WO2016003368A1 - Vecteurs optimisés pour la production de protéines recombinées - Google Patents

Vecteurs optimisés pour la production de protéines recombinées Download PDF

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WO2016003368A1
WO2016003368A1 PCT/SG2015/050176 SG2015050176W WO2016003368A1 WO 2016003368 A1 WO2016003368 A1 WO 2016003368A1 SG 2015050176 W SG2015050176 W SG 2015050176W WO 2016003368 A1 WO2016003368 A1 WO 2016003368A1
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anyone
nucleotide sequence
gene
expression vector
mammalian expression
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Say Kong NG
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Agency For Science, Technology And Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention lies in the field of molecular biology, specifically expression vector engineering technology to increase productivity of recombinant protein synthesis, especially for biopharmaceutical applications.
  • New cell lines derived from high producing clones are desirable for high level recombinant protein production in order to provide large quantities of a given recombinant protein, thus allowing a wide range of possible applications including industrial processes, diagnosis or disease treatment.
  • expression vector engineering technologies are the most timely and convenient method for new cell line development.
  • the primary objective of expression vector engineering technologies is to improve the efficiency and efficacy of generating and isolating high producing clones.
  • the structure of chromatin can be altered by specific DNA elements that maintain the chromatin in an "open" state to increase transcription of the GOI.
  • examples of such elements are the ubiquitous chromatin opening element (UCOE), which is a methylation free CpG island [Benton T et al. Cytotechnology 2002, 38(l-3):43-46], and the matrix attachment regions (MARs) which anchor the chromatin structure to the nuclear matrix during interphase [Mirkovitch J et al.. Cell 1984, 39(l):223-232].
  • UCOE ubiquitous chromatin opening element
  • MARs matrix attachment regions
  • site specific recombination is also used to introduce the GOI into a pre-targeted genomic hotspot of the host cell line, which was previously determined to enable stable and enhanced transcription of a reporter gene.
  • Two site specific recombination systems, Cre and Flp are well established and they are commonly used to insert GOIs into targeted hot spots through their respective cis acting 34 bp loxP sites and 48 bp Flp Recombination Target (FRT).
  • Another expression vector engineering approach is to improve selection stringency. This improves the probability of isolating a high producing clone as the selection marker gene has to be expressed at sufficiently high levels to enable cell survival. As the GOI is likely integrated near the selection marker, this results in the high expression of the GOI. Higher selection stringency has, for example, been achieved by using a mutant neomycin phosphotransferase II (NPT II) selection marker with reduced affinity for the neomycin drug.
  • NPT II mutant neomycin phosphotransferase II
  • selection stringency allows isolating a high producing clone in this approach co-localization of the GOI with the selection marker may be problematic, especially in a gene amplification system.
  • a widely used cell-line for large scale production of recombinant mammalian proteins are Chinese hamster ovary (CHO) cells. These cells are able produce glycoproteins with post-translational modifications compatible to humans [Kim JY, et al.. Appl Microbiol Biotechnol 2012, 93(3):917-930] and have a refractory nature to human viruses [Berting A, et al. Biotechnol Bioeng 2010, 106(4):598- 607]. Moreover, well-established gene amplification systems are available for CHO cells and the cells are able to adapt and grow in serum-free suspension. These characteristics render CHO cells ideal for large scale high-titer cultures in the industry [Kaufman et al.
  • cell line development technologies like expression vector engineering, cell line engineering and clone screening technologies are especially promising.
  • a typical cell line engineering strategy focuses on improving viable cell density (IVCD) and specific protein productivity (qP) of cells.
  • IVCD viable cell density
  • qP specific protein productivity
  • the availability of CHO genome data as well as the advancement of genomics tools and in silico modelling of mammalian systems have also identified target genes with diverse array of functions to improve the titer of biopharmaceuticals. Together with the discovery of genome wide editing tools more of these genes can be validated for their roles in biopharmaceuticals production.
  • Using these cell line development tools as well as bioprocess development and media development the titers of biopharmaceutical production from CHO cells have improved 100-fold since the 1980s.
  • the invention therefore relates to a mammalian expression vector comprising (a) a nucleotide sequence encoding a gene of interest; (b) an IRES nucleotide sequence; and (c) a nucleotide sequence encoding a selection marker, wherein the nucleotide sequence encoding the gene of interest and the nucleotide sequence encoding the selection marker are linked to transcribe a bicistronic mRNA and wherein the IRES nucleotide sequence is located between the nucleotide sequence encoding the gene of interest and the nucleotide sequence encoding the selection marker.
  • the IRES nucleotide sequence is an attenuated IRES nucleotide sequence.
  • the IRES nucleotide sequence is an encephalomyocarditis virus (EMCV) IRES nucleotide sequence.
  • the attenuated IRES nucleotide sequence is an encephalomyocarditis virus (EMCV) IRES nucleotide sequence that is truncated at nucleotide 832 and has an A7 bifurcation loop at nucleotide 770.
  • the EMCV IRES nucleotide sequence comprises an A772C mutation and/or a 709 ⁇ 7 mutation.
