TW201305341A - Production of low fucose antibodies in H4-II-E rat cells - Google Patents

Abstract

The invention concerns the field of cell culture technology. It specifically concerns a rat hepatoma cell, preferably a H4-II-E rat hepatoma cell, carrying a DNA encoding an antibody or Fc-fusion protein and having low fucosylation activity for adding fucose to glycosidic structures such as biantennary glycans, e.g. N-acetylglucosamine. The invention furthermore concerns a method for producing low fucose glycoproteins especially antibodies or Fc-fusion proteins in rat hepatoma cells, preferably in H4-II-E rat hepatoma cells. It further concerns the identification and generation of new host cell lines which are capable of synthetizing glycoproteins with beneficial properties, improving the therapeutic efficacy and/or serum half-life of the product compared to products from commonly used host cell lines.

Description

Production of low trehalose antibodies in H4-II-E rat cells

The present invention relates to the field of cell culture technology. It relates to a method for producing low trehalose glycoproteins, particularly antibodies and Fc-fusion proteins, in H4-II-E rat hepatoma cells. It relates to the identification and production of new host cell lines capable of synthesizing glycoproteins having beneficial properties, enhancing the therapeutic effect and/or serum half-life of the product relative to products from commonly used host cell lines. The present invention relates to the use and optimization of such cell lines, particularly the rat hepatoma cell line H4-II-E, for the expression of recombinant proteins and their use as therapeutic agents for highly active biopharmaceuticals.

The biopharmaceutical market for human therapy continues to grow at a rapid rate, with 270 new biopharmaceuticals evaluated in clinical studies and estimated sales of 30 billion in 2003 (Werner, 2004). Biopharmaceuticals can be produced from different host cell systems, including bacterial cells, yeast cells, insect cells, plant cells, and mammalian cells, as well as cell lines derived from humans. Currently, as eukaryotic cells are able to properly process and translate and recombine recombinant human proteins, more and more biopharmaceuticals are produced. Thus, a key issue affecting the selection of cell lines for use in the manufacturing process is the ability to consistently produce products with uniform post-translational modification patterns (resulting in high biological activity, stability, and batch-to-batch consistency).

The largest part of the anti-system currently licensed for therapeutic applications is manufactured in the Chinese hamster ovary (CHO) cell line. Other production systems are murine lymphoid cells (including NS0 and Sp2/0-Ag 14). These parental cell lines are also the most commonly used in antibody production in clinical trials. In addition, murines and other cell lines such as the human cell line PER.C6 are used.

The treatment of monoclonal antibodies has become one of the main focuses of the biotechnology industry. Although effective monoclonal antibodies approved by the Food and Drug Administration have been available to date, there is still a need for even more effective and compatible drugs to not only reduce manufacturing costs, but also help to apply more Many patients. In addition, the dosage of the optimized therapeutic agent can be reduced, thereby resulting in higher tolerance and lower adverse effects.

Among the five mammalian antibody classes IgA, IgD, IgE, IgG and IgM, the IgG class is mainly used for the treatment, prevention and diagnosis of various diseases. This is due to its advantageous functional properties (eg long half-life in blood) and various effector functions (eg antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)). Among the human IgG classes, subclass (isotype) IgG1 and IgG3 have the highest ADCC and CDC activities, but IgG3 is only 7 to 8 days compared to IgG1 with a half-life of up to 21 days. In view of the above, human IgG1 subclass antibodies are predominantly used if high activity effector functions are required for optimal therapeutic efficacy to remove cells bearing antigen on the surface.

Post-translational modifications are important not only for proper protein folding, intracellular transport, solubility and stability, but also for the biological activity and immunogenicity of secreted proteins. In biopharmaceutical proteins (eg, therapeutic antibodies), post-translational modifications have a specific effect on the therapeutic efficacy, pharmacokinetics, pharmacodynamics, and immunogenicity of the product.

Glycosylation represents the most extensive post-translational modification found in natural secreted proteins as well as in approved biopharmaceuticals. Almost 50% of human proteins are glycosylated. Aspartate (N)- and serine (O)-linked glycans are the two major glycocatelines formed on secreted glycoproteins derived from mammalian cells. The transfer of glycostructure to secreted protein occurs in the endoplasmic reticulum (ER) and Golgi apparatus and represents a complex enzymatic process that is regulated by the activity of numerous genes. Defects in many genes involved in the glycosylation pathway cause congenital conditions with severe medical consequences, which confirms the importance of normal glycosylation.

In eukaryotes, N-linked glycans are attached to proteins in the ER lumen in the form of pre-synthesized oligosaccharides consisting of branched oligosaccharide units (by 3 glucose (Glc), 9 nectars) The composition of sugar (Man) and two N-acetylglucosamine (GlcNAc). These core glycans (Glc ₃ Man ₉ GlcNAc ₂ ) are transferred via a lipid carrier (polysterol-pyrophosphate in the ER membrane) to secreted proteins that are translocated into the ER lumen. The glycan is transferred to the appropriate sequence (i.e., Asn-X-Ser/Thr), wherein any of the amino acids other than proline in the X-linked nascent glycoprotein. The correctly folded glycoprotein is actively transported into the Golgi apparatus, wherein its N-glycan is modified by glucosidase, mannosidase and glycosyltransferase to produce a complex structure containing sialic acid, trehalose and galactose. Glucosidase and mannosidase remove glucose (Glc) and mannose monosaccharide (Man) from glycans at the earliest stages of N-glycan processing. N-Ethyl Glucosaminease then catalyzes the addition of N-acetylglucosamine (GlcNAc) to the mannose attached to the conserved core structure of the N-glycan, which forms a branch on the glycan or The number of branch structures has a decisive role. The trehalose transferase adds trehalose to the N-acetylglucosamine adjacent to the protein and the galactosyltransferase and sialyltransferase add galactose and sialic acid to the ends of the N-glycan branch, respectively. The reaction of these enzymes is generally irreversible, resulting in a stable N-glycosylated protein.

Protein glycosylation can introduce significant heterogeneity by generating core oligosaccharides and performing differential modifications thereto and variable addition of exo-arm sugars.

Incorrect glycosylated or non-glycosylated antibodies have been shown to exhibit uncontrolled functions. Therefore, the selection of a suitable production system capable of consistently generating a product with the desired post-translational modification pattern is critical to successful drug manufacturing and application. The glycoform characteristics of glycoproteins and thus their functional activity can vary significantly depending on the production system. Biopharmaceutical proteins in prokaryotic production systems (e.g., Escherichia coli ) produce non-glycosylated protein products that must be recovered in vitro as dissolved and refolded inclusion bodies. In contrast, the yeast expression system adds a high mannose content sugar side chain, and the glycosylation pattern obtained after production in insect cells is also significantly different from the characteristic mammalian pattern. Plants can differentially glycosylate proteins, but consistently add alpha 1,3-trehalose and beta 1,3-xylose, which are reported to be immunogenic or allergenic in humans.

Different mammalian host cells are capable of differential N-glycan processing. Analysis of the glycoprotein produced shows a heterologous glycoform depending on the tissue or species from which the cell is derived. Therefore, it is important to ensure that the glycosylation pattern that produces the glycoprotein product for clinical use is uniform throughout and throughout the production batch, and also ensures that the beneficial in vivo properties of the antibody are maintained.

Both natural (inherent) antibodies in the body and antibodies produced by recombinant DNA techniques in mammalian host cell lines are covalently attached to oligosaccharides via the evolutionarily conserved Asn297 in the CH2 domain of the IgG-Fc region. Basic. Oligosaccharides are part of the IgG-Fc structure and are absolutely required for effector function. The interaction sites on IgG-Fc for effector ligands (eg, FcyRI, FcyRII, FcyRIII, and C1q) are composed of protein moieties; however, the formation of a basic IgG-Fc protein conformation depends on the presence and chemical composition of the oligosaccharides. Thus, the effector mechanisms mediated through the junction of effector ligands (cleavage mechanisms such as phagocytosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)) may be incorrect or non-glycosylated. IgG is severely damaged or removed.

The human FcγRIIIa receptor is polymorphic and has been shown to have an affinity for the FcγRIIIa-158V (proline) form to be higher than the FcγRIIIa-158F (phenylalanine) form. It was confirmed in vitro that the IgG1 antibody mediates ADCC more efficiently via homozygous FcγRIIIa-158F or hybrid FcγRIIIa-158V/FcγRIIIa-158F cells via cells bearing homozygous FcγRIIIa-158V.

A typical oligosaccharide structure of a normal multi-plant human IgG-Fc is a complex bi-tower structure type. When the glycoprotein has extra sugar residues at the core (trehalose, N-acetylglucosamine (GlcNAc)) (or the outer arm (galactose (Gal) and N-acetylthione (Neu5Ac)) In the Golgi apparatus, the core heptasaccharide of the two branches is subject to variable modification (reviewed by Walsh and Jefferis, 2006).

Serum IgG antibodies from different vertebrate species share a basic two-branched Fc sugar structure, but the structure and composition of the surrounding regions of the sugar chain are different (Hamako et al., 1993). The variability is most likely due to the different activities of glycosyltransferases present in B-lymphocytes of different species and/or the accessibility of each IgG to such transferases. If recombinant DNA technology is used to stably express IgG in host cells that do not naturally secrete IgG, the glycosylation pattern is also defined by the activity of the glycosyltransferase or glycan modifying enzyme present in the cell line. Thus, the glycosylation pattern of recombinant proteins produced in alternative production cell lines is cell type-, tissue- and species-specific and can be significantly different (Raju et al., 2000). Production of the same IgGl antibody in CHO, Y0 myeloma and NSO cells produces three different products that differ in glycosylation patterns and biological activities (Lifely et al, 1995). Thus, it has been well established that activation of effector functions is strongly dependent on the oligosaccharide composition of the antibody molecule and that activation of certain effector functions will be more effective if certain glycosylation patterns are present.

Studies using therapeutic antibodies with modified glycosylation patterns have shown that the lack of α1,6-linked core trehalose increases the binding affinity of IgG1 antibodies to the FcγRIIIa receptor, and thus the ADCC effect mediated by natural killer (NK) cells Up to 100 times.

Due to the high binding affinity of the complement system component to galactosylated Fc-glycans, the galactosylation of Fc glycans increases the activation of the complement system and the complement-dependent cytotoxicity (CDC) ratio is low or non- Antibodies to galactosylated glycoforms are more effective.

Therapeutic antibodies can have different methods of action. Some antibodies, antibody fragments or Fc-fusion proteins are designed to neutralize biomolecules (eg, cytokines) in vivo. In contrast, recombinant antibodies in cancer therapy often recognize proteins on tumor cells and their efficacy is clearly dependent on sensitized effector cells for subsequent antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cells. The toxicity (CDC) mechanism implements killing. However, for other antibody-based therapies, activation of the inflammatory cascade may be detrimental, which can cause undesirable side effects.

Therefore, antibodies produced in commonly used cell lines (eg, CHO, NS0, and SP2/0) do not exhibit the best Fc glycosylation pattern as a challenge.

Antibodies produced in CHO, NS0 or human cell lines show predominantly (approximately 95%) Fc glycosylation features consisting of a dimeric structural core heptasaccharide structure with alpha 1 at the central GlcNAc, 6 connect the trehalose residue.

It has been well established that the presence of alpha 1,6-linked trehalose in the dimeric structural carbohydrate core structure in Fc glycans significantly impairs the potential of antibody activation effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). Another effector function (complement-induced cytotoxicity (CDC)) has been shown to be galactosylation dependent, with higher galactose content enhancing CDC activity. Therefore, a monoclonal antibody having a fucosylated glycan and a low galactose is particularly effective in treating a solid or non-solid tumor, because the therapeutic effect of the antibody therapy in this case mainly depends on the antibody recruiting and activating the tumor. The efficacy of attacking immune cells, apoptosis induction or activation of ADCC or CDC. Thus, antibodies produced in CHO maintain a limited benefit in cancer therapy, and in order to overcome their low activity, patients must be administered at high doses. In contrast, antibodies with altered glycosylation patterns (eg, lacking alpha 1,6-linked core trehalose) should have a significantly enhanced therapeutic effect.

Despite the fact that most licensed therapeutic systems are produced in CHO, NS0 or Sp2/0 cells, it is known that under non-optimal conditions, such cells can produce a number of abnormal glycosyl groups that lack potency or potential immunogenicity. Chemical product. In addition to incorrect processing of oligosaccharides, it is also known that murine cells add sugar residues that are not normally found on human IgG. This structure (eg, Gal-1,3-Gal and N-hydroxyethyl cyanokine (NeuGc)) can trigger an immunogenic or allergic response that is unacceptable for therapeutic agents intended for in vivo delivery to human patients. .

To date, experimental methods for obtaining cell lines capable of producing proteins with favorable glycosylation patterns have focused on the genetic manipulation of genes encoding enzymes of glycosylation machinery, thereby artificially altering glycosylation in existing host cell lines. The nature of the pathway (genetic inactivation or deregulation). The antibodies produced by this method may show a reduced or complete deficiency in the amount of certain sugar structures or linkages. Such genetically modified cell lines can be screened to obtain mutants capable of synthesizing certain desired glycosylation patterns.

Transgenic overexpression of the gene encoding the glycosyltransferase GntIII in CHO cells allows the cell to produce an antibody product with altered glycosylation patterns and a final product with reduced hyphal glycosylation, thereby having a higher Antibodies that modify glycans are 20 to 100 times stronger (Umana et al., 1999). However, it should be considered that such genetically modified host cell lines require active selection to stabilize the effects of genetic alterations. Furthermore, genetic engineering can have undesirable side effects, which increases the regulatory burden of the industrial production process.

The anti-lectin CHO mutant cell line Lec13 (Ripka et al., 1986) lacks an enzyme that converts GDP-mannose into GDP-trehalose due to genetic defects. If the cells are unable to metabolize trehalose from an alternative (e.g., external) source, the cells can be used to produce products with reduced fucosylation and increased ADCC activity (Shields et al, 2002). However, only the mutations contained in the genome of Lec13 cells were maintained after continuous selection with Lens culinaris agglutinin (LCA). However, in terms of adjustment angles, it is highly doubtful to add lectins or other selected substances during large-scale production. Furthermore, Lec13 cells are known to have lower protein yields and only a partial decrease in trehalose content, but are highly variable (Kanda et al., 2006; Shields et al., 2002).

The present invention surprisingly demonstrates that the rat hepatoma cell line H4-II-E is a highly productive biopharmaceutical glycoprotein (eg, antibodies, particularly therapeutic antibodies and Fc-fusion proteins) with excellent production cells with improved glycosylation properties. system.

The invention further sets forth an assay for the suitability of selected non-engineered cells representing various species, tissues, cell types, and differentiation/tumor stages as host cell lines for producing favorable glycosylation patterns and Therefore, the biomedicine that enhances the effector function.

Surprisingly, the rat hepatoma cell line H4-II-E is the only cell line in the rat/hepatocyte-derived cell line that combines a number of advantageous glycan properties for the therapeutic effect of glycoproteins.

The invention is additionally described for the first time that the H4-II-E rat hepatoma cell line can be used as a host cell line for the production of recombinant glycoproteins (e.g., antibodies or Fc-fusion proteins). The present invention demonstrates that H4-II-E rat hepatoma cells can be stably transfected with genetic elements encoding the light chain and heavy chain of an antibody or Fc-fusion protein, and the resulting cells secrete highly active functional antibody molecules to cell culture. In the supernatant, the supernatant can be purified. Recombinant antibodies produced in H4-II-E cells are superior in quality and activity to therapeutic antibodies produced in a conventional manner due to cellular glycosylation ability.

The H4-II-E cell line of the present invention was deposited with the German Collection of Microbial Species (Deutsche Sammlung von Mikroorganismen und) on June 28, 2011 under the Budapest treaty under the accession number DSM ACC3129 (H4-II-E). Zellkulturen GmbH, DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany.

The glycoform pattern of antibodies produced in H4-II-E cells, particularly IgGl antibodies, shows complex dimeric structural glycans that are predominantly free of trehalose and at the same time galactosylation is higher than antibodies normally produced in CHO. Thereby, the biotherapeutic antibodies produced in H4-II-E cells have a high potential to rapidly and efficiently activate antibody-mediated effector functions such as ADCC and CDC.

The invention further clarifies that the H4-II-E rat hepatoma cell line can be cultured in suspension in serum-free medium, which is mandatory when such cell lines are used as a production cell line for biotherapeutics. Surprisingly, after adapting H4-II-E cells to suspension growth in chemically defined medium without animal components and without calcium ions, adherent cell layers were present in H4-II-E cells in serum-containing medium. Growth, viability, and doubling time were not affected by the usual culture mode. In serum-containing adherent cultures and after adaptation to serum-free, animal-free and calcium-free media, the H4-II-E cell culture doubling time is between 24 hours and 32 hours during the adaptation phase. Within the range or within the range of 32 hours to 60 hours. A key aspect of suspension culture of H4-II-E cells with a population doubling time of 24 hours to 32 hours was the use of calcium-free medium. In contrast, serum-free incubation of H4-II-E cells in chemically defined media has previously been described in the literature, but the population doubling time is 68.5 hours (Miyazaki et al., 1991) or 4 days (Niwa et al., 1980). This is too slow to meet the demand for biopharmaceutical production cell lines.

The H4-II-E cell line adapted for suspension growth in serum-free Ca ^2+-free medium described in the present invention was registered under the Budapest Treaty on June 28, 2011 under the accession number DSM ACC3130 (H4-II-Es). Registered at the German Collection of Microorganisms (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany.

The present invention is the first to describe the selection of cell lines derived from different species and tissues which are analyzed for their production to produce a recombination with a favorable glycosylation pattern (reduced haylosylation, presence of terminal NeuAc-residues) The suitability of the protein is related. Analysis has shown that only the rat hepatoma cell line H4-II-E is capable of producing several beneficial glycosylation patterns that can significantly enhance the activity, efficacy and stability of therapeutic proteins, particularly glycoproteins (eg, antibodies).

H4-II-E cells were originally derived from rat hepatocellular carcinoma (REUBER, 1961) and were analyzed as in vivo models for adaptation to the liver. In contrast, the present invention describes for the first time the use of H4-II-E cells as a production system for highly active therapeutic proteins. H4-II-E cells derived from rat liver do not naturally produce antibodies, and H4-II-E cells have not previously been used for recombinant protein production. H4-II-E cells are derived from rat hepatocellular carcinoma (PITOT et al., 1964; REUBER, 1961) and are used exclusively for toxicological analysis and as a model system to study cellular stress responses (eg (Horikoshi et al., 1988; Houser et al., 1992)).

Surprisingly, in particular rat cells and in particular H4-II-E cells produce glycoproteins or glycosylation with a low trehalose content. Anti-systems from blood serum of normal rats are significantly fucosylated compared to other species (eg rabbits or cats) (Raju et al., 2000). Hamako and colleagues analyzed the sugar composition of serum IgG antibodies from different species and found that 92.8% of endogenous antibodies from rat blood serum were fucosylated, which is higher than serum IgG of most other species (Hamako et al. People, 1993).

Therefore, rat H4-II-E cells are expected to produce glycoproteins or glycosylation with high trehalose content rather than low trehalose content.

Unexpectedly, the situation of rat hepatoma cells H4-II-E cells is just the opposite. The present invention shows that the H4-II-E cell line derived from rat liver (for example, cells deposited in DSMZ under accession number DSM ACC3129 (H4-II-E) and deposited under the accession number DSM ACC3130 (H4-II-Es) The cells of DSMZ can be surprisingly distinguished from many other cell lines derived from different species by their ability to produce antibodies with significantly lower levels of trehalose than any other cell line.

Shinkawa and colleagues describe the use of the rat myeloma cell line YB2/0 for antibody production (Shinkawa et al., 2003). The YB2/0 cell line naturally produces a 14-91% reduction in the Fc glycostructure of the fucosylation, thereby producing an antibody production cell line that increases activity by up to 50-fold in the ADCC assay. Therefore, YB2/0 cells are different from H4-II-E cells in that they are known to produce daughter cells. However, as a myeloma cell line, YB2/0 cells are quite sensitive to apoptosis and show low robustness to cell pressure, making this cell line unsuitable for industrial production processes. As reported, in addition, the fucosylation in YB2/0 cells is highly variable, making it difficult to control and maintain trehalylation within certain thresholds. In contrast, the H4-II-E cells described in the present invention surprisingly show that the degree and consistency of de-fucosylation is higher than that reported for YB2/0 cells. Furthermore, H4-II-E cell lines are robust and insensitive to stress or other apoptosis-inducing stimuli, as shown in many toxicity studies using H4-II-E cells as a model system. Direct comparison of the sensitivity of H4-II-E cells and YB2/0 cells to different cell pressures (osmotic pressure, temperature pressure, mechanical pressure, chemical pressure) shows that H4-II-E cells are superior to YB2/0 in all respects. cell. Thus, H4-II-E cells are unexpectedly more suitable than YB2/0 cells for use as host cell lines for industrial production processes.

Another prior method for obtaining a product having a non-fucosylated glycostructure is to target the gene encoding the trehalose transferase 8 (Fut8) in CHO, which can result in the aggregation of secreted proteins (eg, antibody products). Alpha 1,6-trehalylation is completely lost in sugar (Yamane-Ohnuki et al., 2004). Alternative methods of using siRNA knockdown techniques to attenuate the activity of the Fut8 gene allow for the production of partially de-fucosylated antibodies (Mori et al., 2004). These antibodies show 50 to 100 times greater ADCC activation. However, a disadvantage of these strategies, which differ from the use of H4-II-E cells as producer cells, is that genetic engineering of existing host cell lines will certainly affect more cellular processes than glycosylation of only the secreted proteins of interest. In addition to the desired mutating effect, genetically engineered host cell lines that differ from H4-II-E cells may also exhibit some changes, such as reduced yield or instability or unpredictable during, for example, post-production development. problem. In addition, genetically engineered cell lines often require active selection to stabilize the effects of genetic alterations, thereby increasing the regulatory burden of the industrial production process.

In addition to showing reduced fucosylation, H4-II-E cells also showed additional beneficial properties not found in Fut8 deficient CHO cells, such as increased galactosylation and sialylation.

advantage

The following properties of the candidate cell line are believed to be advantageous over existing production cell lines: the cell line should be a natural (naive) host cell line that has not been genetically engineered.

The cell line should be capable of producing a protein with a favorable glycosylation pattern.

The cell line should stably display the desired sugar pattern without selection (eg, lectin resistance).

These cell lines can be cultured in suspension in serum-free medium.

These cell lines are characterized by high robustness and low sensitivity to stress or apoptosis stimuli.

The rat hepatoma cell line H4-II-E described in the present invention combines most of these advantageous properties.

Unlike previous methods for obtaining hypoglycosylated reduced antibodies, which are primarily based on genetic engineering of existing production cell lines, H4-II-E cells are native (ie, without genetic manipulation, mutagenesis or selection). It is suitable for the production of recombinant proteins with favorable glycosylation patterns and their therapeutic applications.

