CRM197 protein variant and application thereof

Abstract

The invention provides a CRM197 protein variant and application thereof. According to the variant, arginine at the 458th site is mutated into proline, proline at the 225th site is mutated into glutamic acid, and glycine at the 510th site is mutated into aspartic acid. The CRM197 protein variant has excellent stability, can stably exist in an environment of 37 DEG C / 50 DEG C and is free of precipitation and degradation loss, the immunogenicity of the CRM197 protein variant is remarkably improved compared with that of CRM197, antigen presenting cells with a higher proportion can be stimulated and activated, and stronger specific humoral immunity, cellular immunity and immune memory can be stimulated in vivo. In addition, the CRM197 protein variant has good anti-tumor activity, and can significantly inhibit the amplification of ovarian tumors. The invention also provides application of the CRM197 protein variant in preparation of diphtheria toxin vaccines, antigen carrier proteins and tumor treatment drugs.

Description

TECHNICAL FIELD

[0001] The application discloses a protein molecule with enhanced stability and immunogenicity, and relates to the fields of vaccines and tumor drugs. BACKGROUND

[0002] CRM197 is a non-toxic mutant of diphtheria toxin derived from the mutation of glycine at the 52nd position to glutamic acid. Compared with diphtheria toxin, the enzyme active site of CRM197 is changed, loses toxicity, but retains immunogenicity. CRM197 has two main applications at present. On the one hand, CRM197 can be used as a carrier protein for bacterial capsular polysaccharide antigens, so that the polysaccharide antigens are converted from non-T cell-dependent antigens to T cell-dependent antigens, and good immune response and immune memory can be generated after vaccination (Sun et al. , 2022). On the other hand, CRM197 can bind to the HB-EGF receptor, hinder mitosis, and inhibit cancer cell proliferation, and has an anticancer effect (Tang et al. , 2019). In addition, CRM197 has immunological cross-reaction with diphtheria toxin and can be used as a potential vaccine for diphtheria (Chai et al. , 2021).

[0003] CRM197 has a stability defect and cannot be stored for a long time in ordinary environments. It needs to be stored in the form of a lyophilized powder by adding trehalose, sucrose, mannitol and other lyophilizing agents, which increases the production cost. In addition, CRM197 is easily degraded in the enzyme environment in vivo, resulting in reduced immunogenicity. Unstable CRM197 is prone to structural disorder during expression and purification, forming unstable aggregates, resulting in low yield of the final CRM197.

[0004] The purpose of the present application is to provide a CRM197 mutant to solve the technical problem of insufficient stability of the prior art CRM197. SUMMARY

[0005] To achieve the above purpose, in a first aspect of the present application, a CRM197 protein mutant is provided, and the amino acid sequence of the CRM197 protein mutant is shown as SEQ ID NO. 1. In the present application, the CRM197 protein mutant is named CRM197-Mut3 (R458P, P225E, G510D), and the mutation sites thereof include: mutating arginine (R) at the 458th position of CRM197 to proline (P), mutating proline (P) at the 225th position to glutamic acid (E), and mutating glycine (G) at the 510th position to aspartic acid (D).

[0006] In a second aspect of the present application, a polynucleotide encoding the CRM197 protein variant is provided. Any polynucleotide sequence designed by one skilled in the art based on the triplet code rule and capable of being translated into the CRM197 protein variant as shown in SEQ ID NO. 1 is a polynucleotide encoding the CRM197 protein variant according to the present application.

[0007] In a preferred embodiment, the polynucleotide has a sequence as shown in SEQ ID NO. 2. The sequence of the polynucleotide provided in the present application is an optimized codon suitable for expression of soluble protein in prokaryotic cells designed by the inventors. Other optimized codons made based on common technical knowledge in the art are also suitable for the present application as long as the amino acid sequence as shown in SEQ ID NO. 1 is ensured.

[0008] In a third aspect of the present application, an expression vector containing the polynucleotide is provided. The expression vector is used for expression of the encoding gene of the CRM197 protein variant. In a specific embodiment of the present application, the expression vector is pET29a. Other vectors known to one skilled in the art, such as prokaryotic expression vectors or eukaryotic expression vectors, can also be used for cloning and expression of the encoding gene according to the present application.

