Engineered EBNA1 with Enhanced Function for Protein Expression in Mammalian Cells

Abstract

Provided are an engineered Epstein-Barr virus nuclear antigen (EBNA1), its coding molecule, a vector containing the same, a mammalian cell expression system, and a target polypeptide recombinantly produced by the foregoing. Also provided are a method for producing the engineered EBNA1, its coding molecule, the vector, and the mammalian cell expression system, and a method for using them in recombinant expression.

Description

Technical Field

[0001] Technical Field The present disclosure relates to genetic engineering and recombinant expression technology. In particular, it relates to modified mammalian cell expression systems, such as CHO and HEK293 cell expression systems, which include vectors or stable cell lines that express an engineered Epstein-Barr virus nuclear antigen 1 (EBNA1) with enhanced function to improve transient gene expression.

Background Art

[0002] Background of the Invention Since the separation of naturally occurring protein products is limited, various techniques and expression systems for the expression of recombinant proteins, such as for clinical and research applications, have been developed. Compared with prokaryotic cell expression systems, mammalian cell expression systems have shown significant advantages in many aspects, such as protein post-translational modification, protein folding quality, and high-yield protein production (Nagesh and Ambuj, 2019). Therefore, mammalian cell expression systems are the most important systems in biopharmaceutical production (e.g., production of antibodies, cytokines, growth factors, etc.) (Jianwei, 2012). Transient expression by plasmid transfection of cells is a technique commonly used for rapid production of recombinant proteins in mammalian cells, and many efforts have been invested to improve transfection efficiency, vector engineering, and cell engineering to increase the titer of protein production (Kishwar, 2013).

[0003] For transient expression in mammalian cells, maintaining a high level of plasmid concentration in the cell nucleus is a major focus in high-level recombinant protein expression. Naturally, some viruses such as Epstein-Barr virus (EBV) adopt different strategies in host cell proliferation to maintain their genomic copy numbers. Epstein-Barr virus nuclear antigen 1 (EBNA1) protein plays an important role in viral genome replication and maintains the viral episome in infected host cells by binding the viral episome to the host chromosome during host cell replication (Wolfgang and Bill, 2013). EBNA1 protein contains two DNA-binding regions: one is the N-terminal AT hook responsible for binding to the host chromosome, and the other is the C-terminal OriP DNA-binding domain responsible for tethering the viral episome (John et al. 2004 and Tohru et al. 2004). The unique structure of EBNA1 ensures efficient propagation of the viral genome to host daughter cells. Not only episomal DNA but also plasmids containing OriP can be maintained at relatively high levels by EBNA1 after transfection (Ann and Bill, 1995). An inducible mammalian expression system based on the EBNA1-OriP interaction has already been developed and is thought to significantly increase the production of recombinant proteins (Francoise et al., 1998 and Olalekan et al., 2014).

[0004] It has been reported that the production level of foreign proteins in mammalian cells is positively correlated with EBNA-1 expression levels (Joo-Hyoung et al., 2017). Therefore, enhanced EBNA1 protein expression levels or enhanced EBNA1 chromosomal binding can further increase recombinant protein expression levels. There is a great need for a recombinant mammalian cell expression system with improved transient gene expression. SUMMARY OF THE INVENTION

[0005] Summary of the Invention The present disclosure discloses a newly engineered EBNA1 and its coding molecule, a vector and a mammalian cell expression system for improving transient gene expression containing the same, and the production and use of the foregoing.

[0006] According to one aspect, the present disclosure provides an engineered EBNA1, provided that, compared with wild-type EBNA1, in the engineered EBNA1, the original G-A rich region is engineered.

[0007] In some embodiments, in the engineered EBNA1, (a) the original G-A rich region is removed; (b) the original G-A rich region is replaced with a linker; and / or (c) the N-terminal fragment containing the original G-A rich region of wild-type EBNA1 (e.g., residues 1 to 336 at the N-terminus) is replaced with an AT hook motif capable of binding to chromosomes during mitosis, such as an AT hook motif derived from human HMG-I / Y (e.g., residues 1 to 92).

[0008] According to another aspect, the present disclosure provides an isolated polynucleotide molecule encoding the engineered EBNA1 described in the present disclosure.

[0009] According to another aspect, the present disclosure provides a vector containing one or more polynucleotide molecules described in the present disclosure.

[0010] According to another aspect, the present disclosure provides an isolated mammalian cell suitable for recombinant expression of a target polypeptide, provided that the mammalian cell contains an engineered EBNA1, provided that the engineered EBNA1 has an enhanced function for recombinant expression compared with wild-type EBNA1 (e.g., wild-type EBNA1 containing the amino acid sequence shown in SEQ ID NO: 1), provided that the original G-A rich region of wild-type EBNA1 is removed or replaced with the engineered EBNA1.

[0011] According to another aspect, the present disclosure provides a method for producing mammalian cells described in the present disclosure, the method comprising introducing a polynucleotide molecule encoding the engineered EBNA1 described in the present disclosure into mammalian cells.

[0012] According to another aspect, the present disclosure provides a method for recombinantly producing a target polypeptide or a method for improving the recombinant expression rate of a target polypeptide, the method comprising:

[0013] (a) culturing the mammalian cells described in the present disclosure under conditions where the target polypeptide can be expressed;

[0014] (b) isolating the target polypeptide; and

[0015] (c) optionally, treating the isolated target polypeptide.

[0016] According to another aspect, the present disclosure provides a product for use in the recombinant expression of a target polypeptide, the product comprising the isolated polypeptide described in the present disclosure, the polynucleotide molecule described in the present disclosure, the vector described in the present disclosure, and / or the mammalian cells described in the present disclosure.

[0017] Other objects, features, advantages, and aspects of the present application will become apparent to those skilled in the art from the following description and the appended claims. However, it should be understood that the following description, the appended claims, and the specific examples are provided by way of illustration and show preferred embodiments of the present application. From reading the following, various changes and modifications within the spirit and scope of the disclosed invention will be readily understood by those skilled in the art.

Brief Description of the Drawings

[0018] Brief Description of the Drawings

[0019] The novel features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which illustrates exemplary embodiments and which utilizes the principles of the present invention. In the accompanying drawings:

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[0034] Detailed Description of the Invention The following description and examples will explain the embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein and can be modified. Those skilled in the art should recognize that there are many changes and modifications to the present invention, and these changes and modifications are included within the scope of the present invention.

[0035] Enhancing the expression titer of recombinant proteins is one of the keys to cost savings in biopharmaceutical production. It has been demonstrated that expressing wild-type EBNA1 protein in mammalian cells can significantly increase the production of many recombinant proteins by transient transfection. The GA-rich linker region and basic region of EBNA1 are essential parts of EBNA1. To enhance the function of EBNA1 in recombinant protein production, EBNA1 was engineered by replacing the G-A rich linker and AT hook motif, and two engineered EBNA1s (e.g., EBNA1_A and EBNA1_B) were produced. Compared with wild-type EBNA1, the engineered EBNA1 with enhanced function significantly enhanced protein production in mammalian cells.

[0036] In particular, EBNA1 is a viral protein necessary for the integrity of the EBV genome in host cells. To enhance transient expression in mammalian cells, the action of tethering the viral genome to the host chromosome via the OriP element is widely used. According to previous studies, the EBNA1 protein contains an N-terminal chromosome-binding domain, a G-A rich region, and a C-terminal OriP-binding domain (Figure 1 (upper), explaining the domain architecture of EBNA1).

[0037] The inventors designed a molecule called EBNA1_A, in which, instead of the original G-A rich region (region range 93 - 325 of EBNA1), a linker with high translation efficiency (amino acid sequence "RGRGGSGGGGSGGAGGGGSGGAGGSGGSGG", SEQ ID NO: 12) was adopted to enhance the translation efficiency of EBNA1 and thus improve the performance of EBNA1 (Figure 1 (middle)).

[0038] Enhancing the chromosomal binding of EBNA1 is another aspect of optimizing its function. EBNA1 has two basic regions, among which the AT hook domain is responsible for chromosomal binding. Some mammalian HMG proteins specifically bind to chromosomes through specific AT hook domains, so the AT hook domain of HMG-I / HMG-Y was selected instead of the original chromosomal binding domain of EBNA1.

[0039] The inventors engineered another molecule called EBNA1_B by binding the AT hook domain derived from HMG-I / HMG-Y and a short linker (protein sequence "

Chemical formula

[0040] Four therapeutic molecules belonging to monoclonal antibodies (OKT3 and Herceptin), bispecific antibodies (emicizumab), and Fc-fusion proteins (dulaglutide), namely OKT3, Herceptin, emicizumab, and dulaglutide, were used to test the effect of engineered EBNA1 on transient expression in CHO cells or HEK293 cells. Wild-type EBNA1, EBNA1_A, and EBNA1_B variants can significantly enhance transient protein expression in mammalian cells several-fold. Also, the EBNA1_A and EBNA1_B variants showed better performance than wild-type EBNA1 in CHO cells. Particularly in the transient transfection system of CHO cells, the EBNA1_A variant achieved an average 1.8-fold increase in titer. In the transient transfection system of HEK293 cells, the effect of EBNA1_A on transient protein expression was also slightly superior to that of wild-type EBNA1, while EBNA1_B was equivalent to wild-type EBNA1 (Figures 6 and 7). Therefore, engineered EBNA1_A and EBNA1_B are more suitable for the transient transfection system of CHO cells. Furthermore, to examine the effect on the expression of target proteins and polypeptides in cell lines, stable cell lines constitutively expressing engineered EBNA1 were prepared. For adalimumab, an exemplary therapeutic protein, a significantly improved expression level was observed, verifying the effect of stable cell lines with engineered EBNA1 on the expression of target proteins.

