SSR4 gene edited CHO cell line as well as preparation method and application thereof

Abstract

The invention discloses an SSR4 gene edited CHO (Chinese Hamster Ovary) cell line as well as a preparation method and application thereof. The preservation number of the cell line is CCTCC (China Center For Type Culture Collection) NO: C2025176. An endogenous strong promoter (CMV) and a resistance selection marker are carried by a lentiviral vector, stable high expression (greater than 5 times) of SSR4 in CHO-S cells is realized, the cell growth rate is not influenced, the processing efficiency of glycoprotein in endoplasmic reticulum is further optimized, the proportion of HM glycotypes is remarkably reduced, excessive intervention of a traditional method on end modification of a carbohydrate chain is avoided, and the method has a good application prospect. Finally, accurate control of antibody drug glycosylation modification and stability improvement among batches are achieved, scientific innovation and industrial practicability are achieved, and a technical foundation is laid for high-quality production of next-generation antibody drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a SSR4 gene edited CHO cell line, a preparation method and application thereof. BACKGROUND

[0002] Chinese hamster ovary (CHO) cells, as the "golden platform" in the field of biopharmaceuticals, play a core role in the production of antibody drugs. Its advantage lies in its ability to complete complex humanized protein post-translational modification, especially glycosylation modification. However, the heterogeneity of glycosylation modification (such as the fluctuation of the proportion of high mannose (HM) glycoforms) has been a key problem restricting the quality of antibody drugs. The abnormal enrichment of HM glycoforms (such as Man5-9GlcNAc2) can significantly reduce the binding affinity of the Fc segment of the antibody to the FcγRIIIa receptor on the surface of immune effector cells, resulting in a decrease in antibody-dependent cellular cytotoxicity (ADCC) effect, and thus affecting the therapeutic effect. At the same time, HM glycoforms are easily recognized by the mannose receptor (such as DC-SIGN) on the surface of antigen-presenting cells, accelerating the clearance rate of the antibody and shortening the half-life of the drug, ultimately threatening the safety and economy of clinical drug use.

[0003] Currently, the regulation strategies for HM glycoforms mainly focus on two types of pathways: one is to overexpress β-1,4-galactosyltransferase (β4GalT) or knock out α-1,6-fucosyltransferase (FUT8) through genetic engineering means to optimize core fucose or galactose modification, but such methods have limited direct regulation effect on HM glycoforms and may cause imbalance of other glycoforms; the other is to indirectly affect the glycosylation process by optimizing cell culture conditions (such as adding manganese ions, uridine or adjusting pH value), but there are large batch-to-batch differences, poor process stability, high production cost and other bottlenecks. In addition, existing technologies focus more on the regulation of end sugar chain modification, while ignoring the intervention of the initial glycosylation process in the endoplasmic reticulum, making it difficult to achieve fine control of the proportion of HM glycoforms.

[0004] In recent years, studies have shown that the protein transport mechanism between the endoplasmic reticulum and the Golgi apparatus has an important influence on the uniformity of glycosylation modification. Among them, SSR4 (Signal Sequence Receptor subunit 4) as a key component of the transport protein particle (TRAP) complex, is involved in the regulation of the directional transport of nascent glycoproteins and the initial step of glycosylation. The loss of function of SSR4 can lead to the retention of incompletely processed glycoproteins in the endoplasmic reticulum, thereby increasing the proportion of HM glycoforms. Based on this, targeted editing of SSR4 provides a potential new idea for regulating HM glycoforms. By precisely intervening the expression or function of the SSR4 gene, it is expected to reduce the formation of HM glycoforms from the source, thereby breaking through the limitations of traditional strategies and laying a foundation for the development of a CHO cell platform with high consistency and low heterogeneity. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a SSR4 gene edited CHO cell line and a preparation method and application thereof, which realizes stable high expression (more than 5 times) of SSR4 in CHO cells by carrying an endogenous strong promoter (CMV) and a resistance screening marker with a lentiviral vector, and does not affect the cell growth rate, thereby optimizing the glycoprotein processing efficiency in the endoplasmic reticulum, significantly reducing the proportion of HM glycosylation, avoiding excessive intervention on the end modification of sugar chains by traditional methods, and finally realizing precise control of glycosylation modification of antibody drugs and improving batch stability.

[0006] The present application is realized by the following technical solutions:

[0007] A SSR4 gene edited CHO cell line, the preservation number of the cell line is CCTCC NO: C2025176.

[0008] The preparation method of the above-mentioned SSR4 gene edited CHO cell line comprises the following steps:

[0009] Step 1) After the 293T cells and CHO-S cells are recovered, they are cultured in a culture medium for one week.

[0010] Step 2) Take the DH5a competent cells, heat shock transform the constructed plasmid, and perform plasmid extraction; the constructed plasmid is PCDH-CMV-Puro-T2A-SSR4.

[0011] Step 3) Use a transfection reagent to transfect the plasmid PCDH-CMV-Puro-T2A-SSR4, pREV, pRRE and pMD2.G into 293T cells, and culture for 24 hours.

[0012] Step 4) After 24 hours, replace the culture medium and continue to culture for 24 hours, collect the culture supernatant, filter, and obtain the lentivirus LV-SSR4.

