CN112458116A

CN112458116A - Construction method of PD-1 knockout CD19CAR-T cell

Info

Publication number: CN112458116A
Application number: CN202011338905.1A
Authority: CN
Inventors: 刘爽; 张青; 张普民
Original assignee: Guangzhou Heavy Pound Biotechnology Co ltd
Current assignee: Guangdong No 2 Peoples Hospital
Priority date: 2020-10-19
Filing date: 2020-11-25
Publication date: 2021-03-09
Anticipated expiration: 2040-11-25
Also published as: CN112458116B

Abstract

The invention discloses a method for constructing a PD-1 gene knockout CD19CAR-T cell, which comprises the following steps: a Nucleofector nuclear technology is adopted, a PiggyBac vector plasmid Piggybac-CAR-CD19 containing a single-chain antibody variable region gene fragment of CD19, a Transposase expression plasmid Piggybac Transposase, an sgRNA expression plasmid pGL3-U6-hPD1sg1 containing a PD1 gene which can be successfully targeted, and a pST1374-NLS-flag-Cas9-ZF plasmid are co-transfected into a T cell, and the PD-1 gene knockout CD19CAR-T cell is obtained. The experiment of the invention proves that: the PD-1 knockout immune cell has higher cell killing capacity than a PD-1 knockout CD19CAR-T cell.

Description

Construction method of PD-1 knockout CD19CAR-T cell

Technical Field

The invention relates to a bioengineering technology, in particular to a novel construction method of a PD-1 knockout CD19CAR-T cell.

Background

CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) is a new adoptive immune Cell therapy developed in recent years, T cells are modified through a genetic engineering means to express Chimeric Antigen receptors, and the Chimeric Antigen receptors can specifically recognize tumor-related Antigen targets, and signals for activating and proliferating the T cells are transmitted into cells after recognition and combination, so that the T cells are activated and proliferated, and the tumor cells are effectively killed. CAR molecules can be broadly divided into 5 generations of evolution: generation I, specific T cell activation; in the II generation, co-stimulation factors are increased, and the cytotoxicity is improved; the III generation has two co-stimulation factors simultaneously, and improves the proliferation capacity and the killing toxicity of the T cells; generation IV, integration of suicide genes, precise regulation, cytokine release (e.g., IL-7, IL-15) activation, etc.; generation V, universal CAR. CAR-T technology has many unique advantages over conventional immune cell therapy, such as CAR's ability to recognize tumor antigens without MHC dependency, and the need to go through APC. Tumor surface protein and lipid antigens can be used as targets; the co-stimulatory molecules are present, and can effectively enhance the proliferation of T cells. Therefore, the technology is considered to be a unique method which is expected to thoroughly overcome human cancers.

Currently, CAR-T immunotherapy has made tremendous progress in many clinical trials. Among them, CAR-T-CD19T has been reported to have significant efficacy against B-cell malignancies positive for the B-associated antigen CD19, such as acute B-lymphocytic leukemia (B-ALL), chronic B-lymphocytic leukemia (B-CLL), B-cell hodgkin's lymphoma (B-HL) and non-hodgkin's lymphoma (B-NHL), and CAR-T-CD19 achieves a Complete remission rate (CR) of up to 90% in relapsed and refractory B-ALL in adults and children. The study report also shows that several clinical trial treatments with different structural designs of CAR-CD19 showed similar good efficacy. Some patients also achieved persistent remission after CAR-T treatment, without the need for additional treatment.

Nevertheless, there are many links to the treatment that need improvement, such as finding better receptors, better vectors, better co-stimulatory molecules, etc., where the safety and effectiveness of the transfection vector is particularly critical for clinical treatment. Nowadays, the main transfection modes of the CAR include lentivirus, retrovirus, electrotransfection and the like, and although the viral vector has better transfection efficiency, the viral vector has the risk of insertional mutation caused by genome integration, so that the transfection result which is equivalent to or even higher than the virus infection efficiency can be obtained by using a non-viral vector in combination with an electrotransfection technology without virus intervention, the influence of random insertion of viral nucleic acid into host cells on the normal growth of the cells and subsequent possible lesions is avoided, and the CAR-T cell transfection method is an important link for improving the safety of CAR-T cell therapy. There is a great need to develop a new method for constructing CD19CAR-T cells

PD-1(programmed death 1) programmed death receptor 1, an important immunosuppressive molecule, is an immunoglobulin superfamily, is a membrane protein of 268 amino acid residues. The primary function of PD1 is to limit effector T cell activity in peripheral tissues. The PD1 ligand expressed by the tumor cells is combined with PD1 of T cells to inhibit the T cells from activating related kinases, thereby inhibiting the activity of effector T cells. The PD1 monoclonal antibody (MDX-1106, MK3475, CT-011, AMP-224 and the like) is used for inhibiting the binding of PD1 ligand and PD1, thereby achieving the effect of treating tumors.

