched sialyltransferase expressed in Leishmania. Figure 49A Muridae ST6GAL1; Figure 49B : Rat ST6GAL1, Figure 49C CstI bacteria containing optimized invertase secretion signals. Figure 49D Murid ST3GAL3. The in vitro reaction of the G2 standard enriched with HA-enriched sialyltransferase is shown, and compared with the commercial standard G2 and sialylated G2S2 (dashed arrows) by RT. Arrows indicate in vitro reactions and asterisks indicate the resulting glycan peaks.

[0185] Figure 50A , 50B 50C, 50D, and 50E: mST6GAL1 expressed and enriched in Leishmania can sialylate galactosylated N-glycans of folded full-length mAbs. Figure 50A Leishmania species expressing ST6 from cell lysates were enriched via HA affinity and tested against the 2AB-labeled G2 standard (determined to be 100%). Figure 50B )) and the effect of MabThera Fc N-glycan ( Figure 50C The activity of the N-glycan was evaluated by RF MS for mAb (light gray) and, after in vitro reactions, using blank lysis buffer (black column) or HA-enriched mST6GAL1 of Mabthera purified from protein A (dark gray column). Figure 50D ).exist Figure 50E The summary shows the total sialylated mono- and dianthin polysaccharides.

[0186] Figure 51 The expression and enrichment of mST6GAL1 in Leishmania can sialylate the galactosylated N-glycans of the folded full-length mAb, as shown on UPLC running by N-glycan RF-MS spectra of the blank reaction superimposed with the ST6 reaction.

[0187] Figure 52A and 52B Glycoengineered strains stably transfected with Sf-Gnt-I and MGAT2 and expressing rituximab in free form produced G0 N-glycans on rituximab. The strains were grown in 1L, and the secreted rituximab was purified from the supernatant using protein A. Figure 52A ), and used for PNGase F and RF-MS analysis ( Figure 52B And semi-quantitative analysis of Man3.

[0188] Figure 53 The diagram illustrates the CMP-Neu5Ac pathway using prokaryotic and eukaryotic enzymes that can be recombinantly added to Leishmania for glycoengineering. 7. Detailed Description of the Invention

[0190] This invention relates to a single-celled kinematoplast eukaryotic host cell that has been modified by the properties of the natural host cell and by a combination of heterologous expression of a group of glycosyltransferases (including N-acetylglucosyltransferase, galactosyltransferase and sialylate) to produce a homogeneous and fully functional customized N-glycan with high site occupancy on therapeutic proteins, thereby becoming a host strain for the production of therapeutic glycoproteins in mammals (e.g., humans).

[0191] This invention provides nucleic acid molecules and combinatorial libraries that can be used to successfully target and express mammalian enzymatic activities (e.g., those involved in N-acetylglucosamine elongation, galactosylation, and sialylation) into intracellular compartments of kinetoplast eukaryotic host cells. It also provides the design of a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins.

[0192] 7.1 Host Cell

[0193] In some embodiments, the present invention provides engineered host cells that can be used to express and target any desired gene involved in glycosylation. The present invention provides eukaryotic host cells modified by heterologous expression of a set of glycosyltransferases, including N-acetylglucosamine transferase, galactosyltransferase, and sialyltransferase, to produce functionally customized and homogeneous N-glycans on proteins, thereby serving as host strains for the production of therapeutic glycoproteins in mammals (e.g., humans).

[0194] This invention also provides engineered host cells that can be used to express and target full-length therapeutic antibodies. These novel host cells synthesize, express, and secrete homogeneous and functionally customized N-glycans on glycoproteins such as erythropoietin or anti-CD20 (rituximab).

[0195] The invention described herein is not limited to the specific enzymes, genes, plasmids, and constructs disclosed herein. Those skilled in the art may use any homologs, variants, and derivatives of genes involved in the synthesis of enzymes in the N-acetylglucosamine transferase, galactosyltransferase, sialyltransferase, and CMP-Sia biosynthetic pathway.

[0196] In specific embodiments, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a heteroglycosyltransferase. In some embodiments, the heteroglycosyltransferase is N-acetylglucosamine transferase; and / or heterogalactosyltransferase; and / or heterosialylate. In some embodiments, this document provides a host cell comprising two or more N-acetylglucosamine transferases. In other embodiments, the host cell comprising heterosialylate further comprises a heterogeneous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0197] In one aspect, this document provides a Leishmania host cell capable of producing glycosylated proteins, wherein the Leishmania host cell comprises (i) a natural or heterologous / recombinant OST; (ii) nucleotides encoding heterologous N-acetylglucosamine transferase, galactosyltransferase, sialyltransferase, and CMP-Sia biosynthetic pathway enzymes or modified forms thereof; and (iii) nucleotides encoding recombinant target proteins and modified forms thereof. In other embodiments, the amino acid sequences of the N-acetylglucosamine transferase, galactosyltransferase, and / or sialyltransferase are derived from the N-acetylglucosamine transferase, galactosyltransferase, or sialyltransferase listed in Table 9 or any functional homolog, isoform, or variant thereof.

[0198] In another embodiment, this document provides a host cell in which one or more endogenous enzymes from the N-glycan biosynthesis pathway are deleted, mutated, and / or functionally inactivated. In other embodiments, the endogenous enzyme in the host cell that is deleted, mutated, and / or functionally inactivated is encoded by an alg gene. In another embodiment, this document provides a host cell in which one or more genes encoding endogenous enzymes from the N-glycan biosynthesis pathway are deleted, mutated, and / or functionally inactivated. In another embodiment, the gene encoding the endogenous enzyme from the N-glycan biosynthesis pathway is deleted, mutated, and / or functionally inactivated using any standard technique known in the art (e.g., by site-specific homologous recombination or random mutation). In some embodiments, the host cell is a Leishmania strain that does not include one or more endogenous enzymes from the N-glycan biosynthesis pathway.

[0199] In some embodiments, the Leishmania host cell is a Leishmania tarentolae cell. In specific embodiments, this document provides a Leishmania tarentolae host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding a heteroglycosyltransferase. In some embodiments, the heteroglycosyltransferase is N-acetylglucosamine transferase; and / or heterogalactosyltransferase; and / or heterosialylate transferase. In some embodiments, this document provides a host cell comprising two or more N-acetylglucosamine transferases. In other embodiments, the host cell comprising heterosialylate transferase also comprises a heterogeneous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0200] In one aspect, this document provides a Leishmania tarentolae host cell capable of producing glycosylated proteins, wherein the Leishmania tarentolae host cell comprises (i) a natural or heterologous / recombinant OST; (ii) nucleotides encoding heterologous N-acetylglucosamine transferase, galactosyltransferase, sialyltransferase, and CMP-Sia biosynthetic pathway enzymes or modified forms thereof; and (iii) nucleotides encoding recombinant target proteins and modified forms thereof. In other embodiments, the amino acid sequences of the N-acetylglucosamine transferase, galactosyltransferase, and / or sialyltransferase are derived from the N-acetylglucosamine transferase, galactosyltransferase, or sialyltransferase listed in Table 9 or any functional homolog, isoform, or variant thereof.

[0201] In another embodiment, this document provides a host cell in which a Leishmania signaling and / or retention sequence is added to N-acetylglucosamine transferase, galactosyltransferase, and / or sialic acid transferase, wherein the signaling sequence targets the N-acetylglucosamine transferase, galactosyltransferase, and / or sialic acid transferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase, galactosyltransferase, and / or sialic acid transferase in the endoplasmic reticulum or Golgi apparatus. In another embodiment, the retention sequence retains the N-acetylglucosamine transferase and / or galactosyltransferase in the endoplasmic reticulum of the host cell. In yet another embodiment, the retention sequence retains the N-acetylglucosamine transferase and / or galactosyltransferase in the cis-membrane sac of the Golgi apparatus compartment of the host cell. In another embodiment, the retained sequence retains N-acetylglucosamine transferase and / or galactosyltransferase in the intermediate membrane sac of the Golgi compartment of the host cell. In another embodiment, the retained sequence retains galactosyltransferase in the trans membrane sac of the Golgi compartment of the host cell. In another embodiment, the retained sequence retains sialyltransferase in the trans membrane sac of the Golgi compartment of the host cell. In another embodiment, the retained sequence retains both sialyltransferase and galactosyltransferase in the trans membrane sac of the Golgi compartment of the host cell. In another embodiment, the signal sequence is processed and removed. In other embodiments, the retained sequence is a cytoplasmic transmembrane-stem (CTS) sequence derived from Leishmania tarentolae proteins. In another embodiment, the CTS sequence is derived from Leishmania tarentolae MAN1, NTPDase 1, or NTPDase 2. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or a functionally active fragment thereof. In another embodiment, the CTS is derived from *Leishmania tarentolae* MAN1. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, or a functionally active fragment thereof. In other embodiments, the preserved sequence comprises a GRIP sequence derived from *Leishmania*, or a functionally active fragment thereof. In another embodiment, the GRIP sequence comprises the sequence of SEQ ID NO: 27, or a functionally active fragment thereof.In other embodiments, the preserved sequence comprises a CTS sequence or a functionally active fragment thereof derived from a Leishmania protein, and a GRIP sequence or a functionally active fragment thereof derived from Leishmania.

[0202] In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding N-acetylglucosamine transferase. In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding galactosyltransferase. In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding sialyltransferase. In other embodiments, the host cell comprising heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0203] In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding N-acetylglucosamine transferase; and (c) a recombinant nucleic acid encoding galactosyltransferase. In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding N-acetylglucosamine transferase; and (c) a recombinant nucleic acid encoding sialyltransferase. In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding sialyltransferase; and (c) a recombinant nucleic acid encoding galactosyltransferase. In other embodiments, the host cell comprising heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0204] In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding N-acetylglucosamine transferase; (c) a recombinant nucleic acid encoding galactosyltransferase; and (d) a recombinant nucleic acid encoding sialyltransferase. In other embodiments, the host cell comprising heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0205] In another embodiment, this document provides a Leishmania tarentolae host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding N-acetylglucosamine transferase. In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding galactosyltransferase. In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; and (b) a recombinant nucleic acid encoding sialyltransferase. In other embodiments, the host cell comprising heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0206] In another embodiment, this document provides a Leishmania tarentolae host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding N-acetylglucosamine transferase; and (c) a recombinant nucleic acid encoding galactosyltransferase. In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding N-acetylglucosamine transferase; and (c) a recombinant nucleic acid encoding sialyltransferase. In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding sialyltransferase; and (c) a recombinant nucleic acid encoding galactosyltransferase. In other embodiments, the host cell comprising heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0207] In another embodiment, this document provides a Leishmania host cell comprising (a) a recombinant nucleic acid encoding a target protein; (b) a recombinant nucleic acid encoding N-acetylglucosamine transferase; (c) a recombinant nucleic acid encoding galactosyltransferase; and (d) a recombinant nucleic acid encoding sialyltransferase. In other embodiments, the host cell comprising heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0208] In some embodiments, the term "heterogeneous" means originating from a different species. For example, a "heterogeneous glycosyltransferase" in a host cell is a glycosyltransferase originating from a species other than the host cell. In some embodiments, the term "heterogeneous" means originating from a different strain. For example, a "heterogeneous glycosyltransferase" in a host cell is a glycosyltransferase originating from a strain other than the host cell. In more specific embodiments, the term "heterogeneous" means originating from a different genus. For example, a "heterogeneous glycosyltransferase" in a host cell is a glycosyltransferase originating from a genus other than the host cell.

[0209] In some embodiments, the glycosyltransferase used with the methods and compositions provided herein is a glycosyltransferase genetically modified from its wild-type gene. In more specific embodiments, such glycosyltransferase is of the same species or strain. In other embodiments, such glycosyltransferase is of a different genus, species, or strain.

[0210] 7.2 Subcellular localization of glycosyltransferases

[0211] In some embodiments, a Leishmania signaling and retention sequence is added to N-acetylglucosamine transferase, galactosyltransferase, and / or sialyltransferase, wherein the signaling sequence targets the N-acetylglucosamine transferase, galactosyltransferase, and / or sialyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase, galactosyltransferase, and / or sialyltransferase in the endoplasmic reticulum or Golgi apparatus. In some embodiments, a Leishmania signaling and retention sequence is added to N-acetylglucosamine transferase and sialyltransferase, wherein the signaling sequence targets the N-acetylglucosamine transferase and sialyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase and sialyltransferase in the endoplasmic reticulum or Golgi apparatus. In some embodiments, a Leishmania signaling and retention sequence is added to N-acetylglucosamine transferase and galactosyltransferase, wherein the signaling sequence targets the N-acetylglucosamine transferase and galactosyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase and galactosyltransferase in the endoplasmic reticulum or Golgi apparatus. In some embodiments, a Leishmania signaling and retention sequence is added to sialyltransferase and galactosyltransferase, wherein the signaling sequence targets the sialyltransferase and galactosyltransferase to the endoplasmic reticulum of the Leishmania host cell, and wherein the retention sequence retains the sialyltransferase and galactosyltransferase in the endoplasmic reticulum or Golgi apparatus.

[0212] In other embodiments, a Leishmania signal and retention sequence are added to N-acetylglucosamine transferase, wherein the signal sequence targets the N-acetylglucosamine transferase to the endoplasmic reticulum of a Leishmania host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase in the endoplasmic reticulum or Golgi apparatus. In other embodiments, the Leishmania signal and retention sequence are fused to the N-terminus of N-acetylglucosamine transferase. In another embodiment, the Leishmania signal and retention sequence are fused to the C-terminus of N-acetylglucosamine transferase. In other embodiments, the Leishmania signal and retention sequence are not fused to the N-terminus of N-acetylglucosamine transferase. In other embodiments, the Leishmania signal and retention sequence are not fused to the C-terminus of N-acetylglucosamine transferase. In other embodiments, the Leishmania signal and reserved sequence are fused into one or more amino acids within the polypeptide of N-acetylglucosamine transferase.

[0213] In other embodiments, a Leishmania signal and retention sequence are added to the galactosyltransferase, wherein the signal sequence targets the galactosyltransferase to the endoplasmic reticulum of a Leishmania host cell, and wherein the retention sequence retains the galactosyltransferase in the endoplasmic reticulum or Golgi apparatus. In other embodiments, the Leishmania signal and retention sequence are fused to the N-terminus of the galactosyltransferase. In another embodiment, the Leishmania signal and retention sequence are fused to the C-terminus of the galactosyltransferase. In other embodiments, the Leishmania signal and retention sequence are not fused to the N-terminus of the galactosyltransferase. In other embodiments, the Leishmania signal and retention sequence are not fused to the C-terminus of the galactosyltransferase. In other embodiments, the Leishmania signal and retention sequence are fused to one or more amino acids within the polypeptide of the galactosyltransferase.

[0214] In other embodiments, a Leishmania signal and retention sequence are added to sialyl transferase, wherein the signal sequence targets the sialyl transferase to the endoplasmic reticulum of a Leishmania host cell, and wherein the retention sequence retains the sialyl transferase in the endoplasmic reticulum or Golgi apparatus. In other embodiments, the Leishmania signal and retention sequence are fused to the N-terminus of the sialyl transferase. In another embodiment, the Leishmania signal and retention sequence are fused to the C-terminus of the sialyl transferase. In other embodiments, the Leishmania signal and retention sequence are not fused to the N-terminus of the sialyl transferase. In other embodiments, the Leishmania signal and retention sequence are not fused to the C-terminus of the sialyl transferase. In other embodiments, the Leishmania signal and retention sequence are fused to one or more amino acids within the polypeptide of the sialyl transferase.

[0215] In another embodiment, the retention sequence retains N-acetylglucosamine transferase and / or galactosyltransferase in the endoplasmic reticulum of the host cell. In another embodiment, the retention sequence retains N-acetylglucosamine transferase and / or galactosyltransferase in the cis-faced membrane sac of the Golgi compartment of the host cell. In another embodiment, the retention sequence retains N-acetylglucosamine transferase and / or galactosyltransferase in the intermediate membrane sac of the Golgi compartment of the host cell. In another embodiment, the retention sequence retains galactosyltransferase in the trans-faced membrane sac of the Golgi compartment of the host cell. In another embodiment, the retention sequence retains sialyltransferase in the trans-faced membrane sac of the Golgi compartment of the host cell. In yet another embodiment, the retention sequence retains both sialyltransferase and galactosyltransferase in the trans-faced membrane sac of the Golgi compartment of the host cell.

[0216] In another embodiment, the signal sequence and / or preserved sequence is a signal sequence or preserved sequence derived from any species of Leishmania. In other embodiments, the signal sequence and / or preserved sequence is a signal sequence or preserved sequence derived from the lizard Leishmania tarentolae.

[0217] In other embodiments, the preserved sequence is a cytoplasmic transmembrane-stem (CTS) sequence derived from a Leishmania tarentolae protein. In another embodiment, the CTS sequence is derived from Leishmania tarentolae MAN1, NTPDase 1, or NTPDase 2. In yet another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or a functionally active fragment thereof.

[0218] In another embodiment, the CTS is derived from Leishmania tarentolae MAN1. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24 or a functionally active fragment thereof. In other embodiments, the CTS sequence has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a CTS derived from Leishmania tarentolae MAN1. In another embodiment, the CTS sequence comprises a sequence or a functionally active fragment thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 24.

[0219] In another embodiment, the CTS is derived from Leishmania tarentolae NTPDase 1. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 25 or a functionally active fragment thereof. In other embodiments, the CTS sequence has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a CTS derived from Leishmania tarentolae NTPDase 1. In another embodiment, the CTS sequence comprises a sequence or a functionally active fragment thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 25.

[0220] In another embodiment, the CTS is derived from NTPDase 2 of *Leishmania tarentolae*. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 26 or a functionally active fragment thereof. In other embodiments, the CTS sequence has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a CTS derived from NTPDase 2 of *Leishmania tarentolae*. In another embodiment, the CTS sequence comprises a sequence or a functionally active fragment thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 26.

[0221] In other embodiments, the preserved sequence comprises a GRIP sequence derived from Leishmania or a functionally active fragment thereof. In another embodiment, the GRIP sequence comprises the sequence of SEQ ID NO: 27 or a functionally active fragment thereof. In other embodiments, the preserved sequence has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a GRIP sequence derived from Leishmania or a functionally active fragment thereof. In another embodiment, the GRIP sequence comprises a sequence or a functionally active fragment thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 27.

[0222] In other embodiments, the preserved sequence comprises a CTS sequence or a functionally active fragment thereof derived from a Leishmania protein, and a GRIP sequence or a functionally active fragment thereof derived from Leishmania. In yet another embodiment, the preserved sequence comprises a CTS sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence derived from a Leishmania protein. or its functionally active fragment, and a GRIP sequence or its functionally active fragment having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a sequence derived from Leishmania.

[0223] In another embodiment, the target protein has been engineered to include a signal sequence from Leishmania. In other embodiments, the signal sequence is a signal sequence from the lizard Leishmania tarentolae. In some embodiments, the signal sequence includes the sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29, or a functionally active fragment thereof. In a specific embodiment, the signal sequence includes the sequence shown in SEQ ID NO: 28, or a functionally active fragment thereof. In another embodiment, the signal sequence comprises a sequence or a functionally active fragment thereof having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 28. In other embodiments, the signal sequence is processed and removed from the target protein.

[0224] 7,3N-acetylglucosamine transferase

[0225] In a specific embodiment, this document provides a hybrid N-acetylglucosamine transferase, wherein the hybrid N-acetylglucosamine transferase comprises (a) a catalytic domain of N-acetylglucosamine transferase not derived from Leishmania; and (b) an amino acid sequence responsible for localization and retention in the endoplasmic reticulum or in the Golgi apparatus compartment of Leishmania.

[0226] In some embodiments, the hybridized N-acetylglucosamine transferase is derived from the lizard leishmania (Leishmania tarentolae).

[0227] In other embodiments, the hybridized N-acetylglucosamine transferase has been engineered to include a signal sequence and at least one retention sequence, wherein the signal sequence targets the N-acetylglucosamine transferase to the endoplasmic reticulum of a Leishmania tarentolae host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase in the endoplasmic reticulum or Golgi apparatus.

[0228] In another embodiment, the hybridized N-acetylglucosamine transferase retains the N-acetylglucosamine transferase in the endoplasmic reticulum. In another embodiment, the hybridized N-acetylglucosamine transferase retains the N-acetylglucosamine transferase in the cisfacial sac of the Golgi apparatus. In yet another embodiment, the hybridized N-acetylglucosamine transferase retains the N-acetylglucosamine transferase in the intermediate sac of the Golgi apparatus.

