CN112795584B

CN112795584B - Nucleic acid resisting GCC, preparation method thereof, immune cell with nucleic acid and application thereof

Info

Publication number: CN112795584B
Application number: CN202010845599.4A
Authority: CN
Inventors: 刘明录; 王立新; 卢永灿; 马洪华; 王亮; 刘敏; 韩国英; 冯建海
Original assignee: Shandong Research Institute Of Adult Cell Industry Technology Co ltd
Current assignee: Shandong Research Institute Of Adult Cell Industry Technology Co ltd
Priority date: 2020-08-20
Filing date: 2020-08-20
Publication date: 2023-01-24
Anticipated expiration: 2040-08-20
Also published as: CN112795584A

Abstract

The nucleic acid for resisting GCC at least comprises a Leader nucleotide sequence, a GCC single-chain antibody nucleic acid artificial sequence, a hinge region nucleotide sequence of CD8, a transmembrane-stimulation structure domain nucleotide sequence, a CD3 zeta signal conduction region nucleotide sequence, a self-cleaving peptide T2A nucleotide sequence, a chemokine IL7 nucleotide sequence and a chemokine CCL19 nucleotide sequence. The invention can improve the pertinence of immune cells, reduce the damage to non-tumor cells and improve the effectiveness and safety.

Description

anti-GCC nucleic acid, preparation method thereof, immune cell with nucleic acid and application of immune cell

Technical Field

The invention relates to the technical field of genes, in particular to a nucleic acid for resisting GCC, a preparation method thereof, an immune cell with the nucleic acid and application thereof.

Background

Colorectal cancer is one of the common digestive tract malignancies, accounting for 10% of all worldwide tumors, accounting for the 3 rd most of the worldwide incidence of malignancies. Although surgery is an effective treatment method, approximately 1/3 of colorectal cancer patients have relapse and distant metastasis after surgery, and the 5-year survival rate is less than 50%. At present, relevant research also shows that colorectal diseases have the trend of increasing and younger year by year, and the serious influence is harmful to human health. Therefore, early detection and early treatment are key to improve the curative effect, prognosis and survival rate of colorectal cancer.

In the malignant tumor treatment method, the cure rate and prognosis of the operation, radiotherapy and chemotherapy and targeted therapy are poor, and the patient is greatly injured, and the adoptive immunotherapy strategy of the chimeric antigen receptor T cells (CAR-T) overcomes the defects of the treatment method, and hopes are brought to the cancer cure. The principle of CAR-T treatment is as follows: through the genetic engineering modification technology, the T cells of the cancer patient separated and collected in vitro express a Chimeric Antigen Receptor (CAR) for identifying tumor specific antigens, and the CAR-T cells are amplified in vitro in large quantities and then are infused into the body of the cancer patient, so that the aim of eliminating tumors is fulfilled. Relevant research shows that CAR-T cell therapy has good effect in treating Acute Lymphoblastic Leukemia (ALL) and lymphoma and other malignant tumors, so CAR-T technology has great application prospect.

Therefore, the development of a nucleic acid for resisting GCC, a preparation method thereof, an immune cell with the nucleic acid and application thereof not only have urgent research values, but also have good economic benefits and industrial application potentials, which is the basis for the achievement of the invention.

Disclosure of Invention

The present inventors have conducted intensive studies to overcome the above-identified drawbacks of the prior art, and as a result, have completed the present invention after having made a great deal of creative efforts.

Specifically, the technical problems to be solved by the invention are as follows: provides a nucleic acid for resisting GCC, a preparation method thereof, an immune cell with the nucleic acid and application thereof, which are used for improving the pertinence of the immune cell, reducing the damage to non-tumor cells and improving the effectiveness and the safety.

In order to solve the technical problems, the technical scheme of the invention is as follows:

in a first aspect, the invention provides an anti-GCC nucleic acid comprising at least a Leader nucleotide sequence, a GCC single-chain antibody nucleic acid artificial sequence, a hinge region nucleotide sequence of CD8, a transmembrane-stimulatory domain nucleotide sequence, a CD3 zeta signaling region nucleotide sequence, a self-cleaving peptide T2A nucleotide sequence, a chemokine IL7 nucleotide sequence, and a chemokine CCL19 nucleotide sequence.

In the present invention, as a preferred embodiment, the transmembrane-stimulatory domain nucleotide sequence is selected from the group consisting of CD8, CD27, CD28, CD137 (i.e., 4-1 BB), CD134 (i.e., OX 40), all or a partial DNA fragment of an ICOS molecule.

