CHIMERIC ANTIGEN RECEPTOR T LYMPHOCYTE FOR TREATING TUMORS, PREPARATION METHOD THEREFOR, AND USE THEREOF

Abstract

Provided are a chimeric antigen receptor T lymphocyte for treating tumors, a preparation method therefor, and the use thereof. The chimeric antigen receptor successively comprises a single-chain antibody against a tumor cell surface antigen, a human hinge transmembrane region, a human intracellular signal domain, a self-cleaving peptide, and the full length of human CD27. The human intracellular signal domain comprises a human intracellular co-stimulatory signal domain and a human intracellular signaling domain. The method for preparing the chimeric antigen receptor T lymphocyte comprises the following steps: introducing the coding gene of the above-mentioned chimeric antigen receptor into a T cell and expressing the coding gene, thereby obtaining the CAR-T cell.

Claims

1. A chimeric antigen receptor, which successively comprising: a single chain antibody fragment (scFv) against tumor associated antigen, a transmembrane region, a intracellular signaling domain, a self-cleaving peptide and a full-length CD27, the above mentioned human intracellular signaling domain includes an intracellular costimulatory signal domain and a signal transduction domain.

2. The chimeric antigen receptor of claim 1, wherein the tumor associated antigen is Siglec-15, BCMA or LILRB4.

3. The chimeric antigen receptor of claim 1, wherein the human hinge transmembrane region is a human CD8 hinge transmembrane region.

4. The chimeric antigen receptor of claim 1, wherein the human intracellular costimulatory signal domain is the intracellular region of human 4-1BB.

5. The chimeric antigen receptor of claim 1, wherein the human intracellular signal transduction domain is the intracellular region of CD3ζ.

6. The chimeric antigen receptor of claim 1, wherein the self-cleaving peptide is a P2A peptide.

7. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises a leading peptide, the scFv against tumor associated antigen, the human hinge transmembrane region, the human intracellular costimulatory signal domain, the human intracellular signal transduction domain, the self-cleaving peptide and the full-length human CD27.

8. The chimeric antigen receptor of claim 7, wherein the leading peptide mentioned is the human CD8 leading peptide.

9. The chimeric antigen receptor of claim 7, wherein the chimeric antigen receptor comprises a leading peptide, a scFv against tumor associated antigen, the human CD8 hinge transmembrane region, the human 4-1BB intracellular region, the human CD3ζ intracellular region, the self-cleaving peptide, the signal peptide, the EGFRt peptide, the self-cleaving peptide, and the full-length human CD27.

10. The chimeric antigen receptor of claim 9, wherein the signal peptide is a CSF2Ra signal peptide.

11. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises sequence of SEQ ID No. 2 or SEQ ID No. 6 or SEQ ID No. 9, or a sequence with 95-99% identify to SEQ ID No. 2 or SEQ ID No. 6 or SEQ ID No. 9.

12. CAR-T cells comprising the chimeric antigen receptor of claim 1 or comprising the isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 1, or, an isolated nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

13. A method of treatment or adjuvant treatment of tumors, comprising administering to the subject an effective amount of the cell of claim 12.

14. The method of claim 13, wherein the tumors include solid tumors or hematological malignancies.

15. The method of claim 14, wherein the solid tumors include Siglec-15-positive solid tumors.

16. The method of claim 15, wherein the Siglec-15-positive solid tumors include glioma, melanoma, bladder cancer, liver cancer, lung cancer, kidney cancer, colorectal cancer, endometrial carcinoma and thyroid cancer.

17. The method of claim 14, wherein the hematological malignancies include BCMA-positive hematological malignancies or LILRB4-positive hematological malignancies.

18. The method of claim 17, wherein the BCMA-positive hematological malignancies include multiple myeloma.

19. The method of claim 17, wherein the LILRB4-positive hematological malignancies include acute monocytic leukemia.

Description

DRAWINGS AND ILLUSTRATIONS THERE OF

[0167] FIG. 1 is the schematic diagram of CAR structure. The upper panel shows the gene structure of S15-CAR, and the lower panel shows the gene structure of S15-CAR-CD27.

[0168] FIG. 2 shows the flow cytometry analysis of the positive rate of EGFR (CAR) on the CD4.sup.+ or CD8.sup.+ CAR-T cells at 3 days after retrovirus transfection of T cells.

[0169] FIG. 3 is the flow cytometry analysis of the positive rate of IFNγ in CD4.sup.+ or CD8.sup.+ CAR-T cells at 3 days after retrovirus transfection of T cells.

[0170] FIG. 4 is the flow cytometry analysis of the positive rate of CD107a in CAR-T cells of CD4.sup.+ or CD8.sup.+ CAR-T cells at 3 days after retrovirus transfection of T cells.

[0171] FIG. 5 shows the CFSE labeling analysis of the lysis rate of target cells after co-culture with CAR-T cells at various E:T ratios.

[0172] FIG. 6 shows the statistical results of tumor volume in mice.

[0173] FIG. 7 shows the statistical results of survival rate of mice.

[0174] FIG. 8 is the schematic diagram of the structure of BCMA-CAR-CD27. ScFv: single chain antibody fragment; Hinge: CD8 hinge region; TM: CD8 transmembrane region.

[0175] FIG. 9 is the flow cytometry analysis showing the rate of Protein L (CAR) positive CD4.sup.+ or CD8.sup.+ CAR-T cells at 3 days after retrovirus transfection of T cells.

[0176] FIG. 10 is the flow cytometry analysis showing the expression level of CD27 on BCMA-CAR-CD27 T, CTR T or BCMA-CAR T cells after retrovirus transfection. MFI: mean fluorescence intensity.

[0177] FIG. 11 shows the CFSE labeling analysis of the lysis rate of target cells after co-culture with CAR-T cells at various E:T ratios.

[0178] FIGS. 12A-12C shows the results of D-luciferin sodium salt imaging after the tail vein injection of CAR-T cells in a tumor xenograft model to observe the residual tumor cells in mice. FIG. 12A refers to the general experimental procedure; FIG. 12B is the statistics of the fluorescein intensity in each group of mice at different time points; FIG. 12C is the sodium salt imaging result of each group of mice.

[0179] FIG. 13 is the schematic diagram of the structure of LILRB4-CAR-CD27. ScFv: single chain antibody fragment; Hinge: CD8 hinge region; TM: CD8 transmembrane region.

[0180] FIG. 14 is the flow cytometry analysis showing the rate of Protein L (CAR) positive CD4.sup.+ or CD8.sup.+ CAR-T cells at 3 days after retrovirus transfection of T cells.

