Cho transcription control elements and uses thereof

Abstract

The present disclosure provides novel transcription control elements comprising an enhancer element operably linked to a mammalian endogenous promoter sequence and optionally a mammalian intron sequence, resulting in strong fusion promoters for constitutive high-level recombinant gene transcription in mammalian cells including CHO cells or CHO-derived cells.

Description

CHO TRANSCRIPTION CONTROL ELEMENTS AND USES THEREOFREFERENCE TO ELECTRONIC SEQUENCE LISTING

[0001] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on December 23, 2024, is named "0132-0322W01.xml" and is 27,937 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety7.FIELD OF THE INVENTION

[0002] The present disclosure relates to transcription control elements for achieving enhanced and controlled expression of recombinant proteins in CHO cells.BACKGROUND

[0003] Recombinant gene transcription in Chinese hamster ovary (CHO) cells is still regularly driven by strong viral promoters such as cytomegalovirus (CMV). While CMV is a potent promoter, its use for recombinant protein production in CHO host cells presents challenges for several reasons including but not limited to (i) the promoter transcriptional activity7may be lower than the theoretical maximum of the CHO host as cytomegalovirus evolved within a different host cell type (e.g. mouse, human), which may have a different transcriptional network to the CHO cells, and (ii) the lifecycle of the parental cytomegalovirus (latency-reactivation), may mean that the promoter is not best suited to long-term constitutive expression in a host cell for bioproduction processes. Therefore, there is a need for improving CHO transcriptional activity' within expression vectors. Furthermore, the options for controlling the expression ratios of multiple product genes from a single expression vector, which may be important for the optimized expression of complex multichain products such as bispecific antibodies, is typically limited to insertion of additional copies of the light and / or heavy chain genes, or use of the vector position effect whereby' the gene order within the transfected expression vector is varied to influence overall product output from the cell. However, in both cases the output can be unpredictable. There is a need for novel transcription control elements to better control gene expression.SUMMARY OF THE INVENTION

[0004] In embodiments, the present invention provides a transcription control element, comprising in order and operably linked: (a) an enhancer nucleotide sequence comprisingabout 200 to about 300 nucleotides of a murine cytomegalovirus (mCMV) sequence: (b) a mammalian endogenous promoter; and (c) optionally a mammalian intron nucleotide sequence, wherein the enhancer nucleotide sequence comprises at least 3, but not more than 6, NFKB binding sites. In some aspects, the mammalian endogenous promoter is a truncated endogenous promoter.

[0005] In a further embodiment, the disclosure provides a transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 10. In further embodiments, the present invention provides a transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 1 1.

[0006] In embodiments, the present disclosure provides a transcription control element, comprising in order and operably linked: (a) an enhancer nucleotide sequence comprising about 300 to about 410 nucleotides of a human cytomegalovirus (hCMV) sequence; and (b) a mammalian endogenous promoter; wherein the enhancer nucleotide sequence comprises at least 2 NFKB binding sites.

[0007] In a further embodiment, the disclosure provides a transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 14. In further embodiments, the present invention provides a transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%. at least 98% or 100% sequence identity to SEQ ID NO: 15.

[0008] In a further embodiment, the disclosure provides a transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity’ to SEQ ID NO: 16.

[0009] In other embodiments, the present disclosure provides an isolated polynucleotide comprising the transcription control element, a vector comprising the transcription control element, a host cell comprising the isolated poly nucleotide or the vector, and a method of expressing a polypeptide of interest in a host cell comprising culturing the host cell under suitable conditions to produce the polypeptide of interest.

[0010] Other features and advantages of the compositions and methods described herein will become more apparent from the following detailed description when read in conjunction with the accompanying drawings.BRIEF DESCRIPTION OF THE FIGURES[OH] The following drawings form part of the present specification and are included to further demonstrate exemplar}7embodiments of certain aspects of the present invention.

[0012] FIG. 1A shows an exemplar}7embodiment of a transcription control element as described herein, comprising in order, and operably linked: an enhancer nucleotide sequence, a mammalian endogenous promoter, and a mammalian intron nucleotide sequence.

[0013] FIG. IB shows a schematic overview of the design of the gene regulator}7units of the transcription control elements described herein, compared to the wild-type murine cytomegalovirus immediate early (mCMV) and the human cytomegalovirus immediate early (hCMV) promoters. Four TATA box-containing endogenous promoters occurring upstream of the Chinese hamster glyceraldehyde-3-phosphate dehydrogenase (chGAPDH) gene, the Chinese hamster 78-kDa glucose-regulated protein (chGRP78) gene, the Chinese hamster calcyclin (chS100a6) gene and the human elongation factor la (hEFl ) gene, and two viral enhancers occurring upstream of the mCMV gene and the hCMV gene were analyzed for transcription factor binding sites. Defined regions of these promoters and enhancers were operably linked to generate seven novel transcription control elements.

[0014] FIG. 2 shows the nucleotide sequence of chGAPDH-mCMV transcription control element with various TFBS (SEQ ID NO: 10).

[0015] FIG. 3 shows the nucleotide sequence of chGRP78-mCMV-intron transcription control element with various TFBS (SEQ ID NO: 11).

[0016] FIG. 4 shows the nucleotide sequence of chS100a6-mCMV transcription control element with various TFBS (SEQ ID NO: 12).

[0017] FIG. 5 shows the nucleotide sequence of hEFla-mCMV transcription control element with various TFBS (SEQ ID NO: 13).

[0018] FIG. 6 shows the nucleotide sequence of hEFl -hCMV transcription control element with various TFBS (SEQ ID NO: 14).

[0019] FIG. 7 shows the nucleotide sequence of short hEFla-hCMV (short) transcription control element (3' hCMV enhancer truncated) with various TFBS (SEQ ID NO: 15).

[0020] FIG. 8 shows the nucleotide sequence of chEFla-hCMV transcription control element with various TFBS (SEQ ID NO: 16).

[0021] FIG. 9 shows a schematic depiction of monoclonal antibody (mAh) expressing GS Xceed® and GS piggyBac® vectors used for comparison of different transcription control elements in CHOK1SV cells. Panel (A): GS Xceed® vector utilizing upstream LC and downstream HC (LC / HC) gene order. Panel (B): GS piggyBac® vector utilizing LC / HC gene order. Panel (C): GS piggy Bac® vector utilizing upstream HC and downstream LC (HC / LC) gene order. SV40E: Simian virus 40 early promoter; GS: glutamine synthetase encoding sequence; LC: antibody light chain encoding sequence; HC: antibody heavy chain encoding sequence; pA: polyadenlyation signal; ITR: piggyBac inverted terminal repeat sequence.

[0022] FIG. 10 shows the expression levels of a monospecific antibody (mAbl) from transcription control elements or wild-tj pe viral promoters following transient transfection of CHOK1SV cells, as described in embodiments herein.

[0023] FIG. 11 shows the expression levels of mAbl from transcription control elements or the wild-type mCMV or hCMV promoter in stable CHOK1SV pools, as described in embodiments herein.

[0024] FIG. 12 shows the expression levels of three mAbs expressed from CHOK1SV cells transiently transfected with GS piggyBac® vectors comprising the transcription control elements or wild-type mCMV in either LC / HC or HC / LC gene order.

