CN110790842A

CN110790842A - FasL-CAR fusion protein, T cell for expressing fusion protein, and preparation method and application thereof

Info

Publication number: CN110790842A
Application number: CN201911167370.3A
Authority: CN
Inventors: 胡以国; 孙媛媛; 潘聪
Original assignee: Guizhou Kahchin Biologic Technology Co Ltd
Current assignee: Guizhou Kahchin Biologic Technology Co Ltd
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2020-02-14
Anticipated expiration: 2039-11-25
Also published as: CN110790842B

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a FasL-CAR fusion protein, a T cell containing the FasL-CAR fusion protein, and a preparation method and application thereof. The T cell expressing the FasL-CAR fusion protein has the property of enhancing the expression of the FasL protein, and the capability of inducing the apoptosis of tumor cells by the FasL/Fas signal pathway of the T cell is enhanced, so that the capability of killing the tumor cells by the CAR-T cell is enhanced, and the possibility of tumor recurrence after treatment is reduced. The FasL-CAR fusion protein and the T cell expressing the FasL-CAR fusion protein can be applied to the drugs for treating tumors, and an effective way for treating cancers can be provided.

Description

FasL-CAR fusion protein, T cell for expressing fusion protein, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a FasL-CAR fusion protein, a T cell for expressing the fusion protein, and a preparation method and application thereof.

Background

A Chimeric Antigen Receptor T Cell (CAR-T) immunotherapy is a high-efficiency and novel tumor immunotherapy, which utilizes the self immune T Cell of a patient to be modified in vitro by a genetic engineering technology and then to be infused back into the body to eliminate the tumor Cell. At present, most of the technologies for modifying and preparing CAR-T cells focus on changing the category of Chimeric Antigen Receptors (CAR) to enhance the ability of T cells to recognize tumor cells, and then using the tumor cell-clearing mechanism of T cells to kill tumor cells. The CAR consists of an intracellular signaling region (e.g., the CD3 zeta-activating region and the CD28/4-1BB costimulatory region), a transmembrane region (e.g., the CD8 transmembrane region), and an extracellular antigen-binding region. The extracellular antigen-binding region has a function of recognizing a specific tumor antigen (TAA), and is formed by connecting a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody against TAA. Once the extracellular antigen-binding domain of the CAR binds to TAA, T cells can be activated by an intracellular signaling domain to perform effector functions, and the activated T cells secrete perforin, granzyme and cytokines which act synergistically to kill tumor cells.

However, the CAR-T cells in the prior art, after recognizing and binding to tumor cells using CAR, only rely on the T cell's own mechanism to clear tumor cells, and their ability to kill tumor cells is low. In the treatment process, a large amount of chimeric antigen receptor T cells need to be delivered to a patient, or the CAR-T cell treatment is repeatedly carried out for multiple times, so that the tumor cells can be eliminated, and the existing CAR-T cell treatment has the disadvantages of poor effect, low efficiency, complex process and high cost; in addition, because the existing CAR-T cell tumor has low lethality, the tumor cells are not completely removed after tumor treatment by the technology, and a large risk of tumor recurrence exists.

Disclosure of Invention

The invention aims to provide a FasL-CAR fusion protein which has the function of enhancing the tumor killing capacity of T cells.

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

a FasL-CAR fusion protein comprising a FasL protein and a CAR protein; the CAR protein comprises a CD3zeta active region, the FasL protein comprises a cytoplasmic region; the cytoplasmic region is linked to the CD3zeta active region.

By adopting the technical scheme, the technical principle is as follows: FasL-CAR fusion proteins include the FasL protein and the CAR protein, both transmembrane proteins. T cells expressing the FasL-CAR fusion protein have stronger tumor killing ability. The CD3 zeta-activating region of the CAR protein is located inside the cell, and the cytoplasmic region of the FasL protein is located inside the cell.

