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WO2024208673A1 - Espaceurs pour constructions d'expression génique - Google Patents

Espaceurs pour constructions d'expression génique Download PDF

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WO2024208673A1
WO2024208673A1 PCT/EP2024/058187 EP2024058187W WO2024208673A1 WO 2024208673 A1 WO2024208673 A1 WO 2024208673A1 EP 2024058187 W EP2024058187 W EP 2024058187W WO 2024208673 A1 WO2024208673 A1 WO 2024208673A1
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expression
seq
ctcf
nucleic acid
expression vector
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Andreas Jonsson
Daniel Ivansson
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Cytiva Sweden AB
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Cytiva Sweden AB
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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
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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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
    • 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
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
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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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/40—Vector systems having a special element relevant for transcription being an insulator
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island

Definitions

  • the present invention relates to expression vector constructs useful in protein production in mammalian host cells. More specifically, the present invention relates to nucleic acid sequences and expression vector constructs comprising spacer elements that can be placed between two or more expression cassettes within an expression vector. The invention also relates to cells and cell lines comprising such constructs, and to a method of recombinantly producing a protein of interest.
  • Plasmids are small circular extra-chromosomal DNA ranging in size from 1 to over 200 kbp and found occurring naturally in several bacteria.
  • a single bacterial cell may contain several different plasmids and hundreds of copies of each of those plasmids residing within it.
  • the most useful property of a plasmid is its ability to replicate autonomously and be stably maintained within a bacterial cell line. This property of plasmids makes them highly suitable as a tool for transferring foreign genetic material into bacterial host cells.
  • a plasmid usually comprises several genetic elements such as origin of replication, replication initiation gene and antibiotic resistance genes. Smaller plasmids generally rely on the replication machinery of the bacterial host cell whereas larger plasmids may carry their own specific replication genes.
  • Genetically engineered artificial plasmid vectors are one of the most commonly used vectors for introducing genes of interest into host cells. Such engineered plasmids are widely used as cloning vectors and are designed to have genetic elements that allow for the insertion of genes of interest within them.
  • replication of the plasmid vector begins within the cell, resulting in an increasing copy number of the plasmid and thereby an increase of the number of the inserted gene of interest.
  • Transient expression of said genes from plasmid can be done in the bacterial host cells but this usually generates low quantities of protein.
  • Expression vectors can be circular DNA or linear DNA fragments that have been engineered to contain the sequence(s) of the genes of interest in the form of expression cassettes.
  • An expression vector typically also contains several other design features or components which are engineered in the expression vector sequence such as regulatory genes that encode promoters and enhancers for effective transcription of the gene(s) of interest to produce stable messenger RNA (mRNA) which can then be translated into a protein in mammalian host cells.
  • mRNA messenger RNA
  • One way to overcome the above limitation and improve the expression of the genes of interest is to integrate the expression vector construct within the genome of the mammalian host cells when the host cells are transfected with said expression vector. This could be done by including restriction endonuclease binding sites into the design of the expression vector to facilitate integration of the expression vector within the genome of the mammalian host cells.
  • Insulators are a class of DNA sequence elements that possess a common ability to protect genes from undesirable signals emanating from their surrounding environment.
  • insulators protect genes by acting as "barriers" that prevent the extension of nearby condensed chromatin domain into a transcriptionally active region that might otherwise silence the gene expression.
  • Some insulators can act both as enhancer blockers and as barriers.
  • HS4 Hydrosensitivity Site 4, HS4
  • cHS4 insulator found at the 5' end of the chicken P- globin locus
  • the cHS4 insulator marks the border between the active euchromatin in the chicken P- globin locus and the upstream heterochromatin region that is highly condensed and inactive.
  • Vector pcDNATM 3.1(+) is a simple monocistronic expression vector and contains an E. coli propagation cassette, a selection marker cassette, and an expression cassette for the protein of interest.
  • nucleic acid sequence represented by:
  • Ni, N 2 , N3, and N 5 each independently is C or G;
  • N 4 is T or G
  • Ng is T or A.
  • SEQ ID NO: 10 represents a CCCTC binding factor (CTCF) binding sequence and is modified in relation to the CTCF-binding sequence of the wild type chicken Hypersensitivity Site 4 (cHS4) insulator.
  • CTCF CCCTC binding factor
  • cHS4 Hypersensitivity Site 4
  • Each of Ni to Ng in SEQ ID NO: 10 represents a nucleic acid substitution at a corresponding locus on the CTCF binding site (SEQ ID NO: 1) of the wild type cHS4 insulator.