[0041] Although not wanting to be bound by theory, the long glycine-alanine rich linker (repeats of G-A) of EBNA1 contains approximately 240 amino acid residues, is highly dynamic, and may impair translation efficiency. Also, the dynamics of the EBNA1 G-A repeats may negatively regulate the binding efficiency to mitotic chromosomes. Therefore, engineering the G-A repeats of EBNA1 can enhance the effect of promoting the expression of recombinant proteins. Also, EBNA1-chromosome binding may be involved in two basic regions containing an AT hook DNA binding motif. The AT hook is a conserved DNA binding motif and is present in various chromosomal proteins. Replacing the AT hook motif of EBNA1 with the AT hook motif of a host chromosomal-related protein may enhance the effect of EBNA1 retaining a plasmid containing foreign OriP in host cells. High mobility group (HMG) proteins are required for various chromosomal events, and some HMGs with AT hooks are specifically localized to chromosomes. Therefore, the AT hook of the HMG protein is a candidate for engineering the AT hook of EBNA1.

[0042] Based on the above disclosure, the inventors provide a new mammalian cell expression system with improved transient gene expression, including a vector or stable cell line expressing engineered EBNA1 with enhanced function, its production and use.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this disclosure belongs. This disclosure can be practiced or tested using any methods and materials similar or equivalent to those described herein, but the preferred methods and materials are described.

[0044] As used herein, the terms "one" or "a" are intended to represent "one or more" (i.e., at least one) of a document grammar object. Unless the context dictates otherwise, singular expressions include plural expressions. For example, "one element" means one element or more than one element.

[0045] "About" means a number, level, value, number of times, frequency, percentage, dimension, size, amount, weight, or length that varies by 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% with respect to a reference number, level, value, number of times, frequency, percentage, dimension, size, amount, weight, or length.

[0046] Unless otherwise indicated, the use of "or" means "and / or".

[0047] As used herein, unless otherwise explained, the terms "comprise", "contain", and "include" should be understood to include a step or element or a group of steps or elements, and do not exclude other steps or elements or a group of steps or elements that do not affect the final result. The terms "comprise", "contain", and "have" can also include the terms "consist essentially of" and "consist of".

[0048] The phrase "consist essentially of" means that the recited elements are necessary or mandatory, but that other elements are optional and may or may not be present, depending on whether they substantially affect the activity or role of the listed elements. The phrase "consist of" means including and being limited to the content following the phrase "consist of". Thus, the phrase "consist of" means that the recited elements are necessary or mandatory and that no other elements are present.

[0049] The term "isolated" means a material that substantially or essentially does not contain the components that are normally associated in its natural state. The said material may be a cell or a macromolecule such as a protein or a nucleic acid. For example, as used herein, an "isolated cell" refers to a cell purified from a cell in its natural state.

[0050] Engineered EBNA1 and its coding nucleotide molecule

[0051] As used herein, the term "wild-type" means the most common genotype of a gene (in the context of a gene) or the most common amino acid sequence found in nature (in the context of a polypeptide or protein), and may be part of a public database (such as the EMBL nucleotide sequence database, NCBI Entrez, ExPasy, the Protein Data Bank, etc.). In the present disclosure, the amino acid sequence of wild-type EBNA1 includes the sequence of SEQ ID NO:1. As used herein, the term "original" refers to part or all of a wild-type molecule.

[0052] As used herein, the term "engineering" means a change in the general sequence of a nucleic acid sequence or an amino acid sequence (such as a gene or a gene product), and this may include the manipulation of nucleic acid or polypeptide molecules by synthetic means (such as recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or a combination of these techniques). Engineered EBNA1 is derived from wild-type EBNA1, but can refer to an analog, fragment, derivative or variant with different structures and functions. For example, it is preferred to express a compound, nucleic acid or protein at a level not expressed by naturally occurring cells or organisms, and enhance the function of EBNA1 in the production of recombinant proteins.

[0053] In some embodiments, wild-type EBNA1 contains B1-GA-B2-C from the N-terminus to the C-terminus, where B1 is the first basic domain; GA is the glycine-alanine rich domain; B2 is the second basic domain; and C is the C-terminal region of EBNA1 containing a nuclear localization signal (NLS), an OriP-DNA binding domain, and an acidic domain.

[0054] In some embodiments, wild-type EBNA1 contains or consists of the amino acid sequence shown in SEQ ID NO: 1, where B1 contains or consists of aa33 to aa84 of SEQ ID NO: 1; GA contains or consists of aa85 to aa328 of SEQ ID NO: 1; B2 contains or consists of aa329 to aa407 of SEQ ID NO: 1; C contains or consists of aa408 to aa641 of SEQ ID NO: 1, and the OriP-DNA binding domain contains or consists of aa461 to aa607 of SEQ ID NO: 1. In some embodiments, wild-type EBNA1 is shown in Figure 1.

[0055] In some embodiments, wild-type EBNA1 is engineered by removing or replacing the region containing the G-A rich linker. In some embodiments, the original G-A rich linker is replaced with a designed-engineered linker. In some embodiments, the engineered EBNA1 contains B1-L-B2-C from the N-terminus to the C-terminus, where B1 is the first basic domain; L is the engineered linker; B2 is the second basic domain; and C is the C-terminal region of EBNA1 containing a nuclear localization signal (NLS), an OriP-DNA binding domain, and an acidic domain.

[0056] In some embodiments, the original G-A rich linker was replaced with a designed linker having the amino acid sequence shown in SEQ ID NO: 12 or a designed linker having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the variant has the amino acid sequence shown in SEQ ID NO: 2 or has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered EBNA1 is EBNA1_A as shown in FIG. 1.

[0057] In some embodiments, the N-terminal portion containing the original G-A rich linker of wild-type EBNA1 is replaced with an AT hook motif derived from another protein or species or replaced with a modified AT hook motif. In some embodiments, the original N-terminal portion of wild-type EBNA1 is replaced with an AT hook motif from a high mobility group (HMG) protein. In some embodiments, the AT hook motif for substitution is connected to the rest of wild-type EBNA1 directly or by a linker (e.g., a linker containing less than 20, 18, 16, 14, 12, 10, 8, 6 amino acid residues).

[0058] In some embodiments, the engineered EBNA1 contains AT-SL-C from the N-terminus to the C-terminus, where AT is an AT-hook motif; SL is a short linker; and C is the C-terminal region of EBNA1 containing a nuclear localization signal (NLS), an OriP-DNA binding domain and an acidic domain.

[0059] In some embodiments, the original N-terminal portion of wild-type EBNA1 is replaced with an AT hook motif having the amino acid sequence shown in SEQ ID NO: 13 and a short linker, or an AT hook motif having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 13 and a short linker. In some embodiments, the variant has the amino acid sequence shown in SEQ ID NO: 3 or at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the engineered EBNA1 is EBNA1_B, as shown in FIG. 1.

[0060] In some embodiments, the engineered EBNA1 with enhanced function, compared to wild-type EBNA1, significantly enhanced protein production in mammalian cells.

[0061] The present invention also provides a nucleotide molecule encoding the engineered EBNA1. Preferably, the nucleotide molecule is codon-optimized to enhance the expression of the engineered EBNA1. The nucleotide molecule can be obtained by engineering the coding sequence of wild-type EBNA1 or synthesized by methods commonly used in the art.

[0062] Target polypeptide

[0063] According to the present disclosure, any target polypeptide can be expressed in mammalian cells. The term "polypeptide" refers to a molecule comprising a polymer of amino acids linked by peptide bonds. Polypeptides include polypeptides of any length, including proteins (e.g., having more than 50 amino acids) and peptides (e.g., having 2 to 49 amino acids). Polypeptides include proteins and / or peptides of any activity, function, or size, and include, for example, enzymes (e.g., kinases, phosphatases), receptors, transporters, bactericidal and / or endotoxin-binding proteins, structural polypeptides, membrane-bound polypeptides, glycopolypeptides, globular proteins, immunopolypeptides, toxins, antibiotics, hormones, growth factors, blood factors, vaccines, viral glycopolypeptides, and the like.

[0064] Based on the teachings described in the present disclosure, the target polypeptide to be expressed may be a subunit or domain of a polypeptide, for example, a heavy or light chain of an antibody or a functional fragment or derivative thereof. Depending on the context, the term "target polypeptide" refers to such a single subunit or domain, or the final protein consisting of the corresponding subunit or domain.

