CN110093359B - Isolable nucleic acids containing CD3 promoter sequences and CAR sequences and applications - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to separable nucleic acid containing a CD3 promoter sequence and a CAR sequence and application thereof. The nucleic acid comprises a CD3 promoter sequence and a CAR sequence, wherein the CD3 promoter sequence is connected with the CAR sequence, the CAR sequence comprises an extracellular region sequence and an intracellular region sequence, the extracellular region sequence is connected with the intracellular region sequence, the extracellular region sequence is an anti-CD 19 immunoglobulin scFV fragment sequence, and the CD3 promoter sequence is SEQ ID NO. 14. The polypeptide coded by the nucleic acid is obtained according to the nucleic acid, a recombinant vector containing the nucleic acid, a recombinant cell containing the recombinant vector and application in pharmacy. The invention utilizes a T cell specific CD3 promoter to regulate the expression of Chimeric Antigen Receptor (CAR), thereby realizing the high-efficiency expression of the CAR.

Description

Isolable nucleic acids containing CD3 promoter sequences and CAR sequences and applications

Technical field

The present invention relates to the field of genetic engineering, specifically to a separable nucleic acid containing a CD3 promoter sequence and a CAR sequence and its application, and in particular to a separable nucleic acid, polypeptide, and recombinant vector containing a CD3 promoter sequence and a CAR sequence. Recombinant cells and their applications.

Background technique

CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy), the full name is Chimeric Antigen Receptor T-Cell Immunotherapy, is a new adoptive immune cell therapy developed in recent years, and is also recognized as a unique method that has the potential to completely conquer human cancer. method. CAR-T refers to extracting T lymphocytes from peripheral blood or umbilical cord blood and modifying them with exogenous genes so that the surface of the T cells expresses a single-chain antibody (scFv) that recognizes tumor-related antigens and a fusion protein of the T cell activation sequence. After expansion, the CAR-T cells are infused back into the patient's body. The CAR-T cells will also proliferate in the patient's body and kill the corresponding specificity in a non-MHC (major histocompatibility complex, major histocompatibility complex) restricted mode. tumor cells with sexual antigens.

However, CAR-T that is reinfused into patients through current methods is prone to gene expression silencing, which limits the application of CAR-T immunotherapy.

Contents of the invention

The present invention aims to solve one of the technical problems in the related art, at least to a certain extent. The present invention utilizes the T cell-specific CD3 promoter to carry the expression of the CAR gene to achieve efficient T cell expansion and killing of tumor cells. Compared with currently commonly used CAR vectors, it can improve the culture and preparation efficiency of CAR-T cells and reduce transgene silencing after T cell colonization.

This application is based on the inventor's discovery and understanding of the following facts and problems:

The extracellular domain of CAR is a single-chain fragment variable antibody (scFv, also known as CD19), which can specifically recognize the surface antigens of tumor cells. The transmembrane region connects the extracellular and intracellular domains, while the intracellular domain contains The co-stimulatory molecules CD3ζ (sometimes also called CD3 directly in the present invention) and 4-1BB (also called CD137, or 41BB), after T cells are activated by contact with specific antigens, show CAR-dependent killing, proliferation and cytokine release, and play a role in to tumor killing effect. Loading T cells with carriers carrying CAR can effectively improve the therapeutic effect of T cells. The principle is based on the special structure of CAR.

Based on the above principles, for example, transfection technology can be used to transfect T cells with viral vectors carrying CAR, which can effectively improve the therapeutic effect of T cells. The CMV promoter is often used in viral vectors, which is a universal mammalian promoter derived from viruses. The genes carried by the CMV promoter are prone to transgene silencing after completion of transgene and cell colonization, resulting in post-colonization CAR-T fails. To address this defect, measures include replacing the CMV promoter with strong promoters such as EF1α or actin. In addition, the French company Theravectys proposed using the human beta-2 microgobulin promoter to start the expression of the CAR gene, because this gene is expressed in APC and T There is strong expression in cells and NK cells, but such strong promoters generally lack lineage specificity of expression, which may cause the expression and stimulatory effect of CAR in other lineages (such as B cells).

The inventor of the present invention discovered during the research process that the expected effector cells of CAR-T are T cells, and T cells are all CD3 positive. According to this characteristic, according to the characteristics of T cells themselves, the CD3 promoter is cloned to carry the expression of the CAR gene, which not only ensures the specificity of CAR expression, but also avoids the occurrence of transgene silencing after T cell colonization, and in comparison Compared with other existing promoters, the promoter required for T cell expression is used to simultaneously start the expression of the C AR gene without introducing any foreign sequences and ensuring high efficiency.

To this end, according to one aspect of the invention, the invention provides an isolable nucleic acid, the nucleic acid has a CD3 promoter sequence and a CAR sequence, the CD3 promoter sequence is connected to the CAR sequence, wherein the The CAR sequence has an extracellular region sequence and a transmembrane/intracellular region sequence, the extracellular region sequence is connected to the transmembrane/intracellular region sequence, and the extracellular region sequence is an anti-CD19 immunoglobulin scFV fragment sequence, The anti-CD19 immunoglobulin scFV fragment sequence is SEQ ID NO: 10, the transmembrane/intracellular region sequence includes at least one selected from the group consisting of CD28 fragment sequence, 41BB fragment sequence and CD3 fragment sequence, and the CD3 initiates The subsequence is SEQ ID NO:14. According to an embodiment of the present invention, the present invention provides a nucleic acid sequence that contains both a CD3 promoter sequence and a CAR sequence. The CD3 promoter sequence can be used to initiate the expression of the CAR sequence, and the sequence can be introduced into T cells. , T cells can express CAR protein, thereby playing a tumor-killing role.

According to some embodiments of the present invention, the transmembrane/intracellular region sequence includes a CD28 sequence fragment, a 41BB sequence fragment and a CD3 sequence fragment, and the CD28 sequence fragment, the 41BB sequence fragment and the CD3 sequence fragment are connected. The transmembrane/intracellular region sequence in the CAR sequence contains a CD28 sequence fragment, a 41BB sequence fragment and a CD3 sequence fragment at the same time. It can express CD28 protein, 41BB protein and CD3 protein at the same time, and can show the tumor killing effect of CAR.

According to some embodiments of the present invention, the CD28 sequence fragment is SEQ ID NO:7, the 41BB sequence fragment is SEQ ID NO:8, and the CD3 sequence fragment is SEQ ID NO:9.

According to some embodiments of the invention, the CAR sequence is SEQ ID NO: 11.

According to some embodiments of the invention, the nucleic acid is DNA.

According to another aspect of the present invention, the present invention provides an isolable polypeptide encoded by the nucleic acid described in the above embodiments, and the polypeptide encodes a CAR protein.

According to another aspect of the present invention, the present invention provides a recombinant vector, which is a vector containing the nucleic acid described in the above embodiments.

According to some embodiments of the invention, the vector is a plasmid or viral vector.

According to some embodiments of the present invention, the viral vector is a retrovirus or a lentiviral vector, and the viral vector is selected from one of pHR-CMV-GFP, pBpLV or pBABE-puro.

According to some embodiments of the present invention, the plasmid is selected from one of pcDNA3.1, pSF-PromMCS-FLuc, or pBR322.

According to another aspect of the present invention, the present invention also provides a method for constructing the recombinant vector described in the above embodiment, including the following steps:

Amplify the CAR sequence and introduce the CAR sequence into the vector to obtain the first recombinant vector containing the CAR sequence,

The CD3 promoter sequence is amplified, and the CD3 promoter sequence is connected to the first recombinant vector to obtain the second recombinant vector, that is, a recombinant vector containing both the CD3 promoter sequence and the CAR sequence is constructed, Wherein, the CAR sequence includes an extracellular region sequence and a transmembrane/intracellular region sequence, the extracellular region sequence is connected to the intracellular region sequence, and the extracellular region sequence is an anti-CD19 immunoglobulin scFV fragment. Sequence, the transmembrane/intracellular region sequence includes at least one selected from the group consisting of CD28 fragment sequence, 41BB fragment sequence and CD3 fragment sequence, and the CD3 promoter sequence is SEQ ID NO: 14.

According to some embodiments of the present invention, the construction method further includes: amplifying the CD28 sequence fragment, the 41BB sequence fragment and the CD3 sequence fragment respectively, preparing a fusion sequence containing CD28, 41BB and CD3, and then amplifying the fusion sequence containing CD28, 41BB and CD3. The fusion sequence of CD3 is introduced into the vector to obtain a third recombinant vector containing the fusion sequence of CD28, 41BB and CD3. The anti-CD19 immunoglobulin scFV fragment sequence is connected to the third recombinant vector to prepare the first recombinant vector. Recombinant vector.

