CN117242175A

CN117242175A - Hypersialylated cells

Info

Publication number: CN117242175A
Application number: CN202280030870.6A
Authority: CN
Inventors: D·布里斯; S·弗雷斯; M·卡穆勒; H·劳克斯; B·C·拉恩; B·沃尔夫
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2021-04-29
Filing date: 2022-04-27
Publication date: 2023-12-15

Abstract

The present invention relates to mammalian cells having increased sialylation activity. Transfection of mammalian cells with coding sequences for sialyltransferases, galactosyltransferases and sialic acid transporters results in hypersialylation of recombinantly expressed glycoproteins.

Description

Hypersialylated cells

Technical Field

The present invention relates to the field of recombinant protein production. Host cells having increased sialylation activity are provided. In particular, the introduction of sialyltransferase genes, galactosyltransferase genes and sialic acid transporter genes into host cells results in hypersialylation of recombinantly expressed glycoproteins. Thus, a protein having a very large amount of sialic acid is produced.

Background

Chinese Hamster Ovary (CHO) cell lines are the most widely used mammalian cell lines for the production of therapeutic proteins and exhibit high productivity in the gram/liter range of antibodies and other therapeutic protein formats. In particular, the expression of recombinant non-antibody therapeutic proteins is of increasing importance.

The hypersialylation of therapeutic proteins has been described in the literature as leading to an extended drug half-life. Hyposialylation (proximal galactose exposure) may result in faster clearance of the protein through an asialoglycoprotein receptor-mediated pathway (bark et al (2009) Journal of Pharmaceutical Sciences [ journal of pharmaceutical science ] 98:3499-3508). Several examples illustrate how hypersalivation improves the overall therapeutic efficacy of important biopharmaceutical proteins (Morell et al (1971) JBC J. Biochem. 246 (5): 1461-7; richards et al (2010) Mol Endocrinol. Mol Endocrinology ]24 (1): 229-39; datta-Mannan et al (2015) Drug Metab Dispos [ drug metabolism and treatment ] 43:1882-90). These include, for example, asparaginase, leptin, luteinizing hormone and cholinesterase. In addition, hypersalivation can lead to reduced immunogenicity of non-human therapeutic proteins by shielding antigenic sites.

Antibodies are an example of therapeutic proteins whose activity is affected by the degree of sialylation. The Fc fragment of an antibody has two conserved N-glycosylation sites at asparagine 297 of the CH2 domain of each heavy chain. Monoclonal antibodies (mabs) produced in mammalian cells have various glycoforms because the attached glycans are modified to varying degrees by core fucosylation, bisecting N-acetylglucosamine addition, galactosylation and sialylation. Glycan composition is important because the presence or absence of a single monosaccharide residue can significantly affect the affinity of a Mab for different fcγ receptors.

Among the various monosaccharides present on Fc glycans, terminal sialic acids are of particular interest. Sialylation of Fc glycans significantly reduces the affinity of mabs for classical Fc receptors, thus inhibiting Antibody Dependent Cellular Cytotoxicity (ADCC), a biological mechanism critical to the efficacy of several anti-cancer antibodies. Furthermore, recent studies of the anti-inflammatory properties of intravenous immunoglobulins (IVIg) have shown that this biological activity may be conferred by the presence of alpha 2, 6-sialic acid residues on Fc glycans (Kaneko et al (2006) Science [ Science ]313:670-3; anthony et al (2008) Science [ Science ] 320:373-6). Thus, enhanced anti-inflammatory activity can be achieved by generating recombinant IgG therapeutics containing alpha 2, 6-linked sialic acid. Unfortunately, CHO cells lack the enzymes responsible for attaching sialic acid in the α2, 6-conformation and only produce glycoproteins with α2, 3-linked sialic acid, and the percentage of these glycoproteins is low.

In view of the above, there is a need in the art to provide host cells, especially CHO cells, with improved α2,6 sialylation activity.

Disclosure of Invention

The inventors found that overexpression of sialyltransferase, galactosyltransferase and sialic acid transporter together in host cells significantly increased the sialylation activity of the cells. Particularly, the use of alpha 2, 6-sialyltransferase, beta 1, 4-galactosyltransferase and CMP-sialic acid transporter in CHO cells produced glycoproteins with significantly increased amounts of alpha 2, 6-linked sialic acid. The introduction of genes for these enzymes via stable transfection with a vector comprising all three coding sequences provides a stable host cell line. Particularly good results were obtained with vectors having an alpha 2, 6-sialyltransferase under the control of a strong promoter, while beta 1, 4-galactosyltransferase and CMP-sialic acid transporter are controlled by a medium strength promoter.

As the inventors show, the proteins produced in such host cells have a significantly increased amount of sialylation, in particular α2, 6-linked sialylation. The inventors can further demonstrate that antibodies with correspondingly high levels of sialylation have reduced immunogenicity. The hypersalivation of antibodies in particular reduces recognition, uptake and presentation by dendritic cells and reduces T cell activation. This translates into reduced formation of anti-drug antibodies, as fewer T helper cells and thus fewer B cells are activated. Thus, increased sialylation of a therapeutic antibody may reduce adverse side effects, particularly those caused by the patient's immune response against the therapeutic antibody.

In view of the above, in a first aspect, the present invention relates to a mammalian cell engineered for increased expression of α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter. In certain embodiments, the mammalian cell comprises

(i) Exogenous nucleic acid encoding an alpha-2, 6-sialyltransferase;

(ii) Exogenous nucleic acid encoding a beta-1, 4-galactosyltransferase; and

(iii) Exogenous nucleic acid encoding a CMP-sialic acid transporter.

In further embodiments, the endogenous genes encoding α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter of the mammalian cell are engineered for increased expression.

In a second aspect, the present invention provides a method for producing a glycosylated polypeptide, the method comprising the steps of:

(a) Providing a mammalian cell according to the first aspect of the invention, the mammalian cell further comprising an expression cassette for recombinant expression of the glycosylated polypeptide;

(b) Culturing the mammalian cell in a cell culture under conditions that allow expression of the glycosylated polypeptide;

(c) Obtaining the glycosylated polypeptide from the cell culture; and

(d) Optionally processing the glycosylated polypeptide.

In certain embodiments of the second aspect, the culture conditions during culturing the mammalian cells do not include a temperature change.

In further embodiments, the method is used to produce an antibody or fragment, derivative or graft thereof, particularly an antibody or fragment, derivative or graft thereof (engraft) having reduced immunogenicity.

In a third aspect, the present invention provides a vector nucleic acid or a combination of at least two vector nucleic acids comprising

(i) A coding sequence for an alpha-2, 6-sialyltransferase;

(ii) A coding sequence for a beta-1, 4-galactosyltransferase; and

(iii) Coding sequence for a CMP-sialic acid transporter.

In a fourth aspect, the present invention provides the use of a vector nucleic acid according to the third aspect of the invention or a combination of at least two vector nucleic acids for transfecting a mammalian cell. The present invention also provides a method for increasing the expression of an alpha-2, 6-sialyltransferase, a beta-1, 4-galactosyltransferase and a CMP-sialic acid transporter in a mammalian cell, the method comprising the step of transfecting the mammalian cell with a vector nucleic acid according to the third aspect of the invention or a combination of at least two vector nucleic acids, and/or the step of engineering endogenous genes encoding the alpha-2, 6-sialyltransferase, the beta-1, 4-galactosyltransferase and the CMP-sialic acid transporter of the mammalian cell for increased expression.

In a fifth aspect, the application provides a method for reducing the immunogenicity of an antibody or fragment, derivative or graft thereof by increasing the amount of sialylation in its glycosylation pattern. In particular, the antibody or fragment, derivative or implant thereof is a therapeutic antibody or fragment, derivative or implant thereof.

Other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples indicating preferred embodiments of the present application are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed application will become apparent to those skilled in the art from a reading of the following.

Definition of the definition

As used herein, the following expressions are generally intended to preferably have the meanings as set forth below, unless otherwise indicated in the context in which they are used.

In addition to its literal meaning, the expression "comprising" as used herein includes and refers specifically to the expressions "consisting essentially of … …" and "consisting of … …". Thus, the expression "comprising" refers to embodiments in which the subject matter "comprising" a specifically listed element does not comprise a further element, and embodiments in which the subject matter "comprising" a specifically listed element may and/or does comprise a further element. Likewise, the expression "having" is to be understood as the expression "comprising", also including and referring in particular to the expressions "consisting essentially of … …" and "consisting of … …". The term "consisting essentially of … …" particularly refers to embodiments in which a subject matter comprises 20% or less, particularly 15% or less, 10% or less, or particularly 5% or less, of additional elements in addition to the specifically listed elements of which the subject matter consists essentially of.

The term "nucleic acid" includes single-and double-stranded nucleic acids, ribonucleic acids, and deoxyribonucleic acids. It may comprise naturally occurring as well as synthetic nucleotides, and may be modified naturally or synthetically (e.g., by methylation, 5 '-and/or 3' -end capping). In particular embodiments, the nucleic acid is double-stranded deoxyribonucleic acid.

The term "expression cassette" particularly refers to a nucleic acid construct capable of initiating and regulating the expression of a coding nucleic acid sequence introduced therein. The expression cassette may comprise promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation. The exact structure of the expression cassette may vary depending on the species or cell type, but typically comprises 5' -untranslated and 5' -and 3' -untranslated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, end-capping sequences, CAAT sequences, and the like. More particularly, the 5' -untranslated expression control sequence comprises a promoter region comprising a promoter sequence for transcriptional control of an operably linked nucleic acid. The expression cassette may also comprise an enhancer sequence or an upstream activator sequence.

According to the invention, the term "promoter" refers to a nucleic acid sequence located upstream (5') of the nucleic acid sequence to be expressed and controls the expression of the sequence by providing recognition and binding sites for RNA-polymerase. A "promoter" may comprise additional recognition and binding sites for additional factors involved in the regulation of gene transcription. Promoters may control transcription of prokaryotic or eukaryotic genes. Furthermore, a promoter may be "inducible", i.e., initiate transcription in response to an inducer, or "constitutive" if transcription is not under the control of an inducer. If no inducer is present, the gene under the control of the inducible promoter is not expressed or is expressed only to a small extent. In the presence of an inducer, the gene is turned on or transcription level is increased. This is typically mediated by the binding of specific transcription factors.

Herein, the term "vector" is used in its most general sense and comprises any intermediate vector of nucleic acids capable of introducing said nucleic acids, for example, into prokaryotic and/or eukaryotic cells and, where appropriate, integrating into the genome. Such vectors are preferably replicated and/or expressed in cells. The vector comprises a plasmid, phagemid, phage or viral genome. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, typically a circular DNA duplex, that can replicate independent of chromosomal DNA. The support according to the invention may be present in circular or linearized form.

The terms "5'" and "3'" are used conventionally to describe nucleic acid sequence features associated with the position of a genetic element and/or the direction of the event (5 'to 3'), such as, for example, transcription by RNA polymerase or translation by ribosome in the 5 'to 3' direction. Synonyms are upstream (5 ') and downstream (3'). Conventionally, DNA sequences, genetic maps, vector cards, and RNA sequences are drawn from left to right in a 5' to 3' direction, or 5' to 3' direction is indicated by an arrow, wherein the arrow points in the 3' direction. Thus, when this convention is followed, 5 '(upstream) indicates a genetic element located toward the left-hand side, and 3' (downstream) indicates a genetic element located toward the right-hand side.

"polypeptide" refers to a molecule comprising a polymer of amino acids linked together by one or more peptide bonds. Polypeptides include polypeptides of any length, including proteins (e.g., having more than 50 amino acids) and peptides (e.g., having 2-49 amino acids). Polypeptides include proteins and/or peptides having any activity or biological activity. The polypeptides may be pharmaceutically or therapeutically active compounds, or research tools for assays and the like. Suitable examples are summarized below.

A target amino acid sequence is "derived from" or "corresponds to" a reference amino acid sequence if it has at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity over its entire length to the reference amino acid sequence. In particular embodiments, the target amino acid sequence "derived from" or "corresponding to" the reference amino acid sequence is 100% homologous, or in particular 100% identical, to the reference amino acid sequence over its entire length. Similarly, a target nucleotide sequence is "derived from" or "corresponds to" a reference nucleotide sequence if the target nucleotide sequence has at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% identity over its entire length to the reference nucleotide sequence. In particular embodiments, a target nucleotide sequence "derived" or "corresponding" to a reference nucleotide sequence has 100% identity to the reference amino acid sequence over its entire length. Preferably the "homology" or "identity" of an amino acid sequence or nucleotide sequence is determined over the entire length of a reference sequence according to the invention.

The term "antibody" particularlyRefers to a protein comprising at least two heavy chains and two light chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V _H ) And a heavy chain constant region (C) _H ) The composition is formed. Each light chain consists of a light chain variable region (V _L ) And a light chain constant region (C _L ) The composition is formed. The heavy chain constant region comprises three or (in the case of IgM or IgE type antibodies) four heavy chain constant domains (C _H1 、C _H2 、C _H3 And C _H4 ) Wherein the first constant domain C _H1 Adjacent to the variable region and can be linked to a second constant domain C by a hinge region _H2 And (5) connection. The light chain constant region consists of only one constant domain. The variable regions can be further subdivided into regions of higher variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs), wherein each variable region comprises three CDRs and four FRs. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The heavy chain constant region may be of any type, such as gamma, delta, alpha, mu or epsilon heavy chains. Preferably, the heavy chain of the antibody is a gamma chain. Furthermore, the light chain constant region may be of any type, such as kappa or lambda type light chains. Preferably, the light chain of the antibody is a kappa chain. The terms "gamma (delta, alpha, mu or epsilon) type heavy chain" and "kappa (lambda) type light chain" refer to an antibody heavy chain or an antibody light chain, respectively, having a constant region amino acid sequence derived from a naturally occurring heavy chain or light chain constant region amino acid sequence, particularly a human heavy chain or light chain constant region amino acid sequence. The antibody may be, for example, a humanized, human or chimeric antibody.

As used herein, the term "antibody" also includes fragments, derivatives and grafts of the antibody. A "fragment or derivative" of an antibody is in particular a protein or glycoprotein derived from said antibody and capable of binding to the same antigen as the antibody, in particular to the same epitope as the antibody. In further embodiments, a "fragment, derivative or graft" of an antibody refers in particular to a polypeptide or protein that comprises one or more Fc regions of the antibody and may or may not comprise an antigen binding region. Thus, a fragment, derivative or graft of an antibody herein generally refers to a functional fragment, derivative or graft, wherein the function of the antibody is to bind antigen and/or interact with Fc receptors. An "graft" of an antibody refers in particular to said antibody in which a heterologous polypeptide is introduced therein or (partially) replaces the CDR sequences of the antibody. Exemplary antibody grafts are described in US 2017/0158747 A1. In particular embodiments, the antibody or fragment, derivative or graft thereof comprises a CH2 domain having an N-glycosylation site comprising an asparagine residue at amino acid position 297 of the heavy chain of the antibody according to Kabat numbering.

The term "glycosylated polypeptide" refers to a polypeptide that carries a carbohydrate chain attached to its polypeptide backbone. Carbohydrate chains are attached to polypeptides, in particular by a cellular glycosylation mechanism. The carbohydrate chain is attached in particular to the glycosylation site of the polypeptide. The term "glycosylation site" particularly refers to an amino acid sequence that can be specifically recognized and glycosylated by a natural glycosylase (particularly a glycosyltransferase, preferably a naturally occurring mammalian glycosyltransferase). Glycosylated polypeptides are in particular polypeptides which carry N-and/or O-glycosylation. N-glycosylation refers to attachment of a carbohydrate chain to an asparagine residue at an N-glycosylation site having the amino acid sequence Asn-Xaa-Ser/Thr/Cys, where Xaa is any amino acid residue. Preferably Xaa is not Pro. O-glycosylation refers to attachment of the carbohydrate chain to serine, tyrosine, hydroxy-lysine or hydroxy-proline residues. In particular embodiments, the term "glycosylated polypeptide" refers to a polypeptide that carries a carbohydrate chain attached to an N-glycosylation site.

In certain embodiments, the term "polypeptide" as used herein refers to a population of polypeptides of the same species. In particular, all polypeptides of a population of polypeptides exhibit the characteristics used to define the polypeptides. In certain embodiments, all polypeptides in the population of polypeptides have the same amino acid sequence. Reference to a specific class of polypeptides, such as antibodies, particularly refers to a population of such antibodies.

The term "sialic acid" particularly refers to any N-or O-substituted derivative of neuraminic acid. It may refer to 5-N-acetylneuraminic acid and 5-N-glycolylneuraminic acid, but preferably refers to only 5-N-acetylneuraminic acid. Sialic acid, in particular 5-N-acetylneuraminic acid, is preferably attached to the carbohydrate chain via an alpha 2, 3-bond or an alpha 2, 6-bond.