In the present invention, as a preferred embodiment, the anti-GCC nucleic acid comprises sequentially arranged nucleic acids

A Leader nucleotide sequence as set forth in SEQ ID NO. 2;

the GCC single-chain antibody nucleic acid artificial sequence as shown in SEQ ID NO. 3;

the hinge region nucleotide sequence of CD8 as set forth in SEQ ID No. 4;

a transmembrane-stimulatory domain nucleotide sequence;

the 4-1BB intracellular domain nucleic acid sequence as set forth in SEQ ID NO. 6;

the nucleotide sequence of the CD3 zeta signaling region as set forth in SEQ ID No. 7;

the self-cleavage peptide T2A nucleic acid artificial sequence as shown in SEQ ID NO. 8;

the chemokine IL7 nucleotide sequence as described in SEQ ID NO. 9;

the chemokine CCL19 nucleotide sequence shown in SEQ ID NO. 10.

In the present invention, as a preferred technical scheme, the transmembrane-stimulating domain nucleotide sequence adopts a CD8 transmembrane region nucleic acid artificial sequence as set forth in SEQ ID NO. 5.

In the present invention, as a preferred technical scheme, the nucleotide sequence of the anti-GCC nucleic acid is shown in SEQ ID NO. 1.

In a second aspect, the present invention provides a method for preparing the nucleic acid, comprising the steps of:

(1) Synthesizing the whole expression frame according to a Leader nucleotide sequence, a GCC single-chain antibody nucleic acid artificial sequence, a CD8 hinge region nucleotide sequence, a transmembrane-stimulating domain nucleotide sequence, a CD3 zeta signaling region nucleotide sequence, a self-cleavage peptide T2A nucleotide sequence, a chemokine IL7 nucleotide sequence, a self-cleavage peptide T2A nucleotide sequence and a chemokine CCL19 nucleotide sequence, and inserting the expression frame into a standard vector pUC to obtain pUC-GCC-CAR;

(2) Performing double enzyme digestion on pUC-GCC-CAR, cutting off the agar part of a GCC-CAR DNA fragment by using agar electrophoresis, treating by using a DNA extraction kit sol solution, passing through a DF column, discarding filtrate, rinsing the DF column, performing air separation, eluting the DF column, and collecting a centrifuge to obtain the purified GCC-CAR DNA fragment.

In a third aspect, the present invention provides an immune cell comprising a plasmid having a nucleic acid as described above.

In the present invention, as a preferred technical solution, the immune cells are selected from autologous or transgenic T cells, NK cells, cytotoxic T lymphocytes or regulatory T cells, memory T cells, bispecific T cells, CIK cells.

In the invention, as a preferable technical scheme, the plasmid is a pLent-GCC-CAR plasmid obtained by inserting a fusion gene fragment GCC-CAR DNA into a lentivirus expression vector pLent-C-GFP.

In the present invention, as a preferred technical scheme, the plasmid is prepared by the following preparation method: the purified GCC-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were ligated in a linker system: 10 × buffer:1 mul; t4 ligase: 1 mul; GCC-CAR DNA fragment: 4 mu l of the solution; linearized pLent-C-GFP DNA fragment: 4 μ l, ligation formation.

In the present invention, as a preferred technical solution, the immune cells are prepared by a method comprising the following steps:

the pLent-GCC-CAR plasmid is subjected to lentivirus packaging firstly, and then immune cells are infected by recombinant lentiviruses.

In a fourth aspect, the invention provides the use of an anti-GCC nucleic acid, which means that the gene can be used in the preparation of a medicament for treating solid tumors. The pharmaceutical form includes, but is not limited to, a kit.

In the present invention, as a preferred technical solution, the solid tumor includes colorectal cancer, gastric cancer, esophageal cancer.

In the present invention, as a preferred embodiment, the kit comprises

(1) Obtaining a vector stably expressing GCC-CAR as described above;

(2) A carrier diluent.

After the technical scheme is adopted, the invention has the beneficial effects that:

in the present invention, guanylyl Cyclase C (GCC) is a polypeptide specifically expressed in intestinal tissues, belongs to a member of the guanylyl cyclase family of receptors, and is an N-linked glycoprotein receptor. The GCC comprises an extracellular N-terminal receptor binding region, a kinase homology regulating region, a C-terminal catalytic region and a carboxyl terminal. GCC has the functions of regulating Na + dynamic balance; stimulating CL-secretion/inhibiting Na + absorption, and regulating intestinal function; regulating the pH value of the intestinal tract and promoting the digestion and absorption of food; regulate the transition and proliferation of intestinal epithelial cells by opening the CNG pathway and inhibiting Ca2+ and Na + exchange; excrete mucus and water, and eliminate pathogenic bacteria in tissues such as intestine and lung. GCC can also be expressed in colorectal cancer cell lines, primary and metastatic colorectal cancers, regulates intestinal epithelial cell transition, and inhibits colorectal cancer cell proliferation and DNA synthesis. In vivo GCC antibodies may regulate proliferation and differentiation of small intestinal mucosal epithelial cells through a relevant mechanism dependent on cyclic guanine phosphate (cGMP) (see fig. 1). In addition, high GCC expression was found in esophageal and gastric adenocarcinoma. Therefore, GCC can be used as a CAR-T target for treating colon cancer, gastric cancer or esophageal cancer, and an effective treatment method is provided for clinical diagnosis and treatment. However, since GCC is simply used as a target of CAR-T for the treatment of colon, gastric or esophageal cancer, the immune cells are vulnerable to non-tumor cell damage.