[0181] FIG. 15 shows the CFSE labeling analysis of the lysis rate of target cells after co-culture with CAR-T cells at various E:T ratios.

[0182] FIGS. 16A-16C shows the results of D-luciferin sodium salt imaging after the tail vein injection of CAR-T cells in a tumor xenograft model to observe the residual tumor cells in mice. FIG. 16A refers to the general experimental procedure; FIG. 16B is the statistics of the fluorescein intensity in each group of mice at different time points; FIG. 16C is the sodium salt imaging result of each group of mice.

EMBODIMENTS

[0183] Unless otherwise specified, the experimental methods presented in the following embodiments are all routine methods.

[0184] Unless otherwise specified, the materials, reagents and so on can all be obtained from commercial sources.

[0185] The detail information of the retroviral vector (MP71) in the following embodiments is documented in“Engels, B., et al., Retroviral vectors for high-level transgene expression in T lymphocytes. Hum Gene Ther, 2003. 14(12): p 0.1155-68.”. It can be obtained by public from Carbiogene Therapeutics Co., Ltd.

Embodiment 1. Preparation of CAR-T Cells Modified by S15-CAR-CD27 Gene

Stage I. Construction of Retroviral Vectors

[0186] 1. Optimization of the full-length cDNA sequence of wild-type human CD27 gene

[0187] The full-length cDNA sequence of the wild-type human CD27 gene is called nature CD27 (nCD27). To make nCD27 more suitable for expression in human cells, the nCD27 sequence was codon-optimized on the website http://sg.idtdna.com/site while remaining the amino acid sequence encoded by nCD27 unchanged, to obtain optimized CD27 (oCD27). The nucleotide sequence of oCD27 is shown at 2719-3501 nt. of SEQ ID NO. 1.

[0188] 2. Design and Synthesis of DNA Expressing CAR Gene

[0189] The gene sequence of S15-CAR-CD27 comprises the encoding gene sequence of the following: human CD8 leading peptide, S15 scFv, human CD8 hinge transmembrane region, the human 4-1BB intracellular region, the human CD3ζ intracellular region, the P2A peptide (recorded as P2A peptide-1), the CSF2Ra signal peptide, the EGFRt peptide, the P2A peptide (recorded as P2A peptide-2), and oCD27. The integrated gene sequence of S15-CAR-CD27 is shown in SEQ ID NO. 1, wherein the encoding gene sequence of the human CD8 leading peptide corresponds to 1-63 nt. of SEQ ID NO. 1, the encoding gene sequence of the S15 scFv corresponds to 64-807 nt. of SEQ ID NO. 1, the encoding gene sequence of the human CD8 hinge transmembrane region corresponds to 808-1014 nt. of SEQ ID NO. 1, the encoding gene sequence of the human 4-1BB intracellular region corresponds to 1015-1155 nt. of SEQ ID NO. 1, the encoding gene sequence of the human CD3ζ intracellular region corresponds to 1156-1491 nt. of SEQ ID NO. 1, the encoding gene sequence of the P2A peptide-1 corresponds to 1492-1569 nt. of SEQ ID NO. 1, the encoding gene sequence of the CSF2Ra signal peptide corresponds to 1570-1635 nt. of SEQ ID NO. 1, the encoding gene sequence of the EGFRt peptide corresponds to 1636-2640 nt. of SEQ ID NO. 1, the encoding gene sequence of the P2A peptide-2 corresponds to 2641-2718 nt. of SEQ ID NO. 1, and the encoding gene sequence of the oCD27 corresponds to 2719-3501 nt. of SEQ ID NO. 1. The amino acid sequence encoded by the S15-CAR-CD27 gene is shown in SEQ ID NO. 2.

[0190] The gene sequence of S15-CAR successively comprises: the encoding gene sequence of the following: human CD8 leading peptide, S15 scFv, the human CD8 hinge transmembrane region, the human 4-1BB intracellular region, the human CD3ζ intracellular region, the P2A peptide, the CSF2Ra signal peptide, and the EGFRt peptide. The gene sequence of S15-CAR is shown in SEQ ID NO. 3, wherein the encoding gene sequence of the human CD8 leading peptide corresponds to 1-63 nt. of SEQ ID NO. 3, the encoding gene sequence of the S15 scFv corresponds to 64-807 nt. of SEQ ID NO. 3, the encoding gene sequence of the human CD8 hinge transmembrane region corresponds to 808-1014 nt. of SEQ ID NO. 3, the encoding gene sequence of the human 4-1BB intracellular region corresponds to 1015-1155 nt. of SEQ ID NO. 3, the encoding gene sequence of the human CD3ζ intracellular region corresponds to 1156-1491 nt. of SEQ ID NO. 3, the encoding gene sequence of the P2A peptide corresponds to 1492-1569 nt. of SEQ ID NO. 3, the encoding gene sequence of the CSF2Ra signal peptide corresponds to 1590-1635 nt. of SEQ ID NO. 3, and the encoding gene sequence of the EGFRt peptide corresponds to 1636-2643 nt. of SEQ ID NO. 3.

[0191] The schematic diagrams of the structure of the main elements of the S15-CAR-CD27 gene and the S15-CAR gene are shown in FIG. 1.

[0192] The full-length DNA expressing S15-CAR-CD27 or S15-CAR can be synthesized by TSINGKE Biotechnology Co., Ltd. The synthesized gene sequence is cloned into pUC57 vetor before sequencing and identification.

[0193] 3. Construction of Retroviral Vector

[0194] The recombinant retroviral vector expressing S15-CAR-CD27 can be constructed by inserting the DNA sequence of the S15-CAR-CD27 shown in SEQ ID NO. 1 between the NotI and EcoRI site of the retroviral vector (MP71).

[0195] The recombinant retroviral vector expressing S15-CAR can be constructed by inserting the DNA sequence of the S15-CAR shown in SEQ ID NO. 3 between the NotI and EcoRI site of the retroviral vector (MP71).

[0196] The control retroviral vector can be made by inserting the gene sequence of the non-S15-targeting CAR shown in SEQ ID NO. 4 between the NotI and EcoRI site of the retroviral vector (MP71), and keep the other sequences of the retroviral vector (MP71) unchanged.

[0197] Stage II. Retrovirus Packaging and Establishment of the Stable Strain for Retrovirus Production

[0198] The recombinant retroviral vectors S15-CAR-CD27 and S15-CAR and the control retroviral vector prepared in Stage I were packaged separately to obtain two retroviruses and the control retrovirus according to the following methods:

[0199] Step 1. Culture of Packaging Cells

[0200] In each 10 cm cell culture dish, deposit 6×10.sup.6 Phoenix Ecotropic (ECO) cells (ATCC, CRL-3214) (less than 20 generations, but not overgrown) and 10 ml of DMEM medium, mix the cells thoroughly, and incubate overnight at 37° C.

