CN116640229B - Construction and application of a low-pH targeted CAR-T cell - Google Patents

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 a low-pH targeted CAR-T cell

Technical field

The invention belongs to the technical field of immune targeted therapy, and in particular relates to the construction and application of a low pH targeted CAR-T cell.

Background technique

CAR-T, the full name is chimeric antigen receptor T-cell, which is chimeric antigen receptor T cell. The first step in CAR-T preparation is to isolate T cells from the patient: collect the patient's peripheral blood mononuclear cells (PBMC cells) through leukapheresis, and then isolate specific T cell subsets, such as CD4+, CD8+, CD25+ or CD62L + T cells; the second step is to transform T cells: generate main specific signals through T cell receptor signals and costimulatory signals such as CD28, 4-1BB or OX40 signals, thereby activating T cells, and then through electroporation, lentivirus Or a retroviral vector, the CAR gene is introduced into the activated T cells for genetic modification, thereby obtaining CAR-T cells that can express the CAR gene; the third step is amplification: culturing in vitro, amplifying a large number of CAR-T cells and then returning them lose. CAR-T therapy is a new type of precise targeted therapy for treating tumors. In recent years, it has achieved good results in clinical tumor treatment through optimization and improvement. It is a very promising therapy that can be precise, fast, efficient, and effective. New cancer immunotherapy approaches that may cure cancer.

Ideally, the target targeted by CAR-T cells is only expressed (or highly expressed) on the surface of tumor cells and not expressed (or expressed at very low levels) on other normal cells. For example, CD-19CAR is the most successful CAR-T treatment method. A large part of its success lies in the fact that CD19 is a very good specific marker for immature B cells. It is only expressed on the surface of immature B cells. Other Not expressed in somatic cells. However, since tumor cells are all produced by the canceration of normal cells, it is actually difficult to find specific markers that only exist on the surface of tumor cells but not on the surface of normal cells. And cancer cells acquire extensive genetic changes when dividing in tumors, including epigenetic regulatory site mutations, gene deletions, gene duplications, and chromosomal rearrangements. These changes are unevenly distributed in a single tumor. Heterogeneity in the expression of specific biomarkers on the cell surface within and between tumors also significantly reduces the effectiveness of drugs targeting specific biomarkers. This is one of the reasons why CAR-T therapy has not yet achieved a breakthrough in solid tumors.

pHLIP (pH-low polypeptide) is a pH-sensitive peptide. A typical pHLIP is a peptide of about 40 amino acids. Under acidic conditions, pHLIP can spontaneously insert into the cell membrane through the transmembrane α-helix, and can transfer polar substances attached to its inserted C-terminus to the cytoplasm of the target cell. This transformation is due to the pHLIP membrane-spanning region Protonation and charge neutralization of aspartic acid residues at acidic pH. pHLIP insertion is dependent on pH value. Studies have shown that pHLIP tends to accumulate in acidic diseased tissues and has high specificity. In solid tumors, due to enhanced glycolytic metabolism (Warburg effect), the action of carbonic anhydrase, and hypoxia/ischemia secondary to increased blood supply, tumors are always acidic during rapid growth. Tumors usually The resulting extracellular pH is 6.2-6.9. The pH on the surface of tumor cells is more acidic (pH 6.0-6.5), while the pH of healthy tissue is 7.4. Therefore, the acidic microenvironment of tumors is selectively targeted. tumor while sparing healthy tissue offers a possibility. At present, in view of the characteristics of pHLIP, although pHLIP has been used in tumor cell targeted therapy, such as connecting phalloidin (a cell-impermeable polar toxin) to the C-terminus of pHLIP to destroy HeLa, JC and The proliferation of M4A4 cancer cells, etc., but there is no treatment method that links pHLIP to T cells.

Contents of the invention

In view of this, the purpose of the present invention is to provide a chimeric antigen receptor, including pHLIP, which can play a targeted recognition role in the activation process of CAR-T cells.