The term "N-glycosylation" refers to the attachment of all glycans to asparagine residues of the polypeptide chain of a protein. These asparagine residues are typically part of an N-glycosylation site having the amino acid sequence Asn-Xaa-Ser/Thr, where Xaa can be any amino acid other than proline. Likewise, an "N-glycan" is a glycan attached to an asparagine residue of a polypeptide chain. The terms "glycan", "glycan structure", "carbohydrate chain" and "carbohydrate structure" are generally used synonymously herein. N-saccharides typically have a common core structure consisting of two N-acetylglucosamine (GlcNAc) residues and three mannose residues, which is of the structure Manα1,6- (Manα1, 3-) Manβ1,4-GlcNAcβ1-Asn, where Asn is an asparagine residue of the polypeptide chain. N-glycans are classified into three different types, i.e., complex glycans, hybrid glycans, and high mannose glycans.

According to the invention, the "sialylation amount" of a polypeptide refers to the amount of glycans comprising at least one sialic acid residue and attached to polypeptide molecules in the population of polypeptides. Consider the number of all glycans carrying sialic acid residues and attached to the polypeptide of interest in the composition. In particular embodiments, the sialylation amount refers to the relative amount of sialylation. The relative amount of sialylation refers to the percentage or range of percentages of glycans attached to polypeptide molecules that are sialylated in the polypeptide population based on the total number of all glycans attached to polypeptide molecules in the polypeptide population.

The cells mentioned herein are in particular host cells. According to the present invention, the term "host cell" relates to any cell that can be transformed or transfected with an exogenous nucleic acid. Mammalian cells, such as human, mouse, hamster, pig, goat, or primate cells, are particularly preferred. Cells can be derived from a variety of tissue types and include primary cells and cell lines. The nucleic acid may be present in the host cell in a single copy or in two or more copies and in one embodiment expressed in the host cell. In certain embodiments, the term "cell" as used herein refers to a population of cells of the same species. In particular, all cells of a cell population exhibit characteristics that are used to define the cell, e.g., they are engineered to increase the expression of a certain gene and/or they produce a polypeptide of interest.

As used herein, "engineered" cells refer to cells that are intentionally altered to obtain different characteristics. Engineering of a cell specifically results in altered expression of one or more genes in the cell. In particular, the genome of the cell is altered, for example by introducing additional genetic information into the cell as an additional plasmid or as part of a chromosome already present in the cell, and/or by deleting part of the genetic information in the cell. In addition, altered expression of genes can also be achieved by controlling transcription and/or translation of genes, for example by altering chromosomal structure, DNA methylation, codon usage, promoters, transcriptional activators and/or repressors, or by introducing interfering nucleic acids such as siRNA. In particular embodiments, engineering refers to genetic engineering.

The term "pharmaceutical composition" or "pharmaceutical formulation" particularly refers to a composition suitable for administration to a human or animal, i.e. a composition containing pharmaceutically acceptable components. Preferably, the pharmaceutical composition comprises the active compound or a salt or prodrug thereof, and a carrier, diluent or pharmaceutical excipient, such as buffers, preservatives and tonicity adjusting agents.

The numbers given herein are preferably understood to be approximate numbers. In particular, the number may preferably be up to 10% high and/or low, in particular up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% high and/or low.

The numerical ranges described herein include numbers defining the ranges. The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. According to one embodiment, a subject matter described herein as comprising certain steps in the case of a method or as comprising certain ingredients in the case of a composition refers to a subject matter consisting of the respective steps or ingredients. The preferred aspects and embodiments described herein are preferably selected and combined, and the specific subject matter resulting from the corresponding combination of the preferred embodiments also falls within the present disclosure.

Detailed Description

The present invention is based on the development of host cells, in particular CHO cells, with high sialylation activity for the production of proteins. The inventors demonstrate that transfection of a host cell with expression cassettes encoding sialyltransferases, galactosyltransferases and sialic acid transporters results in the host cell producing proteins with high amounts of sialic acid. Proteins with a large amount of sialylation generally have a longer circulation half-life. Thus, these host cells are particularly advantageous for the production of therapeutic proteins. In addition to this effect, antibodies with increased sialylation have also proved to be less immunogenic because of their recognition and uptake by dendritic cells and their reduced ability to induce T cell responses.

In view of these findings, in a first aspect, the present invention provides a mammalian cell engineered for increased expression of α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter.

In certain embodiments, the mammalian cell comprises

(i) Exogenous nucleic acid encoding an alpha-2, 6-sialyltransferase;

(ii) Exogenous nucleic acid encoding a beta-1, 4-galactosyltransferase; and

(iii) Exogenous nucleic acid encoding a CMP-sialic acid transporter.

The α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter are generally referred to herein as "glycosylases," which term also refers to each enzyme individually. Furthermore, as used herein, the term "exogenous nucleic acid" refers to the nucleic acids under (i), (ii) and (iii) all together, and also to each of these nucleic acids individually, or to a combination of two of these nucleic acids.

Mammalian cells engineered for increased expression of α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter have in particular higher expression of α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter than the same cells not engineered for said increased expression. Thus, the present invention provides a mammalian cell engineered for increased expression of α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter compared to the same cell not engineered for said increased expression.

In particular, increased expression of the glycosylase results in increased sialylation activity of the mammalian cell. The mammalian cells have higher sialylation activity than the parent cells that have not been genetically engineered as described herein. The mammalian cells are particularly capable of producing proteins with higher sialic acid amounts than the parent cells that have not been genetically engineered. In this respect, "parent cell" particularly refers to a mammalian cell according to the invention before it is engineered. It refers in particular to the same cells as mammalian cells according to the invention, which cells have not been engineered as described herein. The amounts of sialic acid are compared in particular between the same proteins produced under the same conditions. Increased expression of the glycosylase also includes embodiments in which the corresponding glycosylase is expressed in mammalian cells, but not in parent cells.

Hypersialylated host cells

The mammalian cell may be any cell type, and in particular is a cell that can be used to recombinantly produce a protein. The mammalian cells may in particular be rodent cells or human cells. In certain embodiments, the mammalian cells are selected from, but are not limited to, the group consisting of cells derived from: mice such as COP, L, C127, sp2/0, NS-1, at20, and NIH3T3; rats, such as PC12, PC12h, GH3, mtT, YB2/0 and Y0; hamsters such as BHK, CHO and DHFR gene deficient CHO; monkeys such as COS1, COS3, COS7, CV1, and Vero; and humans such as Hela, HEK-293, CAP, retina-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells and HepG2. In a particular embodiment, the mammalian cell is a Chinese Hamster Ovary (CHO) cell. Mammalian cells may be suitable for suspension culture and/or adherent culture, and may be particularly useful for suspension culture.

Mammalian cells are engineered for increased expression of alpha-2, 6-sialyltransferase, beta-1, 4-galactosyltransferase and CMP-sialic acid transporter. This engineering may increase expression of the glycosylase by introducing an exogenous nucleic acid encoding the glycosylase or by upregulating an endogenous nucleic acid encoding the glycosylase. A combination of both options is also possible. For example, exogenous nucleic acid may be introduced into a cell for some glycosylases and up-regulated for other glycosylases. Furthermore, for some or all of the glycosylases, both options may be used simultaneously.

In particular embodiments, the mammalian cell comprises

(i) Exogenous nucleic acid encoding an alpha-2, 6-sialyltransferase;

(ii) Exogenous nucleic acid encoding a beta-1, 4-galactosyltransferase; and

(iii) Exogenous nucleic acid encoding a CMP-sialic acid transporter.

The exogenous nucleic acid is introduced into the mammalian cell in an artificial manner. In particular, they are introduced by transfection. In this regard, transfection may be transient or stable, particularly with stable transfection. Thus, in certain embodiments, the mammalian cell comprises an exogenous nucleic acid stably integrated into its genome.

In certain embodiments, the mammalian cells comprise one or more exogenous expression cassettes comprising exogenous nucleic acid encoding a glycosylase. In some embodiments, each glycosylase is expressed by a separate expression cassette. In other embodiments, two glycosylases are expressed from the same expression cassette, while a third glycosylase is expressed from a separate expression cassette. For example, an α -2, 6-sialyltransferase is expressed by a first expression cassette, and both a β -1, 4-galactosyltransferase and a CMP-sialic acid transporter are expressed by a second expression cassette. In still further embodiments, all three glycosylases are expressed from the same expression cassette. In embodiments in which two or more glycosylases are expressed from the same expression cassette, the expression cassette may further comprise an Internal Ribosome Entry Site (IRES) or coding sequence of a 2A element between coding sequences of different glycosylases. The 2A element is a polypeptide segment fused directly to the polypeptides of the preceding and following glycosylases. The coding sequences are expressed in a single open reading frame, and "self-cleavage" occurs in a co-translational manner.

Each exogenous expression cassette comprises in particular a promoter operably linked to the coding sequence of one glycosylase or a promoter operably linked to the coding sequences of two or three glycosylases. In certain embodiments, the mammalian cell comprises

(i) A first exogenous expression cassette comprising a first promoter operably linked to a coding sequence for an α -2, 6-sialyltransferase;

(ii) A second exogenous expression cassette comprising a second promoter operably linked to a coding sequence for a β -1, 4-galactosyltransferase; and

(iii) A third exogenous expression cassette comprising a third promoter operably linked to a coding sequence for a CMP-sialic acid transporter.

In other embodiments, the mammalian cell comprises

(i) A first exogenous expression cassette comprising a first promoter operably linked to a coding sequence for an α -2, 6-sialyltransferase; and

(ii) A second exogenous expression cassette comprising a second promoter operably linked to a coding sequence for a β -1, 4-galactosyltransferase and a coding sequence for a CMP-sialic acid transporter.

In these embodiments, the second exogenous expression cassette comprises in particular an IRES between the coding sequence of β -1, 4-galactosyltransferase and the coding sequence of CMP-sialic acid transporter.

These expression cassettes typically further comprise mRNA processing and translation signals (generally including Kozak sequences) and mRNA polyadenylation signals. The elements of the expression cassette necessary to enable expression of the coding sequence are known to those skilled in the art. The elements of the expression cassette are specifically selected for expression in mammalian cells.

The promoter used in the expression cassette may be any promoter suitable for driving expression in a mammalian host cell. The promoter may for example be selected from the group consisting of: cytomegalovirus (CMV) promoter, simian virus 40 (SV 40) promoter, ubiquitin C (UBC) promoter, elongation factor 1A (EF 1A) promoter, phosphoglycerate kinase (PGK) promoter, rous Sarcoma Virus (RSV) promoter, choad 3 promoter, murine rosa 26 promoter, pCEFL promoter and optionally a beta-actin promoter (CAGG) coupled to a CMV early enhancer. Specific examples of promoters include the cytomegalovirus early transient promoter, the simian virus 40 early promoter, the human ubiquitin C promoter, the human elongation factor 1 alpha promoter, the mouse phosphoglycerate kinase 1 promoter, the rous sarcoma virus long terminal repeat promoter, and the chicken beta-actin promoter coupled to a CMV early enhancer.

In certain embodiments, the promoter operably linked to the coding sequence of the α -2, 6-sialyltransferase, particularly the first promoter of the above embodiments, is a strong promoter. For example, a strong promoter is a promoter that achieves high expression of a regulatory coding sequence. In this regard, high expression refers to expression that results in a transcript amount of the coding sequence that is at least as high as the transcript amount of a high expression housekeeping gene of a mammalian cell. Suitable examples of highly expressed housekeeping genes are EEF1A1 (eukaryotic translation elongation factor 1A1 gene), ACTB (beta actin gene) and PPIA (peptidyl prolyl isomerase a gene). In certain embodiments, the transcript amount of the coding sequence is at least 1.5-fold or at least 2-fold higher than the transcript amount of a high expression housekeeping gene of a mammalian cell. Transcript amounts may be measured, for example, using next generation sequencing or real-time RT-PCR. The transcript amount may be normalized, for example, with respect to the fragment per kilobase transcript (FPKM) read per million maps, for example as described in the examples. Thus, high expression results in FPKM values that are at least as high expression housekeeping genes (such as EEF1A1, ACTB and PPIA) of mammalian cells, in particular at least 1.5-fold, especially at least 2-fold. In certain embodiments, the first promoter is a strong promoter that achieves high expression of the coding sequence for the α -2, 6-sialyltransferase, resulting in a transcript amount of the coding sequence that is at least as high as the transcript amount of at least one of EEF1A1, ACTB, and PPIA in a mammalian cell.

Suitable promoters operably linked to the coding sequence for α -2, 6-sialyltransferase may be selected from the group consisting of: CMV promoter, EF 1. Alpha. Promoter, RSV promoter, BROAD3 promoter, murine rosa 26 promoter, pCEFL promoter and beta-actin promoter. In a particular embodiment, the promoter operably linked to the coding sequence of the α -2, 6-sialyltransferase, particularly the first promoter of the above embodiment, is a CMV promoter.

In certain embodiments, the promoter operably linked to the coding sequence of β -1, 4-galactosyltransferase and/or the coding sequence of CMP-sialic acid transporter, particularly the second promoter and/or the third promoter of the above embodiments, is a medium strength promoter. In this case, a suitable promoter may be selected from the group consisting of: SV40 promoter, CMV promoter, UBC promoter, EF1A promoter, PGK promoter and CAGG promoter. In particular embodiments, the promoter operably linked to the coding sequence of the β -1, 4-galactosyltransferase and/or the coding sequence of the CMP-sialic acid transporter is an SV40 promoter. In certain embodiments, the promoter operably linked to the coding sequence of β -1, 4-galactosyltransferase and/or the coding sequence of CMP-sialic acid transporter, particularly the second promoter and/or the third promoter of the above embodiments, is selected from the group consisting of: simian Virus 40 (SV 40) promoter, CMV promoter, ubiquitin C (UBC) promoter, elongation factor 1 alpha (EF 1A) promoter, phosphoglycerate kinase (PGK) promoter and beta-actin promoter (CAGG) coupled to CMV early enhancer, especially SV40 promoter.

In a particular embodiment, the promoter operably linked to the coding sequence of the α -2, 6-sialyltransferase, especially the first promoter of the above embodiment, achieves higher expression than the one or more promoters operably linked to the coding sequence of the β -1, 4-galactosyltransferase and/or the coding sequence of the CMP-sialic acid transporter, especially the second promoter and/or the third (if present) promoter of the above embodiment. In this regard, higher expression refers to expression that results in a higher amount of transcripts of the coding sequence of the alpha-2, 6-sialyltransferase than of the coding sequence of the beta-1, 4-galactosyltransferase and/or the CMP-sialic acid transporter. In particular, the expression and/or amount of transcripts is at least 1.5-fold higher, in particular at least 2-fold higher, at least 3-fold higher, at least 5-fold higher or at least 10-fold higher. Transcript amounts can be measured, for example, using next generation sequencing. The transcript amount may be normalized, for example, with respect to the fragment per kilobase transcript (FPKM) read per million maps, for example as described in the examples. Thus, higher expression results in higher FPKM values, in particular at least 1.5 times higher, especially at least 2 times higher, at least 3 times higher, at least 5 times higher or at least 10 times higher. In general, strong promoters in particular lead to a higher FPKM value for the controlled coding sequences compared to medium strength promoters.

In further embodiments, the endogenous genes encoding α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter of the mammalian cell are engineered for increased expression. In these embodiments, the expression of the endogenous glycosylase gene present in the host cell genome is up-regulated to achieve increased glycosylase expression. In these embodiments, the mammalian cells may or may not, and in particular do not, comprise exogenous nucleic acid encoding a glycosylase. Upregulation of endogenous genes results in higher expression of the genes. Upregulation also includes activation of genes that were not expressed prior to cell engineering.

Different techniques for regulating endogenous gene expression are known and can be used to increase the transcriptional levels of α -2, 6-sialyltransferases, β -1, 4-galactosyltransferases and CMP-sialic acid transporters. Non-limiting examples of techniques for up-regulating gene expression include ZFN activators, TALEN activators, and CRISPR activators, which may be designed to selectively bind to the promoter region of a glycosylase gene and, when bound, activate gene expression like a transcription factor. Alternatively, the promoter region of the glycosylase of the host cell may be engineered to insert one or more promoter and/or enhancer elements, resulting in increased expression levels. In further embodiments, chromatin regulation entities are used to change the conformation of the chromatin structure around the loci of the glycosylases to a more transcriptionally active state. Non-limiting examples of such entities include Matrix Attachment Regions (MARs), ubiquitin open elements (UCOEs), STAR elements, and comparable sequence motifs. Similar chromatin regulation capabilities of proxy-CRISPR-based entities are also described, which can also be used to increase expression of glycosylases. In further embodiments, the codon modification is used to increase translation of the host cell's glycosylase. These techniques may also be used in combination with each other.

