CN116640229B

CN116640229B - Construction and application of low-pH targeted CAR-T cells

Info

Publication number: CN116640229B
Application number: CN202310371232.7A
Authority: CN
Inventors: 王一; 蔡博; 郭梅; 胡锴勋; 莫丹; 陈国江; 陈鑫蕊; 李欣阳; 李冰霞; 刘铁强; 刘志青; 黄珊
Original assignee: Fifth Medical Center of PLA General Hospital
Current assignee: Fifth Medical Center of PLA General Hospital
Priority date: 2023-04-10
Filing date: 2023-04-10
Publication date: 2024-01-30
Anticipated expiration: 2043-04-10
Also published as: CN116640229A

Abstract

The invention provides construction and application of a low-pH targeting CAR-T cell, and belongs to the technical field of immune targeting treatment. The Chimeric Antigen Receptor (CAR) of the CAR-T cell comprises pHLIP (pH-low polypeptide), which mainly comprises a CD8 signal peptide, a HiS label, pHLIP, a CD8 hinge region, a CD8 transmembrane structural region, a 4-1BB co-stimulatory signaling region, and intracellular signal regions of CD28 and CD3 zeta in series. The CAR-T cell provided by the invention can realize the expression of pHLIP on the CAR-T cell, thereby realizing targeting identification of solid tumors and avoiding the limitation of traditional tumor marker targeting treatment.

Description

Construction and application of low-pH targeted CAR-T cells

Technical Field

The invention belongs to the technical field of immune targeting therapy, and particularly relates to construction and application of low-pH targeting CAR-T cells.

Background

CAR-T, collectively chimeric antigen receptorT-cell, is a chimeric antigen receptor T cell. CAR-T preparation the first step is to isolate T cells from the patient: peripheral blood mononuclear cells (PBMC cells) of the patient are collected by leukapheresis, and specific T cell subsets, such as cd4+, cd8+, cd25+ or cd62l+ T cells, are isolated; the second step is to engineer T cells: generating main specific signals through T cell receptor signals and co-stimulation signals such as CD28, 4-1BB or OX40 signals, so as to activate T cells, and introducing a CAR gene into the activated T cells through electroporation, lentivirus or retrovirus vectors for genetic modification so as to obtain CAR-T cells capable of expressing the CAR gene; the third step is amplification: in vitro culture, and reinfusion after large number of amplified CAR-T cells. The CAR-T therapy is a novel accurate targeted therapy for treating tumors, achieves a good effect on clinical tumor treatment through optimization and improvement in recent years, and is a novel tumor immunotherapy method which is very promising, can be accurate, rapid and efficient, and can possibly cure cancers.

Ideally, the target to which the CAR-T cell is directed is expressed only on the surface of the tumor cell (or highly expressed), and not on other normal cells (or expressed very low). Such as CD-19CAR, as the most successful CAR-T treatment method, has been largely successful in that CD19 is a very good specific marker for immature B cells, which is expressed only on the surface of immature B cells, and other somatic cells are not. However, since tumor cells are all produced from the cancerous transformation of normal cells, it is actually difficult to find specific markers that are present only on the surface of tumor cells, but not on the surface of normal cells. And cancer cells undergo extensive genetic alterations upon division in tumors, including epigenetic regulatory site mutations, gene deletions, gene duplications, and chromosomal rearrangements, among others, which are unevenly distributed in individual tumors. The heterogeneity of cell surface specific biomarker expression within and between tumors also significantly reduces the effectiveness of drugs targeting specific biomarkers. This is one of the reasons that CAR-T therapy has not yet made a breakthrough in solid tumors.

pHLIP (pH-low polypeptide) is a pH sensitive polypeptide, and a typical pHLIP is a peptide of about 40 amino acids. Under acidic conditions, pHLIP can spontaneously intercalate into cell membranes via transmembrane α -helices and can transfer polar materials attached to its intercalated C-terminal end to the cytoplasm of target cells, this transformation being due to protonation and charge neutralization of aspartic acid residues in the pHLIP membrane transmembrane region at acidic pH. pHLIP insertion is pH dependent, and studies have shown that pHLIP tends to aggregate in acidic diseased tissue with high specificity. In solid tumors, the tumors are always acidic in the rapid growth process under the actions of glycolytic metabolism enhancement (Warburg effect), carbonic anhydrase and hypoxia/ischemia secondary to the increase of blood supply, the extracellular pH value usually generated by the tumors is 6.2-6.9, the pH value on the surfaces of tumor cells is more acidic (pH value is 6.0-6.5), and the pH value of healthy tissues is 7.4, so that the acidic microenvironment of the tumors provides a possibility for selectively targeting the tumors and retaining the healthy tissues. At present, in view of the characteristics of pHLIP, although there have been applications of pHLIP in tumor cell targeted therapies, such as attaching phalloidin (a cell-impermeable polar toxin) to the C-terminus of pHLIP, disrupting proliferation of HeLa, JC and M4A4 cancer cells, etc., there has been no therapeutic means for attaching pHLIP to T cells.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide chimeric antigen receptors, including pHLIP, that can exert targeted recognition during CAR-T cell activation.

