Polypeptides having increased secretion levels and cells comprising same

Abstract

The invention relates to the field of molecular biology, in particular to a polypeptide with an improved secretion level, a cell containing the polypeptide, a pharmaceutical composition containing the cell and application of the pharmaceutical composition. In particular, the polypeptide is a single chain antibody, such as a single chain PD-1 antibody and a single chain TNF-alpha antibody.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a polypeptide with an increased secretion level and a cell containing the polypeptide, a pharmaceutical composition containing the cell and an application thereof. Background Art

[0002] PD-1 is an important negative immune regulator, also known as CD279. PD-1 expressed on the surface of T cells binds to the PD-L1 ligand expressed by tumor cells, which can transmit negative regulatory signals to T cells, thereby inhibiting the proliferation and activation of T cells. TNF-α, also known as tumor necrosis factor-α, is mainly secreted by macrophages and monocytes. It can directly kill tumor cells and promote neutrophil phagocytosis. It has no obvious toxicity to normal cells and also has a strong antiviral function. In recent years, PD-1 and TNF-α have become commonly used targets in tumor treatment.

[0003] The preparation of various PD-1 and TNF-α antibodies is in the ascendant, and one common form is a single-chain antibody fragment (scFv), which is an antibody formed by connecting the heavy chain variable region and the light chain variable region of the antibody through a short peptide (linker). Promoting its expression and secretion in cells has always been one of the important topics studied by technicians in this field, and increasing its secretion amount is of great significance in the clinical stage and actual treatment process. It has been reported in the literature that different signal peptides have different effects on the secretory expression of the same single-chain antibody. At present, it is not possible to predict which signal peptide has the best effect in promoting the secretory expression of a specific single-chain antibody, that is, it is unpredictable.

[0004] Signal peptide, also known as leader peptide, signal sequence, transit peptide, etc., referred to as SP, is located at the N-terminus of the nascent protein, that is, the amino terminus, and is a short peptide chain that guides the transport and secretion of newly synthesized proteins. The C-terminus of the signal peptide is usually the sequence cleavage site of the signal peptide, which can be removed by the action of signal peptidase. Signal peptides play an important role in the transport and secretion of secretory proteins. By promoting transport in cells, proteins are brought to specific destinations in cells, or secreted outside cells. For secretory proteins expressed by exogenous genes, if there is no signal peptide, the secretion effect is poor or not secreted.

[0005] For the same secretory protein, different signal peptides have different secretion effects; for the same signal peptide, when it is used for different secretory proteins, it often shows different secretion effects. Therefore, it is necessary to screen signal peptides for different proteins that need to be secreted. Summary of the invention

[0006] In a first aspect, the present invention provides a polypeptide, whose amino acid sequence comprises a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence is a signal peptide sequence and the second amino acid sequence is a single-chain antibody sequence.

[0007] In some embodiments of the present invention, the first amino acid sequence and the second amino acid sequence are selected from one of the following combinations (1)-(3):

[0008] (1) the first amino acid sequence is as shown in SEQ ID NO:4 and the second amino acid sequence is as shown in SEQ ID NO:

[0009] (2) the first amino acid sequence is as shown in SEQ ID NO: 5 and the second amino acid sequence is as shown in SEQ ID NO: , and

[0010] (3) The first amino acid sequence is shown as SEQ ID NO:3 and the second amino acid sequence is shown as SEQ ID NO:.

[0011] In some embodiments of the present invention, the first amino acid sequence is shown as SEQ ID NO:4 and the second amino acid sequence is shown as SEQ ID NO:. In other embodiments of the present invention, the first amino acid sequence is shown as SEQ ID NO:5 and the second amino acid sequence is shown as SEQ ID NO:. In still other embodiments of the present invention, the first amino acid sequence is shown as SEQ ID NO:3 and the second amino acid sequence is shown as SEQ ID NO:.

