Recombinant lentiviral expression vector suitable for CIK cell and NK cell to express EpCAM antibody, IL-21 and CCL19
By using a recombinant lentiviral vector expressing EpCAM antibody, IL-21, and CCL19 genes in CIK and NK cells, the problems of immunosuppression and short survival time in solid tumor treatment with CAR-T therapy were solved, and the killing ability and immune activation against colorectal cancer were enhanced.
Patent Information
- Application Number
- CN202510982567.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-10-17
AI Technical Summary
CAR-T therapy has problems such as tumor microenvironment immunosuppression, missing or downregulated antigen expression, short survival time and insufficient activation in the treatment of solid tumors.
A recombinant lentiviral expression vector suitable for CIK cells and NK cells was designed, containing EpCAM antibody, IL-21 and CCL19 gene, combined with NKP44 transmembrane and intracellular regions, CD3ζ sequence, 2B4 sequence, DNAM1 sequence and DAP10 sequence, to construct pCDH-21×19EpCAM-CAR lentiviral expression vector, which enhances the specificity and cell activation ability for solid tumors.
It enhances the killing ability of CIK cells and NK cells against colorectal cancer, strengthens immune activation in the tumor microenvironment, prolongs survival time, and provides an effective treatment strategy for solid tumors.
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Figure CN120796394A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application relates to a CAR recombinant lentivirus expression vector suitable for expression of EpCAM antibodies, IL-21 and CCL19 by CIK cells and NK cells, and belongs to the field of genetic engineering and biotechnology. BACKGROUND
[0002] In recent years, the T cell treatment technology using the gene modification technology to express tumor-specific chimeric antigen receptors (CAR) has developed rapidly, and shows good targeting, killing activity and persistence in vitro and clinical trials, thus providing a new effective solution for adoptive cell immunotherapy and showing great application potential and development prospect.
[0003] CIK cells (cytokine induced killer cells) are a non-homogeneous cell population mainly composed of T cells, and the main effector cells are natural killer T cells (NKT cells), which have the dual markers (CD3 + CD56 + ) of NK cells and T cells. Also known as NK cell-like T lymphocytes, they have the strong anti-tumor activity of T lymphocytes and the non-limiting tumor killing advantages of NK cells. At present, many studies have tried to modify CAR targeting CD19, CD33, CD123, HER2, EGFR, CEA, CD28 and other antigens on CIK cells, and various strategies are also being explored to improve the specificity of tumor killing.
[0004] NK cells are one of the two major weapons against tumors in the body in addition to T cells, and have broad-spectrum tumor killing ability. Reduction in the number of NK cells or impairment of their function is associated with the progression of various types of cancer. "Cold tumor" is immune-insensitive, and has little or no MHC class I molecules expressed on the surface, so it is hardly recognized by T cells, but can be recognized and killed by NK cells, and the introduction of this concept improves the status of NK cells in antitumor immunotherapy. At the same time, NK cells have the ability to enhance T cell anti-tumor immune response, and are the basis for T cells to play an anti-tumor function. Many studies have shown that impaired NK cell function is involved in tumor immune escape and promotes the development of colorectal cancer. Therefore, improving and enhancing NK cell function can provide a new way for the treatment of colorectal cancer.
[0005] Epithelial specific adhesion molecule (EpCAM) is a 40 kD type I transmembrane glycoprotein, which is a specific antigen of colorectal cancer and a tumor stem cell antigen. The expression of EpCAM antigen is related to the proliferation and metastasis of tumor cells. Studies have shown that the expression of EpCAM antigen shows a significant upward gradient in normal cells, dysplastic epithelium and tumor tissue. The expression of EpCAM in common digestive system tumors such as gastric cancer and colon cancer is close to 100%. Although it is expressed in normal cells, it is not expressed or has a very low expression rate in important organs. Therefore, if EpCAM is selected as the targeting antigen of CAR, it has a certain safety basis.
