Modified and fully functionally customized glycoproteins

Abstract

To provide a therapeutic agent having extremely uniform humanized glycosylation. [Solution] A composition of a glycosylated target protein produced by a method for producing a glycosylated target protein is provided, wherein the method comprises (i) culturing leishmania host cells and (ii) purifying the target protein from the culture, wherein the leishmania host cells contain recombinant nucleic acids encoding the target protein, and the target protein is an antibody.

Description

[Technical Field]

[0001] (1. Cross-referencing of related applications) This application, filed on June 30, 2017, is incorporated herein by reference in its entirety. We claim the benefit of priority from the pending U.S. Provisional Application No. 62 / 527,466.

[0002] (2. Sequence Listing) This application is filed in ASCII format and is fully incorporated herein by reference. This contains a list. This ASCII copy, created on June 28, 2018, is 14197-005-228_ST25_FIN It is named AL.txt and has a size of 1,065,529 bytes.

[0003] (3. Introduction) This specification describes the production of glycosylated proteins in vitro and in vivo. This is a composition and method for producing glycosylated proteins using host cells. This includes producing. Also described herein are products produced by such methods. This concerns glycosylated proteins and their use. [Background technology]

[0004] (4.Background) Protein glycosylation is a ubiquitous post-translational modification found in all domains of living organisms. The animal system exhibits remarkable complexity, and the glycan structure is a protein structure. From their contributions to folding and quality control, the recognition, stability, action, and Extremely important biological and physiological aspects, including involvement in numerous biological events such as metabolic turnover. It plays a key role (Moremen et al., 2012). Monoclonal antibodies, enzymes, and hormones Such therapeutic glycoproteins are a major product of the biotechnology industry (Lagasse, HA Daniel et al. (2017; Dimitrov (2012)) stated that the effect of glycan heterogeneity is "important product It is increasingly recognized as a "quality characteristic." Of the many characteristics that determine the quality of a product, Griffin is one of them. Cosylation affects the biological activity, serum half-life, and immunogenicity of proteins: It is even considered one of the important properties. Glycans are involved in increasing serum circulating time. Many approved or investigational biologics are designed to maintain therapeutic concentrations over extended periods. It suffers from an insufficient half-life that requires frequent application. Half-life extension strategies are needed to improve This is key to enabling the creation of long-acting therapeutic drugs with controlled pharmacokinetics (Konterm). (Ann, 2016). Glycosylation, for example, reduces the tendency of proteins to aggregate. It appears to improve solubility and stability, and increases cyclic life by preventing protein degradation. Furthermore, N-glycans with different terminal monosaccharides cause recti, which leads to their breakdown. It can be recognized by (Blasko et al., 2013; Varki, 2017). Results Therefore, monitoring and controlling glycosylation is essential for the manufacturing and regulatory authorities of biopharmaceuticals. This is extremely important in this matter (Costa et al., 2014; Eon-Duval et al., 2012; Reusch (and Tejada literature, 2015). For these reasons, glycosylation of the expression platform is It is increasingly being recognized as an important strategy for improving iodine-based pharmaceuticals in many ways (Dicke (r and Strasser, 2015).

[0005] Most protein drugs involve the addition of glycan structures, which affects the properties of the therapeutic protein. Therefore, it is glycosylated. Generally, the number and composition of glycans are related to the protein. Glycans play a crucial role in loading, solubility, and intracellular transport. By blocking the cellular recognition event, immunogenic reactions can be prevented, and different cell recognition events can lead to specific glycans. It depends on the presence of a can structure. Therefore, glycosylation is related to the biological activity of glycoproteins. It has a significant impact on and is carefully controlled during manufacturing to achieve therapeutic efficacy. It is necessary to do so depending on the species, cell type, and physiological state of the host organism that produces the recombinant glycoprotein. The glycosylation patterns shown above are remarkably different. Glycans on proteins are generally structured They are extremely diverse and consist of a series of monosaccharides constructed by various bonds. Even though mature glycans at one glycosylation site are as simple as single sugars, More than 200 sugars potentially modified with phosphate, sulfate, acetate, or phosphorylcholine It may be as complex as a rimer (Stanley, 2011). It is estimated that approximately 700 types of proteins are needed to create sufficient diversity (7,000 It is estimated that there are more than 0 types of structures, but this consists of only 10 types of monosaccharides: fucose (Fuc), galac Tose (Gal), glucose (Glc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine Samine (GlcNAc), glucuronic acid (GlcA), isolonic acid (IdoA), mannose (Man), sialic acid ( It is constructed from sia and xylose (Xyl) (Moremen et al., 2012).

[0006] (Antibody therapy drugs) Antibodies (or immunoglobulins, Igs), particularly IgG antibodies, are part of the most successful therapeutic agents developed in the past decade (e.g., bevacizumab, rituximab, infliximab, a dalimumab, trastuzumab, or cetuximab, among many others). These are highly specific and have a long serum half-life, and they can be expressed by conventional methods in mammalian cultures. The basic structure of an antibody molecule is constructed from two identical heavy polypeptide chains and two identical light polypeptide chains. These chains are linked by disulfide bonds that form a "Y" shaped structure. Human immunoglobulins are classified into five classes, IgG, IgA, IgD, IgE, and IgM, by their heavy chains. IgG and IgA antibodies are found as four subclasses (IgG1 - 4) and two subclasses (IgA1 - 2), respectively. Recognition of a specific antigen is mediated by the antigen-binding fragment (Fab) that includes the variable regions of the light and heavy chains and one constant domain. Effector functions (Figure 1) are initiated by the binding of the fragment crystallizable region (Fc), which corresponds to the other two domains (CH2 and CH3) of the constant region of the heavy chain, to effector proteins such as Fc receptors (FcR). Thus, the Fab fragment is composed of the variable and constant domains of the light and heavy chains, while the Fc fragment is composed entirely of the constant domains of the heavy chain. This Fc domain also

[0007] For example, by changing its binding properties to the ligand or Fc receptor, Alternatively, by further extending its half-life, the clinical success of therapeutic antibodies can be further advanced. To achieve this, antibody engineering techniques are used. A typical technique involves mutation This involves the introduction of a mutation or modification of the glycosylation of the antibody. The introduction of a mutation into the Fc chain is performed in the natural sequence. It has the unique drawback that it no longer functions. Glycosylation of therapeutic proteins is common While this is generally tolerated to extend the circulating half-life, in the case of antibodies, it involves the elimination of Ig from circulation. Previous studies on the effect of glycosylation on velocity have not yielded conclusive results. Furthermore, a more recent understanding suggests that differences in the glycan structure of the Fc portion actually affect the clearance. This has been suggested (Millward et al., 2008; Liu, 2015, 2017). Antibody chain During post-translational modification, enzymes in the endoplasmic reticulum and Golgi apparatus modify the carbohydrate chain of the antibody polypeptide. It can be added to the skeleton. The asparagine at position 297 of the Fc portion of all IgG subclasses can be added. One N-linked glycan is present. Approximately 20% of IgG antibodies have glycans at other locations on the molecule. It contains. Most recombinant antibody drugs contain only a single Fc glycosylation site. It has been modified or selected.

[0008] When the antibody chain is correctly folded and associated, the oligosaccharide at position 297 is CH2 Isolated within an internal space enclosed by domains, the broad non- Covalent interactions exist, resulting in an influence on the three-dimensional structure. The oligosaccharides found at the Asn-297 site are typically fucosylated, branched-type complex oligosaccharides. However, between antibody molecules, there are changes in branching, chain length, and / or the number of carbohydrate moieties. Due to these changes, considerable heterogeneity exists in the carbohydrate structure (glycoform). The structure of the added N-linked oligosaccharide varies considerably depending on the degree of processing. Mannose, and the presence or absence of bisecting GlcNAc and core fucose residues. It can contain complex branched oligosaccharides (Wright and Morrison, 1997). Usually given There is heterogeneous processing of the core oligosaccharide structure added at the glycosylation site, This result shows that even noclonal antibodies exist as multiple glycoforms. Importantly, the main differences in antibody glycosylation occur between antibody-producing cell lines, and these differences are distinct. This can also be observed in a given cell line grown under the same culture conditions. In fact, mammalian N-glyca Each step in enzyme biosynthesis is less than 100% efficient, and some enzymes are involved in the process of transporting the substrate. This competition leads to the production of many different glycoforms in therapeutic protein production. Heterogeneous glycosylation that causes problems is observed. For example, glycans have pharmacokinetic and It can affect biological activity (Ferrara et al., 2011; Elliott et al.) , 2003; Krapp et al., 2003). N-glycans are important for protein folding. This often happens, so either completely remove the N-glycosylation site, or remove it in front of or near the endoplasmic reticulum. These problems can be easily overcome by inhibiting glycosylation within the cytoplasm. No. However, antibodies require N-glycans for proper folding. However (Feige et al., 2010), the problem with glycoforms is that different or inconsistent ef This results in an inhibitor function, which makes it difficult to use antibodies due to therapeutic or regulatory requirements. This can be achieved. By deglycosylating the Fc portion with N-297, the effect of the Fc-containing molecule can be reduced. Loss of function or decreased stability may occur. Importantly, this may be due to the absence of glycerides that are not synthesized in humans. Icoform is allergic and immunogenic, and can develop with repeated therapeutic administration. This can accelerate the plasma clearance of bound antibodies by anti-drug antibodies.

[0009] By modifying the Fc domain, efficacy is improved, therapeutic dosage is reduced, and the overall antibody... Improving clinical outcomes is the next challenge in the development of modified antibodies. cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP) Effector functions such as these, and their impact on the overall half-life of the molecule, are characteristics of antibody therapeutics. The main objective is to improve (Figure 1) (Jennewein and Alter, 2017). The overall market is It is expected to grow rapidly at a healthy annual growth rate of approximately 40% between 2016 and 2026. ru.

[0010] Based on the type of antibody they recognize, Fc receptors (FcRs) are classified into different types: Fc gamma FcγR receptor (which binds to IgG), Fcα receptor (which binds to IgA), Fc ipsilo They are classified into the FcεR receptor (which binds to IgE) and the neonatal Fc receptor (FcRn). Of these, The most important is the "Fc gamma" receptor. The Fc gamma receptor is opsonized micro It is involved in the activation of phagocytosis in organisms. Fc gamma receptors, as a result of their diverse molecular structures, are anti They are further classified into classes according to their affinity for the body. Some of these include FcγRI(C This includes D64), FcγRII (CD32), and FcγRIII (CD16). FcγRIII (CD16) is the most common Natural killer (NK) cells, granulocytes, monocytes, macrophages, and subsets of T cells It is located above. The gene encoding this receptor is FcγRIIIa or FcγRIIIb. RIIIa is an Fc receptor involved in NK cell-mediated ADCC. The Fc alpha receptor is involved in two cell pathways. It consists of external Ig-like domains and has only one subgroup called FcαRI(CD89). It forms parts of both the multi-chain immune recognition receptor (MIRR) and the Ig superfamily. Fc type Silon receptors consist of two types. One of these receptors is the C-type receptor, a low-affinity receptor. One is the cutin receptor FcεRII(CD23), and the other is the high-affinity receptor Ig superfamily. It is a type of FcεRI. Neonatal receptor (FcRn) prevents the lysosomal degradation of antibodies. It is known to play a role in increasing the half-life of therapeutic antibodies. FcRn within the cell binds to IgG that has been internalized by transcytosis. IgG then binds to the cell surface. It is recycled into the surface, released at the blood's pH, and inhibited from lysosomal degradation. Agricosyl The transformation has a significant impact on the effector function, but the interaction between IgG-Fc and FcRn is related to Fc glycation. This is thought to be unrelated to cosylation.

[0011] The glycan determines that the horseshoe-shaped Fc fragment "opens," and the cutting of the Fc glycan This results in a "closed" three-dimensional structure. The "open" IgG-Fc structure interacts with FcγRIII. This is the most advantageous three-dimensional structure, which is fully galactosylated but fucosylated. Observed in IgG-Fc (Krapp et al., 2003). Unique carbohydrate-carbohydrate interaction. This is required for high-affinity binding between FcγRIII and antibodies lacking core fucose. However, Furthermore, core fucosylation is hindered by "fucose collisions," which interfere with high-affinity FcγRIII binding. The fucose residues in the ligosaccharide portion facilitate the binding of therapeutic antibodies to FcγRIIIa (activation of the Fc receptor). It has been shown to cause physical impairment (Figure 2A). Defucosylation enhances ADCC, a function of the Fc receptor. It plays an important role in this. Importantly, (Chung et al., 2012) shows that % fucosylation This demonstrates a linear relationship between and ADCC. This shows that core fucose residues An example of the superior properties of a uniform glycan that does not possess is created. Afucosylated antibodies are α1,6 The core is a structure that inhibits core fucosyltransferase, leading to a decrease in fucosylation. It is produced in cell lines that overexpress GnT-III, which has bisecting GlcNAc added to β-Man. This decrease resulted in approximately 50% afucosylated structures, which are glycoprofiling This was also confirmed by Gazyvaro (registered trademark) (Figure 42).

[0012] To clarify the data so far, (Li et al., 2017; Chen et al., 2017) This new research uses a chemical enzymatic approach to produce a uniform N-glycan, and a 2,6-elongated type Even in the case of branched sialic acid, the FcR affinity is highly maintained, which is an effector like ADCC. —This demonstrated that it leads to improved function. Structures with or without α1,6-linked core fucose. Based on structurally clear and homogeneous glycans, this data demonstrates FcR binding and related downstream effects. This has significantly advanced our understanding of the subject (Li et al., 2017; Sondermann et al., 2013) (Figure 2B). Furthermore, the α2,3-linked sialic acid at the branched end of the N-glycan leads to an increase in FcγR affinity. Therefore, it is due to the lack of Fc receptor function in the activation pathway that inhibitory glialization occurs. It can be assumed that it functions as a can. Therefore, the sialic acid is at the end. This suggests that sialic acid-related effects on anti-inflammatory properties and an increase in the circulating half-life of antibodies are present. Long circulating half-life and (for example, the number of times anti-drug antibodies against potentially immunogenic therapeutic mAbs) It has improved properties such as immune tolerance (for avoidance), but heterogeneous glycosylation, or It is advantageous to obtain antibodies and Fc-containing molecules that do not have drawbacks such as reduced antigen binding. The functionally customized N-glycan is divided for the target downstream effector function. To help children develop.

[0013] Glycosylation processes in the endoplasmic reticulum (ER) and Golgi compartment of eukaryotic cells are involved in recombinant glycotanges. It generates the majority of the heterogeneous glycan structure found on the protein. Its importance in treatment is Because it is recognized, in order to overcome glycan heterogeneity and homogeneous therapeutic glycoproteins To establish in vivo and in vitro glycosylation technologies for the efficient production of glycans, Considerable effort has been made in recent years. By modifying the glycosylation pathway in several expression hosts, Despite progress in producing humanized glycans, there are drawbacks in terms of compatibility and productivity. This can result in loss, or even loss of viability. Importantly, it produces specific, uniform N-glycans. The in vivo system for serotherapy has not been described to date.

[0014] Therefore, having such a uniform and functionally customized specific glycosylation Expression techniques that produce such recombinant therapeutic proteins enhance biological activity It represents a new class of safe, innovative, and next-generation drugs with improved properties.

[0015] (N-glycosylation pathway) The most prominent and best-characterized form of protein glycosylation is glycosylation. The bond between the amide and the side chain of the newly synthesized asparagine protein (N-glycosyl N-glycosylation of proteins is a process in which a conserved molecule is constructed on a lipid carrier. Asn-X-Ser / Thr (here) is exposed on the nascent polypeptide chain of the ligozoxy precursor (Glc3Man9GlcNAc2). So, X is any amino acid other than proline) and by transfer to the consensus sequence, the vesicle It begins in the lumen of the body (ER). This initial glycan transfer reaction involves the heteromeric oligosaccharide. The transferase (OST) complex catalyzes the folding of proteins in the ER. It is supposed to happen before G (Aebi's literature, 2013). Immediately after oligosaccharide transfer, two The terminal glucose residues are cleaved by glucosidases I and II, resulting in Polypeptides having a monoglucosylated glycan structure (Glc1Man9GlcNAc2) are endogenous to the ER. Interaction with the membrane-bound lectin calnexin or its soluble homolog calreticulin. These lectins can act in glycan-dependent protein quality control. It supports protein folding in the kulve and acquires its native three-dimensional structure. The secreted glycoprotein is released from the calnexin / calreticulin cycle, E It exits R and enters the Golgi apparatus. In the Golgi apparatus, the folded glycoprotein undergoes maturation. The ER-derived oligomannose-type N-glycan above undergoes further N-glycan elongation, and as a result This results in the generation of highly diverse complex N-glycans (Stanley, 2011).

[0016] Insects, yeasts, and plants produce N-gluten which is remarkably different from that produced by mammalian cells. To generate lycans. From the first Glc2Man9GlcNAc2 oligosaccharide in the endoplasmic reticulum to Man8GlcNAc2. The processing of these yeasts shows remarkable similarities across species, from yeast to mammals, but Gol Very different processing events occur in di-isomers. For example, yeast has 50 or more Man residues 8-9 While GlcNAc2 can be attached to insect cells, insect cells usually have almost no attachment to it. Remove one or all Man residues to form a portimannose-type Man 3-1 GlcNAc2N-glycan To make it happen. Plant cells also remove Man residues, Man 4-5 GlcNAc2 is produced, resulting in complex GlcNAc or Although it may have Gal modification, in most cases it is a potentially immunogenic β1,2-bound xylamine. The core α1,3-linked fucose (Fuc) residue is added along with the Xyl and insect cells.

[0017] N-glycans of animal glycoproteins typically consist of galactose, fucose, and terminal sialic acid. These sugars are not found on glycoproteins produced in yeast and filamentous fungi. In cellular and other non-human eukaryotic cells, all types of sugar nucleotide precursors (e.g., UDP-N) - Acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid (e.g., UDP-galactose, GDP-fucose) are synthesized in the cytosol and transported to the Golgi apparatus. Therefore, these are then processed by glycosyltransferase ("Gnt"), It is added to gosaccharides.

[0018] (Methods for modifying sugar chains) Genetic and metabolic engineering efforts have led to the development of N-glycan processing in insects, yeasts, and plants. This modification is carried out to modify the pathways, and sialylated complex N-glycans are used in insect cells, yeast, and plant cells. It is produced as a substance, which allows for the modification of cell lines to potentially therapeutically valuable mammals. It has been shown that it can produce glycoproteins (Geisler et al., 2015a; Stras (ser 2016; Hamilton et al., 2006; Jacobs et al., 2009). Mosses, aquatic plants, algae, And other heterologous hosts such as silkworms have been tested for beneficial glycosylation, but these The system is far from optimal (Calow et al., 2016; Cox et al., 2006; Tada et al., 2015). Until now, specific heterologously expressed glycosyltransferases in lower eukaryotes have not been adequately translated. Whether or not it is done, whether or not it has catalytic activity, or whether or not it is localized to the appropriate organelle in the secretory pathway There is no reliable way to predict whether or not this will happen. Furthermore, changes in the glycosylation pathway affect cells. This alters either survival and / or site occupation of glycoproteins, affecting productivity or product quality. This can lead to a decrease.

[0019] Sialic acid (Sia) is an N-acetylnucleotide that is ubiquitous in deuterostomes, from starfish to humans. These are a group of N- or O-substituted derivatives of iraminic acid (Neu5Ac). These compounds are a type of fine-grained compound. It has also been observed in several other organisms, including bacteria, protozoa, and fungi. Pathogenic bacteria and Sialic acid biosynthesis in mammalian cells is well understood. Sialic acid is thought to be primarily a means of escaping the host immune system, but these same sia Acid molecules are involved in protein targeting, cell-cell interactions, cell-substrate recognition, and contact It is also involved in many processes in higher organisms, including maturation (Varki literature, 2017; Vimr (Et., 2004; Schauer, 2000). The presence of sialic acid is observed in vivo in glycoproteins. It affects the half-life of [the substance]. For example, the importance of sialic acid is related to human erythropoietin (hEPO) This has been proven in research. The terminal sialic acid residue on the N-linked glycan of this glycoprotein is It prevents rapid clearance of hEPO from the blood and improves in vivo activity. Galactose The asialylated hEPO (asialo-hEPO), which terminates at a residue, was dramatically reduced in vivo. It has red blood cell production activity. This decrease is due to asia by hepatic asialoglycoprotein receptors. This is caused by increased clearance of low-hEPO (Fukuda et al., 1989). The lack of terminal sialic acid on many therapeutic glycoproteins reduces their in vivo efficacy. Therefore, a more frequent patient administration regimen may be required.

[0020] Generally, the ability to produce mammalian N-glycans and fully humanized N-glycans is a biotechnological skill. It is expected to transform the technology industry, but this is because a series of new organisms have appeared, This is because it produces therapeutic drugs that are beneficial to the health of the human body. The present invention relates to kinetoplasts (Kinetopla Native of the species Leishmania tarentolae (stid) Furthermore, analysis of novel N-glycosylation pathways, discovery of important differences from conserved pathways, and glycans Its use for modification is described. Several methods for glycosylation are presented as examples. .

[0021] This Kinetoplastida glycosylation expression platform is clearly This results in a completely customized N-glycan, based on a simple human portiomannose foundation. Because the modified glycan is built on top of it, it has extremely uniform humanized glycosylation, The production of cheaper, safer, and more consistent therapeutic drugs will significantly advance the field of glycosylation. The specific and remarkably unique N-glycan biosynthesis is novel and has never been done before. It has never been described, and therefore, any other eukaryote such as Pichia or other eukaryotes The methods for modifying glycosylation in organisms are distinguished. Furthermore, shorter than mammalian cells The development deadline and the creation of recombinant strains more quickly are achieved through this simple yet fully functionally customized method. The expression platform has been significantly developed for a wide range of applications for therapeutic proteins. To make someone do it. [Overview of the Initiative]

[0022] (5. Overview) This specification describes N-acetylglucosamine transferase, galactosamine Heteroglycosyltransferase and sialyltransferase are included in heteroglycosyltransferase It is a eukaryotic host cell of the order Kinetoplastes containing spherase. The host cells then process uniform and fully functionally customized N-glycans with high site occupancy. It is possible to produce therapeutic glycoproteins in mammals (e.g., humans). Using the provided host cells, full-length therapeutic antibodies (e.g., anti-CD20 (rituximab)) and others It is possible to express therapeutic proteins (e.g., erythropoietin).

[0023] Also provided herein are mammalian enzyme activities (e.g., N-acetylglucosamine) (Those involved in elongation, galactosylation, and sialylation) are the eukaryotic hosts of kinetoplasts. nucleic acids that can be effectively targeted to intracellular compartments of cells and expressed there. This is a combinatorial library. This process provides modified host cells. Then, using this, any desired gene involved in glycosylation is expressed, and the target It can be set. CMP-sialic acid biosynthesis for the production of sialylated glycoproteins. The design of the formation pathway is also provided.

[0024] In specific embodiments, provided herein are (a) encoding a target protein (b) recombinant nucleic acids that encode heterologous glycosyltransferases These are Leishmania host cells.

[0025] In one embodiment, the heterologous glycosyltransferase is N-acetylglucosa Minttransferase; and / or heterologous galactosyltransferase; and / or heterologous It is a sialyltransferase. In some embodiments, provided herein This refers to host cells containing two or more N-acetylglucosamine transferases. In this embodiment, the host cell containing the heterologous sialyltransferase is CMP-NeuAc It further includes heterologous CMP-Sia biosynthetic pathway proteins that can generate these proteins.

[0026] In another embodiment, provided herein are 1 or more derived from the N-glycan biosynthesis pathway The above endogenous enzymes are deleted, mutated, and / or functionally inactivated. It is a host cell.

[0027] In a further embodiment, N-acetylglucosamine transferase, galactosamine The amino acid sequences of transtransferase and / or sialyltransferase are shown in Table 9. The listed N-acetylglucosamine transferase and galactosyl transferase are A sialyltransferase, or any functional homolog thereof, It is derived from an isoform or a mutant.

[0028] In a further embodiment, a CMP-Sia biosynthesis pathway that can generate CMP-NeuAc The proteins are the CMP-Sia biosynthesis pathway proteins listed in Table 11, or any of these. Functional homologs, isoforms, or variants are present in at least approximately 70%, 71%, 72%, and 73% of each. %, 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 they are 100% identical.

[0029] In another embodiment, the N-acetylglucosamine transferase is GnT-I In another embodiment, the N-acetylglucosamine transferase is GnT-II Yes. In another embodiment, the N-acetylglucosamine transferase is GnT-I and GnT-II. In another embodiment, the galactosyltransferase is B4GA It is LT1. In one embodiment, the N-acetylglucosa is provided herein. The minttransferase is GnT-I and GnT-II, and the galactosyltransferase The host cell is B4GALT1. In another embodiment, the sialyltransfer Ze is 2,6-SiaT or 2,3-SiaT. In one embodiment, provided herein is The N-acetylglucosamine transferase is GnT-I and GnT-II, and the galact The siltransferase is B4GALT1, and the sialyltransferase is 2,6-SiaT or The host cell is 2,3-SiaT. In specific embodiments, the following are provided herein. The N-acetylglucosamine transferase is GnT-I and GnT-II, and the galak The tosyltransferase is B4GALT1, and the sialyltransferase is 2,6-SiaT Alternatively, it is 2,3-SiaT, and the sialyltransferase generates CMP-NeuAc. This is a host cell that further contains heterologous CMP-Sia biosynthesis pathway proteins capable of producing this.

[0030] In one embodiment, the host cell is a Leishmania talentrae cell. In this embodiment, the host cell is any of the strains listed in Table 13.

[0031] In one embodiment, the host cell is a prokaryotic or eukaryotic enzyme as shown in Figure 53. It includes a CMP-Neu5Ac pathway.

[0032] In another embodiment, the leishmania signal and / or retention sequence is N-acetylg Glucosamine transferase, galactosyl transferase, and / or sialyl It is attached to the transferase, where the signal sequence is N-acetylglucosamine Lansferase, galactosyltransferase, and / or sialyltransferase The enzyme is targeted to the endoplasmic reticulum of Leishmania host cells, and the retention sequence is N- Acetylglucosamine transferase, galactosyl transferase, and / or In another embodiment, the sialyltransferase is retained in the endoplasmic reticulum or Golgi apparatus. The retaining sequence is N-acetylglucosamine transferase and / or galactosyl The transferase is retained in the endoplasmic reticulum of the host cell. Another embodiment of the retaining sequence This is N-acetylglucosamine transferase and / or galactosyl transferase. The enzyme is retained in the cis-Golgi compartment of the host cell. In another embodiment, the retaining sequence is N- Acetylglucosamine transferase and / or galactosyl transferase It is retained in the intermediate Golgi compartment of the host cell. In another embodiment, the retaining sequence is galact Syltransferase is retained in the trans-Golgi compartment of the host cell. In another embodiment... The retention sequence then retains sialyltransferase in the trans-Golgi compartment of the host cell. It holds. In another embodiment, the retaining sequence is sialyltransferase and galact Tosyltransferase is retained in the trans-Golgi compartment of the host cell.

[0033] In another embodiment, provided herein are 1 or more derived from the N-glycan biosynthesis pathway The above endogenous enzymes are deleted, mutated, and / or functionally inactivated. It is a host cell.

[0034] In another embodiment, the signal sequence and / or retention sequence may be any leishmania The signal sequence or retention sequence is derived from the species. In a further embodiment, the signal The sequence and / or retained sequence is a signal sequence or derived from Leishmania talentrae. This is a retention sequence.

[0035] In another embodiment, the signal sequence is processed and removed.

[0036] In a further embodiment, the retention sequence is Leishmania talentrae protein It is a cytoplasmic-transmembrane-stem (CTS) sequence derived from . In another embodiment, the CTS sequence is , derived from Leishmania talenttrae MAN1, NTPDase 1, or NTPDase 2. In this embodiment, the CTS sequence is the sequence of sequence number 24, sequence number 25, or sequence number 26. , or comprising these functionally active fragments. In yet another embodiment, the CTS sequence is distributed The array in column number 24 and at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or Its functionally active fragment; sequence of SEQ ID NO: 25 and at least approximately 70%, 71%, 72%, 73%, 74%, 7 5%, 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 10 A sequence or its functionally active fragment that is 0% identical; or at least about 70% identical to the sequence of SEQ ID NO: 26. , 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 It contains sequences or functionally active fragments thereof that are %, 98%, 99%, or 100% identical.

[0037] In another embodiment, the CTS is derived from Leishmania talentrae MAN1. In this embodiment, the CTS sequence includes the sequence of SEQ ID NO: 24 or a functionally active fragment thereof. In a further embodiment, the retaining sequence is a GRIP sequence derived from Leishmania or the It contains a functionally active fragment. In another embodiment, the GRIP sequence is the sequence of SEQ ID NO: 27 or It includes a functionally active fragment of the GRIP sequence. In yet another embodiment, the GRIP sequence is the component of SEQ ID NO: 27 Columns and at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Sequences or their functional activity that are 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Includes a sex fragment. In a further embodiment, the retention sequence is in the leishmania protein. The derived CTS sequence or its functionally active fragment, and the GRIP sequence derived from Leishmania or It contains its functionally active fragment.

[0038] In another embodiment, the target protein is heterogeneous to the Leishmania host cell. be.

[0039] In another embodiment, the target protein includes a signal sequence derived from Leishmania. It has been modified to do so. In another embodiment, the signal sequence is Leishmania. This is a signal sequence derived from Talenttrae. In some embodiments, the signal sequence This includes the sequence of SEQ ID NO: 28 or SEQ ID NO: 29, or their functionally active fragments. In this embodiment, the signal sequence includes the sequence of SEQ ID NO: 28 or a functionally active fragment thereof. In yet another embodiment, the signal sequence is at least about 70% of the sequence of sequence number 28. , 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 Includes a sequence or functionally active fragment thereof that is %, 98%, 99%, or 100%. Other embodiments In this process, the signal sequence is processed and removed from the target protein.

[0040] In another embodiment, the target protein is human interferon-α (INF-α), i Interferon-β (INF-β), Interferon-γ (INF-γ), Interleukin-2 (IL-2) , Chimeric diphtheria toxin-IL-2 (denileukin diphthitox), Interleukin-1 ( IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), tumor Surgery necrosis factor alpha (TNF-α), insulin, plumrintide, growth hormone (GH), Surin-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide -1 agonist (GLP-1), glucagon, growth hormone-releasing hormone (GHRH), secretin, thyroid gland Gastrointestinal stimulant (TSH), human bone morphogenetic protein 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7) ), gonadotropin-releasing hormone (GnRH), keratinocyte growth factor (KGF), platelet-derived growth Factors (PDGF), fibroblast growth factor 7 (FGF7), fibroblast growth factor 20 (FGF20), fibroblast growth long factor 21 (FGF21), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), neurotrophin-3 Human follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), Lutropin-α, Eri Slopoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor The extracellular domain of CTLA4 (e.g., FC-fusion), or the extracellular domain of the TNF receptor It contains the amino acid sequence of the in (e.g., FC-fusion).

[0041] In another embodiment, the target protein is a therapeutic protein. In this embodiment, the target protein is an Fc-fusion protein. In another embodiment, The target protein is an antibody.

[0042] In another embodiment, the target protein is an antibody against a human protein. In the following embodiments, the antibody is adalimumab (Humira); Remicade (infliximab) Absiximab; Rituxan; Basiliximab; Sy nagis (palivizumab); Herceptin (trastuzumab); Mylotarg (gemtuzumab ozogamay) Syn); Campath (Alemtuzumab); Zevalin (Ibritumomab thiuxsetan); Xolair (Oma Rizumab; Bexxar (Toshitumomab-I-131); Erbitux (Cetuximab); Avastin (Bevacizumab) Tysabri (natalizumab); Actemra (tocilizumab); Vectibix (panitumumab); Lucen tis (ranibizumab); Soliris (eculizumab); Cimzia (certolizumab pegol); Simpon i (golimumab); Ilaris (kanakinumab); Stelara (ustekinumab); Arzerra (ofatum) Mab; Prolia (denosumab); Numax (motavizumab); ABThrax (laxibamumab); Benlyst a (belimumab); Yervoy (ipilimumab); Adcetris (brentuximab vedotin); Perjeta (Pertuzumab); Kadcyla (ad-trastuzumab emtansine); or Gazyva (obinutuzumab) It has the amino acid sequence of (b).

[0043] In other embodiments, the antibody is a full-length antibody, Fab, F(ab')2, Scfv, or sdAb. In another embodiment, the target protein includes the amino acid sequence of an enzyme or its inhibitor. In another embodiment, the target protein is factor VII, factor VIII, factor IX, Factor X, Factor XIII, Factor VIIa, Antithrombin III (AT-III), Protein C, Tissue Plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokine Alglucosidase-α, laronidase (α-L-Idro Nidase), Idursulfase (iduronic acid-2-sulfatase), Galsulfase, Agalsidase-β (human α-galactosidase A), botulinum toxin, collagenase, Human DNase-I, hyaluronidase, papain, L-asparaginase, uricase (uric acid Oxidase), glutamate carboxypeptidase (glucarpidase), α1 protea Pancreatic enzyme inhibitors (α1 antitrypsin), lactase, lipase, amylase, pro It contains the amino acid sequences of thease (thease) and adenosine deaminase.