[0012] In some embodiments of the present invention, the first amino acid sequence and the second amino acid sequence are directly connected. In other embodiments of the present invention, there is a protease cleavage site or a direct connection between the first amino acid sequence and the second amino acid sequence, preferably, the protease cleavage site is a furin cleavage site.

[0013] In a second aspect, the present invention provides a polynucleotide encoding the polypeptide of the first aspect.

[0014] In a third aspect, the present invention provides an expression vector comprising the polynucleotide described in the second aspect.

[0015] In a fourth aspect, the present invention provides a host cell comprising the polypeptide described in the first aspect, the polynucleotide described in the second aspect, or the expression vector described in the third aspect. Preferably, the host cell is a mammalian cell.

[0016] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the polypeptide of the first aspect, the polynucleotide of the second aspect, the expression vector of the third aspect, or the host cell of the fourth aspect.

[0017] In a fifth aspect, the present invention provides a method for promoting secretion of a single-chain antibody from a host cell, comprising:

[0018] The polypeptide of the first aspect, the polynucleotide of the second aspect, the expression vector of the third aspect or the host cell of the fourth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The schematic diagram of the aPD1 scFv expression cassette carrying different signal peptides is shown. The strong promoter EF1a is used to replace the CMV promoter of the pcDNA3.1(+) expression vector, and different signal peptides are used to connect aPD1 scFv. aPD1 scFv is fused with a FLAG tag.

[0020] Figure 2 The results of the aPD1 scFv protein secretion level test in Example 2 are shown, wherein: Figure 2 A is a bar chart showing the level of aPD1 scFv-1-FLAG protein in the supernatant detected by ELISA. Figure 2 B is a bar chart showing the level of aPD1 scFv-2-FLAG protein in the supernatant detected by ELISA. ***P<0.001, ****P<0.0001.

[0021] Figure 3 The results of the intracellular expression level detection of aTNF scFv guided by different signal peptides are shown in FIG. Figure 3 A is the flow cytometric peak diagram obtained by flow cytometry detection of aTNF scFv-FLAG protein after transfection of 293T cells with 8 signal peptide verification plasmids. Figure 3 B is a bar chart showing the positive ratio of aTNF scFv-FLAG protein, and the blank group is untransfected 293T cells, serving as a negative control. *P<0.05, **P<0.01.

[0022] Figure 4 The results of the secretion level test of aTNF scFv in the supernatant guided by different signal peptides are shown, among which, Figure 4 A is the band scanning result of WB detection of aTNF scFv-FLAG protein after 293T cells were transfected with 8 signal peptide verification plasmids. Figure 4 B is a bar chart showing the corresponding expression of aTNF scFv-FLAG protein, and the blank group is untransfected 293T cells. DETAILED DESCRIPTION

[0023] the term

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art.

[0025] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values ​​shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0026] Single-chain antibody (scFv) refers to an antibody form formed by connecting the light chain variable region and the heavy chain variable region of the antibody through the hinge region, which retains the ability to bind to the antigen.

[0027] Protease cleavage site refers to the protein structure region that is recognized and cleaved by proteases. Commonly used protease cleavage sites include HRV 3C protease cleavage site, TEV protease cleavage site, enterokinase cleavage site, furin protease cleavage site, etc.

[0028] Furin cutting site (FCS) refers to the position in the protein sequence that can be recognized and cut by furin. Furin is an important protein endonuclease that is expressed in various tissues and cell lines.

[0029] Signal peptide refers to a short peptide chain of 5-30 amino acids in the newly synthesized polypeptide chain that guides the transfer of proteins to the secretory pathway. It is located at the N-terminus of the secretory protein and consists of three parts: the N-terminus is a positively charged alkaline amino terminal; the middle is the main functional region, which is a hydrophobic sequence formed by neutral amino acids; the C-terminus is a negatively charged processing region, which is the cleavage site of the signal peptide. The newly synthesized protein is guided into the endoplasmic reticulum cavity by the signal peptide, and the signal peptide sequence is removed under the action of the signal peptidase, and then the protein continues to be translated, folded and modified at the same time.

