Zeb2-targeting shRNA (short hairpin ribonucleic acid), recombinant vector, cell and application in preparation of medicine for enhancing anti-tumor immune response

Abstract

The invention discloses shRNA (short hairpin Ribonucleic Acid) targeting Zeb2, a recombinant vector, a cell and application of the shRNA in preparation of drugs for enhancing anti-tumor immune response, and belongs to the technical field of research and development of anti-tumor drugs. Experiments prove that the shRNA targeting Zeb2 can reduce the proportion of T cells (Tex, PD1 + Tim3 +), increase the proportion of depleted precursor T cells (Tpex, Ly108 + Tim3-), improve the secretion ability of Granzyme B and TNF-alpha cytokines of CD8 + T cells in tumor tissues, significantly inhibit the tumor growth of skin melanoma mice, increase the infiltration quantity of CD8 + T lymphocytes in the tumor tissues and the anti-tumor ability of the CD8 + T cells, and can be used for preparing anti-tumor drugs for treating skin melanoma. The polypeptide has the potential of being used for preparing drugs for enhancing anti-tumor immune response.

Description

shRNAs targeting Zeb2, recombinant vectors, cells, and their application in the preparation of drugs that enhance antitumor immune responses. Technical Field

[0001] This invention belongs to the field of anti-tumor drug development technology, specifically involving shRNA targeting Zeb2, recombinant vectors, cells, and their application in the preparation of drugs that enhance anti-tumor immune responses. Background Technology

[0002] CD8 + T cells, also known as cytotoxic T cells, are a core functional component of adaptive immunity. They can specifically recognize and kill abnormal target cells such as virus-infected cells and tumor cells, playing a crucial role in anti-tumor immunity. However, multiple inhibitory factors in the tumor microenvironment often lead to dysfunction or exhaustion of these cells. Therefore, genetic engineering techniques are used to target CD8 cells... + Modifying T cells to enhance their cytotoxic activity and persistence has become an important strategy for improving anti-tumor immune efficacy, providing a new direction for the innovative development of clinical tumor immunotherapy.

[0003] The Zeb2 gene (Zinc Finger E-box Binding Homeobox 2), also known as zinc finger homeobox gene 1B (ZFHX1B) or Smad interaction protein 1 (SIP1), is a key transcriptional regulator located on human chromosome 2. Its genomic structure contains 10 exons, encoding a protein belonging to the Zfh1 family, containing 1214 amino acids, and possessing zinc finger and homeo domains. It regulates downstream gene expression by binding to E-box sequences on DNA. As a nuclear transcriptional repressor, Zeb2 plays a central role in embryonic development, cell differentiation, and immune regulation through interaction with activated SMAD proteins. However, there are currently no reports of Zeb2 being associated with tumor immune responses. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide shRNA, recombinant vector, cells, and their application in the preparation of drugs that enhance antitumor immune responses targeting Zeb2.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In the first aspect of the present invention, a shRNA targeting Zeb2 is disclosed, wherein the nucleotide sequence of the shRNA targeting Zeb2 has at least 95% sequence identity with the sequence shown in SEQ ID NO.1.

[0006] Preferably, the nucleotide sequence of the shRNA targeting Zeb2 is shown in SEQ ID NO.1.

[0007] A second aspect of the present invention discloses a recombinant vector containing the nucleotide sequence of the shRNA targeting Zeb2 described above.

[0008] A third aspect of the invention discloses CD8 for silencing Zeb2. + T cells were cultured and CD8 cells were isolated after inoculating a TCR-specific OT1 mouse lymphocyte suspension with the aforementioned retroviral solution targeting Zeb2 shRNA. + T cells are obtained.

[0009] Preferably, the method for preparing the retroviral solution of shRNA targeting Zeb2 is as follows: shRNA targeting Zeb2 and PCL-ECO retroviral vector are co-transfected into 293T cells at a molar ratio of 3:1, the medium is replaced with DMEM containing 10% fetal bovine serum, the cells are cultured for a longer period, the supernatant is collected and filtered to obtain the retroviral solution of shRNA targeting Zeb2.

[0010] A fourth aspect of the present invention discloses shRNA targeting Zeb2, recombinant vectors, or CD8 silencing Zeb2. + Application of T cells in the preparation of drugs that enhance anti-tumor immune responses.

[0011] Preferably, the tumor is a skin melanoma, lung cancer, breast cancer, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, hepatocellular carcinoma, or ovarian cancer.