The invention also aims to provide the low-pH targeting CAR-T cell which can be used for targeting and identifying solid tumors, promoting cancer cell apoptosis and avoiding the limitation of traditional tumor marker targeting treatment.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a chimeric antigen receptor, which comprises pHLIP, wherein the nucleotide sequence of pHLIP is shown as SEQ ID NO. 1.

Preferably, the amino acid sequence of pHLIP is shown in SEQ ID NO. 2.

Preferably, the chimeric antigen receptor consists of a CD8 signal peptide, a HiS tag, pHLIP, a CD8 hinge region, a CD8 transmembrane structural region, a 4-1BB costimulatory signaling region, and intracellular signaling regions of CD28 and CD3 zeta in tandem.

Preferably, the nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 4.

The invention also provides a lentiviral expression vector comprising the nucleotide sequence of the chimeric antigen receptor.

The invention also provides a recombinant lentivirus, which comprises the lentivirus expression vector.

The invention also provides a CAR-T cell which expresses the chimeric antigen receptor; or the nucleotide sequence of the chimeric antigen receptor is integrated in the genome of the CAR-T cell.

The invention also provides application of the chimeric antigen receptor, the recombinant lentivirus or the CAR-T cell in preparing a medicine for treating solid tumors.

Preferably, the solid tumor comprises breast cancer, lung cancer, intestinal cancer, gastric cancer and liver cancer.

The invention has the beneficial effects that:

the invention links pHLIP to T cells for the first time, and constructs a low pH targeting CAR-T cell. The CAR-T cells prepared by the invention can realize the expression of pHLIP, and target and locate the CAR-T cells on the surface of solid tumor according to the characteristic that the tumor microenvironment is acidic, thereby targeting and killing tumor cells and avoiding the limitation of traditional tumor marker targeting treatment.

Drawings

Fig. 1: chimeric antigen receptor structures comprising pHLIP;

fig. 2: an empty chimeric antigen receptor structure;

fig. 3: plasmid enzyme digestion electrophoresis;

fig. 4:293T cell packaging virus and transfection efficiency;

fig. 5: CAR-T cell infection efficiency;

fig. 6: CAR-T cell proliferation activity;

fig. 7: killing ability of CAR-T cells against gastric cancer cell lines;

fig. 8: killing ability of CAR-T cells against breast cancer cell lines;

fig. 9: killing ability of CAR-T cells to colorectal cancer cell lines;

fig. 10: killing ability of CAR-T cells on lung cancer cell lines;

fig. 11: killing ability of CAR-T cells on liver cancer cell lines;

fig. 12: effect of different tumor cells on the expression ratio of CAR-T cell CD69 molecules;

fig. 13: effect of different tumor cells on CAR-T cell IFN-r expression;

fig. 14: effect of different tumor cells on CAR-T cell IL-2 expression;

fig. 15: effect of different tumor cells on CAR-T cell proliferation fold.

Detailed Description

The invention provides a chimeric antigen receptor, which comprises pHLIP (pH-low polypeptide), wherein the nucleotide sequence of pHLIP is GGATGTACAGGCGAGGAT GCTGATGTGCTGCTGGCCCTGGATCTGCTGCTGCTCCCTACCACCTTTC TGTGGGATGCCTACAGAGCCTGGTACATCCCCAATCAAGAAGCCGCC, shown as SEQ ID NO. 1, and the amino acid sequence of pHLIP is GCTGEDADVLLALDLLLLPT TFLWDAYRAWYIPNQEAA, shown as SEQ ID NO. 2.

The chimeric antigen receptor containing pHLIP is prepared by taking a chimeric antigen receptor containing a third-generation CAR structure as a basis, carrying out engineering transformation between a signal peptide of the chimeric antigen receptor and a hinge region, and adding a HiS tag and pHLIP in a connecting way, and can realize the expression of pHLIP on CAR-T cells, so that the CAR-T cells can be targeted and positioned on the surface of solid tumors with acidic microenvironment.