[0013] Step 5) Add the LV-SSR4 obtained in step 4) to the CHO-S cells, replace the culture medium after 24 hours of culture, and continue to culture for 3 days.

[0014] Step 6) Replace the culture medium after 3 days and culture for one week.

[0015] Step 7) Screen the positive CHO-S / SSR4 cells, identify the screened positive cells, and obtain the CHO cell strain stably expressing SSR4 protein, namely the SSR4 gene edited CHO cell line.

[0016] Preferably, the culture medium in step 1) is DMEM+10%FBS; the transfection reagent in step 3) is PEI solution with a final concentration of 60 μg / mL; the culture medium in step 4) is DMEM+1%HEPES; the culture medium in step 6) is CHO-S+1 μg / mL puromycin.

[0017] The CHO cell line as described above, or the CHO cell line prepared by the preparation method as described above, is used in the preparation of an antibody drug.

[0018] Preferably, the drug reduces the proportion of high-mannose glycoforms by overexpressing the SSR4 gene, thereby enhancing antibody-dependent cellular cytotoxicity.

[0019] The CHO cell line as described above, or the CHO cell line prepared by the preparation method as described above, is used in the preparation of an anti-tumor drug.

[0020] The CHO cell line as described above, or the CHO cell line prepared by the preparation method as described above, is used in the preparation of a drug for regulating the modification level of B cell N-glycosylation.

[0021] Preferably, the drug reduces the proportion of high-mannose glycoforms by regulating the formation of the SSR4-DDOST complex, thereby enhancing antibody-dependent cellular cytotoxicity.

[0022] Preferably, the drug further comprises a pharmaceutically acceptable drug delivery carrier.

[0023] The beneficial effects of the present application are as follows:

[0024] (1) The present application first discloses and utilizes the interaction mechanism of the SSR4 gene through its E28 domain with the n-glycan transferase DDOST to directly regulate the processing efficiency of the oligosaccharide chain in the early stage of N-glycosylation. By stable overexpression of SSR4 (>5 times), the mannose trimming process is significantly accelerated, reducing the proportion of high-mannose (HM) glycoforms at the Asn297 site of the antibody to 0.59±0.12 times the wild type level, thereby reducing the accumulation of HM glycoform heterogeneity from the source. Unlike traditional strategies (such as overexpression of β4GalT or knockout of FUT8) which only focus on the end modification of the sugar chain, the present application targets the regulation of the initial stage of glycosylation in the endoplasmic reticulum, thereby achieving precise control of the proportion of HM glycoforms and avoiding the risk of glycoform imbalance, thereby providing a new solution for the glycosylation uniformity of antibody drugs.

[0025] (2) In the SSR4 gene edited CHO cell line of the present application, SSR4 overexpression can enhance antibody function. Firstly, the binding affinity of the antibody produced by the SSR4 edited CHO-S cell to FcγRIIIa receptor is significantly enhanced, and the ADCC activity is increased by 2.38 times (P<0.01); secondly, the complement-dependent cytotoxicity (CDC) activity is simultaneously improved by 1.32 times (P<0.01); thirdly, in the anti-tumor model in mice, the tumor inhibition rate of the antibody in the experimental group reaches 52.51%, which is nearly doubled (P=0.02) compared with the control group (27.58%), proving that SSR4 regulation can significantly enhance the therapeutic potential of the antibody.

[0026] (3) The present application uses a lentiviral vector to carry a CMV strong promoter and a resistance screening marker, realizes stable high expression of SSR4 in CHO-S cells (expression amount > 5 times), and the cell growth rate is not significantly affected (P>0.05), which guarantees the feasibility of large-scale production. Compared with the traditional medium component optimization strategy (such as manganese ion / uridine addition or pH adjustment), the present application directly intervenes in the glycosylation pathway through gene editing means, avoiding the problems of large batch difference, sensitive process parameters, etc., and significantly reducing the production cost and quality control difficulty.

[0027] (4) The SSR4 gene editing strategy of the present application is not only suitable for antibody drug production, but also can be extended to other glycosylation modification dependent biological drugs (such as fusion proteins, enzyme replacement therapy drugs). By reducing the proportion of HM glycoforms, the present application effectively prolongs the in vivo half-life of the antibody drug (reduces the clearance mediated by mannose receptors), while improving the efficacy and safety, providing an efficient and controllable cell platform for the development of antibody drugs in the fields of tumors, autoimmune diseases, etc., which has great clinical transformation significance. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 For the UMAP experimental results in Example 1: A is the total B cell of 7 colorectal cancer single cell data samples; B is the B cell subtype of 7 colorectal cancer single cell data samples; C is the pseudo-time sequence analysis result of the B cell subtype; D is the spatial expression difference of oligosaccharide transferase (OST);

[0029] Figure 2 For the overexpression efficiency of SSR4 of wild type CHO-S cells and CHO-S / SSR4 cells detected by WB (A) and flow cytometry (B) in Example 2;

[0030] Figure 3 For the Trypsin enzyme digestion TIC map of samples mAb01-C (A) and mAb01-CS (B) in Example 4;

[0031] Figure 4Figure 4: Glycoform profile of N299 site of mAb01-C (series 1) and mAb01-CS (series 2) after Trypsin digestion in Example 4;

[0032] Figure 5 Figure 5: TIC of mAb01-C (A) and mAb01-CS (B) after Chymotrypsin digestion in Example 4;

