WO2018158141A1 - Méthode d'expression de protéines - Google Patents

Méthode d'expression de protéines Download PDF

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WO2018158141A1
WO2018158141A1 PCT/EP2018/054459 EP2018054459W WO2018158141A1 WO 2018158141 A1 WO2018158141 A1 WO 2018158141A1 EP 2018054459 W EP2018054459 W EP 2018054459W WO 2018158141 A1 WO2018158141 A1 WO 2018158141A1
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interest
cell
protein
template
dna construct
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Daniel Ivansson
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Ge Healthcare Bio-Sciences Ab
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells

Definitions

  • the present invention relates to an improved method for transient protein expression, for some embodiments in combination with cell line development based on targeted gene integration using the same host cell line, which is generally applicable to production of any therapeutic protein or protein based format that can be produced using mammalian Cell lines and in particular Chinese Hamster Ovary (CHO) cells.
  • CHO Chinese Hamster Ovary
  • the therapeutic protein class includes replacement proteins (insulin, growth factors, cytokines and blood factors), vaccines (antigens, VLPs) and monoclonal antibodies.
  • the by far dominating format is the monoclonal antibodies.
  • Some of the recombinant proteins can be produced in simple microbial cells such as £ coli, but for more complex proteins including the monoclonal antibody class Chinese Hamster Ovary (CHO) cells is the dominating host for production [1].
  • the monoclonal antibody class is projected to continue being the dominating format but with a larger heterogeneity in molecular structure within this class including different multi-specific formats, fusion proteins, alternative scaffolds and antibody drug conjugates (ADCs).
  • ADCs antibody drug conjugates
  • CLD Cell Line Development
  • CHO Choinese hamster ovary
  • a clone adapted to the burden of expressing a foreign protein at very high levels and with maintained good growth characteristics is needed.
  • a CHO host cell is, for example, not a very competent secretor.
  • the CHO genome is highly plastic.
  • By introducing expression of foreign secreted proteins at very high levels an evolutionary pressure towards increased folding and secretory capacity is introduced.
  • By screening many clones cells better adapted for high secretion can be found.
  • the best random integration platforms today can yield high protein titers in a relatively short time period ( ⁇ 3 months) albeit using a very resource intensive workflow. Further, generated cell clones will be different at the genetic and phenotypic level between different cell line development efforts.
  • SDI site-directed integration
  • An alternative approach to increase the speed of protein production is to skip cell line development altogether and use transient protein production.
  • a host cell line is cultured and an expression vector is introduced into cells to achieve expression of the target protein without integration of genetic material into the genome of cells [13].
  • Hurdles for taking this approach into clinical production has been low titers, high costs when scaling up and safety concerns due potential risks for random integration of expression vector DNA into the genome of production cells with unknown effects on the quality and purity of a target protein of interest. With a trend towards reduced volumes of clinical production batches transient production could be feasible if the yields could be increased.
  • one major obstacle is the low protein production competency of CHO host cell lines.
  • the present invention relates to an improved method for transient protein expression which is generally applicable to production of any therapeutic protein that can be produced using mammalian Cell lines and in particular Chinese Hamster Ovary (CHO) cells.
  • the method combines expression construct components improving the post-transcriptional processing of a gene of interest, the introduction of a onetime host cell line selection workflow using a template protein of interest expression construct to generate a production competent cell line and approaches enabling inactivation of the template protein of interest. Further expression and protein production of any desired protein of interest using said production competent cell line for transient protein expression processes with improved yields and efficiencies.
  • the production competent cell line generated can also be used for CLD using targeted integration and hence the same cell line can be used for production of a desired protein of interest using both transient expression processes and production processes utilizing stable production cells generated using fast CLD workflows
  • the invention relates to a method for creating a mammalian cell bank for transient protein production comprising the following steps:
  • protein of interest and template protein refers to a group of proteins sharing a common sequence or structural feature.
  • protein classes include antibodies of the same class (such as IgGl antibodies), fusion proteins sharing at least one conserved domain (such as FC-fusion proteins), or in general proteins sharing a conserved scaffold sequence were sequence variation is introduced only at defined region.
  • the method is a method wherein steps (d) and (e) are omitted and said template protein of interest region comprises inducible promoter(s) for expression of said template protein of interest, and wherein said silencing of said template protein of interest expression is achieved via changing culture conditions to inactivate said inducible promoter(s), and wherein said transient expression of said desired protein of interest is achieved via introduction of a plasmid or an in-vitro generated mRNA set encoding said desired protein of interest into a cell from said cell bank, and wherein said expression is achieved using culture conditions wherein said inducible promoter(s) is inactive.
  • the candidate cells are preferably generated according to any of the following procedures:
  • said top candidate cell Prior to generating said cell bank, said top candidate cell is preferably engineered to increase the genomic stability of said using targeted gene editing methods.
