CN110423767A - Express Chimeric antigen receptor CAR gene and the application of solubility PD-1 - Google Patents

Abstract

The present application discloses a chimeric antigen receptor CAR gene expressing soluble PD-1 and its application. The CAR gene includes an antigen binding domain gene, a transmembrane domain gene, an intracellular domain gene and a soluble PD-1 gene connected in sequence, and the soluble PD-1 is the extracellular segment region of PD-1, preferably The soluble PD-1 includes the amino acid sequence shown in SEQ ID No.1. This application enables CAR-T cells to secrete sPD-1 in the tumor microenvironment, bind to PD-L1 on the surface of tumor cells through sPD-1, and competitively inhibit the binding to transmembrane PD-1 on the surface of CAR-T cells, thereby avoiding CAR-T cells. T cells are inhibited to improve the killing effect of CAR-T cells on tumor cells, so as to achieve the purpose of treating tumors, especially solid tumors. This application provides a new way to improve the effect of CAR-T cells in killing tumor cells.

Description

Chimeric antigen receptor CAR gene expressing soluble PD-1 and its application

technical field

The invention relates to the field of biomedicine, in particular to a chimeric antigen receptor CAR gene expressing soluble PD-1 and its application.

Background technique

Tumor is a disease that seriously threatens human life, and its treatment methods have always been highly concerned by the medical and biological circles. In recent years, with the deepening of people's understanding of the tumor immune microenvironment, a new idea for the treatment of tumors has gradually entered people's field of vision, namely "tumor immunotherapy". In 2013, the US FDA called it "breakthrough therapy". , enough to see its importance, and chimeric antigen receptor T cell therapy (CAR-T) is currently one of the most promising tumor immunotherapy.

The chimeric antigen receptor CAR generally consists of an antigen binding region/extracellular region/transmembrane region and an intracellular signaling region that can activate T cells after binding antigen. CAR-T cells have higher potential because it is activated via an MHC molecule-independent, specific, single-signal pathway. Some in-depth studies in preclinical and clinical trials have shown that CAR-T therapy has achieved encouraging therapeutic effects in the treatment of various cancers, especially with CD19-CAR-T in the treatment of B-cell malignancies. At present, CAR-T cell immunotherapy has achieved significant clinical responses in hematological malignancies. However, the treatment of solid tumors with CAR-T cells is limited by the strong immunosuppressive environment of solid tumor tissue structure, and the lack of ideal targets is another key deficiency in the treatment of solid tumors. Therefore, finding more effective targets to advance CAR-T cell therapy for solid tumors is an important challenge at this stage.

PD-1 is programmed death receptor 1, an important immunosuppressive molecule and a member of the CD28 superfamily. PD-1 is a type I transmembrane protein, which consists of three parts: extracellular segment, transmembrane anchoring region and intracellular signal transduction region. It is mainly induced and expressed on the surface of activated T lymphocytes and B lymphocytes. The ligands of PD-1 are B7-H1 (PD-L1) and B7-DC (PD-L2). The combination of the two inhibits the proliferation and activation of T cells and negatively regulates the response of T cells. They are inhibitory receptors. PD-L1 is often expressed on the surface of tumor cells, which binds to PD-1 on the surface of T cells, inhibits the proliferation and activation of T cells, and achieves immune escape. At present, there are PD-1 and PD-L1 antibodies on the market, which block the inhibitory effect of tumor cells on T cells. In humans, in addition to the transmembrane binding protein PD-1, soluble PD-1 (sPD-1) also exists. sPD-1 retains the extracellular Ig V-Ig C-like domain of the model molecule and can bind to the ligand PD. -L1 and PD-L2. The initial studies on sPD-1 were mostly related to autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis and other autoimmune diseases have been proved to be related to sPD-1. In immune diseases, it is produced by immune cells under the action of pro-inflammatory stimulators. Likewise, sPD-1 has the ability to bind to PD-L1 and function to inhibit T cell activity.

At present, there is no product that co-expresses soluble PD-1 with chimeric antigen receptor CAR to improve the cytotoxicity of CAR-T cells against tumor cells.