When a T cell expressing a FasL-CAR fusion protein (i.e., CAR-FasL-T cell) is used for tumor therapy, the extracellular antigen binding region of the CAR protein recognizes and binds to a specific tumor antigen (TAA) on the tumor cell, and further activates the CD3zeta activation region of the CAR protein, activating the T cell to exert an effector function of tumor killing. When T cells are continuously activated by TAA, the surface of the T cells can express FasL protein, and after the FasL protein is combined with Fas on tumor cells, caspase-8 cascade reaction can be activated, so that the tumor cells are subjected to apoptosis. In the scheme, the CAR-FasL-T cell can express FasL-CAR fusion protein, so that the amount of FasL protein contained in the cell is increased, and the T cell can also synthesize a large amount of FasL protein without the combination of TAA and the T cell, thereby enhancing the induction of a FasL/Fas signal pathway on tumor cell apoptosis and increasing the killing capacity of the CAR-T cell on tumors. In addition, the cytoplasmic region of the FasL protein is connected with the CD3zeta activation region of the CAR protein, and after the FasL protein is activated by corresponding cytokines, the cytoplasmic region of the FasL protein has an activation effect on the CD3zeta activation region, so that the activity of the CD3zeta activation region is further increased, the activation effect on T cells is enhanced, and the killing effect of the T cells on tumor cells is further increased.

Has the advantages that:

(1) the CAR-FasL-T cells of the present protocol may express FasL-CAR fusion proteins, where the CAR protein portion serves to recognize and bind tumor cells, and the FasL protein portion serves to promote apoptosis of tumor cells. FasL protein is not only expressed when T cells are continuously activated by TAA, but also generated along with the expression of FasL-CAR fusion protein, so that the normalized chimeric FasL protein on T cell membranes greatly enhances the efficiency and speed of the FasL protein for inducing tumor cell apoptosis.

(2) In the process of attempting to modify T cells using the FasL gene, the inventors have attempted to prepare T cells expressing only the FasL protein, and T cells expressing the FasL protein and the CAR protein (both proteins are expressed separately, and no fusion protein is formed), but none of them has achieved a desired tumor-killing effect. However, after the cytoplasmic region of the FasL protein is connected with the CD3zeta activation region of the CAR protein (i.e., a fusion protein is formed), the FasL protein can not only perform the function of triggering tumor cell apoptosis, but also can further activate the CD3zeta activation region of the CAR protein in turn, thereby promoting the activation of T cells and further enhancing the tumor killing function of the T cells. In this protocol, fusion of the FasL and CAR proteins can interact and synergistically enhance the tumor killing ability of T cells, resulting in unexpected results.

(3) In the prior art, no attempt has been made to overexpress the FasL protein of T cells, since overexpression of FasL protein alone would result in the situation where the protein is not effectively attached to the T cell membrane, and is then released outside the cell. In addition to tumor cells, normal cells also can be combined into ligand Fas of FasL, and T cells over expressing FasL can cause massive apoptosis of normal cells, bring about greater adverse reaction and great damage to human bodies. The developer therefore does not consider overexpressing the FasL protein, let alone overexpressing the FasL protein simultaneously with other proteins. The FasL protein is connected to the CAR protein to form a fusion protein, the CAR protein is found to be capable of fixing the FasL on a T cell and not forming free FasL, the CAR protein is combined with a tumor cell in a targeted mode, the FasL is brought to the vicinity of the tumor cell by the recognition of a specific surface antigen of the tumor cell through the CAR, the apoptosis of the tumor cell is promoted, the FasL is not combined with a normal cell, and the probability of adverse reaction is reduced.

Further, the CAR protein further comprises a CD28/41BB co-stimulatory region, a CD8 transmembrane region, and an extracellular antigen binding region sequentially linked; the gene sequence of the extracellular antigen binding region is shown as SEQ ID NO. 1; the gene sequence of the CD8 transmembrane region is shown as SEQ ID NO. 2; the gene sequence of the CD28/41BB co-stimulation region is shown as SEQ ID NO. 3; the gene sequence of the CD3 zeta-activating region is shown in SEQ ID NO 4.

By adopting the technical scheme, the protein expressed by the sequence has the capacity of recognizing and combining tumor cells, and can activate T cells to play a tumor killing effect function. The CAR protein comprises an extracellular antigen binding region, a CD8 transmembrane region, a CD28/41BB costimulatory region, and a CD3zeta activating region which are sequentially linked. The CAR protein is embedded on the cell membrane of T cells through the CAR transmembrane region, the CD3 zeta-activating region is located inside the cell, and the extracellular antigen-binding region is located outside the cell.

Further, the FasL protein also comprises a sequence connecting a FasL transmembrane region and an extracellular region; the gene sequence of the FasL protein is shown as SEQ ID NO. 5.