The present invention also aims to provide a low-pH targeted CAR-T cell that can target solid tumors, promote cancer cell apoptosis, and avoid the limitations of traditional tumor marker targeted therapy.

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

The present invention provides a chimeric antigen receptor, which includes pHLIP, and the nucleotide sequence of pHLIP is shown in SEQ ID NO: 1.

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

Preferably, the chimeric antigen receptor is composed of CD8 signal peptide, HiS tag, pHLIP, CD8 hinge region, CD8 transmembrane structural region, 4-1BB costimulatory signaling region, and intracellular signaling regions of CD28 and CD3ζ in series.

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

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

The present invention also provides a lentiviral expression vector, which contains the nucleotide sequence of the above chimeric antigen receptor.

The present invention also provides a recombinant lentivirus, which includes the above-mentioned lentivirus expression vector.

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

The present invention also provides the use of the above chimeric antigen receptor, recombinant lentivirus or CAR-T cells in preparing drugs for treating solid tumors.

Preferably, the solid tumors include breast cancer, lung cancer, intestinal cancer, gastric cancer, and liver cancer.

Beneficial effects of the present invention:

This invention connects pHLIP to T cells for the first time and constructs a low-pH targeting CAR-T cell. The CAR-T cells prepared by the present invention can express pHLIP, and target the CAR-T cells on the surface of solid tumors according to the acidic characteristics of the tumor microenvironment, thereby targeting and killing tumor cells and avoiding traditional tumor markers. Limitations of drug-targeted therapy.

Description of the drawings

Figure 1: Structure of chimeric antigen receptor including pHLIP;

Figure 2: Structure of empty chimeric antigen receptor;

Figure 3: Plasmid digestion electrophoresis pattern;

Figure 4: 293T cells packaging virus and transfection efficiency;

Figure 5: CAR-T cell infection efficiency;

Figure 6: CAR-T cell proliferation activity;

Figure 7: Killing ability of CAR-T cells against gastric cancer cell lines;

Figure 8: Killing ability of CAR-T cells against breast cancer cell lines;

Figure 9: Killing ability of CAR-T cells against colorectal cancer cell lines;

Figure 10: Killing ability of CAR-T cells against lung cancer cell lines;

Figure 11: Killing ability of CAR-T cells against liver cancer cell lines;

Figure 12: The impact of different tumor cells on the expression ratio of CD69 molecules in CAR-T cells;

Figure 13: Effects of different tumor cells on IFN-r expression in CAR-T cells;

Figure 14: Effects of different tumor cells on IL-2 expression of CAR-T cells;

Figure 15: Effects of different tumor cells on the proliferation fold of CAR-T cells.

Detailed ways

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

The present invention is based on a chimeric antigen receptor containing a third-generation CAR structure, carries out engineering transformation between the signal peptide and the hinge region of the chimeric antigen receptor, and joins the HiS tag and pHLIP to prepare a chimeric antigen receptor containing pHLIP. Combined with the antigen receptor, pHLIP can be expressed on CAR-T cells, allowing CAR-T cells to target and locate on the surface of solid tumors with an acidic microenvironment.

The chimeric antigen receptor of the present invention is composed in series of CD8 signal peptide, HiS tag, pHLIP, CD8 hinge region, CD8 transmembrane structural region, 4-1BB costimulatory signaling region, and intracellular signaling regions of CD28 and CD3ζ. Details see picture 1.

The nucleotide sequence of the chimeric antigen receptor of the present invention is shown in SEQ ID NO: 3. In the nucleotide sequence, positions 1 to 63 are CD8 signal peptide sequences, positions 64 to 81 are HiS tag sequences, positions 82 to 195 are pHLIP sequences, and positions 196 to 330 are CD8 hinge region sequences. Positions 331 to 402 are the CD8 transmembrane structural region sequence, positions 403 to 528 are the 4-1BB costimulatory signal transduction region, positions 529 to 651 are the CD28 intracellular signal region sequence, and positions 652 to 987 are the intracellular CD3ζ Signal area sequence.