  • the cell bank for transient protein production may also function as a cell bank for cell line development, wherein cell line development is achieved by a donor DNA construct containing a region coding for a desired protein of interest belonging to the same class as the template protein of interest being introduced via the use of an expression vector and the action of an DNA processing enzyme, and wherein said cell bank is first used for selection of a desired protein of interest via evaluation of candidate desired proteins of interest using transient protein production and/or for producing a desired protein of interest using transient protein production, and wherein said cell bank is then used to generate a stable cell line by cell line development and produce said desired protein of interest at a larger scale.
  • the top candidate cell may additionally also be used to create a cell bank for cell line development, wherein cell line development is achieved by a donor DNA construct containing a region coding for a desired protein of interest belonging to the same class as the template protein of interest being introduced via the use of an expression vector and the action of an DNA processing enzyme, and wherein said cell bank for transient protein production is first used for selection of a desired protein of interest via evaluation of candidate desired proteins of interest using transient protein production and/or for producing a desired protein of interest using transient protein production, and wherein said cell bank for cell line development is then used to generate a stable cell line by cell line development and producing said desired protein of interest at a larger scale.
  • cell line development is achieved by a donor DNA construct containing a region coding for a desired protein of interest belonging to the same class as the template protein of interest being introduced via the use of an expression vector and the action of an DNA processing enzyme
  • said cell bank for transient protein production is first used for selection of a desired protein of interest via evaluation of candidate desired proteins of interest using transient
  • the template protein of interest may be coded by:
  • a single gene of interest such as ones encoding growth factors, blood clotting factors, cytokines, hormones, erythropoietins, albumins, virus proteins, virus protein mimics, bacterial proteins, bacterial protein mimics, domain antibodies, ScFvs, Affibodies, DARPINs, multimerization domains, IgG Fc domains, albumin binding domains, Fc receptor binding domains or fusion proteins based on combinations of the above single gene/mRNA of interest coded groups; or
  • steps (a) to (c) of the method are iterated in the following way prior to generating said cell bank:
  • step (i) in a next iteration the top candidate cell from previous step (c) is used as said recombinant mammalian cell in step (a);
  • step (ii) step (b) to (c) is repeated using a modified template DNA construct having been modified to provide increased expression potential for said template protein of interest compared to earlier iterations; and (iii) repeating (i) and (ii) n times.
  • the cell bank may be based on a final cell comprising a receiving DNA construct version comprising a sequence z2 being unique or rare in the total genome sequence of said cell and two sequences X and Y flanking said receiving DNA construct and being unique or rare in the genome of said cell.
  • said cell bank is based on a final cell comprising a receiving DNA construct version containing two recombinase recognition sequences flanking a first selection marker coding region and wherein said recombinase recognition sequences are both of the same type such as a serine recombinase type such as attP/attB or a tyrosine recombinase type such as Lox, Rox or FRT;
  • said cell bank is based on a final cell comprising a receiving DNA construct version comprising a single recombinase recognition sequence followed by an optional first selection marker coding region and wherein said recombinase recognition sequence is of a type such as a serine recombinase type such as attP/attB or a tyrosine recombinase type such as Lox, Rox or FRT.
  • a serine recombinase type such as attP/attB
  • a tyrosine recombinase type such as Lox, Rox or FRT.
  • the receiving DNA construct may be introduced into said genomic region in the following way: (a) providing a donor DNA construct containing said receiving DNA construct flanked by two sequences X' and Y' being homologous to two corresponding sequences X and Y in a template DNA construct containing cell and wherein said sequences X and Y are unique or rare in the genome of said template DNA construct containing cell and flanking said template DNA construct;
  • nuclease/meganuclease/TALEN or a CRISPR/Cas9 combination, with specificity for said gene editing nuclease sequence is introduced together with said donor DNA construct into said top candidate cell;
  • cells having undergone correct introduction are selected via the use of a selection marker and/or genetic characterization.
  • the mammalian host cell line is preferably a CHO cell line such as CHO DG44, CHO Kl, CHO M, CHO-S or a CHO GS knockout cell line.
  • transient expression of a desired protein of interest is preferably achieved using in-vitro generated mRNA(s) and the amount of in-vitro generated mRNA(s) introduced into cells from said cell bank is controlled so as to closely equal mRNA levels measured during a previous culture for expression of said template protein of interest.