SUMMARY OF THE INVENTION

In view of the defects in the prior art, the purpose of the present invention is to provide a chimeric antigen receptor CAR gene expressing soluble PD-1 and its application. The present invention enables CAR-T cells to secrete sPD-1 in the tumor microenvironment. Through the binding of sPD-1 to PD-L1 on the surface of tumor cells, it competitively inhibits the binding to the transmembrane PD-1 on the surface of CAR-T cells, thereby avoiding the inhibition of CAR-T cells and improving the killing effect of CAR-T cells on tumor cells. , to achieve the purpose of the treatment of tumors, especially solid tumors.

In order to achieve the above purpose, the technical scheme adopted in the present invention is:

The present invention first provides a chimeric antigen receptor CAR gene expressing soluble PD-1. The CAR gene includes an antigen-binding domain (ie, a segment of extracellular single-chain antigen variable region fragment scFV) genes connected in sequence, a transmembrane domain gene, an intracellular domain gene and a soluble PD-1 gene, the soluble PD-1 being the extracellular segment region of PD-1,

Preferably, the soluble PD-1 comprises the amino acid sequence shown in SEQ ID No.1,

More preferably, the sequence of the soluble PD-1 gene is shown in positions 1264-1713 in SEQ ID No.2.

A preferred embodiment, a first signal peptide gene is connected between the intracellular domain gene and the soluble PD-1 gene, so that the extracellular segment of PD-1 is efficiently expressed and secreted into the extracellular space rather than transmembrane expression,

Preferably, the first signal peptide is the signal peptide of the following proteins: CD19, CD20, CD30, CD4, CD8a, CD28, CD137 or a combination thereof;

More preferably, the first signal peptide is a signal peptide of CD8a, and its amino acid sequence is shown in SEQ ID No.3, and more preferably, its gene sequence is shown in positions 1201-1263 in SEQ ID No.2 .

A preferred embodiment, a self-cleaving protein gene is connected between the intracellular domain gene and the first signal peptide gene, preferably, the self-cleaving protein is selected from T2A, P2A, E2A, F2A or a combination thereof, more preferably, the self-cleaving protein is T2A.

The antigen binding domain can specifically bind to a specific ligand on the surface of tumor cells. In this application, the specific ligand is specifically an NKG2D ligand. Preferably, the antigen binding domain is NKG2D Receptor extracellular segment, more preferably, the front end of the antigen binding domain gene includes the second signal peptide gene;

Preferably, the second signal peptide is the signal peptide of the following proteins: CD19, CD20, CD30, CD4, CD8a, CD28, CD137 or a combination thereof; more preferably, the second signal peptide is the signal peptide of CD8a, more preferably Preferably, its amino acid sequence is shown in SEQ ID No.3, and more preferably, its gene sequence is shown in positions 1-63 in SEQ ID No.2;

The NKG2D receptor binds in a "promiscuous" manner to a family of cell surface glycoproteins that share structural homology with MHC class I proteins. It is an activating receptor expressed primarily on cytotoxic immune cells and can induce direct killing of target cells expressing its stress-inducing ligand. The ligands of NKG2D include six members: MICA, MICB, ULBP1-6. DNA damage, oncogene activation, hyperproliferation, etc. are important signs of cancer, and the levels of NKG2D ligands on the cell surface are significantly increased, while these ligands are low expressed on the surface of normal cells. Therefore, the extracellular segment of the NKG2D receptor is designed into the CAR structure, and when it binds to the ligand, it will activate T cells and produce a series of anti-tumor responses.

A preferred embodiment, the transmembrane domain comprises the transmembrane region of the following proteins: CD3ε, CD4, CD8a, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3 or its combination, preferably, CD8a;

A preferred embodiment, the intracellular domain comprises a costimulatory signal molecule and a cytoplasmic signal transduction sequence; preferably, the costimulatory signal molecule is selected from the costimulatory signal molecules of the following proteins: OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1 or a combination thereof, more preferably, 4-1BB; preferably, the cytoplasmic signaling sequence comprises CD3ζ-derived cytoplasmic signaling sequence.

In a preferred embodiment, the chimeric antigen receptor CAR gene sequence expressing soluble PD-1 is shown in SEQ ID No. 2.