The amino acid sequence of the chimeric antigen receptor of the present invention is shown in SEQ ID NO: 4. In the amino acid sequence, positions 1 to 21 are CD8 signal peptide sequences, positions 22 to 27 are HiS tags, positions 28 to 65 are pHLIP sequences, positions 66 to 110 are CD8 hinge region sequences, and positions 111 to 27 are HiS tags. Position 134 is the CD8 transmembrane structural region sequence, positions 135 to 176 are the 4-1BB costimulatory signaling region, positions 177 to 217 are the CD28 intracellular signal region sequence, and positions 218 to 329 are the CD3ζ intracellular signal region sequence. .

The present invention also provides a lentiviral expression vector, which contains the nucleotide sequence of the above chimeric antigen receptor (SEQ ID NO: 3). As an optional embodiment, the present invention can obtain a lentiviral vector plasmid containing the chimeric antigen receptor by performing double enzyme digestion on the base sequence of the chimeric antigen receptor and the lentiviral expression vector and ligating the enzyme digestion products. .

The present invention also provides a recombinant lentivirus, which includes the above-mentioned lentivirus expression vector. The recombinant lentivirus of the present invention is prepared by co-transfecting cells with the above-mentioned lentiviral expression vector and lentiviral packaging plasmid. The preferred method of the present invention uses a three-plasmid virus packaging system.

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

The present invention has no special limitations on the specific preparation method of CAR-T cells, and any conventional CAR-T cell preparation method in the field can be used. As an optional embodiment, the preparation method of the present invention includes the following steps: cloning the chimeric antigen receptor gene fragment into the px330 vector, and then introducing the chimeric antigen receptor gene into the activated vector through lentiviral transfection. Among T cells, CAR-T cells expressing pHLIP were obtained.

The present invention also provides the use of the above-mentioned chimeric antigen receptor, the above-mentioned recombinant lentivirus or the above-mentioned CAR-T cells in the preparation of medicines. In the present invention, the drug preferably includes a drug for immunotherapy of solid tumors, and the solid tumors treated by the drug include but are not limited to breast cancer, lung cancer, intestinal cancer, gastric cancer, and liver cancer.

The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the present invention. In the following examples, unless otherwise specified, all are conventional methods; unless otherwise specified, the materials and reagents used can be obtained from commercial sources.

Example 1

Preparation method of low pH targeted CAR-T cells:

1. Carrier construction

px330 was digested with BbsⅠ and EcoRI, and the insert fragment (SEQ ID NO: 3) was inserted between the BbsⅠ and EcoRI digestion sites to obtain a ligation product; the ligation product was transformed, and clones were picked and sequenced for identification.

2. Preparation of plasmid

Preparation of competent cells

(1) Streak and coat the plate: Use a sterilized inoculation loop to dip a small amount of E.coli DH5α bacterial liquid into a streak on the antibiotic-free LB agar plate to gradually dilute the bacterial liquid. Place upside down in a 37°C bacterial incubator and incubate overnight for 12 to 14 hours.

(2) Pick a single colony and inoculate it into 5 ml of LB medium without antibiotics. 37°C, 250rpm overnight.

(3) Take 2 ml of turbid bacterial liquid and inoculate it into 200 ml of LB medium without antibiotics, and culture it with shaking at 37°C for 2 to 3 hours. Measure OD ₆₀₀ every half hour to reach 0.4~0.6.

(4) Ice-bath the bacterial solution for 20 minutes. Transfer the ice-bathed bacterial solution to a pre-cooled 50ml centrifuge tube. Centrifuge at 4°C, 4000rpm, 10min.

(5) Discard the supernatant. Resuspend the bacterial pellet in 20 ml of pre-chilled _0.1 M CaCl solution. Centrifuge at 4°C, 4000rpm, 10min.

(6) Discard the supernatant. Add 2 ml of pre-cooled 0.1 MCaCl ₂ solution containing 20% sterile glycerol per 50 ml of bacterial solution to resuspend the cell pellet.

(7) Aliquot (50 μl/tube), quickly freeze in liquid nitrogen, and then store at -80°C.