[0044] In another embodiment, the therapeutic protein is abatacept (e.g., Orencia), Flibercept (e.g., Eylea), agalsidase beta (e.g., Fabrazyme), Albig Lutide (e.g., Eperzan), Aldesleukin (e.g., Proleukin), Alefacept (e.g.) For example, Amevive), alglucose (for example, Ceredase), alglucosidase alpha ( For example, LUMIZYME, aliskiren (for example, Tekturna), alpha-1 proteinase inhibitors Factors (e.g., Aralast), alteplases (e.g., Activase), anakins (e.g., Kine ret), anistreplasm (e.g., Eminase), human anthrax immunoglobulin (e.g., ANTH) RASIL), antihemophilic factors (e.g., Advate), antiinhibitor blood coagulation complexes (e.g., F eiba Nf), antithrombin alpha, human antithrombin III, antithymocyte globulin (e.g., anti-thymocyte globulin), anti-thymocyte globulin (equine) (e.g., ATGAM), anti Thymocyte globulin (rabbit) (e.g., ATG-Fresenius), aprotinin (e.g., Trasylol) ), asfotase alpha, asparaginase (e.g., Elspar), Elwinia crista Asparaginase (e.g., Erwinaze) of Erwinia chrysanthemi, Becapreli (e.g., REGRANEX), belatacept (e.g., Nulojix), belatant, bivaliridine ( For example, Angiomax), type A botulinum toxin (for example, BOTOXE), type B botulinum toxin ( For example, Myobloc), brentuximab vedotin (for example, Adcetris), buserelin (for example) , Suprecur), C1 esterase inhibitor (human), C1 esterase inhibitor (recombinant) (for example) (e.g., Ruconest), certolizumab pegol (e.g., Cimzia), choriogonadotropin Fa (e.g., choriogonadotropin alpha), chorionic gonadotropin (human) (e.g., O vidrel), chorionic gonadotropins (recombinant) (e.g., Ovitrelle), coagulation factor ix (e.g., A) Iprolix), coagulation factor VIIa (e.g., NovoSeven), human coagulation factor X (e.g., Coagadex), coagulation Factor XIIIA-subunit (recombinant), collagenase (e.g., Cordase), connecta Rufa, corticotropin (e.g., HPActhar), cosyntropin (e.g., Cortrosyn) Darbepoetin alfa (e.g., Aranesp), defibrotide (e.g., Noravid), Denis Leukin diffitox (e.g., Ontak), decilzine, digoxin immunoFab (sheep) (e.g.) For example, DIGIBIND, Dornase alpha (for example, Pulmozyme), Drotrecogin alpha ( For example, Xigris), dulaglutide, efmoloctocog alpha (for example, ELOCTA), ero Sulfase alpha, Enfuvirtide (e.g., FUZEON), epoetin alpha ( For example, Binocrit), epoetinzeta (for example, Retacrit), eptifibatide (for example, INTEGRILIN), etanercept (e.g., Enbrel), exenatide (e.g., Byetta), IX Factor complex (human) (e.g., AlphaNine), fibrinolysin, also known as plasmin (e.g., Elase), Filgrastim (e.g., NA), Filgrastim-sndz, Folitropin Rufa (e.g., Gonal-F), follitropin beta (e.g., Follistim AQ), galsulf Amethystase (e.g., Naglazyme), gastric factor, gemtuzumab ozogamicin (e.g., Mylotarg ), glucocyte acetate (e.g., Copaxone), recombinant glucagon (e.g., GlucaGen), Glucarpidase (e.g., Voraxaze), Gramicidin D (e.g., Neosporin), Hepatitis B Immunity Epidemic globulin, human calcitonin, human tetanus (Clostridium tetani) toxoid immunoglobulin Robulin, human rabies virus immunoglobulin (e.g., Hyperab human rabies immunoglobulin) (, human Rho(D) immunoglobulin (e.g., Hyp Rho D Inj 16.5%), human serum albumin ( For example, Albuminar, human varicella-zoster immunoglobulin (e.g., Varizig), hyaluronic acid nidase (e.g., HYLENEX), hyaluronidase (human recombinant), ibritumomabuchiuki Cetane (e.g., Zevalin), idulsulfase (e.g., Elaprase), imiglucerase ( For example, Cerezyme), human immunoglobulin, insulin aspart (for example, NovoLog), U Insulin, insulin degludec (e.g., Tresiba), insulin detemir (e.g., LEVEMIR), insulin glargine (e.g., Lantus), insulin glulisine (e.g., API) DRA), insulin lispro (e.g., Humalog), porcine insulin (e.g., Iletin II), Regular insulin (e.g., Humulin R), porcine insulin (e.g., vetsulin), isof Insulin (e.g., Novolin N), interferon alpha-2a, recombinant (e.g., Roferon A), interferon alpha-2b (for example, INTRON A), interferon A Rufacon-1 (e.g., INFERGEN), interferon alpha-n1 (e.g., Wellferon), Interferon alpha-n3 (e.g., Alferon), interferon beta-1a (e.g., Avonex), interferon beta-1b (e.g., Betaseron), interferon gamma-1b (e.g., Actimmune), intravenous immunoglobulin (e.g., Civacir), laronidase (e.g., Aldurazyme, lenograstim (e.g., Granocyte), repiridine (e.g., Refludan), Leuprolide (e.g., Eligard), liraglutide (e.g., Saxenda), lucinactant (e.g.) For example, Surfaxin, Lutropin alfa (e.g., Luveris), Mecasermin (e.g., NA) , menotropin (e.g., Menopur), methoxypolyethylene glycol-epoetin beta (For example, Mircera), metreleptin (for example, Myalept), natural alpha-interferon or These include multiferons (e.g., Intron / Roferon-A), nesiritide (e.g., NATRECOR), and occuli. Plasmin (e.g., Jetrea), oprelbequine (e.g., Neumega), OspA lipoprotein ( For example, Lymerix, oxytocin (for example, Pitocin), palifermin (for example, Kepivanc) e) Pancrelipase (e.g., Pancrecarb), bovine pegademase (e.g., Adagen), PEG Asparagus gauze (e.g., Oncaspar), pegfilgrass (e.g., Neulasta), peg Interferon alpha-2a (for example, Pegasys), pegylated interferon alpha-2b (for example) For example, PEG-Intron, pegylated interferon beta-1a (e.g., Plegridy), and Pegrotica. Ze (for example, (Krystexxa)), Pegvisomant (for example, SOMAVERT), Polactant Alpha ( For example, Curosurf), Plumlintide (for example, Symlin), Preotact (for example, Preota ct E), protamine sulfate (e.g., protamine sulfate injection, USP), human protein S (e.g., For example, human protein S), prothrombin (e.g., Feiba Nf), prothrombin complex ( For example, Cofact), prothrombin complex concentrate (e.g., Kcentra), Rasb uricase ( For example, Elitek, Reteplase (e.g., Retavase), Lilonacept (e.g., Arcalyst) , romiprostim (e.g., Nplate), sacrosidase (e.g., Sucraid), salmon calcium Nin (e.g., Calcimar), salglamostim (e.g., Leucomax), satsumomab pendetide (e.g., OncoScint), seberipase alpha (e.g., Kanuma), secretin (e.g., Se creFlo), cermorelin (e.g., cermorelin acetate), serum albumin (e.g., Albunex) , iodine-labeled serum albumin (e.g., Megatope), simoctocog alfa (e.g., Nuwiq) ), cyplucel-T (e.g., Provenge), somatotropin recombinant (e.g., Nutropin AQ) ), somatropin recombinants (e.g., BioTropin), streptokinases (e.g., Streptase ), susoctocog alpha (e.g., Obizur), taliglucerase alpha (e.g., Elelys o) teduglutide (e.g., Gattex), tenecteplase (e.g., TNKase), teriparath (e.g., Forteo), tessamorelin (e.g., Egrifta), thrombomodulin alpha (e.g., For example, Recomodulin, Simalfacin (for example, Zadaxin), Thyroglobulin, Thyrot Ropin alpha (e.g., Thyrogen), tuberculin purified protein derivatives (e.g., Apris ol), turoctocog alfa (e.g., Zonovate), urofolitropin (e.g., BRAVELLE) ), urokinase (e.g., Kinlytic), vasopressin (e.g., Pitressin), bellaglucate Lase alfa (e.g., Vpriv), absiximab (e.g., ReoPro), adalimumab (e.g., For example, Humira, alemtuzumab (e.g., CAMPATH), alirocumab (e.g., Praluent), Lucitumomab (e.g., CEA-Scan), atezolizumab (e.g., Tecentriq), basilixim (e.g., Simulect), belimumab (e.g., Benlysta), bevacizumab (e.g., Avastin) ), blinatumomab (e.g., Blincyto), brodalumab (e.g., Siliq), canakinumab ( For example, ILARISE, canakinumab (for example, Ilaris), capromab (for example, ProstaScint) , cetuximab (e.g., Erbitux), daclizumab (e.g., Zenapax), daratumumab (e.g., For example, DARZALEX, denosumab (e.g., Xgeva), dinutuximab (e.g., unituxin), Clizumab (e.g., Soliris), efalizumab (e.g., RAPTIVA), elotuzumab (e.g., For example, EMPLICITI, evolocumab (e.g., Repatha), golimumab (e.g., Simponi injection) ibritumomab (e.g., Zevalin), idarucizumab (e.g., Praxbind), inflix Simab (e.g., REMICADE), ipilimumab (e.g., YERVOY), ixekizumab (e.g., T altz), mepolizumab (e.g., Nucala), muromonab (e.g., Orthoclone OKT3), Natalie Zumab (e.g., Tysabri), necitumumab (e.g., Portrazza), nivolumab (e.g., Opd ivo), obilutoxaximab (e.g., Anthim), obinutuzumab (e.g., Gazyva), off Atumumab (e.g., Arzerra), omalizumab (e.g., Xolair), palivizumab (e.g., Synagis), panitumumab (e.g., Vectibix), pembrolizumab (e.g., Keytruda), pe Rutuzumab (e.g., Perjeta), ramucirumab (e.g., Cyramza), ranibizumab (e.g., Lucentis), laxibakuma (e.g., laxibakuma), rituximab (e.g., Rituxa n) secukinumab (e.g., Cosentyx), siltuximab (e.g., Sylvant), tocilizuma (e.g., ACTEMRA), tositumomab (e.g., Bexxar), trastuzumab (e.g., Hercep) amino acids of tin, ustekinumab (e.g., Stellara), or vedolizumab (e.g., Entyvio) Contains acid sequences.

[0045] In a further embodiment, the host cell is (a) Stt3 oligosaccharide transfer It contains (b) an enzyme (OST) and does not have endogenous N-glycan elongation.

[0046] Another embodiment is a method for producing glycosylated target proteins, wherein host cells are cultured. The method includes nourishing and purifying the target protein from the culture.

[0047] Another embodiment includes a composition of glycosylated target proteins.

[0048] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represented; and Asn is the Asn of the N-linked glycosylated consensus sequence in the target protein. The composition supports an oligosaccharide containing (a) an N-linked glycosylated oligosaccharide of the target protein It contains at least approximately 90% to 100% of the census sequence.

[0049] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represented; and Asn is the Asn of the N-linked glycosylated consensus sequence in the target protein. It is a G0-Gn glycan characterized by (a certain) glycosylation on the target protein Approximately 20%~30%, 25%~35%, 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50% ~60%, 55%~65%, 60%~70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, It has 85% to 95%, or 90% to 100%.

[0050] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represented; and Asn is the Asn of the N-linked glycosylated consensus sequence in the target protein. A G0 glycan characterized by (a certain) glycosylation on the target protein About 10~20%, 20%~30%, 25%~35%, 30%~40%, 35%~45%, 40%~50%, 45%~55 %, 50%~60%, 55%~65%, 60%~70%, 65%~75%, 70%~80%, 75%~85%, 80% It has a percentage of ~90%, 85%~95%, or 90%~100%.

[0051] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) The gray circles represent mannose residues, and Asn represents the N-linked type in the target protein. The target protein is a G1-Gn characterized by a glycosylated consensus sequence (Asn). At least approximately 10-20%, 20-30%, 25-35%, 30-40%, and 35% of the qualitative glycosylation. %~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~70%, 65%~75% It has 70%-80%, 75%-85%, 80%-90%, 85%-95%, or 90%-100%.

[0052] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) The gray circles represent mannose residues, and Asn represents the N-linked type in the target protein. The target tan is a G2 glycan characterized by the Asn of the glycosylation consensus sequence. Glycosylation on the protein: at least approximately 10-20%, 20-30%, 25-35%, and 30-40% 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~70%, 65%~ It has 75%, 70%-80%, 75%-85%, 80%-90%, 85%-95%, or 90%-100%.

[0053] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) The gray circles represent mannose residues, and Asn represents the N-linked type in the target protein. The target glycan is a G1 glycan characterized by the Asn of the glycosylation consensus sequence. Glycosylation on the protein: at least approximately 10-20%, 20-30%, 25-35%, and 30-40% 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~70%, 65%~ It has 75%, 70%-80%, 75%-85%, 80%-90%, 85%-95%, or 90%-100%.

[0054] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) The gray circles represent mannose residues, and Asn represents the N-linked type in the target protein. The target glycan is a G1 glycan characterized by the Asn of the glycosylation consensus sequence. Glycosylation on the protein: at least approximately 10-20%, 20-30%, 25-35%, and 30-40% 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~70%, 65%~ It has 75%, 70%-80%, 75%-85%, 80%-90%, 85%-95%, or 90%-100%.

[0055] In another embodiment, glycosylation on a target protein occurs when it is introduced into the target. The pharmacokinetic properties of the target protein are further modified to optimize them. In another embodiment, In this process, glycosylation on the target protein is replaced by sialylation.

[0056] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0057] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0058] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0059] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0060] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0061] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0062] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0063] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0064] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0065] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10 - 20%, 20% - 30%, 25% - 35% of the glycosylation on the target protein , 30% - 40%, 35% - 45%, 40% - 50%, 45% - 55%, 50% - 60%, 55% - 65%, 60% - 70%, 65% - 75%, 70% - 80%, 75% - 85%, 80% - 90%, 85% - 95%, or 90% - 100 %.

[0066] In another embodiment, the composition of the glycosylated target protein has the following structure:

Chemical Formula

[0067] In another embodiment, the composition of the glycosylated target protein has the following structure:

Chemical Formula

[0068] In another embodiment, the N-linked glycosylated consensus sequence is Asn-X-Ser / Thr Yes; here, X is any amino acid other than proline.

[0069] In another embodiment, the glycosylated target protein is secreted into the culture medium, here Therefore, the glycosylation target protein is glycosylated. The glycosylated target protein is purified from the culture medium. In another embodiment, Lycosylated target proteins are cultured by affinity purification or ion exchange chromatography. It is purified from the culture medium. In another embodiment, the glycosylated target protein is FC-domed It contains , and is affinity-purified from the culture medium by protein-A. In another embodiment, Furthermore, glycosylated target proteins contain affinity tags and are purified by affinity.

[0070] In another embodiment, the population of glycosylated target proteins is at least about 90%, 91% It is uniformly 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In this embodiment, the N-sugar site on the target protein occupied by glycosylation 90% to 100% of that.

[0071] In a specific embodiment, provided herein are (a) a leishmania-derived (b) the catalytic domain of N-acetylglucosamine transferase, not (b) leash A hybrid containing amino acid sequences involved in the localization and retention of mania in the Golgi compartment. This is N-acetylglucosamine transferase.

[0072] In one embodiment, the hybrid N-acetylglucosamine transferase is It originates from Leishmania talentrae.

[0073] In another embodiment, the hybrid N-acetylglucosamine transferase is The following has been modified to include a signal sequence and at least one retention sequence, where the The signal sequence transmits the N-acetylglucosamine transferase to Leishmania tarre The intracellular matrix targets the endoplasmic reticulum of the host cell, and the retaining sequence is the N-acetylglucosa The minttransferase is retained in the endoplasmic reticulum or Golgi apparatus.

[0074] In another embodiment, the hybrid N-acetylglucosamine transferase is The N-acetylglucosamine transferase is retained in the endoplasmic reticulum. In another embodiment, Furthermore, the hybrid N-acetylglucosamine transferase is used to transfer the N-acetylglucosamine. Cosamine transferase is held in the cis-Golgi apparatus. In another embodiment, the Hybrid N-acetylglucosamine transferase is used to transfer N-acetylglucosamine Lansferase is held in the intermediate Golgi apparatus. In a further embodiment, the hybrid N-acetylglucosamine transferase is a cytoplasmic-transmembrane-stem (CTS) sequence. There is.

[0075] In certain other embodiments, the N-acetylglucosamine transferase is as set forth in Table 9 the N-acetylglucosamine transferase, or a functional homolog thereof, isoform, or variant.

[0076] In a specific embodiment, provided herein is a hybrid galactosyltransferase comprising (a) the catalytic domain of a galactosyltransferase not from Leishmania; and (b) an amino acid sequence involved in localization and retention in the endoplasmic reticulum or Golgi compartment of Leishmania.

[0077] In another embodiment, the hybrid galactosyltransferase is from Leishmania tarentolae.

[0078] In a further embodiment, the hybrid galactosyltransferase is modified to include a signal sequence that targets the galactosyltransferase to the endoplasmic reticulum of a Leishmania tarentolae host cell and a retention sequence that retains the galactosyltransferase in the endoplasmic reticulum or Golgi apparatus.

[0079] In certain embodiments, the hybrid galactosyltransferase retains the galactosyltransferase in the endoplasmic reticulum. In certain embodiments, the hybrid galactosyltransferase retains the galactosyltransferase in the cis-Golgi apparatus. In another embodiment, the hybrid galactosyltransferase ​​​​​​​​​​​In another embodiment, the galactosyltransferase is held in the intermediate Golgi apparatus. The hybrid galactosyltransferase is said to be the galactosyltransferase The galaxy is held in the trans-Golgi apparatus. In another embodiment, the hybrid galaxy Tosyltransferase is a cytoplasmic-transmembrane-stem (CTS) sequence. Further implementations In this case, the hybrid sialyltransferase is a GRIP sequence. In one embodiment, the hybrid sialyltransferase comprises a CTS sequence and a GRIP sequence. be.

[0080] In another embodiment, the galactosyltransferase is shown in Table 9. Galactosyltransferase, or its functional homolog, isoform, or It originates from a mutant.

[0081] In a specific embodiment, provided herein are (a) a leishmania-derived (b) the catalytic domain of sialyltransferase, as well as (b) vesicles of Leishmania. A hybrid sequence containing amino acid sequences involved in localization and retention in the body or Golgi compartment. It is an allyltransferase.

[0082] In another embodiment, the hybrid sialyltransferase is used in Leishmani It originates from A. talenttrae.

[0083] In one embodiment, the hybrid sialyltransferase is a signal sequence Modified to include, where the signal sequence is the sialyltransferer The ze is targeted to the endoplasmic reticulum of the Leishmania talenttrae host cell, and the retaining sequence The sialyltransferase is retained in the endoplasmic reticulum or Golgi apparatus.

[0084] In one embodiment, the hybrid galactosyltransferase is the galactosyltransferase. The tosyltransferase is held in the endoplasmic reticulum. In another embodiment, the hybrid The sialyltransferase is transferred to the trans-Golgi apparatus. To retain. In another embodiment, the hybrid sialyltransferase is CTS It is a sequence. In a further embodiment, the hybrid sialyltransferase is , a GRIP sequence. In another embodiment, the hybrid sialyltransferase These are the CTS sequence and the GRIP sequence.

[0085] In another embodiment, the hybrid sialyltransferase is shown in Table 9. The listed sialyltransferase, or its functional homolog, isoform, Alternatively, it may originate from a mutant.

[0086] In other embodiments, provided herein are hybrid N-acetylglucosa This is a nucleic acid encoding minttransferase. Further embodiments are described herein. The nucleic acid provided is one that encodes hybrid galactosyltransferase. In one embodiment, a hybrid sialyl transformer is provided herein. It is a nucleic acid that codes for ferase.

[0087] (5.1 Terminology and Abbreviations) When the term "approximately" is used with a number, it means ±1, ±5, or ±10% of the number mentioned. It refers to any number within that range.

[0088] As used herein, the term “subject” means animals (e.g., birds, reptiles, and This refers to mammals. In another embodiment, the subject is a non-primate (e.g., camel, donkey, (Zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and Mammals, including primates (e.g., monkeys, chimpanzees, and humans). One embodiment In this context, the subject is a non-human animal. In some embodiments, the subject is livestock or It is a pet (for example, a dog, cat, horse, goat, sheep, pig, donkey, or chicken). In a specific embodiment, the subject is a human being. The terms "subject" and "patient" are used. These may be used interchangeably in this specification.

[0089] "α[number]", "α[number],[number]", "β[number]", or "β[number],[number]" The abbreviation refers to a glycosidic bond, which is a covalent bond that connects a carbohydrate residue to another base. nd) refers to a glycosidic bond (glycosidic linkage). An α-glycosidic bond is formed between two carbon atoms. A β-glycosidic bond is formed when the two carbon atoms have the same stereochemistry, but the two carbon atoms are different. This occurs when stereochemistry is present. [Brief explanation of the drawing]

[0090] (6. Brief explanation of the drawing) [Figure 1]Figure 1: Antibody combination diversity drives antibody effector function (excerpted from Jennewein and Alter, 2017). Modifying IgG Fc through two changes to the antibody's Fc domain—(i) glycosylation selection (36 options) and (ii) subclass selection (4 subclasses)—created 144 theoretical combinations and associated functional states. Depending on the antibody-glycan combination, many different functional responses can be induced, including anti-inflammatory responses; functional responses such as antibody-dependent cell phagocytosis (ADCP) and antibody-dependent cell-dependent cytotoxicity (ADCC); or induction of inflammatory responses including complement activation and cytokine secretion. Abbreviations: IL, interleukin.

[0091] [Figure 2]Figures 2A, 2B, and 2C: Structural changes of IgG Fc fragments due to glycosylation (excerpted from Sondermann et al., 2013). Figure 2A: A diagram of proposed structural changes within the Fc fragment due to sialylation. The non-galactosylated (G0F) Fc-fragment maintains an open three-dimensional structure that allows for FcγR binding. Triangles represent fucose and low binding. Figure 2B: In fully α2,6-sialized Fc (G2FS2), the α1,3-arm associates with the protein core of the Cγ2 domain, inducing a closed three-dimensional structure. The resulting closed three-dimensional structure of the tertiary-modified Fc fragment exposes the DC-SIGN binding site but blocks the FcγR binding site. According to Figure 2C (Chen et al., 2017; Li et al., 2017), this model can be adapted to have an open conformation on a fucose-deficient, glycosylated N-glycan, enabling higher affinity binding to FcγR. If the fucose-deficient N-glycan is bifurcated with a sialic acid terminus, the conformational change can be open and condensed. The Fc structure is stabilized by two glycan latches, maintaining the proper conformation of Fc, but this is mediated by different glycan residues. Chen's results suggest a role for N-glycosylation in maintaining the structural integrity of Fc and demonstrate the possibility of diverse modes of glycan-glycan interaction regulated by terminal sialic acids.

[0092] [Figure 3]Figures 3A and 3B: Bioinformatics evaluation of endoplasmic reticulum (ER) N-glycan biosynthesis in Leishmania talentlae and comparison with conserved pathways (excerpted from Varki's literature, 2009). Figure 3A: Deficient genes in Leishmania talentlae are indicated by an "x" and a peripheral line. Figure 3B: The enzymes that catalyze each step of biosynthesis were identified mainly from studies of mutants of the budding yeast Saccharomyces cerevisiae. The genes affected by each yeast mutation are known as ALG genes (representing altered glycosylation). Synthesis of dolichol-PP-GlcNAc2Man9Glc3. Dolichol phosphate (Dol-P), located on the cytoplasmic side of the ER membrane (indicated by the red curved line), receives GlcNAc-1-P from UDP-GlcNAc in the cytoplasm to produce Dol-PP-GlcNAc. Dol-PP-GlcNAc is then extended to Dol-PP-GlcNAc2Man5 by the ALG enzyme shown, and then "inverts" across the ER membrane to the lumen side. On the lumen 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 produced on the cytoplasmic side of the ER and "invert" to the lumen side. Homologs present in L. talenttrae are indicated with a checkmark, and deficient homologs are indicated with an "x". Since ALG12, 6, 8, 9, and 10 are absent, and ALG3 is presumed to contain a loss-of-function mutation, the man3 structure is thought to be inverted and directly transferred to the polypeptide without further mannosylation. ALG5 (dritil-phosphate β-glucosyltransferase) is absent. OST consists only of Stt3 (Table 4).

[0093] [Figure 4]Figures 4A, 4B, and 4C: Bioinformatics evaluation of endoplasmic reticulum (ER) N-glycan biosynthesis, trimming, and transfer to protein receptors. Figure 4A: Deficiencies in ALG homologs in biosynthesis, indicated by × marks and peripheral lines, suggest a reduced Man3 precursor. Despite the presumed deficiencies of Man4 or Man5 precursor species (due to deficiencies in ALG3, 9, 11, and 12) and glucosylation precursor species (due to deficiencies in ALG6, 8, and 10, as well as ALG5), the trimming enzymes (GCS1, GNAB, MAN1) shown in Figure 4B are present (deficient enzymes are indicated by peripheral lines). Figure 4C: Only Man3 of the final glycan is thought to be transferred to the N-consensus site of the protein acceptor by Stt3. The folding intermediates Glc1Man9GlcNAc2 are not considered ligands for the lectins involved in quality control.

[0094] [Figure 5]Figure 5: Bioinformatics evaluation of quality control of protein N-glycosylation and protein folding, based on (Moremen et al., 2012). a) During glycoprotein biosynthesis, following the translation of the nascent polypeptide, simultaneous translocation of glycans from lipid-binding intermediates to peptide acceptor sequences occurs via the SEC61 pore and oligosaccharide transferase (OST). One groove in the STT3 subunit of OST scans the Asn-X-Ser / Thr acceptor sequence, while the adjacent groove binds to the glycan donor. b) Glycan trimming by Glc removal occurs immediately after the transfer by the α-β heterodimer (GIsIIα / β) of α-glucosidase I (GIsI) and α-glucosidase II. The folding intermediate containing the Glc1Man9GlcNAc2 structure is a ligand for the lectin calnexin (not present in L. talentlae) or calreticulin (not present in L. talentlae), which functions in complex with ERp57. Further Glc cleavage occurs following dissociation from these lectins. Further chaperone support is provided by the ATP-driven chaperone BiP (also known as GRP78). Correctly folded glycoproteins are packaged for transport to the Golgi apparatus. c) Incompletely folded glycoproteins are recognized by the folding sensor UDP-Glc:glycoprotein glucosyltransferase (UGGT1, not present in L. talentlae). These are then reglucosylated by re-adding Glc residues to the glycan structure and re-incorporated into the calnexin cycle. d) Triangularly misfolded glycoproteins undergo endoplasmic reticulum (ER) discarding via ER trimming (ER α-mannosidase I (ERManI = via the activity of MAN1 present in L. talentlae) or Golgi ManIA, Golgi ManIB, and Golgi ManIC (not shown), followed by ER degradation-enhancing α-mannosidase-like 1 (EDEM1), EDEM2, and EDEM3 (these are homologs of Htm1, also known as Mnl1 in yeast, and none are present in L. talentlae).The trimmed glycan binds to ER lectin OS9 or XTP3B (not shown) and is translocated to the cytosol via the Delrin 1 (DER1), DER2, and DER3 complex (SEL1L complex; Hrd3 in yeast) using the driving force of cytosolic ubiquitin-binding proteins and the ATPase function of balossin-containing proteins (VCP; also known as TER ATPase; also known as Cdc48, Ufd1, or Npl4 in yeast). This peptide is deglycosylated by cytosolic PNGase (not present in L. talenttrae) and degraded by proteasome 149. Homologs not present in L. talenttrae are indicated with an "x", and present homologs are shown in a square box. See also Table 8 for further comparison details.

[0095] [Figure 6] Figure 6: Mammalian N-glycosylation pathway, compared to wild-type Leishmania talentrae (excerpted from Kellokumpu et al., 2016). The schematic diagram shows the sequential processing of N-glycans by glycosyltransferases in the endoplasmic reticulum (ER) and Golgi apparatus. The enzymes involved (glycosyltransferases and glycosidases) are conventionally thought to function individually and sequentially by either adding sugar residues one at a time in a specific order to a growing oligosaccharide chain or removing sugar residues one at a time in a specific order from a growing oligosaccharide chain (left side) ("mammals"). The right side of Figure 6 shows wild-type Leishmania talentrae, which has a reduced precursor and different mature N-glycans compared to the mammalian system.

[0096] [Figure 7] Figure 7: Mammalian N-glycosylation pathway compared to wild-type Leishmania talentlae, excerpted from (Kellokumpu et al., 2016). The schematic diagram of the mammalian pathway complements Figure 6 using a glycosylation process in which the indicated glycans are produced in glycosylated Leishmania.

[0097] [Figure 8] Figure 8: Proposed N-glycosylation biosynthesis pathway in Leishmania talentrae. The N-glycan precursor is constructed as Man3 on the cytoplasmic side of the ER and then inverted into the ER lumen. This Man3 glycan is then directly transferred by Stt3, the only component of the OST complex typically found. The final glycan on the N-sugar site of the acceptor protein is Man3, which then translocates to the cis-Golgi. In the cis-Golgi, heterologous enzymes catalyze glycosyltransferase reactions that construct human bifurcated N-glycans. These are Gnt-I and Gnt-II of mammalian or insect origin, which add the first two GlcNAc residues. The corresponding glycoforms are listed in the box below, along with their textual abbreviations. In the trans-Golgi apparatus, mammalian-derived GalT and SiaT enzymes expressed in Leishmania catalyze the reaction leading to the completion of N-glycans. These N-glycans are homogeneous, fucose-free, but branched, sialylated humanized glycans that mediate different expected functions depending on whether they are linked to α2,3 or α2,6 sialic acid (Neu5Ac).

[0098] [Figure 9]Figure 9: UPLC isolation and MS m / z determination of RF-labeled N-glycans or simulated treatments released by PNGase F or PNGase A from all Leishmania talentrae cell pellets in three different wt strains. (Man)3(GlcNAc)2=Man3 was detected by MS with calculated m / z values ​​of 1222.7166; [M+Na]+ 1244.6985; [M+K]+ 1260.6724. Man3 was observed in all pellet samples with RT values ​​of 9.16; 9.27; 9.34 when released by PNGase F or PNGase A, but not in simulated treatment samples. Simulated treatment samples showed contamination of the strains (middle and bottom panels), for example, from cell wall glycolipids (with polyhexose signatures). At RT 15.76, another peak was observed during the FLR trace, but despite obtaining a good MS spectrum, the compound could not be identified. Normally, not much background is observed during the FLR trace, but considerable background was observed in the MS trace at early RTs (<8 min RT, not shown).

[0099] [Figure 10] Figures 10A and 10B: HPLC separation of 2-AB labeled N-glycan reaction products after in vitro activity assays of SfGnT-I, TbGnT-I, or TbGnT-II. Immunoconcentrates (Figure 10A) or solubilized fractions (Figure 10B) from recombinant L. talenttrae expressing SfGnT-I, TbGnT-I, or TbGnT-II were used as substrates in in vitro assays against free porcimannose (Man3). Washed and 2AB-labeled reaction products were separated by HPLC and compared with standard Man3-2AB, G0-Gn(NGA3-N), and G0(NGA2). The peak at RT 76.7' indicates the activity of heterologous GnT-I enzymes, marked with asterisks.

[0100] [Figure 11]Figures 11A and 11B: HPLC separation of 2-AB labeled N-glycan reaction products after MGAT1 in vitro activity assay. Immunoenriched MGAT1 from recombinant L. talentlae cells expressing MGAT1, including SN or pellets, as well as solubilized fractions and lysates, was used in in vitro assays against: (Figure 11A) 2AB-labeled high mannose (Man5), a commonly used biosynthetic acceptor of MGAT1 in the known N-glycosylation pathway, or (Figure 11B) 2AB-portimannose (Man3) as a substrate mimicking the Man3 acceptor for Golgi extension in the Leishmania N-glycan pathway. Lysates from wt L. talentlae cells were used in these reactions as a negative control. Washed 2AB-labeled reaction products were separated by HPLC and compared to standards. Figure 11A) Man5 or Figure 11B) Man3; G0-Gn (NGA2-N, green). The peaks marked with asterisks indicate the activity of heterologous GnT-I enzymes.

[0101] [Figure 12] Figures 12A and 12B: HPLC separation of 2-AB labeled N-glycan reaction products after MGAT2 in vitro activity assay. Immunoenriched MGAT2 from recombinant L. talenttrae SN or pellet was used in in vitro assays against 2AB-labeled G0-Gn(NGA2-N), a commonly used biosynthetic acceptor for MGAT2 in the known N-glycosylation pathway (Figure 12A), or against 2AB-portion mannose (Man3), a substrate mimicking the Man3 acceptor for Golgi apparatus extension in the Leishmania N-glycan pathway, which is not extended by MGAT2 (Figure 12B). Washed 2AB-labeled reaction products were separated by HPLC and compared with standard Man3, G0-Gn(NGA2-N), and G0(NGA2). Peaks marked with asterisks indicate the activity of heterologous GnT-II enzymes.

[0102] [Figure 13]Figures 13A, 13B, and 13C: HPLC separation of 2-AB labeled N-glycan reaction products after SfGnT-II in vitro activity assay. Crude lysates from recombinant L. talenttrae expressing SfGnT-II were used in in vitro assays against: 2AB-portimannose (Man3) as a substrate mimicking the Man3 acceptor for Golgi extension in the Leishmania N-glycan pathway (Figure 13A); 2AB-labeled G0-Gn(NGA2-N), a commonly used biosynthetic acceptor for Gnt-II reactions in known N-glycosylation pathways (Figure 13B); or 2AB-labeled Man5, which is not a substrate for GnT-II (Figure 13C). Washed 2AB-labeled reaction products were separated by HPLC and compared with standard Man3, G0-Gn(NGA2-N), and Man5. Peaks marked with asterisks indicate the activity of heterologous GnT-II enzymes.

[0103] [Figure 14] Figures 14A, 14B, and 14C: HPLC separation of 2-AB labeled N-glycan reaction products after B4GALT1 in vitro activity assay. Figure 14A: Immunoenriched B4GALT1 from recombinant L. talenttrae was used in an in vitro assay against 2AB-G0-Gn(NGA2-N). Washed 2AB-labeled reaction products were separated by HPLC and compared with standard G0-Gn(NGA2-N), G1(NA2G1), and NA2(G2). Peaks marked with stars indicate the activity of heterologous GalT enzymes. Figure 14B shows the expected reaction results after the B4GALT1 in vitro assay, illustrating the schematic glycoform representation of the composition of a commercially available NGA2-N(G0-Gn) standard and the G1-Gn form on two different branches. Figure 14C shows the expected reaction product when G0(NGA2) is used as a substrate for GalT.

[0104] [Figure 15]Figure 15: MALDI-TOF MS spectra of fully methylated and PNGase F-released wt and glycosylated Leishmania talentrae N-glycans in the range m / z 1000–2200. Glycoform annotations are placed above the m / z peaks.

[0105] [Figure 16] Figure 16: MALDI-TOF / TOF MS fragmentation spectra of identified glycoforms. The fragment ions present in the spectra of wt St10569 at m / z 1171.6 (=Man3) (top), recombinant St11707 expressing SfGnT-I at m / z 1416.7 (=G0-Gn) (middle), and recombinant St12320 co-expressing SfGnT-I and SfGnT-II at m / z 1661.8 (=G0) confirm the presence of fully methylated N-glycans Man3 (top), G0-Gn (middle), and G0 (bottom).

[0106] [Figure 17]Figures 17A, 17B, and 17C: Figure 17A shows m / z determination by MS of RF-labeled N-glycans or simulated treatments released by PNGase F from UPLC-isolated and recombinant glycosylated Leishmania talenttrae cell pellets, with dashed lines representing background peaks observed in the simulated samples. RF-labeled N-glycans were obtained from: 16.260-St11707(SfGnT-I) with PNGase F; 16.261-St11707(SfGnT-I)-simulated; 16.263-St12525(SfGnT-I and SfGnT-II)-PNGase F; and 16.264-St12767(SfGnT-I and MGAT2)-PNGase F and superimposed. Glycan peaks confirmed at m / z are indicated by bold arrows. Figure 17B shows N-glycans derived from St 13065 (MGAT1 and MGAT2), and Figure 17C shows N-glycans from St 13066 (SfGnT-I and B4GalT1). The m / z values ​​were confirmed by the peaks graphically labeled and annotated above: [M+H] m / z=1222 Man3, m / z=1425 G0-Gn, m / z=1628 (G0), and m / z=1587 (G1-Gn). Unlabeled peaks were also observed in the simulated digest and were presumed to contain cell wall glycolipid fragments (dashed arrow, "background").

[0107] [Figure 18] Figure 18: N-glycan elongation of recombinant hEPO expressed in glycosylated leishmania. SfGnT-I, a candidate GnT-I, was recombinantly expressed to elongate the native N-glycan to G0-Gn. The recombinant target protein, human erythropoietin (rhEPO), was expressed alone (St11521) or co-expressed with SfGnT-I (St11895). rhEPO was secreted via the secretory pathway of leishmania and purified from the culture supernatant. Coomassie SDS-PAGE of the two purified EPO samples. Molecular weight changes when GlcNAc elongation is added until high molecular weight is reached.

[0108] [Figure 19]Figure 19: Occupation of rhEPO sites by peptide mapping. Light gray: EPO peptides in the flow-through fraction of the glycopeptide concentration process. Gray: Peptides present in the flow-through fraction and the concentrated glycopeptide fraction. Dark gray: EPO peptides containing N-glycosylation sites N24 and N38, which are found in the deamidated form of the concentrated glycopeptide fraction after deglycosylation by PNGase A+F. This peptide was not detected in its non-glycosylated form. Peptides with N-glycosylation site N83 were not explicitly detected in this study (neither in their non-glycosylated nor deglycosylated forms). N-glycosylation sites are shown in bold. Sequence ID 108 is shown.

[0109] [Figure 20] Figure 20: Glycopeptide of rhEPO containing identified N-glycans. N-glycosylation sites N24 and N38 are indicated. Sequence ID 109 is shown.

[0110] [Figure 21] Figures 21A and 21B: Identification of native secreted glycoproteins. Figure 21A shows the workflow within a rectangular box, with Coomassie SDS-PAGE elution profiles (E1-E5) after ConA enrichment. Major proteins were electrophoresed between 60 and 80 kDa. Elutions were used for N-glycan release and 2AB labeling, and proteins were identified using proteomics techniques with peptide mapping. Figure 21B: Identified proteins are shown along with the number of peptides identified with sequence coverage of >96% probability.

