Anti-cd38 car molecules targeting and inhibiting cd38 and drugs thereof

Abstract

The present application provides an anti-CD38 CAR molecule for shRNA targeted inhibition of CD38, including an anti-CD38 CAR molecule for shRNA targeted inhibition of CD38, characterized by comprising U6 promoter, shRNA targeted inhibition of CD38, EF1 alpha promoter, upstream signal peptide and myc tag for detection in series; CD38 CAR antigen binding region CD38 scfv; CD8 hinge-transmembrane domain; CD28 costimulatory domain and CD3 zeta intracellular signaling domain, CD38 ScFv, CD8 hinge region and transmembrane region, CD28 intracellular region and CD3 zeta intracellular region are connected in series, the shRNA sequence targeted to inhibit CD38 is SEQ ID NO. 1: CCGGCTGAGGATTCATCTTGCACATCTCGAGATGTGCAAGATGAATCCTCAGTTTTTG. The present application successfully constructs an anti-CD38 CAR-T cell for shRNA targeted inhibition of CD38 through the popular shRNA technology of gene research, provides a new method for anti-CD38 CAR-T cell to get out of the "CAR-T cell-autophagy and self-stimulation" cycle, and helps to further enhance the expansion, persistence and function of anti-CD38 CAR-T cell in subsequent research.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and in particular to anti-CD38 CAR molecules and drugs thereof that target and inhibit CD38 using shRNA. Background Art

[0002] The mortality rate of various types of blood cancers, including B-cell non-Hodgkin's lymphoma, multiple myeloma, and relapsed acute lymphoblastic leukemia, has continued to rise in recent years. Despite the development of numerous treatments, including chemotherapy, proteasome inhibitors, immunomodulators, depleting antibodies, and autologous stem cell transplantation, these diseases remain difficult to cure, and most patients ultimately relapse. Chimeric antigen receptor T (CAR-T) cell therapy involves engineering T cells to express a fusion protein consisting of an antigen recognition domain, a costimulatory domain, and a T cell activation domain. These engineered CAR-T cells can specifically recognize and eliminate tumor cells expressing the targeted antigen. This immunotherapy is hailed as a promising future for cancer treatment. With the success of anti-CD19 CAR-T therapy in treating hematologic malignancies caused by B cell abnormalities, research into the treatment of other hematologic malignancies has also begun. The human CD38 antigen is a type II transmembrane glycoprotein widely expressed on various hematologic malignancy cells, including multiple myeloma (MM), B-cell non-Hodgkin's lymphoma, and relapsed acute lymphoblastic leukemia (ALL). Therefore, it serves as a therapeutic target for hematologic malignancies. However, it is also present on the surface of T cells, so these cells may commit suicide during CAR-T cell transduction and culture, thereby affecting the anti-tumor efficacy of anti-CD38 CAR-T cells. Multiple studies have demonstrated that anti-CD38 CAR-T cells exhibit significant cytotoxicity against various hematologic malignancies, but this "suicide" effect, to some extent, compromises the anti-tumor function of CD38-targeted CAR-T cells. Studies have shown that pre-treatment of CD38 CAR-T cells with antibody blocking enhances CAR-T cell proliferation and decreases expression of the depletion factor PD-1.

