Glycosylation modification using Leishmania cells

Abstract

The present application relates to Leishmania cells that have been genetically engineered to reduce or eliminate the formation of O-linked GlcNAc on polypeptides in the Leishmania cells. In the Leishmania cells prior to genetic engineering, the formation of O-linked GlcNAc can be catalyzed by at least one N-acetylglucosamine (GlcNAc) transferase. Also provided herein are methods for preparing polypeptides using the Leishmania cells described herein, and polypeptides produced by the methods provided herein.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 62 / 958,070, filed on January 7, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing The entire sequence listing in computer-readable format (CRF) in ASCII text format is hereby incorporated by reference into this application. This sequence listing text file has a title of "14197-010-228_SEQ_LISTING", a creation date of December 23, 2020, and a size of 1,350,529 bytes.

[0003] 1. Introduction This application relates to Leishmania cells that have been genetically engineered such that the formation of O-linked GlcNAc on polypeptides in the Leishmania cells is reduced or eliminated. In the Leishmania cells prior to such genetic engineering, the formation of O-linked GlcNAc can be catalyzed by at least one N-acetylglucosamine (GlcNAc) transferase. Also provided herein are methods for preparing polypeptides using the Leishmania cells described herein, and polypeptides produced by the methods provided herein.

Background Art

[0004] 2. Background Art A glycoprotein is a complex carbohydrate in which one or more glycans are attached to a polypeptide backbone by covalent bonds, usually via N-linkage or O-linkage. An N-glycan (N-linked glycan, N-[Asn]-linked glycan) is a glycan covalently linked to an asparagine residue of a polypeptide chain and usually contains GlcNAc residues in eukaryotes, and the consensus peptide sequence: Asn-X-Ser / Thr (Varki, Ajit (2009): Essentials of glycobiology. 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) is also involved.

[0005] O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans regulate the structure, stability, and function of proteins and play general and highly specific roles in most biological processes. In eukaryotes, all types of O-glycosylation are initiated by different polypeptide glycosyltransferases, and most glycans are further elongated / branched in the Golgi by sequential monosaccharide additions by different enzymes and / or common enzymes and are capped. O-glycans (O-linked glycans) in vertebrates are often linked to a polypeptide via N-acetylgalactosamine (GalNAc) attached to the hydroxyl group of a serine residue or a threonine residue and can extend into different structural core classes (Joshi, Hiren J et al (2018) Cell 172(3), 632 - 632.e2).

[0006] Mucin-type O-glycosylation is a post-translational modification (PTM) that is expected to occur in more than 80% of the proteins passing through the Golgi apparatus. This PTM is initiated by a family of polypeptide GalNAc transferases (GalNAc-T) that modify the Ser and Thr residues of proteins by adding a GalNAc moiety (Las Rivas, et al. (2019) In Current opinion in structural biology 56, pp. 87-96). Mucins are large glycoproteins in which many O-glycans are clustered (densely) present. There are also several other types of O-glycans (e.g., O-linked fucose, O-linked glucose, or O-linked mannose) (Varki, Ajit (2009): Essentials of glycobiology. 2.ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)).

[0007] O-glycosylation is a common post-translational modification, but its control in the expression of therapeutic biologics remains difficult. Knocking out glycosylation genes to remove unwanted glycans has long been a strategy for obtaining a clean expression host (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)).

[0008] However, since there are up to 20 types of polypeptide GalNAc transferases that synthesize O-GalNAc glycans, it is difficult to manipulate O-GalNAc glycans. As a result, proteins that are naturally found with O-glycans may not be O-glycosylated when expressed in a specific production cell line, and vice versa (Varki, Ajit, et al. (Eds.)(2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor(NY)).

[0009] This problem will be illustrated with some examples: When human O-glycoproteins are expressed in yeast, O-mannosylation can occur at sites where O-GalNAc is present in mammals. Examples of this include the hinge region of Ig and mucin sequences (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)). In general, O-mannosylation will occur exclusively in yeast and fungi. Furthermore, since yeast-type O-glycans are structurally different from human-type O-glycans, concerns regarding immunogenicity arise for O-glycosylated biopharmaceuticals produced by yeast. To address these issues, it is probably the most efficient way to change the protein sequence so that yeast-type O-glycans are completely avoided. However, since the consensus sequence is quite unpredictable, this approach depends on the ability to experimentally determine O-glycosylation sites and is by no means a trivial task. Since yeast requires O-glycosylated proteins to maintain its cell wall integrity and fitness, it has become clear that obtaining deletion mutants will not solve the problem (Wachter, Charlot de;, et al. (2018) Advances in biochemical engineering / biotechnology. DOI: 10.1007 / 10_2018_69).

[0010] Plants do not have the types of O-glycosylation found in other eukaryotes but produce unique O-glycans. Plant O-glycosylation is fundamentally different from mammalian O-glycosylation. In plants, serine residues from extensin and other members of the hydroxyproline-rich glycoprotein family can be glycosylated with a single galactose (Schoberer, Jennifer; Strasser, Richard (2017) Seminars in cell & developmental biology. DOI:10.1016 / j.semcdb.2017.07.005). In plants, the major anchor for O-glycosylation is 4-trans-hydroxyproline (Hyp). Indeed, it has been reported that biopharmaceuticals are subject to prolyl hydroxylation typical of plants without the desired outcome, and then sometimes to arabinosylation without the desired outcome. Prolyl hydroxylation that does not occur in humans not only changes the natural sequence of the protein but also serves as an anchor for O-glycans, and thus such O-glycans can become immunogenic. Plant-derived rhEPO obtained from mosses and Nicotiana benthamiana has been shown to be hydroxylated within the motif SPP (amino acids 147 - 149). P4H1 is a gene that causes prolyl hydroxylation not found in humans in human erythropoietin recombinantly produced in Physcomitrella patens. By targeting P4H1 for knockout, rhEPO no longer undergoes the addition of plant-specific O-glycosylation (Parsons, Juliana; et al. (2013) Scientific reports 3, p. 3019. DOI:10.1038 / srep03019). Some glycosyltransferases have been knocked out in different plants, but a completely clean O-glycosylation-free plant line has not been achieved. Therefore, a suitable system for the manipulation and utilization of different types of mammalian O-glycosylation will only be obtained from plants if the problems associated with hydroxyproline can be solved.It is unclear whether such modifications can be completely removed without affecting viability (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)).

[0011] In insect cells, O-glycosylation occurs in a similar range as in mammalian cells, but the extent to which O-GalNAc glycans are added to the same sites as in mammals has not been explored. Furthermore, at least when using baculovirus expression, the degree of O-glycan processing appears to be incomplete (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)). O-glycans in which GalNAc is directly added to an amino acid are called mucin-type O-glycans and are the most abundant O-glycans found in Drosophila embryos, constituting approximately 90% of the total O-glycan profile (Li, Weidong, et al. (2019) Glycoconjugate journal. DOI: 10.1007 / s10719-019-09867-1).

[0012] In mammalian cells, many different types of O-glycosylation occur, and these play diverse and important biological functions. Recombinant coagulation factors for clinical use have O-GalNAc glycans, O-Fuc glycans, and / or O-Glc glycans, and many other approved drugs (including erythropoietin and Enbrel) also have O-GalNAc-type glycans (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)).

[0013] Importantly, recent reports made in conjunction with the development of high-end analytical tools have pointed out that undesirable and / or elevated levels of O-glycosylation are variably observed in therapeutic proteins, some of which are already approved ((Hashii et al. (2019) Biologicals: journal of the International Association of Biological Standardization. DOI: 10.1016 / j.biologicals.2019.01.005, Plomp et al. (2015) Mol Cell Proteomics 14(5), pp. 1373-1384, Berlec and Strukelj (2013) J Ind Microbiol Biotechnol 40(3-4), pp. 257-274., Spahr, et al. (2013) Protein science: a publication of the Protein Society 22(12), pp. 1739-1753. DOI: 10.1002 / pro.2373, Spahr, et al (2014) mAbs 6(4), pp. 904-914, Stavenhagen et al. (2019) mAbs 11(6), pp. 1053-1063, Wen, et al(2013) Analytical chemistry 85(9), pp. 4805-4812, Zhong, et al. (2013) The Journal Of Biological Chemistry 288(2), pp. 1409-1419).

[0014] In the present application, a recombinant expression host enabling control of O-glycosylation is provided. The approach is based on a custom glycan Leishmania cell line, which lacks the native polypeptide GlcNAc transferase and thus has no ability to O-glycosylate proteins, while still being viable and not subject to any other effects on phenotypes observed in other species. Since unwanted O-glycosylation of recombinant expressed proteins is often a problem, this technology provides a solution for the expression of therapeutic proteins that should not be O-glycosylated. Modification control further improves product uniformity, ensures product consistency, and thus reduces the risk of unwanted immune reactions occurring upon delivery to a subject.

[0015] In the present application, an O-glycosylation-deficient Leishmania host cell is described for providing a consistent therapeutic protein format where the absence of O-glycosylation is required, and unwanted O-glycosylation in sensitive protein regions is further avoided. O-glycosylation was identified and characterized as O-GlcNAc. This also provides insights into the addition of related glycans to O-GlcNAc in Leishmania host cells within a custom glycan platform (International Publication No. WO2019 / 002512A2, which is hereby incorporated by reference in its entirety). SUMMARY OF THE INVENTION

[0016] 3. Summary of the Invention Provided herein are Leishmania cells, methods of preparing polypeptides using the Leishmania cells, and polypeptides produced by the methods.

[0017] In one aspect, provided herein are Leishmania cells genetically engineered such that the formation of O-linked GlcNAc on polypeptides in the Leishmania cells is reduced or eliminated.

[0018] In certain embodiments, the formation of O-linked GlcNAc in Leishmania cells prior to genetic manipulation is catalyzed by at least one N-acetylglucosamine (GlcNAc) transferase.

[0019] In certain embodiments, the gene encoding at least one GlcNAc transferase is functionally inactivated. In certain embodiments, the gene encoding at least one GlcNAc transferase is downregulated. In certain embodiments, the gene encoding at least one GlcNAc transferase is deleted or mutated. In certain embodiments, the gene encoding at least one GlcNAc transferase is overexpressed.

[0020] In certain embodiments, the formation of O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the formation of O-linked GlcNAc in reference Leishmania cells.

[0021] In certain embodiments, at least one GlcNAc transferase is selected from the group consisting of OGNT1, OGNT2, and OGNTL, and their homologous GlcNAc transferases. In certain embodiments, at least one GlcNAc transferase is a GlcNAc transferase homologous to OGNT1, OGNT2, and / or OGNTL. In certain embodiments, the number number of at least one GlcNAc transferase is one, two, or three.

[0022] In certain embodiments, the growth rate of the Leishmania cells is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of the reference Leishmania cells.

[0023] In certain embodiments, the cells are Leishmania tarentolae.

[0024] In certain embodiments, the polypeptide is selected from the group consisting of adalimumab, rituximab, and erythropoietin (EPO).

[0025] In certain embodiments, the Leishmania cells contain a recombinant nucleic acid encoding a heterologous glycosyltransferase. In certain embodiments, the heterologous glycosyltransferase is an N-acetylglucosaminyltransferase, and / or a heterologous galactosyltransferase, and / or a heterologous sialyltransferase. In certain embodiments, the Leishmania cells contain one or more heterologous glycosyltransferases. In certain embodiments, the Leishmania cells contain one or more N-acetylglucosaminyltransferases, and / or one or more heterologous galactosyltransferases, and / or one or more heterologous sialyltransferases.

[0026] In another aspect, a method for preparing a polypeptide is provided herein, the method comprising: (a) culturing the Leishmania cells provided herein under conditions suitable for polypeptide production; and (b) isolating the polypeptide.

[0027] In yet another aspect, a polypeptide produced by the method provided herein is provided herein.

[0028] 3.1 Definitions As used herein, the term "about", when used in connection with a number, unless otherwise specified, refers to a variability of any number within ±1%, ±5%, or ±10% of the recited number.

[0029] As used herein, the term "subject", unless otherwise specified, refers to an animal (e.g., birds, reptiles, and mammals). In another embodiment, the subject is a mammal including non - primates (e.g., camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and primates (e.g., monkey, chimpanzee, and human). In certain embodiments, the subject is a non - human animal. In some embodiments, the subject is a domestic animal or a pet (e.g., dog, cat, horse, goat, sheep, pig, donkey, or chicken). In a particular embodiment, the subject is a human. The terms "subject" and "patient" may be used interchangeably herein.

[0030] As used herein, the term "effective amount" in the context of administering a therapeutic agent (e.g., a composition described herein) to a subject, unless otherwise specified, refers to an amount of the therapeutic agent that has a prophylactic effect and / or a therapeutic effect(s). In certain embodiments, "effective amount" refers to an amount of the therapeutic agent sufficient to achieve one, two, three, four, or more of the following effects: (i) reduction or alleviation of the severity of a disease / disorder or a symptom associated therewith, (ii) reduction of the duration of a disease / disorder or a symptom associated therewith, (iii) prevention of the progression of a disease / disorder or a symptom associated therewith, (iv) induction of regression of a disease / disorder or a symptom associated therewith, (v) prevention of the onset or occurrence of a disease / disorder or a symptom associated therewith, (vi) prevention of recurrence of a disease / disorder or a symptom associated therewith, (vii) reduction of organ failure associated with a disease / disorder, (viii) reduction of the number of hospitalizations of a subject having a disease / disorder, (ix) reduction of the length of hospitalization of a subject having a disease / disorder, (x) extension of the survival period of a subject having a disease / disorder, (xi) elimination of a disease / disorder in a subject, and / or (xii) enhancement or improvement of the prophylactic or therapeutic effect(s) of another treatment.

Table 1

Brief Description of the Drawings

[0031] 4. Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0043] 5. Detailed Description of the Invention This specification provides Leishmania cells, methods for preparing target polypeptides using Leishmania cells, and target polypeptides produced by the methods. In certain embodiments, Leishmania cells are provided that have been modified such that control of O-glycosylation (such as reduction of O-glycosylation or removal of O-glycans) occurs. In such Leishmania cells, fully functionally customized N-glycans can also be uniformly produced on recombinant therapeutic proteins at high site occupancy by a combination of the properties of the host cell and the heterologous expression of a set of glycosyltransferases (including N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase). For example, the Leishmania cells provided herein can be used within a custom glycan platform (International Publication No. WO2019 / 002512A2, which is incorporated herein by reference in its entirety). Also provided herein are nucleic acid sequences and methods that can be used to delete or control O-glycosylation enzymes, such O-glycosylation enzymes being, for example, O-glycosylation enzymes involved in transferring N-acetylglucosamine to serine or threonine residues in a polypeptide).

[0044] This specification provides Leishmania host cells in which the gene encoding the enzyme that catalyzes the formation of O-linked GlcNAc is functionally inactivated. Such genes are described in Section 5.1. Methods for achieving such functional inactivation are described in Section 5.2. The properties of the resulting Leishmania cells are described in Section 5.3. Use of such Leishmania cells as an expression system (such as for therapeutic proteins) is described in Section 5.4. The properties of proteins expressed in the Leishmania host cells provided herein are described in Section 5.5.

[0045] 5.1 Enzymes Catalyzing the Formation of O - linked GlcNAc Provided herein are Leishmania host cells genetically engineered such that the formation of O - linked GlcNAc on polypeptides in Leishmania cells is reduced or eliminated. In certain embodiments, the formation of O - linked GlcNAc in Leishmania cells prior to genetic engineering is catalyzed by at least one N - acetylglucosamine (GlcNAc) transferase. In certain embodiments, the gene encoding the enzyme catalyzing the formation of O - linked GlcNAc is functionally inactivated.

[0046] In certain embodiments, the enzyme catalyzing the formation of O - linked GlcNAc is an N - acetylglucosamine (GlcNAc) transferase. In certain embodiments, the GlcNAc transferase is selected from the group consisting of OGNT1, OGNT2, OGNTL, and homologous GlcNAc transferases thereof. In certain embodiments, the GlcNAc transferase is OGNT1. In other embodiments, the GlcNAc transferase is OGNT2. In still other embodiments, the GlcNAc transferase is OGNTL. In certain embodiments, the GlcNAc transferase is a GlcNAc transferase homologous to OGNT1. In certain embodiments, the GlcNAc transferase is a GlcNAc transferase homologous to OGNT2. In certain embodiments, the GlcNAc transferase is a GlcNAc transferase homologous to OGNTL. In certain embodiments, the GlcNAc transferase is derived from Leishmania tarentolae. In certain embodiments, the GlcNAc transferase is derived from other Trypanosomatida species. Examples of Trypanosomatida GlcNAc transferases are listed in Table 1, which lists one representative genome per species, without limitation. [Table 2] TIFF0007684310000005.tif198165TIFF0007684310000006.tif54165

[0047] In certain embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc is from a species other than Trypanosomatida species. In certain embodiments, the enzyme is human O-GlcNAc transferase (OGT, Uniprot: O15294) and its homologous enzymes. In certain embodiments, O-GlcNAc transferase (OGT; uridine diphosphate-N-acetylglucosamine: polypeptide β-N-acetylglucosaminyltransferase; EC 2.4.1.255) can modify such proteins with beta-linked N-acetylglucosamine (O-GlcNAc) by catalyzing the transfer of a single N-acetylglucosamine from UDP-GlcNAc to serine or threonine residues in cytoplasmic and nuclear proteins. In certain embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc can be a different isoform of OGT. Examples of OGT isoforms include, but are not limited to, (1) the nuclear / cytoplasmic variant, i.e., the full-length variant (ncOGT) (which can be 110 kDa), (2) the short isoform of OGT (sOGT) (which can be 78 kDa), and (3) the variant of OGT targeting mitochondria (mOGT; which can be 90 kDa). In certain embodiments, OGT may be thought to form multimers consisting of one or more 110 kDa subunits and 78 kDa subunits in the nucleus and cytoplasm (Varki, Ajit, et al. (Eds.) (2015): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. 3rd. Cold Spring Harbor (NY)). In certain embodiments, the enzyme that catalyzes the formation of O-linked GlcNAc is human EOGT (Uniprot: Q5NDL2). In certain embodiments, the enzyme modifies such proteins with beta-linked N-acetylglucosamine (O-GlcNAc) by catalyzing the transfer of a single N-acetylglucosamine from UDP-GlcNAc to serine or threonine residues in extracellular proteins.In certain embodiments, the enzyme catalyzes the specific glycosylation of a Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domain.

[0048] In certain embodiments, an enzyme that catalyzes the formation of O-linked GlcNAc can effect transfer in an alpha linkage. In other embodiments, an enzyme that catalyzes the formation of O-linked GlcNAc can effect transfer in a beta linkage.

[0049] In certain embodiments, the formation of O-linked GlcNAc in cells prior to genetic manipulation is catalyzed by at least one of the enzymes described in this section (e.g., one, two, three, four, five, six, seven, eight, nine, or ten of the enzymes described in this section).

[0050] In certain embodiments, the formation of O-linked GlcNAc in cells prior to genetic manipulation is catalyzed by at least one GlcNAc transferase derived from a Trypanosomatida species (e.g., Leishmania tarentolae). In certain embodiments, the formation of O-linked GlcNAc in cells prior to genetic manipulation is catalyzed by at least one GlcNAc transferase (e.g., one, two, three, four, five, six, seven, eight, nine, or ten GlcNAc transferases), one or more of which are derived from a Trypanosomatida species. In certain embodiments, the number of at least one GlcNAc transferase is one, two, or three. In certain embodiments, at least one GlcNAc transferase is selected from the group consisting of OGNT1, OGNT2, OGNTL, and homologous GlcNAc transferases thereof. In certain embodiments, at least one GlcNAc transferase is a GlcNAc transferase homologous to OGNT1, OGNT2, and / or OGNTL.