[0111] [Figure 22]Figures 22A and 22B: Identification of the processed N-terminus of invertase, a native secreted glycoprotein. Figure 22A: The workflow is summarized in a square frame. The supernatant of St10881 was precipitated with (NH4)2SO4 and purified using ConA. ConA elutes E1-E8 are shown in Coomassie SDS PAGE. After size exclusion, the most prominent secreted proteins other than rhEPO were recovered and subjected to EDMAN N-terminal sequencing. Figure 22B shows the sequence of invertase identified by peptide mapping, with the peptide indicated by the light gray mark being identified. The circles indicate the processed N-terminus from EDMAN N-terminal sequencing. [ka] This indicates that sequence number 110 is shown.

[0112] [Figure 23] Figure 23: rhEPO was secreted when it was fused to an invertase-derived invertase secretion sequence. To ensure secretion into the supernatant, EPO was expressed with two different signal peptides for secretory transition. Either immunoblotted SDS-PAGE (left) or Coomassie-stained SDS-PAGE (right) was filled with TCA precipitate supernatant equivalent to 15 OD cells. Both rhEPO mutants, each using five different monoclones, possessing either an LMSAP secretion signal (St11376) or an invertase-derived secretion signal (St11377), were observed in the SNs, migrating to molecular weights corresponding to their N-glycosylation form.

[0113] [Figure 24]Figures 24A and 24B: Domain predictions of native Golgi-localized proteins. The majority of Golgi-localized glycosyltransferases are type II membrane proteins with a short N-terminal cytoplasmic domain, a helical TM-domain of approximately 20 amino acids, a stem domain, and a C-terminal globular catalytic domain within the lumen of the secretory pathway (Kellokumpu et al., 2016). Figure 24A shows a representative example of a secretory signal and N-terminal CTS followed by a globular / catalytic domain, Gnt(Gnt-x) or SfGnT-I. Figure 24B shows native and putative Golgi proteins MAN1, LtaNTPDase 1, and 2 from L. talenttrae, identified by bioinformatics comparison and presented with the predicted CTS domain amino acid sequences below the schematic bars. The GRIP domain was identified in LTAR_110005600.1 (previous nomenclature: LtaP11.0070). Its C-terminal amino acid sequence is shown below the bar. Sequence IDs 24-27 are shown.

[0114] [Figure 25] Figures 25A, 25B, and 25C: Hybrid designs for Gnt domain prediction and improvement of Golgi targeting, retention, and homo- or heterodimerization. Gnt-X is shown as a white bar, which is Sf-Gnt-I, as in the example shown. The dark gray bar is Gnt-Y, which is MGAT1 in the example shown. The numbers below indicate the fusion point of the hybrid design behind the CTS of Gnt-y / MGAT1. Figure 25B shows the N-terminal 90, 110, 130, or 150 amino acids of Gnt-Y / MGAT1 fused as a nucleotide sequence to the nucleotides of Gnt-X / SfGnT-I in N-terminal cleavage form (Δ90, Δ110, Δ130, Δ150 amino acids). The full-length (FL) Gnt-Y (MGAT1) is fused to either the FL Gnt-X / SfGnT-I (top) or the Gnt-X / SfGnT-I (bottom) with the CTS cleaved at the N-terminus, as shown in Figure 25C.

[0115] [Figure 26] Figures 26A and 26B: Growth behavior of recombinant cells expressing rhEPO or SfGnT-I over time, as well as the expression and localization of rhEPO or SfGnT-I. Growth of cells expressing rhEPO (St11357) or SfGnT-I (St11521) was monitored in stationary cultures versus shaking cultures. Growth (OD) and shape are shown graphically over time as shown in Figure 26A. The expression rate and localization of rhEPO or SfGnT-I over time were determined by immunoblotting using anti-Strep antibody in TCA precipitate supernatant (SN) and whole cell extract (WCE) (Figure 26B).

[0116] [Figure 27] Figures 27A and 27B: Localization of SfGnT-I, TbGnT-I, and TbGnT-II. Immunoblot (Figure 27A) detects streptavidin-enriched Strep-tagged Sf-GnT-I from cell lysates precipitated with TCA or solubilized with Triton-X from a culture SN equivalent to 15 OD. TbGnT-I and TbGnT-II derived from Trypanosomes and tagged with HA were detected by immunoblot in the crude fraction and the Triton-X solubilized fraction (Figure 27B).

[0117] [Figure 28] Figures 28A and 28B: Localization of hybrid Gnt mutants of SfGnT-I. Native SfGnT-I, Man1-CTS, or CTS hybrids of LTaNTPDase 1 or 2 were expressed and precipitated from culture SN (Figure 28A) or from crude lysate homogenized from triton-containing (TrX+), triton-free (TrX-), or insoluble (Ins) portions (Figure 28B).

[0118] [Figure 29]Figure 29: Localization and affinity enrichment of MGAT1. HA-tagged MGAT1 was detected by immunoblotting from purified affinity enrichment fractions derived from either recombinant cell culture SN or pellets. Elu represents the eluate used in the in vitro assay. For localization, MGAT1 was detected in lysates homogenized from triton-containing (TrX+), triton-free (TrX-), insoluble portions (Insol. fr.), or residues.

[0119] [Figure 30] Figure 30: Localization and affinity enrichment of SfGnT-II. HA-tagged SfGnT-II was detected by immunoblotting from purified (Purif.) affinity enrichment fractions derived from either recombinant cell culture SN or pellets. Elu is the eluate used in the in vitro assay. For localization, SfGnT-II was detected in lysates homogenized from triton-containing (TrX+), triton-free (TrX-), insoluble portions (Insol. fr.), or residues.

[0120] [Figure 31] Figures 31A and 31B: Localization and affinity enrichment of MGAT2. HA-tagged MGAT2 was detected by immunoblotting from purified (Purif.) affinity enrichment fractions (W, wash; Elu, elution) derived from either recombinant cell culture SN or pellet (Pell). Cells were grown for 96 hours or 72 hours. Elu is the eluate used in the in vitro assay. For localization, SfGnT-II was detected in homogenized lysates from triton-containing (TrX+), triton-free (TrX-), insoluble portion (Insol. fr.), or residue (Figure 31A). Localization and expression levels of MGAT2 in precipitated cell culture SN across various passages are shown by anti-HA immunoblotting (Figure 31B).

[0121] [Figure 32]Figure 32: Customized Fc glycans mediate desired downstream effector functions. Immunoactivating glycans mediate effects such as ADCC, ADCP, and CDC. The glycans shown above are customized using the Leishmania platform for glycosylation of Fc molecules or antibody expression to mediate immunoactivation, immunoinhibition, and / or PK optimization. When a branched glycan terminates with α2,6-linked Neu5Ac, its function is activation; when it terminates with α2,3-linked Neu5Ac, its function is immunoinhibition. Supporting evidence is discussed in (Chen et al., 2017; Li et al., 2017). When a branched N-glycan terminates with sialic acid, regardless of its specific binding, the presence of sialic acid increases the circulating half-life, thereby improving PK. Furthermore, the immunotolerant effect of α2,6-linked Neu5Ac is expected to avoid the development of anti-drug antibodies (ADAs) to potentially immunogenic therapeutics. The glycan that inhibits lower immunity is a core α1,6 fucosylated glycan, a typical glycan derivative (without terminal sialic acid) derived from the CHO cell platform.

[0122] [Figure 33] Figure 33: Amino acid sequence of recombinant rituximab, an anti-CD20 monoclonal antibody. Protein sequences of the light chain (LC) and heavy chain (HC) with modified invertase secretion sequences are shown in bold, and the Fc N-glycosylation site is shown in bold and underlined. Sequence IDs 111 and 112 are shown.

[0123] [Figure 34]Figure 34: Expression cassette for stable integration into Leishmania talentrae containing recombinant rituximab anti-CD20 monoclonal antibody. Related regions: 5'ssu, a 5' homology region for site-specific integration into the ssu locus encoding 18S rRNA via homologous recombination; 5'UTR, a 5' untranslated end repeat containing a splice reader acceptor sequence; a light chain sequence optimized for codon use for Leishmania containing an invertase secretion signal; IR1, a first intergeneric region containing a 3'UTR for polyadenylation and a 5'UTR of downstream genes; a heavy chain sequence optimized for codon use for Leishmania containing an invertase secretion signal; IR2, a second intergeneric region containing a 3'UTR for polyadenylation and a 5'UTR of a downstream resistance marker (sat), followed by its 3'UTR and a schematic diagram of the 3' homology (3'ssu) region for site-specific recombination into the genome. KpnI is a restriction enzyme used to obtain the described expression cassette for transfection linear DNA excised from donor E. coli (E. coli) maintenance plasmids.

[0124] [Figure 35] Figure 35: Expression and purification of full-length monoclonal antibody from recombinant Leishmania talentrae secreting rituximab_LMTB. The workflow is indicated as a square box at the top. The recovered supernatant and dialyzed and 5-fold concentrated Coomassie-stained SDS-PAGE of SN are shown (left). Protein A elution (right) shows two major forms stained by Coomassie: one migrates as FL rituximab on SDS-PAGE under non-reducing conditions ("non"), and the other is a degradation product of approximately 100 kDa. Under reducing conditions ("red"), HC, degradation product, and LC are separated. Hydrophobic interaction chromatography (HIC) elution shows the separation of these two forms when separated under non-reducing or reducing conditions. The SDS-PAGE gel is stained by Coomassie.

[0125] [Figure 36]Figures 36A and 36B: Comparison of rituximab_LMTB purified from recombinant leishmania and Mabthera®, a commercially available product of the same protein expressed in CHO cells. Figure 36A: Coomassie-stained SDS-PAGE shows FL antibodies under non-reducing conditions with or without PNGase F treatment (+) for N-linked glycan release. Figure 36B shows a schematic diagram of the antibody molecule, its domains, and disulfide crosslinks. N-sugar sites are indicated by carbohydrates. The left side of the molecule in the diagram shows the homogeneous Man3 N-glycan of the leishmania-derived antibody, while the right side shows the expected minute heterogeneity of N-glycan from the CHO-expressed antibody.

[0126] [Figure 37] Figure 37: Capillary gel electrophoresis and comparison of rituximab_LMTB purified from recombinant leishmania and Mabthera®, a commercially available product of the same protein expressed in CHO cells. CGE shows FL antibodies under non-reducing conditions with or without PNGase F treatment for N-linked glycan release.

[0127] [Figure 38] Figures 38A and 38B: Size exclusion chromatography and comparison of rituximab LMTB purified from recombinant leishmania (Figure 38B) and Mabthera®, a commercially available product of the same protein expressed in CHO cells (Figure 38A).

[0128] [Figure 39] Figures 39A and 39B: RF-based N-glycan profiling and comparison of rituximab_LMTB (Figure 39B) purified from recombinant leishmania and Mabthera® (Figure 39A), a commercially available product of the same protein expressed in CHO cells. N-glycans were released using PNGase F and labeled with RF. UPLC separation is shown, and m / z values ​​are confirmed for N-glycans annotated above the peak.

[0129] [Figure 40] Figure 40: N-glycan profiling and quantitative comparison using complete methylation of rituximab_LMTB purified from recombinant leishmania and Mabthera®, a commercially available product of the same protein expressed in CHO cells. N-glycans were released using PNGase F and completely methylated. The relative intensity (%) of N-glycans was calculated to establish N-glycan profiles for each spectrum. For this purpose, the sum of the intensities of the deisotized N-glycan peaks was determined and set to 100%. Subsequently, the relative intensity (%) of each glycan was determined in relation to this value. The black bars represent rituximab_LMTB and the gray bars represent Mabthera® (Roche). m / z values ​​are confirmed for N-glycans annotated above the peaks, including confirmation by their fragmentation spectra.

[0130] [Figure 41] Figures 41A and 41B: N-glycan profiling and comparison using complete methylation of rituximab_LMTB (Figure 42B) purified from recombinant leishmania and Mabthera® (Figure 42A), a commercially available product of the same protein expressed in CHO cells. MALDI-TOF MS spectra of fully methylated N-glycans released from Mabthera® by PNGase F at three different acquisitions (Figure 48A) and MALDI-TOF MS spectra of fully methylated N-glycans released from rituximab_LMTB by PNGase F at two independent acquisitions (Figure 42B).

[0131] [Figure 42]Figure 42: Microheterogeneity and quantitative comparison of N-glycans of rituximab_LMTB purified from recombinant leishmania, Mabthera® (a commercial product of the same protein expressed in CHO cells), and the glycosylated third-generation anti-CD-20 Gazyvaro® (Roche). N-glycans were released using PNGase F and fully methylated. The relative intensity (%) of N-glycans was calculated to establish the N-glycan profile for each spectrum. For this purpose, the sum of the intensities of the deisotized N-glycan peaks was determined and set to 100%. Subsequently, the relative intensity (%) of each glycan was determined in relation to this value. The gray bars represent rituximab_LMTB, the dark gray bars represent Mabthera® (Roche), and the light gray bars represent Gazyvaro® (Roche). The m / z values ​​are confirmed for N-glycans annotated above the peaks, including confirmation by their fragmentation spectra.

[0132] [Figure 43]Figures 43A and 43B: Functional studies of rituximab_LMTB purified from recombinant leishmania for antigen binding. Figure 43A shows FACS histograms of Mabthera® or rituximab_LMTB compared to control IgG1κ. The functionality of anti-CD20 antibodies was tested using Raji cells expressing the CD20 antigen. The staining order was FcR blocking at the indicated concentrations, IgG1 blocking, primary antibody Mabthera®, rituximab_LMTB, or negative control IgG1κ, followed by secondary antibody anti-IgG1 APC. After gated, each sample represents the analysis of 10,000–13,000 cells. Arrows indicate the mean fluorescence shift showing the binding of the anti-CD20 antibody to the CD20 antigen on the surface of Raji cells. Figure 43B shows the results for recombinantly expressed CD20 as a GST fusion in E. coli spotted on a membrane at increasing concentrations. Membrane strips were incubated with various concentrations of Mabthera®, rituximab_LMTB, or a control antibody (anti-maltose binding protein, E. coli-derived anti-GroEL heat shock protein, or anti-His IgG antibody), or without a primary antibody. After washing, the secondary antibody was anti-human IgG-HRP.

[0133] [Figure 44] Figure 44: A schematic diagram and summary comparing rituximab_LMTB purified from recombinant leishmania and Mabthera®, a commercially available product of the same protein expressed in CHO cells. The N-terminal sequences of HC and LC, the C-terminal amino acids, and the glycan profile are shown on the right for rituximab_LMTB and on the left for Mabthera®. All disulfide crosslinks shown were identified in both samples.

[0134] [Figure 45]Figure 45: N-glycan profiling and quantitative comparison using complete methylation of various kinetoplasts. N-glycans from Crithidia fasciculata, Crithidia deanei (Angomonas), and Phytomonas davidii were released after delipidation with PNGase F and then completely methylated. The relative intensity (%) of the N-glycans was calculated to establish N-glycan profiles for each spectrum. For this purpose, the sum of the intensities of the deisotized N-glycan peaks was determined and set to 100%. Subsequently, the relative intensity (%) of each glycan was determined in relation to this value. The m / z values ​​were confirmed for all N-glycans annotated above the peak, including confirmation by their fragmentation spectra.

[0135] [Figure 46] Figures 46A and 46B: Localization of hybrid Gnt mutants of SfGnT-I. Native SfGnT-I or hybrids of the SfGnT-I catalytic domain, schematically shown in Figure 46B, were expressed and precipitated from culture SN or from crude lysate (WCE) containing triton X, as shown in Figure 46A.

[0136] [Figure 47] Figures 47A and 47B: Quantification and comparison of N-glycans using RF in Leishmania expressing various Gnt-I strains (Figure 47A) and Leishmania expressing various Sf-Gnt-I hybrids (Figure 47B). Total N-glycans from the cell pellet were released using PNGase F and labeled with RF. The relative quantification of UPLC peaks is shown as the percentage of G0-Gn conversion, representing the in vivo addition of the initial GlcNAc for glycosylation. Error bars show the standard deviation of several independent experiments. m / z values ​​were confirmed for UPLC peaks containing G0-Gn and Man3, but no other peaks differed between the simulated sample and the PNGase F-releasing sample.

[0137] [Figure 48] Figures 48A and 48B show the localization (Figure 48A) and relative activity (Figure 48B) of various sialyltransferases expressed in leishmania. HA-tagged sialyltransferases are detected by anti-HA immunoblotting from affinity-enriched fractions (elus) derived from triton-soluble cell lysates compared to cell culture SNs. Elus are the elutes used in in vitro assays (Figure 48A).

[0138] [Figure 49] Figures 49A, 49B, 49C, and 49D: In vitro activity of HA affinity-enriched sialyltransferase expressed in Leishmania against 2-AB labeled G2 standards. Figure 49A: Mouse ST6GAL1; Figure 49B: Rat ST6GAL1; Figure 49C: Bacterial CstI containing optimized invertase secretion signal; Figure 49D: Mouse ST3GAL3. In vitro reactions of HA-enriched sialyltransferase against G2 standards are shown, with RT comparisons performed against commercially available standard G2 and sialylated G2S2 (dashed arrows). In vitro reactions are indicated by arrows, and the resulting glycan peaks are indicated by asterisks.

[0139] [Figure 50]Figures 50A, 50B, 50C, 50D, and 50E: mST6GAL1 expressed and enriched by Leishmania can sialylate folded full-length mAbs of galactosylated N-glycans. Figure 50A: HA affinity enrichment of Leishmania expressing mouse ST6 from cell lysates, and testing of its activity against a 2AB-labeled G2 standard (Figure 50B) and its activity against MabThera Fc N-glycan (Figure 50C), which have been shown to be 100%. N-glycan composition was evaluated by RF MS (Figure 50D) against mAbs (light gray) and after in vitro reactions of protein A-purified MabThera using either simulated lysate (black bars) or HA-enriched mST6GAL1 (dark gray bars). A summary of all single-chain and branched sialylated glycans is shown in Figure 50E.

[0140] [Figure 51] Figure 51: mST6GAL1 expressed and enriched by Leishmania can sialylate folded full-length mAbs of galactosylated N-glycans, as shown by UPLC analysis, which has an N-glycan RF-MS profile of a simulated reactant overlaid with the ST6 reactant.

[0141] [Figure 52] Figures 52A and 52B: Glycan-modified strains that are stably transfected with Sf-Gnt-I and MGAT2 and express rituximab episomalally generate G0 N-glycans on rituximab. The strains were grown in 1 L of water, and the secreted rituximab was purified from the supernatant using protein A (Figure 52A). The supernatant was then used for PNGase F and RF-MS analysis (Figure 52B), and Man3 was semi-quantified.

[0142] [Figure 53] Figure 53: A schematic representation of the CMP-Neu5Ac pathway, including prokaryotic and eukaryotic enzymes that can be recombinantly added to Leishmania for glycosylation. [Modes for carrying out the invention]

[0143] (7. Detailed explanation) This invention relates to the properties of native host cells, as well as N-acetylglucosamine transfer. A galactosyltransferase and sialyltransferase are included in the 1 By combining heterologous expression of a series of glycosyltransferases, mammals, for example, To create a host strain for the production of therapeutic glycoproteins in humans, It produces uniform and fully functionally customized N-glycans with high site occupancy. This relates to eukaryotic host cells of the order Kinetoplastes that are modified by sea urchins.

[0144] This invention relates to the enzyme activity of mammals (e.g., N-acetylglucosamine elongation, galactosyl (Substances involved in sialylation and sialylation) are placed in the intracellular compartment of the eukaryotic host cell of kinetoplasts. Nucleic acid molecules and combinations that can be used to effectively target and express We provide a natural library. CMP-Sial for the production of sialylated glycoproteins. The design of the acid biosynthesis pathway is also provided.

[0145] (7.1 Host cells) In one embodiment, the present invention expresses any desired gene involved in glycosylation. This provides modified host cells that can be used for targeting. The present invention relates to a host strain for the production of therapeutic glycoproteins in mammals, for example, humans. For example, N-acetylglucosamine transferase, galactosyl transferase , and a series of heterogeneous glycosyltransferases, including sialyltransferase Expression leads to the production of functionally customized and uniform N-glycans on proteins. Provides eukaryotic host cells that have been modified in this way.

[0146] This invention can be used to express and target full-length therapeutic antibodies. The program also provides modified host cells that can be erythropoietin-resistant or anti-CD cells. These novel host cells can be erythropoietin-resistant or anti-CD cells. Homogeneous and functionally customized N-glycans on glycoproteins such as 20 (rituximab) It synthesizes, expresses, and secretes it.

[0147] The inventions described herein include certain enzymes, genes, and plasmids disclosed herein. , and not limited to the use of constructs. Those skilled in the art will know N-acetylglucosamine Transferase, galactosyltransferase, sialyltransferase, and any homologs, mutants, and inducers of genes involved in the synthesis of CMP-Sia biosynthesis pathway enzymes A conductor can be used.

[0148] In certain embodiments, provided herein are (a) encoding a target protein (b) recombinant nucleic acids; and (b) recombinant nucleic acids comprising a heterologous glycosyltransferase. It is a Leishmania host cell. In one embodiment, a heterologous glycosyltransfer -ase is N-acetylglucosamine transferase; and / or heterologous galactosyltran Spherase; and / or heterologous sialyltransferase. In some embodiments, In this specification, two or more N-acetylglucosamine transferers are provided. The host cell contains the enzyme. In another embodiment, the cell contains a heterologous sialyltransferase. The host cell can produce heterologous CMP-Sia biosynthetic pathway proteins that can generate CMP-NeuAc. It also includes.

[0149] In one embodiment, the present invention provides for the production of glycosylated proteins. Leishmania host cells capable of doing so, where said Leishmania host cells are (i) heaven Natural OST or heterologous / recombinant OST; (ii) heterologous N-acetylglucosamine transferase, galact Tosyltransferase, sialyltransferase, and CMP-Sia biosynthesis pathway enzymes , or nucleotides encoding modified versions thereof; and (iii) recombinant target proteins It contains nucleotides that encode modified versions of the target protein and recombinant protein. In one embodiment, the N-acetylglucosamine transferase, galactosyl The amino acid sequences of lanceferase and / or sialyltransferase are shown in Table 9. N-acetylglucosamine transferase and galactosyl transferase are used. ze, or sialyltransferase, or any functional homolog or iso thereof. It originates from a form or variant.

[0150] In another embodiment, provided herein are 1 or more derived from the N-glycan biosynthesis pathway The above endogenous enzymes are deleted, mutated, and / or functionally inactivated. It is a host cell. In a further embodiment, it is deleted in the host cell and mutated Endogenous enzymes that are and / or functionally inactivated are encoded by the alg gene. In another embodiment, provided herein are those derived from the N-glycan biosynthesis pathway. One or more genes encoding an endogenous enzyme are deleted, mutated, and / or are affected. These are host cells that are actively inactivated. Furthermore, in another way, N-glycan biosynthesis The genes (one or more) encoding endogenous enzymes (one or more) derived from the synthesis pathway are included in this technology. Using any of the standard techniques known in the field (e.g., site-directed homologous recombination or randomization) (By mutagenesis), deletion, mutation, and / or functional inactivation In one embodiment, the host cell is one or more of the N-glycan biosynthesis pathways. This is a Leishmania strain that does not contain resident enzymes.

[0151] In one embodiment, Leishmania host cells are Leishmania talentrae cells It is a cell. In a particular embodiment, provided herein are (a) target protein (b) Recombinant nucleic acids encoding heterologous glycosyltransferases; and (b) Recombinant nuclei encoding heterologous glycosyltransferases. These are Leishmania talenttrae host cells containing acid. In one embodiment, heterologous Lycosyltransferase is N-acetylglucosaminetransferase; and / or Heterogalactosyltransferase; and / or heterosialyltransferase. In some embodiments, provided herein are two or more N-acetylglucos The host cell contains samine transferase. In another embodiment, heterologous sialyl Host cells containing transferase can produce heterologous CMP-Sia It also contains biosynthetic pathway proteins.

[0152] In one embodiment, the present invention provides for the production of glycosylated proteins. Leishmania talenttrae host cells capable of this, and here, the Leishmania talenttrae Rentrae host cells are (i) natural OST or heterologous / recombinant OST; (ii) heterologous N-acetylglucosamine Transferase, galactosyltransferase, sialyltransferase , and nucleotides encoding enzymes in the CMP-Sia biosynthesis pathway, or modified versions thereof; (iii) Encoding recombinant target proteins and modified versions of recombinant target proteins It contains the nucleotide. In a further embodiment, the N-acetylglucosamine tran Spherases, galactosyltransferases, and / or sialyltransferases The amino acid sequence of Ze is N-acetylglucosamine transferase, as shown in Table 9. Galactosyltransferase, or sialyltransferase, or these It is derived from any functional homolog, isoform, or variant of [the specified entity].

[0153] In another embodiment, provided herein are leishmania signals and / or The retained sequence is N-acetylglucosamine transferase, galactosyl transferase The signal is attached to the enzyme and / or sialyltransferase, where the signal The sequence is the N-acetylglucosamine transferase, galactosyl transferase The sialyltransferase and / or sialyltransferase target the endoplasmic reticulum of Leishmania host cells. The retaining sequence is set, and the N-acetylglucosamine transferase, galact Siltransferase and / or sialyltransferase in the endoplasmic reticulum or Golgi apparatus It is a host cell that holds the N-acetylg Lucosamine transferase and / or galactosyl transferase into host cells It is retained in the endoplasmic reticulum. In another embodiment, the retaining sequence is the N-acetylglucosamine Lansferase and / or galactosyltransferase are delivered to the cis-Golgi region of the host cell. The image is retained. In another embodiment, the retaining sequence is the N-acetylglucosamine tran Spherase and / or galactosyltransferase into the intermediate Golgi compartment of host cells To retain. In another embodiment, the retaining sequence contains the galactosyltransferase It is retained in the trans-Golgi compartment of the host cell. In another embodiment, the retaining sequence is shea The lyltransferase is retained in the trans-Golgi compartment of the host cell. In another embodiment, The retaining sequence is sialyltransferase and galactosyltransferase It is retained in the trans-Golgi compartment of the host cell. In another embodiment, the signal sequence is , processed and removed. In a further embodiment, the retaining sequence is leash This is a cytoplasmic-transmembrane-stem (CTS) sequence derived from the mania-talentrae protein. In this embodiment, the CTS sequence is Leishmania talentrae MAN1, NTPDase 1, Or derived from NTPDase 2. In another embodiment, the CTS sequence is sequence number 24, sequence number The sequence of sequence number 25 or sequence number 26, or a functionally active fragment thereof, is included in another embodiment. In another embodiment, the CTS is derived from Leishmania talentrae MAN1. The CTS sequence comprises the sequence of SEQ ID NO: 24 or a functionally active fragment thereof. In further embodiments... Furthermore, the retaining sequence includes a GRIP sequence derived from Leishmania or its functionally active fragment. In another embodiment, the GRIP sequence includes the sequence of SEQ ID NO: 27 or a functionally active fragment thereof. In a further embodiment, the retention sequence is CT derived from Leishmania protein. S sequence or its functionally active fragment and GRIP sequence or its functionally active derived from Leishmania Contains sexual fragments.

[0154] Another embodiment provided herein is (a) encoding a target protein Recombinant nucleic acids; and (b) Recombinant nucleic acids encoding N-acetylglucosamine transferase Leishmania host cells containing . In another embodiment, provided herein (a) recombinant nucleic acids encoding a target protein; and (b) galactosyltransferers A leishmania host cell containing recombinant nucleic acid encoding ze. In yet another embodiment... Provided herein are (a) recombinant nucleic acids encoding a target protein; and (b ) Leishmania host cells containing recombinant nucleic acid encoding sialyltransferase Yes. In a further embodiment, a host cell containing a heterologous sialyltransferase is It further includes heterologous CMP-Sia biosynthetic pathway proteins capable of generating CMP-NeuAc.

[0155] Another embodiment provided herein is (a) encoding a target protein Recombinant nucleic acids; (b) Recombinant nucleic acids encoding N-acetylglucosamine transferase; and (c) Leishmania accommodation containing recombinant nucleic acid encoding galactosyltransferase It is a principal cell. In another embodiment, provided herein are (a) a target protein (b) Recombinant nucleic acids encoding N-acetylglucosamine transferase (c) Recombinant nucleic acids; and (c) Leishma containing recombinant nucleic acids encoding sialyltransferase It is a near host cell. In yet another embodiment, provided herein is (a) a target (b) Recombinant nucleic acids encoding target proteins; (b) Recombinant nucleic acids encoding sialyltransferase (c) nucleic acids; and a leash containing recombinant nucleic acids encoding galactosyltransferase Maniac host cells. In a further embodiment, heterologous sialyltransferase is used. Host cells containing this protein can produce CMP-NeuAc, a heterologous CMP-Sia biosynthetic pathway protein. It also includes.

[0156] Another embodiment provided herein is (a) encoding a target protein (b) Recombinant nucleic acids; (c) Recombinant nucleic acids encoding N-acetylglucosamine transferase; Recombinant nucleic acids encoding galactosyltransferase; and (d) sialyltransferase Leishmania host cells containing recombinant nucleic acids encoding the enzyme. Further embodiments In this process, host cells containing heterologous sialyltransferase produce CMP-NeuAc. It further includes heterologous CMP-Sia biosynthetic pathway proteins that can perform this.

[0157] Another embodiment provided herein is (a) encoding a target protein Recombinant nucleic acids; and (b) Recombinant nucleic acids encoding N-acetylglucosamine transferase Leishmania talenttrae host cells containing the above. In another embodiment, this Provided are (a) recombinant nucleic acids encoding a target protein; and (b) galactosyl These are Leishmania host cells containing recombinant nucleic acids that encode lanceferase. Another embodiment provided herein is (a) a set encoding a target protein (b) Recombinant nucleic acids; and (b) Leishma containing recombinant nucleic acids encoding sialyltransferase It is a near-host cell. In a further embodiment, it contains a heterologous sialyltransferase. The host cell can produce heterologous CMP-Sia biosynthetic pathway proteins that can generate CMP-NeuAc. It also includes.

[0158] Another embodiment provided herein is (a) encoding a target protein Recombinant nucleic acids; (b) Recombinant nucleic acids encoding N-acetylglucosamine transferase; and (c) Leishmania containing recombinant nucleic acid encoding galactosyltransferase Talenttrae host cells. In another embodiment, provided herein are (a) (b) Recombinant nucleic acid encoding a target protein; (b) N-acetylglucosamine transferase (c) Recombinant nucleic acids encoding sialyltransferase This includes Leishmania host cells. In yet another embodiment, provided herein (a) Recombinant nucleic acid encoding the target protein; (b) Sialyltransferase (c) Recombinant nucleic acids encoding; and (c) Recombinant nucleic acids encoding galactosyltransferase Leishmania host cells containing [the specified element]. In a further embodiment, heterologous sialiltran Host cells containing spherase can produce heterologous CMP-Sia biosynthesis, which involves generating CMP-NeuAc. It also contains pathway proteins.

[0159] Another embodiment provided herein is (a) encoding a target protein (b) Recombinant nucleic acids; (c) Recombinant nucleic acids encoding N-acetylglucosamine transferase; Recombinant nucleic acids encoding galactosyltransferase; and (d) sialyltransferase Leishmania host cells containing recombinant nucleic acids encoding the enzyme. Further embodiments In this process, host cells containing heterologous sialyltransferase produce CMP-NeuAc. It further includes heterologous CMP-Sia biosynthetic pathway proteins that can perform this.

[0160] In one embodiment, the term "different species" means originating from a different species. For example, The "heterogenetic glycosyltransferase" within the principal cell is derived from a species other than the host cell. It is a lycosyltransferase. In one embodiment, the term "heterogeneous" is used to mean different species. This refers to something derived from a specific strain. For example, "heteroglycosyltransferers" within host cells. "Ze" is a glycosyltransferase derived from a cell line other than the host cell. More specifically In a typical embodiment, the term "different species" means originating from a different genus. For example, host "Hexagram glycosyltransferases" within cells are glycosyltransferases derived from genera other than those of the host cell. It is a cosyltransferase.

[0161] In one embodiment, the method and composition provided herein are used together with the G Cosyltransferase is a gene modified from its wild-type gene to glycosyl It is a lanceferase. In a more specific example, such a glycosyltran The spherases are of the same species or strain. In other embodiments, such Lycosyltransferases belong to the same genus, species, or strain.

[0162] (7.2 Intracellular localization of glycosyltransferases) In some embodiments, the leishmania signal and retention sequence are N-acetylg Glucosamine transferase, galactosyl transferase, and / or sialyl It is attached to the transferase, and here the signal sequence is the N-acetylgluco Samine transferase, galactosyl transferase, and / or sialiltransferase The encephalase was targeted to the endoplasmic reticulum of a Leishmania host cell, and the retention sequence was , the N-acetylglucosamine transferase, galactosyl transferase, and The sialyltransferase is retained in the endoplasmic reticulum or Golgi apparatus. In one embodiment of the application, the leishmania signal and retention sequence are N-acetylglucosamine tran. It is attached to the transferase and sialyltransferase, and here the signal The sequences are N-acetylglucosamine transferase and sialyl transferase. The N-acetyl is targeted to the endoplasmic reticulum of Leishmania host cells, and the retaining sequence is the N-acetyl Glucosamine transferase and sialyl transferase are stored in the endoplasmic reticulum or Golgi apparatus. To be held in place. In some embodiments, the leishmania signal and retention sequence are Addition to N-acetylglucosamine transferase and galactosyltransferase The signal sequence is the N-acetylglucosamine transferase. and galactosyltransferase targets the endoplasmic reticulum of Leishmania host cells. The retaining sequence contains the N-acetylglucosamine transferase and galactosyl The transferase is retained in the endoplasmic reticulum or Golgi apparatus. In some embodiments, The leishmania signal and retention sequence are sialyltransferase and galactosyl It is attached to the transferase, and here the signal sequence is the sialyltrans Ferase and galactosyltransferase are injected into the endoplasmic reticulum of Leishmania host cells. The retaining sequence is configured to support the sialyltransferase and galactosyltransferase. The spherase is retained in the endoplasmic reticulum or Golgi apparatus.

[0163] In another embodiment, the leishmania signal and retention sequence are N-acetylglucosa It is attached to mintransferase, and here the signal sequence is the N-acetylg Lucosamine transferase is targeted to the endoplasmic reticulum of Leishmania host cells. The retaining sequence transfers the N-acetylglucosamine transferase to the endoplasmic reticulum or Golgi apparatus. In another embodiment, the leishmania signal and retention sequence are N-acetylated. It is fused to the N-terminus of luglucosamine transferase. In another embodiment, The leishmania signal and retention sequences are of N-acetylglucosamine transferase. It is fused to the C-terminus. In further embodiments, leishmania signaling and retention The sequence is not fused to the N-terminus of N-acetylglucosamine transferase. In one embodiment, the leishmania signal and retention sequence are N-acetylglucosamine It is not fused to the C-terminus of lanceferase. In another embodiment, leishmania The signal and retention sequences are located within the polypeptide of N-acetylglucosamine transferase. It is fused with one or more amino acids.

[0164] In another embodiment, the leishmania signal and retention sequence are galactosyltran It is attached to the spherase, where the signal sequence is the galactosyltransfer The gelase is targeted to the endoplasmic reticulum of Leishmania host cells, and the retaining sequence is the gas The lactosyltransferase is retained in the endoplasmic reticulum or Golgi apparatus. In other embodiments, The leishmania signal and retention sequence are located at the N-terminus of galactosyltransferase. It is fused to it. In another embodiment, the leishmania signal and retention sequence are fused to It is fused to the C-terminus of lactosyltransferase. In a further embodiment, The leishmania signal and retention sequences are fused to the N-terminus of galactosyltransferase. Not combined. In other embodiments, the leishmania signal and retention sequence are gal It is not fused to the C-terminus of the kutosyltransferase. In other embodiments, Lee The Schmaniah signal and retention sequence are located within the polypeptide of galactosyltransferase. It is fused with one or more amino acids.