[0003] Short hairpin RNA (shRNA) belongs to the category of RNA interference (RNAi). After transduction or transfection of cells, precursor shRNAs are synthesized in the nucleus. The sense and antisense strands form a stem region through base pairing, with the unpaired nucleotides in the middle forming a loop, forming a hairpin structure. After processing by the RNase III enzymes Drosha and DGCR8, shRNAs are transported to the cytoplasm by the protein Exportin-5. Subsequently, shRNAs are cleaved by the RNase III enzymes Dicer and TRBP / PACT, removing the loop sequence and forming the siRNA. After recognition and integration by RISC, the siRNA unwinds and removes one strand from the duplex. The siRNA then recognizes and occupies the target mRNA through its complementary base sequence, leading to its degradation. shRNAs can be stably expressed through transduction and specifically inhibit the expression of target mRNAs, finding widespread applications in therapy, diagnosis, and scientific research. Currently, shRNAs specifically targeting immune checkpoint receptors are co-expressed with CAR-T cells via lentiviral vector transduction to inhibit immune checkpoint receptor expression, suppress the tumor microenvironment, and thereby modulate the immune response. ShRNAs are relatively short, making them easy to design and exhibiting strong inhibitory efficacy and long-lasting efficacy. They are also easily cloned and packaged into expression vectors, making them widely used in the field of gene editing. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides an anti-CD38 CAR molecule that targets and inhibits CD38 with shRNA. The anti-CD38 CAR molecule that targets and inhibits CD38 with shRNA is characterized in that it includes a U6 promoter, a shRNA that targets and inhibits CD38, an EF1α promoter, an upstream signal peptide and a myc tag for detection connected in series; a CD38 CAR antigen binding region CD38scfv; a CD8 hinge-transmembrane domain; a CD28 co-activation domain and a CD3ζ intracellular signaling domain, and the CD38ScFv, the CD8 hinge region and the transmembrane region, the CD28 intracellular region and the CD3ζ intracellular region are connected in series in series. The sequence of the shRNA that targets and inhibits CD38 is SEQ ID NO.1: CCGGCTGAGGATTCATCTTGCACATCTCGAGATGTGCAAGATGAATCCTCAGTTTTTG.

[0005] In one embodiment, the amino acid sequence of the CD38 CAR antigen binding region CD38scfv is SEQ ID NO. 2: SQVQLVQSGGGLVQPGRSLRLPCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGSGSYYNPFYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSQAVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVAWYQQLPGTAPKLLIYNYSQRPSGVPDRFSGSKSGTSSSLAIGGLQSEDEADYYCAAWDDSLNGVVFGGGTKLTVLG.

[0006] In one embodiment, the U6 promoter sequence is SEQ ID NO. 3: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC.

[0007]

[0008] In one embodiment, the upstream signal peptide sequence is SEQ ID NO. 5: EWSWVFLFFLSVTTGVHSDI.

[0009] In one embodiment, the Myc amino acid sequence is SEQ ID NO. 6: EQKLISEEDL.

[0010] In one embodiment, the CD8 amino acid sequence is SEQ ID NO. 7: AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA.

[0011] In one embodiment, the CD28 amino acid sequence is SEQ ID NO. 8: PRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

[0012] In one embodiment, the CD3ζ amino acid sequence is SEQ ID NO. 9: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0013] In one embodiment, the present application provides a medicament for treating tumors, which contains the anti-CD38 CAR molecule of shRNA targeting inhibition of CD38 described above.

[0014] Based on the constructed anti-CD38 CAR-T cells, the present invention constructed an anti-CD38 CAR molecule that targets and inhibits CD38 by RNA interference technology. It was packaged by retroviral vector and successfully transduced into human primary T cells to successfully construct shRNA CD38-anti CD38 CAR-T cells. The experiment found that the expression level of CD38 mRNA in the shRNA2-CD38 CAR-T group cells was significantly lower than that in the CD38 CAR-T group cells, while the difference in CD38 mRNA expression levels between the shRNA1-CD38 CAR-T cell group and the CD38 CAR-T group was not statistically significant. The results showed that the shRNA2 sequence had stronger targeting, RNA interference was successful, and the expression of CD38 in the shRNA2-CD38 CAR-T group cells was inhibited. At the same time, under in vitro culture conditions, the proliferation of the shRNA2-CD38CAR-T group cells was significantly better than that of the CD38 CAR-T group, and the proliferation was accelerated under the stimulation of co-culture with CD38-positive target cells. To validate the ability of shRNA CD38-anti-CD38 CAR-T cells to kill CD38-positive tumor cells, this study used two CD38-positive cell models: Raji-luc and RPMI-8226-luc cells. The results showed that shRNA2-CD38 CAR-T cells were more effective than the CD38 CAR-T group in killing both tumor cell types, a finding also confirmed by IFN-γ assays. Combined with the decreased PD-1 expression in the shRNA2-CD38 CAR-T group compared to the CD38 CAR-T group, this suggests that inhibiting CD38 expression on CAR-T cells can enhance their proliferation and tumor cell killing ability, possibly by reducing T cell suicide and slowing exhaustion.