  • Fig 1 is a general description of a method for generation of an improved cell CI using a single TGOI (target gene of interest) load (Figla) as well as the use of said improved cell for transient expression (Figlb) or targeted CLD generating a protein producing cell C2 (Figlc);
  • Fig 2 is a general description of a method for generation of an improved cell using an iterative workflow in which the expression load is gradually increased and intermittent improved cells are isolated after each increase in expression load until a final improved cell Cn+1 is generated:
  • Fig 3 describes specific implementations of the method described in Fig 1 wherein Fig 3a describes the TGOI under control of an inducible promoter enabling the TGOI to be actively expressed during the improvement workflow; and Fig 3 b describes the TGOI inactive during conditions for transient expression of a desired gene of interest using a plasmid expression vector or a set of synthetic mRNAs;
  • Fig 4 describes a specific implementation for generation of the improved cell CI carrying a template DNA construct T enabling excision of the TGOI via the transient expression of a recombinase
  • Fig 5 describes a special embodiment of the method of Fig 2 utilizing reversible recombinase sites RS2 together with a matching recombinase (Rec-RS2) to perform iterative introductions and excisions of TGOI variants with increasing expression load;
  • Fig 6 describes cell line development using the cell C2 generated according to Fig 4;
  • Fig 7 describes a specific embodiment of the iterative approach described in Fig 2 wherein cassette exchange mediated by a double strand break inducing site specific nuclease is used to exchange between TGOI variants with increasing expression load;
  • Fig 8 describes gene editing approaches for modification of the TGOI containing template recombinant DNA construct T of the improved cell CI to generate a cell C2 containing a desired receiving recombinant DNA construct R compatible with targeted CLD;
  • Fig 9 describes a specific example of a receiving recombinant DNA construct R based on a cassette of two recombinase recognition sequences RS together with different promoter placement alternatives. Further an overview of targeted CLD using such an improved cell C2 together with matching expression vectors EV and recombinase Rec-RS to generate a protein producing cell C3; and Fig 10 describes another specific example of a receiving recombinant DNA construct R based on a single recombinase recognition sequence RS together with different promoter placement alternatives. Further an overview of targeted CLD using such an improved cell C2 together with matching expression vectors EV and recombinase Rec-RS to generate a protein producing cell C3.
  • a unifying concept underlying the invention is the time limited utilization of an actively expressed template protein of interest for guiding the transformation of an initial mammalian host cell line having limited production capacity for a certain class of proteins into a high productivity host cell line that can be used for efficient production of desired proteins of interest using transient protein production methods and were the desired protein of interest belongs to the same class as the template protein of interest.
  • the invention includes methods enabling removal or inactivation of template protein of interest expression before expression of a desired protein of interest is initiated.
  • the current invention offers improvements in that the effects of single or multiple changes can be directly assessed using a protein indicative of the performance for a desired class of proteins and that the expression conditions used for the template protein of interest (mRNA levels and mRNA design) can be reproduced for any desired protein of interest.
  • a key aspect of the invention is the opportunistic utilization of the inherent plasticity of the genome of typical mammalian host cell lines, such as the CHO genome, as an engine for generating epi-genetic and/or genetic changes enabling the desired transformation into a high productivity state and where the continuous high level expression of a template protein of interest during the transformation is used as an inherent selection agent and/or physiological state sensor directing and/or enabling detection of accumulation of positive changes leading towards the desired end goal.
  • the potential for template protein of interest expression acting as an inherent selection agent is based on the fact that a high recombinant expression load imposed on all cells will have an impact on their viability and growth.
  • Cells that do not handle the recombinant expression load well are hypothesized (and this is generally accepted in the field) to be subjected to stress responses (amino acid shortage, charged tRNA shortage, hold up of the ribosomal machinery of recombinant mRNAs, hold up of the folding machinery on recombinant proteins, build-up of soluble or aggregated forms of recombinant protein within cells) reducing viability and growth.
  • stress responses amino acid shortage, charged tRNA shortage, hold up of the ribosomal machinery of recombinant mRNAs, hold up of the folding machinery on recombinant proteins, build-up of soluble or aggregated forms of recombinant protein within cells
  • stress responses amino acid shortage, charged tRNA shortage, hold up of the ribosomal machinery of recombinant mRNAs, hold up of the folding machinery on recombinant proteins, build-up of soluble or aggregated forms of recombinant protein within cells
  • the genome of the improved host cell line is stabilized ("Frozen") using targeted gene editing methods [8] following the desired transformation.
  • the current invention offers potential large improvements.
  • the approach based on utilizing the plasticity of host cell genomes during a pro-longed time period disclosed in the current invention offers a means to sculpture the complete genome to achieve the desired transformation without the need for massive screening to understand and define the changes needed or the need for a large range of costly genome editing reagents.
  • the postulated cost and resource effectiveness of the proposed approach could enable the generation of a range of host cell lines being adapted for the optimal production of different protein/biotherapeutic classes or even sub-groups with different characteristics (such as amino acid frequencies) within a given class.