However, the treatment with the PD1 antibody was only effective in melanoma, and was partially effective in renal cancer and lung cancer. Because treatment of target genes with antibodies is also limited by several factors: (1) the effect of the antibody is only a temporary blocking effect; (2) there are many inhibitory receptors, and there is no strategy for blocking multiple inhibitory receptors simultaneously by using multiple antibodies; (3) effective antibodies are not easily developed; (4) targeting only extracellular targets; (5) antibody drugs are expensive, etc.

The CRISPR-Cas9 gene is knocked out in a quick, simple, convenient and efficient specific targeting manner, and a feasible strategy is provided for realizing tumor immunotherapy by knocking out the PD1 gene in a targeted manner.

Disclosure of Invention

To this end, the invention provides a method for constructing a PD-1 knockout CD19CAR-T cell.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method of constructing a PD-1 knockout CD19CAR-T cell, the method comprising:

determining the gene segment of the variable region of the single-chain antibody of the CD19, and designing and synthesizing a CD19 single-chain antibody;

a target gene formed by fusing a CD8 signal peptide, a CD19 single-chain antibody, a CD8 hinge and transmembrane region, intracellular 4-1BB and intracellular CD3 zeta which are sequentially connected is connected to a pMD 18-Tprime vector;

connecting a target gene to a PiggyBac vector by adopting a method of enzyme digestion, connection and transformation, and transferring a target gene fragment product which is synthesized correctly by sequencing detection into escherichia coli competent TOP10 so as to enable the PiggyBac vector containing the CD19 single-chain antibody variable region gene fragment to be expressed in escherichia coli;

a Nucleofector nuclear technology is adopted, a PiggyBac vector plasmid Piggybac-CAR-CD19 containing a single-chain antibody variable region gene fragment of CD19, a Transposase expression plasmid Piggybac Transposase, an sgRNA expression plasmid pGL3-U6-hPD1sg1 containing a PD1 gene which can be successfully targeted, and a pST1374-NLS-flag-Cas9-ZF plasmid are co-transfected into a T cell, and the PD-1 gene knockout CD19CAR-T cell is obtained.

Preferably, the CD19 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are connected by a connecting peptide, and the heavy chain variable region comprises a heavy chain variable region complementarity determining region 1CDR-H1, a heavy chain variable region complementarity determining region 2CDR-H2 and a heavy chain variable region complementarity determining region 3 CDR-H3;

the light chain comprises a light chain variable region comprising light chain variable region complementarity determining region 1CDR-L1, light chain variable region complementarity determining region 2CDR-L2 and light chain variable region complementarity determining region 3 CDR-L3.

Preferably, the amino acid sequence of the CD19 single-chain antibody is shown in SEQ ID NO. 2.

Preferably, the coding nucleotide sequence of the CD19 single-chain antibody is shown in SEQ ID NO. 1.

Preferably, the CD8 hinge and transmembrane region amino acid sequence is shown as SEQ ID No. 4.

Preferably, the amino acid sequence of intracellular 4-1BB is shown as SEQ ID No. 5.

Preferably, the amino acid sequence of intracellular CD3 zeta is shown in SEQ ID No. 6.

Preferably, the method comprises attaching a single chain antibody fragment scFv specific for the CD19 antigen, myc-tag, a transmembrane hinge from CD8, costimulatory factors CD28 and 4-1BB fragment, and an intracellular signaling CD3 zeta, followed by attaching CD19-CAR to a Piggybab vector.