According to some embodiments of the present invention, the construction method further includes:

The CD28 sequence fragment was amplified using the primer sequences SEQ ID NO: 1 and SEQ ID NO: 2, the 41BB sequence fragment was amplified using the primer sequences SEQ ID NO: 3 and SEQ ID NO: 4, and the 41BB sequence fragment was amplified using the primer sequence SEQ ID NO: 5. and SEQ IDNO:6 were amplified to obtain a CD3 sequence fragment.

According to some embodiments of the present invention, the CD3 promoter sequence is amplified using the primer sequences SEQ ID NO: 12 and SEQ ID NO: 13.

According to some embodiments of the present invention, two of the CD28 sequence fragment, the 41BB sequence fragment and the CD3 sequence fragment partially overlap.

According to some embodiments of the present invention, the anti-CD19 immunoglobulin scFV fragment sequence and the third recombinant vector are digested using the same enzyme, and the anti-CD19 immunoglobulin scFV fragment sequence is connected to the third recombinant vector. on a triple recombinant vector.

According to some embodiments of the present invention, the CD3 promoter sequence and the first recombinant vector are digested using the same enzyme, and the CD3 promoter sequence is connected to the first recombinant vector.

According to another aspect of the present invention, the present invention provides a recombinant cell comprising the above-mentioned recombinant vector.

According to some embodiments of the invention, the recombinant cells are recombinant T cells.

According to another aspect of the present invention, the present invention also provides a method for constructing recombinant cells, including the following steps:

The recombinant vectors described in the above examples are introduced into recipient cells to construct recombinant cells.

According to some embodiments of the present invention, in the above method for constructing recombinant cells, the recipient cells are T cells. The recombinant vector containing the CAR sequence and CD3 promoter sequence is introduced into T cells to construct recombinant T cells. The recombinant T cells can use the CD3 promoter sequence to drive the expression of CAR protein, thereby achieving efficient expression of CAR protein, and because of the CD3 The protein is expressed positively in T cells, so gene silencing can be avoided.

According to another aspect of the present invention, the present invention also provides the use of a CD3 promoter sequence in the field of preparing drugs. The CD3 promoter sequence is SEQ ID NO: 14, and the drug is used to prevent or treat tumors.

According to some embodiments of the present invention, in the above-mentioned uses, the primer sequences used to amplify the CD3 promoter sequence are SEQ ID NO: 12 and SEQ ID NO: 13 respectively.

According to some embodiments of the present invention, in the above-mentioned uses, the drug is a T cell capable of expressing CAR protein (CAR-T cell).

According to another aspect of the present invention, the present invention also provides a drug, which is a T cell (CAR-T cell) capable of expressing CAR protein, and the CAR-T cell contains a CD3 promoter sequence.

According to some embodiments of the present invention, the medicine further includes primer sequences for amplifying the CD3 promoter sequence, which are SEQ ID NO: 12 and SEQ ID NO: 13 respectively.

The beneficial effects achieved by the present invention are: by providing an isolated nucleic acid sequence, the present invention uses the T cell-specific CD3 promoter to regulate the expression of foreign genes (CAR), thereby achieving efficient expression of CAR. On this basis, The virus constructed using this vector and the CAR-T cells cultured and prepared have a CD3 purity of more than 96%, and can still maintain a high proliferation rate on the 15th day of in vitro culture, and can still maintain a high external proliferation rate 14 days after transplantation. source gene (CAR) expression.

Description of the drawings

Figure 1 is a graph of expansion folds of CAR-T cells prepared according to embodiments of the present invention.

Figure 2 is an analysis diagram of surface markers of CAR-T cells prepared according to the embodiment of the present invention.

Figure 3 is a flow cytometry chart showing that T cells prepared according to the embodiment of the present invention were infected with the pHR-CD3p-CD19CAR packaging virus and positive for protein L binding was detected.

Figure 4 is a graph showing the killing efficiency of CAR-T cells against Raji cells under different E:T ratios prepared according to the embodiment of the present invention.

Detailed ways

Embodiments of the invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and are not to be construed as limiting the present invention.

isolable nucleic acid

According to one aspect of the invention, the invention provides an isolable nucleic acid, the nucleic acid includes a CD3 promoter sequence and a CAR sequence, the CD3 promoter sequence is connected to the CAR sequence, wherein the CAR sequence It contains an extracellular region sequence and a transmembrane/intracellular region sequence, the extracellular region sequence is connected to the transmembrane/intracellular region sequence, and the extracellular region sequence is an anti-CD19 immunoglobulin scFV fragment sequence, so The transmembrane/intracellular region sequence includes at least one selected from the group consisting of CD28 fragment sequence, 41BB fragment sequence and CD3 fragment sequence, and the CD3 promoter sequence is SEQ ID NO: 14.

In this article, the term "transmembrane/intracellular" region sequence can include nucleic acid sequence fragments encoding transmembrane region proteins and nucleic acid sequence fragments encoding intracellular region proteins, or it can also mean that the encoded protein has both transmembrane region and intracellular region. In this article, the term "fragment sequence" can also be expressed as "sequence fragment", which refers to part or all of the sequence encoding a certain protein, as long as the function of the protein is retained.

In this article, two nucleic acid sequences are connected not only refers to two nucleic acid sequences being directly connected, that is, the 3' end of one nucleic acid sequence is directly connected to the 5' end of another nucleic acid sequence, but also includes that the two nucleic acid sequences can be connected indirectly. Connection, that is, other nucleic acid sequences can be connected between two nucleic acid sequences, as long as it does not affect the expression of the functions of the two nucleic acid sequences. Moreover, except when describing that the CD3 promoter sequence is connected to other sequences, the CD3 promoter sequence is located at the front end of other sequences, that is, the CD3 promoter sequence is at the front end of the expression of other sequences and is used to regulate the expression of other sequences. Others, for example, when two nucleic acid sequences A and B are connected, the 3' end of A can be connected to the 5' end of B, or the 3' end of B can be connected to the 5' end of A. Specifically, for example, the CD3 promoter sequence and the CAR sequence can be directly connected, that is, the 3' end of the CD3 promoter sequence and the 5' end of the CAR sequence are directly connected; the CD3 promoter sequence and the CAR sequence can also be indirectly connected, that is, CD3 promoter sequence There can be other nucleic acid sequences between the subsequence and the CAR sequence, as long as it does not affect the function of the CD3 promoter and the expression of the CAR protein. For example, when the transmembrane/intracellular region sequence CD28 fragment sequence, 41BB fragment sequence or CD3 fragment sequence of the CAR sequence is connected, it can be in the form of CD28 fragment sequence-41BB fragment sequence-CD3 fragment sequence, or it can be 41BB fragment sequence-CD3 Fragment sequence - a form of CD28 fragment sequence.

In this article, CAR sequences include extracellular region sequences and transmembrane/intracellular region sequences, and can express intracellular polypeptides or proteins and extracellular polypeptides or proteins at the same time. The extracellular region sequence is an anti-CD19 immunoglobulin scFV fragment sequence, and the transmembrane/intracellular region sequence can be one, two or three of the CD28 fragment sequence, 41BB fragment sequence and CD3 fragment sequence.

In this article, the CD28 fragment sequence, 41BB fragment sequence or CD3 fragment sequence can be the full length of the CD28 sequence, 41BB sequence or CD3 sequence in the gene library, or it can be only a part of the sequence length, specifically it can be the full length 40% sequence length, 50% sequence length, 60% sequence length, 70% sequence length, 80% sequence length, 85% sequence length, 90% sequence length, 95% sequence length, as long as the fragment sequence can be complete It is sufficient to express the signal region fragments of CD28 polypeptide or protein, 41BB polypeptide or protein, CD3 polypeptide or protein, that is, it is sufficient to be able to completely express the functional region of each polypeptide or protein. According to some specific embodiments of the present invention, the CD28 fragment sequence is SEQ ID NO:7, and the SEQ ID NO:7 sequence accounts for about 46% of the full length of CD28, and the 41BB fragment sequence is SEQ ID NO:8, and the The CD3 fragment sequence is SEQ ID NO:9.

The term "nucleic acid" used in the present invention can be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA and RNA, and its length is not limited by any Special restrictions.

Recombinant vector

According to another aspect of the present invention, the present invention also provides a recombinant vector containing the above-mentioned nucleic acid. The term "recombinant vector" used in the present invention refers to a genetic vector that contains a specific nucleic acid sequence and can transfer the nucleic acid sequence of interest into a host cell to obtain recombinant cells. According to embodiments of the present invention, the form of the recombinant vector is not particularly limited. According to embodiments of the present invention, it may be at least one of plasmid, phage, artificial chromosome, cosmid (Cosmid), and virus, preferably plasmid and virus. As a genetic carrier, plasmids are simple to operate and can carry larger fragments, making them easy to operate and handle. The form of the plasmid is not particularly limited. It can be either a circular plasmid or a linear plasmid, that is, it can be single-stranded or double-stranded. The virus is easily transfected into recipient cells. Those skilled in the art can make selections as needed. For recombinant vectors used to construct recombinant cells, it is preferred that the nucleic acid is DNA, because DNA is more stable and easier to manipulate than RNA. According to some embodiments of the present invention, the viral vector is a retrovirus or a lentiviral vector, and the viral vector is selected from one of pHR-CMV-GFP, pBpLV or pBABE-puro; the plasmid is selected from pcDNA3. 1. One of pSF-PromMCS-FLuc or pBR322. Viral vectors can be used to transfect recipient cells, thereby expressing the CAR protein in the recipient cells and improving the expression efficiency of the CAR protein.