  • A772C EMCV IRES mutation refers to an EMCV IRES nucleotide sequence wherein the adenosine at position 772 has been replaced by a cytosine.
  • 709 ⁇ 7 EMCV IRES mutation refers to an EMCV IRES nucleotide sequence wherein the nucleotides at positions 709-715 are deleted.
  • the IRES nucleotide sequence is selected from the group of nucleotide sequences consisting of those set forth in (I) SEQ ID Nos. 1-3 (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I).
  • the nucleotide sequence encoding the selection marker is operably linked at its 3 '-end with a nucleotide sequence encoding a naturally occurring PEST sequence.
  • a PEST sequence is a peptide sequence which is rich in proline (P), glutamic acid (E), serine (S), and threonine (T).
  • the naturally occurring PEST sequence is the mouse ornithine decarboxylase (MODC) PEST sequence.
  • the naturally occurring PEST sequence is linked to the 3 '-end of the selection marker in two tandem repeats.
  • the PEST sequence is encoded by a nucleotide sequence as set forth in SEQ ID NO:4.
  • the selection marker is dihydrofolate reductase (DHFR).
  • the DHFR sequence is the mouse DHFR sequence.
  • the gene encoding the DHFR is operably linked at its 3 '-end with a nucleotide sequence encoding a PEST sequence.
  • DHFR is encoded by the nucleotide sequence of SEQ ID NO: 5.
  • the DHFR is a codon de-optimized DHFR. An example of codon de-optimized DHFRs have been described by Westwood et al. [Westwood et al. (2010) Biotechnology Progress, Volume 26, Issue 6, pages 1558-1566].
  • the gene encoding the codon de-optimized DHFR is operably linked at its 3 '-end with a nucleotide sequence encoding a PEST sequence.
  • codon de-optimized DHFR is encoded by the nucleotide sequence set forth in SEQ ID NO:6.
  • the bicistronic mRNA encodes the following nucleotide sequences from 5' to 3': a nucleotide sequence encoding a gene of interest; an IRES nucleotide sequence; and a nucleotide sequence encoding a selection marker.
  • the gene of interest is alpha 1-antitrypsin (A1AT).
  • alpha 1-antitrypsin is human alpha 1-antitrypsin.
  • alpha 1-antitrypsin is encoded by the nucleotide sequence set forth in SEQ ID NO:7.
  • the mammalian expression vector further comprises a mammalian expression promoter such as the cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) promoter, the elongation factor- la (EF-l ) promoter, the human ubiquitin C (Ubc) promoter or the murine phosphoglycerate kinase-1 (PGK) promoter.
  • a mammalian expression promoter such as the cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) promoter, the elongation factor- la (EF-l ) promoter, the human ubiquitin C (Ubc) promoter or the murine phosphoglycerate kinase-1 (PGK) promoter.
  • the mammalian expression promoter is the CMV promoter.
  • said promoter is operably linked to the nucleotide sequence encoding the gene of interest, the IRES and the selection marker.
  • the bicistronic mRNA comprises at its 3 '-end a poly-A (poly- adenosine monophosphate) sequence such as the SV40 poly-A sequence, bovine growth hormone poly-A sequence, the elongation factor- la (EF-la) poly-A sequence, the thymidine kinase poly-A sequence, or synthetic poly-A sequence.
  • the poly-A sequence is a SV40 poly-A sequence.
  • the mammalian expression vector further comprises an intervening (rVS) sequence.
  • the invention relates to a mammalian cell comprising the mammalian expression vector of the invention.
  • the cell is selected from the group comprising or consisting of CHO Kl from Chinese hamster ovary, CHO DukxBl l from Chinese hamster ovary, CHO DG44 from Chinese hamster ovary, HEK293 from human embryonic kidney, BHK from baby hamster kidney, 3T3 from mouse embryo, and COS7 from African Green Monkey kidney.
  • the mammalian cell is deficient in the DHFR gene.
  • the mammalian cell is a Chinese hamster ovary (CHO) cell.
  • the CHO is a CHO-DG44 or CHO-DukxBl l cell.
  • the CHO is a CHO-DG44.
  • the invention relates to the use of the mammalian expression vector of the invention for expressing a gene of interest. In a still further aspect, the invention relates to the use of the mammalian cell of the invention for expressing a gene of interest.
  • the invention relates to a method of expressing a gene of interest, comprising: (a) providing a mammalian cell of the invention; and (b) culturing said mammalian cell under conditions that allow for the expression of said gene of interest.
  • the method of the invention further comprises the step of selecting for the selection marker prior to step (b).
  • the method of the invention further comprises the step of isolating an expression product of the gene of interest.
  • the mammalian cell of the invention is cultured under methotrexate (MTX) selection conditions.
  • the MTX selection conditions comprise MTX concentrations of at least 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 250 nM, or 300 nM.
  • the mammalian cell of the invention is cultured under batch culture, continuous culture or perfusion culture conditions.