  • SEQ ID NO: 10 is referred to as a "modified CTCF-binding sequence".
  • the modified CTCF-binding sequence may be selected from SEQ ID NO: 2 and SEQ ID NO: 3.
  • the nucleic acid sequence may comprise a modified cHS4 core element including said modified CTCF- binding DNA sequence, which is also referred to as a modified footprint II ( Fl I ) of the cHS4 core element.
  • the modified cHS4 core element may, in addition to said modified CTCF-binding DNA sequence, contain footprints I, III, IV and V, which provide binding sites for VEZF1 (Fl, Fill, and FV), and for USF1/USF2 (FIV).
  • the nucleic acid sequence may comprise two HS4 core elements, each comprising a CTCF-binding sequence, at least one of which being a modified CTCF-binding sequence in accordance with the present disclosure (SEQ ID NO: 10).
  • a HS4 double core element at least one of said HS4 core elements may thus comprise a CTCF- binding sequence selected from SEQ ID NO: 2 and SEQ ID NO: 3.
  • one of said HS4 core elements may be represented by SEQ ID NO: 4, which corresponds to the wild type cHS4 core element.
  • one of the HS4 core elements may comprise a CTCF-binding sequence represented by SEQ ID NO: 1 and one of said HS4 core elements may comprise a CTCF-binding sequence selected from SEQ ID NO: 2 and SEQ ID NO: 3.
  • one of said HS4 core elements may comprise a modified CTCF-binding sequence according to SEQ ID NO: 2 and the other of said HS4 core elements may comprise a modified CTCF- binding sequence according to SEQ ID NO: 3.
  • a double core element may contain at least one CTCF-binding motif according to either of SEQ ID NO 2 and 3.
  • a double core element may have a sequence according to SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 (described in more detail hereinbelow).
  • a double core element may have a sequence that is based on any one of SEQ ID NOs: 4-7, but where one of the CTCF-binding motifs has been replaced with a different CTCF-binding motif, such that at least one of the two CTCF-binding motifs is a modified CTCF-binding sequence of the present disclosure.
  • SEQ ID NO: 4 may be modified to contain one CTCF- binding sequence according to SEQ ID NO: 2 or 3, while preserving one wild-type CTCF-binding motif (SEQ ID NO: 1).
  • SEQ ID NO: 5 may be modified to contain one CTCF-binding motif according to SEQ ID NO: 1 or 3, while preserving one CTCF-binding fragment A (SEQ ID NO: 2).
  • one of SEQ ID NOs: 6 or 7 may be modified to contain one CTCF-binding motif according to SEQ ID NO: 1 or 2, while preserving one CTCF-binding fragment B (SEQ ID NO: 3).
  • the nucleic acid sequence may further comprise a spacer sequence of at least 100 nucleotides in length located downstream of a modified cHS4 core element.
  • a spacer sequence of at least 100 nucleotides in length may be located downstream of the two HS4 core elements, i.e. downstream of the double core element, in the 5' to 3' direction.
  • the spacer sequence may have a length of at least 200 nucleotides, and optionally up to 500 nucleotides, such as up to 300 nucleotides.
  • the additional spacer sequence may be a non-coding sequence or a non-functional sequence, and may be a random sequence.
  • the additional spacer sequence should preferably not contain any sequence motifs known to interact with a DNA binding protein.
  • the present invention further provides an expression vector comprising the nucleic acid sequence as described herein, useful for expression of at least two non-identical polypeptides.
  • the expression vector may comprise: a first expression cassette comprising a first coding sequence encoding a first protein of interest, a second expression cassette comprising a second coding sequence encoding a second protein of interest, and the nucleic acid sequence as described above arranged between said first expression cassette and said second expression cassette.
  • the nucleic acid sequence comprising the CTCF-binding motif thus forms a spacer sequence.
  • the expression vector may comprise further expression cassettes or sequences useful for vector propagation, genomic integration, gene expression, gene product processing, selection and/or purification.
  • the expression vector may comprise a third expression cassette comprising a selection marker.
  • the expression vector may further comprise recombinase sites for integration into the genome of a host cell.
  • the expression vector may be provided as a circular vector, or it may be linear or linearized.
  • the first and second proteins of interest typically form part of the same protein complex, which may be a therapeutic protein complex, such as an antibody or an antibody fragment formed of two separate polypeptide chains.
