CN110951695A

CN110951695A - Universal CAR-T cell, preparation method and application thereof

Info

Publication number: CN110951695A
Application number: CN201911407988.2A
Authority: CN
Inventors: 姜舒; 张芸; 熊斌; 王冰; 纪惜銮
Original assignee: Shenzhen Wingor Bio Technology Co ltd
Current assignee: Shenzhen Wingor Bio Technology Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-04-03

Abstract

The invention relates to a universal CAR-T cell, a preparation method and application thereof, wherein the preparation method of the universal CAR-T cell comprises the following steps of (1) respectively carrying out PCR amplification on pCDH, 3gRNA, EF1 α, GPC3CAR-1 and GPC3CAR-2 fragments by using amplification primers P1-P5, then sequentially connecting the obtained amplification fragments, carrying out PCR amplification on a connection product by using a primer P6, carrying out electrophoresis identification on the amplification product, transforming the amplification product to competent cells after the identification is correct, constructing and obtaining pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentiviral plasmid and packaging viral particles, and (2) infecting the amplified T lymphocyte, and introducing Cas9mRNA through electrical transformation to obtain the universal CAR.

Description

Universal CAR-T cell, preparation method and application thereof

Technical Field

The invention relates to CAR-T technology, in particular to a universal CAR-T cell, a preparation method and application thereof.

Background

CAR-T, a chimeric antigen receptor T cell immunotherapy. This is a new type of cell therapy that has been developed for many years, but has only been improved in recent years for clinical use. Similar to other immunotherapy, the basic principle is to use the patient's own immune cells to eliminate cancer cells, but in contrast, this is a cell therapy rather than a drug. The method comprises the steps of carrying out in vitro gene recombination on a single-chain antibody for recognizing tumor-associated antigens and an intracellular signal domain, namely an immunoreceptor tyrosine activation motif to construct a recombinant plasmid, then transfecting the recombinant plasmid into T cells of a patient in vitro by a transfection technology, enabling the T cells of the patient to express tumor antigen receptors, purifying and amplifying the T cells in a large scale after transfection, and obtaining the chimeric antigen receptor T cells (CAR-T cells).

The existing CAR-T cell preparation method has the problem of low killing efficiency caused by immune rejection.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a universal CAR-T cell, a preparation method and application thereof, which can reduce immune rejection and improve the killing efficiency of tumor cells.

In order to solve the technical problems, the invention adopts the technical scheme that:

a method for preparing a universal CAR-T cell, comprising the steps of:

(1) aiming at pCDH, 3gRNA, EF1 α, GPC3CAR-1 and GPC3CAR-2 fragments, respectively, sequentially designing amplification primers P1, P2, P3, P4 and P5, wherein the nucleotide sequences of the amplification primers are sequentially shown as SEQ ID No.1-2, SEQ ID No.3-4, SEQ ID No.5-6, SEQ ID No.7-8 and SEQ ID No. 9-10;

designing a primer P6, wherein the nucleotide sequence is shown as SEQ ID No.11-12 in sequence;

respectively carrying out PCR amplification on pCDH, 3gRNA, EF1 α, GPC3CAR-1 and GPC3CAR-2 fragments by using amplification primers P1, P2, P3, P4 and P5, and then sequentially connecting the amplified fragments obtained after the PCR amplification to obtain a connection product;

adopting a primer P6 to carry out PCR amplification on the ligation product, carrying out electrophoresis identification on the amplification product obtained after PCR amplification, transforming the amplification product into competent cells after successful identification, constructing and obtaining pLenti-3gRNA-EF1 α -GPC3CAR plasmid, and packaging to obtain pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus;

(2) separating T lymphocytes from peripheral blood mononuclear cells, activating and amplifying the separated T lymphocytes, then infecting the amplified T lymphocytes with pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus to obtain GPC3CAR-T cells, and introducing the GPC3CAR-T cells into Cas9mRNA through electric transformation to obtain the universal CAR-T cells.

The invention also relates to the universal CAR-T cell prepared by the preparation method of the universal CAR-T cell.

And the universal CAR-T cell is applied to preparing a medicament for treating or preventing cancer.

The invention has the beneficial effects that:

the pLenti-3gRNA-EF1 α -GPC3CAR plasmid is constructed, pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus is obtained by further packaging, the amplified T lymphocyte is infected by the pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus, Cas9mRNA is further introduced by electric transformation, and detection shows that the PD1 can be effectively knocked out by the obtained universal CAR-T cell, and the universal CAR-T cell has low immune rejection reaction and high tumor cell killing efficiency.