[0012] A fifth aspect of the present invention discloses a kit for inhibiting Zeb2 gene expression, comprising the aforementioned shRNA targeting Zeb2, a recombinant vector, or CD8 silencing Zeb2. + T cells.

[0013] A sixth aspect of the present invention discloses a drug for enhancing anti-tumor immune responses, comprising the above-mentioned shRNA targeting Zeb2, a recombinant vector, or CD8 silencing Zeb2. + T cells, and pharmaceutically acceptable excipients.

[0014] Preferably, the pharmaceutically acceptable excipient is a buffer or stabilizer.

[0015] More preferably, the buffer is PBS or physiological saline, and the stabilizer is glucose, trehalose, sucrose, glycerol, mannitol, or ascorbic acid.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The shRNA targeting Zeb2 provided by the present invention can reduce T cells (Tex, PD1) + Tim3 +The proportion of ) increases the number of exhausted precursor T cells (Tpex, Ly108) + Tim3 - The proportion of CD8 in tumor tissue is increased. + The ability of T cells to secrete Granzyme B and TNF-α cytokines was significantly enhanced, inhibiting tumor growth in mice with cutaneous melanoma and increasing CD8 levels in tumor tissue. + The number of T lymphocytes infiltrating and CD8 + The anti-tumor capacity of T cells. Therefore, this shRNA targeting Zeb2 has the potential to be used in the preparation of drugs that enhance anti-tumor immune responses. Attached Figure Description

[0017] Figure 1 shows the shRNA-Zeb2 plasmid constructed in this invention; Figure 2 shows the RT-qPCR verification of shRNA-Zeb2 activating OT-1 in vitro. + CD8 + Figure 3 shows the knockout efficiency results in T cells; where, ** * P < 0.001; Figure 3 shows the CD8 knockout efficiency results induced by shRNA-Zeb2 in this invention. + Figure 4 shows the results of enhanced T-cell anti-tumor growth capacity; where A is a schematic diagram of tumor model construction, B is the size of melanoma bodies, from left to right on days 10, 11, 12, 13, 14, and 15, and C is the tumor growth curve. ***P < 0.001, ****P < 0.0001; Figure 4 shows the comparison of tumor quality and CD8 between the Mock group and the shRNA-Zeb2 knockdown group of this invention. + The results of the differences in the number of T lymphocyte infiltrations are shown in the figure; where A is the comparison of tumor quality, and B is the comparison of CD8. + Comparison of T lymphocyte infiltration numbers, **P<0.01, ****P<0.0001; Figure 5 shows terminally exhausted T cells (TIM-3) in tumor-infiltrating lymphocytes of the present invention. + PD-1 + ) and exhausted precursor T cells (TIM-3) - Ly108 + The flow cytometry results of ) are shown; where A, terminally exhausted T cells (TIM-3) + PD-1 + A) Flow cytometry diagram; B) Statistical results of graphs A and B, P < 0.0001; C) Exhausted precursor T cells (TIM-3) - Ly108 +Figure 6 shows the flow cytometry results of cytokines GZMB and TNF secreted by tumor-infiltrating lymphocytes (TILs) in the Mock group and shRNA-Zeb2 group of this invention; where A is a flow cytometry diagram of GZMB; B is the statistical result of Figure A; C is a flow cytometry diagram of TNF-α; D is the statistical result of Figure C, and ***P < 0.001. Detailed Implementation

[0018] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0019] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0020] In this article, shRNA targeting Zeb2 and CD8 silencing Zeb2 were discussed. + T cells can be used to prepare drugs that enhance anti-tumor immune responses, wherein the tumors include, but are not limited to, skin melanoma, lung cancer, breast cancer, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, hepatocellular carcinoma, or ovarian cancer.

[0021] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0022] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0023] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0024] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0025] I. Construction of shRNA-Zeb2 plasmid. As shown in Figure 1, shRNA (shRNA-Zeb2, nucleotide sequence shown in SEQ ID NO.1 in Table 1, synthesized by Beijing Qingke Biotechnology Co., Ltd.) targeting the mouse Zeb2 gene (NM_001289521.2) was designed. Primer pairs (Zeb2 upstream primer and Zeb2 downstream primer, nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 in Table 1) for amplification of this fragment were synthesized simultaneously. PCR amplification was performed using the synthesized shRNA-Zeb2 as a template. The amplification system is shown in Table 2. The reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min, to obtain the target shRNA fragment.

