Engineered EBNA1 for functional enhancement of protein expression in mammalian cells

Abstract

Provided are an engineered EB virus nuclear antigen 1 (EBNA1) and a coding molecule thereof, a vector and mammalian cell expression system comprising the EBNA1 and the coding molecule thereof, and a target polypeptide recombined by the aforementioned contents. Also provided are methods of making the engineered EBNA1, and encoding molecules, vectors, and mammalian cell expression systems thereof, and methods of using the same for recombinant expression.

Description

Technical Field

[0001] The present disclosure relates to the field of genetic engineering and recombinant expression technology, and in particular to an altered mammalian cell expression system, such as a CHO and HEK293 cell expression system, comprising a vector or a stable cell line expressing an engineered Epstein-Barr virus nuclear antigen 1 (EBNA1) with enhanced expression function to improve transient gene expression. Background Art

[0002] Given that the isolation of protein products from natural sources is limited, various technologies and expression systems have been developed for recombinant protein expression, such as for clinical and research purposes. Compared with prokaryotic expression systems, mammalian cell expression systems show great 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 for biopharmaceutical production, such as the production of antibodies, cytokines, growth factors, etc. (Jianwei, 2012). Transient expression by transfecting cells with plasmids is a popular technology for rapid production of recombinant proteins in mammalian cells, and a lot of efforts have been invested, including improving transfection efficiency, vector engineering, and cell engineering to enhance protein production titers (Kishwar, 2013).

[0003] For transient expression in mammalian cells, how to maintain a high concentration of plasmids in the nucleus is the main problem for high-level recombinant protein expression. In nature, some viruses, such as Epstein-Barr virus (EBV), use different strategies to maintain their genome copy number during host cell proliferation. The EB virus nuclear antigen 1 (EBNA1) protein plays a key role in viral genome replication and maintains viral episomes in infected host cells by binding the episome to the host chromosome during host cell replication (Wolfgang and Bill, 2013). The EBNA1 protein contains two DNA binding regions: one is the N-terminal AT hook responsible for host chromosome binding, and the other is the C-terminal OriP DNA binding domain responsible for viral episome binding (John et al. 2004 and Tohru et al. 2004). The unique structure of EBNA1 ensures efficient amplification of the viral genome in host daughter cells. In addition to episomal DNA, plasmids containing OriP can also be maintained at a relatively high level by EBNA1 after transfection (Ann and Bill, 1995). A transient mammalian expression system based on the EBNA1-OriP interaction has been developed and approved to significantly increase recombinant protein production (Francoise et al., 1998 and Olalekan et al., 2014).

[0004] It has been reported that the production rate of foreign proteins in mammalian cells is positively correlated with the expression level of EBNA-1 (Joo-Hyoung et al., 2017). Therefore, enhancing the expression level of EBNA1 protein or enhancing EBNA1-chromosome binding may further improve the expression level of recombinant proteins. Currently, there is still an urgent need for recombinant mammalian cell expression systems with improved transient gene expression. Summary of the invention

[0005] Disclosed herein are novel engineered EBNA1 and its encoding molecules, vectors and mammalian cell expression systems comprising improved transient gene expression, and the preparation and use of the aforementioned materials.

[0006] According to one aspect, the present disclosure provides an engineered EBNA1, wherein the original GA-rich region in the engineered EBNA1 is engineered compared to wild-type EBNA1.

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

[0008] According to another aspect, the present disclosure provides isolated polynucleotide molecules encoding the engineered EBNA1 disclosed herein.

[0009] According to another aspect, the present disclosure provides a vector comprising one or more polynucleotide molecules disclosed herein.

[0010] According to another aspect, the present disclosure provides an isolated mammalian cell suitable for recombinant expression of a polypeptide of interest, wherein the mammalian cell comprises an engineered EBNA1, wherein the engineered EBNA1 has enhanced function in recombinant expression compared to wild-type EBNA1 (e.g., wild-type EBNA1 comprises the amino acid sequence of SEQ ID NO: 1), and wherein the original GA-rich region of the wild-type EBNA1 is removed or replaced in the engineered EBNA1.

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

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

[0013] (a) culturing the mammalian cells described herein under conditions that allow expression of the polypeptide of interest;

[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 recombinantly expressing a polypeptide of interest, which comprises an isolated polypeptide disclosed herein, a polynucleotide molecule disclosed herein, a vector disclosed herein, and / or a mammalian cell disclosed herein.

[0017] Through the following description and the appended claims, other purposes, features, advantages and aspects of the present application will become apparent to those skilled in the art. However, it should be understood that although the following description, the appended claims and the specific examples show the preferred embodiments of the present application, they are only given in an exemplary manner. By reading the following content, various changes and modifications made within the spirit and scope of the disclosed invention will be apparent to those skilled in the art.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the invention are described in detail in the appended claims. The features and advantages of the invention may be better understood with reference to the following detailed description, which sets forth illustrative embodiments utilizing the principles of the invention and the accompanying drawings, in which:

[0020] Figure 1 : Structural features of wild-type EBNA1 and ENBA1 variants. Wild-type EBNA1 contains a long GA-rich region flanked by two basic regions ("basic"). The two basic regions contain an AT hook motif that is essential for chromosome binding. The Ori-DNA binding domain directly binds to OriP-DNA and tethers the plasmid or episome containing OriP to the chromosome. The nuclear localization signal (NLS) is responsible for the nuclear transport of the EBNA1 protein. EBNA1_A and EBNA1_B are engineered EBNA1. EBNA1_A contains a designed linker to replace the original GA-rich region, while EBNA1_B contains an AT hook motif and a short linker from human HMG-I / Y to replace the entire N-terminus of EBNA1.

[0021] Figure 2:Therapeutic antibodies or Fc fusion protein drugs were selected as test molecules, including OKT3 (monoclonal antibody), Herceptin (monoclonal antibody), Emicizumab (bispecific antibody) and Dulaglutide (Fc fusion protein). The effects of EBNA1 and engineered EBNA1 on transient expression of test molecules were studied in a CHO cell transient expression system. Plasmids expressing test molecules were co-transfected or not co-transfected with plasmids expressing wild-type (wt) EBNA1 or engineered EBNA1 (EBNA1_A or EBNA1_B), respectively. Significant differences were determined by t-test. * indicates that p value < 0.05 is significantly different.

[0022] Figure 3 :Therapeutic antibodies or Fc fusion protein drugs were selected as test molecules, including OKT3 (monoclonal antibody), Herceptin (monoclonal antibody), Emicizumab (bispecific antibody) and dulaglutide (Fc fusion protein). The effects of EBNA1 and engineered EBNA1 on transient expression of test molecules were evaluated in HEK293 cell transient expression system. Plasmids expressing test molecules were co-transfected with or without plasmids expressing engineered EBNA1. Significant differences were determined by t-test. * indicates a significant difference with p value < 0.05.

[0023] Figure 4 : The percentage of viable CHO cells expressing different test molecules was measured to investigate the cytotoxic effects of engineered EBNA1.

[0024] Figure 5 : The percentage of viable HEK293 cells expressing different test molecules was measured to investigate the cytotoxic effects of engineered EBNA1.

[0025] Figure 6 : Average cell productivity of CHO cells for test molecules was calculated based on titer, cell number and viable cell density. The effect of engineered EBNA1 on transient expression was studied. Significant differences were determined by t-test. * indicates a significant difference with p value < 0.05.

[0026] Figure 7 : The average cell productivity of HEK293 cells for the test molecules was calculated based on titer, cell number and viable cell density. The effect of engineered EBNA1 on transient expression was studied.

[0027] Figure 8: Final product quality of OKT3 co-transfected / not co-transfected with engineered EBNA1 was analyzed by SDS-PAGE. The left panel shows OKT3 protein expressed in CHO cells, and the right panel shows OKT3 protein expressed in HEK293 cells. NR: non-reduced; R: reduced; sample reduction was performed by adding 2 mM DTT.

[0028] Fig. 9 : Final product quality of Herceptin co-transfected / not co-transfected with engineered EBNA1 was analyzed by SDS-PAGE. The left panel shows Herceptin protein expressed in CHO cells, and the right panel shows Herceptin protein expressed in HEK293 cells. NR: non-reduced; R: reduced; samples were reduced by adding 2 mM DTT.

[0029] Fig.10 : Final product quality of Emicizumab co-transfected / not co-transfected with engineered EBNA1 was analyzed by SDS-PAGE. The left panel shows Emicizumab protein expressed in CHO cells, and the right panel shows Herceptin protein expressed in HEK293 cells. NR: non-reduced; R: reduced; samples were reduced by adding 2 mM DTT.

[0030] Fig.11 : Final product quality of dulaglutide co-transfected / not co-transfected with engineered EBNA1 was analyzed by SDS-PAGE. The left panel shows dulaglutide protein expressed in CHO cells, and the right panel shows dulaglutide protein expressed in HEK293 cells. NR: non-reduced; R: reduced; sample reduction was performed by adding 2 mM DTT.

