WO2012095514A1

WO2012095514A1 - Recombinant protein production system

Info

Publication number: WO2012095514A1
Application number: PCT/EP2012/050488
Authority: WO
Inventors: Jérôme COURTETE; Fabienne Guehenneux; Anne ROPART
Original assignee: Vivalis
Priority date: 2011-01-14
Filing date: 2012-01-13
Publication date: 2012-07-19

Abstract

The invention relates to the characterization of the duck Glutamine Synthetase and its use in field of recombinant proteins production.

Description

RECOMBINANT PROTEIN PRODUCTION SYSTEM

FIELD OF THE INVENTION

The present invention relates to the field of recombinant proteins production. Specifically, the present invention refers to a recombinant DNA sequence, vectors and cell lines comprising the same and to methods for the use thereof. Notably, the invention relates to a method for the selection of avian, and more particularly duck, cell clones having an increased expression of a protein of interest, in particular, antibodies.

BACKGROUND OF THE INVENTION

The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including bacterial and eukaryotic hosts. The selection of an appropriate expression system often depends not only on the ability of the host cell to produce adequate yields of the protein in an active state, but, to a large extent, may also be governed by the intended end use of the protein.

Monoclonal antibodies (mAbs) constitute a highly successful class of therapeutic proteins, with applications in various fields such as inflammatory diseases, oncology or infectious diseases. Currently, there are more than 30 antibodies or antibody fragments approved for therapeutic use (Cochet O. and Chartrain M., Med Sci (Paris) 2009; 25: 1078-83). Mammalian cell culture, in particular the Chinese hamster ovary (CHO) cell line, emerged as the expression host of choice for the industrial production of therapeutic glycoproteins because of their capacity for proper protein folding, assembly and post-translational modification.

However, a rapid increase in the number of approved mAbs may cause issues of limited production capacity, high production charges and prohibitive therapeutic costs. As a consequence, access of the general population to such innovative therapies may be delayed. Increasing bioreactors capacity would not reduce sufficiently the cost of production, mainly due to downstream processing becoming a bottleneck. Thus, permanent efforts are dedicated to further improve the production yields achieved by current mammalian systems. Besides such developments, alternative production systems have recently been explored that could produce proteins with enhanced therapeutic indexes and hence with the potential for lower production costs. These alternative systems for mAbs production include: bacteria, yeast, filamentous fungi, insect cells, transgenic plants and animals and were reviewed by Olivier S. and Mehtali M. (Med Sci (Paris) 2009; 25: 1 163-8).

It is generally accepted that the key issues affecting the choice of a cell line for use in a manufacturing process are: the capability to produce high antibody concentrations in the chosen production system, the ability to consistently produce a product of uniform characteristics, and the speed with which a high yielding cell line can be obtained. The availability of a suitable expression system and the importance of post-translational modifications of the recombinant antibodies may also affect this choice.

Over the past ten years, VIVALIS (Nantes, France) established and developed a proprietary avian embryonic derived stem cell platform (EBx cell platform), which involved the isolation of avian, notably chicken or duck, embryonic stem cells and derivation thereof of stable cell lines without genetic, viral or chemical modifications. As described in WO 2003/07660, EBx cell lines are derived following a multi step process permitting the selection of stable cell lines that maintain some of the unique biological properties of ES cells, such as the expression of ES cells specific markers, e.g., telomerase, SSEA-1 , EMA-1 , the ability to indefinitely self-renew in vitro and a long-term genetic stability. EBx cells have demonstrated to be highly susceptible to a very broad range of human and animal viral vaccines and are considered as an alternative for the cost-effective manufacturing of vaccines currently produced in chicken eggs or in primary chicken embryonic fibroblasts (WO 2008/129058).

Such attractive cell growth properties, coupled with their avian origin and their ability to be efficiently genetically engineered, make such cells an attractive platform for the production of mAbs with reduced fucose content and enhanced ADCC activity. Patent application No. WO2008/142124 describes that monoclonal antibody expressed in an avian embryonic derived stem cell line EBx displays a human-like glycosylation pattern. In particular, an increase in the ADCC activity is shown in chicken EB14 cells and duck EB66 cells. The inventors also found that a large proportion of lgG1 antibodies population produced in EBx cells has a common relinked oligosaccharide structure of a bicatenary-type that comprises long chains with terminal GlcNac that are highly galactosylated. Approximately half of lgG1 antibodies population contains the N-linked oligosaccharide structure of bicatenary-type that is non-fucosylated, which confers a strong ADCC activity to antibodies.

Specially interesting is the duck-derived EB66 cell line, which has the capacity to proliferate in stirred-tank bioreactors to high cell densities as suspension cells in serum-free or chemically defined culture media for the production of mAbs at yields beyond 1 g/L. (Olivier S. et al., MAbs 2010; 2(4)). However, productivity of mammalian cell processes has improved dramatically in recent years and modem cell culture processes can achieve antibody concentrations exceeding 5 g/L (R. Winder, Cell culture changes gear, Chem. Ind. (17th October 2005) 18-20).

Hence, there is a need to increase productivity rates in avian cell lines, notably in duck cell lines. The ability of a cell line to achieve high volumetric productivities is believed to result from a combination of characteristics. First, efficient transcription of the antibody genes is achieved by using an appropriately designed expression vector. Secondly, one requires a cell line capable of efficiently translating antibody mRNAs, assembling and modifying the antibody at high rates with minimal accumulation of incorrectly processed polypeptides, and having sufficient secretory capacity for secreting the resulting assembled antibody.

The expression vector systems most frequently used to increase specific production rate by improving transcription of mAbs are the Glutamine Synthetase (GS) Gene Expression System (Birch J.R. and Racher A. J., Advanced Drug Delivery Reviews 2006; 58:671- 685) and those based on dihydrofolate reductase (DHFR) genes (Page MJ, and Sydenham MA, Biotechnology 1991 ; 9:64-68).

The GS system (Lonza Biologies) is based on CHO and NS0 cells and makes use of the metabolic pathway of glutamate and ammonium to glutamine for the selection of recombinant cells. CHO is a cell line expressing endogenous GS and the presence of a GS selective inhibitor, such as Methionine sulphoximine (MSX), in a culture media lacking glutamine will allow survival only of those cell clones having integrated the gene construct containing the GS gene (e.g., US 5,827,739). On the other hand, NSO cells do not present an endogenous GS activity, and are thus auxotroph for glutamine, the absence of glutamine in the culture media will allow only those NSO cells having integrated the GS sequence to survive (e.g. W09106657).

In early expression systems, transcription was generally improved by gene amplification (e.g., WO8704462, US 5,827,739; US 5,770,359; US 5,122,464 and W09106657). Currently, the GS system does not rely upon amplification to achieve high productivities. Instead, the system relies upon insertion of the antibody construct into a transcriptionally active region. Linkage of the antibody construct to the selectable marker gene results in the overproduction of antibody as both genes are integrated into a transcriptionally active locus (Birch J.R. et al., Use of the glutamine synthetase (GS) expression system for the rapid development of highly productive mammalian cell processes, J. Knablein, (Ed.), Modern Biopharmaceuticals, WILEY- VCH Verlag GmbH and Co KGaA, Weinheim, 2005, 809-832; De la Cruz et al., Mol Biotechnol 2006; 34:179-190). The glutamine synthetase (GS) sequence used for both CHO and NSO cells is that of Chinese hamster GS (hamster-GS) which was firstly disclosed by Hayward et al. (Nucleic Acid Res. 1986; 14(2): 999-1008).

WO 97/08307 describes the use of human glutamine synthetase (GS) gene for the amplification of the human erythropoietin (EPO) gene in a quail fibrosarcoma cell line. The amplification using GS gene was used as an alternative to the DHFR system due to the fact that no mutant line of QT defective on the DHFR gene was available. To our knowledge there is no document describing the use of a GS sequence in duck cell lines. The chicken glutamine synthetase gene sequence (chicken-GS) was disclosed by Pu H. and Young A. (Gene 1989; 81 : 169-175) but to our knowledge has never been used up to date to increase transcriptional expression of a recombinant protein.

Since the demand for monoclonal antibodies seems set to increase for the foreseeable future, there is a need for improving the inherent productivity of cell lines. In particular, there is an interest in further developing production systems alternative to mammalian cell lines which can provide certain advantages, such as avian cell lines and notably, duck cell lines. Accordingly, an object of the present invention is to provide a method for increasing the expression yield of a protein of interest (e.g., an antibody) in an avian cell line, e.g., in a duck cell line. In particular, a method for the selection of duck cell clones having an increased expression of a protein of interest is provided.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to an isolated DNA sequence characterised in that

i) comprises the duck glutamine synthetase gene sequence defined in SEQ ID N° 1 , SEQ ID N° 2, SEQ ID N° 3 or SEQ ID N° 4; or

ii) encodes a duck glutamine synthetase protein which comprises the amino acid sequence of SEQ ID N° 5.

The isolated nucleic acid sequence of the first aspect of the invention preferably comprises a sequence selected from the group consisting of SEQ ID N° 1 , SEQ ID N° 2, SEQ ID N° 3 and SEQ ID N° 4, wherein said sequence encodes a protein having a glutamine synthetase activity.

In second aspect, the invention relates to a duck glutamine synthetase protein comprising the amino acid sequence of SEQ ID N° 5.

According to a third aspect of the present invention, there is provided a recombinant DNA vector comprising a recombinant DNA sequence according to the first aspect of the invention.

The present invention also provides according to a fourth aspect of the invention a host cell comprising a vector according to the third aspect of the invention.

In a fifth aspect, the present invention relates to a method for selecting avian, cell clones having integrated in their genome a construct comprising a nucleic acid sequence encoding a nucleic acid sequence encoding the glutamine synthetase (GS) as defined in the first aspect of the invention , wherein said cell clones have the ability to survive in a medium lacking glutamine or in a medium in which the amount of glutamine is progressively depleted, said method comprising the following steps:

a) transfecting an avian cell with a vector comprising a nucleic acid as defined in the first aspect of the invention;

b) culturing the transfected cells obtained in step a) in a selection medium which lacks glutamine or in which the amount of glutamine is progressively depleted.

c) selecting those cell clones having survived to the culturing in the selection medium of step b).

A step of selecting transfected cells obtained in a) and having integrated said vector in their genome may be performed between steps a) and b).

BRIEF DESCRIPTION OF THE FIGURES

Fig.1.- Agarose gel electrophoresis of endogenous duck glutamine synthetase (GS) cDNA obtained by RT-PCR. On the left side of the gel, under "GS" are shown the RT- PCR products specific to the glutamine synthetase (GS) enzyme. On the right side, under "GAPDH" are shown RT-PCR products specific to the glyceraldehyde-3- phosphate dehydrogenase (GAPDH) enzyme, used as loading control. Lanes 1 and 2 correspond to RT-PCR products obtained from Muscovy duck EBx and EB66 cells, respectively. Lanes marked "(+)" correspond to mRNA samples subjected to reverse transcription and those marked "(-)" to those not subjected to reverse transcription, i.e.,"no-RT".