[0015] CD38 has been a popular target in CAR-T research in recent years, and its anti-tumor ability has been verified. Current research focuses on methods to reduce T cell "suicide." This study successfully constructed an anti-CD38 CAR-T cell that inhibits CD38 with shRNA, a popular technique in genetic research. This provides a new approach for anti-CD38 CAR-T cells to break out of the "CAR-T cell-autophagy and self-stimulation" cycle, and will help further enhance the expansion, persistence, and function of anti-CD38 CAR-T cells in subsequent studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a molecular structure diagram of the anti-CD38 CAR that targets and inhibits CD38 with shRNA of the present invention;

[0018] Figure 2 Figures 2A and 2B show the results of the shRNA-CD38 CAR-T cell validation experiment constructed by the present invention, wherein 2A shows the titer of the retroviral vector, 2B shows the transduction efficiency of the CAR-T cells, and 2C shows the relative expression level of CD38 mRNA in the CAR-T cells;

[0019] Figure 3 This is the result graph of CD38 expression level on the surface of Raji-luc or RPMI-8226-luc target cells;

[0020] Figure 4 4A is a graph showing the in vitro proliferation of CAR-T cells of the present invention; 4B is a graph showing the proliferation of CAR-T cells detected by the CFSE method; a: CAR-T cell group stained with CFSE, b: CAR-T cell culture group alone, c: CAR-T and Raji-luc cell co-culture group, d: CAR-T and RPMI-8226-luc cell co-culture group;

[0021] Figure 5 5A is a graph showing the efficiency of CAR-T cells killing target cells and the amount of IFN-γ released by the present invention, wherein 5B is the killing efficiency of CAR-T cells against Raji-luc; 5C is a graph showing the level of IFN-γ in the supernatant after CAR-T cells kill Raji-luc; 5D is a graph showing the level of IFN-γ in the supernatant after CAR-T cells kill RPMI-8226-luc; and

[0022] Figure 6 This is a graph showing the expression level of PD-1 on the surface of CAR-T cells of the present invention. DETAILED DESCRIPTION

[0023] In order to make the technical field personnel better understand the technical solutions in the present application, the present application will be further described below in combination with embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor should belong to the scope of protection of the present application.

[0024] I. Materials and methods

[0025] 1. Cell lines and main reagents

[0026] The retroviral packaging cell line and human multiple myeloma peripheral blood B lymphocyte RPMI-8226-luc were purchased from the American ATCC cell library, and human Burkitt lymphoma cell Raji-luc was purchased from Beijing Weitongda Biotechnology Co., Ltd.

[0027] RPMI 1640 medium, AIM-V medium, fetal bovine serum (FBS) and PBS were purchased from the American Gibco company, FuGene HD transfection reagent was purchased from the American Promega company, lymphocyte separation medium was purchased from Beijing Youyi Zhonglian Biotechnology Co., Ltd., IL-2, CD3 monoclonal antibody (OKT-3) were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., Mlu I enzyme, Not I enzyme, T4 ligase, DH-5α competent, plasmid extraction kit, luciferase reporter gene detection kit, SYBR Green PCR kit, reverse transcription kit were purchased from Beijing Lambolide Technology Co., Ltd., gel recovery kit, RNA extraction kit were purchased from Shanghai Youningwei Company, IFN-γ ELISA kit was purchased from Beijing Bainowei Biological Company, MYC-PE, CD38-APC, CD3-APC, PD-1-PE and other antibodies were purchased from the American BD company.

[0028] 2. Construction of anti-CD38 CAR molecules targeting and inhibiting CD38 by shRNA

[0029] An anti-CD38-CAR with strong affinity was screened through phage display technology. Its structure is a classic second-generation CAR molecule. The CD38 ScFv, CD8 hinge and transmembrane regions, CD28 intracellular domain, and CD3ζ intracellular domain were sequentially connected in series. Then, shRNA sequences targeting CD38 CAR-T were designed through the GPP Web Portal. The two sequences with the highest scores were selected, and a nonsense RNA sequence that did not target any target was designed as a control. The U6 promoter sequence was previously connected in series and synthesized by General Bio. In this way, the anti-CD38-CAR molecule with shRNA targeting CD38 was constructed. It is different from the anti-CD38 CAR with the addition of nonsense RNA sequence. They are named shRNA1-CD38 CAR-T and shRNA2-CD38 CAR-T. The specific molecular structure is shown in [1]. Figure 1 .

