CN116004726B - Genetically modified T cell and preparation method and application thereof - Google Patents

Abstract

The invention provides a genetically modified T cell that can co-express a PD-1 targeted shRNA, IL-24, and NKG2D CAR. The invention utilizes shRNA gene silencing technology to inhibit the expression of PD-1 protein in NKG2Dx24CAR-T cells to prepare PD-1 ^KD NKG2Dx24CAR-T cells can avoid immunosuppression mediated by PD-L1 molecules in tumor microenvironment due to non-expression of PD-1, and can maintain anti-tumor activity for a long time.

Description

Genetically modified T cell and preparation method and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a genetically modified T cell, and a preparation method and application thereof.

Background

Chimeric antigen receptor-modified T cell (CAR-T cell) therapy is a tumor-targeted adoptive cell therapy developed in recent years. The general principle is that T cells express chimeric antigen receptors capable of specifically recognizing tumor antigens by a gene editing method, so that the T cells are endowed with specific killing capacity to tumor cells. CAR-T cells targeting CD19 and BCMA antigens have shown surprising efficacy in hematological tumor therapy. However, CAR-T cell therapy of solid tumors still faces many challenges. The most major factors affecting CAR-T cell efficacy in solid tumor treatment include: 1. lacking tumor-specific antigen, CAR-T cell therapy can produce off-target toxicity; 2. inhibitory tumor microenvironment causes CAR-T cell disability and even apoptosis; 3. the high heterogeneity of solid tumor cells, the inability to recognize single tumor-specific antigens to completely clear tumor cells, and the like; therefore, to overcome the above factors that prevent CAR-T cells from effectively killing solid tumors, T cells must be engineered with a completely new gene editing strategy.

NKG2D is an activating receptor expressed on the surface of human natural killer cells, cd56+ and cd8+ T cells, plays an important role in natural immunity, and is involved in recognition of virus-infected cells and killing of tumor cells by NK cells. Unlike other NKG2 receptors, NKG2D possesses a variety of ligands, including human MHC-class I chain-related molecules (MICA and MICB) and human UL16 binding proteins (ULBPs, also known as human RAET 1). MIC and ULBP are both considered tumor-associated antigens and are widely expressed in tumors of multiple epithelial origin, such as breast, lung, ovarian, colon, glioma, and melanoma. When the CD3 zeta and NKG2D are in tandem design for fusion expression, the ITAM motif of the CD3 zeta subunit is phosphorylated after the NKG2D is combined with a ligand, so that a downstream molecule is further activated, and a first signal of T cell activation is transmitted. At the same time, binding of NKG2D to the ligand induces the charged amino acid residues of the transmembrane region of NKG2D homodimers to link to the transmembrane residues of DAP10 via two salt bridges, forming a hexamer structure, which in turn induces phosphorylation of the yxsm motif in the cytoplasm, and then activates the downstream phosphoinositide 3 kinase (PI 3K) signaling pathway, delivering a second signal for T cell activation. Therefore, the NKG 2D-based CAR-T cell is constructed, so that various tumors can be identified and killed, and the broad-spectrum anti-tumor effect is achieved.

IL-24 is a multifunctional broad-spectrum cancer suppressor gene of IL-10 family, and can promote apoptosis of tumor cells through a plurality of pathways such as JAK/STAT pathway, P38MAPK pathway, PKR pathway, fas/FasL pathway, endoplasmic reticulum stress and the like, and can also inhibit local neovascularization of tumor so as to inhibit tumor growth. In addition, researches report that IL-24 can also enhance sensitivity of radiotherapy and chemotherapy and inhibit invasion and migration of cancer cells, and has no toxic or side effect on normal cells.

Disclosure of Invention

In order to solve the problems, the invention utilizes the shRNA gene silencing technology to inhibit the expression of PD-1 protein in NKG2Dx24CAR-T cells, and designs the PD-1 ^KD NKG2Dx24CAR (structure schematic diagram is shown in figure 1) to prepare PD-1 ^KD NKG2Dx24CAR-T cells which are prevented from being subjected to immunosuppression mediated by PD-L1 molecules in tumor microenvironment due to non-expression of PD-1 can maintain anti-tumor activity for a long time.

In the present invention, "shRNA" refers to short hairpin RNA, translated into "short hairpin RNAs," and shRNA cloned into shRNA expression vectors comprises two short inverted repeats.

In the present invention, "NKG2D" is an activating receptor expressed on the surface of human natural killer cells, cd56+ and cd8+ T cells, and "NKG2D gene" is a nucleotide sequence expressing NKG 2D.

In the present invention, the "CD3 zeta gene" is a gene encoding a CD3 zeta chain, and CD3 has five peptide chains, namely, a gamma chain, a delta chain, an epsilon chain, a zeta chain and a eta chain, which are transmembrane proteins, and the present invention mainly relates to zeta chains.

In one aspect, the invention provides a recombinant plasmid.

The recombinant plasmid comprises:

(1) A shRNA sequence of the targeted silencing PD-1;

(2) Including the tandem sequence of the NKG2D gene, CD3 zeta gene, IL-24 gene and P2A shearing peptide gene.

Preferably, the shRNA sequence is designed according to any one of the siRNA of SEQ ID NO. 1-3.

Further preferably, the shRNA sequence is selected from any one of the following:

(1) Sense strand SEQ ID NO.5, antisense strand SEQ ID NO.6;

(2) Sense strand SEQ ID NO.7, antisense strand SEQ ID NO.8;

(3) Sense strand SEQ ID NO.9, antisense strand SEQ ID NO.10.

Specifically, the NKG2D gene is spliced with the reverse inverted CD3 zeta gene, and the IL-24 gene is connected with the spliced gene in series through the P2A cutting peptide gene sequence.

The NKG2D gene codes an amino acid sequence shown in SEQ ID NO.14;

the CD3 zeta gene codes for an amino acid sequence shown as SEQ ID NO.18;

the IL-24 gene codes an amino acid sequence shown as SEQ ID NO. 20;

the P2A shearing peptide gene codes for an amino acid sequence shown in SEQ ID NO. 16.

Preferably, the NKG2D gene is SEQ ID No.13; the CD3 zeta gene is SEQ ID NO.17; the IL-24 gene is SEQ ID NO.19; the P2A shearing peptide gene is SEQ ID NO.15.