Fig. 2.- Sequence alignment of the two cDNA allelic sequences (SEQ ID N° 3 and SEQ ID N° 4) of the GS enzyme obtained from a Muscovy cell line. The nucleotide differences are highlighted in bold letters.

Fig. 3.- Sequence alignment of the two cDNA allelic sequences of the GS enzyme obtained from a EB66 cell line (SEQ ID N° 1 and SEQ ID N° 2). The nucleotide differences are highlighted in bold letters.

Fig. 4.- Sequence alignment of SEQ ID N° 1 , SEQ ID N° 2, SEQ ID N° 3 and SEQ ID N° 4, showing that there is a 98,8% homology between all sequences. Fig.5.- MSX selection on adherent EB66 cells transfected with plasmids encoding GS enzyme from different species, i.e., duck (GS-duck), chicken (GS-chicken), hamster (GS-CHO) and hamster optimized for expression in duck (GS-CHO duck opti). On the top, Fig 5A and Fig 5C show microscopy photographs of transfected cells at day 13 and 8 post transfection, respectively. On the bottom, Fig 5B and Fig 5D show GIEMSA staining results at day 14 post transfection. A MSX selection pressure of 30 μΜ was applied to the cells in Fig.5A & 5B and of 40 μΜ to those shown in Fig. 5C& 5D. An estimation of cell confluence is mentioned on the top left of each picture.

Fig.6.- GS plasmid integration into the host genome was analyzed by PCR analysis. The agarose gel of Fig.6A shows the results obtained when GS expression is under the control of a weak promoter (SV40 early promoter) and Fig.6B shows the results obtained using a strong promoter (EF1 -HTLV). Lanes 2 to 1 1 of Fig.6A and lanes 2 to 13 of Fig.6B show the PCR-products amplified from surviving clones 14 days post- transfection and in lane 1 are shown the results obtained from a pool of unpicked clones. Endogenous GAPDH was used as loading control. ΈΒ66 WT" and "plasmid" lanes correspond to PCR products from untransfected cells (negative control for plasmid integration) and plasmid only (positive control), respectively.

Fig. 7.- A schematic representation of the plasmids used for the selection with MSX is shown. Fig. 7A shows an example of a basic selection plasmid containing a cassette encoding the glutamine synthetase (GS) enzyme (e.g. containing SEQ ID N° 1) under the control of a promoter (i.e. SV40 early promoter), a kanamycin resistance cassette (KanR), and a col E1 origin for the plasmid replication in prokaryotic cells. A double arrow marks the site of plasmid linearization. Fig. 7B shows an antibody expression plasmid encoding for glutamine synthetase (GS) enzyme under the control of a promoter (i.e., SV40 early promoter), and genes encoding for human antibody light chain and heavy chain cloned in tandem. This plasmid also contains a pUC origin and a kan/neo resistance cassette that encodes the neomycin phosphotransferase protein and provides a resistance to Kanamycin in prokaryotic cells and to Neomycin in eukaryotic cells (nptll resistance cassette). A double arrow marks the site of plasmid linearization. Fig. 7C shows another antibody expression plasmid encoding for glutamine synthetase (GS) enzyme under the control of a promoter (i.e., SV40 early promoter), and genes encoding for human antibody light chain and heavy chain cloned in tandem. It is to be noted that the orientation of the GS cassette is opposite to that in Fig. 7B. This plasmid also contains a pUC origin and a ampicillin resistance cassette for selection in prokaryotic cells. A double arrow marks the site of plasmid linearization.

Fig. 8.- Alignment of duck (SEQ ID N° 5), hamster (SEQ ID N° 8) and chicken (SEQ ID N° 10 ) glutamine synthetase (GS) enzyme protein sequences . The differing amino acids are highlighted in bold letters.

Fig. 9.- Alignment of duck (SEQ ID N° 5) and chicken (SEQ ID N° 10) Glutamine Synthetase (GS) enzyme protein sequences . It was found that chicken and duck protein sequences did only differ in four amino acids which are highlighted in bold letters

Fig. 10.- Monoclonal antibody production of clones selected with MSX. Supernatants of clones selected with 30 μΜ of MSX were collected and analysed by ELISA for antibody production in 96-well and 24-well plates. On the left panel are represented the antibody production levels of clones transfected with plasmids encoding for monoclonal antibody (lgG1) + glutamine synthetase under the control of weak promoter (SV40 early promoter), and on the right panel those transfected with plasmids encoding for monoclonal antibody (lgG1) + glutamine synthetase under the control of strong promoter (promoter EF1 -HTLV).

Fig. 11.- Monoclonal antibody production of clones selected with MSX. Supernatants of clones selected with either 30 or 35 μΜ of MSX were collected and analysed by ELISA for antibody production in 24-well plates. On the left panel are represented the antibody production of clones selected with 30 μΜ of MSX selection pressure, and on the right panel those selected with 35 μΜ of MSX selection pressure.

Fig. 12.- Monoclonal antibody production in 24 well-plates of 200 EB66 cell clones selected for survival to a MSX selection pressure of 35 μΜ. Fig. 13.- Monoclonal antibody production in 50ml_ bioreactors (batch culture) of 24 EB66 cell clones selected for survival to a MSX selection pressure of 35 μΜ.

Fig. 14.- Nucleotide sequence comparison performed between the nucleotide sequence encoding for turkey glutamine synthetase (sequence XM_003208553.1 from NCBI database; (SEQ ID N° 20, indicated as (V)) and the four allelic variants of GS- duck, i.e., SEQ ID N° 1 to SEQ ID N° 4 (sequences (I), (IV) (II) and (III) respectively).

DETAILED DESCRIPTION

The inventors of the present invention wanted to adapt to avian cells, in particular to duck cells, the GS selection process commonly used for increasing expression of heterologous proteins in mammalian cells. For doing so, sequences coding for the GS enzyme belonging to different species were used as selectable dominant marker in the construction of a selection or expression vector, in particular, GS enzyme sequences from hamster (CHO), chicken and duck.

Surprisingly, no or very poor functionality was observed when the chicken-GS sequence, the hamster-GS sequence or the hamster-GS sequence optimized for the expression in duck (hamster-GS opti), were used for the selection of antibody producing clones of a duck cell line, in particular of the EB66 cell line. By contrast, the duck-GS sequence was shown to be functional for the selection of clones having stably integrated the transgene and thus, promising results were obtained.

Therefore, in a first aspect, the invention relates to an isolated DNA sequence characterised in that it i) comprises the duck glutamine synthetase gene sequence defined in SEQ ID N° 1 , SEQ ID N° 2, SEQ ID N° 3 or SEQ ID N° 4; or ii) encodes a duck glutamine synthetase protein which comprises the amino acid sequence of SEQ ID N° 5.

The inventors of the present invention isolated the duck GS sequence from EB66 and Muscovy cells by RT-PCR, as shown in Example 2. EB66 is a duck embryonic-derived cell line established as described in Example 3 of WO 2008/129058 from Pekin duck (Anas platyrhynchos). Two different nucleotide sequences were obtained which are shown in SEQ ID N° 1 and SEQ ID N° 2. However, both sequences translate for the same amino acid sequence (SEQ ID N° 5). Accordingly, in a second aspect, the invention relates to duck glutamine synthetase protein comprising the amino acid sequence of SEQ ID N° 5.

The sequence of the GS gene of a duck from a different genus was also obtained, in particular that of Muscovy EBx cells, duck embryonic-derived cell line established as described in Example 6 of WO 2008/129058 from Muscovy (Cairina moschata), was determined and two allelic forms of the gene were also obtained: SEQ ID N° 3 and SEQ ID N° 4. Both sequences translate for the same amino acid sequence as the two allelic forms obtained from Pekin duck, i.e., SEQ ID N° 5. These duck sequences (i.e., SEQ ID N° 1 , SEQ ID N° 2, SEQ ID N° 3, SEQ ID N° 4 and SEQ ID N° 5) are generically mentioned throughout the specification as the "duck -GS sequence".

Glutamine synthetase (GS) is a universal housekeeping enzyme responsible for the synthesis of glutamine from glutamate and ammonia using the hydrolysis of ATP to ADP and phosphate to drive the reaction. It is involved in the integration of nitrogen metabolism with energy metabolism via the TCA cycle, glutamine being the major respiratory fuel for a wide variety, possible the majority, of cell types. A variety of regulatory signals affect GS levels within cells, for instance glucocorticoid steroids and cAMP, and glutamine in a culture medium appears to regulate GS levels post- translationally via ADP ribosylation. GS from all sources is subject to inhibition by a variety of inhibitors, for example methionine sulphoximine (Msx). A detailed review on Glutamine Synthetase (GS) enzyme is provided in D. Eisenberg et al Biochimica et Biophysica Acta 2000; 1477, 122-145.

Conveniently, the recombinant DNA sequence of this aspect of the invention is cDNA, preferably derived by reverse transcription. However, the recombinant DNA sequence may alternatively or additionally comprise a fragment of genomic DNA.

It will be appreciated that, in accordance with the present invention, a recombinant DNA sequence of the first aspect or a fragment thereof, may be used as a hybridization probe or obtaining GS coding sequences from other species. Moreover, the recombinant DNA sequences of the first aspect of the present invention may be used in medical or diagnostic methods, such as for detecting disease states in which the level of GS in a subject is altered.

However, it is envisaged that the main use of the recombinant DNA sequences of the first aspect of the present invention will be in co-amplification or dominant selectable marker systems employed in recombinant DNA technology. Therefore according to a third aspect of the present invention, there is provided a recombinant DNA vector comprising a recombinant DNA sequence according to the first aspect of the invention.

Vectors

Preferably, the vector is an expression vector capable, in a transformant host cell, of expressing the GS-encoding recombinant DNA sequence, also referred as selection vector. The vector may further comprise a recombinant DNA sequence which encodes the complete amino acid sequence of a protein of interest other than GS (which can be designated as "heterologous protein"). In the preferred case, the vector will also be capable, in the transformant host cell, of expressing the desired protein-encoding recombinant DNA sequence.

Expression vectors contain the necessary elements for the transcription and translation of at least one coding sequence of interest. Methods which are well known to and practiced by those skilled in the art can be used to construct expression vectors containing sequences encoding the proteins and polypeptides of interest, as well as the appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described for example in Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.) and in Ausubel et al. (1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.).