Unlike the proteins produced in the host cell line currently used to produce, the antibodies produced in H4-II-E show a combination of different beneficial glycosylation properties, which significantly enhances the quality of production:

- low seawed glycosylation or lack of trehalose: >80% non-fucosylated dimeric structural glycans

→ Increase the potential of activated effector functions such as ADCC

- High galactosylation : >40% galactosylated di- structural glycans

→ Increase the potential of activated effector functions such as CDC

The antibodies produced in H4-II-E cells thus have the high potential to activate antibody-dependent effector functions such as ADCC and CDC. This activation is associated with binding of the antibody to different Fc receptors. In addition to the higher activation of FcgRIIIa, the antibodies produced in H4-II-E also have the potential to activate the inhibitory receptor FcyRIIb to a lesser extent. This beneficial effect enables antibodies produced in H4-II-E to enhance the immune response mediated by macrophages, since neutrophils, in particular, also exhibit inhibitory receptors. Thus, therapeutic antibodies produced in H4-II-E cells are superior to tumor-specific and other indications in their therapeutic efficiency over conventionally produced antibodies.

- absence of immunogenic residues: lack of Gal-α1,3-Gal-linkage and NeuGc-residue

→ No allergic or immunogenic reactions

Another advantage of lacking a potentially immunogenic glycostructure (eg, Gal-α1,3-Gal-linked sugar or NeuGc-residue) is a glycoprotein (eg, an antibody or Fc-fusion protein) produced in H4-II-E cells . Such structures found in antibodies produced in the mouse myeloma cell line NS0 or SP2/0 can induce undesirable inflammatory responses or immunogenic rejection if applied to sensitive patients.

- sialylation: terminal α2, 3 or α2,6-linked NeuAc-residue

→ Improved serum stability (serum half-life)

The terminal sialylation of the glycans of the antibodies produced in H4-II-E cells has an additional positive effect on the serum stability and catabolic half-life of the therapeutic antibodies.

The H4-II-E rat hepatoma line is not genetically engineered and does not require selection or culture in the presence of lectin to maintain its ability to produce a beneficial glycosylation pattern.

In addition, the H4-II-E cell line can be cultured in suspension in serum-free medium. Even H4-II-E cells (eg, cells deposited with DSMZ under accession number DSM ACC3129 (H4-II-E)) are typically cultured in adherent culture using serum-containing media (also known as American Type Culture). Collection) (ATCC, accession number CRL-1548) or European Collection of Cell Cultures (ECACC, accession number 87031301), can also be confirmed in the present invention, and in serum-containing medium Compared to the growth of adherent cultures, H4-II-E cells can be successfully adapted to suspension growth in serum-free medium without compromising group doubling time of 24 to 32 hours (eg, under the accession number DSM ACC3130 (H4- II-Es) cells deposited in DSMZ).

The doubling time of H4-II-E cells in serum-free medium is 24 to 32 hours. A key factor in achieving constant growth of H4-II-E cells in serum-free medium and such favorable doubling time is the omission of calcium in the medium. If calcium is present in the cell culture medium, the H4-II-E cells will not grow in suspension and will be doubled for a longer period of time, which makes it unabsorbable for biomedical production on a commercial scale.

Another advantage of H4-II-E cells as host cells in biopharmaceutical production is their high robustness and low pressure or apoptosis sensitivity. Other production cell lines (e.g., myeloma cell line YB2/0) also exhibit a beneficial sugar pattern, but are also extremely sensitive to either type of stress and are therefore not suitable for cultivation in large scale production processes.

In addition to the previously mentioned advantages, the therapeutic proteins produced in H4-II-E cells have the following additional beneficial properties:

Higher effectiveness and stability = reduced required dose

Improved patient convenience (reduced treatment (infusion) frequency)

Reduced risk of side effects through reduced circulating dose

Reduced timeline and reduced costs for the supply of clinical and commercial materials

→ In short: reduced treatment costs.

applicability

H4-II-E rat cells can be used to industrially produce therapeutically highly active proteins, preferably antibodies or Fc-fusion proteins, thereby effectively activating effector functions upon administration to a patient.

With a significantly reduced core trehalose content, H4-II-E-producing antibodies or Fc-fusion proteins show higher affinity for the polymorphic receptor FcγRIII (CD16-F158, CD16-V158) and a lower degree of activation inhibition Receptor FcyRIIb. In contrast, unlike the antibodies produced by the fucosylated CHO, the antibodies produced by H4-II-E can not only rapidly and efficiently recruit and activate CD16-positive cells, but also recruit and activate macrophages and neutrophils. Activate the cytotoxic function of the immune system. It is shown that the galactosylation is higher than the antibody produced in a conventional manner, and the product modified and secreted in the H4-II-E cell can additionally bind the component of the complement system (ie, Clq) more effectively and thereby activate to finally kill the target cell. Another cascade.

For a variety of reasons (see the above advantages), H4-II-E cell lines are selected for the production of antibodies or Fc-fusion proteins, particularly those that recognize tumor targets. Enhanced activation of effector functions ADCC and CDC by antibodies or Fc-fusion proteins produced in H4-II-E cells also allows for effective treatment of solid tumors after antibody treatment, which are usually immune to patient immunity The system is effectively attacked.

The improved serum stability of antibodies produced in H4-II-E cells is beneficial in many therapeutic areas, particularly in chronic diseases, where therapeutic substances must currently be delivered to patients frequently and repeatedly.

It is known that post-translational protein modification has a key effect on the biological activity, stability and immunogenicity of the protein. The most common post-translational modifications of secreted proteins are glycosylation and most of the glycoproteins in all approved biopharmaceuticals (including recombinant antibodies or recombinant Fc-fusion proteins).

Since the correct glycosylation pattern is indispensable for therapeutic activity and specificity, most glycoprotein therapeutics are produced in eukaryotic expression systems capable of generating complex spectra of glycosylation patterns. Currently, Chinese hamster ovary (CHO) cells have become standard mammalian host cells for the production of recombinant proteins. However, it is known that different eukaryotic production cell lines produce different Fc glycosylation patterns and thereby have a significant impact on the biological activity of the therapeutic antibodies. In addition, certain certain sugar structures are known to be more effective than others in activating specific downstream effector mechanisms. Therapeutic antibodies with most human-like glycosylation patterns are not particularly effective at activating anti-tumor effector mechanisms, but at the same time, such antibodies are less likely to cause undesirable allergic or immunogenic rejection in patients and therefore Treated as complete. To enhance, for example, cell killing activity induced by anti-cancer antibodies, it is desirable to have different glycosylation patterns. Likewise, altered glycosylation patterns may enhance other antibody-dependent effector functions or may alter serum half-life without compromising the safety or stability of the therapeutic product itself.

In the present invention, different cell lines derived from a variety of species, tissues, and cell lineages are specifically selected and analyzed. The glycans exposed on the cell surface and the enzymatic activity in the cells were examined (Fig. 1). Thereby, each cell line can be given the ability to synthesize certain glycosylation patterns. Selection and analysis showed that the cell line clearly showed differences in its glycosylation ability depending on the species from which it originated and the tissue or cell lineage. Only a few cell lines naturally exhibit low algal glycosylation properties, which have a positive effect on the activation of the antibody-dependent cellular cytotoxic pathway (ADCC). Some cell lines are capable of producing a sialylation structure that enhances the serum stability of the therapeutic protein. Some cell lines produce immunogenic sugar structures (eg, Gal-1,3-Gal and NeuGc) that cause an inflammatory response in humans. Analysis of the present invention shows that the species source or tissue, organ or cell lineage of a given cell line alone is unable to determine the glycosylation ability of the cell. Thus, genetic and epigenetic factors influence the ability of cells to synthesize certain glycosylation patterns. Thus, the data of the present invention demonstrate that it is not possible to predict the glycosylation properties of a cell line based solely on knowledge of the sugar patterns obtained in another cell line derived from the same tissue and/or species (Fig. 1).

The only cell line that exhibits several properties (eg reduced hyalosylation, lack of immunogenic residues, and the presence of alpha-2,6 linked sialic acid) is the rat hepatoma cell line H4-II-E. These properties positively affect the activity and stability of recombinant biotherapeutics, particularly antibodies or Fc-fusion proteins. Selected cell lines H4-II-E were the only cells in the assay that showed no detectable signs of fucosylation in this assay. This is surprising because other rat cell lines and other liver cell lines have different properties. Furthermore, it has been demonstrated that the rat serum anti-system is significantly fucosylated, and according to the literature, other species (such as rabbits or cats) have lower serum levels of trehalose than rats (Raju et al., 2000). .

Sugar structures such as Gal-1,3-Gal and NeuGc are potentially immunogenic. H4-II-E rat cells do not display these potential immunogenic sugar structures. H4-II-E rat cells additionally do not produce antibodies or Fc-fusion proteins with such potential immunogenic sugar structures. In fact, glycostructures such as Gal-1,3-Gal and NeuGc with potential immunogenicity are not present in selected human or rat cell lines, whereas cell lines derived from mice, rabbits and other species are These structures are produced consistently, which induces an immunogenic response in humans. Thus, according to experimental data of the present invention, these potentially immunogenic sugar structures are attached to the secreted glycoprotein by the cells in a species-dependent manner.

To verify that H4-II-E cells are capable of producing secreted glycoproteins with predictive glycosylation properties, stable production is achieved by transfecting the corresponding DNA constructs and subsequently selecting cells that stably integrate the product gene and antibiotic resistance markers. Recombinant antibody H4-II-E cells. The antibody-producing cell population is obtained and cultured as an adherent cell layer. H4-II-E cells were additionally adapted for suspension growth and cultured in serum-free chemically defined medium (Figure 5). The antibody was purified from the cell culture supernatant by protein A chromatography. Fc glycosylation was analyzed and compared to the pattern obtained on antibodies produced in CHO-DG44, CHO-Lec13 mutants and YB2/0 rat myeloma cells. The two branched glycans accounted for the largest part of all four IgG1 formulations. Within the ratio of the two structural glycans, CHO-DG44 cells produced a predominantly (approximately 95%) pre-glycosylated form as previously reported. In contrast, more than 80% of the IgG1 expressed in H4-II-E cells contained trehalose-free twig-structured glycans, which was significantly higher than that produced in the cell line YB2/0 or the CHO mutant Lec13. (figure 2). A detailed analysis of the glycosylation pattern of antibodies produced in H4-II-E cells also showed that >40% glycans were galactosylated, a higher ratio than those obtained when antibodies were produced in CHO-DG44, This CHO-DG44 host cell line is commonly used for the production of biopharmaceutical proteins (Figure 3). In addition, about 8% of antibodies produced in H4-II-E cells carry terminal sialic acid residues, and if a recombinant antibody is produced in CHO-DG44 cells, these terminal sialic acid residues are usually not found (Fig. 4). . The terminal sialic acid residue can have different effects on the activity and stability of the modified antibody. Recent publications have shown that sialic acid inhibits the inflammatory activity of antibodies (Burton and Dwek, 2006; Scallon et al., 2007). Other data show that the absence of sialic acid increases the metabolic rate of the liver in mice, indicating clearance by liver-based receptors (Wright et al., 2000). Sialic acid derivatives (e.g., N-hydroxyethyl ceramide (NeuGc)) have been found to be immunogenic in humans (Noguchi et al., 1995). However, evidence of NeuGc modification on proteins produced in H4-II-E cells could not be detected (Fig. 1). In summary, H4-II-E source antibodies lack potential immunogenic residues.

These performance data confirm the results obtained in the predictive analysis of favorable glycosylation patterns (Figure 1). In conclusion, the rat hepatoma cell line H4-II-E itself is distinguished from other cell lines by the ability to generate several beneficial glycosylation patterns. The glycosylation pattern obtained after antibody production in H4-II-E cells can be expressed as enhanced binding affinity and excellent activity of the antibody to the FcγRIII receptor in the ADCC assay, and in the CDC assay with the complement system component. Strong binding and enhanced activity, strong binding to the neonatal Fc receptor FcRn (positive effect on antibody stability and serum half-life).

In addition to producing beneficial glycoprotein properties, H4-II-E cells are also characterized by high robustness and low sensitivity to stress or apoptosis-inducing stimuli. Accordingly, H4-II-E cells can be adapted to grow in suspension in serum-free medium. H4-II-E cells grow well in different incubation modes and can be cultured in suspension for more than 10 days with high vigor during the feed batch process. In conclusion, H4-II-E cells provide excellent quality for large-scale industrial biotherapeutics with outstanding effector activity and prolonged serum half-life.

definition

The general embodiment "comprising or comprising" encompasses a more specific embodiment "consisting of". In addition, the singular and plural forms are not used in a limiting manner. The terms used in the present invention have the following meanings.

The present invention relates to all rat hepatoma cell lines derived from "Reuber H-35 hepatoma" (REUBER, 1961) and 1964 (PITOT et al., 1964). "Reuber H-35 hepatoma" was induced by chemical carcinogenesis in AxC rats. Cell lines derived from such "Reuber H-35 hepatoma" include, for example, H4-II-E and H4-II-E-C3 cell lines and derivatives or progeny thereof.

More specifically, "H4-II-E cell" means derived from the European Cell Culture Preservation Center (ECACC, catalog number 87031301) or the American Type Culture Collection (ATCC, accession number CRL-1548) or derived from Cells of the rat hepatoma cell line isolated in 1961 (REUBER, 1961) and 1964 (PITOT et al., 1964) were first described in the literature. Specifically, the H4-II-E cell line has cells of ECACC catalog number 87031301 or ATCC number CRL-1548.

The term "H4-II-E cell" additionally means the H4-II-E-C3 cell line (CRL-1600 or HPACC No. 85061112); the H4II cell line (HPACC Nr. 89042702), which is also derived from "Reuber H35 hepatoma". And the same as H4-11-E-C3 (ECACC Cat. No. 85061112); H4-TG cell line (CRL-1578), which is a CRL-1600 derivative with HPRT deficiency and constant expression of amphetamine hydroxylase; H5 Cell line (HPACC, Nr. 94101905), which is also a mesogenic line of H4-II-E-C3 (CRL-1600); and H4-S cell line (HPACC Nr. 89102001), which is infected by VS virus. Murine liver tumor cells.

The term "H4-II-E cells" also encompasses cells originally derived from deposited H4-II-E cells and are progeny originally deposited with H4-II-E cells or cells originally derived from deposited H4-II-E cells. .

Thus, the term "H4-II-E cell" encompasses an unmodified or modified progeny/form of H4-II-E cells. Such unmodified or modified progeny/form of H4-II-E cells are also referred to as "derivatives or progeny" of the originally deposited H4-II-E cells.

The unmodified form of H4-II-E cells is the entire cell produced by the unconstituted subunit of H4-II-E cells. Some examples include: a sub-pure of an unmodified cell line, a purified or fractionated subpopulation thereof.

Modified forms (or derivatives or progeny) of H4-II-E cells confer recombinant proteins, in particular glycoproteins (including antibodies or Fc-fusion proteins), by introducing functional DNA sequences, in particular, to each of the respective starting cells. The potential of all cells produced by the parental H4-II-E cells.

Modified forms (or derivatives or progeny) of H4-II-E cells are all cells produced from the parental H4-II-E cells via mutagenesis or targeted gene modification or gene integration. Such modified H4-II-E cell lines are genetically engineered into H4-II-E cells, which comprise a transgene (eg, a lipid transfer protein CERT, also known as Goodpasture antigen binding protein), a transcription factor (eg, upstream binding factor UBF), or A gene encoding a member of the Sec-1/Munc18 protein family.

Other examples of modified H4-II-E cells knock out or knock down one or more genes (eg, DHFR (dihydrofolate dehydrogenase), GS (glutamate synthase), TIP-5 or SNF 2H) H4-II-E cells. In particular, H4-II-E cells that knock out DHFR or GS can be used in biomedical production due to favorable selection options in the case of recombinant protein expression.

Examples of such auxotrophic cells that knocked out or knocked down the H4-II-E cell line of DHFR or GS. Knockout or knockdown of HH-II-E cells of DHFR are hypoxanthine (H) and thymidine (T) auxotrophs. Knocking or knocking down the GS H4-II-E cell line glutamate auxotrophy. However, there are other auxotrophic H4-II-E cells that can be generated by those skilled in the art.

A modified form (or derivative or progeny) of a H4-II-E cell additionally means any cell produced from a parental H4-II-E cell via adaptation to a particular medium, growth, culture mode or selection of material.

In particular, the term H4-II-E cell line is directed to two deposited cell lines. The H4-II-E cell line of the present invention was deposited with the German Collection of Microbial Species (DSMZ), Inhoffenstrasse 7B, D-38124, on June 28, 2011 under the Budapest Treaty under the accession number DSM ACC3129 (H4-II-E). Braunschweig, Germany.

The H4-II-E cell line adapted for suspension growth in serum-free Ca ^2+-free medium described in the present invention was registered under the Budapest Treaty on June 28, 2011 under the accession number DSM ACC3130 (H4-II-Es). Registered at the German Collection of Microorganisms (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany.

The terms "cell" and "cell line" as used herein, in particular, refer to a cell/presenting cell line and a host cell/host cell line.

The term calcium reduction or preferably calcium-free medium means to define a medium containing 1 μmol/L to 500 μmol/L Ca ²⁺ ions, and more preferably the calcium reduction medium contains 1 μmol/L to 250 μmol/L Ca ²⁺ ions and Even better calcium-reduced or Ca-free medium contains 0 μmol/L to 100 μmol/L or 0.5 μmol/L to 100 μmol/L Ca ²⁺ ions. In AEM medium, addition of 250 μM/L CaCl ₂ or higher CaCl ₂ concentration after 3 days of culture significantly impaired H4-II-E cell growth. In AEM medium supplemented with 250 μM/L CaCl ₂ or higher CaCl ₂ concentration, more than 80% of the cells formed tight aggregates. Calcium-reducing medium can also be obtained by adding EDTA to a medium originally containing calcium, thereby reducing the concentration of free calcium ions in the medium by forming a complex with EDTA. Preferably, EDTA is added at a concentration between 400 μmol/L and 1200 μmol/L or 600 μmol/L to 1000 μmol/L or 700 μmol/L to 900 μmol/L. Preferably, EDTA is added at a concentration of about 800 μmol/L.

An example of a commercially available calcium-free medium is MEM Joklik Modification (Sigma) or MEM Spinner Modification (Sigma). A typical calcium-containing medium for adherent incubation of H4-II-E cells in the presence of serum is Eagle's Minimum Essential Medium (Sigma) containing 1360 μmol/L Ca ²⁺ ions. The serum-free calcium-reducing medium for suspension incubation of H4-II-E cells is AEM (Invitrogen).

Unlike calcium ions, magnesium ions have no effect on the aggregation ratio of rat hepatoma cells/H4-II-E cells. Therefore, rat hepatoma cells/H4-II-E cells aggregated with magnesium (Mg ²⁺ ) ion independence but with calcium (Ca ²⁺ ) ion dependence.

"Glycosylation site" refers to an amino acid residue recognized by a eukaryotic cell as a sugar residue attachment site. The term "glycoside structure" or "glycan" refers to a sugar residue attached to a glycosylation site. Amino acids attached to carbohydrates (e.g., oligosaccharides) are typically aspartic acid (N-linked), serine (O-linked), and threonine (O-linked) residues. The specific attachment site is typically defined by an amino acid sequence referred to herein as a "glycosylation site sequence."

a glycosylation site sequence for N-linked glycosylation - Asn-X-Ser- or -Asn-X-Thr-, wherein X may be any of the amino acids other than proline in the conventional amino acid . The major glycosylation site for O-linked glycosylation - (Thr or Ser)-X-X-Pro- (SEQ ID NO 1), wherein X is any conventional amino acid. The terms "N-linked" and "O-linked" refer to a chemical group that acts as an attachment site between a sugar molecule and an amino acid residue. The N-linked sugar is attached via an amine group; the O-linked sugar is attached via a hydroxyl group. However, not all glycosylation site sequences in the protein must be glycosylated. Some proteins are secreted in both glycosylated and non-glycosylated forms, while others are fully glycosylated at one glycosylation site sequence but contain another unglycosylated glycosylation site. sequence. Therefore, not all glycosylation site sequences present in the polypeptide must be glycosylation sites that actually attach to the sugar. Initial N-glycosylation is inserted into "core carbohydrates" or "core oligosaccharides" during biosynthesis.

In N-linked glycosylation, the carbohydrate moiety is attached to the aspartate residue in the polypeptide chain via GlcNAc. N-linked carbohydrates have different structures, but all contain a common core structure in which the end GlcNAc binding to aspartic acid is referred to as the reducing end and the opposite side is referred to as the non-reducing end:

Those skilled in the art will recognize that, according to the Kabat EU nomenclature (Kabat et al., 1991), for example, murine IgG3, IgG1, IgG2B, IgG2A, and human IgD, IgG3, IgG1, IgA1, IgG2, and IgG4 CH2 domains Each has a single conserved site for N-linked glycosylation at the amino acid residue at position 297. Residues in the antibody domain are numbered in a conventional manner according to the system described by Kabat (Kabat et al., 1991), which refers to the numbering of EU antibodies (Edelman et al., 1969). It should be noted that the Kabat residue designation does not always correspond directly to the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the stringent Kabat number corresponding to the shortened or inserted structural component. The correct Kabat numbering for a given antibody residue can be determined by aligning the homologous residues in the antibody sequence with a "standard" Kabat numbered sequence. Those skilled in the art will appreciate that such practices consist of non-sequential numbering in specific regions of the immunoglobulin sequence such that normalized positions in the reference immunoglobulin family can be normalized.

The most important N-linked carbohydrates are "complex" N-linked carbohydrates. In accordance with the present invention, such complex carbohydrates should have the "two-branched" structure described herein. The core two-branched structure is a typical feature of two-branched oligosaccharides and can be schematically represented as follows.

Since each of the two branched structures may have a halved N-acetylglucosamine (GlcNAc), added to one or two branches on the non-reducing end side, galactose and sialic acid sugar, and added to GlcNAc at the core reducing end. The trehalose, therefore, has a total of 36 structurally complex oligosaccharides that occupy the N-linked Asn 297 site.

It should also be recognized that in a particular CH2 domain, glycosylation at Asn 297 may be due to differences in oligosaccharide chains attached to any Asn 297 residue in the di-chain Fc domain (or post-translational trimming). Symmetrical. For example, although a heavy chain synthesized in a single antibody secreting cell can be homologous in its amino acid sequence, it is usually differentially glycosylated, resulting in a large number of structurally unique immunoglobulin glycoforms (having different Biological activity and biophysical properties). The major types of complex oligosaccharide structures found in the CH2 domain of IgG are shown below.