[0026] Table 1 Sequence List

[0027] Table 2 PCR amplification system

[0028] The MSCV retroviral expression vector (carrying the GFP reporter gene) was double-digested at the corresponding restriction sites using restriction endonucleases XhoⅠ and EcoRⅠ. The digestion system contained 1 μg of MSCV retroviral expression vector, 1 μL each of XhoⅠ and EcoRⅠ, 2 μL of 10×CutSmart Buffer, and ddH2O was added to bring the volume to 20 μL. The mixture was incubated at 37℃ for 2 h to linearize the vector.

[0029] The digested vector and the shDNA fragment amplified by PCR and recovered by agarose gel were mixed at a vector:shDNA fragment molar ratio of 1:3. 1 μL of T4 DNA ligase and 1×T4 DNA ligase buffer were added, and ligation was performed overnight at 16°C. The ligation product was transformed into E. coli DH5α competent cells and plated on LB solid medium containing 100 μg / mL ampicillin. The cells were cultured at 37°C for 14 h. Single colonies were picked and inoculated into LB liquid medium containing 100 μg / mL ampicillin. The cells were cultured at 37°C with shaking at 220 rpm for 12 h. The plasmid was extracted and the size of the insert fragment was verified by double digestion with XhoⅠ / EcoRⅠ. Simultaneously, sequencing confirmed that the sequence was completely identical to SEQ ID NO.1. The successfully constructed shRNA-Zeb2 recombinant plasmid was obtained (Figure 1).

[0030] II. Knock down Zeb2's OT-1+ CD8 + Identification of the reduction efficiency of the Zeb2 gene in T cells: 1. Preparation of retroviral solution. The shRNA-Zeb2 recombinant plasmid obtained in step one and the PCL-ECO retroviral vector were co-transfected into 293T cells at a molar ratio of 3:1. Six hours after transfection, the medium was replaced with DMEM containing 10% fetal bovine serum, and cultured for another 60 hours. The supernatant was collected and filtered through a 0.45 μm filter membrane to obtain a retroviral solution containing shRNA-Zeb2. At the same time, the MSCV retroviral expression vector was packaged using the same method to obtain the Mock group retroviral solution. It was stored at -80℃ for later use.

[0031] 2. Identification of Zeb2 gene reduction efficiency. Peripheral lymph nodes and mesenteric lymph nodes were isolated from 8-12 week old TCR-specific OT1 mice (purchased from Jackson Laboratory). The cells were placed in pre-chilled PBS containing 2% fetal bovine serum, ground, filtered through a 70 μm cell sieve, and the filtrate was collected to obtain a lymphocyte suspension. After removing red blood cells with erythrocyte lysis buffer, the cells were resuspended in RPMI 1640 medium and the cell concentration was adjusted to 1×10⁻⁶. 6Cells / mL. The treated lymphocytes were seeded into 6-well plates coated with 5 μg / mL anti-CD3 antibody and 2 μg / mL anti-CD28 antibody, and cultured overnight at 37℃ in a 5% CO2 incubator after TCR stimulation. The next day, cells were seeded into 24-well plates. In the experimental group (shRNA-Zeb2), 1 mL of 8 μg / mL shRNA-Zeb2 retroviral solution was added to each well; in the control group (WT-Control), 1 mL of 8 μg / mL Mock group retroviral solution was added to each well. Infection was performed by centrifugation at 2500 rpm and 32℃ for 90 min. After infection, RPMI 1640 complete medium containing 30 U / mL IL-2 was added to each well, and the cells were cultured for another 48 h. After culture, cells were collected, resuspended in PBS containing 2% FBS, and screened for GFP-positive cells using flow cytometry. After sorting, the GFP positivity rate was verified to be ≥90%, and OT1 CD8 cells transfected with the Mock vector were obtained. + T cells and OT1 CD8 cells transfected via shRNA Zeb2 vector + T cells were used for subsequent adoptive transfer experiments. Total RNA was extracted from each sorted cell, reverse transcribed into cDNA, and the expression level of Zeb2 mRNA was detected by RT-qPCR to verify the silencing efficiency of shRNA-Zeb2.

[0032] The results are shown in Figure 2. It can be seen that the expression of Zeb2 in the experimental group was significantly lower than that in the control group, indicating that the shRNA-Zeb2 sequence was successfully designed and the RNA interference effect was good.