Suitable expression vectors comprise at least one expression cassette that comprises nucleic acid sequence, preferably DNA sequence, encoding heterologous protein(s) of interest that are operably linked to control element and regulatory sequences. The expression cassette comprises at least a nucleic acid sequence, preferably DNA sequence, encoding heterologous protein(s) of interest that are operably linked to a promoter sequence. "Promoter" as used herein refers to a nucleic acid sequence that regulates expression of a gene. The term "operably linked" as used herein refers to the configuration of coding and control sequences, for example, within an expression vector, so as to achieve transcription and/or expression of the coding sequence. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence and regulating in which tissues, at what developmental time points, or in response to which signals, and the like, a gene is expressed. A coding sequence is operably linked to or under the control of transcriptional regulatory regions in a cell when DNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA that can be translated into the encoded protein. The control sequences need not to be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated or transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence. Such intervening sequences include but are not limited to enhancer sequences which are not transcribed or are not bound by polymerase. The term "expressed" or "expression" as used herein refers to the transcription of a nucleotide sequence into an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of a gene coding sequence and/or to the translation from an RNA nucleic acid molecule into a protein or polypeptide.

As intended herein, a strong promoter is a promoter presenting a strength that is equal or higher than the strength of the EF1 -HTLV promoter. EF1-HTLV promoter is a composite promoter comprising the Elongation Factor-l a (EF-1 a) core promoter (Kim et al. 1990. Gene. 91 (2):217-23.) and the R segment and part of the U5 sequence (R-U5') of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat (Takebe et al. 1988. Mol Cell Biol. 8(1):466-72). The EF-1 a promoter exhibits a strong activity and yields long lasting expression of a transgene in vivo. The R-U5' has been coupled to the EF-1 a core promoter to enhance stability of RNA. Strength of the promoters are compared as follows: a vector for producing a reporter protein (such as the CAT protein, Chloramphenicol Acetyl Transferase) under the control of either the promoter to be tested or the EF1 -HTLV promoter is produced and used to transform cells from a cell line. Cells are cultured such as to express proteins. Total proteins are isolated and the ratio (reporter protein (by weight)) / (total protein (by weight)) is calculated. Said promoter has a strength that is equal or higher than the strength of the EF1-HTLV promoter in said cell line if the ratio obtained for said promoter is higher than the ratio obtained for the EF1-HTLV promoter. In addition to EF1-HTLV, examples of strong promoters include RSV (Rous Sarcoma Virus) and CMV (Cytomegalovirus) promoters.

It should be recognized, however, that the choice of a suitable expression vector and the combination of functional elements therein depends upon multiple factors including for example the type of protein to be expressed. Representative examples of expression vectors include, for example, bacterial plasmid vectors including expression and cloning vectors such as, but not limited to, pBR322, animal viral vectors such as, but not limited to, modified avian adenovirus, measles virus, influenza virus, polio virus, pox virus, retrovirus, and the like and vectors derived from bacteriophage nucleic acid, for example, plasmids and cosmids, artificial chromosomes, such as but not limited to, Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs), and synthetic oligonucleotides like chemically synthesized DNA or RNA. Accordingly, the term "nucleic acid vector" or "vector" as used herein refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule, or any other nucleic acid molecule that can be transfected or transformed into cells and replicate independently of, or within, the host cell genome. A nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together. The nucleic acid molecule can be RNA or DNA. Preferably the nucleic acid molecule is DNA. Certain vectors are capable of autonomous replication in a host cell into which they are introduced, for example, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors. Other vectors, such as non-episomal mammalian vectors, are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Stable expression is generally preferred to transient expression because it typically achieves more reproducible results and also is more amenable to large scale production. Accordingly, for the methods of this invention, the expression vectors of the invention are stably incorporated into the chromosomal DNA of the host cell.

The expression cassettes for use in protein expression systems are designed to contain at least one DNA sequence encoding a recombinant protein of interest operably linked to a promoter sequence, and optionally control element(s) or regulatory sequence(s). Control element or regulatory sequences are necessary or required for proper transcription and regulation of gene expression. These sequences are preferably selected in the group consisting of transcriptional initiation and termination sequences, enhancer, intron, origin of replication sites, polyadenylation sequences, peptide signal and chromatin insulator elements. Regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Enhancer sequences may be located upstream or downstream of promoter region sequences for optimizing gene expression. An "enhancer" is a nucleotide sequence that acts to potentiate the transcription of genes independently of the identity of the gene, the position of the sequence in relation to the gene, or the orientation of the sequence. The vectors of the present invention optionally include enhancers. Examples of enhancers are CMV immediate early enhancer and SV40 early enhancer.

An intronic sequence may be located upstream or downstream of sequence encoding a recombinant protein of interest for optimizing gene expression. According to a preferred embodiment, the intronic sequence is located between the promoter sequence and the sequence encoding recombinant protein of interest. The intronic sequence of the invention is preferably selected in the group consisting of a chimeric intron composed of 5'-donor site from the first intron of human beta-globin gene and the branch and 3'-acceptor site from the intron of an immunoglobulin gene heavy chain variable region.

The promoter sequences of the invention are preferably selected from genes of mammalian or avian origin or from mammalian or avian viral genes. In a preferred embodiment, the promoter sequence is from viral origin, and is selected in the group consisting of human or murine cytomegalovirus (CMV) promoter, avian sarcoma virus (ASV)xLN promoter, early and late promoters from simian virus 40 (SV40) (Fiers et al. (1973) Nature 273:1 13), Rous Sarcoma Virus (RSV) promoter, Herpes Simplex virus (HSV) thymidine kinase promoter, respiratory syncytial virus promoter, MDOT promoter, polyoma virus promoter, adenovirus 2 promoter, bovine papilloma virus promoter, adenovirus major late promoter and functional portions of each of these promoters. According to another embodiment, the promoter sequence is selected in the group consisting of murine phospho-glycerate kinase promoter, murine leukaemia virus (MLV), mouse mammary tumor virus (MMTV), EiF4alpha promoter, chimeric EF1 alpha/HTLV promoter, chimeric CAG promoter (composite promoter that combines human CMV immediate early enhancer and a modified chicken beta-actin promoter and first intron) and avian gene promoters and functional portions of each of these promoters. Among avian gene promoters, the promoter is preferably selected among chicken promoters such as beta-actin promoter, oviduct-specific promoter, ovomucoid promoter, ovalbumin promoter, conalbumin promoter, ovomucin promoter, ovotransferrin promoter, lysozyme promoter, ENS1 gene promoter and functional portions of each of these promoters.

According to another embodiment, promoters may be selected among regulated promoters such promoters that confer inducibility by particular compounds or molecules, e. g., the glucocorticoid response element (GRE) of mouse mammary tumor virus (MMTV) is induced by glucocorticoids (Chandler et al. (1983) Cell 33: 489- 499). Also, tissue-specific promoters or regulatory elements can be used (Swift et al. (1984) Cell, 38: 639-646), if necessary or desired. Non-limiting examples of other promoters which may be useful in the present invention include, without limitation, Pol III promoters (for example, type 1 , type 2 and type 3 Pol III promoters) such as HI promoters, U6 promoters, tRNA promoters, RNase MPR promoters and functional portions of each of these promoters. Typically, functional terminator sequences are selected for use in the present invention in accordance with the promoter that is employed.

In a particular embodiment, the GS-encoding recombinant DNA sequence is under the control a strong promoter, preferably EF1-HTLV. In another particular embodiment, the GS-encoding recombinant DNA sequence is under the control of a weak promoter, preferably SV40 early promoter. In a particularly preferred embodiment, the GS expression vectors of the invention contain the GS gene downstream of a weak promoter such as SV40 early promoter. The use of a weak promoter to regulate GS gene expression should reduce promoter interference thus increasing expression of the protein of interest other than GS, which is preferably under the control of a strong promoter (e.g. human cytomegalovirus or EF1-HTLV).

The expression vector may further comprise at least one expression cassette comprising at least a nucleic acid sequence, preferably a DNA sequence, encoding a selectable marker other than said GS gene operably linked to a promoter sequence capable of effecting expression of said selectable marker in the cell. Such selectable marker may confer resistance to the host cell harbouring the vector to allow their selection in an appropriate selection medium.

In a preferred embodiment, anti-metabolite resistance is used as the basis of selection for the following non-limiting examples of marker genes: DHFR, which confers resistance to methotrexate (Wigler et al. (1980) Proc. Natl. Acad. Sci. USA, 77:357; and O'Hare et al. (Ί 981) Proc. Natl. Acad. Sci. USA, 78:1527); GPT, which confers resistance to mycophenolic acid (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA, 78:2072); neomycin (NEO), which confers resistance to the amino-glycoside G418 (Wu and Wu (1991) Biotherapy, 3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol., 32:573-596; Mulligan (1993) Science, 260: 926- 932; Anderson (1993) Ann. Rev. Biochem., 62:191-21); and Hygromycin B, which confers resistance to hygromycin (Santerre et al. (1984) Gene, 30:147) and puromycine. In a most preferred embodiment, antibiotic resistance genes are used as the basis of selection. According to a preferred embodiment the selectable marker of the invention is neomycin resistance gene. Preferably, the nucleic acid sequence encoding NEO is the neomycin/kanamycin resistance gene of TN5. According to another preferred embodiment the selectable marker of the invention is kanamycin resistance gene. According to another preferred embodiment the selectable marker of the invention is puromycin resistance gene.

Alternatively, such selection systems require that host cells are previously genetically modified to display the appropriate genotype (i.e TK-, HGPRT-, ART-, DHFR-, GPT-, etc.). A number of selection systems can be used, including but not limited to, the Herpes Simplex Virus thymidine kinase (HSV TK), (Wigler et al. (1977) Cell 1 1 :223), hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Szybalska & Szybalski (1992) Proc. Natl. Acad. Sci. USA, 48:202), and adenine phosphor-ribosyl transferase (Lowy et al. 1980 Cell 22: 817) genes, which can be employed in tk-, hgprt-, or art-cells (APRT) respectively. Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant cell clones, and such methods are described, for example, in Ausubel et al. (1993) and Kriegle (1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; in Chapters 12 and 13).