[0031] Fig.12 : Plasmid map of pWX4.1. pWX4.1 contains the OriP element to maintain the function of EBNA1, the ampicillin resistance gene (Amp) for plasmid selection, the pUC ori element for plasmid amplification in E. coli, the cytomegalovirus (CMV) promoter for gene transcription in host cells, the herpes simplex virus (HSV) thymidine kinase polyadenylation signal (TKpA) element for RNA termination and polyadenylation, and a multiple cloning site for inserting the target gene for production.

[0032] Fig.13:Plasmid map of pWX039. pWX039 contains the ampicillin resistance gene (Amp) for plasmid selection, the pUC ori element for plasmid amplification in E. coli, the WXRE1 regulatory element and cytomegalovirus (CMV) promoter for gene transcription in host cells, the herpes simplex virus (HSV) thymidine kinase polyadenylation signal (TKpA) element for RNA termination and polyadenylation, the internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) for the expression of zeocin-resistant protein, and a multiple cloning site for the insertion of target genes for production.

[0033] Fig.14 : Study of protein expression in stable CHO cell lines expressing engineered EBNA1 derivatives (EBNA1_B). Adalimumab was selected to be expressed in different stable cell lines not expressing EBNA1, expressing wild-type EBNA1 and expressing engineered EBNA1. The expression titer of each expression was measured on day 4 and day 7. The stable cell line expressing engineered EBNA1 showed a significant advantage (indicated by *) over the cell line expressing wild-type EBNA1 (titer increased by about 1.8 times). DETAILED DESCRIPTION

[0034] The following description and examples illustrate 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 therefore be changed. It will be appreciated by those skilled in the art that the present invention has many variations and modifications, which are all included within the scope of the present invention.

[0035] Improving the recombinant protein expression titer is one of the most critical points for cost saving in biopharmaceutical production. It has been demonstrated that expressing wild-type EBNA1 protein in mammalian cells significantly improves the yield of a variety of recombinant proteins by transient transfection. The GA-rich linker region and the basic region in EBNA1 are important components of EBNA1. To enhance the function of EBNA1 in recombinant protein production, we engineered EBNA1 by replacing the GA-rich linker and AT hook motifs and generated two engineered EBNA1s (e.g., EBNA1_A and EBNA1_B). Compared with wild-type EBNA1, the functionally enhanced engineered EBNA1 significantly improved protein production in mammalian cells.

[0036] Specifically, EBNA1 is a viral protein that is essential for the integrity of the EBV genome in host cells. Its role is to tether the viral genome to the host chromosome via the OriP element, and this role has been widely used to enhance transient expression in mammalian cells. Based on previous studies, the EBNA1 protein contains an N-terminal chromosome binding domain, a GA-rich region, and a C-terminal OriP binding domain ( Figure 1 (Top), the domain structure of EBNA1 is shown).

[0037] We designed a molecule named EBNA1_A, in which a high translation efficiency linker (amino acid sequence "RGRGGSGGGGSGGAGGGGSGGAGGSGGSGG", SEQ ID NO:12) was used to replace the original GA-rich region (region ranging from 93-325 of EBNA1), thereby achieving better EBNA1 performance by increasing the translation efficiency of EBNA1 ( Figure 1 (Central).

[0038] Enhancing EBNA1's chromosome binding is another aspect of optimizing its function. EBNA1 has two basic regions, of which the AT hook domain is responsible for chromosome binding. Some mammalian HMG proteins specifically bind to chromosomes through a specific AT hook domain, so we chose the AT hook domain of HMG-I / HMG-Y to replace the original chromosome binding domain of EBNA1.

[0039] We combined the AT hook domain from HMG-I / HMG-Y with a short linker (protein sequence

[0040] "MSESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEV PTPKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKKLEK GGGGAGGGGS GGGGAGGGGSGGGGAGGG ”, SEQ ID NO: 13, the underlined part is a short linker) and replaced the entire N-terminal 372 residues of wild-type EBNA1 to engineer another molecule named EBNA1_B ( Figure 1 ,bottom).

[0041] Four therapeutic molecules, OKT3, Herceptin, Emicizumab, and Dulaglutide, were classified as monoclonal antibodies (OKT3 and Herceptin), bispecific antibodies (Emicizumab), and Fc fusion proteins (Dulaglutide), and were used to test the effects of engineered EBNA1 on their transient expression in CHO cells or HEK293 cells. Wild-type EBNA1, EBNA1_A, and EBNA1_B variants significantly enhanced transient protein expression in mammalian cells, up to several folds. In addition, the EBNA1_A and EBNA1_B variants showed better performance than wild-type EBNA1 in CHO cells. In particular, in the CHO cell transient transfection system, the EBNA1_A variant increased the titer by an average of 1.8 times. In the HEK293 cell transient transfection system, EBNA1_A also had a slightly better effect on transient protein expression than wild-type EBNA1, while EBNA1_B was comparable to wild-type EBNA1 ( Figure 6 and Figure 7 ). Therefore, engineered EBNA1_A and EBNA1_B are the first choice for CHO cell transient transfection system. In addition, stable cell lines constitutively expressing engineered EBNA1 were prepared to test the effect of cell lines on the expression of target proteins or polypeptides. For the exemplary therapeutic protein adalimumab, a significant increase in expression level was observed, which demonstrated the effect of stable cell lines with engineered EBNA1 on the expression of target proteins.

[0042] Without being bound by theory, the long glycine-alanine-rich linker (GA repeat) of EBNA1 contains approximately 240 amino acid residues and is highly dynamic, which may impair translation efficiency. In addition, the dynamics of the GA repeat of EBNA1 may also negatively regulate its binding efficiency to mitotic chromosomes. Therefore, engineering the GA repeat of EBNA1 can enhance its role in promoting recombinant protein expression. In addition, EBNA1-chromosome binding may be associated with two basic regions containing AT hook DNA binding motifs. AT hook is a conserved DNA binding motif that is present in various chromosomal proteins. Replacing the AT hook motif of EBNA1 with the AT hook motif of host chromosome-associated proteins can enhance the role of EBNA1 in maintaining exogenous OriP-containing plasmids in host cells. High mobility group (HMG) proteins may be crucial 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 protein for engineering the AT hook of EBNA1.

[0043] Based on the above disclosure, we provide a novel mammalian cell expression system with improved transient gene expression, its preparation and use, including a vector or a stable cell line expressing an engineered EBNA1 with enhanced function.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this document applies. Although any methods and materials similar or equivalent to those described herein can be used to implement or test the present disclosure, preferred methods and materials are described.

[0045] As used herein, the terms "a" or "an" are intended to mean "one or more" (i.e., at least one) of the grammatical object of the article. Unless otherwise defined in the context, singular expressions include plural expressions. For example, "an element" means one element or more than one element.

[0046] "About" or "approximately" means that a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length may vary by 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0047] Unless stated otherwise, "or" means "and / or".

[0048] As used herein, unless otherwise specified, the terms "comprises", "includes" and "containing" will be understood to imply the inclusion of the specified steps or elements or groups of steps or elements, and not to exclude any other steps or elements or groups of steps or elements that do not affect the end result. The terms "comprises", "includes" and "containing" may also include the terms "consisting essentially of" and "consisting of".

[0049] The phrase "consisting essentially of means that the listed elements are required or mandatory, but other elements are optional and may or may not be present depending on whether they materially affect the activity or action of the listed elements. The phrase "consisting of is intended to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present.

[0050] The term "isolated" refers to a material that is substantially or essentially free of components that normally accompany it in its native state. The material can be a cell or a macromolecule, such as a protein or nucleic acid. For example, as used herein, "isolated cells" refers to cells purified from cells in their naturally occurring state.

[0051] Engineered EBNA1 and its encoding nucleotide molecules

[0052] As used herein, the term "wild type" refers to 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), which may be part of a publicly accessible database (e.g., EMBL nucleotide sequence database, NCBI Entrez, ExPasy, protein database, etc.). In the present disclosure, the amino acid sequence of wild type EBNA1 comprises the sequence of SEQ ID NO: 1. As used herein, the term "original" refers to part or all of a wild type molecule.

[0053] As used herein, the term "engineered" refers to an alteration of the normal sequence of a nucleic acid sequence or an amino acid sequence (e.g., a gene or a gene product), which may include manipulation of a nucleic acid or polypeptide molecule by synthetic methods (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or some combination of these techniques). Engineered EBNA1 may refer to an analog, fragment, derivative, or mutant derived from wild-type EBNA1 but differing in structure and function from wild-type EBNA1, e.g., expressing a compound, nucleic acid, or protein at a level not expressed by a naturally occurring cell or organism, preferably with enhanced EBNA1 function in recombinant protein production.

[0054] In some embodiments, wild-type EBNA1 comprises B1-GA-B2-C from N-terminus to C-terminus, wherein B1 is the first basic domain; GA is a glycine-alanine rich domain; B2 is the second basic domain; and C is the C-terminal region of EBNA1, comprising a nuclear localization signal (NLS), an OriP-DNA binding domain, and an acidic domain.

