Chimeric signal peptides for protein production

Abstract

The chimeric signal peptide for protein expression comprises an N region, a hydrophobic region, and a C region, wherein the N region and the C region are from the same signal peptide of a first protein, and the hydrophobic region is from the signal peptide of a second protein, and the first protein is different from the second protein. The first and second proteins can be independently selected from the group consisting of BM40, IL2, HA, insulin, CD33, IFNA2, IgGK leader, AZU, and SEAP.

Description

[Technical Field]

[0001] The present invention relates generally to the preparation of therapeutic proteins. Specifically, the present invention relates to the application of artificial signal peptides to optimize protein expression. [Background technology]

[0002] Developing stable cell lines, such as CHO, that produce high levels of proteins or antibodies has become an industry trend. To date, optimization of mammalian expression systems has primarily focused on downstream processes and vehicle development. However, to meet the needs for high yields, faster development, and lower production costs, optimization of expression vectors and other components must also be considered. When producing secreted proteins, the rate-limiting step affecting protein secretion is the translocation of the protein into the lumen of the endoplasmic reticulum. This is determined by the sequence of the secretion signal, i.e., the signal peptide (SP). Numerous studies have shown that the endogenous SP of the original protein or antibody is usually not optimized, and there is still an unmet need for improvement.

[0003] Signal peptides are short peptides, generally 5 to 30 amino acids long, that are present at the N-terminus of most newly synthesized proteins, and include those that are secreted from cells, present in specific organelles (Golgi apparatus or endoplasmic reticulum), or inserted into the cell membrane.

[0004] Signal peptides consist of an N region, a hydrophobic region (H region), and a C region. The H region of a signal peptide contains a stretch of hydrophobic amino acids (approximately 5–16 residues long) that tends to form a single alpha helix. The N region of many signal peptides contains a short stretch of positively charged amino acids that may help enforce the proper topology of the polypeptide during translocation, known as the positive inside rule. The C region of a signal peptide usually contains a stretch of amino acids recognized for cleavage by a signal peptidase. However, protease cleavage sites are not present in the transmembrane domain, also known as the signal anchor sequence, that acts as the signal peptide.

[0005] Numerous studies have found that most natural signal peptides (SPs) of proteins are not optimal, and optimizing the SP or replacing the natural SP with an alternative SP can lead to increased production of the target protein. For example, L. Kober et al. showed that the natural signal peptides derived from human albumin and human azurocidin (AZU) resulted in better antibody expression (Biotechnol. Bioeng., 2013 Apr;110(4):1164-73. doi:10.1002 / bit.24776). Summary of the Invention

[0006] An embodiment of the present invention relates to a chimeric signal peptide for protein expression, comprising an N region, a hydrophobic region, and a C region, wherein the N region and the C region are from a signal peptide of a first protein, and the hydrophobic region is from a signal peptide of a second protein, wherein the first protein is different from the second protein.

[0007] According to certain embodiments of the invention, the first and second proteins may be independently selected from the group consisting of BM40, IL2, HA, insulin, CD33, IFNA2, IgK, AZU, and SEAP.

[0008] One aspect of the present invention relates to a method for producing a target protein or antibody using the chimeric signal peptide of the present invention. The method according to one embodiment of the present invention comprises transfecting a mammalian host cell with an expression cassette comprising the chimeric signal peptide of the present invention, culturing the mammalian host cell to express the target protein, and harvesting the target protein.