Therefore, through repeated screening experiments and creative labor, the CAR-T co-expressing the immunopotentiator IL7 and the CCL19 anti-GCC is modified on the basis of the conventional CAR-T, so that the conventional CAR-T can express two chemokines of IL7 and CCL19 and can specifically recognize GCC tumor-associated antigens on the surface of colon cancer cells. The interleukin-7 (IL-7) is a cytokine, is secreted by thymocytes, bone marrow stromal cells, small intestine epithelial cells and the like, plays an important role in normal development and maintenance of normal immune function of a human immune system, and can promote proliferation of T cells and maintain stability of the T cells. CCL19 is a member of the CC family of chemokines, and its specific receptor CCR7 is a 7-transmembrane protein distributed on the cell surface. CCL19 has the function of recruiting peripheral T cells and dendritic cells into lymphatic tissues, can inhibit the proliferation, migration and invasion capacity of human colorectal cancer cell strain SW620 cells, has the effect of inhibiting colorectal cancer, and can effectively inhibit solid tumors such as lung cancer, ovarian cancer, fibrosarcoma and the like.

On the basis of the third generation CAR-T technology, GCC is firstly proposed to be used as GCC with high expression quantity of malignant tumors such as colorectal cancer, gastric cancer, esophageal cancer and the like as a target spot, and an immune cell which can express immune enhancement factors IL7 and CCL19 together and resist GCC chimeric antigen receptors is prepared.

The new generation CAR-T vector constructed by the invention is added with immunopotentiating factors IL7 and CCL19, wherein IL7 can promote T cell proliferation and maintain T cell stability, and CCL19 can recruit peripheral T cells and dendritic cells into lymphoid tissues, so that the proliferation and killing capacity of the 7 x 19CAR-T cells constructed by the invention are improved by 2 times compared with that of common CAR-T cells. Moreover, the chemokine provided by the invention can be used for immune cells modified by any CAR technology, so that the clinical effectiveness of the CAR technology is greatly improved.

Drawings

Figure 1 is a schematic diagram of the GCC structure.

FIG. 2 CAR module diagram of co-expression of immunopotentiator IL7 and CCL19 Anti-GCC.

FIG. 3 flow cytometry detects expression of anti-7X 19GCC-CAR vector on the surface of T cells.

FIG. 4 CFSE-labeled 7X 19CAR-T cells and CAR-T cell number comparison graph.

FIG. 5 graph of tumor size change after T lymphocyte treatment in murine models of colorectal cancer.

FIG. 6 is a design drawing of a Leader-scFv (GCC) -CD8-4-1BB-CD3 zeta-T2A-IL 7-T2A-CCL19 recombinant gene according to the present invention.

Detailed Description

The invention is further illustrated by the following specific examples. However, the use and purpose of these exemplary embodiments are only to exemplify the present invention, and do not constitute any limitation in any form to the actual scope of the present invention, and do not limit the scope of the present invention.

Example 1

An anti-GCC nucleic acid comprising at least a Leader nucleotide sequence, a GCC single chain antibody nucleic acid artificial sequence, a hinge region nucleotide sequence of CD8, a transmembrane-stimulatory domain nucleotide sequence, a CD3 zeta signaling region nucleotide sequence, a self-cleaving peptide T2A nucleotide sequence, a chemokine IL7 nucleotide sequence, and a chemokine CCL19 nucleotide sequence. Wherein the transmembrane-stimulating domain nucleotide sequence is selected from the group consisting of CD8, CD27, CD28, CD137 (i.e., 4-1 BB), CD134 (i.e., OX 40), all or a partial DNA fragment of an ICOS molecule.