[0055] In some embodiments, wild-type EBNA1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, wherein B1 comprises or consists of aa 33 to aa 84 of SEQ ID NO: 1; GA comprises or consists of aa 85 to aa 328 of SEQ ID NO: 1; B2 comprises or consists of aa 329 to aa 407 of SEQ ID NO: 1; C comprises or consists of aa 408 to aa 641 of SEQ ID NO: 1, and the OriP-DNA binding domain comprises or consists of aa 461 to aa 607 of SEQ ID NO: 1. In some embodiments, wild-type EBNA1 such as Figure 1 shown.

[0056] In some embodiments, wild-type EBNA1 is engineered by removing or replacing a region containing a GA-rich linker. In some embodiments, the original GA-rich linker is replaced by a designed and engineered linker. In some embodiments, the engineered EBNA1 comprises B1-L-B2-C from N-terminus to C-terminus, wherein B1 is the first basic domain; L is an engineered linker; B2 is the second basic domain; and C is the C-terminal region of EBNA1, comprising a nuclear localization signal (NLS), an OriP-DNA binding domain, and an acidic domain.

[0057] In some embodiments, the original GA-rich linker is replaced with a designed linker having SEQ ID NO: 12 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the variant has SEQ ID NO: 2 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered EBNA1 is Figure 1 EBNA1_A shown.

[0058] In some embodiments, the N-terminal portion of the wild-type EBNA1 containing the original GA-rich linker is replaced by an AT hook motif or a modified AT hook motif derived from other proteins or species. In some embodiments, the original N-terminal portion of the wild-type EBNA1 is replaced by an AT hook motif from a high mobility group (HMG) protein. In some embodiments, the replacement AT hook motif is directly or via a linker (e.g., a linker comprising less than 20, 18, 16, 14, 12, 10, 8, 6 amino acid residues) connected to the remaining portion of the wild-type EBNA1.

[0059] In some embodiments, the engineered EBNA1 comprises AT-SL-C from N-terminus to C-terminus, wherein AT is the AT hook motif; SL is a short linker; and C is the C-terminal region of EBNA1, comprising a nuclear localization signal (NLS), an OriP-DNA binding domain, and an acidic domain.

[0060] In some embodiments, the original N-terminal portion of wild-type EBNA1 is replaced with a short linker having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 13 and an AT hook motif. In some embodiments, the variant has an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the engineered EBNA1 is Figure 1 EBNA1_B shown.

[0061] In some embodiments, the engineered EBNA1 with enhanced function significantly increases protein production in mammalian cells compared to wild-type EBNA1.

[0062] Also provided herein are nucleotide molecules encoding the engineered EBNA1 disclosed herein. Preferably, the nucleotide molecules are codon optimized to improve the expression of the engineered EBNA1. The nucleotide molecules can be obtained by engineering the coding sequence of wild-type EBNA1 by conventional methods in the art, or can also be obtained by synthesis.

[0063] Target peptide

[0064] According to the present disclosure, mammalian cells can express any target polypeptide. The term "polypeptide" refers to a molecule comprising an amino acid polymer linked together by one or more peptide bonds. Polypeptides include polypeptides of any length, including proteins (e.g., having more than 50 amino acids) and peptides (e.g., 2-49 amino acids). Polypeptides include proteins and / or peptides with any activity, function or size, and may include, for example, enzymes (e.g., kinases, phosphatases), receptors, transporters, bactericidal and / or endotoxin binding proteins, structural polypeptides, membrane-bound polypeptides, glycopolypeptides, globular proteins, immune polypeptides, toxins, antibiotics, hormones, growth factors, blood factors, vaccines, viral glycopolypeptides, etc.

[0065] The target polypeptide expressed according to the teachings described herein can also be a subunit or domain of a polypeptide, such as a heavy chain or light chain of an antibody or a functional fragment or derivative thereof. The term "target polypeptide" can refer to such a single subunit or domain or a final protein composed of the corresponding subunit or domain, depending on the context.

[0066] According to some embodiments, the target polypeptide is selected from therapeutic or diagnostic polypeptides. Therapeutic polypeptides and thus polypeptides having therapeutic activity are of particular importance. The term therapeutic polypeptide also includes prophylactic polypeptides, for example for vaccination. The polypeptide may be selected from the group consisting of peptide hormones, interleukins, tissue plasminogen activators, cytokines, growth factors, immunoglobulins, in particular antibodies or functional antibody fragments or variants or derivatives thereof and Fc fusion proteins.

[0067] In some embodiments, the target polypeptide is an immunoglobulin molecule, such as an antibody. The term "antibody" includes naturally occurring antibodies and all recombinant forms of antibodies, such as humanized antibodies, fully human antibodies, and chimeric antibodies. Each heavy chain generally includes a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain generally includes a light chain variable region (VL) and a light chain constant region (CL). However, the term "antibody" also includes other types of antibodies, such as single domain antibodies, heavy chain antibodies (i.e., antibodies consisting of only one or more, particularly two heavy chains) and nano antibodies (i.e., antibodies consisting of only a single monomer variable domain). Nano antibodies can also be connected to form a multivalent structure. As described above, the polynucleotide encoding the target polypeptide can also encode one or more subunits or domains of the antibody as the target polypeptide, such as a heavy chain or a light chain or its functional fragment or derivative. The subunit or domain can be expressed by the same or different expression cassettes.

[0068] Specifically, a "functional fragment or derivative" of an antibody refers to a polypeptide that is derived from an antibody and is capable of binding to the same antigen, particularly the same epitope as the antibody. It has been shown that the antigen binding function of an antibody can be achieved by a fragment of a full-length antibody or its derivative. Examples of fragments or derivatives of antibodies include: (i) a Fab fragment, a monovalent fragment consisting of the variable region and the first constant domain of each of the heavy and light chains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) a Fd fragment, consisting of the first constant domain CH1 of the heavy chain and the variable region; (iv) a Fv fragment, consisting of the heavy and light chain variable regions of a single arm of an antibody; (v) a scFv fragment, a Fv fragment consisting of a single polypeptide chain; (vi) a (Fv)2 fragment, consisting of two Fv fragments covalently linked together; (vii) a heavy chain variable domain; and (viii) a multibody, consisting of a heavy chain variable region and a light chain variable region covalently linked together, such that the binding of the heavy chain variable region and the light chain variable region can only occur between molecules but not within the molecule.

[0069] According to the present disclosure, the production of a target polypeptide is greatly enhanced due to the presence of the engineered EBNA1 of the present disclosure in an expression system (eg, mammalian cells).

[0070] Expression vector

[0071] The molecules encoding the engineered EBNA1 and / or target polypeptides disclosed herein can be cloned into appropriate vectors and introduced into host cells for recombinant expression.

[0072] The term "vector" or "expression vector" used herein for the purpose of the specification and claims refers to a vector that is used as a tool for introducing a desired gene into a cell and expressing it in the cell according to the present invention. As known to those skilled in the art, such vectors can be easily selected from the following groups: plasmids, bacteriophages, viruses and retroviruses. Typically, vectors compatible with the present invention may also contain a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0073] As is known in the art, expression vectors can be used to introduce heterologous polynucleotides into host cells. The polynucleotide can be contained in an expression cassette. The polynucleotides encoding the engineered EBNA1, the target polypeptide and optionally the selectable marker or reporter polypeptide can be located in the same or different expression vectors. If they are located in different expression vectors, the expression vectors are co-transfected into the host cell. Therefore, co-transfection strategies are well known in the art. Introduction into mammalian cells can be achieved, for example, by transfecting one or more suitable expression vectors into the host cell, the expression vectors comprising the polynucleotide encoding the target polypeptide and / or the engineered EBNA1.

[0074] Vectors suitable for use in the present disclosure may include, but are not limited to, plasmids, viral vectors, cosmids, or artificial chromosomes. In some embodiments, for engineered EBNA1, the vector is a vector containing OriP, such as pWX4.1 plasmid. In some embodiments, the vector is a vector for constitutively expressing engineered EBNA1, such as pWX039 plasmid.

[0075] If the heterologous nucleic acid is not inserted into the genome, the heterologous nucleic acid may be lost at a later stage, such as when the cell undergoes mitosis (for transient transfection). In other aspects, the EBNA1_A or EBNA1_B expression vector can be integrated into the genome of the host cell (for stable transfection). Stably transfected EBNA1_A or EBNA1_B cell lines can be used to produce a polypeptide of interest.

[0076] Several suitable methods are known in the prior art for introducing heterologous nucleic acids (e.g., expression vectors) into mammalian host cells, and therefore, no detailed description is required herein. Corresponding methods include, but are not limited to, calcium phosphate transfection, electroporation, lipofection, biolistic and polymer-mediated gene transfer, etc. In addition to traditional random integration-based methods, recombination-mediated methods can also be used to transfer heterologous polynucleotides into the host cell genome. Since corresponding methods are well known in the prior art, no detailed description is required herein. Non-limiting embodiments of suitable vector design are also described subsequently, and reference is made to the corresponding disclosure.

[0077] Mammalian cells and recombinant expression systems

[0078] The present invention also provides mammalian cells that can be used for efficient recombinant expression and corresponding mammalian recombinant expression systems.

