CN107254447B

CN107254447B - Anti AFP CAR-T cell and preparation method and application thereof

Info

Publication number: CN107254447B
Application number: CN201710406296.0A
Authority: CN
Inventors: 刘未斌
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-06-01
Filing date: 2017-06-01
Publication date: 2020-10-16
Anticipated expiration: 2037-06-01
Also published as: CN107254447A

Abstract

The invention provides an Anti AFP CAR-T cell and a preparation method and application thereof, wherein the Anti AFP CAR-T cell expresses SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein in the T cell; in the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein, the SCFV is used for binding the AFP protein, and the amino acid sequence of the SCFV is a first sequence. The cell is obtained by modifying and transforming T cells, can specifically recognize and kill liver cancer, and is suitable for preventing and treating corresponding tumor diseases.

Description

Anti AFP CAR-T cell and preparation method and application thereof

Technical Field

The invention relates to an Anti AFP CAR-T cell and a preparation method and application thereof, belonging to the technical field of gene modified cells and tumor treatment.

Background

Primary liver cancer (HCC) occupies the fifth place of malignant tumor incidence, and the number of deaths per year is about 100 ten thousand worldwide. At present, most of primary liver cancer patients lack effective treatment means, and the total survival rate is only 3% -5%. Although liver transplantation has become an important means for treating primary liver cancer, donor shortage, surgical injury, immune rejection, high cost and other problems constitute major obstacles for the development of liver organ transplantation technology. Therefore, there is a need to explore a new and effective therapeutic approach. With the rapid development of molecular biology, genetic engineering techniques and immunology, immunotherapy-based biotherapy has shown good application prospects.

A Chimeric antigen receptor T cell (CAR-T cell for short) is characterized in that a single-chain variable region (SCFV for short) of a monoclonal antibody capable of specifically recognizing a target antigen is expressed on the surface of the cell by a gene modification means, and is coupled with an activation proliferation signal region in an artificially designed cell through a transmembrane region. The T cells expressing the CAR can directly recognize and combine with tumor-associated antigens on the surface of tumor cells through SCFV, the CAR transmits signals into the T cells, and the activated T cells secrete cytokines including perforin, granzyme, INF-gamma, TNF-alpha and the like, thereby playing a role in killing the tumor cells. Therefore, CAR-T cells are not restricted to Major Histocompatibility Complex (MHC), and the key to their use is to identify a tumor-associated antigen that is highly expressed on the surface of tumor cells and non-expressed or poorly expressed in normal tissues.

Alpha-fetoprotein (AFP) is a glycoprotein synthesized in the liver during fetal period, the level of serum is very low after an adult, and the malignant hepatocyte can restore the synthesis capability thereof, and has higher expression level in 70-80% of primary liver cancer (HCC). Research proves that the mouse hepatoma cell strain Hepa1-6 expressing the mAFP gene is inoculated to a C57BL/6J mouse, and then the mouse is immunized by plasmid DNA inserted with the AFP gene, and the result shows that the therapeutic immune AFP plasmid DNA can induce stronger specific CTL immune response. There have also been studies using immunodominant polypeptide vaccines derived from AFP to treat stage IV HCC patients, 5 of 6 patients showing that the AFP polypeptide is immunogenic in vivo and can stimulate antigen-specific T cells in patients with elevated serum AFP levels. Therefore, the AFP can be used as a specific antigen of the liver cancer cell and can be used as an ideal target point of CAR-T cell anti-tumor treatment.

Disclosure of Invention

The invention provides an Anti AFP CAR-T cell and a preparation method and application thereof, the cell is obtained by modifying and transforming T cells, can specifically recognize and kill liver cancer cells with high AFP expression, and is suitable for preventing and treating corresponding tumor diseases.

The invention provides an Anti AFP CAR-T cell expressing a SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein in a T cell; in the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein, the SCFV is used for binding the AFP protein, and the amino acid sequence of the SCFV is a first sequence which is:

EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYYTTYNPSFQGHVTISIDKSTNTAYLHWNSLAASDTAMYYCARYYVSLVDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPAKVSGSPGQKITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGAKSGNTASLTISGLQAEDEADYYCSSYTTGSRSVFGGGTKLTVLG

the Anti AFP CAR-T cell takes AFP as a target, specifically, the chimeric antigen receptor CAR of the Anti AFP CAR-T cell comprises an extracellular antigen binding region, a hinge region and an intracellular signaling region, wherein the extracellular antigen binding region is SCFV, the hinge region is IgD, and the intracellular signaling region is CD 28-OX 40-CD 3 zeta. Fig. 1 is a schematic design diagram of Anti AFPCAR of the present invention, please refer to fig. 1.