Two-piece structure core (A2G0):

Dichotomous GlcNAc core:

Typical complex type two-structured glycan: A2FG0:

A2G0:

A2FG1:

or

A2G1:

or

A2FG2:

A2G2:

Sialylation of A2FG1:

or

Sialylation of A2G1:

or

Sialylation of A2FG2:

or

or

Sialylation of A2G2:

or

or

In addition to the complex type of oligosaccharide, other examples of N-glycoside linked sugar chains also include high mannose types in which only mannose binds to the non-reducing end of the core structure; heterozygous, wherein the non-reducing end side of the core structure has both a branch of a high mannose N-glycoside linkage sugar chain and a complex N-glycoside linkage sugar chain; and the like.

Trehalose can be found at different sites in the N-glycan tree, but most of the trehalose on the N-glycan attached to the antibody is linked to the terminal GlcNAc at the end of the reducing end with α1,6. The trehalose which is linked to the terminal-reduced GlcNAc by α1,3 is not found in the recombinant protein derived from human, but is at risk of being produced, for example, in plants and maintaining serious immunogenicity. Other possible but rarely found trehalose linkages and branching structures localize α1,3 and α1,4 of GlcNAc or locale 1,2 of the Gal residue with the branch structure (H-antigen, substructure of A and B blood group antigens) .

Most of the sugar chains produced by the H4-II-E cells of the present invention have a "complex dichotomous structure type" and do not contain trehalose which binds N-acetylglucosamine at the reducing end.

The term "low-fucosylation" means that less than 20% of the glycan/glycosidic structure contained in the glycoprotein/containing in the antibody or Fc-fusion protein contains trehalose bound to the terminal-reduced GlcNAc. More preferably, less than 10% or even more preferably less than 5% of all of the composite twig structure glycans contain trehalose bound to the end-reduced GlcNAc of the glycan. Low seawed glycosylation illustrates a range between 0% to 20% trehalylation, 0% to 10% trehalylation, preferably 0% to 5% trehalylation. In particular, low fucosylation illustrates a range between 0.5% to 20% trehalylation, 0.5% to 10% trehalylation, preferably 0.5% to 5% trehalylation. Low seawed glycosylation additionally illustrates a range between 1% to 20% trehalylation, 1% to 10% trehalylation, and 1% to 5% trehalylation.

Therefore, the degree of demyosylation is more than 80%. This means that more than 80% of the glycans do not contain trehalose bound to the terminal-reduced GlcNAc. In a particular embodiment, more than 90% or preferably more than 95% of the glycans do not contain trehalose that binds to the terminal-reduced GlcNAc. Thus, the degree of de-cosamination is in the range of between 80% and 100%, between 90% and 100%, preferably between 95% and 100%. Specifically, the degree of demycosylation is in the range of from 80% to 99.5%, from 90% to 99.5%, preferably from 95% to 99.5%. Preferably, the degree of de-cosamination is in the range of between 80% and 99%, between 90% and 99%, preferably between 95% and 99%.

By "high galactosylation" is meant that more than 40% of all glycan/glycosidic structures contained in the glycoprotein/in the complex antibody or Fc-fusion protein contain one or two non-reducing ends linked to the core structure A galactose residue of the GlcNAc residue at the terminus. More preferably, more than 45% or even more preferably more than 50% of all complex dimeric structural glycans are galactosylated. High galactosylation states between 40% to 100% galactosylation, 45% to 100% galactosylation, preferably 50% to 100% galactosylation or 51% to 100% galactosylation. The range between the two. In particular, high galactosylation states between 40% and 99.5% galactosylation, 45% to 99.5% galactosylation, preferably 50% to 99.5% galactosylation or 51% to 99.5% half. The range between lactosylation. High galactosylation is preferably between 40% and 99% galactosylation, 45% to 99% galactosylation, preferably 50% to 99% galactosylation or 51% to 99% galactosyl The scope between the two.

High galactosylation preferably illustrates the presence of at least one galactose residue (G1) in the glycan/glycosidic structure, more preferably one or two galactose residues (G1 or G2) in the glycan/glycosidic structure. Preferably, high galactosylation means that the 50% glycoside structure contains at least one galactose residue. In particular, high galactosylation means the presence of a G1 or G2 glycoside structure, but almost no G0 glycoside structure.

Therefore, the degree of degalactosylation is less than 60%. This means that less than 60% of all complex glycans do not contain residues linked to GlcNAc at the non-reducing end of the core structure. In a particular embodiment, less than 55% or preferably 50% or less of all complex glycans do not contain a galactose residue attached to the GlcNAc at the non-reducing end of the core structure. Thus, the degree of degalactosylation is in the range of between 60% and 0%, between 55% and 0%, preferably between 50% and 0% or between 49% and 0%. Specifically, the degree of degalactosylation is in the range of from 60% to 0.5%, from 55% to 0.5%, preferably from 50% to 0.5% or from 49% to 0.5%. The degree of degalactosylation is preferably in the range of from 60% to 1%, from 55% to 1%, preferably from 50% to 1% or from 49% to 1%.

High activity with high sialylation or addition of a sialic acid or a neuratic acid residue to a galactosylated glycoside structure (e.g., a two-structured glycan) means that more than 5% of all glycans contain terminal sialic acid residues. More preferably, 5-10% or 0-8% or 1-8% of all Fc-glycans contain sialic acid or ceramide residues. In a particular embodiment, more than 10% or 10%-50% or 10%-45% of all Fc-glycans are sialylated. Thus, the glycan does not contain terminal sialic acid residues to a degree less than 95%, or it is in the range of between 95-90% or 92-100%. In a particular embodiment, the glycan does not contain terminal sialic acid residues to a degree less than 90%, or it is in the range of between 50% and 90% or between 55% and 90%.

The use of H4-II-E cells as "host cells" for the production of recombinant glycoproteins, particularly antibodies or Fc-fusion proteins, is the subject of the present invention. H4-II-E cells are not standard host cells.

Standard "host cells" or common host cells for the production of biopharmaceutical proteins are within the meaning of the present invention (for example) BHK21, BHK TK-, CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1, CHO-DG44, Murine myeloma cells, preferably NS0 and Sp2/0 cells or derivatives/progeny of any of these cell lines. The standard host cell lines are CHO-DG44, CHO-DUKX, CHO-K1 and BHK21 and even better CHO-DG44 and CHO-DUKX cells. The best standard host cell line is CHO-DG44 cells. Examples of rodent and hamster cells are also summarized in Table 1. However, derivatives/progeny of their cells, other mammalian cells (including but not limited to human, mouse, rat, monkey and avian or preferred rodent cell lines) or eukaryotic cells (including However, it is not limited to yeast, insects and plant cells) and is also used as a standard host cell, especially for the production of biomedical proteins. Typically, such cells are capable of expressing and secreting a large number of specific glycoproteins of interest into the culture medium.

Table 1: Eukaryotic Standard Host Cells/Common Production Cell Lines

In general, host cell lines are optimal when established, adapted, and fully cultured under serum-free conditions and optionally in a medium that does not contain any animal-derived protein/peptide. Commercially available media such as the following are exemplary suitable nutrient solutions: Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), lowest Essential medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-Invitrogen), serum-free CHO medium (Sigma) and protein-free CHO medium (Sigma), EX-CELL medium (SAFC), CDM4 CHO and SFM4 CHO (HyClone). Any medium may be supplemented with various compounds as needed. Examples of such compounds are hormones and/or other growth factors (eg, insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (eg, sodium chloride, calcium salts). , magnesium salts, phosphates), buffers (eg HEPES), nucleosides (eg adenosine, thymidine), glutamic acid, glucose or other equivalent energy sources, antibiotics, trace elements. They may also include any other necessary supplements at an appropriate concentration known to those skilled in the art. Preferably, serum-free medium is used in the present invention, but a medium supplemented with a suitable amount of serum can also be used to grow H4-II-E cells and to grow and select stably producing cells. For selection and selection of genetically modified cells expressing the selected gene, a suitable selection agent is added to the culture medium.

To adapt cells grown in serum-containing medium to serum-free growth and to culture cells in single cell suspension culture, serum-free medium is used. To promote suspension growth, a serum-free medium free of calcium ions or calcium is preferred. According to the present invention, H4-II-E cells are adapted to a serum-free and Ca-reduced medium and then the cells are cultured in suspension in the medium. The method of adapting H4-II-E cells to suspension and serum-free medium and suspending the cells in such medium comprises using a serum-free medium without calcium ion (Ca ²⁺ ) or calcium reduction. The term calcium reduction or preferably calcium-free medium means a medium containing 1 μmol/L to 500 μmol/L Ca ²⁺ ions, and more preferably a calcium reduction medium containing 1 μmol/L to 250 μmol/L Ca ²⁺ ions. And even better calcium-reduced or Ca-free medium containing 0 μmol/L-100 μmol/L or 0.5 μmol/L-100 μmol/L Ca ²⁺ ions. An example of a commercially available calcium-free medium is MEM Joklik Modification (Sigma) or MEM Spinner Modification (Sigma). A typical calcium-containing medium for adherent incubation of H4-II-E cells in the presence of serum is Eagle's Minimum Essential Medium (Sigma) containing 1360 μmol/L Ca ²⁺ ions.

The term "host cell" as used in the present invention encompasses cells comprising a heterologous nucleic acid sequence and cells which do not (not yet) comprise a heterologous nucleic acid sequence. In a particular embodiment of the invention, the host cell comprises a heterologous nucleic acid sequence encoding a (recombinant) protein, preferably a glycoprotein of interest, such as an antibody or Fc-fusion protein, expressed by the host cell. To this end, the host cell is cultured under conditions permitting expression of the (recombinant) protein.

The term "protein" is used interchangeably with an amino acid residue sequence or polypeptide and refers to an amino acid polymer of any length. The terms also include proteins that are post-translationally modified via reactions including, but not limited to, glycosylation, acetylation, phosphorylation, or protein processing. Modifications and alterations can be made within the structure of the polypeptide, such as fusion with other proteins, amino acid sequence substitutions, deletions or insertions, while the molecule maintains its biological functional activity. For example, certain amino acid sequence substitutions can be made within a polypeptide or its potential nucleic acid coding sequence and a protein of the same nature can be obtained.

The term "polypeptide" means a sequence having more than 10 amino acids and the term "peptide" means a sequence of up to 10 amino acids in length.

The terms "nucleic acid sequence", "gene of interest" (GOI), "selected sequence" or "product gene" have the same meaning herein and refer to the code "product of interest" or "protein of interest" (also by terminology) The "desired product" refers to a polynucleotide sequence of any length. In a preferred embodiment of the invention, the nucleic acid sequence or gene of interest encodes a glycoprotein, preferably an antibody or an Fc fusion protein. The nucleic acid sequence or gene of interest may be a full length or truncated gene, a fusion gene or a tagged gene, and may be a cDNA, genomic DNA or DNA fragment, preferably a cDNA. It may be in the native sequence, ie in its native form, or may be mutated or otherwise modified as desired. Such modifications include codon optimization, humanization or tagging of the use of optimized codons in the host cell of choice. The selected sequence may encode a secreted polypeptide, a cytoplasmic polypeptide, a nuclear polypeptide, a membrane-bound polypeptide, or a cell surface polypeptide.

The "glycoprotein of interest" includes all proteins, polypeptides, fragments thereof, and peptides which can be expressed in H4-II-E host cells. The desired protein may be, for example, an antibody, an enzyme, a cytokine, a lymphokine, an adhesion molecule, a receptor and derivatives or fragments thereof, and any other polypeptide useful as an agonist or antagonist and/or for therapeutic or diagnostic use. . Examples of desired proteins/polypeptides can also be found below.

In the case of more complex molecules (eg, monoclonal antibodies), the gene of interest encodes one or both of the two antibody chains.

The term "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes as well as myriad immunoglobulin variable region genes.

The term "antibody" as used herein includes a plurality of antibodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies.

The terms "antibody" and "immunoglobulin" are used interchangeably and are used to denote a glycoprotein having the structural properties described above for an immunoglobulin.

The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal antibodies (including agonist antibodies and antagonist antibodies) and antibody compositions having multi-epitope specificity. The term "antibody" specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, as long as the antibodies and fragments contain or are modified to contain The CH2 domain of the immunoglobulin heavy chain constant region may comprise at least a portion of the N-linked glycosylation site. Exemplary antibodies within the scope of the invention include, but are not limited to, anti-CD20, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD52, anti-HER2/neu (erbB2), anti-EGFR, anti-IGF, anti-VEGF, anti- TNFα, anti-IL2 or anti-IgE antibodies.

The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of antibodies that are substantially homologous based on amino acid sequences. Individual antibodies are highly specific and target a single antigenic site. Furthermore, unlike conventional (multi-drug) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. In addition to specificity, mAbs are advantageous because they can be synthesized by cell cultures (fusion tumors, recombinant cells, or the like) that are not contaminated with other immunoglobulins. mAbs include chimeric, humanized, and human antibodies herein.

A "chimeric antibody" is an antibody whose light chain and/or heavy chain genes have been normally engineered by genetically engineering variable regions and constant regions of immunoglobulins, corresponding to different species (eg, mice and humans). The sequences are identical or homologous. Or alternatively, the heavy chain gene belongs to a particular antibody class or subclass, and the remainder of the chain is from another antibody class or subclass of the same species or from another species. It also encompasses fragments of such antibodies as long as the fragments contain or are modified to contain at least one CH2 domain. For example, a variable segment of a gene from a mouse monoclonal antibody can be ligated into a human constant segment, such as gamma 1 and gamma 3 . Thus, a typical therapeutic chimeric anti-system consists of a hybrid protein from a variable domain or antigen-binding domain of a mouse antibody and a constant or effector domain derived from a human antibody (eg, ATCC Accession No. CRL 9688). Tac chimeric antibodies), but other mammalian species can be used.

The term "humanized antibody" as used in the present invention refers to a specific chimeric antibody, immunoglobulin chain or a fragment thereof (for example, Fv, Fab, Fab', F(ab)2 or other antigen-binding sequence of an antibody), as long as the The antibody or fragment contains or is modified to contain a portion of the CH2 domain comprising the constant region of the immunoglobulin heavy chain comprising at least an N-linked glycosylation site and contains minimal sequence derived from a non-human immunoglobulin. To a large extent, humanized anti-system human immunoglobulins (recipient antibodies) in which residues from the complementarity determining regions (CDRs) of the recipient are derived from non-human species such as mice, rats or rabbits (donors) The CDRs of the antibody) are replaced by residues with the desired specificity, affinity and ability. In some cases, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody and the introduced CDR or framework sequences. These modifications are implemented to further improve and maximize antibody performance. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to non-human immunoglobulins, and all or substantially all regions are These are the consensus sequences of human immunoglobulins. The humanized antibody should preferably also contain at least a portion of the immunoglobulin constant region (typically a human immunoglobulin constant region).

Humanized antibodies: comprise human framework regions and one or more CDRs from non-human (usually mouse or rat) antibodies. It may be desirable to modulate the framework amino acid to maintain the antigen binding specificity, affinity and or structure of the domain.

The term "CH2 domain" of the present invention is intended to describe the CH2 domain comprising an N-linked glycosylation site in the immunoglobulin heavy chain constant region. In the definition of immunoglobulin CH2 domain, immunoglobulins may generally be mentioned and, in particular, the domain structure of immunoglobulins as applied to human IgG1 by Kabat, EA may be mentioned (Kabat, 1988; Kabat et al. People, 1991). Thus, an immunoglobulin is typically a heterotetrameric glycoprotein of about 150 kDa, composed of two identical light chains and two identical heavy chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide linkages between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has a regularly spaced intrachain disulfide bridge. Each heavy chain has an amino terminal variable domain (VH) followed by a carboxy terminal constant domain (CH). Each light chain has a variable N-terminal domain (VL) and a C-terminal constant domain (CL).

The amino acid sequence of the constant domain in the heavy chain can be classified into different classes. There are five main categories: IgA, IgD, IgE, IgG, and IgM. The heavy chain constant domains corresponding to different classes of antibodies are referred to as the alpha, delta, epsilon, gamma and mu domains, respectively. The μ chain of IgM contains five domains (VH, CHmu1, CHmu2, CHmu3, and CHmu4). The heavy chain of IgE also contains five domains, while the heavy chain of IgA has four domains. The immunoglobulin class can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known. Wherein IgA and IgM are polymers and each subunit contains two light chains and two heavy chains. The heavy chain of IgG contains a length of polypeptide chain (referred to as the hinge region) between the CHγ1 and CHγ2 domains. The α chain of IgA has a hinge region containing an O-linked glycosylation site and the μ chain and the ε chain do not have a sequence similar to the hinge region of the γ chain and the α chain, however, it contains a constant lack of 4 other chains. Domain.

Thus, the CH2 domain is an immunoglobulin heavy chain constant region domain. The Fc region of an intact antibody typically comprises two CH2 domains and two CH3 domains. According to the invention, the CH2 domain is preferably the CH2 domain of one of the five immunoglobulin classes described above. Preferred are mammalian immunoglobulin CH2 domains, such as primate or murine immunoglobulins, of which primate and especially human immunoglobulin CH2 domains are preferred. Amino acid sequences of the immunoglobulin CH2 domain are known and commonly available to those skilled in the art (Kabat et al., 1991). In the context of the present invention, it is preferred that the immunoglobulin CH2 domain is human IgG and preferably from IgGl, IgG2, IgG3, IgG4, better human IgGl and IgG3 and even better human IgGl. When using the Edelman numbering system (Edelman et al., 1969), the immunoglobulin CH2 domain preferably begins at the amino acid position corresponding to the glutamic acid 233 of human IgG1 and extends to an amine group equivalent to the amine acid 340. Acid (Ellison and Hood, 1982).

As far as human antibody molecules are concerned, mention may be made of the attachment of an N-linked oligosaccharide to the IgG class of the indoleamine side chain at Asn297 in β-4, which is bent toward the inner face of the CH2 domain of the Fc region. A glycoprotein, especially an antibody or Fc-fusion protein of the invention is characterized in that it contains or is modified to contain at least one CH2 domain. The CH2 domain is the CH2 domain of a single N-linked oligosaccharide immunoglobulin with a human IgG CH2 domain. The CH2 domain is preferably the CH2 domain of human IgG1.

"Glycoprotein of interest", "polypeptide of interest", "protein of interest" or "product of interest" are those mentioned above and include all antibodies which are expressed in H4-II-E host cells or Fc-fusion protein. Furthermore, the desired protein or glycoprotein of interest may be, for example, an enzyme, a cytokine, a lymphokine, an adhesion molecule, a receptor and derivatives or fragments thereof, and may be used as an agonist or antagonist and/or have a therapeutic or diagnostic Any other polypeptide of use that is glycosylated. In particular, the desired glycoprotein/polypeptide or protein line of interest (such as, but not limited to) insulin; insulin-like growth factor; hGH; tPA; cytokines, such as interleukin (IL) (eg, IL-1, IL-2, IL) -3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 , IL-16, IL-17, IL-18), interferon (IFN)α, IFNβ, IFNγ, IFNω or IFNτ, tumor necrosis factor (TNF) (eg TNFα and TNFβ, TNFγ, TRAIL), G-CSF, GM-CSF, M-CSF, MCP-1, VEGF; and nanobody. Also included are erythropoietin or any other hormone growth factor and any other polypeptide useful as an agonist or antagonist and/or for therapeutic or diagnostic use. The H4-II-E cells of the invention can be advantageously used to produce antibodies such as: single, multiple, multispecific antibodies or fragments thereof, which comprise a CH2 domain, an Fc and an Fc' fragment, an immunoglobulin Heavy and light chains and their constant fragments. Furthermore, the method of producing a (recombinant) glycoprotein of the present invention can be advantageously used to produce antibodies such as: single, multiple, multispecific antibodies or fragments thereof, which include a CH2 domain, an Fc and an Fc' Fragments, immunoglobulin heavy and light chains and constant fragments thereof, and Fc-fusion proteins.

An "Fc-fusion protein" is defined as a protein comprising or modified to comprise a portion of a CH2 domain comprising an immunoglobulin heavy chain constant region comprising at least a single N-linked glycosylation site. According to the Kabat EU nomenclature (Kabat et al., 1991), this portion is Asn297 in IgG1, IgG2, IgG3 or IgG4 antibodies.

Another portion of the fusion protein can be the entire sequence or any portion of the native or modified heterologous protein sequence or a complete sequence or a combination of any of the native or modified heterologous protein sequences. The immunoglobulin constant domain sequence can be obtained from any immunoglobulin subtype (eg, IgGl, IgG2, IgG3, IgG4, IgA1, or IgA2 subtype) or class (eg, IgA, IgE, IgD, or IgM). Preferentially, it is derived from human immunoglobulins, more preferably from human IgG and even more preferably from human IgGl and IgG3. Examples of Fc fusion proteins include MCP1-Fc, ICAM-Fc, EPO-Fc, scFv fragments, or the like, which are coupled to a CH2 domain comprising an N-linked glycosylation site in the immunoglobulin heavy chain constant region. An Fc-fusion protein can be constructed by genetic engineering by introducing a CH2 domain comprising an N-linked glycosylation site in an immunoglobulin heavy chain constant region into another comprising, for example, other immunoglobulins. The domain, the enzymatically active protein moiety, and the effector domain are expressed in the construct. Thus, an Fc fusion protein of the invention also comprises a single chain Fv fragment linked to a CH2 domain comprising an N-linked glycosylation site in the immunoglobulin heavy chain constant region.

Preferably, the glycoprotein, particularly the antibody or Fc-fusion protein, is recovered/isolated in the form of a secreted polypeptide from the culture medium, or it can be recovered/separated from the host cell lysate if it is expressed without a secretion signal. It is desirable to purify the glycoprotein of interest, particularly an antibody or Fc-fusion protein, from other recombinant proteins and host cell proteins in a manner that results in a substantially homologous preparation of the protein of interest. As a first step, cells and/or particulate cell debris are removed from the culture medium or lysate by, for example, centrifugation or filtration. Thereafter, the glycoprotein of interest, in particular the antibody or Fc-fusion protein, is purified from contaminating soluble proteins, polypeptides and nucleic acids by, for example, fractionation, ethanol precipitation, ammonium sulfate in immunoaffinity or ion exchange columns. Precipitation, reverse phase HPLC, chromatofocusing, Sephadex chromatography, chromatography on cerium oxide or cation exchange resin (eg DEAE), gel filtration or, in particular, protein A affinity chromatography. In general, it is well known in the art to teach those skilled in the art how to purify heterologous proteins expressed by host cells.

By definition, any sequence or gene introduced into a host cell is referred to as a "heterologous sequence" or "heterologous gene" or "transgene" of the host cell, even if the introduced sequence or gene is associated with an endogenous sequence in the host cell or The genes are the same. Thus, a "heterologous" protein is a protein expressed from a heterologous sequence. In the context of the present invention, particularly in the context of protein expression, the term "recombinant" is used interchangeably with the term "heterologous". Thus, a "recombinant" protein is a protein expressed from a heterologous sequence.