Rapid heat-activated transformation of plasmids

(1) Place 50 μl of E.coli DH5α competent cells on ice to thaw, add 1 to 10 ng of plasmid, and mix gently.

(2) Place the mixture of competent cells and plasmid on ice and let it stand for 30 minutes.

(3) Heat-activate the above mixture in a 42°C water bath (90 seconds). After heat shock, quickly place it on ice and let it stand for 2 minutes.

(4) Take an appropriate amount of heat-shocked competent cells and spread them evenly onto an LB agar plate with antibiotics.

(5) Place the LB plate upside down in a 37°C bacterial incubator and incubate for 12 to 16 hours. Many individual bacterial colonies can be seen.

plasmid miniprep

(1) Pick the transformed single colony, inoculate it into 3 to 5 ml of LB medium containing antibiotics, and culture it overnight at 37°C with vigorous shaking.

(2) Centrifuge at 12,000 rpm for 1 minute to collect the bacterial pellet. Add 250μl P1 buffer (50mM Tris Cl, 10mM EDTA, 100μg/ml RNaseA) and shake to mix.

(3) Add 250μl P2 buffer (200mM NaOH, 1% SDS), and mix gently by inverting.

(4) Add 350μl N3 buffer (3.0M NaAC, pH5.5), and mix gently by inverting.

(5) Centrifuge at 13000 rpm for 10 minutes.

(6) Carefully draw the supernatant and add it to the QIAprep spin column. Centrifuge at 12,000 rpm for 1 minute and discard the waste liquid.

(7) Add 0.75ml PE buffer (1.0M NaCl, 50mM MOPS, 15% isopropyl alcohol) to rinse, centrifuge at 12000rpm for 1min, and discard the waste liquid.

(8) Centrifuge at 12,000 rpm for 2 minutes to remove residual rinse liquid.

(9) Add 50μl EB buffer (10mM Tris Cl, pH8.5) and leave it at room temperature for 1 minute. Centrifuge at 12000 rpm for 1 min to elute the plasmid.

Plasmid mid- or max-prep

(1) Pick the transformed single colony, inoculate it into 3 to 5 ml of LB medium containing antibiotics, and culture it for 8 hours at 37°C with vigorous shaking.

(2) Inoculate the bacterial solution into LB medium containing antibiotics at a ratio of 1/500 to 1/1000, and culture it at 37°C with vigorous shaking for 12 to 16 hours.

(3) Centrifuge at 6000g for 15 minutes at 4°C to collect the cells.

(4) Resuspend the bacterial pellet in 4ml (medium extraction) or 10ml (maximum extraction) P1 buffer (50mM Tris Cl, 10mM EDTA, 100μg/ml RNaseA).

(5) Add 4ml (medium extraction) or 10ml (large extraction) P2 buffer (200mM NaOH, 1% SDS), mix gently, and leave at room temperature for 5 minutes.

(6) Add 4ml (medium extraction) or 10ml (large extraction) of pre-cooled P3 buffer (3.0M NaAC, pH5.5), mix gently, and place on ice for 20 minutes.

(7) Centrifuge at 4℃, 20000g, 30min. repeat.

(8) Add 4ml (medium extraction) or 10ml (large extraction) QBTbuffer (750mM NaCl, 50mM MOPS, 15% isopropyl alcohol, 0.15% Triton X-100) to the QIAGEN-tip for column equilibration.

(9) Add the centrifuged supernatant to the equilibrated QIAGEN-tip column. Liquid flows out under the influence of gravity.

(10) Rinse the QIAGEN-tip column with 10ml (medium extraction) or 30ml (large extraction) QC buffer (1.0M NaCl, 50mM MOPS, 15% isopropyl alcohol). repeat.

(11) Use 5ml (medium extraction) or 15ml (maximum extraction) QF buffer (1.25M NaCl, 50mM Tris Cl, 15% isopropyl alcohol) to elute the plasmid.