Further, in the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein, the amino acid sequence of the IgD is a second sequence.

Specifically, the second sequence is:

TFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLAHPSLPPQRLMALREPAAQAPVKLSTNLLASSDPPEAA

further, in the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein, the amino acid sequence of the CD28 is a third sequence.

Specifically, the third sequence is:

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSALLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

further, in the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein, the amino acid sequence of OX40 is a fourth sequence, and the amino acid sequence of CD3 zeta is a fifth sequence.

Specifically, the fourth sequence is:

RRDQRLPPDSHKPPGGGSFRTPIQEEQADAHSTLAKI

specifically, the fifth sequence is:

RVKFSRSAEPPAYQQGQNQLYNELNLGRSEEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

furthermore, in the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein,

the amino acid sequence of the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein is a sixth sequence.

Specifically, the sixth sequence is:

EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTAMYYCARYYVSLVDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSPGQSITISCTGTSSKVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYKSSYTTGSRAVFGGGTKLTVLGTFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRALWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAAFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSAEPPAKQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

further, the Anti AFP CAR-T cells were obtained by infecting T lymphocytes with lentiviruses obtained by infecting 293T cells with a lentivirus expression vector carrying the SCFV-IgD-CD 28-OX 40-CD 3 ζ fusion protein.

The invention also provides a preparation method of any one of the Anti AFP CAR-T cells, which comprises the following steps:

1) construction of a lentiviral expression vector having the same

SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein;

2) infecting 293T cells with a lentiviral packaging plasmid and the lentiviral expression vector to obtain a lentivirus packaged with the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein;

3) isolating a T lymphocyte and infecting said T lymphocyte with said lentivirus to obtain said Anti AFP CAR-T cell expressing said SCFV-IgD-CD 28-OX 40-CD 3 ζ fusion protein within T cells of said Anti AFP CAR-T cell.

In the step 1), the gene of the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein is cloned to a lentivirus expression vector by a genetic engineering means, thereby completing the construction of the lentivirus expression vector. In step 2), 293T cells are infected by using the lentivirus expression vector obtained in step 1), and lentivirus packaged by SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein is obtained. In step 3), step (b) is performed

2) The lentivirus of (1) is expressed in human peripheral blood to isolate collected T lymphocytes to obtain CAR-T cells.

Further, before the step 1), synthesizing and amplifying the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein gene. Specific synthesis and amplification methods can be obtained according to the means of ordinary skill in the art.

Further, the method comprises purifying the lentivirus and infecting the T lymphocyte with the purified lentivirus. Generally, the purification means may be performed using means conventional in the art, such as filtration, ultrafiltration, and the like.

The invention also provides application of any Anti AFP CAR-T cell in preparation of a liver cancer treatment drug, and particularly can be used for treatment of liver cancer with high expression of AFP molecules.

The scheme of the invention has the following advantages:

1. the Anti AFP CAR-T cell uses AFP antibody in the construction of CAR-T cell, and can effectively bind AFP protein.

2. The Anti AFP CAR-T cell preparation method provided by the invention can simply and effectively prepare the Anti AFP CAR-T cell with specific recognition and tumor killing functions.

3. The Anti AFP CAR-T cell has high-efficiency tumor killing activity, can be used for preparing a medicament for treating liver cancer, and particularly can be used for treating liver cancer with high AFP molecule expression by taking AFP molecules as target antigens.