[0229] In other embodiments, the retained sequence is a cytoplasmic transmembrane stem (CTS) sequence. In another embodiment, the CTS sequence comprises the amino acid sequence of MAN1, NTPDase 1, or NTPDase 2. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or a functionally active fragment thereof. In another embodiment, the CTS comprises the amino acid sequence of MAN1. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, or a functionally active fragment thereof. In other embodiments, the GRIP sequence comprises the amino acid sequence shown in SEQ ID NO: 27.

[0230] In another embodiment, the N-acetylglucosamine transferase is GnT-I. In another embodiment, the N-acetylglucosamine transferase is GnT-II. In yet another embodiment, the N-acetylglucosamine transferase is both GnT-I and GnT-II. In some other embodiments, the N-acetylglucosamine transferase is derived from the N-acetylglucosamine transferases listed in Table 9 or their functional homologs, isoforms, or variants. Any N-acetylglucosamine transferase or nucleic acid encoding it known in the art may be used according to the host cells and methods described herein.

[0231] In a specific embodiment, the N-acetylglucosamine transferase is Homo sapiens N-acetylglucosamine transferase 1. In another embodiment, the N-acetylglucosamine transferase is Homo sapiens mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of Homo sapiens. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Homo sapiens N-acetylglucosamine transferase 1 or mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase.

[0232] In a specific embodiment, the N-acetylglucosamine transferase is N-acetylglucosamine transferase 1 of the fall armyworm (Spodoptera frugiperda). In another embodiment, the N-acetylglucosamine transferase is N-acetylglucosamine transferase 2 of the fall armyworm (Spodoptera frugiperda). In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the species Spodoptera frugiperda. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to N-acetylglucosamine transferase 1 or N-acetylglucosamine transferase 2 of the fall armyworm (Spodoptera frugiperda).

[0233] In a specific embodiment, the N-acetylglucosamine transferase is N-acetylglucosamine transferase 1 from Trypanosoma brucei. In another embodiment, the N-acetylglucosamine transferase is N-acetylglucosamine transferase 2 from Trypanosoma brucei. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the species Trypanosoma brucei. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to N-acetylglucosamine transferase 1 or N-acetylglucosamine transferase 2 of Trypanosoma brucei.

[0234] In a specific embodiment, the N-acetylglucosamine transferase is N-acetylglucosamine transferase 2 of the brown rat (Rattus norvegicus). In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the brown rat species (Rattus norvegicus). For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to N-acetylglucosamine transferase 2 of the brown rat (Rattus norvegicus).

[0235] In a specific embodiment, the N-acetylglucosamine transferase is the bonobo (Pan paniscus) mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the bonobo (Pan paniscus) N-acetylglucosamine transferase. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the bonobo (Pan paniscus) mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase.

[0236] In a specific embodiment, the N-acetylglucosamine transferase is the canine (Canis lupus familiaris) mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the canine species (Canis lupus familiaris). For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the canine (Canis lupus familiaris) mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase.

[0237] In a specific embodiment, the N-acetylglucosamine transferase is the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase from Bos taurus. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the Bos taurus species. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase from Bos taurus.

[0238] In a specific embodiment, the N-acetylglucosamine transferase is mannoside acetylglucosamine transferase 2 from the mouse (Mus musculus). In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the mouse (Mus musculus) species. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to mannoside acetylglucosamine transferase 2 from the mouse (Mus musculus).

[0239] In a specific embodiment, the N-acetylglucosamine transferase is the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase of the brown rat (Rattus norvegicus). In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the brown rat species (Rattus norvegicus). For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase of the brown rat (Rattus norvegicus).

[0240] In a specific embodiment, the N-acetylglucosamine transferase is the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase of *Gallus gallus*. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of *Gallus gallus*. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase of *Gallus gallus*.

[0241] In a specific embodiment, the N-acetylglucosamine transferase is the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase of Xenopus tropicalis. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of Xenopus tropicalis. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase of Xenopus tropicalis.

[0242] In a specific embodiment, the N-acetylglucosamine transferase is the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase of zebrafish (Danio rerio). In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the zebrafish species (Danio rerio). For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine transferase of zebrafish (Danio rerio).

[0243] In some embodiments, the N-acetylglucosamine transferase is AgaP_AGAP004397 (GI: 1274542) of Anopheles gambiae. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the species N-acetylglucosamine transferase of Anopheles gambiae. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to AgaP_AGAP004397 (GI: 1274542) of Anopheles gambiae.

[0244] In some embodiments, the N-acetylglucosamine transferase is AgaP_AGAP004397 (GI: 1274542) of *Caenorhabditis elegans*. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of *Anopheles gambiae*. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to AgaP_AGAP004397 (GI: 1274542) of *Anopheles gambiae*.

[0245] In some embodiments, the N-acetylglucosamine transferase is gly-20 (GI: 179562) of *Caenorhabditis elegans*. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the species *Caenorhabditis elegans*. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to gly-20 (GI: 179562) of *Caenorhabditis elegans*.

[0246] In some embodiments, the N-acetylglucosamine transferase is β-1,2-N-acetylglucosamine transferase II from Arabidopsis thaliana. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of Arabidopsis thaliana. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to glyβ-1,2-N-acetylglucosamine transferase II from Arabidopsis thaliana.

[0247] In some embodiments, the N-acetylglucosamine transferase is gly-20 (GI: 179562) of *Caenorhabditis elegans*. In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the species *Caenorhabditis elegans*. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to gly-20 (GI: 179562) of *Caenorhabditis elegans*.

[0248] In some embodiments, the N-acetylglucosamine transferase is the predicted α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase (XP_015280466.1) of the gecko (Gekko japonicus). In some embodiments, the N-acetylglucosamine transferase is an N-acetylglucosamine transferase homologous to the N-acetylglucosamine transferase of the species *Gekko japonicus*. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to XP_015280466.1 of *Gekko japonicus*.

[0249] In other embodiments, this document provides nucleic acids encoding hybridized N-acetylglucosamine transferases.

[0250] 7.4 Galactosyltransferase

[0251] In a specific embodiment, this document provides a hybrid galactosyltransferase comprising (a) a catalytic domain of a galactosyltransferase not derived from Leishmania; and (b) an amino acid sequence responsible for localization and retention in the endoplasmic reticulum or Golgi apparatus compartment of Leishmania.

[0252] In another embodiment, the hybrid galactosyltransferase is derived from the lizard leishmania (Leishmania tarentolae).

[0253] In other embodiments, the hybridized galactosyltransferase has been engineered to include a signal sequence that targets the galactosyltransferase to the endoplasmic reticulum of a Leishmania tarentolae host cell, and the retention sequence retains the galactosyltransferase in the endoplasmic reticulum or Golgi apparatus.

[0254] In some embodiments, the hybridized galactosyltransferase retains the galactosyltransferase in the endoplasmic reticulum. In some embodiments, the hybridized galactosyltransferase retains the galactosyltransferase in the cis-facing sac of the Golgi apparatus. In another embodiment, the hybridized galactosyltransferase retains the galactosyltransferase in the intermediate sac of the Golgi apparatus. In yet another embodiment, the hybridized galactosyltransferase retains the galactosyltransferase in the trans-facing sac of the Golgi apparatus.

[0255] In other embodiments, the retained sequence is a cytoplasmic transmembrane stem (CTS) sequence. In other embodiments, the hybridized galactosyltransferase is a GRIP sequence. In some embodiments, the hybridized galactosyltransferase is a CTS sequence and a GRIP sequence. In another embodiment, the CTS sequence comprises the amino acid sequence of MAN1, NTPDase 1, or NTPDase 2. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or a functionally active fragment thereof. In another embodiment, the CTS comprises the amino acid sequence of MAN1. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, or a functionally active fragment thereof. In other embodiments, the GRIP sequence comprises the amino acid sequence shown in SEQ ID NO: 27.

[0256] In other embodiments, the galactosyltransferase is derived from the galactosyltransferases listed in Table 9 or their functional homologs, isoforms, or variants. Any galactosyltransferase or nucleic acid encoding it known in the art may be used according to the host cells and methods described herein.

[0257] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 (B4GALT1) from Homo sapiens. In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to the galactosyltransferase of Homo sapiens. For example, the galactosyltransferase or the nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from Homo sapiens.

[0258] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from chimpanzee (Pan troglodyte). In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to the galactosyltransferase of the chimpanzee species (Pan troglodyte). For example, the galactosyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from chimpanzee (Pan troglodyte).

[0259] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from rhesus monkeys (Macaca mulatta). In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to a species of galactosyltransferase from rhesus monkeys (Macaca mulatta). For example, the galactosyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from rhesus monkeys (Macaca mulatta).

[0260] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from the canine species *Canis lupus familiaris*. In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to the galactosyltransferase of the species *Canis lupus familiaris*. For example, the galactosyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from the canine species *Canis lupus familiaris*.

[0261] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from bovines (Bos taurus). In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to the galactosyltransferase of the bovine species (Bos taurus). For example, the galactosyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from bovines (Bos taurus).

[0262] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from the mouse (Mus musculus). In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to a species of galactosyltransferase from the mouse (Mus musculus). For example, the galactosyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from the mouse (Mus musculus).

[0263] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from the brown rat (Rattus norvegicus). In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to the galactosyltransferase of the brown rat species (Rattus norvegicus). For example, the galactosyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from the brown rat (Rattus norvegicus).

[0264] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from *Gallus gallus*. In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to the galactosyltransferase of *Gallus gallus*. For example, the galactosyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from *Gallus gallus*.

[0265] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from Xenopus tropicalis. In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to the galactosyltransferase of Xenopus tropicalis. For example, the galactosyltransferase or the nucleic acid encoding it is about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from Xenopus tropicalis.

[0266] In a specific embodiment, the galactosyltransferase is β-1,4-galactosyltransferase 1 from zebrafish (Danio rerio). In some embodiments, the galactosyltransferase is a galactosyltransferase homologous to a species of zebrafish (Danio rerio). For example, the galactosyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-1,4-galactosyltransferase 1 from zebrafish (Danio rerio).

[0267] In other embodiments, this document provides nucleic acids encoding hybridization galactosyltransferases.

[0268] 7.5 Sialyltransferase

[0269] In a specific embodiment, this document provides a hybrid sialyltransferase comprising (a) a catalytic domain of a sialyltransferase not derived from Leishmania; and (b) an amino acid sequence responsible for localization and retention in the endoplasmic reticulum or Golgi apparatus compartment of Leishmania.

[0270] In another embodiment, the hybrid sialyltransferase is derived from the lizard leishmania (Leishmania tarentolae).

[0271] In some embodiments, the hybridized sialyltransferase has been engineered to include a signal sequence that targets the sialyltransferase to the endoplasmic reticulum of a Leishmania tarentolae host cell, and wherein the retention sequence retains the sialyltransferase in the endoplasmic reticulum or Golgi apparatus. In another embodiment, the hybridized sialyltransferase retains the sialyltransferase in the transmembrane sac of the Golgi apparatus.

[0272] In another embodiment, the retained sequence is a CTS sequence. In other embodiments, the retained sequence is a GRIP sequence. In other embodiments, the retained sequence is both a CTS sequence and a GRIP sequence. In another embodiment, the CTS sequence comprises the amino acid sequence of MAN1, NTPDase 1, or NTPDase 2. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or a functionally active fragment thereof. In another embodiment, the CTS comprises the amino acid sequence of MAN1. In another embodiment, the CTS sequence comprises the sequence shown in SEQ ID NO: 24, or a functionally active fragment thereof. In another embodiment, the GRIP sequence comprises the sequence of SEQ ID NO: 27, or a functionally active fragment thereof.

[0273] In another embodiment, the sialyltransferase is 2,6-SiaT or 2,3-SiaT. In some other embodiments, the hybridized sialyltransferase is derived from the sialyltransferases listed in Table 9 or their functional homologs, isoforms, or variants. Any sialyltransferase or nucleic acid encoding it capable of adding one or more sialic acid residues to an N-glycosylated concordance (e.g., Asn-X-Ser(Thr), where X can be any amino acid other than Pro) linked to an N-glycan (e.g., galactose) connected to an Asn residue (or other related residue) can be used, for example, introduced into the host cells described herein. Any sialyltransferase or nucleic acid encoding it known in the art can be used according to the host cells and methods described herein.

[0274] In a specific embodiment, the sialyltransferase is Homo sapiens β-galactosyl α-2,6-sialyltransferase 1. In another specific embodiment, the sialyltransferase is Homo sapiens β-galactosyl α-2,3-sialyltransferase 4. In some embodiments, the sialyltransferase is a sialyltransferase homologous to Homo sapiens sialyltransferases. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Homo sapiens β-galactosyl α-2,6-sialyltransferase 1 or β-galactosyl α-2,3-sialyltransferase 4.

[0275] In a specific embodiment, the sialyltransferase is β-galactoside α-2,6-sialyltransferase 1 from the mouse (Mus musculus). In another specific embodiment, the sialyltransferase is β-galactoside α-2,3-sialyltransferase 3 from the mouse (Mus musculus). In some embodiments, the sialyltransferase is a sialyltransferase homologous to a sialyltransferase from the mouse (Mus musculus). For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-galactoside α-2,6-sialyltransferase 1 or β-galactoside α-2,3-sialyltransferase 3 from the mouse (Mus musculus).

[0276] In a specific embodiment, the sialyltransferase is β-galactosyl α-2,6-sialyltransferase 1 from the brown rat (Rattus norvegicus). In some embodiments, the sialyltransferase is a sialyltransferase homologous to a sialyltransferase from the species *Rattus norvegicus*. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to β-galactosyl α-2,6-sialyltransferase 1 from the brown rat (Rattus norvegicus).

[0277] In a specific embodiment, the sialyltransferase is an α-2,3-sialyltransferase of Campylobacter jejuni. In some embodiments, the sialyltransferase is a sialyltransferase homologous to a species of Campylobacter jejuni. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to an α-2,3-sialyltransferase of Campylobacter jejuni.

[0278] In a specific embodiment, the sialyltransferase is an α-2,3 / 8-sialyltransferase of Campylobacter jejuni. In some embodiments, the sialyltransferase is a sialyltransferase homologous to a species of Campylobacter jejuni. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the α-2,3 / 8-sialyltransferase of Campylobacter jejuni.

[0279] In a specific embodiment, the sialyltransferase is the α-2,3 / 2,6-sialyltransferase of Pasteurella multocida (AAY89061.1). In some embodiments, the sialyltransferase is a sialyltransferase homologous to the sialyltransferase of the species Pasteurella multocida. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the α-2,3 / 2,6-sialyltransferase of Pasteurella multocida.

[0280] In a specific embodiment, the sialyltransferase is α-2,6-sialyltransferase (BAA25316.1) of *Photobacterium damselae*. In some embodiments, the sialyltransferase is a sialyltransferase homologous to sialyltransferases of the species *Photobacterium damselae*. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to α-2,6-sialyltransferase of *Photobacterium damselae*.

[0281] In a specific embodiment, the sialyltransferase is a hypothetical α-2,6-sialyltransferase of *Photobacterium damselae* (WP_005298232.1). In some embodiments, the sialyltransferase is a sialyltransferase homologous to sialyltransferases of *Photobacterium damselae*. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the α-2,6-sialyltransferase of *Photobacterium damselae*.

[0282] In a specific embodiment, the sialyltransferase is a hypothetical α-2,6-sialyltransferase of *Photobacterium leiognathi* (BAF91416.1). In some embodiments, the sialyltransferase is a sialyltransferase homologous to sialyltransferases of *Photobacterium leiognathi*. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the α-2,6-sialyltransferase of *Photobacterium leiognathi*.

[0283] In a specific embodiment, the sialyltransferase is a hypothetical α-2,6-sialyltransferase of *Photobacterium leiognathi* (BAI49484.1). In some embodiments, the sialyltransferase is a sialyltransferase homologous to sialyltransferases of *Photobacterium leiognathi*. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the α-2,6-sialyltransferase of *Photobacterium leiognathi*.

[0284] In a specific embodiment, the sialyltransferase is a hypothetical α-2,6-sialyltransferase of *Photobacterium sp.* (BAF92026.1). In some embodiments, the sialyltransferase is a sialyltransferase homologous to a species of *Photobacterium sp.*. For example, the sialyltransferase or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the α-2,6-sialyltransferase of *Photobacterium sp.*.

[0285] In some embodiments, this document provides nucleic acids encoding hybridization sialyltransferases.

[0286] 7.6 CMP-Sia biosynthetic pathway

[0287] In other embodiments, the host cell containing the heterologous sialyltransferase also contains a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

[0288] In other embodiments, the CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or any of its functional homologs, isoforms, or variants. Any sialic acid biosynthesizer or nucleic acid encoding it known in the art can be used according to the host cells and methods described herein.

[0289] In a specific embodiment, the sialic acid biosynthesizer is a UDP-GlcNAc 2-epiisomerase / N-acetylmnosamine kinase from the mouse (Mus musculus). In another embodiment, the sialic acid biosynthesizer is a CMP-sialic acid transporter from the mouse (Mus musculus). In some embodiments, the sialic acid biosynthesizer is a sialic acid biosynthesizer homologous to a species of sialic acid biosynthesizer from the mouse (Mus musculus). For example, the sialic acid biosynthesizer or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the UDP-GlcNAc 2-epiisomerase / N-acetylmnosamine kinase or the CMP-sialic acid transporter from the mouse (Mus musculus).

[0290] In a specific embodiment, the sialic acid biosynthesizer is a UDP-N-GlcNAc 2-epiisomerase / N-acetylmannosamine kinase from Homo sapiens. In a specific embodiment, the sialic acid biosynthesizer is an N-acetylneuraminic acid phosphatase from Homo sapiens. In another embodiment, the sialic acid biosynthesizer is a Neu5Ac-9-P phosphatase from Homo sapiens. In a specific embodiment, the sialic acid biosynthesizer is a CMP-sialic acid synthase from Homo sapiens. In other embodiments, the sialic acid biosynthesizer is a CMP-Neu5Ac transporter from Homo sapiens. In some embodiments, the sialic acid biosynthesizer is a sialic acid biosynthesizer homologous to that of Homo sapiens. For example, the sialic acid biosynthesizer or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Homo sapiens' UDP-N-GlcNAc2-epiisomerase / N-acetylmannosamine kinase, N-acetylneuraminic acid phosphatase, Neu5Ac-9-P phosphatase, CMP-sialic acid synthase, or CMP-Neu5Ac transporter.

[0291] In a specific embodiment, the sialic acid biosynthesizing enzyme is the UDP-N-GlcNAc 2-epiisomerase / N-acetylmnosamine kinase of the brown rat (Rattus norvegicus). In some embodiments, the sialic acid biosynthesizing enzyme is a sialic acid biosynthesizing enzyme homologous to the sialic acid biosynthesizing enzyme of the brown rat species (Rattus norvegicus). For example, the sialic acid biosynthesizing enzyme or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the UDP-N-GlcNAc 2-epiisomerase / N-acetylmnosamine kinase of the brown rat (Rattus norvegicus). In other embodiments, the UDP-N-GlcNAc 2-epiisomerase / N-acetylmnosamine kinase of the brown rat (Rattus norvegicus) has a point mutation of GNE / MNK from a sialorrhea patient (Son et al., 2011).

[0292] In a specific embodiment, the sialic acid biosynthesizing enzyme is a CMP-sialic acid synthase from *Neisseria meningitidis*. In another embodiment, the sialic acid biosynthesizing enzyme is a UDP-N-acetylglucosamine 2-epiisomerase from *Neisseria meningitidis*. In a specific embodiment, the sialic acid biosynthesizing enzyme is a CMP-sialic acid synthase from *Neisseria meningitidis*. In some embodiments, the sialic acid biosynthesizing enzyme is a sialic acid biosynthesizing enzyme homologous to the sialic acid biosynthesizing enzyme of *Neisseria meningitidis*. For example, the sialic acid biosynthesizer or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to CMP-sialic acid synthase, UDP-N-acetylglucosamine 2-epimerase, or CMP-sialic acid synthase of Neisseria meningitidis.

[0293] In a specific embodiment, the sialic acid biosynthesizer is a CMP-sialic acid synthase from *Escherichia coli* K1. In another embodiment, the sialic acid biosynthesizer is a UDP-N-acetylglucosamine 2-epiisomerase from *Escherichia coli* K1. In a specific embodiment, the sialic acid biosynthesizer is a CMP-sialic acid synthase from *Escherichia coli* K1. In some embodiments, the sialic acid biosynthesizer is a sialic acid biosynthesizer homologous to the sialic acid biosynthesizer of *Escherichia coli* K1. For example, the sialic acid biosynthesizer or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to CMP-sialic acid synthase, UDP-N-acetylglucosamine 2-episomerase, or CMP-sialic acid synthase of Escherichia coli K1.