[0033] Figure 6 Figure 6: Glycoform profile of N299 site of mAb01-C (series 1) and mAb01-CS (series 2) after Chymotrypsin digestion in Example 4;

[0034] Figure 7 Figure 7: TIC of mAb01-C (A) and mAb01-CS (B) after Protease K digestion in Example 4;

[0035] Figure 8 Figure 8: Glycoform profile of N73 site of mAb01-C (series 1) and mAb01-CS (series 2) after Protease K digestion in Example 4;

[0036] Figure 9 Figure 9: Glycoform profile of N299 site of mAb01-C (series 1) and mAb01-CS (series 2) after Protease K digestion in Example 4

[0037] Figure 10 Figure 10: Comparison of N-glycosylation modification of glycosylation site (Asn297) of mAb01-C and mAb01-CS in Example 4;

[0038] Figure 11 Figure 11: Binding activity detection of anti-CLDN6 antibody in Example 5;

[0039] Figure 12 Figure 12: ADCC effect of anti-CLDN6 antibody in Example 5;

[0040] Figure 13 Figure 13: CDC effect of anti-CLDN6 antibody in Example 5;

[0041] Figure 14 Figure 14: In vivo experimental results of anti-CLDN6 antibody in Example 5: A is a linear relationship diagram of tumor volume change over time after treatment; B is an immunological analysis diagram of tumor after treatment;

[0042] Figure 15 Figure 15: Analysis of SSR4 and DDOST action sites in Example 6: A is the experimental result of molecular docking; B is the Co-IP experimental result. DETAILED DESCRIPTION

[0043] The application will be further described in details below with reference to the accompanying drawings and specific examples.

[0044] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art. The experimental methods not specified are conventional methods in the art.

[0045] Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0046] Example 1 Role of SSR4 gene in B cell subtypes

[0047] 1. Experimental procedures

[0048] (1) Data acquisition and preprocessing

[0049] In this example, single-cell RNA sequencing (scRNA-seq) raw data of 7 colorectal cancer (CRC) patients were obtained from the DISCO (deep Integrated human Single-Cell Omics data) database. The scRNA-seq data were analyzed using the read10x function in the Seurat software package. To ensure high data quality, cells with mitochondrial gene proportion > 25% were excluded (to exclude apoptotic / low-quality cells), and cells with gene expression between 200 and 8000 counts were retained (to exclude empty droplets or double cells). Subsequently, normalization was performed using the NormalizeData function (log-standardized gene expression matrix), and high-variation genes were screened using the FindVariableFeatures function (for downstream dimension reduction).

[0050] (2) Cell clustering and subtype annotation

[0051] Linear dimension reduction (standardization of expression matrix) was performed using the ScaleData function, and then principal component analysis (PCA) was performed using the RunPCA function. Significant principal components were selected for further analysis, and unsupervised uniform manifold approximation and projection (UMAP) dimension reduction visualization was performed based on the PCA results using the RunUMAP function. For differential expression analysis, the FindAllMarkers function was used to select significant markers according to specific thresholds, and differential expression genes of each cell cluster were identified, such as adj. P < 0.05.

[0052] (3) Pseudo-time trajectory analysis

[0053] In this embodiment, we use the Monocle3 package (v1.2.9) for pseudo-temporal analysis. First, we extract the gene expression matrix using the GetAssayData function in Seurat, perform dimensionality reduction using the preprocess_cds function, and correct the batch processing effect using the align_cds function. To further illustrate the temporal evolution of cell states, we construct cell developmental trajectories using the learn_graph function. Finally, we use the plot_cell_trajectory function to display the differentiation paths from GCB / memB to PB1 / PB2.

[0054] (4) Validation of key gene expression

[0055] The target gene in this embodiment is signal sequence receptor subunit 4 (SSR4). UMAP spatial mapping of gene expression was used to compare the distribution of SSR4 in B cell subtypes.

[0056] Throughout the analysis, we used R v4.2.0 and Seurat v4.4.0, and fully documented the parameters for each analytical step to ensure the reproducibility of the results.

[0057] 2. Experimental Results

[0058] (1) Identification of B cell subsets

[0059] To investigate the distribution of SSR4 in tumor-infiltrating B cells, we analyzed single-cell data from colorectal cancer patients obtained from the DISCO database, such as... Figure 1 As shown in Figure A. Cell clusters were visualized using UMAP, and B cell subtypes were annotated based on known marker genes, such as... Figure 1 As shown in Figure B, in 7 colorectal cancer samples, total B cells can be subdivided into three subtypes:

[0060] Germinal center B cells (GCB) (marker genes: RGS13, CD79A, STMN1);

[0061] Memory B cells (memB) (marker genes: TNFRSF13B, IGHM, HLA-DQB1);

[0062] Plasma cells (PB) (marker genes: MZB1, JCHAIN).

[0063] (2) B cell developmental trajectory

[0064] like Figure 1 As shown in Figure C, pseudo-time series analysis revealed that both GCB cells and memB cells developed into plasma cells (PB).