  • the bioreactor may include, without limitation, a shake flask, T-flask, petri dish, spinner flask, roller bottle, cell factory, hollow fiber bioreactor, wave bioreactor, stirred tank bioreactor, airlift bioreactor, packed bed bioreactor, fluidized bed bioreactor.
  • the mammalian cell is cultured under batch culture conditions.
  • the batch culture condition is a batch shake flask or stirred tank bioreactor culture condition.
  • the mammalian cell of the invention is cultured under fed-batch culture conditions.
  • the fed-batch culture condition is a fed-batch shake flask or stirred tank bioreactor culture condition.
  • the mammalian cell of the invention is cultured as a suspension culture.
  • the mammalian cell of the invention is cultured under serum-free conditions.
  • Figure 1 schematically depicts the dhfr selection marker attenuation.
  • WT wild type
  • CDD codon deoptimized
  • Figure 2 shows a vector map for vector sets for selection marker destabilization, IRES attenuation or selection marker codon de-optimization.
  • Figure 3 depicts the different vector designs (GOI: gene of interest, i.e. the primary gene that is expressed by the vector).
  • Vectors encoding for hAl AT as GOI start with pA, e.g. pAID has hAl AT, IRESatt (I) and dhfr (D).
  • Vectors comprising a codon de-optimized dhfr are denoted as dhfr*
  • Figure 4 depicts the selection and amplification efficiency of different vectors.
  • Total number of cell pools subjected to -HT selection is 96.
  • Figure 5 shows recombinant human A1AT titers of mini pools derived from different expression vectors in 96 well-plate cultures.
  • Cells were seeded into 96 well plates and allowed to grow for 14 days with one change in culture medium at Day 7.
  • Culture supernatant was then harvested for hAl AT ELISA to determine recombinant protein titer.
  • rhAlAT titers of each cell pool at different stages of selection and MTX amplification were plotted. Titers of the highest producing cell pool and the average titer of all analyzed cell pools were annotated on the graphs.
  • the average titers of each vector set were compared to that of pAID at the same amplification stage and analyzed by one-tail Student's t-test. Results of the analysis were annotated besides the average titers as + for p ⁇ 0.05 and ++ for p ⁇ 0.005.
  • Figure 6 shows recombinant relative average titers and amplification of the cell pools of the mini pools of Figure 5.
  • Relative average titers of each set of cell pools were calculated by dividing the average titers with that of the pAID set.
  • Fold amplification were calculated by dividing the average titers of each set of cell pools with that of the same cell pool set after -HT selection.
  • Figure 7 shows growth and rhAlAT productivities of top 2 cell pools from each vector set.
  • the 2 cell pools that gave the highest titers in 96 well-plate adherent culture with 50 nM MTX were chosen for further amplification to 300 nM MTX.
  • the 50 nM and 300 nM MTX cell pools were then adapted to serum-free suspension culture in shake flasks to evaluate their growth and rhAlAT production profiles.
  • the top producing cell pool in 96-well plate format ( Figure 5) is indicated as Pool 1. Only 1 cell pool from vectors pATD and pAID*p was evaluated as the other did not survive the adaptation to suspension culture or the MTX amplification process respectively.
  • Figure 8 depicts growth and rhAlAT production curves of cell pool 1 from each vector set.
  • Adapted cell pool 1 from each vector set at 50 nM and 300 nM MTX were seeded in 40 ml of serum- free medium at a cell density of 4 x 10 5 cells/ml in 125 ml shake flask on shaker platforms set at 110 rpm in a humidified incubator at 37 °C with 8% CO2.
  • Cell densities and viabilities were determined daily using an automated cell counter and culture supernatant was sampled daily for analysis by ELISA to determine rhAlAT titer.
  • the object of the present invention is to provide novel mammalian expression vectors for high level production of a molecule of interest.
  • the invention relates to a mammalian expression vector comprising (a) a nucleotide sequence encoding a gene of interest; (b) an IRES nucleotide sequence; and (c) a nucleotide sequence encoding a selection marker, wherein the nucleotide sequence encoding the gene of interest and the nucleotide sequence encoding the selection marker are linked to transcribe a bicistronic mRNA and wherein (on said mRNA) the IRES nucleotide sequence is located between the nucleotide sequence encoding the gene of interest and the nucleotide sequence encoding the selection marker.
  • vector refers to a nucleic acid molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed.
  • the four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.
  • the vector itself is generally a nucleic acid sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
  • the purpose of a vector, which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target or host cell.
  • expression vector refers to a vector that is capable of causing expression of the insert in the target or host cell.
  • sequence as used herein in relation to nucleic acids, relates to the primary sequence of nucleic acid molecules.
  • nucleic acid refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or as a double-stranded helix as well as artificial nucleic acid analogs such as peptide nucleic acid, morpholino- and locked nucleic acid, as well as glycol nucleic acid and threose nucleic acid. Each of these artificial nucleic acid analogs is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.
  • the nucleic acid is DNA.
  • the term "gene of interest” refers to the sequence encoding for a peptide, including a polypeptide, or RNA molecule.
  • the gene of interest will be the molecule, which is to be expressed using the novel expression vector for high level expression. In preferred embodiments, it encodes a recombinant polypeptide.