(12) Add 3.5ml (medium extraction) or 10.5ml (large extraction) isopropyl alcohol to the eluted plasmid. After mixing, centrifuge at 4℃, 8000g, 40min. DNA precipitate can be seen at the bottom of the tube.

(13) Discard the supernatant. Add 2 ml (medium extraction) or 5 ml (large extraction) of 70% ethanol to rinse the precipitate. Centrifuge at 4℃, 6000g, 60min.

(14) Discard the supernatant. Dry at room temperature for 5 to 10 minutes. Add TE buffer (10mM Tris Cl, pH8.0) to dissolve the plasmid.

3. Identification of plasmid

Plasmid quality testing

Use NanoPhotometerTM Pearl (IMPLEN) ultra-trace UV-visible spectrophotometer to measure plasmid concentration and purity. Record the plasmid concentration, OD _260/280 , and OD _260/230 ratio. OD _260/280 reaches 1.8~2, and OD _260/230 >2.

Agarose gel electrophoresis detection

(1) Use 1×TAE electrophoresis buffer to prepare a 0.9-1.5% agarose gel and heat it with microwave to dissolve. After cooling to about 55-60°C, add EtBr (ethidium bromide) at a ratio of 1/20000. It can be placed in a 55℃ water bath for short term use.

(2) Pour the prepared agarose gel into a gel-making mold with a comb of appropriate size inserted, and wait for it to solidify at room temperature.

(3) Pull out the comb, mix 100-500ng plasmid with 6× loading buffer, and then add the sample.

(4) Use a voltage of 5 to 7V/cm for electrophoresis.

(5) After electrophoresis, use the LHR gel imaging system (Syngene) to scan the gel and take pictures.

Enzyme digestion identification of plasmids

According to the enzyme cutting pattern of the plasmid, select restriction endonucleases (BbsⅠ, EcoRI) for cutting. Use the size of the specific enzyme digestion fragments generated after enzyme digestion to determine whether the extracted plasmid is correct. The electrophoresis pattern after enzyme digestion is shown in Figure 3.

4. Lentivirus packaging

A second-generation packaging system, a three-plasmid system, is used, which includes a packaging plasmid (psPAX2), an envelope plasmid (pMD2G), a target gene plasmid and a packaging cell (293T cell).

Day1: HEK293T cells in 10cm dishes (6×10 ⁷ /dish) with a confluence of 90% are passaged to 15cm dishes at a ratio of 1:1. The next day, the cell confluence reaches 90-95% (1.5×10 ⁸ /dish) and cultured. The base is Gibico high-glucose DMEM medium (containing 10% FBS).

Day2：

Change the culture medium (containing 10% FBS) 2-3 hours before transfection; prepare the transfection reagent according to the following proportions:

Mix 1 volume μl, Mix 2 volume, DMEM (without FBS) 1000 μl, DMEM (without FBS) 1000 μl, target gene plasmid 25 μg, VGF 190 μl (1 μg/μl), PMD2G 7.5 μg, PSPAX 215 μg.

After Mix 1 and Mix 2 are mixed separately, mix Mix 1 and Mix 2 at room temperature for 5 to 10 minutes, keep at room temperature for 30 minutes, and add to a 15cm dish.

Day3: Replace with fresh culture medium (containing 10% FBS) within 6 to 24 hours.

Day5: Observe the cell status and take pictures for 72 hours. Collect the supernatant culture medium and filter it through a 0.45 μm filter. Add the supernatant culture medium into an ultracentrifuge tube, balance and centrifuge at 25,000 rpm and 4°C for 1.5 h. Discard the supernatant, redissolve in appropriate virus preservation solution, mix and dissolve overnight.

Day6: Collect virus aliquots and measure virus titers.

Determination of recombinant lentivirus titer: integration number method to calibrate the titer of recombinant lentivirus without fluorescence

virus infected cells

HEK293 cells were evenly seeded in a 24-well cell culture plate at 2.5 × 10 ⁵ cells/well 6 h before infection.