Drawings

FIG. 1 is a schematic structural diagram of a recombinant lentiviral plasmid pLenti-3gRNA-EF1 α -GPC3CAR in a method for preparing universal CAR-T cells according to an embodiment of the invention;

FIG. 2 is a map of the recombinant lentiviral plasmid pLenti-3gRNA-EF1 α -GPC3CAR in the method of preparing universal CAR-T cells according to the embodiment of the invention;

FIG. 3 is a ratio graph of PD1 expression rate of GPC3CAR-T cells flow-detected before PMA stimulation in a method of preparing universal CAR-T cells according to an embodiment of the present invention;

FIG. 4 is a ratio graph of PD1 expression rate of GPC3CAR-T cells flow-assayed after PMA stimulation in a method of making universal CAR-T cells according to embodiments of the invention;

FIG. 5 is a ratio chart of PD1 expression rate of flow-testing universal CART cells before PMA stimulation in the preparation method of universal CAR-T cells according to the embodiment of the present invention;

FIG. 6 is a ratio chart of PD1 expression rate of flow-test universal CART cells after PMA stimulation in the preparation method of universal CAR-T cells according to the embodiment of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

The most key concept of the invention is to construct and obtain pLenti-3gRNA-EF1 α -GPC3CAR plasmid.

The invention provides a preparation method of a universal CAR-T cell, which comprises the following steps:

(1) aiming at pCDH, 3gRNA, EF1 α, GPC3CAR-1 and GPC3CAR-2 fragments, respectively, sequentially designing amplification primers P1, P2, P3, P4 and P5, wherein the nucleotide sequences are sequentially shown as SEQ ID No.1-2, SEQ ID No.3-4, SEQ ID No.5-6, SEQ ID No.7-8 and SEQ ID No. 9-10;

adopting a primer P6 to carry out PCR amplification on the ligation product, carrying out electrophoresis identification on the amplification product obtained after PCR amplification, transforming the amplification product into competent cells after correct identification, constructing and obtaining pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus plasmid, and packaging to obtain pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus;

From the above description, the beneficial effects of the present invention are:

the pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus plasmid is constructed, virus particles are packaged, pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus is used for infecting amplified T lymphocytes, the amplified T lymphocytes are further introduced into Cas9mRNA through electric transformation, and detection shows that PD1 can be effectively knocked out by the obtained universal CAR-T cells, and the universal CAR-T cells have the advantages of low immune rejection and high tumor cell killing efficiency.

Further, in the step (1), the competent cell is an e.coli stbl4 competent cell.

Further, in step (1), the PCR amplification reaction system of the amplification primers P1, P2, P3, P4 and P5 on pCDH, 3gRNA, EF1 α, GPC3CAR-1 and GPC3CAR-2 fragments is 10 μ L of 5-fold PrimeSTAR Buffer, 4 μ L of 2.5mMdNTP mix, 1-50ng/μ L of template, 0.5 μ L of DNA polymerase, and 1 μ L of each nucleotide sequence of the amplification primers P1, P2, P3, P4 or P5, and distilled water is added to make the total volume 50 μ L;

in the step (1), the reaction conditions for PCR amplification of the amplification primer P1 are as follows: 10s at 98 ℃, 5s at 55 ℃, 6min at 72 ℃, 30 cycles, 30min at 16 ℃; the conditions for PCR amplification of the amplification primers P2, P3 and P4 are as follows: 10s at 98 ℃, 5s at 55 ℃, 90s at 72 ℃, 30 cycles, 30min at 16 ℃; the conditions for PCR amplification by the amplification primer P5 are as follows: 10s at 98 ℃, 5s at 68 ℃, 60s at 72 ℃, 30 cycles and 30min at 16 ℃.

Further, in step (1), the amplification product obtained was ligated in the reaction system of 100ng pCDH fragment, equimolar amounts of 3gRNA, EF1 α, GPC3CAR-1 and GPC3CAR-2 fragment, 5. mu.L of 2-fold Monad Clone Hi-fusion cloning Mix V2, and distilled water to a total volume of 10. mu.L.

Further, in the step (1), the reaction conditions for connecting the amplification product are as follows: the reaction is carried out for 1h at 50 ℃.