  • peptide relates to two or more amino acids linked by a peptide bond and thus includes dipeptides, oligopeptides, and polypeptides.
  • polypeptide refers to a long, continuous peptide chain, preferably of at least 50 amino acids in length.
  • the term "protein”, as used herein, relates to one or more polypeptides arranged in a biologically functional way.
  • a protein may consist of more than one polypeptide chains, such as an antibody, which consists of two light chains and two heavy chains, with each of the chains being a polypeptide.
  • a protein may be bound to ligands such as coenzymes and cofactors or to another protein or other macromolecule.
  • IRES refers to an internal ribosome entry site, abbreviated IRES, which is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence.
  • RNA sequence for IRES activity relies on a bicistronic reporter construct.
  • an IRES segment is located between two reporter open reading frames in a eukaryotic mRNA molecule (a bicistronic mRNA)
  • a bicistronic mRNA it can drive translation of the downstream protein coding region independently of the 5'-cap structure bound to the 5' end of the mRNA molecule. In such a setup both proteins are produced in the cell.
  • the first reporter protein located in the first cistron is synthesized by the cap-dependent initiation approach while translation initiation of the second protein is directed by the IRES segment located in the intercistronic spacer region between the two reporter protein coding regions.
  • IRES sequence is typically a DNA sequence encoding an RNA IRES sequence.
  • selection marker refers to a gene that confers a trait suitable for artificial selection. This trait can then be used to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell.
  • selectable markers are antibiotic resistance genes, which confer resistance to a specific antibiotic substance with which the cells are treated after transfection.
  • linked means associated in such a way that the gene of interest and the nucleotide sequence encoding the selection marker form a bicistronic mRNA. Accordingly, it means operably linked such that both the gene of interest and the selection marker can be expressed by use of the transcribed bicistronic RNA.
  • bicistronic refers to a nucleotide sequence comprising two cistrons, i.e. comprising two genes.
  • mRNA refers to a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. Following transcription of primary transcript mRNA (known as pre-mRNA) by RNA polymerase, processed, mature mRNA is translated into a polymer of amino acids. As in DNA, mRNA genetic information is in the sequence of nucleotides, which are arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis.
  • the IRES nucleotide sequence is an attenuated IRES nucleotide sequence.
  • the term "attenuated” refers to a gene sequence, which has been altered, e.g. via truncation, mutation or chimerization, in such a way that its original functionality is decreased to a certain extent, for example the protein expression level of the gene is decreased, or the binding affinity for a given binding partner is decreased.
  • IRES sequence this means that the attenuated IRES sequence promotes cap-independent translation to a lesser extent than the native, non-attenuated sequence.
  • the dhfr sequence this means that the attenuated dhfr sequence is expressed at a lower protein level due to reduced transcription, reduced translation or increased protein degradation.
  • an expression vector comprising an attenuated IRES sequence is advantageous as it allows for higher expression of the gene of interest. Without wishing to be bound to any particular theory, it is assumed that this may be due to the fact that the attenuated IRES impedes translation initiation and hence the cells required more gene copies or transcripts of the selection marker and consequently of the gene of interest to survive the selection.
  • the IRES nucleotide sequence is an encephalomyocarditis virus (EMCV) IRES nucleotide sequence.
  • EMCV encephalomyocarditis virus
  • the attenuated IRES nucleotide sequence is an encephalomyocarditis virus (EMCV) IRES nucleotide sequence that is truncated at nucleotide 832 and has an A7 bifurcation loop at nucleotide 770.
  • the A7 bifurcation loop is, for example, described by Hoffman & Palmenberg (J. Virol. 1995 Jul 69(7):4399-406).
  • the EMCV IRES nucleotide sequence comprises a A772C mutation and/or a 709 ⁇ 7 mutation.
  • A772C EMCV IRES mutation refers to an EMCV IRES nucleotide sequence wherein the adenine at position 772 has been replaced by a cytosine.
  • 709 ⁇ 7 EMCV IRES mutation refers to an EMCV IRES nucleotide sequence wherein the nucleotides at positions 709-715 are deleted, n various embodiments, the IRES nucleotide sequence is selected from the group consisting of (I) SEQ ID Nos. 1-3; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I).
  • sequence identity is generally expressed as a percentage and refers to the percent of amino acid residues or nucleotides, as appropriate, that are identical as between two sequences when optimally aligned.
  • sequence identity means the sequence identity determined using the well-known Basic Local Alignment Search Tool (BLAST), which is publicly available through the National Cancer Institute/National Institutes of Health (Bethesda, Maryland) and has been described in printed publications (see, e.g., Altschul et al., J. Mol. Biol, 215(3), 403-10 (1990)).
  • BLAST Basic Local Alignment Search Tool
  • Attenuated IRES nucleotide sequences are advantageous, since they allow for selection of cell clones with higher expression of the gene of interest. It is speculated that this result is achieved, because the lower translation rate of the attenuated IRES forces the cells to require more gene copies or transcripts of the selection marker and consequently of the gene of interest to survive the selection.