  • the first protein of interest may be a first polypeptide chain of an antibody or antibody fragment
  • the second protein of interest may be a second polypeptide chain of an antibody or antibody fragment.
  • the first protein of interest may represent the heavy chain
  • the second protein of interest may represent the light chain of an antibody, such as a monoclonal antibody.
  • the first polypeptide chain may comprise a variable domain of an antibody, such as of an antibody heavy chain
  • the second polypeptide chain may comprise another variable domain of an antibody, such as of an antibody light chain.
  • the first expression cassette and/or the second expression cassette may comprise a eukaryotic promoter sequence, such as a CMV, EFlalpha or SV40 PGK promoter.
  • first expression cassette and/or the second expression cassette may comprise a CMV promoter.
  • the present invention further provides a cell, a cell line, or a cell culture, comprising the expression vector described herein.
  • the cell may be an in vitro growing cell.
  • the cell may be a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, transfected with the expression vector.
  • the expression vector may be integrated, preferably stably integrated, into the cell genome.
  • the present invention further provides a method of recombinantly producing two polypeptides, comprising i) transfecting mammalian cells, such as CHO cells, with an expression vector comprising expression cassettes encoding first and second proteins of interest as described herein, and ii) culturing the transfected cells in cell culture media under conditions enabling expression of the first and second proteins of interest.
  • the method may further comprise purifying the polypeptides from the cell culture, according to known methods.
  • the two polypeptides may be as described above, for instance two polypeptide chains of a monoclonal antibody or of an antibody fragment.
  • peptide and “polypeptide” are used synonymously herein to refer to compounds formed of sequences of amino acids, without restriction as to size.
  • Protein may be used to refer to the larger compounds of this class.
  • wild type or "wt” refers to the typical naturally occurring form.
  • FIG. 1A shows a schematic representation of the core element of the wild type chicken Hypersensitivity Site 4 (cHS4) insulator.
  • FIG. IB shows a schematic representation of the cHS4 double core formed by placing two core elements of the wild type cHS4 insulator one after the other.
  • FIG. 2 shows a map of the known monocistronic plasmid expression vector pcDNATM3.1.
  • FIG. 3 shows a map of the parental/base bicistronic modular expression vector of the present invention in circular configuration for random integration.
  • FIG. 4 shows the parental/base bicistronic modular expression vector of Fig. 3 in linear configuration.
  • FIG. 5 shows a map of the parental/base bicistronic modular expression vector of the present invention in circular configuration for site directed integration.
  • FIG. 6 shows the parental/base bicistronic modular expression vector of Fig. 5 in linear configuration.
  • FIG. 7 shows the flow cytometry data pertaining to cells transfected with expression vector pGE0308 pertaining to experiment 1.
  • the left panel shows a forward scatter to side scatter plot used to gate viable single CHO cells (N-gate).
  • the right panel shows a mTagBFP2 to eGFP plot used to calculate % expression of both eGFP and mTagBFP2 (P-gate) from CHO cells in gate N.
  • FIG. 8 shows a plot with the percentage of expressing recombinant CHO cells from gate P pertaining to experiment 1.
  • the x-axis shows functional Spacer A-D with different length of additional spacer X.
  • C represents a control with only a lOObp spacer X (no double core element).
  • FIG. 9 shows the flow cytometry data pertaining to cells transfected with expression vector pGE0359 pertaining to experiment 2.
  • the left panel shows a forward scatter to side scatter plot used to gate viable single CHO cells (F-gate).
  • the right panel shows a mTagBFP2 to eGFP plot used to calculate % expression of both eGFP and mTagBFP2 (M-gate) from CHO cells in gate F.
  • FIG. 10 shows a plot of the percentage of expressing recombinant CHO cells from gate M pertaining to experiment 2.
  • the x-axis shows functional Spacer A-C with different length of additional spacer.
  • FIG. 11 shows the flow cytometry plots used for calculation of mTagBFP2 and eGFP expression for experiment 3.
  • Left panel shows a forward scatter to side scatter plot used to gate viable cells (A- gate).
  • the middle panel shows a side scatter to mRasberry plot used to gate mRasberry positive cells corresponding to cells having undergone integration at the LP (B-gate).
  • the right panel shows a mTagBFP2 to eGFP plot used to calculate mean expression values based on the fluorescence signals of the main focused population (C-gate).
  • FIG. 12 shows the normalized expression of functional spacer A with two different 200 bp additional spacers (a and P) and with only 200 bp spacer without the double core element.