By adopting the technical scheme, the protein expressed by the sequence has the functions of activating caspase-8 cascade reaction and causing tumor cell apoptosis. The FasL protein is a ligand of the Fas protein, and the FasL protein is a type II transmembrane glycoprotein and comprises a cytoplasmic region (positioned in a cell), a FasL transmembrane region and an extracellular region which are sequentially connected. The FasL protein is embedded on the cell membrane of the T cell through a FasL transmembrane region, a cytoplasmic region is positioned in the cell, and an extracellular region is positioned outside the cell.

Further, a T cell expressing a FasL-CAR fusion protein comprising a FasL protein and a CAR protein; the CAR protein comprises an extracellular antigen binding region, a CD8 transmembrane region, a CD28/41BB costimulatory region and a CD3zeta activation region which are sequentially connected; the FasL protein comprises a cytoplasmic region, a FasL transmembrane region and an extracellular region which are connected in sequence; the cytoplasmic region of the FasL protein is linked to the CD3 zeta-activating region of the CAR protein.

By adopting the technical scheme, the T cell (CAR-FasL-T cell) has the property of enhancing the expression of FasL protein, and the ability of inducing tumor cell apoptosis by the FasL/Fas signal pathway of the T cell is enhanced, so that the ability of killing the tumor cell by the CAR-T cell is enhanced, and the possibility of tumor recurrence after treatment is reduced. In addition, the FasL protein in the expressed FasL-CAR fusion protein not only can play a role in triggering tumor cell apoptosis, but also can further activate a CD3zeta activation region of the CAR protein, so that the activation of T cells is promoted, and the tumor killing function of the T cells is further enhanced.

Further, a method for producing a T cell expressing a FasL-CAR fusion protein, comprising the steps of:

(1) constructing an expression plasmid: connecting the gene of the FasL protein to a lentivirus expression vector connected with the gene of the CAR protein to obtain an expression plasmid; the expression plasmid is used for expressing FasL-CAR fusion protein;

(2) and (3) packaging the virus: co-transfecting 293T cells by using a packaging plasmid and the expression plasmid, then culturing the 293T cells, collecting cell culture medium supernatant, and concentrating the cell culture medium supernatant to obtain virus concentrated solution;

(3) t cell infection: co-infecting T cells with a virus concentrate and a pro-infective agent to obtain the T cells expressing the FasL-CAR fusion protein.

By adopting the technical scheme, the expression plasmid can synthesize FasL-CAR fusion protein in a suitable expression system (such as a protein expression system which transgenically enters T cells and utilizes the T cells); after the 293T cell is co-transfected, a packaged virus can be obtained, the virus can be used for infecting a target T cell, and a vector containing a FasL gene and a CAR gene is transferred into the target T cell.

The FasL-CAR fusion protein formed in the scheme has the functions of originally recognizing and combining tumor cells and starting an apoptosis pathway mediated by FasL-Fas of the tumor cells, and the FasL protein and the CAR protein which are connected together have a synergistic effect, so that the FasL protein can strengthen and activate a CD3zeta activation region of the CAR protein to further activate T cells, and the killing effect of the T cells on the tumor cells is increased. In addition, the scheme for constructing the expression plasmid is simple and easy, only the gene of the FasL protein and the gene of the CAR protein are connected to the same lentiviral vector, and the same promoter on the lentiviral vector is used, so that more complicated design and operation are not needed.

Further, in (1), the lentiviral expression vector is pLVX-IRES-ZsGreen 1; the gene sequence of the FasL-CAR fusion protein is shown as SEQ ID NO 6.

By adopting the technical scheme, after the FasL gene is inserted into the CAR gene, the cytoplasmic region of the FasL protein is connected with the CD3zeta activation region of the CAR protein; pLVX-IRES-ZsGreen1 is a common lentivirus expression vector, is easy to obtain and can be constructed into a stable expression plasmid.

Further, in (2), the method of concentrating the cell culture medium supernatant is: and centrifuging the cell culture medium supernatant to obtain a virus precipitate, and then re-suspending the virus precipitate by using a culture medium to obtain a virus concentrated solution.

By adopting the technical scheme, the infection rate of the virus to the T cells can be improved. Since T cells are difficult to infect, in order to improve the infection efficiency of viruses, the supernatant needs to be centrifuged, and then the viruses are concentrated to obtain a virus concentrated solution.

Further, in (3), the T cells are isolated from human peripheral blood, and the infection promoting agent comprises polybrene and 4-hydroxyethylpiperazine ethanesulfonic acid.