  • the non-targeted sculpturing of the genome can be combined with specific targeted changes introduced by genome editing. Such changes could include modulation/control of glycosylation, further boosting of specific parts of the secretion machinery, introduction of machinery for non-natural amino acids, introduction of machinery for specific post translational protein modifications and the already mentioned changes for stabilizing the genome of a host cell.
  • the same transformed host cell line used for transient protein production of a certain desired protein class can also be used for cell line development using site directed integration to enable production of this protein class using a stable production cell line with identical or at least highly similar phenotype/genotype.
  • transient protein production is a highly attractive concept.
  • Transient expression also offers potential to increase flexibility and efficiency at manufacturing stages. Since the same host cell line can be used for production of multiple products belonging to the same class less complex multi-product facilities utilizing a single common seed train can be designed. Further, due to the increased control over timing and level of intracellular mRNA levels optimal expression profiles allowing the cells to better utilize its resources can be designed, e.g. by separating growth and protein production into two stages. This could also reduce product heterogeneity by minimizing product occupancy in the culture broth and ensuring all product being produced using the same cellular physiological state. Further, the control over timing and levels of different intracellular mRNA species could enable improved performance for production of currently difficult to express protein classes such as certain IgGs, certain multi-chain bi-specific antibodies and certain virus like particles or virus vaccines.
  • protein classes such as certain IgGs, certain multi-chain bi-specific antibodies and certain virus like particles or virus vaccines.
  • transient expression approaches could potentially enable manufacturing of different classes of flu vaccines or other virus or virus like particle based vaccines using e.g. suspension growing CHO cell lines enabling very fast response to e.g. pandemic threats.
  • suspension growing CHO cell lines enabling very fast response to e.g. pandemic threats.
  • the embodiment of the current invention enabling both transient expression and site-directed integration of genes of interest into the same improved host cell line offers an expression platform with major improvements over what is currently available in the art. Using this platform transient expression can be used to enable fast and straightforward developability assessment during pre-clinical stages.
  • the invention will enable increased possibilities to evaluate developability traits (immunogenicity, protein titers, aggregation levels, formulation stability etc.) with high precision for an increased number of protein candidates in each drug development program and with cell lines and conditions nearly identical to ones used in final production and without the data being corrupted by variation coming from differences in the physiological state of cell lines. This can in turn enable even larger cost savings and efficiency increases in drug development by increasing the likelihood of success and reducing the rates of late failures.
  • the similarity in cellular phenotype and expression conditions should also enable the stage for switching between transient expression and stable expression to be highly flexible since comparability of product quality or titers should not be an issue.
  • the switch could be performed during entry into clinical stages, before entry into clinical stage III or first after an increased demand appears after launch of a new drug.
  • FIG. 1 A conceptual general workflow for generating a host cell line with improved properties can be found in Figure la.
  • C an initial mammalian cell (C) carrying a single copy of a template DNA construct T, containing a fully functional template gene of interest (TGOI), at a defined location in the genome (HS) is provided.
  • This cell is then put through a selection workflow (SI) to isolate/select a cell (CI) with highly increased capacity to produce the template protein of interest.
  • the improved CI cell will have a modified genome (Gl) and/or transcriptome compared to the original cell genome (G) and/or transcriptome reflecting multiple accumulated changes in diverse cellular pathways and processes which together give rise to a phenotype with the improved expression capacity.
  • the template DNA construct (T) of the improved cell CI is modified to create a receiving DNA construct (R) lacking the TGOI but containing sequences 0 enabling targeted integration of a desired gene of interest (GOI) from a matching expression vector (EV).
  • This can either be achieved via the use of a DNA modifying enzyme (I) in solitude cutting out the TGOI containing region from T or via the combined use of a DNA modifying enzyme (I) and a donor DNA construct vector (DV).
  • the resulting cell C2 will contain a receiving DNA construct (R) carrying sequences 0 enabling site directed integration of a GOI.
  • This improved cell line C2 can then be used either to enable highly improved performance in transient expression workflows by transfecting the cell with non-integrating Expression plasmids or synthetic mRNA ( Figure lb) or be used in a streamlined CLD workflow ( Figure lc) in which C2 is contacted with an expression vector (EV) containing a matching recombinant construct R' enabling a targeted integration of a desired protein of interest gene by introduction of R' from EV with the aid of a DNA modifying enzyme with specificity for 0 (0) to create a cell C3 maintaining the expression phenotype and genome (Gl) of C2 but now expressing the desired protein of interest.
  • EV expression vector
  • Gl DNA modifying enzyme
  • the genomic location should preferably be a hot spot region, meaning that it supports high transcription of introduced genes and that this transcription is stable over time and reproducible for different genes and different culture conditions. Especially the transcription activity should be high and stable using a serum free culture medium and growth during suspension conditions. Hot spot regions can be identified either via screening approaches, bioinformatics or a combination of these.
  • the current invention builds on that a defined genomic location has been selected and that the sequence of this genomic site is known.
