Viral origin of replication for increasing protein productivity from mammalian cells

Abstract

This disclosure relates to the use of an Epstein-Barr virus origin of replication (oriP) or a functional fragment thereof in a protein expression construct to increase production of a protein of interest in mammalian cells. Also disclosed are protein expression constructs for increasing production of antibodies in mammalian cells, and mammalian cells containing the expression constructs.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 927,833, filed October 30, 2019, which is hereby incorporated by reference in its entirety.

[0002] Field The present disclosure relates to the use of the viral origin of replication (oriP) in protein expression constructs to increase the production of a protein of interest in mammalian cells. Also disclosed are protein expression constructs for increasing the production of antibodies in mammalian cells, and mammalian cells containing the expression constructs.

Background Art

[0003] Introduction Human or animal cells are routinely used in the academic and industrial communities for protein production. Proteins can be produced through transient or stable protein expression. For stable protein expression, generally, a stable pool of cells that can be used for production is first created, and / or the cells in this pool are cloned to identify cell lines that are excellent production strains. In any case, scientists are trying to increase the productivity of cells to reduce production costs.

Prior Art Documents

Non - Patent Documents

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Summary of the Invention

Problems to be Solved by the Invention

[0005] In order to increase protein production from stably expressing cells, several methods are used, for example, modifying the codons of the target gene, modifying the promoter, incorporating elements of scaffold / matrix attachment region (S / MAR) or ubiquitous chromatin opening element (UCOE), improving the cell culture medium and nutrient additives, and improving the selection of highly expressing cells. Other methods are needed to increase protein production from stable pools or cell lines.

Means for Solving the Problems

[0006] Overview The inventors have demonstrated that incorporation of the EBV oriP sequence into an expression plasmid can increase protein production in selected CHO pools and clones even in the absence of the EBV EBNA1 protein.

[0007] To increase protein production, the EBV oriP system of the EBNA1 protein was used in transient transfection of the CHO-3E7 platform. The inventors investigated the use of the EBV oriP system of the EBNA1 protein to increase productivity in stable cell lines. The presence of oriP alone was found to increase the productivity of the stable pool, and the presence of EBNA1 in the cell line did not increase productivity in this context.

[0008] Accordingly, one aspect of the present disclosure is a nucleic acid construct for the expression of a target protein. The nucleic acid construct of the present disclosure comprises: a) at least one expression cassette comprising a DNA sequence encoding a target protein operably linked to a promoter and a transcription termination point; b) a selectable marker; and c) an Epstein-Barr virus (EBV) origin of replication (oriP) or a functional fragment thereof comprising a dyad symmetry (DS) region and a family of repeats (FR) segment. In one embodiment, oriP or a functional fragment thereof has at least 90% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] In one embodiment, the nucleic acid construct further comprises a scaffold attachment region (SAR).

[0010] In one embodiment, the promoter is an inducible promoter, which is optionally a tetracycline response element (TRE), a ponA inducible promoter, or a cumate inducible promoter.

[0011] In one embodiment, the promoter is a constitutive promoter, which is optionally a human ubiquitin C (UBC) promoter, a human elongation factor 1α (EF1A) promoter, a human phosphoglycerate kinase 1 (PGK) promoter, a simian virus 40 early promoter (SV40), a cytomegalovirus immediate early promoter (CMV) promoter, a chicken b-actin promoter linked to the CMV early enhancer (CAG), a hybrid EF1-HTLV promoter, or a Chinese hamster EF1 promoter (CHEF).

[0012] In one embodiment, the selectable marker is a neomycin resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a blasticidin resistance gene, a zeocin resistance gene, or optionally a glutamine synthetase (GS) gene.

[0013] In one embodiment, the expression cassette encodes an antibody or an antibody fragment, or an antibody heavy chain and / or an antibody light chain.

[0014] In some embodiments, the nucleic acid construct encodes two expression cassettes. In one embodiment, one expression cassette encodes an antibody heavy chain and one expression cassette encodes an antibody light chain.

[0015] Another aspect of the present disclosure is a method for producing a target protein, comprising: a) introducing the nucleic acid construct of the present disclosure into a mammalian cell; b) applying a selection pressure to the cell to select a cell carrying the selectable marker; and c) culturing the cell under conditions for producing the target protein. In some embodiments, two different nucleic acid constructs of the present disclosure are introduced into the mammalian cell.

[0016] In one embodiment, one or more nucleic acid constructs are introduced into cells by transfection. In some embodiments, transfection is performed with a transfection reagent, such as a cationic lipid, a non-liposomal reagent, or a cationic polymer. Optionally, the cationic polymer is polyethyleneimine (PEI). In other embodiments, transfection is calcium phosphate transfection or electroporation / nucleofection.