In this embodiment, the anti-GCC nucleic acid includes a CD8 Leader nucleic acid artificial sequence (seq id No. 2), a GCC single-chain antibody nucleic acid artificial sequence (seq id No. 3), a CD8 hinge region nucleic acid artificial sequence (seq id No. 4), a CD8 transmembrane region nucleic acid artificial sequence (seq id No. 5), a 4-1BB co-stimulatory region nucleic acid artificial sequence (seq id No. 6), a CD3 zeta signal region conductive region nucleic acid artificial sequence (seq id No. 7), a self-cleavage peptide T2A nucleic acid artificial sequence (seq id No. 8), a chemokine IL7 nucleic acid artificial sequence (seq id No. 9), a self-cleavage peptide T2A nucleic acid artificial sequence (seq id No. 8), and a chemokine CCL19 nucleic acid artificial sequence (seq id No. 10), which are arranged in sequence.

Example 2

A method for preparing a nucleic acid for the treatment of GCC comprising the steps of:

(1) Synthesizing the whole expression frame according to a Leader nucleic acid artificial sequence, a GCC single-chain antibody nucleic acid artificial sequence, a CD8 hinge region nucleic acid artificial sequence, a CD8 transmembrane region nucleic acid artificial sequence, a 4-1BB costimulation region nucleic acid artificial sequence, a CD3 zeta signal conduction region nucleic acid artificial sequence, a self-cleavage peptide T2A nucleic acid artificial sequence, an IL7 nucleic acid artificial sequence, a self-cleavage peptide T2A nucleic acid artificial sequence and a CCL19 nucleic acid artificial sequence, and inserting the whole expression frame into a standard vector pUC to obtain pUC-GCC-CAR;

(2) Carrying out double enzyme digestion on pUC-GCC-CAR, cutting off an agar part of a GCC-CAR DNA fragment by using agar electrophoresis, treating by using a DNA extraction kit sol solution, passing through a DF column, discarding filtrate, rinsing the DF column, carrying out air separation, eluting the DF column, and collecting a centrifugate to obtain the purified GCC-CAR DNA fragment.

In this embodiment, the more detailed steps are:

(1) The expression cassette was synthesized by Leader nucleic acid artificial sequence, GCC single-chain antibody nucleic acid artificial sequence, CD8 hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, 4-1BB costimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, self-cleaving peptide T2A nucleic acid artificial sequence, IL7 nucleic acid artificial sequence, self-cleaving peptide T2A nucleic acid artificial sequence, CCL19 nucleic acid artificial sequence committing the industrial biotechnology (shanghai) ltd, and inserted into a standard vector pUC, respectively, and was named pUC-GCC-CAR;

(2) The pUC-GCC-CAR vector was digested simultaneously with Fast Digest AsiSI (available from ThermoFisher) and Fast Digest NotI (available from ThermoFisher) at 37 ℃ for 20min. The 100. Mu.l enzyme system is: 10 × buffer:10 mu l of the mixture; 6 mu g of DNA; asiSI enzyme: 3 mu l of the solution; notI enzyme: 3 mul; deionized water to make up the volume. The agar portion containing the GCC-CAR DNA fragment was cut off using agar electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. Mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and sleeved with a Collection Tube (the filtrate was collected). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. Mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, and obtaining the purified GCC-CAR DNA fragment as the liquid in the micro-centrifuge tube.

Example 3

A plasmid containing the gene as described in example 2, which was obtained by inserting the fusion gene fragment GCC-CAR DNA into the lentiviral expression vector pLent-C-GFP, was named 7X 19pLent-GCC-CAR. The plasmid is prepared by the following preparation method: the purified GCC-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were ligated in a linker system: 10 × buffer:1 mul; t4 ligase: 1 mul; GCC-CAR DNA fragment: 4 mu l of the solution; linearized pLent-C-GFP DNA fragment: 4 μ l, ligation formation.

In this example, the plasmid was prepared as follows:

the whole expression cassette was synthesized by Leader nucleic acid artificial sequence, GCC single-chain antibody nucleic acid artificial sequence, CD8 hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, 4-1BB co-stimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, self-cleavage peptide T2A nucleic acid artificial sequence, IL7 nucleic acid artificial sequence, self-cleavage peptide T2A nucleic acid artificial sequence, CCL19 nucleic acid artificial sequence, consortium Biotechnology (Shanghai) Limited and inserted into a standard vector pUC, thus named as-GCC-CAR, while the pUC-GCC-CAR and pLent-C-GFP vectors were subjected to Fast Digest AsiSI (purchased from ThermoFisher) and Fast Digest NotI (purchased from ThermoFisher) double enzyme digestion at 37 ℃ for 20min, respectively. The 100. Mu.l digestion system was: 10 × buffer:10 mu l of the mixture; 6 mu g of DNA; asiSI enzyme: 3 mu l of the solution; notI enzyme: 3 mu l of the solution; deionized water to make up the volume. The agar sites containing the GCC-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were cut off separately by agar electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. Mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and sleeved with a Collection Tube (the filtrate was collected). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. Mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing for 2 minutes at room temperature, centrifuging for 2 minutes at 14000rpm, and obtaining the liquid in the micro-centrifuge tube, namely the purified GCC-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment.