Preferably, the construction process of the sgRNA expression plasmid pGL3-U6-hPD1sg1 plasmid containing the PD1 gene successfully targeted is as follows:

designing and synthesizing an sg RNA oligonucleotide specifically targeting a human PD-1 gene, wherein the target sequence of the sgRNA on the PD-1 gene conforms to the sequence arrangement rule of 5 ' -GGN (19) GG, 5 ' -GN (20) GG or 5 ' -N (21) GG; the target sequence of the sgRNA on the PD-1 gene is located in an exon of the gene; the sgRNA target sequence on the PD-1 gene is located on different common exons in various splicing forms; the sgRNA is unique in the target sequence of the PD-1 gene;

the DNA sequence corresponding to the sg RNA oligonucleotide is shown as SEQ ID NO.7, CCGG is added to the 5 'end of the corresponding DNA sequence, if the sequence has 1 or 2G at the 5' end, 1 or 2G is correspondingly omitted, and Forward oligonucleotide, namely Forward oligo, is synthesized;

the sgRNA obtains a complementary strand of a corresponding DNA sequence, and AAAC is added to the 5' of the complementary strand to synthesize a Reverse oligonucleotide, namely Reverse oligo;

performing paired denaturation and annealing on the forward oligo and reverse oligo of 1 pair of synthesized complementary sgRNA oligonucleotides, and forming double-stranded sgRNA oligonucleotides which can be connected into a U6 eukaryotic expression vector after annealing;

linearized vector pGL3-U6-sgRNA plasmid; connecting the annealed double-stranded sgRNA oligonucleotide with a linearized pGL3-U6-sgRNA plasmid to obtain a pGL3-U6-hPD1sg plasmid;

pGL3-U6-hPD1sg plasmid is transformed into competent bacteria and coated on Amp + plates, and positive clones are selected and identified by a method of sequencing by a universal primer U6; the positive clones were shaken overnight at 37 ℃ and extracted with AxyPrep Plasmid Miniprep Kit to give pGL3-U6-hPD1sg Plasmid.

In the present invention, CAR-T, collectively known as Chimeric Antigen Receptor T-Cell Immunotherapy, is a Chimeric Antigen Receptor T-Cell Immunotherapy.

The invention has the following advantages:

the experiment of the invention proves that: the adopted non-viral vector PiggyBac system has higher safety and effectiveness, the adopted Nucleofector nucleus technology can directly introduce the exogenous gene into the nucleus, the defect that the exogenous gene is possibly subjected to nuclear expression only when the cell division is required in liposome transfection and common electroporation is overcome, and the cell has higher survival rate and higher proliferation efficiency. The PD-1 knockout immune cell has higher cell killing capacity than the PD-1 knockout CAR-CD 19T cell.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a diagram showing the flow detection of expression of CD19CAR-T cell c-Myc (CAR expression) provided by the present invention;

FIG. 2 is a graph of the CD19CAR-T cell killing ability in vitro provided by the present invention;

FIG. 3 is a graph of CD19CAR-Tcell cytokine expression levels provided by the invention;

FIG. 4 is a graph of the proliferation potency of CD19CAR-Tcell provided by the present invention;

FIG. 5 is a graph showing the expression of flow-detected CD19CAR-T cells activating markerCD25/CD 28;

FIG. 6 is a diagram showing the expression of flow detection memory CD19CAR-T cell marker CD45RO/CD62L provided by the present invention.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 construction of pGL3-U6-hPD1sg vector of the present invention example

Design and selection of sgRNA oligonucleotides

1. Design of sgRNA targeting PD1 gene (Homo sapiens programmed cell death 1(PDCD1), RefSeqGene on chromosome 2 NCBI Reference Sequence: NG-012110.1): (1) the PD1 gene may be selected from the 5 '-GGN (19) GG sequence, or the 5' -GN (20) GG or the 5 '-N (21) GG sequence may be excluded if the 5' -GGN (19) GG sequence is not present. (2) The sgRNA targets a site on the PD1 gene at an exon of the gene. (3) The sgRNA targets sites on the PD1 gene on different consensus exons of various splicing forms. (4) Whether the target sequence of the sgRNA is unique is determined in the UCSC database using BLAT or in the NCBI database using BLAST.

2. Selection of sgRNA targeting the PD1 gene: (1) cannot be too close to the ATG initiator, preventing transcription from starting from another downstream ATG to form a truncated gene form, and cannot ensure complete inactivation of the gene; (2) the targeting site of the sgRNA on the PD1 gene is located in the first half of the entire gene, particularly preferably in the functional domain of the gene; (3) sites paired at a certain distance (10-30 bp) are selected. This is advantageous for the formation of specific fragment deletions and also for the reduction of off-target effects.