  • the nucleotide sequence encoding the selection marker is operably linked at its 3 '-end with at least one nucleotide sequence encoding a naturally occurring PEST sequence.
  • PEST sequence refers to a peptide sequence that is rich in proline (P), glutamic acid (E), serine (S), and threonine (T). This sequence is associated with proteins that have a short intracellular half-life; hence, it is hypothesized that the PEST sequence acts as a signal peptide for protein degradation.
  • an expression vector comprising a PEST sequence is advantageous as it allows for higher expression of the gene of interest.
  • the naturally occurring PEST sequence is the mouse ornithine decarboxylase (MODC) PEST sequence.
  • MODC mouse ornithine decarboxylase
  • the naturally occurring PEST sequence is linked to the 3'- end of the selection marker in two tandem repeats.
  • the PEST sequence is encoded by a nucleotide sequence set forth in SEQ ID NO:4.
  • the selection marker is dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • Dihydrofolate reductase is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor, which can be converted to the kinds of tetrahydrofolate cofactors used in 1 -carbon transfer chemistry.
  • the DHFR sequence is the mouse DHFR sequence.
  • the gene encoding the DHFR is operably linked at its 3 '-end with a nucleotide sequence encoding a PEST sequence.
  • the DHFR selection marker is encoded by the nucleotide sequence set forth in SEQ ID NO:5.
  • the DHFR is a codon de-optimized DHFR.
  • Codon de-optimization is possible, because of the redundancy of the genetic code and differences with respect to usage of codons that encode for the same amino acid.
  • a codon is a series of three nucleotides (a triplet) that encodes a specific amino acid residue in a polypeptide chain or for the termination of translation (stop codons). There are 64 different codons (61 codons encoding for amino acids plus 3 stop codons) but only 20 different translated amino acids. The overabundance in the number of codons allows many amino acids to be encoded by more than one codon. Because of such redundancy it is said that the genetic code is degenerate. Different organisms often show particular preferences for one of the several codons that encode the same amino acid.
  • codons help to achieve faster translation rates and higher accuracy. While it is possible to optimize the coding sequence for expression in a given host, it is similarly possible to use codons that are not preferred but still code for the desired amino acid and thus de-optimize a codon.
  • Such a codon de-optimized DHFR is advantageous, since its translation efficiency is reduced. Thus, in order to survive selection, cells harbouring a codon de-optimized DHFR must exhibit higher expression of DHFR and hence of the gene of interest.
  • the gene encoding the codon de-optimized DHFR is operably linked at its 3 '-end with a nucleotide sequence encoding a PEST sequence.
  • codon de-optimized DHFR is encoded by the nucleotide sequence as set forth SEQ ID NO:6.
  • the bicistronic mRNA encodes the following nucleotide sequences from 5' to 3': a nucleotide sequence encoding a gene of interest; an IRES nucleotide sequence; and a nucleotide sequence encoding a selection marker.
  • Such a bicistronic mRNA has the advantage that the selection marker, which ensures that the cells survive the selection process, is only expressed, if the gene of interest is also expressed. Hence, all surviving cell clones must express the gene of interest in substantial quantities.
  • the gene of interest is alpha 1 -antitrypsin.
  • Alpha 1 -antitrypsin or al -antitrypsin is a protease inhibitor belonging to the serpin superfamily. It is generally known as serum trypsin inhibitor. It protects tissues from enzymes of inflammatory cells.
  • alpha 1 -antitrypsin is human alpha 1 -antitrypsin.
  • alpha 1 -antitrypsin is encoded by the nucleotide sequence set forth in SEQ ID NO:7.
  • the mammalian expression vector further comprises a mammalian expression promoter such as the cytomegalovirus (CMV) promoter, the Simian virus 40 (SV40) promoter, the elongation factor- la (EF-l ) promoter, the human ubiquitin C (Ubc) promoter or the murine phosphoglycerate kinase-1 (PGK) promoter.
  • a mammalian expression promoter such as the cytomegalovirus (CMV) promoter, the Simian virus 40 (SV40) promoter, the elongation factor- la (EF-l ) promoter, the human ubiquitin C (Ubc) promoter or the murine phosphoglycerate kinase-1 (PGK) promoter.
  • CMV cytomegalovirus
  • SV40 Simian virus 40
  • EF-l elongation factor- la
  • Ubc human ubiquitin C
  • PGK murine phosphoglycerate kin
  • said promoter is operably linked to the nucleotide sequence encoding the gene of interest, the IRES and the selection marker.
  • operably linked means associated in such a way that the sequences linked in such a way are expressed together. With respect to the promoter this means that it may control expression of the gene of interest, the IRES and the selection marker.
  • the bicistronic mRNA comprises at its 3 '-end a poly-A (poly- adenylation) sequence.
  • Polyadenylation is the addition of a poly(A) tail to a messenger RNA.
  • the poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases.
  • the process of polyadenylation begins as the transcription of a gene finishes, or terminates.