Carry out gradient dilution of the lentivirus and make a total of 3 gradients, that is, each well (500 μl DMEM medium without double antibodies and serum) contains 10 μl, 1 μl, and 0.1 μl virus, shake and mix, and then add to the 24 wells where the cells have been inoculated. In the plate, aspirate the culture medium in the culture plate before adding the virus.

After 18 to 20 hours of infection, the medium in the culture plate was replaced with fresh DMEM complete medium.

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

During the measurement, a set of fluorescent lentiviruses with known TUs were set up as controls to verify the detected values.

5. G-CSF mobilizes peripheral blood hematopoietic stem cells to separate mononuclear cells (using ficoll cell separation medium)

(1) Dilute the blood sample 2 to 4 times with sterile diluent.

(2) First add 15 to 20 ml of separation liquid into a 50 ml conical bottom centrifuge tube, and then slowly add 20 to 30 ml of diluted whole blood to the surface of the separation liquid.

(3) Centrifuge at 400g for 15-30 minutes at room temperature to ensure that the rotor speed decreases smoothly.

(4) Carefully take out the centrifuge tube from the centrifuge and slowly suck off the top layer to avoid contact with the mononuclear cell layer.

(5) Slowly transfer the mononuclear cell layer to another 50ml conical bottom centrifuge tube.

(6) Add sterile washing solution and mix well, centrifuge at 300g for 10 minutes at room temperature, and carefully discard the supernatant to obtain a cell suspension.

(7) Count the number of viable cells using trypan blue staining solution, and filter the cell suspension with a 50 μm cell sieve to obtain G-CSF to mobilize peripheral blood hematopoietic stem cells and mononuclear cells.

The next experiment will be carried out after reaching the following standards: 95±5% of the isolated cells are mononuclear cells; the survival rate of the isolated cells is >90%; 60±20% of the mononuclear cells in the original blood sample can be recovered; 3±2% granulocytes; 5±2% red blood cells

6. G-CSF mobilizes peripheral blood hematopoietic stem cell-derived mononuclear cells to isolate and activate T cells.

(1) Determine the number of mononuclear cells and adjust the cell concentration to 1×10 ⁸ cells/ml.

(2) Put 100 μl of cell suspension (1×10 ⁷ cells) into a new test tube, add 10 μL of negative sorting antibody, stir evenly, protect from light, and incubate on ice for 15 minutes.

(3) Add 20 μl of negative sorting magnetic beads, stir evenly, protect from light, and incubate at 4°C for 15 minutes.

(4) After adding 4 ml of buffer solution to a 5 ml (12 × 75 mm) polypropylene tube, place the test tube in the magnet for 5 minutes, and pour the supernatant into another 15 ml centrifuge tube.

Note: Repeated magnetic separation can improve the yield and has no strong impact on the purity. After the second separation, the yield will increase by 8 to 10%, and each separation can reduce the purity by 1 to 2%.

(5) Add CD3/CD28 activated magnetic beads to the obtained T lymphocytes at a ratio of 3:1, and place them in a 37°C, 5% CO ₂ incubator for overnight culture.

7. Lentivirus infects T cells

(1) After 24 to 72 hours of T cell activation, melt the lentiviral particles at room temperature, mix gently, add the lentiviral particles, and place them in a 37°C, 5% _CO2 incubator for 4 to 6 hours.

(2) Remove most of the supernatant culture medium containing lentiviral particles, add new culture medium, and amplify T cells. After 3 days, transfer to a cell culture bag for culture. The virus packaging and transfection efficiency of 293T cells are shown in Figure 4. From Figure 4, it can be seen that the lentivirus transfection efficiency is above 90%.

Example 2

Experimental group: Use the method of Example 1 to construct low pH targeted CAR-T cells;

Control group: Replace the insert fragment (SEQ ID NO: 3) in step 1 of Example 1 with the empty insert fragment (SEQ ID NO: 5). The other methods are the same as in Example 1 to construct empty CAR-T cells. Chimeric The structure of the antigen receptor is detailed in Figure 2.