[0065] According to some embodiments, the target polypeptide is selected from therapeutic or diagnostic polypeptides. Therapeutic polypeptides, which have therapeutic activity for that purpose, are of particular importance. The term "therapeutic polypeptide" also includes prophylactic polypeptides such as polypeptides used for vaccination. Polypeptides are selected from peptide hormones, interleukins, tissue plasminogen activators, cytokines, growth factors, immunoglobulins, particularly antibodies or functional antibody fragments or variants or derivatives thereof, and Fc-fusion proteins.

[0066] In some embodiments, the target polypeptide is an immunoglobulin molecule, e.g., an antibody. The term "antibody" includes naturally occurring antibodies and all recombinant forms of antibodies such as human-derived antibodies, fully human antibodies, chimeric antibodies, etc. Each heavy chain usually consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain usually consists of a light chain variable region (VL) and a light chain constant region (CL). However, the term "antibody" also includes other types of antibodies, e.g., single-domain antibodies, heavy-chain antibodies (i.e., antibodies consisting of only one or more, especially two heavy chains), and nanobodies (i.e., antibodies consisting of only a single monomeric variable domain). Nanobodies can also be linked to form a multivalent structure. As described above, the polynucleotide encoding the target polypeptide can also encode one or more subunits or domains of an antibody, e.g., a heavy chain or a light chain or a functional fragment or derivative thereof, as the target polypeptide. The said subunits or domains are expressed from the same or different expression cassettes.

[0067] The "functional fragment or derivative" of an antibody particularly refers to a polypeptide derived from an antibody and binding to the same antigen as the antibody, particularly to the same epitope as the antibody. It has been clarified that the antigen-binding function of an antibody can be performed by a fragment of the full-length antibody or a derivative thereof. Examples of antibody fragments or derivatives are: (i) Fab fragment: a monovalent fragment consisting of the variable regions of each heavy and light chain and the first constant domain; (ii) F(ab)2 fragment: a divalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragment: consisting of the variable region of the heavy chain and the first constant domain CH1; (iv) Fv fragment: consisting of the variable regions of the heavy and light chains of one arm of the antibody; (v) scFv fragment: an Fv fragment consisting of one polypeptide chain; (vi) (Fv)2 fragment: consisting of two covalently bound Fv fragments; (vii) heavy chain variable domain; and (viii) multibody: consisting of heavy and light chain variable regions that are covalently bound so that the binding between the heavy and light chain variable regions occurs only intermolecularly and not intramolecularly.

[0068] According to the present disclosure, the presence of the engineered EBNA1 of the present invention in an expression system (e.g., mammalian cells) significantly improves the production amount of the target polypeptide.

[0069] Expression vector

[0070] The engineered EBNA1 of the present disclosure and / or the molecule encoding the target polypeptide can be cloned into an appropriate vector and introduced into a host cell for recombinant expression.

[0071] As used herein, the term "vector" or "expression vector" is used for the purposes of the specification and claims and means a vehicle used to introduce a desired gene into a cell and express it therein according to the present invention. As is well known to those skilled in the art, such vectors are readily selected from plasmids, phages, viruses, retroviruses. Generally, vectors suitable for the present invention include a selectable marker and appropriate restriction sites that facilitate cloning of the desired gene and its entry into and / or replication in eukaryotic or prokaryotic cells.

[0072] As is known in the art, an expression vector can be used to introduce a heterologous polynucleotide into a host cell. The polynucleotide may be included in an expression cassette. The polynucleotide encoding the engineered EBNA1, the target polypeptide, and any selectable marker or reporter polypeptide may be located on the same or different expression vectors. When they are located on different expression vectors, the expression vectors are co-transfected into the host cell. Such co-transfection strategies are well known in the prior art. Introduction into mammalian cells can be achieved, for example, by transfecting one or more appropriate expression vectors containing the polynucleotide encoding the target polypeptide and / or the engineered EBNA1 into the host cell.

[0073] Vectors applicable to the present disclosure include, but are not limited to, plasmids, viral vectors, cosmids, artificial chromosomes. In some embodiments, in the case of engineered EBNA1, the vector is a vector containing OriP, such as the pWX4.1 plasmid. In some embodiments, the vector is a vector used for constitutive expression of engineered EBNA1, such as the pWX039 plasmid.

[0074] When the heterologous nucleic acid is not inserted into the genome, it may be lost later, such as when the cell experiences mitosis (for transient transfection). In some embodiments, the EBNA1_A or EBNA1_B expression vector can integrate into the genome of the host cell (for stable transfection). The stably transfected EBNA1_A or EBNA1_B cell line can be used for the production of the polypeptide of interest.

[0075] Conventionally, several suitable methods are known for introducing heterologous nucleic acids, such as expression vectors, into mammalian host cells, and detailed description is not necessary here. Corresponding methods include, but are not limited to, calcium phosphate transfection, electroporation, lipofection, gene gun and polymer-mediated gene transfer. In addition to the conventional random integration-based methods, recombination-based methods can also be used to transfer heterologous polynucleotides into the host cell genome. Since various methods are well known in the prior art, detailed description is not necessary here. Subsequently, non-limiting embodiments of appropriate vector design were described with reference to appropriate published content.

[0076] Mammalian cells and recombinant expression system

[0077] Also provided are mammalian cells that can be used for efficient recombinant expression and corresponding mammalian recombinant expression systems.

[0078] The mammalian cells of the present disclosure may be selected from, but not limited to, rodent cells, human cells, and monkey cells. Preferred mammalian cells are rodent cells, such as cells derived from hamsters or mice. Rodent cells may be cells selected from the Chinese hamster cell line (e.g., Chinese hamster ovary (CHO) cell line), BHK cell line, NS0 cell line, C127 cell line, mouse 3T3 fibroblast cell line, and SP2 / 0 cell line. Particularly preferred are CHO cells, such as CHO cells derived from CHO-K1.

[0079] In some embodiments, the mammalian cells are derived from human cells and may be selected from, for example, HEK293 cells, MCF-7 cells, PerC6 cells, CAP cells, hematopoietic cells, and HeLa cells. Another option is monkey cells, which may be selected from, for example, COS cells, COS-1, COS-7 cells, and Vero cells.

[0080] According to some embodiments, the mammalian cells are provided as cell clones, cell lines, or cell cultures. Host cell lines are usually available from commercial service institutions such as the American Type Culture Collection or published literature.

[0081] Pharmaceutical composition

[0082] Therapeutic or prophylactic proteins produced by the materials and methods of the present disclosure can be used in the preparation of pharmaceutical compositions for the treatment or prevention of human diseases. The pharmaceutical compositions of the present disclosure typically contain a therapeutically or prophylactically effective amount of the target polypeptide and a pharmaceutically acceptable carrier.

[0083] As used herein, "pharmaceutically acceptable carrier" may be any of a solvent, dispersion medium, coating, antibacterial agent, antifungal agent, isotonic agent, absorption delaying agent, etc. The use of such media and agents with drug active substances is well known in the art. The supplemented active ingredients can also be formulated into the pharmaceutical compositions of the present invention.

[0084] The pharmaceutical composition of the present invention can be administered by any conventional route as long as it can reach the target tissue by this route. This includes oral, nasal, buccal, rectal, vaginal or topical administration. Alternatively, it may be administered in situ, intradermally, subcutaneously, intramuscularly, intraperitoneally, intratumorally, circumferentially, by catheter insertion or by intravenous injection.

[0085] The pharmaceutical composition of the present invention may be administered parenterally or intraperitoneally. The solution of the target protein may be prepared with water appropriately mixed with a surfactant such as hydroxypropyl cellulose. The dispersion can also be prepared in glycerin, liquid polyethylene glycol, or a mixture thereof or in oil. Under normal storage and use conditions, these formulations can contain preservatives to prevent the growth of microorganisms.

[0086] Drug forms suitable for injection include sterile aqueous solutions or dispersions, or sterile powders for temporarily producing sterile injection solutions or dispersions. In many cases, the drug form is sterile, easy to flow, and easy to inject. The drug form is stable under production and storage conditions and can prevent the contaminating effects of microorganisms such as bacteria and fungi.

[0087] Suitable pharmaceutical carriers include, for example, solvents or dispersion media including water, ethanol, polyhydric alcohols (such as glycerin, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, etc., but are not limited thereto. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the desired particle size in a dispersed state, or using a surfactant. Various antibacterial and antifungal agents, such as parahydroxybenzoic acid esters, chlorobutanol, phenol, sorbic acid, thimerosal, etc., can prevent the activity of microorganisms. In many cases, it is preferable to contain an isotonic agent such as sugar or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using a delayed absorption reagent such as aluminum monostearate or gelatin.

[0088] Aseptic injection solutions can be prepared by formulating the required amount of a therapeutic or prophylactic protein in a suitable solvent and, if necessary, adding the various other ingredients described above, followed by filtration sterilization. In general, dispersions are prepared by formulating various sterilized active ingredients in a sterile vehicle containing a basic dispersion medium and the other necessary ingredients. In the case of sterile powders for preparing aseptic injection solutions, a preferred manufacturing method is vacuum drying or lyophilization techniques that produce a powder with the active ingredient and necessary ingredients added from a previously sterile-filtered solution.