[0079] Mammalian cells of the present disclosure may be selected from, but are not limited to, the group consisting of 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 cell lines selected from the group consisting of Chinese hamster cell lines (e.g., Chinese hamster ovary (CHO) cell lines), BHK cell lines, NS0 cell lines, C127 cell lines, mouse 3T3 fibroblast cell lines, and SP2 / 0 cell lines. Particularly preferred are CHO cells, such as CHO-K1 derived CHO cells.

[0080] In some embodiments, the mammalian cell is derived from a human cell, which can be, for example, selected from the group consisting of HEK293 cells, MCF-7 cells, PerC6 cells, CAP cells, hematopoietic cells, and HeLa cells. Another option is a monkey cell, which can be, for example, selected from the group consisting of COS cells, COS-1, COS-7 cells, and Vero cells.

[0081] According to some embodiments, the mammalian cells are provided in the form of cell clones, cell lines or cell cultures. Typically, host cell lines are available from commercial services, such as the American Tissue Culture Collection or from published literature.

[0082] Pharmaceutical composition

[0083] The therapeutic or prophylactic proteins produced by the materials and methods of the present disclosure can be used to prepare pharmaceutical compositions for treating or preventing human diseases. The pharmaceutical compositions of the present disclosure generally include a therapeutically or prophylactically effective amount of a target polypeptide and a pharmacologically acceptable carrier.

[0084] As used herein, "pharmaceutically acceptable carrier" can be any solvent, dispersion medium, coating, antibacterial or antifungal agent, isotonic or absorption delaying agent, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Auxiliary active ingredients can also be incorporated into the pharmaceutical compositions of the present invention.

[0085] The pharmaceutical compositions of the present invention can be administered by any common route, as long as the route can reach the target tissue. This includes oral, nasal, oral, rectal, vaginal or topical administration. Alternatively, administration can be performed by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, intratumoral, cyclic, catheter insertion or intravenous injection.

[0086] Pharmaceutical compositions of the present disclosure may also be administered parenterally or intraperitoneally. Solutions of the target protein may be prepared in water and appropriately mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, or a mixture thereof, or oil. Under common storage and use conditions, these preparations may contain preservatives to prevent the growth of microorganisms.

[0087] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In many cases, the pharmaceutical form is sterile and fluid for easy injection (syringability). The pharmaceutical form can also be stable under the conditions of manufacture and storage, and can prevent the contaminating effects of microorganisms (e.g., bacteria and fungi).

[0088] Suitable pharmaceutical carriers include, but are not limited to, solvents or dispersion media, such as water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), or vegetable oils. For example, appropriate fluidity can be maintained by using a coating (such as lecithin), maintaining the desired particle size in a dispersed state, or by using a surfactant. The action of microorganisms can be prevented by various antibacterial or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugars or sodium chloride. The absorption of the injection composition can be prolonged by using agents that delay absorption (such as aluminum monostearate and gelatin).

[0089] Sterile injections can be prepared by incorporating the desired amount of therapeutic or preventive protein and the various other ingredients described above (as needed) into an appropriate solvent, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile carrier containing a basic dispersion medium and the desired other ingredients. For sterile powders for the preparation of sterile injections, the preferred method of preparation is vacuum drying or freeze drying techniques, which produce a powder of the active ingredient and any other desired ingredients from a previously sterile filtered solution.

[0090] For oral administration, the therapeutic or prophylactic proteins produced by the present invention can be combined with excipients and used in the form of non-ingestible mouthwashes and toothpastes. The therapeutic or prophylactic proteins can also be dispersed in toothpastes, gels, pastes, powders or slurries.

[0091] After formulation, the composition or solution can be administered in a manner compatible with the dosage formulation and in an amount effective for treatment or prevention. The attending physician can determine the dosage regimen based on various factors, such as the role of the protein, the pathological site, the severity of the disease, the patient's age, sex and diet, the severity of any inflammation, the time of administration and other clinical factors. In one example, systemic or injection administration starts with the lowest effective dose and increases the dose over a preselected time course until a positive effect is observed. Subsequently, the dose is gradually increased to a level that produces a corresponding increase in effect while taking into account any adverse effects that may occur. DETAILED DESCRIPTION

[0093] Other embodiments of the present invention are described again below. The present invention also particularly provides the following items:

[0094] 1. An engineered EBNA1, wherein the original GA-rich region in the engineered EBNA1 is engineered compared to wild-type EBNA1.

[0095] 2.1. The engineered EBNA1 as described in item 1, wherein:

[0096] (a) the original GA-rich region is removed;

[0097] (b) the original GA-rich region is replaced by a linker; and / or

[0098] (c) The N-terminal segment comprising the original GA-rich region of wild-type EBNA1 (e.g., residues 1 to residue 336 at the N-terminus) is replaced by an AT hook motif capable of binding to chromosomes during mitosis, such as the AT hook motif from human HMG-I / Y (e.g., residues 1 to residue 92).

[0099] 2.2. The engineered EBNA1 as described in item 1, wherein the wild-type EBNA1 comprises the amino acid sequence of SEQ ID NO:1.

[0100] 3. The engineered EBNA1 as described in item 1, wherein:

[0101] The wild-type EBNA1 comprises from N-terminus to C-terminus:

[0102] B1-GA-B2-C; and

[0103] The engineered EBNA1 comprises from N-terminus to C-terminus:

[0104] (i) B1-L-B2-C; or

[0105] (ii) AT-SL-C,

[0106] Among them, B1 is the first basic domain; GA is a glycine-alanine-rich domain; B2 is the second basic domain; C is the C-terminal region of EBNA1, which contains 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; and SL is a short linker.

[0107] 4.1. The engineered EBNA1 as described in item 2.1, wherein the length of the engineered linker replacing the original GA-rich region is 10-50, 20-40, 30-36 amino acids.

[0108] 4.2. The engineered EBNA1 of item 2.1, wherein the engineered linker comprises (RGRGGSGGGGSGGAGGGGSGGA) n The amino acid sequence of GGSGGSGG, wherein n is 1, 2, 3 or 4.

[0109] 4.3. The engineered EBNA1 as described in item 2.1, wherein the engineered linker comprises RRGRGGSGGGGSGGAGGGGSGGAGGSGGSGG (SEQ ID NO: 12).

[0110] 5. The engineered EBNA1 as described in item 1, wherein:

[0111] (a) the replacement AT hook motif is connected to the original EBNA1 part via a short linker, the length of which is less than 30, less than 25, or less than 20 amino acid residues; and / or

[0112] (b) the short linker comprises the amino acid sequence GGGGAGGGGSGGGGAGGGGGGGGGAGGG (SEQ ID NO: 14); and / or

[0113] (c) the AT hook motif comprises the amino acid sequence MSESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKKLEK (SEQ ID NO: 15); and / or

[0114] (d) the AT hook motif together with the short linker comprises the amino acid sequence SEQ ID NO:13.

[0115] 6. The engineered EBNA1 as described in item 1, wherein the engineered EBNA1 comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0116] 7. An isolated polynucleotide molecule encoding the engineered EBNA1 according to any one of items 1-6.

[0117] 8.1. The isolated polynucleotide molecule according to item 7, wherein the polynucleotide molecule is in a form suitable for introduction into mammalian cells for recombinant expression of the target polypeptide.

[0118] 8.2. The isolated polynucleotide molecule of item 7, wherein the engineered EBNA1 encoding molecule is codon optimized.

[0119] 9. A vector comprising one or more polynucleotide molecules according to any one of items 7 to 8.

[0120] 10. The vector according to item 9, wherein the vector is selected from a plasmid, a viral vector, a cosmid or an artificial chromosome.

[0121] 11.1. The vector according to item 9, wherein the vector is an OriP-containing vector, such as a pWX4.1 plasmid.

[0122] 11.2. The vector according to item 9, wherein the vector is also co-transfected with one or more vectors containing OriP and / or target polypeptide encoding molecules.

[0123] 11.3. The vector according to item 9, wherein the vector is used for constitutive expression of engineered EBNA1, such as pWX039 plasmid.

[0124] 12. An isolated mammalian cell suitable for recombinant expression of a polypeptide of interest, wherein the mammalian cell comprises an engineered EBNA1, wherein the engineered EBNA1 has enhanced function in improving recombinant expression compared to wild-type EBNA1 (e.g., wild-type EBNA1 comprising the amino acid sequence of SEQ ID NO: 1), and wherein an original GA-rich region of the wild-type EBNA1 is engineered in the engineered EBNA1.

[0125] 13.1. The mammalian cell according to item 12, wherein the original GA-rich region:

[0126] (a) removed;

[0127] (b) replaced by an engineered joint; and / or

[0128] (c) The N-terminal segment comprising the original GA-rich region of wild-type EBNA1 (e.g., residues 1 to residue 336 at the N-terminus) is replaced by an AT hook motif capable of binding to chromosomes during mitosis, such as the AT hook motif from human HMG-I / Y (e.g., residues 1 to residue 92).