[0025] FIG. 13 shows the stable pool recovery of cells transfected with mAb-expressing GS piggyBac® vectors containing different transcription control elements or the wild-type mCMV promoter with either LC / HC or HC / LC gene order.

[0026] FIG. 14 shows the mAb expression from cells stably expressing mAbs from GS piggy Bac® vectors containing different transcription control elements or the wild-type mCMV promoter with either LC / HC or HC / LC gene order.

[0027] FIG. 15 shows the LC and HC mRNA transcript levels in cells stably expressing mAbs from GS piggyBac® vectors containing different transcription control elements or the wild-type mCMV promoter.

[0028] FIG. 16 shows the aggregation level in cells stably expressing mAbs from GS piggyBac® vectors containing different transcription control elements or the wild-type mCMV promoter.

[0029] FIG. 17 shows the IgG purity level in cells stably expressing mAbs from GS piggy Bac® vectors containing different transcription control elements or the wild-type mCMV promoter.

[0030] FIG. 18 shows the glycan types in cells stably expressing mAbs from GS piggyBac® vectors containing different transcription control elements or the wild-type mCMV promoter.

[0031] FIG. 19 shows the recombinant mAb2 titers from the top 15-16 clones expressing mAb2 from the hEFla-hCMV promoter with LC / HC gene order (A), mCMV promoter with HC / LC gene order (B), mCMV promoter with LC / HC gene order (C), and chGAPDH- mCMV promoter with LC / HC gene order (D).

[0032] FIG. 20 show s the percent titer change of the mAb2 clones over 60 generations.

[0033] FIG. 21 shows the titer (A), cell-specific productivity (B) and glycan profile (C) of mAb4 from cells stably expressing mAb4 from the transcription control element hEFla- hCMV or the wild-type mCMV promoter.

[0034] FIG. 22 shows the recombinant mAb4 titers from the top 10 clones expressing mAb4 from the hEF 1 a-hCMV promoter with LC / HC gene order (A), hEF 1 a-hCMV promoter with HC / LC gene order (B), mCMV promoter with LC / HC gene order (C), and mCMV promoter with HC / LC gene order (D).

[0035] FIG. 23 show's the percent titer change of the mAb4 clones over 60 generations.

[0036] FIG. 24 show s the titer (A), qP (B), and mRNA level (C) of a bispecific antibody (bsAbl) from cells stably expressing bsAbl from a transcription control element or the wildtype mCMV promoter.

[0037] FIG. 25 shows the product quality of bsAbl produced from cells stably expressing bsAbl from a transcription control element or the wild-type mCMV promoter.

[0038] FIG. 26 shows the titer (A), qP (B) and correctly assembled product (C) of bsAb2 from cells stably expressing bsAb2 from a transcription control element or the wild-type mCMV promoter.

[0039] FIG. 27 shows the product quality of bsAb2 produced from cells stably expressing bsAb2 from a transcription control element or the wild-type mCMV promoter.

[0040] FIG. 28 shows the titer (A), qP (B) and correctly assembled product (C) of bsAB3 from cells stably expressing bsAb3 from a transcription control element or the wild-type mCMV promoter.

[0041] FIG. 29 shows the product quality of bsAb3 produced from cells stable expressing bsAb3 from a transcription control element or the wild-type mCMV promoter.DETAILED DESCRIPTION OF THE INVENTION

[0042] It should be understood that various aspects disclosed herein may be combined in different combinations than the combinations specifically presented in the description and accompanying drawings. It should also be understood that, depending on the example, certain acts or events of any of the processes or methods described herein may be performed in a different sequence, may be added, merged, or left out altogether (e.g., all described acts or events may not be necessary to carry out the techniques).

[0043] Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0044] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0045] The use of the term "or" in the claims is used to mean "and / or," unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or."

[0046] As used herein, the terms "comprising" (and any variant or form of comprising, such as "comprise" and "comprises"), "having" (and any variant or form of having, such as "have" and "has"), "including" (and any variant or form of including, such as "includes" and "include") or "containing" (and any variant or form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps.

[0047] The use of the term "for example" and its corresponding abbreviation "e g." means that the specific terms recited are representative examples and embodiments of the disclosurethat are not intended to be limited to the specific examples referenced or cited unless explicitly stated otherwise.

[0048] As used herein, "about" can mean plus or minus 10% of the provided value. Where ranges are provided, they are inclusive of the boundary values. "About" can additionally or alternately mean either within 10% of the stated value, or within 5% of the stated value, or in some cases within 2.5% of the stated value; or, "about" can mean rounded to the nearest significant digit.

[0049] As used herein, "between" is a range inclusive of the ends of the range. For example, a number between x and y explicitly includes the numbers x and y and any numbers that fall within x and y.

[0050] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary ) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin S., et al, (1990), as modified in Karlin S., et al., (1993), and incorporated into the NBLAST and XBLAST programs. In certain embodiments. Gapped BLAST can be used as described in Altschul SF, et al., (1997). BLAST-2, WU-BLAST-2 (Altschul SF, et al., (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences.

[0051] As used herein the term "operably linked" means associated in such a w ay that a gene regulatory unit, e.g., a promoter, may control expression of the molecule of interest.Elements that are operably linked are aligned together on the same nucleic acid molecule, as described herein.

[0052] In general, the term "promoter" refers to a region of DNA that initiates transcription of a particular gene. As used herein, endogenous promoters for constitutive and efficient expression of transgenes are generally obtained from non-coding regions upstream of a transcription initiation site of genes, that are associated with high and steady expressionthroughout most experimental conditions. The choice of promoter allows for control of the level of expressed protein, such as a gene of interest (GOI).

[0053] As used herein the term "intron" refers to a nucleotide sequence within a gene that is removed by RNA splicing during maturation of the final RNA product. The term intron refers to both the DNA sequence within a gene and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final mature RNA after RNA splicing are exons.

[0054] The terms "5"' and "3'" as used herein refer to the directionality, i.e., the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5'-end and a 3'-end. The relative positions of structures along a strand of nucleic acid, including genes and various protein binding sites, are usually noted as being either upstream (towards the 5'-end) or downstream (towards the 3 '-end). This naming convention is important because nucleic acids can only be synthesized in vivo in the 5'-to-3' direction, as the polymerase that assembles new strands only attaches new nucleotides to the 3 '-hydroxyl ( — OH) group, via a phosphodiester bond.

[0055] As used herein the term "vector" refers to a nucleic acid molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and / or expressed. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multi cloning site, and a selectable marker. The vector itself is generally a nucleic acid sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector, which transfers genetic information to another cell, is typically to isolate, multiply, or express the insert in the target cell. In preferred embodiments, the vector is a plasmid.