  • the template DNA construct T contains a TGOI and optionally gene(s) coding for selection marker(s) (SM(s)).
  • SM(s) selection marker
  • the TGOI design contains genetic elements either outside or inside the coding sequence(s) providing a high level translation power for the corresponding protein to maximize the expression load/potential.
  • Such elements can include strong promoters such as mCMV, hCMV or synthetic promoters [13], 5'-UTR designs providing increased mRNA stability and/or increased translation, 3'-UTR designs providing increased mRNA stability and/or increased translation, signal peptides providing improved secretion properties and optimized sequence stretches in coding regions based on synonymous codon changes.
  • design of these sequence elements are based on TEEs in the 5'-UTR and RESCUE-modification of the coding region [6, 7].
  • Typical mRNA transcriptional levels using a certain promoter at the hot spot region are preferably known so that the mRNA levels during transient transfection conditions can be matched.
  • the generation of an improved cell can either be performed using a single TGOI expression load as outlined in Figure 1 or by an iterative improvement workflow in which the expression load is gradually increased and intermittent improved cells are isolated after each increase in expression load until a final improved cell is generated as outlined in Figure 2.
  • the first step improved cell carrying a recombinant DNA construct T enabling a first TGOI expression load is contacted with exchange vector(s) enabling modification of T including introduction of a new template DNA construct Tlenabling a second higher TGOI expression load.
  • exchange vector(s) enabling modification of T including introduction of a new template DNA construct Tlenabling a second higher TGOI expression load.
  • This enables the selection of a second step improved cell.
  • This gradual increase in expression load can be repeated any number of times until a final improved cell is generated.
  • the expression load can be varied by using different promoter strengths in different TGOI construct generations, by utilizing 5'-UTR and 3'-UTR variants promoting different mRNA stability and/or translational efficiency, by utilizing coding sequences promoting different mRNA stability and/or translational efficiency or by changing the TGOI copy number between different generations.
  • One specific embodiment of this iterative approach is to utilize cassette exchange mediated by a double stand break inducing site specific nuclease (Nz) as outlined in Figure 7.
  • Nz site specific nuclease
  • the template DNA construct T contains a sequence X at the 5'-end, a sequence Y at the 3'-end and an internal sequence z. All sequences X, Y and z are unique or rare in the genome (Gl) of cell CI.
  • Tl contain sequences at the ends that are either identical to or highly similar to the sequences X and Y in T.
  • the site specific nuclease creates a double strand break at z catalyzing cassette exchange between T and Tl via homologous recombination repair mechanisms using Tl as a repair template. If additional cassette exchange reactions are planned Tl should also contain a sequence zl being unique or rare in the genome (Gl) to enable cassette exchange using a second site specific nuclease with specificity for zl.
  • the site specific nuclease can be any type of gene editing solution such as zinc finger nucleases, homing
  • TGOI variants can be introduced at a loxP (RS2) sequence via the aid of the Cre recombinase (Rec- RS2) resulting in an introduced TGOI flanked by two loxP (RS2) sequences.
  • Cre-RS2 Cre/loxP
  • the first approach utilizes the inherent plasticity of the genome of typical mammalian host cell lines used for recombinant protein production.
  • One embodiment of this approach to generate an improved cell line with improved properties is to screen clones from a culture for a desired set of protein production traits and select the top performing clone.
  • Protein production traits could be, but are not limited to: template protein of interest production rate or culture titer, template protein of interest aggregation level, template protein of interest charge heterogeneity, template protein of interest size
  • heterogeneity glycosylation site occupancy and glycosylation profile for the template protein of interest, cell growth characteristics and cell metabolic characteristics, tertiary structure profile for the template protein of interest, template protein of interest self-association tendency, DNA sequence profiles, mRNA profiles, miRNA profiles, proteomic profiles and genomic stability of cells.
  • This can in principle be performed in analogy with current CLD screening approaches used in the field. There initial screens of many clones using simple parallel culture formats and a few measured parameters such as titer and growth are followed by more extensive screening, including protein quality attributes as described above, of a lower amount of selected clones in more predictive culture formats such as shake flasks or bioreactors.
  • a second embodiment is based on directed evolution of the cells via pro-longed culture of the cells with recombinant expression pressure present.
  • the high recombinant expression load imposed on all cells will have an impact on the viability and growth.
  • Cells that do not handle the recombinant expression load well are hypothesized to be subjected to stress responses (amino acid shortage, charged tRNA shortage, hold up of the ribosomal machinery of recombinant mRNAs, hold up of the folding machinery on recombinant proteins, build-up of soluble or aggregated forms of recombinant protein within cells) reducing viability and growth.
  • the TGOI codes for a template protein of interest representing an important class of proteins such as IgGl antibodies or FC-fusion proteins and preferentially a difficult to express protein of this class to promote isolation of the highest possible production competency of the generated host cell line.