The chimeric antigen receptor of the invention consists of a CD8 signal peptide, a HiS label, pHLIP, a CD8 hinge region, a CD8 transmembrane structural region, a 4-1BB costimulatory signaling region, and intracellular signaling regions of CD28 and CD3 zeta in series, and is shown in figure 1 in detail.

The nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3. Among the nucleotide sequences, the 1 st to 63 th positions are CD8 signal peptide sequences, the 64 th to 81 th positions are HiS tag sequences, the 82 nd to 195 th positions are pHLIP sequences, the 196 nd to 330 nd positions are CD8 hinge region sequences, the 331 st to 402 nd positions are CD8 transmembrane structural region sequences, the 403 th to 528 nd positions are 4-1BB costimulatory signal transduction regions, the 529 th to 651 th positions are CD28 intracellular signal region sequences, and the 652 nd to 987 nd positions are CD3 zeta intracellular signal region sequences.

The amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 4. In the amino acid sequence, the 1 st to 21 st positions are CD8 signal peptide sequences, the 22 nd to 27 th positions are HiS labels, the 28 th to 65 th positions are pHLIP sequences, the 66 th to 110 th positions are CD8 hinge region sequences, the 111 th to 134 th positions are CD8 transmembrane structural region sequences, the 135 th to 176 th positions are 4-1BB costimulatory signal transduction regions, the 177 th to 217 th positions are CD28 intracellular signal region sequences, and the 218 th to 329 th positions are CD3 zeta intracellular signal region sequences.

The invention also provides a lentiviral expression vector comprising the nucleotide sequence of the chimeric antigen receptor (SEQ ID NO: 3). As an alternative embodiment, the invention can obtain the lentiviral vector plasmid containing the chimeric antigen receptor by carrying out double enzyme digestion and enzyme digestion product connection on the base sequence of the chimeric antigen receptor and the lentiviral expression vector.

The invention also provides a recombinant lentivirus, which comprises the lentivirus expression vector. The recombinant lentivirus is prepared by co-transfecting cells with the lentivirus expression vector and the lentivirus packaging plasmid, and the invention preferably adopts a three-plasmid virus packaging system.

The specific preparation method of the CAR-T cells is not particularly limited, and the preparation method of the CAR-T cells conventional in the art can be adopted. As an alternative embodiment, the preparation method of the present invention comprises the steps of: cloning the chimeric antigen receptor gene fragment to a px330 vector, and then introducing the chimeric antigen receptor gene into activated T cells through lentiviral transfection to obtain the pHLIP-expressing CAR-T cells.

The invention also provides application of the chimeric antigen receptor, the recombinant lentivirus or the CAR-T cell in preparing medicines. In the present invention, the medicament preferably comprises a solid tumor immunotherapy medicament, wherein the solid tumor treated by the medicament comprises, but is not limited to, breast cancer, lung cancer, intestinal cancer, gastric cancer and liver cancer.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. In the following examples, unless otherwise specified, the methods are conventional; materials, reagents and the like used are commercially available unless otherwise specified.

Example 1

The preparation method of the low-pH targeted CAR-T cell comprises the following steps:

1. vector construction

The px330 is digested with Bbs I and EcoRI, and an insert (SEQ ID NO: 3) is inserted between the digestion sites of Bbs I and EcoRI to obtain a ligation product; the ligation products were transformed and clones were picked for sequencing and identification.

2. Preparation of plasmids

Preparation of competent cells

(1) Scribing and coating a plate: a small amount of E.coli DH5 alpha bacterial liquid is dipped by a sterilized inoculating loop, and streaked on an LB agar plate without antibiotics, so that the bacterial liquid is gradually diluted. The mixture is placed in a bacterial incubator at 37 ℃ for overnight culture for 12 to 14 hours.

(2) Single colonies were picked and inoculated into 5ml LB medium without antibiotics. 37℃at 250rpm overnight.

(3) 2ml of turbid bacterial liquid is inoculated into 200ml of LB culture medium without antibiotics, and the culture is carried out for 2 to 3 hours at 37 ℃. OD was measured every half hour ₆₀₀ So that the temperature reaches 0.4 to 0.6.

(4) The bacterial liquid is ice-bathed for 20min. The ice-bathed bacterial solution was transferred to a pre-chilled 50ml centrifuge tube. Centrifuge at 4℃at 4000rpm for 10min.