[0065] (3) Expression of glycosylation-related genes

[0066] To find the key regulatory molecules in the glycosylation process, we used UMAP to detect the expression differences of oligosaccharyltransferase (OST) family and TRAP family in B cell subtypes. As shown in FIG. 1C, the expression of OST family and TRAP family in B cell subtypes was different. For example, the expression of OST3 in plasma cells was significantly higher than that in other subtypes, suggesting that OST3 may be involved in the glycosylation regulation of plasma cells. As shown in FIG. 1D, the expression of SSR4 in plasma cells was significantly higher than that in other subtypes, suggesting that SSR4 may be involved in the glycosylation regulation of plasma cells, and also implying the potential role of SSR4 in the terminal differentiation of B cells and the process of antibody secretion. Figure 1

[0067] Therefore, in subsequent experiments, we selected CHO cells with high expression of SSR4 to construct cell lines.

[0068] Example 2 Construction of SSR4 gene edited CHO cell line (CHO-S overexpressing SSR4)

[0069] 1. Plasmid extraction

[0070] (1) Take DH5a competent cells and heat shock to transform the constructed plasmid.

[0071] (2) Take 100 mL of overnight culture (12-16 h) and add it to a clean 50 mL centrifuge tube labeled (centrifuge twice), check if the numbers are consistent, and centrifuge at 4500 rpm for 15 min to discard the supernatant.

[0072] (3) Add 10 mL of Rnase A suspension Buffer P1 to the centrifuge tube, and mix the bacterial precipitate thoroughly with a vortex mixer.

[0073] (4) Add 10 mL of Buffer P2 to the centrifuge tube (preheat to 37°C in a water bath if it is winter), immediately gently invert to mix. At this time, the bacterial solution changes from turbid to clear and viscous liquid (if it is not clear and viscous, report immediately and mark on the form).

[0074] (5) Add 10 mL of Buffer P3 to the centrifuge tube, immediately gently invert to mix, at this time white flocculent precipitate appears.

[0075] (6) Centrifuge at 4°C, 4500 rpm for 4 min, filter the supernatant with filter paper into a clean 50 mL centrifuge tube labeled, check if the numbers are consistent.

[0076] (7) Add 10 mL of ER Buffer to the collected filtrate and mix thoroughly.

[0077] (8) Column equilibration: during the centrifugation of the bacterial solution, place the empty column on the centrifuge tube rack and fill the empty column with QBT to equilibrate the filler.

[0078] ​(9) Loading: Load the supernatant after ER Buffer treatment into the equilibrated column under gravity.

[0079] (10) Fill the column with QC, and wash the column twice under gravity.

[0080] (11) Place the column into a clean 50 mL centrifuge tube with a label, and check if the number is consistent. Elute the plasmid with 10 mL Elution Buffer under gravity.

[0081] (12) Add 7 mL isopropanol to the collected filtrate, mix well, centrifuge at 4500 rpm for 15 min at 4°C, carefully pour off the supernatant, and place it upside down on a water absorption paper. Observe the position of the precipitate in time to prevent it from being poured off.

[0082] (13) Add 5 mL 75% ethanol to rinse the precipitate, and centrifuge at 3700 rpm for 10 min. Carefully pour off the supernatant, place it upside down on a water absorption paper, and observe the position of the precipitate in time to prevent it from being poured off. Finally, mark the position of the precipitate.

[0083] (14) Place the centrifuge tube in an ultra-clean bench for 10-20 min to allow the ethanol to evaporate. Add 250-400 μL deionized water to the centrifuge tube, and dissolve the precipitate by blowing with a pipette.

[0084] (15) Transfer the dissolved solution to a clean microfiltration column with a label, filter, and transfer it to a 1.5 mL EP tube. Shake well, and take a sample for concentration detection.

[0085] The constructed plasmid is named PCDH-CMV-Puro-T2A-SSR4, and the nucleotide sequence is shown in SEQ ID NO. 1. The concentration is 1000 ng / μL.

[0086] 2. Lentivirus packaging and infection

[0087] (1) After the 293T cells are recovered, they are cultured in DMEM+10% FBS medium for one week. Take 2.5 x 106 cells, and infect them with 1 mL lentivirus. 6 The cells are inoculated in a T25 culture flask, and cultured for 24 h.

[0088] (2) Prepare the transfection reagent: take 30 μL 1 mg / mL PEI, dilute it with 500 μL opti-MEM to a final concentration of 60 μg / mL. Mix well, and stand for 5 min.

[0089] (3) Prepare the plasmid solution according to the following Table 1.

[0090] Table 1 Plasmid solution preparation information

[0091]

[0092] (4) Slowly add PEI solution dropwise to the plasmid solution, mix thoroughly, and let stand for 10 minutes.

[0093] (5) Take 293T cells that have been cultured for 24 hours, replace them with fresh DMEM + 10% FBS medium, slowly and gently add 1 mL of plasmid-PEI mixture, and culture for 24 hours.

[0094] (6) The next day, the culture medium was changed to DMEM+1%HEPES and cultured for 24 hours.

[0095] (7) The next day, the culture supernatant was collected and filtered through a 0.45 μm filter membrane to obtain lentivirus LV-SSR4. The viral titer was estimated to be greater than 1 × 10⁻⁶ using a semi-quantitative test strip. 7 / mL.

[0096] (8) Take CHO-S cells that have been revived and cultured for one week, and adjust the density to 2×10⁶ cells / year. 5 / mL, take 2×10 5 Cells were seeded in one well of a 6-well plate.