[0030] All publications, patents, and patent applications cited in this specification are herein incorporated by reference, as if each individual publication, patent, or patent application were specifically set forth in its entirety and were individually incorporated herein by reference.

[0031] The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The methods and steps not specifically described in the following examples are all methods and steps known to those skilled in the art, or are operated according to the instructions of the commercially available products. The various experimental reagents used in the examples are all commercially available products, and the same products used in different processes or experimental steps are from the same source. Example

[0032] Some preferred implementations and aspects of the present invention are further described below in conjunction with specific examples, which should not be construed as limiting the scope thereof. Example 1: Construction of anti-PD1 scFv expression plasmids carrying different signal peptides

[0033] In order to improve the secretion level of anti-PD1 scFv (hereinafter referred to as aPD1 scFv), polynucleotide chains encoding different signal peptides were connected with polynucleotide chains encoding aPD1 scFv and cloned into pcDNA3.1(+) expression vector commonly used in mammalian cells to obtain the target gene expression plasmid. It was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The schematic diagram of the aPD1scFv expression frame in the constructed plasmid can be found in Figure 1 The aPD1 scFv was fused with a FLAG tag. In subsequent experiments, the expression level of the aPD1 scFv protein can be reflected by detecting the FLAG tag protein. This example detected the effects of different signal peptides on the secretion levels of two aPD1 scFvs, which were derived from Keytruda monoclonal antibody and WO2018 / 020476A1.

[0034] The amino acid sequence of the signal peptide involved in this example is as follows:

[0035] Artficial SP2:MGKLIFWLVFWLTIFWLGSA(SEQ ID NO:1)

[0036] secrecon:MWWRLWWLLLLLLLLWPMVWA(SEQ ID NO:2)

[0037] Gaussia luciferase:MGVKVLFALICIAVAEA(SEQ ID NO:3)

[0038] Human IgG V:MDWTWRFLFVVAAATGVQS(SEQ ID NO:4)

[0039] Human IgKVIII:MDMRVPAQLLGLLLLWLRGARC(SEQ ID NO:5)

[0040] The nucleotide sequence of the signal peptide involved in this example is as follows:

[0041] Artficial SP2:

[0042] ATGGGCAAGCTGATCTTTTGGCTGGTGTTCTGGCTGACCATCTTCTGGCTGGGCA GCGCC(SEQ IDNO:6)

[0043] secrecon:

[0044] ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTCCTGCTGCTGCTGTGGCCCATGGTGTGGGCC(SEQID NO:7)

[0045] Gaussia luciferase:

[0046] ATGGGCGTGAAGGTGCTGTTTGCCCTGATTTGCATCGCCGTGGCCGAGGCC(SEQ ID NO:8)

[0047] Human IgG V:

[0048] ATGGACTGGACCTGGCGGTTCCTGTTTGTGGTGGCCGCCGCCACCGGCGTGCAGAGC(SEQ ID NO:9)

[0049] Human IgK VIII:

[0050] ATGGACATGAGAGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGAGGGGAGCTAGATGT(SEQ ID NO:10)

[0051] The amino acid sequences of the two aPD1 scFvs are as follows:

[0052] aPD1 scFv-1 (derived from Keytruda monoclonal antibody), the full-length sequence is shown in SEQ ID NO: 37, wherein

[0053] Heavy chain variable region:

[0054] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS(SEQ ID NO:11)

[0055] Hinge Area:

[0056] GGGGSGGGGSGGGGS (SEQ ID NO: 12)

[0057] Light chain variable region:

[0058] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK(SEQ ID NO:13)

[0059] aPD1 scFv-2 (derived from WO2018 / 020476A1), the full-length sequence is shown in SEQ ID NO: 38, wherein

[0060] Heavy chain variable region:

[0061] EVQLVQSGPEVVKPGASVKVSCKASGYTFTSYYIHWVKQAPGKGLEWIGWIFPGDVSTQYNEKFQDKATITVDKSASTAYMQLSSLRSEDTAVYYCTREAYDYAVYWGQGTLVTVSS(SEQ ID NO:14)

[0062] Hinge Area:

[0063] GGGGSGGGGSGGGGS (SEQ ID NO: 15)

[0064] Light chain variable region:

[0065] DIQMTQAPSSLSASVGDRVTITCKASQNVDTNVAWFQQKPGKAPKSLIFSASYRYSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGGGTKLEIK(SEQ ID NO:16)

[0066] The nucleotide sequences of the two aPD1 scFvs are as follows:

[0067] aPD1 scFv-1 (derived from Keytruda monoclonal antibody)

[0068] Heavy chain variable region:

[0069] CAGGTACAACTGGTCCAGTCAGGCGTCGAGGTTAAAAAGCCCGGTGCCTCAGTG AAGGTCTCCTGCAAAGCCAGTGGTTACACCTTCACTAATTACTATATGTATTGGGTCAGACAGGCACCCGGCCAGGGTCTTGAATGGATGGGAGGCATCAATCCCAGCAATGGAGGGACAAACTTCAATGAAAAGTTCAAAAATCGCGTGACCCTCACAACCGACTCCTCCACAACAACAGCATATATGGAACTGAAGTCTCTTCAGTTTGATGACACCGCCGTCTACTACTGCGCCCGACGAGACTATCGGTTCGATATGGGATTCGACTATTGGGGTCAGGGGACGACAGTGACAGTCTCCTCC(SEQ ID NO:17)

[0070] Hinge region:

[0071] GGCGGTGGAGGTTCAGGTGGAGGAGGCTCAGGTGGAGGCGGCTCC(SEQ ID NO:18)

[0072] Light chain variable region:

[0073] GAGATTGTTCTGACCCAATCTCCTGCAACACTGAGCCTGTCCCCCGGAGAGAGG GCAACCCTCTCCTGCCGCGCCAGTAAGGGAGTGTCTACGTCTGGATATTCCTATCTCCATTGGTATCAGCAGAAGCCTGGCCAGGCGCCCAGGCTTCTCATCTACCTTGCTTCCTATCTGGAGTCCGGGGTGCCAGCCCGCTTTTCTGGGTCCGGCTCTGGTACCGACTTCACCCTGACAATTTCCTCACTGGAGCCTGAGGATTTCGCTGTCTACTATTGTCAACATTCCAGAGACCTTCCCCTGACCTTTGGAGGGGGTACAAAAGTGGAGATAAAA(SEQ ID NO:19)

[0074] aPD1 scFv-2:

[0075] Heavy chain variable region:

[0076] GAGGTGCAGCTGGTGCAGAGCGGCCCTGAGGTGGTGAAGCCTGGCGCCTCTGT GAAAGTGAGCTGTAAAGCCTCTGGCTATACCTTTACCTCTTACTACATTCACTGGGTGAAACAGGCCCCTGGCAAGGGCCTGGAGTGGATCGGCTGGATTTTTCCTGGCGATGTGTCTACCCAGTATAATGAAAAGTTTCAGGATAAGGCCACAATTACCGTGGATAAAAGCGCCTCTACCGCTTATATGCAGCTGTCAAGCCTGAGGTCTGAGGATACAGCCGTGTACTATTGTACAAGAGAGGCCTATGATTATGCCGTGTACTGGGGCCAGGGAACACTGGTGACAGTGTCTAGT(SEQ ID NO:20)

[0077] Hinge region:

[0078] GGCGGTGGAGGTTCAGGTGGAGGAGGCTCAGGTGGAGGCGGCTCC(SEQ ID NO:21)

[0079] Light chain variable region:

[0080] GACATCCAGATGACCCAGGCCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCG GGTGACCATCACCTGCAAGGCCTCCCAGAACGTGGACACCAACGTGGCCTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGAGCCTGATCTTCAGCGCCAGCTACAGATACAGCGGCGTGCCCTCCAGATTCAGCGGCTCCGGCAGCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAACTACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG(SEQ ID NO:22)