(5) The supernatant was discarded. 0.1M CaCl pre-chilled with 20ml ₂ The solution resuspended the bacterial pellet. Centrifuge at 4℃at 4000rpm for 10min.

(6) The supernatant was discarded. 2ml of precooled 0.1MCaCl containing 20% of sterile glycerol is added to each 50ml of bacterial liquid ₂ The solution resuspended the cell pellet.

(7) Sub-packaging (50. Mu.l/tube), rapidly freezing in liquid nitrogen, and storing at-80deg.C.

Rapid heat activated transformation of plasmids

(1) Mu.l E.coli DH 5. Alpha. Competent cells were thawed on ice, 1-10 ng plasmid was added and gently mixed.

(2) The mixture of competent cells and plasmid was placed on ice and allowed to stand for 30min.

(3) And (3) performing heat activation (90 s) on the mixed solution in a water bath at 42 ℃, rapidly putting the mixed solution on ice after heat activation, and standing for 2min.

(4) And uniformly coating a proper amount of heat-shocked competent cells on an LB agar plate with antibiotics.

(5) The LB plate is placed in a bacterial incubator at 37 ℃ in an inverted mode for culturing for 12-16 h. Many individual bacterial colonies are visible.

Plasmid miniascape

(1) The single colony after transformation is picked up, inoculated into 3-5 ml LB culture medium containing antibiotics, and cultivated under intense shaking at 37 ℃ for overnight.

(2) The bacterial pellet was collected by centrifugation at 12000rpm, 1min. 250 mu l P buffer (50mM Tris Cl,10mM EDTA,100. Mu.g/ml RNaseA) was added and mixed by shaking.

(3) 250 mu l P2 buffer (200 mM NaOH,1% SDS) was added and gently mixed upside down.

(4) 350 mu l N of buffer (3.0M NaAC, pH 5.5) was added and mixed gently upside down.

(5) Centrifugal 13000rpm,10 min.

(6) The supernatant was carefully aspirated and loaded onto a QIAprep spin column. Centrifuge at 12000rpm,1min, discard the waste.

(7) 0.75ml PE buffer (1.0M NaCl,50mM MOPS,15% isopropyl alcohol) was added for rinsing, 12000rpm,1min centrifuged, and the waste liquid was discarded.

(8) Centrifuge at 12000rpm,2min to remove residual rinse.

(9) Mu.l of EB buffer (10mM Tris Cl,pH8.5) was added and left at room temperature for 1min. The plasmid was eluted by 1min centrifugation at 12000 rpm.

Plasmid middling or macrophyte

(1) The single colony after transformation is picked up and inoculated into 3-5 ml LB culture medium containing antibiotics, and the culture is carried out under intense shaking at 37 ℃ for 8 hours.

(2) Inoculating the bacterial liquid into LB culture medium containing antibiotics according to the proportion of 1/500 to 1/1000, and culturing for 12-16 h under intense shaking at 37 ℃.

(3) The cells were collected by centrifugation at 6000g for 15min at 4 ℃.

(4) Bacterial pellet was resuspended with 4ml (medium extract) or 10ml (large extract) P1 buffer (50mM Tris Cl,10mM EDTA,100. Mu.g/ml RNaseA).

(5) 4ml (medium extract) or 10ml (large extract) of P2 buffer (200 mM NaOH,1% SDS) was added, gently mixed, and left at room temperature for 5min.

(6) 4ml (medium extract) or 10ml (large extract) of pre-chilled P3 buffer (3.0M NaAC, pH 5.5) was added, gently mixed, and left on ice for 20min.

(7) Centrifugation was performed at 20000g for 30min at 4 ℃. Repeating once.

(8) Column equilibration was performed by adding 4ml (medium extract) or 10ml (large extract) of QBTBuffer (750mM NaCl,50mM MOPS,15% isopropyl alcohol, 0.15% Triton X-100) to QIAGEN-tip.

(9) The supernatant after centrifugation was applied to a well equilibrated QIAGEN-tip column. The liquid flows out under the action of gravity.

(10) The QIAGEN-tip column was rinsed with 10ml (medium extract) or 30ml (large extract) QC buffer (1.0MNaCl,50mM MOPS,15% isopropyl alcohol). Repeating once.

(11) The plasmid was eluted with 5ml (medium extract) or 15ml (large extract) QF buffer (1.25M NaCl,50mM Tris Cl,15% isopropanol).

(12) To the eluted plasmid was added 3.5ml (medium extract) or 10.5ml (large extract) of isopropanol. After mixing, centrifugation was performed at 4℃and at 8000g for 40 min. DNA precipitation was visible at the bottom of the tube.