By adopting the technical scheme, the T cells are separated from the human peripheral blood, transformed and then input back to the human body for tumor treatment, so that the rejection reaction of the human body can be reduced, and the treatment effect is enhanced. Polybrene (polybrene) is a polycationic polymer commonly used in DNA transfection experiments of mammalian cells to enhance transfection success, and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) is a zwitterionic organic chemical buffer to maintain optimal pH for transfection.

Further, in (3), the T cells are treated with the T lymphocyte stimulating solution, and then the T cells are co-infected with the virus concentrate and the infection promoting agent; the T lymphocyte stimulating solution contains interleukin 2, a CD3 monoclonal antibody and a CD28 monoclonal antibody.

By adopting the technical scheme, in the culture process, the interleukin 2(IL-2), the CD3 monoclonal antibody (anti-CD3) and the CD28 monoclonal antibody (anti-CD28) in the T lymphocyte stimulating solution can ensure that other cells except the T cells are subjected to apoptosis, only the T cells survive, and the T cells with higher purity can be obtained.

Further, the FasL-CAR fusion protein is applied to the preparation of tumor treatment drugs.

By adopting the technical scheme, the T cell expressing the FasL-CAR fusion protein has stronger tumor cell killing capacity and can prevent tumor recurrence, and the FasL-CAR fusion protein (or the CAR-FasL T cell) can be applied to a medicament for treating tumor, thereby providing an effective way for treating cancer.

Drawings

FIG. 1 is a schematic representation of T cells prepared in example 1;

FIG. 2 is the electrophoretogram of pLVX-CAR-IRES-ZsGreen1 plasmid of example 1 after digestion;

FIG. 3 is an electrophoretogram of pLVX-CAR-FasL-IRES-ZsGreen1 plasmid of example 1 after digestion;

FIG. 4 is a fluorescence image of 293T cells transfected 24h in example 1;

FIG. 5 is a fluorescence image of 293T cells transfected 48h in example 1;

FIG. 6 shows the results of flow cytometry 48h after infection of T cells of example 1;

FIG. 7 shows the flow cytometry results 72h after infecting T cells of example 1;

FIG. 8 shows the results of immunofluorescence assay of Experimental example 1 (using T cells prepared in comparative example 1);

FIG. 9 shows the results of immunofluorescence assay of Experimental example 1 (using T cells prepared in example 1);

FIG. 10 shows the flow cytometry results of Experimental example 2 (using the T cells prepared in example 2);

FIG. 11 shows the flow cytometry results of Experimental example 2 (using the T cells prepared in example 1).

Detailed Description

Example 1: preparation of chimeric antigen receptor T cells that enhance the FasL/Fas signaling pathway

The schematic structure of the T cells (CAR-FasL-T cells) prepared in this example is shown in FIG. 1, in which VH and VL represent the heavy chain variable region and the light chain variable region of an antibody, respectively, the extracellular antigen-binding region includes the heavy chain variable region and the light chain variable region, and FasL represents the FasL protein. The CAR-FasL-T cells were constructed specifically as follows:

(I): constructing an expression plasmid:

the original lentiviral expression vector was pLVX-IRES-ZsGreen1 (product No.: 632187; Taori physicians & Tech Co., Ltd.). The pLVX-IRES-ZsGreen1 was engineered with the insertion of both the CAR gene and the FasL gene. The CAR gene includes an extracellular antigen-binding region gene (SEQ ID NO:1), a CD8 transmembrane region gene (SEQ ID NO:2), a CD28/41BB co-stimulatory region gene (SEQ ID NO:3), and a CD3zeta activating region gene (SEQ ID NO:4) which are sequentially linked. The sequence of the FasL gene is shown in SEQ ID NO: 5.

The CAR gene is inserted into pLVX-IRES-ZsGreen1 to obtain pLVX-CAR-IRES-ZsGreen1 plasmid vector. The specific operation mode is as follows: the synthesized CAR gene was cleaved with EcoR1 and Spe1 restriction enzymes, pLVX-IRES-ZsGreen1 was cleaved with EcoR1 and Xba1 restriction enzymes, and the CAR gene was ligated to pLVX-IRES-ZsGreen 1. Wherein, the CAR gene is followed by an Hpa1 enzyme cutting site, a His tag and a Pac1 enzyme cutting site. The pLVX-CAR-IRES-ZsGreen1 is cut by two restriction enzymes of EcoR1 and Pac1 to identify whether the CAR gene is connected or not, the identification result is shown in FIG. 2, and a band about 1650bp can be seen in agarose electrophoresis (a Lane on the left is DNAladeder, a Lane on the right is a band after plasmid vector cutting, and a band near the lower part of the Lane on the right is the CAR gene), which indicates that the pLVX-CAR-IRES-ZsGreen1 plasmid vector is constructed.