In some embodiments, the tandem sequence is SEQ ID No.21.

Specifically, the shRNA sequence and the tandem sequence are located downstream of different promoters, respectively.

Preferably, the recombinant plasmid has a skeleton of a pLL3.7-U6-EF1a-EGFP plasmid.

Further, the shRNA sequence is positioned downstream of the U6 promoter, and the tandem sequence is positioned downstream of the EF1a promoter.

Furthermore, the insertion enzyme cleavage site of the shRNA sequence is between HpaI and XhoI; the tandem sequence is inserted between Nhe I and EcoR I.

In some embodiments, the recombinant plasmid sequence is SEQ ID No.25.

In another aspect, the invention provides the use of the recombinant plasmid described above in the preparation of lentiviruses or T cells.

Preferably, the T cells are CAR T cells.

In yet another aspect, the invention provides a recombinant lentivirus.

The recombinant lentivirus comprises the recombinant plasmid.

In some embodiments, the recombinant lentivirus is Lenti-PD-1 ^KD NKG2Dx24 recombinant lentiviruses.

In yet another aspect, the invention provides a genetically modified T cell.

The genetically modified T cells co-express the PD-1 targeted shRNA, IL-24 and NKG2DCAR.

Preferably, the genetically modified T cells are prepared by recombinant lentiviruses as described above.

Preferably, the recombinant lentivirus is prepared by transfecting PBMC-derived T cells to obtain genetically modified T cells.

In some embodiments, the genetically modified T cell is PD-1 ^KD NKG2Dx24CAR-T cells.

In still another aspect, the invention provides the use of the recombinant plasmid or recombinant lentivirus or genetically modified T cell described above in the preparation of an antitumor drug.

The antitumor drug comprises the genetically modified T cells.

Or the antitumor drug is prepared by the recombinant plasmid or the recombinant lentivirus.

In yet another aspect, the invention provides an antitumor drug.

The antitumor drug comprises the genetically modified T cells.

Or the antitumor drug is prepared by the recombinant plasmid or the recombinant lentivirus.

In yet another aspect, the invention provides a method of preparing a recombinant plasmid.

The preparation method comprises the step of inserting the shRNA sequence and the tandem sequence into a carrier framework.

Specifically, the preparation method comprises the steps of inserting the shRNA sequence into a carrier framework, and then inserting the tandem sequence into the carrier framework.

In yet another aspect, the invention provides a method of preparing a recombinant lentivirus.

The preparation method comprises the step of transfecting cells with the recombinant plasmid.

Preferably, the recombinant plasmid is a pLL3.7-PD1/sh3-NKG2Dx24 plasmid.

Preferably, the recombinant plasmid is co-transfected with a lentiviral packaging plasmid.

Preferably, the lentiviral packaging plasmid may be psPAX2 and/or pmd2.G.

Further preferably, the molar ratio of the pLL3.7-PD1/sh3-NKG2Dx24 plasmid to the psPAX2 and pMD2.G is 1:1:1.

In yet another aspect, the invention provides a method of preparing a genetically modified T cell.

The preparation method comprises the step of transfecting T cells with the recombinant lentivirus.

Preferably, the T cells are PBMC-derived T cells.

Preferably, the transfection MOI value is 5.

In yet another aspect, the invention provides a method of treating a tumor.

The methods of treatment include administering to a patient the genetically modified T cells described above.

Preferably, the method of treatment comprises administering PD-1 to a patient ^KD NKG2Dx24CAR-T cells.

The invention has the beneficial effects that:

according to the invention, through the strategy of co-expressing shRNA of the targeting PD-1, IL-24 and NKG2D CAR to carry out gene modification on T cells, the T cells can specifically identify and kill various tumor cells, avoid the benefits of immunosuppression of tumor microenvironment and the like, can overcome various difficulties and challenges faced in the process of treating solid tumors by the CAR-T cells, and has important drug development value and wide clinical application prospect.

Specifically, the invention coexpression of IL-24 on NKG2D CAR-T cells (named NKG2Dx24CAR-T cells) can obviously enhance the killing ability of the NKG2D CAR-T cells on various tumors, and still has stronger killing activity on tumor cells with low expression or lack of NKG2D ligand. One of the important reasons for preventing CAR-T cells from continuously and effectively killing tumor cells is that various immunonegative regulation molecules can be expressed in tumor tissues, and can be combined with inhibitory receptors on the surfaces of T cells to induce T cell dysfunction and even apoptosis, so that immune monitoring is avoided. PD-L1 is a ligand molecule capable of mediating immune negative regulation, is expressed in various tumor tissues, and is often up-regulated in some tumor cells under the stimulation of IFNg released by immune cells. PD-L1 on the surface of tumor cells binds to the inhibitory checkpoint molecule receptor PD-1 on the surface of activated T cells (including CAR-T cells prepared in vitro), which can induce T cell depletion. Inhibiting the expression of PD-1 on the surface of T cells can eliminate immunosuppression mediated by the interaction of PD-L1 and PD-1, thereby maintaining the continuous tumor killing capability of the T cells.

Drawings

FIG. 1 is PD-1 ^KD Schematic of NKG2Dx24CAR structure.

FIG. 2 shows the detection of the expression level of PD-1 protein.

FIG. 3 shows the results of agarose gel electrophoresis of recombinant plasmids.

FIG. 4 shows a map of the recombinant plasmid pLL3.7-PD-1/sh3-NKG2Dx 24.

FIG. 5 is a flow cytometry detection of PD-1 ^KD Results of expression of NKG2Dx24CAR-T cells PD-1.

Fig. 6 is a result of detecting expression of NKG2Dx24CAR by flow cytometry.

FIG. 7 is PD-1 ^KD Secretion levels of IL-24 in NKG2Dx24CAR-T cell culture supernatants.

FIG. 8 is PD-1 ^KD IL-2 release levels in NKG2Dx24CAR-T cells and target cell co-culture supernatants.

FIG. 9 is PD-1 ^KD IFNg release levels in NKG2Dx24CAR-T cells and target cell co-culture supernatants.

FIG. 10 is PD-1 ^KD Extent of killing of target cells by NKG2Dx24CAR-T cells.

FIG. 11 is a growth curve of subcutaneous tumor transplantation in mice.