[0056] The terms "host cell" or "host cell line" as used herein include any cells which are capable of growing in culture and either expressing a desired recombinant product protein or reproducing a polynucleotide, e.g., a vector, described herein. In some embodiments, a derived cell is a cell derived from a parental cell line through limiting dilution. In some embodiments, the host cells comprise mammalian cells, e.g., CHO cells, human cells, human kidney cells, HEK293 cells, or HEK293 SF cells. In some embodiments, the host cell is a CHO cell or CHO derived cell. In some embodiments, the host cell is a CHO cell comprising a knockout of its glutathione synthetase (GS) gene, also referred to as a "GS-KO" cell. Insome embodiments, the host cell is a CHOK1SV cell. In some embodiments, the host cell is a CHOK1SV GS-KO cell. In some embodiments, the host cell is a human cell or human derived cell. In some embodiments, the host cell is a human kidney cell. In some embodiments, the human cell is a HEK293 cell. In some embodiments, the human cell is a HEK293 SF cell.

[0057] A polypeptide, antibody, polynucleotide, vector, cell, or composition, which is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition, which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition, which is isolated is substantially pure. In some embodiments, an antibody of the present disclosure is a monospecific antibody. In some embodiments, an antibody of the present disclosure is a bispecific antibody.

[0058] The terms "polypeptide," "peptide." and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.Transcription control elements (TCEs)

[0059] In various embodiments, the present disclosure provides a transcription control element comprising in order and operably linked: an enhancer nucleotide sequence, a mammalian endogenous promoter; and optionally a mammalian intron nucleotide sequence. In some embodiments, the enhancer nucleotide sequence comprises a murine cytomegalovirus (mCMV) sequence. In some embodiments, the enhancer nucleotide sequence comprises a human cytomegalovirus (hCMV) sequence. FIG. 1A shows an exemplary embodiment in which a transcription control element, comprises in order and operably linked the enhancer nucleotide sequence, the mammalian endogenous promoter, and the mammalian intron nucleotide sequence. The transcription control elements describedherein suitably comprise less than 760 base pairs and have increased transcriptional activity, for example, when compared with murine cytomegalovirus (mCMV) promoter. This is also known as a "compact" transcription control element. Transcription control elements with increased transcriptional activity' can be useful for obtaining high yields of recombinant proteins of interest. In some embodiments, the transcription control elements described herein are configured for transgene expression in a mammalian cell. As used herein, "transgene expression" means mediating transcription of a heterologous polynucleotide. In some embodiments, the mammalian cell is a CHO cell, a CHO-derived cell, a human cell, a human kidney cell, a HEK293 cell, or a HEK293 SF cell. In some embodiments, the transcription control elements described herein are suitable for transgene expression in CHO cells and / or CHO derived cells. As used herein, a "heterologous" polynucleotide refers to a polynucleotide that is derived from different species than the host cell, and further encompasses a polynucleotide that is derived from the same species as the host cell but is located at a non-naturally occurring genomic location and / or associated with a non-naturally occurring regulatory element (e.g., a transcriptional control element described herein). Heterologous polynucleotides may produce naturally occurring or synthetic (e.g., engineered) polypeptides, e.g., naturally occurring or engineered antibodies.

[0060] FIG. IB shows a schematic overview of the design of the gene regulatory units of the transcription control elements compared to the wild-type murine cytomegalovirus immediate early (mCMV) and the human cytomegalovirus immediate early (hCMV) promoters. Four TATA box-containing endogenous promoters occurring upstream of the Chinese hamster glyceraldehyde-3-phosphate dehydrogenase (chGAPDH) gene, the Chinese hamster 78-kDa glucose-regulated protein (chGRP78) gene, the Chinese hamster calcyclin (chS100a6) gene and the human elongation factor la (hEFla) gene, and two viral enhancers occurring upstream of the mCMV gene and the hCMV gene were analyzed for transcription factor binding sites. Defined regions of these promoters and enhancers were operably linked to generate seven novel transcription control elements.

[0061] As used herein, an "endogenous" regulatory element, e.g., promoter, refers to a regulatory element that is naturally present in the host cell.

[0062] As used herein, an "enhancer" is a short regulator}' DNA sequence that when bound by specific transcription factors, enhances the transcription of an associated gene. The enhancer may also be referred to herein as the 5’ region of the CMV promoter.

[0063] Various bioinformatic tools were used to scan DNA sequences for transcription factor binding sites (TFBS) and potentially active TFBS were identified within the 5' region of mCMV promoter (FIG. 3), hCMV promoter (FIG. 6), endogenous promoters Chinese hamster glyceraldehyde-3-phosphate dehydrogenase (chGAPDH) (FIG. 2), Chinese hamster 78-kDa glucose-regulated protein (chGRP78) (FIG. 3), Chinese hamster SI 00 calcium binding protein A6 (chS100a6) (FIG. 4), human elongation factor- 1 a (hEF lot) (FIG. 5), and Chinese hamster elongation factor- 1 a (chEF lot) mammalian endogenous promoter (FIG. 8). The 5' region of mCMV or hCMV was operably linked to truncated endogenous (CHO) promoters, effectively increasing TFBS heterogeneity, resulting in very' strong transcription control elements with fairly compact sizes (< 760 base pairs). The level of transcriptional activation mediated by a discrete promoter sequence is largely determined by its unique composition of TFBS, and the availability of cognate TFs (Johari et al., 2019). The 5' region of the mCMV promoter (i.e., the enhancer) comprises several TFBS including 4 NFKB binding sites. The 5' region of hCMV promoter (also known as enhancer) comprises several TFBS including 3 NFKB and 3 CRE binding sites. NFKB is a ubiquitous transcription factor that plays a crucial role in various biological processes such as inflammation, immune responses as well as in regulation of expression of many genes related to cell survival, proliferation and differentiation. In mammals, NFKB comprises five subunits that can bind to promoter regions of target genes as homodimers or heterodimers. The mechanism of transcriptional regulation by NFKB is complex and remains not fully understood (Smale ST, 2012). NFKB is a very strong transcription factor in CHO cells. It w as unexpected that the combination of the murine or human CMV enhancer operably linked to endogenous CHO genome-derived core promoters, which are generally weaker than viral core promoter sequences, would increase recombinant protein expression above that achieved using the standard CMV sequence on its own. It was also unexpected that the transcription control elements described herein wherein the enhancer nucleotide sequence comprises at least 2, but not more than 6, NFKB binding sites provide increased transcriptional activity7. However, increasing NFKB beyond 6 copies (binding sites) did not increase transcriptional activity. In some embodiments, a transcription control element described herein comprises a mCMV enhancer comprising at least 3, but not more than 6 NFKB binding sites. In some embodiments, the mCMV enhancer comprises 3, 4, 5, or 6 NFKB binding sites. In some embodiments, a transcription control element described herein comprises a hCMV enhancer comprising at least 2, but not more than 6 NFKB binding sites. In some embodiments, the hCMV enhancer comprises 2, 3, 4, 5, or 6 NFKB binding sites.