In particular embodiments, the promoter is introduced into an endogenous gene of the corresponding glycosylase such that it is operably linked to the coding sequence of the glycosylase. In certain embodiments, the mammalian cell comprises

(i) A first exogenous promoter operably linked to an endogenous coding sequence for an α -2, 6-sialyltransferase;

(ii) A second exogenous promoter operably linked to an endogenous coding sequence for a β -1, 4-galactosyltransferase;

(i) A third exogenous promoter operably linked to an endogenous coding sequence for a CMP-sialic acid transporter.

Suitable promoters for the different glycosylases are described herein. The same promoters used for the exogenous nucleic acids can in particular also be used for the endogenous nucleic acids. Alternatively or additionally, enhancers may be introduced into endogenous genes of one or more glycosylases. Such enhancers may increase the activity of the endogenous promoter of the glycosylase gene. Introduction of the promoter and/or enhancer into the genome of the mammalian cell may be accomplished by any known genetic engineering method. One exemplary method is to use CRISPR technology.

In particular embodiments, an exogenous nucleic acid encoding a glycosylase and increasing expression of an endogenous nucleic acid encoding the glycosylase may be combined. For example, for one or both glycosylases, the corresponding exogenous nucleic acid is introduced into a mammalian cell, while for the remaining two or one glycosylases, the expression of the corresponding endogenous nucleic acid is increased. In further embodiments, for two or all three glycosylases, the corresponding exogenous nucleic acid is introduced into the mammalian cell and expression of the corresponding endogenous nucleic acid is increased.

The alpha-2, 6-sialyltransferase may be any enzyme capable of attaching sialic acid residues via alpha 2, 6-linkages to terminal galactose residues of complex N-linked oligosaccharides in mammalian cells. In certain embodiments, the α -2, 6-sialyltransferase is derived from chinese hamster (Cricetulus griseus) or human. In particular, the α -2, 6-sialyltransferase is β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1), especially from chinese mice or humans. In some embodiments, the α -2, 6-sialyltransferase has the amino acid sequence of accession number P15907 or the amino acid sequence of SEQ ID NO. 1 or 2 derived from the UniProt database.

The beta-1, 4-galactosyltransferase may be any enzyme capable of attaching a galactose residue via a beta 1, 4-linkage to the terminal GlcNAc residue of a complex N-linked oligosaccharide in a mammalian cell. In certain embodiments, the beta-1, 4-galactosyltransferase is derived from chinese hamster (Cricetulus griseus) or human. In particular, the beta-1, 4-galactosyltransferase is beta-1, 4-galactosyltransferase 1 (B4 GALT 1), especially from Chinese rats or humans. In some embodiments, the α -2, 6-sialyltransferase has the amino acid sequence of accession number P15291 or the amino acid sequence of SEQ ID NO 3 or 4 derived from the UniProt database.

The CMP-sialic acid transporter can be any enzyme capable of transporting a CMP-sialic acid residue into the golgi apparatus of a mammalian cell. In certain embodiments, the CMP-sialic acid transporter is derived from chinese mice or humans. In particular, the CMP-sialic acid transporter is the CMP-sialic acid transporter (SLC 35A 1), especially from the Chinese mouse or human. In some embodiments, the α -2, 6-sialyltransferase has the amino acid sequence of accession number O08520 or P78382 or the amino acid sequence of SEQ ID NO:5 or 6 derived from the UniProt database.

In certain embodiments, the glycosylase is derived from the same species, particularly the same species as the mammalian cells, particularly from chinese mice or humans. For example, in the case of chinese hamster cells (such as CHO), the glycosylase is derived from chinese hamsters. In the case of human cells, the glycosylase is derived from human. In other embodiments, the glycosylase is derived from chinese hamster, and the mammalian cell is not a hamster cell, but, for example, a human cell; alternatively, the glycosylase is derived from a human, whereas the mammalian cell is not a human cell, but is, for example, a CHO cell. In further embodiments, the glycosylase may also be derived from other species, in particular from other mammals, in particular rodents, such as mice and rats, in particular mice (Mus musculus) and brown mice (Rattus norvegicus).

In certain embodiments, the exogenous nucleic acid encoding a glycosylase is present on one vector or a combination of two or three vectors for mammalian cell transformation. In particular, mammalian cells are obtained by transformation with a vector comprising an exogenous nucleic acid or a combination of two or three vectors. In particular embodiments, one vector comprising all three exogenous nucleic acids is used. In particular, the vector or a combination of the two or three vectors comprises an exogenous expression cassette as described herein.

In some embodiments, each vector further comprises at least one selectable marker gene. The selectable marker gene is in particular a mammalian selectable marker gene which allows selection of mammalian host cells comprising said gene and thus allows selection of mammalian host cells comprising the vector.

Non-limiting examples of mammalian selectable marker genes include antibiotic resistance genes, such as conferring resistance to: g418; hygromycin (hyg or hph, commercially available from gaithersboro (gaithsboro) life technologies company (Life Technologies, inc.) in maryland; neomycin (neo, commercially available from gaithersboro (Gaithesboro) life technologies company (Life Technologies, inc.) in maryland); georubicin (Sh Ble, commercially available from Santa George Fabricius Ind, calif.); puromycin (pac, puromycin-N-acetyltransferase available from Palo alto cloning technologies Inc. (Clontech) Calif.), ouabain (oua available from Fabringen Inc. (Pharmingen)) and blasticidin (available from Inboard Inc. (Invitrogen)). Further suitable selectable marker genes include folate receptor genes, such as the folate receptor alpha gene, or genes encoding fluorescent proteins (such as GFP and RFP). Corresponding mammalian selectable marker genes are well known and allow selection of mammalian cells containing the genes and thus allow selection of cells containing the vector. Systems using folate receptor genes are described in WO 2009/080759 and WO 2015/015419. As used herein, the term "gene" refers to a natural or synthetic polynucleotide encoding a functional variant of a selectable marker that provides for the desired resistance. Thus, truncated or mutated versions of the wild-type gene or synthetic polynucleotide are also contemplated, so long as they provide the desired resistance. According to a particular embodiment, the vector comprises a gene encoding the enzyme functional puromycin-N-acetyltransferase (pac) as selectable marker gene.

In some embodiments, mammalian selectable marker genes may be amplifiable and allow for selection of mammalian host cells comprising the vector as well as gene amplification. A non-limiting example of an amplifiable selectable mammalian marker gene is the dihydrofolate reductase (DHFR) gene. Other systems currently in use include glutamine synthetase (gs) systems and histidinol driven selection systems. These amplifiable markers are also selectable markers and thus can be used to select those cells from which the vector was obtained. For an amplifiable system, such as a DHFR system, expression of the recombinant protein may be increased by exposing the cells to certain agents that facilitate gene amplification, such as antifolates (e.g., methotrexate (MTX)), in the case of DHFR systems. An inhibitor suitable for GS to facilitate gene amplification is Methionine Sulfonimide (MSX). Exposure to MSX also results in gene amplification.

The selectable marker gene may be located on the vector upstream, downstream, or between one or more expression cassettes for the glycosylase. In certain embodiments, the selectable marker gene is located on a vector downstream of one or more expression cassettes for the glycosylase. In particular embodiments, the vector comprises a second, different selectable marker gene. In these embodiments, it is preferred that one selectable marker gene is located on the vector downstream of the one or more expression cassettes of the glycosylase and the other selectable marker gene is located on the vector upstream of the one or more expression cassettes of the glycosylase. In embodiments in which a combination of two or three vectors is used, the different vectors specifically comprise different selectable marker genes.

The vector or combination of vectors is particularly suitable for integration into the genome of a mammalian cell. In some embodiments, mammalian cells are stably transfected with the exogenous nucleic acid. In certain embodiments, the vector further comprises a prokaryotic selectable marker gene. The prokaryotic selectable marker may provide resistance to antibiotics such as, for example, ampicillin, kanamycin, tetracycline, and/or chloramphenicol.

In certain embodiments, the mammalian cell further comprises an exogenous expression cassette for recombinant expression of the glycosylated polypeptide. Exogenous expression cassettes for recombinant expression of polypeptides are typically introduced into mammalian cells separately from exogenous nucleic acids encoding glycosylases. For example, mammalian cells are transfected with an additional vector comprising an exogenous expression cassette for recombinant expression of the glycosylated polypeptide. The glycosylated polypeptide may be any glycosylated polypeptide of interest including, inter alia, hormones, cytokines, enzymes, antibodies, fusion proteins, vaccines, coagulation proteins, toxins and growth factors. In certain embodiments, the glycosylated polypeptide is selected from the group consisting of: antibodies and fragments, derivatives or grafts thereof, particularly proteins comprising an antibody Fc region, intact antibodies, and Fc multimers comprising two or more antibody Fc regions. In certain embodiments, the glycosylated polypeptide is a pharmaceutically active polypeptide, such as a therapeutic polypeptide or a diagnostic polypeptide, in particular a therapeutic antibody, a therapeutic antibody fragment, a therapeutic antibody derivative or a therapeutic antibody implant.

Methods for producing glycosylated polypeptides

(c) Obtaining the glycosylated polypeptide from the cell culture; and

(d) Optionally processing the glycosylated polypeptide.

The embodiments, features and examples described herein with respect to mammalian cells are equally applicable to methods of producing glycosylated polypeptides using such mammalian cells.

In certain embodiments, the method further comprises, between steps (a) and (b), the steps of:

(a1) Inoculating a cell culture medium with the mammalian cells to provide a cell culture, and

(a2) Culturing the mammalian cells in the cell culture under conditions that allow for an increase in the number of cells in the cell culture.

Suitable conditions for culturing mammalian cells, increasing their cell numbers, and expressing glycosylated polypeptides depend on the particular mammalian cells, vectors, and expression cassettes used in the method. Suitable conditions can be readily determined by those skilled in the art and are also known in the art for a variety of mammalian cells. In certain embodiments, mammalian cells are transfected with one or more vectors comprising a selectable marker gene. In these embodiments, the culture conditions in step (a 2) and/or (b) may comprise the presence of a corresponding selection agent in the cell culture medium.

In the culture methods used in the art, in particular when culturing CHO cells, a temperature change is performed after the first exponential cell growth phase has been completed, for example when the desired cell density has been reached. For example, when about 10 is reached ⁶ At a cell density of individual cells/ml, the culture temperature was reduced from about 37℃to about 33 ℃. However, the inventors found that keeping the temperature constant, especially at about 36.5 ℃, a higher viable cell density, a higher product concentration and a higher sialylation level was achieved. In addition, the overall process is more robust and less likely to deviate due to the omission of further processing events (lowering of temperature). Thus, in particular embodiments, the method does not include temperature changes during the culturing. According to the invention, a temperature change means a change in the temperature of the cell culture, in particular a decrease of more than 3 ℃, over a duration of at least 1 hour. In particular, small and/or short-term fluctuations in the temperature of the cell culture are not considered as temperature changes. In certain embodiments, the culture conditions during the culturing of the mammalian cells do not include a temperature change of more than 2 ℃. In particular embodiments, the culture conditions during the culturing of the mammalian cells do not include a temperature change of more than 1.5 ℃. In particular embodiments, the culture conditions during the culturing of the mammalian cells do not include a temperature change of more than 1 ℃.

In certain embodiments, the method does not include a temperature set point change during incubation. The temperature set point is a predefined, accurate temperature value that the control system is intended to achieve. Measured value fluctuations due to technical control limitations are possible and are not considered as changes in the temperature set point. Thus, in these embodiments, the temperature set point does not change or does not change by more than 2 ℃, preferably more than 1.5 ℃, more preferably more than 1 ℃ during the incubation.

In particular, the temperature is maintained within a certain range during the culture of mammalian cells. In particular, this means that the temperature change is less than 2 ℃. In certain embodiments, the temperature decreases or changes by no more than 2 ℃, especially no more than 1.5 ℃ or no more than 1 ℃, during the culturing of the mammalian cells. For example, the temperature is maintained in the range from 30 ℃ to 40 ℃, especially from 32 ℃ to 39 ℃ or from 34 ℃ to 39 ℃, especially from 35 ℃ to 38 ℃, such as at about 36.5 ℃. In certain embodiments, the temperature is maintained in the range of 35 ℃ to 38 ℃ during the culturing of the mammalian cells. In a particular embodiment, the temperature is maintained at 35℃during the culturing of the mammalian cells. In particular embodiments, deviations from the desired temperature are allowed to fall outside of the defined range if the deviation duration is less than 1 hour, in particular less than 30 minutes.

Obtaining the glycosylated polypeptide from the cell culture particularly includes isolating the glycosylated polypeptide from the cell culture. Isolation of a glycosylated polypeptide refers in particular to separation of the glycosylated polypeptide from the remaining components of the cell culture. As used herein, the term "cell culture" specifically includes cell culture media and cells. In certain embodiments, the glycosylated polypeptide is secreted by a mammalian cell. In these embodiments, the glycosylated polypeptide is isolated from the cell culture medium. For example, the coding region of an expression cassette for recombinant expression of a glycosylated polypeptide may further comprise a nucleic acid sequence encoding a secretory expressed signal peptide. The separation of glycosylated polypeptides from the cell culture medium may be performed, for example, by chromatography. Suitable methods and means for isolating a polypeptide of interest are known in the art and can be readily applied by those skilled in the art.

The resulting glycosylated polypeptide may optionally be subjected to additional processing steps, such as, for example, additional purification, modification and/or formulation steps, to produce a product of interest having a desired quality and composition. Such additional processing steps and methods are generally known in the art. Suitable purification steps include, for example, affinity chromatography, size exclusion chromatography, anion and/or cation exchange chromatography, hydrophilic interaction chromatography and reverse phase chromatography. Additional steps may include viral inactivation, ultrafiltration, and diafiltration. The modification step may include chemical and enzymatic modification reactions such as coupling of a chemical entity to the glycosylated polypeptide and enzymatic cleavage of the glycosylated polypeptide. The formulation steps may include buffer exchange, formulation component addition, pH adjustment, and concentration adjustment. Any combination of these steps and additional steps may be used.

In certain embodiments, the method for producing a glycosylated polypeptide further comprises the step of providing a pharmaceutical formulation comprising the glycosylated polypeptide as part of step (d) or step (d). Providing a pharmaceutical formulation comprising or formulating a glycosylated polypeptide into a pharmaceutical composition comprises, in particular, exchanging a buffer solution or a buffer solution component of a composition comprising a glycosylated polypeptide. In addition, this step may include lyophilization of the glycosylated polypeptide. In particular, glycosylated polypeptides are transferred into compositions comprising only pharmaceutically acceptable ingredients.

In certain embodiments, the glycosylated polypeptide produced in the method has a higher sialic acid content than the same polypeptide produced in a reference cell under the same conditions, wherein the reference cell is identical to the host cell except that it has not been engineered as described herein. In particular embodiments, the method is used to produce a glycosylated polypeptide having a higher sialic acid content than the same polypeptide produced in a reference cell under the same conditions, wherein the reference cell is identical to the host cell except that it has not been engineered for increased expression of alpha-2, 6-sialyltransferase, beta-1, 4-galactosyltransferase and CMP-sialic acid transporter. In particular, the reference cells were not engineered for increased expression of any of α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter.

In certain embodiments, the sialic acid amount of the glycosylated polypeptide is at least 10 percent greater than the sialic acid amount of the same polypeptide produced in the reference cell. The sialic acid amount is preferably at least 20 percent higher, more preferably at least 30 percent higher, and most preferably at least 40 percent higher.

In further embodiments, the method is used to produce an antibody or fragment, derivative or implant thereof, particularly an antibody or fragment, derivative or implant thereof having reduced immunogenicity.

Thus, in a particular embodiment, the present invention provides a method for producing a glycosylated polypeptide having reduced immunogenicity, the method comprising the steps of:

(c) Obtaining the glycosylated polypeptide from the cell culture; and

(d) Optionally processing the glycosylated polypeptide;

wherein the glycosylated polypeptide is an antibody or a fragment, derivative or graft thereof.

In particular, the glycosylated polypeptide has reduced immunogenicity compared to a reference polypeptide. In this regard, the term "reduced immunogenicity" means that the glycosylated polypeptide is less likely to elicit an immune response against it when administered to a patient than the reference polypeptide.

The reference polypeptide has the same amino acid sequence as the glycosylated polypeptide with reduced immunogenicity, but has a lower sialylation amount. In certain embodiments, the reference polypeptide has the same amino acid sequence as the glycosylated polypeptide with reduced immunogenicity, but does not have any sialylation. Alternatively, 5% or less, in particular 1% or less, of the carbohydrate structures attached to the reference polypeptide are sialylated. Thus, the reference polypeptide in particular has a sialylation amount of 5% or less, preferably 1% or less. In certain embodiments, the sialylation amount of the glycosylated polypeptide is at least 10% greater than the sialylation amount of the reference polypeptide. The sialylation amount is preferably at least 20% higher, more preferably at least 30% higher, and most preferably at least 40% higher.

In particular, the reference polypeptide is produced in the reference cell under the same conditions as the glycosylated polypeptide with reduced immunogenicity, except that the reference cell is not engineered for increased expression of alpha-2, 6-sialyltransferase, beta-1, 4-galactosyltransferase and CMP-sialic acid transporter. In particular, the reference cells were not engineered for increased expression of any of α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter.