Further, in the step (2), the general CAR-T cells are obtained by specifically infecting the T lymphocytes with pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentiviruses on day 3 after T lymphocyte activation, adding polybrene to a final concentration of 5. mu.g/mL to prepare GPC3CAR-T cells, washing GPC3CAR-T cells at least twice with OPTI-MEM on day 4 after infection, then resuspending with OPTI-MEM to adjust the cell density to 1-3 x10⁸Per mL, obtaining a resuspension; 0.1mL of the resuspension solution is mixed with 20 μ g of Cas9mRNA, the mixed solution is used for electric transformation, the cells obtained after electric transformation are transferred to a preheated complete culture medium containing 300IU/mL of IL-2, and the culture and the amplification are continued until the 10 th day.

Furthermore, the invention also relates to the universal CAR-T cell prepared by the preparation method of the universal CAR-T cell;

Referring to fig. 1-6, the embodiments of the present invention are as follows:

the preparation method of the universal CAR-T cell of the embodiment is specifically described as follows:

firstly, a pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus plasmid (shown in figure 1) is constructed.

1. And designing a primer for template amplification.

The primers were designed as specifically shown in Table 1 below. And primers were synthesized by Suzhou Jinzhi Biotechnology, Inc.

TABLE 1

2. And (3) amplifying the template sequence.

And carrying out PCR amplification on the primer template, and identifying the PCR amplified fragment by agarose gel electrophoresis to obtain the correct fragment size. The amplification system and the amplification procedure were as follows:

the PCR reaction system is specifically shown in Table 2 below.

TABLE 2

See table 3 below for PCR reaction procedures.

TABLE 3

3. And (4) carrying out gel recovery and connection on the fragments after the electrophoresis is finished.

The ligation system and ligation procedure were as follows:

see table 4 below for fragment ligation system.

TABLE 4

And (3) connecting procedures: the reaction was carried out at 50 ℃ for 1 h.

Meanwhile, primers are designed to amplify the ligation products, and electrophoresis identification is carried out.

The primer sequences are as follows:

F:TAATAGCAACAGACATAC，(SEQ ID No.11)；

R:AACCAGGATTTATACAAG，(SEQ ID No.12)。

the identification result shows that the connection is successful.

4. Transforming the product to E.coli stbl4 competent cells, shaking the bacteria for about 16h, carrying out bacteria liquid PCR amplification, wherein the identification result shows that the plasmid transformation is successful, carrying out bacteria liquid sequencing, identifying the connection effect, wherein the sequencing result shows that the connection is correct, successfully constructing pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus plasmid (see figure 2), carrying out lentivirus packaging on the recombinant lentivirus plasmid, determining the titer, and storing at-80 ℃ for later use.

Production of Universal CAR-T cells

1. Isolation of T lymphocytes

50mL of peripheral blood is collected under aseptic conditions, and a blood sample is sent to a laboratory for separation of peripheral blood mononuclear cells. Peripheral blood was centrifuged at 2000rpm for l0min and upper autologous plasma was collected. The remaining blood was diluted and blown uniformly with 0.01mol/L PBS at a ratio of 1:1, mononuclear cells were separated using a lymphocyte separation medium, the cells were washed with PBS, the Cell pellet was resuspended in GTT551 serum-free medium containing 10% autologous plasma, and T lymphocytes were separated from peripheral blood mononuclear cells according to the protocol of Pan T Cell Isolation Kit II (Meitianni).

2. Expansion of T lymphocytes

And (3) adding CD3/CD28 antibody coupled magnetic beads into the separated T lymphocyte suspension to activate T cells, wherein the ratio of the CD3/CD28 antibody coupled magnetic beads to the T cells is 1:1-3:1 (preferably 3:1), adding the recombinant human IL-2 into the culture system to enable the final concentration to be 300IU/mL (300IU/mL-500IU/mL), and simultaneously adding the recombinant human IL-7 and the recombinant human IL-15 to enable the final concentration to be 6ng/mL (5ng/mL-8 ng/mL). The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator.

3. Preparation of Universal CAR-T cells

3 days after T lymphocyte activation, the T cells were infected with pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus, Polybrene was added at the time of infection to a final concentration of 5. mu.g/mL, and GPC3CAR-T cells were prepared, and on 4 days after virus infection, GPC3CAR-T cells were washed three times with OPTI-MEM and resuspended in OPTI-MEM to adjust the cell density to 1-3 x10⁸and/mL, 0.1mL is taken and mixed with 20 mu g of Cas9mRNA, the mixed solution is sucked by an electric transfer gun and gently inserted into an electric transfer cup, after the electric transfer is finished, the cells are transferred into a preheated complete culture medium containing IL-2 (the concentration is 300IU/mL), and the culture and the amplification are continued until the 10 th day. Flow cytometry examined CD3, HLA-1, PD1, CAR expression efficiency. Setting GPC3CAR-TCells were control group.