  • Error bar +/- 2 standard deviations.
  • Figure 2 shows a map of the known monocistronic plasmid expression vector pcDNATM3.1 for expression in a variety of mammalian cell lines. This vector was used as the starting point for designing the modular bicistronic expression vector of the present invention.
  • Figure 3 shows a map of the parental/base bicistronic modular expression vector for random integration in circular configuration.
  • the bicistronic expression vector was designed for expression of two polypeptides.
  • the parental/base expression vector comprises a standard
  • E. coli propagation cassette as in Figure 2 (not shown in picture).
  • the selection marker is represented as expression cassette 1.
  • Expression cassette 2 comprises an eGFP encoding gene and expression cassette 3 comprises an mTagBFP2 encoding gene.
  • the two polypeptides expressed are eGFP and mTagBFP2.
  • expression cassettes 2 and 3 have spacer elements between them to isolate gene expression from said cassettes to allow improved expression of the polypeptides of interest.
  • Figure 3 also shows promoters 2 and 3 to improve expression from the expression cassettes 2 and 3 respectively.
  • Figure 4 shows the same parental/base bicistronic modular expression vector in linear configuration.
  • Figure 5 shows a map of the parental/base bicistronic modular expression vector for site directed integration in circular configuration.
  • the bicistronic expression vector was designed for expression of two polypeptides.
  • the parental/base expression vector comprises a standard E. coli propagation cassette as in Figure 2 (not shown in picture).
  • the selection marker gene is represented as expression cassette 1 and have no promotor, but instead an upstream attB2 recombination site.
  • Expression cassette 2 comprises a Fc-eGFP encoding gene and expression cassette 3 comprises a mTagBFP2 encoding gene.
  • the selection marker When the vector is integrated into the unique landing pad integrated in the CHO genome using PhiC31 recombinase, the selection marker will be activated by a promotor from the landing pad.
  • the two polypeptides expressed are eGFP and mTagBFP2.
  • expression cassettes 2 and 3 have spacer elements between them to isolate gene expression from said cassettes to allow improved expression of the polypeptides of interest.
  • Figure 3 also shows promoters 2 and 3 to improve expression from the expression cassettes 2 and 3 respectively.
  • FIG. 6 shows the same parental/base bicistronic modular expression vector in linear configuration.
  • modified versions of the CTCF binding site of the wild type cHS4 double core element were developed and evaluated, including for example, a modified CTCF-binding DNA fragment A (SEQ ID NO: 2) and a modified CTCF-binding DNA fragment B (SEQ ID NO: 3).
  • SEQ ID NO: 2 a modified CTCF-binding DNA fragment A
  • SEQ ID NO: 3 a modified CTCF-binding DNA fragment B
  • Table la The respective nucleic acid sequences of the wild type and the modified CTCF-binding motifs or fragments are shown in Table la. Table la.
  • Table lb Sequence identity between CTCF-binding sequences.
  • modified versions of the entire cHS4 double core element comprising the above-described modified versions of the CTCF binding sites, were developed and evaluated for gene expression. These modified double core elements were used in spacers A-C as outlined below.
  • the modified cHS4 double core element A included the modified CTCF binding DNA fragment A (SEQ ID NO: 2) as its CTCF binding site.
  • the modified cHS4 double core element A is referred to as "spacer A" when used as a spacer element between two expression cassettes (here expression cassettes 2 and 3).
  • the modified cHS4 double core element B included the modified CTCF binding DNA fragment B (SEQ ID NO: 3) as its CTCF binding site.
  • the modified cHS4 double core element B is referred to as "spacer B" when used as a spacer element between two expression cassettes (here expression cassettes 2 and 3).
  • the modified cHS4 double core element C (SEQ ID NO: 7) included the modified CTCF binding DNA fragment B (SEQ ID NO: 3) as its CTCF binding site. Furthermore, the DNA sequences in between the footprints Fl, Fll, Fill, FIV and FV, as well as the DNA sequence in between the two core elements, were different in relation to the Spacer B (SEQ ID NO: 6).
  • the modified cHS4 double core element C is referred to as "spacer C" when used as a spacer element between two expression cassettes (here expression cassettes 2 and 3).
  • spacer D when used as spacer element between two expression cassettes (here expression cassettes 2 and 3).
  • the total length of the spacer elements between the expression cassettes 2 and 3 was also evaluated, and it was found that attaching an additional spacer sequence downstream of the modified cHS4 double core element led to even higher gene expression from the expression cassettes 2 and 3.