[0006] IL-21 is involved in the activation of B cells, T cells, dendritic cells and NK cells, among which CD8 + The activation and proliferation of T cells are particularly noteworthy. In addition to the synergistic effect of IL-15 on T cell proliferation, IL-21 can also stimulate CD8 + T cell differentiation through its own effect. IL-15 acts on naive CD8 + T cells, which can lead to down-regulation of CD28 and CD62L membrane proteins; but IL-21 can prevent the down-regulation of these two proteins, so that they continue to exert their corresponding physiological effects. IL-21 mainly acts on the differentiation process of NK cells, promotes the maturation of NK cells, and activates NK cell lysis activity by up-regulating perforin / granzyme and Fas L. In addition, IL-21 can promote the expression of CD16 in NK cells, high-level expression of perforin, and enhance the ADCC effect. Many preclinical studies have shown that IL-21 has a strong anti-tumor effect, which involves multiple signaling pathways of innate immunity and specific immunity.
[0007] CCL19 is a member of the chemokine CC family, also known as macrophage inflammatory protein 3β and Epstein-Barr virus-induced molecule 1. In recent years, CCL19 has been reported to inhibit a series of solid tumors in vivo, including lung cancer, ovarian cancer, fibrosarcoma, etc. Studies have shown that the necrosis and apoptosis of tumors are related to the promotion of CCL19 to promote dendritic cells and T cells into tumor tissue and promote the maturation of immature T cells to play an immune role.
[0008] Colorectal cancer is one of the common digestive system malignant tumors after gastric cancer and esophageal cancer, and it is very urgent to find and discover safe and effective treatment strategies. Immune cell therapy has been valued since its appearance, especially after it has achieved a shocking effect in the clinical treatment experiment of hematological tumors, researchers have begun to explore the application direction of this technology, and the effect of immunotherapy technology on solid tumors has also become the focus of scientists. However, CAR-T therapy still has many problems in practical application, such as organ toxicity, off-target toxicity, immunosuppression of tumor microenvironment, loss or down-regulation of antigen expression, poor effect on solid tumors, short survival time and the like. SUMMARY
[0009] In view of the problems of immunosuppression of tumor microenvironment, loss or down-regulation of antigen expression, short survival time and insufficient activation of CAR-T therapy, the present application provides a recombinant lentiviral expression vector suitable for high expression of EpCAM antibody, IL-21 and CCL19 of CIK cells and NK cells.
[0010] The first object of the present application is to provide a recombinant lentiviral expression vector suitable for CAR therapy, which comprises:
[0011] pCHD-CMV-MCS-EF1-copGFP-T2A-puro skeleton structure, CAR gene structure, T2A peptide gene structure, IL-21 gene expression structure, P2A peptide gene structure and CCL19 gene expression structure;
[0012] The pCHD-CMV-MCS-EF1-copGFP-T2A-puro skeleton structure comprises the promoter CMV Promoter of the target gene, the reporter gene promoter EF1a Promoter, the marker gene green fluorescent protein GFP and puromycin puro for screening of eukaryotic cells, and the vector map is as shown in Figure 1 The nucleotide sequence is shown as SEQ ID NO. 1.
[0013] The CAR gene structure sequentially from 5 ， end to 3 ， end is: Kozak sequence, CD8 alpha signal peptide sequence, anti-EpCAM-scFv, CD8 alpha hinge region sequence, NKP44 transmembrane region and intracellular region sequence, CD3 zeta sequence, 2B4 sequence, DNAM1 sequence, DAP10 sequence.
[0014] The nucleotide sequence of the T2A peptide gene structure and the glycine-serine-glycine (GSG) sequence structure in front of it is shown in SEQ ID NO. 2.
[0015] The nucleotide sequence of the IL-21 gene expression structure is shown in SEQ ID NO. 3.
[0016] The nucleotide sequence of the P2A peptide gene structure and the glycine-serine-glycine (GSG) sequence structure in front of it is shown in SEQ ID NO. 4.