[0064] The choice of a suitable promoter may, in some embodiments, result in very high and stable transgene expression in mammalian cell cultures. Chinese hamster ovary (CHO) cells are the most widely used platform for the industrial production of therapeutic proteins (Walsh, G., 2018). Several viral-derived promoters, such as human cytomegalovirus (hCMV) or simian virus 40 (SV40) are frequently used for constitutive expression of recombinant proteins in mammalian cells. While these promoters allow high expression rates, they can also lead to constitutive overexpression of the transgene causing cellular toxicity (Romanova and Noll, 2018; Rodova et al., 2013). In addition, viral promoters show cell-cycle dependency (Brown and James, 2016; Brightwell et al., 1997). The CMV, for example, is active primarily in the S phase and presents very low activity in G0 / G1 (Brightwell et al., 1997). There is G0 / G1 synchronization mainly in late exponential and stationary growth phases in CHO-K1 cells cultured in high densify (Tossolini et al., 2018), and the Gl-phase is considered the appropriate stage for increasing the production of recombinant proteins (Kumar et al., 2007). Moreover, several studies showed that CMV promoter could be susceptible to histone modifications (Moritz et al., 2016; Spencer et al.. 2015) or DNA methylation (Brooks et al.. 2004; Brown et al., 2015; Kim et al., 2011; Mariah et al.. 2014; Marx et al., 2021; Moritz et al., 2015), linking these epigenetic modifications to production instability in recombinant CHO cells (Tossolini et al., 2022). A solution to such drawbacks w ould be the use of transcription control elements which function in a coordinated manner with cellular and bioprocess characteristics. For complex multi-chain products such as bispecific antibodies where maximizing transcription alone is less likely to be beneficial, transcription control elements could be utilized to control the relative expression ratios of multiple product genes.

[0065] In some embodiments, the mammalian endogenous promoter of the transcription control element described herein comprises a CHO genome-derived core promoter. Exemplary CHO genome-derived core promoters include, but are not limited to, the promoters shown in FIGS. 2-4 and 8. In some embodiments, the CHO genome-derived core promoter comprises chGAPDH, chGRP78, chS100a6, or combination thereof. In some embodiments, the mammalian endogenous promoter of the transcriptional control element described herein comprises a human genome-derived core promoter. An exemplary’ human genome-derived core promoters includes, but is not limited to, the hEF I a promoter.

[0066] In some embodiments, the transcriptional control element described herein comprises a murine cytomegalovirus (mCMV) enhancer nucleotide sequence ("mCMV enhancer"). Insome embodiments, the mCMV enhancer comprises about 200 to about 300 nucleotides. In some embodiments, the mCMV enhancer comprises about 210 to about 250 nucleotides. In some embodiments, the mCMV enhancer comprises about 210 to about 230 nucleotides. In some embodiments, the mCMV enhancer comprises about 210 to about 250 nucleotides. In some embodiments, the mCMV enhancer comprises about 210 to about 230 nucleotides. In some embodiments, the mCMV enhancer comprises a nucleotide sequence with at least 90%, at least 95%. at least 98%. at least 99%, or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the mCMV enhancer comprises about 210 to about 222 nucleotides of a nucleotide sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the mCMV enhancer comprises about 3 to about 6 NFKB binding sites. In some embodiments, the mCMV enhancer comprises 3, 4, 5, or 6 NFKB binding sites.

[0067] In some embodiments, the mCMV enhancer described herein is operably linked to a CHO genome-derived core promoter. In some embodiments, the CHO genome-derived core promoter comprises chGAPDH, chGRP78, or chS100a6. In some embodiments, the mCMV enhancer is operably linked to a human genome-derived core promoter. In some embodiments, the human genome-derived core promoter comprises hEFla. In some embodiments, a mCMV enhancer is operably linked to a truncated endogenous CHO promoter chGAPDH (SEQ ID NO: 1) ("chGAPDH-mCMV transcription control element"), wherein the chGAPDH-mCMV transcription control element comprises a nucleotide sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity7to SEQ ID NO: 10 (FIG. 2). In some embodiments, the mCMV enhancer is operably linked to a truncated endogenous CHO promoter chS100a6 (SEQ ID NO:4) ("chS100a6- mCMV transcription control element"), wherein the chS100a6-mCMV transcription control element comprises a nucleotide sequence with at least 90%. at least 95%. at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12 (FIG. 4). In some embodiments, the mCMV enhancer is operably linked to a truncated endogenous CHO promoter chGRP78 (SEQ ID NO:2), followed by chGRP78 intron sequence (SEQ ID NO:3) ("chGRP78-mCMV- intron transcription control element"), wherein the chGRP78-mCMV-intron transcription control element comprises a nucleotide sequence with at least 90%. at least 95%. at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 (FIG. 3). In some embodiments, the mCMV enhancer is operably linked to a truncated hEFla promoter (SEQ ID NO:5) ("hEFla-mCMV transcription control element"), wherein the hEFla-mCMVtranscription control element comprises a nucleotide sequence with at least 90%, at least 95%. at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13 (FIG. 5).

[0068] In further embodiments, the transcription control element described herein comprises a human cytomegalovirus (hCMV) enhancer nucleotide sequence ("hCMV enhancer"). In some embodiments, the hCMV enhancer comprises about 2 to about 6 NFKB binding sites. In some embodiments, the hCMV enhancer comprises at least 2 NFKB binding sites. In some embodiments, the hCMV enhancer comprises at least 3 NFKB binding sites. In some embodiments, the hCMV enhancer comprises 2, 3, 4, 5. or 6 NFKB binding sites. In some embodiments, the hCMV enhancer comprises about 300 to about 410 nucleotides. In some embodiments, the hCMV enhancer comprises about 300 to about 320 nucleotides. In some embodiments, the hCMV enhancer comprises about 390 to about 407 nucleotides. In some embodiments, the hCMV enhancer comprises a nucleotide sequence with at least 90%. at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the hCMV enhancer comprises 3 NFKB binding sites. In some embodiments, the hCMV enhancer comprises a nucleotide sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the hCMV enhancer comprises 2 NFKB binding sites. In some embodiments, the hCMV enhancer comprises about 300 to about 320 nucleotides of a nucleotide sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, wherein the hCMV enhancer comprises 2 NFKB binding sites.

[0069] In some embodiments, the hCMV enhancer described herein is fused or operably linked with a truncated hEF la promoter described herein, e.g., wherein the hEFla-hCMV transcription control element comprises a nucleotide sequence with at least 90%. at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14 (FIG. 6). In some embodiments, the hCMV enhancer described herein is fused or operably linked with a truncated hEF la promoter ("hEFla-hCMV transcription control element"), wherein the hEFla-hCMV transcription control element comprises a nucleotide sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15 (FIG. 7). In some embodiments, the hCMV enhancer described herein is fused or operably linked with a truncated chEFla promoter described herein ("chEFla-hCMV transcription control element"), wherein the chEFla-hCMV transcription control element comprises a nucleotide sequence with at least 90%, at least 95%, at least 98%, at least 99%. or 100% sequence identity' to SEQ ID NO: 16 (FIG. 8).

[0070] In a further embodiment, the disclosure provides an isolated polynucleotide comprising a transcription control element described herein.

[0071] In a further embodiment, the disclosure provides a vector comprising a transcription control element described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector further comprises a polynucleotide encoding a polypeptide of interest operably linked to the transcription control element. In some embodiments, the polypeptide of interest is an antibody, or antigen-binding fragment thereof, a fusion protein, an immunoglobulin, an engineered protein, a protein fragment, or an enzyme. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bispecific antibody.