[0017] In one embodiment, protein production is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 250% increased compared to protein production in cells by a nucleic acid construct lacking oriP when cultured under the same conditions.

[0018] In one embodiment, the mammalian cell is an SP2 / 0 cell, an NS / 0 cell, an HT-1080 cell, a PER.C6 cell, an HKB-11 cell, a CAP cell, a Huh-7 cell, a Chinese hamster ovary (CHO) cell, or a human embryonic kidney 293 (HEK293) cell. In one embodiment, the cell does not express EBNA1.

[0019] In one embodiment, the selectable marker is glutamine synthetase (GS) and the selection pressure is the removal of glutamine from the growth medium. In another embodiment, the selection agent is methionine sulfoximine (MSX) selection of cells overexpressing glutamine synthetase. In yet another embodiment, the selectable marker is methotrexate selection of cells expressing dihydrofolate reductase (DHFR).

[0020] In another embodiment, the promoter is an inducible promoter and the conditions for production of the protein of interest include the addition of an inducer.

[0021] In a further embodiment, the nucleic acid is integrated into the genome of a mammalian cell.

[0022] In one embodiment, the method further comprises the steps of collecting mammalian cells and / or cell culture medium containing the target protein, and optionally purifying the target protein from the collected cells and / or cell culture medium.

[0023] In some embodiments, the nucleic acid encodes an antibody fragment, an antibody heavy chain, and / or an antibody light chain. In some embodiments, the target protein is an antibody or an antibody fragment, optionally cetuximab or a fragment thereof.

[0024] A further aspect of the present disclosure is a mammalian cell for increasing the production of a target protein, comprising one or more nucleic acid constructs of the present disclosure. In some embodiments, the cell comprises two different nucleic acid constructs of the present disclosure, each encoding a different target protein. In some embodiments, the target protein is an antibody or an antibody fragment, optionally cetuximab or a fragment thereof.

[0025] In some embodiments, one or more nucleic acid constructs are stably transfected, and optionally, one or more constructs are integrated into the genome of the mammalian cell.

[0026] In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell or a human embryonic kidney 293 (HEK293) cell. In one embodiment, the cell does not express EBNA1.

[0027] The previous section is provided by way of example only and is not intended to limit the scope of the present disclosure or the scope of the appended claims. Additional objects and advantages related to the compositions and methods of the present disclosure will be understood by those skilled in the art in view of the claims, description, and examples of the present invention. For example, the various aspects and embodiments of the present disclosure may be utilized in numerous combinations, all of which are expressly contemplated herein. These additional advantages, objects, and embodiments are expressly included within the scope of the present disclosure. Publications and other materials used herein to clarify the background of the present disclosure and, in certain instances, to provide additional details regarding implementation are incorporated by reference and, for convenience, are listed in the appended References section.

[0028] Drawings Further objects, features, and advantages of the present disclosure will become apparent from the following detailed description in connection with the accompanying drawings that illustrate exemplary embodiments of the present disclosure.

Brief Description of the Drawings

[0029]

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Mode for Carrying Out the Invention

[0030] Description of Various Embodiments The following is a detailed description provided to assist those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the present disclosure. All publications, patent applications, patents, figures, and other references cited herein are hereby expressly incorporated by reference in their entirety.

[0031] I. Definitions As used herein, the following terms may have the meanings ascribed to them unless otherwise specified. However, other meanings known or understood by those skilled in the art are also possible and should be understood to be within the scope of the present disclosure. All publications, patent applications, patents, and other references cited herein are hereby incorporated by reference in their entirety. In case of conflict, this specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0032] When a range of values is provided, each intervening value between the upper and lower limits of the range, to one tenth of the unit of the lower limit, unless clearly separately defined in context, and any other stated value or intervening value in the stated range, is to be understood as being included within the scope of the description. A range from any lower value to any upper value is contemplated. The upper and lower limits of these smaller ranges, which may independently be included in a smaller range, are also included within the scope of the description, while being subject to any specifically excluded limit values within the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of such included limits are also included in the description.

[0033] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0034] All numerical values within the detailed description and claims of this specification are modified by the term "about" or "approximately" of the indicated value, taking into account experimental error and variations expected by one of ordinary skill in the art.

[0035] The phrase "and / or" as used in this specification and the claims is to be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" are to be construed in the same manner, i.e., as "one or more" of the elements so conjoined. Other elements not specifically identified by the "and / or" clause may optionally be present whether or not they are related to those specifically identified elements.