[0089] For oral administration, the therapeutic or prophylactic protein produced according to the present invention can be mixed with excipients and used as an ingestible gargle and dentifrice. The therapeutic or prophylactic protein may be dispersed in a dentifrice, gel, paste, powder, or slurry.

[0090] After formulation, the composition or solution can be administered in a manner compatible with the dosage form and in a therapeutically or prophylactically effective amount. The dosing regimen can be determined by the attending physician based on various factors such as the action of the protein, the pathological site, the severity of the disease, the age, sex, and diet of the patient, the severity of inflammation, the time of administration, and other clinical factors. In one example, systemic or injectable administration is initiated at the lowest effective dose and the dosage is increased over a preselected time course until a positive effect is observed. Thereafter, the dosage increase is limited to a level at which a corresponding increase in effect occurs, taking into account the possible side effects that may occur.

[0091] Specific embodiments

[0092] Next, other embodiments of the present invention will be described. The present invention particularly provides the following items:

[0093] 1. An engineered EBNA1, characterized in that the original G-A rich region has been engineered in the engineered EBNA1 as compared to wild-type EBNA1.

[0094] 2.1. (a) The original G-A rich region has been removed;

[0095] (b) The original G-A rich region has been replaced by a linker; and / or

[0096] (c) The N-terminal fragment containing the original G-A rich region of wild-type EBNA1 (e.g., residues 1 to 336 at the N-terminus) has been replaced by an AT hook motif capable of binding to chromosomes during mitosis, such as an AT hook motif derived from human HMG-I / Y (e.g., residues 1 to 92).

[0097] The engineered EBNA1 according to item 1, characterized in that

[0098] 2.2. The engineered EBNA1 according to item 1, characterized in that wild-type EBNA1 contains the amino acid sequence shown in SEQ ID NO: 1.

[0099] 3. Wild-type EBNA1 contains B1-GA-B2-C from the N-terminus to the C-terminus; and

[0100] The engineered EBNA1 contains

[0101] from the N-terminus to the C-terminus

[0102] (i) B1-L-B2-C; or

[0103] (ii) AT-SL-C ;

[0104] wherein B1 is the first basic domain; GA is the glycine-alanine rich domain; B2 is the second basic domain; C is the C-terminal region of EBNA1 containing the nuclear localization signal (NLS), the OriP-DNA binding domain and the acidic domain; L is the engineered linker; AT is the AT-hook motif; SL is the short linker

[0105] The engineered EBNA1 according to item 1, characterized in that...

[0106] 4.1. The engineered EBNA1 according to item 2.1, characterized in that the length of the engineered linker replacing the original G-A rich region is 10 - 50, 20 - 40, 30 - 36 amino acids.

[0107] 4.2. The engineered EBNA1 according to item 2.1, characterized in that the engineered linker contains the amino acid sequence shown in (RGRGGSGGGGSGGAGGGGSGGA) n GGSGGSGG, where n is 1, 2, 3, or 4.

[0108] 4.3. The engineered EBNA1 according to item 2.1, characterized in that the engineered linker contains RGRGGSGGGGSGGAGGGGSGGAGGSGGSGG (SEQ ID NO: 12).

[0109] 5. (a) The AT hook motif for substitution is linked to the original EBNA1 portion by a short linker that is less than 30, less than 25, less than 20 amino acid residues in length; and / or

[0110] (b) The short linker contains the amino acid sequence shown in GGGGGAGGGGSGGGGAGGGGSGGGGAGGG (SEQ ID NO: 14); and / or

[0111] (c) The AT hook motif contains the amino acid sequence shown in MSESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKKLEK (SEQ ID NO: 15); and / or

[0112] (d) The AT hook motif, together with the short linker, contains the amino acid sequence shown in SEQ ID NO: 13

[0113] The engineered EBNA1 according to item 1, characterized in that...

[0114] 6. The engineered EBNA1 according to item 1, characterized in that it comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0115] 7. An isolated polynucleotide molecule encoding the engineered EBNA1 according to any one of items 1 - 6.

[0116] 8.1. The isolated polynucleotide molecule according to item 7, characterized in that the form of the polynucleotide molecule is suitable for being introduced into mammalian cells and recombinantly expressing the target polypeptide.

[0117] 8.2. The isolated polynucleotide molecule according to item 7, characterized in that the engineered EBNA1 - encoding molecule is codon - optimized.

[0118] 9. A vector comprising one or more polynucleotide molecules according to any one of items 7 - 8.

[0119] 10. The vector according to item 9, characterized in that the vector is selected from plasmids, viral vectors, cosmids, artificial chromosomes.

[0120] 11.1. The vector according to item 9, characterized in that the vector is a vector containing OriP, such as the pWX4.1 plasmid.

[0121] 11.2. The vector according to item 9, characterized in that the vector is further co - transfected with a vector containing OriP and / or a vector containing the target polypeptide - encoding molecule.

[0122] 11.3. The vector is used for the constitutive expression of engineered EBNA1 and is, for example, the pWX039 plasmid, the vector according to item 9.

[0123] 12. The mammalian cell contains engineered EBNA1, provided that the engineered EBNA1 has an enhanced function in recombinant expression compared to wild-type EBNA1 (for example, wild-type EBNA1 containing the amino acid sequence shown in SEQ ID NO: 1) in enhancing recombinant expression, provided that the original G-A rich region of wild-type EBNA1 has been engineered in the engineered EBNA1, an isolated mammalian cell suitable for the recombinant expression of the target peptide.

[0124] 13.1. The original G-A rich region is

[0125] (a) removed;

[0126] (b) replaced with an engineered linker; and / or

[0127] (c) The N-terminal fragment containing the original G-A rich region of wild-type EBNA1 (for example, residues 1 to 336 at the N-terminus) is replaced with an AT hook motif capable of binding to chromosomes during mitosis, such as an AT hook motif derived from human HMG-I / Y (for example, residues 1 to 92).

[0128] The mammalian cell according to item 12, characterized in that.

[0129] 13.2. The wild-type EBNA1 contains the amino acid sequence shown in SEQ ID NO: 1, the mammalian cell according to item 12.

[0130] 14. The wild-type EBNA1 contains B1-GA-B2-C from the N-terminus to the C-terminus; and

[0131] B1-GA-B2-C; and

[0132] Engineered EBNA1 ranges from the N-terminus to the C-terminus

[0133] (i) B1-L-B2-C; or

[0134] (ii) AT-SL-C

[0135] and includes;

[0136] provided that B1 is the first basic domain; GA is the glycine-alanine rich domain; B2 is the second basic domain; C is the C-terminal region of EBNA1 containing the nuclear localization signal (NLS), OriP-DNA binding domain and acidic domain; L is the engineered linker; AT is the AT-hook motif; SL is the short linker

[0137] The mammalian cell according to item 12, characterized in that.

[0138] 15.1. The length of the engineered linker replacing the original G-A rich region is 10-50, 20-40, 30- to 36 amino acids, and the mammalian cell according to item 13 is characterized in that.

[0139] 15.2. The engineered linker includes the amino acid sequence shown in (RGRGGSGGGGSGGAGGGGSGGA) n GGSGGSGG, provided that n is 1, 2, 3, or 4, and the mammalian cell according to item 13 is characterized in that.

[0140] 15.3. The engineered linker includes RGRGGSGGGGSGGAGGGGSGGAGGSGGSGG (SEQ ID NO: 12), and the mammalian cell according to item 13 is characterized in that.

[0141] 16. (a) The AT hook motif for replacement is linked to the original EBNA1 moiety by a short linker that is less than 30, less than 25, less than 20 amino acid residues in length; and / or

[0142] (b) The short linker

[0143] contains the amino acid sequence shown in GGGGGAGGGGSGGGGAGGGGSGGGGAGGG (SEQ ID NO: 14); and / or

[0144] (c) The AT hook motif

[0145] contains the amino acid sequence shown in MSESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKKLEK (SEQ ID NO: 15); and / or

[0146] (d) The AT hook motif, together with the short linker, contains the amino acid sequence shown in SEQ ID NO: 13

[0147] The mammalian cell according to item 13, characterized in that it is so.

[0148] 17.1. The engineered EBNA1 contains the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, the mammalian cell according to item 12.

[0149] 17.2. The engineered EBNA1 is introduced into mammalian cells via an expression vector containing the engineered EBNA1 coding molecule, the mammalian cell according to item 12.

[0150] 18. (a) The engineered EBNA1 coding molecule is codon-optimized; and / or

[0151] (b) The vector is a plasmid containing OriP; and / or

[0152] (c) The vector is the pWX4.1 plasmid or the pWX039 plasmid; and / or

[0153] (d) The vector further contains a polynucleotide encoding a polypeptide of interest; and / or

[0154] (e) The cell further contains one or more isolated vectors carrying a polynucleotide encoding a polypeptide of interest; and / or

[0155] (f) The vector carrying the engineered EBNA1-encoding molecule is co-transfected with a vector containing OriP and / or a polynucleotide encoding a polypeptide of interest; and / or

[0156] (g) The cell constitutively or transiently expresses engineered EBNA1

[0157] The mammalian cell according to item 17, characterized in that.