[0294] In a specific embodiment, the sialic acid biosynthesizing enzyme is GNPE N-acetylglucosamine-6-phosphate 2'-epiisomerase (CAM09378.1) of Campylobacter jejuni. In another embodiment, the sialic acid biosynthesizing enzyme is N-acetylglucosamine-6-phosphate 2'-epiisomerase of Campylobacter jejuni. In a specific embodiment, the sialic acid biosynthesizing enzyme is N-acetylglucosamine-6-phosphate 2'-epiisomerase synthase of Campylobacter jejuni. In some embodiments, the sialic acid biosynthesizing enzyme is a sialic acid biosynthesizing enzyme homologous to the type of N-acetylglucosamine-6-phosphate 2'-epiisomerase. For example, the sialic acid biosynthesizer or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the N-acetylglucosamine-6-phosphate 2'-epiisomerase of Campylobacter jejuni.

[0295] In a specific embodiment, the sialic acid biosynthesizing enzyme is GNPE N-acetylglucosamine-6-phosphate 2'-epiisomerase (AAY27727.1) of *Neisseria meningitidis*. In another embodiment, the sialic acid biosynthesizing enzyme is N-acetylglucosamine-6-phosphate 2'-epiisomerase of *Neisseria meningitidis*. In a specific embodiment, the sialic acid biosynthesizing enzyme is N-acetylglucosamine-6-phosphate 2'-epiisomerase synthase of *Neisseria meningitidis*. In some embodiments, the sialic acid biosynthesizing enzyme is a sialic acid biosynthesizing enzyme homologous to that of *Campylobacter jejuni*. For example, the sialic acid biosynthesizer or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the N-acetylglucosamine-6-phosphate 2'-epiisomerase of Neisseria meningitidis.

[0296] In a specific embodiment, the sialic acid biosynthesizing enzyme is CgNal, an N-acetylneuraminic acid lyase of Corynebacterium glutamicum (NP_601846.1). In another embodiment, the sialic acid biosynthesizing enzyme is CgNal, an N-acetylneuraminic acid lyase of Corynebacterium glutamicum. In a specific embodiment, the sialic acid biosynthesizing enzyme is CgNal, an N-acetylneuraminic acid lyase of Corynebacterium glutamicum. In some embodiments, the sialic acid biosynthesizing enzyme is a sialic acid biosynthesizing enzyme homologous to the sialic acid biosynthesizing enzyme of the same species as CgNal, N-acetylneuraminic acid lyase. For example, the sialic acid biosynthesizer or the nucleic acid encoding it is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to CgNal,N-acetylneuraminic acid lyase of Corynebacterium glutamicum (Ji et al., 2015).

[0297] 7.7 Leishmania and Zooplastic strains

[0298] As used herein, the host cell is a Leishmania cell. In some embodiments, the host cell is a Leishmania tarentolae cell. In other embodiments, the host cell is a strain of Leishmania from Table 13.

[0299] In some embodiments, the host cell is an *Leishmania aethiopica* cell. In some embodiments, the host cell is part of the *Leishmania aethiopica* complex. In some embodiments, the host cell is a *Leishmania aristidesi* cell. In some embodiments, the host cell is a *Leishmania deanei* cell. In some embodiments, the host cell is part of the *Leishmania donovani* complex. In some embodiments, the host cell is a *Leishmania donovani* cell. In some embodiments, the host cell is a *Leishmania chagasi* cell. In some embodiments, the host cell is a *Leishmania infantum* cell. In some embodiments, the host cell is a *Leishmania hertigi* cell. In some embodiments, the host cell is part of the *Leishmania major* complex. In some embodiments, the host cell is a *Leishmania major* cell. In some embodiments, the host cell is a *Leishmania martiniquensis* cell. In some embodiments, the host cell is part of the *Leishmania mexicana* complex. In some embodiments, the host cell is a *Leishmania mexicana* cell. In some embodiments, the host cell is a *Leishmania pifanoi* cell. In some embodiments, the host cell is part of the *Leishmania tropica* complex. In some embodiments, the host cell is a *Leishmania tropica* cell.

[0300] In some embodiments, the host cell belongs to the bodonidae family of kinetid organisms. In specific embodiments, the host cell is a Bodo saltans cell. In some embodiments, the host cell belongs to the ichthyobodonidae family of kinetid organisms. In some embodiments, the host cell belongs to the trypanosomatidae family of kinetid organisms.

[0301] In some embodiments, the host cell belongs to the genus *Blastocrithidia* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Blechomonas* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Herpetomonas* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Jaenimonas* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Lafontella* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Leishmaniinae* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Novymonas* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Partrypanosoma* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Phytomonas* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *Sergeia* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *strigomonadinae* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *trypanosoma* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *wallacemonas* of the family Trypanidae. In some embodiments, the host cell belongs to the genus *blastocrithidia* of the family Trypanidae.

[0302] 7.8 Other engineered Leishmania host cells

[0303] In some embodiments, the host cell used herein is engineered to contain heterologous nucleic acids, such as heterologous nucleic acids encoding one or more carrier proteins and / or heterologous nucleic acids encoding one or more proteins, such as genes encoding one or more proteins. In another specific embodiment, heterologous nucleic acids are introduced into the host cell described herein using the insertion methods provided herein.

[0304] In some embodiments, other modifications may be introduced (e.g., using recombination techniques) into the host cells described herein. For example, host cell nucleic acids (e.g., genes) encoding proteins that may compete or interfere with glycosylation pathways (e.g., competing or interfering with one or more heterologous genes involved in recombination introduced into the host cell) in the host cell background (genome) may be deleted or modified in a manner that renders them inactive / nonfunctional (i.e., the deleted / modified host cell nucleic acids do not encode functional proteins or do not encode proteins in any way). In some embodiments, when nucleic acids are deleted from the genome of the host cells provided herein, they are replaced with desired sequences, e.g., sequences used for glycoprotein production. This replacement may be performed by one or more insertion methods described herein, wherein the heterologous insert DNA inserted into the host cell may replace the function of the gene deleted from the host cell.

[0305] In some embodiments, the host cell provided herein contains a gene deletion, wherein the DNA sequence of interest has been inserted into the host cell genome at the gene deletion site. In a specific embodiment, the host cell provided herein is a Leishmania with a gene deletion.

[0306] 7.9 Introduction of nucleic acids into host cells

[0307] Any method known in the art can be used to introduce nucleic acids (e.g., their gene fragments) into host cells, such as the lizard leishmania (Leishmania tarentolae).

[0308] In some embodiments, a plasmid is used to introduce a heterologous nucleic acid into the host cell described herein. For example, the heterologous nucleic acid is expressed in the host cell via a plasmid (e.g., an expression vector), and the plasmid is introduced into the modified host cell via transfection, infection or electroporation, chemical transformation by thermal shock, natural transformation, phage transduction, or conjugation. In a specific embodiment, the plasmid is introduced into the modified host cell via stable transfection.

[0309] In specific embodiments, linearized heterologous nucleic acids are introduced into the host cells described herein using transfection, infection or electroporation, chemical transformation via thermal shock, natural transformation, phage transduction, or conjugation. In other embodiments, heterologous nucleic acid sites are specifically integrated into the host cell genome via homologous recombination.

[0310] 7.10 Methods for generating glycosylated target proteins

[0311] This article provides a method for generating N-glycosylated target proteins.

[0312] In one embodiment, this document provides a method for generating a glycosylated target protein in vivo using the host cells described herein. In a specific embodiment, this document provides a method for generating a glycosylated target protein, the method comprising (i) culturing the host cells provided herein under conditions suitable for protein production and (ii) isolating the target protein. In a specific embodiment, the host cells comprise (a) a recombinant nucleic acid encoding the target protein; and (b) a recombinant nucleic acid encoding a heteroglycosyltransferase. In some embodiments, the heteroglycosyltransferase is N-acetylglucosamine transferase; or heterogalactosyltransferase; or heterosialylate transferase. In some embodiments, the host cells are Leishmania cells.

[0313] In some embodiments, the target protein produced by the provided host cells is a therapeutic protein, i.e., a protein used in the treatment of a disease or condition. For example, the target protein produced by the host cells provided herein may be an enzyme, a cytokine, or an antibody, wherein the target protein is glycosylated, for example, sialylated. A non-limiting list of target proteins is provided in the following section 7.12.

[0314] 7.11 Methods for culturing cells

[0315] This article provides a method for culturing host cells.

[0316] In one embodiment, the host cells are cultured using any standard culture technique known in the art. For example, cells are typically grown in enriched media such as brain heart infusion, tryptone soybean broth, or yeast extract, all containing 5 μg / ml of ferrous heme chloride. Alternatively, as static or shaking cultures, incubation is performed at 26°C in the dark for 2–3 days. In some embodiments, the recombinant cell line is cultured with suitable selection reagents. A non-limiting list of selection reagents is provided in Table 9.

[0317] 7.12 Target Protein

[0318] According to the methods described herein, any protein known in the art (or a corresponding peptide / polypeptide) can be used as a target protein. Those skilled in the art will readily understand that the nucleic acid sequences of known proteins and newly identified proteins can be readily derived using methods known in the art, and therefore, the introduction of nucleic acids encoding any protein of interest into the host cells provided herein (e.g., via expression vectors, such as plasmids, or site-specific integration via homologous recombination) will be well within the capabilities of those skilled in the art.

[0319] In other embodiments, the target protein comprises the following amino acid sequence: human interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), and chimeric diphtheria toxin-IL-2 (Denileukin-toxin conjugate). Diftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (analexin), tumor necrosis factor-α (TNF-α), insulin, pramlinpeptide, growth hormone (GH), insulin-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide-1 agonist (GLP-1), glucagon, growth hormone-releasing hormone (GHRH), secretin, thyroid-stimulating hormone (TSH), human bone morphogenetic protein 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7), gonadotropin-releasing hormone (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 fertility stimulating hormone (FSH), human chorionic gonadotropin (HCG), luteinizing hormone-α, erythropoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), the extracellular domain of CTLA4 (e.g., FC-fusion protein), or the extracellular domain of the TNF receptor (e.g., FC-fusion protein). In specific embodiments, the target protein used according to the methods described herein and the host cells is an enzyme or an inhibitor. Exemplary enzymes and inhibitors that can be used as target proteins include, without limitation, coagulation factor VII, coagulation factor VIII, coagulation factor IX, coagulation factor X, coagulation factor XIII, coagulation factor VIIa, antithrombin III (AT-III), protein C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, aglucosidase-α, laronidase (α-L-iduronidase), iduronidase (iduronate-2-sulfatase), thioesterase, galactosidase-β (human α-galactosidase A), botulinum toxin, collagenase, human deoxyribonuclease-I, hyaluronidase, papain, asparaginase, uricase (urate oxidase), glutamate carboxypeptidase (glutapeptidase), α1 protease inhibitor (α1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase.

[0320] In specific embodiments, the target proteins used by the methods described herein and the host cells are cytokines. Exemplary cytokines that can be used as target proteins include, without limitation, interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin-IL-2 (Denileukin diftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonists (analexin), and tumor necrosis factor-α (TNF-α).

[0321] In specific embodiments, the target protein used by the methods described herein and the host cells is a hormone or growth factor. Exemplary hormones and growth factors that can be used as target proteins include, without limitation, insulin, pramlinide, growth hormone (GH), insulin-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide-1 agonist (GLP-1), glucagon, growth hormone-releasing hormone (GHRH), secretin, thyroid-stimulating hormone (TSH), human bone morphogenetic protein 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7), gonadotropin-releasing hormone (GnRH), and 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), neurotrophic protein-3, human fertility stimulating hormone (FSH), human chorionic gonadotropin (HCG), luteinizing hormone-α, erythropoietin, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0322] In specific embodiments, the target protein used by the methods described herein and the host cell is a receptor. Exemplary receptors that can be used as target proteins include, without limitation, the extracellular domain of human CTLA4 (e.g., fused to Fc) and soluble TNF receptors (e.g., fused to Fc).

[0323] In other embodiments, the target protein is a therapeutic protein. In other embodiments, the target protein is an approved biological drug. In yet another embodiment, the therapeutic protein comprises the following amino acid sequence: abatacept (e.g., orexina), aflibercept (e.g., elex), β-galactosidase (e.g., Fabrazyme), abirate (e.g., Eperzan), interleukin (e.g., Proleukin), afaspirin (e.g., Amivan), vidarcephalin (e.g., Ceredase), α-glucosidase (e.g., LUMIZYME), aliskiren (e.g., Tecentriq), α-1-protease inhibitor (e.g., Aralast), alteplase (e.g., Activase), anabulin (e.g., Kinetet), aniplasmase (e.g., Eminase), human anthrax immunoglobulin (e.g., ANTHRASIL), antihemophilic factor (e.g., basil), anti-inhibitor coagulant complex (e.g., Feiba). Nf), antithrombin α, human antithrombin III, antithymocyte globulin (e.g., antithymocyte globulin), anti-thymocyte globulin (horse) (e.g., ATGAM), anti-thymocyte globulin (rabbit) (e.g., ATG-Fresenius), aprotinin (e.g., Tessil), asfotase α, asparaginase (e.g., Aspergillus), Erwinia chrysanthemi asparaginase (e.g., Erwinaze), becaprolactone (e.g., REGRANEX), belacept (e.g., Nulojix), belacept, bivalirudin (e.g., Angiomax), botulinum toxin type A (e.g., Botox). Botulinum toxin type B (e.g., Myobloc), bentoximab (e.g., Adcetris), busherin (e.g., Suprecur), C1 esterase inhibitors (human), C1 esterase inhibitors (recombinant) (e.g., Ruconest), pegylated cerolizumab pegol (e.g., Cimzia), human chorionic gonadotropin (e.g., chorionic gonadotropin alpha), human chorionic gonadotropin (e.g., Ovidrel), human chorionic gonadotropin (recombinant) (e.g., Ovitrelle), coagulation factor ix (e.g., Alprolix), coagulation factor VIIa (e.g., NovoSeven), human coagulation factor X (e.g., Coagadex), coagulation factor XIII A-subunit (recombinant), collagenase (e.g., Cordase), Conestarα, adrenocorticotropic hormone (e.g., HPActhar), teicoplanin (e.g., Cortrosyn), dabepoetin α (e.g., Aranesp), defibrinolytics (e.g., Noravid), denileukin-toxin conjugate. diftitox (e.g., Ontak), disiludin, digoxin immune Fab (sheep) (e.g., DIGIBIND), alfa-chain enzyme (e.g., Pulmozyme), trexocin α (e.g., Xigris), dulaglutide, emothrombin α (e.g., ELOCTA), eleoxysulfatase α, enfuviride (e.g., FUZEON), epoetin (e.g., Binocrit), epoetin zeta (e.g., Retacrit), epoetin (e.g., INTEGRILIN), etanercept (e.g., Enbrel), exenatide (e.g., Byetta), factor IX complex (human) (e.g., AlphaNine), plasmin, also known as cytoplasm (e.g., Elase), filgrastim (e.g., NA)), filgrastim-sndz, follicle-stimulating hormone α (e.g., Gonal-F), follicle-stimulating hormone β (e.g., Follistim AQ), sulfonase (e.g., Naglazyme), gastric factor, gemtuzumab oxazolidin (e.g., Merota), glatiramer acetate (e.g., Copaxone), recombinant glucagon (e.g., Novogene (GlucaGen)), glutamate (e.g., Voraxaze), bacitracin D (e.g., Neosporin), hepatitis B immunoglobulin, human calcitonin, human clostridium tetani toxoid immunoglobulin, human rabies virus immunoglobulin (e.g., Hyperab Rabies human immunoglobulin), human Rho(D) immunoglobulin (e.g., Hyp Rho D Inj 16).5%), human serum albumin (e.g., Albuminar), human varicella-zoster immunoglobulin (e.g., Varizig), hyaluronidase (e.g., HYLENEX), recombinant human hyaluronidase, tiuxetan (e.g., Zevalin), idoxurase (e.g., Elaprase), imiglucerase (e.g., Cerezoyme), human immunoglobulin, insulin aspart (e.g., NovoLog), bovine insulin, insulin degludec (e.g., Tresiba), insulin detemir (e.g., LEVEMIR), insulin glargine (e.g., Lantus), insulin lisgludec (e.g., APIDRA), insulin lispro (e.g., Humalog), porcine insulin (e.g., Iletin II), regular insulin (e.g., Humulin). R), porcine insulin (e.g., vetsulin), protamine insulin (e.g., Novolin N), recombinant interferon α-2a (e.g., Roferon A), interferon α-2b (e.g., INTRON) A) Interferon α-con-1 (e.g., INFERGEN), Interferon α-n1 (e.g., Wellferon), Interferon α-n3 (e.g., Alferon), Interferon β-1a (e.g., Avonex), Interferon β-1b (e.g., Betaseron), Interferon gamma-1b (e.g., Actimmune), Intravenous Immunoglobulin (e.g., Civacir), Laronylase (e.g., Aldurazyme), Levofloxacin (e.g., Granocyte), Lepirudine (e.g., Refludan), Levofloxacin (e.g., Eligard), Liraglutide (e.g., Saxenda), Lucisantamine (e.g., Surfaxin), Luteinizing Hormone Alpha (e.g., Luveris), Mecaseemine (e.g., NA)), urinary gonadotropins (e.g., Menopur), methoxy polyethylene glycol-beta-epofetine (e.g., Mircera), metriptine (e.g., Myalept), natural alpha interferon or multiferon (e.g., Intron / Roferon-A), nesiritide (e.g., NATRECOR), plasminogen activator (e.g., Jetrea), interleukin oxytocin (e.g., Neumega), OspA lipoprotein (e.g., Lymerix), oxytocin (e.g., Pitocin), parivamine (e.g., Kepivance), pancreatic lipase (e.g., Pa... ncrecarb), bovine pegaspargase (e.g., Adagen), pegaspargase (e.g., Oncaspar), PEGylated filgrastim (e.g., Neulasta), PEG-interferon α-2a (e.g., Pegasys), PEG-interferon α-2b (e.g., PEG-Intron), PEG-interferon β-1a (e.g., Plegridy), PEGylated uricase (e.g., Krystexxa), pegvisomone (e.g., SOMAVERT), porcine pulmonary phospholipid α (e.g., Curosurf), prumlin peptide (e.g., Symlin), Preotact (e.g., ...). Protamine sulfate (e.g., protamine sulfate injection, USP), human protein S (e.g., human protein S), prothrombin (e.g., Feiba Nf), prothrombin complex (e.g., Cofact), prothrombin complex concentrate (e.g., Kcentra), raburicase (e.g., Elitek), reteplase (e.g., Retavase), linalostat (e.g., Arcalyst), romiplostim (e.g., Nplate), sacrosemases (e.g., Sucraid), salmon calcitonin (e.g., Calcinar), saxaglastin (e.g., Leucomax), satumomab pendiptide (e.g., OncoScint), lysosomal acid lipase α (e.g., Kanuma), secretin (e.g., ... SecreFlo), sermorelin (e.g., sermorelin acetate), serum albumin (e.g., Albunex), iodinated serum albumin (e.g., Megatope), simmotococcal α (e.g., Nuwiq), sipuleucel-T (e.g., Provenge), recombinant growth hormone (e.g., Nutropin AQ), recombinant growth hormone (e.g., BioTropin), streptokinase (e.g., Streptase), susoctocog α (e.g., Obizur), afataglitazone (e.g., Elelyso), teduglutide (e.g., Gattex), tenepidoxetine Enzymes (e.g., TNKase), teriparatide (e.g., Forteo), temorelin (e.g., Egrifta), coagulation regulatory protein α (e.g., Recomodulin), thymosin alpha (e.g., Zadaxin), thyroglobulin, thyroid-stimulating hormone α (e.g., Thyrogen), tuberculin purified protein derivatives (e.g., Aplisol), turoctocog α (e.g., Zonovate), urinary follicle-stimulating hormone (e.g., BRAVELLE), urokinase (e.g., Kinlytic), vasopressin (e.g., Pitressin), vilazinase α (e.g., virazinase). Vpriv), abciximab (e.g., ReoPro), adalimumab (e.g., Humira), alenzumab (e.g., CAMPATH), alicurumab (e.g., Praluent), asimozide (e.g., CEA-Scan), atezolizumab (e.g., Tecentriq), baliximab (e.g., Simulect), belimumab (e.g., Benlysta), bevacizumab (e.g., Avastin), bonatetumab (e.g., Blincyto), brodatumab (e.g., Siliq), canazine (e.g.,ILARISE), cannabinoids (e.g., Ilaris), calorieumab (e.g., ProstaScint), cetuximab (e.g., Erbitux), daclizumab (e.g., Zenapax), daratumumab (e.g., DARZALEX), denosumab (e.g., Xgeva), daclizumab (e.g., unituxin), eculizumab (e.g., Soliris), efalizumab (e.g., RAPTIVA), elutuzumab (e.g., EMPLICITI), evolocumab (e.g., Repatha), golimumab (e.g., Simponi injection). Injection), teimozumab (e.g., Zevalin), edasuzumab (e.g., Praxbind), infliximab (e.g., Remicade), prilimumab (e.g., YERVOY), excimerab (e.g., Taltz), mepolilimumab (e.g., Nucala), morocumab (e.g., ORTHOCLONE) OKT3), nastatinumab (e.g., Tysabri), necitumumab (e.g., Portrazza), nivolumab (e.g., Opdivo), oxocitrazumab (e.g., Anthim), obbituzumab (e.g., Gazyva), ofamumab (e.g., Arzerra), omalizumab (e.g., Xolair), pallizumab (e.g., Synagis), panituzumab (e.g., Vectibix), pembrolizumab (e.g., Keytruda), pertuzumab (e.g., Perjeta), ramucirumab ( For example, Cyramza, ranibizumab (e.g., Lucentis), rexibacumab (e.g., RAXIBACUMAB), rituximab (e.g., Rituxan), secukinumab (e.g., Cosentyx), sulximab (e.g., Sylvant), tocilizumab (e.g., ACTEMRA), tosimomab (e.g., Bexxar), trastuzumab (e.g., Herceptin), uterotumab (e.g., Stelara), or vedolizumab (e.g., Entyvio).