[0051] In certain embodiments, the formation of O-linked GlcNAc in cells prior to being genetically engineered is catalyzed by at least one GlcNAc transferase derived from a species other than Trypanosomatida species (e.g., human). In certain embodiments, the formation of O-linked GlcNAc in cells prior to being genetically engineered is catalyzed by at least one GlcNAc transferase (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GlcNAc transferases), and one or more of such GlcNAc transferases are of human origin. In certain embodiments, the number of at least one GlcNAc transferase is 1, 2, or 3. In certain embodiments, at least one GlcNAc transferase is selected from the group consisting of human O-GlcNAc transferase and human EOGT and their homologous enzymes. In certain embodiments, at least one GlcNAc transferase is an enzyme homologous to human O-GlcNAc transferase and / or human EOGT.

[0052] In certain embodiments, the enzyme catalyzes the formation of O-linked GlcNAc prior to the genetic engineering of Leishmania cells. In certain embodiments, the enzyme still catalyzes the formation of O-linked GlcNAc even after the genetic engineering of Leishmania cells. In certain embodiments, the enzyme does not catalyze the formation of O-linked GlcNAc after the genetic engineering of Leishmania cells.

[0053] 5.2 Method for Genetically Engineering Leishmania Cells Also provided herein is a method for genetically engineering Leishmania cells as described in Section 5.3. In certain embodiments, the method can be used to achieve functional inactivation of a gene encoding an enzyme (described in Section 5.1) that catalyzes the formation of O-linked GlcNAc in Leishmania host cells.

[0054] 5.2.1 Functional inactivation of the gene encoding the enzyme that catalyzes the formation of O-linked GlcNAc In certain embodiments, the gene encoding the enzyme that catalyzes the formation of O-linked GlcNAc can be functionally inactivated. In certain embodiments, the gene encoding at least one GlcNAc transferase is downregulated. In certain embodiments, the gene encoding at least one GlcNAc transferase is deleted. In certain embodiments, the gene encoding at least one GlcNAc transferase is mutagenized.

[0055] In certain embodiments, the gene encoding the enzyme that catalyzes the formation of O-linked GlcNAc can be functionally inactivated using the methods described in the Examples section (Section 6). In certain embodiments, the gene encoding the enzyme that catalyzes the formation of O-linked GlcNAc can be functionally inactivated using any method known in the art. Such methods are, for example, the methods described in International Publication No. WO2019 / 002512A2, which is hereby incorporated by reference in its entirety.

[0056] Examples of mutagenesis methods include, but are not limited to, site-directed mutagenesis using targeted gene editing methods (such as TALEN, ZFN, CRISPR / Cas9, etc.) in combination with a repair backbone for controlled homologous recombination-mediated repair (Zhang, W et al. (2017) mSphere 2(1), Gupta, R. and Musunuru, K. (2014) The Journal of clinical investigation 124(10):4154-4161), transposon mutagenesis (Damasceno, J. et al. (2015) Christopher Peacock (Ed.): Parasite Genomics Protocols, vol. 1201. New York, NY: Springer New York (Methods in Molecular Biology), pp. 235-245), replacement of an endogenous copy in situ using a selectable marker incorporated by homologous recombination (possibly in combination with a mutated gene version) (Roberts, S. (2011) Bioeng Bugs 2(6):320-326); RNA interference (RNAi) (Lye, L. et al. (2010) PLoS Pathog 6(10), e1001161), conditional knockdown using Cre / LoxP or FRT / FLP (Duncan, S. (2017) Molecular and Biochemical Parasitology 216:30-38).

[0057] In certain embodiments, a gene encoding at least one GlcNAc transferase is overexpressed. Such overexpression can be achieved by the following non-limiting exemplary methods. Increasing the gene copy number by introducing additional copies into separate loci (Beverley, S. (1991): Gene amplification in Leishmania. In Annu. Rev. Microbiol. 45, pp. 417-444), high-expression loci (ribosomal DNA loci), or episomal constructs (Lodes, M. et al. (1995) Mol Cell Biol 15(12), pp. 6845-6853. DOI: 10.1128 / mcb.15.12.6845, Boucher, N. (2004) Nucleic Acids Res 32(9):2925-2936), modifying the native UTR adjacent to the coding sequence, introducing additional promoter regions (such as the endogenous PolI promoter or the T7 promoter) in combination with the expression of bacterial T7 polymerase to increase the expression level (Boucher, N. et al. (2002) Molecular and Biochemical Parasitology 119(1):153-158, Gu, P. et al. (2015) Scientific reports 5, p. 9684), introducing multiple copies of the expression construct using transposable elements or recombinase-based systems (such as FRT-FLP or Cre / LoxP) (Duncan, S. et al. (2017) Molecular and Biochemical Parasitology 216, pp. 30-38), integrating mini-chromosomes (Zomerdijk, J. et al. (1992) Nucleic acids research 20(11):2725-2734), as well as and forced chromosomal translocation by CRISPR (Zhang, W. et al. (2017) mSphere 2(1). DOI: 10.1128 / mSphere.00340-16), etc.

[0058] Any method known in the art can be used to manipulate Leishmania cells (e.g., Leishmania tarentolae). In certain embodiments, a plasmid is used to introduce nucleic acid into the host cells described herein. For example, this heterologous nucleic acid is expressed in the host cell by a plasmid (e.g., an expression vector), and introduction of this plasmid into the modified host cell is effected by transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation. In certain embodiments, introduction of the plasmid into the modified host cell is effected by stable transfection.

[0059] In certain embodiments, linearized nucleic acid is introduced into the host cells described herein using transfection, infection, or electroporation, chemical transformation by heat shock, natural transformation, phage transduction, or conjugation. In another embodiment, the heterologous nucleic acid is site-specifically integrated into the host cell genome by homologous recombination.

[0060] In certain embodiments, the genes encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 enzymes that each catalyze the formation of O-linked GlcNAc can be functionally inactivated. In certain embodiments, the genes encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 enzymes that each catalyze the formation of O-linked GlcNAc can be functionally inactivated. In certain embodiments, the genes encoding 3 enzymes that each catalyze the formation of O-linked GlcNAc can be functionally inactivated. In certain embodiments, the genes and / or loci that can be functionally inactivated include, but are not limited to, OGNT1, OGNT2, and OGNTL.

[0061] In certain embodiments, genes encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 GlcNAc transferases, each of which catalyzes the formation of O-linked GlcNAc, can be functionally inactivated. In certain embodiments, genes encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 GlcNAc transferases, each of which catalyzes the formation of O-linked GlcNAc, can be functionally inactivated. In certain embodiments, genes encoding 3 GlcNAc transferases, each of which catalyzes the formation of O-linked GlcNAc, can be functionally inactivated.

[0062] In certain embodiments, genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all enzymes that each catalyze the formation of O-linked GlcNAc can be functionally inactivated. In certain embodiments, genes encoding 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all enzymes that each catalyze the formation of O-linked GlcNAc can be functionally inactivated.

[0063] In certain embodiments, genes encoding at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all GlcNAc transferases that each catalyze the formation of O-linked GlcNAc can be functionally inactivated. In certain embodiments, genes encoding 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of all GlcNAc transferases that each catalyze the formation of O-linked GlcNAc can be functionally inactivated.

[0064] 5.2.2 Cell culture method Disclosed herein is a method for culturing a host cell (e.g., a Leishmania host cell).

[0065] In one embodiment, the host cell is cultured using any of the standard culturing techniques known in the art. For example, the cells are cultured as per usual in a rich nutrient medium such as brain heart infusion, Trypticase soy broth, or yeast extract (all of which contain 5 μg / ml of hemin). Further, the incubation is carried out for 2 - 3 days at 26°C, either as static culture or shaking culture in the dark. In some embodiments, the culture of the recombinant cell line contains an appropriate selection agent. Examples of selection agents are shown in Table 2, but are not limited thereto.

[0066] In certain embodiments, the host cell can be cultured using the methods described in the Assay and Example sections (Sections 5.6 and 6, respectively). Examples of Leishmania strains and the plasmids used as donors for their generation are shown in Table 3, but are not limited thereto.

[0067] 5.3 Leishmania cells Disclosed herein is a Leishmania host cell in which a gene encoding an enzyme that catalyzes the formation of O - linked GlcNAc is functionally inactivated prior to genetic manipulation.

[0068] 5.3.1 Genetically engineered Leishmania cells In certain embodiments, the Leishmania cells used herein are genetically engineered such that the formation of O - linked GlcNAc on polypeptides in the Leishmania cells is reduced or eliminated. In certain embodiments, the formation of O - linked GlcNAc in the Leishmania cells prior to genetic engineering is catalyzed by at least one N - acetylglucosamine (GlcNAc) transferase. In certain embodiments, the gene encoding at least one GlcNAc transferase is functionally inactivated.

[0069] In certain embodiments, the gene encoding at least one GlcNAc transferase is downregulated in Leishmania cells. In certain embodiments, the gene encoding at least one GlcNAc transferase is overexpressed in Leishmania cells.

[0070] In certain embodiments, the Leishmania cells provided herein contain at least one gene deletion. In certain embodiments, the gene encoding at least one GlcNAc transferase is deleted.

[0071] In certain embodiments, the gene encoding at least one GlcNAc transferase is mutagenized.

[0072] In certain embodiments, additional modifications can be introduced (e.g., using recombinant techniques) into the Leishmania cells described herein.

[0073] In certain embodiments, in the genetically engineered Leishmania cells, the formation of O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the formation of O-linked GlcNAc in the reference Leishmania cells. In certain embodiments, in the genetically engineered Leishmania cells, the formation of O-linked GlcNAc is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the formation of O-linked GlcNAc in the reference Leishmania cells. In certain embodiments, the reference Leishmania cells are wild-type. In certain embodiments, the reference Leishmania cells are genetically engineered differently from the genetically engineered Leishmania cells described herein. In certain embodiments, some of the manipulations of the reference Leishmania cells can be the same as the manipulations of the genetically engineered Leishmania cells described herein (e.g., deletion of one or more enzymes that catalyze the formation of O-linked GlcNAc). In certain embodiments, the reference Leishmania cells can contain a recombinant nucleic acid encoding a heterologous glycosyltransferase, and such reference Leishmania cells are, for example, the Leishmania cells described in International Publication No. WO2019 / 002512A2, which is incorporated herein by reference in its entirety. In certain embodiments, the formation of O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the formation of O-linked GlcNAc in wild-type Leishmania cells.In certain embodiments, the formation of O-linked GlcNAc is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the formation of O-linked GlcNAc in wild-type Leishmania cells. In certain embodiments, the formation of O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the formation of O-linked GlcNAc in Leishmania cells comprising a recombinant nucleic acid encoding a heterologous glycosyltransferase. In certain embodiments, the formation of O-linked GlcNAc is reduced by 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the formation of O-linked GlcNAc in Leishmania cells comprising a recombinant nucleic acid encoding a heterologous glycosyltransferase.

[0074] In certain embodiments, the growth rate of the genetically engineered Leishmania cells described herein is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of the reference Leishmania cells. In certain embodiments, the growth rate of the Leishmania cells is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of the reference Leishmania cells. In certain embodiments, the reference Leishmania cells are wild-type. In certain embodiments, the reference Leishmania cells are genetically engineered differently from the genetically engineered Leishmania cells described herein. In certain embodiments, some of the manipulations of the reference Leishmania cells can be the same as the manipulations of the genetically engineered Leishmania cells described herein (e.g., deletion of one or more enzymes that catalyze the formation of O-linked GlcNAc). In certain embodiments, the growth rate of the Leishmania cells is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of wild-type Leishmania cells. In certain embodiments, the growth rate of the Leishmania cells is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of wild-type Leishmania cells.

[0075] 5.3.2 Leishmania and Kinetoplastida Strains In certain embodiments, the cells with reduced or eliminated ability to form O-linked GlcNAc on the polypeptide are Leishmania cells. In certain embodiments, the Leishmania cells are Leishmania tarentolae cells. In certain embodiments, the Leishmania cells are Leishmania aethiopica cells. In certain embodiments, the Leishmania cells are part of the Leishmania aethiopica species complex. In certain embodiments, the Leishmania cells are Leishmania aristidesi cells. In certain embodiments, the Leishmania cells are Leishmania deanei cells. In certain embodiments, the Leishmania cells are part of the Leishmania donovani species complex. In certain embodiments, the Leishmania cells are Leishmania donovani cells. In certain embodiments, the Leishmania cells are Leishmania chagasi cells. In certain embodiments, the Leishmania cells are Leishmania infantum cells. In certain embodiments, the Leishmania cells are Leishmania hertigi cells. In certain embodiments, the Leishmania cells are part of the Leishmania major species. In certain embodiments, the Leishmania cells are Leishmania major cells. In certain embodiments, the Leishmania cells are Leishmania martiniquensis cells. In certain embodiments, the Leishmania cells are part of the Leishmania mexicana species. In certain embodiments, the Leishmania cells are Leishmania mexicana cells. In certain embodiments, the Leishmania cells are Leishmania pifanoi cells. In certain embodiments, the Leishmania cells are part of the Leishmania tropica species.In certain embodiments, the Leishmania cells are Leishmania tropica cells.

[0076] In certain embodiments, cells with reduced or eliminated ability to form O-linked GlcNAc on polypeptides belong to the family Bodonidae of kinetoplast. In certain embodiments, the host cell is a Bodo saltans cell. In certain embodiments, the host cell belongs to the family Ichthyobodonidae of kinetoplast. In certain embodiments, the host cell belongs to the family Trypanosomatidae of kinetoplast. In certain embodiments, the host cell belongs to the family Blastocrithidiidae of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Blechomonadidae of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Herpetomonadidae of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Jaenimonas of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Lafontella of Trypanosomatidae. In certain embodiments, the host cell belongs to the subfamily Leishmaniinae of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Novymonas of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Paratrypanosoma of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Phytomonas of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Sergeia of Trypanosomatidae. In certain embodiments, the host cell belongs to the subfamily Strigomonadinae of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Trypanosoma of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Wallacemonas of Trypanosomatidae. In certain embodiments, the host cell belongs to the family Blastocrithidiidae of Trypanosomatidae.

[0077] 5.4 Use of Leishmania cells as an expression system In certain embodiments, Leishmania cells (such as those described in Section 5.3) with reduced or eliminated ability to form O-linked GlcNAc on polypeptides can be used as an expression system for polypeptides. In certain embodiments, the polypeptide can be a heterologous non-Leishmania protein (such as a therapeutic protein (e.g., an antibody or antibody format)).

[0078] 5.4.1 Compositions comprising host cells In one aspect, compositions comprising the host cells described herein are provided herein, such compositions being, for example, compositions comprising the Leishmania cells described herein. Such compositions can be used in methods for producing the target polypeptides described in Section 5.3. In certain embodiments, the compositions comprising host cells can be cultured under conditions suitable for the production of polypeptides. Thereafter, such polypeptides can be isolated from the host cell-containing compositions using methods known in the art.

[0079] The compositions comprising host cells provided herein can include additional components suitable for the maintenance and survival of the host cells described herein and can further include additional components necessary or advantageous for the production of polypeptides by the host cells. Such additional components are, for example, inducers for inducible promoters (such as arabinose, IPTG, etc.).

[0080] 5.4.2 Method for producing a target polypeptide In one aspect, provided herein is a method for preparing a target polypeptide. In one embodiment, provided herein is a method for producing a target polypeptide in vivo using the host cells described herein. In certain embodiments, provided herein is a method for producing a target polypeptide, the method comprising: (i) culturing the host cells provided herein under conditions suitable for polypeptide production; and (ii) isolating the target polypeptide. In certain embodiments, the host cells comprise: (a) a recombinant nucleic acid encoding the target polypeptide; and (b) a recombinant nucleic acid encoding a heterologous glycosyltransferase. In certain embodiments, the heterologous glycosyltransferase is an N-acetylglucosaminyltransferase, or a heterologous galactosyltransferase, or a heterologous sialyltransferase. In certain embodiments, the host cells are Leishmania cells. In certain embodiments, the Leishmania cells comprise one or more heterologous glycosyltransferases. In certain embodiments, the Leishmania cells comprise one or more N-acetylglucosaminyltransferases, and / or one or more heterologous galactosyltransferases, and / or one or more heterologous sialyltransferases.

[0081] In certain embodiments, the target polypeptide produced by the provided host cells is a therapeutic polypeptide, i.e., a polypeptide used for the treatment of a disease or disorder. For example, the target polypeptide produced by the host cells provided herein can be an enzyme, a cytokine, or an antibody. A list of target polypeptides is shown by way of example in Section 5.5, but is not limited thereto.

[0082] 5.5 Target Polypeptides In certain embodiments, the target polypeptide produced by the provided host cell is a therapeutic polypeptide, i.e., a polypeptide used for the treatment of a disease or disorder. For example, the target polypeptide produced by the host cells provided herein can be an enzyme, a cytokine, or an antibody. In certain embodiments, the target polypeptide is selected from the group consisting of adalimumab, rituximab, and erythropoietin (EPO).

[0083] Any polypeptide known in the art (or a peptide / polypeptide corresponding to such a polypeptide) can be used as the target polypeptide according to the methods described herein. Those skilled in the art will understand that the nucleic acid sequences of known polypeptides as well as those of newly identified polypeptides can be readily deduced using methods known in the art. Thus, it is assumed that it is well within the ability of those skilled in the art to introduce a nucleic acid encoding any desired polypeptide (e.g., via an expression vector (e.g., a plasmid), e.g., by site-specific integration by homologous recombination) into the host cells provided herein.

[0084] In certain embodiments, the target polypeptide is glycosylated (e.g., sialylated). The methods described herein can be used to glycosylate the target polypeptide (this glycosylation can be performed in vivo, e.g., using the host cells provided herein, or in vitro), and such glycosylated target polypeptides have a therapeutic effect (e.g., due to improved pharmacokinetics), and those skilled in the art will further recognize that they can be used for the treatment of subjects having a disease / disorder that will benefit from treatment with such glycosylated (e.g., polysialylated) target polypeptides.

[0085] In certain embodiments, the target polypeptide comprises the amino acid sequence of human interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin-IL-2 (denileukin diftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), tumor necrosis factor alpha (TNF-α), insulin, pramlintide, growth hormone (GH), insulin-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide-1 agonist (GLP-1), glucagon, growth hormone releasing hormone (GHRH), secretin, thyroid stimulating hormone (TSH), human bone morphogenetic polypeptide 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7), gonadotropin releasing hormone (GnRH), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), fibroblast growth factor 7 (FGF7), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), neurotrophin-3, human follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), lutropin-α, erythropoietin, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), the extracellular domain of CTLA4 (e.g., an FC fusion), or the extracellular domain of a TNF receptor (e.g., an FC fusion). In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein is an enzyme or an inhibitor.Examples of enzymes and inhibitors that can be used as target polypeptides include, but are not limited to, Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor VIIa, antithrombin III (AT-III), polypeptide C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, aglucosidase-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 (urate oxidase), glutamate carboxypeptidase (glutcapidase), α1 protease inhibitor (α1 antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase.

[0086] In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein is a cytokine. Examples of cytokines that can be used as target polypeptides include, but are not limited to, interferon-α (INF-α), interferon-β (INF-β), interferon-γ (INF-γ), interleukin-2 (IL2), chimeric diphtheria toxin-IL-2 (denileukin diftitox), interleukin-1 (IL1), IL1B, IL3, IL4, IL11, IL21, IL22, IL1 receptor antagonist (anakinra), and tumor necrosis factor alpha (TNF-α).