[0072] In a further embodiment, the disclosure provides a host cell comprising an isolated polynucleotide described herein or a vector described herein. In some embodiments, the host cell comprises a mammalian cell, a CHO cell or CHO derived cell, a human cell, a human kidney cell, a HEK293 cell, or a HEK293 SF cell. In some embodiments, the host cell comprises a CHO cell or CHO derived cell. In some embodiments, the CHO cell is a CHOK1SV cell. In some embodiments, the CHO cell is a CHOK1SV GS-KO cell.

[0073] In a further embodiment, the disclosure provides a method of expressing a polypeptide of interest in a host cell comprising culturing a host cell described herein under suitable conditions to produce the polypeptide of interest.EMBODIMENTS

[0074] Clause 1. A transcription control element, comprising in order and operably linked: (a) an enhancer nucleotide sequence comprising about 200 to about 300 nucleotides of a murine cytomegalovirus (mCMV) sequence; (b) a mammalian endogenous promoter; and (c) optionally a mammalian intron nucleotide sequence, wherein the enhancer nucleotide sequence comprises at least 3, but not more than 6, NFKB binding sites.

[0075] Clause 2. The transcription control element of clause 1 , wherein the mammalian endogenous promoter is a CHO genome-derived core promoter.

[0076] Clause 3. The transcription control element of clause 2, wherein the CHO genome- derived core promoter is selected from chGAPDH, chGRP78 or chS100a6.

[0077] Clause 4. The transcription control element of clause 3, wherein the CHO genome- derived core promoter is chGAPDH, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 1.

[0078] Clause 5. The transcription control element of clause 3, wherein the CHO genome- derived core promoter is chS100a6, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 4.

[0079] Clause 6. The transcription control element of clause 3, wherein the CHO genome- derived core promoter is chGRP78, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 2.

[0080] Clause 7. The transcription control element of clause 6, wherein the mammalian intron nucleotide sequence is chGRP78, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 3.

[0081] Clause 8. The transcription control element of clause 1, wherein the mammalian endogenous promoter is hEF 1 a.

[0082] Clause 9. The transcription control element of clause 8, wherein the mammalian endogenous promoter is hEF la, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 5.

[0083] Clause 10. The transcription control element of any of clauses 1 to 9, wherein the enhancer nucleotide sequence comprises 210-230 nucleotides of the mCMV sequence.

[0084] Clause 11. The transcription control element of any of clauses 1 to 10, wherein the enhancer nucleotide sequence comprises 5 Nuclear Factor- KB (NFKB) binding sites, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 7.

[0085] Clause 12. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 10.

[0086] Clause 13. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 11.

[0087] Clause 14. A transcription control element, comprising in order and operably linked: (a) an enhancer nucleotide sequence comprising about 300 to about 410 nucleotides of a human cytomegalovirus (hCMV) sequence; and (b) a mammalian endogenous promoter: wherein the enhancer nucleotide sequence comprises at least 2 NFKB binding sites.

[0088] Clause 15. The transcription control element of clause 14, wherein the mammalian endogenous promoter is hEFla.

[0089] Clause 16. The transcription control element of any of clauses 14 or 15. wherein the enhancer nucleotide sequence comprises 3 NFKB binding sites, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 8.

[0090] Clause 17. The transcription control element of any of clauses 14 or 15, wherein the enhancer nucleotide sequence comprises 2 NFKB binding sites, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 9.

[0091] Clause 18. The transcription control element of clause 14. wherein the mammalian endogenous promoter is truncated chEFla.

[0092] Clause 19. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 14.

[0093] Clause 20. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 15.

[0094] Clause 21. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity' to SEQ ID NO: 16.

[0095] Clause 22. The transcription control element of any of clauses 1-21, configured to mediate transcription of a heterologous polynucleotide in CHO cells or CHO derived cells, mammalian cells, human cells, human kidney cells, HEK293 cells or HEK293 SF cells.

[0096] Clause 23. The transcription control element of any of clauses 1-21, configured to mediate transcription of a heterologous polynucleotide in CHO cells or CHO derived cells.

[0097] Clause 24. An isolated polynucleotide comprising the transcription control element of any of clauses 1-21.

[0098] Clause 25. A vector comprising the transcription control element of any of clauses 1- 21.

[0099] Clause 26. The vector of clause 25, wherein the vector is a viral vector.

[0100] Clause 27. The vector of clause 25 or claim 26, comprising a polynucleotide encoding a polypeptide of interest operably linked to the transcription control element.

[0101] Clause 28. The vector of clause 27, wherein the polypeptide of interest is an antibody, or antigen-binding fragment thereof, a fusion protein, an immunoglobulin, an engineered protein, a protein fragment or an enzyme.

[0102] Clause 29. A host cell comprising the isolated polynucleotide of clause 24 or the vector of any one of claims 25-28.

[0103] Clause 30. The host cell of clause 29, wherein the host cell is a CHO cell or CHO derived cell, mammalian cell, human cell, human kidney cell, HEK293 cell or HEK293 SF cell.

[0104] Clause 31. The host cell of clause 29, wherein the host cell is a CHO cell or CHO derived cell.

[0105] Clause 32. A method of expressing a polypeptide of interest in a host cell comprising culturing the host cell of clause 29 or claim 30 under suitable conditions to produce the polypeptide of interest.EXAMPLES

[0106] The different transcription control elements described in embodiments herein were inserted into GS Xceed® or GS piggyBac® antibody expression vectors for comparison of transcription control elements in CHOK1SV GS-KO cells. Each vector utilized the same promoter to drive LC and HC gene expression with the arrangement of the LC and HC in the vectors shown in FIG. 9. In the GS Xceed® vector (panel (A)), an upstream LC and downstream HC (LC / HC) gene order is utilized. In the GS piggyBac® vector, an upstream LC and downstream HC (LC / HC) gene order (panel (B)) or an upstream HC downstream LC (HC / LC) gene order (panel (C)) is utilized.Experimental design and results - transient testing:

[0107] PEI (polyethylenimine)-mediated transfection was utilized for transient expression testing. Prior to transfection, CHOK1SV or CHOK1SV GS-KO cells were centrifuged and resuspended in fresh medium to a density of 1 x 106cells / mL. Aliquots of 20 mL were added to each 125 mL Erlenmeyer flask, followed by 40 pg of GS Xceed® or GS piggyBac® plasmid DNA (FIG. 9) and 100 pL of PEI MAX. Transfected cells were cultured for 4 h at 36.5°C before being shifted to 32°C and cultured for 8 days. Cell viability and viable cell concentration were measured using Vi-CELL® XR and recombinant mAb concentration in cell culture supernatant was measured using Octet® ProA biosensors.

[0108] Results depicting the expression level of a monospecific antibody, mAbl, following transient transfection of CHOK1SV cells (N=6) with the transcription control elements according to FIG. IB in comparison with the wild-type viral promoters mCMV and hCMV, are shown in FIG. 10. PEI-mediated transient expression was performed with mAbl expressing GS Xceed® vectors containing different transcription control elements. Titers of the recombinant mAb were determined as described above. Four out of five transcription control elements exhibited higher or similar transient mAb production compared to the viral promoters.