[0165] In other embodiments, the leishmania signal and retention sequence are sialyltransfer It is attached to the sialyltransferase, where the signal sequence is the sialyltransferase. The vesicle of the Leishmania host cell is targeted, and the retaining sequence is the sialil Lansferase is retained in the endoplasmic reticulum or Golgi apparatus. In another embodiment, leash The maniac signal and retention sequences are fused to the N-terminus of the sialyltransferase. In another embodiment, the leishmania signal and retention sequence are sialyltransfer It is fused to the C-terminus of the gelase. In a further embodiment, leishmaniasigna The sialyltransferase and retention sequences are not fused to the N-terminus. Other implementations In this embodiment, the leishmania signal and retention sequence are of sialyltransferase Not fused to the C-terminus. In other embodiments, the leishmania signal and retention The column is fused to one or more amino acids within the polypeptide of sialyltransferase. ru.

[0166] In another embodiment, the retaining sequence is N-acetylglucosamine transferase and The galactosyltransferase is retained in the endoplasmic reticulum of the host cell. Another embodiment In this, the retaining sequence is N-acetylglucosamine transferase and / or galak Tosyltransferase is retained in the cis-Golgi compartment of the host cell. In another embodiment, The retaining sequence is N-acetylglucosamine transferase and / or galactosyl The transferase is retained in the intermediate Golgi compartment of the host cell. In another embodiment, The retention sequence retains galactosyltransferase in the trans-Golgi compartment of the host cell. In another embodiment, the retention sequence transmits sialyltransferase to the host cell It is held in the Lance Golgi compartment. In another embodiment, the holding array is sialyltrans Ferase and galactosyltransferase are retained in the trans-Golgi compartment of host cells. do.

[0167] In another embodiment, the signal sequence and / or retention sequence may be any leishmania The signal sequence or retention sequence is derived from the species. In a further embodiment, the signal The sequence and / or retained sequence is a signal sequence or derived from Leishmania talentrae. This is a retention sequence.

[0168] In a further embodiment, the retention sequence is Leishmania talentrae protein It is a cytoplasmic-transmembrane-stem (CTS) sequence derived from . In another embodiment, the CTS sequence is , derived from Leishmania talenttrae MAN1, NTPDase 1, or NTPDase 2. In this embodiment, the CTS sequence is the sequence of sequence number 24, sequence number 25, or sequence number 26. , or comprising a functionally active fragment thereof.

[0169] In another embodiment, the CTS is derived from Leishmania talentrae MAN1. In this embodiment, the CTS sequence includes the sequence of SEQ ID NO: 24 or a functionally active fragment thereof. In a further embodiment, the CTS sequence is derived from Leishmania talentrae MAN1. CTS and at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 8% 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical. In another embodiment, The CTS sequence is at least about 70%, 71% of the sequence of Sequence ID No. 24 or its functionally active fragment. , 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 Includes sequences that are %, 99%, or 100% identical.

[0170] In another embodiment, the CTS is derived from Leishmania talenttrae NTPDase 1. In another embodiment, the CTS sequence is the sequence of SEQ ID NO: 25 or its functionally active fragment. Includes. In a further embodiment, the CTS sequence is Leishmania talentrae NTPD - CTS derived from -ze1 and at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% They are identical by %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In one embodiment, the CTS sequence is the sequence of SEQ ID NO: 25 or its functionally active fragment and at least Approximately 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% Includes sequences that are %, 97%, 98%, 99%, or 100% identical.

[0171] In another embodiment, the CTS is derived from Leishmania talentrae NTPDase 2. In another embodiment, the CTS sequence is the sequence of SEQ ID NO: 26 or its functionally active fragment. Includes. In a further embodiment, the CTS sequence is Leishmania talentrae NTPD CTS derived from -ze2 and at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% They are identical by %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In one embodiment, the CTS sequence is the sequence of SEQ ID NO: 26 or its functionally active fragment and at least Approximately 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% Includes sequences that are %, 97%, 98%, 99%, or 100% identical.

[0172] In a further embodiment, the retaining sequence is a GRIP sequence derived from Leishmania or It contains a functionally active fragment of the sequence. In another embodiment, the GRIP sequence is the sequence of SEQ ID NO: 27 or It contains the functionally active fragment. In a further embodiment, the retention sequence is Leishmania. GRIP sequence derived from or its functionally active fragment and 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 They are 100% identical. In another embodiment, the GRIP sequence is the sequence of sequence number 27 or its Functionally active fragments and at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Includes sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0173] In a further embodiment, the retention sequence is a CTS derived from Leishmania protein. Sequence or its functionally active fragment and GRIP sequence or its functional activity derived from Leishmania Includes fragments. In another embodiment, the retention sequence is derived from leishmania protein. The substance or its functionally active fragment and 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 CTS sequences and those derived from Leishmania or their functionally active fragments, and 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%, These are GRIP sequences that are 97%, 98%, 99%, or 100% identical.

[0174] In another embodiment, the target protein includes a signal sequence derived from Leishmania. It has been modified to do so. In another embodiment, the signal sequence is Leishmania. This is a signal sequence derived from Talenttrae. In some embodiments, the signal sequence This includes the sequence of SEQ ID NO: 28 or SEQ ID NO: 29 or its functionally active fragment. Specific implementation In one embodiment, the signal sequence includes the sequence of SEQ ID NO: 28 or a functionally active fragment thereof. In another embodiment, the signal sequence is the sequence of SEQ ID NO: 28 or its functionally active cleavage. One piece and at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Includes sequences that are 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Other embodiments In this process, the signal sequence is processed and removed from the target protein.

[0175] (7.3 N-acetylglucosamine transferase) In certain embodiments, provided herein are (a) of Leishmania origin (b) the catalytic domain of N-acetylglucosamine transferase, not (b) leishma A high amino acid sequence that is involved in the localization and retention of the endoplasmic reticulum or Golgi compartment of the rhynchoplasmic reticulum, It is a hybrid N-acetylglucosamine transferase.

[0176] In one embodiment, the hybrid N-acetylglucosamine transferase is It originates from Leishmania talentrae.

[0177] In another embodiment, the hybrid N-acetylglucosamine transferase is The following has been modified to include a signal sequence and at least one retention sequence, where the The signal sequence transmits the N-acetylglucosamine transferase to Leishmania tarre The intracellular matrix targets the endoplasmic reticulum of the host cell, and the retaining sequence is the N-acetylglucosa The minttransferase is retained in the endoplasmic reticulum or Golgi apparatus.

[0178] In another embodiment, the hybrid N-acetylglucosamine transferase is The N-acetylglucosamine transferase is retained in the endoplasmic reticulum. In another embodiment, Furthermore, the hybrid N-acetylglucosamine transferase is used to transfer the N-acetylglucosamine. Cosamine transferase is held in the cis-Golgi apparatus. In another embodiment, the Hybrid N-acetylglucosamine transferase is used to transfer N-acetylglucosamine Lansferase is retained in the intermediate Golgi apparatus.

[0179] In a further embodiment, the retention sequence is a cytoplasmic-transmembrane-stem (CTS) sequence. In another embodiment, the CTS sequence is an amino acid of MAN1, NTPDase 1, or NTPDase 2. Includes a sequence. In another embodiment, the CTS sequence is sequence number 24, sequence number 25, or It comprises the sequence of sequence number 26, or a functionally active fragment thereof. In another embodiment, the CTS is M It contains the amino acid sequence of AN1. In another embodiment, the CTS sequence is the sequence of SEQ ID NO: 24 or It contains the functionally active fragment. In a further embodiment, the GRIP sequence is the A of SEQ ID NO: 27 Contains a mino acid sequence.

[0180] In another embodiment, the N-acetylglucosamine transferase is GnT-I In another embodiment, the N-acetylglucosamine transferase is GnT-II Yes. In another embodiment, the N-acetylglucosamine transferase is GnT-I and GnT-II. In one other embodiment, the N-acetylglucosamine transfer The enzyme is N-acetylglucosamine transferase listed in Table 9, or its Derived from a functional homolog, isoform, or variant. Any known in the art. N-acetylglucosamine transferase, or nucleic acid encoding it, as specified herein It can be used in accordance with the host cells and methods described.

[0181] In a specific embodiment, the N-acetylglucosamine transferase is human (H This is N-acetylglucosaminyltransferase 1 of omo sapiens. In another embodiment, In this context, the N-acetylglucosamine transferase is human mannosyl(α-1,6-)- It is the glycoprotein β-1,2-N-acetylglucosaminyltransferase. In this context, the N-acetylglucosamine transferase is used to transfer N-acetylglucosamine from the human species. It is homologous to cosamine transferase. For example, the N-acetylglucosamine N-transferase or the nucleic acid encoding it is human N-acetylglucosaminyl Lansferase 1 or mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosa Miniltransferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% , or they are 99% homologous.

[0182] In a specific embodiment, the N-acetylglucosamine transferase is used in the following N-acetylglucosaminyltransferase 1 from the fall armyworm (Spodoptera frugiperda) In another embodiment, the N-acetylglucosamine transferase is used by This is N-acetylglucosaminyltransferase 2 from the armyworm. One embodiment In this context, the N-acetylglucosamine transferase is used in the N- of the fall armyworm species. It is homologous to acetylglucosamine transferase. For example, the N-acetyl Luglucosamine transferase or the nucleic acid encoding it is used in the fall armyworm. N-acetylglucosaminyltransferase 1 or N-acetylglucosaminyltransferase Ferase 2 and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% phase They are the same.

[0183] In a specific embodiment, the N-acetylglucosamine transferase is used in tripartite transfers. N-acetylglucosaminyl transfer of Trypanosoma brucei It is Ze1. In another embodiment, the N-acetylglucosamine transferase is This is N-acetylglucosaminyltransferase 2 from Trypanosoma bruseyi. In one embodiment, the N-acetylglucosamine transferase is used to treat trypanosomes. • It is homologous to the N-acetylglucosamine transferase of the Bursey species. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is a tri Panosoma brusey's N-acetylglucosaminyltransferase 1 or N-acetyl Glucosaminyltransferase 2 and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9 They are 7%, 98%, or 99% homologous.

[0184] In a specific embodiment, the N-acetylglucosamine transferase is used in rats. This is N-acetylglucosaminyltransferase 2 from (Rattus norvegicus). In this configuration, the N-acetylglucosamine transferase is used in rat species N-acetyl It is homologous to glucosamine transferase. For example, the N-acetylgluco Sumintransferase or the nucleic acid encoding it is used in rat N-acetylglucosamine Nyltransferase 1 or N-acetylglucosaminyltransferase 2 and approximately 70%, They are 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0185] In a specific embodiment, the N-acetylglucosamine transferase is a bonobo Mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyl from Pan paniscus It is a transferase. To a more precise extent, the N-acetylglucosamine transfer Ferase is homologous to the N-acetylglucosamine transferase of bonobo species. For example, the N-acetylglucosamine transferase or the nucleus encoding it. The acid is the bonobo mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyl. Lansferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% They are % homologous.

[0186] In a specific embodiment, the N-acetylglucosamine transferase is used in dogs (C Mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylgluco It is a saminyltransferase. In one embodiment, the N-acetylglucosamine The transferase is homologous to the N-acetylglucosamine transferase in canine species. For example, the N-acetylglucosamine transferase or the code for it The nucleic acid involved is canine mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosamin. Lutransferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and They are 99% homologous.

[0187] In a specific embodiment, the N-acetylglucosamine transferase is derived from bovine (B Mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltra from Os taurus It is a transferase. In one embodiment, the N-acetylglucosamine transferase The enzyme is homologous to the N-acetylglucosamine transferase of the bovine species. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is Bovine mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyl transfer It is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the enzyme. be.

[0188] In a specific embodiment, the N-acetylglucosamine transferase is used in mice. This is mannoside acetylglucosaminyltransferase 2 from (Mus musculus). In one embodiment, the N-acetylglucosamine transferase is used in the mouse species N-acetylglucosamine transferase It is homologous to tilglucosamine transferase. For example, the N-acetylg Lucosamine transferase or the nucleic acid encoding it is used in mouse mannoside acetate Luglucosaminyltransferase 2 and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96% They are 97%, 98%, or 99% homologous.

[0189] In a specific embodiment, the N-acetylglucosamine transferase is used in rats. Mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransfer -ase. In one embodiment, the N-acetylglucosamine transferase is It is homologous to the N-acetylglucosamine transferase of rat species. The N-acetylglucosamine transferase or the nucleic acid encoding it is used in rats. Mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferer It is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to Ze.

[0190] In a specific embodiment, the N-acetylglucosamine transferase is used with elderflower Mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosamine from Gallus gallus It is a nilaterose. To a more precise degree, the N-acetylglucosamine The transferase is homologous to the N-acetylglucosamine transferase of chicken species. For example, the N-acetylglucosamine transferase or the code for it The nucleic acid is chicken mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosa Miniltransferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% , or they are 99% homologous.

[0191] In a specific embodiment, the N-acetylglucosamine transferase is used by Netta Mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetate from the African tree frog (Xenopus tropicalis) This is tilglucosaminyltransferase. In one embodiment, the N-acetylg Glucosamine transferase is found in the N-acetylglucosamine tetrapod of the African clawed frog species. It is homologous to the transferase. For example, the N-acetylglucosamine transferase The gelase or the nucleic acid encoding it is found in the mannosyl(α-1,6-)-sugar of the African clawed frog. Protein β-1,2-N-acetylglucosaminyltransferase and approximately 70%, 75%, 80%, They are 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0192] In a specific embodiment, the N-acetylglucosamine transferase is zebra Mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucose from fish (Danio rerio) This is cosaminyltransferase. In one embodiment, the N-acetylglucosamine N-transferase is a type of N-acetylglucosamine transfer enzyme used in zebrafish. It is homologous to -ase. For example, the N-acetylglucosamine transferase or The nucleic acid that codes for it is the zebrafish mannosyl(α-1,6-)-glycoprotein β- 1,2-N-acetylglucosaminyltransferase and approximately 70%, 75%, 80%, 85%, 90%, They are 95%, 96%, 97%, 98%, or 99% homologous.

[0193] In one embodiment, the N-acetylglucosamine transferase is used in Gambia This is AgaP_AGAP004397 (GI: 1274542) of the spotted mosquito (Anopheles gambiae). In one embodiment... In this context, the N-acetylglucosamine transferase is derived from the N-acetylglucosamine of the Anopheles gambier species. It is homologous to cetylglucosamine transferase. For example, the N-acetyl Glucosamine transferase or the nucleic acid encoding it is found in Anopheles gambia. aP_AGAP004397(GI: 1274542) and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% They are 99% or 100% homologous.

[0194] In one embodiment, the N-acetylglucosamine transferase is used in nematodes (Caeno This is AgaP_AGAP004397 (GI: 1274542) of rhabditis elegans. In one embodiment, N-acetylglucosamine transferase is used in nematode species to transfer N-acetylglucosamine. It is homologous to a spherase. For example, the N-acetylglucosamine transfer The enzyme or nucleic acid encoding it is approximately 70% and 75% of the nematode AgaP_AGAP004397 (GI: 1274542). They are homologous by %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0195] In one embodiment, the N-acetylglucosamine transferase is used in nematodes. It is 20 (GI: 179562). In one embodiment, the N-acetylglucosamine transfer The enzyme is homologous to the N-acetylglucosamine transferase of nematode species. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is The nematode gly-20 (GI: 179562) and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, and 98%. , or they are 99% homologous.

[0196] In one embodiment, the N-acetylglucosamine transferase is used in Arabidopsis β-1,2-N-acetylglucosaminyltransferase II from Arabidopsis thaliana In one embodiment, the N-acetylglucosamine transferase is white This is homologous to the N-acetylglucosamine transferase of the thrush species *Tricholoma thaliana*. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is white Approximately 70% and 75% of the gly β-1,2-N-acetylglucosaminyltransferase II from *Trichosanthes thaliana*. They are homologous by %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0197] In one embodiment, the N-acetylglucosamine transferase is used in nematodes. It is 20 (GI: 179562). In one embodiment, the N-acetylglucosamine transfer The enzyme is homologous to the N-acetylglucosamine transferase of nematode species. For example, the N-acetylglucosamine transferase or the nucleic acid encoding it is The nematode gly-20 (GI: 179562) and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, and 98%. , or they are 99% homologous.

[0198] In one embodiment, the N-acetylglucosamine transferase is used in the Japanese gecko. Predicted α-1,3-mannosyl glycoprotein 2-β-N-acetylglucoglycan of Gekko japonicus This is cosaminyltransferase (XP_015280466.1). In one embodiment, the N-A Cetylglucosamine transferase is produced in the Japanese gecko species N-acetylglucosamine. It is homologous to lanceferase. For example, the N-acetylglucosamine trans The ferase or the nucleic acid encoding it is approximately 70% and 75% of that of the Japanese gecko XP_015280466.1. They are 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0199] In other embodiments, provided herein are hybrid N-acetylglucosa It is a nucleic acid that codes for minttransferase.

[0200] (7.4 Galactosyltransferase) In a specific embodiment, provided herein are (a) a leishmania-derived (b) the catalytic domain of galactosyltransferase, not (b) the small leishmania Hybrid gas containing amino acid sequences involved in localization and retention in the cytoplasm or Golgi compartment It is lactosyltransferase.

[0201] In another embodiment, the hybrid galactosyltransferase is used in Leash It originates from Mania Talentrae.

[0202] In a further embodiment, the hybrid galactosyltransferase is sig Modified to include a null sequence, where the signal sequence is the galactosyltra Leishmania talenttrae targets the endoplasmic reticulum of host cells with ferroferase. The retention sequence retains the galactosyltransferase in the endoplasmic reticulum or Golgi apparatus. .

[0203] In one embodiment, the hybrid galactosyltransferase is galactosyltransferase The siltransferase is held in the endoplasmic reticulum. To a more precise extent, the hybrid gas Lactosyltransferase transfers galactosyltransferase to the cis-Golgi apparatus. To retain. In another embodiment, the hybrid galactosyltransferase is The galactosyltransferase is held in the intermediate Golgi apparatus. In another embodiment, The hybrid galactosyltransferase is a galactosyltransferase It is held in a trans-Golgi apparatus.

[0204] In other embodiments, the retaining sequence is a cytoplasmic-transmembrane-stem (CTS) sequence. In one embodiment, the hybrid galactosyltransferase has a GRIP sequence. Yes. In one embodiment, the hybrid galactosyltransferase is CTS The sequence and the GRIP sequence. In another embodiment, the CTS sequence is MAN1, NTPDase 1, and This includes the amino acid sequence of NTPDase 2. In another embodiment, the CTS sequence is SEQ ID NO: 24 , comprising the sequence of Sequence ID No. 25 or Sequence ID No. 26, or a functionally active fragment thereof. Another embodiment In this embodiment, the CTS contains the amino acid sequence of MAN1. In another embodiment, the CTS sequence is , comprising the sequence of Sequence ID No. 24 or its functionally active fragment. In a further embodiment, the GRIP The sequence includes the amino acid sequence of sequence number 27.

[0205] In another embodiment, the galactosyltransferase is shown in Table 9. Galactosyltransferase, or its functional homolog, isoform, or Derived from a mutant. Any galactosyltransferase known in the art or the same The nucleic acid encoding can be used in conjunction with the host cells and methods described herein. Cut.

[0206] In a specific embodiment, the galactosyltransferase is used to transfer human β-1,4-galactosyltransferase. This is lactosyltransferase 1 (B4GALT1). In one embodiment, the galactosyltransferase Galactosyltransferase is homologous to galactosyltransferase in the human species. For example, the galactosyltransferase or the nucleic acid encoding it is human β-1,4-galactosyltransferase 1 and approximately 70%, 75%, 80%, 85%, 90%, 95%, They are 96%, 97%, 98%, or 99% homologous.

[0207] In a specific embodiment, the galactosyltransferase is used in chimpanzee β This is -1,4-galactosyltransferase 1. In one embodiment, the galactosyl The transferase is homologous to the galactosyltransferase in chimpanzee species. For example, the galactosyltransferase or the nucleic acid encoding it is , chimpanzee β-1,4-galactosyltransferase 1 and approximately 70%, 75%, 80%, 85% They are 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0208] In a specific embodiment, the galactosyltransferase is used in rhesus monkeys (Macac This is β-1,4-galactosyltransferase 1 of a mulatta. In one embodiment, The galactosyltransferase is used in galactosyltransferases of the rhesus macaque species. It is homologous to galactosyltransferase. For example, the galactosyltransferase or it is coated The nucleic acids involved are β-1,4-galactosyltransferase 1 from rhesus monkeys and approximately 70% and 75% respectively. They are 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0209] In a specific embodiment, the galactosyltransferase is used to transfer canine β-1,4-galactosyltransferase. This is lactosyltransferase 1. In one embodiment, the galactosyltrans The spherase is homologous to the galactosyltransferase found in canine species. For example, the galactosyltransferase or the nucleic acid encoding it is used in dogs with β-1,4 - Galactosyltransferase 1 and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% They are 98% or 99% homologous.

[0210] In a specific embodiment, the galactosyltransferase is used to transfer bovine β-1,4-galactosyltransferase. This is lactosyltransferase 1. In one embodiment, the galactosyltrans The spherase is homologous to the galactosyltransferase found in bovine species. For example, the galactosyltransferase or the nucleic acid encoding it is derived from bovine β-1,4 - Galactosyltransferase 1 and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% They are 98% or 99% homologous.

[0211] In a specific embodiment, the galactosyltransferase is used to transfer β-1,4- This is galactosyltransferase 1. In one embodiment, the galactosyltransferase 1 is used. The transferase is homologous to the galactosyltransferase of the mouse species. For example, the galactosyltransferase or the nucleic acid encoding it is used in mice. β-1,4-galactosyltransferase 1 and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96% They are 97%, 98%, or 99% homologous.

[0212] In a specific embodiment, the galactosyltransferase is used in rats with β-1,4- This is galactosyltransferase 1. In one embodiment, the galactosyltransferase 1 is used. The transferase is homologous to the galactosyltransferase of the rat species. For example, the galactosyltransferase or the nucleic acid encoding it is used in rats. β-1,4-galactosyltransferase 1 and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96% They are 97%, 98%, or 99% homologous.

[0213] In a specific embodiment, the galactosyltransferase is used in chicken β-1,4 - Galactosyltransferase 1. In one embodiment, the galactosyltransferase The transferase is homologous to the galactosyltransferase of chicken species. Yes. For example, the galactosyltransferase or the nucleic acid encoding it is said to be a type of galactosyltransferase. Approximately 70%, 75%, 80%, 85%, 90%, and 95% of β-1,4-galactosyltransferase 1 of Ri. They are 96%, 97%, 98%, or 99% homologous.

[0214] In a specific embodiment, the galactosyltransferase is used by the tropical clawed frog This is β-1,4-galactosyltransferase 1 of the galactosyltransferase 1. In one embodiment, the galactosyltransferase 1 is used. Tosyltransferase is a galactosyltransferase in the species of the tropical clawed frog. It is homologous to galactosyltransferase. For example, the galactosyltransferase or it is coated The nucleic acid involved is β-1,4-galactosyltransferase 1 from the tropical clawed frog, and approximately 70% They are 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0215] In a specific embodiment, the galactosyltransferase is used in zebrafish This is β-1,4-galactosyltransferase 1. In one embodiment, the galactosyltransferase 1 is used. Syltransferase is a type of galactosyltransferase found in zebrafish species. They are homologous. For example, the galactosyltransferase or the encoding thereof The nucleic acids are approximately 70% and 75% of zebrafish β-1,4-galactosyltransferase 1. They are 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0216] In a further embodiment, a hybrid galactostil is provided herein. It is a nucleic acid that codes for lanceferase.

[0217] (7.5 Siallyltransferase) In a specific embodiment, provided herein are (a) a leishmania-derived (b) the catalytic domain of sialyltransferase, not (b) the endoplasmic reticulum of Leishmania. Or a hybrid shea containing an amino acid sequence involved in localization and retention in the Golgi compartment. It is a lyltransferase.

[0218] In another embodiment, the hybrid sialyltransferase is used in Leishmani It originates from A. talenttrae.

[0219] In one embodiment, the hybrid sialyltransferase is a signal sequence Modified to include, where the signal sequence is the sialyltransferer The ze is targeted to the endoplasmic reticulum of the Leishmania talenttrae host cell, and the retaining sequence This holds the sialyltransferase in the endoplasmic reticulum or Golgi apparatus. In another embodiment, In this case, the hybrid sialyltransferase is sialyltransferase It is held in a trans-Golgi apparatus.

[0220] In another embodiment, the retaining sequence is a CTS sequence. In yet another embodiment, The retaining sequence is the GRIP sequence. In another embodiment, the retaining sequence is the CTS sequence and the GRI It is a P sequence. In another embodiment, the CTS sequence is MAN1, NTPDase 1, or NTPDase 1. It contains the amino acid sequence of Ze2. In another embodiment, the CTS sequence is sequence number 24, sequence number This includes sequence 25, or sequence number 26, or a functionally active fragment thereof. In another embodiment, The CTS contains the amino acid sequence of MAN1. In another embodiment, the CTS sequence is the sequence number. It comprises 24 sequences or functionally active fragments thereof. In another embodiment, the GRIP sequence is sequence number Contains the sequence of No. 27 or its functionally active fragment.

[0221] In another embodiment, the sialyltransferase is 2,6-SiaT or 2,3-SiaT. In another embodiment, the hybrid sialyltransferase is shown in Table 9. The listed sialyltransferases, or their functional homologs and isoforms. , or derived from a mutant. N-glycosylation consensus sequence, for example, Asn-X-Ser(Th r) Asn residue (or other related) in (where X can be any amino acid other than Pro) One or more monosaccharides (e.g., galactose) linked to an N-glycan linked to a chain of residues Any sialyltransferase that can add sialic acid residues or to The nucleic acids used can be used in accordance with the methods described herein, for example, It can be incorporated into host cells as described in the book. Any sialilt known in the art Lansferase or nucleic acid encoding it is used in the host cells and methods described herein. They can be used together.

[0222] In a specific embodiment, the sialyltransferase is used to transfer human β-galactosin D is α-2,6-sialyltransferase 1. In another specific embodiment, the Allyltransferase is used to transfer human β-galactoside α-2,3-sialyltransferase. This is sialyltransferase 4. In one embodiment, the sialyltransferase is used to transfer human sialyl It is homologous to sialyltransferase. For example, the sialyltransferase Or the nucleic acid encoding it is human β-galactoside α-2,6-sialyltransfera -ase 1 or β-galactoside α-2,3-sialyltransferase 4 and approximately 70%, 75%, 80% They are 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0223] In a specific embodiment, the sialyltransferase is used to transfer β-galactose from a mouse. This is side α-2,6-sialyltransferase 1. In another specific embodiment, Sialyltransferase is used in the mouse β-galactoside α-2,3-sialyltransferase. This is sialyltransferase 3. In one embodiment, the sialyltransferase is used in mice. It is homologous to sialyltransferase. For example, the sialyltransferase The enzyme or the nucleic acid encoding it is mouse β-galactoside α-2,6-sialyltran Spherase 1 or β-galactoside α-2,3-sialyltransferase 3 and approximately 70%, 75% They are homologous by %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0224] In a specific embodiment, the sialyltransferase is used to transfer rat β-galactose. The side is α-2,6-sialyltransferase 1. In one embodiment, the sialyl The transferase is homologous to the sialyltransferase of the rat species. For example, the sialyltransferase or the nucleic acid encoding it is a rat β- Galactoside α-2,6-sialyltransferase 1 and approximately 70%, 75%, 80%, 85%, 90% They are 95%, 96%, 97%, 98%, or 99% homologous.

[0225] In a specific embodiment, the sialyltransferase is used to control Campylobacter syltransferase. It is the α-2,3-sialyltransferase of Campylobacter jejuni. In one embodiment, the sialyltransferase is used to transfer Campylobacter jejuni species It is homologous to the sialyltransferase. For example, the sialyltransferase The enzyme or the nucleic acid encoding it is α-2,3-sial of Campylobacter jejuni. Lutransferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and They are 99% homologous.

[0226] In a specific embodiment, the sialyltransferase is used to control Campylobacter syltransferase. It is the α-2,3 / 8-sialyltransferase of Yejuni. In one embodiment, the Allyltransferase is used in Campylobacter jejuni species sialyltransferase It is homologous to the enzyme. For example, the sialyltransferase or the enzyme that co The nucleic acid involved is Campylobacter jejuni's α-2,3 / 8-sialyltransferase. It is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0227] In a specific embodiment, the sialyltransferase is used in Pasteurella murtosa. This is the α-2,3 / 2,6-sialyltransferase of Pasteurella multocida (AAY89061.1). In one embodiment, the sialyltransferase is used in Pasteurella multocida. It is homologous to the sialyltransferase of the species. For example, the sialyltransferase. Spherase or the nucleic acid encoding it is found in Pasteurella multocida's α-2,3 / 2,6-sia Lilyltransferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Alternatively, they are 99% homologous.

[0228] In a specific embodiment, the sialyltransferase is a photobacterium... α-2,6-sialyltransferase (BAA25316.1) from Photobacterium damselae In one embodiment, the sialyltransferase is a photobacterium • It is homologous to the sialyltransferase of the Damsella species. For example, the sialyltransferase. Lilyltransferase or the nucleic acid encoding it is a type of Photobacterium damsella. α-2,6-sialyltransferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9 They are 7%, 98%, or 99% homologous.

[0229] In a specific embodiment, the sialyltransferase is a photobacterium... This is a hypothetical α-2,6-sialyltransferase (WP_005298232.1) of Damsela. In this embodiment, the sialyltransferase is used for Photobacterium damserra It is homologous to the sialyltransferase of the species. For example, the sialyltransferase Ferase or the nucleic acid encoding it is the α-2,6-sia of Photobacterium damsella. Lilyltransferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, Alternatively, they are 99% homologous.

[0230] In a specific embodiment, the sialyltransferase is a photobacterium... A hypothetical α-2,6-sialyl transferer of Photobacterium leiognathi This is ze (BAF91416.1). In one embodiment, the sialyltransferase is fo It is homologous to the sialyltransferase of the species Tobacterium rayognati. For example, the sialyltransferase or the nucleic acid encoding it is photobacterial. α-2,6-sialyltransferase of Lyum rayognati and approximately 70%, 75%, 80%, 85% They are homologous by %, 90%, 95%, 96%, 97%, 98%, or 99%.

[0231] In a specific embodiment, the sialyltransferase is a photobacterium... This is a hypothetical α-2,6-sialyltransferase (BAI49484.1) from Reiognati. In this embodiment, the sialyltransferase is used to control Photobacterium rayognathus. It is homologous to the sialyltransferase of the species Chi. For example, the sialyltransferase The alpha-2 spherase or nucleic acid encoding it is a precursor of Photobacterium rayognathi. ,6-Sialyltransferase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% They are 98% or 99% homologous.

[0232] In a specific embodiment, the sialyltransferase is a Photobacterium genus This is a hypothetical α-2,6-sialyltransferase of the species (BAF92026.1). In this context, the sialyltransferase is used in the sialyltransferase of species of the genus Photobacterium. It is homologous to lanceferase. For example, the sialyltransferase or The nucleic acid that codes for it is α-2,6-sialyltransfera from species of the genus Photobacterium. It is approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to -ze. .

[0233] In one embodiment, a hybrid sialyltransfer is provided herein. It is a nucleic acid that codes for felase.

[0234] (7.6 CMP-Sia biosynthesis pathway) In another embodiment, a host cell containing a heterologous sialyltransferase is subjected to CMP-NeuA It further includes heterologous CMP-Sia biosynthetic pathway proteins that can generate c.

[0235] In a further embodiment, a CMP-Sia biosynthesis pathway that can generate CMP-NeuAc The protein is the CMP-Sia biosynthesis pathway protein listed in Table 11 or any functional protein thereof. homologs, isoforms, or variants are present in at least approximately 70%, 71%, 72%, 73%, and 74% of the original molecule. %, 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 They are 100% identical. Any sialic acid biosynthesis enzyme known in the art or encoding it The nucleic acids can be used in conjunction with the host cells and methods described herein.

[0236] In a specific embodiment, the sialic acid biosynthesis enzyme is obtained from mouse UDP-GlcNAc 2-epime It is an enzyme / N-acetylmannosamine kinase. In another embodiment, the sialic acid enzyme The synthase is the mouse CMP-sialic acid transporter. In one embodiment, The sialic acid biosynthesis enzyme is homologous to the sialic acid biosynthesis enzyme in mouse species. If the sialic acid biosynthesis enzyme or the nucleic acid encoding it is used in mouse UDP-GlcNAc 2-epime Approximately 70% of the enzymes involved in the N-acetylmannosamine kinase or CMP-sialic acid transporter, 7% of the enzymes involved in the N-acetylmannosamine kinase or CMP-sialic acid transporter. They are 5%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous.

[0237] In a specific embodiment, the sialic acid biosynthesis enzyme is derived from human UDP-N-GlcNAc 2-epime It is an enzyme / N-acetylmannosamine kinase. In a specific embodiment, the sia The acid biosynthesis enzyme is human N-acetylneuraminic acid phosphate synthase. In one embodiment, the sialic acid biosynthesis enzyme is human Neu5Ac-9-P phosphatase. In a specific embodiment, the sialic acid biosynthesis enzyme is human CMP-sialic acid synthetase. Yes. In another embodiment, the sialic acid biosynthesis enzyme is used in human CMP-Neu5Ac transporters. It is a ter. In one embodiment, the sialic acid biosynthesis enzyme is used in the sialic acid biosynthesis of the human species. It is homologous to the sialic acid biosynthesis enzyme. For example, the sialic acid biosynthesis enzyme or the nucleus encoding it. The acid is human UDP-N-GlcNAc 2-epimerase / N-acetylmannosamine kinase, N-acetyl Lunoiraminate phosphate synthase, Neu5Ac-9-P phosphatase, CMP-sialic acid synthase Tetase, or CMP-Neu5Ac transporter, and approximately 70%, 75%, 80%, 85%, 90%, 95% They are 96%, 97%, 98%, or 99% homologous.

[0238] In a specific embodiment, the sialic acid biosynthesis enzyme is derived from rat UDP-N-GlcNAc 2-epi It is a melanase / N-acetylmannosamine kinase. In one embodiment, the sialic acid The biosynthetic enzyme is homologous to the sialic acid biosynthesis enzyme in rat species. For example, the sialic acid Alkaline biosynthesis enzymes or nucleic acids encoding them are used in rat UDP-N-GlcNAc 2-epimerase / N-acetylmannosamine kinase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% homology. In other embodiments, rat UDP-N-GlcNAc 2-epime Lase / N-acetylmannosamine kinase is derived from GNE / MNK in patients with sialic aciduria. There are differences (Son et al., 2011).

[0239] In a specific embodiment, the sialic acid biosynthesis enzyme is used by Neisseria meningitidis. It is the CMP-sialic acid synthetase of dis). In another embodiment, the sialic acid biosynthesis enzyme This is meningococcal UDP-N-acetylglucosamine 2-epimerase. Specific embodiments In this case, the sialic acid biosynthesis enzyme is the CMP-sialic acid synthase of Neisseria meningitidis. In one embodiment, the sialic acid biosynthesis enzyme is homologous to the sialic acid biosynthesis enzyme of the Neisseria meningitidis species. It exists. For example, the sialic acid biosynthesis enzyme or the nucleic acid encoding it is found in Neisseria meningitidis. CMP-sialic acid synthetase, UDP-N-acetylglucosamine 2-epimerase, or CMP- Sialic acid synthase and approximately 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or They are 99% homologous.