[0087] In certain embodiments, the target polypeptides used in accordance with the methods and host cells described herein are hormones or growth factors. Examples of hormones and growth factors that can be used as target polypeptides include, but are not limited to, insulin, pramlintide, growth hormone (GH), insulin-like growth factor (IGF1), human parathyroid hormone, calcitonin, glucagon-like peptide-1 agonist (GLP-1), glucagon, growth hormone-releasing hormone (GHRH), secretin, thyroid-stimulating hormone (TSH), human bone morphogenetic polypeptide 2 (hBMP2), human bone morphogenetic protein 7 (hBMP7), gonadotropin-releasing hormone (GnRH), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), fibroblast growth factor 7 (FGF7), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), neurotrophin-3, human follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG), lutropin-α, erythropoietin, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0088] In certain embodiments, the target polypeptides used in accordance with the methods and host cells described herein are receptors. Examples of receptors that can be used as target polypeptides include, but are not limited to, the extracellular domain of human CTLA4 (e.g., fused to Fc) and soluble TNF receptor (e.g., fused to Fc).

[0089] In other embodiments, the target polypeptide is a therapeutic polypeptide. In other embodiments, the target polypeptide is an approved biologic. In another embodiment, the therapeutic polypeptide is abatacept (e.g., Orencia), aflibercept (e.g., Eylea), agalsidase beta (e.g., Fabrazyme), albiglutide (e.g., Eperzan), aldesleukin (e.g., Proleukin), alefacept (e.g., Amevive), alglucerase (e.g., Ceredase), alglucosidase alfa (e.g., LUMIZYME), aliskiren (e.g., Tekturna), alpha-1-proteinase inhibitor (e.g., Aralast), alteplase (e.g., Activase), anakinra (e.g., Kineret), anisoylated plasminogen streptokinase activator complex (e.g., Eminase), human anthrax immune globulin (e.g., ANTHRASIL), antihemophilic factor (e.g., Advate), anti-inhibitor coagulant complex (e.g., FeibaNf), antithrombin alpha, human antithrombin III, antithymocyte globulin (e.g., antithymocyte globulin), antithymocyte globulin (equine) (e.g., ATGAM), antithymocyte globulin (rabbit) (e.g., ATG-Fresenius), aprotinin (e.g., Trasylol), asparaginase alpha, asparaginase (e.g., Elspar), asparaginase Erwinia chrysanthemi (e.g., Erwinaze), becaplermin (e.g., REGRANEX), belatacept (e.g., Nulojix), beractant, bivalirudin (e.g., Angiomax), botulinum toxin type A (e.g., BOTOXE), botulinum toxin type B (e.g., Myobloc), brentuximab vedotin (e.g., Adcetris), buserelin (e.g., Suprecur), C1-esterase inhibitor (human), C1-esterase inhibitor (recombinant) (e.g., Ruconest), certolizumab pegol (e.g., Cimzia), chorionic gonadotropin alpha (e.g., chorionic gonadotropin alpha), chorionic gonadotropin (human) (e.g., Ovidrel), chorionic gonadotropin (recombinant) (e.g., Ovitrelle),Coagulation Factor IX (e.g., Alprolix), Coagulation Factor VIIa (e.g., NovoSeven), Coagulation Factor X Human (e.g., Coagadex), Coagulation Factor XIII A-Subunit (Recombinant), Collagenase (e.g., Cordase), Conestat Alfa, Corticotropin (e.g., H.P.Acthar), Cosyntropin (e.g., Cortrosyn), Darbepoetin Alfa (e.g., Aranesp), Defibrotide (e.g., Noravid), Denileukin Diftitox (e.g., Ontak), Desirudin, Digoxin Immune Fab (Sheep) (e.g., DIGIBIND), Dornase Alfa (e.g., Pulmozyme), Drotrecogin Alfa (e.g., Xigris), Dulaglutide, Emicizumab-Kxwh (e.g., ELOCTA), Erolizumab, Enfuvirtide (e.g., FUZEON), Erythropoietin Alfa (e.g., Binocrit), Erythropoietin Zeta (e.g., Retacrit), Eptifibatide (e.g., INTEGRILIN), Etanercept (e.g., Enbrel), Exenatide (e.g., Byetta), Factor IX Complex (Human) (e.g., AlphaNine), Fibrinolysin (also known as Plasmin) (e.g., Elase), Filgrastim (e.g., N.A.), Filgrastim-sndz, Follitropin Alfa (e.g., Gonal-F), Follitropin Beta (e.g., Follistim AQ), Galsulfase (e.g., Naglazyme), Intrinsic Factor, Gemtuzumab Ozogamicin (e.g., Mylotarg), Glatiramer Acetate (e.g., Copaxone), Recombinant Glucagon (e.g., GlucaGen), Glucarpidase (e.g., Voraxaze), Gramicidin D (e.g., Neosporin), Hepatitis B Immune Globulin, Human Calcitonin, Human Clostridium tetani Toxin Immune Globulin, Human Rabies Virus Immune Globulin (e.g., Hyperab Rabies Immune Globulin Human), Human Rho(D) Immune Globulin (e.g., Hyp Rho D Inj 16.5%), Human Serum Albumin (e.g., Albuminar), Human Varicella-Zoster Immune Globulin (e.g.,Varizig), hyaluronidase (e.g., HYLENEX), hyaluronidase (human recombinant), ibritumomab tiuxetan (e.g., Zevalin), idursulfase (e.g., Elaprase), imiglucerase (e.g., Cerezyme), human immunoglobulin, insulin aspart (e.g., NovoLog), bovine insulin, insulin degludec (e.g., Tresiba), insulin detemir (e.g., LEVEMIR), insulin glargine (e.g., Lantus), insulin glulisine (e.g., APIDRA), insulin lispro (e.g., Humalog), porcine insulin (e.g., IletinII), regular insulin (e.g., HumulinR), porcine insulin (e.g., vetsulin), isophane insulin (e.g., NovolinN), recombinant interferon alpha-2a (e.g., RoferonA), interferon alpha-2b (e.g., INTRON A), interferon alphacon-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), lepirudin (e.g., Refludan), leuprolide (e.g., Eligard), liraglutide (e.g., Saxenda), lucinactant (e.g., Surfaxin), lutropin alpha (e.g., Luveris), mecasermin (e.g., N.A.), menotropin (e.g., Menopur), methoxypolyethylene glycol-epoetin beta (e.g., Mircera), Metreleptin (e.g., Myalept), natural alpha interferon or multiferon (e.g., Intron / Roferon-A), nesiritide (e.g., NATRECOR), ocriplasmin (e.g., Jetrea), oprelvekin (e.g.,Neumega), OspA lipopeptide (e.g., Lymerix), oxytocin (e.g., Pitocin), palifermin (e.g., Kepivance), pancrelipase (e.g., Pancrecarb), bovine pegademase (e.g., Adagen), pegaspargase (e.g., Oncaspar), pegfilgrastim (e.g., Neulasta), peginterferon alfa-2a (e.g., Pegasys), peginterferon alfa-2b (e.g., PEG-Intron), peginterferon beta-1a (e.g., Plegridy), pegloticase (e.g., (Krystexxa)), pegvisomant (e.g., SOMAVERT), poractant alfa (e.g., Curosurf), pramlintide (e.g., Symlin), Preotact (e.g., PreotactE), protamine sulfate (e.g., Protamine Sulfate Injection, USP), human polypeptide S (e.g., human polypeptide S), prothrombin (e.g., FeibaNf), prothrombin complex (e.g., Cofact), concentrated prothrombin complex (e.g., Kcentra), rasburicase (e.g., Elitek), reteplase (e.g., Retavase), rilonacept (e.g., Arcalyst), romiplostim (e.g., Nplate), sacrosidase (e.g., Sucraid), salmon calcitonin (e.g., Calcimar), sargramostim (e.g., Leucomax), satumomab pendetide (e.g., OncoScint), sebelipase alfa (e.g., Kanuma), secretin (e.g., SecreFlo), sermorelin (e.g., sermorelin acetate), serum albumin (e.g., Albunex), iodinated serum albumin (e.g., Megatope), simoctocog alfa (e.g., Nuwiq), sipuleucel-T (e.g., Provenge), recombinant somatropin (e.g., NutropinAQ), recombinant somatropin (e.g., BioTropin), streptokinase (e.g., Streptase), susoctocog alfa (e.g., Obizur), taliglucerase alfa (e.g., Elelyso), teduglutide (e.g., Gattex), tenecteplase (e.g.,TNKase), teriparatide (e.g., Forteo), tesamorelin (e.g., Egrifta), thrombomodulin alpha (e.g., Recomodulin), thymalfasin (e.g., Zadaxin), thyroglobulin, thyrotropin alpha (e.g., Thyrogen), purified polypeptide derivative tuberculin (e.g., Aplisol), tuctocog alfa (e.g., Zonovate), urofollitropin (e.g., BRAVELLE), urokinase (e.g., Kinlytic), vasopressin (e.g., Pitressin), velaglucerase alfa (e.g., Vpriv), abciximab (e.g., ReoPro), adalimumab (e.g., Humira), alemtuzumab (e.g., CAMPATH), alirocumab (e.g., Praluent), arcitumomab (e.g., CEA-Scan), atezolizumab (e.g., Tecentriq), basiliximab (e.g., Simulect), belimumab (e.g., Benlysta), bevacizumab (e.g., Avastin), blinatumomab (e.g., Blincyto), brodalumab (e.g., Siliq), canakinumab (e.g., ILARISE), canakinumab (e.g., Ilaris), capromab (e.g., ProstaScint), cetuximab (e.g., Erbitux), daclizumab (e.g., Zenapax), daratumumab (e.g., DARZALEX), denosumab (e.g., Xgeva), dinutuximab (e.g., unituxin), eculizumab (e.g., Soliris), efalizumab (e.g., RAPTIVA), elotuzumab (e.g., EMPLICITI), evolocumab (e.g., Repatha), golimumab (e.g., Simponi injection), ibritumomab (e.g., Zevalin), idarucizumab (e.g., Praxbind), infliximab (e.g., REMICADE), ipilimumab (e.g., YERVOY), ixekizumab (e.g., Taltz), mepolizumab (e.g., Nucala), muromonab (e.g., ORTHOCLONE OKT3), natalizumab (e.g., Tysabri), necitumumab (e.g., Portrazza), nivolumab (e.g., Opdivo), obinutuzumab (e.g.,Anthim), obinutuzumab (e.g., Gazyva), ofatumumab (e.g., Arzerra), omalizumab (e.g., Xolair), palivizumab (e.g., Synagis), panitumumab (e.g., Vectibix), pembrolizumab (e.g., Keytruda), pertuzumab (e.g., , Perjeta), ramucirumab (e.g., Cyramza), ranibizumab (e.g., Lucentis), raxibacumab (e.g., RAXIBACUMAB), rituximab (e.g., Rituxan), secukinumab (e.g., Cosentyx), siltuximab (e.g., Sylvant), tocilizumab (e.g., ACTEMRA), tositumomab (e.g., Bexxar), trastuzumab (e.g., Herceptin), ustekinumab (e.g., Stelara), or vedolizumab (e.g., Entyvio), and comprises an amino acid sequence of.

[0090] In other embodiments, the target polypeptide is an antibody. In another embodiment, the antibody is adalimumab (Humira), Remicade (infliximab), ReoPro (abciximab), Rituxan (rituximab), Simulect (basiliximab), Synagis (palivizumab), Herceptin (trastuzumab), Mylotarg (gemtuzumab ozogamicin), Campath (alemtuzumab), Zevalin (ibritumomab tiuxetan), Xolair (omalizumab), Bexxar (tositumomab-I-131), Erbitux (cetuximab), Avastin (bevacizumab), Tysabri (natalizumab), Actemra (tocilizumab), Vectibix (panitumumab), Lucentis (ranibizumab), Soliris (eculizumab), Cimzia (certolizumab pegol), Simponi (golimumab), Ilaris (canakinumab), Stelara (ustekinumab), Arzerra (ofatumumab), Prolia (denosumab), Numax (motavizumab), ABThrax (raxibacumab), Benlysta (belimumab), Yervoy (ipilimumab), Adcetris (brentuximab vedotin), Perjeta (pertuzumab), Kadcyla (ado-trastuzumab emtansine), or Gazyva (obinutuzumab), and has the amino acid sequence thereof.

[0091] In other embodiments, the antibody is a full-length antibody, Fab, F(ab’) 2 , Scfv, or sdAb. In other embodiments, the target polypeptide comprises the amino acid sequence of an enzyme or an inhibitor thereof. In another embodiment, the target polypeptide is Factor VII, Factor VIII, Factor IX, Factor X, Factor XIII, Factor VIIa, antithrombin III (AT-III), polypeptide C, tissue plasminogen activator (tPA) and tPA variants, urokinase, hirudin, streptokinase, glucocerebrosidase, aglucosidase-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), glutamic acid carboxypeptidase (glucarpidase), α1 protease inhibitor (α1-antitrypsin), lactase, pancreatic enzymes (lipase, amylase, protease), and adenosine deaminase, including the amino acid sequences of.

[0092] In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein is a receptor. Examples of receptors that can be used as the target polypeptide include, but are not limited to, the extracellular domain of human CTLA4 (e.g., fused to Fc) and soluble TNF receptor (e.g., fused to Fc).

[0093] In another embodiment, the target polypeptide is secreted into the culture medium. In certain embodiments, the target polypeptide is purified from the culture medium. In another embodiment, the target polypeptide is purified from the culture medium by affinity purification or ion exchange chromatography. In another embodiment, the target polypeptide comprises an FC domain and is affinity purified from the culture medium by polypeptide-A. In another embodiment, the target polypeptide comprises an affinity tag and is affinity purified.

[0094] In certain embodiments, the target polypeptide used in accordance with the methods and host cells described herein can be a full-length polypeptide, a truncation thereof, a polypeptide domain, region, motif, or peptide.

[0095] In certain embodiments, the target polypeptide is an Fc fusion polypeptide.

[0096] In certain embodiments, the target polypeptide is a biologic that includes the Fc domain of IgG.

[0097] In certain embodiments, the target polypeptide can be modified. In another embodiment, the target polypeptide is engineered to include a signal sequence from Leishmania. In other embodiments, the signal sequence is processed and removed from the target polypeptide. In another embodiment, the target polypeptide is engineered to include one or more tag(s). In other embodiments, the tag is processed and removed from the target polypeptide.

[0098] 5.5.1 Compositions and / or Formulations Containing Polypeptides In another aspect, compositions (e.g., pharmaceutical compositions) are provided herein that include one or more of the target polypeptides described herein. The compositions described herein are useful for the treatment and / or prevention of diseases / disorders in a subject (e.g., a human subject) (see Section 5.5.2).

[0099] In certain embodiments, the compositions described herein (e.g., pharmaceutical compositions) include a pharmaceutically acceptable carrier in addition to the target polypeptides described herein. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more specifically in humans. The term “carrier” as used herein in the context of a pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle used in the administration of a pharmaceutical composition. Physiological saline as well as aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin.

[0100] In certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for subcutaneous administration, parenteral administration, oral administration, intradermal administration, transdermal administration, colorectal administration, intraperitoneal administration, and rectal administration. In certain embodiments, the pharmaceutical composition can be formulated for intravenous administration, oral administration, intraperitoneal administration, intranasal administration, intratracheal administration, subcutaneous administration, intramuscular administration, topical administration, intradermal administration, transdermal administration, or pulmonary administration.

[0101] In certain embodiments, the compositions described herein can further include one or more buffering agents. Such buffering agents are, for example, phosphate buffers and sucrose phosphate glutamate buffers. In other embodiments, the compositions described herein do not include a buffering agent.

[0102] In certain embodiments, the compositions described herein further comprise one or more salts. Such salts include, for example, sodium chloride, calcium chloride, sodium phosphate, sodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or mixtures of such aluminum salts). In other embodiments, the compositions described herein are salt-free.

[0103] The compositions described herein can be included in a kit, container, pack, or dispenser, together with administration instructions.

[0104] The compositions described herein can be stored until use. For example, the compositions can be stored by freezing (e.g., at about -20 °C or about -70 °C), under refrigerated conditions (e.g., at about 4 °C), or at room temperature.

[0105] 5.5.2 Preventive and Therapeutic Uses In one aspect, provided herein is a method of preventing or treating a disease or disorder in a subject, the method comprising administering to the subject a target polypeptide or a composition thereof described herein. Further provided herein is a method of preventing a disease or disorder in a subject, the method comprising administering to the subject a target polypeptide or a composition thereof described herein.

[0106] In one aspect, provided herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject a target polypeptide or a composition thereof described herein. In another aspect, provided herein is a method of preventing a disease or disorder in a subject, the method comprising administering to the subject a target polypeptide or a composition thereof described herein. In certain embodiments, provided herein is a method of preventing or treating a disease or disorder in a subject, the method comprising administering to the subject a polysialylated target polypeptide produced according to the methods described herein.

[0107] In certain embodiments, a disease or disorder can be caused by the presence of a defective version of a target polypeptide in a subject, the absence of the target polypeptide in the subject, or a decrease in the expression of the target polypeptide in the subject, and can be treated or prevented using the target polypeptide produced using the methods described herein. In certain embodiments, a disease or disorder can be mediated by a receptor to which the target polypeptide produced using the methods described herein binds, or can be mediated by a ligand to which the target polypeptide produced using the methods described herein binds (e.g., the target polypeptide is a receptor for the ligand).

[0108] In certain embodiments, a method for preventing or treating a disease or disorder in a subject comprises administering to the subject an effective amount of the target polypeptide or a composition thereof described herein. In certain embodiments, the effective amount is a therapeutically effective amount that has a prophylactic effect and / or a therapeutic effect(s). In certain embodiments, "effective amount" refers to a therapeutically effective amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reduction or alleviation of the severity of the disease / disorder or a symptom associated therewith, (ii) reduction of the duration of the disease / disorder or a symptom associated therewith, (iii) prevention of the progression of the disease / disorder or a symptom associated therewith, (iv) induction of regression of the disease / disorder or a symptom associated therewith, (v) prevention of the onset or occurrence of the disease / disorder or a symptom associated therewith, (vi) prevention of recurrence of the disease / disorder or a symptom associated therewith, (vii) reduction of organ failure associated with the disease / disorder, (viii) reduction of the number of hospitalizations of a subject having the disease / disorder, (ix) reduction of the length of hospitalization of a subject having the disease / disorder, (x) extension of the survival period of a subject having the disease / disorder, (xi) elimination of the disease / disorder in the subject, and / or (xii) enhancement or improvement of the prophylactic or therapeutic effect(s) of another treatment.

[0109] 5.6 Assay 5.6.1 Strains, Growth Methods, and Genetic Methods A method for culturing host cells is provided in this specification.

[0110] The host cells are cultured using any of the standard culturing techniques known in the art. For example, the cells are cultured as per usual in a rich nutrient medium such as brain heart infusion, Trypticase soy broth, or yeast extract (all of which contain 5 μg / ml of hemin). Further, the incubation is carried out for 2 - 3 days at 26°C, either statically or with shaking in the dark. In some embodiments, the culture of the recombinant cell line contains an appropriate selection agent. In some embodiments, the culture contains biopterin at a final concentration of 10 μM to support growth.

[0111] A list of selection agents is shown as an example in Table 2, but is not limited thereto.