  • This combination of the additional spacer element, herein referred to as "spacer X” or “spacer element X”, and a modified cHS4 double core element (Spacer A, B or C) is referred to as a functional spacer element when used as a spacer between the expression cassettes 2 and 3.
  • additional spacer elements X were constructed and evaluated, comprising, for example, nucleic acid sequences differing in the order of the nucleotides and nucleic acid sequences of different lengths such as 100 bp, 200 bp, 300 bp, 400 and 500 bp, in combination with the modified cHS4 double core elements as described above to evaluate their effect on gene expression.
  • Table 2 shows the various plasmid expression vectors that were constructed based on the parental/base expression vector design as shown in Figure 3 and 5.
  • the expression cassette 2 was configured to encode for eGFP or Fc-eGFP protein with the human cytomegalovirus (hCMV) or chimeric CMV promotor as promotor 2.
  • the expression cassette 3 was configured to encode for mTagBFP2 protein with the mouse cytomegalovirus (mCMV) promotor as promoter 3.
  • a spacer as described above based on the cHS4 double core element was arranged between expression vectors 2 and 3.
  • the selection marker was eighter glutamine synthetase (SMI), TagRFP-T (SM2) or mRaspberry (SM3).
  • SMSI eighter glutamine synthetase
  • SM2 TagRFP-T
  • SM3 mRaspberry
  • Spacer D refers to the wild type cHS4 double core element (SEQ ID NO: 4).
  • plasmid expression vectors pGE0173, pGE0296-pGE0303 and pGE0307-pGE0309 having the general structure as described in connection with Fig. 3 and in Table 2 were linearized and transfected using electroporation into Chinese Hamster Ovary (CHO) cells for random integration within the genome of said CHO cells.
  • the transfected CHO cells were allowed to grow with 25 pM of methionine sulfoximine (MSX). After 2 weeks, cell samples were analysed by flow cytometry for determination of the levels of eGFP and mTagBFP2 expression.
  • Figure 7 shows the flow cytometry data pertaining to cells transfected with expression vector pGE0308 pertaining to experiment 1.
  • the left panel shows selected viable single CHO cells (N-gate) for evaluation and the right panel shows the expression of mTagBFP2 and eGFP from N-gate where % CHO cells with expression of both eGFP and mTagBFP2 (P-gate) can be calculated. All expression vectors in experiment 1 were evaluated in the same way as pGE0308 as described above.
  • plasmid expression vectors pGE0341, pGE0355-pGE0365 having the general structure as described in connection with Fig. 3 were linearized and transfected using electroporation into mammalian CHO cells for random integration within the genome of said CHO cells.
  • the transfected CHO cells were allowed to grow. After 2 weeks, cell samples were analysed by flow cytometry for integration of vector with TagRFP-T and for determination of the levels of eGFP and mTagBFP2 expression.
  • Figure 9 shows a plot of flow cytometry data pertaining to cells transfected with expression vector pGE0359 pertaining to experiment 2.
  • the left panel shows selected viable single CHO cells (F-gate) for evaluation and the right panel shows the expression of mTagBFP2 and eGFP from F-gate where % CHO cells with both expression of eGFP and mTagBFP2 (M-gate) can be calculated. All expression vectors in experiment 2 were evaluated in the same way as pGE0359 as described above.
  • Spacer C with additional 200 bp spacer X shows the best expression with 0.49 % of the CHO cells expressing in gate M.
  • the Spacer C variants provides a generally improved expression compared with Spacers A or B with a chimeric CMV promotor for expression cassette 2.
  • Spacer B with additional 200 bp spacer shows the best improvement.
  • additional spacer sequences were evaluated using two different 200 bp additional spacers sequences for the functional spacer separating the expression cassettes 2 and 3, additional spacer variant a (SEQ ID NO: 8) and additional spacer variant (SEQ ID NO: 9).
  • site directed integration SDI
  • the expression vectors were transfected with electroporation into CHO cells for integration at a landing pad (LP) sequence previously inserted in the CHO cell genome. For each spacer sequence element, duplicate transfections were performed.
  • FIG. 11 shows a plot of the flow cytometry data pertaining to experiment 3. Mean mTagBFP2 and eGFP signals were calculated based on the sub-population of viable and mRasberry-positive cells as shown in Figure 11. Data were then normalized to the donor plasmid (pUP0073) giving the lowest expression level. As shown in Figure 11, the left panel shows a forward scatter to side scatter plot used to gate viable cells (A-gate).