Nucleic acid encoding glycosylase

(i) A coding sequence for an alpha-2, 6-sialyltransferase;

(ii) A coding sequence for a beta-1, 4-galactosyltransferase; and

(iii) Coding sequence for a CMP-sialic acid transporter.

The coding sequence is particularly part of one or more expression cassettes, wherein each expression cassette comprises a promoter operably linked to one or more coding sequences, and wherein the expression cassette is for expression in a mammalian host cell.

The embodiments, features and examples described herein with respect to mammalian cells and exogenous nucleic acids contained therein are equally applicable to vector nucleic acids or combinations of at least two vector nucleic acids.

In particular, each coding sequence may be part of a separate expression cassette, or two or three of these coding sequences may be part of the same expression cassette. For example, the vector nucleic acid or a combination of at least two vector nucleic acids may comprise

(i) A first expression cassette comprising a first promoter operably linked to a coding sequence for the α -2, 6-sialyltransferase;

(ii) A second expression cassette comprising a second promoter operably linked to a coding sequence for the β -1, 4-galactosyltransferase; and

(iii) A third expression cassette comprising a third promoter operably linked to a coding sequence for the CMP-sialic acid transporter.

In another example, the vector nucleic acid or a combination of at least two vector nucleic acids may comprise

(i) A first expression cassette comprising a first promoter operably linked to a coding sequence for the α -2, 6-sialyltransferase; and

(ii) A second expression cassette comprising a second promoter operably linked to the coding sequence for the β -1, 4-galactosyltransferase and the coding sequence for the CMP-sialic acid transporter.

In embodiments in which the expression cassette comprises two or more coding sequences, the expression cassette may further comprise a coding sequence for an IRES or 2A element between the coding sequences.

In certain embodiments, the first promoter achieves higher expression than the second promoter and/or the third promoter (if present). In particular, the promoters described above in relation to expression cassettes used in mammalian cells are also used for expression cassettes of nucleic acids or combinations of nucleic acids. In certain embodiments, the first promoter is a cytomegalovirus promoter (CMV). In certain embodiments, the second promoter and/or the third promoter is selected from the group consisting of: simian virus 40 promoter (SV 40), CMV promoter, ubiquitin C (UBC) promoter, elongation factor 1A (EF 1A) promoter, phosphoglycerate kinase (PGK) promoter and beta-actin promoter (CAGG) coupled to CMV early enhancer, in particular SV40 promoter. In some embodiments, the expression cassette comprises additional elements as described herein, in particular polyadenylation signals.

Glycosylase enzymes are particularly described herein. In particular embodiments, the α -2, 6-sialyltransferase is β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1), particularly derived from chinese mice or humans. In particular embodiments, the beta-1, 4-galactosyltransferase is beta-1, 4-galactosyltransferase 1 (B4 GALT 1), particularly derived from chinese mice or humans. In a particular embodiment, the CMP-sialic acid transporter is a CMP-sialic acid transporter (SLC 35 A1), particularly derived from chinese mice or humans. In certain embodiments, the α -2, 6-sialyltransferase is β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1), the β -1, 4-galactosyltransferase is β -1, 4-galactosyltransferase 1 (B4 GALT 1) of a chinese ground rat, and the CMP-sialic acid transporter is CMP-sialic acid transporter (SLC 35 A1) of a chinese ground rat.

The different expression cassettes may be present on the same nucleic acid or they may be present on separate nucleic acids which together form a combination of nucleic acids. In particular embodiments, one nucleic acid comprises all of the coding sequences for the glycosylase. In particular, one nucleic acid comprises an expression cassette as described herein.

In some embodiments, each vector nucleic acid of the vector nucleic acid or the combination of at least two vector nucleic acids further comprises at least one selectable marker gene. Suitable selectable marker genes are described above with respect to vectors for transformation of mammalian cells. In certain embodiments, the selectable marker gene is an antibiotic resistance gene, such as a puromycin-N-acetyltransferase gene (pac). In particular embodiments, the vector nucleic acid comprises a selectable marker gene located on the nucleic acid downstream of the expression cassette; and optionally a second selectable marker located on the nucleic acid upstream of the expression cassette.

In some embodiments, the vector nucleic acid or a combination of at least two vector nucleic acids is suitable for stably transfecting a host cell, particularly a mammalian host cell, such as a rodent or human cell, particularly a CHO cell. In particular, the vector nucleic acid is a plasmid.

In a fourth aspect, the invention further provides the use of a vector nucleic acid or a combination of at least two vector nucleic acids for transfecting a mammalian cell. In particular, the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

The present invention also provides a method for increasing the expression of an alpha-2, 6-sialyltransferase, a beta-1, 4-galactosyltransferase and a CMP-sialic acid transporter in a mammalian cell, the method comprising the step of transfecting the mammalian cell with a vector nucleic acid or a combination of at least two vector nucleic acids as described herein, and/or the step of engineering endogenous genes encoding the alpha-2, 6-sialyltransferase, the beta-1, 4-galactosyltransferase and the CMP-sialic acid transporter of the mammalian cell for increased expression, as described herein.

The embodiments, features and examples described herein with respect to mammalian cells and exogenous nucleic acids contained therein and vector nucleic acids or combinations of at least two vector nucleic acids are equally applicable to the use of vector nucleic acids or combinations of at least two vector nucleic acids for transfecting mammalian cells and to methods for increasing expression of alpha-2, 6-sialyltransferase, beta-1, 4-galactosyltransferase and CMP-sialic acid transporter in mammalian cells.

Methods for reducing the immunogenicity of antibodies

In a fifth aspect, the invention provides a method for reducing the immunogenicity of an antibody or fragment, derivative or graft thereof, the method comprising the step of increasing the amount of sialylation in the glycosylation pattern of the antibody or fragment, derivative or graft thereof.

In particular, the antibody or fragment, derivative or implant thereof is a therapeutic antibody or fragment, derivative or implant thereof, preferably a therapeutic antibody. In certain embodiments, the antibody or fragment, derivative or graft thereof comprises a CH2 domain having an N-glycosylation site comprising an asparagine residue at amino acid position 297 of the heavy chain of the antibody according to Kabat numbering. In certain embodiments, the step of increasing the sialylation in the glycosylation pattern of the antibody or fragment, derivative or graft thereof comprises increasing the sialylation in the N-glycosylation pattern of the CH2 domain.

As a result of this method, the antibody or fragment, derivative or implant thereof has a higher sialylation amount.

The method may comprise directly increasing the sialylation amount of the antibody or fragment, derivative or graft thereof. In this case, the composition comprising the antibody or fragment, derivative or implant thereof is treated such that sialic acid residues are attached to glycans present on the antibody or fragment, derivative or implant thereof. Suitable means for directly increasing the sialylation amount include, for example, in vitro treatment of an antibody or fragment, derivative or graft thereof with a sialyltransferase such as α -2, 6-sialyltransferase and a sialic acid donor as described herein. Sialyltransferases transfer sialic acid residues to glycans of polypeptides and thereby increase the sialylation of polypeptides.

In further embodiments, the method comprises increasing the sialylation amount of the antibody or fragment, derivative or graft thereof by enriching those antibodies or fragments, derivatives or grafts thereof that carry at least one sialic acid. In the resulting composition of the antibody or fragment, derivative or implant thereof, the relative sialylation amount is higher than the sialylation amount in the composition prior to enrichment. Enrichment of antibodies or fragments, derivatives or grafts thereof carrying at least one sialic acid may be achieved by any suitable means.

An exemplary means for enrichment is chromatography, such as affinity chromatography using a ligand that specifically binds to sialylated glycan structures, e.g., lectin or an antibody specific for sialylated glycan structures. In this respect, suitable lectins are, for example, sambucus nigra (Sambucus nigra) lectins. In these embodiments, the sialylated polypeptide binds to the chromatographic matrix and the non-sialylated polypeptide is washed away. After elution of the bound polypeptide, the sialylation amount increases.

Additional means for enrichment include affinity chromatography using ligands that specifically bind to non-sialylated glycan structures, such as lectins or antibodies specific for non-sialylated glycan structures. In these embodiments, the non-sialylated polypeptide is bound to the chromatographic matrix and the sialylated polypeptide is washed away. The polypeptide obtained from the washing step has a higher sialylation amount than the initial polypeptide.

Additional means for enrichment include methods for separating polypeptides based on their charge. Since sialic acid is negatively charged, sialylated polypeptides can be separated from non-sialylated polypeptides and thus enriched. Exemplary methods include ion exchange chromatography.

In further embodiments, the method comprises increasing the sialylation amount of the antibody or fragment, derivative or implant thereof compared to a reference composition of the antibody or fragment, derivative or implant thereof. In these embodiments, the antibody or fragment, derivative or implant thereof (reference composition) to be sialylated is regenerated using a production method that results in a higher sialylation of the antibody or fragment, derivative or implant thereof. Suitable means for increasing the sialylation amount according to these embodiments include, for example, the production of antibodies or fragments, derivatives or grafts thereof in host cells having higher sialylation activity than the host cells used to produce the reference composition. In certain embodiments, host cells and production methods as described herein may be used.

In certain embodiments, the sialylation amount is increased by at least 10% points. In these embodiments, the relative amount of glycans comprising at least one sialic acid residue (e.g., monosialylated at least on the 3-arm or 6-arm of the glycan core) attached to the antibody or fragment, derivative, or graft in the population of antibodies or fragments, derivatives, or grafts after performing the method is at least 10 percent greater than the population of antibodies or fragments, derivatives, or grafts before performing the method for reducing immunogenicity. The sialylation amount is preferably increased by at least 20 percentage points, more preferably at least 30 percentage points, and most preferably at least 40 percentage points.

In certain embodiments, the antibody or fragment, derivative or graft thereof to be reduced in immunogenicity has a relative sialylation amount of 20% or less, preferably 10% or less, more preferably 5% or less, and most preferably 1% or less. Antibodies or fragments, derivatives or grafts thereof to be reduced in immunogenicity may be produced, for example, in cells not engineered for increased expression of alpha-2, 6-sialyltransferase, beta-1, 4-galactosyltransferase and CMP-sialic acid transporter. In particular, the cells have not been engineered for increased expression of any of α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter. In particular, the antibody or fragment, derivative or graft thereof whose immunogenicity is to be reduced is produced in CHO cells.

An antibody or fragment, derivative or graft thereof having reduced immunogenicity is less likely to elicit an immune response against it, particularly when administered to a patient. In certain embodiments, the recognition and uptake of antibodies or fragments, derivatives, or grafts thereof having reduced immunogenicity by dendritic cells is reduced. In further embodiments, they have a reduced ability to induce a T cell response against the antibody or fragment, derivative or graft thereof. In particular embodiments, the antibody or fragment, derivative or implant thereof has a reduced ability to produce an anti-drug antibody upon administration to a patient.

Particular examples

Hereinafter, specific embodiments of the present invention are described. These embodiments may be combined with further embodiments, features and examples described herein.

Example 1. A mammalian cell comprising

(i) Exogenous nucleic acid encoding an alpha-2, 6-sialyltransferase;

(ii) Exogenous nucleic acid encoding a beta-1, 4-galactosyltransferase; and

(iii) Exogenous nucleic acid encoding a CMP-sialic acid transporter.

Example 2 mammalian cells according to example 1 comprising

Example 3 mammalian cells according to example 1 comprising

Example 4. Mammalian cell according to example 3, wherein the second exogenous expression cassette comprises an Internal Ribosome Entry Site (IRES) between the coding sequence of the β -1, 4-galactosyltransferase and the coding sequence of the CMP-sialic acid transporter.

Example 5. Mammalian cell according to example 3, wherein the second exogenous expression cassette comprises a coding sequence for a 2A element between the coding sequence for the beta-1, 4-galactosyltransferase and the coding sequence for the CMP-sialic acid transporter.

Embodiment 6. The mammalian cell of any one of embodiments 2 to 5, wherein the first promoter is a strong promoter.

Embodiment 7. The mammalian cell according to any of embodiments 2 to 6, wherein the first promoter effects expression of the coding sequence of the α -2, 6-sialyltransferase, which results in a transcript amount of the coding sequence that is at least as high as the transcript amount of highly expressed housekeeping genes of the mammalian cell, such as EEF1A1 (eukaryotic translation elongation factor 1A1 gene), ACTB (β actin gene) and PPIA (peptidyl prolyl isomerase a gene).

Embodiment 8. The mammalian cell according to any of embodiments 2 to 6, wherein the first promoter effects expression of the coding sequence of the α -2, 6-sialyltransferase, which results in a transcript amount of the coding sequence that is at least 1.5 times higher than the transcript amount of a highly expressed housekeeping gene of the mammalian cell, such as EEF1A1 (eukaryotic translation elongation factor 1A1 gene), ACTB (β actin gene) and PPIA (peptidyl prolyl isomerase a gene).

Embodiment 9. The mammalian cell according to any of embodiments 2 to 6, wherein the first promoter effects expression of the coding sequence of the α -2, 6-sialyltransferase, which results in a transcript amount of the coding sequence that is at least 2-fold higher than the transcript amount of a highly expressed housekeeping gene of the mammalian cell, such as EEF1A1 (eukaryotic translation elongation factor 1 α1 gene), ACTB (β actin gene) and PPIA (peptidyl prolyl isomerase a gene).

Embodiment 10. The mammalian cell according to any of embodiments 2 to 9, wherein the first promoter achieves higher expression than the second promoter and/or the third promoter if present.

Embodiment 11. The mammalian cell according to embodiment 10, wherein the expression achieved by the first promoter is at least 3 times higher than the expression achieved by the second promoter and/or the third promoter, if present.

Embodiment 12. The mammalian cell according to any of embodiments 2 to 11, wherein the first promoter is selected from the group consisting of: cytomegalovirus (CMV) promoter, simian virus 40 (SV 40) promoter, ubiquitin C (UBC) promoter, elongation factor 1A (EF 1A) promoter, phosphoglycerate kinase (PGK) promoter, rous Sarcoma Virus (RSV) promoter, choad 3 promoter, murine rosa 26 promoter, pCEFL promoter and optionally a beta-actin promoter (CAGG) coupled to a CMV early enhancer.

Embodiment 13. The mammalian cell of embodiment 12, wherein the first promoter is a Cytomegalovirus (CMV) promoter.

Embodiment 14. The mammalian cell according to any of embodiments 2 to 13, wherein the second promoter and/or the third promoter is selected from the group consisting of: SV40 promoter, CMV promoter, UBC promoter, EF1A promoter, PGK promoter and CAGG promoter.

Embodiment 15. The mammalian cell of embodiment 14 wherein the second promoter and, if present, the third promoter are simian virus 40 (SV 40) promoters.

Embodiment 16. The mammalian cell according to any of embodiments 1 to 15, wherein the α -2, 6-sialyltransferase is β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1), in particular derived from chinese mice or humans.

Embodiment 17. The mammalian cell according to any of embodiments 1 to 16, wherein the beta-1, 4-galactosyltransferase is beta-1, 4-galactosyltransferase 1 (B4 GALT 1), in particular derived from chinese mice or humans.

Embodiment 18. The mammalian cell according to any of embodiments 1 to 17, wherein the CMP-sialic acid transporter is a CMP-sialic acid transporter (SLC 35 A1), in particular derived from chinese mice or humans.

Embodiment 19. The mammalian cell of any one of embodiments 1 to 18, wherein the α -2, 6-sialyltransferase is a β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1) of a chinese ground rat, the β -1, 4-galactosyltransferase is a β -1, 4-galactosyltransferase 1 (B4 GALT 1) of a chinese ground rat, and the CMP-sialic acid transporter is a CMP-sialic acid transporter (SLC 35 A1) of a chinese ground rat.

Example 20. The mammalian cell of example 19, wherein the mammalian cell is a CHO cell.

Embodiment 21. The mammalian cell of any one of embodiments 1 to 18, wherein the α -2, 6-sialyltransferase is human β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1), the β -1, 4-galactosyltransferase is human β -1, 4-galactosyltransferase 1 (B4 GALT 1), and the CMP-sialic acid transporter is human CMP-sialic acid transporter (SLC 35 A1).

Embodiment 22. The mammalian cell of embodiment 21, wherein the mammalian cell is a CHO cell.

Embodiment 23. The mammalian cell of embodiment 21, wherein the mammalian cell is a human cell.

Embodiment 24. The mammalian cell of any one of embodiments 2 to 23, wherein each expression cassette further comprises a polyadenylation signal (pA).

Embodiment 25. The mammalian cell according to any one of embodiments 1 to 24, wherein the mammalian cell is obtained by transformation with a vector comprising the exogenous nucleic acids or a combination of two or three vectors.

Embodiment 26. The mammalian cell according to embodiment 25, wherein the mammalian cell is obtained by transformation with a vector comprising the first expression cassette, the second expression cassette and optionally a third expression cassette.