[0033] 3. Lower Zeb2's CD8 + T-cell inhibition of cutaneous melanoma growth 1. Construction of a melanoma mouse model. As shown in Figure 3A, 25 healthy female C57BL / 6 mice aged 6-8 weeks were acclimatized for 3 days, and then each mouse was subcutaneously injected with 5×10⁻⁶ T cells on the right back. 5 B16-OVA skin melanoma cells in logarithmic growth phase (cell suspension volume 100 μL, resuspended in PBS). After inoculation, the mental state and tumor growth of mice were observed daily. On day 9, all tumor-bearing mice were subjected to whole-body X-ray irradiation with a dose of 3 Gy (irradiation conditions: voltage 100 kV, current 5 mA, irradiation distance 30 cm, dose rate 1 Gy / min). The mice were then fed for another 10 days to stabilize the melanoma model, thus obtaining the melanoma model mouse.

[0034] 2. Melanoma model mice were randomly divided into a blank control group (No ACT), a control group (Mock), and an experimental group (shZeb2), with at least 6 mice in each group. Mice in the blank control group received no treatment, while each mouse in the control group underwent adoptive transfer via the tail vein at a dose of 1×10⁻⁶. 5 OT1 CD8 transfected via a Mock carrier + T cells were adopted by tail vein transfer to each melanoma model mouse in the experimental group, with 1 × 10⁶ cells per mouse. 5 OT1 CD8 transfected with shRNA Zeb2 vector + T cells (cell suspension volume was 100 μL, resuspended in PBS containing 2% fetal bovine serum).

[0035] 3. Monitor the survival status of mice daily after adoptive transfer. Measure the longest diameter (L) and shortest diameter (W) of the tumor daily using calipers. Calculate V using the formula V = (L × W) 2 Tumor volume was calculated by dividing the tumor volume by 2, and the mice were monitored continuously for 15 days. The tumor growth rate and final tumor size were compared between the two groups of mice to assess the effect of CD8 regulation by shRNA Zeb2. + The inhibitory effect of T cells on the growth of cutaneous melanoma was demonstrated. Mice were sacrificed after the experiment, and tumor tissue was dissected, weighed, and recorded. A portion of the tumor tissue was then minced and digested with collagenase IV (final concentration 1 mg / mL) at 37°C for 30 min. After filtration through a 70 μm cell sieve, a single-cell suspension was obtained. Fluorescently labeled anti-CD45, anti-CD3, and anti-CD8 antibodies were added and incubated at 4°C in the dark for 30 min. After washing, the CD8 concentration in the tumor tissue was detected by flow cytometry. + T lymphocytes account for 45% of CD45 + The proportion of immune cells to assess CD8 + Number of T-cell infiltrations.

[0036] The results are shown in Figures 3 and 4. Compared to the Mock group, the OT1 CD8 transfected with the shRNA Zeb2 vector was adopted. + Mice using T cells showed stronger inhibitory effects on tumor growth, with slower tumor growth and smaller tumor size (Figure 3). The tumor mass in the experimental group was significantly smaller than that in the Mock group (Figure 4A), and CD8+ was also more abundant in the tumor tissue. + The number of infiltrating T lymphocytes was significantly increased (Figure 4B). These results indicate that Zeb2 knockdown can effectively increase CD8... + The anti-tumor ability of T cells.

[0037] IV. Zeb2 knockdown leads to CD8 + To investigate the enhanced anti-tumor growth capacity of T cells, and to study the effect of Zeb2 knockdown on CD8... +To investigate the reason for the enhanced anti-tumor growth ability of T cells, the following experiment was conducted: Mouse tumor tissue obtained in step three (Mock transfection and shRNA Zeb2 transfection) was taken and aseptically minced into 1-2 mm pieces. 3 Small pieces of the tumor tissue were added to RPMI 1640 medium containing collagenase IV (final concentration 1 mg / mL) and DNase I (final concentration 50 μg / mL) and digested in a constant temperature shaker at 37°C, 5% CO2, and 150 rpm for 30 min. After digestion, the cells were filtered through a 70 μm cell sieve to obtain a single-cell suspension of tumor tissue. The cell pellet was collected by centrifugation at 1500 rpm for 5 min, resuspended and washed twice with PBS containing 2% fetal bovine serum (FBS), and after verifying cell viability ≥90% by trypan blue staining, the cell concentration was adjusted to 1×10⁶ cells / mL. 6 cells / 100 μL; divide the single-cell suspension into two tubes, one for TILs CD8. + T cell differentiation assay: A mixture of fluorescently labeled antibodies (anti-CD45-APC, anti-CD3-FITC, anti-CD8-PE, anti-PD1-PE-Cy7, anti-Tim3-BV421, and anti-Ly108-Alexa Fluor 647) was added to each tube, with a final concentration of 1 μg / mL for each antibody, and incubated at 4°C in the dark for 30 min. Another tube was used for cytokine secretion assay: PMA (final concentration 50 ng / mL), iomycin (final concentration 1 μg / mL), and GolgiPlug (final concentration 1 μL / mL) were added first, and the tube was stimulated at 37°C in a 5% CO2 incubator for 4 h. Then, anti-CD45-APC, anti-CD3-FITC, and anti-CD8-PE antibodies were added, and the tube was incubated at 4°C in the dark for 30 min. After fixation with a membrane-permeability agent at room temperature for 20 min, and washing twice with permeation buffer, anti-Granzyme B-BV421 and anti-TNF-α-PE-Cy7 antibodies (final concentrations of 1 μg / mL and 1 μL / mL respectively) were added. μg / mL) was incubated at 4℃ in the dark for 30 min; after incubation, both tubes of samples were washed twice with PBS containing 2% FBS, resuspended in 300 μL of PBS containing 2% FBS, and detected by flow cytometry and depleted precursor T cells (Tpex, Ly108). + Tim3 - The proportion of ) and CD3 + CD8 + The proportion of Granzyme B and TNF-α positive cells in T cells was measured. The experiment was repeated three times, and the data were statistically analyzed using FlowJo software.