[0081] The amino acid sequence of the FLAG tag is: DYKDDDDK (SEQ ID NO: 23)

[0082] The nucleotide sequence of the FLAG tag is: GACTACAAGGACGACGACGACAAG (SEQ ID NO: 24) Example 2: Transfection of HEK293T cells with the target plasmid and detection of aPD1 scFv secretion level

[0083] 2.1 Transfection of target gene expression plasmid into HEK293T cell line

[0084] The human embryonic kidney cell line HEK-293T cell line (hereinafter referred to as 293T cells) was used for experimental verification. After 293T cells were digested into single cells with trypsin, they were inoculated in a 24-well plate, with 1.5×10 5 Each group of plasmids was transfected three times independently. When the confluence of 293T cells reached 70%, 400 ng of the target plasmid was transfected into each well using jetPRIME transfection reagent (Polyplus, #114-15). The transfection operation was performed according to the instructions of the reagent. After 12 hours, the culture medium was gently discarded and fresh culture medium was added to continue the culture. The aPD1 scFv expression plasmid without a signal peptide was used as a control.

[0085] 2.2ELISA detection of aPD1 scFv secretion level in 293T cell supernatant

[0086] After transfection for 60 hours, the supernatant was collected into a 1.5 mL EP tube and centrifuged to remove cell debris. The aPD1 scFv-FLAG protein level in the cell supernatant was detected using an ELISA kit (Jianglai Biotechnology, #JL52639). The operation steps were carried out according to the instructions of the kit. The results are shown in Figure 2 .in, Figure 2 A is a bar chart showing the level of aPD1 scFv-1-FLAG protein in the supernatant detected by ELISA. Compared with other types of signal peptides, the signal peptide from Human IgG V can significantly increase the secretion level of aPD1scFv-1 protein.

[0087] Figure 2 B is a bar chart of the aPD1 scFv-2-FLAG protein level in the supernatant detected by ELISA. It can be seen that the five signal peptides tested in the present invention can significantly enhance the secretion of aPD1 scFv-2 protein, with an increase ranging from 20 to 70 times. Compared with other types of signal peptides, the signal peptide from Human IgK VIII has the most significant effect on enhancing the secretion of aPD1 scFv-2 protein, which is enhanced by about 70 times.

[0088] In summary, different aPD1 scFvs are adapted to different signal peptides, and the same signal peptide has different effects on the expression, secretion or binding level of different aPD1 scFvs. Human IgG V can significantly increase the secretion level of aPD1 scFv-1 protein, and Human IgK VIII has the strongest effect on the secretion of aPD1 scFv-2 protein. Example 3: Detection of whether the signal peptide affects the binding of aPD1 scFv to PD-1 protein

[0089] The experimental design of this example is to use aPD1 scFv plasmids carrying different signal peptides to transiently transfect 293T cells expressing PD-1 (referred to as PD-1 293T cells), and then PD-1 293T cells express and secrete aPD1 scFv-FLAG. When aPD1 scFv-FLAG is secreted outside the cell and exists in the cell supernatant, it can bind to the PD-1 protein on the PD-1 293T cell membrane. At this time, the level of aPD1 scFv-FLAG on the PD-1 293T cell membrane can be detected by flow cytometry to reflect the binding of aPD1scFv-FLAG to PD-1.

[0090] 3.1 Construction of HEK293T cell line overexpressing PD-1 protein

[0091] In order to detect whether the aPD1 scFv secreted by the signal peptide can bind to the PD-1 protein normally and function normally, a HEK293T cell line overexpressing the PD-1 protein (referred to as PD-1 293T cells) was constructed using a lentivirus carrying PD1-GFP (purchased from Shanghai Heyuan Biotechnology).