(13) The supernatant was discarded. The precipitate was rinsed by adding 2ml (medium extract) or 5ml (large extract) of 70% ethanol. Centrifuge at 4℃with 600 g for 60 min.

(14) The supernatant was discarded. Drying at room temperature for 5-10 min. The plasmid was dissolved by adding TE buffer (10mM Tris Cl,pH8.0).

3. Identification of plasmids

Quality detection of plasmid

Plasmid concentration and purity were determined using an ultra-low volume ultraviolet visible spectrophotometer of NanoPhotometerTM Pearl (IMPLEN). Record plasmid concentration, OD _260/280 、OD _260/230 Ratio, OD _260/280 Reaching 1.8 to 2 OD _260/230 >2。

Agarose gel electrophoresis detection

(1) Agarose gel of 0.9-1.5% is prepared by using 1 xTAE electrophoresis buffer solution, and is dissolved by microwave heating. EtBr (ethidium bromide) was added in a ratio of 1/20000 after cooling to about 55 to 60 ℃. Can be put in a water bath at 55 ℃ for standby in a short period.

(2) Pouring the prepared agarose gel into a gel preparation mould inserted with a comb with proper size, and standing at room temperature until the agarose gel is solidified.

(3) Pulling out the comb, taking 100-500 ng of plasmid and mixing with 6 x loadingbuffer, and adding the sample.

(4) Electrophoresis was performed using a voltage of 5 to 7V/cm.

(5) After electrophoresis, the gel was scanned using an LHR gel imaging system (Syngene) and photographed.

Restriction enzyme identification of plasmid

Restriction enzymes (Bbs I, ecoRI) were selected for cleavage according to the restriction map of the plasmid. The size of the specific enzyme fragments generated after enzyme digestion is used for determining whether the extracted plasmid is correct, and the electrophoresis diagram after enzyme digestion is shown in FIG. 3.

4. Lentivirus package

A 2-generation packaging system, i.e., a three-plasmid system, was used, which included 1 packaging plasmid (psPAX 2), 1 envelope plasmid (pMD 2G), 1 gene-of-interest plasmid, and 1 packaging cell (293T cell).

Day1: 10cm dishs HEK293T cells with 90% confluency (6X 10) ⁷ /dish) according to 1:1 proportion passage to 15cm dishs, the confluence of cells reaching 90-95% the next day (1.5X10) ⁸ /dish) medium was Gibico high-sugar DMEM medium (10% FBS).

Day2：

Medium (containing 10% fbs) was changed 2-3 hours prior to transfection; the transfection reagents were formulated in the following proportions:

mix 1 volume μl, mix 2 amount, DMEM (no FBS) 1000 μl, target gene plasmid 25 μg, VGF190 μl (1 μg/μl), PMD2G7.5 μg, PSPAX215 μg.

Mix 1 and Mix 2 were mixed separately, and Mix 1 and Mix 2 were mixed after 5 to 10 minutes at room temperature for 30 minutes at room temperature and added to 15cm dish.

Day3: fresh medium (containing 10% FBS) was changed over 6-24 h.

Day5: the cell state was observed for 72h and photographed. Collecting supernatant medium, filtering with 0.45 μm filter membrane, adding the supernatant medium into an overspeed centrifuge tube, balancing, centrifuging, and centrifuging at 25000rpm at 4deg.C for 1.5 hr. The supernatant was discarded, and the mixture was dissolved overnight by re-dissolving the mixture in an appropriate virus-preserving solution.

Day6: and (5) collecting virus split packaging, and measuring the virus titer.

Recombinant lentivirus titer assay: integration method for calibrating recombinant lentivirus titer without fluorescence

Virus-infected cells

6h before infection at 2.5X10 in 24 well cell culture plates ⁵ HEK293 cells were evenly seeded per cell/well.

The lentivirus was subjected to gradient dilution to give 3 gradients, i.e., 10. Mu.l, 1. Mu.l, 0.1. Mu.l of virus were added to 24 well plates inoculated with cells after shaking and mixing in each well (500. Mu.l of serum-free DMEM medium) and the medium was blotted off prior to virus addition.

After 18-20 h of infection, the medium in the plates was replaced with fresh DMEM complete medium.

Cells were collected and genomic DNA was extracted 64-68 hours after infection.

For the assay, a set of lentiviruses with fluorescent known TUs was set as controls to verify the detected values.

5. G-CSF mobilized peripheral blood hematopoietic stem cell mononuclear cell separation (using ficoll cell separation fluid)

(1) The blood sample is diluted 2-4 times with sterile diluent.