The expression vector of the invention may further comprise chromatin insulator elements. Chromatin insulator elements of the invention include boundary elements (BEs), matrix attachment regions (MARs), locus control regions (LCRs), and universal chromatin opening elements (UCOEs). Boundary elements ("BEs"), or insulator elements, define boundaries in chromatin in many cases (Bell & Felsenfeld (1999) Curr Opin Genet Dev 9:191-198) and may play a role in defining a transcriptional domain in vivo. BEs lack intrinsic promoter/enhancer activity, but rather are thought to protect genes from the transcriptional influence of regulatory elements in the surrounding chromatin. The enhancer-block assay is commonly used to identify insulator elements. In this assay, the chromatin element is placed between an enhancer and a promoter, and enhancer-activated transcription is measured. Boundary elements have been shown to be able to protect stably transfected reporter genes against position effects in Drosophila, yeast and in mammalian cells (Walters et al. (1999) Mol Cell Biol 19:3714-3726). Matrix Attachment Regions ("MARs"; also known as Scaffold Attachment Regions or Scaffold/Matrix Attachment Regions ("S/MARs")) are DNA sequences that bind isolated nuclear scaffolds or nuclear matrices in vitro with high affinity (Hart and Laemmli (1998) Curr Opin. Genet Dev 8:519-525). As such, they may define boundaries of independent chromatin domains, such that only the encompassing cis-regulatory elements control the expression of the genes within the domain. MAR elements can enhance expression of heterologous genes in cell culture lines (Kalos and Fournier (1995) Mol Cell Biol 15:198-207)). Locus control regions ("LCRs") are cis-regulatory elements required for the initial chromatin activation of a locus and subsequent gene transcription in their native locations (reviewed in Grosveld 1999, Curr Opin Genet Dev 9: 152-157). The most extensively characterized LCR is that of the globin locus. Ubiquitous chromatin opening elements ("UCOEs", also known as "ubiquitously acting chromatin opening elements") have recently been reported (See WO00/05393). According to a preferred embodiment, the chromatin insulator element of the invention is a MAR element. Preferably, the MAR element is selected among chicken lysozyme 5'MAR elements as described in WO 02/074969 or human MAR elements as described in WO 2005/040377.

As will be appreciated by those skilled in the art, the selection of the appropriate vector, e. g., plasmid, components for proper transcription, expression (promoter, control sequences & regulatory sequence), and isolation of proteins produced in cell expression systems is known and routinely determined and practiced by those having skill in the art.

The expression vectors described herein can be introduced into the host cells by a variety of methods. In particular, standard transfection procedures, well-known from the man skilled in the art may be carried out, such as calcium phosphate precipitation, DEAE-Dextran mediated transfection, electroporation, nucleofection (AMAXA Gmbh, GE), liposome-mediated transfection (using lipofectin® or lipofectamine® technology for example) or microinjection.

Host cell

The present invention also provides according to a fourth aspect of the invention a host cell transformed, transfected, transduced, or the like with a vector according to the second aspect of the invention. The host cell of the invention is transfected with at least one vector comprising at least one GS selection cassette comprising a GS nucleic acid sequence as described in the first aspect of the invention, in the examples such a vector is referred as selection vector. Preferably, said vector is an expression vector also comprising at least one expression cassette comprising a nucleic acid sequence, preferably a DNA sequence, encoding a recombinant protein or polypeptide of interest operably linked to a promoter sequence capable of effecting expression of said protein in the cell. Said selection and expression vectors might further comprise at least one expression cassette comprising at least a DNA sequence encoding a selectable marker operably linked to a promoter sequence capable of effecting expression of said selectable marker in the host cell.

The term "host cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be any eukaryote, such as a mammalian, avian, insect, plant, or fungal cell. In a preferred aspect, the host cell is an avian cell.

The term "avian, "bird", "aves" or "ava" as used herein is intended to have the same meaning, and will be used indistinctly. "Birds" refer to any species, subspecies or race of organism of the taxonomic class « ava ». In a preferred embodiment, "birds" refer to any animal of the taxonomix order:

- "Anseriformes" (i.e duck, goose, swan and allies). The order Anseriformes contains about 150 species of birds in three families: the Anhimidae (the screamers), Anseranatidae (the Magpie-goose), and the Anatidae, which includes over 140 species of waterfowl, among them the ducks, geese, and swans. All species in the order are highly adapted for an aquatic existence at the water surface. All are web- footed for efficient swimming (although some have subsequently become mainly terrestrial).

- "Galliformes" (i.e chicken, quails, turkey, pheasant and allies). The Galliformes is an order of birds containing the chicken, turkeys, quails and pheasants. About 256 species are found worldwide.

- "Columbiformes" (i.e Pigeon and allies). The bird order Columbiformes includes the very widespread doves and pigeons.

Preferably, said avian cells are able to or proliferate indefinitely. The term "indefinitely" refers to cell lines that proliferate in culture beyond the Hayflick limit. Particularly preferred, these cells are avian embryonic derived stem cells, as described in WO03076601 or WO2008142124.

In a preferred embodiment the host cell of the invention is a duck cell. According to the invention, a duck cell is defined as a cell providing from an animal belonging to the Anatidae family. Cells belonging to the Cairina or Anas genus being particularly preferred. Even more preferably, said avian cells belong to the Cairina moschata species, also known as Muscovy duck or to the Anas platyrhynchos species, preferably from the Pekin duck breed.

Advantageously, said duck cells are able to proliferate indefinitely. Suitably, said avian cells are able to proliferate indefinitely in suspension in the absence of animal serum and/or exogenous growth factors. In a preferred embodiment, said duck cell is an embryonic derived cell line established as described for example in WO2008142124 and WO2008129058. In short, the process of establishment of said embryonic derived duck cell lines, so-called duck EBx cells, comprises two steps:

a) isolation, culture and expansion of embryonic stem (ES) cells from duck that do not contain complete endogenous proviral sequences, or a fragment thereof, susceptible to produce replication competent endogenous retroviral particles, more specifically EAV and/or ALV-E proviral sequences or a fragment thereof, in a complete culture medium containing all the factors allowing their growth and in presence of a feeder layer and supplemented with animal serum; optionally, said complete culture medium may comprise additives, such as additional amino acids (i.e glutamine, ...), sodium pyruvate, beta-mercaptoethanol, protein hydrolyzate of non-animal origin (i.e yeastolate, plant hydrolyzates, ...);

b) passage by modifying the culture medium so as to obtain a total withdrawal of said factors, said feeder layer and said serum, and optionally said additives, and further obtaining adherent or suspension duck cell lines, named EBx, that do not produce replication-competent endogenous retrovirus particles, capable of proliferating over a long period of time, in a basal medium in the absence of exogenous growth factors, feeder layer and animal serum.

The modification of the culture medium of step b) of the process of establishment said cell lines, so as to obtain progressive or total withdrawal of growth factors, serum and feeder layer can be made simultaneously, successively or separately. The sequence of the weaning of the culture medium may be chosen among: feeder layer / serum / growth factors;

- feeder layer / growth factors / serum;

serum / growth factors / feeder layer;

serum / feeder layer / growth factors;

- growth factors / serum / feeder layer;

growth factors / feeder layer / serum.

In a preferred embodiment, the sequence of the weaning is growth factors / feeder layer / serum.

Particularly preferred are Pekin duck embryonic-derived EBx cells, such as the cell lines named EB66, EB24, EB26 and Muscovy duck embryonic-derived EBx cell lines. The establishment of EB66, EB24, EB26 and Muscovy duck EBx cell lines is described in examples 3, 4, 5 and 6, respectively, of WO 2008/129058. More preferably, said duck cell line is EB66 cell line.

Other examples of duck cell line are the duck cell lines described in

EP1685243, WO 2007/077256, WO 2009/004016 and DEC99 (Ivanov et al. Experimental Pathology and Parasitology, 4/2000 Bulgarian Academy of Sciences).

The vectors according to the third aspect of the present invention may be used in the co-amplification of non-selected genes. Therefore according to another aspect of the present invention, there is provided a method for co-expressing a recombinant DNA sequence which encodes the complete amino acid sequence of a desired protein other than GS which comprises: either co-transforming a host cell with a vector according to the invention which does not contain a sequence encoding the desired protein, and a second vector comprising said desired protein encoding recombinant DNA sequence; or transforming the host cell with a vector according to the invention which includes both a nucleic acid coding for the GS and a recombinant DNA encoding the desired protein. Cell clone selection method

In a fifth aspect, the invention relates to a method for selecting avian cell clones comprising the following steps: a) transfecting an avian cell with a vector comprising a nucleic acid sequence encoding a protein with a glutamine synthetase activity as defined in a first and second aspects of the invention;

b) culturing the transfected cells obtained in step a) in a selection medium which lacks glutamine or in which the amount of glutamine is progressively depleted; and

c) selecting those cell clones having survived to culturing in the selection medium of step b).

Preferably, the invention relates to a method for the selection of avian cell clones having an increased expression of a heterologous protein of interest. Accordingly, in a preferred embodiment, the present invention relates to a method for selecting an avian, notably duck, cell clones having integrated in their genome a nucleic acid encoding a heterologous protein together with a nucleic acid encoding the glutamine synthetase (GS) sequence as defined in the invention wherein said cell clones have the ability to survive in a medium lacking glutamine or in which the amount of glutamine is progressively depleted, said method comprising the following steps:

a) transfecting an avian cell with a vector comprising both the GS sequence as defined in the first aspect of the invention and a nucleic acid encoding an heterologous protein;

b) culturing the transfected cells in a selection medium which lacks glutamine or in which the amount of glutamine is progressively depleted;

Suitably, the cell clones selected in step c) are those having survived to culturing in the selection medium of step b) for at least 5 days, preferably for at least 7 days. In a preferred embodiment, the cell clones retrieved in step c) are those cell clones maintained in culture for 10 days in the selection medium of step b).

Preferably, this avian cell is a duck cell. The terms avian cell and duck cell have the meaning above defined.

The term "heterologous protein" refers to a protein of interest "other than GS". The term protein includes proteins, protein fragments, protein analogues, polypeptides, oligopeptides, peptides and the like. The term "heterologous" relates preferably to the fact that is not naturally part of the host cell genome. In a specific embodiment of the invention, the term "heterologous protein" may represent a protein that is naturally present in the host cell genome but is other than GS.

An heterologous protein of the invention can include, but is not limited to, a pharmaceutically active protein e.g. growth factors, growth regulators, antibodies, antigens, their derivatives useful for immunization or vaccination and the like, interleukins, insulin, erythropoietin, G-CSF, GM-CSF, hPGCSF, M-CSF, interferons (interferon-alpha, interferon-beta, interferon-gamma), blood clotting factors (e.g. Factor VIII; Factor IX; tPA) or combinations thereof. Preferably, the heterologous protein of the invention is an antibody. The term "antibody" as used herein refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof. The term "antibody" refers to a homogeneous molecular entity, or a mixture such as a polyclonal serum product made up of a plurality of different molecular entities, and broadly encompasses naturally-occurring forms of antibodies (for example, IgD, IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies. The term "antibody" also refers to fragments and derivatives of all of the foregoing, and may further comprises any modified or derivatised variants thereof that retains the ability to specifically bind an epitope.

Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody. A monoclonal antibody is capable of selectively binding to a target antigen or epitope. Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, camelized antibodies, single chain antibodies (scFvs), Fab fragments, F(ab')2 fragments, disulfidelinked Fvs (sdFv) fragments, anti-idiotypic (anti-Id) antibodies, intra-bodies, synthetic antibodies, and epitope-binding fragments of any of the above. The term "antibody" also refers to fusion protein that includes a region equivalent to the Fc region of an immunoglobulin. In a particular embodiment said antibody is a human IgG antibody, suitably an lgG1 antibody. Preferably, said antibody is fully human. A "fully human antibody" is an antibody containing exclusively human sequences. Thus, a fully human antibody shall not induce an immune response when administered to a human recipient. More preferably, said human antibody is a "native human antibody", in which the antibody is naturally occurring in a human, as opposed to a human antibody in which the individual heavy and light chains are isolated from humans but are assembled randomly (i.e. by using library methods such as phage display) creating all forms of natural and unnatural antibodies.

Preferred antibodies within the scope of the present invention include those comprising the amino acid sequences of the following antibodies: anti-HER2 antibodies including antibodies comprising the heavy and light chain variable regions of huMAb 4D5-8 (Carteret ai., Proc. Nati. Acad. Sci. USA, 89: 4285-4289 (1992), U.S. Patent No. 5,725,856) or Trastuzumab such as HERCEPTIN™; anti-CD20 antibodies such as chimeric anti-CD20 "C2B8" as in US Patent No. 5,736, 137 (RITUXAN®), a chimeric or humanized variant of the 2H7 antibody as in US Patent No. 5,721 , 108 or Tositumomab (BEXXAR); anti-IL-8 (St John et ai., Chest, 103: 932 (1993), and International Publication No. WO 95/23865); anti-VEGF antibodies including humanized and/or affinity matured anti-VEGF antibodies such as the humanized antiVEGF antibody huA4.6.1 AVASTIN™ (Kim et ai., Growth Factors, 7: 53- 64 (1992), International Publication No. WO 96/30046, and WO 98/45331); anti-PSCA antibodies (WOO1 I40309); anti-CD40 antibodies, including S2C6 and humanized variants thereof (W000/75348); anti-CDI la (US Patent No. 5,622,700, WO 98/23761); anti-EGFR (chimerized or humanized 225 antibody as in WO 96/40210); anti-CD3 antibodies such as OKT3 (US Patent No. 4,515,893); anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT) and (ZENAPAX) (See US Patent No. 5,693,762); anti- CD4 antibodies such as the cM-7412 antibody (Choy et ai. Arthritis Rheum 39(1): 52- 56 (1996)); antiCD52 antibodies such as CAM PATH- 1 H (Riechmann et ai., Nature 332: 323-337 5 (1988); anti-carcinoembryonic antigen (CEA) antibodies such as hMN- 14 (Sharkey et ai., Cancer Res. 55 (23Suppl): 5935s-5945s (1995); anti-EpCAM antibodies such asl7-IA (PANOREX); anti-Gpllb/llla antibodies such as abciximab or c7E3 Fab (REOPRO); anti-RSV antibodies such as MEDI-493 (SYNAGIS); anti-CMV antibodies such as PROTOVIR; anti-hepatitis antibodies such as the anti-HepB antibody OSTAVIR; anti-human renal cell carcinoma antibody such as ch-G250; anti- human17- IA antibody (3622W94); anti-human colorectal tumor antibody(A33); anti- human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA) antibodies such as SmartlDIO and the anti-HLA DR antibody Oncolym (Lym-I).

The selection medium used in step b) for the selection of cells transfected in step a) having incorporated said vector in their genome is a cell culturing medium lacking glutamine or wherein the amount of glutamine is progressively depleted. Preferably the glutamine in the medium is progressively depleted by dilution with a medium containing aspargine but lacking glutamine.

Typically, "cell culturing medium" (also called "cell culture medium") is a term that is understood by the practitioner in the art and is known to refer to a nutrient solution in which cells, preferably animal or mammalian cells, are grown and which generally provides at least one or more components from the following: an energy source (usually in the form of a carbohydrate such as glucose); all essential amino acids, and generally the twenty basic amino acids, plus cysteine; vitamins and/or other organic compounds typically required at low concentrations; lipids or free fatty acids, e.g., linoleic acid; and trace elements, e.g., inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

Cell culture medium can also be supplemented to contain a variety of optional components, such as hormones and other growth factors, e.g., insulin, transferrin, epidermal growth factor, serum, and the like ; salts, e.g., calcium, magnesium and phosphate, and buffers, e.g. HEPES; nucleosides and bases, e.g. adenosine, thymidine, hypoxanthine; and protein and tissue hydrolyzates, e.g., hydrolyzed animal protein (peptone or peptone mixtures, which can be obtained from animal byproducts, purified gelatin or plant material); antibiotics, e.g. gentamycin; and cell protective agents, e.g. Pluronic polyol (Pluronic F68). Preferred is a cell nutrition medium that is serum-free and free of products or ingredients of animal origin. Duck EBx cells, which establishment is described in WO2008129050 have been adapted to the culture in serum-free conditions.

Commercially available media can be utilized and include, for example, Ham's F12 Medium (Sigma, St. Louis, MO), Dulbecco's Modified Eagles Medium (DMEM, Sigma), VP SFM(lnVitrogen Ref 1 1681 -020, catalogue 2003), Opti Pro (InVitrogen Ref 12309-019, 20 catalogue 2003), Episerf (InVitrogen Ref 10732-022, catalogue 2003), Pro 293 S-CDM (Cambrex ref 12765Q, catalogue 2003), LC17 (Cambrex Ref BESP302Q), Pro CHO 5- CDM (Cambrex ref 12-766Q, catalogue 2003), HyQ SFM4CHO (Hyclone Ref SH30515- 02), HyQ SFM4CHO-Utility (Hyclone Ref SH30516.02), HyQ PF293 (Hyclone ref SH30356.02), HyQ PF Vero (Hyclone Ref SH30352.02), CDM4PERMAb (Hyclone Ref. 25 SH30871), or Excell media (SAFC,

Lenexa, KS), preferably, Excell EBx Gro-I medium (SAFC Biosciences - ref.14530c),

Ex cell 293 medium (SAFC Biosciences ref 14570-1000M), Ex cell 325 PF CHO Protein free medium (SAFC Biosciences ref 14335-1000M), and Ex cell VPRO medium (SAFC Biosciences ref 14560-1000M). To the foregoing exemplary media can be added the above-described supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired, and as would be known and practiced by those having in the art using routine skill.

In a particular embodiment, the method of the invention is performed in an avian, preferably a duck cell line lacking endogenous GS expression. Lack of GS expression can occur naturally or be induced by GS protein function inactivation. It is well known in the art that gene function inactivation can be obtained by acting at two main biological levels: the gene and the messenger RNA (mRNA). To inactivate a gene by Knockout (KO), the gene targeting process is performed with the introduction of mutations or alterations into the desired target genomic sequence by homologous recombination or with a random mutagenesis technique in which the insertion of the DNA element into the endogenous gene leads to transcriptional disruption (Zwambrowicz et al, 2003). In the last decade, new innovative strategies appeared for loss and function studies, notably with the rapid development of RNA interference strategies. The RNA interference (RNAi) technique was described for the first time in 1998 by Fire et al. Subsequent works demonstrated that small exogenous interference RNAs (siRNA) were able to target complementary mRNA and by this way to trigger sequence specific knockdown. This mechanism is driven by an RNA-induced silencing complex (RISC) that will selectively seek out the antisense strand (Ameres et al, 2007). The applications of RNAi are typically mediated through chemically synthesized double-stranded small interfering RNA (siRNA) or vector based short hairpin RNA (shRNA). shRNAs are synthesized in the nucleus of cells, further processed and transported to the cytoplasm, and then incorporated into the RISC for activity (Cullen, 2005).

In a preferred embodiment, the selection method of the invention is performed in an avian, preferably a duck cell line having endogenous GS expression. Duck cell lines belonging to the Cairina moschata or to the Anas platyrhynchos species have an endogenous GS expression. When a duck cell line having endogenous GS expression is used, a GS inhibitor is used (i.e. is added) in said selection culture medium.

The inhibition of glutamine synthetase (GS) has been studied extensively. Inhibitors of GS have been used to establish the kinetic mechanism of the enzyme, to characterize the regulation of GS in vivo and to assess the systemic effect that inhibition of glutamine production has on different organisms. Below is provided a non- exhaustive list of compounds, either natural or synthetic, designed primarily for the inhibition of GS referred in D. Eisenberg et al. (D. Eisenberg et al Biochimica et Biophysica Acta 2000; 1477, 122-145) which can be used in the selection method of the invention:

Methionine sulphoximine, methionine sulfone, phosphinothricin (PPT), tabtoxinine- -lactam, methionine sulfoximine phosphate, alpha-methyl methionine sulfoximine, alpha-ethyl methionine sulfoximine, ethionine sulfoximine, alpha-methyl ethionine sulfoximine, prothionine sulfoximine, alpha-methyl prothionine sulfoximine, gamma-hydroxy phosphinothricin, gamma-methyl phosphinothricin, gamma-acetoxy phosphinothricin, alpha-methyl phosphinothricin, alpha-ethyl phosphinothricin, cyclohexane phosphinothricin, cyclopentane phosphinothricin, tetrahydrofuran phosphinothricin, s-phosphonomethyl homocysteine, s-phosphonomethyl homocysteine sulfoxide, s-phosphonomethyl homocysteine sulfone, 4- (phosphonoacetyl)-L-alpha-aminobutyrate, threo-4-hydroxy- D-glutamic acid, threo-4- fluoro- D,L-glutamic acid, erythro-4-fluoro-D,L-glutamic acid, 2-amino-4- [(phosphonomethyl) hydroxyphosphinyl] butanoic acid, alanosine, 2-amino-4- phosphono butanoic acid, 2-amino-2-methyl-4-phosphono butanoic acid, 4-amino-4- phosphono butanoic acid, 4-amino-4-(hydroxymethylphosphinyl) butanoic acid, 4- amino-4-methyl-4-phosphono butanoic acid, 4-amino-4-(hydroxymethylphosphinyl)-4- methyl butanoic acid, 4-amino-4-phosphono butanamide, 2-amido-4-phosphono butanoic acid, 2-methoxycarbonyl-4-phosphono butanoic acid, methyl 4-amino-4- phosphono butanoate, oxetin, IF7 and IF17 polypeptides, (3-amino-3- carboxypropyl)(phosphonomethyl) phosphinic acid, N-hydroxy-L-2,4-diaminobutyrate, 3-amino-3-carboxypropane-sulfonamide, CBZ-, PA- and PP- amino acid derivatives, 5-hydroxylysine.