[0324] In other embodiments, the target protein is an antibody. In yet another embodiment, the target protein is an anti-human protein antibody.

[0325] In other embodiments, the antibody has the following amino acid sequence: adalimumab (Humira); Remicade (infliximab); ReoPro (abxiximab); Rituxan (rituximab); Simulect (baliximab); Synagis (palizumab); Herceptin (trastuzumab); Mylotarg (gem-tuzumab-olzomicin); Camppath (alenzumab); Zevalin (tiuxeta). n); Xolair (Omarizumab); Bexxar (Tosimozab-I-131); Erbitux (Cetuximab); Avastin (Bevacizumab); Tysabri (Natazumab); Actemra (Tocilizumab); Vectibix (Panitumab); Lucentis (Ranibizumab); Soliris (Eculizumab); Cimzia (PEG-conjugated Certolizumab) pegol); Simponi (golimumab); Ilaris (canazine); Stelara (utecumab); Arzerra (ofumab); Prolia (denomab); Numax (movizumab); ABThrax (recibakumab); Benlysta (belimumab); Yervoy (prelimumab); Adcetris (bentoximab); Perjeta (pertuzumab); Kadcyla (trastuzumab-emtansine conjugate); or Gazyva (obituzumab) amino acid sequences.

[0326] In other embodiments, the antibody is a full-length antibody, Fab, F(ab')2, scFv, or sdAb. In other embodiments, the target protein comprises the amino acid sequence of an enzyme or its inhibitor. In another embodiment, the target protein comprises the following amino acid sequence: coagulation factor VII, coagulation factor VIII, coagulation factor IX, coagulation factor X, coagulation factor XIII, coagulation factor VIIa, antithrombin III (AT-III), protein C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, aglucosidase-α, laronidase (α-L-iduronidase), iduronidase (iduronate-2-sulfatase), thioesterase, galactosidase-β (human α-galactosidase A), botulinum toxin, collagenase, human deoxyribonuclease-I, hyaluronidase, papain, asparaginase, uricase (urate oxidase), glutamate carboxypeptidase (glutapeptidase), α1 protease inhibitor (α1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase.

[0327] In another embodiment, the glycosylated target protein is secreted into a culture medium, and wherein the glycosylated target protein is glycosylated. In some embodiments, the glycosylated target protein is purified from the culture medium. In another embodiment, the glycosylated target protein is purified from the culture medium by affinity purification or ion-exchange chromatography. In another embodiment, the glycosylated target protein contains an Fc domain and is affinity purified from the culture medium by protein-A. In another embodiment, the glycosylated target protein contains an affinity tag and is affinity purified.

[0328] In another embodiment, the glycosylation target protein population is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homogeneous. In other embodiments, 90% to 100% of the N-glycosylation sites on the target protein are occupied by glycosylation. In other embodiments, the target protein used according to the methods described herein and the host cell can be a full-length protein, a truncated version thereof, a domain, region, motif, or peptide.

[0329] In other embodiments, the target protein is an Fc-fusion protein.

[0330] In another embodiment, the target protein may be modified. In another embodiment, the target protein has been engineered to include a signal sequence from Leishmania. In other embodiments, the signal sequence is processed and removed from the target protein. In another embodiment, the target protein has been engineered to include one or more tags. In other embodiments, the tags are processed and removed from the target protein.

[0331] 7.13 Composition

[0332] 7.13.1 Compositions containing host cells

[0333] In one aspect, this document provides compositions comprising the host cells described herein (see Section 7.1). These compositions can be used in methods for producing the glycosylation target proteins described herein, for example, by culturing the compositions comprising host cells under conditions suitable for protein production. Subsequently, the glycosylation target proteins can be isolated from the compositions comprising host cells using methods known in the art.

[0334] Compositions containing the host cells described herein may contain other components suitable for the maintenance and survival of the host cells described herein, and may additionally contain other components required or beneficial for the production of proteins by the host cells, such as inducers of inducible promoters, such as arabinose or IPTG. 7.13.2 Compositions containing glycosylated target proteins

[0335] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% of the N-linked glycosylated concordant sequence of the target protein in the composition having an oligosaccharide comprising the following structure:

[0336]

[0337] The squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0338] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein being a G0-Gn glycan, characterized by any one of the following structures:

[0339] or The squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0340] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as a G0 glycan, characterized by the following structure:

[0341]

[0342] The squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0343] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein being a G1-Gn glycan, characterized by any one of the following structures:

[0344]

[0345] Hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0346] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as a G2 glycan, characterized by the following structure:

[0347]

[0348] Hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0349] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as a G2 glycan, characterized by the following structure:

[0350]

[0351] Hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0352] In another embodiment, the composition of the glycosylated target protein has at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein as a G2 glycan, characterized by the following structure:

[0353]

[0354] Hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0355] In another embodiment, the glycosylation on the target protein is further modified to optimize the pharmacokinetic properties of the target protein upon introduction into the target. In another embodiment, the glycosylation on the target protein is sialylated.

[0356] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0357]

[0358] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0359] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0360]

[0361] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0362] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0363]

[0364] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0365] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0366]

[0367] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0368] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0369]

[0370] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0371] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0372]

[0373] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0374] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0375]

[0376] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0377] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0378]

[0379] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0380] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0381]

[0382] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0383] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0384]

[0385] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0386] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0387]

[0388] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0389] In another embodiment, the composition of the glycosylated target protein is characterized by at least about 10% to 20%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% glycosylation on the target protein, having the following structure:

[0390]

[0391] In this diagram, rhombuses represent sialic acid residues, hollow circles represent galactose residues, squares represent N-acetylglucosamine residues, and gray circles represent mannose residues; and Asn is the Asn of the N-linked glycosylation common sequence in the target protein.

[0392] In some embodiments, in addition to containing the glycosylation target protein described herein (see Section 7.12), the compositions described herein (e.g., pharmaceutical compositions) comprise a pharmaceutically usable carrier. As used herein, the term "pharmaceutically usable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more specifically in humans. In the context of pharmaceutically usable carriers, the term "carrier" as used herein refers to a diluent, adjuvant, excipient, or medium through which a pharmaceutical composition is administered. Salt solutions and aqueous solutions of glucose and glycerol can also be used as liquid carriers, specifically for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. Examples of suitable pharmaceutical carriers are described in EW Martin's "Remington's Pharmaceutical Sciences".

[0393] In some embodiments, the compositions described herein are formulated to suit a predetermined route of administration for the subject. For example, the compositions described herein may be formulated for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration. In specific embodiments, the pharmaceutical compositions may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal, or pulmonary administration.

[0394] In some embodiments, the compositions described herein further comprise one or more buffers, such as phosphate buffer and sucrose phosphate-glutamate buffer. In other embodiments, the compositions described herein do not comprise a buffer.

[0395] In some embodiments, the compositions described herein further comprise one or more salts, such as sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or mixtures of these aluminum salts). In other embodiments, the compositions described herein do not comprise salts.

[0396] The compositions described herein may be included in a kit, container, package, or dispenser along with the instructions for use.

[0397] The compositions described herein may be stored prior to use, for example, by freezing (e.g., at about -20°C or about -70°C); by storing under refrigeration conditions (e.g., at about 4°C); or by storing at room temperature.

[0398] 7.14 Preventive and therapeutic use

[0399] In one aspect, this document provides methods for preventing or treating a disease or condition in a subject, comprising administering to the subject a glycosylation target protein or a combination thereof described herein. This document also provides methods for preventing a disease or condition in a subject, comprising administering to the subject a glycosylation target protein or a combination thereof described herein. 8. Detailed Implementation

[0400] 8.1 Bioinformatics Evaluation of Glycosylation Pathway in Example 1

[0401] First, comparative chromosomal analysis revealed significant differences in the early conserved N-glycan biosynthesis steps in *Leishmania tarentolae* and even its taxonomically related species, *Leishmania major*, as described in Tables 1, 2, 3, 4, 5, 6, and 7. The absence of the *alg* gene indicates a strong reduction in N-glycan precursors and also reduced precursor pruning (Figures 3 and 4) (Varki, 2009). Although ALG3 is present, comparison with human ALG3 revealed a potential missense mutation at conserved residue 127 for glutamine, corresponding to a loss-of-function mutation (R171Q) in human ALG3 (Sun et al., 2005).

[0402] Table 1 Precursor Biosynthesis

[0403]

[0404]

[0405] Table 2 Pruning

[0406]

[0407]

[0408] Table 3 N-Glycan Extension

[0409]

[0410]

[0411] Table 4 Oligosaccharide transferases (OST)

[0412]

[0413]

[0414] Table 5. Precursor Biosynthesis (Neu5Ac)

[0415]

[0416]

[0417] Table 6 Nucleotide-activated sugar synthesis and transport

[0418]

[0419] Table 7 Lipophosphoprotein modifying enzymes

[0420]

[0421] According to such Figure 3B The unexpectedly unique biosynthetic pathways shown, along with the reduction in pruning and endoplasmic reticulum (ER) quality control (Tables 1, 2, and 8), demonstrate the beneficial nature of glycoengineering modifications in terms of high site occupancy, while preserving appropriate folding processes. Figure 5 The companion protein pathway of the present invention for correcting glycoprotein folding is described in more detail in the absence of the normally cyclically incorrectly pruned N-glycan precursor return or unfolded protein to the lectin-mediated (calcified and calreticulin) pathway of the ERAD pathway for proteasome degradation (Moremen et al., 2012), and is also shown in Table 8. Importantly, the OST complex consists only of Stt3, which transports the precursor N-glycan to the protein receptor co-asparagine (Table 4). In the case of Leishmania tarentolae, the transport of a reduced precursor via Stt3 is significantly different from that in any of the other eukaryotes described, indicating the absence of site preference or strict site preference as seen in other eukaryotes containing the OST complex.

[0422] Table 8 ER Quality Control

[0423]

[0424]

[0425]

[0426] As a generally accepted view Figure 6 and 7 Schematic diagrams of N-glycan processing steps occurring in the endoplasmic reticulum and Golgi apparatus of eukaryotic cells are shown, modified from (Kellokumpu et al., 2016). In the lizard leishmania (L. tarentolae), the truncated, reduced precursor is transferred to the protein, and it misses any further N-glycan elongation in the Golgi apparatus during transport along the secretory pathway. This has been concluded by the absence of GlcNAc transferases GnT-I and GnT-II, Gal transferase, and sialyl transferase. Figure 6(Table 6). Furthermore, comparative data showed the absence of a sialic acid biosynthesis step (Table 5). The Golgi-inherent GlcNAc glycosyltransferases from the closely related Trypanosoma brucei, TbGnTI (Damerow et al., 2014) and TbGnTII (Damerow et al., 2016), could not be clearly identified in *L. tarentolae*. Immunofluorescence showed that the C-terminal tagged epitopes TbGnTI and TbGnTII are Golgi-localized (Damerow et al., 2014; Damerow et al., 2016).

[0427] Since nucleotide-activated sugar donors are required for enzymatic reactions catalyzed by glycosyltransferases and must be available within the lumen of the Golgi apparatus, the presence of transmembrane-localized transport proteins was identified using bioinformatics methods. The presence of homologs of UDP-glucose pyrophosphorylase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epiisomerase, and UDP-Gal / UDP-GlcNAc introtransporters, GDP-Man / UDP-GlcNAc introtransporters, UDP-GlcNAc / UDP-GalNAc, GDP-Man introtransporters, and UDP-Glc introtransporters (Roper and Ferguson 2003; Roper et al., 2002; Urbaniak et al., 2006; Capul et al., 2007) was identified (Table 6). UDP-GlcNAc transporters (TcNST1 in *T. cruzi*, TbNST1 to 4 in *T. brucei*, and *L. major*) are present in *L. tarentolae*, while UDP-Gal transporters (TbNST1 and 2 in *T. brucei*, and *L. major*) are also present in *L. tarentolae*. These results indicate that both UDP-GlcNAc and UDP-Gal can be transported into the Golgi apparatus. Therefore, LTAR_180008900.1, LTAR_240008400.1, LTAR_340034000.1 and LTAR_300033500.1 (previous naming schemes: LtaP18.0420, LtaP24.0350, LtaP34.3030 and LtaP30.2670) indicate the availability of GDP-Gal and UDP-GlcNAc in the Golgi apparatus cavity.

[0428] 8.2 Example 2: Method for Engineering Fully Functional-Customized N-Glycan Variants

[0429] Figure 7 and 8 We document the glycoengineering design in the lizard Leishmania (L. tarentolae) for the production of homogeneous and functionally-customized N-glycan extensions for a) desired downstream effects of Fc and Fc receptor interactions, b) enhanced half-life (improved pharmacokinetic PK), and c) anti-inflammatory properties, such as the immunotolerance benefits of 2,6-linked Neu5Ac, to avoid the generation of ADA in potential immunogenic protein therapeutics. Figure 8 Proposals for the biosynthesis of N-glycans in the lizard leishmania (L. tarentolae) are described, demonstrating major and novel differences leading to beneficial properties of the glycoengineered N-glycans. This invention relates to eukaryotic host cells modified to produce functionally customized and homogeneous N-glycans on proteins via heterologous expression of a group of glycosyltransferases, including N-acetylglucosamine transferase, galactosyltransferase, and sialyltransferase.

[0430] 8.3 Experimental evidence of reduced and 100% homogeneous N-glycans in the zooplastic lizard Leishmania tarentolae

[0431] Following the selection of zooplasts of the lizard leishmania (L. tarentolae) based on anticipated beneficial properties, the natural N-glycans of three wild-type (wt) strains (St10569, St10616, and St11262) were analyzed. First, cell granules were denatured and subjected to PNGase A or PNGase F-mediated N-glycan release. Waters GlycoWorks was used for rapid enzymatic release and labeling of N-glycans. TM RapiFluor-MS TM N-Glycans can either be fully methylated or labeled with the released N-glycans.

[0432] By using RapiFluor TM After fluorescent labeling and UPLC separation, we observed only a single N-glycan form corresponding to (Man)3(GlcNAc)2, with a calculated m / z = 1222.7166; [M+Na] + 1244.6985; [M+K] + 1260.6724. Regarding N-glycans, only one homogeneous N-glycan, the so-called oligomannose or Man3 glycan, was identified in all three isolated lizard leishmaniae cells, such as... Figure 9 As shown.

[0433] We also applied full methylation to elucidate the N-glycan profiles of two different weight samples (St10569, St10616) of *Leishmania tarentolae*. Regardless of the method (deglycosylation of trypsin peptides derived from defatted cell granules, or direct deglycosylation of the intact protein after defatting cell granules), the presence of an N-glycan structure was confirmed in both strains. This N-glycan, corresponding to the basic (Man)3(GlcNAc)2 core, was obtained without the N-glycosidase used for deglycosylation. The blank control indicated the presence of cell wall-contaminated glycans, presumably derived from lipophosphoglycosylation, as indicated by the hexose characterization. Although hexose contamination was observed in the MALDI spectra of the extracts, we did not identify any ions potentially corresponding to more complex N-glycans. Notably, no biantennale fucylated polysaccharides (Gal)2(GlcNAc)2(Fuc)1+(Man)3(GlcNAc)2(G2F) (Breitling et al., 2002) with α1,6- or α1,3-linked fucose were found (theoretical total methylation mass [M+Na)). + (2244.1). The spectrum did not contain any ion traces with this m / z value. It is also worth noting that no possible intermediates were observed between the core tissue and other complex fucosylated, N-acetylglucosamine-modified, or galactosylated structures. Precursors to the N-glycosylation pathway in the Golgi apparatus (e.g., Man5GlcNAc2) have also not been identified. This is highly unusual if the biosynthesis of N-glycans in *Leishmania tarentolae* is comparable to that in other organisms. The spectra obtained after deglycosylation with PNGase F or PNGase A were identical. Therefore, there is no indication that the N-glycan spectrum of *Leishmania tarentolae* cellular particles contains N-glycans with α1,3-linked core fucose that will not be released via PNGase F (data not shown).

[0434] To further determine how the N-glycans of *L. tarentolae* differ from those of other zooplasts, genomic analysis and N-glycan profiling were conducted on *Crithidia fasciculata*, *Phytomonas davidi Lafont*, and *Crithidia deanei* (Angomonas). Compared to *L. tarentolae*, *C. fasciculata* also contains mannosyltransferases ALG3, ALG9, and ALG11, which explained the experimentally validated synthesis of high-mannosylglycans (Man5GlcNAc2 to Man...). 11 GlcNAc2)( Figure 45 Furthermore, *C. fasciculata* possesses a calreticulin, a quality-controlling companion protein absent in *L. tarentolae*. For *P. davidi Lafont*, no genomic information was available. N-glycan profiling using PNGase F and PNGase A, along with subsequent full methylation and MALDI-TOF, revealed a high-mannose-type glycan (Man9GlcNAc2) with one or two additional pentoses. Bioinformatic comparisons of glycosylation pathways between *C. deanei* and *L. tarentolae* showed no difference except for the absence of MAN1 in *C. deanei*. However, N-glycan profiling revealed that the core N-glycan (Man3GlcNAc2) was additionally modified by two hexoses and two deoxyhexoses. Figure 45 ).

[0435] In summary, using different experimental methods, only one homogeneous N-glycan with the basic structure (Man)3(GlcNAc)2 was identified in three Leishmania tarentolae wt strains. This homogeneous N-glycosylation via (Man)3(GlcNAc)2 (also known as Man3 or oligomannose) has not been observed in any other eukaryote and represents a novel feature for using this organism as an expression host for therapeutic proteins that retain highly site-occupied homogeneous N-glycosylation and glycoengineered humanized N-glycans.

[0436] 8.4 Identification of heteroglycosyltransferases that elongate natural oligomannose N-glycans in vitro, as described in Example 4.

[0437] To achieve humanized, extended biantennary N-glycans, suitable Gnt was recombinantly expressed in *L. tarentolae* and affinity-enriched from crude lysates, membrane-dissolved fractions, and culture supernatants of *L. tarentolae*. The semi-purified enzymes were tested in vitro for their correct activity with cofactors and associated activated nucleotide sugar donors. Substrates included “free” N-glycans, 2-aminobenzamide (2AB)-labeled N-glycans, or crude lysates from *wt* cells containing only oligomannose (Man3).

[0438] This invention covers the expression of different and suitable glycosyltransferase candidates (Table 9), and analyzes their effects in in vitro assays, demonstrating that all tested heterologous Gnt candidates from insect or mammalian sources, expressed and purified in Leishmania tarentolae cells, can exert their activity on free oligosaccharide substrates or 2AB-labeled substrates in the presence of cofactors and their activating sugar donors (Figures 10-14).

