CN106636155A - Application of CD19-CAR gene sequence to malignant B cell tumors - Google Patents

Abstract

The invention belongs to the technical field of biological treatment of malignant tumors, and in particular relates to a CD19-CAR gene sequence and the application of the sequence in the treatment of malignant B cell tumors. The gene sequence is composed of the scFv fragment of the antibody gene targeting CD19 and the signal domain composed of CD28 and CD3 zeta fragments, with a total of 1635 bases, and its sequence is shown in the sequence table. The sequence can be used to construct a CD19-CAR-BTC carrier, which is applied to the treatment of malignant B-cell tumors. It has been tested in practice that the present invention uses CAR-T cell technology to specifically transform T cells, and further adoptively treats B-cell leukemia and lymphoma cells. Compared with traditional CIK cell immune adoptive therapy, it has strong and specific High degree of characteristics, so it has good promotion and application value.

Description

Application of CD19-CAR gene sequence in malignant B cell tumors

technical field

The invention belongs to the technical field of biological treatment of malignant tumors, and in particular relates to a CD19-CAR gene sequence and the application of the sequence in the treatment of malignant B cell tumors.

Background technique

Tumor biological immunotherapy is the fourth means of tumor treatment after surgery, chemotherapy and radiotherapy. Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell technology (CAR-T cell technology) is a new method of tumor biological immunotherapy that has achieved major breakthroughs in recent years. CAR-T cell technology is to introduce genetically engineered vectors that can specifically recognize tumor antigens into human immune cells (such as killer CD8+ T cells), so that this group of T cells can encode tumor-specific antigens, and then recognize tumor cells on the surface The receptor targets tumor cells for killing. Compared with previous cellular immunotherapy methods, CAR-T cell technology has the following advantages: First, the recognition of CAR-T cells to antigens does not depend on antigen processing and antigen presentation of HLA molecules, and immune escape tumor cells often have low expression The characteristics of HLA and proteasome antigen processing can also be recognized by CAR-T cells, so CAR-T cells have better targeting and killing of tumor cells; second, CAR-T cells can not only recognize cell surface The protein can also recognize the spatial structure of carbon chains and sugar chains on the cell surface, and has the characteristics of recognizing tumor cells more widely; third, in addition to the expression of specific antibodies on the surface of CAR-T cells, there is also the expression of co-stimulatory molecules , the expression of co-stimulatory molecules and the addition of cytokines can effectively promote the survival and activity of T cells, and improve the persistence of immunotherapy to a greater extent in clinical practice. At present, a series of animal experiments and clinical trials have proved the effectiveness and safety of CAR-T cells in the treatment of blood tumors and solid tumors. CAR-T cells have great application potential and development prospects in the clinical treatment of tumors. A weapon for curing tumors.

B-cell malignancies mainly include various types of leukemia and lymphoma. Except for a few patients receiving allogeneic hematopoietic stem cell transplantation, most adult patients with chronic lymphocytic leukemia and mantle lymphoma cannot be cured by the current commonly used clinical treatments. Therefore, the development of new therapeutic methods, especially biological immunotherapy, is extremely important for B cell malignancies.

Contents of the invention

The first purpose of the present invention is to provide a CD19-CAR gene sequence that can specifically recognize CD19 molecules on the surface of B cells after translation; Sequence chimerism into antigen receptor T cells for the treatment of B cell malignancies.

The technical scheme adopted by the present invention is as follows.

A CD19-CAR gene sequence that can specifically recognize the CD19 molecule on the surface of B cells, consisting of the scFv fragment of the CD19 gene and the signal domain composed of CD28 and CD3 zeta fragments, with a total of 1635 bases, and its base sequence is shown in SEQID Shown in NO.1.

Using the CD19-CAR gene sequence that can specifically recognize the CD19 molecule on the surface of B cells, a CD19-CAR-BTC vector can be constructed.

The preparation method of the CD19-CAR-BTC vector comprises the following steps:

(1) PCR amplification of CD19-CAR gene sequence;

During PCR amplification, the primer sequences were designed as follows:

The forward primer sequence is: 5'-GGACTAGTGCCACC ATGGCCTTACCAGTGACCGCCTTGC-3';

The reverse primer sequence is: 5'-CGGAATTCGCGAGGGGGCAGGGCCTGCATGTG-3';

PCR amplification was performed using the pUC-CD19 plasmid carrying the CD19-CAR gene sequence as a template;

(2) Double digestion of the CD19-CAR gene sequence amplified in step (1) with the vector plasmid pSin-EF2-Pur;

(3) Ligate the double digestion product in step (2) to construct the CD19-CAR-BTC vector.