  • the 3'-most segment of the newly made pre-mRNA is first cleaved off by a set of proteins; these proteins then synthesize the poly(A) tail at the RNA's 3' end. In some genes, these proteins may add a poly(A) tail at any one of several possible sites.
  • polyadenylation can produce more than one transcript from a single gene (alternative polyadenylation), similar to alternative splicing.
  • the poly(A) tail is important for the nuclear export, translation, and stability of mRNA. The tail is shortened over time, and, when it is short enough, the mRNA is enzymatically degraded.
  • Non-limiting examples of poly-A sequences include, without limitation, SV40 poly-A sequence, bovine growth hormone poly-A sequence, the elongation factor-la (EF-la) poly-A sequence, the thymidine kinase poly-A sequence, or synthetic poly-A sequence.
  • the poly-A sequence is a SV40 or BGH poly-A sequence. This is advantageous as it stabilizes the mRNA of the gene of interest and hence leads to higher expression levels of the gene of interest.
  • the mammalian expression vector further comprises an intervening (IVS) sequence.
  • intervening sequence refers to any of several families of internal nucleic acid sequences that are not present in the final gene product, including inteins, introns, untranslated sequences (UTR), and nucleotides removed by RNA editing.
  • intron refers to any nucleotide sequence within a gene that is removed by RNA splicing during maturation of the final RNA product. It refers to both the DNA sequence within a gene and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final mature RNA after RNA splicing are exons. Introns are found in the genes of most organisms and many viruses, and can be located in a wide range of genes, including those that generate proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA). When proteins are generated from intron- containing genes, RNA splicing takes place as part of the RNA processing pathway that follows transcription and precedes translation.
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • the invention relates to a mammalian cell comprising the mammalian expression vector of the invention.
  • the cell is selected from the group comprising or consisting of CHO Kl from Chinese hamster ovary, CHO DukxBl l from Chinese hamster ovary, CHO DG44 from Chinese hamster ovary, HEK293 from human embryonic kidney, BHK from baby hamster kidney, 3T3 from mouse embryo, and COS7 from African Green Monkey kidney.
  • the mammalian cell is deficient in the DHFR gene.
  • the mammalian cell is a Chinese hamster ovary (CHO) cell.
  • CHO Chinese hamster ovary
  • these cells are advantageous, because they are able produce glycoproteins with post- translational modifications compatible to humans, have a refractory nature to human viruses, well- established gene amplification systems are available for CHO cells and the cells are able to adapt and grow in serum-free suspension.
  • the CHO cell is a CHO-DG44 or CHO-DukxBl l cell. In more preferred embodiments, the CHO cell is a CHO-DG44 cell.
  • the invention relates to the use of the mammalian expression vector of the invention for expressing a gene of interest.
  • the invention relates to the use of the mammalian cell of the invention for expressing a gene of interest.
  • the invention relates to a method of expressing a gene of interest, comprising: (a) providing a mammalian cell of the invention; and (b) culturing said mammalian cell under conditions that allow for the expression of said gene of interest.
  • the method of the invention further comprises the step of selecting for the selection marker prior to step (b).
  • the method of the invention further comprises the step of isolating an expression product of the gene of interest.
  • the mammalian cell of the invention is cultured under methotrexate (MTX) selection conditions.
  • MTX methotrexate
  • This selection system is based on the fact that the DHFR enzyme catalyses the conversion of folate to tetrahydrofolate. This precursor is necessary in the de novo synthesis of purines, pyrimidine and glycine. Methotrexate (MTX) is a drug structurally resembling folate. MTX binds to DHFR thereby inhibiting the production of tetrahydrofolate. With insufficient levels of DHFR cells are deprived of nucleoside precursors and die. In other words, only cells that have increased copies of the DHFR genes, e.g. from the expression vector described herein, and therefore higher levels of the enzyme survive the treatment with MTX and can be selected.
  • MTX Methotrexate
  • the MTX selection conditions comprise MTX concentrations of at least 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 250 nM, or 300 nM.
  • the mammalian cell of the invention is cultured under batch culture, continuous culture or perfusion culture conditions.
  • the bioreactor may include, without limitation, a shake flask, T-flask, petri dish, spinner flask, roller bottle, cell factory, hollow fiber bioreactor, wave bioreactor, stirred tank bioreactor, airlift bioreactor, packed bed bioreactor, fluidized bed bioreactor.
  • the mammalian cell is cultured under batch culture conditions.
  • the batch culture condition is a batch shake flask or stirred tank bioreactor culture condition.
  • Such culture conditions are advantageous, since they allow for a higher cell density and hence for a higher level of recombinant protein.
  • the mammalian cell of the invention is cultured under fed-batch culture conditions.
  • Fed-batch culture is, in the broadest sense, defined as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run.
  • the advantage of the fed-batch culture is that one can control concentration of fed-substrate in the culture liquid at arbitrarily desired levels (in many cases, at low levels).
  • the fed-batch culture condition is a fed-batch shake flask or stirred tank bioreactor culture condition.
  • the mammalian cell of the invention is cultured under batch shake flask culture conditions.
  • the mammalian cell of the invention is cultured as a suspension culture.