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

[0130] 14. The mammalian cell according to item 12, wherein:

[0131] The wild-type EBNA1 comprises from N-terminus to C-terminus:

[0132] B1-GA-B2-C; and

[0133] The engineered EBNA1 comprises from N-terminus to C-terminus:

[0134] (i) B1-L-B2-C; or

[0135] (ii) AT-SL-C,

[0136] Among them, B1 is the first basic domain; GA is a glycine-alanine-rich domain; B2 is the second basic domain; C is the C-terminal region of EBNA1, which contains 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; and SL is a short linker.

[0137] 15.1. The mammalian cell of item 13, wherein the engineered linker replacing the original GA-rich region is 10-50, 20-40, or 30-36 amino acids in length.

[0138] 15.2. The mammalian cell of item 13, wherein the engineered linker comprises (RGRGGSGGGGSGGAGGGGSGGA) n The amino acid sequence of GGSGGSGG, wherein n is 1, 2, 3 or 4.

[0139] 15.3. The mammalian cell of item 13, wherein the engineered linker comprises RRGRGGSGGGGSGGAGGGGSGGAGGSGGSGG (SEQ ID NO: 12).

[0140] 16. The mammalian cell according to item 13, wherein:

[0141] (a) the replacement AT hook motif is connected to the original EBNA1 part via a short linker, the length of which is less than 30, less than 25, or less than 20 amino acid residues; and / or

[0142] (b) the short linker comprises the amino acid sequence GGGGAGGGGSGGGGAGGGGGGGGGAGGG (SEQ ID NO: 14); and / or

[0143] (c) the AT hook motif comprises the amino acid sequence MSESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKKLEK (SEQ ID NO: 15); and / or

[0144] (d) the AT hook motif together with the short linker comprises the amino acid sequence SEQ ID NO:13.

[0145] 17.1. The mammalian cell of item 12, wherein the engineered EBNA1 comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0146] 17.2. The mammalian cell of item 12, wherein the engineered EBNA1 is introduced into the mammalian cell via an expression vector comprising an encoding molecule of the engineered EBNA1.

[0147] 18. The mammalian cell according to item 17, wherein:

[0148] (a) the engineered EBNA1 encoding molecule is codon optimized; and / or

[0149] (b) the vector is a plasmid containing OriP; and / or

[0150] (c) the vector is pWX4.1 plasmid or pWX039 plasmid; and / or

[0151] (d) the vector further comprises a coding molecule for a target polypeptide; and / or

[0152] (e) the cell further comprises one or more separate vectors, the vectors carrying the coding molecules of the target polypeptide; and / or

[0153] (f) the vector carrying the engineered EBNA1 encoding molecule is also co-transfected with a vector containing an OriP and / or a target polypeptide encoding molecule; and / or

[0154] (g) The cells express the engineered EBNA1 constitutively or transiently.

[0155] 19. A mammalian cell as described in item 12, 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 and enzymes, and is preferably a therapeutically active or diagnostic polypeptide.

[0156] 20. The method of item 20, wherein the target polypeptide is selected from OKT3 (e.g., comprising the amino acid sequence of SEQ ID NO: 4 and / or 5), Herceptin (e.g., comprising the amino acid sequence of SEQ ID NO: 6 and / or 7), Emicizumab (e.g., comprising the amino acid sequence of SEQ ID NO: 8, 9 and / or 10) and dulaglutide (e.g., comprising the amino acid sequence of SEQ ID NO: 11) or a functional chain or fragment thereof.

[0157] 21. The mammalian cell according to item 12, wherein the mammalian cell is selected from human cells, rodent cells or monkey cells.

[0158] 22. The mammalian cell according to item 12, wherein the mammalian cell has one or more of the following characteristics:

[0159] (a) the mammalian cell is a rodent cell derived from a hamster or a mouse; and / or

[0160] (b) the mammalian cell is a rodent cell line selected from the group consisting of a Chinese hamster cell line (e.g., a Chinese hamster ovary (CHO) cell line), a BHK cell line, a NSO cell line, a C127 cell line, a mouse 3T3 fibroblast cell line, and a SP2 / 0 cell line; and / or

[0161] (c) the mammalian cell is derived from a human cell selected from the group consisting of HEK293 cells (eg, HEK293F-S), MCF-7 cells, PerC6 cells, CAP cells, hematopoietic cells, and HeLa cells; and / or

[0162] (d) The mammalian cells are provided in the form of cell culture, cell lines or cell clones.

[0163] 23. A method for producing the mammalian cell of any one of items 12-22, comprising introducing the polynucleotide molecule encoding the engineered EBNA1 of any one of items 7-8 into the mammalian cell.

[0164] 24. The method of item 23, wherein a polynucleotide molecule further encoding a polypeptide of interest, or an isolated polynucleotide molecule encoding a polypeptide of interest, is also introduced into the cell.

[0165] 25. A method for recombinantly producing a target polypeptide or increasing the recombinant expression yield of a target polypeptide, comprising:

[0166] (a) culturing the mammalian cell of any one of items 12 to 22 under conditions that allow expression of the polypeptide of interest;

[0167] (b) isolating the target polypeptide; and

[0168] (c) optionally, treating the isolated target polypeptide.

[0169] 26. A method as described in 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 and enzymes, and is preferably a therapeutically active or diagnostic polypeptide.

[0170] 27. The method of item 25, wherein the target polypeptide is selected from OKT3 (e.g., comprising the amino acid sequence of SEQ ID NO: 4 and / or 5), Herceptin (e.g., comprising the amino acid sequence of SEQ ID NO: 6 and / or 7), Emicizumab (e.g., comprising the amino acid sequence of SEQ ID NO: 8, 9 and / or 10) and dulaglutide (e.g., comprising the amino acid sequence of SEQ ID NO: 11) or a functional chain or fragment thereof.

[0171] 28. A product for recombinant expression of a target polypeptide, comprising the isolated polypeptide of any one of items 1 to 6, the polynucleotide molecule of any one of items 7 to 8, the vector of any one of items 9 to 11 and / or the mammalian cell of any one of items 12 to 22.

[0172] 29. A product comprising a target polypeptide produced using the isolated polypeptide of any one of items 1-6, the polynucleotide molecule of any one of items 7-8, the vector of any one of items 9-11 and the mammalian cell of any one of items 12-22, or produced by the method of any one of items 25-27.

[0173] 30. The product according to claim 29, wherein the product is selected from a drug, a diagnostic or detection kit, and an enzyme preparation.

[0174] The present invention is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those methods or materials described herein can be used for the practice or testing of embodiments of the present invention. Numerical ranges include numerical values ​​that limit the range. The titles provided herein are not limitations of various aspects or embodiments of the present invention, and various aspects or embodiments of the present invention can be understood with reference to the specification as a whole.

[0175] As used in this specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "comprising", "having", "including", and variations thereof are used synonymously and are to be construed as non-limiting. Throughout the specification, where a composition is described as comprising ingredients or materials, it is contemplated that the composition, in an embodiment, may also consist essentially of or consist of any combination of the listed ingredients or materials, unless otherwise stated. References to "the present disclosure", "the invention", and the like include single or multiple aspects taught herein, and the like. Aspects taught herein are encompassed by the term "invention".

[0176] The preferred embodiments described herein are preferably selected and combined, and specific subject matter resulting from corresponding combinations of the preferred embodiments also belongs to the scope of the present disclosure.

[0177] The present invention is further illustrated in the following examples. These examples are for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way.

[0178] Publications cited herein and materials cited therein are specifically incorporated herein by reference in their entirety. Unless otherwise indicated, all reagents are commercially available. Unless otherwise indicated, all parts and percentages are by weight. Unless otherwise indicated, the average value of the results is shown. Unless otherwise defined, the abbreviations used herein are conventional.

[0179] Example

[0180] Materials and methods

[0181] Protein Engineering

[0182] Based on the mechanism of EBNA1 maintaining plasmids in mammalian cells, two strategies were adopted to enhance the function of EBNA1, including replacing the EBNA1 GA-rich repeat sequence with a high translation efficiency linker and replacing the EBNA1 original AT hook with a host AT hook that specifically binds to chromosomes. Two engineered EBNA1s (EBNA1_A and EBNA1_B) were designed.