1. After packaging the virus, infect T cells, and 48 hours later use flow cytometry to detect the T cells:

(1) Dilute the cultured cells with PBS buffer and place them in a centrifuge tube. Centrifuge at 350g for 5 minutes and discard the supernatant. Repeat washing the cells once. Resuspend the cells in PBS buffer and count. The diluted cells are (5~ 10)×10 ⁶ cells/ml, and then add 100 μl of cell suspension ((5～10)×10 ⁵ cells/tube) into each flow cytometry detection tube.

(2) Add an appropriate amount of pre-diluted primary antibody to each flow cytometry detection tube; add the same amount of control reagent as the antibody to the blank tube/well. Then each tube was incubated in an ice bath in the dark for 15 to 20 minutes. When staining with Car-T antibody, incubate for 50 minutes in a 4°C refrigerator.

(3) Add 2 ml of PBS buffer, then centrifuge at 350g for 5 minutes, discard the supernatant, and repeat the washing process twice.

(4) Resuspend the cells in 300 μl PBS buffer or 300 μl 2% paraformaldehyde fixative.

(5) Test on the computer and analyze the results.

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

2. Culture the cells in the control group and the cells in the experimental group in the culture system respectively, and count the results every other day. The results are detailed in Table 1 and Figure 6.

Table 1 Cell proliferation times

Group Control group 1 Control group 2 Experimental group 1 Experimental 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 Figure 6 show that the proliferation activity of T cells does not change after CAR-T cell modification.

3. CAR-T killing ability test

(1) Target cell Cfse staining solution labeling and staining

1) Dilute the target cells with PBS buffer and place them in a centrifuge tube. Centrifuge at 300g for 5 minutes, discard the supernatant, and wash the cells again.

2) Resuspend the cells in PBS buffer and count, dilute the cells to 1×10 ⁷ cells/ml, then add Cfse dye to a final concentration of 2 to 5 μM, and incubate in a 37°C, 5% CO ₂ incubator for 15 minutes.

3) Add 10ml PBS buffer, then centrifuge at 300g for 5 minutes, discard the supernatant, and repeat the washing process twice.

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

The washed target cells and CAR-T cells were resuspended in serum-free 1640 medium and counted at an effect-to-target ratio (target cells: effector cells) of 1:0.25, 1:0.5, 1:1, 1:2, and 1 :5 Add the 96-well round-bottomed cell culture plate and place it in a 37°C, 5% CO ₂ incubator for 4 hours.

(3) Sample staining

1) Aspirate the cells from each culture well into a flow cytometry tube, and centrifuge at 300g for 5 minutes to collect the cells.

2) Wash the cells once with PBS buffer and centrifuge at 300g for 5 minutes. Collect (1~5)×10 ⁵ cells/tube.

3) Aspirate the PBS and add 100 μl 1×Binding Buffer to resuspend the cells.

4) Add 5μl AnnexinV-APC and 10μl 7-AAD to each tube and mix gently.

5) Protect from light and react at room temperature for 10 to 15 minutes.

6) Add 200μl 1×Binding Buffer, mix the sample and detect it with a flow cytometer within 1 hour.

(4) Sample flow cytometry analysis:

AnnexinV-APC is the abscissa, 7-ADD is the ordinate, and the cell apoptosis rate is detected (viable cells have only very low intensity background fluorescence, early apoptotic cells have only strong blue fluorescence, and late apoptotic cells have blue fluorescence). color and red fluorescent double staining).

The killing ability of CAR-T cells against gastric cancer cell lines is shown in Table 2 and Figure 7.

Table 2 Apoptosis rate of gastric cancer cell lines (%)

The killing ability of CAR-T cells against breast cancer cell lines is shown in Table 3 and Figure 8.

Table 3 Apoptosis rate of breast cancer cell lines (%)

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

The killing ability of CAR-T cells against colorectal cancer cell lines is shown in Table 4 and Figure 9.

Table 4 Apoptosis rate of colorectal cancer cell lines (%)

Group Control group 1 Control group 2 Experimental group 1 Experimental group 2 1:0.25 4 4 12 11 1:0.5 3 5 twenty one 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 Control group 1 Control group 2 Experimental group 1 Experimental 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 Figure 11.