[0036] As used in this specification and the claims, "or" is to be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is inclusive, i.e., it includes one or at least one of the elements of the list, but also includes a plurality and optionally additional unlisted items. Only terms that clearly indicate the contrary, such as "only one of" or "exactly one of", or when used in the claims, "consisting of", are considered to refer to including only one element out of a list of numbers or elements within the scope. Generally, the term "or" as used in this specification is to be interpreted as an exclusive term (i.e., when preceded by "either", "only one of", "only one of only", or "exactly one of", it is to be interpreted only as indicating an exclusive option (i.e., "one or the other but not both").

[0037] In the claims and the above specification, all transitional phrases, such as "comprising", "including", "possessing", "having", "containing", "involving", "holding", "being composed of", etc., are to be understood to be open-ended, i.e., they mean including but not limiting. Only the transitional phrases "consisting of" and "consisting essentially of" are to be closed or semi-closed transitional phrases, respectively.

[0038] As used in this specification and the claims, the phrase "at least one" referring to a list of one or more elements means at least one element selected from any one or more of the elements within the list of elements, but does not necessarily include at least one of every element specifically listed within the list of elements, and is to be understood to mean not excluding combinations of elements within the list of elements. This definition also allows for the possibility that elements other than those specifically identified within the list of elements referred to by the phrase "at least one" may optionally exist, whether or not they are related to those specifically identified elements.

[0039] As used herein, the term "about" means plus or minus 10% - 15%, 5 - 10%, or optionally about 5% of the referenced number.

[0040] It should also be understood that in a particular method described herein that includes a plurality of steps or operations, the order of the steps or operations of the method is not necessarily limited to the order in which the steps or operations of the method are described, unless otherwise indicated in the context.

[0041] II. Compositions Incorporation of the EBV oriP sequence into an expression plasmid has been found to increase production of the protein of interest in selected CHO pools and clones in the absence of the EBV EBNA1 protein. Accordingly, nucleic acid constructs useful for increasing expression of the protein of interest are provided herein.

[0042] The term "nucleic acid construct of the present disclosure", as used herein, refers to a nucleic acid construct comprising: a) at least one expression cassette comprising a DNA sequence encoding a protein of interest operably linked to a promoter and a transcription termination point; b) a selectable marker; and c) an EBV oriP or a functional fragment thereof comprising a dyad symmetry (DS) region and a family of repeat (FR) segment.

[0043] As used herein, the term "nucleic acid molecule" and derivatives thereof are intended to include unmodified DNA or RNA or modified DNA or RNA. For example, the nucleic acid molecules or polynucleotides of the present disclosure can be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, which may be single-stranded, more typically double-stranded, or a hybrid molecule containing DNA and RNA that may be a mixture of single-stranded and double-stranded regions. In addition, the nucleic acid molecule can also be composed of a triple-stranded region containing RNA or DNA, or both RNA and DNA. The nucleic acid molecules of the present disclosure may contain one or more modified bases or a DNA or RNA backbone that has been modified for stability or other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA, and thus the term "nucleic acid molecule" encompasses chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning.

[0044] As used herein, the term "operably linked" refers to the relationship between two components that enables the two components to function in the intended manner. For example, when a reporter gene is operably linked to a promoter, the promoter drives the expression of the reporter gene.

[0045] The term "promoter" or "promoter sequence" generally refers to a regulatory DNA sequence to which RNA polymerase binds to initiate transcription of a downstream (i.e., 3') sequence and generate RNA. Suitable promoters may be derived from any organism and may be bound or recognized by any RNA polymerase. Suitable promoters for expression cassettes are considered to be known to those of ordinary skill in the art. In some embodiments, the promoter is an inducible promoter. Examples of inducible promoters include, but are not limited to, the tetracycline response element (TRE) (e.g., Tet-ON or Tet-OFF systems), the ponA inducible expression system (Agilent Technologies), or the cumate inducible promoter, e.g., CuO (System Biosciences). In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters include the human ubiquitin C (UBC) promoter, the human elongation factor 1α (EF1A) promoter, the human phosphoglycerate kinase 1 (PGK) promoter, the simian virus 40 early promoter (SV40) (GenBank accession number J02400.1), the cytomegalovirus immediate early promoter (CMV), the chicken β-actin promoter (CAG) linked to the CMV early enhancer, the EF1-HTLV hybrid promoter, and the Chinese hamster EF1 promoter (CHEF).

[0046] The term "transcription termination site" as used herein generally refers to a polyadenylation signal (pA) that terminates transcription of messenger RNA (mRNA). Suitable pAs may be derived from any organism and are known to those of ordinary skill in the art. Examples of pA signals include, but are not limited to, rabbit β-globin pA (GenBank accession number K03256), SV40 late polyA, hGH polyA, and the strong bovine growth hormone pA (BGHpA) (GenBank accession number M57764.1).