Second, construction of an oligonucleotide double strand of sgRNA

Depending on the sgRNA chosen, adding CCGG at its 5 'end gives a Forward oligonucleotide (Forward oligo) (if the sequence itself already has 1 or 2 Gs at the 5' end, the corresponding omission of 1 or 2 Gs); based on the selected sgRNA, the complementary strand of its corresponding DNA is obtained, and AAAC is added 5' thereto to obtain a Reverse oligonucleotide (Reverse oligo). Synthesizing the forward oligonucleotide and the reverse oligonucleotide respectively, pair-denaturing the forward oligo and reverse oligo of the synthesized sgRNA oligonucleotide, annealing, and forming a double strand which can be connected into a U6 eukaryotic expression vector after annealing, as follows:

Forward oligo:5′-CCGGNNNNNNNNNNNNNNNNNN

Reverse oligo:NNNNNNNNNNNNNNNNNNCAAA-5′

the denaturation and annealing system comprises: 2.5. mu.l forward Oligo (100. mu.M) 2.5. mu.l reverse Oligo (100. mu.M); 1 μ l NEB buffer 2.

Mu.l of sterile water, run in a PCR instrument according to the following touch down program: 95 ℃ for 5 min; 9585 deg.C at-2 deg.C/s; 8525 ℃ at-0.1 ℃/s; hold at 4 ℃.

Construction of sgRNA oligonucleotide plasmid

1. Linearized vector pGL3-U6-sgRNA plasmid

The enzyme digestion system and conditions were as follows: 2 μ g pGL3-U6-sgRNA (400 ng/. mu.l); 1 μ l of CutSmart Buffer; 1 μ l BsaI (NEB, R0535L); adding water to 50 mu l, incubating for 3-4 hours at 37 ℃, shaking at intervals and centrifuging to prevent liquid drops from evaporating to a tube cover. After the enzyme digestion is finished, the enzyme is purified and recovered into 20-40 mu l of sterilized water by using AxyPrep PCR Clean up Kit (AP-PCR-250).

2. The annealed sgRNA oligonucleotide duplex was ligated with the linearized pGL3-U6-sgRNA plasmid to obtain the pGL3-U6-hPD1sg plasmid. 3 μ l, 50 μ M annealing product (double stranded sgRNA oligonucleotide), 1 μ l linearized pGL3-U6-sgRNA plasmid (25 ng/. mu.l), 1 μ l T4 ligation Buffer; 0.5. mu. l T4 ligase (NEB, M0202S), 4.5. mu.l sterile water, incubated for 1 hour at 16 ℃.

3. The ligation product obtained in the above procedure was transformed into DH 5. alpha. competent cells (TransGen, CD201) and plated on Amp + plates (50. mu.g/ml), and clones were picked.

4. Positive clones were identified by sequencing with the universal primer U6.

5. Positive clones were cultured overnight at 37 ℃ with shaking, and the Plasmid was extracted with AxyPrep Plasmid Miniprep Kit (AP-MN-P-250) to obtain pGL3-U6-hPD1sg1 Plasmid.

In the embodiment of the invention, the preparation method of the plasmid pST1374-NLS-flag-Cas9-ZF is disclosed in the literature: shen et al 2013, Generation of gene-modified mica via Cas9/RNA-mediated gene targeting. cell research23,720-723 (doi: 10.1038/cr.2013.46).

Example 2 methods of construction of PD-1 knockout CD19CAR-T cells of the invention

The embodiment of the invention provides a method for constructing a PD-1 knockout CD19CAR-T cell, which comprises the following steps:

according to the characteristics of the PiggyBac vector, the sequence of a single-chain antibody variable region (scFv) gene fragment of CD19 is searched, and a single-chain antibody (scFv) which is composed of a heavy chain variable region fragment and a light chain variable region fragment of an antibody derived from the specificity of a CD19 antigen and connected by an intermediate connecting molecule is designed and synthesized, wherein the intermediate fragment is composed of a connecting and membrane penetrating fragment derived from CD8, and the intramembrane fragment is composed of an effector molecule for transmitting signals, such as CD3 zeta. In the present invention, the CD19CAR is formed by fusing a CD8 signal peptide, a CD19 single-chain antibody, a CD8 hinge and transmembrane region, intracellular 4-1BB (i.e., CD137), and intracellular CD3 zeta, which are linked in this order.