The two DNA fragments were ligated overnight at 16 ℃ to form a 7X 19pLent-GCC-CAR plasmid. The connecting system is as follows: 10 × buffer:1 mul; t4 ligase: 1 mul; GCC-CAR DNA:4 mu l of the solution; linearized pLent-C-GFP DNA:4 μ l.

Example 4

And (3) plasmid purification: coli (DH 5 α) was transformed with the above 7 × 19pLent-GCC-CAR. The method comprises the following specific steps: the plasmid and the competent cells are evenly mixed and incubated on ice for half an hour, then heat shock is carried out at 42 ℃ for 90 seconds, then the mixture is placed on ice for 2min, finally liquid LB culture medium is added and slowly shaken for about 1 hour, then centrifugation is carried out at 3000rpm for 5min, and 100 mul of bacterial liquid is coated on a solid plate containing ampicillin LB. The next day, single colonies were picked and cultured overnight, and pLent-GCC-CAR plasmid was extracted using plasmid extraction and purification kit (purchased from Qiagen) as follows: (1) 1.5ml of the bacterial solution was centrifuged at room temperature 10000 Xg for 1min. (2) The supernatant was removed, 250. Mu.l of solution I (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended. (3) Adding 250 mul of solution II, and gently inverting the centrifuge tube for 4-6 times to obtain clear lysate. Preferably, the incubation is carried out at room temperature for 2min. (4) Add 350. Mu.l of solution III, mix gently by inversion several times until white flocculent precipitate appears, centrifuge at room temperature 10000 Xg for 10min. (5) The supernatant was aspirated with special care and transferred to a clean adsorption column equipped with a 2ml centrifuge tube. It is ensured that there are no aspiration sediment and no cell debris. Centrifugation was carried out at room temperature at 10000 Xg for 1min until the lysate was completely passed through the column. (6) Discarding the filtrate, adding 500. Mu.l Buffer HBC, centrifuging at 10000 Xg for 1min, cleaning the absorption column, and removing residual protein to ensure the purity of DNA. (7) The filtrate was discarded, and the column was washed with 750. Mu.l of Wash Buffer diluted with 100% ethanol and centrifuged at 10000 Xg for 1min. And (8) adding 750. Mu.l of Wash Buffer to clean the absorption column. (9) The column must be centrifuged at 10000 Xg for 2min to ensure that the ethanol is removed. (10) The column was placed into a clean 1.5ml centrifuge tube, 50-100. Mu.l (depending on the desired final concentration) sterile deionized water or TE buffer was added to the filter, and the plasmid DNA was collected by centrifugation at 10000 Xg for 5 min. (11) Agarose gel electrophoresis was performed with a DNA sample (Marker) of a predetermined concentration, and the results were compared to obtain a plasmid concentration of 7X 19pLent-GCC-CAR of 360 ng/. Mu.l.

Example 5

An immune cell comprising a plasmid having a nucleic acid as described above. This example, the 7 × 19pLent-GCC-CAR plasmid purified as in example 4 was first lentivirally packaged and then immune cells were infected with recombinant lentiviruses. The immune cells are selected from autologous or transgenic T cells, NK cells, cytotoxic T lymphocytes or regulatory T cells, memory T cells, bispecific T cells, CIK cells.

The detailed steps are as follows:

(1) Lentiviral packaging and titer detection

The lentivirus packaging cell line 293T was inoculated into a 10cm dish containing DMEM +10% FBS, cultured at 37 ℃ and 5% CO2, and transfected after the adherence rate was 70% to 80%. The recombinant plasmid (about 10. Mu.g) and the empty plasmid (about 10. Mu.g) in example 1 were co-transfected with the lentiviral vector plasmid into 293T cells by calcium phosphate transfection, gently mixed, cultured in a 5% CO2 incubator at 37 ℃ for 12 hours, added with 8mL of 10% FBS-containing DMEM liquid medium, and cultured for up to 48 hours. After 48h, the expression of green fluorescent protein in the cells was observed under an inverted fluorescence microscope. After 72h, the supernatant was collected, cell debris was removed, and after harvesting the virus, the virus was concentrated to obtain concentrated GCC-CAR virus solution, which was stored in a low temperature refrigerator at-70 ℃ for further use. According to Lenti-X ^TM GoStix ^TM The kit (product of Beijing Huaxia ocean technology Co., ltd.) determines the virus titer, and the result shows that the titer of the recombinant lentivirus is 3.12 multiplied by 10 ⁶ pfu/mL。