Drawings

FIG. 1 is a schematic design diagram of an Anti AFP CAR of the present invention;

FIG. 2 is a schematic diagram showing the killing effect of Anti AFP CAR-T cells and uninfected T cells on target cell HepG-2 (liver cancer);

FIG. 3 is a schematic diagram showing the killing effect of Anti AFP CAR-T cells and uninfected T cells on target cell Hep3B (liver cancer);

FIG. 4 is a schematic diagram of the survival time of Anti AFP CAR-T cells, control example and blank example of mice treated with liver cancer (HepG-2) transplanted tumor model mice according to the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Preparation of Anti AFP CAR-T cells of the invention

1. Construction of Lentiviral expression vectors

Through a conventional genetic engineering means, a gene sequence of the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein is synthesized, the gene sequence of the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein is cloned to a lentivirus expression vector, and the SCFV-IgD-CD 28-OX 40-CD 3 zeta lentivirus expression vector pGreen puro-CAR is obtained. Wherein, the sequences of the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein are as described above.

2. Lentiviral packaging

1) The mass fraction of the bovine serum (total bovine serum, abbreviated as: FBS) in 1640 medium to culture 293T cells;

2) 293T cells at 3X 10⁵/cm²The cell confluency is transferred to a culture dish with the diameter of 15cm for culturing for 20 hours, and the cell confluency is ensured to be 80-90% during transfection;

replacing the culture medium with 1640 medium without serum for later use;

3) taking two EP tubes, adding 1ml 1640 into the two EP tubes respectively, and mixing the lentiviral vector pGreen puro-CAR with the pMDLg PRRE, pRSV-Rev and pMD2.G plasmids according to a molar ratio of 1: 1: 1: 1 mixing evenly in one EP tube; adding 150ul Lipo2000 into another EP tube, mixing well and standing for 5 min; adding 1640 mixed with lipo2000 into an EP tube containing plasmids, uniformly mixing to obtain a mixed solution, standing at room temperature for 20min, then dropwise adding the mixed solution into the cell culture dish prepared in the step 2), continuously culturing for 4h, replacing the culture solution with 1640 culture medium containing 10% FBS, continuously culturing for 48h, and collecting cell supernatant to obtain the lentivirus packaged by SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein.

3. Lentiviral purification

Filtering the packaged lentivirus obtained in 2 with 0.22 μm filter membrane, collecting the filtrate in 50ml ultrafiltration tube, and centrifuging at 3000g/min for 45 min; transferring the residual upper layer virus concentrated solution to an EP tube, and storing at-80 ℃ for later use.

4. Isolation of CD8+ T cells

1) Pouring the whole blood into a 50ml centrifuge tube, centrifuging for 20 minutes at room temperature, and controlling the centrifugal force ref to be 700g (common centrifugation);

2) taking the centrifuged lower cell component, and adding Du's phosphate buffer DPBS to 50 ml;

3) respectively adding 25ml of the liquid into 20ml of human lymphocyte separation liquid, centrifuging the centrifuge tube for 15 minutes at room temperature, and controlling the centrifugal force ref to be 800 g;

5) taking the leucoderma cells, adding DPBS, and complementing to 50 ml;

6) centrifuging for 10 minutes, controlling the centrifugal force ref to be 600g, and removing the supernatant to obtain peripheral blood single cells (periphytol blood monunicular cell, abbreviation: PBMCs);

7) CD8+ T cells were sorted using CD8+ T cell magnetic bead sorting kit.

5. Lentiviral vector infection of T lymphocytes

Culturing CD8+ T cells in RPMI 1640 complete culture medium with 10 wt% FBS, and adding anti-CD 3 monoclonal antibody for activation on the first day; adding 150MOI of purified lentivirus (Anti AFP CAR) in 3 on the third day, changing the culture medium into RPMI 1640 complete culture medium containing 50IU/ml recombinant human IL-2 after 16h, and continuously culturing for 10-20 days to obtain Anti AFP CAR-T cells.

Example 2

The Anti AFP CAR-T cells prepared in example 1 were evaluated for their Anti-tumor effect in vitro, comprising the following steps:

to stabilize the watchAFP-expressing hepatoma cell lines HepG-2 and Hep3B as target cells, effector cells were prepared with T cells infected with the lentiviral vector pGreen puro-CAR (i.e., Anti AFP CAR-T cells prepared in example 1) and uninfected T cells, respectively, and the target cells were prepared at a density of 1X 10⁵ Inoculating 100 μ l/ml 96-well plate, adding effector cells to target cells according to the effective target ratio of 5:1, 10:1, 20:1, placing in 5% CO₂Culturing at 37 deg.C for 4 hr, detecting cell activity by WST-1, and calculating killing efficiency.

