CHO-Kl cell line

The current invention is in the field of polypeptide production. More precisely it is reported a new CHO-Kl cell line, CHO-Kl-Gln(-), the generation of such a cell line, and the use of such a cell line in the production of heterologous polypeptides.

Background of the Invention

Expression systems for the production of recombinant polypeptides are well-known in the state of the art and are described by, e.g., Marino, M. H., Biopharm. 2 (1989) 18-33; Goeddel, D.V., et al., Methods Enzymol. 185 (1990) 3-7; Wurm, F., and Bernard, A., Curr. Opin. Biotechnol. 10 (1999) 156-159. Polypeptides for use in pharmaceutical applications are preferably produced in mammalian cells such as

CHO cells, NSO cells, SP2/0 cells, COS cells, HEK cells, BHK cells, PER.C6 ^® cells, or the like. The essential elements of an expression plasmid are a prokaryotic plasmid propagation unit, for example for E.coli, comprising a prokaryotic origin of replication and a prokaryotic selection marker, an eukaryotic selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest each comprising a promoter, a structural gene, and a transcription terminator including a polyadenylation signal. For transient expression in mammalian cells a mammalian origin of replication, such as the SV40 Ori or OriP, can be included. As promoter a constitutive or inducible promoter can be selected. For optimized transcription a Kozak sequence may be included in the 5' untranslated region. For mRNA processing, in particular mRNA splicing and transcription termination, mRNA splicing signals, depending on the organization of the structural gene (exon/intron organization), may be included as well as a polyadenylation signal.

Today CHO cells are widely used for the expression of pharmaceutical polypeptides, either at small scale in the laboratory or at large scale in production processes. Due to their wide distribution and use the characteristic properties and the genetic background of CHO cells is well known. Therefore, CHO cells are approved by regulatory authorities for the production of therapeutic proteins for application to human beings.

But there are still a lot of provisos with respect to the culture medium. By the use of animal-derived components the potential risk of contamination with substances

hazardous to humans, such as viruses or prion proteins, is at hand. Besides the high costs and downstream processing problems another problem of animal-derived components is due to batch-to-batch variations as a natural product making it difficult to obtain it in a constant product quantity and quality.

To overcome these provisos production cell lines are needed which do require fewer animal-derived components for their cultivation.

A super CHO cell line capable of autocrine growth under fully defined protein-free conditions has been reported by Pak, et al., (Pak, S. CO., et al., Cytotechnology 22 (1996) 139-146). This is a CHO-Kl (ATCC CCL 61) cell line expressing transferrin and IGF-I. Morris, A.E., et al. (US 2005/0170462) report a method for the recombinant protein production in cell culture by modulation of the IGF-I signaling pathway. Belaus et al. transfected an IL-3 dependent murine BaF/3 cell with a chimeric ErbB2 ^v→E /IGF-I receptor (Belaus, A., et al., J. Steroid. Biochem. MoI. Biol. 85 (2003) 105-115).

Summary of the Invention

The current invention comprises a CHO-Kl cell, CHO-Kl-Gln(-), characterized in that said CHO-Kl-Gln(-) cell

- is derived from the CHO-Kl cell deposited as ATCC CCL-61,

- grows in suspension, - grows in a polypeptide-free and serum-free cultivation medium,

- requires no glutamine, no insulin, and no growth factors in the cultivation medium for growth,

whereby said CHO-Kl-Gln(-) cell is not genetically modified by molecular biological methods compared to the parent CHO-Kl cell ATCC CCL-61 by the introduction, deletion, or inactivation of a nucleic acid.

In one embodiment said CHO-Kl-Gln(-) cell requires no animal-derived compound for growth.

A second aspect of the current invention is a method for the generation of a CHO- Kl cell according to the invention, characterized in that said method is consisting of the following steps in the following order:

a) providing a CHO-Kl cell ATCC CCL-61,

b) adapting said CHO-Kl cell to growth in suspension in a polypeptide- free, chemical defined medium supplemented with glutamine and optionally supplemented with hypoxanthine and thymidine,

c) adapting said CHO-Kl cell adapted in step b) to growth in suspension in a polypeptide-free, chemical defined medium optionally supplemented with hypoxanthine and thymidine, thereby obtaining said CHO-Kl-Gln(-) cell,

whereby the CHO-Kl-Gln(-) cell grows on a cultivation medium that is a polypeptide-free, chemical defined medium not containing glutamine, insulin or growth factors,

and

whereby said CHO-Kl-Gln(-) cell is not genetically modified by the introduction, deletion, or inactivation of a nucleic acid via molecular biological methods.

Another aspect of the current invention is a CHO-Kl cell obtained by the method according to the invention.

A further aspect of the invention is a method for the recombinant production of a heterologous polypeptide comprising the following steps:

a) providing a CHO-Kl cell according to the invention,

b) providing one or more nucleic acids encoding said heterologous polypeptide,

c) transfecting said CHO-Kl cell of a) with said one or more nucleic acids,

d) cultivating said transfected CHO-Kl cell of step c) in a polypeptide-free, chemical defined medium not containing glutamine, insulin or a growth factor,

e) recovering said heterologous polypeptide from the cultivation medium of said CHO-Kl cell or the CHO-Kl cell, and optionally

f) purifying said heterologous polypeptide by one or more chromatographic steps.

In one embodiment said heterologous polypeptide is an immunoglobulin or an immunoglobulin fragment or an immunoglobulin conjugate or a non- immunoglobulin polypeptide, preferably a therapeutically active polypeptide. In another embodiment the ammonium ion concentration in the cultivation medium is below 0.12 mmol/L during the cultivation. A further embodiment is that said steps c) and d) are performed in the same medium. Still another embodiment is that said steps c) and d) are performed in a polypeptide-free, synthetic chemical defined medium, optionally supplemented with hypoxanthine and thymidine, with the proviso that the cultivation medium does not contain glutamine, either as isolated compound or as part of a peptide or polypeptide, nor insulin nor a growth factor. In another embodiment said cultivation is performed as fed-batch cultivation with the proviso that the feed used in the cultivation does not contain glutamine, either as isolated compound or as part of a peptide or polypeptide, nor insulin nor a growth factor.

Detailed Description of the Invention

Methods and techniques known to a person skilled in the art, which are useful for carrying out the current invention, are described e.g. in Ausubel, F. M., ed., Current Protocols in Molecular Biology, Volumes I to III (1997), Wiley and Sons; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

General chromatographic methods and their use are known to a person skilled in the art. See for example, Chromatography, 5 ^l edition, Part A: Fundamentals and Techniques, Heftmann, E. (ed), Elsevier Science Publishing Company, New York, (1992); Advanced Chromatographic and Electromigration Methods in Biosciences, Deyl, Z. (ed.), Elsevier Science BV, Amsterdam, The Netherlands, (1998);

Chromatography Today, Poole, C. F., and Poole, S. K., Elsevier Science Publishing Company, New York, (1991); Scopes, Protein Purification: Principles and Practice (1982); Sambrook, J., et al. (ed), Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; or Current Protocols in Molecular Biology, Ausubel, F. M., et al. (eds), John Wiley

& Sons, Inc., New York.