[0097] (9) Take 1 mL of LV-SSR4, add a final concentration of 8 μg / mL Polybrene, mix well and add to the above-inoculated CHO-S cells. After culturing for 24 h, change the medium to CHO-S medium.

[0098] (10) After 3 days, the culture medium was changed to CHO-S + 1 μg / mL puromycin and cultured for one week to obtain the SSR4 gene-edited CHO cell line (CHO-S / SSR4 cells).

[0099] (11) Take 1×10 6 CHO-S / SSR4 cells were lysed to extract proteins, and the expression of the target protein was determined by Western blotting.

[0100] WB test results are as follows Figure 2 As shown in Figure A, compared to wild-type CHO-S cells, CHO-S / SSR4 cells exhibited overexpression of the target protein. The basal expression of SSR4 protein was almost nonexistent, indicating a significant overexpression effect. Furthermore, flow cytometry analysis showed that after overexpression, the SSR4 level in the CHO-S cell line increased from 2.1% in the wild-type to 96.1% (…). Figure 2 (B)

[0101] The CHO cell line (CHO-S / SSR4 cells) constructed in this embodiment is deposited at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on June 17, 2025, with accession number CCTCC NO: C2025176, and classified and named as Chinese Hamster Ovary Cells CHO-S / SSR4 (Chineses Hamster Ovary).

[0102] Example 3: Expression and purification of anti-CLDN6 antibody mAb01

[0103] 1. Plasmid extraction

[0104] (1) Take DH5α competent cells and heat shock transform them to construct plasmids.

[0105] (2) Take 100 mL of overnight culture (12-16 h) and add it to a clean 50 mL centrifuge tube (centrifuge twice), check whether the numbers are consistent, centrifuge at 4500 rpm for 15 min and discard the supernatant.

[0106] (3) Add 10 mL of Buffer P1 containing RNase A to the centrifuge tube and suspend the bacterial pellet completely using a vortex mixer.

[0107] (4) Add 10 mL of Buffer P2 to the centrifuge tube (preheat in a 37°C water bath in winter until no precipitate forms), and immediately gently invert to mix. At this point, the bacterial solution will change from turbid to clear and viscous (if it is not clear and viscous, but turbid or white, report it immediately and mark it on the form).

[0108] (5) Add 10 mL of Buffer P3 to the centrifuge tube and immediately and gently invert to mix. At this time, a white flocculent precipitate will appear.

[0109] (6) Centrifuge at 4℃ and 4500rpm for 4min. Filter the supernatant through filter paper into a clean 50mL centrifuge tube that has been labeled. Check that the numbers are consistent.

[0110] (7) Add 10 mL of ER Buffer to the collected filtrate and mix thoroughly by inverting the container.

[0111] (8) Column equilibration: During the centrifugation of collected bacterial culture, the empty column is placed on the centrifuge tube rack and filled with QBT equilibration packing.

[0112] (9) Loading: Pass the supernatant after processing with ER Buffer through a balanced column under gravity.

[0113] (10) Fill the column with QC and wash the column twice under gravity.

[0114] (11) Put the column into the clean 50mL centrifuge tube with label, check the number.

[0115] (12) Add 7mL isopropanol to the collected filtrate, mix well, centrifuge at 4500rpm for 15min at 4℃, carefully pour off the supernatant, and invert the tube on a paper towel, and observe the location of the precipitate to prevent it from being poured off.

[0116] (13) Add 5mL 75% ethanol to rinse the precipitate, centrifuge at 3700rpm for 10min, carefully pour off the supernatant, invert the tube on a paper towel, and observe the location of the precipitate to prevent it from being poured off, and finally mark the location of the precipitate.

[0117] (14) Place the centrifuge tube in an open state on the clean bench for 10-20min to allow the ethanol to evaporate completely. Add 250-400μL deionized water to the centrifuge tube, and use a pipette to blow and dissolve the precipitate.

[0118] (15) Transfer the dissolved solution to a clean microfiltration column with a label, filter bacteria, and then transfer to a 1.5mL EP tube, mix well, and then take a sample for concentration detection.

[0119] The constructed plasmid information is shown in Table 2 (Shanghai Jikai Gene).

[0120] Table 2 Plasmid information

[0121] Plasmid name Plasmid concentration (ng / μL) Heavy chain pCDNA3.4-mAb01V H ]]> 1718 Light chain pCDNA3.4-mAb01V L ]]> 1579

[0122] 2. CHO-S cell transient transfection

[0123] (1) Adjust the density of CHO-S cells to 4x10 6 / mL in 100mL CHO culture medium, and culture at 37℃, 8% CO2, and 110rpm for 24h.

[0124] (2) The next day, adjust the cell density to 6x10 6 / mL in 100mL CHO culture medium.

[0125] (3) Prepare the plasmid solution according to Table 3.

[0126] Table 3 Plasmid solution preparation information

[0127]

[0128]

[0129] (4) Prepare transfection reagent: Take 320 μL ExpiFectamine reagent, dilute in 3.7 mL optiPRO SFM. Mix well, and stand for 3 min.

[0130] (5) Mix the plasmid solution and transfection reagent, invert mix well, and stand for 4 min.

[0131] (6) Take the adjusted cells, slowly and gently add the plasmid-transfection reagent mixture, invert mix well, and culture at 37°C, 8% CO2, 110 rpm for 24 h.