[0240] In a specific embodiment, the sialic acid biosynthesis enzyme is used in CMP-sialic acid synthase of E. coli K1. It is tase. In another embodiment, the sialic acid biosynthesis enzyme is UDP-N-ase from E. coli K1. This is tilglucosamine 2-epimerase. In a specific embodiment, the sialic acid biosynthesis The enzyme is CMP-sialic acid synthase of E. coli K1. In one embodiment, the sialic acid The acid biosynthesis enzyme is homologous to the sialic acid biosynthesis enzyme of the E. coli K1 species. For example, The sialic acid biosynthesis enzyme or the nucleic acid encoding it is the CMP-sialic acid synthetizer of E. coli K1. Approximately 70 times the amount of UDP-N-acetylglucosamine 2-epimerase, or CMP-sialic acid synthase. They are homologous by %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0241] In a specific embodiment, the sialic acid biosynthesis enzyme is Campylobacter jejuni This is GNPE, which is N-acetylglucosamine-6-phosphate 2'-epimerase (CAM09378.1). In another embodiment, the sialic acid biosynthesis enzyme is N-A of Campylobacter jejuni. This is cetylglucosamine-6-phosphate 2'-epimerase. In a specific embodiment, Sialic acid biosynthesis enzyme is N-acetylglucosamine-6-lyx from Campylobacter jejuni. It is a sialic acid 2'-epimerase synthase. In one embodiment, the sialic acid biosynthesis enzyme It is a sialic acid biosynthesis enzyme of the species N-acetylglucosamine-6-phosphate 2'-epimerase. They are the same. For example, the sialic acid biosynthesis enzyme or the nucleic acid encoding it is Pyrobacter jejuni's N-acetylglucosamine-6-phosphate 2'-epimerase and approximately 70 They are homologous by %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0242] In a specific embodiment, the sialic acid biosynthesis enzyme is used in the N-acetylglucosa of Neisseria meningitidis. In another embodiment, this is GNPE, which is mine-6-phosphate 2'-epimerase (AAY27727.1). The sialic acid biosynthesis enzyme is produced by Neisseria meningitidis. It is an enzyme. In a specific embodiment, the sialic acid biosynthesis enzyme is used to synthesize N-acetylated sialic acid in Neisseria meningitidis. It is luglucosamine-6-phosphate 2'-epimerase synthase. In one embodiment, The sialic acid biosynthesis enzyme is a species of N-acetylglucosamine-6-phosphate 2'-epimerase. It is homologous to the sialic acid biosynthesis enzyme. For example, the sialic acid biosynthesis enzyme or it The nucleic acid involved is N-acetylglucosamine-6-phosphate 2'-epimerase from Neisseria meningitidis, and approximately 70 They are homologous by %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0243] In a specific embodiment, the sialic acid biosynthesis enzyme is Corynebacterium glutamate. N-acetylneuraminic acid lyase (NP_601846.1) of Corynebacterium glutamicum It is a certain CgNal. In another embodiment, the sialic acid biosynthesis enzyme is Corynebacterium It is CgNal, the N-acetylneuraminic acid lyase of *Glutamicum*. Specific implementation In one embodiment, the sialic acid biosynthesis enzyme is the N-acetyl of Corynebacterium glutamicum. CgNal is a neuraminic acid lyase. In one embodiment, the sialic acid biosynthesis is carried out. The enzyme is homologous to the sialic acid biosynthesis enzyme of the species CgNal, which is N-acetylneuraminic acid lyase. For example, the sialic acid biosynthesis enzyme or the nucleic acid encoding it is a corine CgNal, the N-acetylneuraminic acid lyase of Bacterium glutamicum, is approximately 70%, 7 They are 5%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous (Ji et al., 2020). 15).

[0244] (7.7 Leishmania species and kinetoplastial species) As used herein, the host cell is a leishmania cell. In one embodiment In this embodiment, the host cell is a Leishmania talenttrae cell. The host cells are the Leishmania strains shown in Table 13.

[0245] In one embodiment, the host cell is Leishmania aethiopica. The host cell is a Leishmania aethiopica cell. In one embodiment, the host cell is a Leishmania aethiopica cell. It is part of the complex. In one embodiment, the host cell is Leishmania aristide These are Leishmania aristidesi cells. In one embodiment, the host cell is a Leishmania aristidesi cell. These are Leishmania deanei cells. In one embodiment, the host cells are It is part of the Leishmania donovani species complex. In one embodiment, the host cell is a Leishmania donova cell. The host cells are Leishmania chagasi cells. In this case, the host cells are Leishmania infantum cells. Yes. In one embodiment, the host cell is Leishmania hertigii. The host cell is a Leishmania major cell. In one embodiment, the host cell is a Leishmania major cell. It is part of the Leishmania major species complex. In one embodiment, the host cell is Leishmania A major cell. In one embodiment, the host cell is Leishmania multi These are Leishmania martiniquensis cells. In one embodiment, host cells The cyst is part of the Leishmania mexicana species complex. In one embodiment, the host cell is a Leishmania mexicana cell. In this embodiment, the host cells are Leishmania pifanoi cells. In one embodiment, the host cell is Leishmania tropica a) It is part of a species complex. In one embodiment, the host cell is Leishmania tropis These are maggot cells.

[0246] In one embodiment, the host cell is a kinetoplast from the bodonidae family. It belongs to the group. In a specific embodiment, the host cell is a Bodo saltans cell. It is a cell. In one embodiment, the host cell is a kinetoplast of the Ichthyobodoceae family (ichth It belongs to the family (yobodonidae). In one embodiment, the host cell is a kinetoplast. It belongs to the family Trypanosomatidae.

[0247] In one embodiment, the host cell is a blastocactis of the Trypanosoma family. It belongs to the rithidia family. In one embodiment, the host cell is a Trypanosomiaceae cell. It belongs to the blechomonas family. In one embodiment, the host cell is It belongs to the herpetomonas family of the Trypanosomataceae family. In this case, the host cell belongs to the Haenimonas family of the Trypanosomataceae family. In one embodiment, the host cell is Lafontella of the Trypanosomataceae family. a) Belongs to the family. In one embodiment, the host cell is a Trypanosoma family member. It belongs to the family Leishmaniinae. In one embodiment, the host cell is It belongs to the nobimonas family of the family Lipanosomataceae. In one embodiment, The host cells belong to the paratrypanosoma family of the Trypanosomataceae family. In one embodiment, the host cell is a phytomonas of the family Trypanosomataceae. It belongs to the s) family. In one embodiment, the host cell is a Trypanosoma sergei. It belongs to the Sergeia family. In one embodiment, the host cell is a trypanosophyte. It belongs to the family strigomonadinae of the family Magnathidae. The host cells belong to the Trypanosoma family of the Trypanosomataceae family. In one embodiment, the host cell is a Trypanosomiaceae Warracemonas (wallacemona It belongs to the family s). In one embodiment, the host cell is a Trypanosoma family member. It belongs to the family blastocrithidia.

[0248] (7.8 Further Modified Leishmania Host Cells) In one embodiment, the host cells used herein are heteronucleotides, for example, one or more. heterogeneous nucleic acids encoding carrier proteins and / or heterogeneous nucleic acids encoding one or more proteins It is modified to include a species nucleic acid, for example, a gene that codes for one or more proteins. In a specific embodiment, a heterologous nucleic acid is inserted using the insertion method provided herein. It is introduced into the host cells described in the detailed document.

[0249] In one embodiment, further modifications are made to the host cells described herein (e.g., group It may be introduced (using substitution techniques). For example, glycosyl that may compete or interfere. It forms part of the chemical pathway (for example, it is involved in glycosylation, which is recombinantly introduced into host cells). Host cells encoding proteins that compete with or interfere with the above-mentioned heterologous genes. Nucleic acids (e.g., genes) are subjected to host cell backgrounds that cause them to become inactive / dysfunctional. It can be deleted or modified in the genome (i.e., it can be deleted / modified) The host cell nucleic acids being modified either do not encode functional proteins or are not proteins at all. (Does not encode) In one embodiment, nucleic acids are used to encode the genotype of a host cell provided herein. When deleting from the molecule, it can be replaced with a desired sequence, such as a sequence useful for glycoprotein production. Replace with. Such replacement is one or more of the insertion methods described herein. This can be the case, and here, the heterologous inserted DNA inserted into the host cell is the host cell The function of the deleted gene can be replaced.

[0250] In one embodiment, the host cells provided herein include a gene deletion, where The target DNA sequence is inserted into the host cell genome at the site of gene deletion. In one embodiment, the host cell provided herein is a Leishma having a gene deletion. It's near.

[0251] (7.9 Introduction of nucleic acids into host cells) Using any method known in the art, nucleic acids (e.g., gene fragments thereof) are introduced into host cells. For example, it can be introduced into Leishmania talentrae.

[0252] In one embodiment, a heterologous nucleic acid is used with a plasmid to form a host as described herein. When introduced into cells, for example, a heterologous nucleic acid is implanted by a plasmid (e.g., an expression vector). The plasmid is expressed in chief cells, and the plasmid can be transfected, infected, or electro-transfected. Electroporation, chemical transformation by heat shock, spontaneous transformation, phage introduction, or It is introduced into modified host cells by conjugation. The plasmid is then introduced into modified host cells by stable transfection. To be admitted.

[0253] In a specific embodiment, linearized heterologous nucleic acids are transfected, infected, Alternatively, electroporation, chemical transformation by heat shock, natural transformation, Introduction into host cells as described herein using phage introduction or conjugation. In a further embodiment, heterologous nucleic acids are recombined with the host cell genome. It is incorporated in a site-specific manner.

[0254] (7.10 Methods for producing glycosylated target proteins) Provided herein is a method for producing an N-glycosylation target protein.

[0255] In one embodiment, the present invention provides for the use of the host cells described herein. This is a method for producing glycosylated target proteins in vivo. Specific embodiments In this specification, a method for producing a glycosylated target protein is provided. (i) to culture the host cells provided herein under conditions suitable for protein production. (ii) a method comprising (ii) isolating the target protein. In a specific embodiment, In this context, the host cell contains (a) recombinant nucleic acids encoding a target protein; and (b) heterologous glycosides. It contains recombinant nucleic acid encoding siltransferase. In one embodiment, the heterogene Glycosyltransferase is N-acetylglucosaminetransferase; or different It is a species galactosyltransferase; or a heterospecies sialyltransferase. In this embodiment, the host cell is a leishmania cell.

[0256] In one embodiment, the target protein produced by the provided host cell is therapeutic A therapeutic protein is a protein used to treat a disease or disorder. For example, The target proteins produced by the host cells provided herein are enzymes, cytoka It can be an in or an antibody, where the target protein is glycosylated, for example , it is sialylated. A non-restrictive list of target proteins is provided in Section 7.12 below. It is being done.

[0257] (7.11 Methods for culturing cells) Provided herein is a method for culturing host cells.

[0258] In one embodiment, host cells are cultured using any of the standard culture techniques known in the art. They are cultured using a brain-heart cell culture medium. For example, all cells contain 5 ug / ml of hemin. In rich media such as infusion, triptychase soy broth, or yeast extract, The culture is performed using conventional methods. Furthermore, incubation is carried out in the dark, either as static or shaking culture. This is carried out at 26°C for 2-3 days. In some embodiments, the culture of recombinant cell lines is suitable It contains a selection agent. A non-exclusive list of selection agents is provided in Table 9.

[0259] (7.12 Target Protein) Any protein known in the art (or the peptide / polypeptide corresponding to said protein) Tide can be used as a target protein in accordance with the method described herein. Those skilled in the art can use the nucleic acid sequences of known proteins and newly identified proteins in this art. It can be easily estimated using methods known in the field, and therefore any target Tan Introducing nucleic acids encoding proteins into host cells provided herein (e.g., expression Vectors, e.g., plasmids, e.g., site-directed integration by homologous recombination It will be easy to see that this is well within the capabilities of a person skilled in the art.

[0260] In other embodiments, the target protein is human interferon-α (INF-α), Taferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL-2), Chimeric diphtheria toxin-IL-2 (denileukin diphthitox), interleukin-1 (IL 1) IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), tumor Necrosis factor alpha (TNF-α), insulin, plumrintide, growth hormone (GH), insulin Phosphorus-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide-1 Agonist (GLP-1), glucagon, growth hormone-releasing hormone (GHRH), secretin, thyroid Glandular tropin-releasing 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 Factors (PDGF), fibroblast growth factor 7 (FGF7), fibroblast growth factor 20 (FGF20), fibroblast growth long factor 21 (FGF21), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), neurotrophin-3 Human follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), Lutropin-α, Eri Slopoietin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor The extracellular domain of CTLA4 (e.g., FC-fusion), or the extracellular domain of the TNF receptor The amino acid sequence of the in (e.g., FC-fusion) is included. In specific embodiments, see herein. The methods described and the target proteins used in conjunction with host cells are enzymes or inhibitors. It is a child. Exemplary enzymes and inhibitors that can be used as target proteins include These are Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor VIIa, and Antitro Electrolyte III (AT-III), protein C, tissue plasminogen activator (tPA), and tPA variants, Urokinase, Hirudin, Streptokinase, Glucocerebrosidase, Alglucosyl Idurase-α, laronidase (α-L-iduronidase), idursulfase (iduronic acid-2- Sulfatase), galsulfase, agalsidase-β (human α-galactosidase A), Botulinum toxin, collagenase, human DNase-I, hyaluronidase, papain, L- Asparaginase, uricase (uric acid oxidase), glutamate carboxypeptide Glucarpidase, α1-protease inhibitor (α1-antitrypsin), lactase, Examples include pancreatic enzymes (lipase, amylase, protease) and adenosine deaminase. However, it is not limited to these.

[0261] In specific embodiments, the methods described herein and the host cells used accordingly The target protein is a cytokine. Examples of cytokines include interferon-α (INF-α) and interferon-β. (INF-β), interferon-γ (INF-γ), interleukin-2 (IL-2), chimeric diphtheria Toxin-IL-2 (denileukin diffutox), interleukin-1 (IL1), IL1B, IL3, I L4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), and tumor necrosis factor AL TNF-α is one example, but it is not limited to these.

[0262] In specific embodiments, the methods described herein and the host cells used accordingly The target proteins used are hormones or growth factors. Examples of hormones and growth factors that can do this include insulin, plumrintide, and growth factors. Long-acting hormone (GH), insulin-like growth factor (IGF1), human parathyroid hormone, calcitonin, Glucagon-like peptide-1 agonist (GLP-1), glucagon, growth hormone-releasing hormone (GH) RH), secretin, thyroid-stimulating hormone (TSH), human bone morphogenetic protein 2 (hBMP2), human bone morphogenetic protein Adult protein 7 (hBMP7), gonadotropin-releasing hormone (GnRH), keratinocyte growth factor (K GF), platelet-derived growth factor (PDGF), fibroblast growth factor 7 (FGF7), fibroblast growth factor 20 (F GF20), fibroblast growth factor 21 (FGF21), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), Neurotrophin-3, human follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), Lutropin-α, erythropoietin, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macula Examples include, but are not limited to, lophage colony-stimulating factor (GM-CSF).

[0263] In specific embodiments, the methods described herein and the host cells used accordingly The target protein is a receptor. Examples of proteins that can be used as target proteins. Typical receptors include the extracellular domain of human CTLA4 (for example, fused to Fc) and Examples include soluble TNF receptors (for example, those fused to Fc), but are not limited to these. I can't.

[0264] In other embodiments, the target protein is a therapeutic protein. In this embodiment, the target protein is an approved biologic. In another embodiment, therapeutic The proteins include abatacept (e.g., Orencia), aflibercept (e.g., Eylea), and Galsidase beta (e.g., Fabrazyme), albiglutide (e.g., Eperzan), Aldes Leukin (e.g., Proleukin), Alefacept (e.g., Amevive), Alglucerase ( For example, Ceredase), alglucosidase alpha (for example, LUMIZYME), aliskiren (for example) For example, Tekturna, alpha-1 proteinase inhibitors (e.g., Aralast), Altepra -ase (e.g., Activase), anakinra (e.g., Kineret), anistreplainase (e.g., Eminase), human anthrax immunoglobulin (e.g., ANTHRASIL), antihemophilic factor (e.g., Adva te), anti-inhibitor blood coagulation complex (e.g., Feiba Nf), antithrombin alpha a. Human antithrombin III, antithymocyte globulin (e.g., antithymocyte globulin) , antithymocyte globulin (horse) (e.g., ATGAM), antithymocyte globulin (rabbit) (e.g., (ATG-Fresenius), aprotinin (e.g., Trasylol), asfotase alpha, aspa Laginase (e.g., Elspar), asparaginase of Elwinia chrysanthemi (e.g., (Erwinaze), becaprelmine (e.g., REGRANEX), beratacept (e.g., Nulojix), Lactant, bivalirudin (e.g., Angiomax), botulinum toxin type A (e.g., BOTOXE) ), type B botulinum toxin (e.g., Myobloc), brentuximab vedotin (e.g., Adce tris), buserelin (e.g., Suprecur), C1 esterase inhibitor (human), C1 esterase ZE inhibitors (recombinant) (e.g., Ruconest), certolizumab pegol (e.g., Cimzia), Choliogonadotropin alpha (e.g., choriogonadotropin alpha), chorionic gonad Tropins (human) (e.g., Ovidrel), chorionic gonadotropins (recombinant) (e.g., Ovitrell) e) Coagulation factor ix (e.g., Alprolix), Coagulation factor VIIa (e.g., NovoSeven), Human coagulation factor X (e.g., Coagadex), coagulation factor XIIIA-subunit (recombinant), collagenase (e.g., C (ordase), conestat alfa, corticotropin (e.g., HPActhar), cosintro Pine (e.g., Cortrosyn), darbepoetin alpha (e.g., Aranesp), defibrotide (e.g., Noravid), Deniloquin diffittox (e.g., Ontak), Decilzine, Digoxin SynimmuneFab (sheep) (e.g., DIGIBIND), Dornase Alpha (e.g., Pulmozyme), D Rotrecogin alpha (e.g., Xigris), dulaglutide, efmoloctocog alpha ( For example, ELOCTA, Erosulfase Alpha, Enfubil Tide (for example, FUZEON) , epoetin alpha (e.g., Binocrit), epoetin zeta (e.g., Retacrit), ep Tifibatide (e.g., INTEGRILIN), etanercept (e.g., Enbrel), exenatide ( For example, Byetta), Factor IX complex (human) (for example, AlphaNine), fibrinolysin, also known as Plasmin (e.g., Elase), filgrastim (e.g., NA), filgrastim-sn dz, follitropin alpha (e.g., Gonal-F), follitropin beta (e.g., Follis Tim AQ), galsulfase (e.g., Naglazyme), gastric factors, gemtuzumab ozogamycyan (e.g., Mylotarg), glucocyte acetate (e.g., Copaxone), glucagon recombinant ( For example, GlucaGen), glucarpidase (for example, Voraxaze), gramicidin D (for example, Neo sporin), hepatitis B immunoglobulin, human calcitonin, human tetanus toxoid immunoglobulin Brin, human rabies virus immunoglobulin (e.g., Hyperab human rabies immunoglobulin) ), human Rho(D) immunoglobulin (e.g., Hyp Rho D Inj 16.5%), human serum albumin (e.g., For example, Albuminar, human varicella-zoster immunoglobulin (e.g., Varizig), hyaluronic acid Hyaluronidase (e.g., HYLENEX), hyaluronidase (human recombinant), ibritumomabuchiukise Tan (e.g., Zevalin), idulsulfase (e.g., Elaprase), imiglucerase (e.g., For example, Cerezyme), human immunoglobulin, insulin aspart (for example, NovoLog), bovine Insulin, insulin degludec (e.g., Tresiba), insulin detemir (e.g., LE) VEMIR), insulin glargine (e.g., Lantus), insulin glulisine (e.g., APIDR) A) Insulin lispro (e.g., Humalog), porcine insulin (e.g., Iletin II), Reg Regular insulin (e.g., Humulin R), porcine insulin (e.g., vetsulin), isofexamine Insulin (e.g., Novolin N), interferon alpha-2a, recombinant (e.g., R Interferon A), interferon alpha-2b (e.g., INTRON A), interferon alpha Acon-1 (e.g., INFERGEN), interferon alpha-n1 (e.g., Wellferon), Terferon alpha-n3 (e.g., Alferon), interferon beta-1a (e.g., Avon) e.g., interferon beta-1b (e.g., Betaseron), interferon gamma-1b (e.g., For example, Actimmune), intravenous immunoglobulin (e.g., Civacir), laronidase (e.g., Al durazyme), lenograstim (e.g., Granocyte), repiridine (e.g., Refludan), roy Prolides (e.g., Eligard), liraglutides (e.g., Saxenda), lucinactants (e.g., (Surfaxin), Lutropin alpha (e.g., Luveris), Mecasermin (e.g., NA), Me Notropin (e.g., Menopur), Methoxypolyethylene glycol-epoetin beta (e.g.) For example, Mircera, metreptin (for example, Myalept), and natural alpha-interferon. Alternatively, multiferon (e.g., Intron / Roferon-A), nesilitide (e.g., NATRECOR), or Cliplasmin (e.g., Jetrea), oprelbequine (e.g., Neumega), OspA lipoprotein Citrates (e.g., Lymerix), oxytocin (e.g., Pitocin), parifermin (e.g., Kep) ivance), pancrelipase (e.g., Pancrecarb), bovine pegademase (e.g., Adagen) , Peg Asparagauze (e.g., Oncaspar), Pegfilgrass (e.g., Neulasta), Peginterferon alpha-2a (e.g., Pegasys), Peginterferon alpha-2 b (e.g., PEG-Intron), pegylated interferon beta-1a (e.g., Plegridy), pegrotyl Ikaze (for example, (Krystexxa)), Pegbisomant (for example, SOMAVERT), Polaktantoa Rufa (e.g., Curosurf), Plumlintide (e.g., Symlin), Preotact (e.g., Preotact E), protamine sulfate (e.g., protamine sulfate injection, USP), human prote In S (e.g., human protein S), prothrombin (e.g., Feiba Nf), prothrombin Complex (e.g., Cofact), prothrombin complex concentrate (e.g., Kcentra), Rasb Uri Case (e.g., Elitek), reteplase (e.g., Retavase), lilonacept (e.g., Arc) alyst), romiprostim (e.g., Nplate), sacrosidase (e.g., Sucraid), sakeca Lucitonin (e.g., Calcimar), salglamostim (e.g., Leucomax), satumomabpen Detide (e.g., OncoScint), seberipase alpha (e.g., Kanuma), secretin (e.g., For example, SecreFlo), cermorelin (e.g., cermorelin acetate), serum albumin (e.g., Al Bunex), iodine-labeled serum albumin (e.g., Megatope), simoctocog alfa (e.g., Nuwiq), Cypreus-T (e.g., Provenge), somatotropin recombinant (e.g., Nut ropinAQ), somatropin recombinant (e.g., BioTropin), streptokinase (e.g., St reptase), susoctocog alpha (e.g., Obizur), taliglucerase alpha (e.g., Elelyso), teduglutide (e.g., Gattex), tenecteplase (e.g., TNKase), te Liparatide (e.g., Forteo), tesamorelin (e.g., Egrifta), thrombomodulin RUFA (e.g., Recomodulin), Simalfasin (e.g., Zadaxin), Thyroglobulin , thyrotropin alpha (e.g., Thyrogen), tuberculin purified protein derivatives (e.g. For example, Aplisol, turoctocog alfa (e.g., Zonovate), urofolitropin (e.g., , BRAVELLE), urokinase (e.g., Kinlytic), vasopressin (e.g., Pitressin), Vera-glucerase alfa (e.g., Vpriv), absiximab (e.g., ReoPro), Adali Mumab (e.g., Humira), alemtuzumab (e.g., CAMPATH), alirocumab (e.g., Pr aluent), alcitumomab (e.g., CEA-Scan), atezolizumab (e.g., Tecentriq), Siliximab (e.g., Simulect), belimumab (e.g., Benlysta), bevacizumab (e.g., For example, Avastin, blinatumomab (e.g., Blincyto), brodalumab (e.g., Siliq), Nakinumab (e.g., ILARISE), canakinumab (e.g., Ilaris), capromab (e.g., Pr (ostaScint), cetuximab (e.g., Erbitux), daclizumab (e.g., Zenapax), darat Mumab (e.g., DARZALEX), denosumab (e.g., Xgeva), dinutuximab (e.g., unit uxin), eculizumab (e.g., Soliris), efalizumab (e.g., RAPTIVA), elotuz Mab (e.g., EMPLICITI), evolocumab (e.g., Repatha), golimumab (e.g., Simpo ibritumomab (e.g., Zevalin), idarucizumab (e.g., Praxbind), i nfliximab (e.g., REMICADE), ipilimumab (e.g., YERVOY), ixekizumab ( For example, Taltz, mepolizumab (for example, Nucala), muromonab (for example, Orthoclone OKT3) ), natalizumab (e.g., Tysabri), nesitumumab (e.g., Portrazza), nivolumab (e.g., For example, Opdivo), obilutoxaximab (for example, Anthim), obinutuzumab (for example, Gazy va), ofatumumab (e.g., Arzerra), omalizumab (e.g., Xolair), palivizumab (e.g., Synagis), panitumumab (e.g., Vectibix), pembrolizumab (e.g., Keyt) ruda), pertuzumab (e.g., Perjeta), ramucirumab (e.g., Cyramza), ranivizumab (For example, Lucentis), laxibakuma (For example, laxibakuma), rituximab (For example) For example, Rituxan, secukinumab (e.g., Cosentyx), siltuximab (e.g., Sylvant), Tocilizumab (e.g., ACTEMA), tocitumomab (e.g., Bexxar), trastuzumab (e.g., For example, Herceptin, ustekinumab (e.g., Stellara), or vedolizumab (e.g., Entyvi Contains the amino acid sequence of (o).

[0265] In another embodiment, the target protein is an antibody. In yet another embodiment, the target Proteins are antibodies against human proteins.

[0266] In further embodiments, the antibody is adalimumab (Humira); Remicade (Inflix). Mab; ReoPro (absiximab); Rituxan (rituximab); Simulect (basiliximab); Synagis (palivizumab); Herceptin (trastuzumab); Mylotarg (gemtuzumab ozogama) Ishin; Campath (Alemtuzumab); Zevalin (Ibritumomab ciuxetan); Xolair (O Malizumab; Bexxar (Tositumomab-I-131); Erbitux (Cetuximab); Avastin (Bevacizumb) Mab); Tysabri (natalizumab); Actemra (tocilizumab); Vectibix (panitumumab); Luc entis (ranibizumab); Soliris (eculizumab); Cimzia (certolizumab pegol); Simp oni (golimumab); Ilaris (kanakinumab); Stelara (ustekinumab); Arzerra (ofatz) Mumab; Prolia (denosumab); Numax (motavizumab); ABThrax (laxibamub); Benly sta (belimumab); Yervoy (ipilimumab); Adcetris (brentuximab vedotin); Perje ta (pertuzumab); Kadcyla (ad-trastuzumab emtansine); or Gazyva (obinutuzumab) It has the amino acid sequence of mab.

[0267] In other embodiments, the antibody is a full-length antibody, Fab, F(ab')2, Scfv, or sdAb. In this embodiment, the target protein includes the amino acid sequence of an enzyme or its inhibitor. In another embodiment, the target protein is Factor VII, Factor VIII, Factor IX, Factor X Factor XIII, Factor VIIa, Antithrombin III (AT-III), Protein C, Tissue Plasma Nogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, Glucocerebrosidase, alglucosidase-α, laronidase (α-L-idronidase) Idursulfase (iduronic acid-2-sulfatase), galsulfase, agaric α-galactosidase A (Human α-galactosidase A), botulinum toxin, collagenase, human DNA -ase-I, hyaluronidase, papain, L-asparaginase, uricase (uric acid oxidizer) α1 protease inhibitors) Glutamate carboxypeptidase (glucarpidase), α1 protease inhibitors Factors (α1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease) ), and the amino acid sequence of adenosine deaminase.

[0268] In another embodiment, the glycosylated target protein is secreted into the culture medium, here Therefore, the glycosylation target protein is glycosylated. The glycosylated target protein is purified from the culture medium. In another embodiment, Lycosylated target proteins are cultured by affinity purification or ion exchange chromatography. It is purified from the culture medium. In another embodiment, the glycosylated target protein is FC-domed It contains , and is affinity-purified from the culture medium by protein-A. In another embodiment, Furthermore, glycosylated target proteins contain affinity tags and are purified by affinity.

[0269] In another embodiment, the population of glycosylated target proteins is at least about 90%, 91% It is uniformly 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In this embodiment, the N-sugar site on the target protein occupied by glycosylation 90% to 100% of the total. In other embodiments, the methods described herein and the host cells are adapted to the following conditions. The target proteins used include full-length proteins, cleavage products, protein domains, and regions. These can be motifs or peptides thereof.

[0270] In another embodiment, the target protein is an Fc-fusion protein.

[0271] In another embodiment, the target protein may be modified. The target protein has been modified to include a signal sequence derived from Leishmania. In one embodiment, the signal sequence is processed and removed from the target protein. In another embodiment, the target protein is modified to include one or more tags. In another embodiment, the tag is processed and removed from the target protein. ru.

[0272] (7.13 Composition) (7.13.1 Compositions containing host cells) In one embodiment, the Specified herein provides a set comprising host cells as described herein. It is a product (see Section 7.1). Such compositions are glycosylated as described herein. It can be used in methods for producing target proteins, for example, in compositions including host cells. It can then be cultured under conditions suitable for protein production. Subsequently, glycosylation target The protein is isolated from the composition containing the host cell using a method known in the art. It is possible.

[0273] Compositions comprising host cells provided herein, and the maintenance of host cells as described herein. It may also contain further components suitable for survival and necessary for protein production by host cells. Further essential or beneficial components, e.g., inducible factors for inducible promoters, e.g., arabino - It can also include IPTG.

[0274] (7.13.2 Compositions containing glycosylated target proteins) In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represented; and Asn is the Asn of the N-linked glycosylated consensus sequence in the target protein. N-linked glycosylated conjugate of target protein in a composition carrying oligosaccharides (including) At least approximately 10-20%, 20-30%, 25-35%, 30-40%, and 35-45% of the census sequence. %, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~70%, 65%~75%, 70% It has a percentage of ~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100%.

[0275] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represented; and Asn is the Asn of the N-linked glycosylated consensus sequence in the target protein. It is a G0-Gn glycan characterized by (a certain) glycosylation on the target protein Approximately 10-20%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45%- 55%, 50%~60%, 55%~65%, 60%~70%, 65%~75%, 70%~80%, 75%~85%, 80 It has percentages of 90%, 85%–95%, or 90%–100%.

[0276] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represented; and Asn is the Asn of the N-linked glycosylated consensus sequence in the target protein. A G0 glycan characterized by (a certain) glycosylation on the target protein About 10~20%, 20%~30%, 25%~35%, 30%~40%, 35%~45%, 40%~50%, 45%~55 %, 50%~60%, 55%~65%, 60%~70%, 65%~75%, 70%~80%, 75%~85%, 80% It has a percentage of ~90%, 85%~95%, or 90%~100%.

[0277] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) The gray circles represent mannose residues, and Asn represents the N-linked type in the target protein. A G1-Gn glycan characterized by one of the following (asn of the glycosylation consensus sequence) and at least about 10-20%, 20-30%, 25-35% of the glycosylation on the target protein. %, 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60% ~70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~10 It has 0%.

[0278] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) The gray circles represent mannose residues, and Asn represents the N-linked type in the target protein. The target tan is a G2 glycan characterized by the Asn of the glycosylation consensus sequence. Glycosylation on the protein: at least approximately 10-20%, 20-30%, 25-35%, and 30-40% 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~70%, 65%~ It has 75%, 70%-80%, 75%-85%, 80%-90%, 85%-95%, or 90%-100%.

[0279] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) The gray circles represent mannose residues, and Asn represents the N-linked type in the target protein. The target tan is a G2 glycan characterized by the Asn of the glycosylation consensus sequence. Glycosylation on the protein: at least approximately 10-20%, 20-30%, 25-35%, and 30-40% 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~70%, 65%~ It has 75%, 70%-80%, 75%-85%, 80%-90%, 85%-95%, or 90%-100%.

[0280] In another embodiment, the glycosylated target protein composition has the following structure: [ka] A G2 glycan characterized by at least about 10-10 of the glycosylation on the target protein 20%, 20%~30%, 25%~35%, 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50 %~60%, 55%~65%, 60%~70%, 65%~75%, 70%~80%, 75%~85%, 80%~90% It has 85% to 95%, or 90% to 100%.

[0281] Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues. The gray circles represent mannose residues, and Asn represents the N-linked type in the target protein. This is the Asn of the glycosylated consensus sequence.

[0282] In another embodiment, glycosylation on a target protein occurs when it is introduced into the target. The pharmacokinetic properties of the target protein are further modified to optimize them. In another embodiment, In this process, glycosylation on the target protein is replaced by sialylation.

[0283] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0284] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0285] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0286] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0287] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0288] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0289] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0290] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0291] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0292] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0293] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0294] In another embodiment, the glycosylated target protein composition has the following structure: [ka] (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents , represents an N-acetylglucosamine residue, the gray circle represents a mannose residue; and Asn is, Characterized by the Asn of the N-linked glycosylation consensus sequence in the target protein. , at least about 10-20%, 20-30%, and 25-35% of the glycosylation on the target protein , 30%~40%, 35%~45%, 40%~50%, 45%~55%, 50%~60%, 55%~65%, 60%~ 70%, 65%~75%, 70%~80%, 75%~85%, 80%~90%, 85%~95%, or 90%~100% %sa.

[0295] In one embodiment, the glycosylated target protein described herein (Section 7.12) In addition to including (see reference), the compositions described herein (e.g., pharmaceutical compositions) are medical Contains a pharmacopoeia-acceptable carrier. Where used herein, "pharmacopoeia-acceptable" means The term is authorized by federal or state regulatory authorities, or is used in animals. More specifically, for use in humans, the United States Pharmacopeia or other generally accepted drugs This means it is listed in the pharmacopoeia. In relation to carriers that are acceptable as medicines, this is true. The term "carrier" as used in this document refers to the diluent in which the pharmaceutical composition is administered. Refers to adjuvants, excipients, or vehicles. Physiological saline solution and aqueous glucose and glycerides. Lyserol solution can also be used, in particular, as a liquid carrier for injectable solutions. Excipients include starch, glucose, lactose, sucrose, gelatin, malt, Rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate Cerol, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol Examples include water and ethanol. For an example of a suitable pharmaceutical carrier, see "Remin" by E.W. Martin. It is described in "Remington's Pharmaceutical Sciences".

[0296] In one embodiment, the compositions described herein are intended to be administered to a subject via a specific route of administration. The formulation is suitable for subcutaneous, non-transcutaneous administration. For example, the compositions described herein are administered subcutaneously, non-transcutaneously. Formulated to be suitable for oral, transdermal, colorectal, intraperitoneal, and rectal administration. It is possible. In a specific embodiment, the pharmaceutical composition is administered intravenously, orally, intraperitoneally, orally, or nasally. It can be formulated for intracavitary, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal, or pulmonary administration. ru.

[0297] In one embodiment, the composition described herein includes one or more buffers, for example It further comprises phosphate buffer and sucrose phosphate buffer. In embodiments thereof, the compositions described herein do not contain buffers.

[0298] In one embodiment, the composition described herein includes one or more salts, for example, sodium chloride. Thorium, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum Aluminum salts (for example, aluminum hydroxide, aluminum phosphate, alum (potassium sulfate) Further comprising aluminum, or a mixture of such aluminum salts. Other embodiments In this specification, the compositions described herein are salt-free.

[0299] The compositions described herein are to be packaged together with the administration instructions in a kit, container, pack, or digital format. It can be included in the dispenser.

[0300] The compositions described herein can be stored before use, for example, Store frozen (for example, at approximately -20°C or approximately -70°C); or under refrigerated conditions (for example, approximately 4 It can be stored at (°C) or at room temperature.

