CA2671825A1 - Highly transcriptionally active cell line for the producion of proteins, notably for therapeutic applications - Google Patents

Abstract

The invention relates to a method for obtaining a cell line, that comprises the steps of integrating into the genome of said cell a unique site for the recognition of a recombinase in an area of high transcription activity of the genome of said cell, and integrating in the genome of said cell, and downstream from the unique recombinase recognition site, a nucleic acid sequence coding for a transcription termination signal. The invention also relates to cell lines obtained by said method.

Description

HIGHLY TRANSCRIPTIONALLY ACTIVE CELL LINE FOR THE
PRODUCTION OF PROTEINS, NOTABLY FOR THERAPEUTIC APPLICATIONS

This invention relates to a process for generating a cell line including, in particular, integration of a sequence encoding a protein of interest at a highly transcriptionally active site, with a view to the production of proteins, notably for therapeutic applications, and to the cell lines generated using this process.

FIELD OF THE INVENTION

As a result of developments in biotechnology, the production of recombinant proteins is now an important activity in the biomedical sector, with numerous applications, both diagnostic and therapeutic. Growing expertise in molecular and cellular engineering is now making it possible to produce large quantities of recombinant proteins in a variety of host cell expression systems.

Various biological systems can be used to generate recombinant proteins: bacteria (1, 2), yeasts and other fungi (35), plants (6), insect cells (7, 8) and mammalian cells (9-12). Mammalian cells are the system most commonly used for the production of complex recombinant proteins intended for therapeutic applications (e. g. monoclonal antibodies) because of their post-translational capacities vis-a-vis the assembly, glycosylation and modification of synthesised recombinant proteins (10, 11).

Unfortunately, the natural productivity of mammalian cells is limited when compared with the expression levels obtained in bacteria or yeast. This is because integration of the expression vector into the host cell genome is a rare event (1/10,000 according to Gorman & Bullock 2000, Ref. 13) in which neither the insertion site nor the number of copies integrated, and thus the resulting expression level can be controlled. Great efforts have been made to improve this type of production system in a series of different approaches: the expression vectors and their method of integration, the cells and their method of culture and the gene amplification (14-26).
All these efforts have focused on cell lines used for industrial-scale production, notably the CHO (14, 25, 27-29) and NSO (22, 30, 31) cell lines, as well as the HEK293 (32, 33) and BHK (34-36) cells. However selection of a highly productive clone is still often a critical step, involving the screening of a great number of transfectants because of the randomness of integration of the expression vector into the host cell genome. Once the expression vector has been integrated into the genome, the level at which the recombinant protein is expressed is strongly dependent on a "position effect", i.e. the genetic environment of the integration locus (37, 38). Thus, insertion of the expression vector at a transcriptionally inactive site in the genome will give low-level expression, or none at all, whereas integration of the vector at a transcriptionally active site could yield high-level expression.

Since most of the genome is in a transcriptionally inactive state, it is usually necessary to screen a large number of transfectants to identify one highly productive clone (22).

BACKGROUND ART
Various strategies have been investigated to control this "position effect". One approach is based on abolishing the position effect using Locus Control Region (LCR) sequences or isolators (39-42. In such a case expression of the transgene is only dependent on the number of integrated copies of the vector and the efficacy of exogenous sequences which regulate expression of the recombinant protein.

Another approach is based on abolishing the position effect by locally modifying the chromatin to enhance transcriptional activity (UCOE) (43).

Alternative strategies have attempted to abolish the randomness of integration by directing vector insertion to a locus in which the transgene will be highly expressed. To do this, the transgene can be targeted to a well-characterised gene (Hollis GF, US 6,750,041) or to sites known to be transcriptionally active (44, 45, Reff MR, US Patent 6,841,383). In this approach, the integration event is controlled and is targeted to a locus where expression of the transgene at a constant, and, if possible high-level is guaranteed. The transfection process is therefore no longer a random event but becomes reproducible, meaning that the screening and characterisation steps are simplified and a comparable level of productivity is obtained from one transfection procedure to another.

The targeted integration of the expression vector can be realised by means of homologous recombination (Reff MR, US
Patent 6,841,383; Hollis GF, US Patent 6,750,041; 46, 47).
However, although this type of recombination is common in yeast and other fungi, it is relatively rare in higher eukaryotes which represents a major obstacle to its exploitation in such organisms (47), As an example, in mammalian cells, the ratio of the frequency of homologous recombination to that of random integration lies between 1/100 and 1/5,000 (48). A complementary approach designed to promote homologous recombination involves creating cleavage sites in the DNA using meganucleases (e.g. I-Sce I) (49).

Another approach exploits recombinases (47, 50), notably the Cre (51, 52) and Flp (53, 54) recombinases. The P1 bacteriophage's Cre-loxP recombination system has been adapted and used to target certain genes in eukaryotic cells (51, 52, Sauer BL, EP 0 220 009). The target integration in the genome of Chinese hamster ovary (CHO) cells (44) using Cre recombinase has been described by way of example (44).
This recombination system is promising in that it ensures reproducible expression from a specific locus in the genome.
However, it only guarantees reproducible expression of a reporter gene without ensuring high-level expression of the gene in question.

In order to obviate this problem, cell lines which can be used as stable expression systems have been created with a FRT recombination site in a transcriptionally active region of the genome (e. g. the Flp-InTM and Invitrogen cell lines).
Integration of the expression vector at the site within the transcriptionally active region is mediated by Flp-FRT
recombination, thereby ensuring high-level expression of the gene of interest. However, the Flp-in cell lines available (293, CHO, BHK, 3T3) are not always optimised for the production of certain recombinant proteins, such as the monoclonal antibodies, because of the cells' endogenous post-translational modification mechanisms. In addition, the genomes of these cell lines contain extraneous sequences designed to make it possible to select individuals in which the recombination site has been integrated into a highly transcribed region.

It would be preferable to have "host" cell lines free of any active sequences (promoters, enhancers or antibiotic resistance-conferring genes) to avoid limiting the ability to use, at a later stage, resistance genes or regulatory sequences (promoters, enhancers, polyA) able to regulate the production of the protein of interest. Finally, the possibility that the reporter gene may be co-expressed with the protein of interest can necessitate complicated checking procedures, especially when it comes to the production of therapeutic proteins which are subject to rigorous regulations, notably governing the molecular engineering of cell lines producing proteins for therapeutic applications.

More recently, Kito et al. (45) adapted the Cre-loxP
approach to select highly productive clones of CHO cells using GFP as the reporter gene; after gene amplification, this resulted in expression levels close to 160 mg/k.
However, as with the abovementioned Flp-In cell lines, this strategy depends on the presence of many active sequences in the cell line to be used as the host for expression.

Since the production of therapeutic recombinant proteins is subject to rigorous regulations governing the molecular engineering of cell lines producing proteins for therapeutic applications, the presence of active sequences in these cells' genomes complicates the registration procedure and prolongs the development process for medicinal products produced in this way.

6, Thus, creating new, highly productive, time-stable cell lines which can be used to produce any protein of industrial interest, notably proteins for therapeutic applications, in a simple process would constitute a major step towards improving recombinant protein production in eukaryotic cells, in particular mammalian cells.

According to the various matters covered by this invention, reference is made to a protein of interest which can be expressed in a cell, either for the purpose of checking integration at a FRT site or, in the invention's global perspective, for the industrial-scale production of such proteins. As presented in this Description and these Claims, this expression means that the cell line produces proteins which may or may not be present in the cell, and over-expresses them at a level above their endogenous expression level.

DEFINITIONS
As a general rule in the Description, Abstract and Claims, the following terms have the following meanings (unless stipulated otherwise):

In the sense of this invention, "nucleic acid sequence"
means a single- or double-stranded oligomer or polymer of nucleotides read from the 5' end towards the 3' end. The term "nucleic acid sequence" may refer to a molecule of DNA or of RNA, or a DNA/RNA hybrid, either of natural or synthetic origin. In the base notation system used in this Application, unless stipulated otherwise, the left end of a single-stranded nucleotide sequence corresponds to its 5' end In the sense of this invention, "DNA molecule" means any single- or double-stranded oligomer or polymer made up of a chain of nucleotides, either of natural or synthetic origin, and comprising, without being limited thereto, a gene, a set of genes, a gene fragment, a mixture of coding and non-coding sequences, regulatory sequence or a sequence complementary to that of a RNA molecule.

In the sense of this invention, "promoter" or "promoter sequence" means a natural or synthesised nucleic acid sequence upstream of the translation initiation codon which is involved in the recognition and binding of RNA polymerase.
A promoter sequence thus helps initiate transcription of a downstream coding sequence. Such promoters are familiar to those skilled in the art and include bacterial, viral, eukaryotic, yeast and mammalian promoters, the choice of the promoter depending on the type of host cell to be used for realising the expression.

In the sense of this invention, "vector" means a vehicle based on nucleic acids, a nucleic acid molecule capable of delivering a nucleic acid, or a DNA molecule capable of autonomous replication in a host cell, e.g. a plasmid, a cosmid, a phagemid, a viral genome (chromosome) or a phage genome, and which can be used to clone DNA molecules.
Depending on the cellular host under consideration and the nature of the vector's constituent nucleic acid sequences, the vector can be stably replicated inside cells either as an independent entity or integrated into the host's genome, and either within the host cell nucleus or in its cytoplasm.

In the sense of this invention, "plasmid" means a circular or possibly cell linearised, independent DNA

molecule which can replicate within a cell. The term plasmid refers to so called "expression plasmids" and the plasmids called "non-expression plasmids". When the plasmid is maintained by a host cell, it can either be stably replicated within the cell as an independent entity, or it may be integrated into the host's genome.

In the sense of this invention, "peptide", "polypeptide"
or "protein" refer to primary sequences of amino acids linked through covalent peptidic bonds. In general, a peptide, which is shorter than a protein, contains a small number of amino acids, typically between 2 and 50. The term polypeptide can cover both peptides and proteins. A peptide, a polypeptide or a protein may be of natural, recombinant or synthetic origin.
In the sense of this invention, "transcription termination signal means a nucleic acid sequence located at the end of a transcribed region and which induces the termination of the transcription of said region by RNA
polymerase. Examples of transcription termination signals relevant to this invention include, but are not limited to, polyadenylation sequences, e.g. the polyadenylation sequence "SV40 early polyadenylation signal", and the bovine Growth Hormone (bGH) polyadenylation sequence.
In the sense of this invention, "polyadenylation sequence" or polyA" means a DNA sequence which triggers both the termination of transcription and polyadenylation of the nascent transcribed RNA molecule. Effective transcript polyadenylation is usually desirable insofar as transcripts without a polyA tail are often unstable and quickly broken down.