Table 3

Table 4

[0112] (i) Plasmid The plasmid was obtained from the pUC57 vector backbone for growth in E. coli and contains an ampicillin selection marker or a kanamycin selection marker. The expression cassette is adjacent to a restriction enzyme recognition site suitable for excision. The composition of the cassette depends on the intended use and is described in each method. The target gene is included as an ORF with codon usage optimized for L. tarentolae. The optimized sequences were manually selected to avoid restriction enzyme recognition sites and to remove repetitive or homopolymeric regions. The generation and sequencing of the plasmid were performed by a gene synthesis provider. The plasmid and the description are shown in the sequence listing.

[0113] For codon usage optimization, the protein sequences were reverse translated into nucleotide sequences using a custom Python3 script that probabilistically selects codons based on the codon usage frequency of L. tarentolae while excluding low-frequency codons (frequency < 10%). Codon usage was calculated using cusp (Rice, et al. (2000) Trends in genetics: TIG 16(6), pp. 276-277) for all annotated L. tarentolae nucleotide coding sequences.

[0114] (ii) DNA construct design for deletion of OGNT Deletion of OGNT for was carried out using three different construct compositions. In all cases, the integration construct includes a 200 bp overlap region within its construct was split into two or more fragments (about 1000 - 2500b p) and to the L. tarentolae genome the complete construct of was generated by enable homologous recombination.

[0115] Replacement with a selectable marker flanked by 5' and 3' untranslated regions (transcription by endogenous PolII). These 5' and 3' untranslated regions provide the defined splice leader acceptor sequence and polyadenylation sequence to the selectable marker. 1.) 5' homologous region for specific integration into the OGNT gene (ideally just after the 5' UTR to remove the entire coding sequence), 2.) intergenic region providing the 5' UTR containing the splice leader acceptor sequence to the target gene, 3.) target gene encoding the selectable marker as an ORF with codon usage optimized for Leishmania (either L. major or L. tarentolae), 4.) intergenic region providing the 3' UTR containing the polyadenylation site to the target gene, 5.) 3' homologous region for site-specific recombination into the OGNT gene (see also variant A in Figure 6).

[0116] Replacement with a selectable marker not adjacent to the untranslated region (transcription by endogenous PolII). 1.) 5' homology region for specific integration immediately downstream of the 5' untranslated region of the OGNT gene; 2.) a gene of interest encoding a selectable marker as an ORF with codon usage optimized for Leishmania (either L. major or L. tarentolae); 3.) 3' homology region for site-specific recombination upstream of the 3' UTR of the OGNT gene (see also variant B in Figure 6).

[0117] Replacement in an expression cassette for glycan modification modification (inverted integration, transcription by PolI). 1.) 5' homology region for specific integration into the OGNT gene; 2.) a promoter region for PolI transcription containing terminal repeats, and a 5' UTR containing a splice leader acceptor sequence for the gene of interest; 3.) a gene of interest encoding a glycosyltransferase or enzyme in the gluconeogenesis pathway as an ORF with codon usage optimized for Leishmania (either L. major or L. tarentolae); 4.) an intergenic region that provides a 3' polyadenylation site to the upstream gene and a splice leader acceptor site to the downstream gene; 5.) a selectable marker as an ORF with codon usage optimized for Leishmania (either L. major or L. tarentolae); 4.) an intergenic region that provides a 3' UTR containing a polyadenylation site to the gene of interest; 5.) 3' homology region for site-specific recombination into the OGNT gene. It is possible to repeatedly arrange some of elements 3 and 4 in front of the selectable marker.

[0118] (iii) DNA construct design for overexpressing OGNT To evaluate the effects of overexpression on three OGNT candidates, plasmids were generated to support stable expression, including a) 5' homologous recombination sites targeting the ssu locus, b) 5' UTR (aprt), c) each 3×HA-tagged OGNT gene, d) intergenic region (CamIR), e) selectable marker gene, f) 3' UTR (dhfr-ts), and g) 3' homologous recombination sites targeting the ssu locus.

[0119] (iv) DNA construct design for expressing hCas9 A plasmid for expressing Cas9 was designed similarly to the above overexpression plasmid using a publicly available Cas9 sequence (Le, et al. (2013) Science 339(6121), pp. 819 - 823) with codon usage optimized for L. tarentolae. Different from the construct for overexpression, the Cas9 expression construct was usually transfected as circular DNA that would remain episomal in L. tarentolae.

[0120] (v) crRNA design For use with SpCas9 (PAM = NGG), crRNAs were designed by EuPaGDT (http: / / grna.ctegd.uga.edu / ) based on the target region (usually the coding sequence of the OGNT gene) and rechecked for on / off-target effects by using blast against the whole genome of L. tarentolae. Next, crRNAs were selected such that the ends of the coding sequence to be replaced would ideally be targeted.

Table 5

[0121] (vi) Transfection method (A) Preparation of DNA Restriction enzyme digestion (12 μg of DNA in a total volume of 240 μL) was carried out using standard restriction enzymes (ThermoFisher, preferably FastDigest) according to the manufacturer's instructions. The restriction enzyme digestion was carried out at 37 °C until completion or overnight, and the DNA purification was carried out by EtOH precipitation (in this EtOH precipitation, 2 volumes of 100% ice-cold EtOH were added to 1 volume of digested DNA, incubated on ice for 30 minutes, and subjected to centrifugation at 17,500 × g for 30 minutes at 4 °C. The pellet was washed with 70% EtOH, dried for a maximum of 15 minutes, and resuspended in ddH2O). To optimally remove circular plasmids, one or two restriction enzymes having recognition sites in the vector backbone were selected, digestion was carried out at 37 °C for 1 hour, and purification was carried out by EtOH as described above. Analysis of the digest was carried out by agarose gel electrophoresis in a 0.7 - 2% agarose gel (TAE buffer). Optionally, extraction from the gel was carried out using NucleoSpin® gel, and undigested plasmids were removed from the preparation by using a PCR Clean-up kit (Macherey & Nagel) according to the manufacturer's instructions.

[0122] (B) Preparation of gRNA for transfection into cell lines constitutively expressing Cas9 For gRNA for CRISPR / Cas9-mediated genome editing, it was assembled from tracrRNA and crRNA (Microsynth) by performing a denaturation step at 95 °C for 5 minutes in a thermal cycler followed by slow cooling at 0.1 °C / second. A total of 10 μg of tracrRNA and 10 μg of crRNA per transfection were used together with integration DNA (1 μg / fragment) serving as a template for homologous recombination repair.

[0123] (C) Preparation of ribonucleoprotein (RNP) complexes for transfection without constitutive Cas9 expression CRISPR / Cas9-mediated genome editing for use For the gRNA, after denaturation at 95°C for 5 minutes in a thermal cycler, slow cooling at 0.1°C / second was performed to assemble equal amounts of tracrRNA and crRNA as described above. mol equal amounts of tracrRNA and crRNA (Microsynth) and assembled from done This assembly was performed individually for each crRNA used, and then equal amounts of different gRNAs were mixed. Next, 122 pmol of recombinant expressed Cas9 protein (i.e., Alt-R® S.p.HiFi Cas9 nuclease V3 (IDT, #1081061)) was added to 360 pmol of the gRNA mixture and incubated at room temperature for 15 minutes to form RNPs. The final volume used for transfection by Nucleofector (see Section E) should not exceed 6 μl. Finally, the RNP mixture was added to the transfection solution containing the repair DNA described below together with 1 μl of Alt-R® Cas9 Electroporation Enhancer (IDT, #1081072). mol

[0124] (D) DNA Preparation for Transfection Linear DNA fragments for integration were mixed for transfection in the required combinations at 1 μg per fragment. When using the CRISPR / Cas9 system, the gRNA was prepared as described above and mixed with the integration fragments. This mixture was concentrated under reduced pressure at 30°C to reduce its volume to a maximum of 2 μl per transfection. For episomal transfection of the Cas9 plasmid, 0.1 - 1 μg of plasmid DNA was used directly for transfection.

[0125] (E) Transfection Using Nucleofector One day before transfection, a dense growth culture of the parental strain was included in fresh medium (BHIH) (containing all antibiotics corresponding to the pre-integrated selectable markers) at a 1:10 dilution and cultured overnight at 26°C.

[0126] Transfection was performed using the 4D-Nucleofector™ Core X together with the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza). For this, the DNA (or DNA / RNA or DNA / RNP) prepared as described above was mixed with 16.4 μl of P3 Primary Cell solution and 3.6 μl of supplement solution. 10 7 An equal volume of the culture containing 10 cells (OD should be approximately 0.3 - 1.0 / ml and the cell shape should be round to drop-shaped) was pelleted by centrifugation at 1800 g for 5 minutes and the supernatant was removed. This cell pellet was resuspended in 20 μl of the above DNA (or DNA / RNA or DNA / RNP) mixture and transfected using a 16-well electroporation strip with Pulse FI-158 (in some examples, FP167, CM150, EO115, DN100, FP158, FB158 were used as alternative pulses). As a negative control, an additional culture was subjected to transfection with ddH2O only.

[0127] 80 μl of fresh medium (BHIH, parental selection marker) was added to each well, and 45 μl of the mixture (in duplicate) was transferred to individual wells of a 96-well culture plate pre-filled with 200 μl of fresh medium. After incubation (recovery) for 24 hours at 26°C in the dark, a new selection marker was added at 50% concentration (pre-selection (see Table 5)). After further incubation for 1 - 2 days, the selection marker was replenished to 100% (main selection (see the table)), and several dilutions (1:2 - 1:10) were performed in 96-well format (final volume 250 μl). The culture was further incubated in the dark at 26°C for up to 7 days. If no growth was observed, the culture medium was replaced (centrifugation at 1800 g for 10 minutes at room temperature) and the culture was incubated again for up to 7 days. This step was repeated as necessary. Before analysis, the growth culture was expanded by dilution in the range of 1:5 - 1:20 to increase the culture volume.

[0128] (F)Transfection using Gene Pulser Xcell (trademark) (Biorad) Transfection (static, 26 °C) Leishmania cultures for transfection were prepared by diluting high-density growth cultures in BHIH 1:10 the day before transfection. of the substance The OD at 600 nm in disposable cuvettes was measured using a photometer, and this OD was adjusted to fall within the range of 0.4 - 1.0 (4 - 6×10 * 7 cells) to optimize efficiency. The cells were in the logarithmic growth phase, as indicated by the presence of a mixed population of round and drop-shaped cells. A higher proportion of round cells was preferred. 10 ml of culture was used for each transfection, and as a negative control for each selectable marker, one culture was always subjected to electroporation with ddH2O. For transfection, the cultures were spun at 1,800×g to It was subjected to room temperature for 5 minutes. The supernatant was removed, and the pellet was resuspended in 5 ml of transfection buffer (200 mM Hepes (pH 7.0), 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM anhydrous dextrose (glucose), filter sterilized (pore size 0.22 μm)). The cells were centrifuged again, and the pellet was resuspended in 400 μl of transfection buffer. 400 μl of the cells were added to the DNA, transferred to a cuvette, and incubated on ice for 10 minutes. Electroporation was performed with Gene Pulser Xcell (trademark) (Biorad) using a low voltage protocol (exponential decay wave: 450 V, 450 uF, 5 - 6 milliseconds, cuvette: d = 2 mm), and immediately placed on ice for exactly 10 minutes afterwards. The entire contents of the cuvette were transferred to 10 ml of BHIH containing no selection marker, and the cells were statically cultured and grown at 26 °C for 20 - 24 hours while aerating in the dark. For the selection of polyclonal cell lines, half the concentration of the selection marker was added, the culture was incubated at 26 °C for 1 - 2 days, and then subcultured at a ratio of 1:10 in 10 ml of BHIH containing the full concentration of the selection marker. The cells were further grown at 26 °C in the dark. If the culture changes to a turbid culture after 7 days, the cells will be subjected to a 5 - minute spin - down at 1,800×g at room temperature, and the pellet will be resuspended in fresh BHIH medium containing the full concentration of the selection marker.

[0129] For the same method, it is described in International Publication No. WO2019 / 002512A2 (this document is incorporated herein by reference in its entirety).

[0130] Clone selection For clone selection, as soon as the liquid culture became turbid, the cells were streaked onto BHIH plates (containing 1.4% agar and the appropriate 100% selection agent). The plates were covered with parafilm and incubated in the dark at 26°C in an inverted state for 7 - 10 days. Single colonies (1 - 2 mm in size) were transferred to 24 - well plates containing 1 ml of BHIH, covered with parafilm, and incubated in the dark at 26°C for approximately 7 - 10 days. Then, 1 ml of the culture was transferred from the 24 - well plate to a flask containing 10 ml of BHI and further statically cultured and grown as usual.

[0131] (vii) PCR analysis and sequence analysis of deletion strains (A) Preparation of gDNA - Isolation of genomic DNA using a tissue kit 2 ml of a dense growth culture of L. tarentolae was pelleted at 1800 g and the supernatant was discarded. This pellet was used for the preparation of genomic DNA using the NucleoSpin® Tissue Kit (Macherey - Nagel). For this, the pellet was resuspended in 200 μl of T1 buffer and further processed according to the manufacturer's instructions until elution. To perform efficient elution, 50 μl of pre - heated (50°C) BE buffer was added to the column and incubated at room temperature for 3 minutes. The eluate was recovered by performing a 1 - minute centrifugation at 11000 g. The yield can be increased by repeating this step and re - loading the eluate.

[0132] (B) Preparation of crude cell extracts for PCR analysis 50 μl of the culture was washed with 1 ml of PBS and pelleted at 1800 g for 5 minutes. The supernatant was removed, the pellet was resuspended in 50 μl of PBS, and boiled at 95°C for 5 minutes while vortexing intermittently. 1 μl was used in the PCR reaction instead of template DNA.

[0133] (C) PCR analysis of OGNT KO OGNT knockout was confirmed by PCR by performing either amplification of the complete locus (OGNT-1, OGNT-2, OGNT-L, or OGNT-1+L) or amplification of a shortened fragment covering the integration site.

[0134] When the wild-type gene is correctly replaced, the amplification product will be much shortened. Therefore, the successful replacement of the wild-type OGNT sequence (OGNT-1 = 3.4 kbp, OGNT-2 = 1.9 kbp, and OGNT-L = 3.4 kbp) with the selectable marker coding sequence (0.4 - 1.0 kbp) (with or without an additional intergenic region (about 0.9 kbp in total)) was usually easily confirmable by the size of the amplification product obtained from PCR targeting the entire native locus. For such PCR, LATaq DNA polymerase (TaKaRa) was used in combination with a buffer for amplification of GC-rich sequences that enables amplification of the long wild-type region (see Table 5). In some cases, it was preferred to amplify the shortened region using primers that bind within the OGNT coding sequence to test for the presence of residual wild-type genes (Table 5). For this, DreamTaq DNA polymerase (Thermo Fisher Scientific) was used. Alternatively, testing that the selectable marker gene was correctly integrated into each OGNT locus can be done by combining primers that bind within the genome, where one primer binds to the selectable marker CDS or intergenic region of the integrated construct and the other primer targets the genome. This was performed, for example, to confirm that the selectable marker cassette was correctly integrated in the heterozygous KO generated in Example 2 (see "OGNT-2 KO (with IR)" in Table 5. The primers are listed in Table 6).

Table 6

Table 7

[0135] (D)OGNT deletion sequencing The genomic DNAs of St16248, St16257, St16249, St16636, St16700, St16702, and St16704 were sequenced by Illumina NextSeq (2×150 bp paired-end sequencing; TruSeq libraries were prepared according to the manufacturer's instructions). The obtained quality-trimmed data consisted of approximately 20 M paired reads per stock. By aligning the reads with the reference sequence using BWA-MEM (Li (2013) (available online at http: / / arxiv.org / pdf / 1303.3997v2)), an average coverage of 186-fold across the entire genome was obtained. The complete absence of reads matching each OGNT region indicates the successful removal of the OGNT gene.

[0136] (viii) Expression analysis by Western blot (A) Sample preparation and expression analysis by Western blot Cells were statically cultured at 26°C for 2 - 3 days to grow (this growth culture was carried out, for example, in 3 ml in a 6-well plate). Analysis of whole cell extracts (WCE) equivalent to an OD value of 0.05 and cell-free culture supernatants equivalent to an OD value of 0.05 was performed by Western blot. For supernatant analysis, the growth culture was subjected to centrifugation at 1800 g at room temperature for 5 minutes, and the cell-free supernatant was transferred to a new tube and mixed with Laemmli dye under reducing or non-reducing conditions. For the cell pellet for WCE, it was washed with 1×PBS, subjected to centrifugation at 1800 g at room temperature again for 5 minutes, and frozen at -80°C for at least 30 minutes. After thawing this pellet at room temperature again, the pellet was dissolved in Leammli (reducing) buffer, boiled again at 95°C for 10 minutes, vortexed vigorously, and then subjected to 4 - 12% bis-tris SDS PAGE at 200 V using MOPS running buffer for 60 minutes. Using the Iblot device over 7 minutes transfer of the gel to a PVDF membrane perform blotting It was carried out. The blocking treatment of the membrane in 10% milk was carried out at room temperature for at least 30 minutes. Polyclonal rabbit anti-HA antibody (H6908, Sigma) was used for overnight incubation at 4°C as a 1:2000 dilution in 1×PBST containing 1% milk. Then, it was subjected to washing with 1×PBST for 5 minutes its The blot was subjected to 3 washes, and then horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (170-6515, BioRad) as a 1:4000 dilution in 1×PBST containing 1% milk using at 30°C while rotating for 3 hours for secondary antibody detection, and then it was subjected to washing with 1×PBST for 5 minutes 3 times, and stained with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (one-component type) (TMBM-1000-01, Surmodics) for colorimetric detection.

[0137] 5.6.2 Expression, purification, and analysis of anti-TNFa "adalimumab" in custom glycan host cells derived from L. tarentolae (i) Large-scale expression and purification The adalimumab expression cassette (containing 5’UTR_adalimumab_LC_intergenic region_adalimumab HC_intergenic region_sat_3’UTR and conferring nourseothricin resistance) integrated into Ssu in St15449 (a recombinant strain based on St10569 (wild type)) was grown in BHIH in a 10 L bioreactor for 50.8 hours and collected at an OD value of 15. The culture was collected, centrifuged at 8000×g for 30 minutes, and the supernatant was filtered through a filter cartridge with pore sizes of 0.45 μm + 0.22 μm (Sartorius, 544307H9--SS--A), and 5 PIC tablets (protease inhibitor cocktail, cOmplete, EDTA-free, Roche, 05056489001) and EDTA (final concentration 5 mM) were added.

[0138] Using a peristaltic pump, the culture supernatant was continuously circulated overnight at 4°C through two 5-ml Protein A columns (HiTrap MabSelect PrismA, GE Healthcare, 17-5498-53). The columns were connected to an NGC system and washed with 10 column volumes (CV) of 1×PBS (pH 7.4). Elution was performed using 0.15 M glycine (pH 2.5) and directly neutralized by adding 150 μl of 1 M Tris (pH 8.5). Fractions containing adalimumab were pooled and subjected to buffer exchange into PBS (pH 6). The adalimumab pool was adjusted with Tris and (NH4)2SO4 and loaded onto two 5-ml HIC (HiTrap Capto Phenyl HP) columns at a flow rate of 1 ml / min, followed by gradient elution using buffer A (20 mM Tris, 1 M (NH4)2SO4, pH 7.0) and buffer B (20 mM Tris, pH 7.0). The fractions were collected and analyzed by SDS PAGE (Coomassie staining) and Western blot. The final pool was subjected to buffer exchange into PBS (pH 6) and stored at -80°C.