[0301] (7.14 Preventive and therapeutic uses) In one embodiment, the Specified herein provides for the prevention or treatment of the disease or disorder in question. A method comprising applying a glycosylated target protein or a set thereof as described herein to the target. A method comprising administering a product. Further provided herein are the disease of interest. or a method for preventing damage, wherein the target is a glycosylated target as described herein. A method comprising administering protein or a composition thereof. [Examples]

[0302] (8. Examples) (8.1 Example 1 Bioinformatics evaluation of the glycosylation pathway) First, Leishmania talentrae is shown in Tables 1, 2, 3, 4, 5, 6, and 7. As noted, even Leishmania major, which is taxonomically close, was early preserved. Comparative genome analysis revealed that the N-glycan biosynthesis process differed significantly. The deficiency of the g gene leads to a significant decrease in N-glycan precursors and also to a decrease in precursor trimming. This is shown (Figures 3 and 4) (Varki, 2009). Although ALG3 is present, the ratio to human ALG3 is low. From comparison, the conserved residue 127 corresponds to the loss-of-function mutation (R171Q) in human ALG3. Potential missense mutations in glutamine have been identified (Sun et al.'s paper). 2005). Table 1. Precursor Biosynthesis [Table 1] TIFF2026067872000041.tif80170 Table 2 Trimmed [Table 2] Table 3 N-glycan elongation [Table 3] TIFF2026067872000044.tif182170 Table 4 Oligosaccharide transferase (OST) [Table 4] TIFF2026067872000046.tif109170 Table 5 Precursor Biosynthesis (Neu5Ac) [Table 5] Table 6 Synthesis and Transport of Nucleotide-Activated Sugars [Table 6] Table 7. Lipophosphoglycan-modifying enzymes [Table 7]

[0303] Following the remarkably unique biosynthetic pathway outlined in Figure 3B, trimming and endoplasmic reticulum (ER) quality are performed. A decline in control (Tables 1, 2, and 8) is incompatible with maintaining a proper folding process. Regarding site occupancy, this suggests beneficial properties of glycosylation modification. Figure 5 is also outlined in Table 8. Typically, improperly trimmed N-glycan precursors are processed in the reverse direction or fork. Unbound proteins are circulated to the ERAD pathway for proteasomal degradation. When the lectin-mediated pathway (calnexin and calreticulin) is absent, the correct sugar This describes the chaperone pathway for protein folding in more detail. (en et al., 2012). Importantly, the OST complex uses the precursor N-glycan as a protein accessory. It consists solely of Stt3 that transfers to Pter's consensus asparagine (Table 4). L. Tallen In the case of Trae, the reduced precursor is Stt3, which is remarkably different from any other described eukaryote. Transplanted by this, site preference or precise part found in other eukaryotes containing the OST complex This suggests that there is no preference for a particular position. Table 8 ER quality control [Table 8] TIFF2026067872000051.tif48170

[0304] The generally accepted view is that Figures 6 and 7 show the endoplasmic reticulum and Golgi apparatus of eukaryotic cells. This diagram shows a schematic of the N-glycan processing process, and the figure is based on the literature by Kellokumpu et al., 201 This is an excerpt from 6). In L. talenttrae, the cleaved and reduced precursor is protein It is transferred to the globulin, and during its transport through the secretory pathway, it undergoes further N-glycation in the Golgi apparatus. Lycan elongation fails. This is due to GlcNAc transferases GnT-I and GnT-II, and Galtran. This is concluded to be due to a lack of spherase and sialyltransferase (Figure 6). (Table 6). Furthermore, comparative data showed the absence of the sialic acid biosynthesis step (Table 5). Related to Trypanosoma burseyi, TbGnTI (Damerow et al., 2014) and TbGnTII (Dam The Golgi-resident GlcNAc glycosyltransferase derived from the literature by erow et al. (2016) is L. It could not be clearly identified by Rentrae. Tb with epitope tagging at the C-terminus. GnTI and TbGnTII have been shown to be localized to the Golgi by immunofluorescence (Damerow et al.). (2014; Damerow et al., 2016).

[0305] Nucleotide-activated sugar donors are catalyzed by glycosyltransferases. Since it is required for elementary reactions and must be available inside the lumen of the Golgi compartment, the membrane The presence of localized transporters was confirmed using bioinformatics techniques. UDP- Glucose pyrophosphorylase, UDP-sugar pyrophosphorylase, UDP-galactose 4-epi Melase, as well as UDP-Gal / UDP-GlcNAc importer, GDP-Man / UDP-GlcNAc importer, UDP-GlcNAc / UDP-GalNAc, GDP-Man importer, and homolog of UDP-Glc importer (Ro Per and Ferguson, 2003; Roper et al., 2002; Urbaniak et al., 2006; Capul The existence of the UDP-GlcNAc transporter (T. cruzi TcN) was confirmed (Table 6). ST1 and T. Bursey TbNST1~4, L. Major) are present in L. Talentrae, UDP-Gal transformer Porter (T. Bursey TbNST 1 and 2, L. Major) is found in L. Talentrae. The results showed that both UDP-GlcNAc and UDP-Gal can be transported into the Golgi lumen. Therefore, LTAR_180008900.1, LTAR_240008400.1, LTAR_340034000.1, and LT AR_300033500.1 (Previous naming scheme: LtaP18.0420, LtaP24.0350, LtaP34.3030, and LtaP30 .2670) suggests the potential for GDP-Gal and UDP-GlcNAc to be utilized within the Golgi lumen.

[0306] (8.2 Example 2 To modify the glycans of fully functionally customized N-glycan mutants) (The method) Figures 7 and 8 show a) desirable downstream effects of the interaction between Fc and Fc receptors, and b) increased half-life (drug c) increased pharmacokinetic (PK), and anti-inflammatory properties (in potentially immunogenic protein therapeutics). For something like the immune tolerance benefit of 2,6-linked Neu5Ac to avoid the development of ADA, L. To produce uniform and functionally customized N-glycan elongation in talentrae This illustrates the design of the glycosylation modification. Figure 8 shows the main and novel properties that result from the glycosylation modification. This document describes the proposed N-glycan biosynthesis of L. talenttrae, highlighting the differences in their characteristics. This invention relates to N-acetylglucosamine transferase and galactosyl transferase. A series of glycosyltransferases, including ze and sialyltransferase. Species expression produces functionally customized and uniform N-glycans on proteins. This concerns eukaryotic host cells that have been modified to do so.

[0307] (8.3 Example 3 Shrinkage of kinetoplasts in Leishmania talentrae, (Experimental evidence of 100% uniform N-glycans) Based on the expected beneficial characteristics, we selected the kinetoplastial organism L. talentrae. After that, three wild-type (wt) strains (St10569, St10616, St11262) were used to analyze their native N-glycans. We analyzed the following: First, we denatured the cell pellet and then examined which of PNGase A or PNGase F was involved. The N-glycan was subjected to release mediated by [unclear]. The released N-glycan was rapidly enzymatically released. For quick and rapid N-glycan labeling, a) fully methylate or b) use Waters GlycoWork Labeled with s(trademark) RapiFluor-MS(trademark) N-Glycan.

[0308] By using RapiFluor™ fluorescent labeling and ULC separation, the inventors have calculated the m / z = 1222.7166; [M+Na] + 1244.6985; [M+K] + 1 corresponding to (Man)3(GlcNAc)2 having 1260.6724 Only one single N-glycan morphology was observed. Regarding N-glycans, three isolated L. In all Talenttrae cells, there is only one uniform N-glycan, known as portmanteau. North or Man3 glycan was identified (Figure 9).

[0309] The inventors have identified two different wt samples of Leishmania talentrae (St10569, St10616) To elucidate the N-glycan profile of ), complete methylation was also applied. This method (degreasing) Deglycosylation of trypsin digested peptides obtained from a crystalline cell pellet or cell pellet Regardless of the direct deglycosylation of intact proteins after the delipidization of Rett, 1 The presence of two N-glycan structures was confirmed in both strains. It corresponds to the basic (Man)3(GlcNAc)2 core. This N-glycan is obtained independently of the N-glycosidase used in deglycosylation. The simulated control is indicated by the polyhexose symbol, probably lipophosphoglycan. The presence of glycans contaminating the cell wall of the ion was observed. The contamination of polyhexoses in the extract was found in MALDI. Although observed in the vector, the inventors believe that this may correspond to more complex N-glycans. No ions were found. In particular, the assumed bifurcation having α1,6 or α1,3 bonded fucose was not found. (Gal)2(GlcNAc)2(Fuc)1+(Man)3(GlcNAc)2(G2F) (Breitling et al. literature) (2002) (Theoretical complete methylation mass [M+Na] of 2244.1 + ) was not found. The spectrum is It did not contain any trace of ions with this m / z value. Core structure and other complex fucosylation, N -Possible intermediates between acetylglucosaminylation and galactosylation structures are observed. It is also worth mentioning that this went unnoticed. N-glycosyl in the Golgi apparatus. None of the precursors to the chemical pathway (e.g., Man5GlcNAc2) were identified. (Leishmania) If N-glycan biosynthesis in Talenttrae is comparable to that of other organisms, then this aspect is truly unique. Yes. The spectrum obtained after deglycosylation with PNGase F or PNGase A is the same. It was one. As a result, the N-glycan pro of Leishmania talentrae cell pellets The file contains an N-glycan having an α1,3-linked core fucose that is not released by PNGase F. There was no indication of its presence (data not shown).

[0310] How does the N-glycan of L. talenttrae differ from the N-glycans of other kinetoplastids? To further clarify this, Critidia fasciculata, Phytomona Phytomonas davidi Lafont and Critizia daeney (A Ngomonas was further investigated using genome analysis and N-glycan profiling. L. tarre Compared to C. fasciculata, C. fasciculata has experimentally proven high mannose glycans. Mannosyltransferases ALG3 and ALG3 explain the synthesis of (Man5GlcNAc2~Man11GlcNAc2). 9, and ALG11 are further contained (Figure 45). Furthermore, C. fasciculata is L. talentrae P. Davidy Raff possesses the missing quality control chaperone, Calreticulin. Regarding the font, genomic information is unavailable. PNGase F and PNGase A and subsequently Complete methylation and N-glycan profiling by MALDI-TOF reveal one or two A high-mannose glycan (Man9GlcNAc2) with additional pentoses has been identified. Bioinformatics comparison of glycosylation pathways in *Deanay* and *L. talenttrae* Furthermore, it became clear that there were no differences except for the absence of MAN1 in C. Deanay. However, N-glycan profiling revealed that the core N-glycan (Man3GlcNAc2) has two hexagons. It was shown that the source and two deoxyhexoses could be further modified (Figure 45).

[0311] In summary, various experimental methods have shown that there is only one basic, uniform N-glycan (Man). 3(GlcNAc)2 was identified in three L. talenttrae wt strains. It is also called Man3 or portimannose. This homogeneous N-glycosylation, possessing (Man)3(GlcNAc)2, is unlike any other true This organism has never been observed in nucleoplasms, and it is being used for therapeutic purposes to achieve uniform N-glycosylation. Humanized N-glycans are used as a protein expression host, maintaining high site occupancy while being converted into sugars. This describes a new feature for modifying the chain.

[0312] (8.4 Example 4 In vitro extension of native porcimannose N-glycan Identification of species glycosyltransferases) To obtain a humanized elongated bifurcated N-glycan, suitable Gnt is recombinant using L. talenttrae. The gene was expressed, and affinity concentrations were obtained from the crude lysate, membrane-solubilized fraction, and culture supernatant of L. talenttrae. It was reduced. The semi-purified enzyme, regarding its appropriate activity, cofactors and related activated nucleotides The study was conducted in vitro using a glucose donor. The substrates were "free" N-glycan and 2-aminobenn. wt cell-derived material containing only zuamide (2AB)-labeled N-glycan or porcimannose (Man3). It was one of the crude lysates.

[0313] Regarding its effects, the present invention relates to the expression and purification of L. talenttrae cells from insects or insects. All heterologous Gnt candidates tested from dairy sources were found to be in the presence of cofactors and their activating sugar donors. This demonstrates that it can exert its activity toward free oligosaccharide substrates or 2AB-labeled substrates. Various suitable glycosyltransferase candidates analyzed by in vitro assays The expression shown in (Table 9) is the target (Figures 10-14). Table 9 GnT Candidates [Table 9] TIFF2026067872000053.tif241170TIFF2026067872000054.tif136170

[0314] GnT-I ("Tb") derived from Trypanosoma bursei, the organism most closely related taxonomically. GnT-I is active against wt lysates containing native portimannose as a substrate. This was not shown (Figure 10). However, the relationship between free porcimannose (Man3) and the substrate was A slight activity (overlapping peaks) was observed. TbGnT-II showed activity toward the Man3 substrate. Furthermore, this allowed us to demonstrate the necessary condition of prior activity of GntI. GnT-I activity is obtained by eluting streptavidin to release the semi-purified or solubilized fraction into the free port. When used in an in vitro assay for simannose, it migrates along with the NGA2-N standard. It was confirmed with a characteristic peak that appeared to be [likely a specific type of peak]. The control was HA eluate from wt lysate or It contained one of the Strep eluates, but did not show any modification to the substrate used. .

[0315] hMGAT1 was purified from lysate + / - TritonX (TrX), and hMGAT1 HA-eluted from the cell pellet. The fractions represent approximately 100% elongation of the 2AB-Man5 substrate (Figure 11A) and approximately 100% elongation of the 2AB-Man3 substrate (Figure 11B). The substrate was shown. The MGAT-I elution fraction derived from the HA concentrate in the supernatant (SN) was found to be either 2AB-Man5 or 2AB-Man3. It exhibited only minimal activity. Lysate derived from wt as a negative control was 2AB-Man5 It did not affect the retention time or modify the standard.

[0316] When rMGAT2 is concentrated from the supernatant or membrane fraction ("pellet"), the NGA2-N standard is G0(NGA2). It was efficiently converted to the NGA2-N type (Figure 12A). As expected, rMGAT-2 converted 2AB-Man3a in vitro to NGA2-N It cannot be extended to this extent (Figure 12B). MGAT2 requires preceding GnT-I activity. NGA2- According to the manufacturer, the N standard contains approximately 50% GlcNAc on the α1,3-linked Man branch and α1,6-linked Man, respectively. It consists of both forms of GlcNAc on the branch. The peaks of NGA2-N and NGA2 are 50 / 50. Subsequently, the in vitro conversion of α1,6 is 100% by rMGAT2.

[0317] SfGnT-II is localized intracellularly and detected in cell lysate + / - 1% (v / v) TritonX, and is found in membranes. While it showed compatibility, it was not non-exclusive (Figure 13). SfGnT-II is a comparison of SfGnT-II with other Gnt candidates that were considered. Unlike nT-I, it was present in small amounts in the SN. In vitro activity assay using the HA fraction I was not conclusive due to background noise. SfGn in lysate T-II is present in relatively low amounts in the crude lysate, which is likely why it is present in such small quantities as NGA2-N. The conversion is shown (Figure 13B). It contains either SN or lysate (+TrX) SfGnT-II HA eluate. The in vitro active sample had a strong background against the pure 2AB standard used as a substrate. It showed fluorescence (not shown). SfGnT-II also requires prior GnT-I activity (Figure 13A), and It does not affect Man5 (Figure 13C).

[0318] In vitro hB4GALT-I activity was evaluated using the substrate NGA2-N (green, 76.8m) (Figure 14A). Treptoavidin-purified hB4GALT-I contains NGA2-N (green) in hypothetically equivalent proportions of α1-3 and α1-6 G1 Converting to -Gn(a) and G1-Gn(b) (Figure 14B); a weak (blue) peak is detected at 76.8m, so the conversion is 100 It was not a percentage. However, a conversion of at least >90% is expected. B4GALT-I sample The "double peak" shows the same pattern as the "double" peak originating from the G1 standard (red). However, because there is less 1×GlcNAc in the structure, the retention time is shorter. Figure 14C shows G2 glycofol This shows the expected in vivo function of B4GALT1 against G0 substrates that induce malformations.

[0319] (8.5 Example 5 Native porcimannose N on native protein in vivo) - Identification of heterologous glycosyltransferases that elongate glycans. Next, recombinant host cells expressing glycosyltransferase were collected, and PNGase F was extracted. The N-glycan was then treated to release it. The released glycan was completely methylated or 2A The protein was labeled with B and its activity against native glycoprotein acceptors was analyzed. GnT-I was expressed. The cells (SfGnT-I, St11707) convert porcimannose to 34% NGA2-N(G0-Gn) in vivo. It was replaced. Since the sample is derived from crude cell extract, it also contains endoplasmic reticulum (ER) localized glycoproteins. This means that these proteins do not pass through the Golgi apparatus to achieve GlcNAc elongation. Cells expressing only GnT-II candidates (not shown) were confirmed by in vitro analysis. As expected, due to the need for prior activity of GnT-I, which is a known finding, porcimannose is extended. This could not be achieved. When GnT-I was co-expressed by recombination with the GnT-II enzyme (SfGnT- Elongation from porcimannose containing two GlcNAcs to the G0 form (I and SfGnT-II, St12320) Confirmed (Figures 15 and 16).

[0320] Analysis by complete methylation and MALDI TOF revealed that cell pellet sample St10569 P16_378 The raw form contained only one N-glycan structure (Man)3(GlcNAc)2(m / z 1171.6). In sample St11707 P16_378 from the genotype ssu::SfGnT-I of *Hymania talenttrae*, The expression of Gnt-I was (Man)3(GlcNAc)2(m / z 1171.6) at relative intensities of 66% and 34%, respectively. This resulted in the presence of both (GlcNAc)1(Man)3(GlcNAc)2(m / z 1416.7). Leishmania ta St12320 derived from the Lentrae St12320 genotype ssu::SfGnT-I;ssu::SfGnT-II (polyclonal) The co-expression of both GnT1 and GnT2 in P16_378 was 48%, 15%, and 37%, respectively. Different N-glycan structures: (Man)3(GlcNAc)2(m / z 1171.6), (GlcNAc)1(Man)3(GlcNAc)2(m / This resulted in the presence of z 1416.7) and (GlcNAc)2(Man)3(GlcNAc)2(m / z 1661.8) (Figure 15).

[0321] Furthermore, ions corresponding to the N-glycan structure were fragmented by MALDI-TOF / TOF MS. The fragment ions present in the spectrum at m / z 1171.6 are N-glycan structure (Man)3. This supports (GlcNAc)2. Fragments present in the spectrum at m / z 1416.7 On supports the N-glycan structure (GlcNAc)1(Man)3(GlcNAc)2. GnT-I is (Ma It catalyzes the transfer of GlcNAc from n)3(GlcNAc)2 to α-1,3 mannose. The inventors have found that the addition is This supports the position of GlcNAc and the predicted [M+Na]+ mass of α-1,3 mannequin with m / z 560.26. Regarding the presence of a cross-ring fragment that identifies the GlcNAc fragment bound to the north: The ion fragmentation spectrum at m / z 1416.7 was then searched. α-1,6 bond The corresponding cross-ring fragment of GlcNAc bound to synmannose has a m / z of 546.25 and It has a mass of 574.28. The cross-ring fragment that identifies the binding of the terminal GlcNAc is spec It was not observed in the clef. The fragment ions present in the spectrum at m / z 1661.8 are This supports the N-glycan structure (GlcNAc)2(Man)3(GlcNAc)2 (Figure 16).

[0322] Direct deglycosylation of intact proteins is used after delipidation of the cell pellet. N-glycan profiling was performed on three different samples of Leishmania talenttrae. As previously observed with wild-type samples analyzed, cell pellet sample St10 The 569 wild type contains only one N-glycan structure, (Man)3(GlcNAc)2 (m / z 1171.6). Sample S In the t11707 genotype ssu::GnT-I, GnT-I expression is (Man)3(GlcNAc)2(m / z 1171.6) and (Glc Both NAc)1(Man)3(GlcNAc)2 (m / z 1416.7) were observed. Gnt-I is the native glycoprotein. It catalyzes the transfer of GlcNAc from the (Man)3(GlcNAc)2 moiety present on the material to α-1,3 mannose. Co-expression of both Gnt-I and Gnt-II in St12320 genotypes ssu::SfGnT-I and ssu::SfGnT-II. are (Man)3(GlcNAc)2(m / z 1171.6), (GlcNAc)1(Man)3(GlcNAc)2(m / z 1416.7), and (GlcN This results in the existence of three different N-glycan types: Ac)2(Man)3(GlcNAc)2(m / z 1661.8). Further expression of Gnt-II leads to the second Gl of (GlcNAc)1(Man)3(GlcNAc)2 into α-1,6 mannose. It catalyzes the transfer of cNAc residues.

[0323] Therefore, these data are used in L. talentlae for its use in glycosylation. This supports the in vivo activity of insect cell-derived Gnt when expressed heterologously. Furthermore, The presence of UDP-GlcNAc in the lumen of L. rugi was also suggested, and activity in native L. talentrae Based on the bioinformatics assessment of the presence of sexualized nucleotide sugar transporters This supports the assumption.

[0324] These findings were obtained using GlycoWorks® RapiFluor-MS®, and are related to St11707 and St1252. N-glycans from five strains were released and labeled, and each strain was either labeled with SfGnT-I alone or with SfGnT-II. This was confirmed when either SfGnT-I was expressed, and it was then confirmed that G0-G was derived from SfGnT-I cells. G0 glycans derived from n and SfGnT-I+SfGnT-II cells were identified in accordance with their m / z ratio (Figure 17A). St13065 co-expresses hMGAT1 and rMGAT2, and has G0 glycans on its native secreted proteins. We induced can formation and demonstrated that both Gnts are active in vivo (Figure 17B).

[0325] Interestingly, some have a combination of Strep-triple HA tags, while others have only Strep tags. Regarding various variants of SfGnt-I that either have a native C-terminus or have a native C-terminus, The activity levels range from 20% to 75% of the total N-glycans, and in vivo positivity This shows the negative effect of the C-terminal tag on the conversion from mannose to NGA2-N(G0-Gn) (Figure). 47B).

[0326] Therefore, the heterogeneous Gnt-I candidates derived from the MGAT1 library shown in Table 9 are C-terminal tagless. The test was conducted using Leishmania expressing recombinant Gnt-I from various species. 3. N-glycan was converted to G0-Gn. The activity was very low, such as 10% of that of muMGAT1 (mouse). The rates indicate either DrMGAT1 (zebrafish) or GjMGAT1 (Japanese gecko). The percentage even exceeded 90% (Figure 47A).

[0327] The glycan obtained from SfGnT-I activity is G0-Gn, in which GlcNAc is attached to the α1,3 Man branch. It extends to glycans. St13066 was created that co-expresses SfGnT-I and hB4GALT1. GlcNAc is In vitro, it acts as an acceptor for B4GALT1 (Figure 14), and this activity is m / z = 1587 The presence of G1-Gn(a) with .63 as a peak at a retention time of 15.32 minutes indicates that Confirmed in vivo (Figure 17C).

[0328] This conclusion was reached through the selection of functional Gnts and furthermore, through bioinformatics evaluation. UDP-GlcNAc and UDP-Ga in the Golgi section of Leishmania talenttrae This supports the availability of l (Table 6). Therefore, in vivo glycosylation is modified, and Novel Leishmani co-expression of Gnt for functionally customized N-glycan production Confirmed in A. talenttrae host cells.

[0329] (8.6 Example 6 Native porcimanno on recombinant human erythropoietin in vivo) (Identification of heterologous glycosyltransferases that elongate N-glycans) Evaluating Gnt activity not only against native glycoproteins or secreted glycoproteins. To evaluate this, the recombinant target protein human erythropoietin (hEPO) was co-expressed with GnT-I. It was designed to be secreted into the supernatant, and therefore, two different signal peptides for secretory transition were attached. EPO was then expressed. N-glycan release and site occupation were detected by peptide mapping and MS. Analysis was performed. When expressed in non-glycosylated cells, homogeneous porcimannose was confirmed on EPO. Although confirmed, EPO co-expressed with GnT-I is determined by N-glycan release and complete methylation. This showed approximately 50% N-glycan elongated with one GlcNAc (Figure 18).

[0330] Site occupancy was analyzed by trypsin-digested peptide mapping (Figures 19 and 20). Both EP The concentrated glycopeptide fraction of the O sample has occupied glycosylation sites N24 and N38. Glycopeptides [ka] It contains the corresponding ion. In contrast, the occupied glycopeptide corresponding to N83 was not detected. It's not possible. Peptide [ka] Both of the glycosylation sites above were occupied by N-glycans. (Wild-type Leishmania) EPO produced in the Leishmania tarentola strain St11521 contains both N-glycosyl It contained only the (Man)3(GlcNAc)2 core structure bound to the fusion site. In strain St11895 Co-expression of EPO and GnT-I is performed by either (Man)3(GlcNAc)2 or (GlcNAc)1(Man)3(GlcNAc)2. This resulted in the occupation of N-glycosylation sites N24 and N38 in both EPO samples. Chido [ka] This is a state in which only one of the two N-glycosylation sites is occupied by an N-glycan. Peptides having N-glycosylation sites N24 and N38 were not found. [21-45] [ka] It was found to be occupied by only two N-glycans. Wild-type Leishmania strain St1 In the case of EPO produced at 1521, the m / z 4588 ion contains two (Man)3(GlcNAc)2N-glycans. It corresponds to the glycopeptides it possesses. In contrast, EPO derived from strain St11895 contains three different glycopeptides. Tydoion: Corresponds to a glycopeptide containing two (Man)3(GlcNAc)2N-glycans, m / z 458 8; For glycopeptides containing one (Man)3(GlcNAc)2 and one (GlcNAc)1(Man)3(GlcNAc)2 Corresponding m / z 4792, and glycopeptides containing two (GlcNAc)1(Man)3(GlcNAc)2 residues. The corresponding m / z was 4994. For the St11521(rhEPO) strain, the wt EPO N-glycosylation site N24 and N38 was occupied solely by (Man)3(GlcNAc)2 core N-glycan. St11895(rhEPO+SfgnT 1) Regarding the strain, co-expression of EPO and GnT1 is in (Man)3(GlcNAc)2 or (GlcNAc)1(Man)3(GlcNAc)2. This resulted in the occupation of both sites by either one. Different N-glycas bound to these sites. No further N-glycopeptide ions with a n-structure were observed.

[0331] After the trypsin digestion of the protein, the inventors identified a peptide ion that could be clearly attributed to it. Since it could not be extracted, a peptide having the N-glycosylation site N83 [77-97] [ka] This could not be analyzed in this study. Therefore, the conclusion regarding the occupancy of site N83 is... It cannot be derived. Deglycosylation by PNGase allows for the clear identification of ions. Deglycosylated peptide having the N-glycosylation site N83 as such [77-97] [ka] The existence of deglycosylated peptides was not revealed. Its mass is 2360.24 Da. The spectra of P2813 of St11895 and P2814 of St11521 are... These contained very faint ions with poor separation, at m / z values ​​of 2361.5 and 2360.01, respectively. It was not possible to identify the ions through fragmentation. Therefore, the present inventors have found that this product in its non-glycosylated or deglycosylated form can be used in this way. No signs of the presence of Petit Do have been found.

[0332] In both peptide mass fingerprints, the inventors identified the N-glycosylation site. Trypsin digest peptides containing N24 and N38 [21-45] [ka] or peptides having an N-glycosylation site N83 [77-97] [ka] It is not found in its non-glycosylation-free form, and 100% N-glycosylation occupancy is indicated. No failures in the cleavage of these peptides were observed. Two of the three N-sugar sites were cleaved. Although it was not confirmed that it was exclusively 100% occupied, the inventors also found that the third part occupied It is presumed that this was present, and this was previously observed in relation to EPO derived from non-glycan-modified cells. It is correlated with the intact protein mass reduction (MS) that was performed.

[0333] (8.7 Example 7 Identification of secretory signals from native secretory proteins) Initially, the signal peptide was described by (Klatt and Konthur, 2012). Obtained from alkaline phosphatase derived from Schmaniac mexicana. Odor specified herein The signal peptide was then purified using ConA and subsequent proteomic methods and EDMAN N-terminal saturation. Obtained from secreted glycoproteins by native host cells identified using quenching. Among the glycoproteins identified by MS and secreted by native host cells... And the most prominent protein is invertase (sucrose hydrolase-like protein) (L TAR_040008100.1 (previous naming system: LtaP04.0290) and GP63 variant LTAR_100010400.1 (previous The naming system is: LtaPcontig00616-1) (Figure 21), and both are N-glycosylation consensus It contained the Man3 portiomanose N-glycan in a uniform manner. EDMAN N-end End sequencing reveals putative secretory signal peptides derived from invertase: [ka] It was identified (Figure 22). [ka] (+It is possible that further amino acids were produced in different cycles: K in the first cycle, S in the second cycle) P in the fourth cycle, E in the fourth cycle). These corresponding nucleotides are coded as hEPO nucleotide codes. By fusing the sequence with the gene in frame, the C-terminus was tagged with His or StrepII. Using host cells that recombinantly express EPO, the secreted recombinant EPO was purified (Figure 23).

[0334] (8.8 Example 8 Targeting and retention in the correct intracellular compartment) As outlined in Figures 6 and 7, the conversion of N-glycans by glycosyltransferase Sequential processing occurs in the endoplasmic reticulum (ER) and the Golgi apparatus (Kellokumpu et al., 2016). The enzymes involved (glycosyltransferase and glycosidase) have traditionally been used in growth Sugar residues are added to the oligosaccharide chain one at a time in a specific order, or the growing oligosaccharide chain is... By removing sugar residues from a sugar chain one at a time in a specific order, they function individually and sequentially. It is thought that this is because the Gnt used for glycosylation is properly localized along the secretory pathway. And it means that it can be improved by retention. Most Golgi-localized Gnts are short N-terminal fins. Spore domain ("C"), helical TM domain ("T") of approximately 20 amino acids, stem domain ("S") ), and next, type II membrane proteins having a C-terminal globular catalytic domain in the lumen of the secretory pathway It is a quality (Figure 24A). Its functional importance for glycosylation is homomer or heteromer - This may be based on their interaction as a medium. The distribution rate and these interactions The protein domain involved can cause changes in enzymatic activity during complex formation (Kello kumpu et al. (2016). Therefore, a) the correct intracellular compartment (intermediate Golgi and transgolgi). (See Figure 8) Improved targeting and retention, and (b) better for glycosylation. Hybrid Gnt was designed to enhance homodimerization and heterodimerization, resulting in enhanced activity. .

[0335] Figure 24A shows the hybrid Gnt design that the inventors suggested for the example shown in Figure 24B. It exhibits the structure of a typical type II membrane protein, and in this design, the native Golgi apparatus The predicted CTS of the protein is inherited in the catalytic domain of heterologous GnT-x, in the following example, SfGnT-I. Genetic fusion occurred. Figure 24B shows the expected Golgi endogenous or related proteins of L. talentrae. This indicates that the secretory signal sequences of Trypanosoma and Leishmania are Gram-positive bacteria. Similar to; does not function in mammals (Al-Qahtani et al., 1998), and the signal peptide sequence is essential. The requirements and cleavage sites appear to differ between trypanosomes and higher eukaryotes. (La Flamme, AC et al., 1995). The composition of the secretory pathway of the Trypanosomataceae family is described elsewhere. This has been reviewed (McConville et al., 2002b). The cell surface protein of T. brusey (GPI) The signaling peptide (of the 'n' type) contains highly conserved signal peptides for secretion (Bohme and Cro). (ss, George AM, 2002). The targeting sequence of the trans-Golgi network is It has been shown to be present in Trypanosoma bursei, and Trypanosoma bursei In this context, the GRIP domain of T. brusey, when fused to the C-terminus of green fluorescent protein (GFP), -TbGRIP) efficiently localized to the Golgi apparatus of transfected COS cells. These GRIP domains worked with T. brusey and Leishmania mexicana, In the quiescent phase of L. mexicana, GRIP transport was reduced. GRIP protein is normally GFP-GRIP. The fusion protein is described as having a coiled-coil region, and these are elongated It is introduced from the elongated GRIP region (McConville et al., 2002a). NTPDase nucleosy Diphosphohydrolase ditriphosphate hydrolyzes nucleotides and parasites, which have a toxic odor. It plays a role and is associated with lipophosphoglycan (LPG) elongation. In L. major, L mNTPDase 1 is localized in the Golgi apparatus, and LmjF.15.0030 has a TM domain, possibly CTS. It has a chief. LmNTPDase 2 has been shown to be secreted. LmjF.10.0170 is predicted. It possesses a signal peptide (Sansom et al., 2014).

[0336] The homolog in L. talenttrae is NTPDase 1 = LTAR_150005200.1 (previous nomenclature) : LtaP15.0020)[Best reverse hit against LmjF.15.0030] and NTPD ase2=LTAR_10 0006700.1 (previous naming system: LtaP10.0140) [highest rev hit for LmjF.10.0170] They were identified as follows: In contrast to L. major, both L. talentlae NTPDases are CTS dormant The inventors have a hybrid sequence of 5 amino acids in the active region and 10 amino acids in the active region. It is set to the CTS region for fusion. The length of the CTS stem region is usually clearly defined. It has not been found, and (Geisler et al., 2015b) used the 13 amino acids following the TM domain. . wt Gnt and hybrid structure (Man1 CTS-SfGnT-I Strep; LtaNTPDase 1 CTS-SfGnT- I Strep; CTS-SfGnT-II (TbGnTI) was heterologously expressed in L. talenttrae, and then its localization was investigated. The analysis was performed using a coarse fractionation experiment. The inventors also predicted GnT-y, in the following example, for human MGAT1. Using the modified CTS domain, recipient GnT-x of various lengths, in the following example, Sf The plasmids were fused to GnT-I (Figure 25). These plasmids (Table 10) were tested for localization (Figure 46). The increasing length of the MGAT1 N-terminal domain is fused to the SfGnt-I catalytic domain, which has a decreasing N-terminus. By using this (Figure 46B), an improvement in cell (membrane) localization was observed (Figure 46A). More specifically, it is derived from native Golgi localization proteins of L. talentrae or from heterologous sources. The hypothesis that testing any different N-terminal retention signals originating from Golgi GnT is being tested is being strengthened. (This is illustrated by using the MGAT1 N-terminal retention signal.) However, N-g The activity for lycan conversion was low, and because a Strep-triple HA tag was used, 90MG1-SfGnT- It was only detected in St13561 expressing the I-3HA-Strep construct, and Strep-triple HA tag The activity of native SfGnT-I with this characteristic is approximately 10% lower compared to Strep-tagged SfGnT-I. It decreased (Figure 46B). Table 10 Hybrid [Table 10]

[0337] First, the present inventors have developed recombinant human erythropoietin having a secreted peptide, or its Recombinant cells expressing one of the SfGnT-I proteins containing active targeting signals The normal growth of the cell line was examined. OD was monitored, and changes in cell shape were observed under a microscope. Follow-up was conducted. Shaking cultures reached high OD, and growth / expression peaked at 72 hours, but at 96 The decrease occurred over time. The resting culture reached maximum growth and expression at OD 1.8 after 96 hours, indicating active production. This persisted. During growth, L. talenttrae cells undergo cell cycle changes (G1>S>G2>M>G1…). The connected morphology was observed (Figure 26A). Expression was observed by separating the crude cell pellet (whole cell extract) and the supernatant. Furthermore, the general localization showed that EPO is mainly secreted and not retained inside the cell. The expected Golgi enzyme Sf-GnT-I was found in the supernatant in a cleaved form and traveled via the secretory pathway. Suitable for potential proteolytic cleavage or retention of it in the Golgi apparatus of L. talentlae. This suggests that CTS is not present (Figure 26B). Furthermore, SfGnT-I is also secreted in SN, but trypanos The enzyme derived from M was found only in the crude fraction and the Triton-X solubilized fraction, and showed membrane and Golgi localization. (Figure 27). TbGnT-I and TbGnT-II were mostly associated with the membrane (Figure 27B). SfGnT-I was In contrast to Tb-derived enzymes, it is sufficiently active in vivo and in vitro, and therefore, The inventors used Sf-GnT-I as a catalytic domain for hybrid design. (Figure 24 and) The CTS N-terminal region described in 25 was gene-fused to the predicted catalytic domain of Sf-GnT-I. Except for the CTS variants described in Figure 24B, TbGnT-I CTS is a functional GnT-I or Gn It could also be used to fuse to the N-terminus of the catalytic domain of the T-II enzyme. The CTS mutant All of this is done to create a functional hybrid GNT that is retained in the Golgi apparatus, and the activity is It is assumed that this is used in the catalytic domains of all Gnt (GnT-I, GnT-II, GalT, SiaT).