For the purification of recombinantly produced heterologous immunoglobulins often a combination of different column chromatography steps is employed. In one embodiment a Protein A affinity chromatography is followed by one or two

additional chromatographic separation steps, e.g. ion exchange chromatographic steps. The final purification step is a so called "polishing step" for the removal of trace impurities and contaminants like aggregated immunoglobulins, residual HCP (host cell protein), DNA (host cell nucleic acid), viruses, and/or endotoxins. For this polishing step often an anion exchange chromatography material in a flow- through mode is used.

Different methods are well established and widespread used for protein recovery and purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).

The term "amino acid" as used within this application denotes the group of carboxy α-amino acids, which directly or in form of a precursor can be encoded by a nucleic acid. The individual amino acids are encoded by nucleic acids consisting of three nucleotides, so called codons or base-triplets. Each amino acid is encoded by at least one codon. The encoding of the same amino acid by different codons is known as "degeneration of the genetic code". The term "amino acid" as used within this application denotes the naturally occurring carboxy α-amino acids and is comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

A "nucleic acid" or a "nucleic acid sequence" or a "nucleic acid molecule", which terms are used interchangeably within this application, refers to a polymeric molecule consisting of individual nucleotides (also called bases) a, c, g, and t (or u in RNA), for example to DNA, RNA, or modifications thereof. This polynucleotide molecule can be a naturally occurring polynucleotide molecule or a synthetic

polynucleotide molecule or a combination of one or more naturally occurring polynucleotide molecules with one or more synthetic polynucleotide molecules. Also encompassed by this definition are naturally occurring polynucleotide molecules in which one or more nucleotides are changed (e.g. by mutagenesis), deleted, or added. A nucleic acid can either be isolated, or integrated in another nucleic acid, e.g. in an expression cassette, a plasmid, or the chromosome of a host cell. A nucleic acid is characterized by its nucleic acid sequence consisting of individual nucleotides. To a person skilled in the art procedures and methods are well known to convert an amino acid sequence, e.g. of a polypeptide, into a corresponding nucleic acid sequence encoding this amino acid sequence. Therefore, a nucleic acid is characterized by its nucleic acid sequence consisting of individual nucleotides and likewise by the amino acid sequence of a polypeptide encoded thereby.

A "polypeptide" is a polymer consisting of amino acids joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 20 amino acid residues may be referred to as "peptides", whereas molecules consisting of two or more polypeptides or comprising one polypeptide of more than 100 amino acid residues may be referred to as "proteins". A polypeptide may also comprise non-amino acid components, such as carbohydrate groups, metal ions, or carboxylic acid esters. The non-amino acid components may be added by the cell, in which the polypeptide is expressed, and may vary with the type of cell. Polypeptides are defined herein in terms of their amino acid backbone structure or the nucleic acid encoding the same. Additions such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term "immunoglobulin" encompasses the various forms of immunoglobulin structures including complete immunoglobulins and immunoglobulin conjugates. The immunoglobulin employed in the current invention is preferably a human antibody, or a humanized antibody, or a chimeric antibody, or a T cell antigen depleted antibody (see e.g. WO 98/33523, WO 98/52976, and WO 00/34317). Genetic engineering of antibodies is e.g. described in Morrison, S.L., et al., Proc.

Natl. Acad Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244; Riechmann, L., et al., Nature 332 (1988) 323-327; Neuberger, M.S., et al., Nature 314 (1985) 268-270; Lonberg, N., Nat. Biotechnol. 23 (2005) 1117-1125. Immunoglobulins may exist in a variety of formats, including, for example, Fv, Fab, and F(ab) ₂ as well as single chains (scFv), bispecific immunoglobulins or diabodies

(e.g. Huston, J.S., et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5879-5883; Bird, R.E., et al., Science 242 (1988) 423-426; in general, Hood et al., Immunology, Benjamin N.Y., 2nd edition (1984); and Hunkapiller, T. and Hood, L., Nature 323 (1986) 15- 16).

The term "complete immunoglobulin" denotes an immunoglobulin which comprises two so called light chains and two so called heavy chains. Each of the heavy and light chains of a complete immunoglobulin contains a variable domain (variable region) (generally the amino terminal portion of the polypeptide chain) comprising binding regions that are able to interact with an antigen. Each of the heavy and light chains of a complete immunoglobulin comprises a constant region

(generally the carboxyl terminal portion). The constant region of the heavy chain mediates the binding of the antibody i) to cells bearing a Fc gamma receptor (FcγR), such as phagocytic cells, or ii) to cells bearing the neonatal Fc receptor (FcRn) also known as Brambell receptor. It also mediates the binding to some factors including factors of the classical complement system such as component

(CIq). The variable domain of an immunoglobulin's light or heavy chain in turn comprises different segments, i.e. four framework regions (FR) and three hypervariable regions (CDR).

The term "immunoglobulin conjugate" denotes a polypeptide comprising at least one domain of an immunoglobulin heavy or light chain conjugated via a peptide bond to a further polypeptide. The further polypeptide is a non-immunoglobulin peptide, such as a hormone, or growth receptor, or antifusogenic peptide, or complement factor, or the like. Exemplary immunoglobulin conjugates are reported in WO 2007/045463.

The term ,,heterologous polypeptide" denotes a polypeptide, which is not naturally produced by a mammalian cell or the host cell. The polypeptide produced according to the method of the invention is produced by recombinant means. Such methods are widely known in the state of the art and comprise protein expression in eukaryotic cells with subsequent recovery and isolation of the heterologous polypeptide, and usually purification to a pharmaceutically acceptable purity. For the production, i.e. expression, of a polypeptide one or more nucleic acid(s) encoding the polypeptide is/are inserted each into an expression cassette by standard methods. Hybridoma cells can e.g. serve as a source of such nucleic acids encoding immunoglobulin light and heavy chains. The expression cassettes may be inserted into an expression plasmid(s), which is (are) then transfected into host

cells, which do not otherwise produce the heterologous polypeptide. Expression is performed in appropriate eukaryotic host cells and the polypeptide is recovered from the cells after lysis or from the culture supernatant.

An "isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e. at least about 80 % pure, at least about 90 % pure, at least about 95 % pure, greater than 95 % pure, or greater than 99 % pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

"Heterologous DNA" or ,,heterologous polypeptide" refers to a DNA molecule or a polypeptide, or a population of DNA molecules or a population of polypeptides, that do not exist naturally within a given host cell. DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e. endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e. exogenous DNA). For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a promoter is considered to be a heterologous DNA molecule. Conversely, a heterologous DNA molecule can comprise an endogenous structural gene operably linked with an exogenous promoter. A peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.