(2) Preparation of T cells

50ml of patient autologous peripheral blood was taken and peripheral blood mononuclear cells were isolated using TBD sample density separation medium (purchased from Tianjin junction Biopsis). With a total volume of 1000IU/mlAfter 24 hours of induction culture in a culture medium (88-551-CM from CORNING corporation) containing recombinant interferon alpha 2a (from Shenyang Sansheng), 1000IU/ml of recombinant interleukin 2 (from Shenyang Sansheng), 50ng/ml of OKT-3 and 5% of autologous plasma of patients were added for further 24 hours of induction culture. Adding liquid at a double ratio every two days, culturing to day 14, detecting CD3 in T cells by flow cytometry ⁺ 、CD56 ⁺ Positive expression rate (CD 3-FITC, CD16/CD56-PE antibody from BECKMAN, A07735). CD3 ⁺ Positive rate>80％，CD3 ⁺ CD56 ⁺ Double positive rate>20% of the cells were regarded as successful T cell induction and were left to stand for viral infection.

(3) Lentiviral-infected T cells and expansion culture of CAR-T cells

After T cells were activated, 1X 10 cells were removed ⁶ Adding concentrated GCC-CAR virus solution into each cell, and uniformly mixing, wherein MOI =6. The transfected T cells were individually tested in association one week later. And detecting the expression of the chimeric antigen receptor by a fluorescence microscope, wherein the GFP-detected positive cells are positive cells expressing the chimeric antigen receptor due to the co-expression of the GFP and the CAR.

FIG. 3 shows the expression of 7X 19GCC-CAR vector constructed according to the present invention on the surface of T cells by flow cytometry. The results showed that 28.9% of the cells were positive for GFP (the lentiviral vector itself expresses GFP protein), indicating that 7 x 19GCC-CAR constructed using the invention is capable of being expressed on the surface of T cells with a transfection efficiency of 28.9%.

Example 6

CFSE-labeled CAR-T cells for detecting cell proliferation

7X 19CAR-T cells and CAR-T cells (anti-GCC CAR-T cells without addition of the immune factors IL7 and CCL 9) were labeled with CFSE (purchased from Invitrogen, USA) and placed at 5% CO ₂ And cultured in an incubator at 37 ℃. Cells were harvested on days 3 and 5/7, respectively, washed 2 times with flow buffer, surface stained, protected from light at 4 ℃ for 30min, washed 2 more times, resuspended, and the number of cells was measured by flow cytometry (FIG. 4).

FIG. 4 shows that by day 7, CAR-T cells expressing IL7 and CCL19 proliferated twice as many as normal CAR-T cells, and therefore, the addition of IL7 and CCL19 to CAR-T cells can promote T cell proliferation and enhance killing ability of CAR-T cells.

Example 7

Evaluation of anti-GCC-CAR T cell killing Effect

1. Killing experiment in vitro

Killing activity assays were performed by inoculating 100 μ L of the prepared 7 × 19CAR-T cells of the invention, CAR-T cells and unloaded lentivirus infected T cells as effector cells, the target cell being colon cancer cell line T84. Adding the effector cells to a 96-well culture plate at a ratio of the number of the effector cells to the number of the target cells of 5% by weight, adding 20ml of CCK-8 to each well after incubation in an incubator at 37 ℃ for 24 hours, and after further incubation for 2 hours, detecting the wavelength of 450nm by a microplate reader to read the OD value, the killing rate = [1- (test group OD value-effector cell control group OD value)/target cell control group OD value ]. Times.100%. T cells infected with unloaded lentivirus served as a control group. The killing efficiency of 7X 19CAR-T cells on GCC expression positive colon cancer cells T84 is as high as 95%, and the killing rate of CAR-T cells is less than 60%. Therefore, the specific killing activity of the 7X 19CAR-T cells is obviously higher than that of the CAR-T cell group and the control group, and the anti-GCC 7X 19CAR-T cells prepared by the invention have the capacity of efficiently killing GCC positive cells.