A heterologous gene sequence can be introduced into a target cell by using a "expression vector", preferably a eukaryotic organism, and even a better mammalian expression vector. Methods for constructing vectors are well known to those skilled in the art and are set forth in various publications. In particular, techniques for constructing suitable vectors (including instructions for functional components (eg, promoters, enhancers, termination signals, and polyadenylation signals, selection markers, origins of replication, and splicing signals) are described in considerable detail. It is outlined in (Sambrook et al., 1989) and references cited therein. Vectors can include, but are not limited to, plasmid vectors, phagemids, cosmids, artificial/minichromosome (eg, ACE) or viral vectors (eg, baculovirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus) , retrovirus, phage). Eukaryotic expression vectors will also typically contain prokaryotic sequences that promote the propagation of the vector in bacteria, such as the origin of replication and the antibiotic resistance genes used to select among the bacteria. A variety of eukaryotic expression vectors containing selection sites for operably linked polynucleotides are well known in the art, and some are commercially available from companies such as: Stratagene, La Jolla, CA; Invitrogen, Carlsbad, CA; Promega, Madison , WI or BD Biosciences Clontech, Palo Alto, CA.

Stabilizing the expression of a glycoprotein, a preferred antibody or an Fc fusion protein in a H4-II-E rat hepatoma cell prior to transfection of the nucleic acid sequence encoding the protein of interest, wherein the nucleic acid sequence is It is operatively linked to at least one regulatory sequence that allows for the representation of the nucleic acid sequence. The cells are transfected with DNA prior to the stable expression of glycoproteins in H4-II-E cells, wherein the gene encoding the protein of interest is functionally linked to a genetic element that controls transcription of the gene.

Such elements or "regulatory sequences" that control gene transcription are, for example, enhancers, promoters, and 5' UTR sequences. Examples include, but are not limited to, SV40 enhancer, CMV enhancer, albumin enhancer, hepatitis B enhancer, aldolase enhancer, Ig enhancer, tyrosinase enhancer, SV40 promoter, EF1-α Promoter, chicken β-actin promoter, CMV promoter, HSV TK promoter, phosphoglycerin kinase (PGK) promoter, polymerase II promoter, ubiquitin C promoter, albumin promoter, α1-antibody Trypsin promoter, α-fetoprotein (AFP) promoter, aldolase promoter, α1 microglobulin promoter, phosphoenolpyruvate carboxykinase promoter, RSV promoter, GAPDH promoter, beta globulin Promoter and MT1 promoter.

Thus, in a preferred embodiment, the H4-II-E cells of the invention comprise an expression vector comprising at least one nucleic acid sequence which is a regulatory sequence necessary for transcription and translation of a nucleic acid sequence encoding a glycoprotein of interest. In a particular embodiment, the expression vector comprises at least one regulatory sequence that permits transcription and translation (expression) of the nucleic acid sequence of interest or encoding of the glycoprotein of interest, preferably an antibody or Fc fusion protein.

"Regulatory sequences" additionally include promoters, enhancers, termination signals, and polyadenylation signals as well as other expression control elements. Both inducible and constitutive regulatory sequences are known in the art to function in a variety of cell types. Transcriptional regulatory elements typically comprise a promoter upstream of the sequence of the gene to be expressed, a transcriptional initiation and termination site, and a polyadenylation signal sequence. The term transcription initiation site refers to a nucleic acid in a construct corresponding to a first nucleic acid that is included in a primary transcript (ie, an mRNA precursor); a transcription initiation site may overlap with a promoter sequence. The term transcription termination site refers to a nucleotide sequence typically displayed at the 3' end of the stretch of interest or a transcribed sequence that causes RNA polymerase to terminate transcription. The polyadenylation signal sequence or the poly A addition signal is a dissociation of a specific site at the 3' end of the eukaryotic mRNA and about 100 to 200 adenine nucleotides are added in the nucleus after transcription at the dissociated 3' end. The sequence of (poly A tail) provides a signal. The polyadenylation signal sequence comprises the sequence AATAAA and the downstream sequence located about 10 to 30 nucleotides upstream of the dissociation site. Various polyadenylation elements are known in the art, such as SV40 late and early poly A or BGH poly A. Translational regulatory elements include a translation initiation site (AUG), a stop codon, and a poly A signal for each of the individual polypeptides to be represented. Some constructs include a nucleosome saccharide entry site (IRES). IRES is defined below. To optimize performance, it may be necessary to remove, add or alter the 5' and/or 3' untranslated portion of the nucleic acid sequence to be expressed in order to eliminate potentially redundant inappropriate replacement translation initiation codons or to interfere at the transcriptional or translational level or Reduce other sequences of performance. Alternatively, a consensus ribosome binding site can be inserted 5' to the start codon to enhance performance. To produce a secreted polypeptide, the selected sequence will typically include a signal sequence encoding a leader peptide that directs the new synthetic polypeptide to and through the ER membrane, wherein the polypeptide can be placed into the secretory pathway. The leader peptide is often, but not exclusively, located at the amino terminus of the secreted protein and is cleaved by signal peptidase after the protein has passed through the ER membrane. The selected sequence will usually (but not necessarily) include its unique signal sequence. If the native signal sequence is absent, the heterologous signal sequence can be fused to the selected sequence. Numerous signal sequences are known in the art and are available from sequence databases such as GenBank and EMBL.

"Promoter" refers to a polynucleotide sequence/nucleic acid sequence that controls the transcription of a operably linked gene or sequence. Promoters include signals for RNA polymerase binding and transcription initiation. The promoter used will be functional in a host cell encompassing the cell type exhibiting the selected sequence, which is a H4-II-E cell in the present invention. A large number of promoters including constitutive, inducible and inhibitory promoters from a variety of different sources are well known in the art (and identified in databases such as GenBank) and can be cloned (for example from, for example, ATCC) Such as storage and other commercial or personal sources) or obtained within the selected polynucleotide. In the case of an inducible promoter, the promoter activity is increased or decreased in response to the signal. For example, a tetracycline (tet) promoter containing a tetracycline operator sequence (tetO) can be induced by tetracycline regulated transactivation protein (tTA). The binding of tTA to tetO can be inhibited in the presence of tet. For other inducible promoters (including jun, fos, metallothionein and heat shock promoters) see, for example, Sambrook et al., 1989. Eukaryotic promoters that have been identified as strong promoters due to their high degree of expression include the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, and the long end of Rous sarcoma virus. Repeat sequences and human cytomegalovirus very early promoter (CMV). Other heterologous mammalian promoters include, for example, actin promoters, immunoglobulin promoters, heat shock promoters. The above promoters are well known in the art.

As used herein, "enhancer" refers to a polynucleotide sequence/nucleic acid sequence that acts on a promoter to enhance transcription of an operably linked gene or coding sequence. Unlike promoters, enhancers have relative orientation and positional independence and have been found to be located within the intron and at the 5' or 3' within the coding sequence itself in the transcription unit. Thus, an enhancer can be placed upstream or downstream of the transcription start site or at a considerable distance from the promoter, but in fact the enhancer can physically and functionally overlap with the promoter. Numerous enhancers from a variety of different sources are well known in the art (and are identified in databases such as GenBank, such as the SV40 enhancer, CMV enhancer, polyoma enhancer, adenovirus enhancer) and can be used to select polynucleotide sequences. (from, for example, a registry such as ATCC and other commercial or personal sources) or obtained within a select polynucleotide sequence. Many polynucleotides comprising a promoter sequence (eg, a common CMV promoter) also contain an enhancer sequence. For example, all of the strong promoters listed above also contain strong enhancers.

The term "operably linked" means that two or more nucleic acid sequences or sequence elements are arranged in a manner that allows them to function in their intended manner. For example, a promoter and/or enhancer is operably linked to a coding sequence if the promoter and/or enhancer acts in a cis manner to control or regulate transcription of the ligated sequence. Typically, but not necessarily, the operably linked DNA sequences are contiguous and, if necessary, ligated to the two protein coding regions or in the context of a secretory leader sequence, are contiguous and in reading frame.

However, although the operably linked promoter is typically located upstream of the coding sequence, it does not have to be contiguous thereto. The enhancer does not have to be contiguous as long as it increases the transcription of the coding sequence. To this end, it can be located upstream or downstream of the coding sequence and even at a distance. A polyadenylation site is operably linked to a coding sequence if the polyadenylation site is located at the 3' end of the coding sequence such that transcription proceeds through the coding sequence to the polyadenylation sequence. Linkages are achieved by recombinant methods known in the art, for example, using PCR methods, by ligation at appropriate restriction sites, or by annealing. If no suitable restriction sites are present, synthetic oligonucleotide linkers or adaptors can be used according to customary practice.

A "transcription unit" defines a region in a construct that contains one or more pseudo-transcribed genes in which genes contained within the segment are operably linked to each other and transcribed from a single promoter, and thus, different genes are at least transcribed. More than one protein or product can be transcribed and expressed from each transcription unit. Each transcription unit will contain the regulatory elements necessary for transcription and translation of any of the selected sequences contained in the unit.

The term "expression" as used herein refers to the transcription and/or translation of a heterologous nucleic acid sequence within a host cell. The degree of expression of the desired product/protein of interest in the host cell can be determined based on the amount of corresponding mRNA present in the cell, or the amount of the desired polypeptide/protein of interest encoded by the sequence selected within the example. For example, mRNA transcribed from a selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to cellular RNA, or by PCR. The protein encoded by the selected sequence can be quantified by various methods, such as by ELISA, by Western blotting, by radioimmunoassay, by immunoprecipitation, by analyzing the biological activity of the protein, borrowing Immunostaining is performed on the protein and then subjected to FACS analysis or by homogeneous time-resolved fluorescence (HTRF) analysis.

A "benefit cassette" defines a region in a body that contains one or more pseudo-transcribed genes in which genes contained within the segment are operably linked to each other and transcribed from a single promoter, and thus, different genes are at least transcribed. More than one protein or product can be transcribed and expressed from each transcription unit. Each transcription unit will contain the regulatory elements necessary for transcription and translation of any of the selected sequences contained in the unit.

The "transfection" of a eukaryotic host cell with a polynucleotide or expression vector to produce a genetically modified cell or transgenic cell can be carried out by any method well known in the art (see, for example, (Sambrook et al., 1989)). Transfection methods include, but are not limited to, liposome-mediated transfection, calcium phosphate co-precipitation, electroporation, nuclear transfection, nuclear nucleoporation, microperforation, polycation (eg, DEAE-dextran) mediated Transfection, protoplast fusion, viral infection and microinjection. Preferably, the transfection is stably transfected. Transfection methods that provide optimal transfection frequencies and performance of heterologous genes in a particular host cell line and type are advantageous. Suitable methods can be determined by routine procedures. For stable transfectants, the construction system is integrated into the genome or artificial chromosome/minichromosome of the host cell or freely positioned to stably maintain in the host cell.

The "selectable marker gene" or "selection marker gene" allows for the specific selection of the gene of the cell containing the gene by adding the corresponding selection agent to the medium. As an illustration, an antibiotic resistance gene can be used as a positive selectable marker. Only cells that have been transformed with this gene are able to grow in the presence of the corresponding antibiotic and are therefore selected. On the other hand, untransformed cells cannot grow or survive under these selective conditions. There are positive, negative and bifunctional selectable markers. Positive selectable markers allow selection and (and therefore) enrichment of transformed cells by conferring resistance to the selection agent or by compensating for metabolic or catabolic defects in the host cell. In contrast, cells that have received a gene for a selectable marker can be selectively eliminated by a negative selectable marker. An example of this is the herpes simplex virus thymidine kinase gene, which is manifested in the elimination of cells that are simultaneously added with acyclovir or gancyclovir. Alternative labels (including amplifiable selectable markers) for use in the present invention include genetically modified mutants and variants, fragments, functional equivalents, derivatives, homologs, and fusions with other proteins or peptides The premise is that the optional marker retains its selective quality. Such derivatives exhibit considerable homology on amino acid sequences in regions or domains that are considered to be selective. The literature describes a number of selectable marker genes (including bifunctional (positive/negative) markers) (see, for example, WO 92/08796 and WO 94/28143). Examples of selectable markers commonly used in eukaryotic cells include the following genes: aminoglycoside phosphotransferase (APH), hygromycine phosphostransferase (HYG), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamate synthase, aspartate synthase; and a gene conferring resistance to neomycin (G418/geneticin) )), puromycin, histamine D, bleomycin, phleomycin and zeocin.

Fluorescence-activated cell sorting (FACS) can be used, for example, on cell surface markers, bacterial beta-galactosidase or fluorescent proteins (eg, from Aequorea victoria and Renilla reniformis or others). Species of green fluorescent protein (GFP) and its variants; red fluorescent protein, from non-bioluminescent species (eg, Discosoma sp . , Anemonia sp .) , Clavularia sp . , the fluorescent protein of Zoanthus sp . and its variants are selected for selection of recombinant cells.

The term "selective agent" refers to a substance that interferes with the growth or survival of H4-II-E cells lacking a particular selectable gene. For example, to select for the presence of an antibiotic resistance gene (eg, APH (aminoglycoside phosphotransferase)) in transfected cells, the antibiotic geneticin (G418) is used.

The term "modified neomycin-phosphotransferase (NPT)" encompasses all mutants described in WO2004/050884, in particular mutant D227G (Asp227Gly) (characterized by the use of aspartic acid at amino acid position 227) (Asp, D) substituted glycine (Gly, G)) and especially preferred mutant F240I (Phe240Ile) (characterized by the replacement of isoleucine with phenylalanine (Phe, F) at position 125 of the amino acid (Ile) , I)).

The invention further comprises a method of making and selecting recombinant H4-II-E cells comprising the steps of: (i) encoding at least one glycoprotein/product of interest and a selection marker, preferably neomycin-phosphotransferase. Genes are transfected into H4-II-E cells, wherein to enhance transcription or expression, at least the gene of interest (or genes) is ligated to enhancers, promoters and 5'-UTR sequences and/or visually The conditional function is linked to at least one TE element (see WO 2008/012142, which is incorporated herein by reference); (ii) cultivating the cells under conditions capable of expressing different genes; and (iii) by selecting a reagent ( Such cells are incubated in the presence of, for example, G418, MTX or MSX) to select such co-integrating genes. Preferably, the transfected cells are cultured in serum-free medium. Preferably, the concentration of G418 is at least 200 μg/mL. However, the concentration can also be at least 400 μg/mL.

Amplable selectable marker genes:

In addition, the cells of the invention may optionally be subjected to one or more gene amplification steps, wherein the cells are incubated in the presence of a selection agent that amplifies the amplifiable selectable marker gene.

It is a prerequisite to additionally transfect H4-II-E cells with a gene encoding an amplifiable selectable marker. It is contemplated that the gene encoding the amplifiable selectable marker will be present on one of the expression vectors of the invention or introduced into the host cell by means of other vectors.

The amplifiable selectable marker gene typically encodes an enzyme required to grow eukaryotic cells under certain incubation conditions. For example, the amplifiable selectable marker gene can encode dihydrofolate reductase (DHFR) or glutamate synthase (GS). In this case, the gene can be amplified if the host cell transfected with the gene is cultured in the presence of the selective agent methotrexate (MTX) or sulfinamide imipenem (MSX).

Table 2 below gives examples of amplifiable selectable marker genes and related selection agents that can be used in accordance with the present invention, as outlined by Kaufman (Kaufman, 1990).

Table 2: Amplable selectable marker genes

According to the invention, the amplifiable selectable marker gene used is preferably a gene encoding a polypeptide having GS or DHFR function.

The terms "transformation or to transform", "transfection or to transfect" as used herein, mean any introduction of a nucleic acid sequence into a cell to produce a genetically modified, recombinant, transformed or transgenic cell. The introduction can be implemented by any method well known in the art. Methods include, but are not limited to, lipofection, electroporation, polycation (eg, DEAE-dextran) mediated transfection, protoplast fusion, viral infection, and microinjection, or may be by calcium, electroshock, vein This is carried out by intra/intramuscular injection, aerosol inhalation or oocyte injection. Transformation can transiently or stably transform host cells. The terms "transfection or to transfect", "transformation or to transform" also mean the introduction of a viral nucleic acid sequence by the natural introduction of a particular virus. The viral nucleic acid sequence does not need to be in the form of a naked nucleic acid sequence, but can be encapsulated in a viral protein envelope. Thus, the term is not only about methods that are commonly known by the terms "transfection or to transfect", "transformation or to transform". Transfection methods that provide optimal transfection frequencies and performance of the introduced nucleic acids are advantageous. Suitable methods can be determined by routine procedures. For stable transfectants, the construction system is integrated into the genome or artificial chromosome/minichromosome of the host cell or freely positioned to stably maintain in the host cell.

Specific embodiment

The invention features a rat hepatoma cell comprising a nucleic acid sequence encoding an antibody or Fc-fusion protein, wherein the nucleic acid sequence is operably linked to at least one regulatory sequence that permits expression of the nucleic acid sequence encoding the antibody or Fc-fusion protein. The invention further describes a rat hepatoma cell characterized by a nucleic acid sequence encoding an antibody or Fc-fusion protein, wherein the nucleic acid sequence is operably linked to at least one nucleic acid sequence which permits expression of the encoding antibody or Fc-fusion protein. Adjust the sequence.

In a specific embodiment, the rat hepatoma cell line H4-II-E cells. In yet another embodiment, the rat hepatoma cell line is deposited in a DSMZ cell with accession number DSM ACC3129 (H4-II-E) or DSM ACC3130 (H4-II-Es).

The invention encloses H4-II-E rat hepatoma cells comprising/characterized with a nucleic acid sequence encoding an antibody or Fc-fusion protein, wherein the nucleic acid sequence is operably linked to at least one of which allows expression of the encoding antibody or Fc- A regulatory sequence of a nucleic acid sequence of a fusion protein.

The present invention describes H4-II-E rat hepatoma cells genetically modified by introducing a nucleic acid sequence of interest/gene encoding a glycoprotein, preferably an antibody or Fc fusion protein operably linked to at least one The regulatory sequence of the nucleic acid sequence/gene of interest encoding the antibody or Fc-fusion protein is allowed to be expressed.

The invention features H4-II-E rat hepatoma cells or derivatives or progeny thereof comprising/characterized with a nucleic acid sequence encoding an antibody or Fc-fusion protein, wherein the nucleic acid sequence is operably linked to at least one of the permits The regulatory sequence of the nucleic acid sequence encoding the antibody or Fc-fusion protein is expressed.

The present invention provides H4-II-E rat hepatoma cells or derivatives or progeny thereof genetically modified by introducing a nucleic acid sequence/gene of interest encoding a glycoprotein, preferably an antibody or an Fc fusion protein, The regulatory sequence is operably linked to at least one nucleic acid sequence/gene of interest that allows expression of the encoding antibody or Fc-fusion protein.

In a specific embodiment, the rat hepatoma cell or the H4-II-E cell line is derived from the European Cell Culture Preservation Center (ECACC, Cat. No. 87031301) or the American Type Culture Collection (ATCC, Accession Number CRL) The cell of -1548) or the cell line is deposited in the cell of the European Cell Culture Preservation Center under the number ECACC, catalog number 87031301, or a derivative or progeny thereof, or the cell line is deposited in the American typical culture under the accession number CRL-1548. The object preservation center ATCC is either a derivative or a progeny thereof. In still another embodiment, the rat hepatoma cell or the H4-II-E cell line has cells of ECACC catalog number 87031301 or ATCC number CRL-1548. In another specific embodiment, the rat hepatoma cell line:

a) cells derived from cells selected from the group consisting of: European Cell Culture Preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession Number CRL-1548), H4-II-E -C3 cell line (CRL-1600 or HPACC No. 85061112 or ECACC catalog number 85061112), H4II cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) And the H4-S cell line (HPACC Nr. 89102001), or

b) deposited in the European Cell Culture Preservation Center under the number ECACC catalog number 87031301 or in the US Type Culture Collection Center ATCC under the accession number CRL-1548, or

c) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E), or

d) cells deposited in the DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es), or

e) a derivative or progeny of any of cells a) or b) or c) or d). In a specific embodiment, the rat hepatoma or the H4-II-E cell line is deposited in a DSMZ cell with accession number DSM ACC3129 (H4-II-E) or DSM ACC3130 (H4-II-Es).

In another specific embodiment, the rat hepatoma cell or the H4-II-E rat hepatoma cell has low algal glycosylation activity for adding trehalose to a structure such as a twig For example, in the glycoside structure such as N-acetylglucosamine.

Specifically, the rat hepatoma cell of the present invention or the H4-II-E rat hepatoma cell is further characterized by: i) the glycoside structure contained in the antibody or the Fc-fusion protein expressed by the cell contains trehalose The degree (or ratio) is less than 20%, 10% or 5% (of all glycan/glycoside structures) or ii) the degree of glycosidic structure contained in the antibody or Fc-fusion protein expressed by the cell is such that the degree of trehalose is ( All glycan/glycosidic structures) between 0% and 20%, 0% to 10%, 0% to 5%, 0.5% to 20%, 0.5% to 10%, 0.5% to 5%, 1% to 20 %, 1% to 10% or between 1% and 5%.

Specifically, the rat hepatoma cell or the H4-II-E rat hepatoma cell of the present invention is further characterized in that less than 20% of the glycan/glycoside structure of the antibody or Fc-fusion protein contains binding to the terminal reduction N - Trehalose of the residue of glucosamine (GlcNAc). Preferably, the de-fucosylated glycoside structure/glycans are N-linked, optimally, for IgGl, IgG2, IgG3 and IgG4 antibodies, according to the Kabat EU nomenclature (Kabat et al., 1991), The glycoside structures/glycans are attached to the amino acid residue at position 297 by N-linked glycosylation.

In a specific embodiment, the rat hepatoma cell or the H4-II-E rat hepatoma cell has high galactosylation activity for adding galactose to a glycan such as a twig (eg N-acetylglucosamine) and other glycoside structures.

Specifically, the rat hepatoma cell or the H4-II-E rat hepatoma cell of the present invention is further characterized by: i) a glycan/glycosidic structure contained in the antibody or Fc-fusion protein expressed by the cell Preferably, the complex glycan/glycosidic structure contains at least one, preferably one or two or one or more galactose residues to a degree (all of the complex glycan/glycosidic structures) of more than 40%, 45% or 50% or ii) the glycan/glycosidic structure, preferably the complex glycan/glycosidic structure contained in the antibody or Fc-fusion protein represented by the cell, containing at least one, preferably one or two or one or more The degree of galactose residues is between 40% to 100%, 45% to 100%, 50% to 100%, 51% to 100%, 40% to 99.5%, of all complex glycan/glycosidic structures, It is in the range of 45% to 99.5%, 50% to 99.5% or 51% to 99.5%, 40% to 99%, 45% to 99%, 50% to 99% or 51% to 99%.

Preferably, the galactosylated glycoside structures/glycans contain one or two galactose residues of N-acetylglucosamine (GlcNAc) preferably attached to the terminal non-reducing sites of the glycoside structures ( G1 or G2). Preferably, for IgGl, IgG2, IgG3 and IgG4 antibodies, the glycoside structure/glycans are N-linked to the amino acid residue at position 297 according to the Kabat EU nomenclature (Kabat et al., 1991).