[0201] Step 2. Transfection of Packaging Cells

[0202] Conduct transfection when the confluence of ECO cells reaches about 50-60%. Add 12.5 μg of target plasmid, 250 μl of 1.25M CaCl.sub.2), 1 ml ddH.sub.2O, with the total volume of 1.25 ml in one tube; add an equal volume of 2×HBS solution to another tube, add the plasmid complex to the 2×HBS solution, vortex and shake for 20 s while adding the plasmid complex to obtain a mixture. Gently add the mixture to the ECO cell culture dish along the side; incubate cells at 37° C. for 6 hours, and replace the medium with pre-warmed fresh complete medium.

[0203] Step 3. Collecting the Crude Retrovirus

[0204] At 48 hours after transfection, collect the supernatant of culture and filter through a 0.45 μm filter to obtain the virus stock, which can be stored in aliquots at −80° C.

[0205] The virus stock obtained from the recombinant retroviral vector S15-CAR-CD27 is referred to as S15-CAR-CD27 retrovirus. The virus stock obtained from the recombinant retroviral vector S15-CAR is referred to as the S15-CAR retrovirus. The virus stock obtained from the control retroviral vector was referred to as the control retrovirus.

[0206] Step 4. Establishment of the Stable Strain for Retrovirus Production

[0207] Transfect PG13 cells (ATCC, CRL-10686) with the virus stock obtained in step 3. At two days after the transfection, the CAR-positive cells were enriched by EGFR antibody (Biolegend) and MACS Anti-APC/PE Micro beads (Miltenyi, 130-090-855). The ratio of CAR-positive cells can be measured by flow cytometry. Dilute the enriched CAR-positive cells into single cells and seed them into a 96-well plate. Collect the supernatant on the 5th day after seeding as the crude retrovirus. The virus titer can be determined through flow cytometry by further transfecting HT1080 cells with these crude retrovirus. Select top three strains with highest virus titer in the 96-well plate and transfer them into a 24-well plate for further expansion and a secondary clonal selection. Collect the supernatant on the 5th day after seeding as the crude retrovirus to transfect HT1080 cells, determine virus titer by flow cytometry. The clone with the highest titer was selected as the stable strain for retrovirus production, and stored in liquid nitrogen. By using such cell strain, crude retrovirus can be prepared on a large scale for gene transduction to prepare CAR-T cells.

Stage III. Preparation of CAR-T Cells

[0208] 1. Thaw an aliquot of the frozen peripheral blood mononuclear cells (PBMC) from healthy donors and adjust the cell density to (1-2)×10.sup.6 cells/ml with RPMI-1640 complete medium containing 10% FBS.

[0209] 2. Use Ficoll-Paque reagent (GE health) and magnetic beads (Miltenyi) to enrich CD3.sup.+ T cells from PBMC, and use clinical-grade Dynabeads Human T Expander CD3/CD28 magnetic beads (Invitrogen) at a ratio of 3:1 for magnetic beads: CD3+ cell volume ratio to activate T cells. 3. On the second day after T cell activation, coat a 6-well plate with Retronectin solution (Takara) at a concentration of 15 μg/ml and seed CD3.sup.+ T cells into these coated plates. Add 1.2 ml Retronectin solution to each well of plate, keep the plate at 4° C. overnight and avoid light exposure for further use.

[0210] 4. At two days after T cell activation, aspirate the coating solution in the 6-well plate, and wash the plate with PBS once.

[0211] 5. Transfection: add the virus stock prepared in step 2 (the culture supernatant of the stable strain with the highest virus titer) into the wells (5-6 ml/well), centrifuge at 32° C., 2000×g for 2 h, discard the supernatant (unbound virus), and add 3 ml of fresh RPMI-1640 complete medium containing hIL-2 (Shanghai Huaxin Biotech Co., Ltd.) (500 U/ml) to each well, with the initial cell density at about 2×10.sup.6 cells/ml.

[0212] 6. After transfection, culture cells at 37° C. and add fresh RPMI-1640 complete medium containing hIL-2 (100 U/ml) to avoid overcrowded of T cells in the wells. The cell density should be maintained at 5×10.sup.5 cells/ml to facilitate cell expansion.

[0213] Collect T cells at 72 hours after transfection with the virus stock and obtain transfected CAR-T cells. The T cells transfected with the S15-CAR-CD27 retrovirus are referred to as S15 CAR-CD27 T cells. The T cells transfected with the S15-CAR retrovirus are referred to as S15 CAR T cells.

[0214] According to above procedures, NO CAR T cells or CTR CAR T cells can be obtained by replacing the virus stock with an equal volume of PBS solution or control retrovirus.

Stage IV. Determination of the Proportion of CAR-T Cells and the Expression Level of CAR Gene by Flow Cytometry

[0215] Since the CAR Gene Contains the EGFRt Fragment, the Expression Level of the CAR gene can be reflected by the expression level of EGFRt. With the S15 CAR-CD27 T cells, S15 CART cells, CTR CAR T cells or No CAR T cells obtained in step 3 as the test cells, the expression level of EGFRt can be determined by FACS method through EGFR antibodies. The procedures are described as follows: centrifuge cells in an EP tube and wash them with FACS buffer (2% (volume fraction) FBS-containing PBS solution), discard the supernatant, resuspend the pellets and add FITC-labeled EGFR antibody (Biolegend). Incubate cells at room temperature and keep away from light for 30 minutes. Wash with FACS buffer again, suspend to obtain resuspended cells. Detect the fluorescence intensity of FITC of resuspended cells by flow cytometry

[0216] Results: As shown in FIG. 2. At 3 days after using the retrovirus prepared in step 2 to transfect T cells, the positive rate of EGFR (CAR) in CD4.sup.+ T cells was between 50% and 80%, and the positive rate of EGFR (CAR) in CD8.sup.+ T cells was between 30%-70%.

Stage V. Determination of the Functional Indexes of CAR-T Cells by Flow Cytometry

[0217] 1. Determination of the Level of IFNγ Expression

[0218] IFNγ is an important indicator reflecting the function of T cells. The higher the expression of IFNγ, the higher the activity of T cells. The expression level of IFNγ in S15 CAR-CD27 T cells, S15 CAR T cells, or CTR CAR T cells obtained in step 3 was detected by intracellular cytokine staining method. Detail procedures are described as follows:

[0219] S15 CAR-CD27 T cells, S15 CAR T cells or CTR CAR T cells were co-cultured with human glioma cell U87-MG (ATCC) at a cell number ratio of 1:1, respectively (2×10.sup.5/well U87-MG), use Golgi Plug reagent (BD bioscience) to inhibit protein transport, and collect cell in 6 hours. The collected cells are performed surface staining first, and then intracellular staining and analyzed by flow cytometry.