[0009] In a third aspect of the present application, a host cell containing the expression vector is provided. The host cell is used for expression to obtain the CRM197 protein variant. The host can be E. coli, Corynebacterium diphtheriae, Bacillus subtilis, Pseudomonas fluorescens, Lactobacillus, yeast, mammalian cells, insect cells, etc. In a specific embodiment of the present application, the host cell is E. coli, which can be E. coli DH5a, E. coli BL21(DE3), E. coli BL21(DE3)pLysS, or E. coli Top10, etc.

[0010] In a fourth aspect of the present application, the CRM197 protein variant is provided for use in preparation of diphtheria toxin vaccine. After immunization of mice with the CRM197 protein variant provided in the present application, the IgG and IgG1 antibody titers produced by the mice are significantly improved compared with the wild type, and the IgG 2a antibody titers after secondary immunization are also significantly improved compared with the wild type. Therefore, the CRM197 protein variant provided in the present application has application prospect in preparation of diphtheria toxin vaccine.

[0011] In a fifth aspect of the present application, the CRM197 protein variant is provided for use as a carrier protein of an antigen. The carrier protein of the present application is a kind of substance that can enhance, prolong or modulate the specific immune response of the body to an antigen, and is usually used together with a vaccine to improve the effect of the vaccine. The results of the spleen lymphocyte restimulation experiment of the present application show that the CRM197 protein variant can stimulate the secretion of cytokines IL-6, TNF-α and IFN-γ, and increase the proliferation rate of lymphocytes. The flow cytometry analysis of spleen lymphocytes shows that the spleen follicular helper T cells (Tfh) and germinal center B cells (GC B) stimulated by the CRM197 protein variant are significantly increased, indicating that the CRM197 protein variant can better activate B cells Figure 8 ). The analysis of CD4 and CD8 memory T cell populations shows that the CRM197 protein variant can stimulate a higher proportion of central memory T cells (Tcm) and effector memory T cells (Tem), showing its ability to trigger stronger T cell immune memory. The above experimental results show the application prospect of the CRM197 protein variant as a carrier protein of a vaccine.

[0012] In a preferred embodiment, the application is the use of the CRM197 protein variant as a carrier protein of a microbial antigen or a tumor antigen. The application can stimulate the host to produce specific protective antibodies against the microbial antigen or the tumor antigen, so as to facilitate the effect of the body against the infection of the microorganism and the prevention or treatment of the tumor. In specific applications, the CRM197 protein variant can be loaded with various types of nanoparticles such as gold, carbon, silica nanoparticles, liposomes, chitosan, polyacrylic acid starch microparticles, etc., and one or more molecular immunoadjuvants such as macrophage colony-stimulating factor, interleukin 2, etc. to increase the immune stimulation effect. The CRM197 protein variant can be used alone, or can be loaded with tumor treatment drugs such as paclitaxel, cisplatin, gemcitabine, etc. or coupled with tumor antigens for the prevention and treatment of various cancers such as ovarian cancer, breast cancer, gastric cancer, lung cancer, oral cancer, neuroblastoma, triple-negative breast cancer, adrenocortical carcinoma, etc.

[0013] In a sixth aspect of the present application, the CRM197 protein variant is provided for use in the preparation of a tumor treatment drug. The CRM197 protein variant provided by the present application is administered intraperitoneally in mice, and the proliferation of ovarian cancer (A2780 cell line) is inhibited, and the tumor volume and weight of the administration group are significantly smaller than those of the PBS control group. Therefore, the CRM197 protein variant provided by the present application has the application prospect of preparing an anti-tumor drug.

[0014] In a seventh aspect of the present application, a preparation method of the CRM197 protein variant is provided, which comprises the following steps: (1) cloning the polynucleotide encoding the CRM197 protein variant into an expression vector; (2) transforming the expression vector obtained in step (1) into host cells, culturing and inducing the expression vector; (3) harvesting the culture solution of the host cells obtained in step (2), and / or harvesting the host cells and lysing the host cells to obtain a cell lysate; (4) obtaining the CRM197 protein variant from the culture solution of the host cells and / or the cell lysate obtained in step (3) through a purification step.