(2) 15-20 ml of separating liquid is firstly added into a 50ml conical bottom centrifuge tube, and then 20-30 ml of diluted whole blood is slowly added to the liquid level of the separating liquid.

(3) And (3) centrifuging for 15-30min at room temperature of 400g, so as to ensure that the rotor speed is steadily reduced.

(4) Carefully remove the centrifuge tube from the centrifuge, slowly suck the uppermost layer, and avoid contacting the mononuclear cell layer.

(5) The mononuclear cell layer was slowly transferred to another 50ml conical bottom centrifuge tube.

(6) After adding sterile washing solution and mixing well, 300g was centrifuged at room temperature for 10min, and the supernatant was carefully discarded to obtain a cell suspension.

(7) The number of living cells was counted in trypan blue dye solution, and the cell suspension was filtered through a 50 μm cell sieve to obtain G-CSF mobilized peripheral blood hematopoietic stem cell mononuclear cells.

After the following criteria were met, the next experiment was performed: separating 95+/-5% of the obtained cells into mononuclear cells; the cell survival rate of the isolated cells is >90%; 60+/-20% of mononuclear cells in the original blood sample can be recovered; 3±2% of granulocytes; 5+ -2% of red blood cells

6. G-CSF mobilization T cell isolation and activation in peripheral blood hematopoietic stem cell-derived mononuclear cells

(1) Determining the number of mononuclear cells and adjusting the cell concentration to 1X 10 ⁸ Individual cells/ml.

(2) Mu.l of the cell suspension (1X 10) ⁷ Individual cells) were placed in a new tube and 10. Mu.L of negative fraction was addedThe antibody was selected, stirred well, protected from light, and incubated on ice for 15 minutes.

(3) Add 20. Mu.l of negative sorting beads, stir well, keep out light, incubate at 4℃for 15min.

(4) After adding 4ml buffer to a 5ml (12X 75 mm) polypropylene tube, the tube was placed in a magnet for 5min and the supernatant was poured into another 15ml centrifuge tube.

Note that: the repeated magnetic separation can improve the yield and has no strong influence on the purity. After the second separation, the yield can be increased by 8-10%, and the purity can be reduced by 1-2% for each separation.

(5) Adding CD3/CD28 activated magnetic beads into the obtained T lymphocyte at a ratio of 3:1, standing at 37deg.C, and 5% CO ₂ Culturing overnight in an incubator.

7. Lentivirus infection of T cells

(1) After T cell activation for 24-72 hours, the lentiviral particles are melted at room temperature, gently mixed, and the lentiviral particles are added and placed at 37 ℃ and 5% CO ₂ Culturing in an incubator for 4-6 hours.

(2) Most of the culture solution containing lentiviral particles is removed, and new culture solution is added to expand T cells. After 3 days, the cells were transferred to a cell culture bag for culturing. The 293T cell packaging virus and the transfection efficiency are shown in FIG. 4, and the lentivirus transfection efficiency is more than 90% as can be seen from FIG. 4.

Example 2

Experimental group: constructing low pH targeted CAR-T cells using the method of example 1;

control group: the empty CAR-T cells were constructed in the same manner as in example 1 except that the insert (SEQ ID NO: 3) in example 1 was replaced with the empty insert (SEQ ID NO: 5), and the chimeric antigen receptor structure was shown in fig. 2.

1. After packaging the virus, T cells were infected and detected 48h later by flow-through:

(1) Diluting the cultured cells with PBS buffer solution, placing into a centrifuge tube, centrifuging for 5min at 350g, discarding supernatant, repeatedly washing the cells once, re-suspending the cells with PBS buffer solution, counting, and diluting the cells to (5-10) x 10 ⁶ Each cell/ml, then each tributary is assayedTo this was added 100. Mu.l of the cell suspension ((5.about.10). Times.10) ⁵ Individual cells/tubes).

(2) Adding a proper amount of pre-diluted primary antibodies into each flow detection tube; control reagents were added in the same amount as the antibody in the blank tube/well. Then each tube is incubated for 15-20 min in a dark ice bath. The Car-T antibody was incubated at 4℃for 50min in a refrigerator during staining.

(3) 2ml PBS buffer was added, then 350g was centrifuged for 5min, the supernatant was discarded, and the washing process was repeated twice.

(4) Cells were resuspended in 300 μl PBS buffer or 300 μl 2% paraformaldehyde fixative.

(5) And (5) detecting and analyzing the result by the machine.