FIG. 12 is a survival curve of mice.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 construction of pLL3.7-PD-1/shRNA-NKG2Dx24 recombinant plasmid vector

Firstly, designing small hairpin RNA (shRNA) for targeted silencing of PD-1, constructing the small hairpin RNA into the downstream of a U6 promoter of a pLL3.7-U6-EF1a-EGFP plasmid, and obtaining the pLL3.7-PD1/shRNA-EF1a-EGFP recombinant plasmid with the optimal PD-1 silencing effect through screening; the NKG2D, CD zeta and IL-24 gene are designed into NKG2Dx24CAR gene through P2A cutting peptide in series, after the NKG2Dx24CAR gene is synthesized completely, the NKG2Dx24CAR gene is constructed to the downstream of the pLL3.7-PD1/shRNA-EF1a-EGFP plasmid EF1a promoter, and the expression of the NKG2Dx24CAR gene is controlled through the EF1a promoter, thereby obtaining the pLL3.7-PD-1 ^KD NKG2Dx24 recombinant plasmid.

Construction of a pLL3.7-PD1/shRNA-EF1a-EGFP recombinant plasmid

And designing the shRNA targeting the PD-1 and constructing the shRNA between Hpa I and XhoI of the pLL3.7-U6-EF1a-EGFP plasmid, so as to obtain the pLL3.7-PD1/shRNA-EF1a-EGFP recombinant plasmid. The method comprises the following specific steps:

1.1 design and Synthesis of PD-1 targeting shRNA sequences

According to BLOCK-iT ^TM RNAi Designer

(https:// rniadesigner. Thermo filter. Com/rniadexpress/design. Do) the PD-1 coding sequences were designed and three top scoring siRNAs were screened (Table 1). The main principle of screening is as follows: (1) the target site must be an open reading frame region; (2) the 5' end starts with G; (3) the content of G+C is 30% -50%; (4) No consecutive 3 or more T's can occur on the target sequence to prevent subsequent shRNA transcription termination. BLAST (NCBI) non-homology queries of the target sequences showed no other homologous sequences. Based on the screened target sequence, a small hairpin RNA (shRNA) interference fragment is connected between HpaI and XhoI of the vector according to the requirements of the pLL3.7-U6-EF1a-EGFP vector, and the shRNA sense strand structure is designed: adding T (rebuilding U6 promoter), interference sequence, loop (TTCAAGAGAGA), interference sequence reverse complementary sequence, termination signal (TTTTTT), and filling in Xho I cleavage site at 5' end; shRNA antisense strand structure: adding A at the 3' end, interfering sequence complementary sequence, loop complementary sequence (AAGTTCTCT), interfering sequence reverse sequence, termination signal complementary sequence (AAAAAA), and filling in Xho I enzyme cutting site. Three PD 1-targeting shRNA sequences were designed as follows (table 2). The designed shRNA sequence is sent to Suzhou gold only intelligent biotechnology Co.

TABLE 1PD1-siRNA sequences

TABLE 2PD1-shRNA sequences

1.2 annealing of PD-1-targeted shRNA fragments to form double strands

The synthesized interfering fragments were diluted to a concentration of 10mM, respectively, and the annealing reaction was prepared as follows. After mixing, the reactants are placed in a PCR instrument and heated at 95 ℃ for 5min, after heating, the samples are taken out and cooled to room temperature, and PD-1-shRNA double-stranded fragments (named as PD1/sh1, PD1/sh2, PD1/sh3 and PD1/shNC respectively) are formed.

1.3pLL3.7-U6-EF1 alpha-EGFP plasmid linearization cleavage

And (3) linearized enzyme digestion of the backbone plasmid: mu.g of pLL3.7-U6-EF 1. Alpha. -EGFP plasmid was taken, and 1. Mu.L of restriction enzyme Hpa I (NEB, #R0105S) and 1. Mu.L of restriction enzyme Xho I (NEB, #R3101S), 5. Mu.L were addedBuffer (NEB, #R0146S), and finally double distilled water was added to bring the whole reaction volume to 50. Mu.L, and the sample was allowed to react at 37℃for 2 hours. After the reaction, the next step of recovering the linearized backbone plasmid is prepared.

1.4 linearized plasmid recovery

The required reagents:

1×TAE：4.84g Tris(solarbio，#T8060-100g)、0.744g Na ₂ EDTA·2H ₂ o (Shanghai, # 10009717) and 1.142mL glacial acetic acid (Guozhi, # 10000218) are fully dissolved in a proper amount of ddH ₂ O is used after the volume is fixed to 1L.

Agarose (Biowest, BY-R0100):

agarose gel recovery kit: a general agarose gel DNA recovery kit (Tiangen, # DP 209-02).

DNA loading:6 XDNA loading (gold full, # GH 101).

Experimental procedure

a.1% agarose gel preparation

Weighing 0.5g agarose, adding 50mL 1 xTAE solution, uniformly mixing, boiling in a microwave oven for 2min, and adding into a gel tank for condensation;

b. agarose gel electrophoresis

Adding DNA loading to the plasmid sample subjected to linearization enzyme digestion in the step 1.3 until the final concentration is 1X, adding the plasmid sample into a gel sample application hole, setting 150V, and carrying out electrophoresis for 15min;

c. linearized plasmid and target tape cutting gel

After electrophoresis, cutting agarose gel containing the target band under an ultraviolet gum cutter, and transferring the agarose gel into a new 1.5mL centrifuge tube for standby;

d. destination strip recovery

The target fragment was recovered according to the agarose gel recovery kit (Tiangen, # DP209-02) procedure, and the concentration was measured and stored at 4℃for further use.

1.5 connections

Taking the example of ligation of PD1/sh1 to a pLL3.7-U6-EF 1. Alpha. -EGFP plasmid vector, pLL3.7-PD1/sh1-EF1a-EGFP was obtained.

Connection kit: ligation high Ver.2 (TOYOBO, # LGK-201).