The FasL gene was then inserted into pLVX-CAR-IRES-ZsGreen1 to obtain pLVX-CAR-FasL-IRES-ZsGreen1 plasmid vector. The specific operation mode is as follows: the synthesized FasL gene is cut by using Hpa1 and Pac1 restriction enzymes, then pLVX-CAR-IRES-ZsGreen1 is cut by using Hpa1 and Pac1 restriction enzymes, and then the FasL gene is connected to pLVX-CAR-IRES-ZsGreen 1. In this, FasL is followed by a His tag. The pLVX-CAR-FasL-IRES-ZsGreen1 is cut by Hpa1 and Pac1 restriction enzymes to identify whether the FasL gene is connected or not, the identification result is shown in FIG. 3, and a band about 870bp (a left lane is a DNA ladder, a right lane is a band after plasmid vector cutting, and a band near the lower part of the right lane is the FasL gene) can be seen in agarose electrophoresis, which indicates that the pLVX-CAR-FasL-IRES-ZsGreen1 plasmid vector is constructed. The complete sequence of the CAR-FasL fusion gene integrated on pLVX-CAR-FasL-IRES-ZsGreen1 is shown in SEQ ID NO 6.

(II): and (3) packaging the virus:

1. 293T cell culture: the culture medium is DMEM culture medium, and 10% FBS is added. When the cells grow until the fusion rate reaches 80-90%, the cells need to be subjected to passage operation to expand the number of the cells and maintain a good growth state of the cells. At 37 deg.C, 5% CO₂And 90% relative humidity.

2. Plate paving: 293T cells are plated in a 10cm dish one night before virus packaging, so that the cell density reaches 80-90% when the virus is packaged. 1h before virus wrapping, the cell culture medium is changed into a DMEM medium (1:4000 added chloroquine), and the DMEM medium is placed into a cell incubator.

3. Plasmid addition: a mixture of viral plasmids and packaging plasmids (two packaging plasmids, psPAX2 and pMD 2G) was prepared, the composition and formulation are shown in Table 1.

Table 1: viral plasmids and packaging plasmid mixtures

4. The mixture of Table 1 was mixed with 500. mu.L of 2 XHBS and added dropwise to a 293T cell culture dish.

5. Liquid changing: the medium was changed to normal medium after 11h and the medium was changed continuously after 24 h.

6. Fluorescence detection: fluorescence was detected 24h and 48h after transfection of 293T cells, and the detection results are shown in FIGS. 4 and 5, which proved successful transfection.

7. Collecting viruses: collecting virus supernatants for three times at 36h, 48h and 60h respectively, and mixing well.

8. And (3) filtering: the supernatant was filtered through a 0.45 μm filter to remove cellular impurities and the like.

9. Freezing and storing the virus: freezing the packaged virus liquid in a refrigerator at the temperature of minus 80 ℃.

(III): isolation culture of primary human T cells

Collecting blood at room temperature, storing in anticoagulation container at room temperature (the storage time is not longer than 4 hr), separating at room temperature, and storing in 4 deg.C refrigerator for a long time. The separation tube (Cat #7121011, Dake, 15 mL/tube) was stored at 4-30 ℃ in the dark. When in use, the separation tubes are taken out, and each separation tube can separate 3mL-5mL of whole blood. The blood was poured into the separation tube and centrifuged at 800g at 20 ℃ for 15 min. The PBMC (human peripheral blood mononuclear cells) cell layer was aspirated, with the PBMC layer below the plasma. Then centrifuging at 1500rpm for 5min, discarding supernatant, adding 1mL of schizophyllic solution, adding 3mL of RPMI1640 medium for neutralization, centrifuging at 1500rpm for 5min, and removing cells. The cells were counted by washing once with 5mL of RPMI1640, and then centrifuged again at 1500rpm for 5min to extract the cells. The components and the proportion of the prepared T lymphocyte stimulating solution are shown in the table 2. Cells were resuspended in the prepared stimulation solution and cultured in six-well plates at 2 mL/well. Observing cell density under the lens, changing the stimulating solution every day, and continuously stimulating for three days. The fourth day the medium was changed to medium containing only IL 2.