[0183] The sequences of wild-type EBNA1, EBNA1_A, and EBNA1_B are shown below:

[0184] Wild type EBNA1(SEQ ID NO:1):

[0185] MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGRGRGGGRPGAPGGSGSGPRRHDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGGAGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAG GGAGGAGAGAGGGAGAGGGAGGGAGAGGGAGGAGGAGAGGGAGAGGAGGAGGAGAGGAGAGGGAGGAGGAGAGGAGGAGAGAGAGAGAGGAGAGGAGGAGGAGAGAGAGGAGGAGAGAGGAGGAGAGAGGAGGAGGAGGAGAGGAGGAGAGGGAGAG GAGAGGGGRGRGGSGGRGRGGSGGRGRGGSGGRRGRGRERARGGSSRERARGRGRGRGEKRPRSPSSQSSSGSSPPRRPPPGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGRRKKGGWFGKHRGQGGSNPKFEN IAEGLRALLARSHVERTTDEGTWVAGVFVYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDDGDEGGDGDEGEEGQE

[0186] EBNA1_A(SEQ ID NO:2):

[0187] MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGRGRGGGRPGAPGGSGSGPRRHDGVRRPQKRPSCIGCKGTHGGTGAGA RGRGGSGGGGSGGAGGGGSGGAGGSGGSGGRGRGGSGGRGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSSGSPPRRPPPGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRAL LARSHVERTTDEGTWVAGVFVYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDEGEEGQE

[0188] EBNA1_B (SEQ ID NO:3):

[0189] MSESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGSKNKGAAKTRKTTTTTPGRKPRGRPKKLEK GGGGAGGGGSGGGGAGGGGSGGGGAGGG GRGRGEKRPRSPSSQSSSSGSPPRRPPPGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRGQGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTWVAG VFVYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESIVCYFMVFLQTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPPMVEGAAAEGDDGDDGDEGGDGDEGEEGQE

[0190] Plasmid preparation

[0191] The genes encoding wild-type EBNA1 and engineered EBNA1 were codon-optimized, synthesized (WuXi Biologics), and cloned into the OriP-containing plasmid pWX4.1 (WuXi Biologics). The pWX4.1 plasmid was constructed by inserting a multiple cloning site, a CMV promoter, and an OriP element on a pUC plasmid backbone containing the Ori replication element (for plasmid replication in E. coli) and an ampicillin resistance gene (Amp, for clonal selection). Fig.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 codon-optimized, synthesized, and cloned into pWX4.1 (WuXi Biologics). The genes encoding the OKT3 light chain and heavy chain were cloned into the sites between SalI and NotI, respectively; the genes encoding the Herceptin light chain and heavy chain were cloned into the sites between XbaI and NotI, respectively; the gene encoding the emicizumab light chain was cloned into the XbaI site; the genes encoding the emicizumab heavy chain were cloned into the sites between SalI and NotI, respectively; and the gene encoding dulaglutide was cloned into the site between SalI and NotI.

[0192] The protein sequence of the test molecule is shown below:

[0193] OKT3 (DrugBank registration number: DB00075)

[0194] OKT3_light chain (SEQ ID NO:4):

[0195] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN RADTAPVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0196] OKT3_Heavy Chain (SEQ ID NO: 5):

[0197] QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0198] Herceptin (DrugBank accession number: DB00072)

[0199] Herceptin_light chain (SEQ ID NO:6):

[0200] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0201] Herceptin_heavy chain (SEQ ID NO:7):

[0202] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0203] Emicizumab (DrugBank accession number: DB13923)

[0204] Emicizumab light chain (SEQ ID NO:8):

[0205] DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0206] Emicizumab heavy chain 1 (SEQ ID NO:9):

[0207] QVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNRYTQKSLSLSP

[0208] Emicizumab heavy chain 2 (SEQ ID NO:10):

[0209] QVQLVQSGSELKKPGASVKVSCKASGYTFTDNNMDWVRQAPGQGLEWMGDINTRSGGSIYNEEFQDRVIMTVDKSTDTAYMELSSLRSEDTATYHCARRKSYGYYLDEWGEGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP

[0210] Dulaglutide (DrugBank accession number: DB09045, SEQ ID NO: 11)

[0211] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0212] Adalimumab (DrugBank accession number: DB00051)

[0213] Adalimumab_light chain (SEQ ID NO: 16):

[0214] DIQMTQSPSSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0215] Adalimumab_heavy chain (SEQ ID NO: 17):

[0216] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0217] Cell culture, transfection, and protein expression

[0218] 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. Suspension culture of CHO-K1 cells (ATCC) and HEK293F (ATCC) was carried out at 36.5°C, 120rpm and 6% CO2 in a humidified orbital shaking incubator (Kuhner). CHO cells were cultured in CD CHO (Thermofisher) medium, and HEK293 cells were cultured in FreeStyle 293 expression medium (Thermofisher). Transfection and protein expression were performed in 24 deep well plates (Kuhner).

[0219] Before transfection of CHO cells, wash the cells and resuspend them in Transpro CD01 (DUONING) to a final cell count of 10-16 x 10 6Cells / ml were added and 2.5 ml of the suspended cells were seeded into each well. Plasmids expressing the test molecules were transfected alone or co-transfected with plasmids expressing wild-type EBNA1 or engineered EBNA1 by mixing with polyetherimide (BIOHUB) at a DNA:PEI molar ratio of 1:2 to 1:5. After transfection, the culture medium was supplemented with 5% CB7a (Hyclone), 0.5% CB7b (Hyclone) and glucose on days 0 and 4, and the culture was incubated in an incubator at 225 rpm, and the temperature was adjusted to 31°C on day 1.

[0220] For HEK293 cell transfection, cells were diluted to 2-3 x 10 cells / mL using Dynamis medium (Thermofisher) one day before transfection. 6 cells / ml. When the cell density reaches 3-6x 10 6 When the cell culture was 100 cells / ml, 2.5 ml of cell culture was inoculated into each well of a 24-well deep-well plate (Kuhner), and the transfection protocol was the same as that of CHO cells described above. After transfection, the cells were fed by adding 5% CB7a (Hyclone), 0.5% CB7b (Hyclone) and glucose on days 1 and 3.

[0221] Transfection of each molecule was repeated twice to ensure data integrity. CHO cells were harvested on day 7 and HEK293 cells were harvested on day 6, and cell viability was measured by Vi-CELL XR (Beckman).

[0222] Protein purification

[0223] The cell culture was harvested by centrifugation at 3000g and 4°C for 30 minutes, and the supernatant was filtered through a 0.22μM filter (Millipore). 200μl of the filtered supernatant from each well was titered, and the remaining supernatant from each well was loaded onto a tip column preloaded with MabSelect SuRe (Cytiva) via a liquid workstation (Hamilton). The protein-loaded tip column was washed with a wash buffer (pH 6.0) containing 25mM sodium acetate, the recombinant protein was eluted with 0.1M sodium acetate (pH 3.0), and the final product was neutralized to pH 6.0 by adding 1M Tris pH 9.0. The sample was loaded and analyzed by SDS-PAGE. Concentration determination of Fc-containing protein and SDS-PAGE

[0224] 200 microliters (200 μl) of filtered supernatant were collected from each well, centrifuged and transferred to Agilent 96-well plates (0.5 mL, round holes, U-shaped, 14 mm). The concentrations of antibodies and Fc fusion proteins were determined by protein A HPLC on an Agilent 1260 Infinity II (Agilent Technologies) with a variable wavelength detector. Two mobile phases were prepared: (A) 50 mM sodium phosphate buffer (PB), 150 mM NaCl, pH 7.0; (B) 100 mM glycine, 150 mM NaCl, pH 2.5. Standards (STD, human IgG4, κ) were first diluted to 2.0 mg / mL using mobile phase A. The antibodies or Fc fusion proteins in the culture medium were quantified by comparing the peak size with the standard curve.

[0225] One microgram (1 μg) of the final product purified from CHO cell culture and HEK293 cell culture was loaded and analyzed by SDS-PAGE (SurePAGE, GenScript) to compare the product quality of each test molecule, including OKT3, Herceptin, Emicizumab, and Dulaglutide.

[0226] Example 1. Effect of engineered EBNA1 on protein expression in CHO cells

[0227] To test the function of engineered EBNA1 in CHO transient transfection system, wild-type EBNA1, EBNA1_A and EBNA1_B were co-expressed with test molecules, respectively. The expression titer, cell viability, average cell productivity and final product quality of each test molecule were analyzed.

[0228] 1.1 Effect of engineered EBNA1 on OKT3 expression in CHO cells

[0229] The average expression titers of OKT3 expressed alone and co-expressed with EBNA1, EBNA1_A and EBNA1_B were 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. In addition, EBNA1_A and EBNA1_B increased the OKT3 expression titer by nearly two times compared to wild-type EBNA1.

[0230] We calculated the average cell productivity and the results also showed that EBNA1_A and EBNA1_B outperformed wild-type EBNA1 in recombinant OKT3 expression ( Figure 6 ).

[0231] We also investigated the cytotoxic effects of EBNA1_A and EBNA1_B and the results showed that the cell viability of cells transfected with EBNA1_A and EBNA1_B was comparable to that of cells not transfected with EBNA1 or transfected with wild-type EBNA1 ( Figure 4 ). SDS-PAGE analysis showed that the final products were of similar quality ( Figure 8 left).

[0232] 1.2 Effect of engineered EBNA1 on the expression of Herceptin in CHO cells

[0233] The average expression titers of Herceptin alone and co-expressed with EBNA1, EBNA1_A and EBNA1_B were 162 mg / L, 367 mg / L, 643 mg / L and 620 mg / L, respectively. Figure 2 Compared with wild-type EBNA1, EBNA1_A and EBNA1_B increased the titer of Herceptin expression in CHO cells by about 1.8-fold.

[0234] The average cell productivity showed that EBNA1_A significantly promoted the expression ( Figure 6 ).