The experimental steps are as follows:

a. reaction system

The following connection reaction system is prepared:

reagent(s)	Volume of
		Ligation high Ver.2	10μL
Linearization carrier	2.5μL
		Gene fragment of interest	7.5μL

The target gene fragment is a PD1-shRNA-1 target fragment formed by annealing in the step 1.2; the linearization carrier is the linearization carrier recovered in the step 1.4; the target gene fragment and the linearization vector are added into the reaction system according to a molar ratio of 10:1.

b. Connection

Mixing uniformly, and then placing at 16 ℃ for reaction for 30min.

c. Storage of

After the reaction is finished, the connection product containing the recombinant lentiviral plasmid pLL3.7-PD1/sh1-EF1a-EGFP is obtained to be used immediately or stored at the temperature of minus 20 ℃ for standby.

The ligation products containing the recombinant lentiviral plasmids pLL3.7-PD1/sh2-EF1a-EGFP, pLL3.7-PD1/sh3-EF1a-EGFP and pLL3.7-PD1/shNC-EF1a-EGFP are obtained by the same method.

1.6 conversion

The required reagents:

DH 5. Alpha. Competent cells (healthy life, # KTSM 101L);

LB liquid medium: 10G tryptone (Sigma-Aldrich, # T9410-250G), 5G yeast extract (Sigma-Aldrich, # T9410-250G), 10G NaCl (Shanghai test, # 10019308) were added to ddH ₂ After O is dissolved, regulating pH7.0 to 1L, sterilizing at 121 ℃ for 15min under high pressure, and cooling to room temperature for use;

LB solid medium: 10G of tryptone (Sigma-Aldrich, # T9410-250G), 5G of yeast extract (Sigma-Aldrich, # T9410-250G) and 10G of NaCl (Shanghai test, # 10019308) are added, the pH is regulated to 7.0, then 10G of agar powder is added, the volume is fixed to 1L, the temperature is 121 ℃ and the temperature is kept at the same time, and the mixture is autoclaved for 15min and then cooled to room temperature for use;

antibiotics: 1g of the corresponding antibiotic powder (sigma) was weighed and dissolved well into 20mL ddH ₂ O,0.22 μm degerming, filtering, washing hair, degerming, split charging and storing at-20 ℃.

The experimental steps are as follows:

a. placing a DH5 alpha competent cell (100 mu L) on ice for thawing, sucking 10 mu L of the connection product obtained in the step 1.5 by a gun head after the competent cell is thawed, adding the connection product into the competent cell, lightly blowing and beating for several times, uniformly mixing, and then carrying out ice bath for 20min;

b. after the ice bath is finished, placing the ice bath in a water bath kettle, and immediately placing the ice bath on ice for 2min after heat shock at 42 ℃ for 90 s;

c. adding 500 mu L of LB liquid medium without antibiotics into the bacterial liquid, transferring to a shaking table at 37 ℃ and incubating for 1h at 200 rpm;

d. absorbing a proper amount of incubated bacterial liquid, and coating the bacterial liquid on LB solid medium containing 100 mug/mL ampicillin;

e, culturing in an incubator at 37 ℃ overnight upside down, and observing that monoclonal colonies appear on the LB solid medium.

1.7 plasmid extraction

The required reagents:

LB liquid medium: 10G tryptone (Sigma-Aldrich, # T9410-250G), 5G yeast extract (Sigma-Aldrich, # 70161-500G), 10G NaCl (Shanghai test, # 10019308) were added with ddH ₂ After O is dissolved, regulating the pH value to 7.0 to 1L, sterilizing at 121 ℃ for 15min under high pressure, and cooling to room temperature for use;

plasmid small extract kit: omega plasmid miniprep kit (Omega, # D6943);

antibiotics: 1g of the corresponding antibiotic powder (sigma) was weighed and dissolved well into 20mL ddH ₂ O,0.22 μm degerming, filtering, washing hair, degerming, split charging and storing at-20 ℃.

The experimental steps are as follows:

a) Selecting a monoclonal colony grown in the LB solid medium in the step 1.6, adding 5mL of LB liquid medium containing 100 mug/mL ampicillin, and culturing overnight (12-16 h) in a bacterial culture shaking table at 37 ℃ and 200 rpm;

b) Taking 1.5-5mL of bacterial liquid, centrifuging for 1min at room temperature 10000 Xg;

c) Removing the supernatant, adding 250 mu L of solution I (containing RNase A), and oscillating by a vortex oscillator until the thalli are completely suspended;

d) 250 μl of solution II was added and the tube was gently inverted 4-6 times to obtain a clear lysate. Preferably, incubation is carried out for 2min at room temperature, and vigorous mixing can cause the chromosomal DNA to be sheared, so that the purity of the plasmid is reduced;

e) Adding 350 μl of solution III, mixing gently and reversely for several times until white flocculent precipitate appears, centrifuging at 10000×g at room temperature for 10min;

f) The supernatant was carefully aspirated and transferred to a clean, well-fitted, 2mL centrifuge tube, absorbent column. To ensure that there are no aspiration sediments and cell debris. Centrifuging at 10000 Xg for 1min at room temperature until the lysate passes through the absorption column completely;

g) Discarding the filtrate, adding 500 μl Buffer HB, centrifuging at 10000×g for 1min, cleaning the absorption column, and removing residual protein to ensure DNA purity;

h) If the next step is not highly demanding of plasmid purity, other screening methods such as enzymatic digestion may be omitted;

i) The filtrate was discarded, and the column was washed with 750. Mu.L Wash Buffer diluted with 100% ethanol and centrifuged at 10000 Xg for 1 min: the Wash Buffer concentrated solution must be diluted with pure ethanol before use, and the method is that if the label is frozen, the label must be restored to room temperature before use;

j) This step is optional: then 750 mu L of Wash Buffer is added to clean the absorption column;

k) Centrifugation of 10000 Xg of the column for 1min is necessary to ensure that ethanol is removed, which can affect the following steps;

l) placing the column in a clean 1.5mL centrifuge tube, adding 50-100. Mu.L (depending on the desired final concentration) of sterile deionized water or TE buffer onto the filter membrane, centrifuging 10000 Xg for 5min;

m) after measuring the concentration, the mixture is stored in a refrigerator at the temperature of minus 20 ℃ for standby.