CD8 a hinge region (SEQ ID No. 3):

FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD；

CD8 a transmembrane region (SEQ ID No. 4): IYIWAPLAGTCGVLLLSLVITLYCNHRN, respectively; 4-1BB (SEQ ID No. 5): RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, respectively;

CD3zeta(SEQ ID No.6)：RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

connecting the synthesized target gene to a pMD 18-T simple vector, connecting the target gene to a PiggyBac vector by adopting a method of enzyme digestion, connection and transformation, detecting whether the sequence of the synthesized target gene is correct by carrying out PCR amplification and sequencing by using a vector specific primer, and then transferring a connection product into escherichia coli competent TOP10 to ensure that the PiggyBac vector containing a target fragment is amplified in escherichia coli in a large quantity.

The specific fragment design synthesis method comprises the following steps: CD19-CAR fragments were synthesized by design, consisting essentially of single chain antibody fragment scFv specific for the CD19 antigen, myc-tag, CD 8-derived transmembrane hinge, costimulatory factor CD28 and 4-1BB fragments, and intracellular signaling CD3 zeta, and CD19-CAR was ligated to Piggybab vector.

After the construction was successful, plasmids were extracted using a large endotoxin-free plasmid draw kit. Taking human serum and lymphocyte separating medium to separate lymphocyte, and adding T lymphocyte culture solution, IFN-gamma, OKT-3 and hIL-2 for expansion culture.

Transfecting the co-transfection plasmids of Piggybab-CAR-CD 19, Piggybab Transposase, pGL3-U6-hPD1sg1 and pST1374-NLS-flag-Cas9-ZF to T cells by using a Nucleofector cell transfection system, collecting the cells after 48 hours, labeling myc-tag fluorescent antibody, and detecting the expression of CD19-CAR by using a flow cytometer.

The invention adopts a Nucleofector nuclear technology to co-transfect a PicgyBac vector plasmid Piggybab-CAR-CD 19 containing a single-chain antibody variable region gene fragment of CD19, a Transposase expression plasmid Piggybab Transposase, a sgRNA expression plasmid pGL3-U6-hPD1sg1 containing a PD1 gene which can be successfully targeted, and a pST1374-NLS-flag-Cas9-ZF plasmid into a T cell, so as to obtain the PD-1 gene knockout CD19CAR-T cell.

The CD19 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are connected through a connecting peptide, and the heavy chain variable region comprises a heavy chain variable region complementarity determining region 1CDR-H1, a heavy chain variable region complementarity determining region 2CDR-H2 and a heavy chain variable region complementarity determining region 3 CDR-H3; the light chain comprises a light chain variable region comprising light chain variable region complementarity determining region 1CDR-L1, light chain variable region complementarity determining region 2CDR-L2 and light chain variable region complementarity determining region 3 CDR-L3.

Specifically, the amino acid sequence of the complementarity determining region 1CDR-H1 in the heavy chain variable region consists of the amino acid residues shown in 26 th to 35 th in the amino acid sequence shown in SEQ ID NO. 2. The amino acid sequence of the complementarity determining region 2CDR-H2 in the heavy chain variable region is composed of the amino acid residues shown in 50 th to 65 th in the amino acid sequence shown in SEQ ID NO. 2. The amino acid sequence of the complementarity determining region 3CDR-H3 in the heavy chain variable region consists of the amino acid residues shown in 98-109 in the amino acid sequence shown in SEQ ID NO. 2. The light chain variable region complementarity determining region 1CDR-L1 amino acid sequence shown in SEQ ID NO.2 amino acid residues 159-168. The light chain variable region complementarity determining region 2CDR-L2 has the amino acid sequence shown in SEQ ID NO.2, and consists of amino acid residues 185-191. The light chain variable region complementarity determining region 3CDR-L3 amino acid sequence shown in SEQ ID NO.2 amino acid residues shown in 224-232. The amino acid sequence of the heavy chain variable region is composed of amino acid residues shown as 1-120 in the amino acid sequence shown as SEQ ID NO. 2. The amino acid sequence of the light chain variable region is composed of amino acid residues shown in 136 th-242 th in the amino acid sequence shown in SEQ ID NO. 2. The amino acid sequence of the connecting peptide is composed of amino acid residues shown as 121-135 in the amino acid sequence shown as SEQ ID NO. 2.

