CN110029091B

CN110029091B - Preparation method of T lymphocyte preparation for relieving inhibition of PD-1 immune check point

Info

Publication number: CN110029091B
Application number: CN201910349259.XA
Authority: CN
Inventors: 王清路
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-04-28
Filing date: 2019-04-28
Publication date: 2021-03-26
Anticipated expiration: 2039-04-28
Also published as: CN110029091A

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a preparation method of a T lymphocyte preparation for relieving inhibition of a PD-1 immune check point. Constructing a single-base gene editing PD-1 transfection system, separating and activating T lymphocytes, co-transfecting the T lymphocytes, screening by a flow cytometer, analyzing by sequencing and identifying. The invention realizes the mutation of the key tyrosine locus codon of the ITSM region gene sequence of human PD-1, which leads to the release of the immunodetection function of PD-1 protein after gene transcription and translation. The PD-1 immune checkpoint disinhibiting activity T lymphocyte preparation improves the tumor cell killing capacity of T lymphocytes, greatly retains the biological activity and other functions of PD-1, and can enhance the long-term tumor immune effect.

Description

Preparation method of T lymphocyte preparation for relieving inhibition of PD-1 immune check point

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a preparation method of a T lymphocyte preparation for relieving inhibition of a PD-1 immune check point.

Background

PD-1(Programmed Death receptor-1) is an important immunosuppressive molecule. The pathway consisting of PD-1 and its ligands PD-L1, PD-L2 plays a crucial role in maintaining peripheral immune tolerance. Tumors and pathogens causing chronic infections can escape from T cell-mediated tumor-specific and pathogen-specific immunity by using the pathway, mainly through the surface to generate PD-L1, and when the PD-L1 is linked to PD-1 protein of T cells of a type of immune cells, the T cells can not find out signals that the tumors attack the tumors to the immune system, namely, the T cells are inactivated. The PD-1-PD-L1 co-inhibition pathway is used for tumor treatment development and achieves remarkable effect, and the remission rate of various types of cancers in multiple clinical trials reaches 20-50%.

PD-1 consists of a single N-terminal immunoglobulin variable region (IgV) -like domain, a stem region that separates the IgV domain from the plasma membrane and comprises approximately 20 amino acids, a transmembrane region, and a cytoplasmic tail comprising a tyrosine-based signaling motif (see fig. 1). PD-1 inhibition is retained when the Immunoreceptor Tyrosine Inhibition Motif (ITIM) tyrosine is changed to phenylalanine (Y223F); however, when the Immunoreceptor Tyrosine Switch Motif (ITSM) was altered (Y248F), these inhibitory effects were lost.

The genome editing (gene editing for short) technology is a genetic engineering technology for targeted modification of a genome by using an artificial nuclease, and is a research hotspot in the field of life science at present. Currently, almost all CAR-T therapy companies are working with gene editing companies, 6 CAR-T therapy enterprises including Nowa, Juno and Cellectis are strongly associated with gene editing companies such as Intellia Therapeutics, Editas Medicine and CRISPR Therapeutics to develop CAR-T therapies that incorporate gene editing technology, especially CRISPR technology.

The CRISPR/Cas9 gene editing system is simple and efficient, but the site-directed modification depends on an inefficient homologous recombination mechanism, so the capability of accurately editing genes needs to be improved. The single base editing technology (BE) can directly edit bases without cutting off DNA and RNA sequences, the editing efficiency reaches over 50 percent, the CRISPR is far exceeded, and almost no adverse side effect exists. The single base editing technology adds an important tool for the research and application of the gene editing technology.

In the prior art, the T lymphocyte preparation for treating tumors has the disadvantages of great side effect, complex preparation process and long period.

Disclosure of Invention

The invention aims to provide a preparation method of a T lymphocyte preparation for relieving the inhibition of a PD-1 immune check point, which simplifies the production process of the cell preparation, improves the speed of obtaining the preparation and has low side effect.