[0009] Other aspects and advantages of the present invention will become apparent from the following description and appended claims. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the efficiency of various common signal peptides in enhancing Herceptin production at different HC / LC ratios. [Figure 2] FIG. 2 shows the efficiency of various common signal peptides in enhancing Avastin production at different HC / LC ratios. [Figure 3A] Figure 3A shows the results of SignalP4.0 prediction of the chimeric CD33_IgK signal peptide. SignalP4.0 predicts C, S, and Y scores. The C score predicts that the signal peptidase should cleave after the alanine (A) and before the glutamic acid (E) in the sequence MPLLLWVLLLWAGALA-EVQLVESGGG (SEQ ID NO: 11). [Figure 3B] Figure 3B shows the results of SignalP-HMM prediction of the chimeric CD33_IgK signal peptide. SignalP-HMM predicts the N, H, and C regions of the signal peptide. Signal-HMM also predicts that the signal peptidase should cleave after the alanine (A) and before the glutamic acid (E) in MPLLLWVLLLWAGALA-EVQLVESGGG (SEQ ID NO: 11). [Figure 3C]Figure 3C shows the results of PrediSi prediction of the chimeric CD33_IgK signal peptide. PrediSi also predicts that the signal peptidase should cleave after the alanine (A) and before the glutamic acid (E) in MPLLLWVLLLWAGALA-EVQLVESGGG (SEQ ID NO: 11). [Figure 3D] Figure 3D shows the results of a BLAST prediction of the chimeric CD33_IgK signal peptide. By comparing sequence homology, BLAST also predicts that the signal peptidase should cleave after the alanine (A) and before the glutamic acid (E) in MPLLLWVLLLWAGALA-EVQLVESGGG (SEQ ID NO: 11). [Figure 4] FIG. 4 shows that both chimeric signal peptides BMCD and CDBM have well-defined signal peptidase cleavage sites, indicating that such signal peptides should likely have their function conserved. [Figure 5] FIG. 5 is a schematic diagram showing the construction of individual heavy and light chain expression vectors containing various signal peptide sequences of the present invention. [Figure 6] Figure 6 shows the results of various chimeric signal peptides of the present invention. Some of these chimeric signal peptides can induce protein expression at levels comparable to or better than AZU. Among the better chimeric signal peptides, BMCD shows the best results. [Figure 7] Figure 7 shows that the BMCD chimeric signal peptide is significantly better than AZU. The expression level with BMCD is approximately 1.5-fold higher than that with AZU. Figure 7 also shows that the proper sequence of the chimeric signal peptide is important. For example, a BMCD mutant lacking the last two residues (LA) is a signal peptide with a weaker expression level. [Figure 8] FIG. 8 shows that the SEAP_AZU chimeric signal peptide increases the production of denosumab by approximately 40%. [Figure 9] FIG. 9 shows that different vectors can affect the efficiency of chimeric signal peptides. [Figure 10A] FIG. 10A shows a procedure illustrating the cell culture and protein production protocols. [Figure 10B] FIG. 10B shows two expression vectors for denosumab production using the AZUd and SEAPAZU signal peptides, respectively. [Figure 10C] FIG. 10C shows the results of denosumab production using the expression vector of FIG. 10B. [Figure 11A] FIG. 11A shows the vector constructs for the production of the fusion protein 3D5R using the native signal peptide, the AZUd signal peptide, and the BMCD signal peptide, respectively. [Figure 11B] Figure 11B shows protein expression levels using the expression vectors of Figure 11A. The level of 3D5R fusion protein produced using the AZUd signal peptide is 2.7-fold higher than that using the native signal peptide, while the level produced using BMCD is 1.9-fold higher than that using the native signal peptide. [Figure 12A] FIG. 12A shows vector constructs for the production of antibodies against T-cell immunoglobulin and mucin domain 3 (TIM3) using the native signal peptide or the BMCD chimeric signal peptide. [Figure 12B] Figure 12B shows the results of protein production using the expression vector of Figure 12A. As shown, the BMCD signal peptide produces higher levels of protein than the native signal peptide. In transient transfections, BMCD production levels are 3-fold higher than with the native signal peptide, while in stably transfected cells, the BMCD signal peptide produces levels 2.6-fold higher than with the native signal peptide. DETAILED DESCRIPTION OF THE INVENTION

[0011] Embodiments of the present invention relate to chimeric signal peptides and their uses that enhance the production of target proteins from eukaryotic cell systems. According to embodiments of the present invention, chimeric signal peptides are designed to enhance the expression and / or secretion of target proteins expressed in eukaryotic host cells. Signal peptides typically contain 5 to 30 amino acids in length and can be divided into an N region, an H region (hydrophobic region), and a C region. The chimeric signal peptides of the present invention can be constructed, for example, by exchanging the hydrophobic region of a first signal peptide with the hydrophobic region of a second signal peptide.

[0012] The chimeric signal peptides of the present invention may be based on known signal peptides. Commonly used signal peptides include those from BM40 (basement membrane protein 40), IL-2 (interleukin 2), HA (hemagglutinin), insulin, CD33, IFNA2 (interferon alpha 2), IgK (immunoglobulin kappa chain), SEAP (secreted alkaline phosphatase), and AZU (azurocidin) proteins. In the following description, the names of these proteins may also be used to refer to their signal peptides (SPs). From these natural signal peptides, better ones may be selected for further optimization. To evaluate the efficiency of these signal peptides in enhancing protein production, expression vectors containing the signal peptides can be constructed and the level of protein expression can be assayed, for example, using ELISA or other assays. The following examples use antibodies as exemplary target proteins. However, those skilled in the art will understand that embodiments of the present invention can be used to express any protein, not just antibodies.