[0439] Table 9 GnT Candidates

[0440]

[0441]

[0442]

[0443] GnT-I (“TbGnT-I”) from the taxonomically related organism *Trypanosoma brucei* showed no activity against wt lysates containing natural oligomannose as a substrate (Fig. 10). However, weak activity (peak overlap) was observed against free oligomannose (Man3) as a substrate. TbGnT-II showed no activity against the Man3 substrate, further indicating that the prior activity of GntI is essential. The activity of insect cell-derived SfGnT-I was confirmed in in vitro assays using either the semi-purified or dissolved fraction eluted with streptavidin, exhibiting distinct peaks co-migrating with the NGA2-N standard. Controls were obtained using HA- or Strep-eluted fractions containing wt lysates, but without any modification to the substrate used.

[0444] hMGAT1 was purified from lysis buffer + / - Triton X (TrX), and the hMGAT1 HA-eluted fraction from cell granules showed ~100% 2AB-Man5 ( ). Figure 11A ) and ~100% 2AB-Man3 ( Figure 11BSubstrate elongation. The HA-enriched MGAT-I eluent from the supernatant (SN) showed minimal activity only for 2AB-Man5 or 2AB-Man3. The lysis buffer from wt, used as a negative control, did not affect the retention time of 2AB-Man5 or the modified standard.

[0445] When enriched from the supernatant or membrane portion (“particles”) HA, rMGAT2 effectively converts NGA2-N standards to the G0(NGA2) form. Figure 12A As expected, rMGAT-2 failed to elongate 2AB-Man3a in vitro to NGA2-N ( Figure 12B MGAT2 requires prior GnT-I activity. According to the manufacturer, the NGA2-N standard comprises two forms of GlcNAc located on α1,3- and α1,6-linked MAN branches, each approximately 50%. The peaks of NGA2-N and NGA2 are 50 / 50, subsequently converted to 100% in vitro via rMGAT2 α1,6.

[0446] SfGnT-II is intracellularly localized and detected in cell lysis buffer + / - 1% (v / v) Triton X, indicating membrane binding; however, this is not exclusive (Figure 13). Unlike another Gnt candidate studied, SfGnT-I, SfGnT-II was present less frequently in SN. In vitro activity assays using the HA fraction were inconclusive due to background noise. SfGnT-II in lysis buffer showed rather low NGA2-N conversion, likely due to the low amount present in crude lysis buffer. Figure 13B In vitro active samples of SfGnT-II HA-eluting fraction with SN or lysis buffer (+TrX) showed strong background fluorescence against 2AB-standard used as substrate (not shown). SfGnT-II also requires prior GnT-I activity (…). Figure 13A And it does not work on Man5 ( Figure 13C ). )

[0447] The substrate NGA2-N (green, 76.8m) was used to evaluate the in vitro activity of hB4GALT-I. Figure 14A streptavidin-purified hB4GALT-I converts NGA2-N (green) into assuming equal proportions of α1-3 and α1-6 G1-Gn(a) and G1-Gn(b). Figure 14B The conversion was not 100%, as a weak (blue) peak was detected at 76.8 nm. However, at least >90% conversion was expected. The "doublet" of the B4GALT-I sample showed the same pattern as the "doublet" from the G1 standard (red); however, its retention time was shorter due to the lower concentration of 1×GlcNAc in the structure. Figure 14C The expected in vivo function of B4GALT1 on the G0 substrate was demonstrated, which resulted in the production of G2 glycoform.

[0448] 8.5 Identification of heteroglycosyltransferases that elongate natural oligomannose N-glycans on natural proteins in vivo, as described in Example 5.

[0449] Next, recombinant host cells expressing glycosyltransferases were collected and treated with PNGase F to release their N-glycans. The released glycans were either fully methylated or 2AB-labeled and their activity against native glycoprotein receptors was analyzed. Cells expressing GnT-I (SfGnT-I, St11707) converted oligomannose in vivo to 34% NGA2-N (G0-Gn). Since the samples were derived from crude cell extracts, endoplasmic reticulum (ER)-localized glycoproteins were also included, indicating that these proteins are not transported via the Golgi apparatus for GlcNAc elongation. Due to the requirement of prior GnT-I activity, cells expressing only GnT-II candidates (not shown) did not elongate oligomannose as expected, a finding confirmed by in vitro analysis. When GnT-I was recombinantly co-expressed with GnT-II enzymes (SfGnT-I and SfGnT-II, St12320), elongation of oligomannose to the G0 form via two GlcNAc receptors was confirmed. Figure 15 and 16 ).

[0450] Full methylation and MALDI TOF analysis revealed that the wild-type cellular particle sample St10569 P16_378 contained only one N-glycan structure: (Man)3(GlcNAc)2 (m / z 1171.6). In the sample St11707P16_378 from the genotype ssu::SfGnT-I of the Leishmania tarentolae St11707, Gnt-I expression resulted in two forms: (Man)3(GlcNAc)2 (m / z 1171.6) and (GlcNAc)1(Man)3(GlcNAc)2 (m / z 1416.7), with relative intensities of 66% and 34%, respectively. In the St12320 P16_378 genotype ssu::SfGnT-I; ssu::SfGnT-II (polyclonal) from the Leishmania tarentolae St12320, co-expression of GnT1 and GnT2 resulted in three different N-glycan structures: (Man)3(GlcNAc)2 (m / z 1171.6), (GlcNAc)1(Man)3(GlcNAc)2 (m / z 1416.7), and (GlcNAc)2(Man)3(GlcNAc)2 (m / z 1661.8), with proportions of 48%, 15%, and 37%, respectively. Figure 15 ).

[0451] Furthermore, fragmentation of ions corresponding to the N-glycan structure was performed using MALDI-TOF / TOF MS. Fragment ions present in the m / z 1171.6 spectrum confirmed the N-glycan structure (Man)3(GlcNAc)2. Fragment ions present in the m / z 1416.7 spectrum confirmed the N-glycan structure (GlcNAc)1(Man)3(GlcNAc)2. GnT-I catalyzes the transfer of GlcNAc to α-1,3-mannose in (Man)3(GlcNAc)2. We searched for the presence of transcyclic fragments in the ion fragment spectra at m / z 1416.7, thus confirming the position of the added GlcNAc. Different fragments of GlcNAc bound to α-1,3-mannose have [M+Na] at m / z 560.26. + Mass. The corresponding transcyclic fragments of GlcNAc bound to α-1,6-linked mannose have masses of m / z 546.25 and 574.28. Transcyclic fragments distinguished by the terminal GlcNAc bond are not visible in the spectrum. Fragment ions present in the m / z 1661.8 spectrum confirm the N-glycan structure (GlcNAc)2(Man)3(GlcNAc)2( Figure 16 ).

[0452] Following defatting of cell granules, direct deglycosylation of the intact protein was used to map the N-glycan profiles of three distinct *Leishmania tarentolae* samples. As observed in previously analyzed wild-type samples, the wild-type cell granule sample St10569 contained only one N-glycan structure: (Man)3(GlcNAc)2 (m / z 1171.6). In sample St11707, genotype ssu::GnT-I expression showed both (Man)3(GlcNAc)2 (m / z 1171.6) and (GlcNAc)1(Man)3(GlcNAc)2 (m / z 1416.7). GnT-I catalyzes the transfer of GlcNAc to α-1,3-mannose, a component of (Man)3(GlcNAc)2 present on the native glycoprotein. In the St12320 genotype ssu::SfGnT-I; ssu::SfGnT-II, co-expression of GnT-I and GnT-II results in three distinct N-glycan types: (Man)3(GlcNAc)2 (m / z 1171.6), (GlcNAc)1(Man)3(GlcNAc)2 (m / z 1416.7), and (GlcNAc)2(Man)3(GlcNAc)2 (m / z 1661.8). Further expression of Gnt-II catalyzes the transfer of the second GlcNAc residue to α-1,6-mannose in (GlcNAc)1(Man)3(GlcNAc)2.

[0453] Therefore, these data confirm the in vivo activity of insect cell-derived Gnt for its use in glycoengineering when heterologously expressed in Leishmania tarentolae. Additionally, the presence of UDP-GlcNAc in the Golgi coelom was demonstrated, confirming the hypothesis of a bioinformatics evaluation of an activated nucleotide sugar transporter present in native Leishmania tarentolae.

[0454] When using GlycoWorks TM RapiFluor-MS TM These findings were confirmed when N-glycans from St11707 and St12525 strains (which, alone or in combination with SfGnT-II, respectively, expressed SfGnT-I) were released and labeled, and G0-Gn from SfGnT-I cells and G0-Gn from SfGnT-I+SfGnT-II cells were identified based on their m / z. Figure 17A St13065 co-expresses hMGAT1 and rMGAT2 and produces G0 glycans on its native secretory proteins, indicating that both Gnt are active in vivo. Figure 17B ).

[0455] Interestingly, in different variants of SfGnt-I with combined Strep-triple HA tags, Strep tags alone, or native C-terminus, the activity for total N-glycans ranged from 20% to 75%, indicating the adverse effect of the C-terminal tag on the in vivo conversion of oligomannose to NGA2-N (G0-Gn). Figure 47B ).

[0456] Therefore, heterologous Gnt-I candidates from the MGAT1 library without any C-terminal tags were tested, as shown in Table 9. Leishmania expressing recombinant Gnt-I from different species converted the natural Man3 N-glycan to G0-Gn. Activity ranged from extremely low efficiencies, such as 10% for muMGAT1 (Muscicapidae), to even greater than 90%, as shown for DrMGAT1 (Danio rerio) or GjMGAT1 (Gekko japonicus). Figure 47A ).

[0457] The glycan derived from SfGnT-I activity led to the formation of G0-Gn glycans, in which GlcNAc was added to the α1,3Man branch. This resulted in St13066, co-expressing SfGnT-I and hB4GALT1. GlcNAc was used as an in vitro receptor for B4GALT1 (Fig. 14), and this activity was confirmed in vivo by the appearance of G1-Gn(a) (m / z = 1587.63, a peak at retention time of 15.32 min). Figure 17C ).

[0458] This supports the selection of functional Gnt, and also supports the availability of UDP-GlcNAc and UDP-Gal in the Golgi apparatus compartment of *Leishmania tarentolae*, as inferred from bioinformatics evaluations (Table 6). Thus, in vivo glycoengineering was confirmed in novel *Leishmania tarentolae* host cells engineered and co-expressed with Gnt for the production of functional-customized N-glycans.

[0459] 8.6 Identification of the heteroglycosyltransferase elongated in vivo on recombinant human erythropoietin by natural oligomannose N-glycan in Example 6

[0460] To evaluate not only the activity of Gnt against native or secreted glycoproteins, the recombinant target protein human erythropoietin (hEPO) was co-expressed with Gnt-I. EPO was expressed with two different signal peptides for secretion translocation to secrete into the supernatant. N-glycan release and site occupancy analyses were performed by peptidomimetics and MS. Homogeneous oligomannose was confirmed on EPO when expressed in non-glycoengineered cells, while N-glycan release and full methylation confirmed that EPO co-expressed with GnT-I showed approximately 50% N-glycan elongation by one GlcNAc (Gnt-I). Figure 18 ).

[0461] Site occupancy was analyzed using trypsin peptide profiling. Figure 19 and 20The enriched glycopeptide portions of both EPO samples contained ions corresponding to occupied glycopeptides [EAENITTGCAEHCSLNENITVPDTK] (SEQ ID NO: 109) with glycosylation sites N24 and N38. Conversely, the unoccupied glycopeptide corresponding to N83 was detectable. The two glycosylation sites on peptide [EAENITTGCAEHCSLNENITVPDTK] (SEQ ID NO: 109) were occupied by N-glycans. EPO produced from wild-type Leishmania tarentola strain St11521 contained only a (Man)3(GlcNAc)2 core structure bound to both N-glycosylation sites. Co-expression of EPO and GnT-I in strain St11895 resulted in N-glycosylation sites N24 and N38 being occupied by either (Man)3(GlcNAc)2 or (GlcNAc)1(Man)3(GlcNAc)2. In both EPO samples, the glycopeptide [EAENITTGCAEHCSLNENITVPDTK] (SEQ ID NO: 109) was not found, where only one of the two N-glycosylation sides was occupied by an N-glycan. Peptides [21-45][EAENITTGCAEHCSLNENITVPDTK] (SEQ ID NO: 109) with N-glycosylation sites N24 and N38 were found to be occupied by only two N-glycans. In the case of EPO produced in wild-type Leishmania strain St11521, the ion at m / z 4588 corresponds to a glycopeptide with two (Man)3(GlcNAc)2 N-glycans. Conversely, EPO from strain St11895 exhibits three distinct glycopeptide ions: m / z 4588 corresponding to an N-glycan with two (Man)3(GlcNAc)2 residues; m / z 4792 corresponding to a glycopeptide with one (Man)3(GlcNAc)2 residue and one (GlcNAc)1(Man)3(GlcNAc)2 residue; and m / z 4994 corresponding to a glycopeptide with two (GlcNAc)1(Man)3(GlcNAc)2 residues. For strain St11521 (rhEPO)wt, EPO N-glycosylation sites N24 and N38 are occupied solely by the (Man)3(GlcNAc)2 core N-glycan. For strain St11895 (rhEPO+SfgnT1), co-expression of EPO and GnT1 resulted in both sites being occupied by either (Man)3(GlcNAc)2 or (GlcNAc)1(Man)3(GlcNAc)2. No other N-glycopeptide ions with different N-glycan structures were observed binding to these sites.

[0462] Since no clearly identifiable peptide ions were found after trypsin digestion of the protein, the peptide with N-glycosylation site N83 [77-97]GQALLVNSSQPWEPLQLHVDK (SEQ ID NO: 156) could not be analyzed in this study. Therefore, it is impossible to draw conclusions about the occupancy of site N83. Deglycosylation by PNGases did not show the presence of the deglycosylated peptide with N-glycosylation site N83 [77-97][GQALLVDSSQPWEPLQLHVDK] (SEQ ID NO: 156) as a clearly identifiable ion. The theoretical monoisotopic mass of the deglycosylated peptide is 2360.24 Da. The spectra of St11895 at P2813 and St11521 at P2814 contain very weakly separated ions at m / z 2361.5 and 2360.01, respectively. It is impossible to identify these ions by fragmentation. Therefore, we have no confirmed indication of the presence of this peptide in its unglycosylated or deglycosylated form.

[0463] In both peptide mass fingerprinting profiles, we did not find the trypsin peptides with N-glycosylation sites N24 and N38 in their unglycosylated free form [21-45]EAENITTGCAEHCSLNENITVPDTK (SEQ ID NO: 109) or the peptide with N-glycosylation site N83 [77-97]GQALLVNSSQPWEPLQLHVDK (SEQ ID NO: 156), indicating 100% N-glycosylation occupancy. No missed cleavage of these peptides was identified. Although only two of the three N-glycosylation sites were identified as 100% occupied, we hypothesize that the third site is occupied, which would be relevant to earlier MS of the intact protein from EPO derived from non-glycoengineered cells.

[0464] 8.7 Example 7 Identification of secretory signals from natural secretory proteins

[0465] Initially, the signal peptide was derived from alkaline phosphatase from *Leishmania mexicana*, as described in (Klatt and Konthur 2012). In this invention, the signal peptide is derived from a glycoprotein secreted by the native host cell, identified by ConA purification followed by proteomics methods and EDMAN N-terminal sequencing. Among the native host cell-secreted glycoproteins identified by MS, the most prominent proteins were invertase (sucrose hydrolase-like protein) (LTAR_040008100.1; previous nomenclature: LtaP04.0290) and the GP63 variant LTAR_100010400.1 (previous nomenclature: LtaPcontig00616-1) (Figure 21), both containing a common N-glycosylation site and possessing a uniform Man3 oligomannose N-glycan. The putative secretion signal peptide from the convertase, DGVPYEx (SEQ ID NO: 157), was identified by EDMAN N-terminal sequencing (Figure 22). DGVPYEx (SEQ ID NO: 157) (with other amino acids possible in different cycles: K in cycle 1, P in cycle 2, and E in cycle 4). Host cells expressing recombinant C-terminal His or StrepII-tagged EPO were used to purify secreted recombinant EPO by fusing these corresponding nucleotides in the boxes with the gene encoding the hEPO nucleotide sequence. Figure 23 ).

[0466] 8.8 Example 8 Targeting and retaining to the correct subcellular compartment

[0467] like Figure 6 and 7 As listed, sequential processing of N-glycans by glycosyltransferases occurs in the endoplasmic reticulum (ER) and Golgi apparatus (kellokumpu et al., 2016). It is generally believed that the involved enzymes (glycosyltransferases and glycosidases) act sequentially and independently, adding or removing sugar residues by traversing an ever-growing oligosaccharide chain at specific times and in a specified order. This suggests that Gnts for glycoengineering can be improved through proper localization and retention along the secretory pathway. Most Golgi-localized Gnts are type II membrane proteins with a short N-terminal cytoplasmic domain (“C”), an approximately 20-amino acid α-helix TM domain (“T”), a stem domain (“S”), and then a C-terminal globular catalytic domain located in the lumen of the secretory pathway. Figure 24ATheir functional importance for glycosylation can also be based on their interactions as homopolymers or heteropolymers. Their universality and these domain-mediated interactions can lead to alterations in enzyme activity resulting from complex formation (Kellokumpu et al., 2016). Therefore, designing hybrid-Gnt for a) improved targeting and retention of the correct subcellular compartments (Golgi intermediate membrane vesicles and trans membrane vesicles, see...) Figure 8 b) and c) lead to better homogeneous and heterodimerization of activity for sugar engineering.

[0468] Figure 24A This shows a typical type II membrane protein structure, which we interpret for use in the design of hybridization Gnt, such as Figure 24B As illustrated in the example below, in the SfGnT-I example, the predicted CTS of the native Golgi protein is fused into the catalytic domain of the heterologous GnT-x. Figure 24B The expected Golgi apparatus retention and binding proteins of *L. tarentolae* are shown. The secretory signal sequences of *Trypanosoma* and *Leishmania* do not function in mammals; they are similar to those of Gram+ bacteria (Al-Qahtani et al., 1998), and the signal peptide sequence requirements and cleavage sites appear to differ from those of trypanosoma and higher eukaryotes (La Flamme, AC et al., 1995). Tissue studies of the trypanomic secretory pathway have been reviewed elsewhere (McConville et al., 2002b). *T. brucei* cell surface proteins (GPI-anchored) contain highly conserved signal peptides for secretion (…). (and Cross, George AM 2002). Target sequences of the Golgi apparatus reverse membrane sac network have been shown to be present in *Trypanosoma brucei*, where the *T. brucei* GRIP domain (GFP-TbGRIP) is efficiently localized to the Golgi apparatus of transfected COS cells when fused to the C-terminus of green fluorescent protein. Furthermore, these GRIP domains function in *T. brucei* and *Leishmania mexicana*, however, GRIP transport is reduced in quiescent *L. mexicana*. GRIP proteins are typically described as having coiled regions; in GFP-GRIP fusion proteins, these are introduced from extended GRIP regions (McConville et al., 2002a). NTPD enzymes are nucleoside triphosphate-diphosphate hydrolases that hydrolyze nucleotides, play a role in parasitic toxicity, and are involved in lipophosphatidylcholine (LPG) elongation. In Leishmania major, LmNTPDase1 is located in the Golgi apparatus, and LmjF.15.0030 possesses a TM domain, which is the presumed CTS motif. LmNTPDase2 is shown to be secreted. LmjF.10.0170 possesses the predicted signal peptide (Sansom et al., 2014).

[0469] Homologs from *L. tarentolae* were identified as NTPDase1 = LTAR_150005200.1 (previous nomenclature: LtaP15.0020) [best reverse hit to LmjF.15.0030] and NTPDase2 = LTAR_100006700.1 (previous nomenclature: LtaP10.0140) [best reverse hit to LmjF.10.0170]. Unlike *L. major*, both *L. tarentolae* NTPD enzymes possess a CTS domain. We designated 5 amino acids as the active region and 10 amino acids as the CTS region for hybridization sequence fusion. The stem length of the CTS is generally indeterminate; (Geisler et al., 2015b) 13 amino acids were used after the TM domain. Heterologous expression of wt Gnt and hybrid structures (CTS of Man1-SfGnT-IStrep; CTS of LtaNTPDase1-SfGnT-I Strep; CTS of TbGnTI-SfGnT-II) in Leishmania tarentolae was performed, followed by analysis of their localization using coarse fractionation assays. We also used the predicted CTS domain of GnT-y in human MGAT1 in the following examples, and fused different lengths to the receptor GnT-x in SfGnT-I in the following examples (Fig. 25). The localization of these plasmids was tested (Table 10) (Fig. 46). The length of the MGAT1 N-terminal domain was increased by fusing to the N-terminus of the reduced SfGnt-I catalytic domain. Figure 46B Improved cell (membrane) localization was observed. Figure 46A This reinforces the hypothesis of testing different N-terminal retention signals from native Golgi localization proteins derived from the lizard Leishmania (L. tarentolae) or from heterologous Golgi GnT (as illustrated by using the N-terminal retention signal of MGAT1). However, due to the Strep-triple HA tag used, the activity for N-glycan conversion was low and detected only in St13561 expressing the construct 90MG1-SfGnT-I-3HA-Strep, and the activity of native SfGnT-I with the Strep-triple HA tag was reduced by approximately 10% compared to the Sf-GnT-I Strep tag. Figure 46B ).