FIG. 2 is a schematic diagram showing the killing effect of Anti AFP CAR-T cells and uninfected T cells on target cell HepG-2 (liver cancer); FIG. 3 is a schematic diagram showing the killing effect of Anti AFP CAR-T cells and uninfected T cells on target cell Hep3B (liver cancer).

The results in fig. 2 and 3 show that T lymphocytes expressing AFP CAR have a strong killing effect on AFP-positive liver cancer cells.

Example 3

The Anti-tumor effect of Anti AFP CAR-T cells prepared in example 1 was evaluated in vivo, comprising the following steps:

taking 15 female nude mice of 6 weeks old, injecting 5x 10 subcutaneous injection at right axilla⁶HepG-2 (liver cancer) cell, when the tumor grows to 60mm³The size and tumor model were randomly divided into 3 groups, i.e., control group, Anti AFP CAR-T cell group, and T cell group, wherein the control group was injected with normal saline 200 ul/time 2 times per week, and the Anti AFP CAR-T cell group was injected with Anti AFP CAR-T cell 1 × 10⁷One time per time, 2 times per week, T cell group tail vein injection T cell 1 × 10⁷2 times per week; and (5) counting the survival state of the mouse within 100 days, and making a survival rate curve. FIG. 4 is a schematic diagram of the survival time of Anti AFP CAR-T cells, control example and blank example of mice treated with liver cancer (HepG-2) transplanted tumor model mice according to the embodiment of the present invention.

The results in fig. 4 show that Anti AFP CAR-T cells can delay the survival of mouse model liver cancer transplantable tumors with high AFP expression relative to uninfected T cells and blanks.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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<120> Anti AFP CAR-T cell and preparation method and application thereof

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Claims

1. An Anti AFP CAR-T cell expressing a SCFV-IgD-CD 28-OX 40-CD 3 ζ fusion protein in a T cell;

in the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein, the SCFV is used for binding the AFP protein, and the amino acid sequence of the SCFV is a first sequence which is:

EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYYTTYNPSFQGHVTISIDKSTNTAYLHWNSLAASDTAMYYCARYYVSLVDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPAKVSGSPGQKITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGAKSGNTASLTISGLQAEDEADYYCSSYTTGSRSVFGGGTKLTVLG；

in the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein, the amino acid sequence of the IgD is a second sequence, the amino acid sequence of the CD28 is a third sequence, and the amino acid sequence of the OX40 is a fourth sequence; the amino acid sequence of CD3 ζ is a fifth sequence;

the second sequence is as follows:

TFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLAHPSLPPQRLMALREPAAQAPVKLSTNLLASSDPPEAA；

the third sequence is as follows:

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSALLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS；

the fourth sequence is as follows:

RRDQRLPPDSHKPPGGGSFRTPIQEEQADAHSTLAKI；

the fifth sequence is as follows:

RVKFSRSAEPPAYQQGQNQLYNELNLGRSEEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

2. the Anti AFP CAR-T cell according to claim 1, wherein in said SCFV-IgD-CD 28-OX 40-CD 3 ζ fusion protein,

the amino acid sequence of the SCFV-IgD-CD 28-OX 40-CD 3 zeta is a sixth sequence which is:

EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTAMYYCARYYVSLVDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSPGQSITISCTGTSSKVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYKSSYTTGSRAVFGGGTKLTVLGTFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRALWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAAFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSAEPPAKQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

3. an Anti AFP CAR-T cell according to any of claims 1-2 characterized in that said Anti AFPCAR-T cell is obtained by infecting a T lymphocyte with a lentivirus obtained by infecting 293T cells with a lentivirus expression vector carrying said SCFV-IgD-CD 28-OX 40-CD 3 ζ fusion protein.

4. A method of preparing Anti AFP CAR-T cells as claimed in any of claims 1 to 3 comprising the steps of:

1) constructing a lentivirus expression vector which has the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein;

5. The method of claim 4, further comprising synthesizing and amplifying the SCFV-IgD-CD 28-OX 40-CD 3 zeta fusion protein gene before the step 1).

6. Use of Anti AFP CAR-T cells according to any of claims 1 to 3 for the preparation of a medicament for the treatment of liver cancer.

7. The use of claim 6, wherein the liver cancer is a tumor with high expression of AFP molecules.