[0047] As used herein, the term "selectable marker" refers to an element in a nucleic acid construct that confers a selective advantage on cells carrying the nucleic acid construct. For example, a selectable marker may encode a protein that is expressed and confers resistance to a particular drug. Alternatively, a selectable marker may encode a protein that is expressed and is essential for cell survival under particular growth conditions. Suitable selectable markers are known to those of skill in the art. Examples of suitable drug-selective markers include, but are not limited to, markers that confer neomycin resistance, hygromycin resistance, blasticidin resistance, zeocin resistance, or puromycin resistance. Such markers are also referred to as resistance genes. Examples of genes required for growth under particular growth conditions include, but are not limited to, glutamine synthetase (GS) (GenBank accession number AY486122.1) and dihydrofolate reductase (DHFR).

[0048] As used herein, the term "oriP" refers to the viral origin of replication found within the Epstein-Barr virus episome that contains the dyad symmetry (DS) region and the family of repeat (FR) segment, or a functional fragment thereof. Epstein-Barr virus (EBV) oriP has 24 EBNA1 binding sites, including four sites within the DS region where replication is initiated and 20 sites within the FR segment. In one embodiment, EBV oriP or a functional fragment thereof has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a functional variant thereof.

[0049] As used herein, the term "functional variant" includes modifications of a nucleic acid sequence disclosed herein that perform substantially the same function in substantially the same manner as the nucleic acid molecules disclosed herein.

[0050] In one embodiment, the present disclosure includes functional variants to the orip nucleic acid sequences disclosed herein. Functional variants include nucleotide sequences that hybridize to the nucleic acid sequences shown above under at least moderately stringent hybridization conditions and optionally under stringent hybridization conditions.

[0051] "At least moderately stringent hybridization conditions" means that conditions promoting selective hybridization between two complementary nucleic acid molecules in solution are selected. The term "at least moderately stringent hybridization conditions" encompasses both stringent hybridization conditions and moderately stringent hybridization conditions. Hybridization can occur to all or part of a nucleic acid sequence molecule. The hybridization portion is typically at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides in length. One of ordinary skill in the art will recognize that the stability of a nucleic acid duplex, or hybrid, is a function of the sodium ion concentration and temperature in a sodium-containing buffer, as determined by Tm (Tm = 81.5°C - 16.6 (Log10 [Na+]) + 0.41(%(G+C) - 600 / I), or a similar equation). Thus, the parameters of the washing conditions that determine hybrid stability are the sodium ion concentration and temperature. To identify molecules that are similar but not identical to a known nucleic acid molecule, it can be assumed that a 1% mismatch results in a decrease in Tm of approximately 1°C. For example, when searching for nucleic acid molecules with greater than 95% identity, the final washing temperature is considered to decrease by approximately 5°C. Based on these considerations, one of ordinary skill in the art will be able to readily select appropriate hybridization conditions. In some embodiments, stringent hybridization conditions are selected. As an example, the following conditions may be employed to achieve stringent hybridization: set Tm - 5°C based on the above formula, hybridization in 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS, followed by washing at 60°C in 0.2x SSC / 0.1% SDS. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. However, it will also be understood that equivalent stringency can be achieved using alternative buffers, salts, and temperatures. Other guidelines regarding hybridization conditions can be found in the following references.Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0052] In another embodiment, the functional variant nucleic acid sequence of oriP comprises a sequence having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, at least 95%, at least 99%, or 100% sequence identity to the oriP of SEQ ID NO: 1 and / or SEQ ID NO: 2 disclosed herein.

[0053] The term "sequence identity," as used herein, refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). Subsequently, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions x 100%). In one embodiment, the two sequences are of the same length. The determination of percent identity between two sequences can also be performed using a mathematical algorithm. A non-limiting example of a mathematical algorithm used for comparing two sequences is the algorithm of Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877, modified from Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268. Such an algorithm is incorporated into the NBLAST program and the XBLAST program of Altschul et al., 1990. The NBLAST nucleotide program parameters can be set, for example, score = 100, wordlength = 12, to perform a BLAST nucleotide search to obtain a nucleotide sequence homologous to the nucleic acid molecules of the present disclosure. The XBLAST program parameters can be set, for example, score = 50, wordlength = 3, to perform a BLAST protein search to obtain an amino acid sequence homologous to the protein molecules of the present disclosure. To obtain a gapped alignment for comparison purposes, Gapped BLAST described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402 can be utilized.Alternatively, iterative searches can also be performed using PSI-BLAST to detect intermolecular distance relationships. When using the BLAST program, the Gapped BLAST program, and the PSI-Blast program, the initial parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the NCBI website). Another non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4: 11-17. The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, incorporates such an algorithm. When using the ALIGN program for amino acid sequence comparison, the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Whether or not gaps are allowed, a method similar to the above can be used to determine the percent identity between two sequences. When calculating the percent identity, typically only exact matches are counted.

[0054] In some embodiments, the nucleic acid construct further comprises a scaffold attachment region (SAR) or a scaffold / matrix attachment region (S / MAR) that is an A / T-rich sequence. The SAR can be derived from any organism and is considered to be known to those skilled in the art. In some embodiments, the SAR contains 750 nucleotides derived from the human interferon α2 upstream scaffold-related region 3, nucleic acid sequence positions 1000-1751 (GenBank accession number U82705.1). In other embodiments, the nucleic acid construct further comprises a ubiquitous chromatin opening element (UCOE) that is a G / C-rich sequence.

[0055] The nucleic acid constructs described herein may include two expression cassettes to enable the expression of two target proteins from the same nucleic acid construct. The additional expression cassettes can include the same or different promoters and / or the same or different pA signals.

[0056] In some embodiments, the nucleic acid construct encodes an antibody fragment, an antibody heavy chain, and / or an antibody light chain. The antibody fragment, antibody heavy chain, and / or antibody light chain may be encoded by separate nucleic acid constructs or may be encoded by two expression cassettes on the same nucleic acid construct.

[0057] As used herein, the term "antibody" is intended to include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, and humanized antibodies. As used herein, the term "antibody fragment" is intended to non-limitingly include Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, Fc fusion proteins, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments, and domain antibodies. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bridges to generate Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, Fc fusion proteins, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques.

[0058] The basic structural unit of an antibody is known to include a tetramer composed of two identical pairs of polypeptide chains, each pair having one light chain ("L") (about 25 kDa) and one heavy chain ("H") (about 50-70 kDa). The amino-terminal portion of the light chain forms the light chain variable domain (VL), and the amino-terminal portion of the heavy chain forms the heavy chain variable domain (VH). The VH domain and the VL domain associate to form the antibody variable region (Fv), which is mainly responsible for antigen recognition / binding. The carboxy-terminal portions of the heavy and light chains associate to form the constant regions, which are mainly responsible for effector functions.

[0059] As used herein, unless otherwise specified, an antibody referred to as comprising "one" specific light chain or "one" specific heavy chain in the singular refers to an antibody in which both light chains or both heavy chains are identical, respectively.

[0060] In some embodiments, the antibody produced is cetuximab, palivizumab, rituximab, trastuzumab or a fragment thereof.

[0061] Also provided are mammalian cells useful for increasing the production of a protein of interest, comprising one or more of the nucleic acid constructs described herein. In some embodiments, the cell comprises two nucleic acid constructs described herein, each construct encoding a different protein of interest. In some embodiments, the protein of interest is an antibody or antibody fragment described herein, optionally cetuximab or a fragment thereof.

[0062] In one embodiment, one or more nucleic acid constructs are stably transfected into mammalian cells. In another embodiment, one or more constructs are integrated into the genome of mammalian cells.

[0063] The mammalian cell can be any mammalian cell. Suitable cells are well known in the art and can include, but are not limited to, SP2 / 0, NS / 0, HT-1080 cells, PER.C6, HKB-11, CAP and HuH-7 human cell lines, Chinese hamster ovary (CHO) cells, and human embryonic kidney 293 (HEK293) cells. In one embodiment, the cell is a CHO cell, optionally a CH0 55E1 cell. In another embodiment, the mammalian cell is a human embryonic kidney 293 (HEK293) cell.

[0064] Epstein-Barr nuclear antigen 1 (EBNA1) is essential for many EBV functions, including gene regulation through positive and negative regulation of viral promoters, extrachromosomal replication, and maintenance of the EBV episomal genome. EBNA1 binds to sequence-specific sites at the EBV viral origin of replication (oriP) within the viral episome. The specific binding ability of EBNA1, as well as its ability to tether EBV DNA to chromosomal DNA, enables EBNA1 to mediate episomal replication and segregation during host cell division. The inventors have found that increased protein production from mammalian cells in the presence of oriP occurs regardless of the presence of the EBNA1 gene. Thus, in one embodiment, the cells do not express EBNA1.

[0065] III. Methods The nucleic acids described herein can be used for increased production of the target protein encoded therein. Thus, one aspect of the present disclosure is a method for increasing production of a target protein, comprising: a) introducing a nucleic acid construct of the present disclosure into a cell; b) applying a selection pressure to the cell to select cells carrying a selectable marker; and c) culturing the cell under conditions for production of the target protein.

[0066] Increased production, as used herein, refers to an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 250% in protein production compared to a protein expressed from a nucleic acid construct lacking oriP under the same conditions.