The term "growth factor" as used within this application denotes a compound that has a positive effect on the growth of a mammalian cell in a cultivation medium. In one embodiment said growth factor is consisting of amino acids residues, i.e. is a peptide, polypeptide or protein. Exemplary growth factors without limitation are transforming growth factor beta (TGF-β) , granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF-9), acidic fibroblast growth factor (aFGF or FGF-I),

basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), activins, bone morphogenic protein, FLT-3 ligand, insulin-like growth factors, neurotrophic factors, sonic hedgehog protein, vascular endothelial growth factor.

Any suitable or desired therapeutic protein for mammalian cell expression can be produced in cell culture using a CHO-Kl-Gln(-) cell according to the present invention. Non-limiting examples of such proteins include cytokines, receptors, soluble receptors, interleukins, growth factors, immunoglobulins, immunoglobulin fragments, immunoglobulin conjugates, and the like.

The term "cell culture," refers to cells growing in suspension or adherent, in roller bottles, flasks, glass or stainless steel cultivations vessels, and the like. Large scale approaches, such as bioreactors, are also encompassed by the term "cell culture". Cell culture procedures for both large and small-scale production of polypeptides are encompassed by the present invention. Procedures including, but not limited to, a fluidized bed bioreactor, shaker flask culture, or stirred tank bioreactor system may be used and operated alternatively in a batch, split-batch, fed-batch, or perfusion mode.

The terms "cell culture medium," and "culture medium" as used interchangeably within the current invention denote a nutrient solution used for growing mammalian cells. Such a nutrient solution generally includes various factors necessary for growth and maintenance of the cellular environment. For example, a typical nutrient solution may include a basal media formulation, various supplements depending on the cultivation type and, occasionally, selection agents. In general, any suitable cell culture medium may be used in the method according to the current invention as long as is does not contain polypeptides, glutamine, insulin or any growth factors.

Proteins which can be produced with a cell according to the invention or a method according to the invention are, e.g., hormones like luteinizing hormone-releasing hormone, thyroid hormone-releasing hormone, somatostatin, prolactin, adrenocorticotropic hormone, melanocyte-stimulating hormone, vasopressin, and derivatives thereof e. g., desmorpessin, oxytocin, calcitonin, parathyroid hormone (PTH) or fragment thereof (e. g. PTH (1-43)), gastrin, secretin, pancreozymin, cholecystokinin, angiotensin, human placenta lactogen, human chorionic gonadotropin (HCG), caerulein and motilin; analgesic substances like enkephalin

and derivatives thereof (see US 4,277,394 and EP 0 031 567), endorphin, daynorphin and kyotorphin; enzymes like e. g. bombesin, neurotensin, bradykinin and substance P; human growth hormone, bovine growth hormone, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, EPO, lipoproteins, alpha- 1 -antitrypsin, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, anti-clotting factors such as Protein C, atrial natriuretic factor, lung surfactant, a plasminogen activator such as urokinase or human urine or tissue-type plasminogen activator (t-PA), thrombin, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), human macrophage inflammatory protein (MlP-1-alpha), a serum albumin such as human serum albumin, mullerian-inhibiting substance, relaxin A- chain, relaxin B-chain, prorelaxin, mouse gonadotropin-associated peptide, a microbial protein, such as beta-lactamase, DNAse, inhibin, activin, renin, receptors for hormones or growth factors, integrin, protein A or D, rheumatoid factors, a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4,-5 or-6 (NT- 3, NT-4, NT-5 or NT-6), insulin-like growth factor binding proteins, CD proteins (cluster of differentiation proteins) such as CD-3, CD-4, CD-8 and CD- 19, osteoinductive factors, immunotoxins, cytokines receptors, interferons such as interferon- alpha,-beta and-gamma, interleukins (ILs), e. g., IL-I to IL-10, superoxide dismutase, T-cell receptors, surface membrane proteins, decay accelerating factor, viral antigen such as, for example, a portion of the AIDS envelope, transport proteins, homing receptors, addressins, regulator proteins, immunoglobulins, chimeric proteins, such as immunoadhesins, and fragments of any of the above listed proteins.

The terms "CHO-Kl cell" or "CHO-Kl" and grammatical equivalents thereof as well as compositions containing this term refer to a CHO-Kl cell into which a nucleic acid, e.g. encoding a heterologous polypeptide, can be or is transfected. As used herein, the expression "cell" includes the subject cell and its progeny. Thus, the words "transfectant" and "transfected cell" include the primary subject cell and cultures derived there from without regard for the number of cell passages (splits) or subcultivations. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transfected cell are included.

The term "expression" as used herein refers to transcription and/or translation processes occurring within a cell. The level of transcription of a nucleic acid sequence of interest in a cell can be determined on the basis of the amount of corresponding mRNA that is present in the cell. For example, mRNA transcribed from a sequence of interest can be quantitated by RT-PCR or by Northern hybridization (see Sambrook, et al., 1989, supra). Polypeptides encoded by a nucleic acid of interest can be quantitated by various methods, e.g. by ELISA, by assaying for the biological activity of the polypeptide, or by employing assays that are independent of such activity, such as Western blotting or radioimmunoassay, using immunoglobulins that recognize and bind to the polypeptide (see Sambrook, et al.,

1989, supra).

An "expression cassette" refers to a construct that contains the necessary regulatory elements, such as promoter and polyadenylation site, for expression of at least the contained nucleic acid in a cell.

A "transfection vector" is a nucleic acid (also denoted as nucleic acid molecule) providing all required elements for the expression of the in the transfection vector comprised coding nucleic acids/structural gene(s) in a host cell. A transfection vector comprises a prokaryotic plasmid propagation unit, e.g. for E. coli, in turn comprising a prokaryotic origin of replication, and a nucleic acid conferring resistance to a prokaryotic selection agent, further comprises the transfection vector one or more nucleic acid(s) conferring resistance to an eukaryotic selection agent, and one or more nucleic acid encoding a polypeptide of interest. Preferably are the nucleic acids conferring resistance to a selection agent and the nucleic acid(s) encoding a polypeptide of interest placed each within an expression cassette, whereby each expression cassette comprises a promoter, a coding nucleic acid, and a transcription terminator including a polyadenylation signal. Gene expression is usually placed under the control of a promoter, and such a structural gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

A "promoter" refers to a polynucleotide sequence that controls transcription of a gene/structural gene or nucleic acid sequence to which it is operably linked. A promoter includes signals for RNA polymerase binding and transcription initiation. The promoter(s) used will be functional in the cell type of the host cell in which expression of the selected sequence is contemplated. A large number of promoters

including constitutive, inducible and repressible promoters from a variety of different sources, are well known in the art (and identified in databases such as GenBank) and are available as or within cloned polynucleotides (from, e.g., depositories such as ATCC as well as other commercial or individual sources). A "promoter" comprises a nucleotide sequence that directs the transcription of an operably linked structural gene. Typically, a promoter is located in the 5' non- coding or untranslated region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee, R.E., et al., MoI. Endocrinol. 7 (1993) 551-560), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, R., Seminars in Cancer Biol. 1 (1990) 47-58), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE/ATF (O'Reilly, M.A., et al., J. Biol. Chem.