The amino acid sequence of the CD19 single-chain antibody is shown in SEQ ID NO.2, and specifically comprises the following steps:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS。

experiments prove that the gene sequence is completely consistent with the designed and synthesized gene sequence. The target genes are as follows: the coding nucleotide sequence of the target gene CD19 monoclonal antibody is shown in SEQ ID NO. 1. Information of SEQ ID NO. 1: (i) sequence features; (A) length: 726bp, (B) type: nucleotide, (C) strand property: single strand, (D) topology: linearity; (ii) molecular type: an oligonucleotide; (iii) description of the sequence: shown as SEQ ID NO. 1.

The nucleotide sequence of the single-chain antibody of the CD19 is shown in SEQ ID NO. 1:

GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA。

in the experiment, lymphocyte separation solution, T lymphocyte culture solution are products of Invitrogen company, Nucleofector Cell transfection reagent is purchased from Lonza company, IFN-gamma, OKT-3 and hIL-2 antibodies are purchased from PeproTech company, Myc-tag antibody is purchased from Celignaling company, endotoxin-free plasmid macroextraction kit and gel recovery kit are purchased from Zymo research company, PCR reagent is purchased from Takara bioengineering (Union) Co., Ltd., various endonucleases and T4 ligase are purchased from NEB company.

Experimental example, functional assay of PD-1 knockout CD19CAR-T cells

1. Killing ability test of PD-1 knockout CD19CAR-T cells

1) Preparing target cells and non-target cells: flow detection of tumor target cell surface antigen expression, and when the antigen expression is low, constructing target cell with over-expressed antigen. At the same time, luciferase needs to be overexpressed on target as well as non-target cells as required for killing detection.

2) Cell plating: take 2X 10⁵Plating target cells or non-target cells in 48-well plate, adding non-transfected T cells (NT cells), CAR-T cells, and gene-edited CD19CAR-T cells at effective target ratio of 2:1, 4:1, 8:1, and 16:1, respectively, and placing at 37 deg.C and 5% CO in a total volume of 300 μ l/well₂The cells were cultured in a cell culture chamber for 24 hours. The control group was cultured without T cells and served as the maximal non-killing value.

3) And (5) taking pictures by a microscope to detect the killing capacity of the T cells.

4) The cultured cells were collected, washed once with PBS, and resuspended in 100. mu.l of PBS. And the cells were transferred to an opaque 96-well plate, 100. mu.l of luciferin was added at a concentration of 150. mu.g/ml, and the fluorescence was detected by a microplate reader.

5) The killing capacity of T cells was calculated: 1-fluorescence value of co-culture of target cell T cells/fluorescence value of target cells cultured alone.

FIG. 2 shows a graph of a PD-1 knockout CD19CAR-T cell killing ability in vitro provided by the invention.

2. PD-1 knockout CD19CAR-T cell cytokine secretion assay (IL-2, TNF, IFN, GM-CSF)

1) Cell plating: take 1X 10⁴The individual target cells or non-target cells were plated in 96-well plates, and non-electroporated T cells (NT cells), CAR-T cells, and gene-edited CD19CAR-T cells were added at an effective target ratio of 2:1, respectively, in a total volume of 200. mu.l/well, placed at 37 ℃ and 5% CO₂The cells were cultured in a cell culture chamber for 24 hours.

2) Aspirate the supernatant 100. mu.l, 1000 rpm, 5 min.

3) Mu.l of the supernatant was transferred to a 1/2area opaque 96-well plate.

4) Detection was performed according to the AlphaLISA kit of PE.

FIG. 3 is a graph showing the expression level of the CD19CAR-Tcell cytokine provided by the present invention.

3. Detection of proliferation ability of PD-1 knockout CD19CAR-T cells

1) The CFSE is gradually diluted along with the cell division in the cell division process by CFSE staining capable of labeling proteins, and the cell proliferation capacity can be roughly predicted according to the CFSE fluorescence signal intensity.

2) Target cells as well as non-target cells were irradiated, 70 Gy.

3) Taking irradiated cells 1X 10⁵One was spread in a 24-well plate.

4) After two washes of T cells in PBS, they were stained with 5. mu.M CFSE in PBS and incubated for 5 minutes in an incubator.

5) After 2 washes with serum-containing medium, CAR-T cells, as well as gene-edited CD19CAR-T cells were added to 24-well plates at a ratio of 10:1 for co-culture with tumor cells for one week in continuous culture, and CFSE fluorescence signals were flow-detected daily starting from 72 hours.