[0067] The nucleic acid construct can be introduced into cells by any suitable method known in the art. In some embodiments, the nucleic acid construct is introduced into cells by transfection, including calcium phosphate transfection and electroporation / nucleofection. Suitable transfection reagents are well known in the art and include, but are not limited to, cationic polymers such as polyethyleneimine (PEI), cationic lipids such as Lipofectamine and related reagents (Invitrogen), and non-liposomal reagents such as Fugene and related reagents (Promega). In some embodiments, the nucleic acid construct is introduced into cells by transfection using PEI.

[0068] A variety of cells can be used in methods for the production of the target protein. Suitable cells are well known in the art and include, but are not limited to, SP2 / 0, NS / 0, HT-1080 cells, PER.C6, HKB-11, CAP and HuH-7 human cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney 293 (HEK293) cells. In some embodiments, the cells are CHO cells, optionally CHO 55E1 cells.

[0069] Epstein-Barr nuclear antigen 1 (EBNA1) is essential for many EBV functions, including gene regulation through positive and negative regulation of viral promoters, episomal replication, and maintenance of the EBV episomal genome. EBNA1 binds to sequence-specific sites at the EBV viral origin of replication (oriP) within the viral episome. The specific binding ability of EBNA1, as well as its ability to tether EBV DNA to chromosomal DNA, enables EBNA1 to mediate episomal replication and segregation during host cell division. EBNA1 has been shown to be able to replicate only extrachromosomal DNA and not DNA integrated into the chromosome. EBNA1 has been shown to activate (transactivate) transcription from transfected templates. As demonstrated herein, the expression of the EBNA1 protein is not required for enhanced protein production in the presence of the oriP sequences of the present disclosure. Thus, in some embodiments, the method is performed in cells that do not express the EBNA1 protein.

[0070] The selection pressure applied to the cells is thought to depend on the selectable marker present in the nucleic acid construct. As used herein, the term "selection pressure" refers to the cell growth conditions that confer a selective advantage in cell viability to cells carrying the selectable marker. Selective growth conditions include, but are not limited to, the addition of a drug or the removal of an essential component for growth. For example, if the selectable marker is an antibiotic resistance gene, the selection pressure is applied by the addition of the antibiotic. As another example, if the selectable marker is glutamine synthetase, the selection pressure is applied by the removal of glutamine from the growth medium. In another example, the selective agent is methionine sulfoximine (MSX) for the selection of cells overexpressing glutamine synthetase. In yet another embodiment, the selectable marker is methotrexate for the selection of cells expressing dihydrofolate reductase (DHFR).

[0071] As described herein, the expression cassette of the nucleic acid construct may include an inducible promoter. Thus, in some embodiments, the conditions for the production of the protein of interest include the addition of an inducer. For example, if the inducible promoter is a cumate-inducible promoter, the conditions for the production of the protein of interest include the addition of cumate to the growth medium.

[0072] As demonstrated herein, the protein produced may be an antibody. The antibody heavy chain and the antibody light chain may be encoded by separate nucleic acid constructs, or may be encoded by two expression cassettes on the same nucleic acid construct. In some embodiments, the antibody is cetuximab.

[0073] In some embodiments, the method further includes the steps of collecting the cells and / or cell culture medium containing the protein of interest, and optionally, purifying the protein of interest from the collected cells and / or cell culture medium. Purification methods are known in the art and are believed to depend on the protein being purified.

[0074] The following non-limiting examples are illustrative of the present disclosure.

Example

[0075] IV. Example (Example 1) Increase in stable protein production from cells by use of the Epstein-Barr virus oriP sequence For example, to generate a stable pool or stable cell line expressing a single-chain protein or an antibody, the gene of interest was cloned into one of four different expression plasmids of the inventors (Table 1).

[0076]

Table 1

[0077] In the case of single-chain proteins, the gene was cloned into either pTT75™ or pTT81™.

[0078] For the antibody situation, two approaches were used.

[0079] a) The heavy chain gene and the light chain gene were cloned into separate pTT75™ or pTT81™ plasmids and then co-transfected into cells. b) The heavy chain gene and the light chain gene were cloned into a single vector, either pTT96™ or pTT109™ plasmid, each controlled by the CR5 promoter. The use of a single plasmid was sufficient for transfection in this case.