Embodiment 27. The mammalian cell of embodiment 25 or 26 wherein each vector further comprises at least one selectable marker gene.

Example 28. Mammalian cells according to example 27, wherein the selectable marker gene is an antibiotic resistance gene that confers resistance to puromycin, G418, hygromycin, neomycin, gecomycin, ouabain, blasticidin, methotrexate (MTX), or Methionine Sulfonimide (MSX).

Embodiment 29. The mammalian cell according to embodiment 27 wherein the selectable marker gene is a folate receptor gene, such as a folate receptor alpha gene, or a gene encoding a fluorescent protein, such as GFP and RFP.

Example 30. The mammalian cell according to example 27, wherein the selectable marker gene is a puromycin-N-acetyltransferase gene (pac).

Embodiment 31. The mammalian cell according to any one of embodiments 27 to 30, wherein one selectable marker gene is located on the vector downstream of the expression cassettes and a second selectable marker, if present, is located on the vector upstream of the expression cassettes.

Example 32 the mammalian cell according to example 1, wherein the mammalian cell is a CHO cell stably transfected with a vector comprising

(i) A first exogenous expression cassette comprising a Cytomegalovirus (CMV) promoter operably linked to a coding sequence for β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1) of a chinese ground rat;

(ii) A second exogenous expression cassette comprising a simian vacuolar virus 40 (SV 40) promoter operably linked to a coding sequence for β -1, 4-galactosyltransferase 1 (B4 GALT 1) of a chinese ground rat; and

(iii) A third exogenous expression cassette comprising a simian vacuolar virus 40 (SV 40) promoter operably linked to a coding sequence of a CMP-sialic acid transporter (SLC 35 A1) of a chinese ground rat.

Example 33. Mammalian cells according to example 32, wherein the vector further comprises the puromycin-N-acetyltransferase gene (pac) as a selectable marker gene.

Example 34. Mammalian cells according to example 33, wherein the selectable marker gene is located on a vector downstream of the expression cassettes.

Example 35. A mammalian cell wherein endogenous genes encoding an α -2, 6-sialyltransferase, a β -1, 4-galactosyltransferase, and a CMP-sialic acid transporter of the mammalian cell are engineered for increased expression.

Example 36. The mammalian cell of example 35, wherein the expression of the endogenous gene is higher as compared to the same cell that has not been engineered for increased expression.

Example 37. Mammalian cells according to examples 35 or 36 wherein expression of endogenous nucleic acids encoding α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter is up-regulated or activated to increase expression.

Embodiment 38. The mammalian cell of any one of embodiments 35 to 37, wherein increased expression is obtained by a ZFN activator, a TALEN activator, a CRISPR activator, or a chromatin regulating entity.

Embodiment 39. The mammalian cell of any one of embodiments 35 to 37, wherein the increased expression is obtained by inserting one or more promoter and/or enhancer elements into genes expressing α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter.

Example 40A mammalian cell according to example 39 comprising

Embodiment 41. The mammalian cell of embodiment 40 wherein the first promoter is a strong promoter.

Embodiment 42. The mammalian cell according to embodiment 40 or 41, wherein the first promoter effects expression of the coding sequence for the α -2, 6-sialyltransferase, which results in a transcript amount of the coding sequence that is at least as high as the transcript amounts of highly expressed housekeeping genes of the mammalian cell, such as EEF1A1 (eukaryotic translation elongation factor 1 α1 gene), ACTB (β actin gene) and PPIA (peptidyl prolyl isomerase a gene).

Embodiment 43. The mammalian cell according to embodiment 40 or 41, wherein the first promoter effects expression of the coding sequence for the α -2, 6-sialyltransferase, which results in a transcript amount of the coding sequence that is at least 1.5 times higher than the transcript amount of highly expressed housekeeping genes of the mammalian cell, such as EEF1A1 (eukaryotic translation elongation factor 1 α1 gene), ACTB (β actin gene) and PPIA (peptidyl prolyl isomerase A gene).

Example 44. The mammalian cell according to example 40 or 41, wherein the first promoter effects expression of the coding sequence for the α -2, 6-sialyltransferase, which results in a transcript amount of the coding sequence that is at least 2-fold higher than the transcript amount of highly expressed housekeeping genes of the mammalian cell, such as EEF1A1 (eukaryotic translation elongation factor 1 α1 gene), ACTB (β actin gene), and PPIA (peptidyl prolyl isomerase a gene).

Embodiment 45. The mammalian cell of any one of embodiments 40 to 44, wherein the first promoter achieves higher expression than the second promoter and/or the third promoter.

Embodiment 46. The mammalian cell of embodiment 45 wherein the expression achieved by the first promoter is at least 3-fold higher than the expression achieved by the second promoter and/or the third promoter.

Embodiment 47. The mammalian cell of any one of embodiments 40 to 46, wherein the first promoter is selected from the group consisting of: cytomegalovirus (CMV) promoter, simian virus 40 (SV 40) promoter, ubiquitin C (UBC) promoter, elongation factor 1A (EF 1A) promoter, phosphoglycerate kinase (PGK) promoter, rous Sarcoma Virus (RSV) promoter, choad 3 promoter, murine rosa 26 promoter, pCEFL promoter and optionally a beta-actin promoter (CAGG) coupled to a CMV early enhancer.

Embodiment 48. The mammalian cell of embodiment 47, wherein the first promoter is a Cytomegalovirus (CMV) promoter.

Embodiment 49. The mammalian cell of any one of embodiments 40 to 48, wherein the second promoter and/or the third promoter is selected from the group consisting of: SV40 promoter, CMV promoter, UBC promoter, EF1A promoter, PGK promoter and CAGG promoter.

Embodiment 50. The mammalian cell of embodiment 49, wherein the second promoter and the third promoter are simian virus 40 (SV 40) promoters.

Embodiment 51. The mammalian cell according to any one of embodiments 1 to 50, wherein the mammalian cell is a CHO cell.

Embodiment 52. The mammalian cell of any of embodiments 1 to 50, wherein the mammalian cell is a human cell.

Embodiment 53. The mammalian cell according to any one of embodiments 1 to 52 further comprising an exogenous expression cassette for recombinant expression of the glycosylated polypeptide.

Embodiment 54. The mammalian cell of embodiment 53 wherein the glycosylated polypeptide is selected from the group consisting of: hormones, cytokines, enzymes, antibodies, fusion proteins, vaccines, coagulation proteins, toxins and growth factors.

Embodiment 55. The mammalian cell of embodiment 53 wherein the glycosylated polypeptide is selected from the group consisting of: antibodies and fragments, derivatives or grafts thereof, particularly proteins comprising an antibody Fc region, intact antibodies, and Fc multimers comprising two or more antibody Fc regions.

Embodiment 56. Mammalian cells according to embodiment 53 wherein the glycosylated polypeptide is an antibody or a protein comprising an antibody Fc region, in particular an Fc multimer comprising two or more antibody Fc regions.

Embodiment 57. The mammalian cell of any one of embodiments 53 to 56, wherein the glycosylated polypeptide is a therapeutic polypeptide or a diagnostic polypeptide.

Example 58 a method for producing a glycosylated polypeptide, the method comprising the steps of:

(a) Providing a mammalian cell according to any one of embodiments 53 to 57;

(c) Obtaining the glycosylated polypeptide from the cell culture; and

(d) Optionally processing the glycosylated polypeptide.

Embodiment 59. The method of embodiment 58, further comprising, between steps (a) and (b), the steps of:

Embodiment 60. The method of embodiment 58 or 59, wherein the temperature is reduced by no more than 2℃during the culturing of the mammalian cells.

Embodiment 61. The method of embodiment 58 or 59, wherein the temperature does not change by more than 2℃during the culturing of the mammalian cells.

Embodiment 62. The method of embodiment 58 or 59, wherein the temperature is reduced by no more than 1.5℃during the culturing of the mammalian cells.

Embodiment 63. The method of embodiment 58 or 59, wherein the temperature does not change by more than 1.5℃during the culturing of the mammalian cells.

Embodiment 64 the method of any one of embodiments 58-63, wherein the culture conditions during the culturing of the mammalian cell do not include a temperature change.

Embodiment 65. The method of any of embodiments 58-63, wherein the culture conditions during the culturing of the mammalian cell do not include a temperature change of more than 2 ℃.

Embodiment 66. The method of any one of embodiments 58-63, wherein the culture conditions during the culturing of the mammalian cell do not include a temperature change of more than 1.5 ℃.

Embodiment 67. The method of any one of embodiments 58 to 63, wherein the culture conditions during the culturing of the mammalian cell do not include a temperature change of more than 1 ℃.

Embodiment 68. The method of any of embodiments 58-63, wherein the temperature set point of the cell culture does not change during the culturing of the mammalian cells.

Embodiment 69. The method of any one of embodiments 58 to 68, wherein during the culturing of the mammalian cell, the temperature is maintained at 35 ℃ or higher.

Embodiment 70. The method of any one of embodiments 58-68, wherein during the culturing of the mammalian cell, the temperature is maintained in the range of 34 ℃ to 39 ℃.

Embodiment 71. The method of any one of embodiments 58 to 68, wherein the temperature is maintained in the range of 35 ℃ to 38 ℃ during the culturing of the mammalian cells.

Embodiment 72. The method of any one of embodiments 58 to 71, wherein the step of obtaining the glycosylated polypeptide comprises isolating the glycosylated polypeptide from the cell culture.

Embodiment 73. The method of any one of embodiments 58 to 72, wherein the glycosylated polypeptide is secreted by the mammalian cell and the glycosylated polypeptide is isolated from the cell culture medium.

Embodiment 74. The method of any one of embodiments 58 to 73, wherein the method comprises step (d) of processing the glycosylated polypeptide.

Example 75. The method of example 74, wherein processing the glycosylated polypeptide comprises additional purification, modification, and/or formulation steps.

Embodiment 76 the method of any one of embodiments 58 to 75, wherein step (d) comprises providing a pharmaceutical formulation comprising the glycosylated polypeptide.

Embodiment 77. The method of any one of embodiments 58 to 76, wherein the glycosylated polypeptide is an antibody or a fragment, derivative or graft thereof.

Example 78. A method for producing a glycosylated polypeptide having reduced immunogenicity, the method comprising the steps of:

(c) Obtaining the glycosylated polypeptide from the cell culture; and

(d) Optionally processing the glycosylated polypeptide;

Embodiment 79. The method of embodiment 78 having any one or more of the features as defined in embodiments 59 to 77.

Example 80. The method of example 78 or 79, wherein the glycosylated polypeptide has reduced immunogenicity as compared to a reference glycosylated polypeptide having the same amino acid sequence but a lower sialylation amount.

Embodiment 81. The method of embodiment 80 wherein the sialylation amount of the reference glycosylated polypeptide is at least 10% lower, preferably at least 20% lower, more preferably at least 30% lower, and most preferably at least 40% lower.

Embodiment 82. The method of embodiment 80 or 81, wherein the reference glycosylated polypeptide has a sialylation amount of 5% or less, preferably 1% or less.

Example 83A vector nucleic acid or a combination of at least two vector nucleic acids comprising

(i) A coding sequence for an alpha-2, 6-sialyltransferase;

(ii) A coding sequence for a beta-1, 4-galactosyltransferase; and

(iii) Coding sequence for a CMP-sialic acid transporter.

Embodiment 84. The vector nucleic acid or the combination of at least two vector nucleic acids of embodiment 83, wherein the coding sequence is part of one or more expression cassettes, each expression cassette comprising a promoter operably linked to the one or more coding sequences, and wherein the expression cassettes are for expression in a mammalian host cell.

Example 85 the vector nucleic acid or the combination of at least two vector nucleic acids of examples 83 or 84, wherein each coding sequence is part of a separate expression cassette or two or three of the coding sequences are part of the same expression cassette.

Embodiment 86 the vector nucleic acid or the combination of at least two vector nucleic acids of any one of embodiments 83 to 85 comprising

Embodiment 87 the vector nucleic acid or the combination of at least two vector nucleic acids of any one of embodiments 83 to 85 comprising

Example 88. The vector nucleic acid or the combination of at least two vector nucleic acids according to example 87, wherein the second exogenous expression cassette comprises an Internal Ribosome Entry Site (IRES) or the coding sequence of a 2A element between the coding sequence of the β -1, 4-galactosyltransferase and the coding sequence of the CMP-sialic acid transporter.

Embodiment 89 the vector nucleic acid or the combination of at least two vector nucleic acids according to any of embodiments 86 to 88, wherein the first promoter achieves higher expression than the second promoter and/or the third promoter if present.

Embodiment 90. The vector nucleic acid or the combination of at least two vector nucleic acids of embodiment 89, wherein expression achieved by the first promoter is at least 3-fold higher than expression achieved by the second promoter and/or the third promoter, if present.

Embodiment 91 the vector nucleic acid or combination of at least two vector nucleic acids of any one of embodiments 86 to 90, wherein the first promoter is a Cytomegalovirus (CMV) promoter.

Embodiment 92. The vector nucleic acid or the combination of at least two vector nucleic acids of any one of embodiments 86 to 91, wherein the second promoter and/or the third promoter is a simian cavitation virus 40 (SV 40) promoter.

Embodiment 93 the vector nucleic acid or the combination of at least two vector nucleic acids according to any one of embodiments 83 to 92, wherein the alpha-2, 6-sialyltransferase is beta-galactoside alpha-2, 6-sialyltransferase 1 (ST 6GAL 1), in particular derived from chinese mice or humans.

Embodiment 94. The vector nucleic acid or the combination of at least two vector nucleic acids according to any one of embodiments 83 to 93, wherein the β -1, 4-galactosyltransferase is β -1, 4-galactosyltransferase 1 (B4 GALT 1), in particular derived from chinese mice or humans.

Embodiment 95. The vector nucleic acid or the combination of at least two vector nucleic acids according to any one of embodiments 83 to 94, wherein the CMP-sialic acid transporter is a CMP-sialic acid transporter (SLC 35 A1), in particular derived from chinese mice or humans.

Example 96 the vector nucleic acid or the combination of at least two vector nucleic acids according to example 83, which is a vector for stably transfecting a host cell, comprising

Embodiment 97 the vector nucleic acid or the combination of at least two vector nucleic acids of any one of embodiments 83 to 96, wherein each expression cassette further comprises a polyadenylation signal (pA).

Embodiment 98 the vector nucleic acid or the combination of at least two vector nucleic acids of any one of embodiments 83 to 97, wherein each vector nucleic acid further comprises at least one selectable marker gene.

Embodiment 99. The vector nucleic acid or a combination of at least two vector nucleic acids according to embodiment 98, wherein the selectable marker gene is an antibiotic resistance gene, such as a puromycin-N-acetyltransferase gene (pac).

Example 100. The vector nucleic acid or combination of at least two vector nucleic acids according to example 98 or 99, wherein one selectable marker gene is located on the nucleic acid downstream of the expression cassettes and a second selectable marker, if present, is located on the nucleic acid upstream of the expression cassettes.

Embodiment 101. The vector nucleic acid or combination of at least two vector nucleic acids of any one of embodiments 83 to 100, for use in stably transfecting a host cell.

Embodiment 102. Use of the vector nucleic acid or the combination of at least two vector nucleic acids according to any of embodiments 83 to 101 for transfecting a mammalian cell.

Embodiment 103. The use of embodiment 102, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

Embodiment 104. A method for increasing expression of an alpha-2, 6-sialyltransferase, a beta-1, 4-galactosyltransferase, and a CMP-sialic acid transporter in a mammalian cell, the method comprising the step of transfecting the mammalian cell with the vector nucleic acid of any one of embodiments 83 to 101 or a combination of at least two vector nucleic acids.

Embodiment 105. The method of embodiment 104, the method comprising the steps of:

(i) Providing a mammalian cell;

(ii) Transfecting the mammalian cell with the vector nucleic acid or the combination of at least two vector nucleic acids according to any one of embodiments 83 to 101;

(iii) Engineered mammalian cells with increased expression of alpha-2, 6-sialyltransferase, beta-1, 4-galactosyltransferase and CMP-sialic acid transporter are obtained.

Embodiment 106. The method of embodiment 105, wherein the engineered mammalian cell is a mammalian cell as defined in any one of embodiments 1 to 34 and 51 to 57.

Example 107A method for increasing expression of an α -2, 6-sialyltransferase, a β -1, 4-galactosyltransferase, and a CMP-sialic acid transporter in a mammalian cell, the method comprising engineering an endogenous gene of the mammalian cell encoding the α -2, 6-sialyltransferase, the β -1, 4-galactosyltransferase, and the CMP-sialic acid transporter for increased expression.

Embodiment 108. The method of embodiment 107, the method comprising the steps of:

(i) Providing a mammalian cell;

(ii) Inserting one or more promoter and/or enhancer elements into the gene expressing α -2, 6-sialyltransferase, the gene expressing β -1, 4-galactosyltransferase and the gene expressing CMP-sialic acid transporter;

Embodiment 109. The method of embodiment 108, wherein the engineered mammalian cell is a mammalian cell as defined in any one of examples 35 to 57.