On the other hand, the present invention also provides a recombinant vector, the recombinant vector contains any of the above CAR genes, the recombinant vector is preferably a lentiviral vector,

In a preferred embodiment, the lentiviral vector used in the present invention is pCDH-NKG2D-sPD-1-CAR, which is connected with SEQ ID No. SEQ ID No. 1 at the SalI and EcoRI restriction sites of the lentiviral expression vector pCDH. .2 CAR gene indicated.

On the other hand, the present invention also provides a recombinant cell or recombinant bacteria, the recombinant cell or the recombinant bacteria contains any of the above CAR genes, or contains the recombinant vector,

The recombinant bacteria are engineering bacteria, and the engineering bacteria can specifically be Escherichia coli,

Preferably, the recombinant cells are immune cells, and more preferably, the recombinant cells are CAR-T cells.

On the other hand, the present invention also provides the application of any of the above-mentioned CAR gene, the recombinant vector, the recombinant cell or the recombinant bacteria in the preparation of a tumor treatment product, and the product may specifically be a reagent, a reagent box or drug.

In the above application, the tumor treatment product is a tumor treatment product with improved killing effect of selectively killing tumor cells.

In the above application, the tumor is a solid tumor, preferably, osteosarcoma U2OS, osteosarcoma MG-3, or renal carcinoma cell 786O, more preferably, osteosarcoma U2OS.

In another aspect, the present invention also provides a method for improving the killing effect of selectively killing tumor cells, comprising the step of administering any one of the above CAR genes, the recombinant vector, or the recombinant cells to target cells.

In the above method, the target cell is a tumor cell expressing PD-L1, preferably, the target cell is derived from osteosarcoma U2OS, osteosarcoma MG-3, or renal cancer cell 786O, more preferably, the target The cells were derived from osteosarcoma U2OS, which highly expressed both PD-L1 and NKG2D ligands.

In the above method, the effector-to-target ratio of the administration is 1:0.5-5, preferably, 1:1-4, more preferably, 1:3.

In the above applications and methods, the increase is relative to the situation in which the soluble PD-1 gene is not linked in the CAR gene or the CAR-T cells do not express or secrete soluble PD-1.

The beneficial effects of the present invention are as follows:

CAR-T technology is an important way to treat tumor cells today. However, the use of CAR-T cells to treat solid tumors is limited by the structure of solid tumors and a strong immune-suppressive environment. The lack of ideal targets is another key deficiency in the treatment of solid tumors. Therefore, finding more effective targets to advance CAR-T cell therapy for solid tumors is an important challenge at this stage. The invention creatively introduces soluble PD-1 into CAR-T cells, so that it has the function of auto-secreting PD-1, and plays the role of reducing tumor cells' inhibition of CAR-T cells. Compared with PD-1 antibody, soluble PD-1 has stronger advantages and effects. First, soluble PD-1 can bind to PD-L1 on the surface of tumor cells to avoid inhibition of T cell activity; second, compared with PD-1 antibody , soluble PD-1 can maintain long-lasting and high-efficiency efficacy at lower levels, so the side effects of soluble PD-1 will be lower than that of PD-1 antibody; finally, because PD-1 antibody needs to be continuously injected, so soluble Small doses of PD-1 are more cost-effective.

In the present invention, the PD-1 extracellular segment sequence is added to the NKG2D-CAR-T plasmid to transform the CAR-T cells, so that they have the function of secreting soluble PD-1, and can reduce the inhibition of solid tumor cells on CAR-T cells. Purpose. Compared with NKG2D-CAR-T cells that do not express soluble PD-1, the transformed NKG2D-sPD-1-CAR-T cells are more than twice as effective in killing tumor cells as the original NKG2D-CAR-T cells.

Description of drawings

Figure 1 is a schematic diagram of the structure of the CAR elements in the vector pCDH-NKG2D-CAR (upper image) and the vector pCDH-NKG2D-sPD-1-CAR (lower image), where NKG2Dex contains a signal peptide at the front end (the signal peptide sequence is in the current Some αCD19-CAR has a significant effect) sequence that expresses the element of the extracellular segment of the NKG2D receptor, CD8a TM is the element that expresses the transmembrane region of the protein CD8a, and 4-1BB is the element that expresses the costimulatory signal molecule of the protein 4-1BB. , CD3ζ is an element that expresses the CD3ζ cytoplasmic signal transduction sequence, T2A is a 2A element from a little brown wing moth virus (TaV) (self-splicing protein, genes linked by T2A can be translated and co-expressed normally after transfection into cells) , sPD-1 is an element expressing soluble PD-1 protein.