Table 2: t lymphocyte stimulating liquid

Components	Storage concentration	Working concentration	Goods number
				Lymphocyte culture medium	/	90％	LONZA X-VIVO 15/04-418Q
FBS	/	10％	Gibco
				Penicillin streptomycin double antibody	100×	100U/mL
IL2(human)	10ug/mL；1000×	10ng/mL	Novoprotein
				Anti-CD3(human)	0.5mg/mL；2500×	0.2ug/mL	Biolegend/317301
Anti-CD28(human)	0.5mg/ml；2500×	0.2ug/mL	Biolegend/302901

(IV) viral infection of T cells

1. Spreading the stimulated T cells in six-well plate at a density of 2-4 × 10⁶One/well, prepare virus infection solution, reagents as table 3:

table 3: viral infection liquid composition

2. Plate centrifugation: 1000g, 37 ℃ for 90 min.

3. After the cell incubator is placed for 8-10h, the cell incubator is changed to a normal culture medium (the culture medium contains IL-2).

4. Fluorescence detection was performed on the transfected T cells at 48h and 72h, and the results are shown in FIGS. 6 and 7, demonstrating successful transfection.

Example 2: preparation of chimeric antigen receptor T cells (non-enhanced FasL/Fas Signaling pathway function, CAR-T cells)

This comparative example is basically the same as example 1, except that the expression vector is not ligated with FasL gene, and is a lentiviral expression vector carrying only the CAR gene (pLVX-CAR-IRES-ZsGreen1 plasmid vector).

Comparative example 1: preparation of empty plasmid-containing T cells

This comparative example is basically the same as example 1, except that the expression vector is a lentiviral expression vector not linked with the FasL gene and the CAR gene (pLVX-IRES-ZsGreen1 plasmid vector).

Experimental example 1: cellular immunofluorescence assay

The experimental method is as follows:

1. the slides were circled with a histochemical pen (the inner periphery was marked with a non-destaining marker) and the cell slides resuspended with a small amount of PBS, dried at 37 ℃ in an incubator and not washed.

2. Fixing with 10 times volume of 4% paraformaldehyde fixing solution at room temperature for 10min, and washing with PBS for 10 min.

3. Membrane breaking: the cells were disrupted with PBS containing 0.2% Triton X-100 for 20min at room temperature, then blocked with PBST solution containing 2% BSA for 50min at room temperature without washing, and the excess blocking solution was spun off and primary antibody was added directly.

4. His primary anti-staining: primary antibody was diluted with PBST containing 1% BSA, blocked overnight (not longer than 16h) in a wet box at 4 deg.C, and then PBST was rinsed for 15 min.

5. Secondary antibody staining: the corresponding secondary antibody was diluted with PBST containing 1% BSA, incubated for 1h at room temperature, and washed with PBST for 15 min. Note that: all subsequent processing steps were performed as dark as possible from the addition of the fluorescent secondary antibody.

6. 5 mu of DAPI is dripped to be incubated for 5min in a dark place, the specimen is stained with nuclei, and then the specimen is soaked and washed for 10min by PBS.

7. And sealing the cover glass by using a sealing fluid containing an anti-fluorescence quencher, and observing and collecting an image under a fluorescence microscope.

The results of the assay are shown in FIG. 8 and FIG. 9, where FIG. 8 is the T cell group (prepared in comparative example 1) and FIG. 9 is the CAR-FasL-T cell group (prepared in example 1). The experimental results show that: the CAR gene is expressed in a large amount in a CAR-FasL-T cell group, and the expression amount in the T cell group is very low, so that the successful construction of the CAR-FasL-T cell is proved, and the FasL can promote the proliferation of the T cell and the expression of CAR protein.

Experimental example 2: tumor cell killing experiment

The experimental method is as follows: daudi cells (human Burkitt's lymphoma cells) and T cells were plated at a ratio of 1:4 in 48-well plates at a Daudi cell density of 1X 10⁵And (4) culturing for 24 h. After 24h, cells were collected and the dead cell ratio was determined by flow.