In a particular embodiment of the invention, the glycan/glycosidic structure is G1 or G2. The glycan/glycoside structure is preferably not G0.

In a specific embodiment, the rat hepatoma cell or the H4-II-E rat hepatoma cell has high sialylation activity for adding a sialic acid or a neuramic acid residue to, for example, a galactosyl group. The structure of glycosides such as dimeric structural glycans.

Specifically, the rat hepatoma cells of the present invention or the H4-II-E rat hepatoma cells are further characterized by: i) the antibody or Fc-fusion protein expressed by the cell (galactosylation) a glycoside structure containing a terminal sialic acid or a ceramide residue to a degree of more than 5% or more than 10% or ii) a (galactosylated) glycoside structure contained in the antibody or Fc-fusion protein expressed by the cell The degree of terminal sialic acid or neuramic acid residues is in the range of 0-8%, 1-8%, 5-10%, 10-50% or 10-45%.

Preferably, the glycoside structure/glycan N-linkage containing a terminal sialic acid or a neuraminic acid residue, optimally, for the IgG1, IgG2, IgG3 and IgG4 antibodies, according to the Kabat EU nomenclature (Kabat Et al., 1991), the glycoside structures/glycans are attached to the amino acid residue at position 297 by N-linked glycosylation.

In a specific embodiment, the rat hepatoma cells of the invention or the H4-II-E rat hepatoma cells are isolated.

In still another embodiment, the rat hepatoma cell of the invention or the H4-II-E rat hepatoma cell is further characterized by a selection marker gene (eg, neomycin-phosphotransferase (NPT)). A resistance gene for puromycin, hygromycin or genomicin or an amplifiable selectable marker gene (for example, dihydrofolate reductase (DHFR) or glutamate synthase (GS)). In a specific embodiment, the NPT is a wild type neomycin-phosphotransferase.

In another specific embodiment of the rat hepatoma cell or H4-II-E rat hepatoma cell of the invention, the regulatory sequence that confers the nucleic acid sequence encoding the antibody or Fc-fusion protein is a) a promoter or b) enhancer or c) 5'-UTR sequence, or d) transcription enhancing (TE) element.

In still another embodiment of the invention, the antibody or Fc fusion protein comprises a glycoside structure comprising the following sugar chains:

In another embodiment of the invention, the antibody or Fc fusion protein comprises a glycoside structure linked to a N-aspartic acid (N-Asn) residue, wherein the glycoside structure comprises the following sugar chain:

Specifically, the glycoside structure comprises the following sugar chains:

Preferably, the N-Asn system N-Asn (297) in the above examples is according to the Kabat EU nomenclature (Kabat et al., 1991).

In a specific embodiment, less than 20%, preferably less than 10% or less than 5% of the GlcNAc residue of the glycan reducing end is bound to trehalose, and more preferably no trehalose is bound to the GlcNAc residue of the reducing end of the glycan, As shown by the following structure:

or

In another embodiment, the rat hepatoma cells or H4-II-E rat hepatoma cells of the invention are adapted to grow in serum-free and calcium-reduced or preferably calcium-free medium.

In yet another embodiment, the rat hepatoma cells or H4-II-E rat hepatoma cells of the invention are adapted to grow in suspension culture. In another embodiment, the rat hepatoma cells or H4-II-E rat hepatoma cells of the invention are additionally adapted to grow in a medium free of protein/peptide of any animal origin. In another specific embodiment, the rat hepatoma cells or H4-II-E rat hepatoma cells of the invention have low sensitivity to apoptosis and/or have cell stress compared to YB2/0 cells. Highly robust.

The invention further describes a method of producing a glycoprotein of interest, characterized by the following steps:

a) providing rat hepatoma cells,

b) adapting the cells of step a) to growth in suspension culture, as appropriate,

c) adapting the cells of step a) and/or step b) to growth in serum-free medium, as appropriate,

d) depending on the condition, the cells of step a) and/or step b) and/or step c) are grown in a calcium-reduced or calcium-free medium,

e) transfecting the optionally adapted rat hepatoma cells with a nucleic acid sequence encoding a recombinant glycoprotein of interest,

f) cultivating the transfected cells of step e) under conditions permitting expression of the glycoprotein of interest,

g) The desired (sugar) protein is isolated and purified as appropriate.

In still another embodiment, the rat hepatoma cell line H4-II-E cells, preferably, the cell line:

i) Cells derived from cells selected from the group consisting of: European Cell Culture Preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession Number CRL-1548), H4-II-E -C3 cell line (CRL-1600 or HPACC No. 85061112 or ECACC catalog number 85061112), H4II cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) And the H4-S cell line (HPACC Nr. 89102001), or

Ii) deposited in the European Cell Culture Preservation Center under the numbered ECACC catalog number 87031301 or in the US Type Culture Collection Center ATCC under the accession number CRL-1548, or

Iii) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E), or

Iv) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-ES), or

v) a derivative or progeny of any of i) or ii) or iii) or iv).

Preferably, the cell line has cells of ECACC catalog number 87031301 or ATCC number CRL-1548. In a particularly preferred embodiment, the rat hepatoma cell or the H4-II-E cell line is deposited in a DSMZ cell with accession number DSM ACC3129 (H4-II-E) or DSM ACC3130 (H4-II-Es).

In a specific embodiment, the medium of step b), c) or d) is additionally free of any animal derived protein/peptide.

In still another embodiment, the method is further characterized in that the transfection step e) comprises introducing into the rat hepatoma cell an expression vector comprising a nucleic acid sequence encoding the glycoprotein of interest, the nucleic acid sequence being operably linked To at least one regulatory sequence that permits expression of the nucleic acid sequence encoding the glycoprotein of interest.

In another embodiment, the method of producing a glycoprotein of interest is further characterized in that the culturing step f) comprises adapting the transfected cells for growth in suspension culture, for growth in serum-free medium, or for adaptation Growth in calcium-reduced or calcium-free medium, or in suspension culture in serum-free and calcium-reduced/calcium-free medium.

In still another embodiment, the method of the invention is further characterized by the glycoprotein antibody or Fc-fusion protein, preferably antibody or Fc-fusion protein of interest, having

a) FcγRIIIa binding activity and preferably ADCC, or

b) complement binding activity and better CDC, or

c) binding activity to the neonatal Fc receptor FcRn and preferably serum stability, in particular long half-life.

The invention further describes methods of producing (recombinant) antibodies or Fc fusion proteins having

a) FcγRIIIa binding activity and/or

b) complement binding activity and / or

c) binding activity to the neonatal Fc receptor FcRn,

The method comprises producing the antibody or Fc fusion protein in a rat hepatoma cell, wherein the rat hepatoma cell is preferably a H4-II-E cell, more preferably, the cell line:

i) Cells derived from cells selected from the group consisting of: European Cell Culture Preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession Number CRL-1548), H4-II-E -C3 cell line (CRL-1600 or HPACC No. 85061112 or ECACC catalog number 85061112), H4II cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) And the H4-S cell line (HPACC Nr. 89102001), or

Ii) deposited in the European Cell Culture Preservation Center under the numbered ECACC catalog number 87031301 or in the US Type Culture Collection Center ATCC under the accession number CRL-1548, or

Iii) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E), or

Iv) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es), or

v) a derivative or progeny of any of i) or ii) or iii) or iv).

Preferably, the cell line has cells of ECACC catalog number 87031301 or ATCC number CRL-1548. In a particularly preferred embodiment, the rat hepatoma cell or the H4-II-E cell line is deposited in a DSMZ cell with accession number DSM ACC3129 (H4-II-E) or DSM ACC3130 (H4-II-Es).

Preferably, the (recombinant) antibody or Fc fusion protein is encoded by a nucleic acid sequence operably linked to at least one regulatory sequence that permits expression of the nucleic acid sequence encoding the antibody or Fc-fusion protein.

In a specific embodiment

i) the antibody or Fc fusion protein of the previous step a) has (increased) antibody-dependent cellular cytotoxicity (ADCC) or

Ii) the antibody or Fc fusion protein of the previous step b) has (increased) complement dependent cytotoxicity (CDC) or

Iii) The antibody or Fc fusion protein of the previous step c) is serum stable. Specifically, iii) means that the terminal sialylation of the glycoside structure produced in H4-II-E cells has a positive effect on the serum stability and catabolic half-life of the therapeutic antibody or Fc-fusion protein. Thus, such antibodies or Fc fusion proteins of iii) have an extended half-life/increased serum stability/prolonged catabolic half-life compared to antibodies or Fc fusion proteins produced in other cells (eg, CHO cells).

Increased ADCC, CDC activity or half-life can be achieved by comparing the individual activity or half-life of antibodies or Fc fusion proteins produced in rat hepatoma cells or H4-II-E rat hepatoma cells with CHO cells (specifically The activity of the corresponding antibody or Fc fusion protein produced in CHO DG44 cells was measured.

The invention further relates to a method for producing an antibody or Fc fusion protein having an promoted ADCC, which comprises introducing a DNA encoding the antibody into a rat liver tumor or a H4-II-E rat liver tumor cell, additionally comprising the cell The antibody is grown and produced.

The invention further relates to a method for producing an antibody or Fc fusion protein having a promoted CDC, which comprises introducing a DNA encoding the antibody into a rat hepatoma cell or a H4-II-E rat hepatoma cell, the method additionally comprising The antibody is grown and produced in the cell.

The invention further relates to a method of producing an antibody or Fc fusion protein having (promoted/improved) serum stability and half-life, especially if compared to an antibody or Fc fusion protein produced in other cells (eg, CHO cells), Including introducing the DNA encoding the antibody into H4-II-E rat hepatoma cells, the method further comprising culturing and producing the antibody in the cell.

Specifically, production means that rat hepatoma cells are cultured and optionally purified and isolated under conditions permitting expression of an antibody or Fc fusion protein, wherein the rat hepatoma cells are preferably H4-II-E cells, More preferably, the cell line:

i) Cells derived from cells selected from the group consisting of: European Cell Culture Preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession Number CRL-1548), H4-II-E -C3 cell line (CRL-1600 or HPACC No. 85061112 or ECACC catalog number 85061112), H4II cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) And the H4-S cell line (HPACC Nr. 89102001), or

Ii) deposited in the European Cell Culture Preservation Center under the numbered ECACC catalog number 87031301 or in the US Type Culture Collection Center ATCC under the accession number CRL-1548, or

Iii) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E), or

Iv) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es), or

v) a derivative or progeny of any of i) or ii) or iii) or iv).

Preferably, the cell line has cells of ECACC catalog number 87031301 or ATCC number CRL-1548. In a particularly preferred embodiment, the rat hepatoma cell or the H4-II-E cell line is deposited in a DSMZ cell with accession number DSM ACC3129 (H4-II-E) or DSM ACC3130 (H4-II-Es).

In a specific embodiment of the method of the present invention, the glycoprotein of interest is further characterized by i) the degree (or proportion) of the glycosidic structure contained in the glycoprotein of interest (eg, antibody or Fc-fusion protein) containing trehalose Is less than 20%, less than 10% or less than 5% of all glycan/glycoside structures or ii) the degree of glycosidic structure contained in the glycoprotein of interest (eg, the antibody or Fc-fusion protein) contains trehalose Between 0% to 20%, 0% to 10%, 0% to 5%, 0.5% to 20%, 0.5% to 10%, 0.5% to 5%, 1% (of all glycan/glycosidic structures) To the range of 20%, 1% to 10% or 1% to 5%. In particular, the glycoprotein of interest (eg, an antibody or Fc-fusion protein) is further characterized by less than 20% glycan/glycoside structure of the glycoprotein of interest (eg, the antibody or Fc-fusion protein) containing binding to terminal reduction Trehalose of the residue of N-acetylglucosamine (GlcNAc). Preferably, the de-fucosylated glycoside structure/glycans are N-linked, optimally, for IgGl, IgG2, IgG3 and IgG4 antibodies, according to the Kabat EU nomenclature (Kabat et al., 1991), The glycoside structures/glycans are attached to the amino acid residue at position 297 by N-linked glycosylation.

In another embodiment of the method of the present invention, the glycoprotein of interest is further characterized by i) a glycan/glycosidic structure contained in the glycoprotein of interest (eg, the antibody or Fc-fusion protein), Preferably, the complex glycan/glycosidic structure contains at least one, preferably one or two or one or more galactose residues to a degree (all of the complex glycan/glycosidic structures) of more than 40%, 45% or 50% Or ii) the glycan/glycosidic structure, preferably the complex glycan/glycosidic structure contained in the glycoprotein of interest (eg, the antibody or Fc-fusion protein) contains at least one, preferably one or two or one or more The degree of galactose residues is between 40% to 100%, 45% to 100%, 50% to 100%, 51% to 100%, 40% to 99.5% (all complex glycan/glycosidic structures) , in the range of 45% to 99.5%, 50% to 99.5% or 51% to 99.5%, 40% to 99%, 45% to 99%, 50% to 99% or 51% to 99%.

Preferably, the galactosylated glycoside structures/glycans contain one or two galactose residues of N-acetylglucosamine (GlcNAc) preferably attached to the terminal non-reducing sites of the glycoside structures ( G1 or G2). Preferably, for IgGl, IgG2, IgG3 and IgG4 antibodies, the glycoside structure/glycans are N-linked to the amino acid residue at position 297 according to the Kabat EU nomenclature (Kabat et al., 1991). In a particular embodiment of the invention, the glycan/glycosidic structure is G1 or G2. The glycan/glycoside structure is preferably not G0.

In still another embodiment of the method of the present invention, the glycoprotein of interest is further characterized by i) a (galactosylated) glycoside structure contained in the glycoprotein (eg, the antibody or Fc-fusion protein) The degree of terminal sialic acid or neuramic acid residues is more than 5% or more than 10% or ii) the (galactosylated) glycoside structure contained in the glycoprotein of interest (eg, the antibody or Fc-fusion protein) contains an end The degree of sialic acid or neuramic acid residues is in the range of 0-8%, 1-8%, 5-10%, 10-50% or 10-45%. Preferably, the glycoside structure/glycans N-linked containing terminal sialic acid or ceramide residues, optimally, for IgG1, IgG2, IgG3 and IgG4 antibodies, according to Kabat EU nomenclature (Kabat Et al., 1991), the glycoside structures/glycans are attached to the amino acid residue at position 297 by N-linked glycosylation.

The invention further clarifies a method of producing a (host) cell for producing a recombinant glycoprotein, comprising:

a) providing rat hepatoma cells,

b) adapting the rat hepatoma cells of step a) to growth in suspension culture, and

c) adapting the rat hepatoma cells of step a) to growth in serum-free medium, and

d) adapting the rat hepatoma cells of step a) to growth in a calcium-reduced or calcium-free medium, and

e) adapting the rat hepatoma cells of step a) as appropriate to growth in a medium free of protein/peptide of any animal origin, and

f) select a single cell pure line as appropriate,

g) Obtain (produce) (host) cells. Preferably, the rat hepatoma cell line H4-II-E cells, more preferably, the cell line:

i) Cells derived from cells selected from the group consisting of: European Cell Culture Preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession Number CRL-1548), H4-II-E -C3 cell line (CRL-1600 or HPACC No. 85061112 or ECACC catalog number 85061112), H4II cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) And the H4-S cell line (HPACC Nr. 89102001), or

Ii) deposited in the European Cell Culture Preservation Center under the numbered ECACC catalog number 87031301 or in the US Type Culture Collection Center ATCC under the accession number CRL-1548, or

Iii) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E), or

Iv) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es), or

v) a derivative or progeny of any of i) or ii) or iii) or iv).

Preferably, the cell line has cells of ECACC catalog number 87031301 or ATCC number CRL-1548. In a particularly preferred embodiment, the rat hepatoma cell or the H4-II-E cell line is deposited in a DSMZ cell with accession number DSM ACC3129 (H4-II-E) or DSM ACC3130 (H4-II-Es).

The invention further clarifies a method of producing a (host) cell for producing a recombinant glycoprotein, comprising:

a) providing H4-II-E rat hepatoma cells,

b) adapting the H4-II-E rat hepatoma cells of step a) to growth in suspension culture, and

c) adapting the H4-II-E rat hepatoma cells of step a) to growth in serum-free medium, and

d) adapting the H4-II-E rat hepatoma cells of step a) to growth in a calcium-reduced or calcium-free medium, and

e) adapting the H4-II-E rat hepatoma cells of step a) to a medium cultured in a protein/peptide free of any animal source, as appropriate, and

f) select a single cell pure line as appropriate,

g) Obtain (host) cells.

In a specific embodiment, the method further comprises:

h) transfecting the H4-II-E rat hepatoma host cell obtained in step g) with a glycoprotein nucleic acid sequence encoding the same, and

i) The transfected cells of step h) are incubated under conditions permitting expression of the glycoprotein of interest.

Preferably, the glycoprotein antibody or Fc fusion protein of interest, the optimal antibody or Fc fusion protein has ADCC and/or CDC and/or serum stability, and/or specifically a long half-life.

Preferably, the anti-system human antibody, humanized antibody, human chimeric antibody or human CDR-grafted antibody.

The invention further relates to a (host) cell produced according to the method of the host cell for producing a recombinant glycoprotein as described above.

The present invention further relates to (rat hepatoma) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es).

The present invention further relates to (rat hepatoma) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E).

The invention further relates to the use of rat hepatoma cells as host cells for biopharmaceutical production or as host cells for the production of (recombinant) glycoproteins, preferably antibodies or Fc fusion proteins. Preferably, the rat hepatoma cell line H4-II-E cells, more preferably, the cell line:

i) Cells derived from cells selected from the group consisting of: European Cell Culture Preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession Number CRL-1548), H4-II-E -C3 cell line (CRL-1600 or HPACC No. 85061112 or ECACC catalog number 85061112), H4II cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) And the H4-S cell line (HPACC Nr. 89102001), or

Ii) deposited in the European Cell Culture Preservation Center under the numbered ECACC catalog number 87031301 or in the US Type Culture Collection Center ATCC under the accession number CRL-1548, or

Iii) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E), or

Iv) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es), or

v) a derivative or progeny of any of i) or ii) or iii) or iv).

Preferably, the cell line has cells of ECACC catalog number 87031301 or ATCC number CRL-1548. In a particularly preferred embodiment, the rat hepatoma cell or the H4-II-E cell line is deposited in a DSMZ cell with accession number DSM ACC3129 (H4-II-E) or DSM ACC3130 (H4-II-Es).

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology, cell culture, immunology, and the like, which are well known in the art. These techniques are fully disclosed in the current literature.

All publications and patents referred to in this specification are indicative of the skill of those skilled in the art. The disclosures of all publications and patent specifications cited herein are hereby incorporated by reference in their entirety in their entirety in their entirety herein The invention as outlined above is more readily understood by reference to the following examples, which are intended to be illustrative only.

experiment Materials and methods DNA construct

Plasmid DNA for stable transfection of different cell lines encodes the IgGl antibody heavy and light chains under the control of viral or ubiquitous promoters, as well as selection markers neo and dhfr.

Cell line and cell culture

The CHO cell line CHO/DG44 lacking dihydrofolate reductase (Urlaub and Chasin, 1980) and the variant CHO cell line Pro-Lec 13.6A lacking endogenous GMD have been previously described (Ripka et al., 1986). The rat fusion tumor cell line YB2/0 and the rat liver tumor cell line H4-II-E were derived from the European Cell Culture Preservation Center (ECACC, Cat. No. 85110501 and 87031301).

The H4-II-E cell line of the present invention was deposited with the German Collection of Microbial Species (DSMZ), Inhoffenstrasse 7B, D-38124, on June 28, 2011 under the Budapest Treaty under the accession number DSM ACC3129 (H4-II-E). Braunschweig, Germany. H4-II-E cells (e.g., cells deposited with DSMZ under accession number DSM ACC3129) were cultured in MEMalpha (Invitrogen) or EMEM containing 5% FCS. The adherent cells were trypsinized with trypsin/EDTA (Invitrogen) every 3 to 4 days and seeded in tissue culture treatment plates or T-flasks (Nunc, Denmark) at a cell density of about 20,000-30,000 cells/cm ² . In the fresh medium.

The H4-II-E cell line adapted for suspension growth in the serum-free, Ca ^2+-free medium of the present invention was deposited on June 28, 2011 under the Budapest Treaty under the accession number DSM ACC3130 (H4-II-Es). Registered at the German Collection of Microorganisms (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany. H4-II-E cells adapted to suspension growth in serum-free, Ca ^2+-free medium were incubated at 37 ° C in an atmosphere containing 5% CO ₂ in a shaking flask (Corning) at 100-120 rpm. For example, the cells deposited in DSMZ with the accession number DSM ACC3130. H4-II-E suspension cultures were subcultured every 3 to 4 days and seeded at a density of 300 000-400 000 cells/mL.

In a surface aerated T-flask (Nunc, Denmark) or shake flask (Corning) at a temperature of 37 ° C and in a 5% CO ₂ atmosphere at 100-120 rpm in an incubator (Thermo, Germany) CHO-DG44 cells and adapted CHO-Lec13 cells, YB2/0 cells and H4-II-E cells were cultured in suspension in serum medium. Suspension cultures were subcultured every 2 to 3 days and seeded at a density of 200 000-300 000 cells/mL cells/mL. Cell concentrations in all cultures were determined by using a hemocytometer. Vitality was assessed by trypan blue exclusion. All suspension cultures were cultured in BI-patent medium or MEM Joklik Modification (Sigma) or MEM Spinner Modification (Sigma).

Predictive analysis of glycosylation properties

Different cell lines were selected and analyzed for the degree of performance of the enzymes involved in the glycosylation machinery. The binding of lectins (carbohydrate-specific binding proteins) of these cell lines to the surface of glycoprotein-containing cells was further analyzed. The values obtained for each cell line were normalized to values obtained for CHO, a conventional production system for CHO-based recombinant proteins and exhibiting a well characterized glycosylation pattern. There are indicators of the glycosylation ability of certain sugar structural cells on the cell surface.

Transfection and isolation of stably transfected cell populations

The guide provided by the manufacturer and then with Lipofectamine ^TM PLUS ^TM reagent (both Invitrogen, Germany), or by nucleofection (Amaxa / Lonza) transfected cell lines.

To isolate stably transfected IgGl producing cell populations, 48 hours later, cells were transfected and plated in the presence of the selective antibiotics G418 and MTX. Surviving cell populations were isolated after approximately 3 weeks of selection and analyzed by ELISA using samples from cell culture supernatants.

Serum-free feed batch culture

The H4-II-E producing cells were adapted to grow in suspension in serum-free medium. For serum-free fed batch culture, the adapted cells were seeded in BI-patented production medium or MEM Spinner Modification (Sigma) at 400,000 cells/ml or 600,000 cells/ml and at 37 ° C and 5% CO _The culture was stirred at 120 rpm under ₂ hours. Culture parameters including pH, glucose, and lactate concentrations were measured daily. The pH was adjusted to pH 7.0 using NaCO ₃ as needed, and glucose was fed to maintain a glucose content of about 4 g/L. Cell density and viability were determined by trypan blue exclusion using an automated CEDEX cell quantification system (Innovatis). The antibody or Fc-fusion protein concentration in the culture supernatant is measured by an enzyme-linked immunosorbent assay (ELISA) specific for the human IgG.