Example 110A method for reducing the immunogenicity of an antibody or fragment, derivative or graft thereof, the method comprising the step of increasing the sialylation in the glycosylation pattern of the antibody or fragment, derivative or graft thereof.

Embodiment 111. The method of embodiment 110, wherein the antibody or fragment, derivative or implant thereof is a therapeutic antibody or fragment, derivative or implant thereof.

Embodiment 112. The method of embodiment 111, the method is used to reduce the immunogenicity of a therapeutic antibody.

Embodiment 113 the method of any one of embodiments 110 to 112, wherein the antibody or fragment, derivative or graft thereof comprises a CH2 domain having an N-glycosylation site comprising an asparagine residue at amino acid position 297 of the antibody heavy chain according to Kabat numbering, and the step of increasing sialylation comprises increasing sialylation in the N-glycosylation pattern of said CH2 domain.

Embodiment 114. The method of any one of embodiments 110 to 113, wherein the step of increasing the sialylation amount comprises treating the antibody or fragment, derivative or implant thereof such that sialic acid residues are attached to glycans present on the antibody or fragment, derivative or implant thereof.

Embodiment 115. The method of embodiment 114, wherein the step of increasing the sialylation amount comprises in vitro treating the antibody or fragment, derivative or graft thereof with a sialyltransferase, such as an α -2, 6-sialyltransferase, and a sialic acid donor.

Embodiment 116 the method of any one of embodiments 110-115, wherein the step of increasing sialylation comprises enriching those antibodies or fragments, derivatives or grafts thereof that carry at least one sialic acid.

Embodiment 117 the method of embodiment 116, wherein the step of increasing the sialylation amount comprises performing one or more of:

(i) Affinity chromatography using ligands that bind specifically to sialylated glycan structures;

(ii) Affinity chromatography using ligands that bind specifically to non-sialylated glycan structures; and

(iii) Ion exchange chromatography.

Embodiment 118 the method of any one of embodiments 110-117, wherein the step of increasing the sialylation comprises producing the antibody or fragment, derivative or implant thereof using a production method that results in a higher sialylation compared to a reference composition of the antibody or fragment, derivative or implant thereof.

Embodiment 119. The method of embodiment 118, wherein the antibody or fragment, derivative or graft thereof is produced in a host cell having higher sialylation activity than the host cell used to produce the reference composition.

Embodiment 120. The method of embodiment 119, wherein the host cell used to produce the antibody or fragment, derivative or graft thereof having reduced immunogenicity is a mammalian cell according to any one of embodiments 1 to 53.

Embodiment 121. The method of embodiment 119 or 120, wherein the antibody or fragment, derivative or graft thereof having reduced immunogenicity is produced by the method according to any one of embodiments 58 to 82.

Embodiment 122. The method of any of embodiments 110-121, wherein the sialylation amount is increased by at least 10% points.

Embodiment 123. The method of any of embodiments 110-121, wherein the sialylation amount is increased by at least 20% points.

Embodiment 124. The method of any of embodiments 110-121, wherein the sialylation amount is increased by at least 30% points.

Embodiment 125. The method of any one of embodiments 110-121, wherein the sialylation amount is increased by at least 40% points.

Embodiment 126 the method of any one of embodiments 110-125, wherein the antibody or fragment, derivative or graft thereof to be reduced in immunogenicity has a relative sialylation amount of 20% or less.

Embodiment 127. The method of any one of embodiments 110 to 125, wherein the antibody or fragment, derivative or graft thereof to be reduced in immunogenicity has a relative sialylation amount of 10% or less.

Embodiment 128 the method of any one of embodiments 110 to 125, wherein the antibody or fragment, derivative or graft thereof to be reduced in immunogenicity has a relative sialylation amount of 5% or less.

Embodiment 129 the method of any of embodiments 110 to 125, wherein the antibody or fragment, derivative or graft thereof to be reduced in immunogenicity has a relative sialylation amount of 2% or less.

Embodiment 130 the method of any one of embodiments 110 to 129, wherein the antibody or fragment, derivative or graft thereof having reduced immunogenicity is less likely to elicit an immune response against it when administered to a patient.

Embodiment 131. The method of any one of embodiments 110 to 130, wherein the recognition and uptake of the antibody or fragment, derivative or graft thereof having reduced immunogenicity by dendritic cells is reduced.

Embodiment 132 the method of any one of embodiments 110 to 131, wherein the antibody or fragment, derivative or graft thereof having reduced immunogenicity induces a T cell response thereto and/or has reduced ability to induce anti-drug antibodies thereto.

Drawings

Fig. 1 shows the experimental design of the hypersalinylation evaluation. Eight different vectors were transfected into three different CHO clones expressing "single arm antibody" molecules.

Fig. 2 schematically shows different vector strategies (left; i=st6gal-I, ii=b4gal1 and iii=slc35a1). The results of the mass spectrometry analysis of the Fc-glycan profile from the single arm antibody pool of protein a purification are listed on the right. The first column shows the vector strategy, the second column shows the transfection performed and how many pools have recovered the selection phase, the other columns show the relative percentages of specific glycans and sialidases.

FIG. 3 shows the productivity (titer) of "non-glycoengineered" control clones and identical clones stably transfected with different sugar vectors.

FIG. 4 schematically shows different vector strategies (transfection in CHO cell lines; left). The relevant glycoprofile data for three Fc fusion proteins ("Fc-trimers") transiently transfected are highlighted on the right. The first column shows the vector strategy, the second column shows the number of transfections performed, and the other columns show the corresponding percentage values for the specific glycosylation (only 2, 6-sialidases are shown).

FIG. 5 schematically shows a different vector strategy transfected in a different CHO clone than in FIG. 4 (left). Relevant glycoprofile data for transiently transfected Fc-trimers are highlighted on the right. The first column shows the vector strategy, the second column shows the transfection performed, and the other columns show the corresponding percentage values for the specific glycosylation (only 2, 6-linked sialidases are shown).

FIG. 6 shows titers of five Fc fusion proteins ("Fc-pentamers") and their variants with point mutations, CHO, and glycoengineered CHO (geCHO).

FIG. 7 shows the glycoprofile of Fc-pentamers (expressed as CHO and geCHO) and variants thereof having point mutations (expressed as geCHO). The first column shows the transfection performed and the other columns show the corresponding percentage values for the specific glycosylation (only 2, 6-sialidases are shown).

FIG. 8 shows the experimental design for determining the relevant factors for glycosylation.

FIG. 9 shows the productivity (titer) of OAA clones stably transfected with different sugar vectors.

Fig. 10 shows sialylation levels (mean and standard deviation of each three transfections) of OAA clones stably transfected with different sugar vectors.

FIG. 11 shows the titer data of Fc-trimer complexes expressed as geCHO (clones).

FIG. 12 shows sialylation levels of Fc-trimer complexes expressed in geCHO (clones).

FIG. 13 shows titer data for two Fc-trimer complexes expressed as parental CHO and geCHO (clones).

FIG. 14 shows sialylation levels of Fc-trimer complexes expressed as parental CHO and geCHO (clones).

Figure 15 shows the titer data (fed-batch) for the different products (2-3 biological replicates each).

FIG. 16 shows the 2, 6-and 2, 3-sialylation levels of the different products (2-3 biological replicates each).

FIG. 17 shows growth (A), viability (B), product concentration (C) and specific productivity qp (D) for process conditions 1 (square) and 2 (round) in a 10L laboratory scale bioreactor.

Figure 18 shows glycan species over time (days 7, 10, 13, and 14) for two different process conditions (left: process condition 2, right: process condition 1). bG0 is the sum of bG0-GlcNac-F (%), bG0-GlcNac (%), bG0-F (%) and bG0 (%). bG1 is the sum of bG1-GlcNac, bG1-F (%), 1.6-bG1 (%) and 1.3-bG1 (%). The mannose species is the sum of M5 (%) and M6 (%). 2.3 sialic acid (formation) is the sum of bG1-2.3-S1 (%), bG2-2.3-S1 (%) and bG2-2.3-S2 (%). Total 2.6 sialic acid (formation) is the sum of bG1-2.3-S1 (%), 1.6bG1-2.6-S1 (%), 1.3bG1-2.6-S1 (%), bG2-2.3-S1 (%), bG2-2.6-S1 (%), bG2-2.3-S2 (%), bG2-2.3/2.6-S2 (%) and bG2-2.6-S2 (%).

FIG. 19 shows viable cell concentration, viability and product concentration (titer) of geCHO parental cells co-cultured with (squares) and without (circles) product in 100mL shake flasks in fed-batch mode over a 14 day culture period.

FIG. 20 shows the average of the polysaccharide species in a 100mL shake flask in fed-batch mode for 14 days of incubation period for the addition of the standard product (Fc-trimer).

FIG. 21 shows FPKM values, a list of housekeeping genes, and average and median values of all expressed genes of the parental CHO cell lines for endogenously expressed St6gal1, B4gal 1 and Slc35a 1.

FIG. 22 shows the FPKM values of exogenously and endogenously expressed St6gal1, B4gal 1 and Slc35a1, the list of housekeeping genes, and the average and median values of all expressed genes of the parental geCHO cell lines.

FIG. 23 shows viability of CHO cells transfected with vector p001 during selection.

Figure 24 shows internalization of mAbX1 variants by IDC measured by indirect FACS method. A: DC internalization of WT, N297A, mannosylation (HiMan) and hypersialylation (HySi) mAbX1 at 60 min; normalized from% mAbX1 at Δ4deg.C and 37deg.C on live IDC, n=6 human donors; statistical analysis was performed by the Kruskal-walli chi-squared test (Kruskal-walli chi-squared test); b: binding of mAbX1 glycovariants to antigen positive Ramos cells.

Figure 25 shows binding and internalization of mAbX1 glycovariants detected by confocal microscopy and real-time imaging. A: quantification of intracellular mAbX1 staining from confocal images after DC uptake of mAbX1 glycovariants at 37℃with HALO ^TM Image analysis software (Akoya)) Calculation ofN=3 images/variants. B: internalization was indicated by kinetics of IDC internalization of mAbX1 glycovariants using an IncuCyte real-time imager (median intensity from 25 human donors: fluorescence signal derived from AF 647-labeled mAbX1 glycovariants).

Figure 26 shows T cell responses to mAbX1 glycovariants. A: proliferation and cd25+ Th cell counts determined from PBMCs of 16 human naive donors primed and challenged with each mAbX1 glycovariant (10 μg/mL); b: response index of T cell assay in the same donor group, response donor: donors with stimulation indices higher than 1.5 (dashed line); c: the SI of the WT responders; d: representative dot plots of proliferating cd25+ Th cells with (+) or without (-) challenge (=priming response) from one human donor.

Examples

Example 1: glycoengineering of CHO cells.

Various cell line engineering strategies for the production of hypersalinated proteins in CHO host cell lines were evaluated. These include overexpression of one, two or three related genes derived from chinese mice:

1: ST6Gal-I: the CHO glycosylation machinery is very similar to that found in human cells, but lacks α -2, 6-sialyltransferase-I activity (ST 6 Gal-I). This gene is responsible for the addition of sialic acid to galactose residues via the alpha-2, 6 linkage.

2: b4galt1: beta-1, 4-galactosyltransferase (B4 galt 1) is responsible for catalyzing the transfer of galactose to ethylene glycol proteins and is therefore used to synthesize complex N-linked oligosaccharides (bG 1 and bG2 increase).

3: overexpression of Slc35a 1: over-expression of CMP-sialic acid transporter (CMPSAT), a nucleotide sugar transporter, can improve sialylation processes in chinese hamster ovary Cells (CHO) by increasing the transport of CMP-sialic acid to the golgi apparatus, resulting in increased CMP-sialic acid endoluminal pool and increased sialylation of the produced protein.

Various promoters that increase 2, 6-sialylation, IRES elements, GFP as a selectable marker, and combinations of these three genes were evaluated. A total of 9 vectors were generated and stably transfected in three CHO clones expressing the "single arm antibody" form (see fig. 1 for experimental setup).

Puromycin was used as a selectable marker for all vector strategies. Fc-glycan profiling was performed by mass spectrometry of a single arm antibody pool purified of protein a. The sugar analysis data for this evaluation experiment are summarized in fig. 2. The MS method is semi-quantitative and is used to rapidly screen pool samples. For the following experiments, a more accurate method of releasing glycans was used, which uses 2-AB labelling and HILIC-FLD chromatography, which can distinguish between 2, 6-linked and 2, 3-linked sialidases.

Surprisingly, the highest sialylation can be achieved using the application strategy of vectors "p003" and "p 006"; both have a strong CMV promoter upstream of ST6Gal-I and expression of additional B4galt1 and Slc35a1 genes (downstream of the medium strength SV40 promoter). The average of total sialylation measured by mass spectrometry was 52.5% and 42.9%, respectively. These data were confirmed using the 2-AB HILIC-FLD method. Furthermore, the use of 2, 3-linked and 2, 6-linked sialoglycan reference standards enables the differentiation of 2, 3-linked and 2, 6-linked sialic acids. 2, 6-linked sialidases are the predominant form (less than 5% 2, 3-linked sialic acid, corresponding to the overall sialylation level of the parental CHO cell).

In the first approach, the transfection vector "p001" produced a non-viable pool, although the number of transfections performed was twice that of the other strategies. This is the only strategy with a strong CMV promoter upstream of B4galt 1. Microarray transcriptomics data (CHO cell line used for this experiment) highlighted that ST6Gal-I was not expressed, B4galt1 was expressed very low, and Slc35a1 was expressed moderately. Thus, we originally expected that "high overexpression" of B4galt1 (using the CMV promoter) was beneficial for high sialylation levels, and unexpectedly "high overexpression" of B4galt1 produced a non-viable pool, while "medium high overexpression" (using the SV40 promoter) did not show negative effects. However, in the second method, the cells survive the transfection/selection phase with vector p 001. Overall, crisis was selected longer than usual, but after 35 days all pools had viability higher than 80% (see fig. 23). Thus, we were able to generate parental cells stably transfected with this plasmid.

The correlation of the over-expressed gene B4galt1 is evident in view of the glycosylation data of vector strategy "p 007". This is the only strategy without over-expression of B4galt 1. The deletion of B4galt1 over-expression resulted in a higher amount of bG0 (38%) compared to other strategies (4% -16%). Galactosyl is a terminal sialylated substrate; thus, the "p007" strategy also resulted in the lowest sialylation compared to the other strategies.

Example 2: productivity of sugar engineered cells.

Another related subject matter is the productivity of a glycoengineered cell line. Thus, it was determined whether overexpression of the three genes had any effect on productivity compared to the non-glycoengineered controls.

As shown in fig. 3, no effect on productivity was detected after stable transfection of single arm antibody clones (control) with different "glycoengineered vectors". Three different single arm antibody expressing clones were used for expression and no positive or negative effect on productivity could be measured in either clone compared to the non-glycoengineered cell line.

Example 3: stable transfection of CHO cell lines.

In the next step, four best vector strategies were stably transfected into the parental CHO cell line clones (strategies p002, p003, p006 and p 008). Each strategy was performed 3 to 5 transfections and stable pools were generated. To assess the ability of these pools to sialylate, fusion proteins containing three Fc fragments ("Fc-trimers") were transiently transfected. The glycoprofile was determined via the 2-AB HILIC-FLD method.

Puromycin was used as a selectable marker for all vector strategies. The results are shown in fig. 4. This data is very relevant to the first evaluation with single arm antibodies. Vector strategies with p003 (strong CMV promoter upstream of ST6Gal-I, and additionally expression of B4galt1 and Slc35a1 genes) resulted in the highest sialylation levels (2, 6 sialylation levels are shown in fig. 4). Most of the glycopatterns are bG2SA (biantennary complex glycans with 2 galactose and at least 1 sialic acid): 32% are bG2S2 (double-antennary complex glycans with 2 galactose and 2 sialic acids) and 29% are bG2S1 (double-antennary complex glycans with 2 galactose and 1 sialic acid). Although all four strategies used the same SV40 promoter upstream of B4galt1, the p003 strategy unexpectedly produced significantly lower amounts of bG0 and bG1 (bi-antennary complex glycans with 0 or 1 galactose, respectively) than the other strategies.

Similar data was also achieved by transfection of the same four vectors in different parental CHO clones, which are less suitable for expression of therapeutic proteins (lower titers) and also show overall lower sialylation levels (see fig. 5). However, the p003 strategy also shows here the highest level of sialylation, and the majority of the glycopatterns are bG2SA (16% are bG2S2 and 20% are bG2S 1).