267 (1992) 19938-19943), AP2 (Ye, J., et al., J. Biol. Chem. 269 (1994) 25728- 25734), SPl, cAMP response element binding protein (CREB; Loeken, M. R., Gene Expr. 3 (1993) 253-264) and octamer factors (see, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre, F. P. and Rousseau, G.G., Biochem. J. 303

(1994) 1-14). Among the eukaryotic promoters that have been identified as strong promoters for high-level expression are the SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus long terminal repeat, Chinese hamster elongation factor 1 alpha (CHEF-I, see e.g. US 5,888,809), human EF-I alpha, ubiquitin, and human cytomegalovirus immediate early promoter (CMV IE). The "promoter" can be constitutive or inducible. An enhancer (i.e., a cis-acting DNA element that acts on a promoter to increase transcription) may be necessary to function in conjunction with the promoter to increase the level of expression obtained with a promoter alone, and may be included as a transcriptional regulatory element. Often, the polynucleotide segment containing the promoter will include enhancer sequences as well (e.g., CMV or SV40).

An "enhancer", as used herein, refers to a polynucleotide sequence that enhances transcription of a gene or coding sequence to which it is operably linked. Unlike promoters, enhancers are relatively orientation and position independent and have been found 5' or 3' (Lusky, M., et al., MoI. Cell Bio., 3 (1983) 1108-1122) to the

transcription unit, within an intron (Banerji, J., et al., Cell, 33 (1983) 729-740) as well as within the coding sequence itself (Osborne, T.F., et al., MoI. Cell Bio., 4 (1984) 1293-1305). Therefore, enhancers may be placed upstream or downstream from the transcription initiation site or at considerable distances from the promoter, although in practice enhancers may overlap physically and functionally with promoters. A large number of enhancers, from a variety of different sources are well known in the art (and identified in databases such as GenBank) and are available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources). A number of polynucleotides comprising promoter sequences (such as the commonly-used

CMV promoter) also comprise enhancer sequences. For example, all of the strong promoters listed above may also contain strong enhancers (see e.g. Bendig, M., M., Genetic Engineering 7 (Academic Press, 1988) 91 -127).

A "nucleic acid conferring resistance to a selection agent" is a nucleic acid that allows cells carrying it to be specifically selected for or against, in the presence of a selection agent. Such a nucleic acid is also denoted as selection marker. Typically, a selection marker will confer resistance to a selection agent (drug) or compensate for a metabolic or catabolic defect in the host cell. A selection marker can be positive, negative, or bifunctional. A useful positive selection marker is an antibiotic resistance gene. This selection marker allows cells transformed therewith to be positively selected for in the presence of the corresponding selection agent, i.e. under selected growth in the presence e.g. of the corresponding antibiotic. A non- transformed cell is not capable to grow or survive under the selective growth conditions, i.e. in the presence of the selection agent, in culture. Positive selection markers allow selection for cells carrying the marker, whereas negative selection markers allow cells carrying the marker to be selectively eliminated. Eukaryotic selection markers include, e.g., the genes for aminoglycoside phosphotransferase (APH) (conferring resistance to the selection agents such as e.g. hygromycin (hyg), neomycin (neomycin phosphotransferase II, neo), and G418), dihydrofolate reductase (DHFR) (conferring resistance to the selection agent methotrexate), thymidine kinase (tk), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (conferring resistance to the selection agent indole), histidinol dehydrogenase (conferring resistance to the selection agent histidinol D), cytidine deaminase, adenosine deaminase and nucleic acids conferring resistance to puromycin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid. Further selection marker nucleic acids are reported e.g. in WO 92/08796 and

WO 94/28143. Prokaryotic selection markers include, e.g. the beta-lactamase gene (conferring resistance to the selection agent ampicillin).

Expression of a gene is performed either as transient or as permanent expression. The polypeptide(s) of interest are in general secreted polypeptides and therefore contain an N-terminal extension (also known as the signal sequence) which is necessary for the transport/secretion of the polypeptide through the cell wall into the extracellular medium. In general, the signal sequence can be derived from any gene encoding a secreted polypeptide. If a heterologous signal sequence is used, it preferably is one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For secretion in yeast for example the native signal sequence of a heterologous gene to be expressed may be substituted by a homologous yeast signal sequence derived from a secreted gene, such as the yeast invertase signal sequence, alpha-factor leader (including Saccharomyces, Kluyveromyces, Pichia, and Hansenula α-factor leaders, the second described in US 5,010,182), acid phosphatase signal sequence, or the C. albicans glucoamylase signal sequence

(EP 0 362 179). In mammalian cell expression the native signal sequence of the protein of interest is satisfactory, although other mammalian signal sequences may be suitable, such as signal sequences from secreted polypeptides of the same or related species, e.g. for immunoglobulins from human or murine origin, as well as viral secretory signal sequences, for example, the herpes simplex glycoprotein D signal sequence. The DNA fragment encoding for such a presegment is ligated in frame, i.e. operably linked, to the DNA fragment encoding a polypeptide of interest.

The first aspect of the current invention is a CHO-Kl cell, denoted as CHO-Kl- Gln(-), which requires no glutamine, no insulin, and no growth factors in the cultivation medium.

The CHO-Kl cell according to the invention is obtained as follows:

providing the CHO-Kl cell deposited as ATCC CCL-61, adapting said CHO-Kl cell to growth in suspension in a polypeptide-free, chemical defined medium supplemented with glutamine, optionally supplemented with hypoxanthine and thymidine, adapting said CHO-Kl cell adapted in the preceding step to growth in suspension in a polypeptide-free, chemical defined medium, optionally supplemented with hypoxanthine and thymidine.

The term "chemical defined medium" denotes a medium comprising only synthetic compounds and which is animal derived component free. Additionally the term "chemical defined medium" denotes a medium which contains in one embodiment less than 0.1 mM glutamine, in another embodiment less than 0.01 mM glutamine and in a further embodiment less than 0.001 mM glutamine. In one embodiment the chemical defined medium is free of glutamine. In one embodiment the chemical defined medium is free of animal derived components and also free of cell hydrolyzates of plant origin. Exemplary synthetic chemically defined media are the CD CHO medium from Invitrogen, or a modified version of the medium reported in WO 02/066603 composed of sodium chloride, 3-5 g/L; potassium chloride, 0.2-

0.4 g/L; HEPES, 5-7 g/L; glucose (dextrose), 3.5-5.5 g/L; biotin, 0.000005-0.000025 g/L; ascorbic acid, 0.002-0.004; pantothenate, 0.002-0.006 g/L; choline, 0.002-0.006 g/L; folate, 0.002-0.006 g/L; inositol, 0.005-0.02 g/L; niacinamide, 0.002-0.006 g/L; pyridoxal, 0.002-0.006 g/L; riboflavin, 0.0002-0.0006 g/L; thiamine, 0.002-0.006 g/L; cyanocobalamin, 0.000005-0.000025 g/L; oxaloacetic acid, 0.1-0.4 g/L; alanine,