[0220] Results: As shown in FIG. 3, compared with that in CTR CAR T cells, the expression levels of IFNγ in S15 CAR T cells and S15 CAR-CD27 T cells were both significantly increased after co-cultured with U87-MG, respectively. The increase of IFNγ was especially high for the CD8.sup.+ T cell subgroup (cytotoxic T cells), the positive rate of IFNγ exceeds 50%.

[0221] 2. Determination of the Level of CD107a Expression

[0222] Lysosome-associated membrane protein 1 (CD107a) is the main component of vesicle membrane protein. When activated T cells differentiate into cytotoxic T-lymphocytes (CTL cells), whose important feature is the high level of cytotoxic particles in the form of vesicles. In the process of CTL cells and NK cells killing target cells, the toxic particles will fuse with the cell membrane (the CD107a molecule will be transported to the cell membrane surface at this time), causing the particle content to be released, and ultimately leading to the death of the target cell. Therefore, the CD107a molecule is a sensitive marker of CTL degranulation, which is directly related to the cytotoxic activity. The expression level of CD107a is detected by flow cytometry to reflect the activity level of T cells. Detail procedures are described as follows:

[0223] S15 CAR-CD27 T cells, S15 CAR T cells or CTR CAR T cells were cocultured with human glioma cell U87-MG (ATCC) in DMEM medium at a cell number ratio of 1:1 (2×10.sup.5 U87-MG/per well). Add APC-labeled anti-CD107a antibody (Biolegend) to the co-culture system and incubate for 1 hour, and then add Golgi Stop reagent (BD), and incubate for another 3 hours before collecting the cells. The collected cells were subjected to surface staining and flow cytometry detection.

[0224] Results: As shown in FIG. 4, compared with that in CTR CAR T cells, the expression level of CD107a in S15 CART and S15 CAR-CD27 T cells had a significant increase after co-culture with target cells. The increase of CD107a was especially high for CD8.sup.+ T cell subgroup (cytotoxic T cells), the positive rate of CD107a reaches 80%-90%.

Stage VI. Determination of the Cytotoxic Effect of CAR-T Cells Genetically Modified by S15-CAR-CD27 on Tumor Cells by CFSE Labeling

[0225] CFSE (CFDA-SE) is a fluorescent staining reagent that can label and visualize living cells. It can easily penetrate cell membranes and covalently bind to intracellular proteins in living cells, which will release green fluorescence after hydrolysis. The CFSE labeling method can be used to label and quantify tumor cells, so as to reflect the cytotoxic effect of CAR-T cells on target tumor cells. Detail procedures are described as follows: equally divide the target cells into two groups, adjust them to the same cell density. Stain the cells with low-concentration or high-concentration of CFSE respectively, in which high-concentration-CFSE-stained target cells and unstained immune cells were co-cultured in a certain proportion. After incubation for a period of time, mix the equal amount of the high-concentration-CFSE-stained target cell tube (along with immune cells) and the low-concentration-CFSE-stained target cell tube stained at a low concentration. Finally, by comparing the percentage of target cells in the CFSE low-concentration labeling group and the CFSE high-concentration labeling group, the lysis rate target cells by CAR T cells can be calculated. Detailed procedures are described as follows:

[0226] 1. Trypsinize the U87-MG cells in the mid-log phase and neutralized with the complete medium. Pipet the cell suspension and transfer to a 15 ml centrifuge tube, wash cells twice with PBS.

[0227] 2. Centrifuge at 300-500 g for 1-5 min, and discard the supernatant. Use PBS to resuspend the cells and adjust the cell density to (1-2)×10.sup.7 cells/ml.

[0228] 3. Divide the above U87-MG cell suspension into two equal parts, one part is marked as CFSE high-labeled cells, and another part is marked as CFSE low-labeled cells. Incubate CFSE low-labeled cells with low-concentration CFSE (Invitrogen, 0.5 μM), and CFSE high-labeled cells with high-concentration CFSE (5 μM). Detailed procedures are described as following: add CFSE dye (Invitrogen) at a predetermined concentration into the tubes, and incubate at 37° C. for 10 min in the dark.

[0229] 4. Stop the staining by adding at least 2 times volume of cold complete medium, and centrifuge at 300-500 g for 5 min.

[0230] 5. Remove the supernatant, collect the cell pellet, and wash cells twice with complete medium.

[0231] 6. Deposite the above stained U87-MG cells into the 96 well plate, in CFSE high-labeled group (CFSE high-labeled cells+ T cells): in each well inoculate U87-MG cells at the density of 5×10.sup.4 cells/100 μl; then add different amounts of CAR-T cells (S15-CAR-CD27 T cells, S15-CAR T cells or CTR CART cells) in the so that the ratios of CAR-T cells to U87-MG cells are 1:1, 1:3, 1:9, 1:27 respectively; in the CFSE low-labeled group (cells just labeled by CFSE): inoculate U87-MG cells (5×104 cells/100 μl) in each well and culture individually, and supplement complete medium in the wells to the same volume with that in the CFSE high-labeled group. CFSE high-labeled cell wells that were not co-cultured with CAR-T cells are set as the control group.

[0232] 7. After 6 hours of culture at 37° C., mix cells in the CFSE high-labeled group and

[0233] CFSE low-labeled group at the ratio of 1:1, and record them as the experimental mix group. In the meantime, collect and mix cells in the control group and CFSE low-labeled group at the ratio of 1:1, and record them as the control mix group.

[0234] 8. Detect the fluorescence value of each group using the FITC channel of flow cytometry (FIG. 2).

[0235] 9. The lysis rate of target cells by T cells (%): two FITC positive peaks, which are CFSE high-labeled and low-labeled peaks respectively, should be detected by the flow cytometry. Measure the proportion of two peakes of the CFSE high-labeled group and the CFSE low-labeled group, respectively. Then, the lysis rate (%) of target cells by T cells should be calculated according to the following formula:

[0236] The lysis rate of target cells by T cells (%)=100%-[(percentage of CFSE high-labeled cells in the experimental mix group %/percentage of low-labeled CFSE cells in the experimental mix group %)/(percentage of CFSE high-labeled cells in the control mix group %)/(percentage of CFSE low-labeled cells in the control mix group %)]×100%.