  • the middle panel shows a side scatter to mRasberry plot used to gate mRasberry positive cells corresponding to cells having undergone integration at the landing pad (B-gate).
  • the right panel shows a mTagBFP2 to eGFP plot used to calculate the mean expression values based on the fluorescence signals of the main focused population (C-gate).
  • C-gate main focused population
  • the mean value and standard deviation for each spacer element were calculated based on the duplicate data.
  • the resulting data is plotted in Figure 12.
  • Experiment 3 shows that the Spacer A with a 200 bp additional spacer has clearly improved expression compared to only a 200 bp spacer (i.e. lacking a cHS4 double core element) and that Spacer A works well with 200 bp additional spacers of different sequences.
  • This example demonstrates successful production of monoclonal antibody (Herceptin) by expression of light and heavy chain expression cassettes separated using functional spacer elements according to embodiments of the present invention.
  • Table 3 Functional spacer designs used in antibody expression vectors.
  • the different expression vectors (Table 3) were linearised and used to transfect CHO-K1 cells by a lipofectamine based method. Two days after transfection, CHO cells were sorted into 96-well plates for static culture using FACS. For each well in the plates, 2000 cells were sorted into 100 pl growth medium supplemented with 25 pM of methionine sulfoximine (MSX). Cells were then grown by static culture in the presence of 25 pM MSX for a total of 26 days. During this static culture period only cells having integrated the expression vector (and hence the GS gene) can divide and multiply in the presence of 25 pM MSX and the absence of L-glutamine.
  • MSX methionine sulfoximine
  • Table 4 Measured titers for the two best performing minipools for each expression vector.
  • both expression vectors containing Spacer A (pGE0349, pGE0352) and expression vectors containing spacer C (pGE0353, pGE0354) resulted in desirably high antibody titers (around or above 1 g/L).
  • the pGE0349 expression vector was compared to a reference expression vector (pGE0164) containing Spacer D as a functional spacer.
  • the reference vector contained no additional Spacer X.
  • Table 5 Comparison of expression vectors pGE0164 and pGE0349.
  • the two expression vectors (Table 5) were linearised and used to transfect CHO-K1 cells by a lipofectamine based method. Three days after transfection, CHO cells were sorted into two 96-well plates for static culture for each expression vector using FACS. For each well in the plates, 5000 cells were sorted into 100 pl growth medium supplemented with 25 pM MSX. Cells were then grown by static culture in the presence of 25 pM MSX for a total of 25 days for pGE0164 and 28 days for pGE0349. During this static culture period only cells having integrated the expression vector (and hence the GS gene) can divide and multiply in the presence of 25 pM MSX and the absence of L- glutamine.
  • Table 6 Median titer of expressing clones using either pGE0164 or pGE0349.
  • the expression vector with the inventive functional spacer element produced antibody titers at least comparable to those of the expression vector including the Spacer D (wild type cHS4 double core).
  • Example 4C Full cell line development
  • pGE0419 modified expression vector was used to perform a full cell line development campaign, including assessment of producer clones using fed-batch cultures in shake flasks.
  • the expression vector pGE0419 contained the following modifications compared to the pGE0349 vector of Examples 4A and 4B: First, the GS gene used as a selection marker was modified by mutation of amino acid number 299 from arginine to Glycine (R299G). Secondly, the promoter driving the Herceptin HC was changed from a chimeric CMV to a mCMV sequence. Lastly, the additional spacer contained 200 bp instead of 100 bp.
  • Example 4B Transfection and minipool generation were performed as described above for Example 4B. However, in this experiment the best performing minipools were selected for further expansion and then used in single cell cloning using FACS. During single cell cloning, single cells were seeded into each well of 96-well static culture plates containing 100 pl cloning media supplemented with 25 pM MSX.
  • Example 4C thus demonstrates successful establishment of a CHO cell line capable of producing a desired antibody at very high levels.
  • This example demonstrates a full cell line development campaign for the production of monoclonal antibody (Herceptin) using site directed integration (SDI) of antibody light and heavy chain expression cassettes separated by functional spacer elements according to embodiments of the present invention.
  • SDI site directed integration
  • a site-directed integration (SDI) approach was used to introduce sequences encoding Herceptin heavy and light antibody chains into a Chinese Hamster Ovary (CHO) cell line (developed in-house) having a landing pad sequence previously inserted in the cell genome.
  • Fig. 13A illustrates the host cell landing pad LP region which was flanked by two full length HS4 sequences.