In a preferred embodiment, said GS inhibitor used in the selection medium is

Methionine sulfoximine (MSX). MSX is a mechanism-based inhibitor of glutamine synthetase (GS). The enzyme phosphorylates MSX, and the phosphorylated form of the drug binds irreversibly to the active site of the enzyme, permanently inactivating it (Cooper et al. J Biol Chem 1976;251 (21):6674-82).

Preferably, MSX is used at a concentration between about 10 μΜ to about 70 μΜ, preferably about 25 μΜ to about 60 μΜ. Advantageously, between about 30 μΜ to about 50 μΜ, preferably between about 35 μΜ to about 45 μΜ, more preferred MSX concentrations being about 35 μΜ and about 40 μΜ.

Suitably, the above MSX concentrations are used when the cell culture medium is changed in a daily basis, however culture medium could also be changed more or less frequently.

The invention also relates to the use of a nucleic acid sequence chosen in the group consisting of SEQ ID N° 1 , SEQ ID N° 2, SEQ ID N°3, SEQ ID N° 4 and nucleic acid sequences encoding SEQ ID N° 5 as a selectable marker in a cell clone selection method, preferably an avian cell clone selection. To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term "about". It is understood that, whether the term "about" is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value.

The following examples further illustrate the invention. They should not be interpreted as a limitation of the scope of the invention.

EXAMPLES

EXAMPLE 1.- Materials and Methods

Cell line and cell maintenance:

EB66 (Vivalis, France) adherent cells were routinely cultured in T-175 static flasks (Sarstedt, Germany) within the EX-CELL EBx-GRO-l medium (SAFC- Biosciences, USA) supplemented with 2.5 mM of L-glutamine (Lonza, USA) at 37°C in humidified atmosphere of 7,5% C0₂. Cells were passaged every two or three days and respectively seeded at 5 or 2,8 x 10⁶ viable cells per flask. Cell number and viable cell percentage were determinated with a malassez hemocytometer (Dutscher, France) and stained with trypan blue (sigma, USA).

Selection and expression vectors:

A) Selection vectors:

Selection plasmids were developed (e.g., Fig. 7A) comprising the following elements:

i) a kanamycin resistance cassette for selection in prokaryotic cells;

ii) a resistance cassette for selection with a GS inhibitor (e.g. MSX) encoding for glutamine synthetase enzyme under the control of SV40 early promoter or EF1- HTLV promoter. The nucleotide sequence (mRNA) encoding the GS enzyme from different animal species was used for comparison purposes:

GS from hamster (GenBank: AF150961 and SEQ ID N° 6), referred as hamster-GS or GS-CHO; GS from hamster (GenBank: AF150961 ) optimized for expression in duck (SEQ ID N° 7 ), referred as hamster-GS opti or GS- CHO duck opti;

GS from chicken (GenBank: S45408) optimized for expression in duck (SEQ ID N° 9), referred as chicken-GS or GS-chicken; and

GS from duck (SEQ ID N° 1), referred as duck-GS or GS-duck. Codon optimization of the hamster and chicken sequences for expression in duck was performed by Geneart (Germany). Plasmid linearization was performed by digestion with restriction enzyme ApaLI (New England Biolabs, UK) into the plasmid replication origin.

B) Expression vectors:

In addition to the resistance cassette for selection with a GS inhibitor (e.g. MSX) encoding for glutamine synthetase enzyme under the control of weak or strong promoter, expression vectors used in the examples further comprise:

iii) A single nptll resistance cassette. The nptll gene encodes the neomycin phosphotransferase protein and provides a resistance to Kanamycin in prokaryotic cells and to Neomycin in eukaryotic cells (Fig. 7B); or a resistance cassette to Ampicillin (Fig. 7C); and iv) Genes encoding a red fluorescent protein (DsRed gene,) or a human lgG1 antibody light and heavy chains, cloned in tandem under the control of a strong promoter.

Plasmid linearization was performed by digestion with restriction enzyme Sfil (New England Biolabs, UK) into the nptll resistance cassette (Fig.7B) or into the ampicillin resistance cassette with restriction enzyme Seal (New England Biolabs, UK, Fig.7C).

Transfection and selection process

Transfection was performed on cells splitting day by nucleofection with Amaxa nucleofector (Amaxa, Germany). Briefly, after enzymatic dissociation, 6 x 10⁶ viable cells were centrifuged (Heraeus ® Multifuge ® 3S Plus, Thermo scientific) at 90g and resuspended with appropriate nucleofection buffer (VVCA-1005 ,Amaxa). 15 to 20 μg of linearized plasmid DNA were added to cell mixture and nucleofection was carried out. Transfection efficiency was verified with the transfection of plasmid encoding for DsRED, which was used as control. After transfection, cells were resuspended in EX- CELL EBx-GRO-l medium (SAFC-Biosciences, USA) depleted of glutamine and seeded into four 100 mm plate (Dutscher, France).

Two days post transfection, control cells (transfected with the DsRed encoding plasmid) were observed with UV microscope (Axiovert 40, Zeiss, Germany) or fixed with paraformaldehyde 4% (Sigma, USA) and analyzed by flow cytometry (Epics XL - Beckam coulter), showing more than 80 percent of fluorescent cells.. Selection with MSX was applied two or three days post transfection according to cell recovery. The selection media used was glutamine-free EX-CELL EBx-GRO-l (SAFC-Biosciences, USA) supplemented with either 30 or 40 μΜ MSX (Millipore, U.S.A.). During the selection process media was changed every day for around 2 weeks until clone picking. Picked clones were seeded into Ultra-Low Attachment 96-well plate (Corning) under a final volume of 100 μί of free-glutamine EX-CELL EBx-GRO-l medium supplemented with the appropriate concentration of MSX. Wells from 96 well-plates were expanded into 24-well plates (Coming) and then subsequently into 6-well plates (Corning). From static 6-well plate culture, few clones were scaled-up into a final volume of 10 ml with the same culture medium in CultiFlask 50 bioreactors (Sartorius, Germany). The culture was then agitated at a rate of 150 rpm at 37°C in the presence of 7,5% C02 (orbital shaker IKA KS 260 basic, Germany).

RT-PCR : mRNA isolation and amplification of duck endogenous GS cDNA sequence

Total RNA isolation from EB66 cell line and Muscovy EBx cells (Vivalis, France) were performed using nucleospin RNA II kit (Macherey-Nagel, Germany). An oligonucleotide with the sequence shown in SEQ ID N°1 1 was used as primer to perform the retrotranscription reaction (RT) and obtain the cDNA of the duck endogenous Glutamine Synthetase (GS) enzyme. Furthermore, a PCR reaction was carried out to amplify the full length cDNA of duck endogenous GS. The sequence of the oligonucleotides used as reverse and forward primers are those shown in Table III, SEQ ID N° 1 1 and SEQ ID N° 12, respectively. The RT-PCR products were cloned into a plasmid using the TOPO® Cloning kit (Invitrogen, U.S.A.). GAPDH was used as loading control. Thus, the presence of similar amounts of genomic DNA or mRNA was controlled by concomitant amplification of endogenous Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Oligonucleotides having the sequences referenced as SEQ ID N° 17 and SEQ ID N° 18 were used as primers for GAPDH amplification, obtaining a PCR product of around 300 bp from cDNA template.

RT-PCR conditions are described in Tables I and II below.

Table I - Retrotranscription reaction (RT) conditions

Table II -PCR conditions

- PCR amplification to determine integration of the GS containing plasmids in the host cell genome

Genomic DNA from EB66 cell clones was extracted using the kit nucleospin blood quick pure (Macherey-Nagel, Germany). 0,5 microgram of the recovered genomic DNA was used as template for the PCR experiments. PCR conditions are as described in Table II above. The following couples of primers were used to prove integration of the transfected plasmids into the host genome: i)SEQ ID N° 13 and SEQ ID N° 14; and ii) SEQ ID N° 15 and SEQ ID N° 16. GAPDH was also used as loading control. Oligonucleotides having the sequences referenced as SEQ ID N° 17 and SEQ ID N° 18 were used as primers for GAPDH amplification, obtaining a PCR product of about 700 bp from genomic DNA of EB66 duck cell line.

The sequences of the oligonucleotides used as primers in the PCR and RT- PCR reactions described above are shown in Table III below.

Table III.- Primer sequences

Quantification of antibody expression by ELISA

Supernatants were collected from either the 96-, 24- or 6-well plates (corning) for secreted antibody quantification using an ELISA assay. Briefly, 96-well plates were coated with anti-human IgG HL (Interchim, France) and blocked with bovine serum albumin (BSA) buffer (Sigma, U.S.A.). Samples were prepared by removing cellular content with centrifugation (Heraeus ® Multifuge ® 3S Plus, Thermo scientific) and the supernatants were used for the assay. The IgG standard and culture supernatants were diluted with appropriate amount of BSA blocking buffer were added into wells. After 30 minutes of incubation at 37°C wells were washed with phosphate buffered saline (PBS) + 0,1 % Tween 20 (Sigma, U.S.A.). Then wells were treated with Goat peroxydase anti-human IgG HL (Interchim, France). Excess of peroxydase-conjugated antibody were removed with a new wash step. The peroxidase substrate (TMB, Sigma, U.S.A.) was then added and the absorption at 450 nm was determined (plate reader Bio-Rad model 680 R-232C, USA) the secreted antibody titer was obtained based on a standard curve.

- GIEMSA staining

Cells were washed twice with Phosphate Buffer Saline (PBS) and were fixed with methanol (VWR, U.S.A.) for 10 minutes at room temperature. Then, cells were dried at room temperature and a Giemsa staining solution prepared with 90% water, 9% Giemsa solution (VWR, U.S.A) and 1 % Wright solution (VWR, U.S.A.) and dropped in each 100mm plate (Dutscher, France). After 20 minutes, plates were washed twice with de-ionized water and observed.

Example 2. Clone selection using the chicken GS sequence

The inventors were willing to provide a GS system adapted for the selection of cell clones having integrated a plasmid of interest in avian cells. The first approach was to use plasmids encoding as selectable marker the chicken-GS sequence which was already available in public databases (GenBank: S45408) optimized for expression in duck (SEQ ID N° 9) by Geneart (Germany).