Efficient cleavage and polyadenylation of mammal messenger RNA require at least two signal elements: an AAUAAA
sequence located 7 to 30 pairs of bases upstream of a processing site, and sequences rich in GU or U located at 3' of the cleavage site.
In the sense of this invention, the expressions "weakly active" or "low efficiency" polyadenylation should be taken to mean a polyadenylation sequence not allowing efficient implementation of transcription termination and polyadenylation of the transcripts. The result is a small quantity of transcripts and/or or significant instability of the transcripts which, for the most part, are broken down too rapidly to allow their translation. More particularly, by "polyadenylation sequence of low efficiency (or weak activity)" should be taken to mean any sequence that allows implementation of transcription termination and polyadenylation of the transcripts at a level lower than or equal to that induced by the "SV40 early polyadenylation signal" polyadenylation sequence. Any polyadenylation sequence making it possible to achieve this objective can be employed in the framework of the invention. More particularly, these sequences can be intact polyadenylation sequences having a weakly active polyadenylation signal for termination of transcription and polyadenylation of the transcripts, such as for example SV40 early polyA or adenovirus Ll polyA (71), or yet again mutated or deleted polyadenylation sequences in order to diminish the level of implementation of transcription termination and the polyadenylation of the transcripts when compared to the non-mutated or not-deleted sequence. We can give as examples of mutation and/or deletion of a polyadenylation sequence, making it possible to decrease the efficiency of the polyadenylation signal when compared to the non-mutated sequence: increasing the distance separating the AATAAA elements and the region rich in GT (66), deletion of late SV40 polyA from one or several nucleotides present upstream of the AATAAA
hexanucleotide (67), deleting certain elements situated 5 between the nucleotides located 13 to 48 nucleotides upstream of the AATAAA sequence (68), deleting sequences rich in GT
downstream of the AATAAA sequence and modifying the space situated between the AATAAA sequence and the region rich in GT
(69), or the mutation or deletion of USE (upstream sequence 10 element) regions located upstream of the AATAAA sequence (70), this list not being limiting.

In the sense of this invention, "isolated" or "purified"
means any modification of the natural state resulting from human intervention. Thus, any object pre-existing in nature which has been modified or extracted from its natural environment is said to be isolated" or "purified". An "isolated" material could be any polynucleotide or any peptide/polypeptide/protein separated from the other molecules in its natural environment, or generated by cloning, amplification and/or chemical synthesis. In addition, a polynucleotide, peptide or protein introduced into an organism by transformation, genetic manipulation or any other method is said to be "isolated" even if it was already present in said organism.

In the sense of this invention, "expression" means the transcription and/or translation of a specific nucleotide sequence which is under the control of a regulatory sequence, e.g. a promoter.

In the sense of this invention, "over-expression" means a per- cell expression level for a given coding sequence 11 ,.

significantly higher (e.g. twice as high but ideally 10 times or even 100 times higher) than the level observed with the corresponding endogenous coding sequence in a cell which has not been transfected with the construction of the invention.

In the sense of this invention, "antibody" means an immunoglobulin molecule WHICH immunologically binds a specific antigen and which may include, depending on the type, polyclonal and/or monoclonal antibodies. This term also covers genetically modified forms such as chimeric antibodies (e.g. humanised mouse or rabbit antibodies) and heteroconjugated antibodies (e.g. bi-specific antibodies).
The term "antibody" also covers any form of antibody that can bind an antigen, including antigen-binding antibody fragments.

In the sense of this invention, "transfection" or "transfect" refer to a process in which cells incorporate exogenous DNA and integrate it into their own genome.
In the sense of this invention, "cell line" means a group of cells descended from the same precursor cell, all with the same genetic characteristics as the parent cell. A
cell line is also characterised by its ability to reproduce stably for several generations in vitro.

In the sense of this invention, "highly transcriptionally active region" means a region of genomic or chromosomal DNA in an organism in which the chromatin structure or the regulatory sequences present significantly enhance transcription of genes in or close to the region. The transcription rate in such a highly active region is usually higher than the average transcription rate generally observed 12 ..

in the organism's genome, and preferably twice as high or advantageously 10 times higher, even more 50 times higher, or sometimes even 100 times higher. By way of example, for the YB2/0 cell line (ATTC CRL 1662), it is considered that a highly transcriptionally active region allows the obtention of a pcd value (picograms of protein per cell per 24 hour day) of at least 5 and preferably at least 10.

In the sense of this invention, "weakly transcriptionally active region" means a region of genomic or chromosomal DNA in an organism which present a chromatin structure or which comprise regulatory sequences which are able to significantly inhibit or completely block the transcription frequence of genes in or close to the region.
The transcription rate in such a region is usually lower than the average transcription rate generally observed in the organism's genome, preferably half as high or advantageously 10 times lower, even more 50 times lower, or sometimes even 100 times lower. The transcription rate in such a weakly active region may be so low that it cannot be measured and it could be nil.

In the sense of this invention, "recombinase" or "site-specific recombinase" refers to an enzyme which acts on two nucleic acid molecules in such a way that they recombine with one another. Recombination is a well-characterised physiological process which involves the cleavage of two nucleic acid molecules with identical or substantially similar (homologous) sequences, followed by recombination of the two molecules in such a way that one region of each of the original molecules is conjugated to the corresponding region of the other molecule. Two types of recombination have been observed. The first type, which corresponds to the 13 , "classic" or "homologous" recombination applies to any pair of molecules with homologous nucleotide sequences which can act as a substrate for a"universal" recombinase. As against this, in the second type, called "site-specific recombination", the homologous molecules have, to be able to act as a substrate for the recombinase, to carry a special nucleotide sequence called "site-specific recombination". A
number of site-specific recombination systems are described in the background art, including that of the E. coli P1 bacteriophage. In particular, the specific sequences and recombinases used can belong to different structural classes, notably the family of the Tn3 transposon resolvase or the lambda bacteriophage integrase family. Among the recombinases which belong to the Tn3 transposon family there is the Tn3 transposon resolvase or the Tn21 and Tn522 transposons (Stark and al., 1992); the Gin invertase of the mu bacteriophage or further the plasmid resolvases such as the RP4 fragment (Abert and al., Mol. Microbiol. 12 (1994) 131). Among the recombinases belonging to the k bacteriophage integrase family, there are the ~. bacteriophage integrase itself (Landy and al., Science 197 (1977) 1147), P22 (Leong and al., J.
Biol. Chem. 260 (1985) 4468), Haemophilus influenza HP1 (Hauser and al. J. Biol. Chem. 267 (1992) 6859), the P1 phage Cre integrase, the pSAM2 plasmid integrase (350341EPA EP 350 341), or further the 211 plasmid FLP recombinase and the E.
coli XerC and XerD recombinases.

In the sense of this invention, "recombination recognition site" means a nucleic acid sequence which can act as a substrate for a recombinase.

In the sense of this invention, "reporter gene" means a polynucleotide with a sequence that encodes a gene product, 14 , usually an enzyme, which can be easily detected and/or quantitated when the construction including the reporter gene sequence is introduced into a cell containing all the factors required for expression of said gene. Examples of reporter genes that could be used in the context of this invention include, but are not limited to, fluorescent proteins such as the maxFP-green protein and derivatives thereof, luciferase, Green fluorescent protein (GFP) and derivatives thereof, or the Reef Coral Fluorescent proteins (RCFP) as well as the beta-galactosidase encoded by the lacZ gene.

In the sense of this invention, "protein of interest"
means any peptide, polypeptide or protein which might be of industrial, therapeutic or prophylactic value. Proteins of interest which can be expressed in cell lines according to this invention could be selected from the following:

- proteins with therapeutic activity, i.e. proteins which have a recognised beneficial physiological effect in humans or animals in any form of recognised disease or pathologically impaired function of said human or animal, including prophylactic treatment modalities; such proteins may be peptides, polypeptides, hormones, enzymes and related species, and preferably polypeptides with an activity chosen from the group which are active vis-a-vis digestive pancreatic, biliary, antiviral, anti-inflammatory, pulmonary, antimicrobial, haematological, neurological, cardiovascular, ophthalmologic, antigenic, cerebral, anti-tumoural, immunostimulatory and immunomodulatory functions; in particularly preferred embodiments, the protein or polypeptide with therapeutic activity is chosen from the group including the insulins;
a growth hormone, including a human growth hormone or a bovine growth hormone; a growth hormone-releasing factor;

15, a parathyroid hormone; a thyroid-stimulating hormone; a folliclestimulating hormone; a luteinising hormone; the interferons such as interferon-alpha, -beta and -gamma or, for example, an interferon 13; a vascular endothelial growth factor (VEGF); receptors for hormones receptors or growth factors; an integrin; an A or D protein; rheumatoid factors; a neutrophic factor such as a bone-derive neutrophic factor (BDNF); a neurotrophin (NT-3, NT-4, NT-5 or NT-6); a nerve growth factor such as NGF-beta; a platelet-derived growth factor (PDGF); a fibroblast growth factor like FGF or bFGF; an epidermal growth factor (EGF); a transformation growth factor (TGF) such as a TGF-alpha and a TGF-beta, including a TGF-beta.l, a TGF-beta.2, a TGF-beta.3, a TGF-beta.4 or a TGF-beta.5; a growth factor resembling type I or type II insulin (IGF-I
and IGF-II), or for example, one of the (1-3)-IGF-I (IGF-I of the brain); a keratinocyte growth factor; growth factor-binding proteins resembling insulin; CD proteins such as a CD-3, a CD-4, a CD-8, or a CD-19; an erythropoietin; bone-inducing factors; immunotoxins; a bone morphogenetic protein (BMP); colony stimulating factors (CSFs), e.g. M-CSF, GM-CSF or G-CSF; ageing accelerating factors; gastric, pancreatic or biliary lipases, elastases, anti-proteases like alpha-1 anti-trypsin;
proteases; oxidases; phytases; chitinases; invertases;
cellulases; xynalases; structural proteins such as collagen; transferrins such as lactoferrin; blood proteins such as haemoglobin or human albumin; blood cofactors, coagulation factors like factor VII, factor VIII, factor IX, factor X, tissue factor, von Willebrand's factor; anti.-coagulati.on factors such as a protein C;
atrial natriuretic factor; renin; calcitonin; glucagon; a pulmonary surfactant; a plasminogen activator such as a 161, urokinase or a tissue-specific plasminogen activator (t-PA); thrombin; thrombopoietin; a haematopoietic growth factor, an alpha- or beta-tumour necrosis factor; an encephalinase; a human macrophage inflammatory protein (MIP-lalpha); a serum albumin such as human serum albumin; a relaxin; a DNase; a cytokine; chemokines such as a RANTES (regulated on activation normally T-cell expressed and secreted); interleukins (ILs), e.g. IL-1 through IL-10; antioxidants such as superoxide dismutase, antibodies, fragments of antibodies and antigens. When the polypeptide or protein is an antibody or a fragment of an antibody, this may be an immunoglobulin molecule, the immunoglobulin heavy chain, essentially complete immunoglobulin molecules and all parts of immunoglobulin that carry a paratope, notably Fab fragments, Fab' fragments, F(ab')2 fragments and Fv fragments, the immunoglobulin light chain and Fv fragments;