[0470] Table 10 Hybrids

[0471] Experiment ID describe plasmid ID Strain ID P16_383_1 1_90MG1-SFGNT-I-3HA-Strep p5825 St13561 P16_383_2 2_110MG1-SFGNT-I-3HA-Strep p5826 St13562 P16_383_3 3_130MG1-SFGNT-I-3HA-Strep p5827 St13563 P16_383_4 4_150MG1-SFGNT-I-3HA-Strep p5828 St13564 P16_383_5 5_MGAT2-Strep-SFGNT1-3xHA p5830 St13566 P16_407_9 9_MGAT2-Strep-MGhybrSFGNT1-3xHA p5831 St13567 P16_383_6 6_SFGNT-I-3xHA-Strep Natural p5829 St13565 P16_463_17 17_Stt3A_d90 SfGnt-I strep p6173 St14295

[0472] First, we investigated the general growth of recombinant cell lines expressing either recombinant human erythropoietin with a secreted peptide or SfGnT-I containing its native targeting signal. OD was monitored, and cell morphology changes were tracked by microscopy. Shaking culture reached higher OD values ​​and peaked at 72 h, but decreased at 96 h. Static culture reached maximum growth and expression (OD 1.8) at 96 h, with prolonged active production. During growth, *L. tarentolae* cells showed morphological changes correlated with cell cycle progression (G1>S>G2>M>G1…). Figure 26A Expression and crude localization of coarse cell granules (intact cell extract) and supernatant showed that EPO was primarily secreted and not retained intracellularly. Similarly, the expected Golgi enzyme Sf-GnT-I was present in the supernatant in a truncated form, suggesting potential proteolytic cleavage via the secretory pathway or unsuitability for retention in the Golgi apparatus of *L. tarentolae* by CTS (Cellular Thyroid Golgi apparatus). Figure 26B Furthermore, although SfGnT-I is also secreted to the SN, the trypanosoma-derived enzyme is only present in the crude fraction and the Triton-X lysate fraction, indicating membrane and Golgi-mediated localization (Fig. 27). TbGnT-I and TbGnT-II are mostly membrane-bound. Figure 27B Unlike Tb-derived enzymes, SfGnT-I exhibits excellent in vivo and in vitro activity; therefore, we used Sf-GnT-I as the catalytic domain in our hybrid design. The CTS N-terminal region gene as shown in Figures 24 and 25 was fused into the predicted Sf-GnT-I catalytic domain. Except... Figure 24B In addition to the aforementioned CTS variants, the TbGnT-I CTS can also be used for N-terminal fusion into the catalytic domain of functional GnT-I or GnT-II enzymes. It is envisioned that all CTS variants be used in the catalytic domains of all active Gnt (GnT-I, GnT-II, GalT, SiaT) to produce functional and Golgi-retaining hybrid Gnt.

[0473] Figure 28 shows the full-length SfGnT-I and the localization of its CTS-hybrid. Although no bands were observed in the empty vector control, SfGnT-I-Strep (St11898) was detected at a truncated size of 50 kDa by anti-Strep immunoblotting in cultured SNs from passage 3 to 6. It degraded at 38 kDa (double band), 30 kDa, and 20 kDa, but no bands were detected in lysis buffer (+ / - TrX) and insoluble cell debris. At 47 kDa, (aStrep)CTS-Man1-SfGnT-I-Strep (St11899) was detected in p3-p6 SNs, and degraded at 38 kDa (double band), 30 kDa, and 20 kDa, but no bands were detected in lysis buffer (+ / - TrX) and insoluble cell debris. Since CTS-LtaNTPDase1-SfGnT-I-Strep (St11900) was not detected in either the membrane portion or the SN, it was either not expressed or completely unstable. In the p5 and p6 SNs, CTS-LtaNTPDase2-SfGnT-I-Strep (St11899) was detected as a weak band at 47 kDa, but no band was detected in lysis buffer (+ / - TrX) or insoluble cell debris. This indicates that the localization has not changed, as SfGnT-I appears to have a more proteolytically accessible site towards the catalytic domain, and a second strategy targeting growth in CTS was designed and tested (Figure 25, Table 10). Figure 46A , Figure 47B Cellular localization was significantly improved in these growing hybrids, supporting the hypothesis (). Figure 47A However, N-glycan conversion cannot be considered, firstly because the activity is adversely affected by the Strep-triple HA tag compared to native SfGntI with SfGntI-Strepo and SfGnt-I Strep-3HA. Figure 47B Furthermore, it is speculated that the N-terminus truncation of 110 amino acids also impairs the catalytic activity of SfGnT-I.

[0474] When natural human Gnt was tested on St12239 cells expressing hMGAT1 3×HA tags in p5 and p6, hMGAT1 was detected in both whole cell extract (WCE) and SN. MGAT1 was also detected in cell debris. Figure 29 This indicates secretion / release, but the in vitro active enzyme is obtained primarily from the membrane portion rather than from SN purification (Figure 11).

[0475] SfGnT-II (ssu::SfGnT-II-3xHA) expressed from p10's St12318 was detected in both WCE and SN, secreted / released at very low levels. Detection of SfGnT-II in TrX- lysis buffer indicated cytoplasmic localization. However, strong detection of SfGNT-II in TrX+ lysis buffer indicated membrane binding. Figure 30 ).

[0476] Relatively strong expression of rMGAT2 was observed in St11897 (ssu::MGAT2-3xHA) cells at p5 and p6, including secretion to the SN, presence in intact cell extracts, and intracellular / cytosolic localization. Strong membrane binding of rMGAT2 was also found in TrX+ lysates and insoluble fractions. Degradation was also observed at approximately 33–35 kDa (Figure 31). rMGAT2 was purified from SN and TrX+ particles for in vitro activity assays (Figure 12).

[0477] Therefore, these novel hybridization strategies are essential to support the retention of heterologous and active Gnt in the Golgi compartment of the lizard Leishmaniae (L. tarentolae).

[0478] 8.9 Example 9 Sialic acid biosynthesis and sialylation of biantennary N-glycans

[0479] In mammals and bacteria, Neu5Ac undergoes both anabolism and catabolism via different pathways (Angata and Varki, 2002). Two main types of enzymes are involved in the formation of Neu5Ac. N-acetylneuraminic acid lyases (Neu5Ac lyases) participate in the catabolism of sialic acid by catalyzing the reversible cleavage of Neu5Ac into N-acetylmannosamine (D-ManNAc) and pyruvate. At high concentrations of D-ManNAc and pyruvate, the equilibrium can be shifted to Neu5Ac synthesis. Neu5Ac lyases from *E. coli*, combined with glucosamine 2-epiosome activity, can be used for the large-scale production of Neu5Ac from D-GlcNAc. Alternatively, Neu5Ac synthases, such as NeuB, can catalyze the condensation of ManNAc on phosphoenolpyruvate (PEP) and directly participate in the biosynthesis of sialic acid (reviewed in Tanner, 2005). CgNal is an N-acetylneuraminic acid lyase from *Corynebacterium glutamicum* used to produce Neu5Ac. It catalyzes the reversible Neu5Ac aldol condensation from ManNAc and pyruvate, but favors Neu5Ac synthesis over cleavage (Ji et al., 2015). The initiating step in the mammalian sialylation pathway is the biosynthesis of the activated glyconucleotide precursor CMP-Neu5Ac. To achieve the production of this precursor from the endogenously present metabolite UDP-GlcNAc, at least four enzymes can be used: (1) GNE, a bifunctional enzyme that catalyzes the conversion of UDP-GlcNAc to ManNAc and the phosphorylation of ManNAc to ManNAc-6-phosphate; (2) NANS, which condenses ManNAc-6-phosphate with phosphoenolpyruvate, resulting in Neu5Ac-9-phosphate; (3) a specific phosphatase acting on Neu5Ac-9-phosphate; and (4) CMAS that activates the predominant sialic acid obtained in the nucleus to CMP-Neu5Ac (Castilho et al., 2010). Furthermore, bacteria NanE or GNPE (Geisler and Jarvis 2012) can also facilitate the formation of the precursor ManNAc-6-phosphate from GlcNAc-6-phosphate. Figure 53 ).

[0480] Suitable sialyltransferases (from Table 9) can be recombinantly expressed in *L. tarentolae* and affinity-enriched from crude lysates, membrane lysates, and culture supernatants of *L. tarentolae*. The semi-purified enzymes ( Figure 49AIn vitro tests were conducted to determine their correct activity with cofactors and the activated glyconucleotide precursor CMP-Neu5Ac. Figure 48A The relative activities of sialyltransferases expressed in *L. tarentolae* to 2AB-labeled G2 substrates are shown in relation to the transfer of one sialic acid (= monoantennae sialylation) or two sialic acids (biantennae sialylation) to a biantennae-galactosylated substrate. The determination of the bonds on the 2AB-labeled glycans was evaluated by retention time comparison (Fig. 49), confirming α2,6 or α2,3-linked sialic acids. Figure 48B The relative amounts in this screening assay are shown. In the next method, the activity of the primary candidate (murretic mST6GAL1) against the fully folded monoclonal antibody (MabThera) was tested. Figures 50A-50E Although mST6 was extracted from the lysis buffer and affinity enriched (…). Figure 50A This resulted in 100% biantennary α2,6 sialylation of the 2AB-labeled G2 substrate. Figure 50B However, the same enzyme preparation primarily sialylates one branch (~14%) and the two antennae are almost undetectable (0.4%). Figure 50C and 50D The HA-eluted portion of mST6 converted (sialylated) 14.2% of total N-glycans (~25% of total galactosylated N-glycan conversion); the HA-eluted portion of rSAP+mST6 converted (sialylated) 15.36% of total N-glycans (~26.5% of total galactosylated N-glycan conversion). Beads of mST6 captured by HA-tag (uneluted) converted (sialylated) 20.13% of total N-glycans (~34.7% of total galactosylated N-glycan conversion). 2 μM dCTP appeared to inhibit (or increase) sialyltransferase activity in the HA-eluted portion of mST6, resulting in only 4.12% total N-glycan conversion (sialylation) (~6.9% of total galactosylated N-glycan conversion). Figure 50D In summary, mST6 can sialylate galactosylated N-glycans of MabThera in vitro in either free (eluting) or captured (bead form), and rSAP has a slight positive effect on ST-transformation, while dCTP inhibits ST-transformation.

[0481] Host cells can be engineered to express a functional CMP-sialic acid (CMP-Sia) biosynthetic pathway. Mammalian and / or bacterial biosynthetic pathways can be used in *L. tarentolae* host cells (Table 11), as previously demonstrated in various other organisms such as *Pichia*, insect cells, and plants (Aumiller et al., 2003; Hamilton et al., 2006; Castilho et al., 2010). Once CMP-NeuAc is available in the Golgi apparatus of *L. tarentolae* host cells, specific sialyltransferases can transfer sialic acid to a receptor substrate (e.g., β1,4-galactosylated biantennary N-glycan). Table 9 shows mammalian and bacterial sialyltransferases. Figure 53 The expected cellular localization of the biosynthetic pathway is shown.

[0482] Table 11 Sialic Acid Biosynthesis

[0483]

[0484]

[0485] A method is provided for engineering the CMP-Sia biosynthetic pathway into non-human eukaryotic cells. The method involves the cloning and expression of several enzymes of mammalian, bacterial, or both originating from a lizard leishmania (L. tarentolae) host cell lacking endogenous sialylation. The engineered CMP-Sia biosynthetic pathway is useful for the in vivo production of sialylated glycolipids, O-glycans, and N-glycans. Therefore, this is useful for assisting the production of sialylated therapeutic glycoproteins in non-human host cells lacking endogenous sialylation.

[0486] α2,3- or α2,6-sialyltransferases in humans cap galactose residues with sialic acid in the trans-Golgi apparatus and the trans-Golgi membrane sac network (TGN), resulting in the production of the mature form of the glycoprotein. To engineer this processing step into lower eukaryotic host cells and other host cells that naturally lack sialyltransferase activity, the following can be introduced into the host cells: (1) α2,3- or α2,6-sialyltransferase activity and (2) adequate supply of CMP-N-acetylneuraminic acid in the late Golgi apparatus. To obtain sufficient α2,3- or α2,6-sialyltransferase activity in the secretory pathway (e.g., late Golgi apparatus), for example, the catalytic domain of a known sialyltransferase (e.g., from mammalian or bacterial origin) can be directed to the secretory pathway in lower eukaryotic host cells. Similarly, transport proteins can be engineered to allow the transport of CMP-N-acetylneuraminic acid to the same location in the secretory pathway (e.g., the late Golgi apparatus). Therefore, to ensure an adequate supply of substrate to the corresponding glycosyltransferase, the production of CMP-sialic acid can be engineered in these host cells. All analyses can be performed as described in Examples 4 and 5 above.

[0487] 8.10 Example 10 Expression of anti-CD20 rituximab in Leishmania tarentolae

[0488] As discussed previously and Figure 1 As illustrated in Figure 2, the improvement of antibody efficacy, reduction of therapeutic dose, and enhancement of overall clinical performance through alterations in Fc glycans have led to the design of glycan libraries selected for different properties. Figure 32 To test the concept in potential applications, it was chosen as... (Roche) Rituximab, a commercially available anti-CD20 monoclonal antibody, serves as a test case for its known ADCC mode of action. Figure 33 The amino acid sequence, signal peptide, and the Asn 297 glycosyl site discussed are shown. Figure 34 The expression cassette integrated into the host cell of the lizard leishmania (L. tarentolae) is shown. The resulting cell line St12427 was grown to harvest the antibody molecule secreted into the culture supernatant, which was named "rituximab_LMTB". Purification was performed in two steps using the filtered supernatant: first, protein A capture purification, followed by hydrophobic interaction column purification. Figure 35 ).

[0489] Figure 36A The comparison between rituximab_LMTB and a commercially available contrast agent from Roche was shown. A comparison. Under non-reducing conditions, rituximab_LMTB showed similarity to... Similar banding patterns, but with varying intensities. Degradation product traces were observed in both samples. Proteins digested with PNGaseF showed... The heterogeneity in it is higher than that of rituximab-LMTB, as the former has a longer glycan. Figure 36B The expected differences were observed. Capillary gel electrophoresis confirmed these observations. Figure 37 ).

[0490] Using the MAbPac SEC-1 size exclusion chromatography (SEC) column, which is specific for the separation and identification of monoclonal antibodies, the aggregate formation of rituximab_LMTB was determined to be 5.8%, compared to... It is 0.3% (Figure 38).

[0491] Then, use Waters GlycoWorks TM RapiFluor-MS TM The procedure (Figure 39) involves PNGaseF release and full methylation followed by MALDI-TOF ( Figure 40 41) Perform N-glycan profiling to determine the comparison with rituximab_LMTB. The heterogeneity of N-glycans and their fucose levels. Indeed, rituximab_LMTB has 100% homogeneous Man3 glycans, compared to antibodies produced in CHO. There are more than 8 types of sugar forms in it. Figure 40 The quantitative comparison based on full methylation shown reveals the predominant fucosylation structure, which is suboptimal for Fc-mediated receptor function in ADCC. Indeed, this is true for third-generation anti-CD20 antibodies engineered for both ADCC protein engineering and glycoengineering. Roche has improved the quality of fucoidan-free N-glycans ( Figure 42 ). The fully methylated N-glycan profiles of rituximab produced from Leishmania tarentolae and from Roche The spectra are significantly different. The main structures are equal N-glycans G0B, G0BF, G1B, and G1BF. Compared to 94%, The fucosylation level was 52%. In other words, compared with... 48% and Compared to only 6%, rituximab-LMTB produced in *Leishmania tarentolae* is 100% unfucosylated. However, compared to... 14 structures and Compared to the eight structures produced, rituximab produced from *Leishmania tarentolae* showed only one structure corresponding to Man3GlcNAc2. A commercially available contrast agent produced in CHO cells... The fully methylated N-glycan spectrum showed eight structures, among which the typical fucosylated biantennary structures G0F and G1F were the main structures.

[0492] This strongly supports the concept of homogeneous and functionally customized N-glycans in the lizard Leishmania tarentolae for the engineering of therapeutic recombinant antibodies or Fc-containing glycosides.

[0493] In addition, FACS staining was used to determine the CD20 antigen binding capacity on Raji cells. Figure 43A And it used the dot blot analysis of recombinant CD20 spot blots cultured with specified antibodies. Figure 43B In both assays, rituximab_LMTB and It binds equally to its antigen (CD20). In FACS, both antibodies emit a clear dose-dependent signal relative to the IgG1 control antibody, however, the signal of Mabthera appears to be somewhat stronger.

[0494] Figure 44 Furthermore, illustrative quality assessments implemented are shown, which are also listed in Table 12.

[0495] Table 12 Quality Standards for Rituximab

[0496]

[0497] Figure 52 shows the in vivo glycan extension from Man3 to G0 in the Leishmania tarentolae strain (St13418) stably expressing glycosyltransferases SfGnt-I Strep and MGAT2-HA; and the free expression of rituximab from pLMTB5026. The secreted rituximab was then purified from 2-day cultures in 1L scale using protein A. Analysis of 20 μg of purified rituximab_LMTB by PNGase F and RF-MS showed successful in vivo glycan engineering for the secreted target protein (full-length monoclonal antibody).

[0498] 8.11 Materials and Methods

[0499] 8.11.1 Plant, Growth and Genetic Methods

[0500] Generally, unless otherwise specified, the strains (Table 13) are cultured in brain heart extract containing 5 μg / ml thioheme (BHIH) at 26°C, in the dark, statically or with shaking, for 2–3 days. Recombinant cell line cultures contain appropriate selection reagents (Table 14).

[0501] Table 13 plants

[0502]

[0503]

[0504]

[0505] Table 14 Reagent Selection

[0506]

[0507] Transfection to generate stable cell lines is performed as described below to integrate the heterologous gene of interest. The expression cassette contains 1.) a homologous site for site-specific integration via homologous recombination, 2.) a 5' untranslated terminal repeat containing a splicing leader receptor sequence, 3.) the gene of interest as an ORF codon optimized for Leishmania (L. major or L. tarentolae), 4.) an intergenic region containing a 3' UTR for polyadenylation sequences and a 5' UTR for downstream genes; the downstream genes, for example, 5.) resistance markers, followed by 6.) their 3' UTR and 7.) a 3' homologous region for site-specific recombination into the genome.

[0508] For integration into the genome, 5–10 μg of donor plasmid DNA is digested with a side-linked restriction endonuclease to excise the expression cassette from the vector backbone. Restriction digestion is performed at 30 °C until complete or 0 / n, and the DNA is purified by EtOH precipitation (2 volumes of 100% ice-cold EtOH are added to 1 volume of digested DNA, incubated on ice for 30 min, and centrifuged at 17500 × g for 30 min at 4 °C). The precipitate particles are washed with 70% EtOH, dried for a maximum of 15 min, and resuspended in ddH₂O. For optimized removal of circular plasmids, one or two restriction endonucleases with recognition sites on the vector backbone are selected, and digestion is performed at 37 °C for approximately 1 h, followed by purification with EtOH as described above.

[0509] Transformation of *E. coli* DH5α with 100 ng of heat-shocked digested DNA, an undigested control, and a ddH2O control was performed to check for the presence of still intact plasmid DNA. Chemocompetent DH5α cells, thawed on ice for 10–15 min, were carefully mixed with 100 ng of digested plasmid DNA, incubated on ice for 25–30 min, followed by heat shock at 42 °C for 90 sec and incubation on ice for 5 min. 1 ml of LB or SOC medium was added, and the mixture was incubated at 37 °C for 1 h. Aliquots were then plated onto LB agar plates containing ampicillin and incubated upside down at 37 °C.