The positive rates in the control flow detection tubes are respectively 3%, 4%, 5%, 2% and 5%, and the positive rates in the experimental flow detection tubes are respectively 36%, 44%, 39%, 47% and 56%, and FIG. 5 shows that pHLIP-related genes can be normally expressed on the surfaces of T cells to obtain CAR-T cells.

2. The control group cells and the experimental group cells were cultured in the culture system, respectively, and the results were counted every other day, and the results are shown in Table 1 and FIG. 6.

TABLE 1 cell proliferation fold

Group of	Control group 1	Control group 2	Experiment group 1	Experiment group 2
					0D	1	1	1	1
2D	0.8	0.9	0.6	0.5
					4D	1.2	1.3	1	1.1
6D	2.4	2.6	2.2	2.1
					8D	5	5.2	4.8	5.8
10D	15	18	14	17

Table 1 and fig. 6 show that CAR-T cell engineering does not alter T cell proliferation activity.

3. CAR-T killing ability detection

(1) Target cell Cfse dye liquor labeling staining

1) Target cells were diluted with PBS buffer and placed in a centrifuge tube, 300g was centrifuged for 5min, the supernatant was discarded, and the cells were washed repeatedly.

2) Cells were counted after being resuspended in PBS buffer and diluted 1X 10 ⁷ Each cell/ml, then adding Cfse dye solution to a final concentration of 2-5 mu M, standing at 37 ℃ and 5% CO ₂ Incubate in incubator for 15min.

3) 10ml PBS buffer was added, then 300g was centrifuged for 5min, the supernatant was discarded, and the washing process was repeated twice.

(2) Co-culture of cfse+ target cells and effector cells

The washed target cells and CAR-T cells were resuspended and counted in serum-free 1640 medium, respectively, and added to 96-well round-bottomed cell culture plates at an effective target ratio (target cells: effector cells) of 1:0.25, 1:0.5, 1:1, 1:2, 1:5, placed at 37℃with 5% CO ₂ Culturing in an incubator for 4 hours.

(3) Sample staining

1) Cells from each well were aspirated into the flow tube and collected by centrifugation at 300g for 5min.

2) The cells were washed 1 time with PBS buffer and centrifuged at 300g for 5min. Collecting (1-5) x 10 ⁵ Cells/tubes.

3) The PBS was pipetted off and 100. Mu.l of 1 Xbinding Buffer were added to resuspend the cells.

4) Mu.l of Annexin V-APC and 10. Mu.l of 7-AAD were added to each tube, and gently mixed.

5) And (3) carrying out reaction at room temperature for 10-15 min in dark place.

6) 200 μl of 1 Xbinding Buffer was added, and after mixing, the sample was examined with a flow cytometer over 1 hour.

(4) Sample flow cytometry analysis:

annexin V-APC is on the abscissa, 7-ADD is on the ordinate, and the apoptosis rate is detected (living cells have only very low intensity background fluorescence, early apoptotic cells have only stronger blue fluorescence, and late apoptotic cells have blue and red fluorescence double staining).

The killing capacity of CAR-T cells against gastric cancer cell lines is shown in table 2 and fig. 7.

TABLE 2 apoptosis Rate of gastric cancer cell lines (%)

The killing capacity of CAR-T cells against breast cancer cell lines is shown in table 3 and fig. 8.

TABLE 3 apoptosis Rate of breast cancer cell lines (%)

Group of	Control group 1	Control group 2	Experiment group 1	Experiment group 2
					1:0.25	5	4	10	12
1:0.5	8	7	21	18
					1:1	12	14	34	38
1:2	16	15	52	56
					1:5	20	17	70	72

The killing capacity of CAR-T cells against colorectal cancer cell lines is shown in table 4 and fig. 9.

TABLE 4 apoptosis Rate of colorectal cancer cell lines (%)

Group of	Control group 1	Control group 2	Experiment group 1	Experiment group 2
					1:0.25	4	4	12	11
1:0.5	3	5	21	20
					1:1	5	6	31	32
1:2	8	10	43	44
					1:5	11	12	57	59

The killing ability of CAR-T cells against lung cancer cell lines is shown in table 5 and figure 10.

TABLE 5 apoptosis Rate of lung cancer cell lines (%)

Group of	Control group 1	Control group 2	Experiment group 1	Experiment group 2
					1:0.25	3	5	16	19
1:0.5	6	7	25	28
					1:1	9	7	54	51
1:2	13	11	67	70
					1:5	17	14	88	85

The killing ability of CAR-T cells against liver cancer cell lines is shown in Table 6 and FIG. 11.