The results of the experiments are shown in FIG. 10 and FIG. 11, where FIG. 10 is the CAR-T cell group (prepared in example 2) and FIG. 11 is the CAR-FasL-T cell group (prepared in example 1). Experimental results show that the killing capacity of the CAR-FasL-T cell group on cell tumor cells is far greater than that of the CAR-T cell group, the enhancement effect is not achieved by singly enhancing the expression of CAR protein or singly enhancing the expression of FasL, and the CAR protein and the FasL protein are mutually influenced in the CAR-FasL fusion protein, so that the apoptosis signal cascade is expanded, and the anti-tumor effect is synergistically enhanced. In addition, the CAR protein can immobilize FasL on the T cell surface, so that FasL is not released outside the cell, reducing non-specific killing. And the T cells can target the FasL/Fas interaction on the tumor cells through the combination of the CAR and the specific surface antigen of the tumor cells, thereby enhancing the anti-tumor effect.

The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

SEQUENCE LISTING

<110> Biotech Co., Ltd of Kanzhou Cheng Ping, Guizhou

<120> FasL-CAR fusion protein, T cell for expressing fusion protein, preparation method and application thereof

<130>2019.11.14

<160>6

<170>PatentIn version 3.5

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<400>5

cagcagccct tcaattaccc atatccccag atctactggg tggacagcag tgccagctct 60

ccctgggccc ctccaggcac agttcttccc tgtccaacct ctgtgcccag aaggcctggt 120

caaaggaggc caccaccacc accgccaccg ccaccactac cacctccgcc gccgccgcca 180

ccactgcctc cactaccgct gccacccctg aagaagagag ggaaccacag cacaggcctg 240

tgtctccttg tgatgttttt catggttctg gttgccttgg taggattggg cctggggatg 300

tttcagctct tccacctaca gaaggagctg gcagaactcc gagagtctac cagccagatg 360

cacacagcat catctttgga gaagcaaata ggccacccca gtccaccccc tgaaaaaaag 420

gagctgagga aagtggccca tttaacaggc aagtccaact caaggtccat gcctctggaa 480

tgggaagaca cctatggaat tgtcctgctt tctggagtga agtataagaa gggtggcctt 540

gtgatcaatg aaactgggct gtactttgta tattccaaag tatacttccg gggtcaatct 600

tgcaacaacc tgcccctgag ccacaaggtc tacatgagga actctaagta tccccaggat 660

ctggtgatga tggaggggaa gatgatgagc tactgcacta ctgggcagat gtgggcccgc 720

agcagctacc tgggggcagt gttcaatctt accagtgctg atcatttata tgtcaacgta 780

tctgagctct ctctggtcaa ttttgaggaa tctcagacgt ttttcggctt atataagctc 840

taa 843

<210>6

<211>2493

<212>DNA

<213> Artificial sequence

<400>6

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaagtgc agctgctcga gtctggggct gagctggtga ggcctgggtc ctcagtgaag 120