[0510] Leishmania cultures for transfection were prepared by diluting the culture, which had been statically and densely grown at 26°C, 1:10 in BHIH the day before transfection. OD was measured at 600 nm using a spectrophotometer in individually used cuvettes, and for optimal efficiency, the range was 0.4–1.0 (4–6 × 10⁷ cells). Cells should be in the log-phase, indicated by a mixed population of non-rounded, drop-shaped cells. More rounded cells were preferred. 10 ml of culture was used per transfection, and electroporation was performed on each culture, with ddH₂O used as a negative control for each selection marker. For transfection, the culture was centrifuged at 1800 × g for 5 min at RT. After removing the SN, the precipitate was resuspended in 5 ml of transfection buffer (200 mM Hepes pH 7.0, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose, anhydrous (glucose), filtered through a 0.22 μm sterile filter). The cells were centrifuged again, and the precipitate was resuspended in 400 μL of transfection buffer. 400 μL of cells were added to the DNA and transferred to a cuvette, then incubated on ice for 10 min. Gene Pulser Xcell was used. TM (Biorad), using a low-voltage protocol, exponential wave decay: 450V, 450uF, 5-6ms, cuvette: d=2mm for electroporation. Immediately place precisely on ice for 10 min. Transfer all contents of the cuvette to 10ml BHIH without any selection markers, and allow cells to grow statically at 26°C, protected from light, and aerated for 20-24 h.

[0511] For selection of polyclonal cell lines with subsequent clonal selection, add half-concentration of the selection marker and incubate the culture at 26°C for 1–2 days. Passage the cells at a 1:10 ratio in 10 ml of BHIH + full concentration of the selection marker. Assign new cell numbers and passage number 0. Label the passage number of the transfected cells for all subsequent experiments. Grow the cells further at 26°C in the dark. If the culture becomes turbid after 7 days, centrifuge the cells at 1800 × g for 5 min at RT, and resuspend the pellet in fresh BHIH medium containing the full concentration of the selection marker.

[0512] For clonal selection, once the liquid culture becomes turbid, streak cells onto BHIH plates (containing 1.4% agar) with appropriate 100% selection reagent. Seal the plates with sealing film and protect from light, incubating upside down at 26°C for 7–10 days. Transfer individual colonies (1–2 mm in size) to 24-well plates containing 1 ml of BHIH, seal with sealing film, and incubate at 26°C in the dark for approximately 7–10 days. Then, transfer 1 ml of culture from the 24-well plate to 10 ml of BHIH in a flask and continue static growth as before.

[0513] Integration of the targeted genomic sites was confirmed by PCR and sequencing.

[0514] 8.11.2 Plasmids

[0515] The plasmid was derived from the pUC57 vector backbone used for E. coli propagation and contained an ampicillin selection marker. The expression cassette was flanked by restriction sites suitable for excision. The expression cassette contained: 1.) a homologous site for site-specific integration via homologous recombination; 2.) a 5' untranslated terminal repeat containing a splicing leader receptor sequence; 3.) the gene of interest for an ORF codon-optimized for Leishmania (L. major or L. tarentolae); 4.) an intergenic region containing a 3' UTR for polyadenylation and a 5' UTR for downstream genes, such as, for example, 5.) a resistance marker, followed by 6.) its 3' UTR and 7.) a 3' homologous region for site-specific recombination into the genome. The plasmid was generated by a gene synthesis provider and sequenced. Use the following codon usage table to optimize the codons of *Leishmania* for *L. major* from http: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=347515 or *L. tarentolae* from http: / / genomes.urv.es / OPTIMIZER / :

[0516] GCA: 22.791; GCC: 29.220; GCG: 34.130; GCT: 19.986; TGC: 13.986; TGT: 6.059; GAC: 30.120; GAT: 17.49 9; GAA: 15.444; GAG: 43.653; TTC: 17.287; TTT: 12.657; GGA: 8.405; GGC: 27.204; GGG: 11.971; GGT: 14.2 74; CAC: 19.637; CAT: 8.559; ATA: 3.929; ATC: 17.100; ATT: 10.524; AAA: 18.838; AAG: 26.306; CTA: 6.29 3; CTC: 23.191; CTG: 32.564; CTT: 14.172; TTA: 2.836; TTG: 12.837; ATG: 23.282; AAC: 20.217; AAT: 7.91 2; CCA: 13.041; CCC: 12.627; CCG: 20.543; CCT: 10.758; CAA: 9.847; CAG: 30.402; AGA: 3.857; AGG: 6.04 6; CGA: 8.962; CGC: 26.322; CGG: 12.218; CGT: 12.622; AGC: 22.575; AGT: 9.528; TCA: 10.537; TCC: 16.05 7; TCG: 18.475; TCT: 12.894; ACA: 14.345; ACC: 16.949; ACG: 21.677; ACT: 9.877; GTA: 7.998; GTC: 16.92 4; GTG: 35.086; GTT: 10.582; TGG: 10.776; TAC: 18.379; TAT: 5.571; TAA: 0.366; TAG: 0.560; TGA: 0.719;

[0517] Manually plan and optimize sequences to avoid the loss of restriction sites and duplicate or homopolymer extensions. Table 15 lists the plasmids and their descriptions.

[0518] Table 15 Plasmids

[0519]

[0520]

[0521]

[0522] 8.11.3 Gnt Candidate

[0523] Tables 9 and 10 list Gnt and other Gnt hybrids.

[0524] 8.11.4 N-Glycan spectra using Rapifluor (RF) labeling and MS

[0525] (a) Sample preparation

[0526] Samples were prepared according to the Waters application notes: "Quality control and Automation Freiendly GlycoWorks RapiFluor-MS N-Glycan Sample Preparation". Briefly, for purified protein samples, the sample volume was 10 μl, 1.5 mg / ml (15 μg), or as precipitate particles, 10 × 8 cells. 3% RapiGest was added, and the mixture was incubated at 100°C for 3 min, followed by RT for 3 min. Then, 10 μl of PNGase F (Sigma, diluted 30 μl PNGase + 220 μl water) was added and incubated at 50°C for 5 min. After RT for 3 min, 10 μl of RFMS (9 mg in 110 μl anhydrous DMF) was added and incubated at RT for 5 min. 360 μl of ACN was added, and the sample was washed with SPE, eluting with 3 × 30 μl, and then mixed to 90 μl. Before injecting 10 μl via full-loop injection (4× overflow factor), dilute the sample with 100 μl of DMF and 210 μl of ACN.

[0527] (b) Column Setting

[0528] Use Glycan BEH Amide column (Waters); Liquid chromatography (HPLC) was performed on a WatersAcquity UPLC system (1.7 μm, 2.1 mm × 150 mm). N-glycan separation was achieved using a gradient of 20% aqueous solution of 0.1% formic acid (FA) (buffer A) and 80% acetonitrile solution of 0.1% FA (buffer B), reaching 27% of buffer A in 3 min, and then 37% of buffer A in 32 min, at a flow rate of 0.5 mL / min. Detection was performed at 215 nm using a UV detector.

[0529] The UPLC was directly connected to the Waters Q-TOF Synapt HDMS in ESI mode for mass determination. Mass was acquired in positive ion mode between m / z 300 and 3500 using a lock mass spray (leucine enkephalin) for lock mass correction.

[0530] ACQUITY UPLC Glycan BEH Amide 1.7μm, 2.1×150mm; Buffer: A: 50mM AmFor(H2O) pH 4.5, B: CAN; Flow rate: 0.5ml / min; Temperature: 45℃; Injection volume: 10μl (full loop injection); Gradient: 80-73% B (3'), 73-63% B (32') (total 55min); LC method: ESI_RFMS_mAB_55_FLR; Synapt settings: 20161006_uba284_esi_RFMS); MS method: ESI_RFMS_mAB_300_3500_vpos_55; Cap voltage: 3kV; Cone voltage: 80V; Source temperature: 120℃; Desolvation gas temperature: 350℃; Desolvation gas flow rate: 800l / h; Locked mass: Leucine enkephalin, 1ng / ul, flow rate 4uL / min; Fluorescence detection: Ex 265 / Em 425nm (RapiFluor-MS) (2Hz).

[0531] 8.11.5 N-glycan profiles of crude cell precipitate particles by full methylation and MALDI-TOF

[0532] (a) Sample preparation

[0533] The precipitate containing 10*8 cells was defatted by shaking with chloroform / methanol (2:1) for 30 min. After centrifugation, a second defatting step was performed with chloroform-methanol-water (40:20:3) while shaking for 30 min. After centrifugation, the defatted cell particles were dried under a nitrogen stream.

[0534] By containing 0.1% in 200 μL Proteins were extracted from cell granules by sonication in 50 mM ammonium bicarbonate buffer (SF (WATERS) and 10 mM DTT) for 3 × 90 sec. The proteins were denatured at 56 °C for 45 min and alkylated at room temperature by inhalation with iodoacetamide (50 mM) for 60 min in the dark. The samples were then incubated at 37 °C with 10 μg trypsin / Lys-C mixture (promega mass spectrometry grade) for 16 h to obtain digestible proteins.

[0535] (b) Glycosidase digestion and full methylation of N-glycans

[0536] After inactivating trypsin / Lys-C at 95°C for 5 min, the sample was aliquoted into two equal parts. One aliquot was deglycosylated with 20 units of PNGase F (Promega#175715), while the second aliquot was deglycosylated with 15 μL of PNGase A (Roche#10472620) adjusted to pH 5 using 200 mM acetate buffer. Hydrolytic surfactants were removed by adding 0.5% TFA to the digested protein sample and incubating at 37°C for 30 min, followed by centrifugation at 15000 × g for 10 min. The trypsin peptide was loaded onto a SEP PACK C18 200 mg column, and the fraction containing N-glycans was collected.

[0537] N-Glycans were purified on an Ultra Clean SPE Carbograph (ALLTECH). The SPE was equilibrated with 0.1% TFA and washed with 0.1% TFA before loading N-glycans released by PNGase. After elution with 3 mL of 25% acetonitrile and 0.1% TFA, the N-glycans were lyophilized before full methylation. Full methylation of the lyophilized sample was performed over 40 min using approximately 25 mg of sodium hydroxide, 500 μl of DMSO, and 300 μl of ICH3. After quenching the reaction with 1 mL of water, 3 × 500 μl of chloroform was used for the extraction of the fully methylated glycans. The chloroform phase was washed with an equal volume of water and then dried. The reaction product was loaded onto a C18 SepPak 200 mg (WATERS) and eluted in 2 mL of 80% acetonitrile, then lyophilized before MALDI-TOF MS analysis.

[0538] (c) MALDI-TOF analysis of N-glycans

[0539] The purified fully methylated glycan was dissolved in 20 μl of a 50:50 methanol / water solution. 2 μl of undiluted and half-diluted N-glycan were mixed with 2 μl of 2,5DHB (LaserBiolabs) matrix solution (10 mg / ml, 50:50 methanol / water solution). Positive ion reflectance MALDI-TOF mass spectra were acquired using an Autoflex III mass spectrometer (Bruker). Spectra were obtained through accumulation of 4000 shots and calibrated using an external standard (Pepmix4 LaserBiolabs). The accelerating and reflecting electrode voltage conditions were set as follows: voltage 10.3 × 1954 V, 80% laser irradiance.

[0540] 8.11.6 Purification of affinity-tagged heterologous Gnt for in vitro activity assays

[0541] The HA purification procedure is shown below. First, intracellular expression analysis of the target HA-tagged Gnt enzyme was performed on recombinant cell cultures expressing heterologous Gnt. 1 × 10⁻⁶ cells were harvested. 9 Cells were centrifuged (2000×g / 5min) to separate cell pellet particles for affinity purification. The particles were resuspended in 1 mL of extraction buffer containing 1% (v / v) Triton (25 mM Tris pH 7.5, 100 mM NaCl, 1% v / v Triton X-100, EDTA-free protease inhibitor [Roche], 1 mM PMSF). The resuspended cells were sonicated on ice to avoid any kind of overheating! Sonication was performed for 7 cycles at 3 steps, 20 seconds, 70% power input [Bandelin Sonopuls]. The cells were then vigorously mixed for 10 seconds on a vortex mixer. The cell suspension was centrifuged at 13000×g for 1 h at 4°C. The supernatant (lysis buffer) was carefully removed and used for purification. The remaining solid fraction was resuspended in 1×Laemmli and used for SDS-PAGE analysis. The lysis buffer was mixed with cold PBS (containing 1× protease inhibitor tablets) at a 1:2 ratio and incubated with 100 μL of anti-HA magnetic beads [Thermo Scientific] at RT, 600 rpm for 30 min. HA-magnetic beads were harvested in 2 mL tubes using a magnetic rack [Thermo Scientific] and washed twice with 2 mL of ice-cold TBS. Elution was performed with 70 μL of elution buffer (2 mg / mL HA peptide [Thermo Scientific] in TBS). The eluted fraction was analyzed on SDS-PAGE, followed by anti-HA Western blotting and in vitro activity assays.

[0542] The StrepTactin purification procedure is shown below: Intracellular expression analysis of the target Strep-tagged Gnt enzyme was performed on recombinant cell cultures expressing heterologous Gnt. 1 × 10⁻⁶ cells were harvested. 9Centrifuge cells (2000×g / 5min) to separate cell pellet particles for affinity purification. Resuspend the particles in 1mL extraction buffer containing 1% (v / v) Triton. Sonicate the resuspended cells on ice to avoid any kind of overheating! Perform sonication [Bandelin Sonopuls] for 7 cycles at 3 steps, 20 seconds, 70% power input. Vortex vigorously for 10 seconds. Centrifuge the cell suspension at 13000×g for 1 h at 4°C. Carefully remove the supernatant (lysis buffer) and use it for purification. Resuspend the remaining solid fraction in 1× Laemmli and use it for SDS-PAGE analysis. Mix the lysis buffer with cold PBS (containing 1× protease inhibitor tablet) at a 1:5 ratio and incubate with 100μL StrepTactin sepharose [VWR] at RT, 600rpm for 30 min. A 5-fold dilution was performed in PBS to reduce the interference of cell / culture medium biotin on the purification efficiency of StrepTactin. StrepTactin sepharose was collected in 2 mL tubes by centrifugation (2000 × g / 1 min) and washed twice with 2 mL of cold TBS. Elution was performed with 70 μL of elution buffer (2.5 mM dethiobiotin in 1 × TBS). The eluted fraction was analyzed on SDS-PAGE, followed by anti-Strep Western blotting and in vitro activity assays.

[0543] 8.11.7 In vitro glycosyltransferase activity assay, 2-aminobenzamide (2AB) labeling and / or polysaccharide washing and HPLC separation

[0544] To determine the in vitro activity of HA-purified or streptavidin-purified GNTs, use freshly prepared reagents added in the following order to a 1.5 ml tube on ice: 1.50 ng of a suitable 2AB-labeled receptor glycan (e.g., 2AB-Man3a [Prozymes]) or 500 ng of a suitable unlabeled receptor glycan (e.g., Man3a [Prozymes]); 10 mM MgCl2; 10 mM MnCl2; 1.2 mM activated sugar (e.g., UDP-GlcNAc or UDP-Gal [Sigma Aldrich]). Adjust the volume to 50 μl using TBS buffer (pH 8.0, 25 mM Tris, pH 7.5, 100 mM NaCl); add 20 μL of HA-enriched or streptavidin-enriched GNT enzyme, or, for the corresponding blank control, eluent only, water. Afterward, briefly centrifuge the mixture and incubate at RT, 600 rpm, o / n. For unlabeled substrates, the glycans were purified from the reaction mixture using a Sep-Pak C18 classic column [Waters] prior to HPLC run. The samples were labeled with 2-AB according to (Bigge et al., 1995). Glycan washing was performed using the paper disc method as described in (Merry et al., 2002). In the case of 2AB-labeled substrates, PTFE washing was performed prior to HPLC run for in vitro assays.

[0545] The in vitro reaction for sialyl transferase using 2AB standards (Luley-Goedl et al., 2016) comprised 4 μL of 2AB-G2 (substrate sugar) containing 40 ng; 20 μL of HA-enriched protein SiaT; 24 μL of 100 mM MES pH 6.5 (final concentration 50 mM MES pH 6.5); 1 μL of Triton X-100 solution (in 100 mM MES) to a final concentration of 0.1% (v / v) of Triton X-100; and 1 μL of 3.75 mM CMP-Neu5Ac (dissolved in dH2O) stock solution (final concentration 0.75 mM). The reaction was carried out at 37 °C for 12 h at 400 rpm, cooled at 4 °C, and frozen at -20 °C until PTFE washing and HPLC analysis. MabThera was used as a substrate for further reactions.

[0546] The in vitro activity assay for sialyl transferase using MabThera antibody employed 50 μg (5 μL) of commercially available MabThera (Roche, in formulation buffer), 20 μL of HA-enriched protein SiaT (containing 40 μg HA peptide, in 100 mM MES, derived from elution buffer); 22 μL of 100 mM MES pH 6.5 (final concentration ~100 mM MES pH 6.5); and 1 μL of Triton X-100 solution (in 100 mM MES) to a final concentration of 0.1% (v / v). Initially, 1.5 mM CMP-Neu5Ac was added, and after 6 h, an additional 1.5 mM CMP-Neu5Ac enhancer was added to obtain a final concentration of 3 mM CMP-Neu5Ac. The reaction was carried out at 37 °C for 12 h at 400 rpm, cooled at 4 °C and frozen at -20 °C until N-glycans were released by PNGaseF and labeled by RF-MS (N-glycans were labeled with Rapifluor (RF) and MS according to 6.11.4).

[0547] Based on (Royle et al., 2002), but modified to a three-solvent system, using a GlycoSep-N normal-phase column, the separation of 2-AB-labeled glycans was performed by HPLC. Solvent A was a solution of 10 mM ammonium formate (pH 4.4) in 80% acetonitrile. Solvent B was a solution of 30 mM ammonium formate (pH 4.4) in 40% acetonitrile. Solvent C was 0.5% formic acid. The column temperature was 30 °C, and 2-AB-labeled glycans were detected by fluorescence (λex = 330 nm, λem = 420 nm). The gradient conditions were a linear gradient from 100% A to 100% B at a flow rate of 0.4 mL / min over 160 min, followed by a gradient from 100% B to 100% C over 2 min, with the flow rate increased to 1 mL / min. The column was washed with 100% C for 5 min, then returned to 100% A within 2 min and run at 100% A and 1 ml / min for 15 min. The flow rate was then restored to 0.4 ml / min for 5 min. The sample was injected into water.

[0548] 8.11.8 Expression and purification of rhEPO from unengineered and glycoengineered cell lines

[0549] For both strains, 2 × 200 ml were cultured at 26 °C and grown in BHIH with shaking at 140 rpm for 72 h. The culture was harvested and centrifuged at 2000 × g for 45 min. The SN was harvested for ammonium sulfate precipitation. Briefly, the SN was precipitated at RT for 45 min by passing (NH4)2SO4 (40% w / v) at 4 °C before centrifugation at 11000 × g for 1 h. The SN was removed and resuspended, and the remaining brown / black particles were dissolved in 50 ml of 1 × PBS pH 7.4. Dialyzed at 4 °C with 5 L PBS for 2 h and 5 L dialysis o / n. 50 ml of the dialyzed sample was loaded into a 6 ml StrepTactin column. Washed with 20 CV 1 × PBS pH 7.4 at 0.5 ml / min. The protein was eluted in 1 ml fractions with 10 CV 2.5 mM dethiobiotin in 1 × PBS. The mixture was collected and precipitated at -20°C with 4.5 mL of cold acetone (-20°C) added to the eluent. The sample was centrifuged at 8000 g for 2 h, and the precipitate particles were dissolved in 500 μL ddH₂O. 8.11.9 Determination of N-glycosylation site occupancy and site-specific glycosylation

[0550] (a) Peptide mass fingerprint of rhEPO

[0551] Will SF was used to enhance the enzymatic digestion of protein in 50 mM ammonium bicarbonate. 180 μg of protein, corresponding to 250 μL of each sample, was prepared by Vivaspin 500PES (5 kDa) and four centrifugations (15000 × g; 10 min, 4 °C) in 50 mM ammonium bicarbonate buffer, pH 8. The final protein concentration was approximately 3 mg / mL.

[0552] according to The SF procedure (WATERS) involves placing the protein at 0.1%. Denaturation in SF, reduction by DTT (5 mM) at 60 °C for 45 min, and alkylation by iodoacetamide (15 mM) at room temperature in the dark for 45 min. The sample was incubated at 37 °C with 6.4 μg trypsin / Lys-C mixture (mass spectrometry grade (PROMEGA V507A#201254)) for 16 h to obtain the digested protein. Hydrolytic surfactants were removed by adding 0.5% TFA to the digested protein sample and incubating at 37 °C for 30 min, followed by centrifugation at 15000 g for 10 min. All trypsin peptides were purified in a solution of 0.1% TFA in water using SEP Pack C18, 200 mg (Waters). The elution volume (2 mL 70% acetonitrile, 0.1% TFA) was completely lyophilized.