Figure 2 shows the electrophoresis results of colony PCR amplification of sPD-1. Among them, samples 1, 3, 5, 6, and 8 were positive, and the rest were negative.

Figure 3 is a schematic diagram of the structure of the vector pCDH-NKG2D-sPD-1-CAR.

Figure 4 shows the results of the positive rate of CAR-T cells detected by flow cytometry after APC-hNKG2D antibody staining.

Figure 5 shows the results of RT-PCR identification of NKG2D-sPD-1-CAR-T cells, in which 1 is the result of uninfected T cells, 2 is the result of NKG2D-sPD-1-CAR-T cells, and 3 is the molecular weight standard .

Figure 6 shows the results of ELISA identification of NKG2D-sPD-1-CAR-T cells.

FIG. 7 is a graph showing the screening results of PD-L1 on the surface of tumor cells.

Figure 8 shows the results of killing tumor cells by NKG2D-CAR-T and NKG2D-sPD-1-CAR-T cells.

Figure 9 is a bar graph showing the results of tumor cell killing by NKG2D-CAR-T and NKG2D-sPD-1-CAR-T cells, wherein the three bar graphs in each group in the 1:3 group and the 1:1 group are from the left To the right represent T cells, NKG2D-CAR-T cells and NKG2D-sPD-1CAR-T cells in order.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Acquisition of human PD-1 gene extracellular segment sequence

The CDS region of the extracellular segment sequence of the PD-1 gene was found on the website https://www.ncbi.nlm.nih.gov/pubmed/, and the PD-1 protein was found on the website https://www.uniprot.org/ the extracellular segment region. Use SnapGene software to design and synthesize primers in the company (the primers contain the signal peptide sequence of CD8a), using human T cell cDNA as a template, amplify the extracellular segment of the PD-1 gene by PCR, and sequence the amplified product The signal peptide gene sequence containing CD8a shown in positions 1201-1263 in SEQ ID No.2 is obtained and connected to the PD-1 extracellular segment gene sequence shown in positions 1264-1713 in SEQ ID No.2, and its amino acid sequences are respectively As shown in SEQ ID No.3 and SEQ ID No.1.

Example 2. Construction of pCDH-NKG2D-sPD-1-CAR vector

The constructed vector PCDH-NKG2D-CAR (to connect the CAR element shown in the upper figure in Figure 1 between the EcoRI and BamHI restriction sites of the vector pCDH) was digested with the fast cutting enzymes BamHI and SalⅠ. The vector PCDH-NKG2D-CAR after double restriction digestion is connected with the PCR amplification product (containing sPD-1 sequence) of Example 1 and the T2A gene fragment (sequence shown in positions 1141-1200 of SEQ ID No. 2), The recombinant vector was obtained, and the recombinant vector was transformed into E. coli. After colony PCR and sequencing identification, it was determined that the pCDH-NKG2D-sPD-1-CAR vector (containing the CAR element shown in the lower figure in Figure 1) was obtained.

The above-mentioned recombinant vector pCDH-NKG2D-sPD-1-CAR is connected between the SalI and EcoRI restriction sites of the lentiviral expression vector pCDH. CAR gene),

In the CAR gene shown in SEQ ID No.2, the 1-63rd position is the CD8a signal peptide gene sequence, the 64th-471st position is the NKG2D receptor extracellular region gene sequence, and the 472nd-1140th position is the transmembrane region of the protein CD8a Gene, co-stimulatory signal molecule gene of protein 4-1B B and CD3ζ cytoplasmic signal transduction sequence gene, the 1141-1200 position is the T2A gene sequence, the 1201-1263 position is the CD8a signal peptide gene sequence, the 1264-1713 position is the Soluble PD-1 (extracellular region of PD-1) gene sequence.