The preparation method of the T lymphocyte preparation for relieving the inhibition of the PD-1 immune checkpoint comprises the following steps:

(1) construction of Single-base Gene-editing PD-1 transfection System

Designing a gRNA target sequence according to an ITSM region gene sequence of human PD-1, and cloning the gRNA target sequence into an expression vector to obtain a gRNA target expression vector plasmid;

gRNA target sequence is 5'-GTGGCATACTCCGTCTGCTC-3' or 5'-TGGCATACTCCGTCTGCTCA-3';

mixing the gRNA target expression vector plasmid and the non-shearing DNA gene editing plasmid to obtain a single-base gene editing PD-1 transfection system;

(2) t lymphocyte isolation and activation

Separating, culturing, activating, propagating and collecting T lymphocytes;

(3) cotransfection of T lymphocytes

Adding an electrotransformation liquid and the single base gene editing PD-1 transfection system obtained in the step (1) into the T lymphocyte for cotransfection, continuing to culture after the transfection is finished, and collecting cells;

(4) flow cytometer screening, sequencing analysis and identification

Separating the collected cells after the transfection by using a flow cytometer, carrying out expanded culture on the separated monoclonal cells, extracting a genome, carrying out sequencing analysis and identification, and continuing the expanded culture to obtain the T lymphocyte preparation with the PD-1 immune check point relieved from inhibition.

The gRNA target sequence in step (1) is preferably 5'-GTGGCATACTCCGTCTGCTC-3'.

The expression vector in the step (1) is pHBLV-U6-gRNA-EF1-ZsGreen expression vector.

The non-cutting DNA gene editing plasmid in the step (1) is xCas9(3.7) -ABE (7.10), and xCas9(3.7) -ABE (7.10) is provided by Hanhengzheng Biotechnology (Shanghai) Co., Ltd.

xCas9(3.7) -ABE (7.10) expresses TadA and nCas9 genes and linker and NLS nuclear localization signals, and can be used for programming the conversion of A: T base pairs into adenine of G: C base pairs in tyrosine of non-sheared DNA single base editing ITSM.

The molar ratio of the gRNA target expression vector plasmid to the non-sheared DNA gene editing plasmid in the step (1) is 1: 1.

The electrotransformation liquid in the step (3) comprises the following components in percentage by volume:

no calcium magnesium PBS 90%

Serum-free medium RPMI 164010%;

the addition amount of EDTA is 30g/L and the addition amount of sucrose is 200mmol/L, based on the total volume of calcium-magnesium-free PBS and serum-free culture medium RPMI 1640 being 100%.

The electrotransformation liquid in the step (3) is prepared by mixing calcium-magnesium-free PBS with serum-free medium RPMI 1640, adding EDTA and sucrose, dissolving, and filtering for sterilization.

The continuous culture time in the step (3) is 48 h.

And (4) the flow cytometer screening method in the step (4) is to wash the cells twice by using PBS after the cells are transfected for 48 hours, and screen GFP positive T lymphocytes by the flow cytometer, namely to obtain a population immune cell preparation with high gRNA expression.

The preparation method of the T lymphocyte preparation for relieving the inhibition of the PD-1 immune checkpoint comprises the following specific steps:

(1) construction of Single-base Gene-editing PD-1 transfection System

Two gRNA target sequences are designed according to the gene sequence of the ITSM region of human PD-1: 5'-GTGGCATACTCCGTCTGCTC-3' and 5'-TGGCATACTCCGTCTGCTCA-3', one of the gRNA target sequences is selected to be cloned into an expression vector to obtain a gRNA target expression vector plasmid;

scoring the two gRNA sequences according to a CRISPR-DO website, wherein the score of the gRNA target sequence 5'-GTGGCATACTCCGTCTGCTC-3' is the highest, so that the gRNA target sequence 5'-GTGGCATACTCCGTCTGCTC-3' is preferably cloned into an expression vector;

(2) t lymphocyte isolation and activation

Separating, culturing, activating, propagating and collecting T lymphocytes;

(3) cotransfection of T lymphocytes

Adding an electrotransformation liquid into the T lymphocytes and the single-base gene editing PD-1 transfection system (namely two plasmids, a gRNA target expression vector plasmid and a non-shearing DNA gene editing plasmid) obtained in the step (1) for cotransfection, continuously culturing after the transfection is finished, and collecting cells;

(4) flow cytometer screening, sequencing analysis and identification

Separating the monoclonal cells with green fluorescent protein from the collected cells after the transfection is finished by using a flow cytometer, respectively carrying out amplification culture on the separated monoclonal cells, extracting a genome for sequencing analysis and identification, and continuously carrying out amplification culture on the cells meeting the requirements to obtain the T lymphocyte preparation with the suppression removed from the PD-1 immune check point.