[0013] Using an antibody as the target protein, two vectors containing the antibody heavy and light chains, respectively, are cloned. Alternatively, an expression vector containing both the heavy and light chains may be used. To facilitate assays, these vectors may also contain tags (e.g., red and green fluorescent tags, respectively). A test signal peptide is incorporated before the translation initiation codon ATG of the antibody heavy or light chain. The efficiency of antibody production is then evaluated by cotransfection of these vectors into appropriate host cells. Several signal peptides known to support efficient protein production, such as albumin (ALB), azurocidin (AZU), H5L1, and H7N1, can be used as positive controls.

[0014] For protein production, constructs containing the signal peptides of the present invention can be constructed in any suitable expression vector suitable for a protein expression system (e.g., E. coli, yeast cells, CHO, etc.). For example, in our previous work, suitable expression vectors J1.0delta and J1.0.2 (based on pTCAE8.3) were constructed for the production of Herceptin and Avastin. The signal peptide of each vector is AZU (azurocidin) or Ori (original). To find a better signal peptide, AZU and Ori in separate vectors containing the nucleic acid sequences of the heavy or light chain, respectively, can be replaced with other signal peptides. In one example, a total of 32 vectors were constructed, each containing the light or heavy chain of an antibody (e.g., Herceptin or Avastin) with a different signal peptide.

[0015] These expression vectors were transiently transfected into CHO cells (e.g., DXB11 cells or CHOK1 (CCL61-S1) cells) at different heavy chain (HC) to light chain (LC) ratios (1:1 or 1:4) to assess transfection / expression efficiency, and antibody expression levels were assessed using ELISA.

[0016] As shown in Figure 1, constructs using Herceptin as the target protein and having BM40 and CD33 as signal peptides yielded yields comparable to or better than the positive control using AZU as the signal peptide. In addition, Figure 1 shows that titers could reach 8-9 μg / ml at a 1:1 HC / LC ratio and 4-5.5 μg / ml at a 1:4 HC / LC ratio. Therefore, in this example, a higher yield was obtained with a 1:1 HC / LC ratio.

[0017] Next, we applied different signal peptides to produce Avastin. As shown in Figure 2, constructs with the BM40, CD33, and IgK signal peptides yielded higher yields than constructs with the original signal peptide (ORI). Protein expression levels with the BM40, CD33, and IgK signal peptides were comparable to or better than those of the positive control (AZU). Different transfection ratios (HC / LC = 1:1 or 1:4) resulted in different yields. The titer with HC / LC = 1:1 was 200-250 ng / ml, while the titer with HC / LC = 1:4 reached 300-550 ng / ml. Therefore, in this example, a ratio of 1:4 produced more protein.

[0018] Based on these and similar studies, several signal peptides were found to have similar or better ability to enhance protein expression compared to the positive control AZU. These signal peptides were selected for further optimization. These selected signal peptides include the CD33, IgK, BM40, AZU, and SEAP signal peptides.

[0019] As mentioned above, signal peptides can be divided into three regions: the N region, the H region (hydrophobic region), and the C region. The hydrophobic region (H region) of a signal peptide forms a helix that is important for interaction with the signal recognition particle (SRP). Such interaction enables SRP to bind to the nascent protein and promotes its translocation across the ER membrane. Because the H region plays such an important role, we decided to investigate whether altering the H region could improve the efficiency of signal peptides.

[0020] One approach of the present invention is to create chimeric signal peptides in which the H region of a first signal peptide is replaced with the corresponding H region of a second signal peptide. To perform such H region exchanges, we first analyzed the signal peptides and identified the boundaries and signal protease cleavage sites of each region. Numerous methods / programs, such as SignalP4.0, SignalP-HMM, PrediSi, and Signal Blast, are available in the art for such analyses.