0.015-0.035 g/L; asparagine, 0.01-0.035 g/L; arginine, 0.06-0.10 g/L; aspartate, 0.02- 0.04 g/L; cysteine, 0.3-0.5 g/L; cystine, 0.05-0.2 g/L; glutamate, 0.06-0.09 g/L; glycine, 0.02-0.04 g/L; histidine, 0.03-0.05 g/L; isoleucine, 0.05-0.25 g/L; leucine, 0.05-0.25 g/L; lysine, 0.05-0.25 g/L; methionine, 0.02-0.04 g/L; phenylalanine, 0.055-0.075 g/L; proline, 0.03-0.05 g/L; serine, 0.03-0.055 g/L; threonine, 0.07-0.15 g/L; tryptophan, 0.005-0.025 g/L; tyrosine, 0.05- 0.15 g/L; valine, sodium selenate, 0.0000005-0.000060 g/L; magnesium sulfate, 0.05-0.2 g/L; potassium chloride, 0.15- 0.45 g/L; sodium phosphate, 0.075-0.2 g/L; potassium nitrate, 0.00005- 0.00009 g/L; calcium chloride, 0.08-0.25 g/L; sodium pyruvate 0.05-0.4 g/L; linoleic acid, 1-100 mg/L; ethanolamine, 5-25 μg/L; sodium bicarbonate, 1-5 g/L; ferric citrate, 1-10 mg/L; Pluronic F68,0.2-2 g/L; sodium hydroxide, 0.3-0.9 g/L; mycophenolic acid, 0.1-2 mg/L; hypoxanthine, 2-5 mg/L; xanthine; 10-200 mg/L; sodium bicarbonate 1.5-4.5 g/L, or are based on commercially available media, e. g. from Sigma/ Aldrich (product numbers S2772, S2897 and S8284 (www. sigma-Aldrich. com)), or Life Technologies, Rockville, MD (www. lifetech. com), or JRH Biosciences, Lenexa, KS

(www. jrhbio. com) and modified according to the provisos of the current invention to provide a medium useful in the current invention, or Iscoves modified media (Iscove et al., J. Exp. Med. 147 (1978) 923-933; Iscove, et al., Exp. Cell Res. 126 (1980) 121-126), or Dulbecco's Modified Eagle's Medium (Dulbecco and Freeman, Virology 8 (1959) 396-397; Smith et al.,, J. D., Freeman, G., Vogt, M. and

Dulbecco, R., Virology 12 (1960) 185-196; Morton, In Vitro 6 (1970) 89; Rutzky

and Pumper, In Vitro 9 (1974) 468), or Ham's F-12/Dulbecco's Modified Eagle's Medium (Barnes and Sato, Analyt. Biochem. 102 (1980) 255-270).

The CHO-Kl cell ATCC CCL-61 as obtained from the ATCC is an adherent growing cell adapted to growth in a cultivation medium which is a 1:1 (w/w) mixture of Dulbecco's Modified Eagle Medium and Ham's F12 Medium

(DMEM/F12) supplemented with 2 mM glutamine and 10 % (v/v) fetal Bovine serum (FCS) under standard humidified conditions (95 % humidity, 37 ⁰ C, 5 % CO ₂ ).

In the first step the cells are adapted to growth in suspension. Therefore the cells are detached by enzymatic treatment with trypsin or Accutase and collected by centrifugation. The collected cells are seeded in a synthetic polypeptide-free, chemical defined medium containing hypoxanthine and thymidine as well as 2 mM L-alanyl-L-glutamine. This cultivation in one embodiment is performed at small scale in a cultivation vessel volume of 50 ml to 250 ml. In one embodiment the inoculation cell density is 3xlO ⁵ cells/ml. In one embodiment the cells are every 3 to

4 generations, i.e. every 3 to 5 days, splitted into fresh medium. In one embodiment the cells are splitted for a total of 35 to 60 generations or 12 to 20 splits. In one embodiment the doubling time of the cell adapted in the first step is between 25 and 30 hours.

In the second step the cell derived from the first step is adapted to growth in suspension in the synthetic polypeptide-free, chemical defined medium containing hypoxanthine and thymidine but without L-alanyl-L-glutamine. Therefore the cells are passaged over 25 to 33 generations or 23 to 35 days. The synthetic polypeptide- free, chemical defined medium of the second step does not contain glutamine or a peptide or polypeptide containing glutamine. In one embodiment the doubling time of the cell obtained in the second step is between 22 and 25 hours. In one embodiment the maximum viable cell density of the cell obtained in the second step is 6-7xlO ⁶ cells/ml.

It has surprisingly been found that a CHO-K l-Gln(-) cell according to the invention has improved properties compared to the parent cell line ATCC CCL-61.

Additionally with the exclusion of glutamine from the cultivation medium of the CHO-Kl-Gln(-) cell the formation of ammonium ions and ammonia during the cultivation process is reduced. Thus, in one embodiment the ammonium ion concentration in the cultivation medium is below 0.12 mmol/L, in a further

embodiment below 0.1 mmol/L. It has also been found that the concentration of lactate in the cultivation medium is maintained at a constant level. Thus, in one embodiment according to the invention the lactate generation/accumulation in the cultivation medium during the cultivation of a cell according to the invention is reduced. In another embodiment the lactate concentration is below 2.5 g/L in the cultivation medium. Further with the cell according to the invention it is now possible to use the same cultivation medium in all steps required for the generation of a production cell line, i.e. from the first transfection till the large scale fermentation. The employed synthetic polypeptide-free, chemically defined medium has only to be supplemented with the required selection agents. Therefore, no adaptation step to a new/different medium is required during the entire process, saving time and costs. Also, in one embodiment if the cultivation is performed as fed-batch cultivation the employed feed is glutamine-free, insulin-free, growth- factor-free and polypeptide-free. It was now also surprisingly found that such a useful cell can be obtained by a sequence of adaptation steps and does not require a genetic modification of the cell e.g. by the introduction of additional genes encoding enzymes in order to convert unwanted, toxic metabolic side product(s) into non-harmful compounds. It has also been found that with a CHO-Kl-Gln(-) cell according to the invention the pH value in the cultivation vessel is maintained in the pH range of pH 6.8 to pH 7.2.

Therefore the second aspect of the current invention is a method for the generation of a CHO-Kl-Gln(-) cell line, comprising the following steps in the following order:

- providing the CHO-K 1 cell ATCC CCL-61 ,

- adapting said CHO-Kl cell to growth in suspension in a polypeptide-free, chemical defined medium supplemented with glutamine, optionally additionally supplemented with hypoxanthine and thymidine,

adapting said CHO-Kl cell adapted in the preceding step to growth in suspension in a polypeptide-free, chemical defined medium, optionally supplemented with hypoxanthine and thymidine, and thereby obtaining the CHO-Kl-Gln(-) cell line.