[0132] (7) The next day, add 600 μL CHO Enhancer and 24 mL Feed, continue to culture for 5 days, and then perform antibody purification.

[0133] 3. Antibody purification

[0134] (1) Centrifuge the expression supernatant of CHO-S cells or the CHO-S / SSR4 cells prepared in Example 1.

[0135] (2) Protein A affinity chromatography purification:

[0136] Load the Protein A filler into the column.

[0137] Balance the chromatography column: 1x DPBS, flow rate 1 mL / min, 20 mL.

[0138] Load: flow rate 1 mL / min.

[0139] Rinse: 1x PBS, flow rate 1 mL / min, 20 mL.

[0140] Elution: 0.1M Gly solution (pH=3.0), 1 mL / min, 500 μL / tube. Collect a total of 10 tubes. Add 1M Tris-HCL (pH=8.5).

[0141] Adjust the pH to neutral.

[0142] (3) Ultrafiltration: transfer the collected effective eluent to an ultrafiltration tube, centrifuge at 12000 rpm, 4°C for 5 min, and discard the centrifugate.

[0143] (4) Add 10 times the volume of DPBS to the ultrafiltration tube, centrifuge at 12000 rpm, 4°C for 5 min, and discard the centrifugate.

[0144] (5) Repeat step (4) 3 times.

[0145] (6) Invert the ultrafiltration tube, centrifuge at 12000 rpm, 4°C for 3 min, and collect the antibody solution.

[0146] (7) Read the absorbance value at 280 nm using a NanoDrop instrument.

[0147] The antibody concentration was detected by NanoDrop, and the results are shown in Table 4 below.

[0148] Table 4 Antibody purification results

[0149]

[0150]

[0151] As can be seen from Table 4, the transfection does not affect the expression of the antibody.

[0152] Example 4 Chromatographic separation of protein samples and identification of glycosylation modification by high performance liquid chromatography-mass spectrometry (LC-MS)

[0153] 1. Sample preparation

[0154] (1) Take 10 μg of each of the two antibody samples mAb01-C and mAb01-CS prepared in Example 2, and transfer enzyme digestion buffer to make the volume 100 μL.

[0155] (2) Add DTT to a final concentration of 10 mM, and reduce at 56°C for 1 h;

[0156] Add IAM to a final concentration of 20 mM, and react at room temperature in the dark for 40 min;

[0157] Add DTT to a final concentration of 20 mM to neutralize the unreacted IAM.

[0158] (3) For the first set of samples, add 5 μL of Chymotrypsin enzyme (concentration 0.5 μg / μL), and incubate in a 30°C constant temperature incubator for 16 h;

[0159] For the second set of samples, add 5 μL of Trypsin enzyme (concentration 0.25 μg / μL), and incubate in a 37°C constant temperature incubator for 16 h;

[0160] For the third set of samples, add 2 μL of Proteinase K enzyme (concentration 1 μg / μL), and incubate in a 37°C constant temperature incubator for 8 h.

[0161] (4) After desalination by a C18 desalination column, dry by vacuum centrifugation, and redissolve the peptide fragments in 0.1% formic acid water before loading onto the machine.

[0162] 2. Equipment parameters

[0163] Chromatographic separation: mobile phase A is 0.1% formic acid in water; mobile phase B is 0.1% formic acid in 80% acetonitrile.

[0164] Liquid gradient setting: 0-1 min, 8%-12% B; 1-9 min, 12%-15% B; 9-31 min, 15%-25% B; 31-38 min, 25%-36% B; 38-45 min, 36%-60% B; 45-53 min, 60%-95% B; 53-60 min, 95% B; 60-70 min, 95%-8% B.

[0165] Flow rate: 0.3 μL / min.

[0166] Chromatographic column: 75 μm x 25 cm, NanoViper C18 2 μm, 100 A, column temperature 60 °C.

[0167] Mass spectrum acquisition: Full MS: Resolution (120000), AGC target (Standard), Maximum IT (20 ms), Number of scan ranges (1), Scan range Spectrum (300-1800 m / z), Data type (Profile); dd-MS2: Resolution (15000), AGC target (Standard), Maximum IT (22 ms), NCE stepped NCE (30).

[0168] 3. Software analysis

[0169] The sample glycosylation modification information was analyzed using BioPharma Finder 5.1 software.

[0170] Data processing parameters: Protease (Trypsin / Chymotrypsin / Protease K); Variable Modification (Deamidation, Oxidation, Glycosylation); Fixed Modification (Carbamidomethylation); Mass Accuracy (20 ppm).