[0092] 3.2 Transfection of target gene expression plasmid into HEK293T cell line overexpressing PD-1 protein

[0093] PD-1 293T cells were digested into single cells using trypsin and seeded in 24-well plates at a rate of 1.5 × 10 5 Each group of plasmids was transfected three times independently. When the confluence of 293T cells reached 70%, 250 ng of the target plasmid was transfected into each well using jetPRIME transfection reagent. The transfection operation was performed according to the instructions of the reagent. After 12 hours, the culture medium was gently discarded and fresh culture medium was added to continue the culture.

[0094] 3.3 Flow cytometry detection of aPD1 scFv binding on the surface of PD-1 293T cells

[0095] At 60h after transfection, PD-1 293T cells were digested with trypsin and collected into 1.5mL EP tubes. The cells were incubated with FLAG flow cytometry antibody (Biolegend, #637310) and then tested on the flow cytometer. The test results showed that after expression and secretion, aPD1 scFv carrying different signal peptides could bind normally to PD-1 protein on the cell surface, and there was no significant difference in binding ability. Example 4: Construction of anti-TNF-α scFv expression vectors carrying different signal peptides

[0096] 8 secretory signal peptides were connected to the N-terminus of anti-TNF-αscFv (hereinafter referred to as aTNF scFv). The nucleotide sequence encoding the above structure was inserted into the multi-cloning site of the miniphrneo-CMV-MCS-poly A expression vector disclosed in the prior art (see prior art CN113462721A), and 8 aTNF scFv expression plasmids guided by different signal peptides were constructed, and then transfected into human embryonic kidney cells (HEK-293T cells) to detect the expression and secretion of the target protein aTNF scFv. A FLAG tag was fused to the 3' end of aTNF scFv, and subsequent experiments reflected the expression level of aTNF scFv protein by detecting the FLAG tag.

[0097] The amino acid sequence of the signal peptide involved in this example is as follows:

[0098] Artficial SP2:MGKLIFWLVFWLTIFWLGSA(SEQ ID NO:1);

[0099] secrecon:MWWRLWWLLLLLLLLWPMVWA(SEQ ID NO:2);

[0100] Gaussia luciferase(Gluc):MGVKVLFALICIAVAEA(SEQ ID NO:3);

[0101] Mouse Ig Kappa:METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 25);

[0102] Human IgG V: MDWTWRFLFVVAAATGVQS (SEQ ID NO: 4);

[0103] Human IgKVIII:MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 5);

[0104] Ig heavy chain signal peptide 7(H7):MEFGLSWVFLVALFRGVQC(SEQ ID NO:26);

[0105] Igκ light chain signal peptide 1 (L1): MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO: 27).

[0106] The nucleotide sequences of some signal peptides involved in this example are as follows:

[0107] Mouse Ig Kappa:

[0108] ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCGAC(SEQID NO:28)

[0109] Ig heavy chain signal peptide 7(H7):

[0110] ATGGAGTTCGGCCTGTCCTGGGTGTTCCTGGTGGCCCTGTTCAGAGGCGTGCAGTGC (SEQ ID NO: 29)

[0111] Igκlight chain signal peptide 1(L1):

[0112] ATGGACATGAGAGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGAGCGGCGCCAGATGC (SEQ ID NO: 30)

[0113] The aTNF scFv (derived from Adalimumab monoclonal antibody) used in this example has a full-length sequence as shown in SEQ ID NO: 39, wherein

[0114] Heavy chain variable region:

[0115] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGT LVTVSS(SEQ ID NO:31)

[0116] Hinge Area:

[0117] GGGGSGGGGSGGGGS (SEQ ID NO:32)

[0118] Light chain variable region:

[0119] DIQMTQSPSSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK(SEQ ID NO:33)

[0120] The aTNF scFv nucleotide sequence used in this example is as follows:

[0121] Heavy chain variable region:

[0122] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCCGGCAGGTCCCT GAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAATGGGTCTCAGCTATCACTTGGAATAGTGGTCACATAGACTATGCGGACTCTGTGGAGGGCCGATTCACCATC TCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGATACGGCCGTATATTACTGTGCGAAAGTCTCGTACCTTAGCACCGCGTCCTCCCTTGACTATTGGGGCCAAGGTACCCTGGTCACCGTCTCGAGT(SEQ IDNO:34)

[0123] Hinge Area:

[0124] GGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCG (SEQ ID NO:35)

[0125] Light chain variable region:

[0126] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGACAGAG TCACCATCACTTGTCGGGCAAGTCAGGGCATCAGAAATTACTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTG GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTACAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGGTATAACCGTGCACCGTATACTTTTGGCCAGGGGACCAAGGTGGAAATCAAA(SEQ ID NO:36)

[0127] Conventional molecular biology techniques were used to construct the target gene expression vector, which was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing was correct, and aTNF scFv expression plasmids carrying 8 different signal peptides were obtained for subsequent experiments. Example 5: Transfection of HEK-293T cell line and detection of intracellular expression level of aTNF scFv

[0128] 5.1 Transfect the target plasmid into HEK-293T cell line

[0129] Human embryonic kidney cell line HEK-293T cell line (Wuhan Pronocell Life Science Co., Ltd., #CL-0005, hereinafter referred to as 293T cells) was cultured and grown to a confluence of about 80%. The culture medium (Gibco, #11965092) was discarded, and the cells were gently washed twice with DPBS. Then, the cells were digested with 0.05% trypsin (Gibco, #15050065) for 1-2 minutes. The digestion solution was discarded and the cells were resuspended with culture medium. 1×10 cells were inoculated into each well of a 12-well plate. 5 When the confluence of 293T cells reached 80%, 1 μg of the plasmid to be verified was transfected into each well using jetPRIME transfection reagent (Polyplus, #114-15), and the transfection operation was carried out according to the instructions; after 12 hours, fresh culture medium was replaced and culture was continued. The transfection experiment was repeated 3 times for each group of plasmids.

[0130] 5.2 Flow cytometry detection of intracellular expression level of aTNF scFv

[0131] 60h after transfection, the culture medium was discarded, 293T cells were digested with trypsin and collected into 1.5mL EP tubes with culture medium, washed once with DPBS, and stained with Zombie Red live and dead dye (BD biosciences, #423110) for 30 minutes in the dark. After washing once with DPBS, the cells were fixed and permeabilized according to the instructions of the membrane permeabilization kit (BD biosciences, #554714). After fixation and permeabilization, the cells were washed once, stained with FLAG flow cytometry antibody (Biolegend, #637310), washed twice with DPBS and then tested on the machine. The results are shown in Figure 3 shown.

[0132] like Figure 3 As shown in the results, aTNF scFv was expressed to a certain extent in 293T cells transfected with 8 signal peptides, and the effects of different types of signal peptides in guiding the intracellular expression of aTNF scFv were similar. However, according to the prior art, for secretory proteins, it is impossible to reflect the level of secretion based on their intracellular expression. Example 6: Detection of the secretion level of aTNF scFv directed by different signal peptides

[0133] According to the prior art, for secretory proteins, it is impossible to judge the secretion level based on their intracellular expression, and some proteins with low intracellular expression levels have high secretion levels, which is unpredictable. Therefore, it is necessary to detect the expression of aTNF scFv guided by different signal peptides in the culture supernatant to determine the optimal secretory signal peptide suitable for the expression of aTNF scFv.

[0134] 6.1 Transfection of target gene expression plasmid into HEK-293T cell line

[0135] The cultured 293T cells were digested with 0.05% trypsin and 1×10 5 Cells / well were inoculated in a 12-well plate; when the confluence of 293T cells reached 80%, 1 μg of the plasmid to be verified was transfected into each well using jetPRIME transfection reagent, and the transfection operation was carried out according to the instructions; after 12 hours, fresh culture medium was replaced and cultured. Each group of plasmids was independently repeated 3 times.