[0109] Results depicting the expression levels of three monospecific antibodies, mAbl, mAb2, and mAb3, with the transcription control elements according to FIG. IB in comparison with the wild-type mCMV promoter are shown in FIG. 12. Panels A, B, and C show the results for mAbl, mAb2, and mAb3, respectively. CHOK1SV cells were transiently transfected as described above with GS piggyBac® vectors containing the different transcription control elements with either LC / HC or HC / LC gene order (see FIG. 9), and antibody expression level was measured (N=5). As shown in FIG. 12, mCMV with HC / LC gene order exhibited higher transient mAbl and mAb3 production compared to its LC / HC counterpart and to the transcription control elements. The transcription control elements exhibited higher or similar transient mAb production compared to the mCMV promoter with LC / HC gene order.Experimental design and results - stable pool testing:

[0110] General Protocol.

[0111] Stably expressing CHOK1SV GS-K.0 pools were generated by transfecting 10xl06cells with 40 pg of GS piggyBac® plasmid DNA (see FIG. 9) and 4 pg of piggyBac transposase mRNA using Bio-Rad® Gene Pulser Xcell. Electroporated cells were resuspended in 10 mL protein-free medium containing 1% HT in TPP TubeSpin® Bioreactor 50. At 24 h post-transfection, transfected cells were centrifuged at 200 xg for 5 min and cell pellets were resuspended in 10 mL protein-free medium containing 1% HT and 50 pM MSX to select for stable transfectants. The stably transfected pools were cryopreserved in protein- free medium containing 7.5% DMSO when the viability of pools recovered above 96%. Cell viability and viable cell concentration were measured using Vi-CELL® XR. Production of each stably transfected pool was determined in 250 mL Erlenmeyer flask fed-batch cultures. Cells were seeded at 0.5 xlO6cells / mL and cultured for 12 days at 36.5°C with a shift to 33°Cat Day 6. Recombinant mAb concentration in cell culture supernatant was measured using Octet® ProA biosensors. LC and HC mRNA levels at Day 9 were measured using Applied Biosystem SYBR® Green qPCR. Purified samples were subjected to gel permeation chromatography (GPC) for percent of aggregates, non-reducing Caliper CE-SDS for impurities, and Gly-X for glycan types.

[0112] Experiment 1.

[0113] Stable expression of a monospecific antibody, mAbl, in CHOK1SV GS-KO cells from the wild-type mCMV or hCMV promoters, or from transcription control elements chEFla-hCMV, hEFla-hCMV, and hEFla-hCMV with a shortened 3' hCMV enhancer was tested. CHOK1SV GS-KO cells were electroporated with mAbl expressing GS piggyBac® vectors containing the different transcription control elements with LC / HC gene order. Recombinant mAbl titers were determined in 12-day shake flask fed-batch cultures at 36.5°C with a shift to 33°C at Day 6. Results are shown in FIG. 11 (N=3). Transcription control elements chEFla-hCMV and hEFla-hCMV exhibited 4 fold and 1.5 fold-increase in mAbl production compared to the mCMV and hCMV promoter, respectively. Transcription control elements hEFla-hCMV with a shortened 3' hCMV enhancer exhibited slightly lower mAbl production compared to the hCMV promoter. qP: Cell-specific productivity.

[0114] Experiment 2,

[0115] CHOK1SV GS-KO cells were electroporated with GS piggyBac® vectors expressing one of three monospecific antibodies, mAbl, mAb2, or mAb3, containing the different transcription control elements as described in Experiment 1 or the wild-type CMV promoter, with either LC / HC or HC / LC gene order. Stable pool recovery of the CHOK1SV GS-KO cells was tested. Cell viability and viable cell concentration (VCC) were measured at Day 5 post-transfection. Results are shown in FIG. 13 (N=5) - mAbl : panels A, B; mAb2: panels C, D; mAb3: panels E, F. The transcription control elements exhibited faster stable pool recovery rates compared to the mCMV promoter.

[0116] Further, the expression levels for the stable CHOK1SV GS-KO pools expressing mAbl, mAb2, or mAb3 were compared. The average titer of pools generated using each promoter w as determined, and results are shown in FIG. 14 (N=3). Transcription control element hEFla-hCMV with both LC / HC and HC / LC gene orders, as well as mCMV promoter with HC / LC gene order, exhibited 3 to 5 fold-increase in mAbl (FIG. 14, panels A, B), mAb2 (FIG. 14, panels C, D) and mAb3 (FIG. 14. panels E, F) production compared tothe mCMV promoter with LC / HC gene order. Transcription control elements chGAPDH- mCMV and chGRP78-mCMV exhibited similar mAh production compared to the mCMV with LC / HC order.

[0117] Further, the LC and HC mRNA transcript levels during stable fed batch production of the CHOK1 SV GS-KO cells stably expressing mAbl , mAb2, or mAb3 were measured using Applied Biosystem SYBR® Green qPCR. Cells were sampled at Day 9, and mRNA levels were quantified by qPCR. Results are shown in FIG. 15 (N=3). Transcription control element hEFla-hCMV with both LC / HC and HC / LC gene orders as well as mCMV promoter with HC / LC gene order, exhibited 1.6 to 3.3 fold-increase in mAbl (FIG. 15, panel A), mAb2 (FIG. 15, panel B), and mAb3 (FIG. 15, panel C) LC mRNA level, and 3.5 to 7.7 foldincrease in HC mRNA level. Transcription control elements chGAPDH-mCMV and chGRP78-mCMV exhibited comparable LC and HC mRNA levels compared to the mCMV with LC / HC gene order.

[0118] Further, mAb aggregation in stable pools of the CHOK1SV GS-KO cells stably expressing mAbl, mAb2, or mAb3 was measured by gel permeation chromatography (GPC). Results are shown in FIG. 16. The transcription control elements exhibited similar mAb aggregation levels compared to the mCMV promoter.

[0119] Further, IgG purity level in stable pools of the CHOK1SV GS-KO cells stably expressing mAbl, mAb2, or mAb3 was measured by non-reducing Caliper CE-SDS. Results are shown in FIG. 17. The transcription control elements exhibited similar impurity levels compared to the mCMV promoter.

[0120] Further, glycan types in stable pools of the CHOK1SV GS-KO cells stably expressing mAbl, mAB2, or mAb3 were determined by Gly-X. Results are shown in FIG. 18. The transcription control elements exhibited highly comparable glycan profde compared to the mCMV promoter.

[0121] Experiment 3,

[0122] A further monospecific antibody, mAb4, w as stably transfected into CHOK1SV GS- KO cells with the hEFla-hCMV transcriptional control element or wild-type mCMV promoter. Recombinant mAb4 titer, cell-specific productivity’, and glycan profile in the CHOK1SV GS-KO pools were determined in 15-day shake flask fed-batch cultures at 36.5°C with a shift to 33°C on Day 6 (N=3). Results of the measured titer, cell-specific productivity (qP), and glycan profile are shown in panels A, B, and C of FIG. 21, respectively. hEFla-hCMV with LC / HC and HC / LC gene orders exhibited 1.81 to 2.22 fold-increase in production compared to the mCMV promoter, and comparable glycan profile as analyzed in two stable pools.

[0123] Experiment 4: Bispecific Antibodies.