[0017] The nucleotide sequence of the CCL19 gene expression structure is shown in SEQ ID NO. 5.
[0018] In an embodiment of the present application, the nucleotide sequence of the CMV Promoter of the target gene is shown in SEQ ID NO. 6; the nucleotide sequence of the EF1a Promoter of the reporter gene is shown in SEQ ID NO. 7; the nucleotide sequence of the marker gene green fluorescent protein GFP of the eukaryotic cell screening is shown in SEQ ID NO. 8; and the nucleotide sequence of the puromycin puro is shown in SEQ ID NO. 9.
[0019] In an embodiment of the present application, the nucleotide sequence of the Kozak sequence is shown in SEQ ID NO. 10; the nucleotide sequence of the CD8a signal peptide sequence is shown in SEQ ID NO. 11; the nucleotide sequence of the anti-EpCAM-scFv is shown in SEQ ID NO. 12; the nucleotide sequence of the CD8a hinge region sequence is shown in SEQ ID NO. 13; the nucleotide sequence of the NKP44 transmembrane region and intracellular region sequence is shown in SEQ ID NO. 14; the nucleotide sequence of the CD3 zeta sequence is shown in SEQ ID NO. 15; the nucleotide sequence of the 2B4 sequence is shown in SEQ ID NO. 16; the nucleotide sequence of the DNAM1 sequence is shown in SEQ ID NO. 17; and the nucleotide sequence of the DAP10 sequence is shown in SEQ ID NO. 18.
[0020] The second object of the present application is to provide a construction method of the above-mentioned lentivirus recombinant expression vector, which comprises the following steps:
[0021] S1. In the synthesis of CAR gene + IL-21 + CCL19 full-length DNA sequence, Nhe1 and Not1 enzyme cutting sites are added at both ends, and the sequence is connected to the pCHD-CMV-MCS-EF1-copGFP-T2A-puro empty vector through the two enzyme cutting sites, to construct a pCDH-21x19EpCAM-CAR lentiviral recombinant expression vector.
[0022] S2. The E. coli containing the recombinant expression vector is streaked on an ampicillin-resistant LB plate and cultured at 37°C overnight. Several single colonies are picked for sequencing.
[0023] S3. The single colony with correct sequencing results is shaken and cultured, and the plasmid is extracted using an endotoxin-free plasmid extraction kit. After detecting the purity and concentration, it is stored at -20°C.
[0024] The third object of the present application is to provide a recombinant microbial cell of the above-mentioned lentiviral recombinant expression vector.
[0025] In an embodiment of the present application, the recombinant microbial cell is a recombinant E. coli.
[0026] In an embodiment of the present application, the recombinant microbial cell is an E. coli DH5α host, and the above-mentioned pCDH-21x19EpCAM-CAR lentiviral recombinant expression vector is transformed into the host cell.
[0027] The fourth object of the present application is to provide a transgenic cell of the above-mentioned lentiviral recombinant expression vector.
[0028] In an embodiment of the present application, the transgenic cell is a transgenic pCDH-21x19EpCAM-CAR-CIK cell.
[0029] In an embodiment of the present application, the transgenic cell is a transgenic cell obtained by a method of infecting a CIK cell with a lentivirus.
[0030] In an embodiment of the present application, the transgenic cell is a transgenic pCDH-21x19EpCAM-CAR-NK cell.
[0031] In an embodiment of the present application, the transgenic cell is a transgenic cell obtained by a method of infecting a NK cell with a lentivirus.
[0032] The fifth object of the present application is to provide a construction method of the above-mentioned transgenic cell, which comprises the following steps:
[0033] S1. The pCDH-21x19EpCAM-CAR lentivirus recombinant expression vector is transfected into HEK-293FT cells by using a transfection reagent polyethyleneimine (PEI), and the supernatant collected 48 h and 72 h after transfection is concentrated by using a PEG-8000 solution and centrifugation to obtain packaged pCDH-21x19EpCAM-CAR lentivirus particles.