[0139] (ii) Fed-batch fermentation for the analysis of deletion strains The OGNT deletion strains (St16704 (OGNT-1 / L KO) and St16636 (OGNT-1 / 2 / L KO)) were subjected to fed-batch fermentation in a bio-t® xpac multi-bioreactor system. After the cells were grown in BHIH (2 / 3 of the total fermentation volume) for 50 hours, the concentrate (56 g / L yeast extract, 32 g / L soy peptone, 28.8 g / L glucose, 15 mg / L hemin; 1 / 3 of the total fermentation volume) was fed at a constant rate. After 94 hours, the culture was collected and subjected to centrifugation at 3220×g for 30 minutes at 4°C. As described below, 50 ml of the supernatant was used for the small-scale concentration of adalimumab by Protein A.

[0140] (iii) Small-scale expression and purification of adalimumab The host cells were grown as required for 48 hours at 26 °C with shaking at 140 rpm and 50 ml of culture in BHIH. The culture was collected and subjected to centrifugation at 1800×g for 10 minutes at room temperature. The culture supernatant was filtered through a filter with a pore size of 0.22 μm (Steriflip, SCGP00525), and a 1:100 dilution of EDTA (0.5 M pH 8) was added to each row. to the well The culture supernatant of each strain was incubated batchwise for 4 hours at room temperature with 100 μl of protein A resin (Protein A - Sepharose 4B Fast Flow, Sigma Aldrich, P9424) per Falcon tube. rotate After treatment with the protein A resin, the samples were subjected to centrifugation at 500×g for 5 minutes, flow-through part (FT) the supernatant was discarded, and the resin was transferred to a spin column. Washing was performed 3 times using 500 μl of buffer A (20 mM Na 2 HPO 4 (pH 7.2), 150 mM NaCl (pH adjusted to 7.20 with HCl)) (5 CV). For this washing, centrifugation at 1000×g was performed for 1 minute at room temperature between each step. Elution was performed using 100 μl of buffer B (0.1 M acetic acid, 100 mM NaCl (pH adjusted to 3.20 with 1 M NaOH)) for 100 μl of resin (performed in several CVs). For this elution (e.g., 3 times with 1 CV and 1 time with 0.5 CV), centrifugation at 1000×g was performed for 1 minute at room temperature between each step.

[0141] The elution fractions were pooled and quickly neutralized by adding 100 mM Tris - HCl (1 M pH 8). Then, the pooled eluate was subjected to buffer exchange into PBS (pH 6) using a 7K ZebaSpin desalting column (2 ml) and optionally concentrated using an Amicon 30K concentrator (0.5 ml).

[0142] Next, the samples were subjected to analysis (such as O - HexNac quantification) as described below.

[0143] 5.6.3 Expression, purification, and analysis of LMSAP-rhEPO-Strep in custom glycan host cells derived from L. tarentolae The host cells were grown for 72 h at 26 °C in 1 L of yeast extract medium culture while shaking at 140 rpm. The culture was subjected to centrifugation at 8000 g for 30 min at 4 °C, and the supernatant was collected and subjected to precipitation with 40% ammonium sulfate (45 min at room temperature). The precipitate was collected by centrifugation at 11000 g for 1 h at 4 °C and then resuspended in 50 ml of PBS (pH 7.4). This sample was subjected to dialysis against 5 l of PBS (pH 7.4) for 2 h (SpectraPor™ dialysis membrane, MWCO 3.5 kDa) and further dialysis against another 5 l of PBS (pH 7.4) overnight at 4 °C. After filtering the sample through a 0.22-μm pore size filter (Steriflip, SCGP00525), the sample was subjected to recirculation loading onto a 1-ml StrepTrap™ HP (StrepTrap™ HP, GE Healthcare, 29-0486-53) column overnight at 4 °C. After washing with 20 CV of PBS (pH 7.4), LMSAP-rhEPO-Strep was eluted using PBS (pH 7.4) containing 2.5 mM desthiobiotin (5 CV, fraction volume 0.5 ml) and concentrated using an AmiconUltra-0.5 concentrator.

[0144] Thereafter, the sample was subjected to glycopeptide analysis.

[0145] 5.6.4 Analysis (i) SDS PAGE and capillary gel electrophoresis For Coomassie, 10 μg was used, and for WB, 2.5 μg was used to perform SDS PAGE under reducing or non-reducing conditions. The separation in this SDS PAGE was carried out on a 4→12% gel using MOPS buffer for 55 minutes. The determination of protein purity was performed by Coomassie-stained SDS-PAGE using 10 μg of protein sample and compared with a BSA standard curve. The quantification of impurities was performed by ImageQuant. Capillary gel electrophoresis (CGE) was carried out using the Agilent Protein 230 kit (5067-1518) according to the protocol.

[0146] (ii) Analytical SEC MAbPac SEC-1 (4×300 mm) is a size exclusion chromatography (SEC) column specially designed for the separation and characterization of monoclonal antibodies (mAbs). This column was used according to the manufacturer's recommended conditions (temperature: 30 °C; eluent: PBS (50 mM NaPO4, 300 mM NaCl) (pH 6.8); elution: isocratic elution for 30 minutes; flow rate: 0.2 mL / min; detection: 215 nm; injection volume: 5 μL (equivalent to 5 μg of protein)).

[0147] (iii) Identification of O-HexNAc Intact monoclonal antibodies were analyzed by mass spectrometry. In this mass spectrometry, the latest measuring instrument (Orbitrap FTMS), data processing by SpectroSwiss, and data analysis (bioinformatics) tools were used. In addition to intact measurement, the antibody was subjected to reduction treatment with TCEP or cleavage treatment with an enzyme (IdeS) to generate Fd subunit, LC subunit, and Fc / 2 subunit, and these subunits were analyzed using the same measuring instrument.

[0148] (iv) Sample preparation for analyzing the subunits obtained by IdeS To generate mAb subunits of approximately 25 kDa each, digestion of the mAb with IdeS (FabRICATOR, Genovis, Lund, Sweden) was carried out in the formulation buffer. One unit of IdeS was added per μg of mAb and left to react at 37 °C for 30 minutes. Subsequently, the mAb was denatured and reduced by incubating with 6 M GdnCl and 30 mM TCEP for 30 minutes at room temperature. Finally, the reaction was stopped by exposing the solution to 1% TFA to acidify it. For analysis, the sample was diluted with 0.1% aqueous FA to a final concentration of 1 μg / μl.

[0149] (v) Mass spectrometry, bioinformatics, and data processing for intact subunit analysis. Standard settings for Orbitrap-based intact protein mass measurements were used (triple LC-MS experiment). In these standard settings, the following were used:

[0150] - Analytical HPLC (Dionex) (analysis time 30 minutes)

[0151] - Analytical column (Waters), C4 Aquity, BEH, 90 μl / min, 60 °C

[0152] - Q Exactive HF FTMS (Thermo)

[0153] - FTMS Booster (SpectroSwiss), advanced data acquisition system

[0154] - Full MS resolution: 30,000 at m / z 200 for intact mAb and 60,000 at m / z 200 for subunit analysis

[0155] - Charge target number (number of ions): AGC value 3e6

[0156] For the mass spectral deconvolution of intact mAbs, Intact Mass (Protein Metrics) was used. For the deconvolution of mass spectra in subunit analysis, the MASH Suite software tool (open access, Wisconsin University, Ying Ge Group) was used. At the resolution used, it was possible to obtain isotopically resolved data. For the processing of time-domain signals and mass spectra, the Peak-by-Peak software tool (SpectroSwiss) was used. In the method used, it was possible to obtain spectral data with a wide dynamic range.

[0157] (vi) Identification of O-HexNAc positions by peptide mapping To identify the positions of HexNAc modifications observed in the Fd subunit, a bottom-up proteomics approach using trypsin digestion and chymotrypsin digestion as well as HCD fragmentation and ETD fragmentation was implemented. In this approach, SpectroSwiss was used with the latest measuring instrument (Orbitrap FTMS), initially without targeting specific targets and then additionally targeting the most promising peptides. A standard trypsin digestion protocol using approximately 5 μg of protein (concentration approximately 1 μg / μl) was used. The sample was reduced by adding 6 μl of 50 mM TEAB containing 2 M urea and 1.5 μl of 20 mM TCEP, alkylated with 1.5 μl of 80 mM chloroacetamide (incubated at room temperature for 1 hour), and then subjected to digestion at 37 °C for 2 hours using 0.2 μg of trypsin. The digestion was stopped using 10 μl of 0.2% TFA, the sample was centrifuged, and the supernatant was used for injection.

[0158] Standard settings for Orbitrap-based bottom-up proteomics were used (triple LC-MS experiment). In these standard settings, the following were used:

[0159] - Nano HPLC (Dionex) (analysis time 140 minutes)

[0160] - Q Exactive Plus FTMS using HCD as the MS / MS method

[0161] - Full MS resolution: 70,000 at m / z 200

[0162] - Charge target number (number of ions): AGC value of 3e6

[0163] - MS / MS resolution: 17,500 at m / z 200

[0164] - Charge target number (number of ions): AGC value of 1e5

[0165] - MS / MS method: HCD at 27 NCE

[0166] - Data protein-dependent Dependent analysis: top 10

[0167] - Dynamic exclusion: 60 seconds

[0168] Scaffold (Proteome Software Inc.) was used for data analysis. The following parameter set was used in this analysis:

[0169] Variable modifications: +1 for NQ (deamidation), +16 for M (oxidation), +42 for N (acetylation)

[0170] Digestive enzyme: Trypsin

[0171] Maximum number of cleavage failures: 2

[0172] Scaffold: Version Scaffold_4.8.3

[0173] Protein classification guideline: Classification throughout the experiment using both peptide weight and protein weight

[0174] Peptide threshold: Minimum 90.0%

[0175] Protein threshold: minimum 95.0%, minimum number of peptides 1

[0176] Peptide FDR: 0.2% (decoy)

[0177] Protein FDR: 9.2% (decoy)

[0178] For more detailed data analysis, specifically PTM analysis, Peaks software was used.

[0179] For the processing of time-domain signals and mass spectra, the Peak-by-Peak software tool (SpectroSwiss) was used. In the method used, it was possible to obtain spectral data with a wide dynamic range.

[0180] (vii) Characterization of O-HexNAc by monosaccharide analysis using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) For the confirmation of O-glycosylation and the determination of the amount of O-HexNAc found on T225 of the heavy chain of adalimumab, in combination with several purification steps, FabRICATOR (IdeS) (Genovis AB, Lund, Sweden) and GingisKHAN (Kgp) (Genovis AB, Lund, Sweden) were used according to the product manuals to generate 14-amino acid glycopeptides.

[0181] Briefly described, adalimumab 2 mg purified from commercially available Humira or L. tarentolae was first incubated with PNGaseF (50 U added, 3 h at 37 °C) to remove N-glycans, thereby reducing the possibility of interference by N-glycan-derived monosaccharides during monosaccharide analysis. Next, this PNGaseF-treated sample was incubated overnight at 37 °C with FabRICATOR (2000 U) in PBS (pH 7.1). This digest was subjected to a batch CaptoL purification step (3 h at room temperature) for purification to remove the Fc / 2 fragment. The remaining F(ab)2 fragment was concentrated using a Pierce PES 3 kDa column and placed back into buffer using a ZebaSpin column containing 0.1 M Tris ( 0. containing 1 M NaCl, pH 8.0). This sample placed back into buffer was incubated overnight at 37 °C with GingisKHAN (2000 U) in the presence of 2 mM cysteine. After reducing the sample with TCEP (20 mM) for 20 minutes, a 14-amino acid peptide containing the O-HexNAc site was purified using a 10 kDa Pierce PES column (the LC and Fd fragments are retained). The presence of the 14-amino acid peptide with O-HexNAc modification and the 14-amino acid peptide without O-HexNAc modification was confirmed by MALDI. During the MALDI measurement, the subunits (LC, Fc / 2, Fd) were not identified.

[0182] The monosaccharides, which are seen as O-glycosylation on the 14-amino acid peptide ("10k pass-through"), were analyzed by HPAEC-PAD and the monosaccharide peaks were identified by comparison with commercially available monosaccharide standards. Monosaccharides were liberated from this bioconjugate by TFA hydrolysis (treatment at 121 °C for 2 h with 2 M TFA). Commercially available monosaccharide standards used (GlcNAc, GalNAc, and ManNAc) were also treated with TFA to mimic the situation of the monosaccharides liberated from the mAb (GlcN, GalN, and ManN after TFA hydrolysis). The obtained non-derivatized monosaccharides were separated on a CarboPac PA1 column 4 × 250 mm (Thermo Fisher Scientific) by elution with NaOH / NaOAc and then detected by pulsed amperometric detection (PAD). The identity of the sugar was able to be determined by overlaying the monosaccharides liberated from the mAb with the corresponding monosaccharide standards.

[0183] (viii) Characterization of O-HexNAc by MS fragmentation The group of Manfred Wuhrer at Leiden University (Medical Center (LUMC), Center for Proteomics and Metabolomics, Leiden, The Netherlands) performed LC-MS / MS experiments to identify the nature of O-HexNAc, more specifically, to determine whether O-HexNAc corresponds to N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc). It has previously been demonstrated that GalNAc and GlcNAc can be discriminated by using the pattern of oxonium ions obtained in MS fragmentation (Halim et al. (2014) J Proteome Res 13(12), pp. 6024-6032). In particular, the ratio of the intensity of the ion found at m / z 138.055 to the intensity of the ion found at m / z 144.066 is very different for GalNAc and GlcNAc. Unfortunately, for peptides with N-acetylmannosamine (ManNAc), such information is currently not available. The modification site was a threonine in the peptide (SEQ ID NO: 227) (modified Thr is underlined) obtained from the heavy chain of the antibody by trypsin digestion. An antibody that does not have this specific modification (Humira as a control Ab) produced in CHO cells was also available. Trypsin digestion was performed and the two samples were subjected to a series of LC-MS / MS analyses, followed by manual data analysis to search for specific glycopeptides. [Chemical formula] (SEQ ID NO: 227) It has been identified as the threonine in which the modified Thr is underlined. An antibody that does not have this specific modification (Humira as a control Ab) produced in CHO cells was also available. Trypsin digestion was performed and the two samples were subjected to a series of LC-MS / MS analyses, followed by manual data analysis to search for specific glycopeptides.

[0184] To obtain glycopeptides, in-gel trypsin digestion was performed on the HC band of Humira (control Ab) and the HC band of LMTB adalimumab with O-HexNAc modification (HexNAc Ab) after SDS-PAGE under reducing conditions (loading 10 μl containing 10 μg protein), and these were analyzed in duplicate using LC-MS / MS method on an Orbitrap Fusion Lumos mass spectrometer.

[0185] For digestion in the gel, the in-house digestion robot system was used. In this procedure, the gel plugs were first washed with 400 μl of 25 mM NH4HCO3 and dehydrated with acetonitrile. Next, protein reduction and alkylation were performed (using DTT (10 mM) and iodoacetamide (50 mM), respectively). Then, trypsin was added and the protein was digested overnight. The next day, the tryptic digested peptides were collected and lyophilized. Before LC-MS / MS analysis, the tryptic digested peptides were reconstituted in 30 μl of 0.1% formic acid, and 1 μl of it was used for each individual LC-MS / MS analysis.

[0186] LC-MS / MS analysis was performed on an Orbitrap Fusion Lumos mass spectrometer (Thermo). Separation of the tryptic digested peptides was performed using water + 0.1% formic acid as solvent A and acetonitrile + 0.1% formic acid as solvent B. This separation was performed using a linear gradient with the solvent B content increased from 10% to 40% over 40 minutes at 250 nl / min. The peptides eluted by this gradient were analyzed by mass spectrometry on an Orbitrap Fusion Lumos instrument (Thermo). First, the peptides were analyzed in a discovery-type MS / MS experiment. For this purpose, MS scans were recorded at a resolution of 120000. Furthermore, the AGC target value was 4 * 10 5 , and the injection time was set to 50 milliseconds. The mass was dynamically excluded for 10 seconds from detection, including those with charge states of 2 - 5. The intensity threshold was 5 * 10 4 For data-dependent MS / MS fragmentation (cycle time, master scan interval 3 seconds), high-energy collisional dissociation (HCD) was used with a collision energy of 32%. For MS / MS scans, the orbitrap resolution was set to 30000 and the AGC target value was 5 * 10 4 It was set to * 10 5 and the maximum injection time was set to 200 milliseconds. For fragmentation, the HCD collision energy was set to 32%, 37%, and 42%. Furthermore, an MS / MS scan with a CID collision energy of 35% was also performed. Data analysis was carried out manually.

[0187] (ix) Confirmation that O-HexNAc is absent in the KO cell line To confirm the absence of O-HexNAc on adalimumab in the KO strain, in addition to MALDI measurement, HCD fragmentation using the latest measuring instrument (Orbitrap FTMS) in a targeted approach and peptide mapping were performed using tryptic digests (see the section on identification of O-HexNAc positions by peptide mapping).

[0188] (x) Quantification of O-glycosylation on glycopeptides by MALDI-TOFMS Regarding the quantification of O-glycosylation with O-HexNAc at T225 on the heavy chain of adalimumab, FabRICATOR (IdeS) (Genovis AB, Lund, Sweden) and GingisKHAN (Kgp) (Genovis AB, Lund, Sweden) were used consecutively to generate 14-amino acid glycopeptides. For sample digestion, the sample was placed again under the buffer of 0.1 mM Tris buffer (pH 8.0), and approximately 10 μg (10 μl) of the sample was incubated with 0.5 μl of FabRICATOR (approx. 30 U) at 37 °C for 1 hour. Then, 1 μl of GingisKHAN (approx. 10 U) and 2 mM reducing agent (cysteine) were added and incubated at 37 °C for 3 hours. For the reduction of the disulfide bridge in the hinge region, 0.5 μl of 1 M TCEP was added (final concentration 50 mM) and incubated at room temperature for 60 minutes.

[0189] After desalting the sample using a C18 ZipTip pipette tip (Millipore) according to the manufacturer's instructions, on a ground steel target plate in which α-cyano -4-Hydroxycinnamic acid (HCCA; containing 5 mg / ml HCCA in 40% acetonitrile with 0.1% trifluoroacetic acid content ) spot on the matrix was used. MALDI-TOF MS was performed by operating an UltrafleXtreme matrix-assisted laser desorption / ionization-time of flight / time of flight (MALDI-TOF / TOF) mass spectrometer (Bruker) in reflector positive ion mode. The instrument was operated with flexControl 3.4 (Bruker), and at that time, the laser intensity was set to 30 - 40%, the mass range was m / z 700 - 3500, the detector gain was 2×, and the sampling rate was 2.5 GS / sec. 3000 laser shots performed in 500-shot steps were integrated. The signal / noise cutoff value was set to 5, and smoothing and baseline correction using the TopHat algorithm were performed to process the spectrum with flexAnalysis 3.4 (Bruker). The selection of target peaks was performed manually considering the non-glycosylated and O-glycosylated versions of the same peptide. The relative abundance of O-glycosylated peptides was determined by integrating the areas of H + ions and Na + ions and associating them with the integrated value of the intensities of all identified peptide forms.

[0190] (xi) HILIC-UPLC-MS analysis of purified protein and N-glycans released from the cell surface Enzymatic release of N-glycans from purified proteins was performed using Rapid PNGase F (New England Biolabs) as recommended by the supplier. 8 μl of sample (15 μg of protein) was mixed with 2 μl of Rapid buffer and 1 μl of Rapid PNGase F. The mixture was incubated at 50 °C for 10 minutes and then at 90 °C for 1 minute. Enzymatic release of N-glycans from the cell surface was performed using PNGase F (New England Biolabs). 50 mg of cells were resuspended in Glyco buffer 2 and incubated with 1 μl of PNGase F at 37 °C, 650 rpm for 1 hour. The cells were pelleted by centrifugation and 75 μl of the supernatant was centrifugally dried in a SpeedVac concentrator. The glycan was resuspended in 10 μl of water. After glycan release, the glycan was directly labeled with procainamide as previously reported (Behrens, et al. (2018) Glycobiology 28(11), pp. 825-831). Briefly, 1 μl of acetic acid, 8 μl of procainamide stock solution (550 mg / ml in DMSO), and 12 μl of sodium cyanoborohydride stock solution (200 mg / ml in H 2 O) were mixed with the released glycan. The sample was incubated at 65 °C for 60 minutes and purified using an LC-PROC-96 purification plate (Ludger Ltd) according to the manufacturer's instructions.