1.8 identification of recombinant plasmids

1. Mu.g of the recombinant plasmid extracted in step 1.7 was taken and 1. Mu.L of restriction enzyme Hpa I (NEB,

#R0105S) and 1. Mu.L of restriction enzyme Xho I (NEB, #R3101S), 5. Mu.LBuffer (NEB, #R0146S), was finally added with double distilled water to bring the whole reaction volume to 50. Mu.L, and incubated in a 37℃water bath for 1 hour. Then the identification is carried out by 1% agarose gel electrophoresis, a target band of about 70bp is cut off, the recombinant plasmid with correct enzyme digestion is sent to Jin Weizhi biotechnology limited company for sequencing comparison to be consistent with the shRNA sequence of the target gene targeting PD1, and the successful preparation is proved

The pLL3.7-PD1/sh1-EF1a-EGFP, pLL3.7-PD1/sh2-EF1a-EGFP, pLL3.7-PD1/sh3-EF1a-EGFP and pLL3.7-PD1/shNC-EF1a-EGFP recombinant plasmids.

1.9 screening recombinant plasmid with optimal PD-1 expression inhibition effect

1) Transfection

a) To verify the efficiency of four different PD-1 targeted shRNA targeted silencing of PD-1 we constructed 293A cells stably expressing PD-1-EGFP fusion proteins, designated 293A-PD1 cells. 293A-PD1 cells were plated at 5X 10 the day prior to transfection ⁴ Wells were seeded into 24-well plates and placed in 5% CO ₂ Incubation overnight in an incubator at 37 ℃;

b) Transfection was started after cell confluence reached around 70%. Adding 2 mu g of the pLL3.7-PD1/sh1-EF1a-EGFP, pLL3.7-PD1/sh2-EF1a-EGFP, pLL3.7-PD1/sh3-EF1a-EGFP or pLL3.7-PD1/shNC-EF1a-EGFP recombinant plasmid obtained in the step 1.8 into a 1.5mL centrifuge tube containing 50 mu L jet PRIME buffer, vortex shaking for 10 seconds, centrifuging to enable tube wall liquid to sink into the bottom of the tube;

c) 2. Mu.L of jet prime@regent (Polyplus, cat.) was added to each tube in the 1.5mL centrifuge tube of step b): 21Y0910L 3), vortex shaking for 10s, centrifuging to enable the tube wall liquid to sink into the tube bottom, and incubating for 10min at room temperature;

d) Dripping the four transfection mixtures prepared in the step c) into 24-well plate cells inoculated on the previous day, wherein the final volume of each well is 0.5mL, and placing the mixture in 5% CO ₂ Incubating in an incubator at 37 ℃;

e) After 4 hours, the transfection wells were replaced with fresh DMEM medium containing 10% FBS and incubation was continued for 48 hours.

2) WB detection

a) Collecting 293-PD1 cells transfected by the four recombinant plasmids in the step 1), adding 100 mu L of lysis solution containing 1% NP-40 (XW 0901645901, national drug group) and 1% PMSF (solabio, R0010) into RIPA (solabio, R0010), mixing by vortex for 30s, and standing on ice for 20min;

b) Centrifuging the sample at 12000rpm at 4deg.C for 10min, and transferring the supernatant to a new centrifuge tube;

c) Adding 6 Xprotein loading to working concentration (1X), heating at 100deg.C for 6min to denature protein, and directly using sample in SDS electrophoresis or preserving in-20deg.C refrigerator;

d) Respectively taking 30 mu L of each of the four cell lysates, and adding samples to 10% SDS-PAGE,120V and 90min to complete electrophoresis;

e) Proteins in SDS-PAGE were transferred to PVDF membrane by constant flow of 350mA for 120 min; placing the PVDF film after film transfer in a sealing liquid containing 5% of skimmed milk, and sealing overnight at 4 ℃;

f) The blocked PVDF membrane was cut into two parts, and 10mL of antibody containing TIM3 (3G 11, antibody purified from mouse ascites, 1:5000 dilution) and 10mL of GAPDH antibody (cat# 39-8600,Thermo fisher,1:5000) were placed into each part, and incubated for 1h with shaking at room temperature;

g) The primary incubation-resistant PVDF membrane was washed 4 times with TBST for 5 minutes each;

h) Putting 10mL of goat anti-mouse IgG secondary antibody (Zhonghua gold bridge, ZB-2305) containing horseradish peroxidase mark into the washed PVDF membrane, and incubating for 50min at room temperature;

i) TBST is washed for 3 times, each time for 5min;

j) After addition of the ECL-sensitized developing solution, a photograph was taken with a Technical imaging system (Shanghai Technical Co., # Tanon-4600 SF).

3) Analysis of results

293A-PD1 cells are cell lines stably expressing human PD-1. The molecular weight of the PD-1-EGFP fusion protein is about 70kDa, and the PD1/shRNA targets to silence the PD-1 gene, so that the brightness of a protein band of a WB result near 70kDa can be predicted to be reduced compared with a control group. WB results showed that PD1/sh2/3 significantly reduced expression of the target protein, with PD1/sh3 having the highest silencing efficiency for the target gene (fig. 2). Thus, we selected the pLL3.7-PD1/sh3-EF1a-EGFP plasmid for subsequent experiments.

Construction of pLL3.7-PD1/sh3-NKG2Dx24 recombinant plasmid

Nucleotide and amino acid sequences encoding human NKG2D, IL-24 and CD3 ζ were queried at NCBI website and were referenced from literature: systematic comparison of 2, apeptides for cloning muLti-genes in a polycistronic vector scientific Reports volume7, arc number 2193 (2017); then splicing the NKG2D gene with the reverse inverted CD3 zeta gene, and connecting the IL-24 gene with the spliced gene in series through P2A cutting peptide gene sequence, thereby designing the NKG2Dx24 polycistronic gene. The NKG2Dx24 gene synthesized by the whole genes is used as a template, the upstream and downstream specific primers of the NKG2Dx24 gene are designed, the upstream primers and the downstream primers respectively comprise homologous arm sequences of the upstream and downstream of the cleavage site of the pLL3.7-PD1/sh3-EF1a-EGFP plasmid Nhe I, the amplified target gene fragment is connected between Nhe I and EcoR I of the pLL3.7-PD1/sh3-EF1a-EGFP recombinant plasmid obtained in the step 1.9 by a homologous recombination biological method, and the recombinant plasmid pLL3.7-PD1/sh3-NKG2Dx24 is obtained. The specific method comprises the following steps:

the nucleotide sequence for coding NKG2D is SEQ ID NO.13;

the NKG2D amino acid sequence is SEQ ID NO.14;

the nucleotide sequence of T2A is SEQ ID NO.15;

the amino acid sequence of T2A is SEQ ID NO.16;

the nucleotide sequence of CD3 zeta is SEQ ID NO.17;

the CD3 zeta amino acid sequence is SEQ ID NO.18;

the nucleotide sequence for coding IL-24 is SEQ ID NO.19;

the amino acid sequence SEQ ID NO.20 encoding IL-24.