[0237] For example, in mixed cells of the experimental group, the proportion of CFSE high-labeled cells was 42.5%, while the proportion of CFSE low-labeled cells was 57.5%; the proportion of CFSE high-labeled cells in mixed cells of the control group was 49.5%, and the proportion of CFSE low-labeled cells was 51.5%; therefore, the lysis rate of target cells by T cells (%)=100%−(42.5%/57.5%)/(49.5%/51.5%)×100%.

[0238] Results: as shown in FIG. 5 and Table 2, after co-culture with S15 CAR-CD27 T cells, the lysis rate of target cells U87-MG reached 80% at the E:T ratio of 1:1; the lysis rate remained on about 20% at the E:T ratio of 1:27.

TABLE-US-00002 TABLE 2 The lysis rate of target cells by CAR-T cells (%) S15 CAR-CD27 S15 CAR CTR CAR T Cells T Cells T Cells E:T 1:1 85.63801027 87.20338913 10.47173548 ratio 1:3 68.27473587 59.38989916 12.28141664 1:9 34.73851586 32.26043245 8.727444664 1:27 18.26043245 19.26043245 6.209480257

Stage VII. Determination of the Cytotoxic Effect of CAR-T Cells Genetically Modified by S15-CAR-CD27 Using a Tumor Xenograft Model In Vivo

[0239] Experimental materials: B-NDG severe combined immunodeficiency (SCID) mice weighing 18-22 g of 5-6 weeks old (Biocytogen Biotech Co., Ltd.).

[0240] Experimental groups: The experimental mice are randomly divided into 3 groups with 5 mice in each group.

[0241] S15-CAR-CD27 T: Inject U87-MG cells (PBS as solvent) intravenously into the B-NDG mice through the tail vein, the injected amount is 0.3 mL (contain 2×10.sup.6 tumor cells). Five days after inoculation, mice are injected with the S15-CAR-CD27 T cell (PBS as solvent) solution prepared in Embodiment 1 through the tail vein. The injected amount of S15 CAR-CD27 T cells is 0.2 ml (contain 5×10.sup.6 S15 CAR-CD27 T cells).

[0242] S15 CAR T: Inject U87-MG cells (PBS as solvent) intravenously into the B-NDG mice through the tail vein, the injected amount is 0.3 mL (contain 2×10.sup.6 tumor cells). Five days after inoculation, mice are injected with the S15 CAR T cell (PBS as solvent) solution prepared in Embodiment 1 through the tail vein. The injected amount of S15 CART cells is 0.2 ml (contain 5×10.sup.6 S15 CAR T cells).

[0243] CTR CAR T: Inject U87-MG cells (PBS as solvent) intravenously into the B-NDG mice through the tail vein, the injected amount is 0.3 mL (contain 2×10.sup.6 tumor cells). Five days after inoculation, mice are injected with the CTR CAR T cell (PBS as solvent) solution prepared in Embodiment 1 through the tail vein. The injected amount of CTR CART cells is 0.2 ml (contain 5×10.sup.6 CTR CAR T) Experimental method: Within 42 days after the CAR-T cell injection, measure the tumor diameter of each mouse every three days. Count and plot the tumor diameter at each time point. Within 90 days after the CAR-T cell injection, check the number of surviving mice and draw the survival curve.

[0244] Results: as shown in FIG. 6 and FIG. 7, compared with the S15 CAR-T control group, the U87-MG cell residues in the S15 CAR-CD27 T group are significantly reduced. The results show that S15 CAR-CD27 T cells are more effective in killing U87-MG tumor cells.

Embodiment 2. Preparation of CAR-T Cells Genetically Modified by BCMA-CAR-CD27

I. Construction of Retroviral Vectors

[0245] 1. Design and Synthesis BCMA-CAR-CD27 Gene

[0246] The gene sequence of BCMA-CAR-CD27 comprises the DNA encoding gene sequence of the following: the human CD8 leading peptide, BCMA scFv, the human CD8 hinge transmembrane region, the human 4-1BB intracellular region, the human CD3ζ intracellular region, the P2A peptide, and oCD27.

[0247] The integrated gene sequence of BCMA-CAR-CD27 is shown in SEQ ID NO. 5, wherein the encoding gene sequence of the human CD8 leading peptide corresponds to 1-63 nt. of SEQ ID NO. 5, the encoding gene sequence of BCMA scFv corresponds to 64-792 nt. of SEQ ID NO. 5, the encoding gene sequence of the human CD8 hinge transmembrane region corresponds to 793-999 nt. of SEQ ID NO. 5, the encoding gene sequence of the human 4-1BB intracellular region corresponds to 1000-1140 nt. of SEQ ID NO. 5, the encoding gene sequence of the human CD3ζ intracellular region corresponds to 1141-1476 nt. of SEQ ID NO. 5, the encoding gene sequence of the P2A peptide corresponds to 1477-1554 nt. of SEQ ID NO. 5, and the encoding gene sequence of oCD27 corresponds to 1555-2337 nt. of SEQ ID NO. 1. The amino acid sequence encoded by the BCMA-CAR-CD27 gene sequence is shown in SEQ ID NO. 6. The schematic diagram of the structure of the main elements of the BCMA-CAR-CD27 gene is shown in FIG. 8.

[0248] The gene sequence of BCMA-CAR shown in SEQ ID NO. 7 comprises the encoding gene sequence of the following: the human CD8 leading peptide, BCMA scFv, the human CD8 hinge transmembrane region, the human 4-1BB intracellular region, and the human CD3ζ intracellular region.

[0249] The full-length gene sequence expressing BCMA-CAR-CD27 or BCMA-CAR can be synthesized by TSINGKE Biotechnology Co., Ltd. The synthesized gene sequence is cloned into pUC57 vector for sequencing and identification.

[0250] 2. Construction of Retroviral Vector

[0251] The recombinant retroviral vector BCMA-CAR-CD27 can be obtained by inserting the gene sequence of the BCMA-CAR-CD27 shown in SEQ ID NO. 5 between the NotI and EcoRI restriction site of the retroviral vector (MP71), and keeping other part of the retroviral vector (MP71) unchanged.

[0252] The recombinant retroviral vector BCMA-CAR can be obtained by inserting the gene sequence of the BCMA-CAR between the NotI and EcoRI restriction site of the retroviral vector (MP71) and keeping other part of the retroviral vector (MP71) unchanged. The full-length gene sequence of BCMA-CAR corresponds to 1-1476 nt. of SEQ ID No: 5.

[0253] The control retroviral vector in step I of Embodiment 1 is used as a non-BCMA-targeting control retroviral vector.