[0030] Among them, CD38-shRNA1 (SEQ ID NO.10):

[0031] CCGGCCTCACATGGTGTGGTGAATTCTCGAGAATTCACCACACCATGTGAGGTTTTTG

[0032] CD38-shRNA2 (SEQ ID NO. 1):

[0033] CCGGCTGAGGATTCATCTTGCACATCTCGAGATGTGCAAGATGAATCCTCAGTTTTTG

[0034] 3. Construction of pMFG-CD38 shRNA-MYC-CD38-CAR plasmid vector

[0035] After the construction of the anti-CD38 CAR molecule targeting CD38 with shRNA was completed, the sequence and vector were double-digested with Mlu I and Not I. The digested fragment and vector were recovered by gel electrophoresis and purified. The fragments were then ligated with T4 ligase at 16°C for 1 hour. Transformation was performed using a DH-5α competent cell culture medium, and single colonies were selected and added to LB medium containing ampicillin and cultured on a shaker at 37°C for 12–16 hours. Plasmid DNA was extracted, and after verification of correct enzyme digestion, sequencing was performed. The plasmid vectors that were sequenced correctly were named pMFG-MYC-CD38-CAR, pMFG-shRNA1 CD38-MYC-CD38-CAR, and pMFG-shRNA2 CD38-MYC-CD38-CAR.

[0036] 4. Primary T Cell Extraction

[0037] 10 mL of peripheral blood was drawn from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. The cells were resuspended in AIM-V complete medium (supplemented with 10% FBS and 1% penicillin-streptomycin solution). OKT-3 and 100 U / mL IL-2 were added to stimulate T cell activation and proliferation at a final concentration of 100 ng / mL. The cells were cultured aseptically in a cell culture incubator at 37°C and 5% CO2. Thereafter, the cells were passaged every 48 h using AIM-V complete medium containing 100 U / mL IL-2.

[0038] 5. CAR-T Cell Preparation

[0039] After 48 hours of culture, primary T cells were divided into four groups: untransduced T cells, CD38 CAR-T cells, shRNA1-CD38 CAR-T cells, and shRNA2-CD38 CAR-T cells. The pMFG-MYC-CD38-CAR, pMFG-shRNA1 CD38-MYC-CD38-CAR, and pMFG-shRNA2 CD38-MYC-CD38-CAR plasmid vectors were packaged into retroviral vectors and transduced into human primary T cells, resulting in CD38 CAR-T cells, shRNA1-CD38 CAR-T cells, and shRNA2-CD38 CAR-T cells. Untransduced T cells were not treated and passaged normally. MYC antigen expression was detected by flow cytometry using a MYC-PE antibody 48 hours after transduction. Transduction efficiency (i.e., MYC expression rate) was calculated as: number of MYC-PE-positive cells / total number of viable cells × 100%.

[0040] 6. Detection of relative expression level of CD38 mRNA in CAR-T cells by RT-qPCR

[0041] 24 h after transduction, 5×10 6 RNA was extracted from live cells and verified for integrity by electrophoresis. The RNA was then reverse-transcribed into cDNA and subjected to RT-qPCR using SYBR Green. The RT-qPCR protocol employed a two-step reaction with 40 cycles of 95°C for 2 min, 95°C for 15 s, and 60°C for 30 s.

[0042] GAPDH primer sequences: upstream is CATGTTCGTCATGGGTGTGAACCA (SEQ ID NO. 11), downstream is ATGGCATGGACTGTGGTCATGAGT (SEQ ID NO. 12);

[0043] CD38 primer sequence: upstream is CGCCCCTAGGAAGAGAGGCAGAAAGAAGCT (SEQ ID NO. 13), downstream is ATCGATGCGGCCGCTCATTATC (SEQ ID NO. 14). GAPDH was used as an internal reference gene, and the relative expression level of CD38 mRNA was calculated by 2 -ΔΔCt The relative expression level of CD38 mRNA was calculated by the method.