Accordingly, several transfection series were performed to test the chicken-GS transfection cassette. EB66 cells were transfected in the same experiment with selection plasmids encoding chicken-GS only or encoding both chicken-GS and an lgG1 antibody, as shown in Figures 7A and 7B, respectively, using the method described in Example 1. Transfected cells were submitted to a MSX selection pressure between 10μΜ to 50μΜ in a EX-CELL EBx-GRO-l (SAFC-Biosciences, USA) culture medium deprived in glutamine. No positive results were obtained, as the minimal selection pressure used resulted in cell death for both transfected and untransfected control cells. Transfection efficacies as well as plasmid sequences were verified and were found to be correct (results not shown). Furthermore, a selection of the cell culture medium optimization was also assayed unsuccessfully. EXAMPLE 3.- Amplification of full length endogenous glutamine synthetase cDNA from EB66 and Muscovy EBx cells

Whole RNA extract from EB66 cells and Muscovy EBx cells were submitted to a GS specific RT-PCR using the primers and conditions described in Example 1. Then, PCR products were cloned in the plasmid pCR®4-TOPO® with the TOPO® TA Cloning® Kit for Sequencing (Invitrogen, U.S.A) and sent for sequencing (Millegen, France).

Oligonucleotides with sequences defined in SEQ ID N° 1 1 and SEQ ID N° 12, were used. These were designed based on chicken glutamine synthetase (GS) sequence (GenBank: S45408), to amplify specifically the mRNA and the full-length cDNA of duck endogenous GS enzyme.

RT-PCR results are shown in Figure 1. No PCR product was amplified for the control sample, i.e., "no-RT" control. Conversely, for RT (+) samples, specific amplification products were obtained. DNA fragments of both Muscovy duck EBx cells (1 ) and EB66 cells (2) seem to have the same length (around 1 100 bp) close to the expected size for the full length cDNA of duck GS enzyme (1 122 bp). RT-PCR on GAPDH was used as loading control and specific primers (SEQ ID N° 17 and SEQ ID N° 18) were used. As expected samples submitted to reverse transcription (+) amplified only the cDNA of GAPDH (PCR product of around 300 bp). Some GAPDH gene amplification is observed on lane (-) for EB66 cells (2), but this is explained by a contamination of the RNA samples with some genomic DNA (PCR product of around 700 bp). Duck GS specific PCR products were then cloned into the pCR®4-TOPO®, plasmid and sent for sequencing (Millegen, France). Sequencing results confirmed that the full length cDNA of duck endogenous GS enzyme was amplified according to a strong sequence identity with known sequences of GS from other species. The obtained results show two allelic forms of endogenous GS enzyme cDNA sequence in duck, both in EB66 cells (SEQ ID N° 1 and SEQ ID N° 2) and in Muscovy duck EBx cells (SEQ ID N° 3 and SEQ ID N° 4). The differences between Muscovy and EB66 allelic sequences are shown in Fig.2 and Fig.3, respectively. Furthermore, the alignment of the four obtained allelic sequences show that there is a 98,8% sequence identity (Fig. 4).

However, the obtained duck GS nucleic acid sequences from EB66 cells (SEQ ID N° 1 and SEQ ID N° 2) and from Muscovy duck EBx cells (SEQ ID N° 3 and SEQ ID N° 4) encode the same protein sequence (SEQ ID N° 5), showing that the GS protein sequence is well conserved among different species from duck (Anatidae family). Once duck GS sequence was identified, its amino acidic sequence was compared with GS sequences already available through public databases for other species such as chicken, hamster, rat, human, and Taurus (Table IV).

Table IV.- Interspecies comparison of GS protein sequence

In particular, it was found that duck GS protein sequence presented 89,5 % of sequence identity with hamster GS protein sequence and 98,9 % of sequence identity with chicken sequence (Fig.8). Accordingly, chicken and duck GS amino acidic sequences have the highest degree of sequence identity. However, it was found that the duck GS amino acid sequence (SEQ ID N° 5) and that of chicken (GenBank: S45408) only differed in four punctual mutations. Interestingly, the amino acid residues in these 4 differing positions were common to duck and hamster GS enzyme (Fig.9). EXAMPLE 4.- Selection of EB66 clones transfected with a GS selection cassette by the use of a GS inhibitor in a glutamine-free culture medium

EB66 adherent cells were transfected with GS selection plasmids (Fig. 7A) containing two selection cassettes: the kanamycin resistance cassette and the GS selection cassette encoding the glutamine synthetase enzyme from duck, hamster or chicken, respectively. After transfection cells were resuspended in glutamine-free EX- CELL EBx-GRO-l medium. Two days after transfection, the L-methionine sulfoximine (MSX) selection pressure was applied on cells. Media change was performed every day during the selection process. Fourteen days post transfection, plates were submitted to GIEMSA staining and some clones were picked and expanded to 6-well plates. After cell amplification, dry pellets from clones were kept aside for the later investigation of plasmid integration into the host genome.

Transfection with chicken-GS, duck-GS and hamster-GS in parallel resulted in significant differences in the number of selected cell clones. In particular, cells transfected with chicken-GS, as well as untransfected cells did not survive when submitted to a selection pressure of 30 μΜ of MSX in glutamine-free media (Fig. 5A and Fig. 5B). By contrast in the same conditions, cells survived when transfected with duck-GS and a few well defined clones were also observable from cells transfected with hamster-GS. Accordingly, best results were obtained when selection vectors containing a duck-GS sequence were used for the selection of duck cell lines, notably in EB66 cells, in the presence of a GS inhibitor in a glutamine-free culture medium.

Example 5.- Optimization of hamster-GS sequence for expression in duck cells

The genetic code is the set of rules by which information encoded in genetic material (DNA or mRNA sequences) is translated into proteins (amino acid sequences) by living cells. The code defines how sequences of three nucleotides, called codons, specify which amino acid will be added next during protein synthesis. With some exceptions, a three-nucleotide codon in a nucleic acid sequence specifies a single amino acid. Because the vast majority of genes are encoded with exactly the same code, this particular code is often referred to as the canonical or standard genetic code, or simply the genetic code.

It is well known in the art however, that one amino acid residue can be encoded by more than one codon. This is commonly known as the degeneracy of the genetic code. The codons encoding one amino acid may differ in any of their three positions. For example the amino acid glutamic acid is specified by GAA and GAG codons (difference in the third position), the amino acid leucine is specified by UUA, UUG, CUU, CUC, CUA, CUG codons (difference in the first or third position), while the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, AGC (difference in the first, second, or third position) (Watson JD, Baker TA, Bell SP, Gann A, Levine M, Oosick R. (2008). Molecular Biology of the Gene. San Francisco: Pearson/Benjamin Cummings).

Codon optimization is mainly based on the preferential usage of certain codons in a specific organism, commonly referred to as codon usage bias. It is strongly correlated with corresponding tRNA abundances and expression levels (Ikemura T et al (1981), J Mol Biol 151 :389-409; Dong H et al (1996) J Mol Biol 260:649-663.). Codon frequency usage tables for different species are available through public databases such as the NCBI (NCBI-GenBank Flat File Release 160.0 [June 15 2007]). See table V below for the codon usage in duck (Anas platyrhynchos [gbvrt]: 151 CDS's (45582 codons)) wherein the fields show the frequency: per thousand for each triplet.

Table V. Codon usage frequency in duck

uuu 14.9 UCU 13.5 UAU 10.5 UGU 8.1 uuc 21.9 UCC 16.2 UAC 21 .1 UGC 14.8

UUA 5.4 UCA 9.9 UAA 1.6 UGA 1.1

UUG 1 1.1 UCG 3.9 UAG 0.6 UGG 14.2

CUU 1 1.3 ecu 12.4 CAU 9.6 CGU 5.4 cue 19.8 ccc 19.7 CAC 16.7 CGC 10.4

CUA 5.4 CCA 13.8 CAA 12.9 CGA 4.3

CUG 40.7 CCG 6.7 CAG 32.6 CGG 7.9

AUU 15.2 ACU 13.2 AAU 15.6 AGU 10.4

AUC 24.4 ACC 21 .4 AAC 23.7 AGC 24.2

AUA 7.6 ACA 15.2 AAA 25.8 AGA 10.9

AUG 23.2 ACG 7.3 AAG 34.5 AGG 12

GUU 10.7 GCU 21 .1 GAU 21 .8 GGU 1 1

GUC 16.7 GCC 23.8 GAC 26.7 GGC 21.5

GUA 6.8 GCA 18 GAA 27.4 GGA 16.4

GUG 29 GCG 7.9 GAG 38.7 GGG 19.7

However, the simple sequence optimization strategy of backtranslating an amino acid sequence by using the most frequently used synonymous codon for each amino acid has been superseded by the development of advanced algorithms, which take into account multiple criteria to calculate a near optimal solution for the experimental requirements. Some of these criteria are the following: codon optimization and G/C content adaptation, inhibition of internal splicing and premature polyadenylation, prevention of creation of stable RNA secondary, introduction/avoidance of immunomodulatory CpG motifs, avoidance of direct DNA repeats and thereby recombination events, domain shuffling, epitope scrambling and protein chimarization, obstruction of formation of stable dsRNAs with host transcriptom and avoidance of possible RNAi viable genes. Geneart (Germany) has developed proprietary software which applies a sophisticated algorithm which will optimize a gene sequence by considering all relevant transcription and translation optimization parameters in a single operation and deliver a DNA optimized for maximum performance in a host cell from a particular species. Hence, carefully designed synthetic genes are expected to reach the highest level of gene expression in a particular cell system (Fath et al (201 1), PLoS One; 6(3): e17596). Accordingly, in a further experiment, in addition to the chicken-, hamster- and duck-GS sequences, a plasmid encoding for the hamster-GS sequence with a codon usage optimized for expression in duck cells (hamster-GS opti) was also used.

Cells were submitted to a selection pressure of 40 μΜ of MSX in a media depleted of glutamine (Fig. 5C and Fig. 5D). Cells survived to MSX selection pressure when transfected with sv40 GS-Duck or EF1 -HTLV GS duck, by contrast all cells were dying in all other conditions. For the hamster-GS opti sequence, even if during selection the codon optimization seemed to have a positive affect (Fig. 5C) at the end of the selection process only cells transfected with duck GS survived (Fig. 5D). Therefore, unexpectedly hamster-GS opti sequence was also found not to allow the selection of duck cell clones having incorporated the selection vector.

These experiments show the interest in using duck-GS sequence in the selection of clones of a duck cell line, in comparison with the GS enzyme coming from other species. Furthermore, it should be noted that only those cells transfected with duck-GS were able to survive to a selection pressure of 40 μΜ of MSX. On the other hand, the difference of survival rate between cells transfected with duck-GS under a weak (SV40 early) or a strong (EF1-HTLV) promoter, seems to suggest that cell survival is dependent of the level of duck-GS expression in presence of MSX selection pressure.