- cosmetically active proteins and polypeptides which, according to the legislation in many countries are those which exclusively act at the epidermis, i. e. compounds which do not penetrate down into the lower layers or, in other words, have no effect on the dermis or basal cells.
Such proteins or polypeptides are familiar to those skilled in the art as such, a few examples of which are ceramides, keratides, moisturising agents, antibacterial agents and related compounds;

- nutraceutically active proteins or polypeptides, i.e.
compounds which are identical or similar to those commonly found in the human or animal diet, and which can be found whole or in part in foodstuffs or fractions thereof, and which have a beneficial effect on health; as examples of the types of compound that are covered by this part of the invention, mention can be made of modified or derived 17.
phenylalanine ammonia lyase (PAL), allergens, e.g. birch, poplar, grasses, superoxide, dismutase (SOD) and related compounds.

In the sense of this invention, "high antibiotic dose"
means any antibiotic dose of at least 1/gk, or advantageously of at least 4g/P and preferably of 8g/Y

One aspect of this invention provides for a process for generating a cell line comprising at least one cell, involving the following steps:

- incorporation into the genome of the said cell of a unique recombinase recognition site in a highly transcriptionally active region of genome of the said cell; and - incorporation into the genome of said cell, downstream of the unique recombinase recognition site, of a nucleic acid sequence encoding a transcription termination signal.
Preferably, the nucleic acid sequence encoding a transcription termination signal is a sequence encoding a polyadenylation signal. More preferred still, the nucleic acid sequence encoding a transcription termination signal is a sequence that encodes at least part of a weakly active polyadenylation signal, such as the SV40 early polyadenylation signal or the adenovirus L1 polyadenylation signal or any other weakly active polyadenylation signal such as defined here above According to another particularly advantageous embodiment of this invention, the cell or cells of the cell line used in the processes of the invention is a mammalian or an avian cell.
The original cell (i.e. prior to modification) is to be chosen from the group including: rat myeloma cell lines, notably YB2/0 18 , (ATCC CRL-1662) and IR983F, human myelomas like Namalwa or any other human cell such as PERC6, CHO cell lines (notably CHO-K, CHOLec, CHO-Lec1, CHO Pro-5, CHO dhfr-, CHO Lec13) or other cell lines chosen from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2/0-Ag 14, P3X63Ag8.653 and EBx.

According to a preferred form of the process of this invention, the unique recombinase recognition site is incorporated in a series of steps including at least:
- integration of two nucleic acid sequences both encoding a recombinase recognition site;

- integration of a nucleic acid sequence encoding a reporter gene between the two sequences which both encode a recombinase recognition site;

- integration of a nucleic acid sequence encoding a protein of interest between the two sequences which both encode a recombinase recognition site; and - integration of a nucleic acid sequence encoding a selection marker, preferably a gene conferring resistance to an antibiotic, between the two sequences which both encode a recombinase recognition site.

In this case, the recombinase recognition sequence could be the loxP and/or FRT sequence. The protein of interest could for example be an antibody or a fragment of an antibody.

It is also preferred to select only highly productive cells which have incorporated a single copy of all the above-mentioned sequences. More preferred still, only cells with a pcd (picograms of protein per cell per 24 hour day) of at least 5, or preferably of at least 10, or further of at least 19.
20, 30, 30, 50, 80 or 100 are selected.

Preferably, and subsequently, all the above-mentioned sequences can be excised through the action of a recombinase on the cell. In this case, recombinase activity can be induced by coexpression of said recombinase in the cell from a vector carrying a nucleic acid sequence encoding said recombinase.

According to another preferred embodiment of this invention, the above-mentioned series of steps could also include selection of cells from which all the above-mentioned nucleic acid sequences have been excised, carrying a unique intact recombinase recognition site.
Furthermore, the series of steps can, more preferably still, include incorporation of a nucleic acid sequence encoding the thymidine kinase of Type I Herpes simplex (HSV1-TK). In this case, cells can be selected by adding ganciclovir to the culture medium. Indeed, this selection method would ensure that, if ganciclovir is present in the medium, only those cells in which the HSV1-TK nucleic acid sequence has been deleted will survive.

The above-mentioned series of steps also include, in a particularly preferred manner, transfection of the selected cell line with an expression vector carrying a nucleic acid sequence encoding a protein or polypeptide of interest and a nucleic acid sequence encoding a recombinase recognition site directly downstream of a nucleic acid sequence encoding a selection marker, preferably one conferring resistance to an antibiotic, without any polyadenylation sequence. In this case, said expression vector is incorporated at the unique recombinase recognition site, by virtue of the action of said recombinase which is induced or introduced at the moment of transfection. The selection of cells carrying the expression vector integrated at the target site can also be 5 advantageously carried out by testing for expression of the protein or polypeptide of interest. Advantageously the cells could also be selected for their resistance in the presence of a high antibiotic concentration, particularly for their resistance to antibiotic doses of at least lg/ l, or 2 g/ 1 or 10 preferably 4g/1 or 8 g/f.

In an especially preferred manner, said protein or polypeptide of interest is a therapeutic protein or polypeptide, notably chosen from the group with activity in 15 digestive, pancreatic, biliary, antiviral, anti-inflammatory, pulmonary, antimicrobial, haematological, neurological, cardiovascular, ophthalmologic, antigenic, cerebral, anti-tumoural, immunostimulatory and immunomodulatory functions.

20 The steps of the above-described process at the various stages yield one or more cell lines with particularly advantageous properties. In consequence, another object of this invention is a cell line with a unique recombinase recognition site stably integrated in a highly transcriptionally active region of the genome of the said cell and, directly downstream of said unique recombinase recognition site, a nucleic acid sequence encoding a transcription termination signal. Preferably, the stably integrated nucleic acid sequence encoding a transcription termination signal is a sequence encoding a polyadenylation signal. This nucleic acid sequence encoding a transcription termination or polyadenylation signal is a sequence encoding all or part of a weakly active polyadenylation signal like 21 , the SV40 early polyadenylation signal or any other polyadenylation signal modified to alter its efficiency. As said above, the cell line is preferably mammalian in origin.

The cell line, as described here above, can therefore also advantageously include:

- two nucleic acid sequences both encoding a recombinase recognition site;

- at least one nucleic acid sequence encoding a protein of interest between the two sequences which both encode a recombinase recognition site;

- at least one nucleic acid sequence, encoding a selection marker, preferably a gene conferring resistance to an antibiotic, lacking in polyadenylation sequence, located between the two sequences each coding a recombinase recognition site, this sequence coding for a selection marker being located directly upstream of the recombinase recognition site, itself located directly upstream of the low polyadenylation sequence described earlier.
Preferably, as stated above, a single copy of all the above-mentioned sequences are integrated together into the genomes of the cell line cells. In a still more preferred manner, each cell line cell has a pcd of at least 5 or advantageously of at least 10.

The cell line can also, and in a highly preferred manner, overexpress a protein or polypeptide of interest chosen from the group of proteins or polypeptides of interest with digestive pancreatic, biliary, antiviral, anti-inflammatory, pulmonary, antimicrobial, haematological, neurological, cardiovascular, ophthalmologic, antigenic, cerebral, anti-22, tumoural, immunostimulatory and immunomodulatory activities.
According to yet another object of this invention, the cell line is that identified by the reference YGM-1/10G10 registered under Application Number CNCM 1-3704, (cell line filed on December 18 2006 at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15.

According to another object of the present invention, the cell line is that identified by the reference YGM-2/3G5 registered under Application Number CNCM 1-3885, (cell line filed on December 19, 2007 at the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15.

Yet another object of this invention provides for an isolated nucleic acid molecule including a fragment of nucleic acid identified by the number SEQ ID NO: 1. This molecule represents or indicates a highly transcriptionally active site, and can therefore be used for the integration of the other abovementioned sequences. Obviously the SEQ. ID NO 1 sequence could also be used-directly or in a complementary way or by hybridisation to create a highly transcriptionally active site in the genome of an appropriate cell. This could be achieved by means of a vector carrying a nucleic acid sequence as identified in the listing of sequences by the number SEQ ID NO: 1.

Yet another particularly preferred object of this invention provides for a production process for at least one protein or polypeptide of interest, characterised in that a cell line as described above is cultured in order to express 23, said protein or polypeptide of interest, followed by at least one step in which said protein of interest is harvested. The cell lines such as described and identified by their registration reference number given here above are perfectly convenient for carrying this type of process. In this case, the protein or polypeptide of interest is preferably chosen from the group of proteins or polypeptides of interest which are active vis-a-vis digestive pancreatic, biliary, antiviral, antiinflammatory, pulmonary, antimicrobial, haematological, neurological, cardiovascular, ophthalmologic, antigenic, cerebral, anti-tumoural, immunostimulatory and immunomodulatory functions. More preferred still, the protein or polypeptide of interest is an antibody or a fragment of an antibody.

DRAWINGS
Figure 1: Figure 1 is a representation of the pTV1 targeting vector.
Figure 2: Figure 2 is a representation of the pFlpe recombinase expression vector.
Figure 3: Figure 3 is a representation of the pT125FRT
expression vector.
Figure 4: Figure 4 is a representation of the T125-IG24 vector.
Figure 5: Figure 5 outcell lines the mechanism underlying deletion or excision of the targeting vector by the Flp recombinase.
Figure 6: Figure 6 outcell lines the mechanism underlying reintegration of the expression vector by the Fip recombinase.