[0338] The localization of the entire SfGnT-I and its CTS-hybrid is shown in Figure 28. Empty vector pair Although the band is not visible, SfGnT-I-Strep (St11898) is detected by anti-Strep immunoblotting. Culture samples from 3 to 6 subculturings showed SN values ​​of 38 kDa (double band), 30 kDa, and 20 kDa degradation products. It was detected at a fragmented size of 50 kDa, but lysate (+ / -TrX) and insoluble cell residues were also detected. No band was detected. CTS-Man1-SfGnT-I-Strep(St11899) is 47kDa p3~p6 It was detected in the SN, as well as in the decomposition products at 38kDa (double band), 30kDa, and 20kDa (aStrep). No bands were detected in lysate (+ / -TrX) or insoluble cell residue. CTS-LtaNTP D-ase 1-SfGnT-I-Strep (St11900) was not detected in either the membrane fraction or the SN. And it was neither expressed nor stable at all. CTS-LtaNTPDase 2-SfGnT-IS trep(St11899) was detected as a faint band in the 47kDa p5 and p6 signal-to-noise ratio, but the lysate (+ No bands were detected in / -TrX) and insoluble cell residue. This suggests that SfGnT-I is more responsive. It appears that there are sites near the medium domain that are easily utilized for protein degradation, leading to an increase in CTS. When we designed and tested a second strategy aimed at adding to the market, we showed that the localization did not change (Figure 25). (Table 10, Figure 46A, Figure 47B). Intracellular localization was significantly improved in these increased hybrids. The hypothesis was supported (Figure 47A). However, firstly, the native SfGntI was converted to SfGntI-Stre Compared to po and SfGnt-I Strep-3HA, Strep-triple has a negative effect on activity. Secondly, for the HA tag (Figure 47B), the cleavage of the N-terminus of 110 amino acids is also likely SfGnT- Since the catalytic activity of I was impaired, N-glycan conversion cannot be considered.

[0339] We tested St12239 cells from p5 and p6 expressing native human Gnt and hMGAT1 tagged with 3×HA. When tested, hMGAT1 was detected in both whole cell extract (WCE) and SN. MGAT1 was found in cell residue. However, it was detected (Figure 29). This indicates secretion / release, but in vitro active enzymes are primarily It was purified from the membrane fraction but not from the SN (Figure 11).

[0340] SfGnT-II (ssu::SfGnT-II-3×HA) expressed from St12318 of p10 was detected in WCE and SN. It was secreted / released at low levels. SfGnT-II was detected in TrX-lysate and in the cytoplasm. Localization was shown. However, SfGNT-II was strongly detected in TrX+ lysate and associated with the membrane. This was shown (Figure 30).

[0341] The relative strong expression of rMGAT2 from St11897 in p5 and p6 (ssu::MGAT2-3×HA) leads to rMGAT2 in SN. The secretion of AT2, its presence in whole cell extracts, and the intracellular / cytosolic localization of rMGAT2 are also linked. It was observed that rMGAT2 is strongly bound to the membrane in the TrX+ lysate and insoluble fraction. Degradation at approximately 33-35 kDa was also observed (Figure 31). rMGAT2 was subjected to in vitro activity testing. For use with SN and TrX + Refined from pellets (Figure 12).

[0342] Therefore, these novel hybrid strategies are in the Golgi section of L. Talentrae. This is necessary to support the retention of heterogeneous and active Gnt.

[0343] (8.9 Example 9 Sialic acid biosynthesis and sialylation of branched N-glycans) In mammals and bacteria, the assimilation and catabolism of Neu5Ac occur through different pathways (Ang (Ata and Varki, 2002). Using two major classes of enzymes, Neu5Ac is formed. This can be done. N-acetylneuraminic acid lyase (Neu5Ac lyase) is a reversible reaction that can convert Neu5Ac By catalyzing the cleavage of N-acetylmannosamine (D-ManNAc) and pyruvate, It is involved in the catabolism of alic acid. At high concentrations of D-ManNAc and pyruvate, the equilibrium is set to the synthesis of Neu5Ac. It can be made to work. By coupling glucosamine 2-epimerase activity, it can be derived from E. coli. Neu5Ac lyase can be used for large-scale Neu5Ac production from D-GlcNAc. Alternatively, Neu5Ac synthases such as NeuB perform the condensation of ManNAc to phosphoenolpyruvate (PEP). It can be used as a catalyst and is directly involved in the biosynthesis of sialic acid (Tanner's text). (Reviewed in 2005). CgNal is used by Corynebacterium gluconate for the production of Neu5Ac. This is an N-acetylneuraminic acid lyase derived from Tamicum, which contains ManNAc and pyruvate It catalyzes the reversible aldol condensation of Neu5Ac, but not the cleavage of Neu5Ac, which is advantageous for synthesis. (Ji et al., 2015). The first step in the mammalian sialylation pathway is activated sugar This is the biosynthesis of the nucleotide precursor CMP-Neu5Ac. To achieve the production of this precursor, at least four enzymes are used: (1) ManNA from UDP-GlcNAc GN is a bifunctional enzyme that catalyzes the conversion to c and the phosphorylation of ManNAc to ManNAc-6-phosphate. E;(2)ManNAc-6-phosphate and phosphoenolpyruvic acid are condensed to produce Neu5Ac-9-phosphate. (3) NANS that cause (4) Neu5Ac-9-phosphate specific phosphatase; and (5) as a result The action of CMAS, which activates the resulting primary sialic acid in the nucleus to CMP-Neu5Ac, can be utilized. (Castilho et al., 2010). Furthermore, bacterial NanE or GNPE (Geisler and Jarvis, 2010). 12) is even more favorable for the formation of precursor ManNAc-6-phosphate from GlcNAc-6-phosphate. It is possible (Figure 53).

[0344] A suitable sialyltransferase (Table 9) is recombinantly expressed in L. talenttrae, and L. talenttrae Affinity can be concentrated from the crude lysate, membrane-solubilized fraction, and culture supernatant of *Centrae*. Subsequently, the semi-purified enzyme (Figure 49A) was used to determine its appropriate activity, and the cofactors and activated sugar nucleotides were used. The test was performed in vitro using the otiide precursor CMP-Neu5Ac. Figure 48A shows one sialic acid (= simple (Chain sialylation) or two sialic acids (branched sialylation) are converted to a branched galactosylated substrate. Regarding the transfer of L. talenttrae-expressed sialyl to 2AB-labeled G2 substrates, This shows the relative activity of the enzyme. By comparing the retention times (Figure 49), the enzyme is superior to that of the 2AB-labeled glycan. The bond determination was evaluated and confirmed to be either α2,6-linked sialic acid or α2,3-linked sialic acid. Figure 48B shows the relative amounts in this screening assay. In the following method, the main A monoclonal antibody (MabThera) that completely folds a candidate (mouse mST6GAL1) Its activity against (Figures 50A-50E) was tested. Extracted from lysate and affinity concentrated. The mST6 (Figure 50A) resulted in 100% bifurcated α2,6 sialylation of the 2AB-labeled G2 substrate, but ( Figure 50B), the same enzyme preparation mainly sializes one branch (~14%), while the bibranch is almost... Almost undetectable (0.4%) (Figures 50C and 50D). HA eluate from mST6 was 14% of the total N-glycans. 2% converted (sialylation) (~25% conversion of total galactosylated N-glycans); HA of rSAP+mST6 The eluted product is 15.36% of the total N-glycans converted (sialized) (total galactosylated N-glycans). (~26.5% conversion). Beads that capture mST6 via HA-tags (those that have not been eluted) 20.13% of all N-glycans were converted (sialized) (approximately 34.7% of all galactosylated N-glycans) (Conversion). 2 μM dCTP inhibits the sialyltransferase activity of HA eluate from mST6 (and It appears to enhance sialidase activity, and converts 4.12% of all N-glycans (sialylation). ) only resulted in (~6.9% conversion of all galactosylated N-glycans) (Figure 50D). And mST6 exists either in a free state (eluted) or a captured state (bead form), MabThera can sialize galactosylated N-glycans in vitro, and rSAP is dCTP has a slightly positive effect on ST conversion, while dCTP inhibits ST conversion.

[0345] Modifying host cells to express the functional CMP-sialic acid (CMP-Sia) biosynthesis pathway. This can be done. It has been previously carried out in a variety of other organisms such as Pichia, insect cells, and plants. Thus, mammalian biosynthesis and / or bacterial biosynthesis (Table 11) are utilized in L. talenttrae host cells. It can be used (Aumiller et al., 2003; Hamilton et al., 2006; Castilho et al.) References, 2010). If CMP-NeuAc becomes available in the Golgi apparatus of L. talenttrae host cells, then specific A typical sialyltransferase accepts sialic acid as an acceptor substrate (e.g., β1,4-galactate). It can be transferred to tosylated bifid N-glycans. Table 9 shows mammalian and bacterial sial Figure 53 shows the transferase and the expected intracellular localization of the biosynthetic pathway. . Table 11 Sialic acid biosynthesis [Table 11]

[0346] A method is provided to modify the CMP-Sia biosynthesis pathway to one used in non-human eukaryotic cells. The method involves mammalian origins and bacterial origins in L. talentlae host cells lacking endogenous sialylation. This includes cloning and expression of several enzymes from the source, or both. Modified CMP-Sia The biosynthetic pathway produces sialylated glycolipids, O-glycans, and N-glycans in vivo. This is useful for non-human host cells that lack endogenous sialylation. It is useful in promoting the production of sialylated therapeutic glycoproteins.

[0347] α2,3- or α2,6-sialyltransferase is used in human trans-Golgi and trans In the Golgi network (TGN), sialic acid is used to seal galactose residues, and the mature form This process generates glycoproteins. By modifying this process, naturally occurring sialyltran To introduce spherase activity into inferior eukaryotic host cells and other host cells, the following is required. (1) α2,3- or α2,6-sialyltransferase activity and (2) CMP-N-acetylneurase A sufficient supply of sic acid can be incorporated into the late Golgi apparatus of the host cell. Secretory pathway (for example) Therefore, in order to obtain sufficient α2,3- or α2,6-sialyltransferase activity in the late Golgi For example, known sialyltransferases (e.g., those of mammalian or bacterial origin) The catalytic domain of (of) can be directed to the secretory pathway of lower eukaryotic host cells. Similarly, Modify the lanceporter to secrete CMP-N-acetyl into the same location in the secretory pathway (e.g., late Golgi). This enables the transport of neuraminic acid. As a result, the corresponding glycosyltran To ensure a sufficient supply of substrates to spherase, metabolic production of CMP-sialic acid It can be modified and introduced into these host cells. All analyses were performed in Examples 4 and 5. It can be done as described.

[0348] (8.10 Example 10: Anti-CD20 "rituximab" expression in L. talentrae) As previously discussed and schematically shown in Figures 1 and 2, efficacy is improved and therapeutic dosage is increased. This reduces the risk and improves the overall clinical outcomes of antibodies through modification with Fc glycans. As a result, the design of the glycan repertoire has been selected based on a variety of characteristics (Figure 32). To test the concept in its application, we used anti-CD2, which is commercially available as MabThera® (Roche). Rituximab, a monoclonal antibody, was used as a test case for its known ADCC mechanism of action. I selected it. Figure 33 shows the amino acid sequence, signal peptide, and the Asn 297 sugar moiety considered. Figure 34 shows the expression cassette incorporated into L. talenttrae host cells. The resulting cell line St12427 was grown, and a substance called "rituximab_LMTB" was added to the culture supernatant. The released antibody molecules were recovered. Using the filtered supernatant, first, protein A capture and purification was performed, and then The purification was performed using a two-step process involving hydrophobic interaction columns (Figure 35).

[0349] Figure 36A shows a comparison between rituximab_LMTB and the commercially available comparison substance MabThera® from Roche. This indicates that, under non-reducing conditions, rituximab_LMTB exhibits a somewhat different intensity, M It showed a band pattern similar to abThera®. Traces of degradation products were present in both samples. Observed: Proteins digested by PNGase F were more effective than rituximab_LMTB. The era(registered trademark) contained significantly elongated glycans, suggesting greater heterogeneity. The differences observed are shown in Figure 36B. Capillary gel electrophoresis revealed these observations. This supported the result (Figure 37).

[0350] Rituximab (LMTB) aggregate formation is particularly important for the isolation and characterization of monoclonal antibodies. Using the MAbPac SEC-1 size exclusion chromatography (SEC) column, MabThera ( Compared to 0.3% for registered trademarks, the figure was determined to be 5.8% (Figure 38).

[0351] Next, the Waters GlycoWorks® RapiFluor-MS® procedure (Figure 39) or PNGase F release and N-glycan profiles using either complete methylation or subsequent MALDI-TOF (Figures 40 and 41) Filing was performed to compare the N-glycan of MabThera® with rituximab_LMTB. We addressed the heterogeneity of the fucose levels in biferous iodine. In fact, rituximab_LMTB is produced in CHO. Compared to the eight glycoforms found in the antibody MabThera®, 100 It had a % uniform Man3 glycan. The quantitative comparison based on complete methylation shown in Figure 40 is It exhibits a largely fucosylated structure, which is not optimal for the Fc-mediated receptor function of ADCC. In fact, for ADCC, not only are glycosylation modifications being performed, but protein modifications are also being carried out. Gazyvaro® (Roche), a 20-generation anti-CD20 antibody, is effective in increasing the amount of afucosylated N-glycans. It is added (Figure 42). Gazyvaro® fully methylated N-glycan profiling is, Rituximab produced in Leishmania talentrae and MabThera (registered trademark) manufactured by Roche It differs significantly from the standard profile. The main structure is bisecting N-glycan G0B. These are G0BF, G1B, and G1BF. The fucosylation levels of Gazyvaro (registered trademark) are the same as those of MabThera (registered trademark). Compared to 94% of the trademarked product, it is 52%. That is, produced in Leishmania talentrae The rituximab LMTB used was 48% of Gazyvaro® and only a small amount of MabThera®. Compared to 6%, it is not 100% fucosylated. However, Leishmania talenttra The rituximab produced in E has 14 structures found in Gazyvaro(registered trademark) and MabThera( Compared to the eight structures found in (registered trademark), it shows only one structure corresponding to Man3 GlcNAc2. The fully methylated N-glyca of the commercially available comparative substance MabThera® produced in CHO cells. The profiling revealed that G0F and G1F, typical fucosylated bifurcated structures, are the main structures. The eight structures it possesses were shown.

[0352] This is for Leishmania talentrae for therapeutic recombinant antibodies or Fc-containing molecules. I strongly support the concept of homogeneous and functionally customized N-glycan sugar chain modification. .

[0353] Furthermore, FACS staining was used to determine the CD20 antigen binding ability in Raji cells (Figure 43A), and recombinant CD2 Dot plot analysis (Figure 43B) involves spotting 0 and incubating it with the displayed antibody. In both assays, rituximab_LMTB performed to the same extent as MabThera®. Both antibodies were bound to the antigen (CD20). Both antibodies showed clear dose-dependent effects, surpassing the IgG1 control antibody in FACS. While it produced a residual signal, the Mabthera signal appeared slightly stronger.

[0354] Figure 44 further illustrates the schematic quality assessments that were conducted, which are also listed in Table 12. Table 12 Rituximab Quality Standards [Table 12]

[0355] Figure 52 shows stable expression of glycosyltransferases SfGnt-I Strep and MGAT2-HA; Leishmania talenttrae expresses rituximab episomatically from pLMTB5026. This shows in vivoglycan elongation from Man3 to G0 in strain (St13418). Next, secretion Rituximab was purified using protein A from a 1L scale culture for 2 days. 20 ug of purified rituximab was obtained. Rituximab LMTB was analyzed using PNGase F and RF-MS, and this was used to analyze glycans in vivo. The modification was shown to be successful with full-length monoclonal antibodies of secreted target proteins.

[0356] (8.11 Materials and Methods) (8.11.1 Strains, growth, and genetic methods) Unless otherwise indicated, the strain (Table 13) contains Brain Heart Infusion containing 5 ug / ml hemin. In a BHIH culture medium, in the dark at 26°C, as a static culture or shaking culture, for 2-3 days using a conventional method. The cultures of the recombinant cell lines were allowed to grow. The cultures contained appropriate selective agents (Table 14). Table 13 Strains [Table 13] TIFF2026067872000069.tif244170TIFF2026067872000070.tif146170 Table 14 Selective Agents [Table 14]

[0357] Transfection to create a stable cell line is performed as follows to obtain the desired heterogeneity The gene was incorporated. The expression cassette is for 1) site-specific integration by homologous recombination. 1. Homologous sites, 2.) 5' untranslated terminal repeats containing splice reader acceptor sequences, 3.) Codon usage is optimized for Leishmania (either L. major or L. talenttrae). 4.) For example, the target gene as an ORF, the 3'UTR of the polyadenylated sequence and the 5 downstream gene 'Intergenetic region containing UTR, 5.) resistance marker, followed by, 6.) its 3'UTR, and 7 It contains a 3' homology region for site-specific integration into the genome.

[0358] For integration into the genome, 5-10 ug of donor plasmid DNA is digested with adjacent restriction enzymes. Then, the expression cassette was excised from the vector skeleton. Restriction digestion was completed or heated at 30°C. The DNA was purified by EtOH precipitation (2 volumes of 100% ice-cold EtOH were added to 1 volume of digested DNA). (Then, incubated on ice for 30 minutes, and centrifuged at 17,500 × g for 30 minutes at 4°C). The pellets were washed with 70% EtOH, dried for up to 15 minutes, and resuspended in ddH2O. Cyclomorphization To optimize the removal of the plasmid, one or two plasmids having recognition sites in the vector backbone are used. One restriction enzyme was selected, digested at 37°C for approximately one hour, and then purified using EtOH as described above.

[0359] Transformation of E. coli DH5α with 100 ng of digested DNA, undigested control, and ddH2O control The procedure involved heat shock to determine whether intact plasmid DNA remained. A plasmid containing 100 ng of chemically competent DH5α, thawed on ice for 10-15 minutes. Carefully mix with DNA, incubate on ice for 25-30 minutes, then heat at 42°C for 90 seconds. Shock-treated and incubated on ice for 5 minutes. Add 1 ml of LB or SOC medium and incubate at 37°C for 1 minute. Incubated for 2 hours. Then, the aliquots were plated onto a LB containing ampicillin. They were then placed upside down and incubated at 37°C.

[0360] Prepare the leishmania culture for transfection at a static state at 26°C. The day before infection, the procedure was performed using a 1:10 dilution of high-density grown cultures in BHIH. OD was used. Measurements were taken at 600 nm using a photometer in a sacrificial cuvette, and for optimal efficiency, the values ​​were set to 0.4~1.0 (4~6 × The area was set to 10*7 cells. The cells should be in the logarithmic phase, and this means they are round in shape and This is shown by a mixed population of teardrop-shaped cells. Rounder-shaped cells were preferred. 10 ml of culture is used for one transfection, and one culture is used for each selected mark. As a negative control for Kerr, electroporation with ddH2O was always performed. For this purpose, the culture was spun at 1800 × g for 5 minutes in RT. The SN was removed and the pellet was placed in 5 ml. Transfection buffer (200 mM Hepes pH 7.0, 137 mM NaCl, 5 mM KCl, 0.7 mM The solution was resuspended in Na2HPO4, 6 mM glucose (anhydrous glucose, sterilized and filtered at 0.22 μm). The cells were centrifuged again, and the pellet was resuspended in 400 µl of transfection buffer. 400 µl of cells were added to the DNA, transferred to a cuvette, and incubated on ice for 10 minutes. Low voltage protocol, exponential decay: 450V, 450uF, 5~6ms, cuvette: d=2mm, G Electroporation was performed using ene Pulser Xcell (trademark) (Biorad). Immediately afterwards, Place on ice for 10 minutes. Dilute the entire contents of the cuvette with 10 ml of BHIH without the selection marker. The cells were then transferred to a container, aerated, and grown in a quiescent state in the dark at 26°C for 20-24 hours.

[0361] Selection at half concentration for subsequent selection of polyclonal cell lines by clonal selection. Add the marker, incubate the culture at 26°C for 1-2 days, and add 10 ml of BHIH+ at maximum concentration. Among the selected markers, subgeneration was performed at a ratio of 1:10. A new strain number was assigned, and the number of subgenerations was set to 0. Subsequently... For all experiments, the passage number of the transfected strain was recorded. The cells were kept in the dark at 26°C. They were then allowed to grow. After 7 days, if the culture was turning cloudy, the cells were refrigerated for 5 minutes. During this time, the pellets were spun at 1800 × g and then transferred to a new BHIH medium containing the maximum concentration of the selective marker. It was resuspended.

[0362] For clonal selection, as soon as the liquid culture becomes cloudy, plate the cells in a BHIH plate (1.4%). Streaked with agar (containing) and a suitable 100% selective agent. The plate was sealed with Parafilm. The cells were then inverted and incubated in the dark at 26°C for 7-10 days. A single colony (1-2 mm) was observed. Transfer the (size) to a 24-well plate containing 1 ml of BHIH, seal it with Parafilm, The culture was incubated in the dark at 26°C for approximately 7-10 days. Afterward, 1 ml of the culture was converted to 24-wellpray. The cells were transferred from the container to 10 ml of BHI in a flask and grown further statically as usual.

[0363] Integration at the targeted genomic region was confirmed by PCR and sequencing.

[0364] (8.11.2 Plasmid) The plasmid was obtained from the pUC57 vector skeleton for E. coli growth, and the ampicillin partial mer It contained KAR. The expression cassette is adjacent to a restriction site suitable for excision. The cassette contains: 1.) homologous sites for site-specific integration by homologous recombination, and 2.) splices. 5' untranslated end repeats containing leader acceptor sequences, 3.) Leishmania (L. major) The target gene as an ORF optimized for codon use for either L. talenttrae or L. talenttrae. 4.) For example, an intergenetic region containing the 3'UTR of a polyadenylated sequence and the 5'UTR of a downstream gene. 5.) resistance markers, followed by 6.) their 3'UTR, and 7.) site-specific combinations into the genome. It contains a 3' homology region for inclusion. This plasmid is supplied by gene synthesis suppliers. It was created and sequenced. http: / / www.kazusa.or.jp / codon / cgi-bin / showco L. Major can be found at don.cgi?species=347515 or at http: / / genomes.urv.es / OPTIMIZER / The use of codons optimized for leishmania, derived from either L. talentrae, is The following codon usage table is used:

[0365] [ka]

[0366] The optimized sequence avoids restriction sites and deletions of repeats or homopolymer stretches. Therefore, the plasmids were manually curated. Plasmids and descriptions can be found in Table 15. Table 15 Plasmids [Table 15] TIFF2026067872000074.tif241170TIFF2026067872000075.tif43170

[0367] (8.11.3 Gnt candidate) Gnt and further Gnt hybrids are listed in Tables 9 and 10.

[0368] (8.11.4 N-glycan profiling using Rapifluor (RF) labeling and MS) (a) Sample preparation Waters Application Note: GlycoWorks RapiFluo is suitable for quality control and automation. Sample preparation using r-MS N-Glycan (Quality control and Automation-Friendly GlycoWorks R) Samples were prepared according to the apiFluor-MS N-Glycan Sample Preparation procedure. A brief description follows. The sample volume is 10 μl, and in the case of purified protein samples, it is 1.5 mg / ml (15 μg) or pellet. The cells were 10*8. 3% RapiGest was added at 100°C for 3 minutes, then RT for 3 minutes, followed by 10 μl of P. Add NGase F (Sigma, diluted 30 μl PNGase + 220 μl water) and incubate at 50°C for 5 minutes. Incubated. After 3 minutes on RT, 10 μl of RFMS (9 mg in 110 μl of anhydrous DMF) was added, and RT continued for 5 minutes. The sample was incubated for 1 minute. 360 μl of ACN was added, and the sample was eluted using SPE with 3 × 30 μl of solution. The solution was washed by cleanup and pooled up to 90 μl. 10 μl was then injected using a complete loop injection. Previously (4 × overfill factor), the sample was diluted with 100 μl of DMF and 210 μl of ACN.

[0369] (b) Column setting Liquid chromatography was performed using a Waters Acquity UPLC system, using Glycan BEH amide chromatography. The procedure was performed using a Waters sieve (130 Å, 1.7 μm, 2.1 mm × 150 mm). Separation of N-glycans was performed using a 20% solution. 0.1% formic acid (FA) (Buffer A) in water and 0.1% FA (Buffer A) in 80% acetonitrile Starting from B), 27% of buffer A is reached in 3 minutes, and then 37% of buffer A is reached within 32 minutes. The procedure was performed using a gradient at a flow rate of 0.5 mL / min. Detection was performed using a UV detector at 215 nm.

[0370] For mass determination in ESI mode, the UPLC was directly connected to a Waters Q-TOF Synapt HDMS. Lock mass spray (leucine enkephalin) is used for lock mass correction, and the mass is positively adjusted. I obtained this in IV-ion mode with a m / z of 300-3500.

[0371] ACQUITY UPLC Glycan BEH amide 130 Å, 1.7 μm, 2.1 × 150 mm; Buffer: A: 50 mM AmF or(H2O) pH 4.5, B: CAN;Flow rate: 0.5ml / min;Temperature: 45℃;Injection volume: 10μl (complete loop injection); Gradient: 80-73%B in 3 minutes, 73-63%B in 32 minutes (total 55 minutes), LC method ESI_RFMS_mAB_55_FLR; Sy napt settings: 20161006_uba284_esi_RFMS), MS method ESI_RFMS_mAB_300_3500_vpos_55; Pump voltage: 3kV; Cone voltage: 80V; Source temperature: 120℃; Melting temperature: 350℃; Dissolved gas: 800l / h; Lock mass: Leucine enkephalin, 1 ng / µl at a flow rate of 4 µL / min; Fluorescence detection: Ex 265 / Em 425 nm ( RapiFluor-MS (2Hz).

[0372] (8.11.5 Complete methylation and MALDI-TOF of N-glycan profiling of crude cell pellets) G) (a) Sample preparation A pellet of 10*8 cells was treated with chloroform / methanol for 30 minutes while shaking. Delipidation was performed using a 2:1 ratio. After centrifugation, a second delipidation was performed using chloroform methanol water (40:20:3). The delipidation process was carried out for 30 minutes while shaking. After centrifugation, the delipidated cell pellet was It was dried under a nitrogen stream.

[0373] The cell pellet contains proteins with 0.1% Rapidest® SF (WATERS) and 10 mM DTT. Extraction by sonication for 3 × 90 seconds in 200 μl of 50 mM ammonium bicarbonate buffer. The protein was denatured at 56°C for 45 minutes, and then treated with iodoacetamide (50 mM) in the dark at room temperature. The sample was alkylated for 60 minutes. Then, the sample was subjected to mass spectrometry of 10 μg of trypsin / Lys-C mix, etc. The protein was incubated with PROMEGA at 37°C for 16 hours to obtain digested protein.

[0374] (b) Glycosidase digestion and complete methylation of N-glycans After inactivating trypsin / Lys-C at 95°C for 5 minutes, the sample was divided into two aliquots. One aliquot was deglycosylated with 20 units of PNGase F (Promega #175715), and two were... Prepare aliquots of the eye by adjusting the pH to 5 with 15 μl of PNGase A (Roc) in 200 mM acetate buffer (pH 5). Deglycosylation was performed using he #10472620. The four samples were incubated at 37°C for 16 hours. By adding 0.5% TFA to the processed protein sample, hydrolyzable surfactants are removed. The mixture was incubated at 37°C for 30 minutes and centrifuged at 15,000 × g for 10 minutes. Trypsin digestion. The peptide was packed into a SEP PACK C18 200 mg column, and a flow-through containing N-glycan was passed through it. I collected it.

[0375] N-glycans were purified using an Ultra Clean SPE Carbograph (ALLTECH). The SPE was then fermented in 0.1% TFA. After balancing, the N-glycan released by PNGase was packed into the sample and washed with 0.1% TFA. 3 ml After elution with 25% acetonitrile and 0.1% TFA, the N-glycan was freeze-dried, and then... Complete methylation was performed using approximately 25 mg of sodium hydroxide, 500 μl of DMSO, and 300 μl of ICH3. Complete methylation was performed on the lyophilized sample for 40 minutes. The reaction mixture was quenched with 1 ml of water. Next, 3 × 500 μl of chloroform was used to extract the fully methylated glycan. Chloroform phase The product was washed with an equal amount of water and then dried. The reaction product was packed into 200 mg C18 SepPak (WATERS). The sample was then eluted in 2 ml of 80% acetonitrile, lyophilized, and analyzed by MALDI-TOF MS. .

[0376] (c) MALDI-TOF analysis of N-glycans Purified fully methylated glycan was solubilized in 20 μl of 50:50 methanol / water. 2 μl Undiluted N-glycan and 1 / 2 diluted N-glycan were mixed in 2 μl of 2,5 DHB (LaserBiolabs) matrix. Mixed with Rix solution (10 mg / ml 50:50 methanol / water). Positive ion reflect Ron-MALDI-TOF mass spectra were obtained using an Autoflex III mass spectrometer (Bruker). The spectrum was acquired by accumulating 4000 shots and calibrated using an external standard (Pepmix4 LaserBiolabs). The acceleration and reflector voltage conditions were set as follows: voltage 10.3 × 1954V, 80% User.

[0377] (8.11.6 Purification of affinity-tagged heterologous Gnt for in vitro activity assays) The HA purification procedure was as follows: A culture of recombinant cells expressing heterologous Gnt was used to purify the target HA. We first analyzed the intracellular expression of tagged Gnt enzymes. 1 × 10 9 Individual cells are collected and centrifuged. The cell pellet was then separated for affinity purification (2000 × g / 5 min). ) 1 mL extraction buffer containing Triton (25 mM Triton, pH 7.5, 100 mM NaCl, 1% v / v Triton) n X-100 was resuspended in EDTA-free protease inhibitor [Roche] (1 mM PMSF). The suspended cells were sonicated on ice to avoid any kind of overheating. The Delin Sonopuls was run in 3 steps, 20 seconds each, at 70% power input, for 7 cycles. The cells were vigorously stirred for 10 seconds. The suspension of destroyed cells was heated at 13,000 × g for 1 hour at 4°C. The heart was separated. The supernatant (lysate) was carefully removed and used for purification. The remaining solid fraction was 1 × L The lysate was resuspended in aemmli and used for SDS-PAGE analysis. The lysate was chilled in cold PBS (1× protease). Mix with (including inhibitor tablets) in a 1:2 ratio, and add 100 μL of anti-HA magnetic beads [Thermo Scientific] Both were incubated at RT, 600 rpm for 30 minutes. HA magnetic beads were placed in a magnetic rack [Thermo Sc]. The solution was collected in a 2 mL tube using [ientific] and washed twice with 2 mL of ice-cold TBS. The eluate was collected in 70 μL of [a solution]. Elution was performed using elution buffer (2 mg / mL HA peptide in TBS [Thermo Scientific]). The fractions were analyzed by SDS-PAGE, followed by anti-HA WB analysis, and used in in vitro activity assays.

[0378] The StrepTactin purification procedure was as follows: A culture of recombinant cells expressing the heterologous Gnt was analyzed for intracellular expression of the target Strep-tagged Gnt enzyme. 1×10 9 cells were harvested and centrifuged (2000×g / 5 min) to separate the cell pellet for affinity purification. The pellet was resuspended in 1 mL of extraction buffer containing 1 (v / v) Triton. The resuspended cells were sonicated on ice to avoid any kind of overheating. Sonication [Bandelin Sonopuls] was performed in 3 steps for 20 s at 70% power input and 7 cycles. Vortexed vigorously for 10 s . The suspension of disrupted cells was centrifuged at 13'000×g for 1 h at 4 °C. The supernatant (acetate) was carefully removed and used for purification. The remaining solid fraction was resuspended in 1×Laemmli and used for SDS-PAGE analysis. The lysate was mixed 1:5 with cold PBS (containing 1× protease inhibitor tablets) and incubated with 100 μL of StrepTactin sepharose [VWR] at RT, 600 rpm for 30 min . To reduce the interference effect of biotin in the cells / media on the effect of StrepTactin purification, 5-fold dilution was performed in PBS. The StrepTactin sepharose was recovered in a 2 mL tube by centrifugation (2000 ×g / 1 min) and washed twice with 2 mL of cold TBS. Elution was performed using 70 μL of elution buffer (2.5 mM desthiobiotin in 1×TBS). The elution fraction was analyzed by SDS-P AGE, then anti-Strep WB and used for in vitro activity assay. (8.11.7 In vitro glycosyltransferase activity assay, 2-amino of glycan

[0379] (8.11.7 In vitro glycosyltransferase activity assay, 2-amino of glycan (Benzamide (2AB) labeling and / or washing and HPLC separation) Add the newly prepared reagents to a 1.5 ml tube on ice in the following order to perform HA purification or stoking. In vitro activity assays were performed on leptavidin-purified GNTs: 1. 50 ng of appropriate 2AB-labeled alpha Acceptor glycan (e.g., 2AB-Man3a[Prozymes]) or 500 ng of suitable unlabeled acceptor - Glycan (e.g., Man3a[Prozymes]); 10mM MgCl2; 10mM MnCl2; 1.2mM activated sugar (e.g., (For example, UDP-GlcNAc or UDP-Gal [Sigma Aldrich]). TBS buffer (pH 8.0, 25mM Tris pH 7. 5. Adjust the volume to 50 μL using 100 mM NaCl; 20 μL of HA concentrate or streptavian Add din-concentrated GNT enzyme, or a corresponding simulated control of water, e.g., elution buffer. Only was added. The mixture was then centrifuged for a short time and incubated at RT, 600 rpm on / off. For unlabeled substrates, use the Sep-Pak C18 Classic cartridge [Waters] before performing HPLC. The glycan was purified from the reaction mixture using [method / method]. The sample was prepared according to 2-A (Bigge et al., 1995). Labeled with B. Glycan washing was performed using the filter paper disc method described in (Merry et al., 2002). Purification was performed. For 2AB-labeled substrates in in vitro assays, PTFE washing was performed before HPLC. They did that.

[0380] In vitro reaction of sialyltransferase using 2AB standard (Luley-Goedl et al. literature) (2016) 40 ng of 2AB-G2 (substrate sugar), 20 μL of HA-enriched protein SiaT; 24 μL of 100 mM MES p 1 μL of TritonX-100 solution up to H 6.5 (final 50 mM MES pH 6.5); final 0.1% (v / v) TritonX-100. (In 100 mM MES), and 1 μL of 3.75 mM CMP-Neu5AC (dissolved in dH2O) stock solution (final 0.75 mM) It contained 4 μL. The reaction was carried out at 37°C for 12 hours at 400 rpm, cooled to 4°C, and PTFE washing was performed. The solution was frozen at -20°C until purification and HPLC analysis. Further reactions were carried out using MabThera as a substrate. They did that.

[0381] The in vitro activity assay of sialyltransferase using MabThera antibody is performed using 50 μg. g (5 μL) of commercially available MabThera (Roche, in formulation buffer), 20 μL of HA-enriched protein SiaT( The elution buffer contains 40 μg of HA peptide in 100 mM MES; 22 μL of 100 mM MES ES pH 6.5 (final ~100 mM MES pH 6.5); final 0.1% (v / v) TritonX- 1 μL up to TritonX-100 A 100 solution (in 100 mM MES) was used. 1.5 mM CMP-Neu5Ac was added first, and after 6 hours, further... 1.5 mM CMP-Neu5Ac was added to the mixture to obtain a final 3 mM CMP-Neu5Ac solution. The reaction was carried out at 37°C for 12 minutes. The process was carried out at 400 rpm for several hours, cooled to 4°C, and N-glycan release was performed using PNGase F, followed by RF-MS labeling (6.1 1.4 Up to -20°C (according to Rapifluor (RF) labeling and N-glycan profiling using MS) It was frozen.

[0382] The separation of 2-AB labeled glycans was performed using a GlycoSep-N normal-phase column, according to the method described by Royle et al. (2002). The procedure was performed by HPLC using [specific solvent], but was modified to a three-solvent system. Solvent A is 10 [units of solvent] in 80% acetonitrile. The solution was mM ammonium formate, pH 4.4. Solvent B was 30 mM ammonium formate in 40% acetonitrile. The solution was mononium, pH 4.4. Solvent C was 0.5% formic acid. The column temperature was 30°C, and the 2-AB column was used. Labeled glycans were detected by fluorescence (λex=330nm, λem=420nm). The gradient condition was 0.4 ml / At a flow rate of 10 minutes, 100% A to 100% B is converted over 160 minutes, then the flow rate is increased to 1 ml / min for 2 minutes, 10 The gradient was linear from 0%B to 100%C. The column was returned to 100%C for 5 minutes and then to 100%A for 2 minutes. The process was run at 100% A with a flow rate of 1 ml / min for 15 minutes, and then the flow rate was reduced to 0.4 ml / min for 5 minutes. The sample was injected in water.