[0021] For example, the SignalP server, hosted by the Center for Biological Sequence Analysis at the Technical University of Denmark, predicts the presence and location of signal peptide cleavage sites in amino acid sequences. Methods include cleavage site prediction and signal peptide / non-signal peptide prediction based on a combination of several artificial neural networks. Additionally, the server provides methods for predicting N-regions, H-regions, and C-regions.

[0022] Using the CD33_IgK chimeric signal peptide, MPLLLWVLLLWAGALA-EVQLVESGGG (SEQ ID NO: 11) as an example, SignalP4.0 predicts C, S, and Y scores. The C score predicts that the signal peptidase should cleave after the alanine (A) and before the glutamic acid (E) in the sequence MPLLLWVLLLWAGALA-EVQLVESGGG (SEQ ID NO: 11). The S score indicates the likelihood that the sequence is a signal peptide, with a higher score meaning that it is more likely to be a signal peptide. The Y score is based on a combination of the C and B scores. The Y score further confirms that the peptidase should cleave before the glutamic acid residue (see Figure 3A).

[0023] A similar program, SignalP-HMM (based on the hidden Markov model, Henrik Nielsen and Anders Krogh, in Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB6), AAAI Press, Menlo Park, CA, pp. 122-130 (1998)), predicts the N-, H-, and C-regions as well as the signal peptidase cleavage site (Figure 3B). The program also predicts that the peptidase cleaves before the glutamate, consistent with the prediction of SignalP4.0.

[0024] Another program, PrediSi (Prediction of Signal Peptides, Karsten Hiller et al., "Prediction of Signal Peptides and Their Cleavage Positions," Nucleic Acids Res., 2004, July 1, 32::W375-W379, doi:10.1093 / nar / gkh378), also predicts that peptidases cleave between alanine (A) and glutamic acid (E) (Figure 3C). Finally, the BLAST program can be used to predict the cleavage site by homology comparison with other known signal peptide cleavage sites (Figure 3D).

[0025] Any of these methods can be used to predict signal peptides and their corresponding N, H, and C regions. They generally produce consistent results. Even if there are slight discrepancies, the precise boundaries between the N, H, and C regions are not critical to the present invention. As noted above, the H region is a hydrophobic stretch that favors α-helix formation. The M domain of the signal recognition particle (SRP) is known to bind signal peptides with many sequence variations in the H region, including synthetic peptides with poly-leucine (poly-Leu) for the H region (Kendall et al., "Idealization of the hydrophobic segment of the alkaline phosphatase signal peptide," Nature, 1986;321:706-708). Therefore, exchanging one α-helix for another should not affect the precise boundaries for the α-helix.

[0026] According to embodiments of the present invention, chimeric signal peptides can be created by designing H region exchanges based on the above-described prediction methods or other similar methods. For example, the H region of a first signal peptide can be exchanged with the H region of a second signal peptide. Various chimeric signal peptides can be created based on promising signal peptides described above, such as the CD33, IgK, BM40, AZU, and SEAP signal peptides.

[0027] In this description, the following notation will be used for chimeric signal peptides: the first signal peptide providing the N and C regions is listed first, followed by the name of the second signal peptide providing the H region. For example, the BM40_CD33 (or BM-CD or BMCD) chimeric signal peptide contains the N and C regions from BM40 and the H region from CD33. Similarly, the CD33_BM40 (or CD_BM or CDBM) chimeric signal peptide contains the N and C regions from CD33 and the H region from BM40. Other examples include CD33_IgK (CD33 with the hydrophobic region of the IgK leader), IgK_CD33 (IgK leader with the hydrophobic region of CD33), SEAP_AZU (SEAP with the hydrophobic region of AZU), and AZU_SEAP (AZU with the hydrophobic region of SEAP). The nucleic acid and peptide sequences for some of these chimeric signal peptides are shown in Table 1 below. [Table 1] The underlined portion indicates the hydrophobic region in the chimeric signal peptide.

[0028] The following sections illustrate embodiments of the present invention using a limited number of specific chimeric signal peptides as examples, however, those skilled in the art will understand that these limited examples are for illustrative purposes only and are not meant to limit the scope of the invention.

[0029] These chimeric signal peptides can first be analyzed in silico using the SignalP server (or other similar services) to confirm that they can still function as signal peptides and that they still contain a signal peptidase cleavage site. The following examples illustrate embodiments of the invention using BMCD and CDBM chimeras as signal peptides; however, the descriptions are equally applicable to other chimeric signal peptides of the invention.