II. Retrovirus Packaging and Establishment of the Stable Strain for Retrovirus Production

[0254] After the recombinant retroviral vectors or control vectors were constructed, recombinant retrovirus that express BCMA-CAR-CD27 or BCMA-CAR, or control retrovirus can be obtained respectively following procedures provided below:

[0255] Step 1. Culture of Packaging Cells

[0256] In each 10 cm cell culture dish, deposite 0.6×10.sup.6 Phoenix Ecotropic (ECO) cells (ATCC, CRL-3214) (less than 20 generations, but not overgrown) and at the density of 10 ml of DMEM medium, mix the cells thoroughly, and incubate overnight at 37° C.

[0257] Step 2. Transfection of Packaging Cells

[0258] Transfection can be performed when the confluence of ECO cells reaches about 90%; Add 12.5 μg of target plasmid, 250 μl of 1.25M CaCl.sub.2), 1 ml ddH.sub.2O, with the total volume of 1.25 ml in one tube; add an equal volume of 2×HBS solution to another tube, add the plasmid complex to the 2×HBS solution, vortex and shake for 20 s while adding the plasmid complex to obtain a mixture and gently add the above-mentioned mixture into the ECO cell culture dish along the side; incubate cells at 37° C. for 6 hours, and replace the medium with pre-warmed fresh complete medium.

[0259] Step 3. Collecting the Crude Retrovirus

[0260] At 48 hours after transfection, collect the supernatant of culture and filter through a 0.45 μm filter to obtain the virus stock, which can be stored in aliquots at −80° C. The virus stock obtained from the recombinant retroviral vector BCMA-CAR-CD27 is referred to as BCMA-CAR-CD27 retrovirus. The virus stock obtained from the recombinant retroviral vector BCMA-CAR is referred to as the BCMA-CAR retrovirus.

[0261] Step 4. Establishment of the Stable Strain for Retrovirus Production

[0262] Transfect PG13 cells (ATCC, CRL-10686) with the virus stock obtained in step 3. At two days after the transfection, the CAR-positive cells were enriched by EGFR antibody (Biolegend) and MACS Anti-APC/PE Micro beads (Miltenyi, 130-090-855). The ratio of CAR-positive cells can be measured by flow cytometry. Dilute the enriched CAR-positive cells into single cells and seed them into a 96-well plate. Collect the supernatant on the 5th day after seeding as the crude retrovirus. The virus titer can be determined through flow cytometry by further transfecting HT1080 cells with these crude retrovirus. Select top three strains with highest virus titer in the 96-well plate and transfer them into a 24-well plate for further expansion and a secondary clonal selection. Collect the supernatant on the 5th day after seeding as the crude retrovirus to transfecting HT1080 cells, determine virus titer by flow cytometry. The clone with the highest titer was selected as the stable strain for retrovirus production and stored in liquid nitrogen. By using the cell strain, crude retrovirus can be prepared on a large scale for gene transduction to prepare CAR-T cells.

III. Preparation of CAR-T Cells

[0263] 1. Thaw an aliquot of the frozen peripheral blood mononuclear cells (PBMC) from healthy donors and adjust the cell density to (1-2)×10.sup.6 cells/ml with RPMI-1640 complete medium containing 10% FBS.

[0264] 2. Use Ficoll-Paque reagent (GE health) and magnetic beads (Miltenyi) to enriched CD3.sup.+ T cells from PBMC, and use clinical-grade Dynabeads Human T Expander CD3/CD28 magnetic beads (Invitrogen) at a ratio of 3:1 for magnetic beads: CD3+ cell volume ratio to activate T cells.

[0265] 3. On the second day after T cell activation, coat a 6-well plate with Retronectin solution (Takara) at a concentration of 15 μg/ml and seed CD3.sup.+ T cells into these coated plates. Add 1.2 ml Retronectin solution to each well of plate, keep the plate at 4° C. overnight and avoid light exposure for further use.

[0266] 4. At two days after T cell activation, aspirate the coating solution in the 6-well plate, and wash the plate with PBS once.

[0267] 5. Transfection: add the virus stock prepared in step II (the culture supernatant of the stable strain with the highest virus titer) into the wells (5-6 ml/well), centrifuge at 32° C., 2000×g for 2 h, discard the supernatant (unbound virus), and add 3 ml of fresh RPMI-1640 complete medium containing hIL-2 (Shanghai Huaxin Biotech Co., Ltd.) (500 U/ml) to each well, with the initial cell density at about 2×10.sup.6 cells/ml, continue to culture for one day.

[0268] 6. After transfection, culture cells at 37° C. and add fresh RPMI-1640 complete medium containing hIL-2 (100 U/ml) to avoid overcrowded of T cells in the wells. The cell density should be maintained at 5×10.sup.5 cells/ml to facilitate cell expansion.

[0269] Collect T cells at 72 hours after transfection with the virus stock, obtaining the CAR-T cells transfected with retrovirus The T cells transfected with the BCMA-CAR-CD27 virus stock are referred to as S15 CAR-CD27 T cells. The T cells transfected with the BCMA-CAR virus stock are referred to as S15 CAR T cells.

[0270] According to above procedures, CTR T cells or Exb T cells can be obtained by replacing the virus stock with an equal volume of PBS solution or control retrovirus.

IV. Determination of the Proportion of CAR-T Cells and the Expression Level of CAR Gene by Flow Cytometry

[0271] 1. The Proportion of CAR-Positive T Cells and the CAR Gene Expression

[0272] Since the light chain of the anti-BCMA scFv is a κ subtype that can bind to Protein L, the expression level of the CAR gene can be reflected by the expression level of biotin-labeled Protein L(PL) bound to CAR-T cells. The procedures of detecting CAR gene expression are described as follows:

[0273] Centrifuge and Collect two kind of CAR-T cells and CAR-T cells (control) prepared in the Stage III at 72 h post transfection, and wash them with 1% BSA-containing PBS solution once, discard the supernatant, resuspend the pellets and add Biotin-labeled protein L antibody (Biolegend). Incubate cells at room temperature and keep away from light for 30 minutes. Wash again with 1% PBS solution with 1% BSA and resuspend. Then add PE-labeled avidin (Streptavidin) (Sigma). Incubate cells at room temperature and keep away from light for 10 minutes, Wash again with 1% PBS solution with 1% BSA and resuspend, detect the fluorescence intensity of PE by flow cytometry.

[0274] Results: as shown in FIG. 9, at 3 days after transfection by the retrovirus prepared in the step III the positive rate of PL (CAR) in both CD4+ T cells and CD8+ T cells reach 80%.