[0553] (b) Glycopeptide enrichment methods

[0554] The peptide was dissolved in 40 μL of ultrapure water, and according to The glycopeptide enrichment kit specification 72103-3 (Novagen) specifies that 30 μl should be used for glycopeptide enrichment.

[0555] Add 30 μL of sample to 150 μL of ZIC Glycocapture resin and collect the flow-through fraction containing unbound peptides. Elute the glycopeptides with 225 μL of ZIC elution buffer. Allow the flow-through fraction and eluted sample to evaporate completely in a speed vac. Resuspend the glycopeptides in 15 μL of MilliQ water. After purification on a Zip Tips MillIPORE C18 to improve the signal-to-noise ratio, dry the glycopeptide solution in 50% methanol on a Maldi plate and add 1 μL of CHCA (LaserBiolabs) matrix solution (7 mg / mL in 50% acetonitrile).

[0556] (c) Deglycosylation of the enriched portion

[0557] 5 μL of the suspended glycopeptide was adjusted to 50 mM sodium acetate buffer, and 1 μL of PNGase F (Promega V483A#226517) and 1 μL of PNGase A (Sigma#01000353) were added for deglycosylation over 16 hours at 37 °C. After preparation on a Zip Tips MILLIPORE C18 (SOP P17 / 2), the deglycosylated peptide was mixed with CHCA (1:1) (LaserBiolabs) matrix solution (7 mg / mL in 50:50 acetonitrile / water, 0.1% TFA).

[0558] The flow fraction was prepared and analyzed using CHCA (1:1) (LaserBiolabs) matrix solution (7 mg / mL, in 50:50 acetonitrile / water, 0.1% TFA).

[0559] (d) MALDI-TOF MS analysis

[0560] Peptides, deglycosylated glycopeptides, and occupied glycopeptides in the flow-through fraction were analyzed by MALDI-TOF MS in positive ion reflectance and linear modes. Linear model MALDI mass spectra were acquired on a MALDI-TOF / TOF Autoflex III (Bruker Daltonics) spectrometer under the following conditions: 14.3 × 2904 V, 49% laser, 6000 shots. Positive ion reflectance MALDI mass spectra were also acquired on a MALDI-TOF / TOF Autoflex III (Bruker Daltonics) spectrometer (SOP 44 / 1) using the RP850-3500 Bruker method under the following conditions: 40 × 2160 V, 97% laser, 5000 shots.

[0561] 8.11.10 Peptide profiles for the identification of secreted proteins from the lizard leishmania (Leishmania tarentolae)

[0562] St10569 cells were cultured at 26°C with shaking (140 rpm) in 400 ml BHIH for 2 days until the OD reached 3.4. The supernatant was harvested by centrifugation at 2000×g and stored at -80°C until use. SN was thawed at 4°C and incubated on RT with a mixture of protease inhibitor tablets (Roche, EDTA-free) for 4 h.

[0563] Ammonium sulfate (NH4)2SO4 precipitation was performed to precipitate secreted proteins. Briefly, 40 g of (NH4)2SO4 was added stepwise to SN (initial percentage concentration 10%). After adding 160 g, the sample became turbid and a final 40 g was added. The sample was incubated at RT with stirring for 40 min. Centrifuged at 11000×g for 30 min at 4 °C. The precipitated particles were then placed in 20 ml of 1× binding buffer (1×TBS + 1 mM CaCl2 + 1 mM MnCl2) (“1 stResuspended in SN. Centrifuged at 4000×g for 15 min at 4 °C, the particles contained only protein and no BHIH residue. Small black particles were observed and resuspended in 10 ml of 1× binding buffer (1×TBS + 1 mM CaCl2 + 1 mM MnCl2). The SN and resuspended particles were dialyzed in 1×TBS at 12-14 K MWCO at 4 °C to remove (NH4)2SO4. The buffer was replaced with freshly prepared 1×TBS and dialyzed again at 12-14 K MWCO at 4 °C for 2.5 h. A 0.7 ml ConA column was used to prepare a bead-to-sample ratio of 1:50 (1.4 ml slurry). 1 mM CaCl2 + 1 mM... MnCl2 was added to the sample of the second SN and the volume was filled to 35 ml with 1× binding buffer for “resuspending particles” sample. ConA slurry was added to the sample and rotated at RT for 4 h. For all the following steps, centrifugation was performed at 1000×g for 5 min at RT. The flow-through fraction (FT) was collected and 100 μl was taken for SDS PAGE. The slurry was washed 4× with 2CV binding buffer and the washed fraction was collected. Protein was eluted with 6CV, of which 1CV (700 μl) was used. The first two eluted fractions were incubated in elution buffer (Vectorlab) for 10 min.

[0564] Peptide profiling was performed using the following procedure: 5 μl of sample was mixed with 40 μl of buffer (10 mM Tris / 2 mM CaCl2, pH 8.2) and 5 μl of trypsin (100 ng / μl in 10 mM HCl), and microwaved at 60 °C for 30 min. The sample was then dried, dissolved in 20 μl of 0.1% formic acid, diluted 1:2 with 0.1% FA, and transferred to an autosampler vial for LC / MS / MS. 1 μl of sample was injected for measurement.

[0565] Using Mascot(swiss prot and uniref(all species)), the database of the following FASTA files is searched in the search program.

[0566] TriTrypDB-26_LmajorFriedlin_AnnotatedProteins.fasta

[0567] TriTrypDB-26_LtarentolaeParrotTarII_AnnotatedProteins.fasta

[0568] In addition, a search was performed on conventional databases containing proteomes of known Leishmania and Trypanosoma species:

[0569] Uniprot: Trypanosoma brucei brucei (strain 927 / 4GUTat10.1); Trypanosoma cruzi; Trypanosoma cruzi marinkellei; Trypanosoma vivax (strain Y486); Trypanosoma cruzi Dm28c; Trypanosoma rangeli SC58; Trypanosoma congolense (strain IL3000); Trypanosoma cruzi (strain CL Brener); Trypanosoma brucei gambiense (strain MHOM / CI / 86 / DAL972); Leishmania infantum; Leishmania major; Leishmania brasiliensis Leishmania braziliensis; Leishmania mexicana (strain MHOM / GT / 2001 / U1103); Leishmania donovani (strain BPK282A1).

[0570] TriTrypDB (http: / / tritrypdb.org / tritrypdb / ): Leishmania major friedlin; Leishmania tarentolae Parrot Tar II; Trypanosoma brucei Lister 427; Trypanosoma cruzi Dm28c.

[0571] Total protein: 155,504

[0572] The search results are aggregated using Scaffold (if present, trypsin, keratin, other common contaminants, and decoy hits are hidden).

[0573] 8.11.11 N-terminal sequencing for identifying signal peptides

[0574] For N-terminal sequencing of signal peptides degraded by EDMAN, Con A-enriched secreted proteins were further purified using a SEC 10 / 300 Superdex GL. To do this, 24 mL of CV Con A elution fraction was mixed and concentrated to 500 μL in 1×PBS pH 7.4 using a 3K MWCO Amicon filter. An SEC column was loaded at a flow rate of 0.5 mL / min, and aliquots were collected in 0.5 mL volumes. Several peaks observed at very low mA were observed and mixed. Mixing was performed in a manner that allowed for the separation of different peaks / proteins. Ultracentrifugation was performed using an Amicon 3K MWCO to concentrate the sample volume. The sample was loaded onto an SDS-PAGE plate and blotted on a PDVF membrane, stained with Ponceau S, and the protein bands were cut and sequenced using EDMAN sequencing.

[0575] 8.11.12 Evaluation by rhEPO expression and secretion of different signal peptides

[0576] Recombinant cell lines St 11376 and St 11377 were grown at 26°C, 140 rpm, and a 1:40 ratio in 5 × 400 ml BHIH for 3 days (72 h). The supernatant was harvested after centrifugation at 2000 × g for 40 min. The supernatant corresponding to 15 OD was used for trichloroacetic acid (TCA) precipitation as follows: The supernatant was cooled on ice, 100% TCA was added to a final concentration of 10%, and the mixture was incubated on ice for 15–30 min. It was then centrifuged at 11000 × g for 30 min at 4°C, washed with 1 mL of ice-cold acetone, and centrifuged again at 11000 × g for 30 min at 4°C. The precipitate particles were resuspended in 1 × Laemmli (30 μL) and the pH was adjusted by adding 5 μL of 1 M TrisHCl (pH 8.0) (the color indicator changed from yellow to blue). The supernatant containing the secreted Strep-tagged rhEPO TCA precipitate was used for Western blotting and Coomassie Brilliant Blue analysis. 15 OD SN TCA was loaded and run on a 10% Bis-Tris gel at 200 V using MOPS run buffer for 60 min. For anti-Strep immunoblotting and Coomassie Brilliant Blue staining, 2 μg Darpin LL was used as a loading control. The gel was blotted at 20–25 V using iblot. TM The membrane was blotted with (Invitrogen) P0 and then blocked in 10% milk powder. Detection was performed for 1 h at 30°C using a polyclonal anti-Strep-tag II antibody (rabbit, Abcam ab76949) at a dilution of 1:1000, followed by detection for 1 h at 30°C using a secondary antibody, goat anti-rabbit HRP (Biorad, 170-6515). TMB was used for colorimetric detection. Coomassie brilliant blue staining was performed by staining with o / n and destaining with ddH2O.

[0577] 8.11.13 Bioinformatics prediction and hybrid design of CTS regions of Golgi localization proteins

[0578] The method used for TM and signal peptide prediction is: 1) http: / / phobius.sbc.su.se / Phobius( 2004), A Combined Transmembrane Topology and Signal Peptide Prediction Method; 2) HMM-based prediction of signal peptides and transmembrane topology (claiming to have a lower false positive rate than TMHMM and SignalP); and 3) SignalP. HMM was trained on four classes of proteins: transmembrane proteins with / without signal peptides, and non-transmembrane proteins with / without signal peptides.

[0579] 8.11.14 Purification of affinity-tagged heterologous Gnt for subcellular isolation

[0580] Harvest cells expressing HA- or Strep-tagged GNT enzymes (1 × 10⁻⁶) 9The supernatant (corresponding to 50 OD) was centrifuged (4000×g / 10 min) to separate the SN from the cell particles. As shown below, the supernatant (corresponding to 5 or 10 OD) was used for TCA precipitation: the supernatant was cooled on ice, 100% TCA was added to a final concentration of 10%, and incubated on ice for 15–30 min. The mixture was then centrifuged at 11000×g for 30 min at 4 °C, washed with 1 mL of ice-cold acetone, and centrifuged again at 11000×g for 30 min at 4 °C. The precipitate particles were resuspended in 1×Laemmli (~30 μL) and 5 μL of 1M TrisHCl (pH 8.0) was added to adjust the pH (the color indicator changed from yellow to blue). The supernatant from the TCA precipitation was used for Western blotting analysis. Resuspend the particles in 1 mL of extraction buffer (ExB, 125 mM Tris pH 7.5, 100 mM NaCl, EDTA-free protease inhibitor [Roche], 1 mM PMSF) with or without 1% (v / v) Triton X-100. Sonicate the resuspended cells on ice [Bandelin Sonopuls] to avoid any kind of overheating. Sonicate for 7 cycles at 70% power input for 10 seconds, followed by vigorous mixing on a vortex mixer for 10 seconds. Rotate the lysed cell suspension at 13000 × g for 1 h at 4 °C. Carefully remove the supernatant (lysis buffer) and use it as a direct loading for immunoblotting analysis (with 1 × Laemmli). Using a cell homogenizer [Sigma-Aldrich], the remaining particles (+ / - Triton X) were dissolved in 500 μL of ExB supplemented with 1% (v / v) Triton X-100 for better homogenization. The cell suspension was then centrifuged at 13000 × g for 1 h at 4 °C. The soluble fraction (with 1 × Laemmli) was used as a direct loading for Western blotting analysis (referred to as the "insoluble fraction"). Optionally, the remaining particles (referred to as cell debris) were resuspended in 1 × Laemmli and used for Western blotting analysis. After Western blotting with an appropriate detection antibody, all fractions were analyzed on SDS-PAGE. The membrane binding of Gnt can be interpreted based on the presence and intensity of Western blotting bands in different fractions (e.g., supernatant, lysis buffer + / - Triton X, cell debris).

[0581] The SDS-PAGE conditions are as follows: Heat the sample in 1×Laemmli dye solution to 95°C for 10 min; run the 10% Bis-Tris gel at 200V for 60 min using MOPS buffer. Then, run the gel at 20-25V using iblot. TMThe membrane was blotted with (Invitrogen) P0 and then blocked in 10% milk powder. Detection was performed at 30°C for 1 h using a 1:1000 dilution of polyclonal anti-Strep-tag II antibody (rabbit, Abcam ab76949) or a 1:1000 dilution of rabbit anti-HA-tag antibody (Sigma, H6908). Detection was then performed at 30°C for 1 h using a 1:1000 or 1:2000 dilution of a secondary antibody, goat anti-rabbit IgG HRP (Biorad, 170-6515). TMB was used for colorimetric detection.

[0582] 8.12 Expression, purification, and analysis of anti-CD20 rituximab in Leishmania tarentolae (L. tarentolae)

[0583] (a) Expression

[0584] Five 1000 mL cultures of St12427 (p13, p5026) were incubated at 26 °C in BHIH with shaking at 140 rpm for 72 h. The cultures were harvested and centrifuged at 2000 × g for 15 min. Four tablets of a protease inhibitor mixture (Roche) and EDTA (1 mM final concentration) were added to 5000 mL of medium SN. The 5000 mL medium SN was filtered through a 0.22 μm filter before concentration with Vivaflow 200 (50000 Da). Vivaflow was blocked with 100 mL of BSA (2 g / L), and the membrane was washed with 1 L of ddH2O after blocking. The SN was concentrated 5-fold to 1000 mL at a Vivaflow flow rate of 600 mL / min.

[0585] (b) Purification

[0586] At 4°C, the culture medium was continuously loaded offline overnight into a 10 mL protein A column using a peristaltic pump. The column was connected to an NGC system and washed with 8 CV 1×PBS pH 7.4. Elution was performed with 0.15 M glycine pH 2.5 and neutralized directly by adding 150 μL 1 M Tris pH 8.5. The mixture containing rituximab (10 mL) was adjusted with 0.7 g (NH4)2SO4 and loaded into 1 mL HIC (PhenylSepharose GE) at a flow rate of 1 mL / min. Elution was performed with a gradient of buffer A 20 mM Tris, 1 M (NH4)2SO4 pH 7.0, and buffer B 20 mM Tris pH 7.0. A 1000 μL fraction was collected and analyzed by SDSPAGE and Western blotting stained with Coomassie Brilliant Blue.

[0587] (c) Analysis

[0588] SDS-PAGE and capillary gel electrophoresis:Under reducing or non-reducing conditions, 10 μg was used for Coomassie Brilliant Blue staining and 2.5 μg for Western blotting. Separation was performed on a 4–12% gel using MOPS buffer and SDS-PAGE for 55 min. Protein purity was determined by SDS-PAGE with Coomassie Brilliant Blue staining using 10 μg protein samples and compared with a BSA standard curve. Impurities were quantified using ImageQuant. Capillary gel electrophoresis (CGE) was performed using the Agilent Protein 230 kit (5067-1518) according to protocol.

[0589] Analytical SEC: MAbPac SEC-1 (4×300mm) is a size exclusion chromatography (SEC) column designed for the separation and identification of monoclonal antibodies (mAbs) and used according to the manufacturer's recommendations (temperature: 30°C; eluent: PBS 50mM NaPO4, 300mM NaCl pH 6.8; elution: isocratic, 30 min; flow rate: 0.2 mL / min; detection: 215 nm; injection volume: 5 μL, corresponding to 5 μg of protein).

[0590] 8.12.2 N-glycan profiles of monoclonal antibody samples

[0591] (a) Glycosidase digestion and full methylation of N-glycans:

[0592] IgG (200 μg / 40 μl) was suspended in 200 μl of 50 mM phosphate buffer (pH 7.5) and denatured for 10 min at 90 °C in 0.5% sodium dodecyl sulfate (SDS Sigma L4509) and 1% β-mercaptoethanol. After adding 22 μl of NP40 to a final reaction volume of 484 μl, the protein was deglycosylated by enzymatic digestion at 37 °C in phosphate buffer (pH 7.5) for 15 h using 15 units of PNGase F (PROMEGA#226517ref V483A). Deglycosylation was controlled by electrophoresis. During the 10 min interval, 6.5 μl of the pre- and post-deglycosylated samples were heated at 70 °C for 10 min and loaded onto... MES buffer (Invitrogen NP0002) + antioxidant Novex Bis-Tris 4-12% gel, 1.0 mm, 12 w (Invitrogen). Run at constant 200 V for 35 min. During 1 H, the gel was stained with Simply Blue Safestain (Invitrogen) and destained in water.

[0593] N-glycans were purified on Hypercarb Hypersep 200 mg (Thermo Fisher). SPE was equilibrated with 0.1% TFA and washed with 0.1% TFA before loading N-glycans released by PNGase. After elution with 3 mL of 25% acetonitrile and 0.1% TFA, the N-glycans were lyophilized before full methylation. Full methylation of the lyophilized sample was performed over 40 min using approximately 25 mg of sodium hydroxide, 500 μl of DMSO, and 300 μl of ICH3. After quenching the reaction with 1 mL of water, 3 × 500 μl of chloroform was used for the extraction of the fully methylated glycans. The chloroform phase was washed with an equal volume of water and then dried. The reaction product was loaded onto C18SepPak 200 mg (WATERS) and eluted with 2 mL of 80% acetonitrile, and lyophilized before MALDI-TOF MS analysis.

[0594] 8.12.3 MALDI-TOF analysis of N-glycans:

[0595] The purified fully methylated glycan was dissolved in 20 μl of a 50:50 methanol / water solution. 2 μl of undiluted and half-diluted N-glycan were spotted with 1 μl of CHCA (LaserBiolabs) matrix solution (7 mg / ml, 50:50 acetonitrile / water). Positive ion reflectance MALDI-TOF mass spectra were acquired using an Autoflex III mass spectrometer (Bruker). Spectra were obtained through accumulation of 4000 shots and calibrated using an external standard (Pepmix4 LaserBiolabs). The accelerating and reflecting electrode voltage conditions were set as follows: voltage 14.7 × 2008 V, 80% laser irradiance.

[0596] The EXPAZY GlycoMod tool was used to determine the monoisotopic mass [M+Na]. ...

Claims

1. A Leishmania host cell comprising: a. a recombinant nucleic acid encoding a target protein; and b. Recombinant nucleic acid encoding a heterologous glycosyltransferase.

2. The host cell according to claim 1, wherein the heterologous glycosyltransferase is N-acetylglucosamine transferase; and / or a heterologous galactosyltransferase; and / or a heterologous sialyltransferase.

3. The host cell of claim 2, wherein the host cell comprises two or more N-acetylglucosamine transferases; and / or two or more heterologous galactosyltransferases; and / or two or more heterologous sialyltransferases.

4. The host cell of any one of claims 1 to 3, wherein the host cell comprising a heterologous sialyltransferase further comprises a heterologous CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc.

5. The host cell according to any one of claims 1 to 4, wherein one or more endogenous enzymes from the N-glycan biosynthetic pathway have been deleted, mutated and / or functionally inactivated.

6. The host cell according to any one of claims 1 to 4, wherein the Leishmania cell is a Leishmania tarentolae cell.

7. according to the host cell described in any one in claim 2 to 6, the amino acid sequence of wherein said N-acetylglucosamine transferase, galactosyltransferase and / or sialyltransferase is derived from N-acetylglucosamine transferase, galactosyltransferase or sialyltransferase listed in Table 9, or its functional homologue, isoform or variant.

8. The host cell of any one of claims 1 to 7, wherein a Leishmania signal and / or retention sequence is added to the N-acetylglucosamine transferase, galactosyltransferase and / or sialyltransferase, wherein the signal sequence targets the N-acetylglucosamine transferase, galactosyltransferase and / or sialyltransferase to the endoplasmic reticulum of a Leishmania host cell, and wherein the retention sequence retains the N-acetylglucosamine transferase, galactosyltransferase and / or sialyltransferase in the Golgi apparatus.

9. The host cell of any one of claims 4 to 8, wherein the CMP-Sia biosynthetic pathway protein capable of producing CMP-NeuAc is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CMP-Sia biosynthetic pathway proteins listed in Table 11 or is any functional homolog, isoform or variant thereof.

10. The host cell according to any one of claims 1 to 9, wherein the N-acetylglucosamine transferase is GnT-I.

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