[0030] As shown in Figure 4, both chimeric signal peptides, BMCD and CDBM, have well-defined signal peptidase cleavage sites, indicating that such chimeric signal peptides are likely to preserve their function. All other chimeras, CDIgK, IgKCD, SEAPAZU, and AZUSEAP, were found to have similarly conserved signal peptidase cleavage sites.

[0031] These chimeric signal peptides are then tested for their ability to act as signal peptides and to assess their ability to support and enhance protein expression / secretion. Testing is performed using the exemplary construct shown in Figure 5.

[0032] As shown in Figure 5, the chimeric signal peptide can be cloned into an expression vector for the antibody heavy chain (HC) and another expression vector for the antibody light chain (LC) at the signal peptide (SP) position. In this example, the expression vectors are under the control of an appropriate promoter (e.g., a CMV promoter) and contain an internal ribosome entry site (IRES) sequence for ribosome entry. The HC vector and the LC vector can be tagged with enhanced green fluorescent protein (EGFP) and sea anemone red fluorescent protein (DsRed), respectively, to facilitate analysis. The two expression vectors are then co-transfected into host cells (e.g., CHO cells) for protein production. The amount of protein produced can be analyzed by any suitable method, such as ELISA.

[0033] Using Herceptin as the target protein, Figure 6 shows the results from various chimeric signal peptides (e.g., CD-IgK, IgK-CD, SEAP-AZU, AZU-SEAP, BM-CD, and CD-BM). Indeed, some chimeric signal peptides can confer protein expression at levels comparable to or better than that of AZU (the positive control). Among the better chimeric signal peptides, BMCD shows the best results.

[0034] The above results are from the cotransfection of two separate vectors for the production of Herceptin HC and LC chains. An alternative method is to construct an expression vector that produces both HC and LC from the same vector. To test whether a vector expression system using one vector is more effective, the BMCD-containing HC vector and the BMCD-containing LC vector are subcloned into an appropriate expression vector (e.g., J2.0-PDSZ based on pTCAE8.3). The expression of Herceptin using this vector is tested in CHO cells. As shown in Figure 7, the BMCD chimeric signal peptide is significantly better than AZU (positive control). The expression level by BMCD is approximately 1.5-fold higher than that of AZU.

[0035] Figure 7 also demonstrates the importance of proper sequence of the chimeric signal peptide. For example, a BMCD deletion mutant (SEQ ID NO: 12) lacking the last two residues (LA) loses some signal peptide function, presumably because this deletion prevents signal peptide cleavage. The C region of a signal peptide is known to contain a more polar carboxyl terminus, where positions -3 and -1 (counting from the signal peptidase cleavage site) usually contain small aliphatic residues such as alanine and are rarely aromatic or charged residues. Therefore, deletion of leucine-alanine from the C terminus of BMCD may impair signal peptidase cleavage, thereby preventing protein secretion.

[0036] While the above results use Herceptin as the target protein / antibody, other proteins (e.g., Avastin, denosumab, etc.) have also been tested and found to have similar results. For example, Figure 8 shows that the SEAP_AZU chimeric signal peptide increases the production of denosumab by approximately 40%.

[0037] In addition to the chimeric signal peptide itself, the context / environment of these chimeric signal peptides can also affect the efficiency of protein production. That is, different vectors can further enhance the efficiency of these chimeric signal peptides. For example, we found that expression vector J2.0.2 produced approximately twice the level of denosumab compared to the J2.0 vector. Similarly, Avastin was also produced better using the J2.0.2 vector. Meanwhile, vector J2.0 yielded higher yields for Herceptin and Tim3 antibodies. As shown in Figure 9, the J2.0.2 vector has different promoters for the HC and LC chains. Therefore, the ability of chimeric signal peptides to enhance protein production can be further increased by selecting a better vector.

[0038] Figures 10A-10C show another example of using the J2.0.2 vector to express denosumab using the SEAP_AZU chimeric signal peptide. Figure 10A shows the cell culture and protein expression procedure. Briefly, CHO cells (3 x 10 5 1000 cells / ml) are seeded into batch culture flasks. After 5-6 days of batch culture, the supernatant can be collected and analyzed for protein production using ELISA or HPLC. Figure 10B shows the J2.0.2-PDSB-Deno AZUd and J2.0.2-PDSB-Deno SEAPAZUFigure 10 shows the vector constructs for the SEAPAZU chimeric signal peptide (SEAPAZU). These vectors contain the dihydrofolate reductase (DHFR) gene, which facilitates transfection of cell lines derived from CHO cells lacking the DHFR gene. Figure 10C shows denosumab production on days 5 and 6 for both vectors. Production levels were similar between these vectors, indicating that the SEAPAZU chimeric signal peptide is as effective as the positive control AZU.