Another set of experiments was performed with different sample proteins. Two stabilization pools transfected with p003 (see fig. 4) were mixed and then stably transfected with a vector encoding a fusion protein comprising five Fc fragments ("Fc-pentamer"). The same procedure was performed with variants of Fc-pentamers having point mutations in the Fc portion (single amino acid mutations in the Fc portion are known to enhance galactosylation and sialylation). In addition, this Fc-pentamer was also transfected in the parental CHO cell clone. After one selection step, the productivity is determined and glycan analysis is performed.

Very high productivity can be measured for both cell lines and all constructs. Titers of Fc-pentamers and variants with point mutations were comparable in geCHO (see fig. 6).

Sugar analysis of the Fc-pentamer showed that no 2, 6-sialylation was detected in the parental CHO cell clone (as expected) (fig. 7). In contrast, the Fc-pentamer expressed in geCHO and the variant are 2, 6-sialylated. The overall 2, 6-linked sialylation of the Fc-pentamer was lower (about 30% of the total 2, 6-linked sialylation) compared to transient transfection of the Fc-trimer in the same pool (about 60%). This may be due to more complex molecules (pentamers than trimers) or lack of selection pressure during the selection phase of the Fc-pentamers (only the selection agent for Fc-pentamers is applied, but no selection agent for the p003 construct (puromycin)) is applied). However, point mutant variants show high sialylation up to more than 60%. In all of the above examples, the distribution of bG2S2 and bG2S1 is about the same, while the Fc-pentamer has less than 1% bG2S2 and 21% bG2S1. In contrast, the variants had 40% bG2S2 and 13% bG2S1.

Up to now, it can be clearly shown that vector strategies with strong promoters upstream of ST6Gal-I (e.g. CMV promoter) and additionally over-expression of B4galt1 and Slc35a1 genes with medium strength promoters (e.g. SV40 promoter) without any additional elements (e.g. IRES) produce the highest sialylation. The second highest sialylation was detected by a similar vector method but with an IRES element downstream of ST6GAL-1 followed by the GFP cassette. Direct comparison of the CMV promoter upstream of ST6Gal-I with the SV40 promoter upstream of ST6Gal-I shows that ST6Gal-I expression driven by the CMV promoter results in significantly higher sialylation.

In addition, over-expression of B4galt1 is necessary to increase sialylation. Sialic acid is attached to terminal galactose residues of the N-glycan structure. Higher B4galt1 activity increases the amount of such galactose residues and thus provides more attachment sites for sialic acid. Thus, the increased sialylation activity provided by overexpression of the α -2, 6-sialyltransferase ST6GAL-1 and the CMP-sialic acid transporter Slc35a1 is further improved by overexpression of the β -1, 4-galactosyltransferase B4galt 1. Application of the p007 strategy (over-expression of ST6GAL-1 only and over-expression of B4GAL 1) resulted in the lowest sialylation level.

Example 4: correlation of sialic acid transporter.

Slc35a1 is necessary to increase the transport of CMP-sialic acid to the Golgi apparatus, resulting in an increase in the pool in the CMP-sialic acid chamber. In p002 and p008, with a weaker SV40 promoter upstream of ST6Gal-I, we speculate that intracavity CMP-sialic acid is not yet a limiting factor, and thus whether this gene expression has no effect. However, with a very strong CMV promoter upstream of ST6Gal-I, intracavity CMP-sialic acid may become a limiting factor, and overexpression of Slc35a1 may become beneficial. To evaluate this factor, a comparison shown in fig. 8 was made.

Thus, no differences in productivity (titer) were detected for any sugar strategy (see fig. 9). FIG. 10 shows the sialylation levels of different strategies (overexpression of one, two or three "glycoengineered genes" (including overexpression of ST6Gal-I using SV40 or CMV promoters)). Overexpression of ST6Gal-I using only the SV40 promoter resulted in the lowest sialylation level (see fig. 10). Additional overexpression of B4galt1 or B4galt1 plus Slc35a1 resulted in increased sialylation levels. This is likely due to increased galactose as a substrate for further sialylation (bG 0 levels between 31% -34% if only ST6Gal-I is expressed and to 14% -20% if B4galt1 or B4galt1 plus Slc35a1 is additionally expressed). There is no difference if Slc35a1 expresses or does not indicate that sialic acid is not restricted in the golgi.

In contrast to the same vector strategy using the SV40 promoter, the use of a strong CMV promoter upstream of ST6Gal-I only overexpresses ST6Gal-I or overexpresses ST6Gal-I plus B4galt1 does not result in any increased sialylation. Surprisingly, a significant increase in sialylation was detected once Slc35a1 was additionally expressed alongside ST6Gal-I (downstream of CMV promoter) and B4galt1 (see fig. 10). It appears that the amount of sialic acid in the golgi will limit high sialylation as long as Slc35a1 is overexpressed.

Example 5: expression of different products in sugar engineered CHO cell lines.

Parental CHO and geCHO pools (stably transfected with vectors encoding ST6Gal-I (downstream of CMV promoter), B4galt1 and Slc35a 1) were evaluated with different projects up to pool levels, as shown in fig. 5.

Four Fc-trimeric protein constructs (with different Fc-multimerization domains, linker lengths and amino acid exchanges of the three constructs) were expressed in the geCHO clone. Expression of the constructs without amino acid exchange resulted in the highest titres and the lowest sialylation (titres were almost 2-fold higher but total sialylation levels were 10% lower) (fig. 11 and 12). Amino acid exchanges are known to have enhanced effector functions. However, the titres of all constructs are in a good range and the sialylation level is unexpectedly high. These data highlight that the geCHO clone is capable of good productivity and high sialylation levels for this complex molecule.

Furthermore, two Fc-trimeric protein constructs (the only difference being the linker length) were expressed in the parental CHO and geCHO clones. Both constructs have amino acid exchanges. The expression levels of CHO and geCHO were comparable (CHO showed higher titres for one Fc-trimer construct and geCHO for the other construct) (fig. 13 and 14). The overall sialylation level of the construct with short linker was about 65% and the overall sialylation level of the construct with long linker was about 75% (if expressed in the geCHO clone) (the amount of 2, 3-sialylation level was below 5%). In cynomolgus monkey and mouse studies, higher sialylation amounts were shown to be more effective and produced longer half-lives.

The 2, 6-sialylation ability of the optimal geCHO clone (stably transfected with vectors encoding ST6Gal-I (downstream of CMV promoter), B4galt1 and Slc35a1 (downstream of SV40 promoter)) was further evaluated with various different therapeutic protein forms up to pool levels. The protein evaluated was one Fc wild-type IgG antibody (mAb 1), another IgG antibody with an Fc wild-type form, an Fc half-life extended form, or a DAPA silenced form (mAb 2), and an Fc fusion protein (DAPA form). The titer of fed-batch and sialylation levels are shown in fig. 15 and 16.

The example of mAb2 shows that Fc structure also plays a role in the extent of 2, 6-sialylation. mAb2 WT showed the lowest degree of 2, 6-sialylation (53% sialylation), the half-life extended form showed a slight increase in 2, 6-sialylation (60% sialylation), and the DAPA form showed a significant increase in 2, 6-sialylation (79% sialylation). DAPA forms contain key point mutations that cancel Fc receptor (fcγr, fcR) binding, thereby eliminating antibody directed cytotoxicity (ADCC) effector function. The DAPA mutant set is thought to affect the conformation of the Fc domain in some way and thus alter Fc glycosylation by opening the Fc structure around the N297 site. For example, enzymes like galactosyltransferases or sialyltransferases may better access the N-linked glycosylation site. In addition, the half-life extended form with mutations in the constant domain CH2 may induce minor conformational changes, leading to a more open "horseshoe" -Fc and better entry of the enzyme into glycosylation sites N297 and N297'.

Example 6: cell culture process with sugar engineered CHO cell line.

Cell culture production processes for the geCHO cell line were developed to produce highly sialylated Fc-multimers. The production process involves thawing the cells in expansion medium (including puromycin and MTX) and dividing the cells twice at a rhythm of 4:3:4 (4 days 3 days 4 days) before growing them in a 10L laboratory scale production bioreactor. The process conditions applied can be seen in table 1.

Table 1:summary of production process conditions for studying the effect of geCHO cells expressing recombinant proteins on growth, product concentration and sialylation degree.

Growth and productivity data for process 1 and process 2 are shown in fig. 17. Under process condition 1, cells grew to 19e6 viable cells/mL, which was higher than process condition 2 (maximum cell density 15e6 VCD/mL). After 14 days of culture, the viability of the cells under process condition 1 was 20% higher than that of the cells under process condition 2 (79% vs 59% viability). The product concentration at process condition 1 (4 g/L) was 34% higher than that at process condition 2 (2.65 g/L). The higher viable cell density and product concentration at process condition 1 produced a specific productivity 36% (33.34 pg/VC/d versus 24.44 pg/VC/d) higher than process condition 2.

In addition to growth and product formation, the degree of sialylation of the product is of paramount importance. In fig. 18, sialylation levels over time for two test process conditions are compared (days 7, 10, 13 and 14; condition 2 is shown on the left and condition 1 is shown on the right). Under both process conditions, sialylation levels were identical (50%) at day 7. By day 14, sialylation levels unexpectedly decreased to 40.4% and 27.4% under process conditions 1 and 2, respectively. Thus, under process condition 1, the sialylation level was more stable and decreased to a lower extent than under process condition 2. The final sialylation degree under process condition 1 was 47% higher than that under process condition 2.

Externally expressed sialidases or neuraminidases (Neu 1, neu2, neu3 and Neu4, see Smutova et al (2014) PLoS ONE 9 (9): e 106320) are thought to reduce the overall sialylation level of the expressed molecules. Thus, the following labeling experiments were performed:

shake flask experiments (500 ml,100ml working volume, 200rpm,5% co) were performed using cells of the parental geCHO cell line master cell bank ₂ ) Duplicate and process condition 1 (see table 1 above) was applied. Polishing material for the product (Fc-trimer) was added to a set of shake flasks on day 0. The other flask was not added with any product, which served as a reference.

The viable cells, viability, and product concentration (titer) of the experimental setup are shown in fig. 19. There was no significant difference in viable cell density and viability between the two settings. The product concentration remained unchanged over a 14 day incubation period, although viability was reduced at the end of incubation. Thus, it can be concluded that the product has no effect on cell growth and viability.

Fig. 20 shows the sialylation level of the product from shake flask 1. On days 0, 7, 10 and 14, the 2.6-sialylation levels were 68.7%, 70.0%, 70.8% and 70.4%, respectively, and thus the 2.6-sialylation levels did not change over time. Under these culture parameters, neuraminidase appears to have no effect on sialylation pattern.

Summarizing:

by growing cells at a constant temperature of 36.5 ℃ the cells grew to a higher cell number, the product concentration was higher, and the sialylation level was unexpectedly 47% higher, compared to the same procedure where the culture temperature was changed to 33 ℃ once the culture reached a high cell density. In addition, process condition 1 increases the simplicity of the production process, since one piece less process events (no culture temperature change) are required to be considered. Thus, the process is less likely to deviate and is therefore more robust.

It can also be shown that the reduced sialylation level is not due to externally expressed sialidase activity in the medium. The product was unexpectedly stable over the incubation period.

Example 7: expression level of the introduced glycosylase.

To determine the gene expression levels of the exogenous expression genes St6gal1 (downstream of the CMV promoter), B4galt1 (downstream of the SV40 promoter) and Slc35a1 (downstream of the SV40 promoter) and thus the strength of the corresponding promoters, next generation sequencing (transcriptomics) was performed. Sequencing libraries were prepared using the TruSeq strand total RNA sample preparation of Illumina and Ribo-Zero Gold, and sequenced in double-ended mode (2x76+8) with a 76bp read on HiSeq 2500. Sequence reads were then aligned with the gcf_000223135.1_crigri_1.0 reference genome using STAR (version 2.5.2a), and transcript counts at the gene level were normalized to FPKM (fragments of transcripts per kilobase per million map reads).

In fig. 21 and 22, the gene expression values of 12 representative endogenous expression housekeeping genes are shown as FPKM. In addition, the average and median of all expressed genes (18,516 genes) are also shown. The gene expression level of housekeeping genes varied between about 20 and 4000 FPKM. The average value of all expressed genes was 30FPKM, and the median was about 6FPKM. In addition, these figures also show the expression values of endogenously expressed St6gal1, B4gal 1 and Slc35a 1. The values shown in figure 21 are for parental CHO and the values shown in figure 22 are for geCHO (clone selected for MCB).

The average and median gene expression of these housekeeping genes and all expressed genes were very similar for the geCHO cell line (derived from the parental CHO cell line). In addition, we also measured the gene expression of the exogenously expressed genes St6gal1, B4gal 1 and Slc35a 1. The highest expressing gene was St6gal1 downstream of the CMV promoter. The gene expression of exogenous B4galt1 and Slc35a1 (both downstream of the SV40 promoter) was 16-fold lower than that of St6gal1 driven by the CMV promoter. In general, the gene expression value of St6gal1 (downstream of the CMV promoter) was the highest of all expressed genes (the expression was almost 3-fold higher compared to the highest expressing housekeeping gene), highlighting that the CMV promoter is driving very strong gene expression. In contrast, the B4galt1 and Slc35a1 genes downstream of the SV40 promoter were in a similar range to the housekeeping genes expressed at medium intensity (FPKM values of about 300-1800). The gene expression value of endogenous B4galt1 was 55-fold lower than that of exogenous B4galt1, and the gene expression of endogenous Slc35a1 was 15-fold lower than that of exogenous Slc35a 1. As expected, endogenous St6gal1 was not expressed in CHO.

Example 8: recognition and uptake of hypersialylated antibodies by dendritic cells.

FACS-based assays

Binding and internalization of model antibodies (mAbX 1) produced in parental CHO (WT) or geCHO (HySi) was assessed on Immature Dendritic Cells (IDC) using FACS-based assays. As a further control, mAbX1 with a large amount of high mannose glycans (HiMan) and mAbX1 in which the glycosylation site was removed by the N297A mutation (N297A) were used.

For FACS-based internalization assays, IDC (1.5 x 10 ⁵ Sample/sample) with 10. Mu.g/mL unlabeled mAbX1 glycovariants in binding buffer (HBS (Hepes buffered saline) +1mM CaCl ₂ 、1mM MgCl ₂ 、1mM MnCl ₂ ) Incubate at 4 ℃ (for binding) or 37 ℃ (for internalization) for 15, 30, 60 and 120min. IDC was washed to remove excess free mAbX1. The residual amount of surface bound mAbX1 was then detected using 10. Mu.g/mL FITC-labeled anti-mAbX 1. After another washing step, the stained IDC was fixed (1% paraformaldehyde) and measured on an Attune NxT flow cytometer. Initially staining antigen positive Ramos cells was performed to confirm that the indirect staining protocol was effective and that mAbX1 glycovariants bound equally to specific antigen.

The difference in fluorescence signals at 4 ℃ and 37 ℃ was calculated and scaled using the plosis () function in R and expressed as a percentage of internalization.

Mannosylation of mAbX1 significantly increased recognition and internalization (median internalization=84.6%) compared to WT (median internalization=3.6%). In contrast, hypersialylation of mAbX1 (median internalization=0.2%) reduced recognition and internalization compared to WT (fig. 24A).

Altered glycosylation did not affect Fab mediated binding to the target antigen, as all mAbX1 glycovariants showed equal binding to antigen positive Ramos cells (fig. 24B).

In summary, the data show that recognition of IDC can be affected by modifying antibody glycosylation, where high sialylation reduces IDC recognition of the antibody.

Confocal microscopy

The FACS-based internalization assay indicates only residual mAbX1 cell surface binding. To demonstrate that mAbX1 was absorbed into cells, binding and internalization of fluorescent dye-labeled mAbX1 glycovariants by IDC was assessed by confocal microscopy.

IDC is set to 3x10 ⁵ Individual cells were seeded in 300 μl of differentiation medium/chamber of 8-well chamber slides and incubated at 37 ℃ and 5% co ₂ Incubate overnight. On the next day, binding buffer (HBS+1 mM CaCl) containing AF647 conjugate of mAbX1 glycovariant at 20. Mu.g/mL was used ₂ ，1mM MgCl ₂ ) The medium was replaced and incubated at 4 ℃ (refrigerator) and 37℃with 5% CO ₂ Incubate for 120min. IDC was immobilized (4% paraformaldehyde) and permeabilized (0.1% triton X-100 in PBS). After a blocking step with 2% BSA in PBS, IDCs were incubated with labeled antibodies to LAMP-1, EEA1 and Rab7, followed by incubation with secondary donkey anti-rabbit-AF 488. DAPI (4', 6-diamidino-2-phenylindole) was added as a nuclear stain. The stained IDC was mounted in FluoSafe reagent (Calbiochem corporation) and covered with a glass slide. The cured slide was imaged on Olympus FV3000 at 40x magnification. Using HALO ^TM Image analysis software (Akoya Biosciences company) quantified internalized mAbX 1.

Confocal images obtained from IDC incubated at 4 ℃ showed that mAbX1 was localized only at the cell surface that is unique to the expected loop structure of the fluorescent signal. mAbX 1-derived fluorescence (stained with DAPI) was detected in the cytoplasm adjacent to the nucleus at 37℃and no longer detected on the surface.