[0044] 7. Microscopic counting method for detecting the proliferation ability of CAR-T cells

[0045] The day of extracting PBMC was recorded as day 0, and the cells were counted and passaged once at 48 h, so that the cell density was maintained at 1×10 6 The single proliferation multiple was the ratio of the cell density at counting to the final cell density at the last treatment, and the proliferation multiple was the product of the single proliferation multiples. The single proliferation multiple and the proliferation multiple at day 0 were both defined as 1. The proliferation multiples of CAR-T cells cultured in vitro for 0-14 d were calculated, and the proliferation curve was drawn.

[0046] 8. CFSE staining method for detecting the proliferation ability of CAR-T cells when co-cultured with target cells

[0047] Raji-luc and RPMI-8226-luc cells were detected for the positive rate of cell surface CD38 by flow cytometry using CD38-APC antibody. The two kinds of target cells were diluted to a density of 4×10 4 μL, 100 μL was inoculated in the experimental wells of a 96-well plate. CAR-T cells were taken, and CFSE (diluted to 10 μmol / mL with AIM-V medium) was used for 37°C dark staining for 30 min. The cell density was adjusted to 1×10 4 μL, and 100 μL was added to the wells with target cells, i.e., the effector-target ratio was 1:4. After 24 h of continuous culture, T cells were labeled with CD3-APC staining, and T cells cultured alone were used as a control. The proliferation of cells after co-culture was compared by the strength of CFSE signal. The weaker the CFSE signal (the more obvious the left shift), the stronger the proliferation ability.

[0048] 9. Luciferase method for chemiluminescence detection of the killing ability of CAR-T cells on target cells

[0049] Raji-luc and RPMI-8226-luc cells were prepared into cell suspensions, and experimental plating was performed according to the method of 1.8. CAR-T cells were co-cultured with Raji-luc and RPMI-8226-luc cells at different effector-target ratios (1:1, 1:2, 1:4, 1:8, 4×10 4The cells were co-cultured with the target cells (1:1) containing only Raji-luc cells or RPMI-8226-luc cells, and the wells containing only culture medium were used as the maximum release wells, and the wells containing only culture medium were used as blank wells. After 12 hours, 100 μL of the prepared luciferase substrate was added to each well and incubated at room temperature in the dark for 3 minutes. The chemiluminescence mode (LUM) was set to all-white ELISA plates, and the plate was shaken for 5 seconds. The PMT was set to 500. The killing efficiency was calculated as follows: killing efficiency = 1-(apoptosis rate of experimental wells - apoptosis rate of blank wells) / (apoptosis rate of maximum release wells - apoptosis rate of blank wells) × 100%.

[0050] 10. ELISA to detect IFN-γ levels in the supernatant after CAR-T cells kill target cells

[0051] CAR-T cells were co-cultured with Raji-luc or RPMI-8226-luc cells for 12 h, with effector-target ratios of 1:1 and 1:4 (4×10 4 After the cells were collected, they were centrifuged at 400×g for 5 min, and the supernatant was aspirated. The IFN-γ level in the supernatant was detected by ELISA.

[0052] 11. Detection of PD-1 expression level in CAR-T cells by flow cytometry

[0053] The CAR-T cell suspension was collected by centrifugation at 400 g for 5 minutes. The cells were rinsed once with PBS and stained with CD3-APC and PD-1-PE for 60 minutes at room temperature in the dark. After rinsing with PBS, the cells were resuspended in PBS and PD-1 levels were measured by flow cytometry.

[0054] 12. Statistical Processing

[0055] The above experiments were repeated three times. GraphPad Prism 8 statistical software was used to process the data. The comparison among the groups was performed by one-way analysis of variance, and P < 0.05 or P < 0.01 indicated that the difference was statistically significant.

[0056] 2. Results

[0057] 1. Successful construction of shRNA-CD38 CAR-T cells

[0058] Virus titer test results ( Figure 2 A) shows that the titers of CD38 CAR, shRNA1-CD38 CAR and shRNA2-CD38 CAR retroviral vectors were all 1×10 7 Copies / mL, higher titers can be used for T cell transduction experiments. Figure 2In A, H0, H1, H2, H3, and H4 represent the cell culture supernatant as viral vectors harvested for 5 consecutive days after successful packaging cell preparation, i.e., H0-H4.

[0059] Flow cytometry results ( Figure 2 B) shows that the transduction efficiencies of CD38 CAR-T, shRNA1-CD38 CAR-T, shRNA2-CD38 CAR-T, and untransduced T cells were 60.3%, 67%, 57.4%, and 0.098%, respectively. These results demonstrate that CAR molecules are successfully expressed on the surface of T cells.

[0060] RT-qPCR test results ( Figure 2 C) shows that the expression level of CD38 mRNA in the shRNA2-CD38 CAR-T group was only 0.303±0.139, significantly lower than the expression level of 1.0±0.023 in the CD38 CAR-T group (P<0.01). The expression level of CD38 mRNA in the shRNA1-CD38 CAR-T group was 1.520±0.647, which was not statistically different from the CD38 CAR-T group (P>0.05). These results indicate that the shRNA2 sequence was successfully designed and the RNA interference effect was good.

[0061] 2. Raji-luc and RPMI-8226-luc cells highly express CD38 on their surface

[0062] Flow cytometry results ( Figure 3 ) showed that the CD38 positivity rates on the surface of Raji-luc and RPMI-8226-luc cells were 94.6% and 92.0%, respectively. These results indicate that these two hematologic tumor cell lines express a high proportion of CD38 and can serve as target cells for anti-CD38 CAR-T cells.

[0063] 3. Enhanced in vitro proliferation of shRNA-CD38 CAR-T cells

[0064] Microscope counting method experimental results ( Figure 4 A) showed that compared with the CD38 CAR-T group, the cell proliferation fold in the shRNA2-CD38 CAR-T group was significantly increased (P<0.05), while the cell proliferation fold in the shRNA1-CD38 CAR-T group had no statistically significant difference (P>0.05).

[0065] Table 1 In vitro proliferation of CAR-T cells

[0066]

[0067] CFSE proliferation assay results (Figure 4 B) shows that the FITC signal of the co-culture group is obviously left-shifted compared with the non-co-culture group. The CAR-T cells proliferate faster under the stimulation of target cells, indicating that they can be activated by target cells.

[0068] 4. The killing ability of shRNA-CD38 CAR-T cells on target cells is enhanced

[0069] Results of luciferase experiment Figure 5 A, B) show that when the target cells are Raji-luc or RPMI-8226-luc, the killing efficiency of the shRNA2-CD38 CAR-T group is statistically different from that of the CD38 CAR-T group (both P<0.05), while the killing efficiency of the shRNA1-CD38 CAR-T group is not statistically different (P>0.05). The results show that the killing ability of the shRNA2-CD38 CAR-T group on target cells is stronger.

[0070] Table 2 Killing effect of CAR-T cells on Raji-luc cells

[0071]

[0072] Table 3 Killing effect of CAR-T cells on RPMI-8226-luc cells

[0073]

[0074] 5. The level of IFN-γ in the supernatant after the shRNA-CD38 CAR-T cells kill target cells is higher

[0075] Results of ELISA detection Figure 5 C, D) show that when the target cells are Raji-luc or RPMI-8226-luc, the release amount of IFN-γ of the shRNA2-CD38 CAR-T group is significantly higher than that of the CD38 CAR-T group (P<0.01), while the difference of the shRNA1-CD38 CAR-T group is not statistically significant (P>0.05). The results show that the release level of IFN-γ after the shRNA2-CD38 CAR-T group kills target cells is higher.

[0076] Table 4 Release level of IFN-γ (pg / ml) of CAR-T cells killing RPMI-8226-luc cells

[0077]

[0078] Table 5 IFN-γ release level of CAR-T cells killing Raji-luc cells (pg / ml)

[0079]

[0080] 6. Decreased PD-1 expression on the surface of shRNA-CD38 CAR-T cells

[0081] Flow cytometry results ( Figure 6 ) showed that compared with the CD38 CAR-T group, the PD-1 expression level in the shRNA2-CD38 CAR-T group was significantly decreased (P<0.05), approaching that of untransduced normal T cells (P>0.05). However, the PD-1 expression level in the shRNA1-CD38 CAR-T group was not statistically different from that in the CD38 CAR-T group (P>0.05). These results indicate that the shRNA2-CD38 CAR-T group exhibited lower levels of CAR-T cell exhaustion than the CD38 CAR-T group.

[0082] Table 6 PD-1 expression levels in CAR-T cells

[0083]

[0084] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0085] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

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