[0039] While the above examples demonstrate the production of various antibodies, embodiments of the present invention can also be used to produce other proteins (such as fusion proteins of interest). Figure 11A shows expression vector constructs for the production of the fusion protein 3D5R. The three constructs contain the native signal peptide (SP, MRVPAQLLGLLLLWLPGARC, SEQ ID NO: 10), AZUd (SEQ ID NO: 9), and BMCD (SEQ ID NO: 5), respectively. Figure 11B shows the results of protein expression. The AZUd signal peptide, which lacks seven amino acid residues from the C region and was found to be as good as the AZU signal peptide, was clearly more efficient in producing and secreting the 3D5R fusion protein, with production levels 2.7-fold higher than those of the native signal peptide. Similarly, the chimeric signal peptide BMCD was also highly efficient, producing levels 1.9-fold higher than those of the native signal peptide.

[0040] Figures 12A and 12B show another example of the production of T-cell immunoglobulin and mucin domain 3 (TIM3) using the J2.0 vector and the BMCD signal peptide. Figure 12A shows vector constructs containing either the native signal peptide (ori) or the BMCD signal peptide. Figure 12B shows the production of TIM3 after 3 days of transient transfection or after 6 days of phase I selection in batch culture of transfected cells. In both transfection-expression systems, BMCD produces 3-fold and 2.6-fold higher protein expression levels compared to vectors using the native signal peptide.

[0041] The above examples demonstrate that the chimeric signal peptides of the present invention can effectively enhance the production and secretion of recombinant proteins, including antibodies. Enhancement was observed in transient transfections and in stably transfected cells in batch culture.

[0042]

[0023] Embodiments of the present invention may be implemented in any suitable manner known in the art. The following description provides some examples. Those skilled in the art will appreciate that these examples are for illustrative purposes only, and that other variations and modifications are possible without departing from the scope of the present invention.

[0043] Cell culture and media The Chinese hamster ovary (CHO) cell line DXB11 was obtained from Dr. Lawrence Chasin at Columbia University. The DHFR-negative CHO DXB11 cell line (also known as DUX-B11 and DUKX) was historically the first CHO cell line used for large-scale production of heterologous proteins and is still used for the production of many complex proteins (C.S. Kaas et al., "Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy," BMC Genomics, 16, Article No. 160 (2015)). Cell culture was performed in an incubator at 37°C and 95% humidity under 5% CO2. The cell culture medium contained Hyclone and mixed medium (50% CDFortiCHO and 50% ActiCHO). Cell counts and viability analyses were performed after trypan blue staining using an automated cell counter TC10 (Bio-Rad, USA).

[0044] The transfection construct contains a puromycin resistance gene. Stable pools were selected based on puromycin resistance. Once they were single clones, no selection was required.

[0045] Vector constructs In an embodiment, each HC or LC peptide fragment containing a signal peptide sequence can be amplified using PCR and incorporated into an expression vector using any technique known in the art, such as the highly efficient In-Fusion® HD enzyme (Clontech, TakaraBioUSA, Mountain View, CA). To construct an expression vector of the present invention, the selected signal peptide sequence is cloned into an appropriate vector (e.g., pcDNA3.1 or pTCAE-type vector) containing the protein to be expressed (e.g., the HC or LC chain of a target antibody). The vector also contains an IRES sequence controlling a reporter gene (e.g., EGFP and DsRed). Using the CD33 signal peptide as an example, CMV-HC CD33 -IRES-EGFP and CMV-LC CD33 -IRES-DsRed was obtained. Other signal peptides can be constructed similarly. For example, the following signal peptide sequences (Table 2) were constructed into expression vectors in a similar manner: [Table 2-1] [Table 2-2]

[0046] The construction of these vectors may use any conventional cloning technique. In certain embodiments, overlap PCR techniques are used, such as PCR amplification and ligation of amplified overlapping fragments. These techniques are routine and conventional.