The application of the CD19-CAR-BTC vector in B-cell malignancies, when applied, its specific application steps are as follows:

(1) T cell culture and transformation, collecting peripheral blood from patients with B cell malignant tumors, separating their T cells, and using T high-efficiency culture technology to rapidly proliferate T cells in vitro;

(2) Using lentiviral vector transfection technology, transfect CD19-CAR-BTC vector to construct chimeric antigen receptor T cells;

(3) Infuse the antigen receptor T cells constructed in step (2) into the blood of patients with B-cell malignancies, so as to achieve the purpose of curing the patients with B-cell malignancies.

Since the CD19 molecule is only expressed on the surface of B cells and not expressed in normal cells other than the B cell lineage, it is useful in the treatment of B lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. It is a better specific recognition antigen molecule in malignant tumors.

The present invention designs and constructs the CD19-CAR gene sequence that can specifically recognize the CD19 molecule on the surface of malignant tumors of B cells after expression. The CD19-CAR-BTC vector constructed by using the CD19-CAR gene sequence can specifically recognize Recognizes B-cell malignancy-related cells and makes them apoptotic, so as to achieve the purpose of cure.

In order to verify the technical effect of the CD19-CAR gene sequence provided by the present invention, the inventors used CAR-T cell technology to specifically transform T cells, and further compared the technical effect of traditional CIK cell immune adoptive therapy. The results are shown in Under all the conditions of effect-to-target ratio, the death rate of Raji cells co-cultured with CD19-CAR T cells was high, which proved that CD19-CAR T cells had a stronger killing effect on Raji cells than CIK, and had the characteristics of strong efficacy and high specificity , so it has good promotion and application value.

Description of drawings

Figure 1 is a schematic diagram of the sequence structure of the CD19-CAR gene that specifically recognizes the CD19 molecule on the surface of B cells;

Figure 2 is the detection result of the transfection efficiency by flow cytometry; the upper left picture is the total number of cells entering the cytometer, the upper right picture is the detection result, and the lower picture is the detection and judgment data;

Figure 3 is a comparison of the cytotoxicity of CD19-CAR T cells on Raji cells and the killing effect of traditional CIK cells (co-cultured for 4 hours).

detailed description

The present invention is further explained below in conjunction with embodiment.

Example 1

As shown in Figure 1, the CD19-CAR gene sequence that can specifically recognize the CD19 molecule on the surface of B cells provided by the present invention consists of the scFv fragment of the antibody gene targeting CD19 and the signal domain composed of CD28 and CD3 zeta fragments , with a total of 1635 bases, and its sequence is shown in the sequence listing (SEQ ID NO.1).

The sequence was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; before specific use, the sequence had been provided by the sequence synthesis provider to link the sequence with the plasmid pUC-CD19.

Example 2

According to the CD19-CAR gene sequence provided in Example 1, the construction of the CD19-CAR-BTC vector specifically includes the following steps.

(1) PCR amplification of CD19-CAR gene sequence

First, the primer sequences for PCR amplification were designed as follows:

The forward primer sequence is: 5'-GGACTAGTGCCACC ATGGCCTTACCAGTGACCGCCTTGC-3';

The reverse primer sequence is: 5'-CGGAATTCGCGAGGGGGCAGGGCCTGCATGTG-3';

Using the pUC-CD19 plasmid carrying the CD19-CAR gene sequence as a template, a 50 μL PCR reaction system was designed as follows:

Forward primer, 0.75µL (concentration: 10µmol/mL);

Reverse primer, 0.75µL (concentration: 10µmol/mL);

PrimeSTAR Max Premix (Bao Bioengineering (Dalian) Co., Ltd.), 25µL;

pUC-CD19 plasmid template carrying CD19-CAR gene sequence, 1.5µL (500ng/µL);

ddH2O, ₂₂ µL;

Reaction program: 94°C pre-denaturation for 3 minutes; 98°C, 10s, 55°C, 5s, 72°C, 10s, 35 cycles; 4°C extension for 10min.

Perform electrophoresis on the PCR amplified product, and recover the amplified product for future use.

(2) Carry out double digestion of the CD19-CAR gene sequence amplified in step (1) and the vector plasmid pSin-EF2-Pur, respectively;

The 20μL double enzyme digestion system is designed as follows:

10X M Buffer (Bao Bioengineering (Dalian) Co., Ltd.), 2µL;

Spe I enzyme (Bao Bioengineering (Dalian) Co., Ltd.), 1 µL;

EcoR I enzyme (Bao Bioengineering (Dalian) Co., Ltd.), 1 µL;

CD-19 CAR sequence (or pSin-EF2-Pur plasmid (Addgene)) amplified in step (1), 1 µg;

ddH ₂ O, make up to 20 µL;

Enzyme digestion at 37°C for 2h.

After digestion, the bands were separated by agarose gel electrophoresis, and the gel extraction kit (MiniBEST Agarose GelDNA Extraction Kit, Treasure Bioengineering (Dalian) Co., Ltd.) was used to recover the digested CD19-CAR sequence and linear pSin-EF2 respectively - Pur plasmid.

(3) Ligation of digested products

Ligate the digested product recovered in step (2), and design the 10 μL ligation system as follows:

DNA mixture (recovered product in step (2), molar ratio, pSin-EF2-Pur:CD19-CAR=1:5), 5 µL;

Solution I (Bao Bioengineering (Dalian) Co., Ltd.), 5µL;

Ligate overnight at 4°C.

(4) Screening of positive clones

Add 1 microliter of Solution II (Bao Bioengineering (Dalian) Co., Ltd.) to the ligation product in step (3), and then transform Escherichia coli DH5α competent cells;

The transformed strains were cultured in agarose medium containing ampicillin (1mg/100mL) at 37°C for 16h for resistance screening;

Select positive cloned strains for PCR identification and double enzyme digestion identification (refer to the above content for enzyme digestion system and PCR identification), and further sequence identification for the correctly identified strains, after further culturing the correctly identified strains, the plasmid can be extracted For standby, the extracted plasmid is the recombinant CD19-CAR-BTC vector carrying the CD19-CAR sequence.

Example 3

In order to test the specific application effect of the CD19-CAR-BTC vector prepared in Example 2 in the treatment of B-cell malignancies, the inventors conducted further in vitro tests, which are briefly introduced as follows.

(1) Collection of T cells

Collect 10 mL of peripheral blood from a healthy volunteer (provided by the First Affiliated Hospital of Zhengzhou University), centrifuge at 1500 rpm for 10 min (or according to the situation: 1500-2000 rpm, centrifuge for 7-10 min), discard the supernatant;

Rinse once with PBS buffer, discard the supernatant;

The precipitated cells were resuspended with 30 mL of PBS buffer, placed on 15 mL of lymphocyte separation medium, subjected to density gradient centrifugation, and mononuclear cells were collected and washed three times with PBS buffer for later use.

(2) Transfection of T cells

First, the helper cell 293T cells (ATCC cell bank) used for packaging and producing viruses were inoculated in a 6-well plate at a confluence of 85% (80-90% confluence can also be selected according to the situation), without antibiotics DMEM+10%FBS Culture medium, cultivated overnight at 37°C;

Secondly, 2 μg of the CD19-CAR-BTC vector prepared in Example 2 + 1.5 μg psPAX2 (Intrivogen) + 1.5 μg pMD2.G (Intrivogen) were transferred into 293T cells with Lipofectamin 2000 transfection reagent (Invitrogen) , transfected for 48 hours;

Third, after transfection, filter the cell suspension with a 0.45 μm filter membrane (Millopore Company), add PEG6000 to the cell culture supernatant at a ratio of 1 mL PEG6000 per 4 mL of cell supernatant, mix again, and place at 4 °C for 2 day, centrifuged at 8000 rpm for 30 minutes, and collected the virus pellet;

Fourth, inoculate the suspended T cells collected in step (1) at a concentration of 2×10 ⁵ /mL into a 6-well plate, add polybrene (polybrene, Sigma) to a concentration of 6 μg/mL, and at the same time add the above For the virus precipitate prepared in the third step, the amount of the virus precipitate was 100 µL of the virus solution with a titer of 10 ⁶ , mixed thoroughly, and incubated at 37°C for infection.

After 48 hours of infection, by flow cytometry (BD company FASCanto Flow cytometry) to detect GFP fluorescence to determine transfection efficiency. The principle is mainly to use fluorescently labeled CD19 molecules as bait to screen T cells that have expressed anti-CD19 antibody molecules on the cell surface (T cells that have been successfully transfected with CD19-CAR vectors will express anti-CD19 antibodies on the cell surface), calculate The formula is:

Transfection efficiency = number of fluorescently labeled cells/total number of cells entering the flow cytometer.

The test results are shown in Figure 2. Analysis of Figure 2 shows that the cells shown in phase P1 in the upper left picture of Figure 2 are the total number of living cells entering the flow cytometer, and the cells in P1 are further screened according to the fluorescence of the bound CD19 molecule, and the cells shown in phase Q4 are For the T cells successfully transfected with the CD19-CAR vector marked by the CD19 molecule, flow cytometry data analysis showed that the ratio of Q4 cells to the total number of living cells was 51.2%, that is, the transfection efficiency was 51.2%.

Fifth, the T cells transfected with CD19-CAR-BTC vector and the Raji cell line, Ramos cell line and K562 cell line of lymphoma cells in the tumor cells (the cell lines were all from the ATCC cell bank) were compared at a ratio of 0.5:1, respectively. , 2:1 and 10:1 volume ratio (the design principle of the volume ratio is based on the design principle of the same effect-to-target ratio) mixed culture for 48 hours (cultivated in a cell incubator at 37°C and 10% carbon dioxide); set GFP at the same time Control (GFP control refers to the GFP-BTC vector constructed by inserting GFP into the plasmid pSin-EF2-Pur instead of the CD19-CAR sequence when constructing the CD19-CAR-BTC vector).

The apoptosis ratio of tumor cells in different cell lines was detected by flow cytometry, and the specific detection results are shown in the table below:

.

It can be seen from the data in the above table that T cells after CD19-CAR-BTC transfection have good selectivity for CD19-positive B cells. Specifically, T cells transfected with CAR-CD19-BTC carrier had a significantly higher killing ability on CD19-positive cells Raji and Ramos than T cells transfected only with GFP control (increased apoptosis rate); while in CD19 Among negative K562 cells, the killing ability of CAR-CD19-BTC cells to K562 was not significantly different from that of control cells. Therefore, it can be shown from the above data that the CD19-CAR gene sequence and CD19-CAR-BTC vector provided by the present invention have good application prospects in the treatment of B cell malignant tumors.

In the prior art, the general steps followed in the treatment of B-cell malignancies by using chimeric antigen receptor T cell technology (CAR-T cell technology) are as follows:

(1) T cell culture and transformation, collecting peripheral blood from patients with B cell malignant tumors, separating their T cells, and using T high-efficiency culture technology to rapidly proliferate T cells in vitro;

(2) Use the lentiviral vector transfection technology to transfect the corresponding viral vector to construct chimeric antigen receptor T cells;

(3) Infuse the antigen receptor T cells constructed in step (2) into the blood of patients with B-cell malignancies, so as to achieve the purpose of curing the patients with B-cell malignancies.

Referring to the prior art method to detect the killing effect of CD19-CAR T cells on lymphoma, using traditional CIK cells as a control, CD19-CAR T cells and traditional CIK cells were respectively in the same ratio (100:1, 50: 1, 10:1, 5:1) were co-cultured with the lymphoma cell line Raji cells for 48 hours (cultivated in a cell incubator at 37°C and 10% carbon dioxide), and the killing process of Raji cells was detected by using the LDH cytotoxicity detection kit The activity of the marker lactate dehydrogenase produced in the CD-19 CAR T cells and CIK cells was compared to the killing effect of the Raji cell line. The experimental results are shown in Figure 3.

In Figure 3, the abscissa is the ratio of T cells transfected with CD19-CAR-BTC vector or traditional CIK cells to lymphoma Raji cells (effect-to-target ratio), and the ordinate is the ratio of killed Raji cells (toxicity rate ). When the effect-to-target ratio is 100:1, about 38% of Raji cells are killed by traditional CIK, about 62% of Raji cells are killed by CD19-CAR T cells, and the ratio of Raji cells killed by CD19-CAR T cells Significantly higher than the ratio of Raji cells killed by conventional CIK. Also at the effect-to-target ratios of 50:1, 10:1 and 5:1, the ratio of Raji cells killed by CD19-CAR T cells was higher than that of Raji cells killed by traditional CIK. This result proves that the killing effect of CD19-CAR T cells on tumor cells is better than that of traditional CIK cells. The CD19-CAR gene sequence provided by the present invention has the characteristics of strong potency and high specificity, and has better application value.

SEQUENCE LISTING

<110> The First Affiliated Hospital of Zhengzhou University

<120> Application of CD19-CAR gene sequence in malignant B cell tumors

<130> none

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1635

<212>DNA

<213> CD19-CAR

<400> 1

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtga aactgcagga gtcaggacct ggcctggtgg cgccctcaca gagcctgtcc 120

gtcacatgca ctgtctcagg ggtctcatta cccgactatg gtgtaagctg gattcgccag 180

cctccacgaa agggtctgga gtggctggga gtaatatggg gtagtgaaac cacatactat 240

aattcagctc tcaaatccag actgaccatc atcaaggaca actccaagag ccaagttttc 300

ttaaaaatga acagtctgca aactgatgac acagccattt actactgtgc caaacattat 360

tactacggtg gtagctatgc tatggactac tggggtcaag gaacctcagt caccgtctcc 420

tcaggtggag gcggttcagg cggaggtggc tctggcggtg gcggatcgga catccagatg 480

acacagacta catcctccct gtctgcctct ctgggagaca gagtcaccat cagttgcagg 540

gcaagtcagg acattagtaa atatttaaat tggtatcagc agaaaccaga tggaactgtt 600

aaactcctga tctaccatac atcaagatta cactcaggag tcccatcaag gttcagtggc 660

agtgggtctg gaacagatta ttctctcacc attagcaacc tggagcaaga agatattgcc 720

acttactttt gccaacaggg taatacgctt ccgtacacgt tcggaggggg gactaagttg 780

gaaataacac gggctgatgc tgcaccaact gtatccatct tccccaccatc cagtaataag 840

cccaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 900

ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgagggggg 960

ctggacttcg cctgcaaaat tgaagttatg tatccctcctc cttacctaga caatgagaag 1020

agcaatggaa ccattatcca tgtgaaaggg aaacaccttt gtccaagtcc cctatttccc 1080

ggaccttcta agcccttttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 1140

ttgctagtaa cagtggcctttattattttc tgggtgagga gtaagaggag caggctcctg 1200

cacagtgact acatgaacat gactccccgc cgccccgggc ccaccccgcaa gcattaccag 1260

ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1320

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1380

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1440

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1500

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1560

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1620

gccctgcccc ctcgc 1635

Claims (5)

1. A CD19-CAR gene sequence that can specifically recognize CD19 molecules on the surface of B cells, characterized in that the gene sequence consists of a scFv fragment of an antibody gene targeting CD19 and a signal domain consisting of CD28 and CD3 zeta fragments , a total of 1635 bases, and its sequence is shown in SEQ ID NO.1. 2. The CD19-CAR-BTC vector constructed by utilizing the CD19-CAR gene sequence of claim 1 that can specifically recognize the CD19 molecule on the surface of B cells. 3. The preparation method of the CD19-CAR-BTC carrier described in claim 2, is characterized in that, comprises the following steps: (1) PCR amplification of CD19-CAR gene sequence; (2) Double digestion of the CD19-CAR gene sequence amplified in step (1) with the vector plasmid pSin-EF2-Pur; (3) Ligate the double digestion product in step (2) to construct the CD19-CAR-BTC vector. 4. The method for preparing the CD19-CAR-BTC vector according to claim 3, characterized in that, in step (1), during PCR amplification, the primer sequence is designed as: The forward primer sequence is: 5'-GGACTAGTGCCACC ATGGCCTTACCAGTGACCGCCTTGC-3'; The reverse primer sequence is: 5'-CGGAATTCGCGAGGGGGCAGGGCCTGCATGTG-3'. 5. The application of the CD19-CAR-BTC vector according to claim 2 in malignant B cell tumors.