Adherent H4-II-E can be used to produce materials in a fixed feed batch culture in serum-free medium prior to suspension adaptation. H4-II-E IgG1-producing cells were grown to confluence in Hyperflasks (Corning) in MEMalpha containing 5% FCS. The medium is then replaced with serum-free BI-patented medium or serum-free commercial medium (eg, HyClone SFM4 (Thermo Fisher Scientific)). After 13 days of culture, the antibody was purified from the culture medium using MabSelect (Amersham) and stored in 10 mM citrate / 0.15 M NaCl (pH 6.0).

CHO-DG44, CHO-Lec13 and YB2/0 IgG1 producing cells were seeded in BI-patent producing medium at 300,000 cells/ml and the culture was stirred at 120 rpm at 37 ° C and 5% CO ₂ , CO ₂ later It decreased to 2% as the number of cells increased. Culture parameters including pH, glucose, and lactate concentrations were measured daily. The pH was adjusted to pH 7.0 using NaCO ₃ as needed, and glucose was fed to maintain a glucose content of about 4 g/L. The BI-patented feed solution was added every 24 hr. Cell density and viability were determined by trypan blue exclusion using an automated CEDEX cell quantification system (Innovatis).

The concentration of the antibody in the culture supernatant was measured by an enzyme-linked immunosorbent assay (ELISA) specific for the human IgG.

ELISA

Quantification of IgG molecules in cell culture supernatants was performed via a sandwich ELISA technique. ELISA plates were coated overnight at 4 °C using goat anti-human IgG Fc fragment antibody (Dianova, Germany). After washing and blocking the plates with a 1% BSA solution, samples were added and incubated for 1.5 hours. After washing, a detection antibody (alkaline phosphatase-conjugated goat anti-human kappa light chain antibody) was added and incubated with 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma, Germany) as a substrate. To perform colorimetric detection. After incubation for 20 min in the dark, the reaction was stopped and the absorbance was measured immediately at 405/492 nm using an absorbance reader (Tecan, Germany). The concentration is calculated from the standard curve present on each plate.

Purification of IgG1 from cell culture supernatant

Recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences) and stored in 10 mM citrate / 0.15 M NaCl (pH 6.0). The concentration of the purified antibody was measured by protein A-HPLC.

Analysis of glycosylation patterns

To elucidate the structure and composition of Fc-glycosylation of IgG produced in different cell lines, glycans were released from purified antibodies after reduction by enzymatic digestion with PNGase F. The glycan is purified, fluorescently labeled with 2-aminobenzamide (2-AB) and used with exoglycosidases (eg alpha-mannosidase, neuraminidase, beta-galactosidase, Fractionation was carried out on HPLC columns before and after treatment with α-galactosidase, β-hexosaminidase and α-trehalosidase. Calculate the percentage of non-trehalose-based twig-structured glycans and other glycoside structures from the ratio of chromatographic peak areas before and after digestion with exoglycosides and characterize the sugar structure and composition And quantitative verification.

FcγRIIIa-binding assay

BIAcore T100 instruments and CM5 sensor wafers (BIACORE, Uppsala, Sweden) were used to measure the binding kinetics of IgGl and FcgRIIIa produced in different cell lines as follows. Soluble recombinant FcgRIIIa was immobilized onto a BIAcore sensor wafer. Purified IgG1 was diluted in HBS-EP buffer (0.01 M HEPES, 0.15 M NaC1, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) at 6 different concentrations (from 4.17 nM to 133.3 nM), and Each diluted IgGl was injected onto the surface of the receptor capture sensor at a flow rate of 5 mL/min. The experiments were carried out using HBS-EP as a running buffer at 25 °C. A buffer solution without sample IgG1 was injected onto the surface of the receptor capture sensor as a blank control. Soluble FcgRIIIa and IgG1 bound to the sensor surface were removed by injecting 7.5 mM HCl for 30 s at a flow rate of 10 mL/min. Data obtained by injection of IgG1 were corrected for blank controls prior to data analysis. Affinity (KD) of FcgRIIIa was calculated by steady state analysis using BIAcore T100 Kinetic Evaluation Software (BIACORE).

FcγRIIb-binding assay

The binding kinetics of IgGl and FcgRIIIb produced in different cell lines were measured using a BIAcore T100 instrument and a CM5 sensor wafer (BIACORE, Uppsala, Sweden) as follows. Soluble recombinant FcgRIIIa was immobilized onto a BIAcore sensor wafer. Purified IgG1 was diluted in HBS-EP buffer (0.01 MHEPES, 0.15 M NaC1, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) at 6 different concentrations (from 4.17 nM to 133.3 nM) and A flow rate of 5 mL/min injected each diluted IgGl onto the surface of the receptor capture sensor. The experiments were carried out using HBS-EP as a running buffer at 25 °C. A buffer solution without sample IgG1 was injected onto the surface of the receptor capture sensor as a blank control. Soluble FcgRIIb and IgGl bound to the sensor surface were removed by injecting 7.5 mM HCl for 30 s at a flow rate of 10 mL/min. Data obtained by injection of IgG1 were corrected for blank controls prior to data analysis. Affinity (KD) for FcgRIIb was calculated by steady state analysis using BIAcore T100 Kinetic Evaluation Software (BIACORE).

ADCC analysis

ADCC analysis was performed by lactate dehydrogenase (LDH) release assay using human peripheral blood mononuclear cells (PBMC) prepared from healthy donors by Lymphoprep (AXIS SHIELD, Dundee, UK) as effector cells. Aliquots of target tumor cells (human Burkitt's lymphoma cell line Ramos or HER2-positive breast cancer cell lines expressing human CD20) to a 96-well U-shaped substrate (stored in Incubate with contiguous dilutions of antibody (50 μL) at an E/T ratio of 20/1 in the presence of PBMC (100 μL) in 10,000 cells in 50 μl/well. Supernatant LDH activity was measured using a non-radioactive cytotoxicity assay kit (Promega, Madison, WI) after incubation for 4 h at 37 °C. Calculate the specific cell lysis % from the sample activity according to the following formula: specific dissolution [%] = 100 × (ES _{E -} S _T ) / (MS _T ), where E is the experimental release amount (from the antibody and effector cells) The activity of the supernatant in the supernatant of the target cells cultured together, the spontaneous release of the S _E in the presence of effector cells (the activity in the supernatant from the effector cells using the medium alone), the S _T- target cells The spontaneous release amount (activity from the supernatant above the target cells cultured with the medium alone), and the maximum release amount of the M-type target cells (the activity released from the target cells dissolved with 9% Triton X-100).

Clq-binding analysis

Purified human complement Clq was used to investigate the ability of each purified IgG to bind to the Clq component of complement by flow cytometric analysis. The human Burkitt's lymphoma cell line Ramos or HER2-positive breast cancer cell line expressing human CD20 was adjusted to 2×10 ⁶ cells/mL and in anti-human CD20 in PBS containing 1% (w/v) BSA. Serial dilutions of IgG or anti-human HER2 were incubated for 30 min. After washing with PBS containing 1% (w/v) BSA, purified human complement Clq (Biogenesis Ltd, Poole, UK) was added at a final concentration of 20 mg/mL and IgG was conjugated to the cells at 37 °C. Combined for 30 min. The cells were then washed and incubated with luciferin isothiocyanate conjugated to human Clq (Acris Antibodies GmbH, Hiddenhausen, Germany) for 30 min. The stained cells were analyzed by flow cytometry using a FACSCalibur.

CDC analysis

CDC activity was determined by LDH analysis. Briefly, the human CD20 target human Burkitt's lymphoma cell line Ramos or HER2-positive breast cancer cell line, 2-fold diluted human serum complement (Sigma-) in a 96-well flat bottom plate (Greiner) at 37 °C. Aldrich) and serial dilutions of anti-human CD20 or anti-human HER2-IgG1 were incubated for 3 h. Cell proliferation LDH reagent (Roche Diagnostics, Basel, Switzerland) was added to each well (15 μL/well) and incubated at 37 ° C for 30 min. The absorbance in each well was measured at 492 nm using a microplate reader (Tecan, Germany) and expressed as relative absorbance units (RAU) as a viable cell number index. CDC% was calculated according to the following formula: CDC activity [%] = 100 x (RAU _{background -} RAU _test ) / RAU _background .

FcRn-binding assay

The recombinant soluble human FcRn-b2 microglobulin complex was expressed in CHO/DG44 cells and purified from the culture supernatant by Ni-NTA chromatography (Qiagen). The kinetics of the human IgGl-FcRn interaction was measured using a BIAcore T100 instrument and a CM5 sensor wafer. Anti-human b2-microglobulin monoclonal antibody (Abeam, Cambridge, UK) was immobilized onto the wafer using an amine coupling kit (BIACORE). The complex was captured by immobilization of an anti-b2-microglobulin antibody by injecting a soluble FcRn-b2 microglobulin complex at a flow rate of 5 mL/min. A buffer solution without the complex was injected onto the surface of the antibody capture sensor as a blank control. Each purified IgG was diluted in 5 different concentrations (from 4.17 nM to 66.7 nM) to HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) adjusted to pH 6.0. Each diluted IgGl was injected onto the composite capture sensor surface or blank at a flow rate of 5 mL/min. Soluble FcRn and IgGl bound to the surface of the sensor were removed by injecting 7.5 mM HCl for 1 min at a flow rate of 60 mL/min. These experiments were carried out using HBS-EP+ as a running buffer at 25 °C. Data analysis was performed using data obtained by subtracting blanks. Apparent association rate constants (ka), dissociation rate constants (kd), and binding affinities (KD) were calculated using a BIAcore T100 evaluation software by a binary fit model.

Pharmacokinetic analysis of mice

For the purified anti-human CD20 IgG1 produced in CHO or H4-II-E cells, 20 mg of IgG1 was injected into the tail vein of 13-week-old female ddY mice (Charles River Laboratories). Peripheral blood samples were taken from the tail vein at 0.083 h (5 min), 0.5 h, 1 h, 6 h, 24 h, 60 h, 120 h, 216 h, 312 h, and 384 h, and as previously An ELISA specific for human IgG1 is described to measure the antibody concentration in plasma. The serum half-life of IgG1 administered was calculated by eliminating the slope of the β-phase.

Instance Example 1: Predictions show significant differences in the relative amounts of glycostructures on secreted proteins between cell lines

Cell lines derived from different species and derived from different tissues or cell lineages within the species were selected and analyzed for surface structure and enzymatic activity (Figure 1). Based on the results of the analysis, each cell line can be assigned the ability to synthesize certain glycosylation patterns. The results of the analysis show that the cell line differs in its glycosylation properties depending on the species from which it is derived and the tissue or cell lineage. Only a few cell lines naturally show low algal glycosylation ability. Some cell lines potentially produce sialylated structures and several cell lines form immunogenic sugar structures (Gal-1,3-Gal and NeuGc). The source of the species and the tissue, organ or cell lineage of a given cell line cannot fully determine the glycosylation ability of the cell. Both species source and tissue cell lineage can affect the ability of cells to synthesize certain glycosylation patterns. However, it is not possible to fully predict the glycosylation properties of cell lines based solely on the knowledge of the sugar patterns obtained in another cell line derived from the same tissue and/or species (Fig. 1).

The rat hepatoma cell line H4-II-E can be distinguished from all selected and analyzed cell lines in its high potential to generate antibodies with favorable saccharide properties and is therefore selected for further evaluation.

Cell line H4-II-E All selected and analyzed cell lines were the only ones in this analysis that did not show detectable signs of fucosylation. The surprising reason for this is that other rat cell lines do not accumulate beneficial glycochemical properties in the same way as other hepatic cell lines, such as reduced haylosylation, lack of immunogenic residues, or the presence of alpha-2,6 linked saliva. Acid (Figure 1).

In particular, rat cell H4-II-E produces a glycosylation line with a low trehalose content, which is due to the significant fucosylation of the anti-system from rat blood serum, while other species (eg rabbit or The intrinsic antibody in cats has a low trehalose content.

Unlike trehalose content, there may be a species-dependent propensity to generate potentially immunogenic sugar structures (eg, Gal-1,3-Gal and NeuGc) in certain cell lines. None of the human or rat cell lines selected and analyzed showed such residues, and consistent with published data, cell lines derived from mice, rabbits, and other species consistently produced such structures, and potentially in humans. An immunogenic response is induced (Jenkins et al., 1996; Raju et al., 2000).

Example 2: Antibodies produced in the rat hepatoma cell line H4-II-E showed significantly reduced fucosylation of FC glycans compared to other known production cell lines.

To verify the predicted glycosylation properties produced in H4-II-E cells, transfection is performed by DNA constructs encoding the light and heavy chains of IgG1 antibodies and subsequently also present on transfected DNA constructs. The resistance marker is subjected to antibiotic selection to generate H4-II-E cells that produce a stable IgGl antibody. The IgGl producing cell population was obtained and adapted to grow in serum-free chemically defined medium.

The IgG1 anti-system produced in H4-II-E cells and purified from the cell culture supernatant by protein A chromatography was complete and produced discrete bands of heavy and light chains after electrophoretic separation (data not shown) . The structure and composition of Fc glycans on Protein A purified IgG1 antibody produced in H4-II-E cells were analyzed and generated in CHO-DG44, CHO-Lec13 mutants and YB2/0 rat myeloma cells. The glycosylation patterns obtained on the same antibodies were compared. In all four IgGl antibody preparations from different host cell lines, the dimeric structural glycans accounted for the largest portion of all measured Fc glycans. For the antibodies produced in YB2/0 cells alone, the proportion of other structures, which are predominantly incomplete heteropolysaccharides or high mannose structures, also accounted for a significant portion of approximately 23% (Figure 2). In terms of the ratio of the two branched structural glycans, CHO-DG44 cells were mainly reported (about 95%) to produce a fucosylated structure as previously reported. Unlike other cells, antibodies produced in H4-II-E cells clearly carry most of the non-fucosylated dimeric structural glycans. More than 80% of the IgG1 expressed in H4-II-E cells contained non-trehalose dimeric structural glycans, which was significantly higher than that produced in the cell line YB2/0 or the CHO mutant Lec13 (Fig. 2). YB2/0 and CHO-Lec13 cells also produced trehalose-free glycans. However, the percentage difference between the form containing no trehalose and the form containing trehalose was not as significant as the antibody produced by H4-II-E (Fig. 2). After initial selection and analysis of the favorable glycan structure, the IgG1 antibody produced in H4-II-E cells, although not genetically engineered, clearly showed the highest proportion of non-fucosylated dimeric structural glycans, thereby confirming the prediction (Fig. 1). For the known correlation of low algae glycosylation to enhance ADCC activation, H4-II-E cells are superior to other host cell lines in producing antibodies with high FcγRIII-dependent effector functional activity.

Example 3: Antibodies produced in the rat hepatoma cell line H4-II-E showed significantly increased galactosylation of FC glycans compared to CHO-producing antibodies

A detailed analysis of the glycosylation pattern of IgG1 produced in H4-II-E cells showed that the content of galactosylated glycoforms was increased compared to the antibody produced in CHO-DG44, which is commonly used in CHO-DG44 lines. Host cell line produced by biomedical proteins (Figure 3). Among the IgG1 antibodies produced in H4-II-E, >40% of the dimeric structural glycans were galactosylated, which was significantly higher than that expressed in CHO cells (Fig. 3). Increased galactosylation of the two structural glycans enhances the potential of the antibody to activate the complement system. This combined with the reduced fucosylation of Fc glycans produced in H4-II-E cells allows H4-II-E-producing antibodies to be higher on both types of antibody-dependent effector functions (ADCC and CDC). effectiveness. In addition to enhancing the Fc-binding and activation of the components of the complement system, galactosylation of the dimeric structural glycans is also the basis for further modification of the glycans at the terminal positions.

Example 4: Unlike antibodies produced by CHO, antibodies produced in the rat hepatoma cell line H4-II-E show detectable sialylation of FC glycans

It can be found that the sialic acid or neuraminic acid residue is attached to the previous galactose residue at the end of the branched structure of the complex N-glycan with α-2,3 or α-2,6. In terms of sialylation, the results obtained after analyzing the glycostructure of the IgG1 antibody produced in H4-II-E cells were consistent with the predicted glycostructure (Fig. 1). About 8% of the antibodies produced in H4-II-E cells harbor terminal sialic acid residues that can be dissociated by treatment of the released glycans with exoglycosidase neuraminidase (Fig. 4). Unlike antibodies produced in H4-II-E cells, and consistent with the predicted glycosylation pattern, antibodies produced in CHO-DG44 cells do not carry terminal sialic acid residues (Figure 4). The terminal sialic acid residue can have different effects on the activity and stability of the modified antibody. Recent publications have shown that sialic acid inhibits the inflammatory activity of antibodies (Burton and Dwek, 2006; Scallon et al., 2007). Other data show that the absence of sialic acid increases the metabolic rate of the liver in mice, indicating clearance by liver-based receptors (Wright et al., 2000). Sialic acid derivatives (e.g., N-hydroxyethyl cyanokinin (NeuGc)) have been found to be immunogenic in humans (Noguchi et al., 1995), however, proteins produced in H4-II-E cells cannot be detected. Evidence of NeuGc modification (Figure 1).

Example 5: H4-II-E rat hepatoma cells adapted for suspension growth in serum-free medium

H4-II-E cells cultured in serum-containing medium (EMEM (EBSS) + 2 mM glutamic acid + 1% non-essential amino acid + 10% fetal bovine serum (FBS)) were grown as adherent cell layers. For industrial production of biopharmaceutical proteins, H4-II-E cells are grown in serum-free medium, preferably in chemically defined animal-free medium, and in suspension culture. In order to adapt H4-II-E cells to suspension culture in serum-free medium, cells were first adherently expanded in serum-containing medium showing an average doubling time of 30 hours. The cells were then separated and dissociated by treatment with trypsin/EDTA, harvested by centrifugation, washed thoroughly with calcium and magnesium-free Dubec's phosphate buffered saline (DPBS) and at 200,000 cells/ml to 600,000 cells/ The density of ml is suspended in serum-free BI patented medium (which is specifically designed for suspension culture of H4-II-E cells) or commercial AEM (Invitrogen) or PEM (Invitrogen) medium. The key aspect of H4-II-E cells in which the suspension culture group is doubling from 24 hours to 32 hours is to use a medium without calcium or calcium reduction. The cell suspension was incubated in a shaking flask (Corning) at 37 ° C, 100-120 rpm and 5% CO ₂ . Cells were passaged every 3 to 4 days and suspended in fresh medium. After several passages, H4-II-E cells were constantly grown in the form of single cell suspensions with a doubling time of 24 hours to 32 hours. H4-II-E cells grow well in different incubation modes and can be cultured in suspension for more than 10 days with high vigor during the feed batch process. In standard CHO optimized media, H4-II-E cells reached a maximum density of 8,000,000 cells/ml, which was further improved using H4-II-E optimized media (Figure 5). In conclusion, adapted H4-II-E cells are well suited as host cells (lines) for large-scale biopharmaceutical protein production.

Example 6: H4-II-E cells have low sensitivity to apoptosis and/or high robustness to cell stress compared to YB2/0 cells

Unlike YB2/0 cells, H4-II-E cells show high robustness to cell pressure induced by high temperature, low or high permeability, pH change, mechanical stimulation or treatment with chemicals or drugs. H4-II-E rats in the complete medium (serum-free BI medium (without Ca) for H4-II-E cells, RPMI + 10% FCS for YB2/0 cells) Hepatoma cells and YB2/0 rat myeloma cells were exposed to a pressure source below a certain dose (pressure time shown in brackets): +42 ° C (2 hours, see Figure 6A), exposure to low or high salt concentrations (24 Hours, see Figure 6B), exposure to 2 μg/ml or 5 μg/ml puromycin (48 hours, see Figure 6C): After temperature stimulation, cells were in complete medium at 37 ° C, 5% CO ₂ The cells were cultured for 24 hours, and then the number of cells and viability were analyzed by trypan blue exclusion staining. After cell stress or apoptosis-inducing stimuli, H4-II-E cells showed significantly higher robustness and viability than YB2/0 cells (Fig. 6). The low sensitivity and high robustness of H4-II-E cells to cell stressors make H4-II-E an excellent system for large-scale biopharmaceutical production processes, where high cell viability and survival are required during long culture periods. .

Example 7: Single cell suspension incubation of H4-II-E cells requires serum-free medium with reduced CA ²⁺ or no CA ²⁺

The key aspect of H4-II-E cells in which the suspension culture group is doubling from 24 hours to 32 hours is to use a medium without calcium or calcium reduction. Two calcium-reduced or calcium-free media were identified by analyzing the Ca-concentration using Hitachi 917 (Roche) (Fig. 7A). Both calcium-reducing medium AEM and Ca-free BI medium allowed single cell suspensions to culture H4-II-E cells (Fig. 7B, C). If AEM was supplemented with 1 mM CaCl ₂ (Fig. 7C) or if cells were seeded in Ca-containing BI medium (Fig. 7E), the cells formed large aggregates and the growth of single cell suspension was blocked. The reduction of free calcium ions in the calcium-containing medium can also be achieved by adding EDTA to the medium to reduce aggregation of H4-II-E cells in the medium supplemented with EDTA (Fig. 7F).

Example 8: H4-II-E cell aggregation has CA ²⁺ concentration dependence and MG ²⁺ independence

In order to solve the problem that whether the effect of calcium ion on H4-II-E cell aggregation can also be achieved by other divalent cations usually present in the cell culture medium, the suspension culture of H4-II-E cells is subjected to an increase. Concentration of CaCl ₂ or MgCl ₂ (Figure 8). If 50 μMol/L CaCl _{2 was} added to the CaBI-free medium, an increase in H4-II-E cell aggregation was observed. The aggregation ratio and tightness of the cell aggregates were further increased as the concentration of CaCl ₂ added to the Ca-free medium was increased ( FIG. 8A ). In contrast, the addition of an equal concentration of MgCl ₂ to the medium had no significant effect on single cell suspension or cell aggregation (Fig. 8B). In AEM medium, addition of 250 μM/L CaCl ₂ or higher CaCl ₂ concentration after 3 days of culture significantly impaired H4-II-E cell growth. In AEM medium supplemented with 250 μM/L CaCl ₂ or higher CaCl ₂ concentration, more than 80% of the cells form tight aggregates, which are undesirable during cell culture fermentation. Unlike CaCl ₂ , increasing the concentration of MgCl ₂ had no effect on the aggregation ratio of H4-II-E cells.