The invention constructs a specific and high-efficiency PD-1 gene immunoreceptor tyrosine inhibition motif ITSM tyrosine single-base gene editing PD-1 transfection system which can be used for immune cell preparation production for tumor treatment and prevention.

After a single-base gene editing PD-1 transfection system electrically transfects T lymphocytes, GFP positive cells are screened by using a flow cytometer, and the screened T lymphocytes with the disinhibition of PD-1 immune check points after sequencing identification can be used for human body feedback prevention or tumor treatment.

The invention utilizes bioinformatics technology to obtain a gRNA target sequence for specifically and efficiently identifying a target site, and then clones the sequence into an pHBLV-U6-gRNA-EF1-ZsGreen expression vector by Hanhengshen Biotech (Shanghai) Limited company to successfully construct an expression vector capable of expressing the gRNA with the target sequence.

According to the invention, on the basis of constructing a gRNA expression vector and xCas9(3.7) -ABE (7.10), the expression vector is transferred into activated T lymphocytes by using an electrotransfection technology, an optimized electrotransfection buffer solution is adopted for electrotransfection, the transformation efficiency is high, the cell death rate is low, and the T lymphocyte population with the activity of relieving inhibition of a PD-1 immune checkpoint is more effectively obtained.

The gRNA sequences provided by the invention are unique to editing the tyrosine in the ITSM motif. Because the NGG base determines the sequence and the recognizable target point of the gRNA, and the ABE (adenine editor) edits A base in the sequence corresponding to the gRNA to have a certain window (4-6 bases), the situation that no other sequence can change tyrosine in ITSM motif of PD-1 gene except the gRNA sequence provided by the invention is determined.

The invention adopts single base gene editing technology to carry out point-to-point gene editing on tyrosine in ITSM on PD-1 in T lymphocyte, and blocks signal path of PD-1 for inhibiting T lymphocyte, thereby activating capability of T lymphocyte for attacking tumor cell, and the method greatly retains biological activity and other functions of PD-1, realizes immunotherapy of tumor, and reduces side effect of immunotherapy. The method has relatively simple cell preparation process, can realize the release of T cells from the immunosuppression of tumor cells only by changing the key amino acid-tyrosine in the immunoreceptor tyrosine inhibition motif ITSM, has short production period, and simultaneously retains other functions of PD-1 protein, thereby avoiding some unknown side effects and being more suitable for the national conditions and common people patients.

The invention has the following beneficial effects:

the invention firstly determines a gRNA target sequence capable of realizing specificity and high-efficiency recognition of ITSM motif of target site human PD-1, then introduces T lymphocyte by a double-plasmid co-electrotransfection method, screens GFP positive cell group by using a flow cytometer and performs sequencing identification to obtain a T lymphocyte preparation with the PD-1 immune checkpoint being relieved. The invention realizes the mutation of the key tyrosine locus codon of the ITSM region gene sequence of human PD-1, which leads to the release of the immunodetection function of PD-1 protein after gene transcription and translation. The PD-1 immune checkpoint disinhibiting activity T lymphocyte preparation improves the tumor cell killing capacity of T lymphocytes, greatly retains the biological activity and other functions of PD-1, and can enhance the long-term tumor immune effect.

Drawings

FIG. 1 is the structure of human PD-1.

FIG. 2 is a gRNA expression vector map.

FIG. 3 is an xCas9(3.7) -ABE (7.10) plasmid map.

Figure 4 is a schematic diagram of xCas9(3.7) -ABE (7.10) editing.

FIG. 5 is activated T lymphocytes.

FIG. 6 is a sequencing analysis of ITSM single base mutations in GFP positive cells.

Detailed Description

The present invention is further described below with reference to examples.

The experimental procedures, in which specific conditions are not specified, in the following examples are generally carried out under the conditions described in the conventional protocols in the protocols commonly used in the art, such as "molecular cloning laboratory Manual" (third edition, scientific Press, 2005) or under the conditions recommended by the reagent manufacturers.

Example 1

(1) Construction of Single-base Gene-editing PD-1 transfection System

Design of gRNA target sequence

Obtaining a PD-1 whole gene sequence (EF064716.1) from NCBI, finding out a gene sequence corresponding to an ITSM region, designing a gRNA target sequence based on a CRISPR-DO website, then selecting a proper target sequence 5'-GTGGCATACTCCGTCTGCTC-3' (20bp length) according to set parameters, then synthesizing the gRNA target sequence by Hanhengshen Biotechnology (Shanghai) Limited company, and adding a sticky end to form a double-stranded oligo sequence.