[0171] 4. Analysis results

[0172] The sample mAb01-C and mAb01-CS were subjected to three kinds of enzyme digestion, and the mass spectrum data was analyzed, as follows:

[0173] (1) N-glycosylation modification - Trypsin digestion

[0174] The samples mAb01-C and mAb01-CS were subjected to Trypsin enzyme digestion, the TIC graph of the Trypsin enzyme digestion of the sample mAb01-C is shown in FIG. 2A, and the TIC graph of the Trypsin enzyme digestion of the sample mAb01-CS is shown in FIG. 2B. Figure 3 The TIC graph of the Trypsin enzyme digestion of the sample mAb01-C is shown in FIG. 2A, and the TIC graph of the Trypsin enzyme digestion of the sample mAb01-CS is shown in FIG. 2B. Figure 3 The mass spectrometry data analysis showed that the N73 and N299 sites were N-glycosylated, and the T301 site was O-glycosylated. The total glycosylation modification ratio of the N73 site of the samples mAb01-C and mAb01-CS was 100.00%, and one glycoform A1S1F was identified. The total glycosylation modification ratio of the N299 site of the samples mAb01-C and mAb01-CS was 100.00% and 99.48%, respectively, and multiple glycoforms were identified. The glycoform ratio distribution graph is shown in FIG. 3. Figure 4 The TIC graph of the Trypsin enzyme digestion of the sample mAb01-C is shown in FIG. 2A, and the TIC graph of the Trypsin enzyme digestion of the sample mAb01-CS is shown in FIG. 2B.

[0175] (2) N-glycosylation modification-Chymotrypsin enzyme digestion

[0176] The samples mAb01-C and mAb01-CS were subjected to Chymotrypsin enzyme digestion, the TIC graph of the Chymotrypsin enzyme digestion of the sample mAb01-C is shown in FIG. 4A, and the TIC graph of the Chymotrypsin enzyme digestion of the sample mAb01-CS is shown in FIG. 4B. Figure 5 The TIC graph of the Chymotrypsin enzyme digestion of the sample mAb01-C is shown in FIG. 4A, and the TIC graph of the Chymotrypsin enzyme digestion of the sample mAb01-CS is shown in FIG. 4B. Figure 5 The mass spectrometry data analysis showed that the N299 site was N-glycosylated. The total glycosylation modification ratio of the N299 site of the samples mAb01-C and mAb01-CS was 100.00%, and multiple glycoforms were identified. The glycoform ratio distribution graph is shown in FIG. 5. Figure 6

[0177] (3) N-glycosylation modification-Protease K enzyme digestion

[0178] The samples mAb01-C and mAb01-CS were subjected to Protease K enzyme digestion, the TIC graph of the Protease K enzyme digestion of the sample mAb01-C is shown in FIG. 6A, and the TIC graph of the Protease K enzyme digestion of the sample mAb01-CS is shown in FIG. 6B. Figure 7 The TIC graph of the Protease K enzyme digestion of the sample mAb01-C is shown in FIG. 6A, and the TIC graph of the Protease K enzyme digestion of the sample mAb01-CS is shown in FIG. 6B. Figure 7 The mass spectrometry data analysis showed that the N73 and N299 sites were N-glycosylated. The total glycosylation modification ratio of the N73 site of the samples mAb01-C and mAb01-CS was 100.00%, and multiple glycoforms were identified at the N73 site. The glycoform ratio distribution graph is shown in FIG. 7. Figure 8 ​As shown; for samples mAb01-C and mAb01-CS, the total glycosylation modification ratios at the N299 site were 93.99% and 95.94%, respectively, and multiple glycoforms were identified. The glycoform ratio distribution is shown in the figure. Figure 9 As shown.

[0179] (4) Statistical results

[0180] like Figure 10 As shown, SSR4 overexpression significantly reduced the high-mannose (HM, Man5-9) glycoform at Asn297 of mAb01-CS, which was 0.59 ± 0.12 times that of the control group (mAb01-C). * P < 0.05), while there was no significant difference in the levels of fucose (Fuc) and sialylation (Neu) (ns).

[0181] The experimental results of this embodiment show that high expression of SSR4 can significantly reduce the high mannose modification level of the antibody.

[0182] Example 5: Functional detection of anti-CLDN6 antibody mAb01

[0183] 1. Cellular level binding activity detection

[0184] Human CLDN6 overexpressing cell line (HEK293T-hCLDN6) was trypsinized, centrifuged to remove supernatant, and placed in FACS buffer (PBS + 2% FBS) for serial dilution of the test antibody (CLDN6 antibody). Cells were incubated at 4°C for 1 h, centrifuged to remove supernatant, washed twice, and then incubated at 4°C for 0.5 h with a second antibody. Cells were washed twice with FACS buffer and then resuspended. Flow cytometry (BD, #C6+) was used to measure and read the experimental cells. Data analysis was performed using GraphPad, with the x-axis representing the logarithm of antibody concentration and the y-axis representing the fold change in mean fluorescence intensity. The EC50 of the antibody was fitted to a curve.

[0185] like Figure 11 As shown, SSR4 overexpression does not alter the antibody affinity level.

[0186] 2. Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0187] Jurket-Luc cells and HEK293T-hCLDN6 cells were used as effector and target cells, respectively, and co-cultured with anti-hCLDN6 antibody (OE-SSR4, mAb01-CS) and anti-hCLDN6 control antibody (mAb01-C). The antibody-mediated killing effect was detected by fluorescence assay.

[0188] like Figure 12 As shown, SSR4 overexpression can significantly enhance the ADCC level of the antibody (** P < 0.01).

[0189] 3. Complement-dependent cytotoxicity (CDC) assay

[0190] Human serum and HEK293T-CLDN6 cells were used as effector and target cells, respectively. Anti-cldn6 antibody (mAb01-CS) and anti-cldn6 control antibody (mAb01-C) were added and co-cultured. CDC was detected by CCK8 assay.