[0015] In a preferred embodiment, the polynucleotide in step (1) is further linked to a TrxA tag-encoding polynucleotide, the culture solution of the host cells is harvested in step (2), and the CRM197 protein variant is obtained from the culture solution of the host cells through a purification step in step (3).

[0016] In the application of the present application, other tags such as His tag, Etag tag, Flag tag, GST tag, MBP tag, etc. can also be used. A non-tagged protein expression scheme can also be used. The CRM197 protein variant provided by the present application can be expressed in a secreted form, a soluble form, or an inclusion body form.

[0017] The CRM197 protein variant provided by the present application improves the stability of the protein, significantly improves the immunogenicity, and can stimulate a higher proportion of antigen-presenting cells, thereby triggering stronger specific humoral immunity, cellular immunity and immune memory in vivo, showing its application prospect in preparing diphtheria toxin vaccine and as an antigen carrier protein. In addition, the CRM197 protein variant has higher anti-tumor activity and can inhibit tumor growth, showing its application prospect in preparing anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 . CRM197-OA / Mut3 protein expression identification; Figure 2 . CRM197-OA / Mut3 protein purification results; Figure 3 . CRM197-SD / OA / Mut3 stability analysis; Figure 4 . CRM197-SD / OA / Mut3 toxicity detection; Figure 5 . DC2.4 cell activation detection; Figure 6 . Antibody titer detection; Figure 7. Cytokine and lymphocyte proliferation rate detection; Figure 8 . Follicular helper T cell and germinal center B cell detection; Figure 9 . CD4 and CD8 memory T cell population analysis; Figure 10 . Mouse tumor volume and weight measurement. DETAILED DESCRIPTION

[0019] The present application will be further described below in connection with specific embodiments, and its advantages and features will become more apparent from the description. However, these embodiments are only exemplary and do not constitute any limitation on the protection scope defined by the claims of the present application.

[0020] Example 1. Preparation and identification of CRM197-Mut3 CRM197-Mut3 is a CRM197 variant designed by the present application, which specifically includes three mutation positions: arginine (R) at position 458 is mutated to proline (P), proline (P) at position 225 is mutated to glutamic acid (E), and glycine (G) at position 510 is mutated to aspartic acid (D), which is specifically represented as CRM197-Mut3 (R458P, P225E, G510D), and the complete amino acid sequence is shown in SEQ ID NO. 1.

[0021] 1. Preparation of CRM197-Mut3 CRM197-Mut3 is loaded into pET29a vector (provided by Zhongmeihe Biological Co., Ltd.) and codons are optimized, the optimized codon sequence is shown in SEQ ID NO. 2, and a solubility-promoting tag TrxA (SEQ ID NO. 3) is introduced for co-expression in E. coli BL21 (DE3).

[0022] Expression conditions: 1:100 inoculation, 37°C, 220 rpm culture for 16 h, then 1:100 inoculation into new LB culture medium, 37°C, 220 rpm culture until the bacterial solution reaches OD600 0.6-0.8 (about 2.5 h), then add 2 mM IPTG, 28°C, 220 rpm induction for 4 h.

[0023] 2. Identification of CRM197-Mut3 (1) SDS-PAGE and WB identification The expressed bacterial liquid was collected, centrifuged at 12000 x g for 20 minutes, and the bacterial precipitate was collected. Then, 1 ml of PBS was added to resuspend the bacterial precipitate, and the resuspended bacterial liquid was transferred to a new 1.5 ml centrifuge tube. The resuspended bacterial liquid was placed on ice for ultrasonic crushing, 60 Hz, ultrasonic for 5 seconds, stop for 5 seconds, a total of 10 minutes. The crushed bacterial supernatant was collected by centrifugation at 12000 x g for 10 minutes, and the crushed bacterial precipitate was resuspended with 1 ml of PBS. The whole bacterial liquid, the crushed bacterial supernatant, and the crushed bacterial precipitate after ultrasonic crushing were subjected to SDS-PAGE and WB identification.