In one embodiment the cultivation medium required for said CHO-K l-Gln(-) cell is free of glutamine, insulin and growth factors, and said CHO-Kl-Gln(-) cell is not

genetically modified by the introduction, deletion, or inactivation of a nucleic acid by molecular biological methods or by the introduction, deletion, or inactivation of a nucleic acid encoding an enzyme not required for the expression, secondary modification, or secretion of a heterologous polypeptide.

A further aspect of the current invention is a CHO-Kl cell obtained with a method according to the current invention.

Another aspect of the current invention is a method for the recombinant production of a heterologous polypeptide comprising the following steps:

providing a CHO-Kl-Gln(-) cell according to the invention, - providing one or more nucleic acids encoding said heterologous polypeptide, transfecting said CHO-Kl -Gln(-) cell with said one or more nucleic acids, cultivating said transfected CHO-Kl-Gln(-) cell in a polypeptide-free, chemical defined medium not containing glutamine, insulin or a growth factor, recovering said heterologous polypeptide from the cultivation medium or the cells, and

optionally purifying said heterologous polypeptide by one or more chromatographic purification steps.

In one embodiment said heterologous polypeptide is an immunoglobulin or an immunoglobulin fragment or an immunoglobulin conjugate or a therapeutically active polypeptide.

The method according to the invention is suited for the production of a secreted heterologous polypeptide in large scale, i.e. industrially. The cultivation of a cell for the production of a desired polypeptide in large scale generally consists of a sequence of individual cultivations, wherein all cultivations except the final, i.e. the large scale, cultivation, i.e. the last one in the sequence, are performed until a certain cell density is reached in the culture vessel. If the predetermined cell density is reached the entire cultivation or a fraction thereof is used to inoculate the next cultivation vessel, which has a larger volume, up to 100 times the volume of the preceding cultivation. All cultivations which serve as a basis for at least one further

cultivation in a larger volume are denoted as "seed train fermentation". Only in the large scale cultivation, i.e. in the cultivation which is not intended to serve as the basis for a further cultivation in a larger volume, which is also denoted as "main fermentation", is the endpoint of the cultivation determined depending on the concentration of the produced secreted heterologous immunoglobulin in the cultivation medium or the cultivation time. The term "large scale" as used within this application denotes the final cultivation of an industrial production process. In one embodiment a large scale cultivation is performed in a volume of at least 100 1, in another embodiment of at least 500 1, in a further embodiment of at least 1000 1 up to a volume of 25,000 1. In one embodiment the final, i.e. large scale, cultivation medium does not contain a eukaryotic selection agent.

In one embodiment the cultivation of said transfected CHO cell is performed in the presence of eukaryotic selection agent(s) in a volume of less than 500 liter and the cultivation of said transfected CHO cell is performed in the absence of eukaryotic selection agent(s) in a volume of 500 liter or more and the recovering of the heterologous polypeptide is from the cultivation medium without said eukaryotic selection agents. In a further embodiment the cultivation is comprising sequential cultivations with increasing cultivation volume up to a final cultivation volume, whereby the cultivations are performed in the presence of eukaryotic selection agent(s) up to a cultivation volume of 1 % (v/v) of the cultivation volume of the final or main cultivation, and in the absence of all eukaryotic selection agents in a cultivation volume of more than 1 % (v/v) of the cultivation volume of the final cultivation. In a further embodiment said cultivation comprises sequential seed train cultivations with increasing cultivation volume, whereby each of the seed train cultivations is performed in the presence of eukaryotic selection agent(s) and the main fermentation is performed in the absence of all eukaryotic selection agents. In one embodiment the cultivation of said transfected CHO cell is performed in the presence of eukaryotic selection agent(s) in the seed train fermentations and the cultivation of said transfected CHO cell is performed in the absence of eukaryotic selection agents in the main fermentation and the recovering of the heterologous polypeptide is from the main cultivation medium not containing eukaryotic selection agent(s). In these embodiments the eukaryotic selection agent(s) is(are) added during the seed train cultivations and omitted during the production phase (main fermentation culture) of said CHO cell. The term "production phase" denotes the cultivation of a CHO cell in a large volume, i.e. the main fermentation, after which the produced heterologous polypeptide is recovered.

In another embodiment of the method according to the invention the productivity of said CHO cell is over 40 generations not less than 70 % and not more than 130 % of the productivity after 10 generations of cultivation as split-batch cultivation. In an embodiment the productivity of said CHO cells is over 60 generations not less than 50 % and not more than 150 % of the productivity after 10 generations of cultivation as split-batch cultivation. The productivity of said CHO cell is at least 1.5 g/1 of said heterologous immunoglobulin within 21 days as fed-batch cultivation in another embodiment. In one embodiment the specific productivity of the CHO cell obtained with the method according to the invention is more than 1 μg/10 ⁶ cells/d, more than 5 μg/10 ⁶ cells/d, or more than 10 μg/10 ⁶ cells/d. In one embodiment the secreted heterologous immunoglobulin is a completely processed secreted heterologous immunoglobulin. The term "completely processed secreted heterologous immunoglobulin" denotes an immunoglobulin i) which is secreted to the cultivation medium and whose signal sequences has been cleaved, ii) which comprises an antigen binding region, iii) which has secondary modifications, such as attached saccharides or polysaccharides, and/or correctly formed disulfide bonds.

In one embodiment of the invention the heterologous immunoglobulin is an anti- CD4 antibody-conjugate. In another embodiment the heavy chain variable domain of said anti-CD4 antibody in said conjugate comprises a CDR3 with an amino acid sequence selected from SEQ ID NO: 01, 02, or 03. In a further embodiment the light chain variable domain of said anti-CD4 antibody in said conjugate comprises a CDR3 with an amino acid sequence selected from SEQ ID NO: 04, 05, or 06. In a further embodiment said anti-CD4 antibody in said conjugate comprises a heavy chain variable domain with an amino acid sequence selected from SEQ ID NO: 07, 08, or 09. In still a further embodiment said anti-CD4 antibody in said conjugate comprises a light chain variable domain with an amino acid sequence selected from SEQ ID NO: 10, 11, or 12.

A mammalian cell usable for the large scale production of therapeutics, i.e. polypeptides intended for the use in humans, has to fulfill distinct criteria. Amongst others are these that it has to be cultivatable in serum-free medium. Serum is a mixture of multitude of compounds. Normally bovine serum has been used for the cultivation of mammalian cells. With the arising problem of transmissible diseases from one species to another the use of serum and other non-defined mammal- derived components has to be avoided. The term "non-defined mammal-derived component" as used within this application denotes components which are derived

from a mammal, especially preferred from a cow, a pig, a sheep, or a lamb, and whose composition can be specified to less than 80 % (w/w), preferably to less than 90 % (w/w). A "defined mammal-derived component" is a component that is obtained from a mammal, especially preferred from a cow, a pig, a sheep, or a lamb, and whose composition can be specified to more than 95 % (w/w), preferably to more than 98 % (w/w), most preferably to more then 99 % (w/w). An example of a defined mammal-derived component is cholesterol from ovine wool, and galactose from bovine milk.