[0080] These plasmids contain the CR5 cumate-inducible promoter (owned by the National Research Council of Canada) combined with the rabbit β-globin polyadenylation signal (pA) (GenBank accession number K03256) [1]. All of these plasmids are derived from the pTT(trademark) expression vector [2] and contain the glutamine synthetase gene (GenBank accession number AY486122.1) from HEK293-6E cells (human fetal kidney 293 cell clone 6E expressing EBNA1) as an amplifiable mammalian selectable marker under the control of the constitutive SV40 promoter (GenBank accession number J02400.1), combined with the strong bovine growth hormone polyadenylation signal (BGHpA) (GenBank accession number M57764.1) [3]. The cDNA encoding the glutamine synthetase gene (GS) was synthesized by reverse transcription / amplification using the ThermoScript(trademark) RT-PCR System & Platinum(registered trademark) Taq DNA Polymerase Kit (Invitrogen, USA), and the mRNA was isolated from HEK293-6E cells using the Micro FastTrack(trademark) mRNA Isolation Kit (Invitrogen, USA). A scaffold attachment region (SAR) conferring enhanced and persistent transgene expression was inserted upstream of the SV40 promoter [4]. The SAR sequence synthesized by the GeneArt(trademark) gene synthesis service contains 750 nucleotides derived from the human interferon α2 upstream scaffold-associated region 3, nucleic acid sequence positions 1000-1751 (GenBank accession number U82705.1). The pMB1 ori sequence and the ampicillin sequence are derived from the pcDNA3.1 vector (Thermo Fisher Scientific, USA).

[0081] EBNA1 is essential for many EBV functions, including gene regulation through positive and negative regulation of viral promoters, episomal replication, and maintenance of the EBV episomal genome. Studies have shown that these functions are regulated by phosphorylation of ten specific sites on EBNA1. In the absence of phosphorylation, the replication and transcriptional activities of this protein are significantly reduced. EBNA1 binds to sequence-specific sites at the viral origin of replication (oriP) within the viral episome. oriP has 24 EBNA1 binding sites, including four sites within the dyad symmetry region (referred to as DS) where replication is initiated, and 20 sites within the family of repeat segments (referred to as FR). The specific binding ability of EBNA1, as well as its ability to tether EBV DNA to chromosomal DNA, enables EBNA1 to mediate episomal replication and segregation during host cell division. EBNA1 also interacts with some viral promoters through several mechanisms and contributes to the transcriptional regulation of EBNA1 itself and other EBNA (2 and 3), as well as the Epstein-Barr virus latent membrane protein 1 (LMP1). EBNA1 has been shown to be able to replicate only extrachromosomal DNA and not DNA integrated into the chromosome.

[0082] The transient CHO-3E7 protein production system relies on CHO cells that express a codon-optimized truncated EBNA1 protein [5]. Epstein-Barr nuclear antigen 1 (EBNA1) has been shown to activate transcription (transactivate) from transfected templates, but there is debate about its ability to activate transcription from chromosomally integrated templates [6]. To examine whether EBNA1 can transactivate regions of integrated oriP-containing plasmid DNA in CHO cells, stable CHO cells that stably express EBNA1 were used, along with stable expression of cetuximab from plasmids that either contain or do not contain the EBV oriP. 55E1A cell line was generated. oriP, which contains two functional components, the dyad symmetry (DS) element and the family of repeats (FR), derived from Epstein-Barr virus (EBV) (GenBank accession number V01555.2) [7], was introduced downstream of the antibody expression cassette (CR5 promoter) and upstream of the ampicillin resistance gene. The map of the pTT109™ plasmid is shown in Figure 1A. Methods for cell culture, transfection, selection, induction of protein expression, and purification are essentially described in [3] and [8].

[0083] In contrast to the situation observed in CHO-3E7 cells, the presence of EBNA1 in CHO 55E1 cells did not significantly increase cetuximab production when using oriP-containing plasmids (compared to non-oriP plasmids), suggesting that EBNA1 does not efficiently transactivate the oriP-containing plasmid DNA integrated in CHO 55E1 cells. Furthermore, two pools generated using oriP-containing plasmids significantly increased cetuximab productivity compared to the pool generated with a non-oriP plasmid in non-EBNA1 CHO 55E1 cells (Figure 2).

[0084] Upon further examination of this matter, it was found that by incorporating the oriP sequence derived from Epstein-Barr virus after the region encoding the target gene in the CR5-based expression plasmid, a stable cell pool with increased productivity could almost always be obtained. 93 percent (26 out of 28) of the pools prepared using plasmids containing oriP had equal or increased productivity compared to the control pools prepared using plasmids without oriP (see Figure 3). Over the entire 28 experiments, productivity increased by an average of 55% with a standard deviation of 52% (median 53%). The improvement due to the presence of oriP was similar regardless of whether a single promoter approach (pTT81(trademark) vs. pTT75(trademark)) or a dual promoter approach (pTT109(trademark) vs. pTT96(trademark)) was used. In addition, single cell cloning was performed on two pools expressing antibodies, one prepared by transfection with an oriP-containing plasmid (Group 1) and the other prepared by transfection with a plasmid without oriP (Group 2). The clones from the oriP pool (Group 1) had increased productivity (Figure 4). The 288 Group 1 clones had an average productivity of 1067 mg / L, while the 288 Group 2 clones had 608 mg / L (a 76% increase). From this, when only the top 96 producing strains in one typical cloning project were retained for the next use, the 96 producing strains in Group 1 had an average productivity of 1566 mg / L, while for Group 2 it was 1006 mg / L (a 56% increase).