The present invention will be described below with reference to specific examples. It should be noted that these examples are only illustrative and should not be construed as limitations of the present invention. If not otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and can be carried out with reference to the third edition of "Molecular Cloning Experiment Guide" or related products. The reagents and products used are also available. Commercially acquired. Various processes and methods that are not described in detail are well-known conventional methods in the art. The source and trade name of the reagents used, as well as if it is necessary to list their components, are indicated when they first appear. The same reagents used thereafter will be used unless otherwise specified. Descriptions are the same as those first indicated.

Among them, the reagents, instruments and manufacturers used in the present invention are as follows:

T cells: prepared by induction from human umbilical cord blood mononuclear cells.

Raji cells: purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai.

K562 cells: purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai.

NOD/SCID mice: purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

293FT cells: purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai.

Human peripheral lymphocyte isolation medium: Tianjin Haoyang Company.

DEPC (purchased from Sigma), TRIzol (Invitrogen), RNA extraction related reagents (Sinopharm Chemical Reagent Co., Ltd.), ReverTra Ace qRCR RT Master Mix (purchased from TOYOBO), Taq DNApolymerase (Bio Labs), Reverse Transcriptase M-MLV kit (TaKaRa Company).

Cell Counting Kit-8 (CCK-8) cell proliferation-toxicity detection kit: DOJINDO, product number: #CK04-1000.

Lactate dehydrogenase release detection kit, LDH Cytotoxicity Assay Kit/LDH Release AssayKit: Biyuntian, product number: #C0017.

Antibodies: CD3-APC, CD8-PerCPCy5.5, and CD4-APC were purchased from eBioscience.

Embodiment 1

(1) Construction of fusion sequence and recombinant vector pHR-CMV-CD19CAR

The inventor constructed the fusion sequence CD19CAR, namely antiCD19-CD28-41BB-CD3 fusion sequence, and the recombinant vector pHR-CMV-CD19CAR. The specific steps are as follows:

1. Isolation of human peripheral blood lymphocytes

1) Extract 2 mL of peripheral blood from volunteers and add EDTA to a final concentration of 1.2 mg/mL for anticoagulation.

2) Add an equal volume (2mL) of PBS and mix well to obtain a cell suspension.

3) Add 4 mL of human lymphocyte separation solution that has been pre-equilibrated to room temperature in a 15 mL centrifuge tube. Slowly add the cell suspension on top of an equal amount of human lymphocyte separation solution. Be gentle and avoid shaking. Do not destroy the dividing line between the two. .

4) Centrifuge at the lowest acceleration and 1800 rpm for 25 minutes. After centrifugation, it can be seen that the liquid in the centrifuge tube is divided into 4 layers. From top to bottom, they are: clear buffer layer, turbid and cloudy lymphocyte layer, and clearer lymphocyte separation fluid. layer and layer of red blood cells;

5) Gently insert the dropper into the lymphocyte layer and rotate it to gently aspirate the lymphocyte layer. Transfer it to a 5mL centrifuge tube, fill it with PBS, mix by pipetting, and centrifuge at 1800 rpm for 5 minutes. Use the dropper to discard the supernatant, collect the cells, and separate. out lymphocytes.

2. Extraction and preparation of human lymphocyte cDNA

First, follow the steps below to extract RNA. Note that the pipette tips and EP tubes specially used for RNA extraction need to be treated with DEPC water in advance and soaked for more than 24 hours.

1) Collect an appropriate amount of cells into a 1.5ml EP tube, wash once with PBS, add 1ml Trizol to each tube of cells, pipette and grind to completely homogenize the cells.

2) Add 0.2 ml of chloroform, shake up and down for 15 seconds to mix thoroughly, let stand at room temperature for 10 minutes, then centrifuge at 4°C and 12,000 rpm for 15 minutes.

3) Carefully take out the EP tube. The liquid is divided into three layers from top to bottom, namely the clear water phase layer, the turbid protein layer, and the pink phenol/chloroform layer. Aspirate the upper aqueous phase and transfer it to a new 1.5 mL centrifuge tube.

4) Add 0.5 ml of pre-cooled isopropyl alcohol, mix gently by inverting, let stand at room temperature for 10 minutes, then centrifuge at 4°C and 12,000 rpm for 15 minutes.

5) Carefully discard the supernatant and keep the white flocculent precipitate at the bottom of the tube. Add 1 ml of 75% ethanol (75% absolute ethanol + 25% DEPC water), mix gently, and centrifuge at 4°C and 12,000 rpm for 15 min to wash the RNA.

6) Carefully discard the supernatant and place the centrifuge tube in a fume hood for 5-10 minutes until the white flocculent precipitate becomes transparent.

7) Add 10-30 μL DEPC water to the centrifuge tube to dissolve the RNA pellet. Use a UV spectrophotometer to measure the RNA concentration and absorbance. When OD260/OD280 is between 1.8-2.0, it means the RNA is of good purity and can be used for downstream reverse transcription experiments. The extracted RNA is at risk of being degraded by RNase, so it can be stored at -70°C and used within half a year.

Then perform the reverse transcription experiment according to the following steps, including the following steps:

a) Take 800ng of RNA template, add nuclease-free water to 16 μL, heat denature at 65°C for 5 minutes, and quickly cool on ice.

b) On ice, add 4 μL of reverse transcription PCR mix (ReverTra Ace qRCR RT Master Mix), mix the reaction solution gently, and centrifuge.

c) Proceed as follows

Top cover temperature: LID=104℃,

Reverse transcription reaction: 37℃15min, 50℃5min,

Enzyme inactivation reaction: 98℃5min,

Cool to 4°C.

d) After the reaction is completed, the resulting cDNA solution is stored at -20°C and can be used directly or diluted for PCR amplification reactions.

3. PCR amplification of CD28-41BB-CD3 fusion sequence

The following primers were artificially synthesized:

Use lymphocyte cDNA as a template, pair primers in pairs, use primers EcoRI-CD28-F and 4-1BB-CD28-R to amplify the CD28 coding fragment, use primers CD28-4-1BB-F and CD3-ζ-4- 1BB-R was amplified to obtain the 4-1BB coding fragment, and primers 4-1BB-CD3-ζ-F and KpnI-CD3-ζ-R were used to amplify the CD3-ζ coding fragment. Since each primer has set overlapping sequences, the CD28 coding fragment and the 4-1BB coding fragment are first mixed, and then denatured, annealed, and extended again to obtain the fusion sequence of CD28 and 4-1BB (CD28-4-1BB). The CD28-4-1BB fragment and the CD3-ζ fragment are mixed, denatured, annealed, and extended again to obtain the CD28-4-1BB-CD3-ζ fusion sequence (also called CD28-41BB-CD3 fusion sequence in the present invention, or CD28 -41BB-CD3 fusion fragment, all represent the sequence fragment).

The amplified CD28 sequence fragment is SEQ ID NO:7:

ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCC GGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

The amplified 41BB sequence fragment is SEQ ID NO:8:

CTCCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA

The amplified CD3 sequence fragment is SEQ ID NO:9:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT

The specific PCR reactions and fragment splicing reactions involved are as follows:

1) Configure the following system in a dedicated PCR tube, centrifuge and mix thoroughly

The PCR reaction system is as follows (total volume is 20.0 μL):

2) Place the sample in the real-time quantitative PCR instrument and proceed as follows,

Top cover temperature: LID=104℃,

Pre-denaturation: 95℃ 30s,

Amplification cycle: 95℃ for 30s, 58℃ for 30s, 72℃ for 30s, repeat 25-40 cycles according to the primer efficiency.

Enzyme inactivation reaction: 72℃5min,

Cool to 4°C.

3) Recovery and purification of PCR fragments: Use Axygen's AxyPrep PCR cleaning kit and follow the operating steps to complete the recovery and purification of PCR fragments.

4) PCR fragment splicing:

Configure the reaction solution according to the following system (total volume is 20.0 μL):

Place the sample in the real-time quantitative PCR machine and proceed as follows,

Top cover temperature: LID=104℃,

Pre-denaturation: 95℃ 30s,

Amplification cycle: 95℃ for 30s, 58℃ for 30s, 72℃ for 30s, repeat 25 cycles,

Enzyme inactivation reaction: 72℃5min,

Cool to 4°C.

The spliced fragments are then recovered by the kit and used for vector construction.