[0275] 2. Determination of CD27 Expression Level

[0276] The expression level of CD27 was determined by flow cytometry. The procedures are described as follows: centrifuge and collect the CAR-T cells and CTR T cells prepared in step III after 72 hours of transfection, respectively.

[0277] Results: as shown in FIG. 10, the expression level of CD27 in BCMA CAR-CD27 T cells was significantly higher than that in CTR T and BCMA CAR T cells. The results indicate that CD27 can be highly express on the surface of CAR-T cells.

V. Detection of the Cytotoxic Effect of T Cells Genetically Modified by BCMA-CAR-CD27 on Tumor Cells by CFSE Labeling

[0278] The procedure of CFSE labeling was similar to the methods described in Embodiment 2, effector cells are the BCMA-CAR-CD27 T cells, BCMA-CAR T cells, or CTR T cells prepared in embodiment 4, the target cells are human multiple myeloma cells RPMI8226, and keep other step unchanged to determine the cytotoxic effect of T cells genetically modified by BCMA-CAR-CD27 on tumor cells.

[0279] The results are shown in FIG. 11 and Table 3: after co-culture with BCMA CAR-CD27 T cells, the cell lysis rate of RMPI-8226 reached over 80% at E:T ratio of 3:1; when the E:T ratio was 1:3, the cell lysis rate of RMPI-8226 remained on about 20%.

TABLE-US-00003 TABLE 3 Cell Lysis Rate of target cells by CAR-T cells (%) BCMA-CAR-CD27 BCMA-CAR CTR T Cells T Cells T Cells E:T 3:1 78.16955684 80.00963391 20.83044316 ratio 1:3 50.47976879 57.70712909 15.1734104 1:3 18.76685934 24.08477842 7.803468208

VI. Determination of the Cytotoxic Effect of T Cells Genetically Modified by BCMA-CAR-CD27 Using a Tumor Xenograft Model In Vivo

[0280] Experimental materials: B-NDG SCID mice weighing 18-22 g of 5-6 weeks old (Biocytogen Biotech Co., Ltd.).

[0281] Experimental groups: The experimental materials are randomly divided into 4 groups with 6 mice in each group.

[0282] Experimental procedure: Inject Daudi-Luc cells (Shanghai Meixuan Biotechnology Co., Ltd., MXC193) (PBS as solvent) intravenously into the B-NDG mice through the tail vein, by inoculation amount 0.3 ml (contain 2×10.sup.6 tumor cells). Five days after inoculation, mice are injected with the BCMA-CAR-CD27 T cell solution prepared in step III of this embodiment (PBS as solvent) through the tail vein. The inoculation amount of BCMA CAR-CD27 T cells is 0.2 ml (contain 5×10.sup.6 CAR′ T cells).

[0283] BCMA CAR T: Inject Daudi-Luc cells (PBS as solvent) intravenously into the B-NDG mice through the tail vein, the injected amount is 0.3 mL (contain 2×10.sup.6 tumor cells). Five days after inoculation, mice are injected with the BCMA CAR T cell (PBS as solvent) solution prepared in Embodiment 2 through the tail vein. The injected amount of BCMA CAR T cells is 0.2 ml (contain 5×10.sup.6BCMA CAR T cells).

[0284] Exb T: Inject Daudi-Luc cells (PBS as solvent) intravenously into the B-NDG mice through the tail vein, the injected amount is 0.3 mL (contain 2×10.sup.6 tumor cells). Five days after inoculation, mice are injected with the Exb T cell (PBS as solvent) solution prepared in Embodiment 2 through the tail vein. The injected amount of Exb T cells is 0.2 ml (contain 5×10.sup.6 Exb T cells).

[0285] Experimental method: At 7 days, 14 days and 21 days after inoculation, intraperitoneally inject 3 mg of D-luciferin into each mice for sodium salt-base imaging and measure the number of residual tumor cells and the fluorescein intensity (photon density).

[0286] Results: as shown in FIGS. 12A-12C, compared that in mice of BCMA CAR T group, the number of residual human lymphoma cells in mice of BCMA CAR-CD27 T group are significantly reduced. The results indicated that BCMA CAR-CD27 T cells are more effective in killing tumors.

Embodiment 3 Preparation of CAR-T Cells Modified by LILRB4-CAR-CD27 Gene

I. Construction of Retroviral Vectors

[0287] The DNA sequence of LILRB4-CAR-CD27 comprises the DNA sequence of the following: human CD8 leading peptide, LILRB4 scFv, human CD8 hinge transmembrane region, the human 4-1BB intracellular region, the human CD3ζ intracellular region, the P2A peptide, and oCD27. The integrated DNA sequence of LILRB4-CAR-CD27 is shown in SEQ ID NO. 8, wherein the human CD8 leading peptide corresponds to 1-63 nt. of SEQ ID NO. 8, LILRB4 scFv corresponds to 64-807 nt. of SEQ ID NO. 8, the human CD8 hinge transmembrane region corresponds to 808-1014 nt. of SEQ ID NO. 8, the human 4-1BB intracellular region corresponds to 1015-1155 nt. of SEQ ID NO. 8, the human CD3ζ intracellular region corresponds to 1156-1491 nt. of SEQ ID NO. 8, the P2A peptide corresponds to 1492-1569 nt. of SEQ ID NO. 8, and oCD27 corresponds to 1570-2349 nt. of SEQ ID NO. 8. The amino acid sequence encoded by the LILRB4-CAR-CD27 gene is shown in SEQ ID NO. 9.

[0288] The DNA sequence of LILRB4-CAR comprises the DNA sequence of the following: human CD8 leading peptide, S15 scFv, the human CD8 hinge transmembrane region, the human 4-1BB intracellular region, the human CD3ζ intracellular region. The DNA sequence of LILRB4-CAR corresponds to 1-1494 nt. of SEQ ID NO. 10.

[0289] The schematic diagrams of the structure of the main elements of the LILRB4-CAR-CD27 gene and the LILRB4-CAR gene are shown in FIG. 13.

[0290] The full-length DNA expressing LILRB4-CAR-CD27 or LILRB4-CAR can be synthesized by TSINGKE Biotechnology Co., Ltd. The synthesized DNA is cloned into pUC57 vector before sequencing and identification.

[0291] The recombinant retroviral vector expressing LILRB4-CAR-CD27 or LILRB4-CAR can be constructed by inserting the DNA sequence of the CAR into the retroviral vector (MP71).

II. Retrovirus Packaging and Establishment of the Stable Strain for Retrovirus Production

[0292] After the recombinant retroviral vectors or control vectors were obtained, recombinant retrovirus expressing LILRB4-CAR-CD27 or LILRB4-CAR, or control retrovirus can be collected respectively by the procedures provided in step II of Embodiment 1.