  • the cells were grown in ActiPro medium containing 6 mM L-Glutamine at all times, except for transfections and cloning.
  • pUP0295 and pUP0296 Two donor vectors, pUP0295 and pUP0296 (50:50 mix) were used for transfection together with a PhiC31 plasmid to facilitate integration into the landing pad.
  • the donor vector design is shown in Fig. 13B.
  • Each of the vectors pUP0295 and pUP0296 contained either the light chain (LC) encoding sequence in the first cassette and the heavy chain (HC) encoding sequence in the second cassette, as shown in Fig 13B, or vice versa.
  • Each cassette was under the control of a mCMV promoter.
  • the expression cassettes were separated by a spacer consisting of Spacer A (SEQ ID NO: 5) fused to a 200 bp additional spacer (SEQ ID NO: 8).
  • All donor vectors further contained a marker for SDI (eGFP) and a marker for all inserted events, both random and SDI (CD8).
  • the CD8 can be stained with a CD8- R667 Ab for cell sorting and flow cytometry analysis to evaluate plasmid insertion.
  • Cre excision of fluorescent markers and bulk sort Following expansion after the last selection marker positive bulk sort, the cells were transfected with Cre recombinase mRNA to remove the LoxP flanked portion of the plasmid with the selection markers.
  • Fig.l3C illustrates the landing pad genomic region of the transfected cells after excision of the plasmid backbone and selection markers. The cells were analyzed with flow cytometry 7 days after transfection to confirm that the Cre excision had gone well. Results showed that >97 % of the cells had lost the eGFP fluorescent marker. Thereafter a last bulk sort was made to purify the eGFP and CD8 negative population before cloning. After 3-4 days of expansion, the cells were analyzed by flow cytometry and confirmed ready for cloning as the pool had ca 99.9 % eGFP negative and 99.6 % CD8 negative cells.
  • Cloning and titer evaluation Ten 96-well plates were single cell sorted using the UP.SIGHT cell printer (Cytena). The plates were kept in a static incubator for expansion for 16 days before the cells were ready for titer screen. The titer screens were done using a Biacore 8K instrument (Cytiva), and the titer was normalized to confluence. Thereafter, the top 192 clones per campaign were selected for expansion in 96-deep well plates. Expansion and titer evaluation: After 2 passages and 11-12 days in expansion, a death run was seeded for each plate. After 9-10 days in culture, the death runs were ready for Ab titer evaluation using Biacore 8K instrument (Cytiva). 14 clones were selected for expansion in shake flasks.
  • the Ab titer and metabolites were measured with Cedex Bio HT Analyzer (Roche).
  • the clones lasted for 10-17 days in Fed Batch.
  • the titers for the regular Fed Batch ranged between 0.5-7.8 g/L (up to 17 days).
  • Table 8 reports Ab titer at day 14 for the top 5 Herceptin expressing clones.
  • the Examples 1-5 demonstrate that the spacer elements disclosed herein offer the potential to provide improved cell lines for high expression of a protein of interest, in particular where two proteins are expressed simultaneously, such as is the case of mAbs and other antibody fragments or variants incorporating both light chain and heavy chain components.

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Abstract

L'invention concerne une séquence d'acide nucléique comprenant une séquence de liaison CTCF modifiée représentée par : TGCAGTACCTCCCTN1N2N3CCAGCAGGN4GGCAN5N6AGN7GAAN8GGTGAACTGGAGT (SEQ ID NO : 10) où N1, N2, N3 et N5 sont chacun indépendamment C ou G ; N4 est T ou G ; N6 et N7 sont chacun indépendamment C ou T ; et N8 est T ou A. Chacun des N1 à N8 représente une substitution d'acide nucléique au niveau d'un locus correspondant du site de liaison au CTCF (SEQ ID NO : 1) de l'isolateur de type sauvage du site d'hypersensibilité 4 du poulet (cHS4). La présente invention concerne également des vecteurs d'expression et des cellules recombinées incorporant la séquence modifiée de liaison au CTCF, ainsi qu'un procédé associé de production d'au moins deux polypeptides.