[0158] 19. The mammalian cell according to item 12, characterized in that the polypeptide of interest is selected from glycoproteins, antibodies, non-IgG proteins, Fc fusion proteins, Fab fragments, protein complexes, peptidases, signal peptides, nanobodies, growth factors, hormones, cytokines, blood factors, enzymes, and is preferably a therapeutically active or diagnostic polypeptide.

[0159] 20. The target polypeptide is selected from OKT3 (for example, including the amino acid sequence shown in SEQ ID NO: 4 and / or 5), Herceptin (for example, including the amino acid sequence shown in SEQ ID NO: 6 and / or 7), Emicizumab (for example, including the amino acid sequence shown in SEQ ID NO: 8, 9 and / or 10) and Dulaglutide (for example, including the amino acid sequence shown in SEQ ID NO: 11) or its functional chain or fragment. The mammalian cell according to item 12, characterized in that.

[0160] 21. The mammalian cell according to item 12, characterized in that the mammalian cell is selected from human cells, rodent cells or monkey cells.

[0161] 22. The mammalian cell has one or more of the following characteristics:

[0162] (a) The mammalian cell is a rodent cell derived from hamster or mouse; and / or

[0163] (b) The mammalian cell is a rodent cell line selected from Chinese hamster cell line (for example, Chinese hamster ovary (CHO) cell line), BHK cell line, NS0 cell line, C127 cell line, mouse 3T3 fibroblast cell line, and SP2 / 0 cell line; and / or

[0164] (c) The mammalian cell is derived from human cells selected from HEK293 cells (for example, HEK293F-S), MCF-⑦ cells, PerC6 cells, CAP cells, hematopoietic cells and HeLa cells; and / or

[0165] (d) The mammalian cell is provided as a cell culture, cell line or cell clone.

[0166] The mammalian cell according to item 12, characterized in that.

[0167] 23. A method for producing mammalian cells according to items 12 - 22, characterized by comprising introducing a polynucleotide molecule encoding engineered EBNA1 according to item 7 or 8 into mammalian cells.

[0168] 24. The method according to item 23, further characterized by introducing a polynucleotide molecule encoding a target polypeptide, or an isolated polynucleotide molecule encoding a target polypeptide, into the cells.

[0169] 25. (a) Culturing mammalian cells according to any one of items 12 - 22 under conditions where the target polypeptide can be expressed;

[0170] (b) Isolating the target polypeptide; and

[0171] (c) Optionally, treating the isolated target polypeptide A method for recombinantly producing a target polypeptide or increasing the recombinant expression rate of a target polypeptide, characterized by the above.

[0172] 26. The method according to item 25, wherein the target polypeptide is selected from glycoproteins, antibodies, non - IgG proteins, Fc - fusion proteins, Fab fragments, protein complexes, peptidases, signal peptides, nanobodies, growth factors, hormones, cytokines, blood factors, enzymes, and preferably is a therapeutically active or diagnostic polypeptide.

[0173] 27. The method according to item 25, wherein the target polypeptide is selected from OKT3 (for example, comprising the amino acid sequences shown in SEQ ID NO: 4 and / or 5), Herceptin (for example, comprising the amino acid sequences shown in SEQ ID NO: 6 and / or 7), Emicizumab (for example, comprising the amino acid sequences shown in SEQ ID NO: 8, 9 and / or 10), and Dulaglutide (for example, comprising the amino acid sequence shown in SEQ ID NO: 11), or a functional chain or fragment thereof.

[0174] 28. A product for recombinant expression of a target polypeptide, comprising the isolated polypeptide according to any one of items 1-6, the polynucleotide molecule according to any one of items 7-8, the vector according to any one of items 9-11, and / or the mammalian cell according to any one of items 12-22.

[0175] 29. A product comprising a target polypeptide produced using the isolated polypeptide according to any one of items 1-6, the polynucleotide molecule according to any one of items 7-8, the vector according to any one of items 9-11, the mammalian cell according to any one of items 12-22, or produced by the method according to any one of items 25-27.

[0176] 30. The product according to item 29, wherein the product is selected from drugs, kits for use in diagnosis or testing, and enzyme preparations.

[0177] The present invention is not limited by the method examples and material examples disclosed herein, and any methods and materials similar to or equivalent to the methods and materials described herein can be used in the implementation or testing of the embodiments of the present invention. Numerical ranges include the numerical values defining the ranges. The titles provided here are not limited to various aspects or embodiments of the present invention, and various aspects or embodiments of the present invention can be read with reference to the entire specification.

[0178] Unless otherwise expressly indicated in context, as used in the specification and claims, the singular forms "a," "one," and "the" include plural aspects. The terms "comprising," "having," "including," and variations thereof are used synonymously and are to be construed as non-limiting. Throughout this specification, when a composition is described as including a component or material, unless otherwise specified, the composition in an embodiment may consist essentially of any combination of the components or materials, or may consist of any combination of the components or materials. References to "the disclosure" or "the invention" etc. include one or more aspects of the teachings of this specification. The term "invention" covers aspects of the teachings of this disclosure.

[0179] It is preferred to select and combine the preferred embodiments described herein, and the specific subject matter resulting from the corresponding combinations of the preferred embodiments also belongs to this disclosure.

[0180] The present invention is further illustrated by the following examples. These examples are provided for illustrative purposes only and should in no way be construed as limiting the scope or content of the present invention.

[0181] Publications and the materials referenced therein as cited herein are hereby incorporated by reference in their entirety. Unless otherwise specified, all reagents are commercially available. Unless otherwise specified, all parts and percentages are by weight. Unless otherwise explained, average values of the results are given. Unless otherwise defined, the abbreviations used herein are common abbreviations.

Modes for Carrying Out the Invention

[0182] Examples

[0183] Materials and methods

[0184] Protein Engineering

[0185] [[ID=**30**]] Based on the mechanism by which EBNA1 maintains plasmids in mammalian cells, there are two ways to enhance EBNA1 function, including replacing the G-A rich repeats of EBNA1 with highly efficient translation linkers and replacing the original AT hook of EBNA1 with a host AT hook specific for chromosomal binding. Two engineered EBNA1s (EBNA1_A and EBNA1_B) were designed.

[0186] The sequences of wild-type EBNA1, EBNA1_A, and EBNA1_B are as follows:

[0187] Wild-type EBNA1 (SEQ ID NO: 1): MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGRGRGRGGGRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGGAGAGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAGGGAGAGGAGGAGGAGAGGAGAGGGAGGAGGAGAGGAGAGGAGAGGAGAGGAGGAGAGGAGGAGAGGGAGGAGAGGGAGGAGAGGAGGAGAGGAGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRGGSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSGSPPRRPPPGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDEGEEGQE

[0188] EBNA1_A (SEQ ID NO:2):

Chem.

[0189] EBNA1_B (SEQ ID NO:3):

Chem.

[0190] Preparation of plasmid

[0191] The genes encoding wild-type EBNA1 and engineered EBNA1 were each codon-optimized, synthesized (WuXi Biologics), and cloned into the plasmid - pWX4.1 (WuXi Biologics) containing OriP. By inserting a multiple cloning site, CMV promoter, and OriP element, the pWX4.1 plasmid was constructed on a pUC plasmid backbone containing an Ori replication element (for plasmid amplification in Escherichia coli) and an ampicillin resistance gene (Amp for clone selection) (Figure 12). The genes encoding wild-type EBNA1, EBNA1_A, and EBNA1_B were cloned into the SalI and NotI sites, respectively. Commercial antibodies and Fc-fusion proteins, including OKT3 (monoclonal antibody), Herceptin (monoclonal antibody), Emicizumab (bispecific antibody), and Dulaglutide (Fc-fusion protein), were selected to test the effects of EBNA1 and engineered EBNA1. The protein sequences of the test molecules were obtained from DrugBank. The genes encoding these antibodies and Fc-fusion proteins were each codon-optimized, synthesized, and cloned into pWX4.1 (WuXi Biologics). The genes encoding the light and heavy chains of OKT3 were cloned into the sites between SalI and NotI, respectively; the genes encoding the light and heavy chains of Herceptin were cloned into the sites between XbaI and NotI, respectively; the gene encoding the light chain of Emicizumab was cloned into the XbaI site; the gene encoding the heavy chain of Emicizumab was cloned into the sites between SalI and NotI, respectively; the gene encoding Dulaglutide was cloned into the sites between SalI and NotI.