  • the cells multiply while suspended in a suitable medium.
  • suspension cultures are homogenous, very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture), can be standardized at large scale, and automation and cleaning procedures are well established.
  • Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood.
  • the mammalian cell of the invention is cultured under serum-free conditions.
  • Serum-free culture has several advantages such as lower risk with regard to a contamination with bacteria, fungi or virus, better defined and reproducible formulations allowing more comparable research results and fulfilment of legal conditions for the production of medical products. [00120] It is understood that all embodiments disclosed herein in relation to the vectors of the invention are similarly applicable to the host cells, uses and methods, described herein, and vice versa.
  • Human alpha 1-antitrypsin (hAlAT) precursor sequence (UniProt Identifier P01009-1, Accessed on 16 May 2012) was codon-optimized for Chinese Hamster Ovary expression (Genscript, Piscataway, NJ).
  • Attenuated encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) sequence (Clontech, Palo Alto, CA) was modified by deleting 7 nucleotides at position 709 to obtain IRESatt709, and by an A-» C point mutation at position 772 to obtain IRESatt772 [Hoffman MA, Palmenberg AC: Revertant analysis of J-K mutations in the encephalomyocarditis virus internal ribosomal entry site detects an altered leader protein. J Virol 1996, 70(9): 6425-6430].
  • Murine dhfr sequence from pSV2-dhfr was modified by manual codon de-optimization to maximize the occurrence of de-optimized codon pairs in the first 120 amino acids of the sequence, based on codon usage database: http://www.kazusa.or.jp/codon/ [ Nakamura Y, Gojobori T, Ikemura T: Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res 2000, 28(1):292] ( Figure 1).
  • hAlAT, IRESatt709, IRESatt772, codon-deoptimized dhfr (dhfr*), codon-deoptimized dhfr with PEST sequence (dhfr*-PEST), and murine dhfr with 2 tandem PEST sequences (dhfr-PEST-PEST) were synthesized by Genscript (Piscataway, NJ).
  • Expression vectors were purified with NucleoBond® Xtra Midi EF (MACHEREY-NAGEL) according to manufacturer's instructions and dissolved in sterilized nucleases, proteases and pyrogen free biotechnology grade water (1st Base). The DNA quality and concentration was analysed using NanoDrop 2000 spectrophotometer (Thermo Scientific).
  • Suspension CHO-DG44 cells (GibcoTM Catalog number 12609-012, Invitrogen, Carlsbad, CA) adapted to HyQ PF-CHO (Hyclone, Logan, UT) with 4 n M L-glutamine (Invitrogen), 0.1% Pluronic® F-68 (Invitrogen) and lx HT supplement (Invitrogen) were cultivated in 125 ml disposable Erlenmeyer flasks (Corning, Acton, MA) on shaker platforms set at 110 rpm in a humidified incubator at 37 °C with 8% CO2. The cells were passaged every 3 to 4 days, and cell densities and viabilities were determined using an automated cell counter, Vi-Cell XR (Beckman Coulter, Fullerton, CA), according to manufacturer's instructions.
  • suspension CHO-DG44 cells were seeded at a density of 3 x 10 5 cells/ml into CD DG44 Medium (Life Technologies) with 8 niM L-glutamine (Invitrogen) and 0.18% Pluronic® F-68 (Invitrogen) in a 250 ml shake flask 3 days before transfection.
  • 5 million cells were pelleted at 90 xg for 10 min at room temperature. 2 ⁇ g of pMax-GFP (Amaxa, Gaithersburg, MD) was included as a positive control.
  • the cell pellet was resuspended in the vector solution mix consisting of 2 ⁇ g of expression vector mixed with 100 ⁇ of the supplemented NucleofectorTM Cell Line SG solution. This was transferred into a NucleocuvetteTM and electroporated with the AmaxaTM 4D-NucleofectorTM using pulse code DT-137 (Lonza). After 2 min, 500 ⁇ of pre-warmed CD DG44 media was added, followed by incubation in a static incubator at 37 °C with 5% CO2 (Sanyo) for 10 min, before the cells were transferred into 1.5 ml of prewarmed CD DG44 medium in a 24 well suspension culture plate. After 24 hours, cell pellets were harvested by centriiugation at 6000 xg for 10 min, washed once with sterile phosphate buffer saline (PBS) and stored immediately in a -80°C freezer for future analysis.
  • PBS sterile phosphate buffer saline
  • the two highest producing cell pools from each vector at 50 nM MTX were selected for further MTX amplification to 300 nM MTX.
  • the 50 and 300 nM MTX pools were adapted to serum-free suspension culture in HyQ PF-CHO with 50 and 300 nM MTX respectively.
  • Selected cell pools at 300 nM MTX were single cell cloned and adapted to serum-free suspension culture. Characterization of rhAlAT producing cells
  • Cells adapted to serum free culture were characterized by seeding them in 40 ml of serum- free medium at a cell density of 4 x 10 5 cells/ml in 125 ml shake flask on shaker platforms set at 110 rpm in a humidified incubator at 37 °C with 8% CO2.