[0191] like Figure 13 As shown, SSR4 overexpression can significantly enhance the antibody's CDC level ( ** P < 0.01).

[0192] 4. Results Analysis

[0193] In humanized pMC-reconstructed NSG (M-NSG) mice carrying Caco2-CLDN6 tumors, mAb01-CS showed better therapeutic efficacy, with a tumor growth inhibition rate of 52.51%, compared to 27.58% for mAb01-C (P = 0.02). Figure 14 (A). Immunological analysis of the tumor after treatment showed enhanced T cell infiltration in the mAb01-CS group. Figure 14 (B) indicates that SSR4-mediated glycosylation enhances the anti-tumor immune response.

[0194] Example 6: Alphafold2 predicts the interaction between human SSR4 and DDOST proteins.

[0195] SignalP-5.0 was used to predict the signal peptide, and the sequence other than the signal peptide was retained for structural prediction. Protonation was first performed under neutral conditions (pH=7) using the h++3 online server. Then, UCSFChimera software was used to remove heteroatoms and water molecules from the crystal structure, leaving only the protein structure, and Amber14SB charges were assigned. Molecular docking was then performed using the professional protein-protein docking tool HDOCK, and the empirical iterative scoring function ITScorePP was used for molecular docking and configuration scoring. Negative values ​​indicate molecular binding. The larger the absolute value, the stronger the binding ability. The maximum output configuration number for docking was set to 100, and the first 10 configurations were scored, with confidence scores used for confidence analysis. If the docking score is greater than 0.7, it indicates reliable docking and a high probability of molecular binding. The configuration with the first interconnection score and confidence score was selected from the interconnection configurations for subsequent analysis. Three-dimensional mapping analysis was performed using PyMOL 2.04, and two-dimensional interaction analysis was performed using Maestro, with statistical analysis of interaction type, distance, and number.

[0196] The three-dimensional structures of SSR4 and DDOST were predicted using Alphafold2. Molecular docking studies showed that their interactions mainly occurred in the extracellular domain ( Figure 15 The docking score was -266.39 kcal / mol, with a confidence level of 0.9217 (Table 6). A fully mutant plasmid predicting the binding site was constructed, and Co-IP experiments confirmed that the E28 site in SSR4 is a DDOST-specific binding site. Figure 15 (B)

[0197] Table 6 Extracellular domain binding and docking score

[0198]

[0199]

[0200] The molecular docking experiments in this embodiment predicted the target sites of SSR4 and DDOST, indicating that the CHO cell line (CHO-S / SSR4 cells) in Example 1 can enhance antibody-dependent cytotoxicity (ADCC) by regulating the formation of the SSR4-DDOST complex and reducing the glycoform ratio of high-mannose immunoglobulin (HM).

[0201] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A CHO cell line with SSR4 gene editing, characterized in that, The cell line has the accession number CCTCC NO: C2025176.

2. The method for preparing an SSR4 gene-edited CHO cell line according to claim 1, characterized in that, Includes the following steps: Step 1) After resuscitating 293T cells and CHO-S cells, culture them in culture medium for one week; Step 2) Take DH5α competent cells, transform them with heat shock to construct the plasmid, and extract the plasmid; the constructed plasmid is PCDH-CMV-Puro-T2A-SSR4; Step 3) Transfect plasmids PCDH-CMV-Puro-T2A-SSR4, pREV, pRRE, and pMD2.G into 293T cells using transfection reagents and culture for 24 hours; Step 4) After 24 hours, change the culture medium and continue culturing for another 24 hours. Collect the culture supernatant, filter it, and obtain lentivirus LV-SSR4. Step 5) Add the LV-SSR4 obtained in step 4) into CHO-S cells, culture for 24 hours, change the culture medium, and continue culturing for 3 days. Step 6) After 3 days, change the culture medium and incubate for one week; Step 7) Screen for transfected positive CHO-S / SSR4 cells, identify the screened positive cells, and obtain a CHO cell line that stably expresses SSR4 protein.

3. The method for preparing an SSR4 gene-edited CHO cell line according to claim 2, characterized in that, Step 1) The culture medium is DMEM + 10% FBS; Step 3) The transfection reagent is PEI solution with a final concentration of 60 μg / mL; Step 4) The culture medium is DMEM + 1% HEPES; Step 6) The culture medium is CHO-S + 1 μg / mL puromycin.

4. The use of the CHO cell line as described in claim 1, or the CHO cell line prepared by the method described in claim 2 or 3, in the preparation of antibody drugs.

5. The application according to claim 4, characterized in that, The drug enhances antibody-dependent cytotoxicity by overexpressing the SSR4 gene and reducing the proportion of high-mannose glycoforms.

6. The use of the CHO cell line as described in claim 1, or the CHO cell line prepared by the method described in claim 2 or 3, in the preparation of antitumor drugs.

7. The use of the CHO cell line as described in claim 1, or the CHO cell line prepared by the method described in claim 2 or 3, in the preparation of drugs that regulate the N-glycosylation modification level of B cells.

8. The application according to claim 7, characterized in that, The drug enhances antibody-dependent cytotoxicity by regulating the formation of the SSR4-DDOST complex and reducing the glycoform ratio of high-mannose immunoglobulins.

9. The application according to any one of claims 4-8, characterized in that, The drug also includes pharmaceutically acceptable drug delivery carriers.

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