2. In vivo killing experiment

A male Wistar rat model (purchased from Guangzhou university of traditional Chinese medicine) with the age of 4 months is selected to be raised in an animal room (the room temperature is 23 +/-2 ℃, the humidity is 50% +/-10%), DMH is prepared into a solution with the concentration of 4mg/ml by using normal saline, the pH value is adjusted to be 6.5 by using 1mol/L NaHCO3 solution, and the mixture is filtered and sterilized for later use. Mice were intraperitoneally injected under sterile conditions at a dose of 21mg/kg once a week for 20 weeks to obtain colorectal cancer mice. Dividing the colorectal cancer mice into 4 groups, wherein the A group is the 7X 19GCC-CAR T cell treatment group prepared in example 3; panel B was the GCC-CAR T cell treatment panel prepared in example 3; group C was the untransfected T lymphocyte treatment group prepared in example 3; group D was normal saline control group. Group A was cultured on 7X 19GCC-CAR T cells to day 14 and 16, and injected into tail vein 2X 10 ⁶ Cells, group B, were cultured on GCC-CAR T cells to day 14 and 16, and injected in tail vein at 2X 10 ⁶ CellsGroup C was cultured on untransfected T lymphocytes to day 14 and day 16, and injected into tail vein at 2X 10 ⁶ Cells, group D, and groups a, B, and C were treated for the same time, and the tail vein was injected with the same volume of saline. After one month of continuous observation after treatment, neck-pulled sacrifice was performed at 7d, 14d, 21d and 28d, respectively, and mouse model tumor tissue of colorectal cancer was taken out and mouse model tumor size was calculated.

FIG. 4 is a graph of the change in tumor size after completion of treatment with murine model T lymphocytes, the size of the tumors in treatment groups A and B was reduced significantly in the placebo D group compared to the 7X 19GCC-CAR T cell treatment group A and the GCC-CAR T cell treatment group B, but the tumors in treatment group A were reduced significantly compared to treatment group B, and the tumors in group A eventually disappeared almost completely. Therefore, the improved 7X 19GCC-CAR T cells have strong killing effect on colorectal cancer cells, and the killing effect is remarkably superior to that of common CAR-T cells.

Therefore, the 7X 19GCC-CAR T cells provided by the invention have stronger killing capacity on tumors.

Example 8

Use of anti-GCC nucleic acid in the form of a kit for treating solid tumors including rectal, gastric, and esophageal cancer, the kit comprising

(1) Obtaining a vector stably expressing GCC-CAR as described above;

(2) A carrier diluent;

(3) Instructions for use, the instructions comprising a method as in example 7.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.

Sequence listing

<110> Shandong Xingyi Biotechnology Ltd

<120> nucleic acid against GCC, preparation method thereof, immune cell having the nucleic acid and use thereof

<130> 2018

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gggaaggggc tggagtggat tggggaaatc aatcatcgtg gaaacaccaa cgacaacccg 240

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tgtcagcagt ataaaacctg gcctcggacg ttcggccaag ggaccaacgt ggaaatcaaa 780