atttcctgca aggcttctgg ctatgcattc agtagctact ggatgaactg ggtgaagcag 180

aggcctggac agggtcttga gtggattgga cagatttggc ctggagatgg tgatactaac 240

tacaatggaa agttcaaggg taaagccact ctgactgcag acgaatcctc cagcacagcc 300

tacatgcaac tcagcagcct acgatctgag gactctgcgg tctattcttg tgcaagacgg 360

gagactacga cggtaggccg ttattactat gctatggact actggggcca agggaccacg 420

gtcaccggtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc agagctcgtg 480

ctcacccagt ctccagcttc tttggctgtg tctctagggc agagggccac catctcctgc 540

aaggccagcc aaagtgttga ttatgatggt gatagttatt tgaactggta ccaacagatt 600

ccaggacagc cacccaaact cctcatctat gatgcatcca atctagtttc tgggatccca 660

cccaggttta gtggcagtgg gtctgggaca gacttcaccc tcaacatcca tcctgtggag 720

aaggtggatg ctgcaaccta tcactgtcag caaagtactg aggatccgtg gacgttcggt 780

ggagggacca agctggaaat aaaacgttct agaaccacga cgccagcgcc gcgaccacca 840

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtcccggcca 900

gcggcggggg gcgcagtgca cacgaggggg ctggacaagc ccttttgggt gctggtggtg 960

gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat tattttctgg 1020

gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 1080

cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 1140

cgctcctctg ttgttaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1200

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1260

gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1320

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1380

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 1440

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1500

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1560

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1620

catcaccatc accatcacta agttaaccag cagcccttca attacccata tccccagatc 1680

tactgggtgg acagcagtgc cagctctccc tgggcccctc caggcacagt tcttccctgt 1740

ccaacctctg tgcccagaag gcctggtcaa aggaggccac caccaccacc gccaccgcca 1800

ccactaccac ctccgccgcc gccgccacca ctgcctccac taccgctgcc acccctgaag 1860

aagagaggga accacagcac aggcctgtgt ctccttgtga tgtttttcat ggttctggtt 1920

gccttggtag gattgggcct ggggatgttt cagctcttcc acctacagaa ggagctggca 1980

gaactccgag agtctaccag ccagatgcac acagcatcat ctttggagaa gcaaataggc 2040

caccccagtc caccccctga aaaaaaggag ctgaggaaag tggcccattt aacaggcaag 2100

tccaactcaa ggtccatgcc tctggaatgg gaagacacct atggaattgt cctgctttct 2160

ggagtgaagt ataagaaggg tggccttgtg atcaatgaaa ctgggctgta ctttgtatat 2220

tccaaagtat acttccgggg tcaatcttgc aacaacctgc ccctgagcca caaggtctac 2280

atgaggaact ctaagtatcc ccaggatctg gtgatgatgg aggggaagat gatgagctac 2340

tgcactactg ggcagatgtg ggcccgcagc agctacctgg gggcagtgtt caatcttacc 2400

agtgctgatc atttatatgt caacgtatct gagctctctc tggtcaattt tgaggaatct 2460

cagacgtttt tcggcttata taagctctaa taa 2493

Claims

1. A FasL-CAR fusion protein, wherein the FasL-CAR fusion protein comprises a FasL protein and a CAR protein; the CAR protein comprises a CD3zeta active region, the FasL protein comprises a cytoplasmic region; the cytoplasmic region is linked to the CD3zeta active region.

2. A FasL-CAR fusion protein according to claim 1, wherein said CAR protein further comprises an extracellular antigen binding region, a CD8 transmembrane region, and a CD28/41BB co-stimulatory region, sequentially linked; the gene sequence of the extracellular antigen binding region is shown as SEQ ID NO. 1; the gene sequence of the CD8 transmembrane region is shown as SEQ ID NO. 2; the gene sequence of the CD28/41BB co-stimulation region is shown as SEQ ID NO. 3; the gene sequence of the CD3 zeta-activating region is shown in SEQ ID NO 4.

3. A FasL-CAR fusion protein according to claim 2, further comprising sequentially joining a FasL transmembrane region and an extracellular region; the gene sequence of the FasL protein is shown as SEQ ID NO. 5.

4. A T cell expressing a FasL-CAR fusion protein, wherein the FasL-CAR fusion protein comprises a FasL protein and a CAR protein; the CAR protein comprises an extracellular antigen binding region, a CD8 transmembrane region, a CD28/41BB costimulatory region and a CD3zeta activation region which are sequentially connected; the FasL protein comprises a cytoplasmic region, a FasL transmembrane region and an extracellular region which are connected in sequence; the cytoplasmic region of the FasL protein is linked to the CD3 zeta-activating region of the CAR protein.

5. The method of claim 4, comprising the steps of:

6. The production method according to claim 5, wherein in (1), the lentiviral expression vector is pLVX-IRES-ZsGreen 1; the gene sequence of the FasL-CAR fusion protein is shown as SEQ ID NO 6.

7. The production method according to claim 4, wherein in (2), the method of concentrating the cell culture medium supernatant is: and centrifuging the cell culture medium supernatant to obtain a virus precipitate, and then re-suspending the virus precipitate by using a culture medium to obtain a virus concentrated solution.

8. The method according to claim 4, wherein in (3), the T cells are isolated from human peripheral blood, and the pro-infection agent comprises polybrene and 4-hydroxyethylpiperazine ethanesulfonic acid.

9. The method according to claim 8, wherein in (3), the T cells are treated with the T lymphocyte stimulating solution and then co-infected with the virus concentrate and the pro-infection agent; the T lymphocyte stimulating solution contains interleukin 2, a CD3 monoclonal antibody and a CD28 monoclonal antibody.

10. Use of a FasL-CAR fusion protein according to any of claims 1 to 3 for the preparation of a medicament for the treatment of tumors.