TABLE 6 apoptosis Rate of liver cancer cell lines (%)

As can be seen from tables 2-6 and figures 7-11, low pH targeted CAR-T cells can kill tumor cell lines of different origins in a dose dependent manner, whereas control T cells hardly kill tumor cells.

4. Activation ability of tumor cells to CAR-T cells

(1) Control and experimental T cells were co-cultured with different tumor cells in vitro at 1:1 target ratio for 4h, and then the cells were subjected to flow assay to investigate the expression ratio of CD69 molecules, and the results are shown in table 7 and fig. 12.

TABLE 7 influence of different tumor cells on the expression ratio of CAR-T cell CD69 molecules (%)

Group of	Control group 1	Control group 2	Experiment group 1	Experiment group 2
					Colorectal cancer	5	7	55	70
Stomach cancer	8	9	67	77
					Lung cancer	12	14	45	48
Breast cancer	11	14	37	46
					Liver cancer	6	8	71	83

Table 7 and fig. 12 illustrate that different tumor cells can stimulate low pH targeted CAR-T cells to express CD69 molecules, while control T cells cannot be stimulated to express CD69 molecules. CD69 molecules are expressed after T cells are activated, suggesting that different tumor cells may activate low pH targeted CAR-T cells.

(2) The control group T cells and the experimental group T cells are respectively co-cultured with different tumor cells for 4 hours in vitro according to the 1:1 effective target ratio, and cytokines in culture supernatants are detected, and the results are shown in tables 8-9 and figures 13-14.

TABLE 8 Effect of different tumor cells on CAR-T cell IFN-r expression (pg/ml)

TABLE 9 Effect of different tumor cells on CAR-T cell IL-2 expression (pg/ml)

Group of	Control group 1	Control group 2	Experiment group 1	Experiment group 2
					Colorectal cancer	10	12	550	540
Stomach cancer	11	14	640	660
					Lung cancer	14	18	760	770
Breast cancer	14	16	450	460
					Liver cancer	14	15	890	980

Tables 8-9 and figures 13-14 demonstrate that different tumor cells can stimulate low pH targeted CAR-T cells to secrete cytokines, while not stimulating control T cells to secrete cytokines. It is demonstrated that different tumor cells can activate low pH targeted CAR-T cells, promoting the release of cytokines IFN-r and IL-2.

(3) Control and experimental T cells were co-cultured with different tumor cells in vitro at 1:1 target ratio for 48h, respectively, and then the number of T cells was examined, and the results are shown in table 10 and fig. 15.

TABLE 10 Effect of different tumor cells on the proliferation fold of CAR-T cells

Group of	Control group 1	Control group 2	Experiment group 1	Experiment group 2
					Colorectal cancer	1.8	1.9	2.4	2.6
Stomach cancer	1.4	1.5	3.3	3.5
					Lung cancer	1.6	1.6	3.4	3.1
Breast cancer	1.8	1.5	2.8	2.6
					Liver cancer	2.1	2.2	3	3.2

Table 10 and fig. 15 demonstrate that different tumor cells can stimulate proliferation of low pH targeted CAR-T cells, but not control T cells. It is demonstrated that different tumor cells can activate low pH targeted CAR-T cells, promoting proliferation thereof.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The chimeric antigen receptor is characterized by comprising pHLIP, wherein the nucleotide sequence of the pHLIP is shown as SEQ ID NO. 1;

the chimeric antigen receptor consists of a CD8 signal peptide, a HiS tag, pHLIP, a CD8 hinge region, a CD8 transmembrane structural region, a 4-1BB costimulatory signaling region, and intracellular signaling regions of CD28 and CD3 zeta in tandem.

2. The chimeric antigen receptor according to claim 1, wherein the nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID No. 3.

3. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 4.

4. A lentiviral expression vector comprising the nucleotide sequence of the chimeric antigen receptor of claim 2.

5. A recombinant lentivirus comprising the lentivirus expression vector of claim 4.

6. A CAR-T cell expressing the chimeric antigen receptor of any one of claims 1-3; or the CAR-T cell has integrated into its genome the nucleotide sequence of the chimeric antigen receptor of claim 2.

7. Use of the chimeric antigen receptor of any one of claims 1 to 3, the recombinant lentivirus of claim 5, or the CAR-T cell of claim 6 in the manufacture of a medicament for treating a solid tumor, wherein the solid tumor is breast cancer, lung cancer, intestinal cancer, gastric cancer, or liver cancer.