[0191] Analysis of procainamide-labeled N-glycans was performed by hydrophilic interaction chromatography-ultra performance liquid chromatography-mass spectrometry (HILIC-UPLC-MS) using an Acquity UPLC System (Waters) capable of fluorescence detection connected to a Synapt G2-Si mass spectrometer (Waters). Separation of glycans was performed using an Acquity BEH amide column (130 Å, 1.7 μm, 2.1 mM × 150 mM; Waters) with 50 mM ammonium formate (pH 4.4) as solvent A and acetonitrile as solvent B. This separation was performed using a linear gradient with the solvent B content changed from 72% to 55% over 40 minutes at a flow rate of 0.5 ml / min. Fluorescence detection was performed with an excitation wavelength of 310 nm and a detection wavelength of 370 nm. A Synapt G2-Si mass spectrometer equipped with a Zspray electrospray ion source was used for mass detection in positive resolution mode. The following parameters were used for this detection: scan range: m / z 300 - 3500, scan time: 1 second, capillary voltage: 2.2 kV, ion source temperature: 120 °C, and sampling cone voltage: 75 V. MassLynx 4.2 (Waters) was used for data acquisition. Data processing and analysis were performed using Unifi 1.9.4.053 (Waters). Glucose unit assignments were made using a fifth-order polynomial distribution curve based on the retention times of a procainamide-labeled dextran ladder (Ludger Ltd). Glycan structures were assigned based on their m / z values and their retention times and subjected to matching against an N-glycan library constructed previously. For individual samples, UPLC was combined with a Synapt HDMS mass spectrometer using equivalent settings.

[0192] For a small number of samples, labeling was performed using a Waters RapiFluor labeling kit (used mostly in accordance with the Waters instruction: <<Quality control and Automation Friendly GlycoWorks RapiFluor-MS N-Glycan Sample Preparation>>) and analyzed using the same equipment as for procainamide-labeled glycans.

[0193] (xii) Analysis of EPO glycopeptides (A) Determination of N-glycosylation site occupancy and site-specific glycosylation By using Rapigest® SF, the enzymatic digestion of proteins in 50 mM ammonium bicarbonate was enhanced. A 50 mM ammonium bicarbonate buffer (pH 8) containing 180 μg of protein (equivalent to 250 μL of each sample) was prepared by performing four centrifugations (15,000×g; 10 minutes, 4 °C) using Vivaspin 500 PES (5 kDa). The final protein concentration was approximately 3 mg / ml.

[0194] The protein was denatured in 0.1% Rapigest® SF according to the protocol of Rapigest® SF (WATERS), reduced with DTT (5 mM) at 60 °C for 45 minutes, and alkylated with iodoacetamide (15 mM) in the dark at room temperature for 45 minutes. The sample was incubated with 6.4 μg of trypsin / Lys-C Mix, Mass Spec Grade (Promega V507A #201254) at 37 °C for 16 hours to obtain digested proteins. 0.5% TFA was added to the digested protein sample, incubated at 37 °C for 30 minutes, and centrifuged at 15,000 g for 10 minutes to remove the hydrolyzable surfactant. All tryptic digestion peptides were purified using SEP Pack C18, 200 mg (Waters) with 0.1% aqueous TFA solution. The elution volume of 2 mL (70% acetonitrile containing 0.1% TFA) was completely dried by lyophilization.

[0195] The peptide was dissolved in 40 μL of ultrapure water, and 30 μl was used for glycopeptide enrichment according to the protocol of ProteoExtract® Glycopeptide Enrichment Kit 72103-3 (Novagen).

[0196] 30 μL of the sample was added to 150 μL of ZIC Glycocapture resin, and the flow-through containing unbound peptides was collected. The glycopeptides were eluted using 225 μL of ZIC elution buffer. The flow-through sample and the elution sample were completely evaporated in a speed vac. The glycopeptides were resuspended in 15 μL of MilliQ water. After purification with Zip Tips MILLIPORE C18 to improve the signal / noise ratio, a 50% methanol solution containing the glycopeptides was dried on a maldi plate, and 1 μl of CHCA (LaserBiolabs) matrix solution (7 mg / mL in 50% acetonitrile) was overlaid.

[0197] 5 μL of the suspended glycopeptides was included in 50 mM sodium acetate buffer, 1 μL of PNGaseF (Promega V483A #226517) and 1 μL of PNGaseA (Sigma #01000353) were added, and deglycosylation was carried out at 37 °C for 16 hours. After preparation with Zip Tips MILLIPORE C18 (SOP P17 / 2), the deglycosylated peptides were mixed (1:1) with CHCA (LaserBiolabs) matrix solution (7 mg / mL in 50:50 acetonitrile / water with 0.1% TFA content).

[0198] The flow-through fraction was prepared and analyzed (1:1) using CHCA (LaserBiolabs) matrix solution (7 mg / mL in 50:50 acetonitrile / water with 0.1% TFA content).

[0199] The peptides, deglycosylated glycopeptides, and occupied glycopeptides in the flow-through fraction were analyzed by MALDI-TOF MS mass spectrometry in positive mode reflectron and linear mode. Acquisition of the linear mode MALDI mass spectrum was performed using a MALDI-TOF / TOF Autoflex III (Bruker Daltonics). The acquisition conditions were as follows: 14.3×2904V, laser output 49%, number of shots 6000. Acquisition of the positive ion reflectron MALDI mass spectrum was performed using a MALDI-TOF / TOF Autoflex III (Bruker Daltonics) (SOP 44 / 1). The acquisition conditions using the RP850-3500 Bruker method were as follows: 40×2160V; laser output 97%, number of shots 5000.

[0200] (B) Sample preparation and analysis of EPO-O-glycosylated peptides by LC-MS For the analysis of EPO O-glycopeptides, filter-aided sample preparation (FASP) for purified proteins was applied using trypsin digestion. An Amicon 30kd centrifugal filter unit (Millipore, catalog number UFC503096) was prepared by washing twice with water. In this preparation, centrifugation was performed at 10,000g for 10 minutes each time, and the flow-through part was discarded. 100 μg of purified EPO sample was added to this Amicon filter, and ABC buffer (0.05 M NH 4 HCO 3 aqueous solution (pH 8.0)) was added to make 400 μl. The filter was centrifuged at 10,000g for 10 minutes, washed once with 400 μl of ABC buffer, centrifuged again, and the flow-through part was discarded. All further centrifugation steps were performed at 10,000g for 10 minutes.

[0201] Subsequently, the protein was reduced with 100 μl of ABC buffer containing 50 mM DTT at 37 °C for 30 minutes. Then, the filter was centrifuged, washed with 400 μL of ABC buffer, and centrifuged again. Next, the protein was alkylated with 100 μl of ABC buffer containing 65 mM IAA in the dark at room temperature for 1 hour. Then, the filter was centrifuged and washed three times with 400 μl of ABC buffer. In this washing step, the filter was centrifuged each time, and the flow-through fraction was discarded.

[0202] The column was transferred to a new collection tube. In this tube, 2 μl of trypsin (1 μg / μl) and 98 μl of ABC buffer were added to hEPO (wild type) to make the enzyme:protein ratio 1:20. For hEPO from triple OGNT-KO, an additional 1.5 μl of trypsin (1 μg / μl) and 98.5 μl of ABC buffer were added to maintain the enzyme:protein ratio at 1:20. The samples were incubated at 37 °C overnight (16 hours) without shaking. The peptides were collected by centrifugation, and the flow-through fraction was transferred to a low-binding tube. 400 μl of H2O was added to the Amicon, rinsed by pipetting, centrifuged, and the flow-through fraction was transferred to the same low-binding tube. Next, 400 μl of 10% AcN was added to the Amicon, carefully rinsed by pipetting, and centrifuged to collect the remaining flow-through fraction in the same low-binding tube. The samples were dried overnight at room temperature in a SpeedVac. The samples were resuspended with 25 μl of 5% acetonitrile + 0.1% formic acid, and 10 μl of this was injected into the LC-MS.

[0203] Trypsin-digested peptides were analyzed using an Acquity UPLC system (Waters) capable of UV detection connected to a Synapt G2-Si mass spectrometer (Waters). Peptides were separated using an Acquity UPLC Peptide BEH column (130 Å, 1.7 μm, 2.1 mM × 150 mM; Waters) with 0.1% aqueous formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B. This separation was carried out using a linear gradient with the content of solvent B changing from 0 to 38% over 120 minutes. A Synapt G2-Si mass spectrometer equipped with a Zspray electrospray source was used for mass detection in the positive resolution mode. At that time, the following parameters were used: scanning range: m / z 50 - 2000; scanning time: 0.5 seconds; cone voltage: 37 V. MSe was used for fragmentation, and this fragmentation was carried out using a trap of 4 V and a transfer cell of 2 V for low-energy scans, and a gradient collision energy of 15 - 45 V in the trap cell for high-energy scans. Data analysis was carried out using Byonic and Byologic (Proteinmetrics).

Example

[0204] 6. Example 6.1 Identification of O-HexNAc Modifications on rhEPO and Recombinant IgG1 6.1.1 rhEPO A custom glycan platform has been developed for producing functionally customized recombinant glycoprotein drugs, and compositions and methods for producing glycoproteins in vivo are described (International Publication No. WO2019 / 002512A2, which is hereby incorporated by reference in its entirety). Recombinant human erythropoietin (rhEPO) contains three N-glycosylation sites and one O-glycosylation site. In L. tarentolae, the latter site is modified during expression, with one HexNAc added to serine 126. Examples of this modification include those occurring in custom glycan host cells (St11521 and St11895) (International Publication No. WO2019 / 002512A2, for example, Figures 19 and Method Section 8.11.9: Determination of N-glycosylation site occupancy and site-specific glycosylation). In both examples (St11521 and St11895), the mass of the peptide ion found at m / z 1668.892 was consistent with that of a trypsin-digested O-glycopeptide (position [117-131][EAISPPDAASAAPLR] (SEQ ID NO: 237) with one attached HexNAc residue). The EPO O-glycopeptide was present as a peptide occupied by one HexNAc residue in both the flow-through fraction and the enriched glycopeptide fraction. This peptide was not detected in its non-glycosylated form (Figure 1).

[0205] 6.1.2 Rituximab Recombinant expression of human IgG1 antibodies (such as rituximab and adalimumab) was also carried out in custom glycan host cells derived from L. tarentolae. The purified mAbs were characterized in detail for their (post-translational) modifications (such as glycosylation). In rituximab, several variants at the N-terminus occurred due to suboptimal cleavage of the secretion peptides located upstream of the heavy chain (HC) and light chain (LC) sequences (International Publication No. WO2019 / 002512A2, for example, Table 12 and Figure 44), and some additional hints were obtained from subunit analysis, which indicated that approximately 20% of the total mAb had additional modifications consistent with the mass of HexNAc (data not shown). Due to the extremely complex interpretation of MS data caused by the heterogeneity of the samples, rituximab was not further analyzed using high-end MS methods.

[0206] 6.1.3 Adalimumab (i) Identification Next, the heavy and light chains of anti-TNF alpha IgG1 (adalimumab) together with the optimized secretion peptide were co-expressed in L. tarentolae custom glycan host cells. Adalimumab was purified from the supernatant by Protein A and HIC and used for characterization of the recombinant mAb preparation (such as intact protein), subunit analysis, and peptide mapping by MS.

[0207] In the MS measurement of intact antibodies, a mass consistent with the mass predicted by N-glycosylation of HC by Man3 was observed. On the other hand, there were additionally two dominant peaks, and these peaks were consistent with the masses with one or two additional HexNAc (Figure 2A). In addition to the intact measurement, the antibody was subjected to reduction treatment with TCEP or cleavage treatment with an enzyme (IdeS) to generate Fd subunit, LC subunit, and Fc / 2 subunit. By using the reduced antibody, it was revealed that this modification was present in the heavy chain of the antibody. No HexNAc modification was observed in the light chain (Figure 2B). In the analysis of the subunits obtained by IdeS, it was shown that this additional HexNAc was localized in the Fd subunit of adalimumab, suggesting that one or two heavy chains were modified with the additional HexNAc glycosylation event (Figure 2C). Such analysis indicated that the additional HexNAc modification was unexpectedly localized within HC, more precisely within the Fd portion.

[0208] To identify the position of the additional HexNAc modification observed on the Fd subunit of adalimumab, a bottom-up proteomics approach using trypsin digestion and chymotrypsin digestion as well as HCD fragmentation and ETD fragmentation was carried out using state-of-the-art Orbitrap FTMS instrumentation, initially without targeting specific targets and then targeting the most promising peptides additionally. From these results, it was clearly shown that the threonine in the peptide of the hinge region of the heavy chain was HexNAc-modified:

Chemical Structure

[0209] Therefore, it was confirmed that O-glycosylation in adalimumab is located at Thr225 within the hinge region of the antibody.

[0210] (ii) Characterization By subjecting the HexNAc modifications found in adalimumab produced by LimmaTech Biologics' custom glycan platform to special analysis, the entity was determined, more specifically, whether it corresponded to N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), or N-acetylmannosamine (ManNAc). For this purpose, two different approaches were used. First, monosaccharide analysis by high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) was performed, and second, monosaccharide analysis by MSLC-MS / MS analysis was performed.

[0211] (A) Monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) To approach the entity by different methods, adalimumab was first characterized for O-HexNAc by monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). For the identification of the nature of the O-HexNAc found on T225 of the heavy chain of adalimumab, in combination with several purification steps, FabRICATOR (IdeS) and GingisKHAN (Kgp) were used according to the product manuals to generate a 14-amino acid glycopeptide.

[0212] TFA was used for the hydrolysis of HexNAc from the mAb. During this TFA treatment, N-Ac was lost and each amino sugar was generated. During HPAEC, the standards (GlcNAc, GalNAc, and ManNAc) were also treated with TFA, and these standards were well chromatographically separated. In the hydrolyzed adalimumab sample, three peaks were observed at low intensity, and two of these peaks were also present in the buffer control and in the hydrolyzate of commercially available Humira® (which has no O-HexNAc). The third peak (RT 9.767 min) in the adalimumab sample was not present in the control and eluted at a similar RT as the TFA-treated GlcNAc standard, although there was a slight shift in the RT (RT 9.95). When the treated GlcNAc standard was added to the hydrolyzed adalimumab sample, this slight shift was shown to be due to the matrix difference between the hydrolyzed sample and the standard, and thus it was possible to assume that the HexNAc present in adalimumab was GlcNAc (Figs. 3A - 3B).

[0213] (B) The entity of O-HexNAc characterized by MS fragmentation To investigate the nature of O-HexNAc, a targeted approach was applied. In this method, additional MS / MS events of an ion were induced when a characteristic HexNAc oxonium ion at m / z 204.087 was shown in the first MS / MS scan of the ion. It has been previously demonstrated that it is possible to discriminate between GalNAc and GlcNAc by using this pattern of oxonium ions (Halim et al. (2014) J Proteome Res 13(12), pp. 6024-6032). In particular, the ratio of the intensity of the ion found at m / z 138.055 to the intensity of the ion found at m / z 144.066 is very different between GalNAc and GlcNAc.

[0214] First, a specific tryptic digest peptide (THTCPPCPAPELLGGPSVFLFPPKPK) (SEQ ID NO: 227) with a HexNAc modification was identified. The calculated m / z of this peptide is 762.6397 ([M+4H] 4+ )). This peptide (eluting around 29.2 minutes) was found in the adalimumab sample but not in the commercial control (Humira®) sample, confirming that this single HexNAc modification is specific to adalimumab from a custom glycan host cell. As an internal reference, a species found at m / z 1467.0175 ([M+3H] 3+ ) was used. This species eluted around 35.2 minutes in all samples.

[0215] Subsequently, glycopeptides were searched for each analysis by examining the presence of characteristic HexNAc oxonium ions found at m / z 204.087 in high collision dissociation (HCD) MS / MS data. It was also possible to observe tryptic digest glycopeptides containing conserved N-glycosylation sites in the heavy chain.

[0216] Next, the sample was analyzed using a more targeted LC-MS / MS method. In this method, when a characteristic HexNAc oxonium ion at m / z 204.087 was shown in the first MS / MS scan of the ions, additional MS / MS events of the ions were induced. For these additional MS / MS experiments, different collision energies were used (the normalized collision energy (NCE) of HCD was set to 32%, 37%, and 42%), and the number of ions selected for fragmentation was increased.

[0217] In previous experiments using electron transfer dissociation (ETD), it was demonstrated that HexNAc is present on Thr-3 in the tryptic peptide (THTCPPCPAPELLGGPSVFLFPPKPK+HexNAc) (SEQ ID NO: 227) containing HexNAc. When HCD is used for fragmentation, ions that have lost HexNAc (as neutral loss ions) are mainly observed. To examine the identity of HexNAc, the relative intensity of the HexNAc oxonium ion (Table 7) in the low mass region of the MS / MS spectrum was evaluated. [Table 8]

[0218] Figure 4A shows the pattern of the HexNAc oxonium ion for the tryptic peptide (THTCPPCPAPELLGGPSVFLFPPKPK) (SEQ ID NO: 227) (m / z 762.641) containing HexNAc, which was obtained with an NCE of 32%. (The patterns with NCEs of 37% and 42% are not shown.) A similar pattern was also observed in the fragmentation spectrum of the tryptic peptide (SCDKTHTCPPCPAPELLGGPSVFLFPPKPK) (SEQ ID NO: 239) (containing HexNAc) with a cleavage failure number of 1 (data not shown).

[0219] Previously, it has been demonstrated that adalimumab contains a uniform glycan on the conserved N-glycosylation site within the tryptic digest peptide EEQYNSTYR (SEQ ID NO: 228). The fragmentation spectrum of this peptide with a glycan structure composed of GlcNAc2Man3 was also analyzed (m / z 1041.4221 [M+2H] 2+ ). As expected, the pattern of HexNAc oxonium ions in this MS / MS spectrum (the intensity of the ion at m / z 144 is lower compared to the intensity of the ion at m / z 138) is consistent with the pattern of GlcNAc (Figure 4B). Furthermore, the integrated value of the intensities of the ions found at m / z 138.055 and m / z 168.066 and the intensities of the ions found at m / z 126.055 and m / z 144.066 (the above-mentioned ones) was 3.8 in this spectrum.

[0220] As a reference for the peptide with GalNAc (measured using the same method with the same instrument as in the previous experiments), the pattern of HexNAc oxonium ions of a CD43-derived peptide (MoxYTTSITSDPK) (SEQ ID NO: 229) with three GalNAcs is shown in Figure 4C.

[0221] Based on the observation pattern shown in Figure 4A, particularly the lower intensity of the ion found at m / z 144 compared to the intensity of the ion found at m / z 138, it is highly likely that the HexNAc on this peptide in the adalimumab sample is GlcNAc. This is further supported by the numerical value obtained by dividing the integrated intensity values of the ions found at m / z 138.055 and m / z 168.066 by the integrated intensity values of the ions found at m / z 126.055 and m / z 144.066. In the paper by Halim et al. (Halim et al. (2014) J Proteome Res 13(12), pp. 6024 - 6032), it was shown that for peptides with GlcNAc, this value is 2 or more. For the spectrum shown in Figure 4A, this value is 2.6. In contrast, the value found in the spectrum for GalNAc shown in Figure 4C is 0.6, which is consistent with the study by Halim et al.