[0383] (8.11.8 Expression and purification of rhEPO from unmodified and glycosylated cell lines) Two 200 ml cultures of each strain were grown in BHIH at 26°C for 72 hours while shaking at 140 rpm. The culture was collected and centrifuged at 2000 × g for 45 minutes. SN was used for ammonium sulfate precipitation. It was recovered. In short, SN was precipitated with (NH4)2SO4 (40% W / V) at RT for 45 minutes. Then, it was centrifuged at 11,000 × g for 1 hour at 4°C. The SN was removed and the mixture was resuspended, leaving the remaining brown / black The pellet was dissolved in 50 ml of 1 × PBS pH 7.4. It was then incubated with 5 L of PBS for 2 hours, and then in 5 L of PBS... Dialysis was performed at 4°C. 50 ml of the dialyzed sample was packed into a 6 ml StrepTactin column. 2 Washing was performed with 0 CV of 1×PBS pH 7.4 at 0.5 ml / min. 2.5 mM desthio in 1×PBS was added to the 1 ml fraction. The protein was eluted using 10 CV of biotin. The fraction was pooled, and the pooled volume was 4.5 ml. Cold acetone (-20°C) was added to the eluate and o / n precipitation was performed at -20°C. The sample was subjected to a 2-hour fractionation at 8000g. The core was separated, and the pellet was dissolved in 500 µl of ddH2O.

[0384] (8.11.9 Determination of N-glycosylation site occupation and site-specific glycosylation) (a) Peptide mass fingerprint of rhEPO Using Rapigest® SF, enzyme-mediated protein synthesis in 50 mM ammonium bicarbonate Enhanced quality digestion. 180 μg of protein corresponding to 250 μL of each sample was processed using Vivaspin 500 PES. (5kDa) and four centrifugations (15,000 × g; 10 min, 4°C) yielded a 50 mM ammonium bicarbonate bath. The solution was prepared in a solution at pH 8. The final protein concentration was approximately 3 mg / ml.

[0385] The protein was processed according to the Rapidest® SF protocol (WATERS) using 0.1% Rapidest ( (Registered Trademark) Denatured in SF, reduced with DTT (5 mM) at ...

Claims

1. a. Recombinant nucleic acids encoding target proteins; and b. Recombinant nucleic acids encoding heterologous glycosyltransferases Leishmania host cells, including:

2. The aforementioned heterologous glycosyltransferase is N-acetylglucosamine transfer ZE; and / or heterologous galactosyltransferase; and / or heterologous sialyltransferase The host cell according to claim 1, which is an enzyme.

3. The host cell contains two or more N-acetylglucosamine transferases; and / or two or more heterologous galactosyltransferases; and / or two or more heterologous sialyltransferases. A host cell according to claim 2, comprising -ase.

4. The host cells containing the aforementioned heterologous sialyltransferase produce CMP-NeuAc. The description according to any one of claims 1 to 3, further comprising heterologous CMP-Sia biosynthetic pathway proteins. The host cell.

5. One or more endogenous enzymes derived from the N-glycan biosynthesis pathway are deleted or mutated. and / or functionally inactivated, host cells according to any one of claims 1 to 4.

6. The leishmania cells are leishmania talentrae cells, according to claims 1 to 4. Either of the host cells described in item 1.

7. The aforementioned N-acetylglucosamine transferase, galactosyl transferase, and / or the amino acid sequence of sialyltransferase is the same as that of N-acetyl listed in Table 9. Luglucosamine transferase, galactosyl transferase, or sialyl Lutransferase, or any functional homolog, isoform thereof, A host cell according to any one of claims 2 to 6, derived from a mutant.

8. The leishmania signal and / or retention sequence is the N-acetylglucosamine trans Ferases, galactosyltransferases, and / or sialyltransferases The signal sequence is added to the N-acetylglucosamine transferase. galactosyltransferase and / or sialyltransferase are used in the The endoplasmic reticulum of the Schmaniac host cell is targeted, and the retaining sequence is the N-acetylg Glucosamine transferase, galactosyl transferase, and / or sialyl A host cell according to any one of claims 1 to 7, which holds the transferase in the Golgi apparatus. 。

9. The CMP-Sia biosynthetic pathway proteins capable of generating the aforementioned CMP-NeuAc are listed in Table 11. CMP-Sia biosynthetic pathway proteins, or any functional homologs or isophores thereof. At least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, and 77% of the variants are either the same or a variant. 、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、 Claims that are 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. A host cell as described in any one of items 4 to 8.

10. The N-acetylglucosamine transferase is GnT-I, any one of claims 1 to 9. The host cells described in item 1.

11. The N-acetylglucosamine transferase is GnT-II, any one of claims 1 to 9. The host cells described in item 1.

12. Claims 1 to 1, wherein the N-acetylglucosamine transferase is GnT-I and GnT-II. A host cell as described in any one of item 9.

13. The galactosyltransferase is B4GALT1, as described in any one of claims 1 to 9. The host cell of the host.

14. The N-acetylglucosamine transferases are GnT-I and GnT-II, and the Gala The host tissue according to any one of claims 1 to 9, wherein the custosyltransferase is B4GALT1. Cell.

15. The sialyltransferase is 2,6-SiaT or 2,3-SiaT, any one of claims 1 to 9. The host cells described in item 1.

16. The N-acetylglucosamine transferases are GnT-I and GnT-II, and the Gala The custosyltransferase is B4GALT1, and the sialyltransferase is 2,6-S A host cell according to any one of claims 1 to 9, which is iaT or 2,3-SiaT.

17. The N-acetylglucosamine transferases are GnT-I and GnT-II, and the Gala The custosyltransferase is B4GALT1, and the sialyltransferase is 2,6-S It is iaT or 2,3-SiaT, and the sialyltransferase causes CMP-NeuAc to be produced. Any one of claims 1 to 9 further comprises a heterologous CMP-Sia biosynthesis pathway protein capable of performing the above. The host cells described.

18. The retaining sequence is the N-acetylglucosamine transferase and / or galactosamine The host according to claim 8, which retains the transferase in the cis-Golgi compartment of the host cell. cell.

19. The retaining sequence is the N-acetylglucosamine transferase and / or galactosamine The host according to claim 8, which retains the transferase in the intermediate Golgi compartment of the host cell. cell.

20. The retaining sequence transfers the galactosyltransferase to the transgolgia region of the host cell. The host cell according to claim 8, which is retained in a compartment.

21. The retaining sequence transfers the sialyltransferase to the trans-Golgi compartment of the host cell. The host cell according to claim 8, which is retained in

22. The retaining sequence is the sialyltransferase and galactosyltransferase The host cell according to claim 8, wherein the host cell retains the host cell in the trans-Golgi compartment.

23. The signal sequence and / or retention sequence are derived from Leishmania talenttrae. A host cell according to any one of claims 8 or 10 to 22, wherein the host cell is a null sequence or a retention sequence.

24. The host cell according to claim 23, wherein the signal sequence is processed and removed.

25. The aforementioned retaining sequence is derived from leishmania talentraeprotein and is a cytoplasmic-transmembrane-sequence A host cell according to any one of claims 8 or 10 to 23, wherein the sequence is a TEM (CTS) sequence.

26. The aforementioned CTS sequence is Leishmania talentrae MAN1, NTPDase 1, or NTPDase 2 A host cell according to claim 25, derived from the host cell according to claim 25.

27. The CTS sequence is sequence number 24, sequence number 25, or sequence number 26, or a functional thereof. A host cell according to claim 25, comprising a sequence of active fragments.

28. The aforementioned CTS sequence is at least approximately 70%, 71%, 72%, 73%, 74%, and 75% of the sequence of sequence number 24. ％、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 sequences or their functionally active fragments; at least about 70%, 71% identical to the sequence of SEQ ID NO: 25 、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 A sequence or its functionally active fragment that is 99% or 100% identical; or the sequence of Sequence ID No. 26 Columns and at least approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% 、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、 Sequences or their functional activity that are 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. A host cell according to claim 25, comprising a sex fragment.

29. The host cell according to claim 25, wherein the CTS is derived from Leishmania talentrae MAN1.

30. The host according to claim 25, wherein the CTS sequence comprises the sequence of sequence number 24 or a functionally active fragment thereof. cell.

31. The retaining sequence includes a GRIP sequence derived from Leishmania or a functionally active fragment thereof. A host cell as described in any one of the requirements 20-22.

32. The host according to claim 31, wherein the GRIP sequence comprises the sequence of sequence number 27 or a functionally active fragment thereof. cell.

33. The aforementioned GRIP sequence is at least approximately 70%, 71%, 72%, 73%, 74%, and 75% of the sequence of sequence number 27. ％、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% The host cell according to claim 31, comprising a sequence or a functionally active fragment thereof that is % identical.

34. The retained sequence is a CTS sequence derived from Leishmania protein or a functionally active cleavage thereof. Claims 20 to include a fragment and a GRIP sequence or functionally active fragment thereof derived from Leishmania. A host cell as described in any one of item 22.

35. The aforementioned CTS sequence is Leishmania talentrae MAN1, NTPDase 1, or NTPDase 2 A host cell according to claim 34, derived from the host cell according to claim 34.

36. The CTS sequence includes the sequence of sequence number 24, sequence number 25, or sequence number 26, and the GRIP sequence The host cell according to claim 34, wherein the column comprises the sequence of sequence number 27 or a functionally active fragment thereof.

37. The host cell according to claim 34, wherein the CTS sequence is derived from Leishmania talentrae MAN1. Cell.

38. The CTS sequence includes the sequence of sequence number 24, and the GRIP sequence includes the sequence of sequence number 27 or its mechanism. A host cell according to claim 34, comprising a potent active fragment.

39. Claims 1 to 38, wherein the target protein is heterogeneous to the Leishmania host cell. A host cell as described in any one of the following items.

40. The target protein has been modified to include a signal sequence derived from Leishmania. The host cell according to claim 39.

41. The aforementioned signal sequence derived from Leishmania is a signal derived from Leishmania talentrae. The host cell according to claim 40, which is a sequence.

42. The aforementioned signal sequence is the arrangement of SEQ ID NO: 28 or SEQ ID NO: 29 or their functionally active fragments. A host cell according to claim 39, comprising a row.

43. The signal sequence comprises the sequence of sequence number 28 or a functionally active fragment thereof, according to claim 39. host cell.

44. The aforementioned signal sequence is at least approximately 70%, 71%, 72%, 73%, and 74% of the sequence of sequence number 28. 、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 The host cell according to claim 39, comprising a sequence or a functionally active fragment thereof that is 100% identical.

45. Claim 41, wherein the signal sequence is processed and removed from the target protein. Host cells as described in any one of items ~44.

46. The aforementioned target proteins are human interferon-α (INF-α) and interferon-β (INF -β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin Syn-IL-2 (denileukin diffittox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), tumor necrosis factor alpha (TNF- α), insulin, plumrintide, 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 release Hormones (GnRH), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), fibroblasts Fibroid growth factor 7 (FGF7), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), above Skin growth factor (EGF), vascular endothelial growth factor (VEGF), neurotrophin-3, human follicular stimulating hormone Mon (FSH), human chorionic gonadotropin (HCG), Lutropin-α, erythropoietin, granules Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), CTLA4 The extracellular domain of the TNF receptor (e.g., FC-fusion), or the extracellular domain of the TNF receptor (e.g., FC-fusion) A host cell according to any one of claims 39 to 45, comprising the amino acid sequence of a combined (combined) host cell.

47. The accommodation according to any one of claims 39 to 45, wherein the target protein is a therapeutic protein. Principal cell.

48. The host cell according to any one of claims 39 to 45, wherein the target protein is an antibody.

49. The host cell according to claim 48, wherein the antibody is an antibody against a human protein.

50. The aforementioned antibodies include adalimumab (Humira); Remicade (infliximab); and ReoPro (abscisic). Simab; Rituxan; Simulect; Synagis; Herceptin (trastuzumab); Mylotarg (gemtuzumab ozogamicin); Campath (alem) Tuzumab); Zevalin (ibritumomab thiuxetane); Xolair (omalizumab); Bexxar ( Citumomab-I-131); Erbitux (Cetuximab); Avastin (Bevacizumab); Tysabri (Natalin) Zumab; Actemra (tocilizumab); Vectibix (panitumumab); Lucentis (ranibizumab); Soliris (eculizumab); Cimzia (certolizumab pegol); Simponi (golimumab); Ilar is(canakinumab); Stelara(ustekinumab); Arzerra(ofatumumab); Prolia(deno Sumab; Numax (motavizumab); ABThrax (laxibamub); Benlysta (belimumab); Yer voy (ipilimumab); Adcetris (brentuximab vedotin); Perjeta (pertuzumab); Ka The amino acid sequence of dcyla (ad-trastuzumab emtansine) or Gazyva (obinutuzumab) A host cell according to claim 48 or 49, having the following characteristics.

51. The aforementioned antibody is a full-length antibody, Fab, F(ab') 2 Any of claims 48 to 50, which is Scfv or sdAb The host cells described in item 1.

52. Claims 39 to 45, wherein the target protein comprises the amino acid sequence of an enzyme or its inhibitor. Either of the host cells described in item 1.

53. The aforementioned target proteins are Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, and Factor VIIa, antithrombin III (AT-III), protein C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosi Dase, alglucosidase-α, laronidase (α-L-iduronidase), idursulf Agalsidase (iduronate-2-sulfatase), galsulfase, agalsidase-β (human α- Galactosidase A), botulinum toxin, collagenase, human DNase-I, hyaluronic acid Calcium oxidase, papain, L-asparaginase, uricase (uric acid oxidase), glutamic acid Carboxypeptidase (glucarpidase), α1 protease inhibitor (α1 antitri Pancreatic enzymes (lipase, amylase, protease), and adenosine The host cell according to claim 52, comprising the amino acid sequence of a deaminase.

54. The aforementioned therapeutic protein is abatacept (e.g., Orencia), aflibercept (e.g., Eylea), agalsidase beta (e.g., Fabrazyme), albiglutide (e.g., Eperza) n) Aldesleukin (e.g., Proleukin), Alefacept (e.g., Amevive), Alg Lucerase (e.g., Ceredase), alglucosidase alpha (e.g., LUMIZYME), ant Skiren (e.g., Tekturna), alpha-1-proteinase inhibitors (e.g., Aralast), Alteplase (e.g., Activase), Anakinra (e.g., Kineret), Anistreplar Eminase (e.g., eminase), human anthrax immunoglobulin (e.g., ANTHRASIL), antihemophilic factor ( For example, Advate), anti-inhibitor blood coagulation complex (e.g., Feiba Nf), anti- Thymocyte alpha, human antithrombin III, antithymocyte globulin (e.g., antithymocyte Globulin), anti-thymocyte globulin (horse) (e.g., ATGAM), anti-thymocyte globulin (horse) Heron (e.g., ATG-Fresenius), aprotinin (e.g., Trasylol), asphotaseal Fa, asparaginase (e.g., Elspar), Elwinia chrysanthemiasparaginase Ze (e.g., Erwinaze), becaprelmine (e.g., REGRANEX), beratacept (e.g., Nul Ojix), belactant, bivalirudin (for example, Angiomax), type A botulinum toxin (for example) For example, BOTOXE, type B botulinum toxin (e.g., Myobloc), brentuximab vedotin (e.g., For example, Adcetris), Buserelin (for example, Suprecur), C1 esterase inhibitor (human), C1 Esterase inhibitors (recombinant) (e.g., Ruconest), certolizumab pegol (e.g., , Cimzia), coliogonadotropin alpha (e.g., coliogonadotropin alpha), Human chorionic gonadotropin (e.g., Ovidrel), recombinant chorionic gonadotropin (e.g.) For example, Ovitrelle, coagulation factor ix (e.g., Alprolix), coagulation factor VIIa (e.g., NovoSeven), Human coagulation factor X (e.g., Coagadex), coagulation factor XIIIA-subunit (recombinant), collagen -ase (e.g., Cordase), Conestat alfa, Corticotropin (e.g., HPActhar) ), cosintropin (e.g., Cortrosyn), darbepoetin alpha (e.g., Aranesp), Defibrotide (e.g., Noravid), denileukin diffitox (e.g., Ontak), deci Ludin, Digoxin ImmunoFab (sheep) (e.g., DIGIBIND), Dornase Alpha (e.g., Pulmozyme), Drotrecogin alpha (e.g., Xigris), Dulaglutide, Efmoloct Cog Alpha (e.g., ELOCTA), Erosulfase Alpha, Enfubil Tide (e.g., For example, FUZEON), epoetin alpha (for example, Binocrit), epoetin zeta (for example, Re Tacrit), Eptifibatide (e.g., Integrilin), Etanercept (e.g., Enbrel), Exenatide (e.g., Byetta), Factor IX complex (human) (e.g., AlphaNine), fibril Noridine, also known as plasmin (e.g., Elase), filgrastim (e.g., NA), phi Lugrastim-sndz, follitropin alpha (e.g., Gonal-F), follitropin beta (e.g., Follistim AQ), galsulfase (e.g., Naglazyme), gastric factors, gemz Mabuozogamicin (e.g., Mylotarg), glutillamer acetate (e.g., Copaxone), glucosozogamicin Kagon recombinants (e.g., GlucaGen), glucarpidases (e.g., Voraxaze), Gramicidal Hepatitis B immunoglobulin (e.g., Neosporin), human calcitonin, human tetanus bacteria Xoid immunoglobulins, human rabies virus immunoglobulins (e.g., Hyperab human rabies) Disease immunoglobulins), human Rho(D) immunoglobulin (e.g., Hyp Rho D Inj 16.5%), human Serum albumin (e.g., Albuminar), human varicella-zoster immunoglobulin (e.g., Vari) zig), hyaluronidase (e.g., HYLENEX), hyaluronidase (human recombinant), ibri Tsumomabuchiukisetan (e.g., Zevalin), Izurusulfase (e.g., Elaprase), I Miglucerase (e.g., Cerezyme), human immunoglobulin, insulin aspart (e.g., (e.g., NovoLog), bovine insulin, insulin degludec (e.g., Tresiba), insulin Detemir (e.g., LEVEMIR), insulin glargine (e.g., Lantus), insulin glug Lysine (e.g., APIDRA), insulin lispro (e.g., Humalog), porcine insulin (e.g., For example, Iletin II, regular insulin (e.g., Humulin R), porcine insulin (e.g., vetsulin), isophenes (e.g., Novolin N), interferon alpha-2a , recombinant (e.g., Roferon A), interferon alpha-2b (e.g., INTRON A), Interferon alpha-con-1 (for example, INFERGEN), interferon alpha-n1 (for example) For example, Wellferon, interferon alpha-n3 (Alferon), interferon ve Interferon beta-1a (e.g., Avonex), interferon beta-1b (e.g., Betaseron), inter Ferron-gamma-1b (e.g., Actimmune), intravenous immunoglobulin (e.g., Civacir), Lonidase (e.g., Aldurazyme), lenograstim (e.g., Granocyte), repiridine (e.g.) For example, Refludan, leuprolide (e.g., Eligard), liraglutide (e.g., Saxenda), Lucinactant (e.g., Surfaxin), Lutropin alpha (e.g., Luveris), Mechacel Min (e.g., NA), menotropin (e.g., Menopur), methoxypolyethylene glyco Lu-epoetin beta (e.g., Mircera), metreleptin (e.g., Myalept), natural alcohol Fine interferon or multiferon (e.g., Intron / Roferon-A), nesilitide (For example, NATRECOR), ocriplasmin (for example, Jetrea), oprelbequin (for example, Neu mega), OspA lipoprotein (e.g., Lymerix), oxytocin (e.g., Pitocin), par Fermin (e.g., Kepivance), Pancrelipase (e.g., Pancrecarb), Usipegade Marsage (e.g., Adagen), Peg Asparagauze (e.g., Oncaspar), Pegfil Glass (e.g., Neulasta), pegylated interferon alpha-2a (e.g., Pegasys), pegylated in Taferon alpha-2b (for example, PEG-Intron), pegylated interferon beta-1a (for example) Plegridy), Pegrotica (e.g., (Krystexxa)), Pegvisomant (e.g., SOM) AVERT), Polactant Alpha (e.g., Curosurf), Plumlintide (e.g., Symlin) , Preotact (e.g., Preotact E), protamine sulfate (e.g., protamine sulfate injection) Injectable fluid, USP), human protein S (e.g., human protein S), prothrombin (e.g., Fei ba Nf), prothrombin complex (e.g., Cofact), prothrombin complex concentrate (e.g., For example, Kcentra, Rasb uricase (e.g., Elitek), reteplase (e.g., Retavase), Lonacept (e.g., Arcalyst), romiplostim (e.g., Nplate), sacrosidase (e.g., For example, Sucraid, salmon calcitonin (e.g., Calcimar), salgramostim (e.g., Leu comax), satsumomab pendetide (e.g., OncoScint), seberipase alpha (e.g., Ka numa), secretin (e.g., SecreFlo), cermorelin (e.g., cermorelin acetate), blood Clear albumin (e.g., Albunex), iodine-labeled serum albumin (e.g., Megatope), simulant Cthutocogalpha (e.g., Nuwiq), Cyprusel-T (e.g., Provenge), Somatotoro Recombinant iontoplasm (e.g., NutropinAQ), somatropin recombinant (e.g., BioTropin), st Leptokinase (e.g., Streptase), susoctocog alpha (e.g., Obizur), Talig Lucerase alpha (e.g., Elelyso), teduglutide (e.g., Gattex), tenectep Lase (e.g., TNKase), teriparatide (e.g., Forteo), tessamorelin (e.g., Egrif) ta), thrombomodulin alpha (e.g., Recomodulin), simalfasin (e.g., Z adaxin, thyroglobulin, thyrotropin alpha (e.g., thyrotropin), tuberculin Purified protein derivatives (e.g., Aplisol), turoctocog alpha (e.g., Zonovate), Urofolitropin (e.g., BRAVELLE), urokinase (e.g., Kinlytic), vasopress Syn (e.g., Pitressin), bellaglucerase alfa (e.g., Vpriv), absiximab (e.g., ReoPro), adalimumab (e.g., Humira), alemtuzumab (e.g., CAMPATH) , alirocumab (e.g., Praluent), alsitumomab (e.g., CEA-Scan), atezolizum (e.g., Tecentriq), basiliximab (e.g., Simulect), belimumab (e.g., Benl) (ysta), bevacizumab (e.g., Avastin), blinatumomab (e.g., Blincyto), Brodal Mab (e.g., Siliq), canakinumab (e.g., ILARISE), canakinumab (e.g., Ilaris) , capromab (e.g., ProstaScint), cetuximab (e.g., Erbitux), daclizumab ( For example, Zenapax, daratumumab (for example, DARZALEX), denosumab (for example, Xgeva), d Nutuximab (e.g., unituxin), eculizumab (e.g., Soliris), efalizumab (e.g., For example, RAPTIVA, elotuzumab (e.g., EMPLICITI), evolocumab (e.g., Repatha), Golimumab (e.g., Simponi injection), ibritumomab (e.g., Zevalin), idarucizuma (e.g., Praxbind), infliximab (e.g., REMICADE), ipilimumab (e.g., Y ERVOY), ixekizumab (e.g., Taltz), mepolizumab (e.g., Nucala), muromonab ( For example, ORTHOCLONE OKT3), natalizumab (for example, Tysabri), nesitumumab (for example, Po (e.g., rtrazza), nivolumab (e.g., Opdivo), obilutoxaximab (e.g., Anthim), obi Nutuzumab (e.g., Gazyva), ofatumumab (e.g., Arzerra), omalizumab (e.g., Xolair, palivizumab (e.g., Synagis), panitumumab (e.g., Vectibix), pemb Lolizumab (e.g., Keytruda), pertuzumab (e.g., Perjeta), ramucirumab (e.g., Cyramza), ranibizumab (e.g., Lucentis), laxibakuma (e.g., laxibakuma) Rituximab (e.g., Rituxan), secukinumab (e.g., Cosentyx), siltuximab (e.g., Sylvant), tocilizumab (e.g., ACTEMA), tositumomab (e.g., Bexxar) , trastuzumab (e.g., Herceptin), ustekinumab (e.g., Stellara), or vedol A host cell according to claim 47, comprising the amino acid sequence of zumab (e.g., Entyvio).

55. The target protein is an Fc-fusion protein, according to any one of claims 39 to 54. host cell.

56. The host cells a. Contains Stt3 oligosaccharide transferase (OST), and b. Lacking endogenous N-glycan elongation, A host cell according to any one of claims 1 to 55.

57. The host cell is any strain listed in Table 13, as per any one of claims 1 to 56. The host cell of the host.

58. The host cell possesses the CMP-Neu5Ac pathway, which has the prokaryotic or eukaryotic enzyme shown in Figure 53. A host cell according to any one of claims 1 to 57, including the host cell according to any one of claims 1 to 57.

59. A method for producing a glycosylated target protein, wherein the method is as described in any one of claims 1 to 47. This includes culturing the host cells and purifying the target protein from the culture. The aforementioned method.

60. A composition of glycosylated target protein obtained by the method described in claim 59.

61. At least one of the N-linked glycosylation consensus sequences of the target protein in the composition Approximately 90% to 100% of each has the following structure: 【Chemistry 1】 (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represent; and Here, Asn is the Asn of the N-linked glycosylation consensus sequence in the target protein. A composition of glycosylated target protein according to claim 60, which supports an oligosaccharide containing (a). 。

62. Glycosylation on the target protein of at least about 20% to 30%, 25% to 35%, 30% 40％、35％～45％、40％～50％、45％～55％、50％～60％、55％～65％、60％～70％、65 % to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% are below Structure below: 【Chemistry 2】 (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represent; and Here, Asn is the Asn of the N-linked glycosylation consensus sequence in the target protein. The glycosylated target protein according to claim 60, which is a G0-Gn glycan characterized by (a composition.

63. Glycosylation on the target protein of at least about 20% to 30%, 25% to 35%, 30% 40％、35％～45％、40％～50％、45％～55％、50％～60％、55％～65％、60％～70％、65 % to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% are below Structure below: 【Transformation 3】 (Here, the squares represent N-acetylglucosamine residues, and the gray circles represent mannose residues) Represent; and Here, Asn is the Asn of the N-linked glycosylation consensus sequence in the target protein. A set of glycosylated target proteins according to claim 60, which is a G0 glycan characterized by (a) Finished product.

64. Glycosylation on the target protein of at least about 20% to 30%, 25% to 35%, 30% 40％、35％～45％、40％～50％、45％～55％、50％～60％、55％～65％、60％～70％、65 % to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% are below Structure below: 【Chemistry 4】 (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) This represents, and the gray circles represent mannose residues; and Here, Asn is the Asn of the N-linked glycosylation consensus sequence in the target protein. A G1-Gn glycan characterized by (a), the glycosylated target protein according to claim 60 composition.

65. Glycosylation on the target protein of at least about 20% to 30%, 25% to 35%, 30% 40％、35％～45％、40％～50％、45％～55％、50％～60％、55％～65％、60％～70％、65 % to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% are below Structure below: 【Transformation 5】 (Here, the white circles represent galactose residues, and the squares represent N-acetylglucosamine residues.) This represents, and the gray circles represent mannose residues; and Here, Asn is the Asn of the N-linked glycosylation consensus sequence in the target protein. The glyco according to claim 60, which is a G2 or G1 glycan characterized by one or more of the following: A composition of silation target proteins.

66. When glycosylation on the target protein is introduced into the target, the target protein The glycosylated target 65 described above has been further modified to optimize its pharmacokinetic properties. A protein composition.

67. The glycosylation on the target protein is sialylated, according to claim 66. A composition of silation target proteins.

68. Glycosylation on the target protein of at least about 20% to 30%, 25% to 35%, 30% 40％、35％～45％、40％～50％、45％～55％、50％～60％、55％～65％、60％～70％、65 % to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% are below Structure below: 【Transformation 6】 (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents The gray circles represent N-acetylglucosamine residues, and the gray circles represent mannose residues; and Here, Asn is the Asn of the N-linked glycosylation consensus sequence in the target protein. A glycosylated target protein according to claim 67, characterized by one or more of the following: composition.

69. Glycosylation on the target protein of at least about 20% to 30%, 25% to 35%, 30% 40％、35％～45％、40％～50％、45％～55％、50％～60％、55％～65％、60％～70％、65 % to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, or 90% to 100% are below Structure below: 【Transformation 7】 (Here, the diamond represents a sialic acid residue, the white circle represents a galactose residue, and the square represents The gray circles represent N-acetylglucosamine residues, and the gray circles represent mannose residues; and Here, Asn is the Asn of the N-linked glycosylation consensus sequence in the target protein. A glycosylated target protein according to claim 67, characterized by one or more of the following: composition.

70. The aforementioned N-linked glycosylation consensus sequence is Asn-X-Ser / Thr; Here, X is any amino acid other than proline. The composition according to any one of claims 61 to 65, 68, or 69.

71. The glycosylated target protein is secreted into the culture medium, and here the glycosylated target The target protein is glycosylated as described in any one of claims 52 to 61, The method described in item 59.

72. The method according to claim 71, wherein the glycosylated target protein is purified from the culture medium.

73. The glycosylated target protein is purified by affinity purification or ion exchange chromatography. The method of claim 72, wherein the culture medium is purified.

74. The glycosylated target protein contains an FC domain and is cultured by protein-A. The method according to claim 73, wherein affinity purification is performed from a nutrient medium.

75. Claim 73, wherein the glycosylated target protein contains an affinity tag and is purified by affinity. Method of description.

76. Glycosylated target produced by the method according to any one of claims 59 or 71-75 A composition containing protein, wherein the population of glycosylated target proteins is at least about 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% uniform, Recording composition.

77. Glycosylated target produced by the method according to any one of claims 59 or 71-75 A composition containing protein, wherein 90% to 100% of the N-sugar moieties on the target protein are glycosylated. The composition, which is occupied by sylation.

78. a. Contact of N-acetylglucosamine transferase not derived from Leishmania Medium domain; and b. Amino acid combinations involved in the localization and retention of Leishmania in the endoplasmic reticulum or Golgi compartment. column A hybrid N-acetylglucosamine transferase containing this enzyme.

79. The hybrid according to claim 78, wherein the Leishmania is Leishmania talentrae. N-acetylglucosamine transferase.

80. The N-acetylglucosamine transferase is a signal sequence and at least one Modified to include a retention sequence, where the signal sequence is the N-acetylglucosa Minttransferase targets the vesicles of the Leishmania talenttrae host cell. The retaining sequence is set, and the N-acetylglucosamine transferase is transferred to the vesicle. The hybrid N-acetylglucosamine according to claim 78 or 79, which is held in the body or in the Golgi apparatus. Transferase.

81. The retaining sequence retains the N-acetylglucosamine transferase in the cis-Golgi apparatus. The hybrid N-acetylglucosamine transferase according to claim 78.

82. The retaining sequence retains the N-acetylglucosamine transferase in the intermediate Golgi apparatus. The hybrid N-acetylglucosamine transferase according to claim 78.

83. Any one of claims 78 to 82, wherein the retaining sequence is a cytoplasmic-transmembrane-stem (CTS) sequence. The described hybrid N-acetylglucosamine transferase.

84. The aforementioned N-acetylglucosamine transferase amino acid sequence is shown in Table 9. N-acetylglucosamine transferase, or its functional homolog, isoform N-acetylglucosa according to any one of claims 78 to 83, derived from or a variant of Minttransferase.

85. a. Catalytic domain of galactosyltransferase not derived from Leishmania. ;and b. Amino acid combinations involved in the localization and retention of Leishmania in the endoplasmic reticulum or Golgi compartment. column A hybrid galactosyltransferase containing [the specified ingredient].

86. The hybrid according to claim 85, wherein the Leishmania is Leishmania talentrae. Galactosyltransferase.

87. The galactosyltransferase has been modified to include a signal sequence, Thus, the signal sequence transmits the galactosyltransferase to the Leishmania tarre The intracellular matrix targets the endoplasmic reticulum of the host cell, and the retaining sequence is the galactosyl The hybrid according to claim 85 or 86, which holds lanceferase in the endoplasmic reticulum or Golgi apparatus. Galactosyltransferase.

88. The aforementioned retention sequence holds the galactosyltransferase in the cis-Golgi apparatus. A hybrid galactosyltransferase as described in item 87.

89. The aforementioned retention sequence holds the galactosyltransferase in the intermediate Golgi apparatus. A hybrid galactosyltransferase as described in item 87.

90. The retaining sequence holds the galactosyltransferase in the transgolgi apparatus. The hybrid galactosyltransferase according to claim 87.

91. Any one of claims 87 to 89, wherein the retaining sequence is a cytoplasmic-transmembrane-stem (CTS) sequence. The described hybrid galactosyltransferase.

92. The hybrid shear according to any one of claims 87 to 89, wherein the retaining sequence is a GRIP sequence. Lilyltransferase.

93. The retaining sequence is a CTS sequence and a GRIP sequence, according to any one of claims 87 to 89. Brid sialyltransferase.

94. The galactosyltransferase amino acid sequence is the one shown in Table 9. Siltransferase, or its functional homologs, isoforms, or variants A galactosyltransferase according to any one of claims 87 to 93, derived from.

95. a. Catalytic domain of sialyltransferase not derived from Leishmania; Bini b. Amino acid combinations involved in the localization and retention of Leishmania in the endoplasmic reticulum or Golgi compartment. column A hybrid sialyltransferase containing [a specific type of sialyltransferase].

96. The hybrid according to claim 95, wherein the Leishmania is Leishmania talentrae. sialyltransferase.

97. The sialyltransferase has been modified to include a signal sequence, where The signal sequence transmits the sialyltransferase to the Leishmania talentrae. It targets the endoplasmic reticulum of the host cell, and the retaining sequence is the sialyltransfera The hybrid sialyl according to claim 95 or 96, which holds the sialyl in the endoplasmic reticulum or Golgi apparatus. Transferase.

98. The aforementioned retention sequence holds the sialyltransferase in the trans-Golgi apparatus. A hybrid sialyltransferase as described in item 97.

99. The hybrid sialyltrans according to claim 97 or 98, wherein the retaining sequence is a CTS sequence. Ferase.

100. The hybrid sialyltrans according to claim 97 or 98, wherein the retaining sequence is a GRIP sequence. Ferase.

101. The hybrid shear according to claim 97 or 98, wherein the retaining sequence is a CTS sequence and a GRIP sequence. Lilyltransferase.

102. The sialyltransferase amino acid sequence is shown in Table 9. Derived from encephalase, or its functional homolog, isoform, or mutant. or the hybrid sialyltransferase according to any one of claims 89 to 101.

103. The CTS signal includes the amino acid sequence of MAN1, NTPDase 1, or NTPDase 2 CTS. or the hybrid according to any one of claims 83, 91, 93, 99, or 101.

104. The CTS signal includes the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO:

26. or the hybrid according to any one of claims 83, 91, 93, 98, or 101.

105. Claims 83, 91, 93, 99, or 101, wherein the CTS signal comprises the amino acid sequence of MAN1 CTS. A hybrid as described in any one of the following items.

106. Claims 83, 91, 93, 99, or 101, wherein the CTS signal comprises the amino acid sequence of SEQ ID NO:

24. A hybrid as described in any one of the following items.

107. The GRIP sequence comprises the amino acid sequence of SEQ ID NO: 27, according to claim 92, 93, 100, or 101 A hybrid as described in one of the items.

108. Hybrid N-acetylglucosamine transfer according to any one of claims 78 to 84 A nucleic acid that codes for an enzyme.

109. A hybrid galactosyltransferase according to any one of claims 85 to 94 nucleic acids that are involved in the process.

110. A hybrid sialyltransferase according to any one of claims 95 to 102 Nucleic acid that does something.

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