[0192] The protein sequences of the test molecules were as follows:

[0193] OKT3 (DrugBank accession number: DB00075)

[0194] OKT3 light chain (SEQ ID NO:4): QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0195] OKT3 heavy chain (SEQ ID NO:5): QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0196] Herceptin (DrugBank registration number: DB00072)

[0197] Herceptin light chain (SEQ ID NO:6): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0198] Herceptin heavy chain (SEQ ID NO:7): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0199] Emicizumab (DrugBank registration number: DB13923)

[0200] Emicizumab light chain (SEQ ID NO:8): DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0201] Emicizumab heavy chain_1 (SEQ ID NO:9): QVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNRYTQKSLSLSP

[0202] Emicizumab heavy chain_2 (SEQ ID NO:10): QVQLVQSGSELKKPGASVKVSCKASGYTFTDNNMDWVRQAPGQGLEWMGDINTRSGGSIYNEEFQDRVIMTVDKSTDTAYMELSSLRSEDTATYHCARRKSYGYYLDEWGEGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP

[0203] Dulaglutide (DrugBank registration number: DB09045, SEQ ID NO: 11) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0204] Emicizumab (DrugBank registration number: DB00051)

[0205] Emicizumab light chain (SEQ ID NO: 16) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0206] Emicizumab heavy chain (SEQ ID NO: 17) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0207] Cell culture, transfection and protein expression

[0208] The above test molecules (including OKT3, Herceptin, Emicizumab, and Dulaglutide) were transfected into CHO cells and HEK293 cells to verify the effects of EBNA1 and engineered EBNA1, respectively. Suspension cultures of CHO-K1 cells (ATCC) and HEK293F (ATCC) were performed in a humidified orbital shaker incubator (Kuhner) at 36.5 °C, 120 rpm, and 6% CO2. CHO cells were maintained in CD CHO (Thermofisher) medium, and HEK293 cells were maintained in FreeStyle 293 Expression Medium (Thermofisher). Transfection and protein expression were carried out in 24-deep well plates (Kuhner).

[0209] Before transfection of CHO cells, the cells were washed and suspended in Transpro CD01 (DUONING), and 2.5 ml of suspended cells were plated in each well until the final cell number reached 10 - 16×10 6 cells / ml. When the DNA:PEI molar ratio was 1:2 - 1:5, the plasmid expressing the test molecule was transfected alone or co-transfected with the plasmid expressing wild-type EBNA1 or engineered EBNA1 by mixing with polyetherimide (BIOHUB). After transfection, the medium was supplemented with 5% CB7a (Hyclone), 0.5% CB7b (Hyclone), and glucose on day 0 and day 4, and the cultures were incubated in the incubator at 225 rpm, and the temperature was changed to 31 °C on day 1.

[0210] For the transfection of HEK293 cells, on the day before transfection, the cells were diluted to 2 - 3×10 6 cells / ml using Dynamis medium (Thermofisher). The cell density was 3 - 6×10 6When the cell density reached [[X]] cells / ml, 2.5 ml of the cell culture was plated into each well of a 24-well deep well plate (Kuhner), and the transfection protocol was as described above for the transfection of CHO cells. After transfection, feeding was performed on day 1 and day 3 by adding 5% CB7a (Hyclone), 0.5% CB7b (Hyclone), and glucose.

[0211] Transfection of each molecule was repeated twice to ensure data integrity. CHO cells were harvested on day 7, HEK293 cells were harvested on day 6, and cell viability was detected using a Vi-CELL XR (Beckman).

[0212] Protein purification

[0213] The cell culture was harvested by centrifugation at 3000 g for 30 minutes at 4°C, and the supernatant was filtered through a 0.22 μM filter (Millipore). Titration was performed on 200 μl of the filtered supernatant from each well, and the remaining supernatant from each well was loaded onto a tip column pre-packed with MabSelect SuRe (Cytiva) using a liquid workstation (Hamilton). The tip column loaded with the protein was washed with a wash buffer containing 25 mM sodium acetate (pH 6.0), and the recombinant protein was eluted with 0.1 M sodium acetate (pH 3.0). The final product was neutralized to pH 6.0 by adding 1 M Tris (pH 9.0). Samples were loaded and analyzed by SDS-PAGE.

[0214] Concentration identification and SDS-PAGE of proteins containing Fc

[0215] Note: The value of [[X]] in the translation of is not provided in the original text, so it remains as a placeholder.From each well, 200 microliters (200 μl) of the filtered supernatant was collected, centrifuged, and transferred to an Agilent 96-well plate (0.5 mL, round well, U-shaped, 14 mm). The concentrations of the antibody and Fc-fusion protein were measured by protein A high performance liquid chromatography on an Agilent 1260 Infinity II (Agilent Technologies) with a variable wavelength detector. Two mobile phases were made: (A) 50 mM sodium phosphate buffer (PB), 150 mM sodium chloride, pH 7.0; (B) 100 mM glycine, 150 mM sodium chloride, pH 2.5. First, the standard (STD, human IgG4, Kappa) was diluted to 2.0 mg / mL using mobile phase A. The antibody or Fc-fusion protein in the medium was quantified by comparing the peak size with the calibration curve.

[0216] One microgram (1 μg) of the final product purified from CHO cell cultures and HEK293 cell cultures was loaded and analyzed by SDS-PAGE (SurePAGE, GenScript) to compare the product quality of each measured molecule (including OKT3, Herceptin, Emicizumab, and Dulaglutide).

Example

[0217] Example 1 Influence of engineered EBNA1 on protein expression in CHO cells

[0218] To test the function of engineered EBNA1 in a CHO transient transfection system, wild-type EBNA1, EBNA1_A, and EBNA1_B were co-expressed with each test molecule respectively. The expression titer, cell viability, average cell production, and final product quality of each test molecule were analyzed.

[0219] 1.1 Effect of Engineered EBNA1 on OKT3 Expression in CHO Cells

[0220] The average expression titers expressed alone or together with EBNA1, EBNA1_A, and EBNA1_B of OKT3 are 60 mg / L, 120 mg / L, 232 mg / L, and 224 mg / L, respectively (Figure 2). Both engineered EBNA1s significantly increased the expression level of OKT3 in CHO cells. Also, EBNA1_A and EBNA1_B enhanced the expression titer of OKT3, which is nearly twice that of wild-type EBNA1.

[0221] As a result of calculating the average cell production, it was also found that EBNA1_A and EBNA1_B are superior to wild-type EBNA1 in recombinant OKT3 expression (Figure 6).

[0222] In addition, as a result of examining the cytotoxic effects of EBNA1_A and EBNA_B, it was shown that the cell viability of cells transfected with EBNA1_A and EBNA1_B is equivalent to that of cells not transfected with EBNA1 and cells transfected with wild-type EBNA1 (Figure 4). SDS-PAGE analysis showed that the quality of the final products is similar (left panel of Figure 8).

[0223] 1.2 Effects of Engineered EBNA1 on Herceptin Expression in CHO Cells

[0224] The average expression titers expressed alone or together with EBNA1, EBNA1_A, and EBNA1_B of Herceptin are 162 mg / L, 367 mg / L, 643 mg / L, and 620 mg / L, respectively (Figure 2). Compared with wild-type EBNA1, the Herceptin expression of EBNA1_A and EBNA1_B in CHO cells showed a titer enhancement of about 1.8 times.

[0225] The average cell production indicates that EBNA1_A significantly promotes the expression (Figure 6).

[0226] EBNA1_A and EBNA1_B also show low cytotoxicity against Herceptin expression (Figure 4). The finally purified product showed the same quality by SDS-PAGE (left panel of Figure 9).

[0227] 1.3 Effects of Engineered EBNA1 on Emicizumab Expression in CHO Cells

[0228] The average expression titers of Emicizumab expressed alone or together with EBNA1, EBNA1_A, and EBNA1_B were 110 mg / L, 180 mg / L, 330 mg / L, and 333 mg / L, respectively (Figure 2). EBNA1_A and EBNA1_B are superior to wild-type EBNA1 in Emicizumab expression.

[0229] EBNA1_A and EBNA1_B also improved the average cell production of this test molecule (Figure 6).

[0230] Live cell analysis and SDS-PAGE analysis showed that EBNA1_A and EBNA1_B have no side effects on cell viability and protein quality (left panels of Figures 4 and 10).

[0231] 1.4 Effects of Engineered EBNA1 on Dulaglutide Expression in CHO Cells

[0232] The average expression titers of Dulaglutide expressed alone or together with EBNA1, EBNA1_A, and EBNA1_B were 151 mg / L, 353 mg / L, 623 mg / L, and 537 mg / L, respectively (Figure 2).

[0233] Similar to other test molecules, Dulaglutide expression was significantly enhanced by EBNA1_A and EBNA1_B.

[0234] EBNA1_A also significantly improved the average cell production (Figure 6). Also, EBNA1_A and EBNA1_B show low cytotoxicity and do not affect the quality of the protein (left panels of Figures 4 and 11).

[0235] Consideration

[0236] As a result, it was found that both EBNA1_A and EBNA1_B were superior in expression in CHO cells compared to wild-type EBNA1. In addition, EBNA1_A and EBNA1_B had no obvious cytotoxicity to cell viability and no side effects on protein quality.

[0237] Example 2 Influence of engineered EBNA1 on protein expression in HEK293 cells

[0238] To test the function of engineered EBNA1 in the HEK293 transient transfection system, wild-type EBNA1, EBNA1_A, and EBNA1_B were co-expressed with each test molecule, respectively. The expression titer, cell viability, average cell production, and quality of the final product of each test molecule were analyzed.

[0239] 2.1 Effect of Engineered EBNA1 on OKT3 Expression in HEK293 Cells

[0240] The average expression titers of OKT3 expressed alone or together with EBNA1, EBNA1_A, and EBNA1_B were 60 mg / L, 66 mg / L, 76 mg / L, and 77 mg / L, respectively (Figure 3).