In one embodiment the polypeptide-free, chemical defined media is the CD CHO medium available from Invitrogen Corp., or the ProCHO4 medium available from

Gibco, or the protein free medium HyQ SFM4CHO available from Hyclone. In one embodiment said polypeptide-free, chemical defined cultivation medium is derived from Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle medium (DMEM), RPMI 1640, Iscove's modified Dulbecco's medium (IMDM), or NCTC 109 cultivation medium (all available from Lonza Inc., USA).

In another embodiment of the method according to the invention is the method beginning with the first transfection and ending with the recovery of the heterologous polypeptide performed in the same medium. The term "in the same medium" denotes within the current application that beginning with the first transfection and ending with the recovery of the heterologous polypeptide from the main fermentation cultivation medium the same medium is used. This denotes that in all steps new medium of the same composition is employed. This does not denote that the same additives have to be added to the medium in all steps, i.e. the medium may be supplemented with different additive in different steps of the method. Additives are compounds that are added to a medium in total to less than

20 % (w/w), in one embodiment to less than 15 % (w/w), in another embodiment to less than 10 % (w/w). In one embodiment the medium used in the method according to the invention is the same medium in all steps and is a medium suitable for the large scale production of the heterologous polypeptide.

The heterologous polypeptide can be recovered from the cultivation medium with chromatographic methods known to a person of skill in the art. Therefore in one embodiment the method according to the invention comprises the final step of purifying said heterologous polypeptide with one or more chromatographic steps.

For example, for the expression of a heterologous immunoglobulin the vector with which the CHO-Kl-Gln(-) cell is transfected comprises a nucleic acid conferring resistance to a eukaryotic selection agent, comprises a nucleic acid encoding the light chain of said heterologous immunoglobulin and/or a nucleic acid encoding the heavy chain of said heterologous immunoglobulin. If the vector comprises only a nucleic acid encoding either the light chain of said immunoglobulin or the heavy chain of said immunoglobulin said CHO cell is also transfected in each step by another vector comprising a nucleic acid encoding the corresponding other chain of said immunoglobulin.

The invention further comprises a CHO-Kl cell cultivated in a polypeptide-free, chemical defined medium. Also encompassed is a heterologous polypeptide expressed from such a cell line. Another aspect of the current invention is a composition comprising the CHO-K l-Gln(-) cell according to the invention.

In one embodiment said heterologous polypeptide is selected from an immunoglobulin, an immunoglobulin fragment, an immunoglobulin conjugate, a soluble receptor, a transmembrane protein, a cytoplasmic protein, a soluble protein, an extracellular protein, a fusion protein, or any fragment or portion thereof.

In one embodiment said cultivating said transfected CHO-Kl-Gln(-) cell is performed for a time sufficient to express said heterologous polypeptide. In another embodiment said time is 14 to 21 days.

Another aspect of the current invention is a CHO-Kl cell derived from a parental CHO-Kl cell, in one embodiment from CHO-Kl ATCC CCL-61, wherein said derived cell line has a doubling time in a polypeptide-free, chemical defined medium not containing glutamine of not more than 120 % of the doubling time of said parental cell line in a serum- and glutamine-containing medium.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Description of the Figures

Figure 1 Viable cell density obtained in a) CD_CHO_HT_Gln medium and b) CD_CHO_HT medium of three cultivations each.

Figure 2 Lactate concentration in the cell culture supernatant in a) CD_CHO_HT_Gln medium and b) CD_CHO_HT medium of three cultivations each.

Figure 3 Ammonium-ion concentrations in the cell culture supernatant in a) CD_CHO_HT_Gln medium and b) CD_CHO_HT medium of three cultivations each. Figure 4 Annotated plasmid map of plasmid 6311.

Examples

Materials & Methods

General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National

Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered according to EU numbering (Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991)).

Recombinant DNA techniques:

Standard methods were used to manipulate DNA as described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.

Gene synthesis:

Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The 100 - 600 bp long gene segments, which are flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned into the pCR2.1 -TOPO-TA cloning vector (Invitrogen Corp., USA) via A-overhangs or pPCR-Script Amp SK(+) cloning vector (Stratagene Corp., USA). The DNA sequence of the subcloned gene fragments were confirmed by DNA sequencing.

Protein determination:

Protein concentration was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

Antibody titer determination:

Antibody titers were determined either by anti-human Fc ELISA or by Protein A chromatography using the autologous purified antibody as a reference.

Analysis of Metabolites:

The concentration of glucose, lactate, glutamine and ammonium is analyzed using the Bioprofile 100 plus Analyzer (Nova Biomedical).

SDS-PAGE

LDS sample buffer, fourfold concentrate (4x): 4 g glycerol, 0.682 g TRIS-Base, 0.666 g TRIS-hydrochloride, 0.8 g LDS (lithium dodecyl sulfate), 0.006 g EDTA (ethylene diamin tetra acid), 0.75 ml of a 1% by weight (w/w) solution of Serva Blue G250 in water, 0.75 ml of a 1% by weight (w/w) solution of phenol red, add water to make a total volume of 10 ml.

The culture broth containing the secreted antibody was centrifuged to remove cells and cell debris. An aliquot of the clarified supernatant was admixed with 1/4 volumes (v/v) of 4xLDS sample buffer and 1/10 volume (v/v) of 0.5 M 1,4- dithiotreitol (DTT). Then the samples were incubated for 10 min. at 70 ⁰ C and protein separated by SDS-PAGE. The NuPAGE® Pre-Cast gel system (Invitrogen Corp.) was used according to the manufacturer's instruction. In particular, 10% NuPAGE ^® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MOPS running buffer was used.

Western blot

Transfer buffer: 39 mM glycine, 48 mM TRIS-hydrochloride, 0.04% by weight (w/w) SDS, and 20% by volume methanol (v/v).

After SDS-PAGE the separated antibody chains were transferred electrophoretically to a nitrocellulose filter membrane (pore size: 0.45 μm) according to the ,,Semidry- Blotting-Method" of Burnette (Burnette, W.N., Anal. Biochem. 112 (1981) 195-

203).

Example 2

Generation of a CHO-Kl-Gln(-) cell

The parent CHO-Kl cell was obtained from ATCC (No. CCL-61) as frozen stock. The generation of this CHO-Kl cell is reported by Puck, T.T., et al. in J. Exp. Med. 108 (1958) 945-956, and Kao, F.T., and Puck, T.T., in Proc. Natl. Acad. Sci. USA 60

(1968) 1275-1281.

A CHO-Kl-Gln(-) host cell was derived from the deposited CHO-Kl cell line by pre-adaptation to growth in suspension culture in polypeptide-free, chemical defined, animal component- free, and glutamine-free CD CHO medium (10743- Oil, Invitrogen Corp., USA) containing hypoxanthine-thymidine-supplement

(11067-030, Invitrogen Corp., USA). This medium is designated in the following as CD_CHO_HT medium.