[0024] Take the prepared CRM197-OA (to distinguish from the commercially available CRM197, the CRM197 expressed under the same conditions as CRM197-Mut3 is named CRM197-OA), whole bacterial liquid, crushed bacterial supernatant, and crushed bacterial precipitate of CRM197-Mut3, each 15 μl, add 5 μl of 4x protein loading buffer (Solebo, Cat #P1015) and mix well. Take two pieces of SDS-PAGE precast gel (GenScript, Cat M00668), one for Coomassie blue staining and one for WB identification. Load 10 μl of each gel, pour into the electrophoresis liquid (GenScript, Cat M00138), set the voltage to 160 V, and electrophorese for 40 minutes. After electrophoresis, one gel was placed in an eStain LG protein staining instrument (GenScript) for Coomassie blue staining and decolorization, and then the gel was placed in a gel chemiluminescence imager (VIBER, Fusion FX EDGE) for imaging. The other gel was placed in an eBlot™ L1 rapid wet transfer instrument (GenScript) for membrane transfer. After membrane transfer, the membrane was taken out and placed in an eZwest™ automatic protein blotting system (GenScript). 10 ml of 5% skimmed milk powder blocking solution (BD, REF 232100), 10 ml of 1 / 2000 diluted Anti-Diphtheria Toxin antibody (Abeam, ab151222), and 10 ml of 1 / 10000 diluted goat anti-rabbit IgG H&L (HRP) (Abeam, ab205718) were added to the eZwest™ automatic protein blotting system. The system program settings are shown in Table 1: Table 1. Automatic protein blotting system program settings

[0025] After running, the membrane was taken out, WB substrate developer (Millipore, Cat: WBKLS0100) was added for development, and the membrane was placed in a gel chemiluminescence imager for imaging and photographing.

[0026] CRM197-OA, CRM197-Mut3 were first purified by nickel column, then further purified by anion column. The purification conditions are as follows: Nickel column purification (Ni FF, Cytiva); Equilibration buffer (A buffer): 20 mM PB, 20 mM imidazole, pH 8.0; Elution buffer (B buffer): 20 mM PB, 400 mM imidazole, pH 8.0; 4 CV (column volume) of equilibration buffer was used to equilibrate the column, and then the sample was loaded. After the end of loading, 4 CV of equilibration buffer was used to equilibrate the column again, and finally 100% B buffer was used to elute and collect the protein.

[0027] Anion column purification (Q Sepharose Fast Flow, Cytiva); Equilibration buffer (A buffer): 20 mM PB, pH 8.0; Elution buffer (B buffer): 20 mM PB, 0.5 M NaCl, pH 8.0; 4 CV of equilibration buffer was used to equilibrate the column, and then the sample was loaded. After the end of loading, 4 CV of equilibration buffer was used to equilibrate the column again, and 35% B buffer was used to elute and collect the protein.

[0028] The experimental results show that CRM197-OA and CRM197-Mut3 are expressed in soluble form in E. coli Figure 1 . After nickel column and anion column purification, pure CRM197-OA and CRM197-Mut3 can be obtained Figure 2 .

[0029] (2) Stability evaluation CRM197-SD (commercial CRM197), CRM197-OA, and CRM197-Mut3 were diluted to 0.1 mg / ml, and then each group of proteins was divided into 4 tubes, 200 μl / tube, and placed in 4℃, 25℃, 37℃, and 50℃ environments for 48 hours. Whether the proteins precipitated or not was observed, and SDS-PAGE was used to identify whether the proteins were degraded and lost.

[0030] Compared with commercial CRM197 (CRM197-SD, expressed by diphtheria) and CRM197 (CRM197-OA) expressed under the same conditions, CRM197-Mut3 can exist stably at 37℃ / 50℃ environment without degradation, and the protein does not precipitate. CRM197-SD and CRM197-OA appear protein degradation and aggregation precipitation after 48h at 37℃, and the protein is almost completely lost at 50℃ Figure 3 .