The glycosylation-dependent binding and internalization pattern of mAbX1 observed by FACS can be summarized as: non-glycosylated mAbX1 showed lower binding and internalization relative to WT, mannosylated mAbX1 showed the strongest binding and internalization (fig. 25A). Interestingly, hypersalinated mAbX1 showed a strong binding signal comparable to mannosylated mAbX1 at 4 ℃. However, only very weak internalization was detected for HySi at 37 ℃. This observation suggests that HySi is recognized by lectin receptors that are different from mannosylated mAbX 1. Such unknown receptors appear to cause strong interactions but do not lead to internalization of the binding ligand and thus may exert inhibitory functions.

Next, the effect of mAbX1 glycosylation on endosomal routing was assessed. For this purpose, IDC was stained with lysosome-labeled LAMP-1 after internalization of the fluorochrome-labeled mAbX 1. mAbX1 was detected in lysosomal compartments and this effect was closely related to glycosylation pattern. Consistent with the strongest internalization of mannosylated mAbX1, this glycovariant resulted in the most prominent pathway selection into lysosomes. In contrast, hypersalinated mAbX1 exhibited weaker or undetectable co-localization with lysosomes. N297A shows similar or slightly higher co-localization as LAMP-1. EEA1 and Rab7 were used as markers for assessing potential glycosylation-related differences to pathway selection in early and late endosomes, respectively, in addition to LAMP-1. Obvious co-localization with both markers was detected for HiMan, weaker co-localization was detected for WT and N297A, slightly stronger co-localization was detected for N297A, and barely detectable co-localization was detected for HySi. At the test time point (2 h), co-localization of mannosylated mAbX1 with early endosomes appears to dominate. For other glycovariants, no difference in pathway selection into early or late endosomes could be observed.

Taken together, the data show that mAbX1 glycosylation determines the pattern of surface binding and intracellular uptake by IDC. In addition, hypersalivation was shown to reduce the pathway selection of mAbX1 into the degradation pathway.

Real-time imaging

Based on use of an IncuCyte analyzerLive cell image analyzers (Sartorius) imaging in real time established another method of determining internalization over time. The principle is based on the detection of intracellular fluorescent signals resulting from internalization of fluorescent dye-labeled mAbX1 glycovariants.

IDC is 1x10 ⁵ Well/100. Mu.L was seeded into clear flat bottom 96-well plates and incubated at 37℃and 5% CO ₂ Incubate overnight. On the next day, after the plates were kept in the refrigerator for 10min, the medium was replaced with medium containing 10 μg/mL AF647 conjugated mAbX1 glycovariant. After 30min incubation in the refrigerator, the supernatant was replaced with warmed medium and immediately placed in the IncuCyte analyzer. Images were acquired at 20x magnification, once every 20 minutes for the first 5 hours and once every hour thereafter, for up to 24 hours. Internalization was determined by quantification of intracellular fluorescence. To this end, a mask was created on the phase object and the fluorescent object using Basic analyzer mode to capture IDC and internalized mAbX1, respectively. Cumulative intensity (RCU x μm2)/well is reported.

In the same donor, results obtained from the indirect FACS assay and the IncuCyte assay are similar. Measurement of the glycosylation-dependent internalization of mAbX1 by IDC by the IncuCyte assay confirmed that at all measurement time points, mannosylation increased internalization and hypersalinylation decreased internalization (fig. 25B).

If the slope of each glycovariant is compared, a kinetic difference can be observed. The slope is a measure indicating the rate of internalization per time unit. HiMan exhibits the highest slope (0.273), followed by N297A (0.06) and WT (0.04). HySi exhibits the lowest slope (0.02). Thus, hiMan shows the highest internalization rate/rate, while HySi shows the lowest internalization rate/rate.

Example 9: recognition of hypersialylated antibodies by human T cells.

The next question is whether glycosylation-mediated effects on internalization observed for mAbX1 are associated with T cell activation. Thus, internalization and T cell activation were studied in the same donor set (naive donor).

PBMCs (peripheral blood mononuclear cells) were isolated from buffy coats donated by primary human subjects in a berni (Bern) donor center according to local ethical practices. The isolated PBMC were treated with 5. Mu.M CellTrace in a water bath (37 ℃) ^TM Violet (CTVio, life tech) staining for 20min. After incubating CTVio-doped cells with platelet-free autologous plasma for 5min (RT), excess CTVio was removed by centrifugation at 360g for 5 min. CTVio negative PBMCs were used as compensation controls and FMO (fluorescence minus one) controls. CTVio+PBMC at 1X 10 ⁶ Individual cells/mL were inoculated into 24-well plates in X-Vivo (Lonza) plus 5% platelet-free autologous plasma and stimulated (primed) with 1 and 10 μg/mL of the corresponding mAbX1 glycoconjugate, 5-30 μg/mL KLH (keyhole limpet hemocyanin, sameifer-tech (Thermo Scientific)), 0.5 μg/mL tetanus toxoid (TT, enzoie) or medium for 5 days. On day 5, the DC-PBMC co-cultures (1:10) were re-stimulated (stimulated) with 1 and 10. Mu.g/mL mAbX1 glycoconjugate, 5-30. Mu.g/mL KLH, 0.5. Mu.g/mL TT or medium and incubated in the presence of 5U/mL IL-2 for an additional 4 days. On day 9, the stimulated cells were harvested and stained with surface markers CD3, CD4, CD25, CD137, including viability dye (zembie aqua, biolegend). Stained cells were measured on an Attune NxT flow cytometer using constant volume termination conditions for all samples. FMO controls were used to distinguish positive from negative populations. Counts of proliferating and activating Th cells were determined at the time of priming and priming, and expressed as Stimulation Index (SI), indicating the priming response relative to the priming response. Donors exhibiting SI higher than 1.5 were designated as T cell responders.

5 of the 16 donors (31%) showed WT-specific responses (fig. 26B). Mannosylated mAbX1 enhanced the increased T cell response compared to WT, 7 responders (43%) out of 16 test donors. The hypersalinated mAbX1 enhanced the reduced T cell response compared to WT, with 4 responders (25%) out of 16 test donors. Notably, if T cells were primed and stimulated with HySi, the count of proliferating cd25+ Th cells was significantly reduced compared to all other mAbX1 glycovariants (fig. 26A and 26D). Non-glycosylated mAbX1 exhibited similar or slightly higher T cell activation (47%) compared to mannosylated mAbX 1. Donors were dissected in WT responders. All WT responders did not respond to hypersalinated mAbX1 (fig. 26C), while few WT responders reacted to HiMan and N297A. The positive control Tetanus Toxoid (TT) induced strong T cell activation (determined as CD25 upregulation) and proliferation, indicating good assay performance.

Taken together, these data demonstrate that T cell responses can be enhanced or inhibited if the fructosylation pattern is altered. Although mannosylation enhanced the T cell response compared to WT, hypersalinated mAbX1 was not recognized by WT responder T cells. This suggests that sialylation of mAbX1 does lead to reduced antigen presentation efficiency, does not provide a co-stimulatory signal to activate pre-existing WT-reactive T cells or/and promotes Treg polarisation.

Sequence listing

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Sequence listing

<110> Noval Co., ltd (Novartis AG)

<120> hypersialylated cells

<130> PAT059101-WO-PCT

<150> US 63/181,746

<151> 2021-04-29

<150> US 63/181,739

<151> 2021-04-29

<160> 6

<170> patent In version 3.5

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Gln Asp Pro Lys Gln Asp Ser Gln Val Leu Ser His Ala Arg Val Thr

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Leu Asn Met Asn Lys Tyr Lys Val Ser Tyr Lys Gly Pro Gly Pro Gly

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Val Lys Phe Ser Ala Glu Ala Leu Arg Cys Arg Leu Arg Asp Arg Val

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Asn Val Ser Met Ile Glu Ala Thr Asp Phe Pro Phe Asn Thr Thr Glu

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Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg

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His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala

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Lys Glu Thr Gly Ser Leu Gly Arg Phe Lys Ala Ser Leu Ser Glu Asn

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Val Leu Gly Ser Pro Lys Glu Leu Met Lys Leu Ser Val Pro Ser Leu

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Val Tyr Ala Val Gln Asn Asn Met Ala Phe Leu Ala Leu Ser Asn Leu

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Asp Ala Ala Val Tyr Gln Val Thr Tyr Gln Leu Lys Ile Pro Cys Thr

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Ala Leu Cys Thr Val Leu Met Leu Asn Arg Thr Leu Ser Lys Leu Gln

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Trp Val Ser Val Phe Met Leu Cys Gly Gly Val Ile Leu Val Gln Trp

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Lys Pro Ala Gln Ala Thr Lys Val Val Val Glu Gln Ser Pro Leu Leu

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Gly Phe Gly Ala Ile Ala Ile Ala Val Leu Cys Ser Gly Phe Ala Gly

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Val Tyr Phe Glu Lys Val Leu Lys Ser Ser Asp Thr Ser Leu Trp Val

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Arg Asn Ile Gln Met Tyr Leu Ser Gly Ile Val Val Thr Leu Val Gly

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Thr Tyr Leu Ser Asp Gly Ala Glu Ile Lys Glu Lys Gly Phe Phe Tyr

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Gly Phe Ser Ala Ala Ala Ala Ile Val Leu Ser Thr Ile Ala Ser Val

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Tyr Thr Arg Thr Ser Asp Lys Glu Leu Tyr Phe Ser Thr Thr Ala Val

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Cys Ile Thr Glu Val Ile Lys Leu Leu Leu Ser Val Gly Ile Leu Ala

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Lys Glu Thr Gly Ser Leu Gly Arg Phe Lys Ala Ser Leu Arg Glu Asn

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Val Leu Gly Ser Pro Lys Glu Leu Leu Lys Leu Ser Val Pro Ser Leu

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Val Tyr Ala Val Gln Asn Asn Met Ala Phe Leu Ala Leu Ser Asn Leu

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Asp Ala Ala Val Tyr Gln Val Thr Tyr Gln Leu Lys Ile Pro Cys Thr

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Ala Leu Cys Thr Val Leu Met Leu Asn Arg Thr Leu Ser Lys Leu Gln

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Trp Val Ser Val Phe Met Leu Cys Ala Gly Val Thr Leu Val Gln Trp

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Gly Leu Tyr Thr Ser Val Val Val Lys Tyr Thr Asp Asn Ile Met Lys

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Gly Phe Ser Ala Ala Ala Ala Ile Val Leu Ser Thr Ile Ala Ser Val

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Val

Claims

1. A mammalian cell engineered for increased expression of an α -2, 6-sialyltransferase, a β -1, 4-galactosyltransferase, and a CMP-sialic acid transporter.

2. The mammalian cell of claim 1, comprising

(i) Exogenous nucleic acid encoding an alpha-2, 6-sialyltransferase;

(ii) Exogenous nucleic acid encoding a beta-1, 4-galactosyltransferase; and

(iii) Exogenous nucleic acid encoding a CMP-sialic acid transporter.

3. The mammalian cell of claim 2, comprising

4. The mammalian cell of claim 1, wherein endogenous genes of the mammalian cell encoding α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter are engineered for increased expression.

5. The mammalian cell of claim 4, comprising

6. The mammalian cell of claim 3 or 5, wherein the first promoter is a strong promoter.

7. The mammalian cell of any one of claims 3, 5, and 6, wherein the first promoter is a Cytomegalovirus (CMV) promoter.

8. The mammalian cell of any one of claims 3 and 5 to 7, wherein the second promoter and/or the third promoter is selected from the group consisting of: simian Virus 40 (SV 40) promoter, CMV promoter, ubiquitin C (UBC) promoter, elongation factor 1 alpha (EF 1A) promoter, phosphoglycerate kinase (PGK) promoter and beta-actin promoter (CAGG) coupled to CMV early enhancer, especially SV40 promoter.

9. Mammalian cell according to any one of claims 1 to 8, wherein the α -2, 6-sialyltransferase is β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1), in particular derived from chinese ground mouse or human; the beta-1, 4-galactosyltransferase is beta-1, 4-galactosyltransferase 1 (B4 GALT 1), in particular derived from chinese mice or humans; and the CMP-sialic acid transporter is a CMP-sialic acid transporter (SLC 35 A1), particularly derived from chinese mice or humans.

10. The mammalian cell according to any one of claims 1 to 9, wherein the mammalian cell is a rodent cell or a human cell, in particular a Chinese Hamster Ovary (CHO) cell.

11. The mammalian cell of any one of claims 1 to 10, further comprising an exogenous expression cassette for recombinant expression of the glycosylated polypeptide.

12. A method for producing a glycosylated polypeptide, the method comprising the steps of:

(a) Providing a mammalian cell according to claim 11;

(c) Obtaining the glycosylated polypeptide from the cell culture; and

(d) Optionally processing the glycosylated polypeptide.

13. The method according to claim 12, wherein the culture conditions during the culture of the mammalian cells do not comprise a temperature change exceeding 2 ℃, in particular not exceeding 1 ℃.

14. The method of claim 12 or 13, wherein the temperature is maintained in the range of 35 ℃ to 38 ℃ during the culturing of the mammalian cells.

15. The method of any one of claims 12 to 14, wherein step (d) comprises providing a pharmaceutical formulation comprising the glycosylated polypeptide.

16. The method of any one of claims 12 to 15, wherein the method is for producing a glycosylated polypeptide having reduced immunogenicity, and wherein the glycosylated polypeptide is a therapeutic antibody or a fragment, derivative or graft thereof.

17. A vector nucleic acid or a combination of at least two vector nucleic acids comprising

(i) A coding sequence for an alpha-2, 6-sialyltransferase;

(ii) A coding sequence for a beta-1, 4-galactosyltransferase; and

(iii) Coding sequence for a CMP-sialic acid transporter.

18. The vector nucleic acid or the combination of at least two vector nucleic acids according to claim 17, comprising

(i) A first expression cassette comprising a first promoter operably linked to a coding sequence for said α -2, 6-sialyltransferase;

(ii) A second expression cassette comprising a second promoter operably linked to a coding sequence for said β -1, 4-galactosyltransferase; and

19. The vector nucleic acid or the combination of at least two vector nucleic acids of claim 18, wherein

(i) The first promoter is a cytomegalovirus promoter (CMV); and/or

(ii) The second promoter and/or the third promoter is selected from the group consisting of: simian virus 40 promoter (SV 40), CMV promoter, ubiquitin C (UBC) promoter, elongation factor 1A (EF 1A) promoter, phosphoglycerate kinase (PGK) promoter and beta-actin promoter (CAGG) coupled to CMV early enhancer, in particular SV40 promoter.

20. The vector nucleic acid or the combination of at least two vector nucleic acids according to any one of claims 17 to 19, wherein the α -2, 6-sialyltransferase is β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1), in particular derived from chinese mice or humans; the beta-1, 4-galactosyltransferase is beta-1, 4-galactosyltransferase 1 (B4 GALT 1), in particular derived from chinese mice or humans; and the CMP-sialic acid transporter is a CMP-sialic acid transporter (SLC 35 A1), particularly derived from chinese mice or humans.

21. Use of the vector nucleic acid or the combination of at least two vector nucleic acids according to any one of claims 17 to 20 for transfecting a mammalian cell, in particular a Chinese Hamster Ovary (CHO) cell.

22. A method for increasing expression of an α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and a CMP-sialic acid transporter in a mammalian cell, the method comprising the step of transfecting the mammalian cell with the vector nucleic acid of any one of claims 17 to 20 or a combination of at least two vector nucleic acids, and/or the step of engineering endogenous genes encoding the α -2, 6-sialyltransferase, β -1, 4-galactosyltransferase and CMP-sialic acid transporter of the mammalian cell for increased expression.

23. A method for reducing the immunogenicity of an antibody or fragment, derivative or graft thereof, the method comprising the step of increasing the amount of sialylation in the glycosylation pattern of the antibody or fragment, derivative or graft thereof.

24. The method of claim 23, wherein the step of increasing sialylation comprises one or more of:

(i) Treating the antibody or fragment, derivative or implant thereof with a sialyltransferase and a sialic acid donor such that sialic acid residues are attached to glycans present on the antibody or fragment, derivative or implant thereof;

(ii) Enriching those antibodies or fragments, derivatives or grafts thereof carrying at least one sialic acid;

(iii) The antibodies or fragments, derivatives or grafts thereof are produced using production methods that result in substantial sialylation.

25. The method of claim 23 or 24, wherein the immunogenicity of the antibody or fragment, derivative or graft thereof is reduced compared to a reference antibody or fragment, derivative or graft thereof having the same amino acid sequence and a relative sialylation amount of 5% or less, and wherein the step of increasing the sialylation amount comprises producing the antibody or fragment, derivative or graft thereof in a mammalian cell according to any one of claims 1 to 11 and/or using the method according to any one of claims 12 to 16.