[0222] In summary, such complementary data clearly identified the presence of O - linked GlcNAc at threonine 225 (EU numbering) within the IgG1 hinge region of the heavy chain of adalimumab produced by the custom glycan host cell.

[0223] 6.2 Example 2 - Bioinformatics screening and analysis of OGNT candidate genes in Leishmania tarentolae In eukaryotes, all types of O - glycosylation are initiated by different polypeptide glycosyltransferases, and most glycans are further elongated / branched in the Golgi by sequential monosaccharide additions by different enzymes and / or common enzymes and undergo capping (Joshi, et al (2018) Cell 172(3), 632 - 632.e2). O - glycans (O - linked sugar chains) in vertebrates are often linked to polypeptides via N - acetylgalactosamine (GalNAc) added to the hydroxyl group of a serine residue or a threonine residue and can extend into different structural core classes.

[0224] A major problem in identifying O-glycosyltransferases (referred to as "OGT" or "OGNT") is that there does not appear to be a unique identifying feature that generally distinguishes OGT from normal glycosyltransferases (referred to as "GT"). It may be possible to rely on homology with known O-glycosyltransferases.

[0225] In humans, OGTs that transfer GalNAc, Man, Fuc, Glc, xylose, and GlcNAc are widely known (Bennett et al. (2012) Glycobiology 22(6), pp. 736-756), but no homologs were found in Leishmania tarentolae.

[0226] Furthermore, several exogenous OGT candidates in KEGG were further evaluated and screened against the L. tarentolae genome. 1) For KEGG ortholog K09667 (protein O-GlcNAc transferase), several matches were obtained from human OGT (Uniprot: O15294) representing this family, but these were considered to be due to accidental coincidence of their secondary structures. The relevant active protein domain is PF13844 (GT family 41 described by PFAM), but no match to this domain was found in L. tarentolae. 2) For K18134 (protein O-GlcNAc transferase), one match (LTAR_110011600) showing the same GT family domain was present in the L. tarentolae genome in human EOGT (Uniprot: Q5NDL2) representing this family, but since there was no reverse hit to the human enzyme, this was not further explored. Human EOGT transfers a single N-acetylglucosamine from UDP-GlcNAc to serine or threonine residues in a protein and specifically glycosylates the Thr residue located between the 5th and 6th conserved cysteines of the folded EGF-like domain. 3) For COLGALT2 (human Gal OGT that transfers beta-galactose to hydroxylysine residues on collagen), two matches (LTAR_130014000, LTAR360049600) were also present. The reverse hit matched back to COLGALT2. However, what they share is the GT25 family domain, and this domain is very broadly defined.

[0227] Importantly, homologs of OGNT-1, OGNT-2, and OGNT-L were identified as candidates. These OGNT-1, OGNT-2, and OGNT-L are characterized Trypanosoma cruzi GlcNAc O-glycosyltransferases (Heise, et al. (2009) Glycobiology 19(8), pp. 918-933). Based on these T. cruzi sequences, homologs were identified and confirmed by reciprocal best hits (phmmer v3.1b; Table 8 and Figure 5A).

Table 9

[0228] Therefore, we focused on closely related Trypanosoma enzymes that are well characterized biochemically for GlcNAc transfer. The biosynthesis of O-glycans in mucins in T. cruzi is initiated by the addition of GlcNAc residues from UDP-GlcNAc (activated sugar donor) to serine or threonine residues in proteins by uridine diphospho-N-acetylglucosamine:polypeptide-α-N-acetylglucosaminyltransferase (ppGlcNAc transferase) (Previato, et al (1998) Journal of Biological Chemistry 273(24), pp. 14982-14988). This reaction represents the main difference between O-glycosylation in T. cruzi and in mammals. In mammals, the orthologous enzyme is UDP-GalNAc transferase, which transfers N-acetylgalactosamine (GalNAc) from UDP-GalNAc to peptides (Hagen (2002) Glycobiology13(1), 1R-16). This difference may be due to the fact that T. cruzi does not have the ability to interconvert UDP-GalNAc and UDP-GlcNAc (Roper, et al (2002) Proc Natl Acad Sci U S A 99(9), pp. 5884-5889). The T. cruzi enzyme, like most glycosyltransferases, is active for requires divalent metal cationic ions, with Mn2+ being the most effective. This is in contrast to the lack of metal ion dependence shown by human cytoplasmic O-GlcNAc transferase. Most strikingly, while the T. cruzi enzyme adds GlcNAc to hydroxylated amino acids via an alpha linkage, the anomeric specificity of the cytoplasmic enzyme is beta (Previato, et al (1998) Journal of Biological Chemistry 273(24), pp.14982-14988). In T. cruzi, the TcOGNT-2 gene encoding ppGlcNAc transferase activity has also been reported. Enzymatic analysis has shown high levels of UDP-GlcNAc transferase activity and UDP-GlcNAc hydrolase activity, with both activities being specific for UDP-GlcNAc (Heise, et al.(2009) Glycobiology 19(8), pp.918-933). Peptide selectivity and optimal pH are equivalent to those of the native GlcNAc transferase activity previously reported in the microsomal fraction of T. cruzi (Previato, et al(1998) Journal of Biological Chemistry 273(24), pp.14982-14988).

[0229] The sequence features of the OGNT homologs identified in the L. tarentolae genome were also evaluated using sequence alignment with human GalNAcT1 (UniProt ID Q10472), but no significant results were obtained for OGNT-1. On the other hand, for the evaluation of OGNT-2 with human GalNAcT1, catalytic residues for Mn2+ coordination and UDP-GalNAc binding appear to be conserved in OGNT-2. Structure prediction was performed using Phyre for the three OGNT candidates, and the hits obtained for OGNT-1 and OGNT-2 include different members of the UDP-GalNAc polypeptide GalNAc transferase family (GalNAc T1, GalNAc T2, GalNAc T4, GalNAc T10, and PGANT9). 223 residues (35% of the full sequence) of OGNT-2 were modeled with 99.0% confidence by a single top-scoring template. No significant hits were identified for OGNT-L.

[0230] The following regions were identified by InterProScan:

[0231] OGNT-2 (638 amino acids, 72.2 kDa): IPR021067; family: GlcNAc (PF11397 aa160 - 316) (GT-A the folding is common doing). Phobius (a combined prediction program for transmembrane (TM) and signal peptide (SP)) ( aa 1 - 28 cytoplasmic, 29 - 50 TM, 51 - 638 non-cytoplasmic).

[0232] OGNT-1 (1136 amino acids, 124.8 kDa): IPR021067; family: GlcNAc (PF11397 aa742 - 1121) (GT-A the folding is common doing). Phobius (1 - 302 non-cytoplasmic, 303 - 325 TM, 326 - 1136 cytoplasmic). OGNT-1 also has a trace connecting to the nucleotide-diphospho-sugar transferase (IPR029044) superfamily.

[0233] OGNT-L (1,136 amino acids, 124.4 kDa): IPR021067; Family: GlcNAc (PF11397) (sharing the GT-A fold). Phobius (1 - 20 cytoplasmic, 21 - 42 TM, 43 - 1,136 non-cytoplasmic).

[0234] Furthermore, the available proteomes of 96 isolates from the TriTryp database (release 44) were analyzed to identify OGNT homologs in other Kinetoplastida. In the search for OGNT variants, T. cruzi OGNT was used as a reference, and hits were tested by reciprocal best hits against the L. tarentolae genome, and significant hits were obtained for 47 Kinetoplastida (date September 2019). For each species, one representative isolate was selected, and a total of 81 sequences were used to construct a phylogenetic tree by multiple sequence alignment using T-coffee and RAxML (Figures 5B and 5C).

[0235] Next, the phenotypic effects or lethality upon deletion of O-glycosylation enzymes were also considered. Interestingly, it was possible to map the fitness costs associated with RNAi in Trypanosoma brucei by using a high-throughput phenotyping method called RNA interference target sequencing (i.e., RIT-seq) (Alsford et al. (2011) Genome research 21(6), pp. 915 - 924). In T. brucei, RNAi against OGNT-2 (Tb927.5.2350) and OGNT-1 (Tb927.2.2400) is associated with loss of fitness in individual samples, which implies that these enzymes are not essential, but the double knockout suggests that it may be potentially harmful to the cell. OGNT-L (Tb927.2.2380) has been reported to be inactive and does not seem to be essential in T. brucei. Therefore, single knockouts and especially heterozygous mutations were expected to give viable mutants in L. tarentolae cells.

[0236] 6.3 Example 3 - Control of O-HexNAc levels on mAbs by target-directed deletion. Methods and compositions of knockout (KO) host cells OGNT candidates in Leishmania tarentolae were deleted by homologous recombination of DNA constructs into the coding sequences of each gene and the adjacent untranslated regions. Since OGNT-L and OGNT-1 are arranged in tandem on chromosome 2 (Figures 5A and 6), it was also possible to target both ORFs simultaneously. For this, an integration construct was synthesized that ultimately replaced the OGNT coding sequence (CDS) and included a selectable marker that could be used for the selection of positive transfectants. Three different versions of the integration construct were used:

[0237] Replacement with a selectable marker adjacent to the 5' and 3' untranslated regions (transcription by endogenous PolII). These 5' and 3' untranslated regions provide the defined splice leader acceptor sequence and polyadenylation sequence to the selectable marker.

[0238] Replacement with a selectable marker not adjacent to the untranslated regions (transcription by endogenous PolII). Such constructs are provided with the splice leader acceptor sequence and polyadenylation sequence from the 3'UTR and 5'UTR of the endogenous OGNT.

[0239] Replacement with an expression cassette for glycan modification modification. This expression cassette is integrated in the reverse direction and comprises a promoter region for PolI transcription and an untranslated region that provides a splice leader addition and polyadenylation sequence to the gene(s) of interest.

[0240] The above genetic integration construct is divided into two DNA fragments within the coding sequence of the selectable marker, and a 200 bp overlap for homologous recombination is left between these two fragments. This was necessary to avoid the occurrence of false positive transfectants due to the expression of the selectable marker from the plasmid (episome).

[0241] DNA fragments for transfection were prepared by digesting the cloning plasmid with restriction enzymes and mixing the fragments in appropriate combinations. Transfection was performed standardly using 4D-Nucleofector (trademark)_Core_X.

[0242] By performing transfection of the DNA fragment for replacing the OGNT-2 gene by homologous recombination, positive transfectants capable of growing in each selectable antibiotic were generated. PCR analysis of the crude DNA preparation revealed that while one allele of OGNT-2 was replaced by the selectable marker construct, the other wild-type allele remained (heterozygous KO, data not shown). Attempts to create homozygous knockout by increasing the selectable marker concentration or by transfecting a second construct with a different selectable marker were unsuccessful. In high-throughput RNAi screening, it has been shown that the homologous gene of T. brucei is not lethal under certain conditions but potentially has a growth defect, so the achievement of knockout of homologous OGNT may have been complicated by the impaired growth of the transfectants.

[0243] Therefore, since the efficiency of double-strand break formation by CRISPR / Cas9 is high and homologous recombination repair is assumed to be the main DNA repair mechanism in Leishmania (Zhang and Matlashewski (2015) MBio 6(4), e00861, Duncan et al (2017) Molecular and Biochemical Parasitology 216, pp.30-38), an attempt was made to achieve homozygous knockout by this mechanism. This mechanism has been used previously in other Leishmania species (Beneke, et al (2017) R.Soc.open sci.4(5), p.170095, Ishemgulova (2018) PloS one 13(2), e0192723, Peng, et al (2015) mBio 6(1), p.409, Fernandez-Prada, et al (2018) International Journal for Parasitology:Drugs and Drug Resistance 8(2), pp.165-173). For this, a 3×flag-hCas9 expression construct containing a nuclear localization signal (Le, et al.(2013) Science 339(6121), pp.819-823) was transfected into St10569 as an episome. Subsequently, the resulting St15392 strain was transfected with a mixture of a guide RNA (gRNA) and a DNA fragment for replacing each OGNT gene by integration of a selectable marker (adjacent to IR). To confirm the absence of the OGNT gene in these strains (St16248 OGNT-2 KO; St16257 OGNT-1 KO; St6249 OGNT-L KO), these strains were subjected to sequencing on an Illumina NextSeq. High genomic coverage was achieved, and therefore the absence of reads matching each OGNT region was ensured, indicating that the OGNT gene was successfully and completely removed in all three strains (Figure 7A).By using PCR analysis instead of sequencing, homozygous knockout of each OGNT gene can also be confirmed (see Example 6 and the representative figure in Fig. 9D).

[0244] To analyze which of these genes would be sufficient to block O-glycosylation of the adalimumab hinge region, individual knockout strains co-expressing adalimumab from the ssu locus were engineered. Furthermore, cell lines were created in which the tandemly arranged OGNT-L gene and OGNT-1 gene were simultaneously replaced by a selectable marker integration construct. For this, homologous recombination sites targeting the 5'-end of OGNT-L and the 3'-end of OGNT-1 were combined with gRNA.

[0245] From the obtained cell lines (St16478 OGNT-2 KO + adalimumab, St16700 OGNT-1 KO + adalimumab, St16702 OGNT-L KO + adalimumab, St16704 OGNT-1+L KO + adalimumab), adalimumab was concentrated by protein A affinity chromatography and subjected to MALDI-based relative O-HexNAc quantification. For the preparation for MALDI-based relative O-HexNAc quantification, adalimumab was subjected to double digestion with FabRICATOR and GingisKHAN. In this double digestion, a 14aa peptide with the sequence THTCPPCPAPELLG (SEQ ID NO: 230) containing the O-glycosylation site of the IgG hinge region is generated.

[0246] The MALDI spectra obtained after double digestion with Fabricator / GingisKHAN were analyzed, the areas of the H+ ions and Na+ ions of the monoisotopic masses were integrated, and the relative abundances of the non-glycosylated and O-glycosylated versions of the same peptide were determined by correlating with the integrated intensities of all identified peptides. MALDI can be considered suitable for the relative quantification of modified and unmodified peptides, but the quality of the crystals formed between spots on the target varies, so the absolute intensities and noise levels obtained can differ between samples. Therefore, samples for which low levels of O-HexNAc are expected due to changes in the signal-to-noise level in some cases positive definitive confirmation become will obtain, and for spots that weak are is certain not recognized there is a possibility . Therefore, for some of the KO strains, the O-HexNAc levels were also determined by targeted glycopeptide analysis or subunit analysis. Table 3 summarizes the results obtained.

[0247] For OGNT-2 KO, it was revealed that 15.8% of the hinge region peptides were still O-HexNAc modified, while it was impossible to detect O-HexNAc modification on the antibodies prepared from the OGNT-1 KO cell line and the OGNT-L KO cell line. On the other hand, in the samples obtained from the OGNT-1+L double KO cell line, 0.3% of the peptides still had O-HexNAc modification. To further evaluate the amount of O-HexNAc modification by a more sensitive method, the purified antibodies of OGNT-1 KO and OGNT-1+L KO were further analyzed by peptide mapping. These measurements revealed that in both strains, the remaining O-HexNAc modified hinge regions of these strains were almost undetectable (about 0.04 - 0.05%).

[0248] Since there was a possibility that the remaining OGNT-2 gene in the OGNT-1+L KO cell line was involved in the remaining modification, a triple KO cell line was generated by transfection of the construct for simultaneous knockout of OGNT-1+L into the previously generated OGNT-2 KO strain (St16478). In the obtained strain St16636, it was confirmed by Illumina NextSeq that all three OGNT genes were knocked out (Figure 7B).

[0249] In Figure 8A, the MALDI spectra obtained from double digestion with FabRICATOR / GingisKHAN are shown for wild-type background and triple KO, respectively. In the wild-type background, a 14aa peptide without modification and the peptide with HexNAc modification were observed, while for the KO strain, only the 14aa peptide without modification was present. Furthermore, confirmatory targeted glycopeptide analysis was performed to confirm the absence of O-HexNAc modification. In Figure 8B, the selected ion chromatograms (SIC) of the peptide THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 227) (+2 * carbamidomethylation) (with or without O-HexNAc) are shown for the triple KO strain. These peptides were subjected to trypsin digestion and measured using a very sensitive ESI-LC-MS method (Orbitrap FTMS system). The SIC of the peptide without HexNAc modification showed a good signal, while it was impossible to confirm the signal for the modified peptide. This supports the hypothesis that when all three OGNT candidate genes are knocked out, the unwanted O-glycosylation on IgG can completely disappear. No other O-HexNAc modifications were found in the peptide data search. For adalimumab expressed in a custom glycan host cell without OGNT knockout, the peptide THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 227) (+2 * For carbamidomethylation, signals were obtained in its SIC for both those with and without the O-HexNAc modification.

[0250] For the generation of all the strains mentioned so far, an integration construct was used that incorporated a selection marker adjacent to an additional untranslated region to correctly and reliably cause expression. Next, it was tested whether it was possible to omit such additional untranslated regions and whether it was possible to express the selection marker under the control of the native OGNT UTR. For this, an integration construct containing only the homologous recombination site for the 5' intergenic region of OGNT-L and the selection marker coding sequence adjacent to the homologous recombination site for the 3' intergenic region of OGNT-1 was transfected into a strain (St16569) that co-expresses 3×flag-hCas9 and adalimumab. It was confirmed by PCR that the OGNT-1 gene and the OGNT-L gene were correctly replaced in the resulting strain (St16770), and the antibody prepared from this strain was analyzed by MALDI. As seen in the OGNT-1+L KO cell line (St16704) generated with the additional intergenic region, it was revealed that the O-HexNAc-modified hinge peptide contained in the strain without such an intergenic region was present in extremely small amounts (0.2 - 0.3%). This supports the fact that it is possible to omit the incorporation of the additional intergenic region for the creation of the OGNT KO cell line.

[0251] In the CRISPR / Cas9-based knockout approach, since homozygous knockout of individual genes or genes arranged in tandem was reproducibly generated, next, gRNAs and homologous recombination constructs targeting the 5' and 3' ends of OGNT-2, as well as the 5' end of OGNT-L and the 3' end of OGNT-1, were included in the transfection of the co-expression strain of adalimumab and 3×flag-hCas9 (St16872, cas9 episome) to attempt to create knockout of all three candidate genes at once. In this example, the same selection marker was used to target both loci, and an integration construct without additional intergenic regions was used. Viable clones were successfully obtained from this transfection, and PCR confirmed that all OGNT genes were replaced by the selection marker in these clones. When the adalimumab purified from this St17127 strain was subjected to MALDI-based analysis, it was confirmed that all three OGNT genes were functionally knocked out because no detectable HexNAc modification was present.

[0252] In summary, the generation of a complete OGNT-deficient cell line in which O-glycans are completely absent in recombinant adalimumab made such host cells ideal for producing recombinant antibodies or antibody formats without unwanted modifications.

Table 10

[0253] 6.4 Example 4 - Control of O-HexNAc Levels on mAb by Overexpression of OGNT To evaluate the effect of overexpression on the three OGNT candidates, DNA fragments were generated that contained each of the 3×HA-tagged OGNT genes and the selection marker "ble" flanked by intergenic regions (aprt 5’UTR, CamIR, dhfr-ts 3’UTR) to support stable expression. The ends of the constructs included homologous recombination sites for the ssu locus, which is known to support high-level protein expression.