[0124] Pools of CHOK1SV GS-KO cells stably expressing a recombinant bispecific antibody (bsAbl) were generated from transfection of GS piggyBac® vectors containing the hEFla- hCMV transcription control element or the wild-type mCMV promoter with eitherLC / HC 1 / HC2 (LHH), HC1 / LC / HC2 (HLH), or HC1 / HC2 / LC (HHL) gene order (N=3). FIG.24 shows results of recombinant bsAbl titer (panel A), qP (panel B), and mRNA level (panel C) in these stable pools. The transcription control element hEFla-hCMV with LC / HC 1 / HC2 (LHH), HC1 / LC / HC2 (HLH), and HC1 / HC2 / LC (HHL) gene orders, exhibited up to 1.72 foldincrease in bsAbl production compared to the mCMV promoter, with an overall increase in HC mRNA levels.

[0125] FIG. 25 shows the product quality, including correct assembly (panel A), aggregation level (panel B), and glycan profile (panel C) of bsAbl from the stable pools described above in FIG. 24. The transcription control element hEFla-hCMV exhibited lower levels of misassembled product, highly comparable aggregation, and highly comparable glycan profile compared to the mCMV promoter.

[0126] Pools of CHOK1SV GS-KO cells stably expressing another bispecific antibody (bsAb2) from the hEFla-hCMV transcription control element or the wild-ty pe mCMV promoter were generated as described above. FIG. 26 shows the results (N=3) of recombinant bsAb2 titer (panel A), qP, (panel B). and correctly assembled product (panel C) . The transcription control element hEFla-hCMV with LC1 / LC2 / HC1 / HC2 (LLHH) andHC 1 / LC2 / LC 1 / HC2 (HLLH) gene orders, exhibited up to 1.29 to 1.58 fold-increase in bsAb2 production compared to the mCMV promoter, with comparable percentage of correctly assembled product as analyzed in two stable pools.

[0127] FIG. 27 shows the product quality, including IgG purity (panel A), aggregation level (panel B), and glycan profile (panel C), of bsAb2 from the stable pools described above in FIG. 26. The transcription control element hEFla-hCMV exhibited similar impurity level, aggregation level, and glycan profile compared to the mCMV promoter.

[0128] Pools of CHOK1SV GS-KO cells stably expressing another bispecific antibody (bsAb3) from the hEFla-hCMV transcription control element or the wild-ty pe mCMVpromoter were generated as described above. FIG. 28 shows the results (N=3) of recombinant bsAb3 titer (panel A), qP (panel B), and correctly assembled product (panel C). The transcription control element hEFla-hCMV with LC1 / LC2 / HC1 / HC2 (LLHH) and HC1 / LC2 / LC1 / HC2 (HLLH) gene orders, exhibited 1.16 to 2.89 fold-increase in bsAb3 production compared to the mCMV promoter, with comparable percentage of correctly assembled product as analyzed on two stable pools.

[0129] FIG. 29 shows the product quality, including IgG purity (panel A), aggregation level (panel B), and glycan profile (panel C), of bsAb3 from the stable pools described above in FIG. 28. The transcription control element hEFla-hC MV exhibited similar impurity level, aggregation level, and glycan profile compared to the mCMV promoter.Experimental design and results - clonally-derived cell lines:

[0130] Clonally-derived cell lines were generated from the stable pools using Beacon® Optofluidic system and expanded in protein- and MSX-free medium. The cell lines were cryopreserved in protein-free medium containing 7.5% DMSO. Recombinant mAb titers were determined in Ambr®15 fed-batch cultures. Cells were seeded at 0.5xl06cells / mL and cultured for 12 days at 36.5°C with a shift to 33°C at Day 6. Cell viability and viable cell concentration were measured using Vi-CELL® XR and recombinant mAb concentration in cell culture supernatant was measured using Octet® ProA biosensors. Stability of productivity' for the cell lines was assessed by passaging the cell lines in Erlenmeyer flasks and MSX-free medium for 60 generations from RCB (research cell bank). The early and late- generation cell lines were revived concurrently and recombinant mAb titers were determined in Ambr®15 fed-batch cultures. Cells were seeded at 0.5xl06cells / mL and cultured for 15 days at 36.5°C with a shift to 33°C at Day 6.

[0131] Experiment 5,

[0132] Antibody production titers were compared in the top 15-16 clones with hEFla- hCMV, chGAPDH-mCMV, or wild-type mCMV promoter for expressing mAb2 (see Experiment 2 and FIG. 19). Clones were derived from the stable pools generated as described above in Experiment 2, using Beacon® Optofluidic System. Recombinant mAb2 titers were determined in 12-day Ambr®15 fed-batch cultures at 36.5°C with a shift to 33°C at Day 6. FIG. 19 shows the results from clones harboring hEFla-hCMV promoter with LC / HC gene order (panel A), mCMV promoter with HC / LC gene order (panel B), mCMV promoter with LC / HC gene order (panel C), and chGAPDH-mCMV promoter with LC / HC gene order(panel D). Clones harboring the hEFla-hCMV promoter exhibited significantly higher average titer compared to the mCMV promoter. Clones with an asterisk (*): duplicate vessels.

[0133] A total of 40 clones expressing mAb2 were passaged in Erlenmeyer shake flasks for 60 generations. FIG. 20 depicts the percent titer change in these clones over 60 generations. Clones harboring the hEFla-hCMV promoter exhibited superior overall long-term expression stability- (<30% change) titer compared to the mCMV promoter. Clones harboring the chGAPDH-mCMV with LC / HC gene order exhibited similar overall expression stability compared to the mCMV with LC / HC gene order. Clones with an asterisk (*): duplicate vessels.

[0134] Experiment 6,

[0135] FIG. 22 shows the expression levels in the top 10 clones for expressing mAb4 derived from the stable pools as described in Experiment 3 (see FIG. 21), harboring hEFla-hCMV promoter with LC / HC gene order (panel A), hEFla-hCMV promoter with HC / LC gene order (panel B), mCMV promoter with LC / HC gene order (panel C), and mCMV promoter with HC / LC gene order (panel D). Clones harboring the hEFla-hCMV promoter exhibited significantly higher average titer compared to the mCMV promoter.

[0136] A total of 40 clones expressing mAb4 were passaged for 60 generations as described herein. FIG. 23 depicts the percent titer change in these clones after 60 generations. Clones harboring the hEFla-hCMV promoter exhibited superior overall long-term expression stability- (<30% change) titer compared to the mCMV promoter. Clones with an asterisk (*): duplicate vessels.REFERENCES1. Altschul, S. F., & Gish, W. "