DETAILED DESCRIPTION

A first object of the invention relates to a process 24, based on the insertion of a DNA molecule of interest at a target site in the genome of a mammalian cell. This process includes a first step of generating a cell line which has a unique recombinase recognition site integrated in a highly transcriptionally active region of its genome. This first step includes the following steps:

1) Integration in a mammalian cell of a first nucleic acid, referred to as the "targeting vector", containing (i) two sequences in tandem corresponding to recombinase recognition sites, between which there is a reporter gene, a gene encoding a protein similar to the protein of interest, a selector gene permitting amplification and a gene conferring resistance to an antibiotic, but no polyadenylation sequence; and (ii) a polyadenylation site located downstream of the second recombinase recognition site, i.e. 5' of the antibiotic resistance gene;

2) the selection of "highly productive" cells carrying a single copy of the targeting vector;

3) excision of the targeting vector by means of the recombinase, and 4) the selection of cells to make cell lines which have lost the targeting vector, keeping only one intact, unique integrated recombinase recognition site.
By virtue of the process of the invention, the cells making up the cell lines selected at Step 4 all carry a unique recombinase recognition site integrated in a highly transcriptionally active region of their genomes.
One of the characteristics of the mammalian cell used in Step 1) of the process is that, before modification by the process of the invention, it does not carry any sequence 25 , similar to that of the recombinase recognition site integrated in the course of implementation of the process of the invention. As a result, the recombinase recognition site integrated in the genome of the cell line cells of the invention is unique, since no other identical sequence is found in the genome of cells in said cell line.

Integration of a first nucleic acid in a mammalian cell, as undertaken in Step 1) of the process, can be achieved using any of the ways known to those skilled in the art. By way of example, mention can be made of: the calcium phosphate (CaPO4) precipitation method in which the precipitated DNA is "incorporated" by the cells by phagocytosis; alternatively, the lipofection method which involves coating the DNA to be integrated in lipid vesicles which can fuse with the host cell membrane; another possible method is that of electroporation in which administering an electric shock to the cell leads to integration of the DNA of interest; another possible method is that of pronuclear microinjection.

The targeting vector used in the first step of the process of the invention consists of a nucleic acid molecule containing two recombinase recognition sites. These sites are identical both to one another and to the unique site in the cell line generated by means or the process of the invention.
These two sites flank a DNA fragment which contains a reporter gene, a gene encoding a protein similar to the proteins of interest, a gene conferring resistance to an antibiotic, and a selector gene which permits gene amplification. The targeting vector also includes all the sequences necessary for expression of the genes inserted between the two recombinase recognition sites. Among these 26 .

necessary sequences, mention can be made, without any limitation, of promoter, enhancer and polyadenylation sequences.

Integration of the targeting vector in the genome of the transfected cell occurs in a random fashion. The targeting vector could therefore insert in a region which is inactive, relatively inactive, moderately active or highly active vis-a-vis transcription. The cell lines in which the targeting vector has been inserted in a highly transcriptionally active region are selected on the basis of the expression or over-expression of a reporter gene.

Expression of this reporter gene-the product of which is readily detected and analysed-constitutes a means of assessing the level of transcriptional activity of the surroundings in which said reporter gene is integrated: if transcriptional activity at the insertion locus is low, the activity of the reporter gene will be low; in contrast, if transcriptional activity at the insertion locus is high, the activity of the reporter gene will be high.

The gene encoding a protein similar to the protein of interest, located downstream of the reporter gene, makes it possible to determine the capacity of the cell containing the transgenes to secrete proteins, a parameter which cannot be determined using a fluorescent protein expressed inside the cell. Any protein which can be readily detected can be used for this purpose. Among the proteins that can be used in the context of this invention, mention can be made, without this constituting any limitation, of the products of the genes for immunoglobulins, growth factors, interleukins, stimulatory factors, kinases, coagulation factors, alpha-antitrypsin and 27, albumin.

The antibiotic resistance gene makes it possible to select for transformed cells carrying the targeting vector.
Selection is undertaken by placing the transformed cells in contact with the relevant antibiotic so that only those cells which have incorporated the targeting vector survive. The antibiotic resistance gene is followed by a polyadenylation sequence ("polyA") which plays an important role in stabilising the corresponding mRNA molecules (56-63). Any polyadenylation sequence known to those skilled in the art which permits expression of the antibiotic resistance gene can be used, although is preferably used a weakly active polyA sequence Advantageously, the polyadenylation sequence is a weak polyadenylation sequence, i.e. a relatively ineffective polyadenylation sequence. In consequence, if the targeting vector has been inserted in a transcriptionally inactive region, the antibiotic resistance gene will not be expressed at a high enough level to enable the cell to survive when exposed to the antibiotic. In contrast, if the insertion locus is in a highly transcriptionally active region, the "weakness" of the polyadenylation sequence will not prevent expression of the product of the resistance gene and the corresponding cells will be resistant to the antibiotic. The use of such a weak polyadenylation sequence, especially when the selection pressure is strong (at high antibiotic concentrations), will substantially reduce the number of clones that have to be screened and will contribute to identifying clones that may be "highly productive" for the proteins of interest.
By way of example of a weak polyadenylation sequence, 28 .

mention can be made of the "SV40 early polyadenylation signal" a signal which is used in certain commercially available expression vectors (64-66).
Advantageously, the cells can also be selected for their resistance in the presence of a strong concentration of antibiotic. Combination of these two selection tools, in other words use of a weakly active polyadenylation sequence and the use of a strong concentration of antibiotic in the medium makes this manner of selection a particularly advantageous means for selecting cells in which the transgene has been integrated at the desired place, in other words at the recombinase recognition site, without selecting those cells in which the recognition site for recombinase has been integrated into a region inducing a lower transcriptional activity. In effect, the fact that the polyadenylation sequence only is weakly active has the effect that the gene of resistance to the antibiotic situated directly upstream of this site is only expressed to a small degree. Thus, if the concentration of antibiotic added in the medium is high (for example greater than or equal to 1 g/l, or yet again greater than or equal to 2 g/l, or to 4 g/l, or greater than or equal to 8 g/l), only the cells that have integrated the transgene in a region allowing sufficiently strong expression of the gene of resistance to the antibiotic, and this despite the low efficiency or weak activity of the polyadenylation sequence, survive. This manner of selection which simultaneously implements the weak activity of the polyadenylation sequence and the addition of a strong concentration in antibiotic to the cell culture medium, has several advantages: less screening of clones becomes possible since clones that have integrated the transgene elsewhere in the genome than at a recombinase recognition site die under the effect of the strong concentration in antibiotic present in the medium. Moreover, the selected clones have greater capacities for production when compared to a method of selection performed with processes of the prior art.

Advantageously, the targeting vector is cell linearised using a restriction enzyme before transfection into the chosen cells. In order to prevent conjugation of multiple copies of the targeting vector before integration into a cell's genomic DNA, the restriction enzyme used should create blunt ends. Moreover, only small quantities of the targeting vector are used in the transfection procedure. These specific conditions allow to substantially reduce the number of copies of the vectors which will be integrated into the cellular genome. In practice, for satisfactory excision, it is important that only a single copy of the targeting vector be integrated into the genome of the transfected cell, so that excision subsequently occurs between the only two recombinase recognition sites present. Similarly, the process of the invention can only be successfully implemented if a single highly transcriptionally active region has been targeted.

The targeting vector also includes a polyadenylation sequence downstream of the second recombinase recognition site. This polyadenylation sequence is designed for use for the later integration of a different vector carrying the gene for a protein of interest. Thus, as will be described in detail below, this polyadenylation sequence will make it possible to select cells which have integrated the second vector at the recombinase recognition site. Advantageously, this polyadenylation sequence is relatively ineffective and can be considered as a "weak polyadenylation sequence"; one example is the SV40 early polyadenylation sequence called "SV40 early polyadenylation signal"

301, Advantageously, the targeting vector carries a gene which permits implementation of a gene amplification mechanism. This gene could for example, be the gene for dihydrofolate reductase (DHFR) or glutamine synthethase (GS), or metabolic enzymes which are essential to cell survival.
When transfected cells, which have integrated the targeting vector, are cultured with increasing concentrations of a specific inhibitor of the relevant enzyme, (e.g. methotrexate for DHFR and methionine sulfoximine for GS), only those in which the vector has been multiplied will express enough DHFR
or GS to survive (23,30) . The extent of gene amplification obtained varies, essentially depending on the region of the genome into which the vector has been integrated. The integration region of the targeting vector is therefore chosen for its "amplification capacity" in order to guarantee that it will be subsequently possible to amplify expression of the transgene of interest.

In addition, in another particular embodiment of the invention, the targeting vector carries, between the two recombinase recognition sites, the gene for the thymidine kinase of Type I Herpes simplex virus (HSV1-TK). Cell selection in Step 4) is based on adding ganciclovir to the culture medium: cells from which the targeting vector has not been excised will be killed by the ganciclovir while those in which the targeting vector has been excised will survive. It is worth noting that any suicide gene based on using any potentially toxic "prodrug" other than HSVl-TK could be used in this Step. Mention could be made, for example, of the CodA
and Fcy genes with the prodrug 5fluorocytosine (5-FC); this list is not limiting.

The recombinase recognition site used in the context of this invention can correspond to any site known to those skilled in the art. By way of example, mention can be made of the loxP and the FRT sites. In one embodiment of the invention, the loxP and FRT sites are used at the same time.
By way of example, this embodiment can be used to insert an expression vector using FRT, then remove this vector's selector gene after integration in the genome using loxP.

The reporter gene encoding a protein similar to a protein of interest may correspond to any gene encoding a secreted protein which is identical or similar to a protein of therapeutic or industrial value. By way of example, mention can be made of genes encoding antibodies, growth factors, interleukins, stimulatory factors, kinases, coagulation factors, alpha-1 antitrypsin or albumin, this list being non-limiting.

The additional expression of a protein similar to a protein of interest as well as the protein encoded by the reporter gene makes it possible, in Step 2), to select cells not only on the basis of the level of expression of the reporter gene but also on the basis of the cells' secretory capacities.
Advantageously, the protein of interest is an antibody.
In this case, it would be particularly advantageous to include in the vector the genes encoding both the heavy chain and the light chain of said antibody. In addition, if the protein of interest is an antibody, it would be useful, at Step 2), to select antibody-producing cells on the basis of the rate or the form of the foreseen glycosylation.

32, Advantageously, the original mammalian cell used in the process of the invention is chosen from: rat myeloma cell lines, notably YB2/0 (ATCC CRL-1662) and IR983F, human myelomas like Namalwa or any other human cell such as PERC6, CHO cell lines (notably CHO-K, CHO-LeclO, CHO-Lecl, CHO Pro-5, CHO dhfr-, CHO Lec13, avian cell lines or other cell lines such as Wil-2, Jurkat, Vero, Molt-4, COS-7, K6H6 and P3X63Ag8.653.
In a particularly advantageous manner, the YB2/0 cell line is used.