Table 6 Apoptosis rate of liver cancer cell lines (%)

As can be seen from Tables 2 to 6 and Figures 7 to 11, low pH targeted CAR-T cells can kill tumor cell lines of different origins in a dose-dependent manner, while T cells in the control group can hardly kill tumor cells.

4. The ability of tumor cells to activate CAR-T cells

(1) In vitro, T cells in the control group and T cells in the experimental group were co-cultured with different tumor cells at a 1:1 target ratio for 4 hours, and then flow cytometry was performed on the cells to explore the expression ratio of CD69 molecules. The results are shown in Table 7 and Figure 12.

Table 7 Effects of different tumor cells on the expression ratio of CD69 molecules in CAR-T cells (%)

Group Control group 1 Control group 2 Experimental group 1 Experimental 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 Figure 12 illustrate that different tumor cells can stimulate low-pH targeted CAR-T cells to express CD69 molecules, but cannot stimulate control T cells to express CD69 molecules. CD69 molecule is a molecule expressed by T cells after activation, indicating that different tumor cells can activate low-pH targeted CAR-T cells.

(2) In vitro, the T cells of the control group and the T cells of the experimental group were co-cultured with different tumor cells at a 1:1 target ratio for 4 hours, and the cytokines in the culture supernatant were detected. The results are shown in Tables 8 to 9 and Figure 13 to 14.

Table 8 Effects of different tumor cells on IFN-r expression of CAR-T cells (pg/ml)

Table 9 Effects of different tumor cells on IL-2 expression of CAR-T cells (pg/ml)

Group Control group 1 Control group 2 Experimental group 1 Experimental 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 to 9 and Figures 13 to 14 show that different tumor cells can stimulate low pH targeted CAR-T cells to secrete cytokines, but cannot stimulate control T cells to secrete cytokines. This shows that different tumor cells can activate low-pH targeted CAR-T cells and promote the release of their cytokines IFN-r and IL-2.

(3) In vitro, T cells in the control group and T cells in the experimental group were co-cultured with different tumor cells at a 1:1 effect-to-target ratio for 48 hours, and then the number of T cells was detected. The results are shown in Table 10 and Figure 15.

Table 10 Effects of different tumor cells on CAR-T cell proliferation fold

Group Control group 1 Control group 2 Experimental group 1 Experimental 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 Figure 15 show that different tumor cells can stimulate the proliferation of low-pH targeted CAR-T cells, but cannot stimulate the proliferation of control T cells. This shows that different tumor cells can activate low pH targeted CAR-T cells and promote their proliferation.

The above are only the preferred embodiments of the present invention. It should be pointed out that those of ordinary skill in the art can also make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (7)

1. A chimeric antigen receptor, characterized in that the chimeric antigen receptor includes pHLIP, and the nucleotide sequence of the pHLIP is as shown in SEQ ID NO: 1; The chimeric antigen receptor is composed in series of CD8 signal peptide, HiS tag, pHLIP, CD8 hinge region, CD8 transmembrane structural region, 4-1BB costimulatory signaling region, and intracellular signaling regions of CD28 and CD3ζ. 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, characterized in that the vector contains the nucleotide sequence of the chimeric antigen receptor according to claim 2. 5. A recombinant lentivirus, characterized in that the recombinant lentivirus contains the lentiviral expression vector of claim 4. 6. A CAR-T cell, characterized in that the CAR-T cell expresses the chimeric antigen receptor described in any one of claims 1 to 3; or the genome of the CAR-T cell has the right to integrate The nucleotide sequence of the chimeric antigen receptor described in claim 2. 7. Use of the chimeric antigen receptor according to any one of claims 1 to 3, the recombinant lentivirus according to claim 5 or the CAR-T cell according to claim 6 in the preparation of drugs for the treatment of solid tumors. , characterized in that the solid tumor is breast cancer, lung cancer, intestinal cancer, gastric cancer or liver cancer.