  • Cell densities and viabilities were determined daily using an automated cell counter, Vi-Cell XR (Beckman Coulter), according to manufacturer's instructions, until culture viability dropped below 50%.
  • Culture supernatant was sampled daily for analysis by ELISA to determine rhAlAT titer, and for biochemical analysis using BioProfile 100 Plus (Nova Biomedical).
  • A1AT samples were diluted in the range of 1:10000, 1:20000, 1 :50000 and 1 :75000 so that they fall within the range of the standard curve and were assayed in duplicates.
  • 0.3 ⁇ g/ml HRP conjugate secondary antibody in sample diluent buffer was used and 3,3',5,5'-Tetramethylbenzidine (TMB), super slow for ELISA (T5569, Sigma, USA) served as the substrate.
  • TMB 3,3',5,5'-Tetramethylbenzidine
  • T5569 3,3',5,5'-Tetramethylbenzidine
  • the absorbance at 450 nm was read using the ASYS UVM340 microplate reader (Biochrom, UK).
  • P was determined according to Equation 4 where p is the rhAlAT titer obtained from
  • IVC was determined by trapezium rule according to Equation 5.
  • IVC t IVC t _ + 0.5 X i t + X V t X t — Equation 5
  • the 7 newly designed expression vectors expressing hAlAT can be classified into 3 sets ( Figure 3).
  • the second vector set consists of pAI709Dp and pAI772Dp.
  • the third vector set i.e. vectors pAID* and pAID*p, incorporated a codon de-optimized dhfr selection marker ( Figure 1) to evaluate the use of codon de- optimization as a strategy to reduce selection marker expression levels.
  • This strategy of codon deoptimization affects a different aspect of gene expression when compared to the attenuated IRES and PEST.
  • modified expression vectors with recombinant human alpha-1 antitrypsin were transfected into CHO-DG44 cells, which were subsequently plated in 96 well plates for selection in a HT-deficient culture medium. 30 randomly picked cell pools for each vector from these 96 well plates were then subjected to sequential MTX amplification at 10 nM and 50 nM concentrations. The selection and amplification efficiencies are listed in Figure 4.
  • the pAI772Dp and pAIDpp cell pools produced the most rhAlAT with maximum titers of up to 1,3 g/1 and 1.15 g/1, respectively. . These are the highest recombinant protein titers reported from shake flask batch culture of stable mammalian cell pool. Moreover, also the other cell lines produced remarkable expression rates.

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Abstract

La présente invention concerne un vecteur d'expression de mammifère comprenant (a) une séquence nucléotidique codant pour un gène d'intérêt; (b) une séquence nucléotidique IRES; et (c) une séquence nucléotidique codant pour un marqueur de sélection, la séquence nucléotidique codant pour le gène d'intérêt et la séquence nucléotidique codant pour le marqueur de sélection étant liées pour transcrire un ARNm bicistronique et ladite séquence nucléotidique IRES étant située sur ledit ARNm entre la séquence nucléotidique codant pour le gène d'intérêt et la séquence nucléotidique codant pour le marqueur de sélection, ainsi que des cellules comprenant ledit vecteur et ses méthodes d'utilisation.
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CN107338267A (zh) * 2016-05-03 2017-11-10 中国科学院深圳先进技术研究院 双顺反子表达载体及其构建方法与应用
WO2018150345A1 (fr) * 2017-02-16 2018-08-23 De Vita Bio Life Sciences Vecteur d'expression
CN112135909A (zh) * 2018-05-24 2020-12-25 国立大学法人北海道大学 新载体及其应用
WO2022165001A1 (fr) 2021-01-29 2022-08-04 Merck Sharp & Dohme Llc Compositions d'anticorps anti-récepteur 1 de mort programmée (pd-1) et procédés d'obtention des compositions les contenant
WO2024075112A1 (fr) * 2022-10-06 2024-04-11 Kamada Ltd. Protéine alpha 1 antitrypsine (aat) humaine recombinante hautement fucosylée ayant une activité immunomodulatrice et compositions la contenant

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107338267A (zh) * 2016-05-03 2017-11-10 中国科学院深圳先进技术研究院 双顺反子表达载体及其构建方法与应用
WO2018150345A1 (fr) * 2017-02-16 2018-08-23 De Vita Bio Life Sciences Vecteur d'expression
CN112135909A (zh) * 2018-05-24 2020-12-25 国立大学法人北海道大学 新载体及其应用
EP3805396A4 (fr) * 2018-05-24 2022-03-23 National University Corporation Hokkaido University Nouveau vecteur et utilisation associée
WO2022165001A1 (fr) 2021-01-29 2022-08-04 Merck Sharp & Dohme Llc Compositions d'anticorps anti-récepteur 1 de mort programmée (pd-1) et procédés d'obtention des compositions les contenant
WO2024075112A1 (fr) * 2022-10-06 2024-04-11 Kamada Ltd. Protéine alpha 1 antitrypsine (aat) humaine recombinante hautement fucosylée ayant une activité immunomodulatrice et compositions la contenant

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