[0241] All engineered EBNA1 slightly enhanced the OKT3 expression level in HEK293 cells. In addition, the expressions of EBNA1_A and EBNA1_B were slightly superior to that of wild-type EBNA1. Analysis of the average cell production also showed that EBNA1_A and EBNA1_B were slightly improved (Figure 7).

[0242] When EBNA1_A and EBNA1_B were expressed, no significant cytotoxic effect was observed (Figure 5). Protein quality was also not affected by EBNA1_A and EBNA1_B (right panel of Figure 8).

[0243] 2.2 Effects of Engineered EBNA1 on Herceptin Expression in HEK293 Cells

[0244] The average expression titers expressed alone or together with EBNA1, EBNA1_A, and EBNA1_B are 314 mg / L, 524 mg / L, 732 mg / L, and 538 mg / L, respectively (Figure 3). EBNA1_A is clearly superior to EBNA1, and EBNA1_B is equivalent to EBNA1. However, in the analysis of average cell production, the performance of EBNA1_A and EBNA1_B was shown to be better (Figure 7).

[0245] Cell viability and protein quality are not affected by EBNA1_A and EBNA1_B (right panels of Figures 5 and 9).

[0246] 2.3 Effects of Engineered EBNA1 on Emicizumab Expression in HEK293 Cells

[0247] The average expression titers expressed alone or together with EBNA1, EBNA1_A, and EBNA1_B are 164 mg / L, 337 mg / L, 404 mg / L, and 366 mg / L, respectively (Figure 3). EBNA1_A is clearly superior to EBNA1, and EBNA1_B is equivalent to EBNA1. The improvement in the average cell production of EBNA1_A and EBNA1_B is equivalent to that of wild-type EBNA1 (Figure 7).

[0248] When this test molecule is expressed, cell viability and protein quality are also not affected by EBNA1_A and EBNA1_B (right panels of Figures 5 and 10).

[0249] 2.4 Effects of Engineered EBNA1 on Dulaglutide Expression in HEK293 Cells

[0250] The average expression titers expressed alone or together with EBNA1, EBNA1_A, and EBNA1_B are 321 mg / L, 727 mg / L, 946 mg / L, and 702 mg / L, respectively (Figure 3). EBNA1_B shows an effect similar to wild-type EBNA1, and EBNA1_A is much superior to EBNA1. EBNA1_A and EBNA1_B showed slight dominance over EBNA1 (Figure 7).

[0251] When this test molecule is expressed, cell viability and protein quality are not affected by EBNA1_A and EBNA1_B (right panels of Figures 5 and 11).

[0252] Consideration

[0253] As a result, it was found that EBNA1_A showed a dominant effect on protein expression in HEK293 cells, and EBNA1_B corresponded to wild-type EBNA1 in the HEK293 transient expression system. In addition, EBNA1_A and EBNA1_B have no obvious cytotoxicity to cell viability and no side effects on protein quality.

[0254] Example 3 Construction of a stable cell line constitutively expressing engineered EBNA1 and its influence on protein production

[0255] To further investigate the effect of engineered EBNA1 on the protein production of the target recombinant protein in mammalian cells, a stable cell line constitutively expressing engineered EBNA1 was prepared, and the effect of the cell line on the expression of the target protein and polypeptide was detected.

[0256] 3.1 Construction of a stable cell line with engineered EBNA1

[0257] For stable transfection of mammalian cells, the gene encoding engineered EBNA1 (EBNA1_B) was cloned into the pWX039 plasmid (Figure 13) (WuXi Biologics). CHO cells were transfected with 1.5 - 2.0×10 6Grow to a cell density of cells / ml and collect 10 7 cells, resuspend them in 300 μl of electroporation buffer. Dilute 5 μg of the plasmid containing the engineered EBNA1 gene in the electroporation buffer and mix it with the cells. Perform transfection using a Bio-Rad electroporator and transfer the cells to pre-warmed CD CHO medium (Hyclone). Select stable cells with engineered EBNA1 by adding 400 μg / ml of zeocin. Select stable cell clones expressing EBNA1 by limiting dilution.

[0258] 3.2 Express the target protein using stable cells with engineered EBNA1

[0259] Clone the gene encoding the target protein, adalimumab, into the pWX4.1 vector and transfect it into the above stable cell line. Further culture the cell line under conditions where the target protein can be expressed.

[0260] As a result, it was found that a cell line constitutively expressing engineered EBNA1 was successfully constructed and significantly improved the expression of the recombinant protein (the titer increased by about 1.8 times compared to the cell line expressing wild-type EBNA (Figure 14).

[0261] The scope of the present invention is not limited to the embodiments disclosed herein, and these embodiments are intended as a single illustration of various aspects of the present invention, and any functionally equivalent embodiments are within the scope of the present invention. In addition to what is described herein, various modifications to the compositions and methods of the present invention will be apparent to those skilled in the art from the foregoing description and teachings and are also intended to be within the scope of the present invention. Such modifications or other embodiments can be implemented without departing from the true scope and spirit of the present invention.

[0262]

Table 1

[0263] References

[0264] Nagesh, T., Ambuj, S., 2019. Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Front Bioeng Biotechnol. 7, 420.

[0265] Jianwei, Z., 2012. Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv. 30(5), 1158 - 1170.

[0266] Kishwar, K., 2013. Gene expression in Mammalian cells and its applications. Adv Pharm Bull. 3(2), 257 - 263.

[0267] Wolfgang, H., and Bill, S., 2013. Replication of Epstein - Barr viral DNA. Cold Spring Harb Perspect Biol. 5(1), a013029.

[0268] John, S., Maki, U., Samantha, W., Christopher, O., Jaap, M., Ashok, A., 2004. The amino terminus of Epstein-Barr Virus (EBV) nuclear antigen 1 contains AT hooks that facilitate the replication and partitioning of latent EBV genomes by tethering them to cellular chromosomes. J Virol. 78(21), 11487-11505.

[0269] Tohru, D., Ayumi, K., Masatoshi, F., Yutaka, S., Hiroki, I., Tatsuya, T., 2004. In vivo dynamics of EBNA1-oriP interaction during latent and lytic replication of Epstein-Barr virus. J Biol Chem. 279(52), 54817-54825.

[0270] Ann, K., and Bill, S., 1995. Plasmid maintenance of derivatives of oriP of Epstein-Barr virus. J Virol. 69(2), 1280-1283.

[0271] Francoise, L., Volker, P., Annie, B., Martine, T., Nathalie, S., Albine, B., Georg, S., Eric, J., Karola R., 1998. Up to 100-fold increase of apparent gene expression in the presence of Epstein-Barr virus oriP sequences and EBNA1: implications of the nuclear import of plasmids. J Virol. 72(7), 6181-6185.

[0272] Olalekan, D., Jessica, S., Greg, D., Diane, H., Gary, P., William, H., Ray, F., 2014. A high-yielding CHO transient system: coexpression of genes encoding EBNA-1 and GS enhances transient protein expression. Biotechnol Prog. 30(1), 132-141.

[0273] Joo-Hyoung, L., Sung-Min, L., Sun-Hye, P., Jeong-Ki, M., Gyun, L., Yeon-Gu, K., 2017. Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells. Process Biochem. 55, 182-186.

Claims

1. Engineered EBNA1, wherein the original G-A rich region has been engineered in the engineered EBNA1 as compared to wild-type EBNA1.

2. (a) The original G-A rich region has been removed; (b) The original G-A rich region has been replaced with a linker; and / or (c) The N-terminal fragment containing the original G-A rich region of wild-type EBNA1 (e.g., residues 1 to 336 at the N-terminus) has been replaced with an AT hook motif capable of binding to chromosomes during mitosis, such as an AT hook motif derived from human HMG-I / Y (e.g., residues 1 to 92); and / or provided that wild-type EBNA1 contains the amino acid sequence shown in SEQ ID NO: 1 The engineered EBNA1 according to claim 1, characterized in that.

3. Wild-type EBNA1 contains B1-GA-B2-C from the N-terminus to the C-terminus; and Engineered EBNA1 contains (i) B1-L-B2-C from the N-terminus to the C-terminus; or (ii) AT-SL-C from the N-terminus to the C-terminus; provided that B1 is the first basic domain; GA is the glycine-alanine rich domain; B2 is the second basic domain; C is the C-terminal region of EBNA1 containing a nuclear localization signal (NLS), an OriP-DNA binding domain and an acidic domain; L is an engineered linker; AT is an AT-hook motif; SL is a short linker The engineered EBNA1 according to claim 1, characterized in that.

4. The length of the engineered linker replacing the original G-A rich region is 10-50, 20-40, 30-36 amino acids; and / or The engineered linker contains RGRGGGSGGGGGSGGAGGGSGGAGGGSGGGSGG (SEQ ID NO: 12) The engineered EBNA1 according to claim 2, characterized in that. The engineered linker comprises the amino acid sequence shown in (RGRGG SGGGGG SGGAG GGGGG SGGAG) ｎ GG SGG SGG, where n is 1, 2, 3, or 4; and / or

5. (a) The AT hook motif for substitution is linked to the original EBNA1 portion by a short linker having less than 30, less than 25, less than 20 amino acid residues in length; and / or (b) The short linker contains the amino acid sequence shown in GGGGGAGGGGGGGGGAGGGGGAGGGGGAGGGG (SEQ ID NO: 14); and / or ​ ​ (c) The AT hook motif comprises the amino acid sequence shown in MSESSSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGSKNKGAAKTRKT TTTTPGRKPRGRPKKLEK (SEQ ID NO: 15); and / or (d) The AT hook motif, together with a short linker, comprises the amino acid sequence shown in SEQ ID NO: 13 The engineered EBNA1 according to claim 1, characterized in that it is as described above.