Example 9: Anti-CD20 IGG1 antibody produced in H4-II-E rat hepatoma cells binds to FC receptors CD16-V158 and CD16-F158 (FCGRIIIA) with higher affinity than anti-CD20 IGG1 produced in CHO

The anti-CD20 IgG1 antibody expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the anti-CD20 IgG1 antibody was generated by transfection with a DNA construct encoding the heavy chain (SEQ ID NO: 2) and the light chain (SEQ ID NO: 3) of the anti-CD20 IgG1 antibody. . The stable anti-CD20 production cell line was isolated by selecting an antibiotic resistance marker and analyzing the anti-CD20 expression of the cell supernatant of the viable cells by ELISA. Anti-CD20 producing cells were grown in serum-free fed batch culture and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

BIAcore analysis was used to measure the binding kinetics of anti-CD20 IgG1 and FcγRIIIa receptors CD16-V158 and CD16-F158 produced in H4-II-E cells and CHO cells. The anti-CD20 IgG1 produced in H4-II-E cells showed significantly higher affinity with the two variants of FcγRIIIa compared to the anti-CD20 IgG1 produced in CHO cells (Fig. 9).

Example 10: Anti-CD20 IGG4 antibody produced in H4-II-E rat hepatoma cells binds to FC receptors CD16-V158 and CD16-F158 (FCGRIIIA) with higher affinity than anti-CD20 IGG4 produced in CHO

The anti-CD20 IgG4 antibody expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the anti-CD20 IgG4 antibody was generated by transfection with a DNA construct encoding the heavy chain (SEQ ID NO: 4) and the light chain (SEQ ID NO: 5) of the anti-CD20 IgG4 antibody. . The stable anti-CD20 production cell line was isolated by selecting an antibiotic resistance marker and analyzing the anti-CD20 expression of the cell supernatant of the viable cells by ELISA. Anti-CD20 producing cells were grown in serum-free fed batch culture and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

Binding kinetics of anti-CD20 IgG4 and FcγRIIIa receptors CD16-V158 and CD16-F158 produced in H4-II-E cells and CHO cells were measured using BIAcore analysis. Anti-CD20 IgG4 produced in H4-II-E cells showed significantly higher affinity than the two variants of FcyRIIIa compared to anti-CD20 IgG4 produced in CHO cells.

Example 11: Anti-CD20 IGG1 antibody produced in H4-II-E rat hepatoma cells activates in vitro ADCC more efficiently than anti-CD20 IGG1 antibody produced in CHO

The anti-CD20 IgG1 antibody expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the anti-CD20 IgG1 antibody was generated by transfection with a DNA construct encoding the heavy chain (SEQ ID NO: 2) and the light chain (SEQ ID NO: 3) of the anti-CD20 IgG1 antibody. . The stable anti-CD20 production cell line was isolated by selecting an antibiotic resistance marker and analyzing the anti-CD20 expression of the cell supernatant of the viable cells by ELISA. Anti-CD20 producing cells were grown in serum-free fed batch culture and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

ADCC analysis was performed by lactate dehydrogenase (LDH) release assay using human peripheral blood mononuclear cells (PBMC) as effector cells. Aliquots of target tumor cells (human Burkitt's lymphoma cell line Ramos expressing human CD20) were dispensed into 96-well U-shaped bottom plates (10,000 cells in 50 μl/well) and in PBMC ( 100 μL) was incubated with a serial dilution of purified anti-CD20 IgG1 (50 μL) produced in H4-II-E cells or CHO cells at an E/T ratio of 20/1. After incubation at 37 ° C for 4 h, the supernatant LDH activity was measured. The % specific cytolysis was calculated and if the anti-CD20 IgG1 line used was produced and purified from H4-II-E cells but not from cell culture supernatants of CHO cells, the value was significantly higher, indicating H4-II-E production. The potential of IgG1 to induce ADCC in effector cells was significantly higher (Fig. 10).

Example 12: Anti-HER2 IGG4 antibody produced in H4-II-E rat hepatoma cells activates in vitro ADCC more efficiently than anti-HER2 IGG4 antibody produced in CHO

The anti-HER2 IgG4 antibody expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The stable anti-HER2 production cell line was isolated by selecting an antibiotic resistance marker and analyzing the anti-HER2 expression of the cell supernatant of the viable cells by ELISA. Anti-HER2 producing cells were grown in serum-free fed batch cultures and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

ADCC analysis was performed by lactate dehydrogenase (LDH) release assay using human peripheral blood mononuclear cells (PBMC) as effector cells. An aliquot of the HER2-positive breast cancer cell line was dispensed to a 96-well U-shaped bottom plate (10,000 cells in 50 μl/well) and an E/T ratio of 20/1 in the presence of PBMC (100 μL) Incubation was carried out with serial dilutions (50 μL) of purified anti-HER2 IgG4 produced in H4-II-E cells or CHO cells, respectively. After incubation at 37 ° C for 4 h, the supernatant LDH activity was measured. The specific cell lysis is calculated and if the anti-HER2 IgG4 line is produced and purified from H4-II-E cells but not from cell culture supernatants of CHO cells, the value is significantly higher, indicating H4-II-E production. The potential of IgG4 to induce ADCC in effector cells is significantly higher.

Example 13: Anti-CD20 IGG1 antibody produced in H4-II-E rat hepatoma cells binds to components of the complement system with higher affinity than the anti-CD20 IGG1 antibody produced in CHO

The anti-CD20 IgG1 antibody expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the anti-CD20 IgG1 antibody was generated by transfection with a DNA construct encoding the heavy chain (SEQ ID NO: 2) and the light chain (SEQ ID NO: 3) of the anti-CD20 IgG1 antibody. . The stable anti-CD20 production cell line was isolated by selecting an antibiotic resistance marker and analyzing the anti-CD20 expression of the cell supernatant of the viable cells by ELISA. Anti-CD20 producing cells were grown in serum-free fed batch culture and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

The ability of each purified IgGl binding complement Clq component was investigated by flow cytometric analysis using purified human complement Clq. Human Burkitt's lymphoma Ramos cells expressing human CD20 were incubated with serial dilutions of anti-human CD20 IgG1 produced in H4-II-E cells or CHO, respectively. After washing with PBS containing 1% (w/v) BSA, purified human complement Clq was added at a final concentration of 20 mg/mL and bound to IgG1 of the bound cells for 30 min at 37 °C. The cells were then washed and incubated with luciferin isothiocyanate-conjugated antibodies against human Clq. The stained cells were analyzed by flow cytometry using a FACSCalibur. The anti-CD20 IgG1 produced in H4-II-E cells showed a much stronger binding to the complement component Clq compared to the anti-CD20 IgG1 produced in CHO cells.

Example 14: Anti-CD20 IGG1 antibody produced in H4-II-E rat hepatoma cells showed enhanced in vitro complement activation compared to anti-CD20 IGG1 antibody produced in CHO cells

The anti-CD20 IgG1 antibody expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the anti-CD20 IgG1 antibody was generated by transfection with a DNA construct encoding the heavy chain (SEQ ID NO: 2) and the light chain (SEQ ID NO: 3) of the anti-CD20 IgG1 antibody. . The stable anti-CD20 production cell line was isolated by selecting an antibiotic resistance marker and analyzing the anti-CD20 expression of the cell supernatant of the viable cells by ELISA. Anti-CD20 producing cells were grown in serum-free fed batch culture and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

CDC activity was determined by WST-1 analysis. Briefly, the human CD20 target human Burkitt's lymphoma cell line Ramos, 2-fold diluted human serum complement (Sigma-Aldrich), and H4-, respectively, in a 96-well flat bottom plate (Greiner) at 37 °C Serial dilutions of anti-human CD20 produced in II-E cells or CHO cells were incubated for 1 h. Cell proliferation WST-1 reagent was added to each well (15 μL/well) and incubated for 6 h at 37 °C. The absorbance in each well was measured at 450 nm using a microplate reader (Tecan, Germany) and expressed as a relative absorbance unit (RAU) as a viable cell number index. CDC% was calculated according to the following formula: CDC activity [%] = 100 x (RAU _{background -} RAU _test ) / RAU _background . If the anti-CD20 IgG1 line is produced in H4-II-E but not in CHO cells, the CDC activity measured in the assay is significantly higher.

Example 15: Anti-CD20 IGGI antibody produced in H4-II-E rat hepatoma cells binds to neonatal FC receptor FCRN with higher affinity than anti-CD20 IGG1 antibody produced in CHO.

The anti-CD20 IgG1 antibody expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the anti-CD20 IgG1 antibody was generated by transfection with a DNA construct encoding the heavy chain (SEQ ID NO: 2) and the light chain (SEQ ID NO: 3) of the anti-CD20 IgG1 antibody. . The stable anti-CD20 production cell line was isolated by selecting an antibiotic resistance marker and analyzing the anti-CD20 expression of the cell supernatant of the viable cells by ELISA. Anti-CD20 producing cells were grown in serum-free fed batch culture and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

Binding of anti-CD20 IgGl produced in H4-II-E cells or CHO cells to the neonatal Fc receptor FcRn was measured using BIAcore analysis. The recombinant soluble human FcRn-b2 microglobulin complex was expressed in CHO/DG44 cells and purified from the culture supernatant by Ni-NTA chromatography (Qiagen). Anti-human b2-microglobulin monoclonal antibody (Abeam, Cambridge, UK) was immobilized onto a BIAcore T100 CM5 sensor wafer using an amine coupling kit (BIACORE). The complex is captured by immobilization of an anti-b2-microglobulin antibody by injection of a soluble FcRn-b2 microglobulin complex. Each purified anti-CD20 IgG1 was diluted at 5 different concentrations (from 4.17 nM to 66.7 nM) to HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant) adjusted to pH 6.0. In each of P20), each diluted IgG1 was injected onto the composite capture sensor surface or blank at a flow rate of 5 mL/min. Soluble FcRn and IgGl bound to the surface of the sensor were removed by injecting 7.5 mM HCl for 1 min at a flow rate of 60 mL/min. These experiments were carried out using HBS-EP+ as a running buffer at 25 °C. Data analysis was performed using data obtained by subtracting blanks. Apparent association rate constants (ka), dissociation rate constants (kd), and binding affinities (KD) were calculated using a BIAcore T100 evaluation software by a binary fit model. The anti-CD20 IgG1 produced in H4-II-E cells showed a much stronger binding to the neonatal Fc receptor FcRn compared to the anti-CD20 IgG1 produced in CHO cells (Fig. 11).

Example 16: MCP1-FC-fusion protein produced in H4-II-E rat hepatoma cells binds to FC receptors CD16-V158 and CD16-F158 with higher affinity than MCP1-FC-fusion protein produced in CHO ( FCGRIIIA)

The MCP1-Fc fusion protein expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the MCP1-Fc fusion protein was generated by transfection with the DNA construct encoding SEQ ID NO: 6. The stable MCP1-Fc fusion protein producing cell line was isolated by selecting an antibiotic resistance marker and analyzing the MCP1-Fc expression of the cell supernatant of the viable cells by ELISA. MCP1-Fc-producing cells were grown in serum-free fed batch cultures and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

BIAcore analysis was used to measure the binding kinetics of MCP1-Fc and FcγRIIIa receptors CD16-V158 and CD16-F158 produced in H4-II-E cells and CHO cells. MCP1-Fc produced in H4-II-E cells showed significantly higher affinity than the two variants of FcyRIIIa compared to anti-CD20 IgG1 produced in CHO cells.

Example 17: EPO-FC-containing FC-fusion protein produced in H4-II-E rat hepatoma cells binds to FC receptor CD16 with higher affinity than EPO-FC-containing FC-fusion protein produced in CHO- V158 and CD16-F158 (FCGRIIIA)

The expression vector containing the nucleic acid sequence of EPO fused to the nucleic acid sequence of IgG1 Fc in the framework was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the EPO-Fc fusion protein was generated by transfection with the DNA construct encoding SEQ ID NO: 7. A cell line producing a stable Fc fusion protein comprising EPO-Fc was isolated by selecting an antibiotic resistance marker and analyzing the Fc-fusion protein expression of the cell supernatant of the viable cells by ELISA. Fc-fusion protein producing cells were grown in serum-free fed batch culture and EPO-Fc protein was purified from serum-free culture supernatant by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

BIAcore analysis was used to measure the binding kinetics of EPO-Fc-containing Fc fusion proteins produced in H4-II-E cells and CHO cells to the FcγRIIIa receptors CD16-V158 and CD16-F158. The EPO-Fc-containing Fc fusion protein produced in H4-II-E cells showed significantly higher affinity to the two variants of FcyRIIIa compared to the anti-CD20 IgG1 produced in CHO cells.

Example 18: Anti-CD20 IGG1 antibody produced in H4-II-E rat hepatoma cells binds FC receptor CD32A and CD32B with lower affinity than anti-CD20 IGG1 antibody produced in CHO

The anti-CD20 IgG1 antibody expression vector was used to stably transfect CHO cells and H4-II-E cells, respectively. The H4-II-E cell line producing the anti-CD20 IgG1 antibody was generated by transfection with a DNA construct encoding the heavy chain (SEQ ID NO: 2) and the light chain (SEQ ID NO: 3) of the anti-CD20 IgG1 antibody. . The stable anti-CD20 production cell line was isolated by selecting an antibiotic resistance marker and analyzing the anti-CD20 expression of the cell supernatant of the viable cells by ELISA. Anti-CD20 producing cells were grown in serum-free fed batch culture and recombinant antibodies were purified from serum-free culture supernatants by protein A-affinity chromatography using MabSelectTM (Amersham Biosciences).

BIAcore analysis was used to measure the binding kinetics of anti-CD20 IgG1 to FcγRIIa and FcγRIIb produced in H4-II-E cells and CHO cells. Anti-CD20 IgG1 produced in H4-II-E cells showed significantly higher affinity to both variants of FcγRIIa and FcγRIIb compared to anti-CD20 IgG1 produced in CHO cells.

Reference list

1. Burton, DR and Dwek, RA (2006). Immunology. Sugar determined antibody activity. Science 313 , 627-628.

2. Edelman, GM, Cunningham, BA, Gall, WE, Gottlieb, PD, Rutishauser, U. and Waxdal, MJ (1969). The covalent structure of an entire gammaG immunoglobulin molecule. Proc. Natl. Acad. Sci. US A 63 , 78-85.

3. Ellison, J. and Hood, L. (1982). Linkage and sequence homology of two human immunoglobulin gamma heavy chain constant region genes. Proc. Natl. Acad. Sci. US A 79 , 1984-1988.

4. Fallaux, FJ, Bout, A., van, dV, I, van den Wollenberg, DJ, Hehir, KM, Keegan, J., Auger, C., Cramer, SJ, van, OH, van der Eb, AJ , Valerio, D. and Hoeben, RC (1998). New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses. Hum. Gene Ther. 9 , 1909-1917.

5. Hamako, J., Matsui, T., Ozeki, Y., Mizuochi, T. and Titani, K. (1993). Comparative studies of asparagine-linked sugar chains of immunoglobulin G from eleven mammalian species. Comp Biochem. B 106 , 949-954.

6. Horikoshi, N., Tashiro, F., Tanaka, N. and Ueno, Y. (1988). Modulation of hormonal induction of tyrosine aminotransferase and glucocorticoid receptors by aflatoxinB1 and sterigmatocystin in Reuber hepatoma cells. Cancer Res. 48 ,5188 -5192.

7. Houser, WH, Raha, A. and Vickers, M. (1992). Induction of CYP1A1 gene expression in H4-II-E rat hepatoma cells by benzo[e]pyrene. Mol. Carcinog. 5 , 232-237.

8. Jenkins, N., Parekh, RB and James, DC (1996). Getting the glycosylation right: implications for the biotechnology industry. Nat. Biotechnol. 14 , 975-981.

9. Kabat, EA (1988). Antibody complementarity and antibody structure. J. Immunol. 141 , S25-S36.

10. Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S. and Foeller, C. (1991). Sequences of Proteins of Immunological Interest. U. S. Department of Health and Human Services, Natl. Inst. of Health, Bethesda.

11. Kanda, Y., Yamane-Ohnuki, N., Sakai, N., Yamano, K., Nakano, R., Inoue, M., Misaka, H., Iida, S., Wakitani, M., Konno , Y., Yano, K., Shitara, K., Hosoi, S. and Satoh, M. (2006). Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC. Biotechnol. Bioeng. 94 ,680 -688.

12. Kaufman, RJ (1990). Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol. 185 , 537-566.

13. Lifely, MR, Hale, C., Boyce, S., Keen, MJ and Phillips, J. (1995). Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions. Glycobiology 5 , 813-822.

14. Miyazaki, M., Bai, L., Taga, H., Hirai, H., Sato, J. and Namba, M. (1991). Expression of liver-specific functions and secretion of a hepatocyte growth factor by a Newly established rat hepatoma cell line growing in a chemically-defined serum-free medium. Res. Exp. Med. (Berl) 191 , 297-307.

15. Mori, K., Kuni-Kamochi, R., Yamane-Ohnuki, N., Wakitani, M., Yamano, K., Imai, H., Kanda, Y., Niwa, R., Iida, S. , Uchida, K., Shitara, K. and Satoh, M. (2004). Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnol. Bioeng. 88 , 901-908.

16. Niwa, A., Yamamoto, K., Sorimachi, K. and Yasumura, Y. (1980). Continuous culture of Reuber hepatoma cells in serum-free arginine-, glutamine- and tyrosine-deprived chemically defined medium. In Vitro 16 , 987-993.

17. Noguchi, A., Mukuria, CJ , Suzuki, E. And Naiki, M. (1995). Immunogenicity of N-glycolylneuraminic acid-containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. J. Biochem. 117 , 59-62.

18. PITOT, HC, PERAINO, C., MORSE, PA, Jr. and POTTER, VR (1964). HEPATOMAS IN TISSUE CULTURE COMPARED WITH ADAPTING LIVER IN VIVO. Natl. Cancer Inst. Monogr 13 , 229-245.

19. Raju, TS, Briggs, JB, Borge, SM and Jones, AJ (2000). Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 10 , 477-486.

20. REUBER, MD (1961). A transplantable bile-secreting hepatocellular carcinoma in the rat. J. Natl. Cancer Inst. 26 , 891-899.

21. Ripka, J., Adamany, A . , And Stanley, P. (1986). Two Chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose. Arch. Biochem. Biophys. 249, 533-545 .

22. Sambrook, J., Fritsch, d. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor: Cold Spring Harbor Laboratory Press).

23. Scallon, BJ, Tam, SH, McCarthy, SG, Cai, AN and Raju, TS (2007). Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely affect functionality. Mol. Immunol. 44 , 1524-1534.

24. Shields, RL, Lai, J., Keck, R., O'Connell, LY, Hong, K., Meng, YG, Weikert, SH and Presta, LG (2002). Lack of fucose on human IgG1 N- linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733-26740.

25. Shinkawa, T., Nakamura, K., Yamane, N., Shoji-Hosaka, E., Kanda, Y., Sakurada, M., Uchida, K., Anazawa, H., Satoh, M., Yamasaki , M., Hanai, N. and Shitara, K. (2003). The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity J. Biol. Chem. 278 , 3466-3473.

26. Umana, P., Jean-Mairet, J. and Bailey, JE (1999). Tetracycline-regulated overexpression of glycosyltransferases in Chinese hamster ovary cells. Biotechnol. Bioeng. 65 , 542-549.

27. Urlaub, G. and Chasin, LA (1980). Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. US A 77 , 4216-4220.

28. Urlaub, G., Kas, E., Carothers, AM and Chasin, LA (1983). Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell 33 , 405-412.

29. Walsh, G. and Jefferis, R. (2006). Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol. 24 , 1241-1252.

30. Werner, RG (2004). Economic aspects of commercial manufacture of biopharmaceuticals. J. Biotechnol. 113 , 171-182.

31. Wright, A., Sato, Y., Okada, T., Chang, K., Endo, T. and Morrison, S. (2000). In vivo trafficking and catabolism of IgG1 antibodies with Fc associated carbohydrates of differing structure Glycobiology 10 , 1347-1355.

32. Yamane-Ohnuki, N., Kinoshita, S., Inoue-Urakubo, M., Kusunoki, M., Iida, S., Nakano, R., Wakitani, M., Niwa, R., Sakurada, M. , Uchida, K., Shitara, K. and Satoh, M. (2004). Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for playing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng. 87 , 614-622.

Sequence table description

SEQ ID NO 1: O-linked amino acid sequence of a glycosylation site

SEQ ID NO 2: Amino acid sequence of the anti-CD20 IgG1 mAb heavy chain

SEQ ID NO 3: Amino acid sequence of the anti-CD20 IgG1 mAb light chain

SEQ ID NO 4: Amino acid sequence of the anti-CD20 IgG4 mAb heavy chain

SEQ ID NO 5: Amino acid sequence of the anti-CD20 IgG4 mAb light chain

SEQ ID NO 6: amino acid sequence of MCP1-Fc fusion protein

SEQ ID NO 7: amino acid sequence of EPO-Fc fusion protein

<110> Dessert Bailingjia Ingelheim International Co., Ltd.

<120> Production of low trehalose antibodies in H4-II-E rat cells

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<141> 2011-09-26

<150> 10180321.1; 11156848.1

<151> 2010-09-27;2011-03-03

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<170> PatentIn version 3.3

<210> 1

<211> 4

<212> PRT

<213> Labor

<220>

<223> o-linked glycosylation site sequence

<220>

<221> X

<222> (1)..(1)

<223> Thr or ser

<220>

<221> X

<222> (2)..(3)

<223> Any of the conventional amino acids: Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, Val

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<223> Amino acid sequence of the anti-CD20 IgG1 mAb heavy chain

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<220>

<223> Amino acid sequence of the anti-CD20 IgG1 mAb light chain

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<210> 4

<211> 449

<212> PRT

<213> Labor

<220>

<223> Amino acid sequence of the anti-CD20 IgG4 mAb heavy chain

<400> 4

<210> 5

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<220>

<223> Amino acid sequence of the anti-CD20 IgG4 mAb light chain

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<223> Amino acid sequence of MCP1-FC fusion protein

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Figure 1: Predictive relative amounts of glycostructures on glycoproteins produced in different cell lines.

Analysis of the sugar properties of established cell lines derived from different species and different tissues within such species. Estimating the protein produced in these cell lines by measuring the enzymatic activity in cells and analyzing the structure on the cell surface, trehalose, α-2,6 sialylation structure, N-acetylthioneamine (NeuAc) ), the relative content of galactose-1,3-galactose (Gal-1,3-Gal) and N-hydroxyacetamidine neurone (NeuGc). The values obtained were normalized to the results obtained in the Chinese hamster ovary cell line (CHO) and plotted as a predicted relative content of the individual sugar structures.

Abbreviations: ha, hamster; mo, mouse; gp, guinea pig; rab, rabbit; go, goat; sh, sheep; hu, human; ch, chicken; du, duck; te, testicle; ov, ovary; pa, pancreas ;ki, kidney; li, liver; carc, hepatoma/cancer; eo, esophagus; lu, lung; br. canc, breast cancer; co.carc, colon cancer; my, myeloma; pr.ly, primary lymphoblast Like cells; pr. bm, bone marrow stem cells; emb. fib, embryonic fibroblasts.