[0034] S2. The CIK cells or NK cells in a good growth state for 3-5 d are inoculated into a 12-well plate at a density of 2x10 6 / well, and 300 μl / well of the concentrated lentivirus particles and 4 ng / ml of polybrene are added. After low-speed centrifugation at 150 g for 4 h at room temperature (26℃), the cells are cultured in a 37℃, 5% CO2 incubator.
[0035] S3. The infection efficiency is observed by fluorescence microscopy and detected by flow cytometry 48 h after infection.
[0036] The application also protects the pCDH-21x19EpCAM-CAR lentivirus recombinant expression vector, the recombinant microbial cells thereof, the pCDH-21x19EpCAM-CAR-CIK cells and the pCDH-21x19EpCAM-CAR-NK cells in the application in the fields of biology, medicine and medical treatment. Advantages of the application
[0037] The application constructs a CAR vector taking the EpCAM gene as a target according to the characteristics that the EpCAM gene is expressed in nearly 100% of colorectal cancer and is not expressed or is expressed at a very low level in important tissues and organs. In order to solve the problems of a short survival time, insufficient activation and tumor microenvironment immunosuppression in the CAR-T therapy, the IL-21 and CCL19 genes are added in the vector design. In order to enhance the specificity of the vector to solid tumors, the activation and co-stimulation ability of CIK cells and NK cells, the NKP44 transmembrane region and intracellular region sequence, the CD3ζ sequence, the 2B4 sequence, the DNAM1 sequence and the DAP10 sequence are used for the structure of the CAR in the vector design. The application successfully constructs the pCDH-21x19EpCAM-CAR lentivirus expression vector for the solid tumor colorectal cancer and the corresponding transgenic CIK cells and transgenic NK cells, and further lays a foundation for the research on the application of CAR technology in the treatment of solid tumors. BRIEF DESCRIPTION OF DRAWINGS
[0038] Figure 1 pCHD-CMV-MCS-EF1-copGFP-T2A-puro vector map.
[0039] Figure 2pCDH-21x19EpCAM-CAR lentivirus vector transfected HEK-293FT cells, A: bright field image of pCDH-21x19EpCAM-CAR lentivirus recombinant expression vector transfected (50x); B: fluorescence image of pCDH-21x19EpCAM-CAR lentivirus recombinant expression vector transfected (50x).
[0040] Figure 3 pCDH-21x19EpCAM-CAR lentivirus particles infect CIK cells and NK cells, A: bright field image of pCDH-21x19EpCAM-CAR lentivirus particles infecting CIK cells (50x); B: fluorescence image of pCDH-21x19EpCAM-CAR lentivirus particles infecting CIK cells (50x); C: bright field image of pCDH-21x19EpCAM-CAR lentivirus particles infecting NK cells (50x); D: fluorescence image of pCDH-21x19EpCAM-CAR lentivirus particles infecting NK cells (50x). DETAILED DESCRIPTION
[0041] Material description in the example:
[0042] 1. Strain information: E. coli DH5a competent, commercial strain.
[0043] 2. Vector information: plasmid pCHD-CMV-MCS-EF1-copGFP-T2A-puro empty vector, PsAX2, OG are commercial vectors.
[0044] 3. DMEM high-sugar medium, fetal bovine serum (FBS), opti-MEM medium, PBS buffer, polyethylene (PEI), PEG-8000, polycondensate are commercial products.
[0045] 4. LB medium formula: 10 g / L peptone, 5 g / L yeast powder, 10 g / L Nacl; solid medium plus 20 g / L agar powder.
[0046] Example 1 Design and gene sequence synthesis of CAR
[0047] Find the required gene sequence in NCBI, the CAR gene structure sequence is from 5, end to 3, end in turn: Kozak sequence, CD8a signal peptide sequence, anti-EpCAM-scFv, CD8a hinge region sequence, NKP44 transmembrane and intracellular region sequence, CD3z sequence, DNAM1 sequence, DAP10 sequence, IL-21 and CCL19 sequence. After arranging the full-length DNA sequence of CAR gene, it is synthesized by the company.