At Step 2) are selected highly productive cells producing the protein of interest at a rate of over 5 pcd (picograms of protein per cell per 24 hours). Advantageously, the production rate of the cells of interest is over 10 pcd.
In a particularly advantageous manner, the rate of production of the cells of interest is over 15 pcd, and more particularly over 20 pcd. Advantageously, this production rate is between 5 and 50 pcd, or more particularly between 10 and 30 pcd.

The number of copies integrated in the original mammalian cell can be estimated using any method known to those skilled in the art. By way of example, mention can be made of the quantitative Polymerisation Chain Reaction (PCR) assay.

At the targeting vector excision step (Step 3), the recombinase mediates a recombination event at both recombinase recognition sites and induces excision of the targeting vector from the cellular genome, while a recombinase recognition site remains in the genome. The excision mechanism implemented in the process of the invention leads to the removal of all the nucleotide sequences between the two recombinase recognition sites of the integrated targeting vector. The recognition site remaining in the highly transcriptionally active region of the genome is still in the same orientation as the original recognition site in the integrated targeting vector. In addition, after excision of the targeting vector, a polyadenylation sequence is left in the mammalian cell downstream of the remaining recombinase recognition site. The integrity of the remaining recombinase recognition site could be checked using any method known to those skilled in the art, e. g. PCR (Polymerisation Chain Reaction) followed by sequencing of the amplified DNA sequence.

By way of example, the recombinase could be expressed in the cell through transient transfection of a vector encoding said recombinase, with which the cell would have been transfected after Step 2.

It is worth noting that, if the recombinase recognition site is the loxP site, the Cre recombinase is used. In contrast if the recognition site is FRT, the recombinase used is Flp. Advantageously, in the context of this invention, the recombinase used is Flpe, a recombinase derived from Flp which is more active in the culture conditions used to grow mammalian cells (55).

The excision step therefore removes all the active sequences that might affect subsequent expression of future proteins of interest to be inserted in the highly transcriptionally active region using the FRT recombination sequence left in the genome or the transfected cells.
Therefore no active transgenic elements are left in the cell line after excision of the targeting vector.

Cell lines generated in the first step of the process of the invention which carry a unique recombinase recognition site inserted in a highly transcriptionally active region of their genome ("highly productive cell lines") can be used to produce any protein of interest. Such a protein could subsequently be produced by targeting the unique recombination site with a vector carrying the sequences required for the transcription of a protein of interest imported on said vector. Such an application of the highly productive cell line of the invention will be described in detail below.

Another object of the invention concerns a process as defined above including a second step designed to insert a DNA molecule of interest into a cell line generated in the first step of the process of the invention. This second step includes the following stages:

5) transfection of a cell line generated in Step 4) with an expression vector carrying a gene encoding a protein of interest, a gene permitting gene amplification and a gene conferring resistance to an antibiotic, without any polyadenylation sequence, this vector being localised directly upstream of a recombinase recognition site 6) insertion of said expression vector at a unique recombinase recognition site, mediated by said recombinase;

7) the selection of cells carrying the expression vector integrated at the target site by assaying for expression of the protein of interest. Advantageously, selection is performed in the presence of a strong dose of antibiotic in order to discourage random integrations.

The expression vector used in the reintegration step of the process includes a gene coding for a protein of interest, a gene permitting gene amplification, together with all the 5 sequences necessary for expression of the coding sequences present in the vector, i.e. promoters, enhancers and polyadenylation sequences. This expression vector also comprises a gene conferring resistance to an antibiotic with its promoter, without any polyadenylation sequence, this 10 vector being localised directly upstream of a recombinase recognition site The antibiotic resistance gene carried on said expression vector does not have a polyadenylation sequence.
15 The antibiotic resistance gene must therefore be positioned at the end of the vector so that it is inserted directly upstream of the polyadenylation sequence left in the cell's genome after excision of the targeting vector. Optionally, the antibiotic resistance gene is the same as that on the 20 targeting vector so that cell selection can be carried out using the same concentration of antibiotic as was used to select the highly productive cells in Step 2).
In this case, selection is carried out by exposing the cells to the antibiotic corresponding to the gene conferring 25 resistance.

The recombinase mediates insertion of the expression vector in the cellular genome at the unique recombination site left after Step 4). To do this, the recombinase 30 recognition site in the expression vector is identical to the one integrated in the cellular genome in the first part of the process.

As described above, if the recombinase recognition site is the loxP site, the Cre recombinase is used; if the recognition site is FRT, the recombinase used is Flp.
Moreover any way of getting the recombinase to work in the cell can be implemented in Step 6) of the process of the invention. By way of example, the recombinase could be produced in a cell transfected with a vector carrying a gene encoding said recombinase.

Cells which have not integrated the expression vector at the recombinase recognition site will die consequently due to the high concentrations of antibiotic used. Indeed the antibiotic resistance gene does not have a polyadenylation sequence downstream of its coding sequence will therefore only be effectively expressed if the gene is inserted close to the polyadenylation sequence left in the cellular genome after Steps 3) and 4).

Advantageously, the protein of interest resulting from expression of the DNA of interest in the expression vector can be any protein of therapeutic or industrial value, e. g.
antibodies, coagulation factors, cytokines growth factors, enzymes or hormones, this list being non-limiting. In addition, the gene encoding the protein of interest can be the same as that carried on the targeting vector, or it can be a gene encoding a different protein.

Another object of the invention concerns a cell line carrying a unique recombinase recognition site integrated in a highly transcriptionally active region of the genome, said cell line having integrated a single copy of a transgene at said recombinase recognition site, said cell line being stable over time and able to be obtained in Step 4) of the 371, process of the invention.

Advantageously, this cell line is the YGM-1/10G10 cell line (application number CNCM 1-3704 submitted on December 18 2006 to the Collection Nationale de Culture de Microorganismes (CNCM). , The YGM-1/10G10 cell line is generated by implementation of the process of the invention starting from YB2/0 cell (ATCC CRL-1662) at Step 1) of the process. This YGM-1/10G10 cell line possesses the following characteristics: no active sequences (promoters, selector gene, antibiotic resistance gene), a recombinase recognition site, stable culture parameters and a stable integration site. In addition, this cell line advantageously has a rate of production of the protein of interest of over 5 pcd (picograms of protein per cell per 24 hour day).
Advantageously, the rate of production of the cells of interest is over 10 pcd. In a particularly advantageous manner, the rate of production of the cells of interest is over 15 pcd, and more particularly 20 pcd. Advantageously, the production rate is between 5 and 50 pcd, or more particularly between 10 and 30 pcd.

Thus, an object of the invention is a new expression cell line, YGM-1/10G10, devoid of any active transgenic sequence, derived from the YB2/0 cell line, in which integration of the expression vector can be controlled and directed to a region favourable for transcription by using a recombinase.

According to a further object of the invention, this cell line is the YGM-2/3G5 cell line (application number CNCM
1-3885 submitted on December 19 2007 to the Collection Nationale de Culture de Microorganismes (CNCM). The YGM-2/3G5 38, cell line is generated by implementation of the process of the invention starting from YB2/0 (ATCC CRL-1662) at Step 1) of the process. This YGM-2/3G5 cell line possesses the following characteristics: no active sequences (promoters, selector gene, antibiotic resistance gene), a recombinase recognition site, stable culture parameters and a stable integration site. In addition, this cell line advantageously has a rate of production of the protein of interest of over 5 pcd (picograms of protein per cell per 24 hour day).
Advantageously, the rate of production of the cells of interest is over 10 pcd. In a particularly advantageous manner, the rate of production of the cells of interest is over 15 pcd, and more particularly 20 pcd. Advantageously, the production rate is between 5 and 50 pcd, or more particularly between 10 and 30 PCD.

Thus, an object of the invention is a new expression line, YGM-2/3G5, devoid of any active transgenic sequence, derived from the YB2/0 cell line, in which integration of the expression vector can be controlled and directed to a region favourable for transcription by using a recombinase.

The new YGM-1/10G10 and YGM-2/3G5 cell lines present the following properties and advantages:
- Reproducible constant, high level expression with each transfection procedure as a result of guaranteed integration at the same site;
- No active transgenic sequences (promoters, resistance genes, transcription enhancers) are present to distort the properties of the original YB2/0 cell line, thus facilitating exploitation following subsequent integration of an expression vector carrying the gene for a protein of interest;

391.
- Savings in terms of both time and resources because of the reduction in the number of transfectants that has to be screened;

The possibility of gene amplification (e.g. using a DHFR
methotrexate system) afforded by the selection of an "amplifiable" integration site.

Advantageously, the cell line generated in the process of the invention is stable over time for a period of at least three months, i.e. about 80 generations.

Another object of the invention concerns a nucleic acid molecule isolated from the SEQ ID NO: 1 sequence including a fragment of nucleic acid with the SEQ ID NO: 4 sequence, said fragment being capable of enhancing the expression of a recombinant protein of interest when incorporated into an expression vector or the highly transcriptionally active region including the SEQ ID NO: 1 sequence or a fragment of nucleic acid which is at least 80% homologous with the SEQ ID
NO: 1 sequence, said fragment being capable of enhancing the expression of a recombinant protein of interest when incorporated in an expression vector.

The nucleic acid sequence corresponding to the nucleic acid molecule isolated from the SEQ ID NO: 1 sequence including a nucleic acid fragment capable of enhancing the expression of a recombinant protein of interest when incorporated in an expression vector, is the following:

40, 1651 C"ITT"TTGATA ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA

2351 TACGGTTCCT GGCCTTT'I'GC TGGCCTTTTG CTCACATGGC TCGACAGATC

411, 4001 AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAA.TA CATTCAAATA

The nucleic acid molecule isolated from the SEQ ID NO: 1 sequence including a nucleic acid fragment capable of enhancing the expression of a recombinant protein of interest when incorporated in an expression vector with the sequence presented above possesses the following characteristics (positions are given according to the numbering of nucleotides in the sequence, said numbering system having been detailed above):
- from Position 1 to Position 1024: genomic sequence;

- from position 2631 to Position 2678: Frt site;
- from Position 1025 to Position 4419: sequence of the targeting vector left in situ after deletion.