The pHBLV-U6-gRNA-EF1-ZsGreen plasmid (figure 2) is cut by restriction enzyme, the cut product is subjected to agarose gel electrophoresis to detect the cutting effect, the target vector band is cut from the gel after the agarose gel electrophoresis, and the gel is recovered to obtain the linearized expression vector.

The gRNA target sequence was ligated to the linearized pHBLV-U6-gRNA-EF1-ZsGreen vector using T4 ligase, transformed (competent cells: DH 5. alpha.), a single colony was picked after transformation, shaken at 37 ℃ 250 rpm for 14 hours, and the bacterial solution was sequenced.

And (4) sequencing and verifying correct positive cloning, and arranging plasmid miniextraction to obtain a gRNA target expression vector plasmid.

The xCas9(3.7) -ABE (7.10) plasmid (fig. 3) deaminates the a base in the tyrosine of ITSM to convert it to inosine, which is read and copied as G, thus effecting a to G mutation in the tyrosine of ITSM (fig. 4).

And mixing the gRNA target expression vector plasmid and the non-shearing DNA gene editing plasmid to obtain a single-base gene editing PD-1 transfection system, wherein the molar ratio of the gRNA target expression vector plasmid to the non-shearing DNA gene editing plasmid is 1: 1.

(2) T lymphocyte isolation and activation

①CD³⁺Antibody coated plate

Add 2 ml/well PBS buffer to 6 well plates, then add CD³⁺Antibody to a final concentration of 10. mu.g/ml, incubated at 37 ℃ CO₂Incubations were performed for 2 hours, followed by PBS aspiration, washing of the plates 2 times with fresh pre-warmed PBS, and 1 time with serum-free RPMI-1640 medium.

② separation and culture of mature T lymphocyte

1. 1mL of fresh blood from a healthy person was drawn through the brachial artery and immediately added to a 1.5mL centrifuge tube containing 10. mu.L of heparin anticoagulant. To maintain lymphocyte viability, isolation was performed immediately after blood collection.

2. Blood or plasma was diluted by adding 3mL of PBS or 0.9% NaCl. The blood dilution can reduce the agglutination of red blood cells and improve the yield of lymphocytes.

3. 4mL of the lymphocyte separation medium was added to the bottom of the centrifuge tube and the temperature was raised to room temperature.

4. 4ml of the diluted blood sample is sucked by a Pasteur glass pipette and is slowly spread on the lymphocyte separation solution along the wall of the tube without disturbing the interface of the liquid layer.

5. Centrifuging with a horizontal rotor at 2000r/min or 700 Xg for 20min at room temperature of 20 ℃. Storing for more than 2h, centrifuging for 30 min.

6. After centrifugation, the bottom of the tube is red blood cells, the middle layer is separation liquid, and the uppermost layer is plasma. Between the plasma layer and the separation fluid is a thin, dense white membrane containing mononuclear cells (including lymphocytes and monocytes). The layer of mononuclear cells was directly inserted with a pipette and the layer was pipetted into another tube.

7. The isolated lymphocytes were diluted with 10ml Hank's solution, centrifuged at 250 Xg for 10min and the supernatant discarded. Washing was repeated 2 times to remove platelets and anticoagulant. Finally, the cells were counted on a 10-fold counting plate diluted with PBS and trypan blue staining confirmed cell viability > 95%.

③ T lymphocyte activation and reproduction

Resuspending RPMI-1640 Whole Medium in step 7Precipitation of T lymphocytes, transfer to (r) CD³⁺Antibody coated 6-well plates, in CO₂The incubator was incubated for 24 hours, and then interleukin-2 (final concentration 1000U/ml) was added to stimulate cell growth, and the results are shown in FIG. 5.

(3) Cotransfection of T lymphocytes

The collected T lymphocytes are washed once with PBS, and then resuspended to a final concentration of 5-10X 10 by adding 400. mu.l of optimized electrotransfer solution (formula: calcium-magnesium-free PBS 90% (V/V), EDTA 30g/L, serum-free medium RPMI 164010% (V/V), sucrose 200mmol/L)⁶cell/ml, and then the single-base gene-editing PD-1 transfection system constructed in (1) was added to a final concentration of 40. mu.g/ml. 0.4cm electric shock cup, 280V voltage, electric shock 20 ms. After the electrotransfection is finished, adding a preheated RPMI-1640 whole culture medium, placing the mixture into a carbon dioxide incubator for continuous culture, and collecting cells after 48 hours.