  • the culture of the cells is performed using conditions highly similar to a platform process defined for production of protein for clinical phases or commercial purposes to enable the adaptation through directed evolution to be directly compatible with these conditions.
  • This could for example mean using a bioreactor fed-batch culture with defined culture medium, feed medium and process parameters.
  • Pro-longed culture in this format could for example be achieved by inoculation of next generation cultures using a fraction of the culture from the previous culture.
  • Prolonged culture could also be achieved in a chemostat reactor or a perfusion culture, potentially repeated multiple times using seeding of cells from a previous culture stage.
  • a selection marker such as Neomycin resistance, a DHFR gene or a GS gene
  • Neomycin resistance a selection marker
  • a DHFR gene or a GS gene a selection marker that is used together with culture conditions that put a strong selection pressure for the presence of an active selection marker.
  • Another potential selection marker design could utilize a genetic circuit coupling cellular survival directly to expression of the TGOI.
  • Such a genetic circuit could be based on non-native miRNAs binding both to a sequence stretch of TGOI mRNA and a sequence stretch on a selection marker gene such as NeoR, GS or DHFR. This is to further ensure, in addition to the use of a transcription hot spot region, that the expression construct is not silenced during culture leading to the enrichment of cells that are not expressing the template protein of interest.
  • This approach has the potential to generate superior protein production clones as compared to approaches based on mere screening of clones. Typically in screening approaches a first culture is performed to select a first set of clones from.
  • Directed evolution and screening can also be combined and preferentially at least one final step including screening of production traits should be included.
  • Intermediate screening steps in a workflow based on directed evolution can be used to further ensure that the rare event of clones having managed to silence the SM/TGOI does not lead to such cells being enriched in cultures. If the workflow starts with a clone screening and selection step a range of medium to high performance clones should preferably be selected for a round of culturing to ensure a large genetic diversity.
  • a clone or a pool of cells isolated from any of these workflows is used to create a master cell bank (MCB) of a final improved host cell line.
  • MBC master cell bank
  • the final host cell line having accumulated genetic and/or epigenetic changes compared to the initial host cell line and recombinant mammalian host cell.
  • phenotypic diversity could also be artificially increased between selection/screening rounds by use of chemicals such as epigenetic de-regulators or by radiation increasing mutation rates.
  • a second approach based on targeted engineering can also be used to generate the final host cell line for CLD.
  • a cell (C) according to Figure la being subjected to a defined TGOI expression load is subjected to targeted changes to the genome (G) via the use of genome editing enzymes (such as Zinc finger nucleases, meganucleases, TALENs, CRISPR/Cas9 variants) and recombinant nucleic acid donor constructs to knock-out genetic functionality or add novel genetic functionality.
  • genome editing enzymes such as Zinc finger nucleases, meganucleases, TALENs, CRISPR/Cas9 variants
  • recombinant nucleic acid donor constructs to knock-out genetic functionality or add novel genetic functionality.
  • the evaluation of protein production traits is performed using culture conditions highly similar to a platform process defined for production of proteins for clinical phases or commercial purposes to enable a fit to these conditions. This could for example mean using a bioreactor fed-batch culture with defined culture medium, feed medium and process parameters.
  • the method according to the present invention enables several major advantages. First, the presence of a TGOI with controlled expression properties that can be reproduced for any GOI of the same class following CLD enables evaluation of targeted changes to be performed in conditions that are predictive of the intended final use.
  • the continuous presence of an expression load during the engineering workflow reduces the risk of loss of functionality due to genetic instability.
  • screening of natural genetic diversity and targeted changes can be combined in any form together with conditions predictable to the final use to generate the final improved host cell line.
  • the instability of the host cell genome is first used to enable generation of an improved host cell via multiple genetic and/or epigenetic changes throughout the genome that would likely be difficult to generate using targeted engineering alone.
  • the instability of the genome is reduced either via directed evolution/selection or via targeted engineering. Research is currently underway to define engineering targets enabling stabilization of the genome of for example CHO cells [8].
  • the isolation/selection steps can also be repeated multiple times using a gradually increased expression load as outlined in Figure 2.
  • the rationale for a stepwise increase in the expression load is to enable a gradual move towards a higher performance phenotype. If the initial expression load is too high a low number of clones will be capable of matching this expression load and show up as competent producers during screening and hence a low number of clones can be passed on to a second round of growth and screening. This leads to an early potentially detrimental reduction of phenotypic diversity. There is a high risk for a low survival frequency and very low growth of cultured cells again leading to an inefficient sampling of clonal variation and slow accumulation of improved phenotypes. The initial clonal variation might not even be sufficient to enable survival of any cells at all. By gradually increasing the expression load cells can gradually adapt and an increased genetic variation can be sampled and taken forward in each iteration.
  • a template protein of interest enables selection/evolution of protein production traits matching the combined demands of the specific culture conditions and a high level recombinant expression pressure.
  • the cassette exchange approach to CLD or the use of transient expression processes enables the conditions experienced by the cells during production of a desired protein of interest to be highly similar to the conditions used during generation of the host cell line.
  • Utilization of pre-adapted cells has also been proposed for targeted integration based CLD [10]. In this approach it is proposed that a cell line generated using random integration CLD and displaying desired protein production traits should be selected as a source for generating a final host cell line.
  • the genomic location is identified (must be a single site) and the recombinant constructs are cut out using gene editing based homologous recombination and exchanged for a construct carrying a selection marker flanked by recombinase sequences.
  • the genomic site is treated with a recombinase to cut out the selection marker and leave a single recombinase site flanked by a promoter.
  • This host cell line can then be used for targeted integration of a second expression construct. In this approach there is not a match between the expression load provided by the multiple copies of the first expression construct and the single copy of a second expression construct following CLD.
  • the properties of the host cell line are not likely to be fully suitable to the new conditions. This mismatch can be further increased if the culture conditions are different between the initial cell line and the second cell line. Most importantly no mention of the possibility to use this host cell line for improved transient protein production is made.
  • the current invention offers multiple improvements over this approach in that the selection/evolution of traits can be better matched between host cell line and the cell line producing the desired protein of interest using transient protein expression processes or using a stable cell line based on CLD using targeted integration.
  • the increased sampling of diversity possible by directed evolution and the possibility to add targeted modifications as described in the current invention has the potential to generate production clones with superior protein production traits.
  • the TGOI is under control of an inducible promoter enabling the TGOI to be actively expressed during the improvement workflow (Figure 3a) but inactive during a conditions for transient expression of a desired gene of interest using a plasmid expression vector or a set of synthetic mRNAs ( Figure 3b).
  • This implementation is only applicable for transient expression and not for complementing targeted CLD for expression of a desired gene of interest (GOI).
  • the improved cell CI carrying a receiving DNA construct R is generated according to Figure 4 using a recombinase (Rec-RS2) to excise a TGOI region being flanked by reversible recombinase target sequences RS2.
  • CLD using this cell is outlined in Figure 6.
  • the cell CI containing one irreversible recombinase recognition sequence (RSI) and one reversible recombinase recognition sequence (RS2) is contacted with a recombinase with specificity for RS1/RS1' and a matching Expression Vector (EV) containing a recombinant DNA construct R' with a matching irreversible recombinase recognition sequence RSI' a desired gene of interest (GOI) and a selection marker (SM2).
  • a cell or a pool of cells having undergone correct modification is selected using the selection marker (SM2).
  • the recombinases used can be of any reversible and irreversible type such as attP/attB/PhiC31 as an irreversible system and loxP/Cre as an reversible system.
  • the template recombinant DNA construct T of the improved cell CI is modified to a receiving recombinant DNA construct R via the use of gene editing approaches as outlined in Figure 8.
  • a cassette exchange between T and T' occurs via homologous repair mechanisms catalyzed by a double strand break at z.
  • the two sequences X' and Y' are identical or highly homologous to the sequences X and Y in the genome Gl.
  • the resulting recombinant DNA construct R contains sequences 0 enabling targeted integration of a GOI into the hot spot region HS.
  • the site specific nuclease can be based on any gene editing solution such as zinc finger nucleases, homing endonucleases, TALENs or CRISPR/Cas9 variants or other CRISPR systems.
  • One approach utilizes a receiving DNA construct design R in which an optional SM gene(s) are flanked by two recombinase recognition sequences (RS) and an expression vector design in which a GOI(s) and an optional second SM gene(s) are flanked by matching recombinase recognition sequences (RS').
  • R receiving DNA construct design
  • RS' recombinase recognition sequences
  • a cassette exchange between R and R' is achieved.
  • a cell C2 having undergone the correct exchange only can be selected via the difference in SMs between R and R'.
  • the resulting recombinant DNA construct E contains recombined recombinase recognition sequences RC. Depending on the recombinase system used these can either be different from RS and RS' and differ between the 5' and 3' sequences (as for
  • AttP/attB/PhiC31 or be identical to RS/RS' (as for loxP/Cre).
  • the recombinase recognition sequences used can be of any type such as a serine recombinase type such as attP/attB or a tyrosine recombinase type such as Lox, Rox or FRT together with matching recombinases such as PhiC31, Cre, Dre, or Flp.
  • Different examples of this approach based on varying the promoter placement for R and R' are outlined in Figure 9.
  • the receiving DNA construct R contains a single recombinase recognition sequence RS followed by an optional SM gene(s) and the matching expression vector EV a single matching recombinase recognition sequence RS' followed by a GOI(s) and a SM gene(s) in any order.