[0038] The results are shown in Figures 5 and 6. The shRNAZeb2 group showed exhausted T cells (Tex, PD1) + Tim3 +The proportion of ) decreased (A and B in Figure 5), while the proportion of exhausted precursor T cells (Tpex, Ly108) decreased. + Tim3 - The proportion of CD8+ was increased (C and D in Figure 5). These results indicate that Zeb2 may be a key factor promoting the differentiation of T cells from a reversible precursor state to a terminal exhaustion state. shRNA levels in Zeb2-treated tumor tissues showed increased CD8+ levels. + The secretion of Granzyme B and TNF-α cytokines by T cells was significantly increased (Figure 6), indicating that knockdown of Zeb2 increased CD8 levels in tumor tissue. + The ability of T cells to secrete cytokines.

[0039] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A shRNA targeting Zeb2, characterized in that, The nucleotide sequence of the shRNA targeting Zeb2 has at least 95% sequence identity with the sequence shown in SEQ ID NO.

1.

2. The shRNA targeting Zeb2 according to claim 1, characterized in that, The nucleotide sequence of the shRNA targeting Zeb2 is shown in SEQ ID NO.

1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleotide sequence of the shRNA targeting Zeb2 as described in claim 1 or 2.

4. Silence Zeb2's CD8 + T cells, characterized by... CD8 cells were isolated by inoculating a TCR-specific OT1 mouse lymphocyte suspension with a retroviral solution targeting Zeb2 shRNA as described in claim 1 or 2, followed by culturing and isolation. + T cells are obtained.

5. The CD8 of silencing Zeb2 according to claim 4 + T cells, characterized by... The method for preparing the retroviral solution of shRNA targeting Zeb2 is as follows: shRNA targeting Zeb2 and PCL-ECO retroviral vector are co-transfected into 293T cells at a molar ratio of 3:

1. The cells are then replaced with DMEM medium containing 10% fetal bovine serum and cultured for a further period. The supernatant is collected and filtered to obtain the retroviral solution of shRNA targeting Zeb2.

6. The shRNA targeting Zeb2 as described in claim 1 or 2, the recombinant vector as described in claim 3, or the CD8 silencing Zeb2 vector as described in claim 4. + Application of T cells in the preparation of drugs that enhance anti-tumor immune responses.

7. The application according to claim 6, characterized in that, The tumors are skin melanoma, lung cancer, breast cancer, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, hepatocellular carcinoma, or ovarian cancer.

8. A kit for inhibiting Zeb2 gene expression, characterized in that, Includes the shRNA targeting Zeb2 as described in claim 1 or 2, the recombinant vector as described in claim 3, or the CD8 silencing Zeb2 as described in claim 4. + T cells.

9. A drug for enhancing anti-tumor immune responses, characterized in that, Includes the shRNA targeting Zeb2 as described in claim 1 or 2, the recombinant vector as described in claim 3, or the CD8 silencing Zeb2 as described in claim 4. + T cells, and pharmaceutically acceptable excipients.

10. A drug for enhancing antitumor immune responses according to claim 9, characterized in that, The pharmaceutically acceptable excipients are buffers or stabilizers.

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