The results show that: CAR + cells were 90. + -. 4%, with CD3-HLA1-PD 1-cells accounting for 38. + -.5%.

4. Universal CART cell function detection

General-purpose CART cells and GPC3CAR-T cells the PD1 expression rate of CART cells was flow-tested 3 days after stimulation with 1:800 PMA.

The specific detection method of the PD1 expression rate of the CART cell comprises the following steps: collecting cell suspension, centrifuging, removing supernatant at 1000rpm for 10min, washing cell precipitate with 4 deg.C pre-cooled 0.01mol/L PBS for 2 times at 1000rpm for 10min, re-suspending the cell precipitate with 200 μ LPBS, counting cells, and collecting 1 × 10⁶The individual cells were resuspended in 0.1mL of pre-cooled 0.01mol/L PBS, PD1 fluorescent antibody was added in the dark, incubated for 30min in the dark at 4 ℃, 2mL PBS was added, L000 rpm, L0min, the cells were washed to remove unbound antibody, the supernatant was removed, the cell pellet was resuspended in PBS, checked at 4 ℃ and examined by flow cytometry. The results are shown in FIGS. 3-6.

In particular, FIG. 3 shows GPC3CAR-T cell (pre-PMA stimulation) PD1 expression rates; FIG. 4 shows GPC3CAR-T cell (post PMA stimulation) PD1 expression rates; FIG. 5 shows the expression rate of PD1 in general CART cells (before PMA stimulation); FIG. 6 shows the expression rate of PD1 in general CART cells (after PMA stimulation).

By detecting and according to the graphs of 3-6, the expression rate of PD1 of GPC3CAR-T cells is up-regulated from 3.1% to 20.2% (P <0.05) after stimulation, and the expression rate of PD1 of general CART cells is up-regulated from 2.87% to 4.32% (P >0.05), the PD1 knockout is proved to be effective.

After the universal CART cells and GPC3CAR-T cells were stimulated with 1:800PMA for 3 days, the universal CART cells and GPC3CAR-T cells were co-cultured with GPC3+ HepG2-PDL1 stable cell lines for 24h, respectively, and cell killing efficiency and INF-gamma levels were measured.

The specific method for detecting the cell killing efficiency comprises the following steps: adjusting the density of effector cells (universal CART cells or GPC3CAR-T cells) and target cells (GPC3+ HepG2-PDL1 stable cell line) to be 1X10⁶Per mL, according to the effective target ratio of 1: 1. 3: 1. 6:1 adding effector cell suspension and target cell suspension into 96-well plate with total volume of 200uL, placing at 37 deg.C and 5% CO₂CulturingAfter 24h of culture in a box, adding 20 mu L of CCK-8 into each hole, continuing to incubate for 2h, detecting by using an enzyme labeling instrument, and reading the OD value at 450nm, wherein the killing rate is [ 1- (experiment group OD value-effector cell group OD value)/target cell group OD value ] 100%. The experiment is divided into a blank control group, a target cell group, an effector cell group and an experimental group (a universal CART cell group and a target cell mixed group, and GPC3CAR-T cells and a target cell mixed group).

The specific method for detecting the cytokine INF-gamma comprises the following steps: taking out the enzyme label plate coated with the antibody, setting a TMB (3,3',5,5' -Tetramethylbenzidine) blank developing hole, and sequentially adding 0.1mL of standard substance diluted according to a certain multiple and a sample diluted by a sample diluent. The plate was covered with a lid and reacted at 37 ℃ for 90 min. After the reaction, the liquid in the ELISA plate is blotted by an automatic plate washing machine. Biotin anti-human INF-gamma antibody working solution was added to each well in an amount of 0.1mL in turn (except for TMB blank color-developing wells), reacted at 37 ℃ for 60min, and washed 3 times with 0.01M PBS. ABC working solution was added to each well in an amount of 0.1mL in turn (except for TMB blank color developing wells), reacted at 37 ℃ for 30min, and washed 5 times with 0.01M PBS. Adding TMB color developing solution into each well in sequence at 90 μ L, reacting at 37 deg.C in dark for 20-25min, adding TMB stop solution into each well in sequence at 0.1mL, wherein the blue color changes to yellow immediately, and measuring OD value at 450nm with microplate reader. And subtracting the OD value of the blank hole from the OD value of the sample, drawing by taking the OD value and the concentration of the standard substance as XY axes, searching the INF-gamma concentration on a standard curve, multiplying the INF-gamma concentration by the dilution factor, and calculating the INF-gamma concentration in the sample.

The results show that: when the effective target ratio is 6:1, the killing rate of the universal CART cell to GPC3+ HepG2-PDL1 cells is 94 +/-3%, the killing rate of the GPC3CAR-T cell to GPC3+ HepG2-PDL1 cells is 20 +/-1.2%, and the two groups have obvious difference (P < 0.05). After the general-purpose CART cells and GPC3 CART cells are co-cultured with GPC3+ HepG2-PDL1 cells for 24h, INF-gamma levels are respectively (25300 +/-374) pg/mL and (4000 +/-185) pg/mL, and the two groups have significant difference (P < 0.05).

In conclusion, the preparation method of the universal CAR-T cell provided by the invention has the advantages of effective PD1 knockout, low immune rejection and high tumor cell killing efficiency.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Jun Yingguan Biotech Co., Ltd

<120> general type CAR-T cell, preparation method and application thereof

<130>2019

<160>12

<170>PatentIn version 3.5

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<213> Artificial sequence

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<213> Artificial sequence

<400>2

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<210>3

<211>37

<212>DNA

<213> Artificial sequence

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cgctattacc atggagggcc tatttcccat gattcct 37

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<211>44

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<400>4

agtccttttg tatgaattac tcaaggactg aacctccaac tagt 44

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<211>42

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<213> Artificial sequence

<400>5

ggaggttcag tccttgagta attcatacaa aaggactcgc cc 42

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<213> Artificial sequence

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<400>10

ccagaggttg attttagcga gggggcaggg cctg 34

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Claims

1. A method for producing a universal CAR-T cell, comprising the steps of:

(2) separating T lymphocytes from peripheral blood mononuclear cells, activating and amplifying the separated T lymphocytes, then infecting the amplified T lymphocytes with pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentivirus to obtain GPC3CAR-T cells, and introducing Cas9mRNA through electric transformation to obtain the universal CAR-T cells.

2. The method for producing a universal CAR-T cell according to claim 1, wherein the competent cell in the step (1) is e.

3. The method for preparing a universal CAR-T cell according to claim 1, wherein in step (1), the PCR amplification reaction system of the amplification primers P1, P2, P3, P4 and P5 is: mu.L of 5-fold PrimeSTAR Buffer, 4. mu.L of 2.5mM dNTP mix, 1-50 ng/. mu.L of template, 0.5. mu.L of DNA polymerase, and 1. mu.L of each nucleotide sequence of amplification primers P1, P2, P3, P4, or P5, and distilled water was added to a total volume of 50. mu.L;

4. The method for preparing a universal CAR-T cell according to claim 1, wherein the ligation reaction is performed in step (1) using 100ng of pCDH fragment, equimolar amounts of 3gRNA, EF1 α, GPC3CAR-1 and GPC3CAR-2 fragments, 5. mu.L of 2-fold Monad Clone Hi-Fusion Cloning Mix V2, and distilled water to a total volume of 10. mu.L.

5. The method for producing a universal CAR-T cell according to claim 4, wherein in the step (1), the reaction conditions for ligation of the ligation product are as follows: the reaction is carried out for 1h at 50 ℃.

6. The method for preparing a universal CAR-T cell according to claim 1, wherein the step (2) of infecting the expanded T lymphocytes with pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentiviruses comprises infecting the T lymphocytes with pLenti-3gRNA-EF1 α -GPC3CAR recombinant lentiviruses on day 3 after T lymphocyte activation, adding polybrene to a final concentration of 5 μ g/mL during infection, and preparing GPC3CAR-T cellsOn day 4 after staining, GPC3CAR-T cells were washed at least twice with OPTI-MEM, and then resuspended with OPTI-MEM at a cell density of 1-3 x10⁸Per mL, obtaining a resuspension; and mixing 0.1mL of the heavy suspension with 20 mu g of Cas9mRNA, performing electric transformation by using the mixed solution, transferring the cells obtained after the electric transformation into a preheated complete culture medium containing 300IU/mL of IL-2, and continuously culturing and amplifying until the 10 th day.

7. The universal CAR-T cell produced according to the method of any one of claims 1 to 6.

8. The use of a universal CAR-T cell according to claim 7 for the preparation of a medicament for the treatment or prevention of cancer.