[0031] (3) Toxicity analysis VeroE-6 cells were diluted to a concentration of 1 x 10 5 cells / ml and inoculated into a 96-well plate at 100 μl / well. After 24 hours of incubation at 37°C in a 5% CO2incubator, 2.34 μg / ml-600 μg / ml of CRM197-SD, CRM197-OA, CRM197-Mut3 and diphtheria toxoid (DTd) were added to the culture wells in sequence at 100 μl / well. The cells were incubated at 37°C in a 5% CO2incubator for another 48 hours, and three replicate wells were set for each group. After 48 hours of incubation, CCK-8 solution was added to the culture wells at 10 μl / well, and the cells were incubated at 37°C for 2 hours. After the incubation, the plates were placed in an enzyme labeler, and the absorbance values of each culture well at 450 nm and 630 nm were measured. According to the absorbance values OD 450-630 , the cell survival rate was calculated. Cell survival rate = (absorbance value of sample well - average of blank well) / (average of control well - average of blank well) x 100%. According to the cell survival rate, the IC 50 values (half-inhibitory concentration) of CRM197-SD, CRM197-OA and CRM197-Mut3 were calculated using Graphad prism 9 software.

[0032] Toxicity evaluation showed that CRM197-Mut3 was safe and had an IC 50 value of 13.33 μg / ml for VeroE-6 cells. The IC 50 values of CRM197-SD and CRM197-OA were 10.82 μg / ml and 15.71 μg / ml, respectively, which were comparable to that of CRM197-Mut3. CRM197-Mut3 did not have a virulence recovery, and its toxicity was weaker than that of diphtheria toxoid. Figure 4

[0033] Example 2. Immunostimulatory effect of CRM197-Mut3 on DC2.4 cells DC2.4 cells (mouse dendritic cells) were inoculated into a 24-well plate at a density of 5 x 10 5 cells / well (1 ml of culture system) and incubated at 37°C in a 5% CO2incubator for 24 hours. CRM197-SD, CRM197-OA, CRM197-Mut3, LPS (lipopolysaccharide) and PBS were added to the culture wells to stimulate the cells, and the final concentration of each drug was 20 μg / ml. Three replicate wells were set for each group. After 48 hours of incubation at 37°C in a 5% CO2incubator, the stimulated DC2.4 cells in each group were collected by trypsin digestion, washed with PBS twice, counted, and 2 x 10 6 ​The cells were placed in 500 μl centrifuge tubes, CD16 / 32 blocking solution (Biolegend, 101301) was added for blocking for 20 minutes, washed with PBS for 2 times, and PE-CD11b (Biolegend, 101207), APC-CD86 (Biolegend, 105012), APC-CD80 (Biolegend, 104714), APC-MHC I (Biolegend, 116518), and APC-MHC II (Biolegend, 107614) antibodies were added, and incubated in the dark for 30 minutes. After washing with PBS for 2 times, the cells were screened through a mesh, and flow cytometry detection was performed.

[0034] CRM197-SD, CRM197-OA, and CRM197-Mut3 stimulated DC2.4 cells (mouse dendritic cells) in vitro, respectively. The results showed that the CD11b + MHC Ⅱ + The proportion of the cell population was 10.3%, 10.6%, and 19.7%, respectively. The CD11b + MHC Ⅱ + The proportion of the cell population was significantly increased compared with the CRM197-SD and CRM197-OA groups. The CD11b + CD86 + The proportion of the cell population was 10.7%, 11.6%, and 21.2%, respectively. The CD11b + MHCⅡ + / CD86 + The proportion of the cell population was significantly higher than that of the CRM197-SD and CRM197-OA groups, indicating that CRM197-Mut3 could activate more antigen-presenting cells and exhibit higher immunogenicity Figure 5 .

[0035] Example 3. Immune stimulation effect of CRM197-Mut3 on mice CRM197-SD, CRM197-OA, CRM197-Mut3, and PBS, with Al(OH)3 adjuvant, were subcutaneously inoculated into 4-week-old NIH female mice at a dose of 5 μg / mouse, with 8 mice per group. Three immunizations were performed, with the first immunization designated as day 0. Immunizations were repeated on days 0, 14, and 28. Blood was collected from the eyeglass frame on days 14, 28, and 42, and serum was isolated for ELISA to determine antibody titers after each immunization. On day 48, spleens of four mice in each group were randomly isolated, and splenic lymphocytes were extracted for drug-induced splenic lymphocyte restimulation. Lymphocyte proliferation and cytokine secretion after restimulation were measured. On the 72nd day, the remaining 4 mice in each group were killed, the spleens were separated, and splenic lymphocytes were extracted. The proportion of follicular helper T cells (Tfh), germinal center B cells (GC B), and CD4 and CD8 memory T cell populations in the spleen were analyzed by flow cytometry.