EXAMPLE 6.- Determination of plasmid integration into the host genome

To demonstrate plasmid integration into the host genome after selection with MSX, EB66 cells were transfected with plasmids encoding a duck GS enzyme under the control of a weak (SV40 early) or strong (EF1-HTLV) promoter (i.e. vector shown in Fig. 7A) and then submitted two days later to a selection pressure of 40 μΜ of MSX in glutamine-free EX-CELL EBx-GRO-l medium (SAFC-Biosciences, USA). Fourteen days post transfection 10 or 12 survival clones of each transfection were picked and transfer in 96-well plates. Clones were expanded to 6-well plates and dry cell pellets prepared. Genomic DNA was extracted from those dry pellets as described in Example 1 and PCR reactions were carried out to determine whether the transfected plasmids were integrated into the host genome. Results are shown in Fig.6. PCR analysis after genomic DNA extraction shows that all clones transfected with a plasmid encoding for duck glutamine synthetase enzyme under the control of a strong promoter (EF1-HTLV) have integrated in their genome the plasmid (13 positives clones out of 13; Fig.6B). In the case of a plasmid where duck GS is under the control of a weak promoter (SV40 early) similar results were obtained (9 positives clones out of 10, Fig.6A).

The obtained results show that almost all clones surviving to MSX selection pressure have integrated in their genome the linearized plasmid encoding for an exogenous duck glutamine synthetase enzyme.

Accordingly, these results show that a selection process based on glutamine metabolism using a sequence encoding for duck GS enzyme (SEQ ID N°5) in the selection/expression plasmids may be successfully used to select duck cell lines having stably integrated a heterologous protein. .

EXAMPLE 7.- Selection of EB66 cells antibody producer clones

To show that clones selected with MSX are antibody producers, the EB66 cells were transfected with expression plasmids encoding the light and heavy chains of an lgG1 monoclonal antibody under the control of a strong promoter and the duck GS enzyme under the control of a weak promoter (SV40 early) or a strong promoter (EFI- HTLV, see Fig.7B). Two days post-transfection cells were submitted to a selection pressure of 30 μΜ of MSX in glutamine-free EX-CELL EBx-GRO-l medium (SAFC- Biosciences, USA). Fourteen days post-transfection some survival clones were picked and transfered in 96-well plates. Clones were expanded to 24-well plates and supernatants samples were taken and analyzed by ELISA for antibody production during the scale-up.

Fig.10 shows that most of the clones transfected with plasmids encoding for the duck GS enzyme (SEQ ID N°5) under the control of a strong promoter are antibody producers. By contrast, none of the clones transfected with GS under the control of a weak promoter were identified as antibody producers. Without wishing to be bound by theory, it has been hypothesized that the selection pressure might have an impact in the heterologous protein expression and thus the particular MSX concentration used in this experience might not be adapted when protein expression is under the control of SV40 early promoter, thus not allowing the selection of cell clones.

This experiment shows the selection of protein (i.e., antibody) producer clones from a duck cell line using a selection process based on glutamine metabolism. According to the fact that more producer clones are obtained with plasmids encoding duck GS under a strong promoter, it seems that the level of expression of the selection cassette could be important in order to select monoclonal antibody producer clones. Furthermore, a high level of exogenous duck glutamine synthetase appears to favor not only cell clones survival to MSX selection but also to promote the monoclonal antibody production. EXAMPLE 8.- Selection of antibody producer EB66 cell clones with vectors having a different orientation of the GS cassette

To verify the results obtained in Example 5 a similar experiment was carried out where the plasmids used as expression vectors comprised a GS selection cassette with the same orientation as the antibody molecule light and heavy chains cassettes and where the nptll selection cassette was replaced by an ampicillin resistance cassette (see Fig. 7C). EB66 cells were transfected with expression plasmids having the above-mentioned features and encoding a monoclonal antibody lgG1 under the control of a strong promoter and duck GS enzyme under the control of a weak promoter (SV40 early) or strong promoter (EF1-HTLV). Two days post- transfection, the cells were submitted to a selection pressure of 30 μΜ or 35 μΜ of MSX in glutamine-free EX-CELL EBx-GRO-l medium (SAFC-Biosciences, USA). Nine or twelve days post-transfection (depending on cell clone growth) ten of the clones having survived to the selection pressure were picked for each condition and transferred in 96-well plates. Clones were expanded to 24-well plates and supernatant samples were analyzed by ELISA for antibody production. No antibody production was obtained with a GS cassette under the control of SV40 early promoter (data not shown). Figure 11 shows the antibody production obtained following the selection by MSX of clones transfected with plasmid encoding for lgG1 and the selection cassette GS under the control of strong promoter (EF1- HTLV). It can be observed that the median antibody production of those clones selected with 35 μΜ of MSX is almost double (0,86 μg mL) of that of clones selected with 30 μΜ of MSX (0,45 μg mL). Thus, under the tested conditions, the stringency of the MSX selection pressure has an impact on the final level of antibody expression of the selected clones. Notably, a higher selection pressure was associated with an increased antibody production of selected clones.

All these data confirm that it is possible to select clones producing antibodies in an avian cell line using a selection system based on the metabolism of glutamine, in particular using a GS selection vector containing a GS duck sequence

EXAMPLE 9.- EB66 cell antibody producer clones selection and scale-up

To test the robustness of the duck GS clone selection system, a selection of EB66 cell antibody producer clones was carried out, similar to that of Example 6 but with a statistical dimension. Briefly, cells were transfected with a plasmid encoding for duck GS under the control of a strong promoter and an lgG1 antibody light and heavy chains also under the control of a strong promoter. 48 hours later, transfected cells were submitted to a MSX selection pressure of 35 μΜ in EX-CELL EBx-GRO-l medium (SAFC-Biosciences, USA) depleted of glutamine and supplemented with 2 mM of Glutamic acid. 200 clones were picked-up for antibody production determination.

All the clones were subcultured from 96-well to 24-well plates. The antibody production displayed in Figure 12 shows antibody production levels per clone in 24- well plates. As shown in Figure 12, most of the clones selected were antibody producers, showing the efficiency of the selection process. Clones were classified based on antibody production levels obtained in 24- well plates and the fifty (50) best antibody producer clones were scaled-up to 6-well plates and finally 24 clones were cultured in 50ml_ disposable bioreactors (cultiflask 50; Sartorius Stedim Biotech, Germany). A batch culture (i.e., no addition of nutrient feeds) was performed under the above described culture conditions to assess the antibody production rates of the clones selected with the duck GS clone selection system (see Figure 13). Briefly, clones were maintained in culture for 12 days without any fed batch feed. Supernatant from cell culture was harvested and an ELISA assay was performed to assess antibody production. Under these conditions, all clones but one were considered to be antibody producers, showing the reliability of this system to select stable antibody producer clones, as some of the selected clones were shown to maintain antibody molecule expression along the different steps of the scale-up.

Overall, these data proofs the feasibility of selecting antibody producer cell clones with the duck GS system and the ability to apply this system in scale-up experiments in order to select the best antibody producer cell clone in avian cells, in particular in EB66 cells.

EXAMPLE 10.- Duck and turkey sequences comparison

Recently, the inventors find out that turkey (Meleagris gallopavo) glutamine synthetase (GS) amino acid sequence was available through public databases (sequence UPI0001C99696, ENSEMBL database; (SEQ ID N° 19). Upon sequence comparison, it was found that turkey and duck amino acid sequences have a 100% sequence identity.

Furthermore, the nucleotide sequence comparison was also performed between the nucleotide sequence encoding for turkey glutamine synthetase (sequence XM_003208553.1 from NCBI database; (SEQ ID N° 20) and the four allelic variants of GS-duck, i.e., SEQ ID N° 1 to SEQ ID N° 4 (Fig. 14). At nucleotide level, it was found that turkey and duck cDNA sequences are highly similar (94% of sequence identity) but punctual mutations were uncovered which might affect the GS enzyme expression level, particularly in duck cells.

Claims

An isolated nucleic acid sequence characterised in that it comprises a sequence selected from the group consisting of SEQ ID N° 1 , SEQ ID N° 2, SEQ ID N° 3 and SEQ ID N° 4 wherein said sequence encodes a protein having a glutamine synthetase activity.

A vector comprising at least one expression cassette comprising at least one nucleic acid sequence of claim 1.

A vector according to claim 2, wherein said vector further comprises at least one expression cassette comprising at least a nucleic acid sequence encoding a heterologous protein.

4. A vector according to any one of claims 2 or 3 wherein the expression of the nucleic acid sequence as defined in claim 1 is under the control of a strong promoter.

5. A host cell comprising a vector according to any of claims 2 to 4.

6. A method for selecting avian cell clones comprising the following steps:

a) transfecting an avian cell with a vector comprising a nucleic acid sequence encoding a protein with a glutamine synthetase activity wherein said nucleic acid sequence is (i) a nucleic acid sequence as defined in claim 1 , or (ii) a nucleic acid sequence encoding SEQ ID N° 5;

b) culturing the transfected cells obtained in step a) in a selection medium which lacks glutamine or in which the amount of glutamine is progressively depleted;

c) selecting those cell clones having survived to culturing in the selection medium of step b);

wherein a glutamine synthetase inhibitor is added in said selection medium when said avian cell has an endogenous glutamine synthetase activity.

7. The method of claim 6 wherein the transfected vector of step a) further comprises at least one expression cassette comprising at least a nucleic acid sequence encoding a heterologous protein.

8. The method of any of claims 6 or 7, wherein the cell clones selected in step c) are those having survived to culturing in the selection medium of step b) for at least 5 days.

9. The method of any of claims 6 to 8 wherein said glutamine synthetase inhibitor added in said selection medium when said avian cell has an endogenous glutamine synthetase activity is L-methionine sulphoximine and is used at a concentration between 10 μΜ to 70 μΜ.

10. The method of any of claims 6 to 9 wherein said avian cell is a duck cell.

1 1. The method of claim 10 wherein said duck cell is able to proliferate indefinitely in suspension in the absence of animal serum and/or exogenous growth factors.

12. The method of any of claims 10 or 1 1 wherein said duck cell is an embryonic- derived duck cell line.

13. The method of any of claims 10 to 12 wherein the transfected vector in step a) further comprises at least one expression cassette comprising at least a nucleic acid sequence encoding a selectable marker operably linked to a promoter sequence capable of effecting expression of said selectable marker in the host cell where said selectable marker is other than the nucleic acid sequence defined in claim 1 or a nucleic acid sequence encoding SEQ ID N° 5.

14. The method of any of claims 7 to 13 wherein said heterologous protein is an antibody, preferably an IgG antibody, more preferably a human IgG antibody. Use of a nucleic acid sequence as defined in claim 1 or a nucleic acid sequence encoding SEQ ID N° 5 as a selectable marker in a cell clone selection method.