6. The engineered EBNA1 according to claim 1, characterized in that it comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

3.

7. An isolated polynucleotide molecule encoding the engineered EBNA1 according to any one of claims 1 - 6.

8. The form of the polynucleotide molecule is suitable for introduction into mammalian cells and for recombinant expression of the polypeptide of interest; and / or The engineered EBNA1 coding molecule is codon-optimized The isolated polynucleotide molecule according to claim 7, characterized in that it is as described above.

9. A vector comprising one or more polynucleotide molecules according to any one of claims 7 - 8.

10. The vector is selected from plasmids, viral vectors, cosmids, artificial chromosomes. The vector according to claim 9, characterized in that it is as described above.

11. The vector is a vector containing OriP, such as the pWX4.1 plasmid; and / or The vector is further co-transfected with a vector containing OriP and / or the polypeptide coding molecule of interest; and / or The vector is used for constitutive expression of the engineered EBNA1, such as the pWX039 plasmid The vector according to claim 9, characterized in that it is as described above.

12. An isolated mammalian cell comprising engineered EBNA1, provided that the engineered EBNA1 has an enhanced function in recombinant expression as compared to wild-type EBNA1 (e.g., wild-type EBNA1 comprising the amino acid sequence shown in SEQ ID NO: 1) in enhancing recombinant expression, provided that the original G-A rich region of wild-type EBNA1 has been engineered in the engineered EBNA1, for recombinant expression of a target peptide suitable for.

13. The original G-A rich region is (a) removed; (b) replaced with an engineered linker; and / or (c) the N-terminal fragment comprising the original G-A rich region of wild-type EBNA1 (e.g., residues 1 to 336 of the N-terminus) is replaced with an AT hook motif capable of binding to chromosomes during mitosis, such as an AT hook motif from human HMG-I / Y (e.g., residues 1 to 92); and / or Wild-type EBNA1 comprises the amino acid sequence shown in SEQ ID NO: 1 The mammalian cell according to claim 12, characterized in that.

14. Wild-type EBNA1 comprises from N-terminus to C-terminus B1-GA-B2-C; and Engineered EBNA1 comprises from N-terminus to C-terminus (i) B1-L-B2-C; or (ii) AT-SL-C comprising; provided that B1 is the first basic domain; GA is the glycine-alanine rich domain; B2 is the second basic domain; C is the C-terminal region of EBNA1 comprising a nuclear localization signal (NLS), an OriP-DNA binding domain and an acidic domain; L is an engineered linker; AT is an AT-hook motif; SL is a short linker The mammalian cell according to claim 12, characterized in that.

15. The length of the engineered linker replacing the original G-A rich region is 10-50, 20-40, 30-36 amino acids; and / or The engineered linker comprises the amino acid sequence shown in (RGRGG SGGGGS GGAGGGG SGGAG), where n is 1, 2, 3, or 4; and / or ｎ GGGSGGGSGG, provided that n is 1, 2, 3, or 4; and / or The engineered linker comprises RGRGGGSGGGGGSGGAGGGSGGAGGGSGGGSGG (SEQ ID NO: 12) The mammalian cell according to claim 13, characterized in that.

16. The AT hook motif for replacement is linked to the original EBNA1 moiety by a short linker that is less than 30, less than 25, less than 20 amino acid residues in length; and / or The short linker comprises the amino acid sequence shown in GGGGGAGGGGSGGGGAGGGGSGGGGAGGGG (SEQ ID NO: 14); and / or The AT hook motif comprises the amino acid sequence shown in MSESSSKSSQPLASKQEKDGTEKRRGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGSKNKGAAKTRKT TTTTPGRKPRGRPKKLEK (SEQ ID NO: 15); and / or The AT hook motif, together with the short linker, comprises the amino acid sequence shown in SEQ ID NO: 13 The mammalian cell according to claim 13, characterized in that.

17. The engineered EBNA1 comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3; and / or The engineered EBNA1 is introduced into mammalian cells via an expression vector comprising the engineered EBNA1 coding molecule The mammalian cell according to claim 12, characterized in that.

18. The engineered EBNA1 coding molecule is codon-optimized; and / or The vector is a plasmid containing OriP; and / or The vector is the pWX4.1 plasmid or the pWX039 plasmid; and / or The vector further comprises a coding molecule for a polypeptide of interest; and / or The cell further comprises one or more isolated vectors carrying a coding molecule for a polypeptide of interest; and / or The vector carrying the engineered EBNA1 coding molecule is co-transfected with a vector further comprising OriP and / or a coding molecule for a polypeptide of interest; and / or The cell constitutively or transiently expresses the engineered EBNA1 The mammalian cell according to claim 17, characterized in that.

19. The mammalian cell according to claim 12, wherein the target polypeptide is selected from glycoprotein, antibody, non-IgG protein, Fc fusion protein, Fab fragment, protein complex, peptidase, signal peptide, nanobody, growth factor, hormone, cytokine, blood factor, enzyme, and is preferably a therapeutic active or diagnostic polypeptide.

20. The mammalian cell according to claim 12, wherein the target polypeptide is selected from OKT3 (for example, including the amino acid sequence shown in SEQ ID NO: 4 and / or 5), Herceptin (for example, including the amino acid sequence shown in SEQ ID NO: 6 and / or 7), Emicizumab (for example, including the amino acid sequence shown in SEQ ID NO: 8, 9 and / or 10) and Dulaglutide (for example, including the amino acid sequence shown in SEQ ID NO: 11) or a functional chain or fragment thereof.

21. The mammalian cell according to claim 12, wherein the mammalian cell is selected from human cells, rodent cells or monkey cells.

22. The mammalian cell has one or more of the following characteristics: (a) The mammalian cell is a rodent cell derived from hamster or mouse; and / or (b) The mammalian cell is a rodent cell line selected from the Chinese hamster cell line (for example, Chinese hamster ovary (CHO) cell line), BHK cell line, NS0 cell line, C127 cell line, mouse 3T3 fibroblast cell line, and SP2 / 0 cell line; and / or (c) The mammalian cell is derived from human cells selected from HEK293 cells (for example, HEK293F-S), MCF-7 cells, PerC6 cells, CAP cells, hematopoietic cells and HeLa cells; and / or (d) The mammalian cell is provided as a cell culture, cell line or cell clone The mammalian cell according to claim 12, characterized in that.

23. A method for producing the mammalian cell according to claims 12-22, comprising introducing a polynucleotide molecule encoding the engineered EBNA1 according to claim 7 or 8 into the mammalian cell.

24. The method according to claim 23, further comprising introducing a polynucleotide molecule encoding the target polypeptide, or a separated polynucleotide molecule encoding the target polypeptide, into the cell.

25. Culturing the mammalian cells according to any one of claims 12 - 22 under conditions that allow the expression of the target polypeptide; Isolating the target polypeptide; and Optionally, treating the isolated target polypeptide A method for recombinantly producing a target polypeptide or increasing the recombinant expression rate of a target polypeptide, characterized by the above. **Claim 26** The target polypeptide is selected from glycoproteins, antibodies, non - IgG proteins, Fc fusion proteins, Fab fragments, protein complexes, peptidases, signal peptides, nanobodies, growth factors, hormones, cytokines, blood factors, enzymes, and is preferably a therapeutically active or diagnostic polypeptide. The method according to claim 25 is characterized by this. **Claim 27** The target polypeptide is selected from OKT3 (for example, including the amino acid sequences shown in SEQ ID NO: 4 and / or 5), Herceptin (for example, including the amino acid sequences shown in SEQ ID NO: 6 and / or 7), Emicizumab (for example, including the amino acid sequences shown in SEQ ID NO: 8, 9 and / or 10) and Dulaglutide (for example, including the amino acid sequence shown in SEQ ID NO: 11) or its functional chain or fragment. The method according to claim 25 is characterized by this. **Claim 28** A product for the recombinant expression of a target polypeptide, comprising the isolated polypeptide according to any one of claims 1 - 6, the polynucleotide molecule according to any one of claims 7 - 8, the vector according to any one of claims 9 - 11, and / or the mammalian cells according to any one of claims 12 - 22. **Claim 29** A product comprising the isolated polypeptide according to any one of claims 1 - 6, the polynucleotide molecule according to any one of claims 7 - 8, the vector according to any one of claims 9 - 11, the mammalian cells according to any one of claims 12 - 22, or the target polypeptide produced by the method according to any one of claims 25 - 27. **Claim 30** The product is selected from drugs, kits for use in diagnosis or testing, and enzyme preparations. The product according to claim 29 is characterized by this.

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