[0235] EBNA1_A and EBNA1_B also showed low cytotoxicity to Herceptin expression ( Figure 4 The final purified products showed the same mass on SDS-PAGE ( Fig. 9 left).

[0236] 1.3 Effect of engineered EBNA1 on emicizumab expression in CHO cells

[0237] The average expression titers of emicizumab alone and co-expressed with EBNA1, EBNA1_A and EBNA1_B were 110 mg / L, 180 mg / L, 330 mg / L and 333 mg / L, respectively. Figure 2 ). The expression effects of EBNA1_A and EBNA1_B on emicizumab were better than those of wild-type EBNA1.

[0238] EBNA1_A and EBNA1_B also enhanced the average cellular production rate of the tested molecule ( Figure 6 ).

[0239] Live cell assays and SDS-PAGE analysis showed that EBNA1_A and EBNA1_B had no adverse effects on cell viability and protein quality ( Figure 4 and Fig.10 left).

[0240] 1.4 Effect of engineered EBNA1 on dulaglutide expression in CHO cells

[0241] The average expression titers of emicizumab alone and co-expressed with EBNA1, EBNA1_A and EBNA1_B were 151 mg / L, 353 mg / L, 623 mg / L and 537 mg / L, respectively. Figure 2 ).

[0242] Similar to the other tested molecules, EBNA1_A and EBNA1_B significantly enhanced dulaglutide expression.

[0243] EBNA1_A also significantly enhanced the average cell productivity ( Figure 6 In addition, EBNA1_A and EBNA1_B showed low cytotoxicity and did not affect protein quality ( Figure 4 and Fig.11 left).

[0244] in conclusion

[0245] The results showed that both EBNA1_A and EBNA1_B performed better than wild-type EBNA1 in CHO cells. In addition, EBNA1_A and EBNA1_B had no obvious cytotoxicity on cell viability and no side effects on protein quality.

[0246] Example 2. Effect of engineered EBNA1 on protein expression in HEK293 cells

[0247] To test the function of engineered EBNA1 in HEK293 transient transfection system, wild-type EBNA1, EBNA1_A and EBNA1_B were co-expressed with test molecules, respectively. The expression titer, cell viability, average cell productivity and final product quality of each test molecule were analyzed.

[0248] 2.1 Effect of engineered EBNA1 on OKT3 expression in HEK293 cells

[0249] The average expression titers of OKT3 expressed alone and co-expressed with EBNA1, EBNA1_A and EBNA1_B were 60 mg / L, 66 mg / L, 76 mg / L and 77 mg / L, respectively. Figure 3 ).

[0250] All engineered EBNA1s slightly enhanced OKT3 expression levels in HEK293 cells. In addition, EBNA1_A and EBNA1_B performed slightly better than wild-type EBNA1. Average cell productivity analysis also showed that EBNA1_A and EBNA1_B brought a slight enhancement ( Figure 7 ).

[0251] No significant cytotoxic effects were observed when EBNA1_A and EBNA1_B were expressed ( Figure 5). Protein quality is also not affected by EBNA1_A and EBNA1_B ( Figure 8 right).

[0252] 2.2 Effect of engineered EBNA1 on the expression of Herceptin in HEK293 cells

[0253] The average expression titers of Herceptin alone or co-expressed with EBNA1, EBNA1_A, and EBNA1_B were 314 mg / L, 524 mg / L, 732 mg / L, and 538 mg / L, respectively. Figure 3 EBNA1_A performed significantly better than EBNA1, while EBNA1_B was comparable to EBNA1. Nevertheless, analysis of average cell productivity showed that EBNA1_A and EBNA1_B performed better ( Figure 7 ).

[0254] EBNA1_A and EBNA1_B did not affect cell viability and protein quality ( Figure 5 and Fig. 9 right).

[0255] 2.3 Effect of engineered EBNA1 on emicizumab expression in HEK293 cells

[0256] The average expression titers of emicizumab alone or co-expressed with EBNA1, EBNA1_A, and EBNA1_B were 164 mg / L, 337 mg / L, 404 mg / L, and 366 mg / L, respectively. Figure 3 EBNA1_A performed significantly better than EBNA1, while EBNA1_B was comparable to EBNA1. The average cell productivity enhancement of EBNA1_A and EBNA1_B was comparable to that of wild-type EBNA1 ( Figure 7 ).

[0257] EBNA1_A and EBNA1_B also did not affect cell viability and protein quality when expressing the test molecule ( Figure 5 and Fig.10 right).

[0258] 2.4 Effect of engineered EBNA1 on dulaglutide expression in HEK293 cells

[0259] The average expression titers of dulaglutide expressed alone or co-expressed with EBNA1, EBNA1_A, and EBNA1_B were 321 mg / L, 727 mg / L, 946 mg / L, and 702 mg / L, respectively. Figure 3EBNA1_B showed similar effects to wild-type EBNA1, while EBNA1_A was significantly superior to EBNA1. EBNA1_A and EBNA1_B showed slightly significant differences in EBNA1 ( Figure 7 ).

[0260] EBNA1_A and EBNA1_B did not affect cell viability and protein quality when expressing the test molecules ( Figure 5 and Fig.11 right).

[0261] in conclusion

[0262] The results showed that EBNA1_A showed a dominant effect on protein expression in HEK293 cells, while EBNA1_B was comparable to wild-type EBNA1 in the HEK293 transient expression system. In addition, EBNA1_A and EBNA1_B had no obvious cytotoxicity on cell viability and no side effects on protein quality.

[0263] Example 3. Construction of a stable cell line constitutively expressing engineered EBNA1 and its effect on protein production Influence

[0264] To further investigate the effects of engineered EBNA1 on protein production of target recombinant proteins in mammalian cells, we generated stable cell lines constitutively expressing engineered EBNA1 to examine the effects of the cell lines on the expression of target proteins or peptides.

[0265] 3.1 Construction of stable cell lines carrying engineered EBNA1

[0266] The gene encoding the engineered EBNA1 (EBNA1_B) was cloned into the pWX039 plasmid ( Fig.13 ) (WuXi Biologics) for stable transfection of mammalian cells. CHO cells were grown to a cell density of 1.5 to 2.0 x 10 6 cells / ml, collect 10 7 cells and resuspended in 300 μl electroporation buffer. Dilute 5 μg of the plasmid containing the engineered EBNA1 gene into the electroporation buffer and mix with the cells. Transfection was performed using a Bio-Rad electroporator and the cells were transferred to pre-warmed CDCHO medium (Hyclone). Stable cells carrying the engineered EBNA1 were selected by adding 400 μg / ml zeocin. Stable cell clones expressing the engineered EBNA1 were selected by limiting dilution. 3.2 Expression of target protein using stable cells carrying engineered EBNA1

[0267] The gene encoding the target protein adalimumab was cloned into the pWX4.1 vector and transfected into the above stable cell line. The cell line was further cultured under conditions that allowed the expression of the target protein.

[0268] The results showed that a cell line constitutively expressing the engineered EBNA1 could be successfully constructed, and the expression of the recombinant protein was significantly increased (the titer was increased by about 1.8 times compared to the cell line expressing wild-type EBNA) ( Fig.14 ).

[0269] The present invention is not limited to the scope of the embodiments described herein, which are intended only to illustrate individual aspects of the present invention, and any functional equivalents are also within the scope of the present invention. Based on the foregoing and teachings, in addition to the contents described herein, various modifications to the compositions and methods of the present invention are obvious to those skilled in the art, and are also intended to fall within the scope of the present invention. Such modifications or other embodiments may be implemented without departing from the true scope and spirit of the present invention.

[0270] Information about sequences in the sequence listing

[0271]

[0272] References

[0273] Nagesh, T., Ambuj, S., 2019. Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Front Bioeng Biotechnol. 7, 420.

[0274] Jianwei, Z., 2012. Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv. 30(5), 1158-1170.

[0275] Kishwar, K., 2013. Gene expression in Mammalian cells and its applications. Adv Pharm Bull. 3(2), 257-263.

[0276] Wolfgang, H., and Bill, S., 2013. Replication of Epstein-Barr viral DNA. Cold Spring Harb Perspect Biol. 5(1), a013029.

[0277] 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.

[0278] 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.

[0279] Ann, K., and Bill, S., 1995. Plasmid maintenance of derivatives of oriP of Epstein-Barr virus. J Virol. 69(2), 1280-1283.

[0280] 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.

[0281] 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.

[0282] 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. An engineered Epstein-Barr virus nuclear antigen 1 (EBNA1), wherein: Compared to wild-type EBNA1, the original GA-rich region in the engineered EBNA1 is engineered.

2. The engineered EBNA1 of claim 1, wherein: (a) the original GA-rich region is removed; (b) the original GA-rich region is replaced by a linker; and / or (c) the N-terminal segment comprising the original GA-rich region of wild-type EBNA1 (e.g., residues 1 to residue 336 at the N-terminus) is replaced by an AT hook motif capable of binding to chromosomes during mitosis, such as the AT hook motif from human HMG-I / Y (e.g., residues 1 to residue 92); and / or The wild-type EBNA1 comprises the amino acid sequence of SEQ ID NO:

1.