2.1 Gene Synthesis

The NKG2Dx24 gene fragment is synthesized by the full gene of Jin Weizhi Biotechnology Co, the nucleotide sequence of the gene is SEQ ID NO.21, and the corresponding amino acid sequence is SEQ ID NO.22.

2.2pLL3.7-PD1/sh3-EF1 alpha-EGFP plasmid linearization cleavage

And (3) linearized enzyme digestion of the backbone plasmid: 1. Mu.g of pLL3.7-PD1/sh3-EF 1. Alpha. -EGFP plasmid was added with 1. Mu.L of restriction enzyme Nhe I (NEB, #R313131S) and 1. Mu.L of restriction enzyme EcoR I (NEB, #R3101S), 5. Mu.LBuffer (NEB, #B7204), and finally double distilled water was added to bring the whole reaction volume to 50. Mu.L, and the sample was left to react at 37℃for 2 hours. Gel electrophoresis and gel recovery of linearized plasmids after the reaction.

2.3 target fragment amplification

Designing an upstream primer and a downstream primer of NKG2Dx24 specificity, wherein the 5 'end of the upstream primer comprises 20bp basic groups homologous with the upstream 20bp basic groups of the enzyme cutting site of the pLL3.7-U6-EF1 alpha-EGFP plasmid Nhe I, the 3' end of the downstream primer comprises 20bp basic groups homologous with the downstream 20bp basic groups of the enzyme cutting site of the pLL3.7-U6-EF1 alpha-EGFP plasmid EcoRI (the upstream primer sequence of NKG2Dx24 is SEQ ID NO.23 and the downstream primer sequence of NKG2Dx24 is SEQ ID NO. 24), amplifying the gene synthesized in the step 2.1 by using KOD-Plus-Neo (TOYOBO, # KOD-401) reagents to obtain G2Dx24 gene fragments respectively comprising the homologous arm sequences of the upstream and EcoR I of the pLL3.7-U6-EF1a-EGFP plasmid Nhe I, and recovering the gene fragments into gel fragments with the expected size of 168 bp. The reaction system is as follows:

component (A)	Volume of
		10x Buffer for KOD–Plus-Neo	5μL
2mM dNTPs	5μL
		25mM MgSO 4	2μL
10 pmol/. Mu.L of upstream primer	1.5μL
		10 pmol/. Mu.L downstream primer	1.5μL
Template DNA	1μL
		PCR grade water	33μL
KOD-Plus-Neo(1.0U/μL)	1μL

2.4 linearization plasmids and methods for recovery of gene fragments of interest refer to section 1.4.

2.5 homologous recombination

The NKG2DxIL-24 gene fragment synthesized by the gene in the step 2.1 is connected with the linearization plasmid recovered in the step 2.3 through homologous recombination.

The required reagents:

connection kit: minerva Super Fusion Cloning Kit kit (Yu Heng Su, #M2026-50T);

the experimental steps are as follows:

a. reaction system

Component (A)	Volume of
		Super Fusion Cloning Mix(2×)	5μL
Linearization vector	1μL
		Target gene fragment	4μL

* : and 2.1, adding the target gene fragment synthesized by the gene in the step 2.1 and the linearization vector recovered by linearization in the step 2.3 into a reaction system according to a molar ratio of 3:1.

b. Connection

Mixing uniformly, and then placing at 50 ℃ for reaction for 30min.

c. After the reaction is finished, the connection product containing the recombinant plasmid pLL3.7-PD-1/sh3-NKG2Dx24 is obtained and immediately transformed or stored at the temperature of minus 20 ℃ for standby.

2.6 transformation methods refer to section 1.6.

2.7 plasmid extraction methods refer to section 1.7.

2.8 recombinant plasmid identification methods refer to section 1.8.

The recombinant plasmids extracted in step 2.7 were used for identification, and restriction enzymes were XhoI (NEB, #R0146S) and EcoRI (NEB, #R3101S).

The agarose gel electrophoresis result is shown in FIG. 3, wherein 1 is recombinant plasmid without enzyme digestion; 2: xhoI/EcoRI double-restriction recombinant plasmid; 3 is Marker, the cut band is about 2300bp, the correctly cut recombinant plasmid is sent to Jin Weizhi biotechnology limited company for sequencing comparison and is consistent with the sequence of the target gene NKG2Dx24, and the successful preparation of the pLL3.7-PD-1/sh3-NKG2Dx24 recombinant plasmid is proved, and the plasmid map is shown in figure 4. The sequence of the recombinant plasmid pLL3.7-PD-1/sh3-NKG2Dx24 is shown as SEQ ID No.25.

Example 2PD-1 ^KD Preparation of NKG2Dx24CAR T cells

1.Lenti-PD-1 ^KD NKG2Dx24 recombinant lentivirus preparation

1) Transfection

a) 293T cells were grown at 7X 10 ⁵ The density of individual cells/well was inoculated into 6-well plates containing 2mL of lentiviral packaging medium. Placing at 37deg.C, 5% CO ₂ Cells were incubated overnight under conditions to ensure that cell densities reached 95-99% confluence at transfection.

All plasmids were diluted to 1. Mu.g/. Mu.L with Opti-MEM (Thermo, # 31985070).

A pipe A: mu.L of Lipofectamine 3000 (Thermo, #L 3000015) was diluted in 250. Mu.L of serum-free Opti-MEM medium and vortexed for 10s.