[0254] adalimumab band expressing strain ( not only the ssu locus St15449) was transfected with linearized constructs (p5932, p5933, p5934). The resulting strains expressed either OGNT-2_3×HA (St15884), OGNT-1_3×HA (St15885), or OGNT-L_3×HA (St15886) as expected. done This could be confirmed by Western blot. Next, adalimumab was concentrated from these strains by protein A affinity chromatography, and the O-HexNAc levels were quantified by subunit analysis. The O-HexNAc modification levels on adalimumab in the OGNT-L overexpression strain were almost at wild-type levels, but for both the OGNT-1 overexpression strain and the OGNT-2 overexpression strain, the O-glycosylation that occurred on the adalimumab hinge region was reduced. This effect was very prominent in the OGNT-1 overexpression strain, and the amount of O-HexNAc decreased to one-tenth and became 1.9% compared with adalimumab from the wild-type control (Table 4). Although further analysis has not been carried out, since the protein primary sequence contains a THT repeat region, it can be speculated that this effect occurred due to the regulation by auto-glycosylation. Alternatively, another hypothesis can be based on Heise (Heise, et al. (2009) Glycobiology 19(8), pp. 918-933). According to Heise's explanation, OGNT-1 has not only transferase activity but also hydrolysis activity for UDP-GlcNAc. When the peptide concentration decreases (decrease in protein substrate), the degradation of UDP-GlcNAc occurs. When OGNT-1 is overexpressed while the protein substrate concentration remains unchanged, the OGNT-1 activity shifts to the hydrolysis of UDP-GlcNAc, and it can be expected that UDP-GlcNAc in the cell may be depleted. However, these hypotheses require further investigation.

[0255] In summary, the modification of the OGNT variant expression level can be used to adjust and reduce the O-glycosylation level in L. tarentolae.

Table 11

[0256] 6.5 Example 5 - The phenotype of the OGNT deletion strain is not affected - Growth in a bioreactor To analyze whether there is an impact on the phenotypes of the OGNT-deficient strains (St16704 (OGNT-1 / L KO) and St16636 (OGNT-1 / 2 / L KO)), these OGNT-deficient strains were subjected to fed-batch fermentation. Both strains grew stably in the bioreactor and reached is the most OD values of 18 and 20 to at the end of fermentation. This is completely equivalent to the case where strains with wild-type OGNT genotypes (such as St12427) were grown under the same conditions. Furthermore, it was possible to isolate the full-length antibody by protein A concentration from the supernatant after fermentation, and this full-length antibody was subjected to MALDI-based relative O-HexNAc quantification. In both samples, it was impossible to detect O-HexNAc-modified peptides (Table 9). Therefore, the knockout of OGNT depletes O-HexNAc modification regardless of the culture scale and has no effect on growth during fermentation.

[0257] 6.6 Example 6 - There is no phenotypic impact due to OGNT deletion and OGNT overexpression on N-glycan engineering Overexpression

[0258] To determine whether OGNT overexpression affects other strain modifications (such as N-glycan engineering), plasmid pLMTB5933 linearized with SmiI was transfected into a strain (St15882, "Adalimumab G0") expressing adalimumab and glycosyltransferases (sfGnt1, rnMGAT2, and drMGAT1) to integrate the OGNT-1 overexpression construct into the ssu locus (St16292, "Adalimumab G0 OGNT-1 OE"). The resulting strain and the parental strain were cultured and grown in 50 ml shake flasks, and the secreted antibody was concentrated by protein A affinity chromatography. The samples were subjected to N-glycan analysis (RFMS) as previously described, and almost identical N-glycan profiles were shown. Therefore, overexpression of OGNT-1 does not affect the expression and function of glycosyltransferases that modify N-glycans on adalimumab (Figure 9A).

[0259] Knockout

[0260] To evaluate the effect of OGNT deletion on the expression and function of N-glycan modifying enzymes, all three OGNT candidates (1 / 2 / L) were knocked out in a strain with a background expressing adalimumab and glycosyltransferases (sfGntI, rnMGAT2, drMGAT1) by CRISPR / Cas9 mediation. Adalimumab concentrated with protein A was prepared from the obtained strain St17863 ("Adalimumab-G0, OGNT-1 / 2 / L KO") and the parental strain St17607 ("Adalimumab-G0"), and subjected to N-glycan analysis (PC labeling), showing an almost indistinguishable N-glycan profile (Figure 9B). It was impossible to detect O-HexNAc by MALDI-based analysis on the antibody purified from the St17863 strain. Therefore, the absence of putative O-glycosyltransferase does not affect N-glycan glycan modification. The same was observed for the glycan-modified strain expressing sfGntI, drMGAT1, rnMGAT2, and hsB4GalT1 for N-glycan conversion up to G2 in the comparison between St17318 ("Adalimumab-G2, OGNT-1 / 2 / L KO") and the parental strain St15451 ("Adalimumab-G2") (Figure 9C). Therefore, the galactosylation of N-glycan is also not affected by OGNT knockout. In both examples, the absence of the gene encoding OGNT was evaluated by PCR (see the representative example in Figure 9D).

[0261] 6.7 Example 7 - Replacement of OGNT with an expression cassette for glycan modification In the previous examples, the OGNT coding sequence was replaced by a selection marker with the intention of removing the OGNT function. As a result, it became clear that the expression of the selection marker from this chromosomal locus could be induced by utilizing the natural intergenic region. To evaluate whether it is also possible to use the OGNT locus for cell functionalization, i.e., cell functionalization by expressing a glycan modification construct, linear fragments obtained from plasmids (pLMTB8223, pLMTB8381, pLMTB8301, pLMTB8234, pLMTB8629, pLMTB8238, pLMTB8287, pLMTB8383, pLMTB8384, pLMTB8229) were transfected into an adalimumab-expressing strain (St15449). By this multiple-fragment assembly (described in the provisional application entitled "Engineered Leishmania Cells" with the same filing date as this specification), the natural OGNT-1 / L locus on chromosome 2 was targeted for expression constructs for drMGAT1, drMGAT2, gjMGAT1, and agMGAT1. The resulting St17846 strain was subjected to N-glycan analysis based on adalimumab purified with protein A, and a nearly homogeneous G0 glycan was detected (92%) (Figure 10A). Furthermore, the integration of the construct into the OGNT locus was confirmed by PCR covering the selection marker including the 3' end of the introduced construct (Figure 10B), and the relative O-HexNAc content was determined to be 7% by MALDI for the hinge region peptide obtained from adalimumab concentrated with protein A from this strain (see Table 9). In summary, these data support the fact that although one of the two alleles encoding OGNT-1 and OGNT-L was successfully replaced by a functional expression cassette for glycan modification, thereby reducing O-HexNAc modification, the disappearance of O-HexNAc modification was not achieved.

[0262] 6.8 Example 8 - OGNT deletion blocks O-glycosylation on erythropoietin In contrast to the unexpectedly O-glycosylated IgG hinge region, the native O-glycosylation sites of rhEPO were completely modified with O-HexNAc upon expression in wild-type L. tarentolae (see Example 1). To also evaluate whether the modification of this highly occupied native site is controllable by targeted knockout of L. tarentolae OGNT, the Cas9 expression cassette was transfected into the St17785 strain expressing LMSAP-rhEPO-Strep (St17785), and then all three OGNTs were simultaneously knocked out as described in the previous examples (St17917). The St18143 strain was obtained upon single clone selection. This St18143 strain was shown by PCR to be positive for replacement of all OGNT genes by the selection marker neo (OGNT-1 / 2 / L KO).

[0263] Next, LMSAP-rhEPO-Strep was concentrated from the cell culture supernatant of St18143 by ammonium sulfate precipitation and StrepTrap affinity purification and then analyzed by targeted peptide mapping by LC-MS. The unglycosylated form of this peptide containing the native rhEPO O-glycosylation site was readily detected, while no evidence was found for the presence of a mass corresponding to the peptide with O-HexNAc modification (Figure 11), and no evidence was found for the presence of oxonium ions due to O-HexNAc in the expected elution time range (data not shown). This supports that these three enzymes are the true O-glycosyltransferases of L. tarentolae and that these three enzymes are knockout-able. Thus, by utilizing a custom glycan platform, unwanted O-glycosylation on mammalian target proteins can be blocked.

[0264] 6.9 Example 9 - Improved method for performing triple knockout of OGNT via CRISPR / Cas9 The above-described method for knocking out Leishmania OGNT based on CRISPR / Cas9 has been shown to be highly efficient. On the other hand, this procedure requires two sets of transfections to introduce constitutively expressed 3×flag-hCas9 and generate actual knockouts in any given target strain. Since this requires the use of two different selectable markers, the possibility of further modifying the cell line is limited. Additionally, the modification of the host cell's protein composition due to constitutive expression of Cas9 may not be desirable for stable cell lines. For these reasons, we attempted knockout generation in L. tarentolae using ribonucleoprotein complexes (RNPs). The above-described CRISPR guide RNAs targeting OGNT-1, OGNT-2, and OGNT-L were complexed with recombinantly produced Cas9 protein (Alt-R® S.p.HiFi Cas9 nuclease, catalog number 1081061, Integrated DNA Technologies, Inc.) and directly transfected into cells together with the same DNA repair construct used above to CRISPR-mediated replace all three OGNT genes with the same selectable marker without using additional untranslated regions.

[0265] So far, the only report of high knockout efficiency in Leishmania (major) was using SaCas9 / sgRNA RNPs (Soares Medeiros, et al (2017) mBio 8:e01788-17), and there was no report on SpCas9. However, we were able to reproducibly achieve complete knockout of all three OGNT genes in a single transfection using SpCas9 / gRNA RNPs.

[0266] The examples presented herein demonstrate that triple OGNT knockout occurs in the St19462 strain, which already contains two expression constructs encoding glycosyltransferases (drMGAT1(2×), drMGAT2, rnMGAT1, rnMGAT2(3×), hsMGAT1, hsMGAT2, and sfGntI). Thus, the resulting St19855 strain will have the ability to perform N-glycan modification to G0, while natural O-glycosylation will be lost (Figure 12A). The absence of the OGNT gene was revealed by comparing with the wild-type strain (St18344) in a PCR analysis searching for the open reading frames of OGNT-1, OGNT-2, and OGNT-L in the obtained cell line St19855 (Figure 12B).

[0267] Similarly, the cell line St20097 was created by replacing all three OGNTs RNP-mediatedly with a selection marker for geneticin resistance. From the comparison of the N-glycan profiles released from the surface proteins of L. tarentolae of the parental cell line St19462 and the triple OGNT knockout cell line St20097, it was demonstrated that the efficiency of N-glycan conversion to G0 is not affected by the OGNT knockout introduced by the RNP transfection method (Figure 12C). This supported the reproducibility of this method.

[0268] Next, the adalimumab expression construct was transfected into the knockout cell line St19855 (Figure 12A). The antibody purified from the resulting cell line (St19915) was subjected to the above FabRICATOR / GingisKHAN double digestion. From the MALDI spectrum shown in Figure 12D, it was confirmed that there is no O-HexNAc modification on the 14aa peptide.

[0269] To evaluate how far the cell line with OGNT triple KO by RNP transfection can be further manipulated, the knockout cell line St19855 was further modified by transfection with another glycan modification module containing several copies of MGAT1 (drMGAT1, gjMGAT1, agMGAT1) and another copy of MGAT2. The resulting cell line St19931 (Figure 12A) was further transfected with an adalimumab expression construct to obtain cell line St19998 in order to analyze its glycan modification ability. By comparing the profiles of N-glycans released from the antibodies (Fc) produced in cell lines St19915 and St19998, it was clearly demonstrated that further improvement of the starting strain St19855 was achieved by transfection with an additional glycan modification module. The reason for this improvement is that 100% conversion to G0 occurred in St19998, while only 96% of the N-glycans were completely converted in St19915 (Figure 12E). This supported the fact that the resulting cell line was still fully modifiable after the removal of OGNT by CRISPR / Cas9-mediated RNP transfection. Furthermore, these OGNT-deficient O-glycosylation-deficient strains completely retained their growth ability and target protein expression ability.

[0270] To evaluate whether cell lines with alternative N-glycan phenotypes and no O-glycosylation can be established using RNP transfection, in addition to the G0 variants shown previously, the following cell lines were generated: A) St19955 (with triple OGNT replacement with a zeocin resistance-conferring selectable marker in wild-type L. tarentolae St18344) and St20107 (with triple OGNT replacement with a geneticin resistance-conferring selectable marker in wild-type L. tarentolae St18344) (these strains produce Man3 N-glycans homogeneously), and B) St19885 (St19855, a G0 N-glycan-producing strain in which all three OGNTs were replaced with a zeocin resistance marker, transfected with a galactosyltransferase expression cassette) and St20201 (St20097, a G0 N-glycan-producing strain in which all three OGNTs were replaced with a geneticin resistance marker, transfected with a galactosyltransferase expression cassette) (these strains convert their surface N-glycans to G2 homogeneously). Table 11 shows a summary of the resulting strains and their characteristics. This further confirmed the reproducibility of the RNP-mediated OGNT removal procedure and the fact that this method is available at any point in the cell line manipulation process and does not interfere with other modifications. Importantly, the O-glycosylation-deficient N-glycan-modified cell lines completely retained their proliferative ability, viability, and even target protein expression ability.

Table 12

[0271] In summary, the RNP-mediated knockout procedure combined with the introduction of an antibiotic resistance marker is as efficient as using constitutively expressed Cas9. On the other hand, in this knockout procedure, all that is required is the expression of a single selectable marker, and further time is saved by avoiding the second transfection step, and ultimately the retention of the Cas9 expression construct is also avoided, so this method is preferable. Since this method has been shown to be very efficient in removing three OGNT genes at once, it can be a useful tool for knocking out other targets as well.

[0272] 7. Equivalents The scope of the viruses, nucleic acids, methods, host cells, and compositions disclosed herein is not limited by the specific embodiments described herein. Indeed, various modifications of such viruses, nucleic acids, methods, host cells, and compositions will be apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to be included within the scope of the appended claims. This application provides an invention in the following aspects. (Aspect 1) The Leishmania cell genetically engineered such that the formation of O-linked GlcNAc on the polypeptide in the Leishmania cell is reduced or eliminated. (Aspect 2) The Leishmania cell according to Aspect 1, wherein the formation of the O-linked GlcNAc in the Leishmania cell before being genetically engineered is catalyzed by at least one N-acetylglucosamine (GlcNAc) transferase. (Aspect 3) The Leishmania cell according to Aspect 2, wherein the gene encoding the at least one GlcNAc transferase is functionally inactivated. (Aspect 4) The Leishmania cell according to any one of Aspects 2 to 3, wherein the gene encoding the at least one GlcNAc transferase is downregulated. (Aspect 5) The Leishmania cell according to any one of Aspects 2 to 3, wherein the gene encoding the at least one GlcNAc transferase is deleted or mutated. (Aspect 6) The Leishmania cell according to Aspect 2, wherein the gene encoding the at least one GlcNAc transferase is overexpressed. (Aspect 7) The Leishmania cell according to any one of aspects 1 to 6, wherein the formation of the O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the formation of the O-linked GlcNAc in the reference Leishmania cell. (Aspect 8) The Leishmania cell according to any one of aspects 2 to 7, wherein the at least one GlcNAc transferase is selected from the group consisting of OGNT1, OGNT2, and OGNTL, and their homologous GlcNAc transferases. (Aspect 9) The Leishmania cell according to aspect 8, wherein the at least one GlcNAc transferase is a GlcNAc transferase homologous to OGNT1, OGNT2, and / or OGNTL. (Aspect 10) The Leishmania cell according to any one of aspects 2 to 9, wherein the number of the at least one GlcNAc transferase is one, two, or three. (Aspect 11) The Leishmania cell according to any one of aspects 1 to 10, wherein the growth rate of the Leishmania cell is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of the reference Leishmania cell. (Aspect 12) The Leishmania cell according to any one of aspects 1 to 11, wherein the Leishmania cell is Leishmania tarentolae. (Aspect 13) The Leishmania cell according to any one of aspects 1 to 12, wherein the polypeptide is selected from the group consisting of adalimumab, rituximab, and erythropoietin (EPO). (Aspect 14) The Leishmania cell according to any one of aspects 1 to 13, wherein the Leishmania cell contains a recombinant nucleic acid encoding a heterologous glycosyltransferase. (Aspect 15) The Leishmania cell according to aspect 14, wherein the Leishmania cell contains one or more heterologous glycosyltransferases. (Aspect 16) The Leishmania cell according to any one of aspects 14 and 15, wherein the heterologous glycosyltransferase is N-acetylglucosaminyltransferase and / or heterologous galactosyltransferase and / or heterologous sialyltransferase. (Aspect 17) A method for preparing a polypeptide, the method comprising: (a) culturing the Leishmania cell according to any one of aspects 1 to 16 under conditions suitable for polypeptide production; and (b) isolating the polypeptide. (Aspect 18) A polypeptide produced by the method according to aspect 17.

[0273] In this specification, various publications, patents, and patent applications are cited, and the disclosures of such are incorporated by reference in their entirety.

Table 13

Claims

**Claim 1** The Leishmania cell genetically engineered such that the formation of O-linked GlcNAc on polypeptides in the Leishmania cell is reduced or eliminated, wherein the formation of O-linked GlcNAc in the Leishmania cell prior to genetic engineering is catalyzed by at least one N-acetylglucosamine (GlcNAc) transferase selected from the group consisting of OGT1, OGT2, and OGTL, and their homologous GlcNAc transferases, and the gene encoding the at least one GlcNAc transferase is downregulated, functionally inactivated, deleted, mutagenized, or overexpressed, said Leishmania cell. **Claim 2** The Leishmania cell according to claim 1, wherein the gene encoding the at least one GlcNAc transferase is downregulated, functionally inactivated, deleted, or mutagenized. **Claim 3** The Leishmania cell according to claim 1 or 2, wherein the gene encoding the at least one GlcNAc transferase is functionally inactivated. **Claim 4** The Leishmania cell according to claim 1 or 2, wherein the gene encoding the at least one GlcNAc transferase is downregulated. **Claim 5** The Leishmania cell according to claim 1 or 2, wherein the gene encoding the at least one GlcNAc transferase is deleted or mutagenized. **Claim 6** The Leishmania cell according to claim 1, wherein the gene encoding the at least one GlcNAc transferase is overexpressed. **Claim 7** The Leishmania cell according to any one of claims 1 to 6, wherein the formation of the O-linked GlcNAc is reduced by at least 5%, 7%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the formation of the O-linked GlcNAc in wild-type Leishmania cells. **Claim 8** The number of said at least one GlcNAc transferase is one, two, or three, the Leishmania cell according to any one of claims 1 to 7.

9. The growth rate of said Leishmania cell is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the growth rate of wild-type Leishmania cells, the Leishmania cell according to any one of claims 1 to 8.

10. The Leishmania cell is Leishmania tarentolae, the Leishmania cell according to any one of claims 1 to 9.

11. The polypeptide is selected from the group consisting of adalimumab, rituximab, and erythropoietin (EPO), the Leishmania cell according to any one of claims 1 to 10.

12. The Leishmania cell contains a recombinant nucleic acid encoding a heterologous glycosyltransferase, the Leishmania cell according to any one of claims 1 to 11.

13. The Leishmania cell contains one or more heterologous glycosyltransferases, the Leishmania cell according to claim 12.

14. The heterologous glycosyltransferase is N-acetylglucosamine transferase, and / or heterologous galactosyltransferase, and / or heterologous sialyltransferase, the Leishmania cell according to claim 13.

15. A method for preparing a polypeptide, comprising: (a) culturing the Leishmania cell according to any one of claims 1 to 14 under conditions suitable for polypeptide production, and (b) isolating said polypeptide, said method.

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