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 840

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 900

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 960

ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 1020

aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1080

tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1140

gcccccgcgt acaagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1200

gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1260

agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1320

gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1380

taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1440

ccccctcgcg aaggccgagg gagcctgctg acatgtggcg atgtggagga aaacccagga 1500

ccaatgttcc atgtttcttt taggtatatc tttggacttc ctcccctgat ccttgttctg 1560

ttgccagtag catcatctga ttgtgatatt gaaggtaaag atggcaaaca atatgagagt 1620

gttctaatgg tcagcatcga tcaattattg gacagcatga aagaaattgg tagcaattgc 1680

ctgaataatg aatttaactt ttttaaaaga catatctgtg atgctaataa ggaaggtatg 1740

tttttattcc gtgctgctcg caagttgagg caatttctta aaatgaatag cactggtgat 1800

tttgatctcc acttattaaa agtttcagaa ggcacaacaa tactgttgaa ctgcactggc 1860

caggttaaag gaagaaaacc agctgccctg ggtgaagccc aaccaacaaa gagtttggaa 1920

gaaaataaat ctttaaagga acagaaaaaa ctgaatgact tgtgtttcct aaagagacta 1980

ttacaagaga taaaaacttg ttggaataaa attttgatgg gcactaaaga acacgaaggc 2040

cgagggagcc tgctgacatg tggcgatgtg gaggaaaacc caggaccaat ggccctgcta 2100

ctggccctca gcctgctggt tctctggact tccccagccc caactctgag tggcaccaat 2160

gatgctgaag actgctgcct gtctgtgacc cagaaaccca tccctgggta catcgtgagg 2220

aacttccact accttctcat caaggatggc tgcagggtgc ctgctgtagt gttcaccaca 2280

ctgaggggcc gccagctctg tgcaccccca gaccagccct gggtagaacg catcatccag 2340

agactgcaga ggacctcagc caagatgaag cgccgcagca gttag 2385

<210> 2

<211> 57

<212> DNA

<213> Homo sapiens

<400> 2

atggactgga tttggagaat cctcttcttg gtgggagcag cgactggtgc ccactcc 57

<210> 3

<211> 780

<212> DNA

<213> Homo sapiens

<400> 3

atggactgga tttggagaat cctcttcttg gtgggagcag cgactggtgc ccactcccag 60

gtgcagctac agcagtgggg cgcaggactg ttgaagcctt cggagaccct gtccctcacc 120

tgcgctgtct ttggtgggtc cttcagtggt tactactgga gctggattcg ccagccccca 180

gggaaggggc tggagtggat tggggaaatc aatcatcgtg gaaacaccaa cgacaacccg 240

tccctcaaga gtcgagtcac catatcagta gacacgtcca agaaccagtt cgccctgaag 300

ctgagttctg tgaccgccgc ggacacggct gtttattact gtgcgagaga acgtggatac 360

acctacggta actttgacca ctggggccag ggaaccctgg tcaccgtctc ctcaggaggt 420

gggggcagtg gtggcggggg atctggaggt ggaggttccg aaatagtgat gacgcagtct 480

ccagccaccc tgtctgtgtc tccaggggaa agagccaccc tctcctgcag ggccagtcag 540

agtgttagca gaaacttagc ctggtatcag cagaaacctg gccaggctcc caggctcctc 600

atctatggtg catccaccag ggccactgga atcccagcca ggttcagtgg cagtgggtct 660

gggacagagt tcactctcac catcggcagc ctgcagtctg aagattttgc agtttattac 720

tgtcagcagt ataaaacctg gcctcggacg ttcggccaag ggaccaacgt ggaaatcaaa 780

<210> 4

<211> 135

<212> DNA

<213> Homo sapiens

<400> 4

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 5

<211> 72

<212> DNA

<213> Homo sapiens

<400> 5

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 6

<211> 126

<212> DNA

<213> Homo sapiens

<400> 6

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 7

<211> 336

<212> DNA

<213> Homo sapiens

<400> 7

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 8

<211> 54

<212> DNA

<213> Homo sapiens

<400> 8

gaaggccgag ggagcctgct gacatgtggc gatgtggagg aaaacccagg acca 54

<210> 9

<211> 531

<212> DNA

<213> Homo sapiens

<400> 9

atgttccatg tttcttttag gtatatcttt ggacttcctc ccctgatcct tgttctgttg 60

ccagtagcat catctgattg tgatattgaa ggtaaagatg gcaaacaata tgagagtgtt 120

ctaatggtca gcatcgatca attattggac agcatgaaag aaattggtag caattgcctg 180

aataatgaat ttaacttttt taaaagacat atctgtgatg ctaataagga aggtatgttt 240

ttattccgtg ctgctcgcaa gttgaggcaa tttcttaaaa tgaatagcac tggtgatttt 300

gatctccact tattaaaagt ttcagaaggc acaacaatac tgttgaactg cactggccag 360

gttaaaggaa gaaaaccagc tgccctgggt gaagcccaac caacaaagag tttggaagaa 420

aataaatctt taaaggaaca gaaaaaactg aatgacttgt gtttcctaaa gagactatta 480

caagagataa aaacttgttg gaataaaatt ttgatgggca ctaaagaaca c 531

<210> 10

<211> 294

<212> DNA

<213> Homo sapiens

<400> 10

atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60

agtggcacca atgatgctga agactgctgc ctgtctgtga cccagaaacc catccctggg 120

tacatcgtga ggaacttcca ctaccttctc atcaaggatg gctgcagggt gcctgctgta 180

gtgttcacca cactgagggg ccgccagctc tgtgcacccc cagaccagcc ctgggtagaa 240

cgcatcatcc agagactgca gaggacctca gccaagatga agcgccgcag cagt 294

Claims

1. A nucleic acid that is resistant to GCC, comprising:

which comprises sequentially arranged

A Leader nucleotide sequence as set forth in SEQ ID No. 2;

a hinge region nucleotide sequence of CD8 as set forth in SEQ ID No. 4;

the CD8 transmembrane region nucleic acid artificial sequence as set forth in SEQ ID NO. 5;

the chemokine IL7 nucleotide sequence as described in SEQ ID NO. 9;

the chemokine CCL19 nucleotide sequence as set forth in SEQ ID NO. 10;

the nucleotide sequence of the anti-GCC nucleic acid is shown in SEQ ID NO. 1.

2. A recombinant immune cell characterized by: a plasmid comprising the anti-GCC nucleic acid of claim 1.

3. The recombinant immune cell of claim 2, wherein: the immune cells are selected from autologous or transgenic T cells and NK cells.

4. The recombinant immune cell of claim 3, wherein: the immune cell is a cytotoxic T lymphocyte, regulatory T cell, memory T cell, bispecific T cell, or CIK cell.

5. Use of an anti-GCC nucleic acid according to claim 1 for the preparation of a medicament for the treatment of colon cancer.