The colony PCR is to amplify the sequences shown in positions 1201-1713 in SEQ ID No.2 after picking positive monoclonal colonies to check whether the plasmid transformed into E. coli has been successfully connected with sPD-1-CAR2, The size of the band obtained by agarose electrophoresis was as expected (Figure 2), about 500bp. The plasmids were extracted from the monoclonal colonies with bands and sequenced for identification, and finally the target recombinant vector pCDH-NKG2D-sPD-1-CAR was obtained (Figure 3). G2D-CAR2-T2A-SIPD1 in Figure 3 is the CAR element shown in the lower figure in Figure 1.

Example 3, virus packaging

1. Plasmid transfection

The recombinant vector (the vector pCDH-NKG2D-sPD-1-CAR or the vector PCDH-NKG2D-CAR in Example 2) was sent into the 293T cells as the destination vector by the three-plasmid transfection system, and the helper plasmid psPAX2: the helper plasmid pMD2. G: The mass ratio of target carrier=5:3:3 was added into Opti-MEM medium, 50ul PEI solution was added, and after repeated pipetting and beating 20 times, the mixture was allowed to stand at room temperature for 20 min to obtain a DNA/PEI mixture. Take 1ml of DNA/PEI mixture and slowly drop it into the 293T petri dish laid on the previous day, mix gently, incubate in a 37°C incubator, replace with fresh medium after 6-8 hours, and place it in a 37°C incubator to continue incubation.

2. Virus collection and concentration

48h after plasmid transfection, collect the supernatant, add 10ml of fresh Opti-MEM medium and continue to culture for 72h, collect the supernatant again, mix it with the supernatant collected at 48h, and place it in a 4°C refrigerator for later use; 4°C, Centrifuge at 4000g for 10min to remove cell debris; filter the obtained supernatant with a 0.45μm filter; transfer the filtered virus supernatant into an ultracentrifuge tube, centrifuge at 25,000 rpm for 2h, dilute with 1/100 of the supernatant volume of PBS, and repeat. After pipetting, it was transferred to a closed centrifuge tube at 4°C overnight to obtain a virus solution; the virus solution was divided into appropriate volumes, stored at -80°C, and 200 μl of the virus solution was taken for titer determination.

3. Determination of virus titer

Digest 293T cells, count after centrifugation, prepare cell suspension with serum-containing medium, adjust the cell density to 2×10 ⁵ /ml, add 0.5ml of cell suspension to each well of a 24-well culture plate; use complete medium Dilute the virus solution obtained in step 2 according to the following ratios: 1:3, 1:9, 1:27, respectively, add 100 μl of the virus solution stock solution and the virus solution diluted in different proportions to the 24-well plate that has been seeded with cells After 16 h, the infection supernatant was discarded, and 0.5 ml of fresh complete medium was added; after 48 h, the target gene expression of infected cells was detected by flow cytometry after staining with NKG2D antibody. To calculate the titer, titer=2×10 ⁵ ×infection efficiency×dilution factor.

Result: After collecting and concentrating in step 2, the titer of the virus solution (lentivirus NKG2D-sPD-1-CAR) obtained by transfecting the vector pCDH-NKG2D-sPD-1-CAR was T=1.89×10 ⁹ TU/mL, The titer of the virus solution (lentivirus NKG2D-CAR) obtained by transfecting the vector PCDH-NKG2D-CAR was T=1.87×10 ⁹ TU/mL.

Example 4. Preparation and detection of CAR-T cells

1. Preparation of CAR-T cells

The two lentiviral NKG2D-CAR and NKG2D-sPD-1-CAR prepared in Example 3 were added to the medium containing human T cells at the ratio of MOI (effect-target ratio) = 5:1 (the cell density at the time of infection). 1×10 ⁶ cells/mL), centrifuged to change the medium after 16 h, and added fresh complete medium to continue the culture to obtain two CAR-T cells, named NKG2D-CAR-T and NKG2D-sPD-1-CAR respectively. -T.

2. Flow cytometry

After two CAR-T cells were cultured for two days, the cells were collected, RNA was extracted, and the expression of sPD-1 was detected by RT-PCR. The positive rate of CAR-T cells was detected by flow cytometry after staining with APC-hNKG2D antibody. Results: As shown in Figure 4, the positive rates of the two CAR-T cells were 80.3% and 86.3%, respectively, and the results of the uninfected T cells (negative control UT-T) were 1.22%.