And B, pipe B: the recombinant lentiviral plasmid pLL3.7-PD1/sh3-NKG2Dx24 was added to 250. Mu.L of serum-free Opti-MEM medium in a molar ratio of 1:1:1 with lentiviral packaging plasmids psPAX2 and pMD2.G, and 6. Mu. L P3000 reagent was added and vortexed for 10s.

b) Preparation of liposome-DNA complexes: the tube A mixture was transferred to tube B and thoroughly mixed and incubated at room temperature for 10min.

c) Before adding the complex, 1mL of the medium was removed per well to make the total volume per well 1mL, and then 500. Mu.L of the liposome-DNA complex was added to each well, and the plate was gently shaken to make its distribution uniform. The plates were placed at 37℃with 5% CO ₂ Incubate under conditions for 6 hours.

d) After 6 hours of transfection, the medium containing the liposome-DNA complex was carefully aspirated from each well and the aspirated medium was treated with 10% bleaching solution and disposed. 2mL of pre-warmed DMEM (Hyclone, # SH30022.01; FBS, # FSP 500) with 10% FBS was replaced with fresh medium. The plates were returned to the incubator at 37℃with 5% CO ₂ Incubation in the incubator was continued.

e) After 24 hours of transfection, 2mL of cell supernatant was collected per well, loaded into a 15mL conical tube and stored at 4 ℃. 2mL of fresh medium of pre-warmed DMEM (Hyclone, # SH30022.01; FBS, # FSP 500) containing 10% FBS was added. The plates were placed at 37℃with 5% CO ₂ The conditions continued to incubate.

f) After 24 hours (about 48 hours of transfection), 2mL of cell supernatant was collected from each well and mixed with the first supernatant collected to give a total volume of 4mL of supernatant collected.

g) Cell debris was removed by centrifugation at 2,000rpm for 10 minutes at room temperature. Collecting and transferring supernatant, discarding cell precipitate, and filtering with 0.45 μm filter membrane to obtain Lenti-PD-1 ^KD The NKG2Dx24 recombinant slow virus suspension is preserved in a refrigerator at-80 deg.c. The preparation of a large amount of lentiviruses is amplified in equal proportion according to the method.

2) Lentivirus titer detection

a) 293T cells were grown at 1X 10 ⁵ Density of wells/Density of wells was inoculated in 6-well plates containing 10% FBS in DMEM medium in 1mL total volume at 37℃at 5% CO ₂ Cells were incubated overnight under conditions.

b) Respectively taking Lenti-PD-1 ^KD NKG2Dx24 lentivirus 10. Mu.L, 5. Mu.L, 2.5. Mu.L, 1.25. Mu.L, 0.625. Mu.L, 0.3125. Mu.L to wells was adjusted to a total volume of 500. Mu.L/well, and polybrene (Sigma-Aldrich, # TR-1003-G) was added to adjust its final concentration to 6. Mu.g/mL. 37 ℃,5% CO ₂ After culturing for 24 hours, the liquid is changed.

c) Third day: flow cytometry

i. Cell counts were collected according to 1X 10 ⁵ The individual cells/tubes were sampled and aliquoted, and 10. Mu.L of 20. Mu.g/mL ROR1-hFC recombinant protein (novoprotein, # Q01973 (CU 96)) was added and incubated on ice for 20min;

after washing twice with staining buffer, 10. Mu.L of PE-crosslinked anti-human igG Fc secondary antibody (ebioscience, # 12-4998-82) was added to each tube and incubated on ice for 20min at a concentration of 100. Mu.g/mL;

after washing twice with staining buffer, flow cytometry was analyzed and the viral titer was calculated as: viral titer = (number of initial cell inoculations x positive cell proportion) ×10 ³ Volume of virus added (μl).

T cell activation

Experimental materials: dynabeads T-Activater CD3/CD28 (Thermo # 11131D) 2mL volume, 4×10 ⁷ mL Dynabeads in PBS, pH7.4; peripheral blood mononuclear cells (Shanghai Miaoshun Co.).

The method comprises the following steps:

a) Preparing a CAR-T complete medium: IL-2, IL-7 and IL-15 were added to the X-vivo 15 medium to a final concentration of 300U/mL,5ng/mL,10ng/mL, respectively;

b) Taking 2X 10 ⁷ PBMC,1500rpm,5min, after one wash pass add pre-warmed CAR-T complete medium and seed into T75 flasks, placed in CO ₂ An incubator;

c) 160. Mu.L of CD3/CD28 Dynabeads was added to 1.5mL of sterilized EP tube (6.4X10) ⁶ beads)；

d) Cleaning CD3/CD28 Dynabeads: placing a 1.5mL sterilization EP tube containing Dynabeads on a magnetic separation frame, waiting for 1 minute, and allowing the beads to be fully adsorbed on the wall of a centrifugal tube; carefully sucking the liquid in the centrifuge tube by using a 1mL pipetting gun, taking out a 1.5mL sterilization EP tube, adding 1mL CAR-T culture medium, and blowing up and down for several times to ensure that the beads are fully and uniformly mixed; repeating the steps for 2 times;

e) Taking 1mL of PBMC cell suspension from a T75 culture bottle, taking the centrifugal tube out of the magnetic separation frame in the 1.5mL sterilization EP tube containing the beads, and gently blowing up and down for several times by using a 1mL pipettor to mix the beads and the cells uniformly;

f) Transferring the above beads/PBMC mixture into T75 culture flask, mixing cells with beads with gentle shaking culture plate, and placing CO ₂ Culturing overnight in an incubator.

T cell infection

a) After counting the activated T cells from the previous day, the cell density was adjusted to 1.2X10 ⁶ /mL。

b) In a 6-well plate, 6X 10 wells are added per well ⁵ Individual cells.

c) The Lenti-PD-1 prepared in the step 3 is subjected to ^KD After dissolving the NKG2Dx24 recombinant lentivirus in a water bath at 37 ℃, the number of added viruses was 1.8X10 as calculated by MOI=5 ⁶ /well. Polybrene was added simultaneously to a final concentration of 6. Mu.g/mL and the final medium volume per well was 1mL. Mixing, adding 37 deg.C and 5% CO ₂ And (5) incubating in an incubator.

d) After 5 days of enlarged culture, PD-1 is prepared ^KD NKG2Dx24CAR T cells. Detection of PD-1 by flow cytometry ^KD NKG2Dx24CAR T cell surface PD-1 and CAR expression profile. At the same time, the method comprises the steps of,detection of PD-1 by Elsia ^KD Secretion levels of IL-24 in NKG2Dx24CAR T-cell supernatants. The results show that PD-1 ^KD NKG2Dx24CAR-T cell PD-1 expression level is extremely low, PD-1 prepared from peripheral blood mononuclear cells of three different donors ^KD NKG2Dx24CAR-T cells, both with CAR positive rates above 35% and up to more than 60% (shown in fig. 5 and 6). PD-1 ^KD The NKG2Dx24CAR-T cell culture supernatant contained high levels of IL-24 secretion (FIG. 7). Demonstration of successful preparation of PD-1 ^KD NKG2Dx24CAR-T cells.