4. Construct antiCD19-CD28-41BB-CD3 fusion sequence vector

1) The CD28-41BB-CD3 fusion fragment was double digested by EcoRⅠ and KpnⅠ, and inserted into the pHRs1-Cla lentiviral vector digested and purified by the same enzyme to establish the pHR-CD28-41BB-CD3 vector. The steps for enzyme digestion system, agarose gel recovery, and recombinant plasmid construction are as follows.

(1) Enzyme digestion system

(2) Recover DNA fragments from agarose gel

a) Agarose gel electrophoresis, cut out the required fragments under UV light and place them in a 1.5mL EP tube;

b) Add 3 volumes of Buffer DE-A for the enzyme digestion system and mix evenly;

c) Add 0.5 volumes of Buffer DE-A to Buffer DE-B and mix evenly;

d) Aspirate the mixed solution in step (2), transfer it to a DNA preparation tube (place it in a 2ml centrifuge tube), centrifuge at 12,000×g for 1 minute, and discard the filtrate;

e) Put back into the 2ml centrifuge tube, add 500μl Buffer W1, centrifuge at 12,000×g for 30s, discard the filtrate;

f) Place the preparation tube back into the 2ml centrifuge tube, add 700μL Buffer W2, centrifuge at 12,000×g for 30 seconds, and discard the filtrate; wash once more with 700μL Buffer W2 in the same way and centrifuge at 12,000×g for 1 minute (washing with Buffer W2 twice can ensure The salt is completely removed, eliminating the impact on the enzyme digestion reaction);

g) Place the preparation tube back into the 2ml centrifuge tube and centrifuge at 12,000×g for 1 minute;

h) Place the preparation tube in a clean 1.5 ml centrifuge tube (provided in the kit), add 25-30 μl Elution buffer or deionized water to the center of the preparation membrane, let stand at room temperature for 1 min, centrifuge at 12,000×g for 1 min to elute the DNA ( Heating the Elutionbuffer or deionized water to 65°C will improve elution efficiency).

(3)Connect

Take 5 μL of the recovered fragment (100-600ng), add 2 μL of the recovered pHRS1-Cla vector (20-200ng), add 7 μl of solution I in the TaKaRa Quick Connection Kit, and ligate at 16°C for 120 minutes.

(4)Transformation of target plasmid

a) Dilute the target plasmid to 100ng/μL, add 1μL to an EP tube containing 30μl E. coli competent cell suspension (use 5μL for ligation product), mix gently, place on ice for 30 minutes, heat at 42°C Shock for 90 seconds, then place on ice for 2 minutes.

b) Add 800 μl of antibiotic-free liquid LB culture medium to each tube, and shake in a 37°C shaking box at 150 rpm for 45 minutes.

c) Add 200-300 μl bacterial solution to each agar plate, use a sterile elbow glass tube to evenly apply the bacterial solution on the surface of the agar plate containing antibiotics, place it horizontally at 37°C for 20 minutes, and then invert it at 37°C for 12 hours.

d) In a sterile large test tube, add 5 ml of LB medium and add corresponding antibiotics according to bacterial resistance.

e) Observe the colonies on the surface of the agar plate, select independent, plump and moderately sized colonies, pick them with a clean small pipette tip, put the small pipette tip into the corresponding large test tube, and put the test tube into the gas bath constant temperature shaking box. Fix firmly and shake at 200rpm and 37℃ for 12-14 hours.

(5) Extraction of plasmid

Follow the steps for purifying plasmid DNA by centrifugation in the product instructions of the plasmid miniprep kit. The specific steps are as follows:

a) Depending on the turbidity of the bacterial solution, take 1-4ml of the bacterial solution in the above large test tube, centrifuge at 12000g for 1 minute, and discard the supernatant.

b) Add 250 μl Buffer S1 to suspend the bacterial pellet, shake gently, and suspend evenly. There should be no small bacterial clumps.

c) Add 250 μL Buffer S2, mix gently and thoroughly by turning it up and down 4-6 times to fully lyse the bacteria until a clear solution is formed. This step should not exceed 5 minutes.

d) Add 350 μL Buffer S3, mix gently and thoroughly by turning it up and down 6-8 times, and centrifuge at 12000g for 10 min.

e) Aspirate the centrifugation supernatant in step 4 and transfer it to a preparation tube, centrifuge at 12000g for 1 minute, and discard the filtrate.

f) Place the preparation tube back into the centrifuge tube, add 500 μL Buffer W1, centrifuge at 12000g for 1 min, and discard the filtrate.

g) Place the preparation tube back into the centrifuge tube, add 700 μL Buffer W2, centrifuge at 12,000 g for 1 min, and discard the filtrate; repeat washing once with 700 μL Buffer W2 in the same way, and discard the filtrate.

h) Place the preparation tube back into the 2mL centrifuge tube and centrifuge at 12000g for 1 minute.

i) Move the preparation tube into a new 1.5 ml centrifuge tube, add 60-80 μl of Eluent preheated to 65°C in the center of the membrane of the preparation tube, let stand at room temperature for 1 minute, and centrifuge at 12000g for 1 minute.

j) Measure the concentration of plasmid using a spectrophotometer.

(5) Enzyme digestion identification of recombinant plasmid

Digest the recombinant plasmid according to the aforementioned double enzyme digestion system. After agarose gel electrophoresis, the correct recombinant plasmid can be cut out and the connected fragments can be stored at -20°C for subsequent experiments.

2) Refer to the literature (Construction and characterization of a functional CD19specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma.Nicholson IC, et al., Mol Immunol, 1997) to synthesize the following antiCD19 immunoglobulin scFv fragment (SEQ ID NO: 10 ), adding BamHI and EcoR I enzyme cutting sites and protective bases to its 5' and 3' ends respectively. The synthesized fragment was double digested by BamHI and EcoR I, and inserted into pHR- that had been digested and purified with the same enzyme. Among the CD28-41BB-CD3 vectors, the construction of the pHR-antiCD19-CD28-41BB-CD3 (pHR-CMV-CD19CAR) vector was completed. The steps for enzyme digestion system, agarose gel recovery, and recombinant plasmid construction are the same as above.

The sequence of the synthetic antiCD19 immunoglobulin scFv fragment is SEQ ID NO:10::

CGGGATCCATGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATTATT GCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAATGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCG ACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCATTCC AGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAGAATTCC

The corresponding CAR sequence SEQ ID NO:11 is as follows:

CGGGATCCATGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATTATT GCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAATGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCG ACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCATTCC AGAATCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAGAATTCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCT TTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCACCACCACGCGACTTCGCAGCCTATCGCTCCCTCCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAAGA GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGCCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTTAAGGTACCACA

(2) Construction of recombinant vector pHR-CD3p-CD19CAR

Using the recombinant vector pHR-CMV-CD19CAR constructed in step (1), further construct the recombinant vector pHR-CD3p-CD19CAR. The specific method is as follows:

1. Cloning of CD3 promoter (CD3p)

1) Extraction of total DNA from human umbilical cord blood mononuclear cells

Use Tiangen Biochemical Technology Company's blood genomic DNA extraction kit (DP318) to extract total DNA from human umbilical cord blood mononuclear cells. The specific steps are as follows:

a) Process blood materials: Take 200 μL of blood sample, add 400 μL of cell lysis solution CL, mix by inverting, centrifuge at 10,000 rpm for 1 min, aspirate the supernatant, and leave the nucleus to precipitate.

b) Add 20μl Proteinase K solution and mix well.

c) Add 200 μL of buffer GB, mix thoroughly by inverting, and place at 56°C for 10 minutes. Invert and mix several times during this period. The solution should be clear.

d) Add 200 μL of absolute ethanol and mix thoroughly by inverting. At this time, flocculent precipitation may appear.

e) Add the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (put the adsorption column CB3 into the collection tube),

f) Centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CB3 into the collection tube.

g) Add 500 μL buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column CB3 into the collection tube.

h) Add 600 μl of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column CB3 into the collection tube.

i) Repeat step 7.

j) Centrifuge at 12,000 rpm for 2 minutes and discard the waste liquid. Place the adsorption column CB3 at room temperature for a few minutes to completely dry out the remaining rinse liquid in the adsorption material.

k) Transfer the adsorption column CB3 into a 1.5ml centrifuge tube, drop 50-200μl elution buffer TB into the middle of the adsorption membrane, leave it at room temperature for 2-5min, centrifuge at 12,000rpm for 2min, and collect the solution into the centrifuge tube. That is, extracted genomic DNA.

2) Amplification of human CD3 promoter

Using the total DNA extracted from human umbilical cord blood mononuclear cells as a template, use the following primers to amplify the CD3 promoter of about 1.3 kb (located on chromosome 11). The upstream of the primer contains a Sac I enzyme cutting site, and the downstream primer adds a BamHI enzyme for cutting. site for further molecular cloning.

Fw： GAGCTCCTGGCCTCAAGC (SEQ ID NO:12)

Rw: GGATCC CCTGGAGGGGCCATGAAAAAT (SEQ ID NO: 13)

Among them, GAGCTC is the Sac I enzyme cleavage site, and GGATCC is the BamHI enzyme cleavage site.

The PCR reaction system is as follows (total volume is 20.0 μL):

Place the sample in the real-time quantitative PCR machine and proceed as follows,

Top cover temperature: LID=104℃,

Pre-denaturation: 95℃ 30s

Amplification cycle: 95℃ for 30s, 58℃ for 30s, 72℃ for 30s, repeat 35 cycles

Enzyme inactivation reaction: 72℃5min

Cool to 4°C.

The amplified CD3 promoter sequence SEQ ID NO:14 is:

GAGCTCCTGGCCTCAAGCAATCTGCCTGCTTTGCCTCCCAAAGTACCATATCTGGCGAATTTCTTATATTATTTCTTACACTCCTCGTTCTCCCTGTTACCTGCCGTGCTGGATTTTCCATTTCTCTTCCTCATCTCTGGACTTCAAACATGCTGCTGCCTCTATCTGGAATGCTTTCCACTCCACCTGGTTCATTGCTGTTCCTCCATCAAATTTCAGCTTAGATGTCACCTGCTCAGGGGGACCTTCTCTTCCCCATCCCACCACC CCAGACTATCTTAGATCCCTCTATCACTCCCACAGCACCTGAACGTCTTTTTTCTTAGCATCAATCTCCTGAAGTTATCTGGGGGGTTTTTTAGAGATGGGATCTCACTATGTTGCCTAGGCTGGAGCGCAGTGGCTATTCACAGGCACAATCACACTCACTGCAGTCTCGAACCCCTGGGCTCAGTCAGTCCTCCTGCCTCAGCCTCCTGAGTAGCTGGGAATACAGGTGTACACCATCATGCGGGGCATGGTTATT GGTTATGAGGTTATCTGTTTAATCTCCACATTTCCTTTTTTATCCAAGAGGACTATTTATCTTGTTTCTCCTGTATTACTAGCACCCAGCATATAACTACAGTACAGTAGACAGAAAATATGTATTAAATAAGTGAATTTCATCAAAATGTTTTCTAGAGTCCCAGCTCTGTAATCTGCAACTTAGATAACCTGCCTGAGCCTTAGTTTGCTCAAGTATAAAATGGAAATAAAAATAAAAATAATAACTTACCTTAGAGGGTCGTTTTGA GGATTAAATAAGATATATCAGAAAAGCTCTTAGAACGGTGCTTGGATATAGTAAGCATTTGATTAATGCATGCTTAAACATAAAGAAAGGTTACATCAATTCACACTCATACCAGCAGACTTTGACACTATCACTCTTTACCCTGTTCAACATTGAGTCTTGTGTTTTTAAATATTTTTGTAAAGAATATAGGTAAAAAGTGGCATTTTTTCTTTGGATTTAATTCTTATGGATTTAAGTCAACATGTATTTTCAAGCCACAAGTTTTTGT TAATAAGATGGCTGCACCCTGCTGCTCCATGCCAGATCCACCACACAGAAAGCAAATGTTCAGTGCATCTCCCTCTTCCTGTCAGAGGAAGGAAGACCCCGCAATGTGGAGGCATATTGTATTACAATTACTTTTAATGGCAAAAACTGCAGTTACTTTTGGTGCCAACCTACTACATGGTCTGGACAGCTAAATGTCATGTATTTTTCATGGCCCCTCCAGGGGATCC

3) The amplified fragment is ligated into the T vector

Prepare the ligation reaction according to the above system, incubate at 4°C overnight, then transform, select the positive colonies and extract the plasmid for the next step of vector construction.

2. Construction of lentiviral vector pHR-CD3p-CD19CAR

The recombinant vector pHR-CMV-CD19CAR prepared in step (1) contains Sac I and BamH I sites before and after the CMV promoter respectively. Therefore, the recombinant vector pHR-CMV-CD19CAR is double-digested using Sac I and BamH I enzymes. At the same time, the CD3 promoter prepared in the previous steps and connected to the T vector was also double digested with Sac I and BamH I enzymes, and the pHR-CD19CAR vector fragment and CD3 promoter fragment were recovered through agarose gel electrophoresis respectively, and further connected and transformed. , the lentiviral vector pHR-CD3p-CD19CAR was obtained. The methods involved in enzyme digestion, DNA fragment recovery, ligation, E. coli competent transformation, and plasmid extraction are the same as before.

Embodiment 2

1. Virus packaging

Mix lentiviral packaging plasmid and expression plasmid, transfect them into packaging cells 293FT with liposomes, change the medium 12 hours after transfection, collect the culture supernatant 48 hours later, and concentrate by high-speed centrifugation to obtain a virus stock solution.

The specific methods are as follows:

(1) Preparation of DNA-Lipofectamine ^TM 2000 complex

① Take a sterile 5 ml tube and add 1.5 mL of serum-free Opti-MEM, then add 4.2 μg pLP1, 2 μg pLP2, 2.8 μg Plp/VSVG and 5 μg pHR-CD3p-CD19CAR in sequence, mix gently and dilute.

② Take another 5mL tube, add 1.5mL of serum-free Opti-MEM medium, and add 42μL Lipofectamine ^TM 2000 (mix gently before use), mix it gently with Opti-MEM, and let it stand at room temperature for 5 minutes ( Lipofectamine ^TM 2000 should be mixed gently before pipetting in this process).

③After incubating at room temperature for 5 minutes, add the diluted plasmid to the diluted Lipofectamine ^TM 2000, mix gently, and incubate at room temperature for 20 minutes (this process should not exceed 20 minutes).

(2) During the formation of the DNA-Lipofectamine ^TM 2000 complex, use trypsin to digest 293T cells, count the cells, and resuspend the cells in growth medium containing serum (not containing antibiotics) to achieve a density is 1.2×10 ⁶ cells/mL.

(3) After the DNA-Lipofectamine ^TM 2000 complex is incubated for 20 minutes, add the complex to a cell culture dish containing 5 mL of growth medium (the medium cannot contain antibiotics).

(4) Add 5ml of 293T cell suspension (6 × 10 ⁶ cells in total) to the culture dish containing DNA-Lipofectamine ^TM 2000 complex and growth medium, gently move the culture dish back and forth to mix thoroughly, and then add the cells Place in a 37°C 5% _CO2 incubator overnight.

(5) After 12 hours, replace the medium containing DNA-Lipofectamine ^TM 2000 complex with 10 ml of complete medium containing sodium pyruvate. VSVG glycoprotein will fuse 293T to form multinucleated syncytia. This is a normal phenomenon and does not affect the production of lentivirus.

(6) 48 to 72 hours after plasmid transfection, collect the supernatant in a 15 ml sterile centrifuge tube. There is not much difference in the yield of virus supernatants collected 48 or 72 hours after transfection (the viruses that should be collected during this process are infectious, and care should be taken to protect them).

(7) After virus collection, centrifuge at 4°C and 3000rpm for 15 minutes, discard cell debris, and collect the supernatant. Then filter using a 0.45 μm filter.

(8) If the virus needs to be concentrated, use a Beckman SW41t rotor to centrifuge at 4°C and 26,000 rpm for 90 minutes to collect the precipitate. The virus is then resuspended in the culture medium used to transfect the cells. The collected virus can be aliquoted and stored at -80°C.

2. Isolation and culture of T cells

Use human lymphocyte separation fluid to isolate mononuclear cells from peripheral blood or umbilical cord blood for subsequent experiments. The specific method is as follows.

(1) Isolation of human umbilical cord blood mononuclear cells

1) Aseptically collect a portion of cord blood (100-150mL) from a full-term fetus after the umbilical cord is cut off, and use sodium citrate/heparin for anticoagulation.

2) Mix the cord blood with physiological saline in a ratio of 1:1, then mix it with 6.0% hydroxyethyl starch (HESpan) in a ratio of 4:1, and let it sit at room temperature for 30 minutes until the red blood cells naturally settle to the limit. Separately, precipitate red blood cells, collect the upper liquid and place it in a 50mL centrifuge tube. Use a 1mL pipette at the last 0.8cm to slowly suck it out.

3) Centrifuge the collected liquid at 1800 rpm for 15 minutes. After centrifugation is completed, discard the upper liquid.

4) Dilute the precipitate to 10 ml with physiological saline, and use Ficoll (which needs to be placed at room temperature in advance) to separate mononuclear cells (MNC). Take 5 ml of human lymphocyte separation liquid and add it to a 10 ml centrifuge tube. Slowly add the blood to be separated to the upper layer of the lymphocyte separation liquid along the tube wall so that the volume ratio of blood to separation liquid is about 1:1, 1800 rpm Centrifuge horizontally for 25-30 minutes.

5) After centrifugation, the contents of the tube are divided into three layers. Aspirate the mononuclear cells (white liquid) in the upper and middle liquid interface layer, transfer it to a centrifuge tube, dilute it with more than 5 times of physiological saline, centrifuge at 1800 rpm for 10 minutes, and discard. The supernatant was washed twice.

6) After the final centrifugation, aspirate the supernatant, resuspend the cells with culture medium, take 1 drop of cell suspension to count, and adjust the cell concentration to 0.5 ~ 1×10 ⁶ /mL; place it at 37°C and culture in 5% CO ₂ box culture.

(2) Culture and expansion of T cells

1) Collect cells again every 2 to 3 days, centrifuge, discard the supernatant, and replace with fresh culture medium. At the same time, count the cell suspension concentration and maintain the cell concentration at 0.5-1×10 ⁶ /ml.

2) Generally, a large amount of rehydration or subculture is required after 5 to 7 days.

3) When passaging, one bottle can be passed directly to two bottles without centrifugation.

4) The cells were cultured until the 10th to 14th day, and the cells were collected by centrifugation. The expression of CD3, CD4, and CD8 was detected by flow cytometry, and the survival rate of the cells was analyzed by trypan blue staining.

Among them, the composition of the T cell culture medium is: X-VIVO 15 ^TM serum-free cell culture medium (Lonza), adding 5% inactivated human AB serum, 2mM L-glutamine, 20mM HEPES and 50U/ mL of rhIL-2.

3. In vitro preparation and culture of CAR-T cells

(1) Separate T cells cultured for 2 days, centrifuge, resuspend in fresh culture medium, adjust the cell density to 0.6x10 ⁶ /ml, add the prepared lentivirus (MOI is 1-5), mix and culture in CO2 box culture.

(2) After incubating the cells with the virus for 24 hours, discard the supernatant, add the virus again for infection for 24 hours, then centrifuge, and continue culturing with fresh medium.

(3) The prepared CAR-T cells continue to be cultured and expanded for 9-14 days. The concentration of T cells is maintained at 1.0×10 ⁶ cells/ml. The growth of the cells is regularly observed, mainly observing the changes in their morphology, number and proliferation. , cell debris and impurities, add liquid every 2-3 days for culture.

CAR-T cells prepared with the pHR-CMV-CD19 CAR vector and CAR-T cells prepared with the pHR-CD3p-CD19 CAR vector were respectively prepared according to the above method, and then the prepared CAR-T cells were subjected to cell viability according to the method described in Example 3. , purity and functional testing.

Embodiment 3

(1) Analyze the expansion efficiency of CAR-T cells using CCK-8 detection method

CCK-8 (Cell Counting Kit) is a kit based on WST-8 (chemical name: 2-(2-methoxy-4-niphenyl)-3-(4-niphenyl)-5- (2,4-disulfobenzene)-2H-tetrazole monosodium salt) is widely used in rapid and highly sensitive detection kits for cell proliferation and cytotoxicity. The principle is that in the presence of electronic coupling reagents, WST- 8 can be reduced by dehydrogenase in mitochondria to produce a highly water-soluble orange-yellow formazan product. The depth of the color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is then measured at a wavelength of 450nm using a microplate reader to reflect the number of living cells. The present invention uses CCK-8 to detect the expansion efficiency of CAR-T cells prepared by pHR-CMV-CD19CAR and pHR-CD3p-CD19CAR vectors respectively. Specific steps are as follows:

(1) Collect cells by centrifugation (CAR-T cells prepared with pHR-CMV-CD19CAR vector and CAR-T cells prepared with pHR-CD3p-CD19CAR vector) respectively, and resuspend 1×10 ³ cells in 200 μL fresh culture medium. , and place the suspension in a 96-well plate, inoculate 8 duplicate wells in each group, and repeat a total of 4 96-well plates.

(2) After mixing the cells evenly, place them for static culture at 37°C and 5% _CO2 .

(3) On the 0th day, the 5th day, the 7th day and the 14th day of culture, randomly select a set of duplicate wells, add 20 μL CCK-8 solution to each well, and shake gently to mix.

(4) Incubate the well plate for 1 hour under the culture conditions of 37°C and 5% _CO2 .

(5) After the incubation, place the cells on a shaking mixer and shake for 10 minutes.

(6) Use an enzyme-linked instrument to measure: the measurement conditions are the light absorption value when the OD value is 450nm and the reference wavelength is 600nm.

(7) Perform enzyme-linked detection at the same time point every day.

(8) After the detection is completed, perform data statistics and analysis.

The experimental results are shown in Figure 1. The abscissa represents the number of days of cell culture, the ordinate represents the expansion fold, CMVp represents the CAR-T cells prepared by the pHR-CMV-CD19CAR vector, and CD3p represents the pHR-CD3p-CD19CAR vector. CAR-T cells. As can be seen from the results in Figure 1, lentiviruses packaged with pHR-CMV-CD19CAR and pHR-CD3p-CD19CAR were used to infect T cells. On the 14th day of culture, T cells could be expanded more than 45 times, and using The pHR-CD3p-CD19CAR vector infects T cells, which can achieve more than 60-fold expansion of T cells on the 14th day of culture. At the same culture time, using the pHR-CD3p-CD19CAR vector to infect T cells, compared with using pHR-CMV- The CD19CAR vector infects T cells with a higher amplification factor.

(2) Flow cytometry analysis of T cell differentiation efficiency using CD3, CD4, and CD8 antibodies

T cells are divided into helper T cells, suppressor T cells, cytotoxic T cells, etc. Different classification methods can be used to distinguish different T cells, among which cell surface markers are a commonly used method. Among them, CD3 is a marker common to all T cells. CD4 and CD8 are the most commonly used markers to further classify T cells. Generally speaking, CD4 positivity represents helper T cells, and CD8 positivity represents cytotoxic T cells. Because the T cells in CAR-T are actually a mixed system, mixed with multiple types of T cells, the expression ratio of CD3 is used to judge the purity of the prepared T cells, and CD4 and CD8 are used to judge the subtype of the prepared T cells. . Therefore, CD3, CD4 and CD8, as surface markers of T lymphocytes, can be used to reflect the status of CAR-T cells. The present invention analyzes the differentiation efficiency of T cells through flow cytometry technology. Specific steps are as follows:

(1) Take the T cells that have been cultured and wash them twice with calcium-magnesium-free PBS. Resuspend 1×10 ⁵ cells in 100 μL PBS buffer and add them to the corresponding FCM tubes.

(2) Add 5 μL of each of the monoclonal antibodies to be detected, CD3, CD4, and CD8, and incubate in the dark at 4°C for 30 minutes. Shake every 10 minutes to fully contact the cells with the antibodies.

(3) Wash twice with PBS, resuspend in 400 μL of PBS, and detect using flow cytometer FASCSCalibur (BDBiosciences).

The experimental results are shown in Figure 2. Among them, CMVp represents the CAR-T cells prepared by the pHR-CMV-CD19CAR vector, and CD3p represents the CAR-T cells prepared by the pHR-CD3p-CD19CAR vector. The CAR-T cells prepared by the pHR-CMV-CD19CAR and pHR-CD3p-CD19CAR vectors can achieve high-purity T cell preparation after 10 days of culture and expansion. The CAR-T cells prepared by the pHR-CD3p-CD19CAR vector are relatively pure ( CD3 ⁺ %>96%), and the proportion of CD8 positive cells was above 80%.

(3) Trypan blue staining experiment to analyze cell viability

When cells are damaged or dead, trypan blue can penetrate the denatured cell membrane and combine with the disintegrated DNA to color it. Living cells can prevent the dye from entering the cells, so it can be used to identify dead cells and living cells and analyze cell viability. . The present invention uses trypan blue staining experiment to analyze the cell viability of CAR-T cells. Includes the following steps:

(1) Use physiological saline to prepare 0.4% trypan blue dye solution.

(2) CAR-T cells cultured for 10 days were pipetted and mixed, put 2 drops of cell suspension into a 1.5mL EP tube, add 2 drops of 0.4% trypan blue solution, and mix.

(3) Within 3 minutes of adding the dye solution, draw 10uL of the cell-stain solution mixture, add it to the cell counting plate, inspect under a microscope, and count the number of viable cells. Under high-power microscope examination, living cells are not stained and have strong refraction; dead cells are stained blue and slightly enlarged in size. The number of viable cells should be above 95%. Multiply the calculated cell density by 2 to get the actual cell density.

Experimental results showed that CAR-T cells prepared with pHR-CMV-CD19CAR and pHR-CD3p-CD19CAR vectors still maintained high cell viability (>88%) after 10 days of culture and expansion, and there was no significant difference between the two groups.

(4) Flow cytometry to detect the expression of active components of CAR

The flow cytometry method is the same as above. Protein L is an immunoglobulin-binding protein that can bind to the immunoglobulin kappa light chain without affecting antigen binding. The present invention utilizes the binding ability of protein L to antibody light chain kappa, uses protein L-biotinylated as the primary antibody and PE-Streptavidin (PE-streptavidin) as the secondary antibody to detect the extracellular antiCD19scFv region in the CAR structure.

The experimental results are shown in Figure 3. The abscissa in Figure 3 (Protein L-PE) represents the antibody Protein L captured by the fluorescent dye PE, and curve 1 represents the CAR cells obtained after transfecting T cells with lentivirus prepared from the pHR-CD3p-CD19CAR vector. The curve 2 represents T cells that have not been transferred into the pHR-CD3p-CD19CAR vector. As can be seen from Figure 3, the lentivirus prepared by the pHR-CD3p-CD19CAR vector was transfected into T cells. After 48 hours, the cells were collected for labeling antibody analysis. Obvious protein L binding positivity, that is, the expression of antibody scFv, was detected, proving the CAR structure. Get effective expression.

(5) Cells were co-cultured with Raji and K562 cells, and LDH release and cytotoxicity were analyzed using a lactate dehydrogenase cytotoxicity detection kit.

K562 cells do not express CD19 and can be used as negative effector cells of antiCD19-CAR-T. Raji cells express CD19 on their surface and can be used as positive effector cells of antiCD19-CAR-T. K562 cells and Raji cells can be used to detect the killing effect of the prepared CAR-T cells on Raji cells. The specific preparation process is as follows:

(1) Use a 96-well flat-bottom culture plate with 12 experimental wells, 12 pure effector cell wells, 12 pure target cell wells, and blank wells.

(2) Add 1×10 ⁴ /100 μL of K562 and Raji cells to the experimental well and pure target cell well respectively, and culture for 24 hours.

(3) Add 100 μL of CAR-T cells cultured for 10 days to each of the experimental wells and the pure effector cell wells to make the effect-to-target ratio (E:T) 10:1, 5:1, and 1:1 respectively; in the pure effector cells Add 100 μL of serum-free medium to each of the cell wells and pure target cell wells.

(4) In the LDH release test, after the effector cells and target cells are co-incubated for 48 hours, take out the cell culture plate from the cell culture incubator, add 10% of the original culture medium volume of LDH release reagent to each well, and mix by pipetting several times. Mix well and continue incubating in the cell culture incubator for 1 hour.

(5) After the action of the LDH releasing reagent is completed, centrifuge the cell culture plate at 400g for 5 minutes in a multi-well plate centrifuge. Take 120 μL of the supernatant from each well and add it to the corresponding well of a new 96-well plate, and then perform sample measurement. The absorbance was measured at 490 nm, and 600 nm was used as the reference wavelength for dual-wavelength measurements.

The experimental results are shown in the figure. Figure 4 is a graph showing the killing efficiency of CAR-T cells against Raji cells under different effect-to-target ratios (E:T ratio). The abscissa represents different effect-to-target ratios (CAR-T cells:Raji cells), and the ordinate represents is cytotoxicity (that is, it represents the killing rate of CAR-T cells against Raji cells), CMVp represents CAR-T cells prepared with the pHR-CMV-CD19CAR vector, and CD3p represents CAR-T cells prepared with the pHR-CD3p-CD19CAR vector. . As can be seen from Figure 4, both the CAR-T cells prepared with pHR-CMV-CD19CAR and pHR-CD3p-CD19CAR vectors showed a relatively obvious killing effect on CD19 ⁺ cells, and under the same effect-to-target ratio, compared with CAR-T cells prepared with pHR-CMV-CD19CAR vector and CAR-T cells prepared with pHR-CD3p-CD19CAR vector showed more efficient CD19 ⁺ cell killing effect.

(6) Cells were transplanted into NOD/SCID mice, and peripheral blood was isolated 14 days later to analyze the long-term survival in vivo and the stability of exogenous gene expression.

NOD/SCID mice have both innate immune deficiencies and a deficiency of T lymphocytes and B lymphocytes. The prepared CAR-T cells were transplanted into NOD/SCID mice, and then isolated from the periphery on the 14th day after transplantation. Blood can be used to analyze the survival of CAR-T cells in mice and the stability of exogenous gene expression in animals. The specific experimental steps are as follows:

(1) Before the start of the experiment, 100 μL of peripheral blood of mice from each group was taken, and the red blood cells were lysed with red blood cell lysis solution and used as a blank control group for flow cytometry analysis;

(2) NOD/SCID mice were irradiated with 2.2Gy and used as recipients of transplanted cells.

(3) Label the CAR-T cells cultured for 10 days with the fluorescent dye CFSE, and retain a sample of the labeled CAR-T cells for flow cytometry analysis;

(4) Inject CFSE-labeled CAR-T cells into the above irradiated NOD/SCID mice through the tail vein, and mark the 1st, 3rd, 7th, 10th and 14th days after injection. Open the tail vein, collect 50 μL of peripheral blood, lyse red blood cells with red blood cell lysis solution, and conduct flow cytometry analysis to analyze the proportion of CFSE-positive cells and the proportion of human IgG F(ab')2 fragment-positive cells in CFSE+ cells.

Experimental results showed that 14 days after transplantation, about 2% of CFSE-positive cells could still be detected in peripheral blood, and the proportion of CFSE-positive cells (transplanted CAR-T cells) in the experimental group transfected with pHR-CD3p-CD19CAR was 2.7%. higher than the pHR-CMVp-CD19CAR transfection group (2.2%). According to protein L affinity detection, the CAR-positive proportions of CFSE-positive cells in the two groups were 56% (pHR-CD3p-CD19CAR group) and 45% (pHR- CMVp-CD19CAR group), it can be seen that the CD3 promoter can maintain high expression of foreign genes (CAR).

Therefore, the CAR-T cells prepared by the pHR-CD3p-CD19CAR vector in the present invention express highly efficient exogenous gene expression and can be sustained and stable. Compared with the CAR-T cells prepared by pHR-CMVp-CD19CAR, the effect is better. To be significant. The CAR-T cells prepared by the pHR-CD3p-CD19CAR vector of the present invention have a CD3 purity of more than 96%, and can still maintain a high proliferation rate on the 15th day of in vitro culture, and can still maintain a high growth rate 14 days after transplantation. CAR expression

In addition, the terms “first” and “second” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.

In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.

SEQUENCE LISTING

<110> South China Institute of Biomedicine, Institute of Military Medicine, Academy of Military Sciences, Chinese People's Liberation Army

<120> Isolable nucleic acids containing CD3 promoter sequences and CAR sequences and their applications

<130>PIDC3175503

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> DNA

<213> Artificial sequence

<400> 1

ccggaattca ttgaagttat gtatcctcc 29

<210> 2

<211> 43

<212> DNA

<213> Artificial sequence

<400> 2

gtttaacaac agagaaacgg agggagcgat aggctgcgaa gtc 43

<210> 3

<211> 43

<212> DNA

<213> Artificial sequence

<400> 3

gacttcgcag cctatcgctc cctccgtttc tctgttgtta aac 43

<210> 4

<211> 41

<212> DNA

<213> Artificial sequence

<400> 4

gctcctgctg aacttcactc tttcttcttc tggaaatcgg c 41

<210> 5

<211> 41

<212> DNA

<213> Artificial sequence

<400> 5

gccgatttcc agaagaagaa agagtgaagt tcagcaggag c 41

<210> 6

<211> 31

<212> DNA

<213> Artificial sequence

<400> 6

tgtggtacct taagggggca gggcctgcat g 31

<210> 7

<211> 321

<212> DNA

<213> Artificial sequence

<400> 7

attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60

catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120

tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 180

tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240

atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300

gacttcgcag cctatcgctc c 321

<210> 8

<211> 126

<212> DNA

<213> Artificial sequence

<400> 8

ctccgtttct ctgttgttaa acggggcaga aagaaactcc tgtatatatt caaacaacca 60

tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 120

gaagaa 126

<210> 9

<211> 333

<212> DNA

<213> Artificial sequence

<400> 9

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttggggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cct 333

<210> 10

<211> 867

<212> DNA

<213> Artificial sequence

<400> 10

cgggatccat ggacatccag atgacacaga ctacatcctc cctgtctgcc tctctgggag 60

acagagtcac catcagttgc agggcaagtc aggacattag taaatattta aattggtatc 120

agcagaaacc agatggaact gttaaactcc tgatctacca tacatcaaga ttacactcag 180

gagtcccatc aaggttcagt ggcagtgggt ctggaacaga ttatctctc accattagca 240

acctggagca agaagatatt gccacttact tttgccaaca gggtaatacg cttccgtaca 300

cgttcggagg ggggactaag ttggaaataa cacgggctga tgctgcacca actgtatcca 360

tcttcccacc atccagtaat ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg 420

gttctgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca cagagcctgt 480

ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc tggattcgcc 540

agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa accacatact 600

ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag agccaagttt 660

tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt gccaaacatt 720

attactacgg tggtagctat gctatggact actggggtca aggaacctca gtcaccgtct 780

cctcattcca gaatgcgcta ttagttcgtt acaccaagaa agtaccccaa gtgtcaactc 840

caactcttgt agaggtctca gaattcc 867

<210> 11

<211> 1658

<212> DNA

<213> Artificial sequence

<400> 11

cgggatccat ggacatccag atgacacaga ctacatcctc cctgtctgcc tctctgggag 60

acagagtcac catcagttgc agggcaagtc aggacattag taaatattta aattggtatc 120

agcagaaacc agatggaact gttaaactcc tgatctacca tacatcaaga ttacactcag 180

gagtcccatc aaggttcagt ggcagtgggt ctggaacaga ttatctctc accattagca 240

acctggagca agaagatatt gccacttact tttgccaaca gggtaatacg cttccgtaca 300

cgttcggagg ggggactaag ttggaaataa cacgggctga tgctgcacca actgtatcca 360

tcttcccacc atccagtaat ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg 420

gttctgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca cagagcctgt 480

ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc tggattcgcc 540

agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa accacatact 600

ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag agccaagttt 660

tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt gccaaacatt 720

attactacgg tggtagctat gctatggact actggggtca aggaacctca gtcaccgtct 780

cctcattcca gaatgcgcta ttagttcgtt acaccaagaa agtaccccaa gtgtcaactc 840

caactcttgt agaggtctca gaattcattg aagttatgta tcctcctcct tacctagaca 900

atgagaagag caatggaacc attatccatg tgaaagggaa acacctttgt ccaagtcccc 960

tatttcccgg accttctaag cccttttggg tgctggtggt ggttggtgga gtcctggctt 1020

gctatagctt gctagtaaca gtggccttta ttatttctg ggtgaggagt aagaggagca 1080

ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc acccgcaagc 1140

attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccctc cgtttctctg 1200

ttgttaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt atgagaccag 1260

tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa gaaagagtga 1320

agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag ctctataacg 1380

agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt ggccgggacc 1440

ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac aatgaactgc 1500

agaaagataa gatggcggag gcctacagtg agattggggat gaaaggcgag cgccggaggg 1560

gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac acctacgacg 1620

cccttcacat gcaggccctg cccccttaag gtaccaca 1658

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence

<400> 12

gagctcctgg cctcaagc 18

<210> 13

<211> 27

<212> DNA

<213> Artificial sequence

<400> 13

ggatcccctg gaggggccat gaaaaat 27

<210> 14

<211> 1302

<212> DNA

<213> Artificial sequence

<400> 14

gagctcctgg cctcaagcaa tctgcctgct ttggcctccc aaagtaccat atctggcgaa 60

tttcttatat tatttcttac actcctcgtt ctccctgtta cctgccgtgc tggattttcc 120

atttctcttc ctcatctctg gacttcaaac atgctgctgc ctctatctgg aatgctttcc 180

actccacctg gttcattgct gttcctccat caaatttcag cttagatgtc acctgctcag 240

ggggaccttc tcttccccat cccaccccag actatcttag atcccttcta tcactcccac 300

agcacctgaa cgtctttttt cttagcatca atctcctgaa gttatctggg gggtttttta 360

gagatgggat ctcactatgt tgcctaggct ggagcgcagt ggctattcac aggcacaatc 420

acactcactg cagtctcgaa cccctgggct cagtcagtcc tcctgcctca gcctcctgag 480

tagctgggaa tacaggtgta caccatcatg cggggcatgg ttatggtta tgaggttatc 540

tgtttaatct ccacatttcc ttttttatcc aagaggacta tttatcttgt ttctcctgta 600

ttactagcac ccagcatata actacagtac agtagacaga aaatatgtat taaataagtg 660

aatttcatca aaatgttttc tagagtccca gctctgtaat ctgcaactta gataacctgc 720

ctgagcctta gtttgctcaa gtataaaatg gaaataaaaa taaaaataat acttacctta 780

gagggtcgtt ttgaggatta aataagatat atcagaaaag ctcttagaac ggtgcttgga 840

tatagtaagc atttgattaa tgcatgctta aacataaaga aaggttacat caattcacac 900

tcataccagc agactttgac actatcactc tttaccctgt tcaacattga gtcttgtgtt 960

tttaaatatt tttgtaaaga atataggtaa aaagtggcat tttttctttg gatttaattc 1020

ttatggattt aagtcaacat gtattttcaa gccaacaagt tttgttaata agatggctgc 1080

accctgctgc tccatgccag atccaccaca cagaaagcaa atgttcagtg catctccctc 1140

ttcctgtcag agctttataga ggaaggaaga ccccgcaatg tggaggcata ttgtattaca 1200

attactttta atggcaaaaa ctgcagttac ttttgtgcca acctactaca tggtctggac 1260

agctaaatgt catgtatttt tcatggcccc tccaggggat cc 1302

Claims (6)

1. A recombinant vector, characterized in that the recombinant vector is a vector containing isolable nucleic acid; The vector is a pHRs1-C1a lentiviral vector; The nucleic acid is a CD3 promoter sequence and a CAR sequence, and the CD3 promoter sequence is connected to the CAR sequence, wherein the CAR sequence has an extracellular region sequence and a transmembrane/intracellular region sequence, and the extracellular region sequence is connected to the transmembrane/intracellular region sequence, The extracellular region sequence is an anti-CD19 immunoglobulin scFV fragment sequence, and the anti-CD19 immunoglobulin scFV fragment sequence is SEQ ID NO: 10, The CD3 promoter sequence is SEQ ID NO: 14; The transmembrane/intracellular region sequence includes a CD28 sequence fragment, a 4-1BB sequence fragment and a CD3 sequence fragment, and the CD28 sequence fragment, the 4-1BB sequence fragment and the CD3 sequence fragment are connected; The CD28 sequence fragment is SEQ ID NO:7, The 4-1BB sequence fragment is SEQ ID NO:8, The CD3 sequence fragment is SEQ ID NO: 9; The CAR sequence is SEQ ID NO: 11. 2. The recombinant vector according to claim 1, wherein the nucleic acid is DNA. 3. The method for constructing a recombinant vector according to any one of claims 1 to 2, characterized in that it includes the following steps: Amplify the CAR sequence and introduce the CAR sequence into the vector to obtain the first recombinant vector containing the CAR sequence, Amplify the CD3 promoter sequence and connect the CD3 promoter sequence to the first recombinant vector to obtain the second recombinant vector, that is, construct a recombinant vector containing both the CD3 promoter sequence and the CAR sequence, Wherein, the CAR sequence includes an extracellular region sequence and a transmembrane/intracellular region sequence, and the extracellular region sequence is connected to the transmembrane/intracellular region sequence, The extracellular region sequence is an anti-CD19 immunoglobulin scFV fragment sequence, and the anti-CD19 immunoglobulin scFV fragment sequence is SEQ ID NO: 10, The CD3 promoter sequence is SEQ ID NO: 14; Amplify the CD28 sequence fragment, the 41BB sequence fragment and the CD3 sequence fragment respectively to prepare a fusion sequence containing CD28, 41BB and CD3, and then introduce the fusion sequence containing CD28, 41BB and CD3 into the vector to obtain a fusion sequence containing CD28, 41BB and CD3. The third recombinant vector of CD3 fusion sequence, The anti-CD19 immunoglobulin scFV fragment sequence is connected to the third recombinant vector to prepare the first recombinant vector. 4. The construction method according to claim 3, further comprising: The CD28 sequence fragment was amplified using the primer sequences SEQ ID NO: 1 and SEQ ID NO: 2, the 41BB sequence fragment was amplified using the primer sequences SEQ ID NO: 3 and SEQ ID NO: 4, and the 41BB sequence fragment was amplified using the primer sequences SEQ ID NO: 5 and SEQ ID NO: 4. SEQ ID NO:6 was amplified to obtain a CD3 sequence fragment; The CD3 promoter sequence was amplified using the primer sequences SEQ ID NO:12 and SEQ ID NO:13; Among the CD28 sequence fragment, the 41BB sequence fragment and the CD3 sequence fragment, two sequence fragments partially overlap; Use the same enzyme to digest the anti-CD19 immunoglobulin scFV fragment sequence and the third recombinant vector, and connect the anti-CD19 immunoglobulin scFV fragment sequence to the third recombinant vector; The CD3 promoter sequence and the first recombinant vector are digested using the same enzyme, and the CD3 promoter sequence is connected to the first recombinant vector. 5. A recombinant cell, characterized in that the recombinant cell contains the recombinant vector according to any one of claims 1 to 2; The recombinant cells are recombinant T cells. 6. The method for constructing recombinant cells according to claim 5, characterized in that it includes the following steps: Introduce the recombinant vector according to any one of claims 1 to 2 into a recipient cell to construct a recombinant cell; The receptor cells are T cells.