3. Identification of sPD-1 secretion

Count the NKG2D-sPD-1-CAR-T cells and virus-uninfected T cells obtained in step 1, and plate them in a 96-well plate at a density of 1×10 ⁶ cells/mL. After culturing for 24 h, the cells were taken to extract RNA, and the RNA was reversely transcribed into cDNA using a reverse transcription kit. The cDNA was used as a template for PCR amplification, and the mRNA expression levels of the sPD-1 gene in the three cells were compared. The results are shown in Figure 5, NKG2D-sPD-1-CAR-T cells highly expressed sPD-1 mRNA.

Two CAR-T cells, NKG2D-CAR-T and NKG2D-sPD-1-CAR-T, and uninfected T cells were inoculated into the human osteosarcoma cell line U2OS according to the effect-target ratio of 1:3, and no tumor was added. For the negative control containing only NKG2D-sPD-1-CAR-T, the cells were co-cultured in a 37°C incubator for 24 hours, and the supernatant culture medium was taken to test the expression level of sPD-1 with an ELISA kit. Results: As shown in Figure 6a, compared with NKG2D-CAR-T and uninfected T cells, NKG2D-sPD-1-CAR-T cells highly expressed sPD-1, and NKG2D-sPD-1-CA R-T group The expression of sPD-1 was 94.19 μg/mL; as shown in Figure 6b, the addition of the human osteosarcoma cell line U2OS could significantly increase the secretion of sPD-1.

Example 5. Selection of CAR-T target cells and research on killing function of two CAR-T cells

By referring to the literature, antibodies were used to screen tumor cells with high expression of NKG2D ligand and PD-L1. The PD-L1 expression was detected by staining tumor cells with PD-L1 antibody. After staining at room temperature for 15 minutes, flow cytometry was performed. The positive rate of surgical detection was determined. The results are shown in Figure 7. According to the expression of PD-L1, the CAR-T target cell line was finally determined to be the human osteosarcoma cell line U2OS.

The target cell human osteosarcoma cell line U2OS was labeled with the fluorescent dye CFSE, and the two CAR-T cells, NKG2D-CAR-T and NKG2D-sPD-1-CAR-T, and uninfected T cells were divided according to the effect-target ratio of 1:3. , 1:1 inoculated into the target cells U2OS, each group set up two replicates, each well was supplemented to 200ul; the culture plate after mixing the cells was placed in a 37°C incubator for 24h; after 24h, all cells in each well were collected. , the cells were transferred to a flow tube, stained with the apoptosis antibody Annexin V for 15 min at room temperature, and the apoptosis ratio of the target cells was detected by flow cytometry.

Results: As shown in Figure 8 and Figure 9, with the increase of the effector-target ratio, NKG2D-sPD-1-CAR-T had better effect on target cell U2OS than uninfected T cells and NKG2D-CAR-T. Killing effect.

When the effector-target ratio was 1:3, the apoptotic ratio of target cells treated with NKG2D-CAR-T was 22.3%, and that of target cells treated with NKG2D-sPD-1-CAR-T was 52.5%.

To sum up, it can be found by RT-PCR in this application that the mRNA of PD-1 in NKG2D-sPD-1-CAR-T cells is highly expressed compared to T cells, and the increased expression of sPD-1 can be directly detected by ELISA, that is, It can be considered that the plasmid of interest has been constructed. Using the constructed CAR-T cells to co-culture with osteosarcoma cells that highly express NKG2D ligand and PD-L1 ligand, it can be observed that NKG2D-sPD-1-CAR-T has a higher effect-target ratio after 24 hours of killing. The better killing effect was in line with the experimental expectations; compared with the killing effect of NKG2D-CAR-T, NKG2D-sPD-1-CAR-T was significantly improved. At the same time, it is worth noting that under the stimulation of tumor cells, NKG2D-sPD-CAR-T cells secreted sPD-1 significantly increased, which indicates that the cells need to be further stimulated in the tumor microenvironment to exert their effect, compared with systemic injection. PD-1 antibody has lower toxicity and side effects, and has the advantage of being safer and more efficient.