[0027] Local alignment statistics," Methods in enzymology Vol. 266, pp. 460-480. (1996).2. Altschul, S. F. et al., "Gapped BLAST and PSI-BLAST: anew generation of protein database search programs," Nucleic acids research, 25( T), 3389-3402 (1997).3. Brightwell, G. et al., "Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector," Gene. 194, 115-123 (1997).4. Brooks, A.R. et al., "Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle," J. Gene Med. 6, 395-404 (2004).Brown, A. J. et al., "NFKB, CRE and YY1 elements are key functional regulators of CMV promoter-driven transient gene expression in CHO cells," Biotechnol. J. 10, 1019-1028 (2015). Brown. A. J., James, D.C. "Precision control of recombinant gene transcription for CHO cell synthetic biology," Biotechnol. Adv. 34, 492-503 (2016). Karlin, S., & Altschul, S. F. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proceedings of the National Academy of Sciences, 87(6), 2264-2268, (1990). Karlin, S., & Altschul, S. F. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proceedings of the National Academy of Sciences. 90(12). 5873-5877, (1993). Kim, M. et al., "A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies," Biotechnol. Bioeng. 108, 2434- 2446 (2011). Kumar. N. et al., "Proliferation control strategies to improve productivity and survival during CHO based production culture." Cytotechnology. 53. 33-46 (2007). Mariah, J.H.M. et al., "Yang, insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells," Biotechnol. Prog. 30, 523-534 (2014). Marx, N. et al.. "Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3 A3L entails distinct subsequent histone modification changes in CHO cells," Metab. Eng. 66, 268-282 (2021). Moritz, B. et al., "CMV promoter mutants with a reduced propensity to productivity loss in CHO cells," Sci. Rep. 5 (2015). Moritz, B. et al., "High levels of histone H3 acetylation at the CMV promoter are predictive of stable expression in Chinese hamster ovary cells," Biotechnol. Prog. 32, 776-786 (2016). Rodova, M. et al., "CMV promoter is repressed by p53 and activated by JNK pathway," Plasmid. 69(3), 223-230 (2013). Romanova, N., Noll, T. "Engineered and Natural Promoters and Chromatin- Modifying Elements for Recombinant Protein Expression in CHO Cells," Biotechnology Journal, 1700232: 1-13. (2018). Smale, S. T. "Dimer-specific regulatory mechanisms within the NFKB family of transcription factors," Immunological reviews, 246(1), 193-204 (2012).18. Spencer, S. et al., "Stability of single copy transgene expression in CH0K1 cells is affected by histone modifications but not by DNA methylation," J. Biotechnol. 195, 15-29 (2015).19. Tossolini, I. et al., "Characterization of cellular states of CHO-K1 suspension cell culture through cell cycle and RNA-sequencing profiling," J. Biotechnol. 286, 56-67 (2018).20. Tossolini, I. et al., "Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures," Plasmid, 119, 102620 (2022).21. Walsh, G., "Biopharmaceutical benchmarks 2018," Nat. Biotechnol. 36, 1136-1145 (2018).22. Yusuf, B. et al.. "CHO genome mining for synthetic promoter design." Journal of Biotechnology, 294, 1-13, (2019).

[0137] While the disclosed methods have been described in connection with what is presently considered to be the most practical and preferred embodiments, it is to be understood that the methods encompassed by the disclosure are not to be limited to the disclosed embodiments, but on the contrary', is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0138] All publications, patents, patent applications, internet sites, and accession numbers / database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number / database sequence were specifically and individually indicated to be so incorporated by reference.

[0139] Table 1 shows the different elements used for generation of the transcription control elements as well as the tested combinations.TABLE 1: SEQUENCES

Claims

CLAIMSWhat is claimed is:

1. A transcription control element, comprising in order and operably linked:(a) an enhancer nucleotide sequence comprising about 200 to about 300 nucleotides of a murine cytomegalovirus (mCMV) sequence;(b) a mammalian endogenous promoter; and(c) optionally a mammalian intron nucleotide sequence, wherein the enhancer nucleotide sequence comprises at least 3, but not more than 6, NFKB binding sites.

2. The transcription control element of claim 1, wherein the mammalian endogenous promoter is a CHO genome-derived core promoter.

3. The transcription control element of claim 2, wherein the CHO genome-derived core promoter is selected from chGAPDH, chGRP78 or chS100a6.

4. The transcription control element of claim 3, wherein the CHO genome-derived core promoter is chGAPDH, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 1.

5. The transcription control element of claim 3, wherein the CHO genome-derived core promoter is chS100a6, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 4.

6. The transcription control element of claim 3, wherein the CHO genome-derived core promoter is chGRP78, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 2.

7. The transcription control element of claim 6, wherein the mammalian intron nucleotide sequence is chGRP78, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 3.

8. The transcription control element of claim 1, wherein the mammalian endogenous promoter is hEFla.

9. The transcription control element of claim 8, wherein the mammalian endogenous promoter is hEFla, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 5.

10. The transcription control element of any of claims 1 to 9, wherein the enhancer nucleotide sequence comprises 210-230 nucleotides of the mCMV sequence.

11. The transcription control element of any of claims 1 to 10, wherein the enhancer nucleotide sequence comprises 5 Nuclear Factor- KB (NFKB) binding sites, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 7.

12. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%. at least 98% or 100% sequence identity to SEQ ID NO: 10.

13. A transcription control element, comprising a nucleotide sequence with at least 90%. at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 11.

14. A transcription control element, comprising in order and operably linked:(a) an enhancer nucleotide sequence comprising about 300 to about 410 nucleotides of a human cytomegalovirus (hCMV) sequence; and(b) a mammalian endogenous promoter; wherein the enhancer nucleotide sequence comprises at least 2 NFKB binding sites.

15. The transcription control element of claim 14, wherein the mammalian endogenous promoter is hEFla.

16. The transcription control element of any of claims 14 or 15, wherein the enhancer nucleotide sequence comprises 3 NFKB binding sites, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 8.

17. The transcription control element of any of claims 14 or 15, wherein the enhancer nucleotide sequence comprises 2 NFKB binding sites, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity' to SEQ ID NO: 9.

18. The transcription control element of claim 14, wherein the mammalian endogenous promoter is truncated chEFla.

19. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 14.

20. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity' to SEQ ID NO: 15.

21. A transcription control element, comprising a nucleotide sequence with at least 90%, at least 95%, at least 98% or 100% sequence identity to SEQ ID NO: 16.

22. The transcription control element of any of claims 1-21. configured to mediate transcription of a heterologous polynucleotide in CHO cells or CHO derived cells, mammalian cells, human cells, human kidney cells, HEK293 cells or HEK293 SF cells.

23. The transcription control element of any of claims 1-21, configured to mediate transcription of a heterologous polynucleotide in CHO cells or CHO derived cells.

24. An isolated polynucleotide comprising the transcription control element of any of claims 1-21.

25. A vector comprising the transcription control element of any of claims 1-21.

26. The vector of claim 25, wherein the vector is a viral vector.

27. The vector of claim 25 or claim 26, comprising a polynucleotide encoding a polypeptide of interest operably linked to the transcription control element.

28. The vector of claim 27, wherein the poly peptide of interest is an antibody, or antigenbinding fragment thereof, a fusion protein, an immunoglobulin, an engineered protein, a protein fragment or an enzyme.

29. A host cell comprising the isolated polynucleotide of claim 24 or the vector of any one of claims 25-28.

30. The host cell of claim 29, wherein the host cell is a CHO cell or CHO derived cell, mammalian cell, human cell, human kidney cell, HEK293 cell or HEK293 SF cell.

31. The host cell of claim 29, wherein the host cell is a CHO cell or CHO derived cell.

32. A method of expressing a polypeptide of interest in a host cell comprising culturing the host cell of claim 29 or claim 30 under suitable conditions to produce the polypeptide of interest.

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