[0048] Construction of pCDH-21x19EpCAM-CAR lentivirus expression vector
[0049] Nhe1 and Not1 enzyme cutting sites were added at both ends of the full-length DNA sequence of the CAR gene when it was synthesized, and the sequence was connected to the pCHD-CMV-MCS-EF1-copGFP-T2A-puro empty vector through the two enzyme cutting sites to construct the pCDH-21x19EpCAM-CAR lentivirus recombinant expression vector. The vector was transformed into E. coli DH5a competent cells, and after transformation, it was plated on ampicillin-resistant LB solid medium plates and incubated at 37°C overnight. Several single colonies were picked and sent to the company for sequencing. The single colonies with correct sequencing results were cultured in LB medium, the plasmid was extracted using an endotoxin-free plasmid extraction kit, and the purity and concentration were detected before being stored at -20°C.
[0050] Packaging of pCDH-21x19EpCAM-CAR lentivirus expression vector
[0051] HEK-293FT cells were seeded in 10mm culture dishes at a density of 1-2x10 6 The cells were incubated at 37°C in a 5% CO2 incubator overnight. Fresh preheated DMEM high glucose medium was used to replace the medium before virus packaging, and the cells were incubated for another 2h. 20μg of pCDH-21x19EpCAM-CAR lentivirus vector and 10μg of each lentivirus packaging plasmid PsAX2+OG were transfected into HEK-293FT cells (0h) under the mediation of polyethyleneimine (PEI). The PEI solution was dissolved in a 60°C water bath for 5min, and then the three plasmids and PEI solution (plasmid:PEI ratio 1:9) were added to opti-MEM medium, the total volume of each tube was 1.5ml, and the solution was mixed gently and incubated at room temperature for 5min. The opti-MEM medium containing PEI was slowly added to the plasmid-containing culture medium, mixed gently, and incubated at room temperature for 20min. The mixed solution was gently added dropwise to the 10mm culture dish, mixed gently, and incubated at 37°C in a 5% CO2 incubator for 6h. The medium was then replaced with DMEM high glucose medium containing 10% FBS, and the cells were incubated for 24h. The fluorescence was observed under an inverted fluorescence microscope and the transfection efficiency was calculated (see Table 1). Figure 2 ).
[0052] Concentration of pCDH-21x19EpCAM-CAR lentivirus particles
[0053] The supernatant of the HEK-293FT transfected cells collected at 48h and 72h was centrifuged at 4℃, 3000rmp / min, low speed for 10min, and the supernatant was further removed of cell debris by using 0.45μM filter membrane. The filtered supernatant was mixed with the prepared 5×PEG-8000 solution, and placed in a 4℃ refrigerator, and mixed every 30min. After 3h, the mixture was placed in a 4℃ refrigerator overnight, and the next day, the mixture was centrifuged at 4℃, 4500rmp / min, low speed for 30min, and the supernatant was discarded. PBS buffer was added to resuspend the lentivirus particles at a concentration of 100 times, and 300ul / tube was aliquoted in centrifuge tubes and stored in a -80℃ refrigerator for standby.
[0054] Example 5 Determination of the titer of pCDH-21×19EpCAM-CAR lentivirus
[0055] One day before the experiment, HEK-293FT cells were inoculated in a 12-well plate at a density of 1×10 5 After 24h of culture in a carbon dioxide incubator, the concentrated virus solution was inoculated in a 12-well plate at a volume of 0.01μl, 0.1μl, 1μl, and 2μl, and 4μg / ml of polybrene was added, and 3 replicate wells were set for each group. After 24h of cell culture, the cells were trypsinized and washed twice with PBS buffer, and a 500μl cell suspension was prepared with PBS buffer and filtered into a flow tube for flow cytometry detection. According to the volume of the added virus solution, the positive rate of cell transfection was determined by flow cytometry to calculate the virus titer. The calculation formula is: virus titer = cell number × positive rate percentage / added virus volume (TU / mL).