[0085] To evaluate whether this effect is also found in the original full-length oriP sequence derived from EBV, another plasmid containing the full-length oriP sequence (pTT153, shown in Fig. 1E) was constructed, and the stable pool productivity of the antibody was compared using two different nutrient addition regimens (R1 and R2). For R1, a commercially available cell culture nutrient additive was added at 1.5%, 5%, 5%, 7.5%, 5%, 5%, and 7.5% of the culture volume on days 0, 3, 5, 7, 10, 12, and 14 after induction, respectively. For R2, another commercially available cell culture nutrient additive was added at 5%, 5%, 10%, 15%, 10%, 10%, and 7.5% of the culture volume on days 0, 3, 5, 7, 10, 12, and 14 after induction, respectively. In the plasmid pTT153 containing the full-length sequence or oriP, stable pool productivity increased compared to pTT96 that does not contain the oriP sequence (Fig. 5). The increase in productivity by pTT153 was comparable to that obtained using the short oriP sequence (pTT109, shown in Fig. 1A).

[0086] Taken together, the data suggest that antibodies using two plasmids containing oriP, such as the pTT81(trademark) and pTT109(trademark) plasmids, confer an increase in pool productivity, which means that it is more likely that clones with improved productivity will be selected. This increased productivity is also observed when using the full-length sequence of EBV's oriP.

[0087] Table 2: Sequences

[0088] Mini oriP (SEQ ID NO: 1)

Chemical formula

[0089] oriP (SEQ ID NO: 2)

Chemical formula

[0090] References TIFF0007692905000004.tif125170

Claims

1. A mammalian cell for increasing the production of a target protein, wherein the cell contains a nucleic acid construct, the cell does not express Epstein-Barr virus nuclear antigen 1 (EBNA1), and the nucleic acid construct a) at least one expression cassette containing a DNA sequence encoding the target protein operably linked to a promoter and a transcription termination point, b) a selectable marker, and c) Epstein-Barr virus (EBV) oriP or a functional fragment thereof containing a dyad symmetry (DS) region and a family of repeat (FR) segment and contains, the mammalian cell is a Chinese hamster ovary (CHO) cell, the increased production is relative to the production of the target protein by control cells that lack the EBV oriP or fragment thereof in the nucleic acid construct. A mammalian cell.

2. The mammalian cell according to claim 1, wherein the cell contains two nucleic acid constructs, each nucleic acid construct is as defined in claim 1, and each nucleic acid construct encodes a different target protein.

3. The mammalian cell according to claim 1, wherein the functional fragment of oriP contains a sequence having at least 95% identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

4. The mammalian cell according to claim 1, wherein the nucleic acid construct contains a scaffold attachment region (SAR).

5. The mammalian cell according to any one of claims 1 to 4, wherein one or more of the nucleic acid constructs are integrated into the genome.

6. A method for producing a target protein in a mammalian cell, the method comprising: a) introducing a nucleic acid construct containing a selectable marker into the mammalian cell, b) applying a selection pressure to the cells to select cells carrying the selectable marker, and c) culturing the cells under conditions for production of the target protein comprising wherein the mammalian cell does not express Epstein - Barr virus nuclear antigen 1 (EBNA1), and the nucleic acid construct comprises i) at least one expression cassette comprising a DNA sequence encoding the target protein operably linked to a promoter and a transcription termination point, and ii) Epstein - Barr virus (EBV) oriP or a functional fragment thereof comprising a dyad symmetry (DS) region and a family of repeat (FR) segment comprising, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell method.

7. The method according to claim 6, wherein two different nucleic acid constructs are introduced into the cell, and each nucleic acid construct is as defined in claim 6.

8. The method according to claim 6, wherein the nucleic acid construct comprises a scaffold attachment region (SAR).

9. The method according to claim 6, wherein the oriP functional fragment comprises a sequence having at least 95% identity to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

10. The method according to claim 6, wherein protein production is increased by at least 20% compared to protein production in cells by a control nucleic acid construct lacking the oriP or a functional fragment thereof when cultured under the same conditions.

11. The method according to any one of claims 6 to 10, wherein the nucleic acid construct is integrated into the genome of the mammalian cell.

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