[0036] ELISA antibody titer determination: CRM197-SD, CRM197-OA, and CRM197-Mut3 were diluted to 2 μg / ml using ELISA coating buffer (0.05 mol / L carbonate buffer, pH 9.6). 100 μl / well of the diluted antibodies were added to a 96-well plate and incubated overnight at 4°C. The plates were washed three times with 200 μl / well of PBST. Blocked the plates were then added with 100 μl / well of 1% BSA and incubated at 37°C for 1 hour. The plates were washed with PBST and serum was diluted. 200 μl / well of the diluted mouse serum was added to the first column of the 96-well plate, followed by a two-fold serial dilution. The plates were incubated at 37°C for 1 hour. The plate was washed with PBST and then HRP--anti-mouse-IgG (Abcam, ab97265), HRP--anti-mouse-IgG1 (Abcam, ab97240), and HRP--anti-mouse-IgG were added at a dilution of 1 / 50000. 2a Antibody (Abcam, ab97245) was added at 100 μl / well and incubated at 37°C for 1 hour. The plate was washed with PBST and TMB colorimetric solution (Solybol, PR1200) was added at 100 μl / well to the 96-well plate. After incubation at room temperature in the dark for 10 minutes, ELISA stop solution (2 M H2SO4) was added at 50 μl / well to the 96-well plate. The OD values ​​were read on a microplate reader. 450-630 The absorbance value is positive if it is greater than 2.1 times the absorbance value of the negative serum control well. The maximum serum dilution factor corresponding to the positive well is the antibody titer, which is expressed as a logarithm with base 10.

[0037] The antibody titers in the mice were detected after immunizing the mice with CRM197-SD, CRM197-OA, and CRM197-Mut3. The IgG antibody titers of the CRM197-Mut3 group were 5.2 times, 4 times (first immunization), 3.2 times, 1.4 times (second immunization), and 3.1 times, 2 times (third immunization) of those of the CRM197-SD and CRM197-OA groups, respectively. The IgG1 antibody titers of the CRM197-Mut3 group were 3.4 times, 3.2 times (first immunization), 2.6 times, 2.6 times (second immunization), and 2.5 times, 1.5 times (third immunization) of those of the CRM197-SD and CRM197-OA groups, respectively. The IgG2a antibody titers of the CRM197-Mut3 group were 1.2 times, 1.7 times (first immunization), 4.1 times, 5.2 times (second immunization), and 2.6 times, 2.4 times (third immunization) of those of the CRM197-SD and CRM197-OA groups, respectively. The antibody levels of the three groups of the CRM197-Mut3 group were significantly higher than those of the CRM197-SD and CRM197-OA groups, and the immunogenicity was stronger. 2a Figure 6

[0038] Example 4. Immunization effect of CRM197-Mut3 on spleen lymphocyte restimulation 1. Isolation and culture of mouse spleen lymphocytes The mouse spleen lymphocyte isolation kit (Solebo, Cat: P8860) was used to isolate the spleen lymphocytes according to the operation instruction of the kit. The spleen lymphocytes were inoculated into a 24-well plate at a concentration of 2×10 5 After 12 h of culture at 37°C in a 5% CO2 incubator, the spleen lymphocytes were stimulated with CRM197-SD, CRM197-OA, CRM197-Mut3, and PBS at a final concentration of 20 μg / ml in accordance with the corresponding groups, and the culture medium was collected after 24 h and 48 h of culture at 37°C in a 5% CO2 incubator for detection of cytokines.