[0056] Example 6 Infection of CIK cells and NK cells with pCDH-21×19EpCAM-CAR lentivirus particles
[0057] The well-grown CIK cells and NK cells cultured for 3-5d were inoculated in a 12-well plate at a density of 2×10 6 Each well was added with 300μl of concentrated virus and 4ng / ml of polybrene, and the plate was centrifuged at 150g, low speed for 4h at room temperature (26℃), and then cultured in a 37℃, 5% CO2 incubator. After 48h of infection, the infection efficiency was observed and calculated by fluorescence microscopy. Figure 3
[0058] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.
Claims
1. A recombinant lentiviral expression vector, characterized in that: The CAR gene structure, IL-21 gene expression structure and CCL19 gene expression structure were connected to the vector pCHD-CMV-MCS-EF1-copGFP-T2A-puro backbone structure to obtain the recombinant lentiviral expression vector pCDH-21×19EpCAM-CAR; the nucleotide sequence of the CAR gene structure is shown in SEQ ID NO.10-18, the nucleotide sequence of the IL-21 gene expression structure is shown in SEQ ID NO.3; the nucleotide sequence of the CCL19 gene expression structure is shown in SEQ ID NO.
5.
2. The recombinant lentiviral expression vector according to claim 1, wherein The CAR gene structure, IL-21 gene expression structure and CCL19 gene expression structure were ligated between the multiple cloning sites Nhe1 and Not1 of the vector pCHD-CMV-MCS-EF1-copGFP-T2A-puro and expressed at a high level under the mediation of the CMV promoter.
3. The recombinant lentiviral expression vector according to claim 1, wherein The nucleotide sequence of the T2A peptide gene structure connecting the CAR gene structure and the IL-21 gene expression structure and the preceding glycine-serine-glycine (GSG) sequence structure is shown in SEQ ID NO.
2.
4. The recombinant lentiviral expression vector according to claim 1, wherein The nucleotide sequence of the P2A peptide gene structure connecting the IL-21 gene expression structure and the preceding glycine-serine-glycine (GSG) sequence structure is shown in SEQ ID NO.
4.
5. A recombinant microbial cell, characterized in that Contains the recombinant lentiviral expression vector according to any one of claims 1 to 4.
6. The recombinant microbial cell according to claim 5, wherein The recombinant microbial cell uses Escherichia coli DH5α as a host.
7. The recombinant microbial cell according to claim 6, wherein The preparation method of the recombinant microbial cells is to connect the CAR gene structure, the IL-21 gene expression structure and the CCL19 gene expression structure between the multiple cloning sites Nhe1 and Not1 of the vector pCHD-CMV-MCS-EF1-copGFP-T2A-puro to obtain the recombinant plasmid pCDH-21×19EpCAM-CAR, and transform the recombinant plasmid into Escherichia coli DH5α.
8. A eukaryotic transgenic cell, characterized in that Contains the recombinant lentiviral expression vector according to any one of claims 1 to 4.
9. The eukaryotic transgenic cell according to claim 8, wherein The eukaryotic transgenic cells are CIK cells and NK cells as the objects of gene editing.
10. The eukaryotic transgenic cell according to claim 9, wherein The pCDH-21×19EpCAM-CAR recombinant lentiviral expression vector was packaged and concentrated into lentiviral particles, and then infected with CIK cells and NK cells to obtain transgenic cells.
11. Use of the recombinant lentiviral expression vector according to any one of claims 1 to 4, or the recombinant microbial cell according to any one of claims 5 to 7, or the eukaryotic transgenic cell according to any one of claims 8 to 10 in the fields of biology, medicine, and healthcare.