The adherent growing CHO-Kl cell line ATCC CCL-61 was expanded in DMEM/F12 medium (Invitrogen Corp.; 1:1 mixture of Dulbecco's Modified Eagle Medium and Ham's F 12 Medium) supplemented with 2 mM glutamine and 10 %

FCS (Gibco; catalog no.: 26400; USA) in T-tissue culture flasks and standard humidified conditions (95 %, 37 ⁰ C and 5 % CO ₂ ).

The cells were detached by enzymatic treatment with Accutase, a cell detachment solution prepared from an invertebrate (shrimp, PAA Laboratories; catalog no.: Ll 1-007) and seeded in CD_CHO_HT medium containing 2 mM ultra-glutamine

(L-Alanyl-L-glutamine; Cambrex; BE17-605E/U1) in a 125 ml Erlenmeyer flask under shaking (110-130 rpm). This medium is designated in the following CD_CHO_HT_Gln medium. Every 3-4 days the cells were splitted into fresh CD_CHO_HT_Gln medium (inoculum concentration approximately 3xlO ⁵ cells/ml) over a period of about 60 days. Thereafter, the cells were passaged over a period of about 30 days using glutamine-free CD_CHO_HT medium.

The obtained cell is adapted to growth in suspension culture and to growth in the absence of glutamine, insulin, and growth factors in a synthetic polypeptide-free, chemically defined cultivation medium not containing animal-derived components.

In Figure 1 the viable cell density obtainable with the cell after the adaptation to growth in suspension in CD_CHO_HT_Gln medium (first step) and after adaptation to growth in suspension in CD_CHO_HT medium (second step) is

shown. It can be seen that in the glutamine-free medium (Figure Ib)) a higher cell density can be obtained. At day 7 the pH value of the CD_CHO_HT_Gln cultivation medium is 6.7, whereas the pH value at day 7 of the CD_CHO_HT medium is between pH 6.8 and pH 7.0.

In Figure 2 the lactate concentration in the cell culture supernatant from cultivation with the cell after the adaptation to CD_CHO_HT_Gln medium and after adaptation to CD_CHO_HT medium is shown. It can be seen that in the glutamine-free medium (Figure 2b)) a reduced lactate production can be observed.

In Figure 3 the ammonium ion concentration in the cell culture supernatant from cultivation with the cell after the adaptation to CD_CHO_HT_Gln medium and after adaptation to CD_CHO_HT medium is shown. It can be seen that in the glutamine-free medium (Figure 3b)) a reduced ammonium ion concentration can be observed.

Table 1: Comparison of cell doubling time.

Example 3

Expression vector for expressing an anti-CD4 antibody conjugate

An example (monoclonal) antibody which can be expressed using a cell line according to the invention is an antibody against the human CD4 surface receptor (anti-CD4 antibody) which is conjugated to two to eight antifusogenic peptides. Such an antibody and the corresponding nucleic acid sequences are for example reported in PCT/EP2008/005894 or SEQ ID NO: 01 to 12.

A genomic human kappa-light chain constant region gene segment (C-kappa) was added to the light chain variable region of the anti-CD4 antibody of SEQ ID NO: 11, whereas a human gamma 1-heavy chain constant region gene segment (Cm- Hinge-CH ₂ -CH ₃ ) was added to the heavy chain variable region of the anti-CD4 antibody of SEQ ID NO: 08. The expression plasmid 6311 comprises an anti-CD4 antibody γl -heavy chain, which is joint at the last but one C-terminal amino acid, i.e. the C-terminal lysine residue of the heavy chain is removed, with a nucleic acid encoding an antifusogenic peptide of SEQ ID NO: 13 via the peptidic glycine-serine linker of SEQ ID NO: 14, and a anti-CD4 antibody κ-light chain, and a nucleic acid conferring resistance to the selectable marker neomycin. An annotated plasmid map is shown in Figure 4.

a) Heavy chain expression cassette

The transcription unit of the anti-CD4 antibody conjugate heavy chain is composed of the following elements:

- the immediate early enhancer and promoter from the human cytomegalovirus (CMV IE),

- a 5'-untranslated region (5' UTR), the coding sequence for the anti-CD4 antibody gamma 1-heavy chain conjugate including a signal peptide in an intron-exon gene structure, - the SV 40 early poly A signal sequence.

b) Light chain expression cassette

The transcription unit of the anti-CD4 antibody conjugate light chain is composed of the following elements:

the immediate early enhancer and promoter from the human cytomegalovirus (CMV IE),

- a 5'-untranslated region (5' UTR), the coding sequence for the anti-CD4 kappa-light chain in an intron-exon gene structure, the SV 40 early poly A signal sequence.

c) Expression plasmids

For the expression and production of the anti-CD4 antibody conjugate the light and heavy chain expression cassettes were placed on a single expression vector (light chain upstream of heavy chain). Three identical expression vectors were generated differing only in the selectable marker gene included, in particular, a neomycin resistance gene, a puromycin resistance gene, and a hygromycin resistance gene.

The expression vectors contain beside the light and heavy chain expression cassette the following elements:

an origin of replication allowing for the replication of the plasmid in E. coli taken from pUC 18 ( pUC origin ) , a beta-lactamase gene which confers ampicillin resistance in E. coli.

Example 4

Transfection and selection of a CHO cell line expressing an anti-CD4 antibody conjugate

The cell obtained in Example 2 was propagated in CD_CHO_HT medium in

Erlenmeyer flasks (50 or 125 ml) under shaking (110-130 rpm) using 30-50 % of the nominal volume as working volume and standard humidified conditions (95 %, 37 ⁰ C and 5 % CO ₂ ). Every 3-4 days the cells were splitted into fresh medium (inoculum cell density of approximately 3xlO ⁵ cells/ml). The cells were harvested by centrifugation in the exponential growth phase, washed once in sterile Phosphate

Buffered Saline (PBS) and resuspended in sterile PBS.

Prior to transfection the plasmid 6311 was linearized within the β-lactamase gene (E. coli ampicillin resistance gene) using the restriction endonuclease enzyme Pvul. The cleaved DNA was precipitated with ethanol, dried under vacuum and dissolved in sterile PBS at a concentration of about 1 μg DNA/μl. For transfection, the CHO host cells were electroporated with 20-50 μg linearized plasmid DNA per approximately 0.9xl0 ⁷ cells in a total volume of 200-300 μl PBS at room temperature. The electroporations were performed with a Gene Pulser XCeIl electroporation device (Bio-Rad Laboratories) in a 2 mm gap cuvette, using a square wave protocol with a single 160 V pulse for 15 ms. After transfection, the cells were plated out in CD_CHO_HT medium (10 ⁴ cells per 100 μl medium per well of a 96-well culture plate). After 24 h, 100 μl of CD_CHO_HT medium supplemented with 2-fold concentration of Geneticin " (G418: 1400 μg/ml) was

added to each well without replacing the original medium and plates. After every 7 days, the CD_CHO_HT_G418 selection medium was replaced. After 2-3 weeks of incubation, the antibody concentration was analyzed with an ELISA assay specific for human IgGi in the culture supernatants.