[0047] Transfection of CHO cells The constructs can be transfected into host cells using any suitable method and setup known in the art. For example, CHOK1 cells were cultured in 6-well plates. Each well contained 1 x 10 6Cells are seeded in 3 mL of Hyclone™ HyCell™ CHO medium (GE Healthcare) containing 8 mM GlutaMAX™ (Thermo Fischer). Transfection of vectors (e.g., pcDNA3.1) containing the LC and HC of the Herceptin construct can be carried out using any suitable reagent known in the art, such as the lipophilic drug FreeStyle MAX™ (Thermo Fischer). For example, Herceptin HC BM40CD33 , Herceptin LC BM40CD33 and transfection reagent Freestyle MAX™ (Thermo Fixher) were added separately to OptiPRO™ SFM (Thermo Fixher) to prepare vector solutions as shown in Table 3. These solutions were allowed to stand for 5 minutes and mixed thoroughly before adding to the transfection reagent. The resulting solutions were allowed to stand for 20 minutes before transfection into cells. Cells were evaluated 3 days after transfection. [Table 3]

[0048] Protein expression For protein / antibody production in test CHO cells, antibody / protein expression constructs may be obtained from commercial sources or may be prepared according to procedures known in the art and transfected into test CHO cells for transient expression of the antibody or protein. The transfected CHO cells are cultured for an appropriate period (e.g., 3 days) to produce the target protein. Alternatively, transfection may produce a stable cell line that can be used to produce the protein in conventional or batch culture. Any method known in the art that facilitates the selection of stable transfectant cell lines may be used. For example, a vector containing the DHFR gene and CHO cells lacking the DHFR gene may be used. Only CHO cells containing the transfected vector should survive in the defined medium.

[0049] Protein expression levels can be assessed using any appropriate method, such as ELISA, HPLC, or other methods. If the expressed protein has enzymatic activity (e.g., SEAP), activity can also be used to assess protein expression levels. For example, for SEAP, the GreatEscAPe™ Chemiluminescence Kit may be obtained from Clontech. Prepare 1x dilution buffer by diluting 5x dilution buffer 1:5 with ddH2O. To assess protein expression levels, transfer 25 μl of cell culture medium from transfected or mock-transfected cells to a 96-well microtiter plate. If desired, the plate can be sealed and frozen at -20°C for later analysis. Add 75 μl of 1x dilution buffer to each sample in the 96-well microtiter plate. Seal the plate with adhesive aluminum foil or a regular 96-well lid, and incubate the diluted samples at 65°C for 30 minutes using a heat block or water bath.

[0050] The samples were cooled on ice for 2-3 minutes and allowed to equilibrate to room temperature. 100 μl of SEAP substrate solution was added to each sample. The samples were incubated at room temperature for 30 minutes before reading. The chemiluminescent signal was detected and recorded using a 96-well plate reader luminometer (e.g., CLARIOstar®). The antibody titer in the culture supernatant was determined by ELISA. The cell-specific productivity (Qp) was calculated by dividing the titer by the integrated area under the curve of the daily viable cell density.

[0051] While embodiments of the present invention have been described with respect to a limited number of embodiments, those skilled in the art, having the benefit of this disclosure, will appreciate that other embodiments may be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the present invention should be limited only by the appended claims.

Claims

1. 1. A chimeric signal peptide for protein expression in a mammalian host cell, comprising an N region, a hydrophobic region (H region), and a C region, wherein the N region and the C region are due to a signal peptide of a first protein, and the hydrophobic region (H region) is due to a signal peptide of a second protein, wherein the first protein is different from the second protein, and the chimeric signal peptide comprises the amino acid sequence of SEQ ID NO:

5.

2. 2. An isolated DNA encoding the chimeric signal peptide for protein expression in a mammalian host cell according to claim 1, comprising the DNA sequence set forth in SEQ ID NO:

25.

3. 10. An expression vector for producing a target protein from a mammalian host cell, comprising a promoter operably linked to the isolated DNA of claim 2, wherein the promoter is linked in-frame to the DNA encoding the target protein.

4. 1. A method for producing a target protein, comprising: a) transfecting a mammalian host cell with the expression vector of claim 3 comprising DNA encoding the target protein; b) culturing the mammalian host cells to express the target protein; c) harvesting the target protein; A method for providing the above.

5. 5. The method of claim 4, wherein the mammalian host cell is a CHO cell.

6. The method of claim 4 , wherein the target protein is an antibody.

Images