  • the R' construct is introduced into the cell and simultaneously changing the relative position of the original R construct so that the resulting E recombinant DNA construct is a combination of R and R'.
  • Different examples of this approach based on varying the promoter placement for R and R' are outlined in Figure 10.
  • the TGOI/GOI could contain a single gene of interest coding for (or mRNA set encoding) proteins such as growth factors, blood clotting factors, cytokines, hormones, erythropoietins, albumins, virus proteins, virus protein mimics, bacterial proteins, bacterial protein mimics, domain antibodies, ScFvs, Affibodies, DARPINs, multimerization domains, IgG Fc domains, albumin binding domains, Fc receptor binding domains or fusion proteins based on combinations of the above single gene of interest coded groups.
  • proteins such as growth factors, blood clotting factors, cytokines, hormones, erythropoietins, albumins, virus proteins, virus protein mimics, bacterial proteins, bacterial protein mimics, domain antibodies, ScFvs, Affibodies, DARPINs, multimerization domains, IgG Fc domains, albumin binding domains, Fc receptor binding domains or fusion proteins
  • the TGOI/GOI could also contain two or more genes of interest coding for (or an mRNA set encoding) proteins such as monoclonal antibodies based on naturally occurring scaffolds, bi-specific antibodies based on naturally occurring scaffolds, Fabs, virus like particles, native virus particles, multiple chain proteins based on association of two or more different protein chains selected from the list of single gene coded proteins above.
  • the TGOI (or mRNA set) of the host cell line and the GOI (or mRNA set) used for protein production of a desired protein of interest encodes proteins belonging to the same protein class.
  • the TGOI (or mRNA set) represents a hard to express protein of that protein class.
  • a single copy of TGOI and GOI is used or the mRNA levels are designed to match between host cell line generation and conditions for production of a desired protein of interest.
  • genetic elements such as promoter(s), 5'-UTR(s), signal peptide(s), design principle for synonymous nucleotide encoding in the coding region and 3'-UTR(s), used in the TGOI and GOI (or mRNA set) are identical (for transient expression during generation of the host cell line and use of stable cell lines for production of a desired protein of interest the mRNA amounts in said mRNA should be designed to match typical levels generated using a certain promoter at said genomic region in said host cell line).
  • a TGOI construct design (or mRNA set design) enabling a very high expression load is used, such as genetic elements having previously been shown to support an expression rate of a recombinant protein in excess of 40, 60 or 80 picogram of protein per cell and day or a final culture titer of 1, 3 or 5 g/l.
  • multiple final host cell lines having utilized different TGOIs (or mRNA sets) of the same protein class but with different amino acid ratios are available and the specific cell line used for CLD or transient protein production using a specific GOI (or mRNA set) is selected based on closest match between amino acid ratios of the template protein of interest (encoded by TGOI) and the desired protein of interest (encoded by GOI).
  • the TGOI (or mRNA set) and GOI can represent identical proteins at the amino acid sequence level but being encoded by different nucleotide sequences.
  • multiple final host cell lines having utilized identical TGOIs (or mRNA sets) but selected/derived to display a specific protein quality profile, such as a specific glycol- profile, are available.
  • the specific cell line used for CLD or transient protein production using a specific GOI (or mRNA set coding for the desired protein of interest) is selected based on closest match between desired protein quality profile and available protein quality profiles.

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Abstract

La présente invention concerne une méthode améliorée d'expression de protéine transitoire qui est généralement applicable à la production d'une quelconque protéine thérapeutique qui peut être produite à l'aide de lignées cellulaires de mammifère et en particulier de cellules d'ovaire de Hamster chinois (CHO). La méthode combine des composants de construction d'expression améliorant le traitement post-transcriptionnel d'un gène d'intérêt, l'introduction d'un processus de sélection de lignée de cellules hôtes en une seule fois à l'aide d'une construction d'expression de protéine matrice d'intérêt pour générer une lignée cellulaire compétente pour la production et des approches permettant l'inactivation de la protéine matrice d'intérêt. L'invention concerne en outre l'expression et la production de protéines de n'importe quelle protéine d'intérêt souhaitée à l'aide de ladite lignée cellulaire compétente pour la production pour des processus d'expression de protéines transitoires avec des rendements et une efficacité améliorés. Dans un mode de réalisation associé préféré de l'invention, la lignée cellulaire compétente pour la production générée peut également être utilisée pour CLD à l'aide d'une intégration ciblée et, par conséquent, la même lignée cellulaire peut être utilisée pour la production d'une protéine d'intérêt souhaitée en utilisant à la fois des processus d'expression transitoire et des processus de production utilisant des cellules de production stables générées à l'aide de processus CLD rapides.
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