[0039] 2. Spleen lymphocyte restimulation proliferation experiment The experiment was divided into two batches, and the cell proliferation was detected after 24 h and 48 h of drug stimulation. The spleen lymphocytes were inoculated into a 96-well plate at a concentration of 5×10 4 ​​The amount of cells / well was inoculated into a 96-well plate, and after 12 h of culture at 37°C in a 5% CO2 incubator, the corresponding groups were sequentially added with CRM197-SD, CRM197-OA, CRM197-Mut3, and PBS at a final concentration of 20 μg / ml to stimulate lymphocytes, and after 24 h and 48 h of culture in the incubator, CCK-8 solution was added to the culture wells at 10 μl / well, and incubated for 2 h in a 37°C incubator. After incubation, the culture plate was placed in a microplate reader, and the absorbance values of each culture well at 450 nm and 630 nm were measured, and the absorbance value OD 450-630 The cell survival rate was calculated. Cell survival rate = (sample well absorbance value - blank well mean) / (control well mean - blank well mean) x 100%.

[0040] The results of the CRM197-SD, CRM197-OA, and CRM197-Mut3 spleen lymphocyte restimulation experiment showed that after 24 h of stimulation, the secretion amounts of IL-6 in the CRM197-SD, CRM197-OA, and CRM197-Mut3 groups were 19.4 pg / ml, 22.43 pg / ml, and 37.6 pg / ml, respectively, the secretion amounts of TNF-α were 10.53 pg / ml, 18.62 pg / ml, and 23.9 pg / ml, respectively, and the secretion amounts of IFN-γ were 1.94 pg / ml, 6.92 pg / ml, and 13.56 pg / m, respectively. After 48 h of stimulation, the secretion amounts of IL-6 in the CRM197-SD, CRM197-OA, and CRM197-Mut3 groups were 36.05 pg / ml, 40.8 pg / ml, and 63.05 pg / ml, respectively, the secretion amounts of TNF-α were 16.45 pg / ml, 20.55 pg / ml, and 24.2 pg / ml, respectively, and the secretion amounts of IFN-γ were 7.06 pg / ml, 8.71 pg / ml, and 30.33 pg / m, respectively. The secretion amounts of the cytokines IL-6, TNF-α, and IFN-γ in the CRM197-Mut3 group were significantly higher than those in the CRM197-SD and CRM197-OA groups Figure 7 ).

[0041] After 24 h of stimulation, the lymphocyte proliferation rates in the CRM197-SD, CRM197-OA, and CRM197-Mut3 groups were 4.61%, 19.0%, and 40.15%, respectively. After 48 h of stimulation, the lymphocyte proliferation rates in the CRM197-SD, CRM197-OA, and CRM197-Mut3 groups were 23.67%, 45.72%, and 59.87%, respectively. The lymphocyte proliferation rate in the CRM197-Mut3 group was significantly higher than that in the CRM197-SD and CRM197-OA groups Figure 7 ).

[0042] 3. Flow cytometry analysis of lymphocyte populations: Antibodies used in flow cytometry are shown in Table 2.

[0043] Table 2. List of antibodies used in flow cytometry analysis of lymphocyte populations

[0044] 1. Dilute the above antibodies according to the antibody instructions, and mix with True-Stain Monocyte Blocker (Biolegend, 426102) and Brilliant Stain Buffer (BD, 563794) to form a staining antibody mixture;

[0045] 2. Take 2 x 10 6 cells into a flow tube, centrifuge at 400 x g for 5 min, discard the supernatant, and resuspend the cells with 200 μl DPBS (containing 1% FBS); 3. Add 2 μl of Fc receptor blocker anti-mouse CD16 / 32 (BD, 553141), mix well, and incubate at room temperature for 10 min in the dark; 4. Add the premixed staining antibody mixture, mix well, and incubate at room temperature for 25 min in the dark, centrifuge at 400 x g for 5 min, and discard the supernatant; 5. Add 2 ml of DPBS (containing 1% FBS) to wash the cells, centrifuge at 400 x g for 5 min, and discard the supernatant; 6. Add cell viability dye Zombie NIR™ Fixable Viability Kit (Biolegend, 423105), mix well, and incubate at room temperature for 10 min; 7. Add 2 ml of DPBS (containing 1% FBS) to wash the cells, centrifuge at 400 x g for 5 min, and discard the supernatant, and wash twice; 8. Resuspend the cells with 0.2 ml of DPBS (containing 1% FBS), and use the Aurora spectral flow cytometer (Cytek) to obtain the sample; 9. Use FlowJo_v10.8.1 software for data analysis Spleen lymphocyte flow analysis showed that the proportion of follicular helper T cells (Tfh) in the spleen of CRM197-SD, CRM197-OA, and CRM197-Mut3 groups was 9.01%, 9.33%, and 11.48%, respectively. The proportion of germinal center B cells (GCB) in the spleen of CRM197-SD, CRM197-OA, and CRM197-Mut3 groups was 0.99%, 0.96%, and 1.46%, respectively. The proportion of Tfh cells and GCB cells in the CRM197-Mut3 group was significantly increased compared with the CRM197-SD and CRM197-OA groups, indicating that Mut3 can better activate B cells Figure 8 ).