(4) Flow cytometer screening, sequencing analysis and identification

After T lymphocytes are electrotransfected for 48 hours, the cells are collected and washed for 2 times by PBS, and the cells are placed in 1ml of PBS buffer solution to separate GFP positive cells by a flow cytometer, and monoclonal cells are obtained.

Respectively expanding and culturing the separated monoclonal cells to 1-2 × 10⁶Extracting genome by cell, carrying out PCR by using an identifying primer (PD 1F: 5'-GACATTTCAGAGGGGGACATTG-3'; PD 1R: 5'-CATGTACCTCCCACTCCTGT-3'), connecting a PCR product with a T vector after recovering the PCR product, then transforming escherichia coli competence, selecting monoclonal bacteria to carry out sanger sequencing analysis, and finding that in a single-base gene editing experiment, effective point-to-point mutation of changing A into G on two sites occurs in 44.7% of cells; 48.6% of the cells underwent efficient point-to-point mutation of A to G at a single site, which resulted in tyrosine alteration and loss of PD-1 inhibition in ITSM, whether at a single or both sites; FIG. 6 shows the results of mutation of one of the monoclonal cells; sequencing results of 15 ligation products show that only one ligation is not subjected to point-to-point mutation, so that tyrosine in ITSM of human PD-1 edited by single-base gene can obtain enough number of T lymphocyte populations with the immunosuppression of PD-1 immune checkpoints, and the cell populations only blockPD-1 inhibits the signaling pathway of T lymphocytes and does not alter the rest, so that the cell population can achieve the same immune efficacy as the wild type T lymphocytes.

In conclusion, the T lymphocyte population with the disinhibition of the PD-1 immune check point constructed by the invention is a novel immune cell preparation and can be used for preventing and treating tumor patients.

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Claims

1. A method for preparing a preparation of T lymphocytes with disinhibition of PD-1 immune checkpoint, comprising the steps of:

(1) construction of Single-base Gene-editing PD-1 transfection System

(2) t lymphocyte isolation and activation

Separating, culturing, activating, propagating and collecting T lymphocytes;

(3) cotransfection of T lymphocytes

(4) flow cytometer screening, sequencing analysis and identification

Separating the collected cells after the transfection by using a flow cytometer, carrying out expanded culture on the separated monoclonal cells, extracting a genome, carrying out sequencing analysis and identification, and continuing the expanded culture to obtain a T lymphocyte preparation with the suppression removed from the PD-1 immune check point;

the plasmid without shearing DNA gene editing in the step (1) is xCas93.7-ABE 7.10.

2. The method for preparing a PD-1 immune checkpoint disinhibiting T lymphocyte preparation according to claim 1, characterized in that in step (1) the gRNA target sequence is 5'-GTGGCATACTCCGTCTGCTC-3'.

3. The method for preparing a PD-1 immune checkpoint disinhibiting T lymphocyte preparation as claimed in claim 1, characterized in that the expression vector in step (1) is pHBLV-U6-gRNA-EF1-ZsGreen expression vector.

4. The method for preparing a preparation of T lymphocytes having disinhibition at PD-1 immune checkpoint as claimed in claim 1, wherein the molar ratio of gRNA target expression vector plasmid to non-splicing DNA gene editing plasmid in step (1) is 1: 1.

5. The method for preparing a preparation of T lymphocytes having disinhibition at a PD-1 immune checkpoint as claimed in claim 1, wherein in step (3) the electrotransfer composition is as follows, in volume percent:

no calcium magnesium PBS 90%

Serum-free medium RPMI 164010%;

6. The method for preparing a preparation of T lymphocytes having disinhibition at PD-1 immune checkpoint as claimed in claim 1 or 5, wherein the electrotransfer in step (3) is prepared by mixing calcium-magnesium-free PBS with serum-free medium RPMI 1640, adding EDTA and sucrose, dissolving, and then sterilizing by filtration.

7. The method for preparing a preparation of T lymphocytes with disinhibition of PD-1 immune checkpoint as claimed in claim 1, characterized in that the incubation period in step (3) is 48 h.