The nucleic acid sequence corresponding to the nucleic acid fragment capable of enhancing the expression of a recombinant protein of interest when incorporated in an expression vector is the following (SEQ ID NO: 4):

I TGGAAACAGA AACTAAATAG AGACATAGAG AAAATGAACA GAAGTTATGA

Another object of the invention concerns a vector including the SEQ ID NO: 1 nucleic acid sequence.

Another object of the invention is a cell line carrying in its genome a unique recombinase recognition site integrated in a highly transcriptionally active region, said cell line having integrated a single copy of a transgene at said recombinase recognition site, said cell line being stable over time as well as being possible to generate using the process of the invention.

Such a cell line carries two sequences in tandem corresponding on either side to recombinase recognition sites and a gene encoding a protein of interest and a gene conferring resistance to an antibiotic, located immediately upstream of the downstream recombinase recognition site, with the antibiotic resistance gene's polyadenylation sequence immediately downstream of this recognition site to the antibiotic.
Advantageously this cell line has, prior to gene amplification, a productivity from said transgene of between 5 and 50 pcd (picograms of protein per cell per 24 hours) Advantageously, this cell line expresses said transgene in a stable fashion for a period of at least three months.
Another object of the invention concerns a production process for proteins of interest, in which the cell line of the invention expressing a protein of interest is cultured in such a way that said protein of interest is expressed and can be harvested.

Another object of the invention is a vector for inserting a DNA molecule of interest into the genome of a mammalian cell ("targeting vector"), carrying two recombinase recognition sites located either side of a reporter gene, a gene encoding a protein of interest, a selector gene permitting gene amplification and a gene conferring resistance to an antibiotic, and a polyadenylation sequence downstream of the second recombinase recognition site.
Advantageously, the antibiotic resistance gene's polyadenylation sequence is a weak polyA which results in higher production rates after screening.
Advantageously, this vector carries, between the two recombinase recognition sites, a gene encoding the thymidine kinase of Type I Herpes simplex virus (HSV1-TK).

44 :

Another object of the invention relates to a system of vectors for inserting a DNA molecule of interest into a target site in the genome of a mammalian cell, including at least the following constituents:
- a targeting vector as described above, - an expression vector carrying a recombinase recognition site downstream of a gene encoding a protein of interest and a gene conferring resistance to an antibiotic, the recombinase recognition site being located directly upstream of the gene conferring resistance to antibiotics.

Another object of the invention concerns the use of the above-mentioned vector system in a process for inserting a DNA molecule of interest into a target site in the genome of a mammalian cell, including the following steps:
- transfection of a mammalian cell with said targeting vector;
- the selection of "highly productive" cells in which a single copy of the targeting vector has been integrated;
- excision of the targeting vector using a recombinase;
- the selection of cells from which the targeting vector has been excised and which possess an intact recognition site;
- transfection of the cells selected in Step 4) with said expression vector;
- integration of the expression vector at the intact recognition site using the recombinase;
- the selection of cells carrying the expression vector integrated at the target site by testing for expression of the protein of interest. Advantageously, selection will be performed in the presence of a strong dose of 45, antibiotic in order to discourage random integrations.
Other aspects and advantages of the invention will be described in the following examples which are presented for the purposes of illustration and which are not to be taken as limiting the scope of the invention.

EXAMPLES
Example 1: Construction of vectors used to generate the cell line in which a unique recombinase recognition site is integrated in a highly transcriptionally active region a. Targeting vector pTV1 (see Figure 1):
- Transcription units: the targeting vector pTV1 has been constructed (SEQ ID NO: 2) (Fig. 1) with the following transcription units:
1- the maxFP-Green transcription unit (a fluorescent reporter gene) containing (in order) the minimum CMV
promoter (the promoter of the early human cytomegalovirus gene without either its activating part or enhancer), the gene encoding the maxFPTM-Green protein (Evrogen) and the late SV40 (Simian Virus 40) polyadenylation sequence (polyA) 2- the transcription unit of the heavy chain of the anti-D
antibody in the order of a RSV promoter (Long Terminal Repeat of the Rous Sarcoma Virus), an artificial intron derived from the pCi-neo vector the heavy chain sequence of anti-D immunoglobulin and the bGH (Bovine Growth Hormone) polyA sequence, 3- the transcription unit of the light chain of the anti-D
antibody containing the same components as the antibody heavy chain transcription unit apart from the immunoglobulin light chain sequence and the bGH polyA
sequence.

46 ~

4- the neo transcription unit containing the SV40 promoter, the neomycin resistance gene and the sequence of the SV40 early polyadenylation signal which has "weak" polyA activity, 5- the DHFR (dihydrofolate reductase) transcription unit containing the SV40 promoter, the DHFR selector gene modified by directed mutagenesis in order to abolish the Scal restriction site (which mutation is silent in the gene product) and its polyA site, 6- the HSV1-TK transcription unit containing the SV40 promoter, a suicide gene encoding the thymidine kinase of Type 1 Herpes Simplex virus (HSV1-TK) and the "SV40 early polyA" sequence.

- Synthesis or Frt recombination sites:

The Frt recombination sites are synthesised by PCR using the following primers:

Sense primer (SEQ ID NO: 5):

5'-ACAGCTGTCGACTGAAGTACCTATTCCGAAGTTCCTATTCTCTAGAAAGT-3' Anti-sense primer (SEQ ID NO: 6):
5'-CGTCCGGATATCTAAGATCTGAAGTTCCTATACTTTCTAGAGAATAGGAA-3' The resultant PCR product contains the Frt site (in bold italics) as well as restriction sites (undercell lined) permitting subsequent cloning in the pTV1 targeting vector pTV1 (SEQ ID NO: 7):

AGACGTCT CGACT GAAGTACCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC
SalI Frt site AGATCTTAGATATCCGGACG
BglII EcoRV

A first Frt site (Frtl) was cloned upstream of the maxFP-47, Green transcription unit on the pTV1 vector. A second Frt site (Frt2) was cloned in the same sense as Frtl between the HSV1-tk gene and its polyadenylation site the "SV40 early po1yA".

The nucleic acid sequence corresponding to the targeting vector pTV1 is the following (SEQ ID NO: 2):

48, 51, 8051 AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTT"TGTCCAA ACTCATCAAT

9151 AAGACTAACA GGAAGATGCT T1'CAAGTTCT CTGCTCCCCT CCTAAAGCTA

9551 TTC,CTTCAGA ATTGCTAAGT TTTTTGAGTC ATGCTGTGTT TAGTAATAGA

9751 GTGTCTGCTA TTAATAACTA TGCTCAAAAA TTGTGTACCT TTAGCTTI"I'T

10001 AGCATCACAA ATTTCACP.AA TAAAGCATTT TTTTCACTGC ATTCTAGTTG

13101 CT"T"I"I"I'TGCG GCATTTTGCC TTCCTGTTTT TGCTCACCCA GAAACGCTGG

The pTVl targeting vector, the sequence of which is shown above (SEQ ID NO: 2) possesses the following characteristics (positions are given according to the numbering of nucleotides in the sequence, said numbering system having been detailed above):
- from Position 1 to Position 48: FRT recombinase site;
- from Position 63 to Position 144: minimum CMV promoter;
- from Position 478 to Position 1176: maxFP-green protein;
- from Position 1352 to Position 1402: late SV40 55, polyadenylation signal;
- from Position 1888 to Position 2283: RSV LTR;
- from Position 2521 to Position 3951: heavy chain (H) Ig anti-D=
- from Position 3958 to Position 4184: bGH polyadenylation signal;
- from Position 4431 to Position 4826: RSV LTR;
- from Position 5064 to Position 5771: bGH Kappa light chain (K) Ig anti-D;
- from Position 5778 to Position 6004: bGH polyadenylation signal;
- from Position 6658 to Position 6983: SV40 promoter;
- from Position 7019 to Position 7813: sequence encoding neo phosphotransferase;
- from Position 7987 to Position 8117: SV40 early polyadenylation signal;
- from Position 8301 to Position 8496: SV40 promoter;
- from Position 8595 to Position 9158: DHFR coding sequence;
- from Position 9965 to Position 10019: late SV40 polyadenylation signal;
- from Position 10092 to Position 10394: SV40 promoter;
- from Position 10442 to Position 11572: HSV-TK coding sequence;
- from Position 11584 to Position 11631: FRT recombinase site;
- from Position 11974 to Position 12104: SV40 early polyadenylation signal (SV40 early polyA);
- from Position 12996 to Position 13926: ampicillin resistance gene.

b. pEFrat-FLPe vector (SEQ ID NO: 3, Fig. 2):

The gene encoding the Flp recombinase was PCR-amplified 56, from the pOG4-FLPe vector (55) using the following primers:
Sense primer (SEQ ID NO: 8):
51- ATCTGGCTAGCCGCCACCATGCCACAATTTGATATATTAT-3' The undercell lined sequence is the NheI restriction site and the beginning of the FLPe gene is in bold with the initiator ATG in italics.

Anti-sense primer (SEQ ID NO: 9):

51- TGTCATCTAGATTATTATATGCGTCTATTTATGT-3' The undercell lined sequence is the XbaI restriction site. The end of the coding sequence is shown in bold with the Stop codon in italics.

The resultant PCR FLPe product was cloned between the rat EF1-alpha promoter and a bGH polyadenylation sequence at the NheI and XbaI sites, in order to obtain the final pEFrat-FLPe expression vector.

The nucleic acid sequence of the pEFrat-FLPe vector is as follows (SEQ ID N 3):

901 GAGCl'CAAAA TGGAGGACGC GGCAGCCCGG TGGAGCGGGC GGGTGAGTCA

1251 CCACCATGCC ACAATTTGAT )"=TATTATGTA AAACACCACC TAAGGTCCTG

1451 AATTCGCTGA GTTTCGATAT TGTCAACAAA. TCACTCCAGT TTAAATACAA

58, 4451 C.CTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT

The pEFrat-FLPe vector, the sequence of which is presented above (SEQ ID NO: 3), possesses the following characteristics (positions are given according to the numbering of nucleotides in the sequence, said numbering system having been detailed above):

- from Position 1 to Position 1243: rat EF-lalpha promoter;
- from Position 1256 to Position 2527: Flpe coding sequence;
- from position 2537 to Position 2763: bGH
polyadenylation signal;
- from Position 3020 to Position 3543: Col El replication origin;
- from Position 4887 to Position 4027: ampicillin resistance gene.

c. pT125-FRT reintegration vector (Fig. 3):

The pT125-FRT reintegration vector is derived from the T125-IG24 vector (Fig. 4) in which the CMV promoters have been substituted by RSV promoters and the neomycin resistance gene's polyadenylation sequence has been deleted and replaced by a Frt site inserted in the same direction as the Frt sites in the pTV1 vector.
Example 2: targeting of regions of high transcriptional activity in the YS2/0 cell line a. Transfection of the YB2/0 cell line with the pTV1 vector The rat cell line YB2/0 (ATCC # CRL-1662) was grown in EMS
medium (Invitrogen, ref. 041-95181M) supplemented with 5%
foetal bovine serum (FBS). 5 million cells were electroporated (BioRad electroporator, model 1652077) using an Optibuffer transfection kit (Thermo Electron).
For each transfection, 5lig of pTVl vector cell linearised using the ScaI restriction enzyme were used.
Electroporation was undertaken at 230 volts and 950 microfarads in a 0.5ml cuvette. The contents of each electroporation cuvette were then divided between 10 96-well plates at a density of 5,000 cells per well. Three days after transfection, the cells were transferred to a selective medium of EMS supplemented with 5% FBS and 2mg/m? G418 (Invitrogen, ref. 10131-027).

b. Fluorescence screening for G418-resistant clones 10 and 15 days after transfection, resistant clones were identified on the basis of the fluorescence of the maxFPTM-Green protein maxFPTM-Green (excitation at 10 482 nm, Emission at 502 nm). All the resistant clones were first screened in a fluorescence plate reader (VICTOR3, Perkin Elmer). All fluorescent clones were then run in a flow cytometer to measure each cell's fluorescent intensity and the homogeneity of the clones.
15 The results were analysed using free WinMDI 2.8 (http://facs.scripps.edu) software. All clones with a homogenous fluorescent peak and a mean fluorescent intensity (MFI) of over 500 were sorted and transferred to 24-well plates for amplification.
c. ELISA screening and copy number determination The cells were maintained in 24-well plates and supernatants were assayed (ELISA) to estimate anti-D
antibody production. All clones with a productivity of more than 10 pcd (picograms per cell per 24 h) were then subjected to semi-quantitative PCR to estimate the number of copies of the pTV1 vector. The copy number estimate was made using a ABI PRISM 7000 machine using CELL LINE sequences (the number of which is stable in clones generated from the YB2/0 cell line) as the standardising control. Clones found to contain a single copy were then subjected to Southern blotting to confirm the copy number.

d. Methotrexate (MTX) gene amplification Gene amplification tests using MTX (Sigma, ref. M8407) were carried out on highly productive clones carrying a single copy in order to estimate the amplification capacities of the locus targeted by the pTV1 vector. MTX
concentrations of 25 nM and over were added to the EMS
medium (+ 5% FBS) . After 15 days of culture in the selective medium, resistant clones were subjected to ELISA to check that productivity had increased and to quantitative PCR in order to check that the copy number had increased. The productivity of the 3G11 clone (9.7 pcd) had risen to 22 pcd after a single amplification cycle. Identically the productivity of the 35H4 cloid (11.4 pcd) has risen of approximately 3 times after an amplification cycle Example 3: Excision of the PTV1 targeting vector using the F1p recombinase; generation of the YGM-1/10G10 and YGM-2/3G5 cell lines (Fig. 5) = YGM-1/10G10 cell line Highly productive clones 8A10 and 3G11 (6.6 pcd and 9.7 pcd respectively before amplification) carrying single copies of the pTVl vector were selected to generate a YGM cell line by deleting the targeting vector.

Clone 8A10 was first amplified in T75 flasks in EMS
medium (5% FBS) with 2mg/mk G418. The day before transfection, the G418 was removed from the culture medium.

An aliquot of 5~ig of the non-linearised pEFrat-FLPe vector resuspended in 175 e serum-free EMS together with 25ut Superfect was incubated at room temperature for 10 62, minutes before mixing with 1 ml EMS (5% FBS). This mixture was added to 2x106 PBS-washed cells and diluted to 2.5x105 cells/mk in EMS medium (5% FBS). After 4 hours incubation at 37 C, the cells were transferred to EMS medium (5% FBS).
After 24 hours of culture, the cells were dispensed into 96-well plates at a density of 5,000 cells per well.

The cells were transferred to selective medium two days after transfection in EMS medium (5% FBS) supplemented with 4 uM ganciclovir (Invivogen, ref. sud-gcv).

After 15 days of culture, surviving clones tested in by flow cytometry to check for the absence of fluorescence, by ELISA for the absence of anti-D immunoglobulin, and by PCR
for complete excision of the pTV1 vector using the following primers:
5 PTV1 (SEQ ID NO: 10): 5'-CCTATGGAAAAACGCCAGCAAC-3' 3 PTV1 (SEQ ID NO: 11): 5'-CCTTAGAAAGCGGTCTGTGAAA-3' Of the excised clones, clone 10G10 was chosen to constitute the YGM-1/10G10 expression cell line and checked for stability of the integration site over a period of three months.
= YGM-2/3G5 cell line Clones 35H4(1)2G2 (13.3 pcd), a highly productive derivative of the cloid 35H4 (11.4 pcd before amplification) carrying single copies of the pTV1 vector, was selected to generate a YGM cell line by deleting the targeting vector.
Clone 35H4(1)2G2 was first amplified in T75 flasks in RPMI medium (5% FBS) with lmg/mf G418). Two sub-culture before transfection the G418 was removed from the culture 63 .
medium.

An aliquot of 25 -pg of the non-linearised pEFrat-FLPe vector resuspended in 500 k serum-free RPMI together with 75~iP Fugene HD (Roche) was incubated at room temperature for minutes. 120 uf of this mixture was added in each well of 6-well plates containing 6xl05 cells/mi RPMI medium (5% FBS).
After 24 hours of culture, the cells were dispensed into 96-well plates at a density of 1,000 cells per well.
The cells were transferred to selective medium three days after transfection in RPMI medium (5% FBS) supplemented with 4 ~iM ganciclovir (Invivogen, ref. sud-gcv).

After 15 days of culture, surviving clones tested in by flow cytometry to check for the absence of fluorescence, by ELISA to check the absence of anti-D immunoglobulin, and by PCR for complete excision of the pTV1 vector using the following primers:

DEL REV (SEQ ID NO: 14): 5'-TGGTATGGCTGATTATGATCCTC-3' DEL FOR 3 (SEQ ID NO: 15): 5'-CCTTTTGCTCACATGGCTCGAC-3' Of the excised clones, clone 35H4(1)2G2 was chosen to constitute the YGM-2/3G5 expression line and checked for stability of the integration site over a period of three months.

Example 4: Re-integration of an expression vector in the YGM-1/10G10 and YGM-2/3G5 cell lines (Fig. 6) = YGM-1/10G10 cell line A vector encoding the same antibody as was used in the screening step (anti-D antibody) was re-integrated in order to check the reproducibility of expression levels after re-integration at the Frt site in YGM-1 (clone 10G10).

The clone 10G10 generated by excision of the TV1 targeting vector TV1 which carries a single Frt recombination site and a polyadenylation sequence corresponding to the "SV40 early polyA", was amplified in T75 flasks in EMS medium (5% FBS)-Cotransfection was carried out with 10 ~ig of non-linearised pEFrat-FLPe vector and 5~ig of non-linearised pT125-FRT reintegration vector. 5 million cells were electroporated in a 0.5 ml cuvette (BioRad electroporator, model 1652077) using an Optibuffer transfection kit (Thermo Electron) with the following parameters: 230 volts and 950 microfarads.

The cells were then dispensed into ten 96-well plates at a density of 5,000 cells per well.

The cells were transferred to selective medium two days after transfection in EMS medium (5% FBS) supplemented with 2mg/mk G418.

After 15 days of culture, surviving clones were amplified in 24-well plates and screened by PCR to check for re-integration at the Frt site of the YGM-1 cell line.

For this screening the m5NEO-2 and the SV40polyA-lrev primers were used:
m5NEO-2 primer SEQ ID NO: 12):
5'-GATGCCTGCTTGCCGAATA-3' SV40polyA-lrev primer SEQ ID N0: 13):

5'CCTTAGAAAGCGGTCTGTGAAA-3' Clones with random integration in the genome of the YGM-1 cell line were rejected. Clones in which the pT125-FRT
vector was integrated at the Frt site of the YGM-1 cell line were subjected to ELISA in order to measure productivity of 5 the anti-D T125 antibody.

Clone 21B10 (5.5 pcd) was chosen to check for stable re-integration over a period of three months.

10 = YGM-2/3G5 cell line A vector encoding the same antibody as was used in the screening step (anti-D antibody) was re-integrated in order to check the reproducibility of expression levels after re-integration at the Frt site in YGM-2 (clone 35H4(2)3G5).

The clone 35H4(2)3G5 generated by excision of the TV1 targeting vector TV1 which carries a single Frt recombination site and a polyadenylation sequence corresponding to the SV40 early polyadenylation signal, was amplified in T75 flasks in RPMI medium (5% FBS).

Cotransfection was carried out with 4 pg of non-linearised pEFrat-FLPe vector and 2jig of non-linearised pT125-FRT reintegration vector.

The two non-linearised vectors diluted in 300 ~if serum-free EMS together with 18 k Fugene HD (Roche) were incubated at room temperature for 15 minutes. 75 uf of this mixture was added in each well of 6-well plates containing 6x105 cells/ml EMS medium (5% FBS). After 24 hours of culture, the cells were dispensed into 96-well plates at a density of 1,000 cells per well.

66, The cells were then dispensed into 10 96-well plates at a density of 1,000 cells per well.

The cells were transferred to selective medium two days after transfection in EMS medium (5% FBS) supplemented with 3mg/ml G418.

After 12 days of culture, surviving clones were amplified in P24-well plates and screened by PCR to check for re-integration at the Frt site of the YGM-2 cell line.

For this screening the following primers were used:
- checking of the 5'integration DEL FOR 3 (SEQ ID NO: 15): 5'-CCTTTTGCTCACATGGCTCGAC-3' 3FRT3 (SEQ ID NO: 16): 5'-TTGTCTCATGAGCGGATACA-3' - checking of the 3'integration DEL REV (SEQ ID NO: 14): 5'-TGGTATGGCTGATTATGATCCTC-3' m-5-NEO-2 (SEQ ID NO: 12): 5'-GATGCCTGCTTGCCGAATA-3' Thanks to this screening, transfectants resistant to G418 but showing random integrations into the genome of the YGM-2 line have been eliminated. Among transfectants having integrated the pT125-FRT vector into the Frt site of the YGM-2 line, the 19D1, 25E5, 30A5 and 20F11 cloids were ELISA tested in order to evaluate their productivity in Anti-D antibody.
Productivities were obtained for these 4 clones (7.1 pcd; 7.0 pcd; 7.1 pcd and 7.7 pcd respectively) are homogeneous and of the same order of magnitude as that observed for the 35H4 cloid or the 35H4(1)2G2 parental clone (11.4 pcd and 13.3 pcd respectively) bearing witness to the value of this strategy of targeting in order to obtain powerful producers in reproducible fashion. Genetic stability of these cloids was 67 .

studied, after cloning, over a three month period.

Example 5: identification of the integration site in the YGM-1 cell line (clone 10G10) by reverse PCR assay The integration site was sequenced after reverse-PCR
amplification. Briefly, DNA from the 8A10 (containing the targeting vector) and 1OG10 (deleted YGM-1 cell line) clones was digested using various restriction enzymes and the resultant restriction fragments were religated to one another using a T4 ligase. Reverse PCR was then carried out using anti-sense primers for sequences within the targeting vector.
The resultant PCR products have, at their 5' and 3' ends, targeting vector sequences and, in their middle segment, an unknown sequence of variable length (depending on which restriction enzyme was used) corresponding to the integration region directly next to the targeting vector. Sequencing of the various PCR products yields the sequence of the highly transcriptionally active integration site (SEQ ID NO: 1).

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Claims (45)

1. Process to generate a cell line comprising at least one cell, including the following steps:
- integration into a highly transcriptionally-active region of the genome of said cell, of a unique recombinase recognition site; and - integration into the genome of said cell, downstream of the unique recombinase recognition site, of a nucleic acid sequence encoding a transcription termination signal.

2. Process according to claim 1, in which the nucleic acid sequence encoding a transcription termination signal is a sequence encoding a polyadenylation signal.

3. Process according to claim 1, in which the nucleic acid sequence encoding a selection marker also includes a weakly active polyadenylation sequence.

4. Process according to any one of claims 1 to 3, in which the nucleic acid sequence encoding a transcription termination signal is a sequence encoding, whole or part of the SV40 early polyadenylation signal or the adenovirus Li polyadenylation signal.

5. Process according to any one of claims 1 to 3, in which the nucleic acid sequence encoding a transcription termination signal is a mutated or deleted sequence of a polyadenylation signal, said mutation and/or deletion making it possible to reduce the efficiency of the polyadenylation signal compared to the non-mutated sequence being selected from the group comprising increasing the distance separating the AATAAA elements and the region rich in GT, deleting SV40 late polyA from one or several nucleotides present upstream of the AATAAA
hexanucleotide, deleting certain elements located between nucleotides 13 to 48 upstream of the AATAAA sequence, deleting sequences rich in GT downstream of the AATAAA
sequence accompanied by modification of the space located between the AATAAA sequence and the region rich in GT, or mutating or deleting USE (upstream sequence element) regions located upstream of the AATAAA sequence.

6. Process according to claim 1, in which the cell is a mammalian cell.

7. Process according to claim 1, in which integration of the unique recombinase recognition site is achieved in a series of steps, including at least:
- integration of two nucleic acid sequences both encoding a recombinase recognition site;
- integration of a nucleic acid sequence, encoding a reporter gene, between the two sequences which both encode a recombinase recognition site;
- integration of a nucleic acid sequence encoding a protein of interest, between the two sequences which both encode a recombinase recognition site; and - integration of a nucleic acid sequence, encoding a selection marker, preferably a gene conferring resistance to an antibiotic, between the two sequences which both encode a recombinase recognition site.

8. Process according to claim 1, in which the only cells selected are highly productive cells that have integrated a single copy of the set of sequences specified in claim 7.

9. Process according to claim 8, in which only those cells with a pcd (pg of protein/cell/day) of 5 or over, or preferably of 10 or over are selected.

10. Process according to claim 7, in which the series of steps also includes excision of the set of sequences specified in claim 5 by the action of a recombinase on the cell.

11. Process according to claim 7, in which the action of a recombinase is obtained by co-expression of said recombinase in the cell by means of a vector carrying a nucleic acid sequence encoding said recombinase.

12. Process according to claim 7, in which the series of steps also includes the selection of cells from which the set of nucleic acid sequences specified in claim 7 has been excised, and having an intact recombinase recognition site.

13. Process according to claim 7, in which the series of steps also includes incorporation of a nucleic acid sequence encoding the thymidine kinase of Type 1 Herpes simplex virus (HSV1-TK).

14. Process according to claim 13, in which the selection of cells prior to excision is performed by adding ganciclovir to the culture medium.

15. Process according to any one of claims 7 to 14, in which the selection of cells is achieved by adding an antibiotic to the medium at a dose greater than or equal to 1 g/l, preferably greater than or equal to 2 g/l, 4 g/l or yet again 8 g/l.

16. Process according to claim 1, in which the recombinase recognition sequence is the loxP and/or FRT
sequence.

17. Process according to claim 7, in which the protein of interest is an antibody or an antibody fragment.

18. Process according to claim 1, in which the cell is a mammalian cell chosen from the group comprising: rat myeloma lines, notably YB2/0 (ATCC CRL-1662) and IR983F, human myelomas like Namalwa or any other human cell such as PERC6, CHO lines (notably CHO-K, CHO-Lec10, CHO-Lec1, CHO
Pro-5, CHO dhfr-, CHO Lec13) or other lines chosen from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653.

19. Process according to claim 7, in which the series of steps also includes transfection of the selected cell line with an expression vector carrying a nucleic acid sequence encoding a protein or polypeptide of interest and a nucleic acid sequence encoding a recombinase recognition site directly downstream of a nucleic acid sequence encoding a selection marker, preferably conferring resistance to an antibiotic, without any polyadenylation sequence.

20. Process according to Claim 19, in which said expression vector is inserted at the unique recombinase recognition site through the action of said recombinase.

21. Process according to claim 19, in which cells carrying expression vector integrated at the target site are selected for by assaying for expression of the protein of interest and by adding a high antibiotic dose in the culture medium.

22. Process according to claim 7, in which said protein or polypeptide of interest is a therapeutic protein or polypeptide, notably chosen from the group which are active vis-à-vis digestive, pancreatic, biliary, antiviral, anti-inflammatory, pulmonary, antimicrobial, haematological, neurological, cardiovascular, ophthalmologic, antigenic, cerebral, anti-tumoral, immunostimulatory and immunomodulatory functions.

23. Cell line generated according to the process of any of claims 1 through 22.

24. Cell line carrying, stably integrated into its genome, a unique recombinase recognition site in a highly transcriptionally active region of the genome of said cell and, directly downstream of said unique recombinase recognition site, a nucleic acid sequence encoding a transcription termination signal.

25. Cell line according to claim 24, in which the nucleic acid sequence encoding a transcription termination signal is a sequence encoding a polyadenylation signal, notably a weakly active polyadenylation signal.

26. Cell line according to claim 24, in which the nucleic acid sequence encoding a transcription termination signal is a sequence encoding at least partly the SV40 early polyadenylation signal.

27. Cell line according to claim 24, in which the cell is a mammalian cell.

28. Cell line according to claim 24, in which the cell is also carrying:
- two nucleic acid sequences both encoding a recombinase recognition site;
- at least one nucleic acid sequence encoding a protein of interest between the two sequences which both encode a recombinase recognition site; and - at least one nucleic acid sequence, encoding a selection marker, preferably a gene conferring resistance to an antibiotic between the two sequences which both encode a recombinase recognition site, said sequence encoding a selection marker being located directly upstream of the nucleic acid sequence encoding said transcription terminating signal.

29. Cell line, in which all the sequences of claim 28 are integrated together in one single copy.

30. Cell line according to claim 29, in which the cells have a pcd (pg of protein/cell/day) value of 10 or over 10.

31. Cell line in which the cells carry only one intact recognition recombinase site.

32. Cell line according to claim 24, in which the recombinase recognition sequence is the loxP and/or FRT
sequence.

33. Cell line according to claim 28, in which the protein of interest is an antibody or an antibody fragment.

34. Cell line modified according to claim 24, in which the original cell prior to modification is a mammalian cell, chosen from the group which comprises: rat myeloma lines notably YB2/0 (ATCC CRL-1662) and IR983F, human myelomas like Namalwa or any other human cell such as PERC6, CHO lines (notably CHO-K, CHO-LeclO, CHO-Lec1, CHO Pro-5, CHO dhfr-, CHO Lec13) or other lines chosen from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, BHK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653.

35. Cell line according to claim 28, which is carrying a nucleic acid sequence encoding a recombinase recognition site downstream of a nucleic acid sequence encoding a protein of interest and a nucleic acid sequence encoding a selection marker, preferably conferring resistance to an antibiotic, without any polyadenylation sequence.

36. Cell line according to either of Claims 24 or 28, over-expressing a protein or polypeptide of interest chosen from the group of proteins or polypeptides of interest which are active vis-à-vis digestive, pancreatic, biliary, antiviral, anti-inflammatory, pulmonary, antimicrobial, haematological, neurological, cardiovascular, ophthalmologic, antigenic, cerebral, anti-tumoral, immunostimulatory and immunomodulatory functions.

37. Cell line, identified by reference number YGM-1/10G10, registered under application number CNCM I-3704.

38. Cell line, identified by reference number YGM-2/3G5, registered under application number CNCM I-3885.

39. An isolated nucleic acid molecule including a nucleic acid fragment identified by the number SEQ ID NO: 1.

40. A vector carrying a nucleic acid sequence identified in the sequence listing by the number SEQ ID NO:
1.

41. Process for producing at least one protein or polypeptide of interest, characterised in that a cell line according to any of claims 24 through 38 is cultured in such a way that said protein of interest is expressed, followed by at least one step in which said protein of interest is harvested.

42. Process for producing at least one protein or polypeptide of interest according to claim 41, characterised in that it is based on a cell line according to claim 37 or 38.

43. Process according to either of claims 41 or 42, in which the protein or polypeptide of interest is chosen from the group of proteins or polypeptides of interest which are active vis-à-vis digestive, pancreatic, biliary, antiviral, anti-inflammatory, pulmonary, antimicrobial, haematological, neurological, cardiovascular, ophthalmologic, antigenic, cerebral, anti-tumoral, immunostimulatory and immunomodulatory functions.

44. Process according to claim 43, in which the protein or polypeptide of interest is an antibody or antibody fragment.

45. A vector comprising the sequence SEQ ID NO: 2.