[0046] CD4 and CD8 memory T cell population analysis showed that the proportion of CD4 central memory T cells (CD4 + Tcm) in the CRM197-SD, CRM197-OA, and CRM197-Mut3 groups was 8.83%, 8.73%, and 14.7%, respectively. The proportion of CD4 effector memory T cells (CD4 + Tem) was 21.08%, 21.85%, and 26.25%, respectively. The proportion of CD8 central memory T cells (CD8 + Tcm) was 19.43%, 20.78, and 28.15%, respectively. The proportion of CD8 effector memory T cells (CD8 + Tem) was 12.33%, 12.5%, and 17.58%, respectively. The CRM197-Mut3 group had a higher proportion of central memory T cells (Tcm) and effector memory T cells (Tem), and CRM197-Mut3 could stimulate stronger T cell immune memory Figure 9 ).

[0047] Example 5. Evaluation of the proliferation inhibition effect of CRM197-Mut3 on ovarian cancer A2780 ovarian cancer cells were subcutaneously inoculated into BALB / c Nude mice at 1×10 6 cells / mouse. When the tumor grew to about 100 mm 3 , drug administration was started. CRM197-SD, CRM197-OA, and CRM197-Mut3 were intraperitoneally injected at 1 mg / mouse / week, and continuous administration was performed for 4 weeks. The long diameter and short diameter of the tumor were measured every week, the tumor volume was calculated, and the tumor was removed and weighed at the 4th week, with 4 mice in each group.

[0048] After 4 weeks of continuous administration, the tumor volume of the PBS, CRM197-SD, CRM197-OA, and CRM197-Mut3 groups was 1301.63 mm 3 , 703.3 mm 3, 348.29 mm 3 , 270.87 mm 3 , the tumor weights were 0.67 g, 0.48 g, 0.32 g, 0.29 g, respectively. The tumor volume and weight of the CRM197-Mut3 group were significantly lower than those of the PBS control group, and were better than those of the CRM197-SD and CRM197-OA groups, indicating that CRM197-Mut3 had good anti-tumor activity and could inhibit tumor proliferation Figure 10 .

Claims

1. A CRM197 protein variant, characterized in that, The amino acid sequence of the CRM197 protein variant is shown as SEQ ID NO.

1.

2. A polynucleotide encoding the CRM197 protein variant of claim 1.

3. The polynucleotide of claim 2, wherein, The sequence of the polynucleotide is shown as SEQ ID NO.

2.

4. An expression vector containing the polynucleotide of claim 2 or 3.

5. A host cell containing the expression vector of claim 4.

6. Use of the CRM197 protein variant of claim 1 in the preparation of a diphtheria toxin vaccine.

7. Use of the CRM197 protein variant of claim 1 as a carrier protein of an antigen.

8. Use of the CRM197 protein variant of claim 1 in the preparation of a tumor treatment drug.

9. The method for preparing the CRM197 protein variant according to claim 1, characterized in that: The method comprises the following steps: (1) cloning the polynucleotide of claim 2 into an expression vector; (2) transforming the expression vector obtained in step (1) into a host cell culture and inducing the expression vector; (3) harvesting the culture solution of the host cell obtained in step (2), and / or harvesting the host cell and lysing the host cell to obtain a cell lysate; (4) obtaining the CRM197 protein variant from the culture solution of the host cell and / or the cell lysate obtained in step (3) through a purification step.

10. The method of claim 9, wherein, In step (1), the polynucleotide is further connected with a TrxA tag encoding polynucleotide, in step (2), the culture solution of the host cell is harvested, and in step (3), the CRM197 protein variant is obtained from the culture solution of the host cell through a purification step.

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