Contents not described in detail in this specification belong to the prior art known to those skilled in the art. The above descriptions are merely examples of the present application, and are not intended to limit the present application. Various modifications and variations of this application are possible for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the scope of the claims of this application.

SEQUENCE LISTING

<110> East China Normal University Shanghai Bangyao Biotechnology Co., Ltd.

<120> Chimeric antigen receptor CAR gene expressing soluble PD-1 and its application

<130> JH-CNP190581

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<170> PatentIn version 3.5

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Claims (10)

1. A chimeric antigen receptor CAR gene expressing soluble PD-1, characterized in that, the CAR gene comprises an antigen binding domain gene, a transmembrane domain gene, an intracellular domain gene and a soluble domain gene that are sequentially connected. PD-1 gene, the soluble PD-1 is the extracellular segment region of PD-1, Preferably, the soluble PD-1 comprises the amino acid sequence shown in SEQ ID No.1, More preferably, the sequence of the soluble PD-1 gene is shown in positions 1264-1713 in SEQ ID No.2. 2. The CAR gene according to claim 1, wherein a first signal peptide gene is connected between the intracellular domain gene and the soluble PD-1 gene, Preferably, the first signal peptide is the signal peptide of the following proteins: CD19, CD20, CD30, CD4, CD8a, CD28, CD137 or a combination thereof; More preferably, the first signal peptide is a signal peptide of CD8a, and its amino acid sequence is shown in SEQ ID No.3, and more preferably, its gene sequence is shown in positions 1201-1263 in SEQ ID No.2 . 3. The CAR gene according to claim 2, wherein a self-cleaving protein gene is connected between the intracellular domain gene and the first signal peptide gene, preferably, the self-cleaving The protein is selected from T2A, P2A, E2A, F2A or a combination thereof, more preferably, the self-cleaving protein is T2A. 4. The CAR gene according to any one of claims 1-3, wherein the antigen binding domain can specifically bind to NKG2D ligands, preferably, the antigen binding domain is an NKG2D receptor cell. The outer segment, more preferably, the front end of the antigen binding domain gene includes the second signal peptide gene; Preferably, the second signal peptide is the signal peptide of the following proteins: CD19, CD20, CD30, CD4, CD8a, CD28, CD137 or a combination thereof; more preferably, the second signal peptide is the signal peptide of CD8a, more preferably Preferably, its amino acid sequence is shown in SEQ ID No.3, and more preferably, its gene sequence is shown in positions 1-63 in SEQ ID No.2; The transmembrane domain includes the transmembrane region of the following proteins: CD3ε, CD4, CD8a, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3 or a combination thereof, preferably, CD8a; The intracellular domain includes a costimulatory signal molecule and a cytoplasmic signal transduction sequence; preferably, the costimulatory signal molecule is selected from the costimulatory signal molecules of the following proteins: OX40, CD28, CD30, CD40, CD70, CD134, 4 -1BB, PD1, Dap10, CDS, ICAM-1 or a combination thereof, more preferably, 4-1BB; preferably, the cytoplasmic signaling sequence comprises a cytoplasmic signaling sequence derived from CD3ζ. 5. The CAR gene according to any one of claims 1-4, wherein the sequence of the chimeric antigen receptor CAR gene expressing soluble PD-1 is shown in SEQ ID No.2. 6. A recombinant vector, characterized in that: the recombinant vector contains the CAR gene according to any one of claims 1-5, and the recombinant vector is preferably a lentiviral vector. 7. A recombinant cell or recombinant bacteria, characterized in that: the recombinant cell or the recombinant bacteria contain the CAR gene described in any one of claims 1-5, or the recombinant vector described in claim 6, Preferably, the recombinant cells are immune cells, and more preferably, the recombinant cells are CAR-T cells. 8. The application of the CAR gene according to any one of claims 1-5, the recombinant vector according to claim 6, or the recombinant cell or recombinant bacteria according to claim 7 in the preparation of a tumor treatment product. 9 . The application according to claim 8 , wherein the tumor treatment product is a tumor treatment product with improved killing effect of selectively killing tumor cells. 10 . The use according to claim 8 or 9, wherein the tumor is a solid tumor, preferably, osteosarcoma U2OS, osteosarcoma MG-3, renal carcinoma cell 786O, more preferably, osteosarcoma U2OS.