III. Preparation of CAR-T Cells

[0293] After the recombinant retrovirus or control retrovirus were obtained, T cells expressing LILRB4-CAR-CD27, or LILRB4-CAR, or CTR CAR were prepared by following the procedures provided in step III of Embodiment I.

[0294] NO CAR T cells can be obtained by replacing the virus stock thereof with an equal volume of PBS solution, and by following the above procedures.

IV. Determination of the Expression Level of CAR Gene on Transfected T Cells by Flow Cytometry

[0295] The detection of biotin-labeled protein L (PL) is used to reflect the expression of CAR gene. The procedures of detecting PL by flow cytometry are described in step IV of Embodiment 2.

[0296] Results: as shown in FIG. 14, at 3 days after transfection, the positive rates of PL (CAR) in CD3+ T cells are between 40-50%.

V. Detection of the Cytotoxic Effect of LILRB4-CAR-CD27 T Cells on Tumor Cells by CFSE Labeling

[0297] The procedure of CFSE labeling was similar as described in the step VI of Embodiment 1, except for difference of the target cells and effector CAR-T cells. In this embodiment, effector cells are the LILRB4-CAR-CD27 T cells, LILRB4-CAR T cells, or NO CAR T cells prepared in the step III of these embodiment. The target cells are human AML cells THP-1.

[0298] The results are shown in FIG. 15 and Table 4: after co-culture with LILRB4-CAR-CD27 T cells or LILRB4-CAR T cells, the cell lysis rate of THP-1 reached nearly 90% at E:T ratio of 1:1; when the E:T ratio was 1:27, the cell lysis rate of THP-1 remained about 30%.

TABLE-US-00004 TABLE 4 Cell Lysis Rate of target cells by CAR-T cells (%) LILRB4-CAR-CD27 T LILRB4-CAR T NO CAR T E:T 1:1 89.16201 94.16537 18.86584 ratio 1:3 64.94319 74.93448 9.30364 1:9 37.89472 48.16738 6.82762 1:27 31.52405 28.37413 5.02689

VI. Determination of the Cytotoxic Effect of LILRB4-CAR-CD27 T Cells Using a Tumor Xenograft Model In Vivo

[0299] Experimental materials: B-NDG SCID mice weighing 18-22 g of 5-6 weeks old (Biocytogen Biotech Co., Ltd.); human AML cell line transfected with Luciferase MV-4-11-Luc (Labcorp).

[0300] Experimental groups: The experimental materials are randomly divided into 3 groups with 5 mice in each group.

[0301] Experimental procedure: Inject MV-4-11-Luc cells (PBS as solvent) intravenously into the B-NDG mice through the tail vein, at the concentration of 2×10.sup.6 cells/0.3 ml. Five days after inoculation, mice are injected with 5×10.sup.6/0.2 ml LILRB4-CAR-CD27 T or LILRB4 CAR T or NO CAR T cell solution prepared in step III of this embodiment (PBS as solvent) through the tail vein. The concentration of T cells for inoculation is 5×10.sup.6 CAR′ T cells/0.2 ml.

[0302] At 7 days, 14 days and 21 days after inoculation, intraperitoneally inject 3 mg of D-luciferin sodium salt into each mice and measure the number of residual tumor cells by quantifying the fluorescein intensity (photon density).

[0303] Results: as shown in FIGS. 16A-16C, compared that in mice of LILRB4-CAR T group, the number of residual human AML cells in mice of LILRB4-CAR-CD27 T group are significantly reduced. The results indicated that LILRB4-CAR-CD27 T cells are more effective in killing tumors.

INDUSTRIAL APPLICATIONS

[0304] This disclosure provided a new CAR design that use CD27 as the costimulatory signal for the first time, which make the CD27 signaling pathway can be activated independent of the CAR signaling in an uncoupled manner. Based on our current knowledge, the intracellular domain, but not the full-length of CD28, 4-1BB or OX40, are usually used as the costimulatory signaling domain of the CAR gene.

[0305] The main reason for using the intracellular domain instead of the full-length of the costimulatory receptor is that the length of CAR gene is limited for viral packaging; on the other hand, the simultaneous activation of the costimulatory signal and the ITAM signal through a tandam expression pattern was shown to be beneficial for full activation of T cells.

[0306] However, subsequent studies have shown that constitutive activation of one costimulatory signal, such as CD28, is more likely to cause T cell exhaustion, which will be devastating for CAR-T cell therapy. As a matter of fact, the type and the time point of co-stimulatory signal received by T cells determine whether T cells can be fully activated.

[0307] For example, CD27 and CD28 are both highly expressed in primitive T cells, while their expression are downregulated in terminally differentiated T cells, indicating that both of these signals play important functions in the early stages of T cell response. On the contrary, other co-stimulatory receptors, such as CD137 and CD134, are highly expressed in activated T cells, and they play an important role in resisting T cell exhaustion caused by apoptosis. Activating any one of these costimulatory signals constitutively cannot replace the functions of the others.

[0308] Based on the above hypothesis, some studies tandemly connect two or more intracellular signal domains of costimulatory receptors, which is the design principle of the third generation of CAR. However, recent studies show that the anti-tumor effect of the third-generation CAR-T cells is not good enough compared with that of the second-generation CAR-T cells, possibly because the tandem expression of multiple costimulatory signal domains may conflict with each other, thus inhibit effective transmission of the signals.

[0309] Based on much preliminary studies, the inventors of this invention found that by activating the CD27 signal of CAR-T cells, the differentiation of T cells into effector T cells and memory T cells after antigen stimulation can be significantly promoted, which is vital for the long-term survival and secondary response of CAR-T cells in vivo. Thus the inventors uncoupled the two costimulatory signals, so that they can be activated separately in the presence of antigens.

[0310] More specifically, a gene-optimized human full-length CD27 gene fragment (oCD27) is added at the C-terminal of the CAR, and separate it with P2A peptide; oCD27 is designed to co-express with the CAR gene comprising a 4-1BB costimulatory domain; these two products will be cleaved by the P2A self-cleaving peptide released from the ribosome; the released CD27 is transported to the cell surface, where it can be activated by CD70 and other ligands. The main feature of this design is to achieve uncoupled activation of the two costimulatory signal pathways of 4-1BB and CD27.

[0311] Apart from anti-BCMA and anti-Siglec-15 scFv, other anti-TAA scFv can also be applied in preparing the CAR-T cells provided in this disclosure. These CAR-T cells therefore target the corresponding TAAs and can be applied in treating different types of solid carcinomas or hematological malignancies.