Figure 2: IgGl antibodies produced in CHO, Lec13, YB2/0 and H4-II-E cells differ in their glycosylation pattern: fucosylation.

The structure and composition of Fc glycans of IgG1 antibodies produced in CHO-DG44 cells, CHO-Lec13 mutants, YB2/0 rat myeloma cells and H4-II-E rat hepatoma cells were analyzed. The glycan is released from the purified antibody after reduction by enzymatic digestion with PNGase F. The glycan was purified and fluorescently labeled with 2-aminobenzamide (2-AB) and before and after treatment with exoglycosidase alpha-trehalosidase or other exoglycosidases in HPLC column Fractionation is performed on. Most sugar structures are non-sialylated bi-glycans. Calculation of the fucosylated and non-fucosylated dimeric structural glycans and other glycoside structures (sialylated glycans, high mannose structures or hybrid glycans) from the ratio of chromatographic peak areas before and after digestion with exoglycosides The ratio of ).

Figure 3: Sugar pattern of IgG1 antibodies produced in CHO and H4-II-E: galactosylation

The structure and composition of Fc glycans of IgG1 antibodies produced in CHO-DG44 cells and H4-II-E rat hepatoma cells were analyzed. The glycan is released from the purified antibody after reduction by enzymatic digestion with PNGase F. The glycan was purified, fluorescently labeled with 2-aminobenzamide (2-AB) and before and after treatment with exoglycosidase β-galactosidase or other exoglycosidases, in HPLC tubes Fractionation was carried out on the column. The percentage of galactosylated versus non-galactosylated bi-structured glycans was calculated from the ratio of chromatographic peak areas before and after exo-glycoside digestion.

Figure 4: Sugar pattern of IgG1 antibodies produced in CHO and H4-II-E: sialylation

The structure and composition of Fc glycans of IgG1 antibodies produced in CHO-DG44 cells and H4-II-E rat hepatoma cells were analyzed. The glycan is released from the purified antibody after reduction by enzymatic digestion with PNGase F. The glycan was purified, fluorescently labeled with 2-aminobenzamide (2-AB) and applied to the HPLC column before and after treatment with exoglycosidase neuraminidase or other exoglycosidases Perform fractionation. The percentage of sialylated di-branched glycans was calculated from the ratio of chromatographic peak areas before and after digestion with exo glycosides.

Figure 5: Adapting H4-II-E cells to suspension growth in serum-free medium

(A) Adherent growth of H4-II-E cells in MEMalpha medium containing 10% FCS. (B) Suspension culture of H4-II-E cells after growth in serum-free Ca-free medium in shake flasks. (C) Growth curves of H4-II-E cultures in BI SFM medium inoculated at two different inoculum cell densities. Cultures were incubated in shake flasks at 37 ° C, 5% CO ₂ and 120 rpm. The viable cell concentration was measured at the indicated time points.

Abbreviations: BI-SFM, Boehringer Ingelheim serum-free calcium-free medium; FCS, fetal bovine serum; VCC, viable cell concentration; 4 × 10 ⁵ cells / ml = 400,000 cells / ml; 6 × 10 ⁵ cells / ml = 600,000 cells/ml.

Figure 6: Low sensitivity of H4-II-E cells to apoptosis and high robustness to cellular stress

(A) H4-II-E cells (black bars) after heating the cell suspension containing equal cell numbers to 42 ° C for 2 hours and then incubated at 37 ° C, 5% CO ₂ for 22 hours Relative living cell density and viability of YB2/0 cells (grey bar graph). Control cells were incubated for 24 hours at 37 ° C, 5% CO ₂ . After heat stress, H4-II-E cells showed significantly higher viable cell density and viability. (B) H4-II-E cells and YB2/0 after incubation for 24 hours at 37 ° C, 5% CO ₂ in medium diluted with demineralized water (low ionic strength) or 10×PBS (high ionic strength) Relative living cell density and viability of cells. Control cells were cultured in undiluted medium. After incubation under low or high permeability, H4-II-E cells showed significantly higher viable cell density and viability. (C) H4-II-E cells and YB2/0 cells after treatment with cell suspension containing equal cell number for 48 hours at 37 ° C, 5% CO ₂ with 2 μg/ml or 5 μg/ml puromycin Relative living cell density and vigor. Control cells were cultured for 48 hours in a puromycin-free medium at 37 ° C, 5% CO ₂ . H4-II-E cells showed significantly higher viable cell density and viability after drug treatment.

Figure 7: Single cell suspension incubation of H4-II-E cells for Ca ²⁺ reduced or no Ca ²⁺ medium.

(A) Hitachi 917 (Roche) was used to analyze the Ca content of two media suitable for suspension incubation of H4-II-E cells. AEM medium containing very low Ca concentration and no Ca form of BI patent medium, both suitable for single cell suspension incubation of H4-II-E cells (B, D). (B)-(E) Inoculation of H4-II-E cells adapted to suspension growth in AEM or in BI (Ca-free) medium at a density of 3 x 10 ⁵ cells/ml = 300,000 cells/ml Store in a 12-well plate in the medium with or without CaCl ₂ added. Microscopic analysis of growth morphology and cell aggregation was performed 3 days after inoculation. (B) H4-II-E cells were grown in suspension in a single cell format in AEM medium. (C) H4-II-E cells formed large aggregates in AEM supplemented with 1 mM = 1 mMol/L CaCl ₂ . (D) H4-II-E cells were grown in suspension in a single cell format in a Ca BI-free medium. (E) H4-II-E cells formed large aggregates in BI medium (containing 1.38 mM = 1.38 mMol/L Ca according to the analysis in (A)). (F) Inoculation of H4-II-E cells adapted to suspension growth in BI (Ca-free) medium at a density of 3 × 10 ⁵ cells/ml = 300,000 cells/ml in BI stored in a 12-well plate The medium (containing Ca) was added to the culture in the amount of EDTA indicated. Microscopic analysis of growth morphology and cell aggregation was performed 3 days after inoculation. It should be noted that the dose-dependent, EDTA-mediated consumption of free Ca ^{2+ in} the medium reduces cell aggregation and increases the proportion of suspended single cells.

Figure 8: Ca ²⁺ concentration-dependent and Mg ²⁺ independent aggregation of suspended H4-II-E cells.

(A) Inoculation of H4-II-E cells adapted to suspension growth in BI (Ca-free) medium at a density of 4 × 10 ⁵ cells/ml = 400,000 cells/ml in BI stored in a 12-well plate (Ca-free) medium and the indicated amount of CaCl _{2 was} added to the culture. Microscopic analysis of growth morphology and cell aggregation was performed 2 days after inoculation. Note the concentration-dependent, Ca ²⁺ -mediated cell aggregation of suspended H4-II-E cells. (B) Inoculation of H4-II-E cells adapted to suspension growth in BI (Ca-free) medium at a density of 4 × 10 ⁵ cells/ml = 400,000 cells/ml in BI stored in a 12-well plate (Ca-free) medium and the indicated amount of MgCl _{2 was} added to the culture. Microscopic analysis of growth morphology and cell aggregation was performed 2 days after inoculation. It should be noted that the cells are suspended in a single cell form independently of the Mg ²⁺ concentration. (C) Inoculation of H4-II-E cells adapted to suspension growth in AEM medium at a density of 3 × 10 ⁵ cells/ml = 300,000 cells/ml in AEM medium stored in a shaking flask and An amount of CaCl ₂ or MgCl _{2 was} added to the culture. The culture was incubated at 37 ° C, 5% CO ₂ and 120 rpm. Live cell density and cell aggregation ratio were analyzed using the CEDEX Cell Quantification System (Innovatis). It should be noted that if the CaCl ₂ concentration in the medium is higher than 100 μM, the viable cell density of the culture is significantly decreased. In contrast, increasing the concentration of MgCl _{2 has} no effect on viable cell density. Furthermore, it should be noted that the cell aggregation ratio increases as the CaCl ₂ concentration increases and reaches saturation when the CaCl ₂ concentration is >250 μM. In contrast, increasing the concentration of MgCl ₂ had no effect on the aggregation ratio of H4-II-E cells.

Figure 9: Binding affinities of IgG1 and FcγRIIIa produced in CHO and H4-II-E.

BIAcore T100 instruments and CM5 sensor wafers (BIACORE, Uppsala, Sweden) were used to measure the binding kinetics of IgGl and FcyRIIIa produced in different cell lines as follows. Soluble recombinant FcyRIIIa was immobilized onto a BIAcore sensor wafer. Purified IgG1 was diluted in HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) at 6 different concentrations (from 4.17 nM to 133.3 nM), and Each diluted IgGl was injected onto the surface of the receptor capture sensor at a flow rate of 5 mL/min. The experiments were carried out using HBS-EP as a running buffer at 25 °C. A buffer solution without sample IgG1 was injected onto the surface of the receptor capture sensor as a blank control. Soluble FcγRIIIb and IgG1 bound to the sensor surface were removed by injecting 7.5 mM HCl for 30 s at a flow rate of 10 mL/min. Data obtained by injection of IgG1 were corrected for blank controls prior to data analysis. Affinity (KD) for FcyRIIIa was calculated by steady state analysis using BIAcore T100 Kinetic Evaluation Software (BIACORE). It should be noted that the binding affinity of IgG1 produced in the H4-II-E cell line to the receptor is higher than that produced in CHO.

Figure 10: Effector function of IgG1 produced in CHO and H4-II-E: ADCC.

ADCC analysis was performed by lactate dehydrogenase (LDH) release assay using human peripheral blood mononuclear cells (PBMC) prepared from healthy donors by Lymphoprep (AXIS SHIELD, Dundee, UK) as effector cells. Aliquots of target tumor cells (human Burkitt's lymphoma cell line Ramos expressing human CD20) were dispensed into 96-well U-shaped bottom plates (10,000 cells in 50 μl/well) and in PBMC ( Incubate with serial dilutions of antibody (50 μL) at an E/T ratio of 20/1 in the presence of 100 μL). Supernatant LDH activity was measured using a non-radioactive cytotoxicity assay kit (Promega, Madison, WI) after incubation for 4 h at 37 °C. The specific cell lysis % was calculated from the sample activity according to the following formula: specific dissolution [%] = 100 × (ES _{E -} S _T ) / (MS _T ). FU stands for fluorescent unit.

It should be noted that IgG1 produced in H4-II-E has stronger ADCC activation than IgG1 produced in CHO.

Figure 11: Binding affinities of IgG1 and FcRn produced in CHO and H4-II-E.

The kinetics of the human IgGl-FcRn interaction was measured using a BIAcore T100 instrument and a CM5 sensor wafer. Anti-human b2-microglobulin monoclonal antibody (Abeam, Cambridge, UK) was immobilized onto the wafer using an amine coupling kit (BIACORE). The complex was captured by immobilization of an anti-b2-microglobulin antibody by injecting a soluble FcRn-b2 microglobulin complex at a flow rate of 5 mL/min. A buffer solution without the complex was injected onto the surface of the antibody capture sensor as a blank control. Each purified IgG was diluted in 5 different concentrations (from 4.17 nM to 66.7 nM) to HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) adjusted to pH 6.0. Each diluted IgGl was injected onto the composite capture sensor surface or blank at a flow rate of 5 mL/min. Soluble FcRn and IgGl bound to the surface of the sensor were removed by injecting 7.5 mM HCl for 1 min at a flow rate of 60 mL/min. These experiments were carried out using HBS-EP+ as a running buffer at 25 °C. Data analysis was performed using data obtained by subtracting blanks. Apparent association rate constants (ka), dissociation rate constants (kd), and binding affinities (KD) were calculated using a BIAcore T100 evaluation software by a binary fit model. It should be noted that the degree of binding of the antibody produced in H4-II-E to FcRn is comparable to that produced in CHO.

(no component symbol description)

Claims (29)

A rat hepatoma cell comprising a nucleic acid sequence encoding an antibody or an Fc-fusion protein, wherein the nucleic acid sequence is operably linked to at least one regulatory sequence that permits expression of the nucleic acid sequence encoding the antibody or Fc-fusion protein. The rat hepatoma cell of claim 1, wherein the cell line is a H4-II-E cell. The rat hepatoma cell of claim 1 or 2, wherein the cell line: a) is derived from a cell selected from the group consisting of: European Collection of Cell Cultures (ECACC, catalogue) No. 87031301), American Type Culture Collection (ATCC, accession number CRL-1548), H4-II-E-C3 cell line (CRL-1600 or HPACC number 85601112 or ECACC catalog number 85061112), H4II cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) and H4-S cell line (HPACC Nr. 89102001), or b) numbered ECACC Catalog No. 87031301 is deposited with the European Cell Culture Preservation Center or with cells deposited with the American Type Culture Collection ATCC under the accession number CRL-1548, or c) deposited with DSMZ under the accession number DSM ACC3129 (H4-II-E) Cells of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, or d) cells deposited in the DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es), or e) a) or b) or c) or d) a derivative or progeny of any of the cells. The rat hepatoma cell of claim 1 or 2, wherein the cell line is deposited in a cell of DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E) or wherein the cell line is registered with a number DSM ACC3130 (H4-II-Es) is deposited in the cells of the DSMZ (German Microbial Culture Preservation Center). The rat hepatoma cell of claim 1 or 2, wherein a) the glycoside structure contained in the antibody or the Fc-fusion protein expressed by the cell contains trehalose to a degree of less than 20%, 10% or 5%, or b) the glycoside structure contained in the antibody or Fc-fusion protein expressed by the cell contains trehalose to a degree ranging from 0% to 20%, 0% to 10%, 0% to 5%, 0.5% to 20% , in the range of 0.5% to 10%, 0.5% to 5%, 1% to 20%, 1% to 10% or 1% to 5%. The rat hepatoma cell of claim 1 or 2, wherein a) the glycoside structure contained in the antibody or the Fc-fusion protein expressed by the cell contains at least one galactose residue in an amount of more than 40%, 45% or 50%, or b) the degree to which the glycoside structure contained in the antibody or Fc-fusion protein expressed by the cell contains at least one galactose residue is between 40% and 100%, 45% to 100%, 50% to 100%, 51% to 100%, 40% to 99.5%, 45% to 99.5%, 50% to 99.5% or 51% to 99.5%, 40% to 99%, 45% to 99%, 50% to 99% Or in the range of from 51% to 99%, wherein the glycoside structures preferably contain one or two, preferably attached to the N-acetylglucosamine (GlcNAc) at the terminal non-reducing end of the glycoside structure. A galactose residue (G1 or G2). The rat hepatoma cell of claim 1 or 2, wherein a) the glycoside structure contained in the antibody or the Fc-fusion protein expressed by the cell contains a terminal sialic acid or a neuramic acid residue of more than 5% or More than 10%, or b) the degree of glycosidic structure contained in the antibody or Fc-fusion protein expressed by the cell containing terminal sialic acid or a tranexamic acid residue is between 0 and 8%, 1% to 8%, Between 5% and 10%, 10% to 50% or 10% to 45%. The rat hepatoma cell of claim 1 or 2, further comprising a selectable marker gene, such as neomycin-phosphotransferase (NPT); for puromycin, hygromycin or gem A resistance gene for zeocin; or an amplifiable selectable marker gene, such as dihydrofolate reductase (DHFR) or glutamate synthase (GS). The rat hepatoma cell of claim 1 or 2, wherein the regulatory sequence that allows expression of the nucleic acid sequence encoding the antibody or Fc-fusion protein is a) a promoter, or b) an enhancer, or c) 5'-UTR sequence. The rat hepatoma cell of claim 1 or 2, wherein the antibody or Fc fusion protein comprises a glycoside structure linked to a N-aspartic acid (N-Asn) residue, wherein the glycoside structure comprises the following sugar chain: The rat hepatoma cell of claim 1 or 2, wherein the antibody or Fc fusion protein comprises a glycoside structure linked to a N-aspartic acid (N-Asn) residue, wherein the glycoside structure comprises the following sugar chain: The rat hepatoma cell of claim 10, wherein the N-Asn is preferably N-Asn (297) according to the Kabat EU nomenclature. The rat hepatoma cell of claim 11, wherein the N-Asn is preferably N-Asn (297) according to the Kabat EU nomenclature. A rat hepatoma cell according to claim 1 or 2, wherein the cell is adapted to grow in a serum-free and calcium-reduced or preferably calcium-free medium. The rat hepatoma cell of claim 1 or 2, wherein the cell is adapted to grow in suspension culture. The rat hepatoma cell of any one of claims 1 or 2, wherein the cell has low sensitivity to apoptosis and/or high robustness to cell pressure as compared to YB2/0 cells. A method of producing a glycoprotein of interest, characterized by the steps of: a) providing a rat hepatoma cell, b) adapting the cell of step a) to growth in suspension culture, as appropriate, c) optionally The cells of step a) and/or step b) are adapted to grow in serum-free medium, d) the cells of step a) and/or step b) and/or step c) are optionally reduced in calcium or calcium-free medium. Medium growth, e) transfection of the optionally adapted rat hepatoma cell with a nucleic acid sequence encoding a recombinant glycoprotein of interest, f) cultivating the transfection of step e) under conditions permitting expression of the glycoprotein of interest Cells, g) The glycoprotein of interest is isolated and purified as appropriate. The method of claim 17, wherein the rat hepatoma cell line H4-II-E cells, preferably, the cell line: a) is derived from a cell selected from the group consisting of cells: European cell culture preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession Number CRL-1548), H4-II-E-C3 Cell Line (CRL-1600 or HPACC No. 85061112 or ECACC Catalog No. 85061112), H4II Cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) and H4-S cell line (HPACC Nr. 89102001), or b) numbered ECACC catalog No. 87031301 is deposited with the European Cell Culture Preservation Center or with cells deposited with the American Type Culture Collection Center ATCC under the accession number CRL-1548, or c) deposited with DSMZ under the accession number DSM ACC3129 (H4-II-E) Cells of the Culture Collection Center, or d) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es), or e) a) or b) or c) or d a derivative or progeny of any of the cells, optimally, the cell line is registered under the accession number DSM ACC3129 (H4-II- E) Cells deposited in DSMZ (German Microbial Culture Preservation Center) or cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es). The method of claim 17 or 18, wherein the medium of steps b), c) and/or d) is additionally free of protein/peptide of any animal origin. The method of claim 17 or 18, wherein the transfection step e) further comprises introducing into the rat hepatoma cell an expression vector comprising a nucleic acid sequence encoding the glycoprotein of interest, the nucleic acid sequence being operably linked to at least A regulatory sequence that allows expression of the nucleic acid sequence encoding the glycoprotein of interest. The method of claim 17 or 18, wherein the glycoprotein antibody or Fc-fusion protein of interest preferably has the following antibody or Fc-fusion protein a) FcγRIIIa binding activity and preferably ADCC, or b) complement binding activity And preferably CDC, or c) binding activity to the neonatal Fc receptor FcRn and preferably serum stability. A method for producing a (recombinant) antibody or an Fc-fusion protein, the (recombinant) antibody or Fc-fusion protein having a) FcγRIIIa binding activity and/or b) complement binding activity and/or c) and neonatal Fc receptor FcRn The binding activity, the method comprising producing the antibody or Fc fusion protein in a rat hepatoma cell, wherein the rat hepatoma cell is preferably a H4-II-E cell, more preferably, the cell line: i) Cells from cells selected from the group consisting of: European Cell Culture Preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession No. CRL-1548), H4-II-E-C3 cells Department (CRL-1600 or HPACC No. 85061112 or ECACC Catalog No. 85061112), H4II cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) and H4- S cell line (HPACC Nr. 89102001), or ii) deposited in the European Cell Culture Preservation Center under the number ECACC catalog number 87031301 or cells deposited in the American Type Culture Collection Center ATCC under the accession number CRL-1548, or iii) Registration number DSM ACC3129 (H4-II-E) registered in DSMZ (Germany Cell of the Biological Bacterial Preservation Center), or iv) cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es), or v) i) or ii) or iii) or Iv) a derivative or progeny of any of the cells, optimally, the cell line is deposited in the cell of DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3129 (H4-II-E) or under the accession number DSM ACC3130 (H4-II-Es) is deposited in the cells of DSMZ (German Microbial Culture Preservation Center). The method of claim 22, wherein i) the antibody or Fc fusion protein of claim 22 a) has antibody-dependent cellular cytotoxicity (ADCC) or ii) the antibody or Fc fusion protein of claim 22 b) has complement Dependent Cytotoxicity (CDC) or iii) The antibody or Fc fusion protein of claim 22 c) is serum stable. A method of producing a host cell for producing a recombinant glycoprotein, comprising: a) providing a rat hepatoma cell, b) adapting the rat hepatoma cell of step a) to growth in suspension culture, and c Adapting the rat hepatoma cells of step a) to growth in serum-free medium, and d) adapting the rat hepatoma cells of step a) to growth in calcium-reduced or calcium-free medium, and e) The rat hepatoma cells of step a) are optionally adapted to grow in a medium free of protein/peptide of any animal origin, and f) a single cell pure line is selected as appropriate, g) obtaining a host cell. The method of claim 24, wherein the rat hepatoma cell line H4-II-E cells, preferably, the cell line: i) is derived from a cell selected from the group consisting of cells: European cell culture preservation Center (ECACC, Cat. No. 87031301), American Type Culture Collection (ATCC, Accession Number CRL-1548), H4-II-E-C3 Cell Line (CRL-1600 or HPACC No. 85061112 or ECACC Catalog No. 85061112), H4II Cell line (HPACC Nr. 89042702), H4-TG cell line (CRL-1578), H5 cell line (HPACC, Nr. 94101905) and H4-S cell line (HPACC Nr. 89102001), or ii) numbered ECACC catalog No. 87031301 is deposited in the European Cell Culture Preservation Center or in cells deposited with the American Type Culture Collection Center ATCC under the accession number CRL-1548, or iii) deposited in DSMZ under the accession number DSM ACC3129 (H4-II-E) Cells of the Culture Collection Center, or iv) cells deposited with DSMZ (HZII-E-S) in DSMZ (German Microbial Culture Preservation Center), or v) i) or ii) or iii) or iv a derivative or progeny of any of the cells, optimally, the cell line is numbered DSM ACC312 9 (H4-II-E) cells deposited in DSMZ (German Microbial Culture Preservation Center) or cells deposited in DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es). The method of claim 24 or 25, further comprising h) transfecting the host cell obtained according to step g) of claim 24 with a nucleic acid sequence encoding a glycoprotein of interest, and i) permitting expression of the The transfected cells of step h) are incubated under the condition of glycoprotein, wherein the glycoprotein of interest is preferably an antibody or Fc fusion protein, an antibody or Fc fusion optimally having ADCC and/or CDC and/or serum stability. protein. A cell produced according to the method of any one of claims 24 to 26. A (rat hepatoma) cell deposited with DSMZ (German Microbial Culture Preservation Center) under the accession number DSM ACC3130 (H4-II-Es). Use of a rat hepatoma cell according to any one of claims 1 to 16, 27 and 28 as a host cell for biomedical production.