PCT/EP2024/058187 2023-04-05 2024-03-27 Espaceurs pour constructions d'expression génique Pending WO2024208673A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010036976A2 (fr) * 2008-09-25 2010-04-01 Transgenrx, Inc. Nouveaux vecteurs pour la production d'anticorps
WO2010046493A2 (fr) * 2008-10-23 2010-04-29 Université de Lausanne Vecteurs de transfert de gène comprenant au moins une molécule d'adn isolée ayant des propriétés d'isolateur et/ou d'arrêt et procédés pour identifier ceux-ci
US20110294114A1 (en) * 2009-12-04 2011-12-01 Cincinnati Children's Hospital Medical Center Optimization of determinants for successful genetic correction of diseases, mediated by hematopoietic stem cells
WO2014140218A1 (fr) * 2013-03-13 2014-09-18 Fundación Pública Andaluza Progreso Y Salud Isolateur pour améliorer les vecteurs de transfert de gènes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010036976A2 (fr) * 2008-09-25 2010-04-01 Transgenrx, Inc. Nouveaux vecteurs pour la production d'anticorps
WO2010046493A2 (fr) * 2008-10-23 2010-04-29 Université de Lausanne Vecteurs de transfert de gène comprenant au moins une molécule d'adn isolée ayant des propriétés d'isolateur et/ou d'arrêt et procédés pour identifier ceux-ci
US20110294114A1 (en) * 2009-12-04 2011-12-01 Cincinnati Children's Hospital Medical Center Optimization of determinants for successful genetic correction of diseases, mediated by hematopoietic stem cells
WO2014140218A1 (fr) * 2013-03-13 2014-09-18 Fundación Pública Andaluza Progreso Y Salud Isolateur pour améliorer les vecteurs de transfert de gènes

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ALI RAMEZANI: "Performance and safety-enhanced lentiviral vectors containing the human interferon-β scaffold attachment region and the chicken (3-globin insulator", BLOOD, vol. 101, 2003, pages 4717 - 4724, XP002435838, DOI: 10.1182/blood-2002-09-2991
JAZAYERI SEYEDEH HODA ET AL: "Vector and Cell Line Engineering Technologies Toward Recombinant Protein Expression in Mammalian Cell Lines", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, HUMANA PRESS INC, NEW YORK, 3 February 2018 (2018-02-03), pages 986 - 1003, XP036554247, ISSN: 0273-2289, Retrieved from the Internet <URL:https://link.springer.com/article/10.1007/s12010-017-2689-8> [retrieved on 20240528], DOI: 10.1007/S12010-017-2689-8 *
KAZUHIDE YAHATA: "cHS4 Insulator-mediated Alleviation of Promoter Interference during Cell-based Expression of Tandemly Associated Transgenes", JOURNAL OF MOLECULAR BIOLOGY, 1 November 2007 (2007-11-01), United Kingdom, pages 580 - 590, XP093167632, ISSN: 0022-2836, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S0022283607012430/pdfft?md5=272b4d3ecca4f119290f07d7f47f51b4&pid=1-s2.0-S0022283607012430-main.pdf> [retrieved on 20240528], DOI: 10.1016/j.jmb.2007.09.054 *
KRITIKA GUPTA: "Vector-related stratagems for enhanced monoclonal antibody production in mammalian cells", BIOTECHNOLOGY ADVANCES., 1 December 2019 (2019-12-01), GB, pages 107415, XP093166188, ISSN: 0734-9750, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S0734975019301053?via%3Dihub> [retrieved on 20240523], DOI: 10.1016/j.biotechadv.2019.107415 *
O MAKSIMENKO ET AL: "Regulatory Elements in Vectors for Efficient Generation of Cell Lines Producing Target Proteins", ACTA NATURAE, 1 July 2015 (2015-07-01), Russia (Federation), pages 15, XP055370514, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610161/pdf/AN20758251-26-015.pdf> [retrieved on 20240528] *
QIN JYZHANG LCLIFT KLHULUR IXIANG AP ET AL.: "Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter", PLOS ONE, vol. 5, no. 5, 2010, pages e10611, XP055151992, DOI: 10.1371/journal.pone.0010611
RAMEZANI A ET AL: "Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 101, no. 12, 15 June 2003 (2003-06-15), pages 4717 - 4724, XP002435838, ISSN: 0006-4971, DOI: 10.1182/BLOOD-2002-09-2991 *
SAUNDERS FAY ET AL: "Chromatin Function Modifying Elements in an Industrial Antibody Production Platform - Comparison of UCOE, MAR, STAR and cHS4 Elements", PLOS ONE, vol. 10, no. 4, 7 April 2015 (2015-04-07), pages e0120096, XP055867398, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4388700/pdf/pone.0120096.pdf> [retrieved on 20240527], DOI: 10.1371/journal.pone.0120096 *

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