3. The engineered EBNA1 of claim 1, wherein: The wild-type EBNA1 comprises from N-terminus to C-terminus: B1-GA-B2-C; and The engineered EBNA1 comprises from N-terminus to C-terminus: (i) B1-L-B2-C; or (ii) AT-SL-C, Among them, B1 is the first basic domain; GA is a glycine-alanine-rich domain; B2 is the second basic domain; C is the C-terminal region of EBNA1, which contains 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; and SL is a short linker.

4. The engineered EBNA1 of claim 2, wherein: The length of the engineered linker replacing the original GA-rich region is 10-50, 20-40, 30-36 amino acids; and / or Wherein, the engineered linker comprises an amino acid sequence (RGRGGSGGGGSGGAGGGGSGGA) n GGSGGSGG, wherein n is 1, 2, 3 or 4; and / or Wherein, the engineered joint comprises RGRGGSGGGGSGGAGGGGSGGAGGSGGSGG (SEQ ID NO: 12).

5. The engineered EBNA1 of claim 1, wherein: (a) the replacement AT hook motif is connected to the original EBNA1 part via a short linker, the length of which is less than 30, less than 25, or less than 20 amino acid residues; and / or (b) The short linker comprises an amino acid sequence GGGGAGGGGSGGGGAGGGGGGGGGAGGG (SEQ ID NO: 14); and / or (c) the AT hook motif comprises an amino acid sequence MSESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPT PKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKKLEK(SEQ ID NO:15); and / or (d) the AT hook motif together with the short linker comprises the amino acid sequence SEQ ID NO:

13.

6. The engineered EBNA1 of claim 1, wherein: The engineered EBNA1 comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

3.

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

8. The isolated polynucleotide molecule of claim 7, wherein: The polynucleotide molecule is in a form suitable for introduction into mammalian cells for recombinant expression of the target polypeptide; and / or Wherein, the engineered EBNA1 encoding molecule is codon optimized.

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

10. The vector according to claim 9, wherein The vector is selected from a plasmid, a viral vector, a cosmid or an artificial chromosome.

11. The vector according to claim 9, wherein The vector is a vector containing OriP, such as pWX4.1 plasmid; and / or wherein the vector is also co-transfected with a vector containing an encoding molecule of OriP and / or a target polypeptide; and / or Wherein, the vector is used for constitutive expression of engineered EBNA1, such as pWX039 plasmid.

12. An isolated mammalian cell suitable for recombinant expression of a target polypeptide, wherein: The mammalian cell comprises an engineered EBNA1, wherein the engineered EBNA1 has enhanced function in improving recombinant expression compared to wild-type EBNA1 (e.g., wild-type EBNA1, comprising the amino acid sequence of SEQ ID NO: 1), and wherein the original GA-rich region of the wild-type EBNA1 is engineered in the engineered EBNA1.

13. The mammalian cell according to claim 12, wherein The original GA-rich region: (a) removed; (b) replaced by an engineered joint; and / or (c) the N-terminal segment comprising the original GA-rich region of wild-type EBNA1 (e.g., residues 1 to residue 336 at the N-terminus) is replaced by an AT hook motif capable of binding to chromosomes during mitosis, such as the AT hook motif from human HMG-I / Y (e.g., residues 1 to residue 92); and / or The wild-type EBNA1 comprises the amino acid sequence of SEQ ID NO:

1.

14. The mammalian cell of claim 12, wherein: The wild-type EBNA1 comprises from N-terminus to C-terminus: B1-GA-B2-C; and The engineered EBNA1 comprises from N-terminus to C-terminus: (i) B1-L-B2-C; or (ii) AT-SL-C, Among them, B1 is the first basic domain; GA is a glycine-alanine-rich domain; B2 is the second basic domain; C is the C-terminal region of EBNA1, which contains 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; and SL is a short linker.

15. The mammalian cell according to claim 13, wherein The length of the engineered linker replacing the original GA-rich region is 10-50, 20-40, 30-36 amino acids; and / or The engineered linker comprises an amino acid sequence (RGRGGSGGGGSGGAGGGGSGGA) n GGSGGSGG, wherein n is 1, 2, 3 or 4; and / or The engineered linker comprises RRGRGGSGGGGSGGAGGGGSGGAGGSGGSGG (SEQ ID NO: 12).

16. The mammalian cell of claim 13, wherein: (a) the replacement AT hook motif is connected to the original EBNA1 part via a short linker, the length of which is less than 30, less than 25, or less than 20 amino acid residues; and / or (b) The short linker comprises an amino acid sequence GGGGAGGGGSGGGGAGGGGGGGGGAGGG (SEQ ID NO: 14); and / or (c) the AT hook motif comprises an amino acid sequence MSESSSKSSQPLASKQEKDGTEKRGRGRPRKQPPVSPGTALVGSQKEPSEVPT PKRPRGRPKGSKNKGAAKTRKTTTTPGRKPRGRPKKLEK(SEQ ID NO:15); and / or (d) the AT hook motif together with the short linker comprises the amino acid sequence SEQ ID NO:

13.

17. The mammalian cell according to claim 12, wherein The engineered EBNA1 comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; and / or Wherein, the engineered EBNA1 is introduced into the mammalian cell via an expression vector comprising an encoding molecule of the engineered EBNA1.

18. The mammalian cell of claim 17, wherein: (a) the engineered EBNA1 encoding molecule is codon optimized; and / or (b) the vector is a plasmid containing OriP; and / or (c) the vector is pWX4.1 plasmid or pWX039 plasmid; and / or (d) the vector further comprises a coding molecule for a target polypeptide; and / or (e) the cell further comprises one or more separate vectors, the vectors carrying the coding molecules of the target polypeptide; and / or (f) the vector carrying the engineered EBNA1 encoding molecule is also co-transfected with a vector containing an OriP and / or a target polypeptide encoding molecule; and / or (g) The cells constitutively or transiently express the engineered EBNA1.

19. The mammalian cell according to claim 12, 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 and enzymes, and is preferably a therapeutically active or diagnostic polypeptide.

20. The mammalian cell of claim 12, wherein The target polypeptide is selected from OKT3 (e.g., comprising the amino acid sequence of SEQ ID NO: 4 and / or 5), Herceptin (e.g., comprising the amino acid sequence of SEQ ID NO: 6 and / or 7), Emicizumab (e.g., comprising the amino acid sequence of SEQ ID NO: 8, 9 and / or 10), dulaglutide (e.g., comprising the amino acid sequence of SEQ ID NO: 11), adalimumab (e.g., comprising the amino acid sequence of SEQ ID NO: 16 and / or 17) or a functional chain or fragment thereof.

21. The mammalian cell of claim 12, wherein The mammalian cell is selected from a human cell, a rodent cell or a monkey cell.

22. The mammalian cell of claim 12, wherein The mammalian cell has one or more of the following characteristics: (a) the mammalian cell is a rodent cell derived from a hamster or a mouse; and / or (b) the mammalian cell is a rodent cell line selected from the group consisting of a Chinese hamster cell line (e.g., a Chinese hamster ovary (CHO) cell line), a BHK cell line, a NSO cell line, a C127 cell line, a mouse 3T3 fibroblast cell line, and a SP2 / 0 cell line; and / or (c) the mammalian cell is derived from a human cell selected from the group consisting of HEK293 cells (e.g., HEK293F-S), MCF-7 cells, PerC6 cells, CAP cells, hematopoietic cells, and HeLa cells; and / or (d) The mammalian cells are provided in the form of cell culture, cell lines or cell clones.

23. A method for producing the mammalian cell of any one of claims 12-22, comprising introducing the polynucleotide molecule encoding the engineered EBNA1 of any one of claims 7-8 into the mammalian cell.

24. The method of claim 23, wherein: A polynucleotide molecule that also encodes a polypeptide of interest, or an isolated polynucleotide molecule that encodes a polypeptide of interest, is also introduced into the cell.

25. A method for recombinantly producing a target polypeptide or increasing the recombinant expression yield of a target polypeptide, comprising: (a) culturing the mammalian cell according to any one of claims 12 to 22 under conditions that allow expression of the polypeptide of interest; (b) isolating the target polypeptide; and (c) optionally, treating the isolated target polypeptide.

26. The method of claim 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 and enzymes, and is preferably a therapeutically active or diagnostic polypeptide.

27. The method of claim 25, wherein: The target polypeptide is selected from OKT3 (e.g., comprising the amino acid sequence of SEQ ID NO: 4 and / or 5), Herceptin (e.g., comprising the amino acid sequence of SEQ ID NO: 6 and / or 7), Emicizumab (e.g., comprising the amino acid sequence of SEQ ID NO: 8, 9 and / or 10) and dulaglutide (e.g., comprising the amino acid sequence of SEQ ID NO: 11) or a functional chain or fragment thereof.

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

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

30. The product of claim 29, wherein: The product is selected from the group consisting of a drug, a diagnostic or detection kit, and an enzyme preparation.

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