[0136] 6.2 Western blotting (WB) to detect the secretion level of aTNF scFv in the supernatant

[0137] 60 h after transfection, the culture medium was discarded and RIPA lysis buffer (Beyotime, P0013B) supplemented with protease inhibitors was added to the well plate. The cells were blown off and transferred to EP tubes. The cells were lysed and ultrasonically disrupted on ice. The total cell protein was collected after centrifugation. After the protein was quantified, 4×LDS sample buffer (Thermo Fisher Scientific, NP0007) was added and denatured at 99°C for 10 min.

[0138] The denatured proteins were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane, which was blocked with 5% skim milk powder. The membrane corresponding to the size of aTNF scFv protein was cut out and stained with primary antibodies (Cell Signaling Technology, 14793S) and secondary antibody (Jackson, 111-035-144), the membrane was scanned and photographed under a gel electrophoresis imaging analyzer, and the gray value of the protein band was analyzed using ImageJ software. Figure 4 shown.

[0139] from Figure 4 It can be seen that even though the expression levels of aTNF scFv carrying 8 different signal peptides in cells are similar, their secretion levels into the supernatant vary greatly. Among them, Gluc has a significant advantage in enhancing the secretion level of aTNF scFv, achieving an unexpected technical effect of enhancing the secretion of aTNF scFv.

[0140] All disclosures and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the present invention, multiple modifications and variants of the described method and composition of the present invention are apparent to those skilled in the art. Although the present invention has been described by specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, those multiple variants of the described mode for implementing the present invention that are apparent to those skilled in the relevant art are intended to be included in the scope of the appended claims.

Claims

1. A polypeptide, whose amino acid sequence comprises a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence is a signal peptide sequence and the second amino acid sequence is a single-chain antibody sequence.

2. The polypeptide according to claim 1, wherein the first amino acid sequence and the second amino acid sequence are selected from one of the following combinations (1)-(3): (1) the first amino acid sequence is as shown in SEQ ID NO:4 and the second amino acid sequence is as shown in SEQ ID NO: As shown in 37, (2) the first amino acid sequence is as shown in SEQ ID NO:5 and the second amino acid sequence is as shown in SEQ ID NO:38, and (3) The first amino acid sequence is shown as SEQ ID NO:3 and the second amino acid sequence is shown as SEQ ID NO:

39.

3. The polypeptide according to claim 2, wherein the first amino acid sequence is shown as SEQ ID NO:4 and the second amino acid sequence is shown as SEQ ID NO:

37.

4. The polypeptide according to claim 2, wherein the first amino acid sequence is shown as SEQ ID NO:5 and the second amino acid sequence is shown as SEQ ID NO:

38.

5. The polypeptide according to claim 2, wherein the first amino acid sequence is shown as SEQ ID NO:3 and the second amino acid sequence is shown as SEQ ID NO:

39.

6. The polypeptide according to any one of claims 1 to 5, wherein the first amino acid sequence and the second amino acid sequence are directly linked.

7. The polypeptide according to any one of claims 1 to 5, wherein a protease cleavage site exists between the first amino acid sequence and the second amino acid sequence.

8. The polypeptide of claim 7, wherein the protease cleavage site is a furin cleavage site.

9. A polynucleotide encoding the polypeptide according to any one of claims 1 to 8.

10. An expression vector comprising the polynucleotide according to claim 9.

11. A host cell comprising the polypeptide according to any one of claims 1 to 8, the polynucleotide according to claim 9 or the expression vector according to claim 10.

12. The host cell according to claim 11, wherein the host cell is a mammalian cell.

13. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 8, the polynucleotide according to claim 9, the expression vector according to claim 10 or the host cell according to claim 11.

14. A method for promoting secretion of a single-chain antibody from a host cell, comprising: Construct the polypeptide according to any one of claims 1-8, the polynucleotide according to claim 9, the expression vector according to claim 10 or the host cell according to claim 11.

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