Example 3PD-1 ^KD Application of NKG2Dx24CAR-T cells

1.PD-1 ^KD NKG2Dx24CAR-T cell antigen-dependent cytokine release enhancement

1) To detect PD-1 ^KD Killing ability of NKG2Dx24CAR-T cells on target cells positive for various NKG2D ligands we first screened four NKG2DL positive cell lines, PANC-1, PC-3, THP-1, HCT116, as target cells.

2) HCT116 cells were cultured according to 2X 10 ⁴ Wells were seeded in 96-well plates and 3 replicate wells were seeded per target cell.

3) Taking prepared T cells, NKG2D CAR-T cells and PD-1 ^KD NKG2Dx24CAR-T cell count at an effective target ratio of 10:1 to adjust the cell density.

4) 2X 10 of the additive is added to each hole ⁵ Effector cells were suspended into step 1) wells containing target cells, with a final medium volume of 200 μl per well.

5)37℃，5％ CO ₂ Incubation in incubator.

6) After 24 hours 100 μl of supernatant was harvested for cytokine detection and the remaining supernatants were harvested for LDH release levels.

The results show that PD-1 ^KD The NKG2Dx24CAR-T cells co-cultured with the target cells released higher levels of IL-2 and IFNg (FIGS. 8 and 9), and the target cells were killed to a much higher extent than the control group (FIG. 10). The above results indicate that PD-1 ^KD The NKG2Dx24CAR-T cells have the strongest specific recognition and killing capacity to target cells and have wide tumor killing capacity.

2.PD-1 ^KD NKG2D CAR-T cell obviously inhibits tumor growth and prolongs the survival period of mice

To verify PD-1 ^KD In vivo anti-tumor Activity of NKG2D CAR-T cells we followed HCT116 cells by 2X 10 ⁶ The amount of the drug is subcutaneously injected to the right side of a 6-8-week-old NSG mouse with severe immunodeficiency, the tumor volume of the mouse is measured by a vernier caliper every 3 days, and when the tumor volume reaches 100mm ³ Each mouse was given a 5 x 10 intravenous injection at the tail ⁶ Mock-T or PD-1 ^KD NKG2D CAR-T cells, tumor volume was measured continuously, and tumor growth curves and survival curves of mice were made. We found that PD-1 compared to the control group ^KD NKG2DCAR-T cells significantly inhibited tumor growth and effectively increased survival in mice (shown in fig. 11 and 12).

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (15)

1. A recombinant plasmid, comprising:

(1) A shRNA sequence of the targeted silencing PD-1;

(2) Tandem sequences including the NKG2D gene, CD3 zeta gene, IL-24 gene, P2A scissoring peptide gene;

the sense strand of the shRNA is SEQ ID NO.9, and the antisense strand is SEQ ID NO.10; the NKG2D gene is spliced with the reverse inverted CD3 zeta gene, and then the IL-24 gene is connected with the spliced gene in series through a P2A shearing peptide gene sequence;

the NKG2D gene is SEQ ID NO.13; the CD3 zeta gene is SEQ ID NO.17; the IL-24 gene is SEQ ID NO.19; the P2A cleavage peptide gene is SEQ ID NO.15;

the shRNA sequence and the tandem sequence are respectively positioned at the downstream of different promoters.

2. The recombinant plasmid of claim 1, wherein the tandem sequence is SEQ ID No.21.

3. The recombinant plasmid of claim 1, wherein the backbone of the recombinant plasmid is a pll3.7-U6-EF1a-EGFP plasmid.

4. The recombinant plasmid of claim 3, wherein the shRNA sequence is located downstream of the U6 promoter and the tandem sequence is located downstream of the EF1a promoter.

5. The recombinant plasmid of claim 4, wherein the insertion cleavage site of the shRNA sequence is between HpaI and XhoI; the tandem sequence is inserted between Nhe I and EcoR I.

6. The recombinant plasmid according to claim 5, wherein the sequence is SEQ ID NO.25.

7. Use of the recombinant plasmid according to any one of claims 1-6 for the preparation of lentiviruses or T cells.

8. The use of claim 7, wherein the T cells are CAR T cells.

9. A recombinant lentivirus comprising the recombinant plasmid of any one of claims 1-6.

10. A genetically modified T cell, wherein a PD-1-targeted shRNA, IL-24, and NKG2D CAR are co-expressed; is prepared by the recombinant lentivirus of claim 9.

11. Use of the recombinant plasmid of any one of claims 1-6 or the recombinant lentivirus of claim 9 or the T cell of claim 10 in the preparation of an anti-tumor medicament.

12. An anti-neoplastic agent comprising the recombinant plasmid of any one of claims 1-6 or the recombinant lentivirus of claim 9 or the T cell of claim 10.

13. A preparation method of a recombinant plasmid is characterized by comprising a cascade of NKG2D gene, CD3 zeta gene, IL-24 gene and P2A shearing peptide gene and an shRNA insertion carrier skeleton;

the sense strand of the shRNA is SEQ ID NO.9, and the antisense strand is SEQ ID NO.10; the NKG2D gene is spliced with the reverse inverted CD3 zeta gene, and then the IL-24 gene is connected with the spliced gene in series through a P2A shearing peptide gene sequence;

the NKG2D gene is SEQ ID NO.13; the CD3 zeta gene is SEQ ID NO.17; the IL-24 gene is SEQ ID NO.19; the P2A cleavage peptide gene is SEQ ID NO.15;

the shRNA sequence and the tandem sequence are respectively positioned at the downstream of different promoters.

14. A method for preparing a recombinant lentivirus comprising transfecting a cell with the recombinant plasmid of any one of claims 1-6.

15. A method of preparing a genetically modified T cell comprising transfecting a T cell with the recombinant lentivirus of claim 9.