As shown in fig. 4, a PD-1 knockout CD19CAR-T cell proliferative capacity assay provided herein.

4. Detection of PD-1 knockout CD19CAR-T cell activation, memory cell formation and differentiation

1) Taking target cells 5X 10⁵Spreading in T25 culture flask, adding 10 × 10⁶The individual T cells were co-incubated for 72 hours. T cells co-cultured without target cells were used as controls.

2) Flow detection of expression of T cell activated marker CD25/CD 28:

a. t cells were collected and washed once with 1ml PBS.

b. Add 100. mu.l of antibody mixer and incubate for 20 min on ice in the dark.

c. The antibody color matching scheme comprises anti-c-myc-BV421, anti-CD25 APC-Cy7, anti-CD28-APC

d. Wash once by adding 1ml PBS.

e. 500 μ l PBS was resuspended.

f. And (5) detecting on a flow type computer.

3) Flow detection of expression of memory T cell marker CD45RO/CD 62L:

the process is as follows:

CD8a-APC,Myc-BV421,CD45RO,CD62L

4) flow detection of the expression of T cell exhausted marker PD1/Lag3/Tim 3: the process is as follows:

anti-c-myc-BV421,anti-PD1 PE,anti-Tim3 PerCP-efluor710,anti-Lag3 APC。

as shown in fig. 1, the flow assay provided by the present invention detects expression of c-Myc (CAR expression) of PD-1 knockout CD19CAR-T cells; FIG. 6 is a diagram showing the expression of PD-1 knock-out CD19CAR-T memory cell marker CD45RO/CD62L in flow detection provided by the invention.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Guangzhou heavy-pound Biotech Co., Ltd

<120> method for constructing PD-1 knockout CD19CAR-T cell

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gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

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aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctca 726

<210> 2

<211> 242

<212> PRT

<213> Artificial Sequence

<400> 2

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 3

<211> 55

<212> PRT

<213> Artificial Sequence

<400> 3

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp

50 55

<210> 4

<211> 28

<212> PRT

<213> Artificial Sequence

<400> 4

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn

20 25

<210> 5

<211> 47

<212> PRT

<213> Artificial Sequence

<400> 5

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40 45

<210> 6

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 6

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 7

cacctaccta agaaccatcc 20

Claims

1. A method of constructing a PD-1 knock-out CD19CAR-T cell, comprising:

2. The method of constructing a PD-1 gene knockout CD19CAR-T cell of claim 1,

the CD19 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are connected through a connecting peptide, and the heavy chain variable region comprises a heavy chain variable region complementarity determining region 1CDR-H1, a heavy chain variable region complementarity determining region 2CDR-H2 and a heavy chain variable region complementarity determining region 3 CDR-H3;

3. The method of constructing a PD-1 gene knockout CD19CAR-T cell of claim 1,

the amino acid sequence of the CD19 single-chain antibody is shown in SEQ ID NO. 2.

4. The method of constructing a PD-1 gene knockout CD19CAR-T cell of claim 1,

the coding nucleotide sequence of the CD19 single-chain antibody is shown in SEQ ID NO. 1.

5. The method of constructing a PD-1 gene knockout CD19CAR-T cell of claim 1,

the CD8 hinge and transmembrane region amino acid sequence is shown as SEQ ID No. 4.

6. The method of constructing a PD-1 gene knockout CD19CAR-T cell of claim 1,

the amino acid sequence of the intracellular 4-1BB is shown as SEQ ID No. 5.

7. The method of constructing a PD-1 gene knockout CD19CAR-T cell of claim 1,

the amino acid sequence of the intracellular CD3 zeta is shown as SEQ ID No. 6.

8. The method of constructing a PD-1 gene knockout CD19CAR-T cell of claim 1,

the method comprises attaching a single chain antibody fragment scFv, myc-tag, a transmembrane hinge from CD8, costimulatory factors CD28 and 4-1BB fragments, and an intracellular signaling CD3 zeta, with specificity for the CD19 antigen, followed by attaching CD19-CAR to a Piggybab vector.

9. The method of constructing a PD-1 gene knockout CD19CAR-T cell of claim 1,

the construction process of the sgRNA expression plasmid pGL3-U6-hPD1sg1 plasmid containing the PD1 gene which can be successfully targeted comprises the following steps: