CD38-CAR-T for treating AML malignancy and application thereof

Abstract

The application discloses a nucleotide sequence of CD38-CAR-T for treating AML malignant tumors, a vector, a host cell and application of the nucleotide sequence, the vector and the host cell in preparation of an antitumor drug. Through design and screening of various structures, the application overcomes technical prejudice, and the prepared CAR-T cell has good specific recognition function and effective in-vitro killing capacity on tumor cells, and prolongs the expansion time of the CAR-T cell and the mode of curative effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a CD38-CAR-T and its applications. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem / progenitor cells. It is a highly heterogeneous group of diseases that can result from the malignant transformation of hematopoietic progenitor cells at different stages of normal myeloid cell differentiation and development, including all non-lymphocyte-derived acute leukemias. It is characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Clinical manifestations include anemia, bleeding, infection and fever, organ infiltration, and metabolic abnormalities. Most cases are acute and severe, with a poor prognosis, and can often be life-threatening if not treated promptly.

[0003] Currently, the main treatment options are chemotherapy and hematopoietic stem cell transplantation (HSCT). Chemotherapy has relatively poor efficacy. Allogeneic HSCT is currently considered a cure for AML; however, most patients relapse after HSCT, and once relapsed, treatment options are extremely limited, with a poor prognosis and an overall one-year survival rate of less than 20%. There is currently no effective, unified treatment plan for relapsed or refractory AML. Specific, targeted immunotherapy (CAR-T therapy) offers new treatment opportunities for these intractable diseases.

[0004] CAR-T therapy has achieved significant clinical efficacy in the treatment of refractory relapsed acute B-lymphoblastic leukemia (r / r B-ALL), but its efficacy is poor in the treatment of refractory relapsed acute myeloid leukemia (r / r AML). Although researchers have explored targets such as CD33 and CD123, there is a risk of "off-tumor, on-target" reactions because these antigen markers are also expressed in normal hematopoietic cells.

[0005] CD38, widely expressed in various hematologic malignancies, is a membrane-bound glycoprotein that catalyzes the synthesis and degradation of cyclic adenosine diphosphate ribose (cADPR). cADPR, a nucleotide metabolite, participates in calcium mobilization from intracellular calcium stores by acting on ryanodine receptors (RyRs). Numerous studies have found that CD38 / cADPR-mediated Ca2+ signaling and Ca2+ release via RyRs channels play crucial roles in the regulation of Ca2+ homeostasis. CD38 / cADPR / RyRs-mediated Ca2+ signaling also participates in many pathological and physiological processes. Furthermore, CD38 is strongly expressed on immunosuppressive cells, making it a potential specific target for these cells.

[0006] Currently, two CD38 monoclonal antibodies (Daratumumab and Isatuximab) have been approved for marketing, showing some therapeutic efficacy in clinical trials. However, their approved indications are both multiple myeloma, and there are no published treatment results for AML, so their efficacy in AML remains unclear. Statistics show that 90% of multiple myeloma (MM) patients express high levels of CD38, and CD38 expression is closely related to patient survival. Besides MM, CD38 is also highly expressed in various hematological malignancies such as acute myeloid leukemia (AML) and acute B-lymphoblastic leukemia (B-ALL). CD38 and hematological malignancies are potential therapeutic targets for various hematological malignancies, and clearing immunosuppressive cells presents a potential application area.

[0007] CD38 is also expressed on T cells, and its inhibitory effect during culture can lead to culture failure. The poor efficacy of CAR-T in AML treatment is also related to the AML tumor microenvironment. Constructed CAR-T cells must meet the requirements of clinical treatment, overcome the inhibitory factors of the AML tumor microenvironment, and be sufficiently activated upon re-exposure to the antigen. Summary of the Invention

[0008] The purpose of this invention is to address the aforementioned deficiencies of the prior art by selecting several more potent structural combinations from a variety of options, which can meet the needs of clinical amplification and treatment. At the same time, the CAR structure can inhibit CD38 protein expression, achieving better culture and treatment results.

[0009] A-DU-BB28 and A-DU-BB40 are CAR-T drugs, especially those with stronger CAR expression, prepared by inhibiting CD38 antigen expression.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] The first objective of this invention is to provide an amino acid sequence of CD38-CAR-T, selected from at least one of the following sequences: SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24.

[0012] The present invention also provides a nucleotide sequence encoding CD38-CAR-T, selected from at least one of the following sequences: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.

[0013] A second object of the present invention is to provide a CAR-T drug comprising the CD38-CAR-T and a pharmaceutically acceptable carrier.

[0014] A third objective of this invention is to provide an application of the CD38-CAR-T in the preparation of antitumor drugs.

[0015] Furthermore, the tumor is a malignant hematologic malignancy. Acute myeloid leukemia is preferred.

[0016] The present invention also provides a vector comprising the nucleotide sequence encoding CD38-CAR-T. In some embodiments, the vector is a recombinant lentiviral vector.

[0017] The present invention also provides a host cell comprising the nucleotide sequence encoding CD38-CAR-T or the vector. As used herein, the term "host cell" should be interpreted broadly and may refer to a prokaryotic or eukaryotic cell; it may be any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0018] The advantages of this invention are:

[0019] 1. This invention has discovered that, in the past, those skilled in the art believed that CAR-38 required gene knockout or gene expression interference to prevent CAR-T cell autolysis, or that antibodies or proteins needed to be added during preparation to inhibit this phenomenon. Therefore, this invention, through the design and screening of various structures, has identified a method for preparing CAR-T cells without the above methods, extending their expansion time and efficacy. This invention overcomes the aforementioned technical biases by employing techniques that were previously abandoned due to such biases, thereby solving the technical problem.

[0020] 2. This invention experimentally investigated the feasibility of preparing CAR gene-modified T cells that specifically recognize CD38 using conventional CAR-T production processes, and the feasibility of using CD38-negative CAR-38 to treat CD38+ AML. The results showed that CAR-38 has specific recognition function and effective in vitro killing ability against CD38+ tumor cells. Therefore, the feasibility of manufacturing clinical-grade CAR-T cells targeting CD38 was verified, and CAR-T drugs prepared using CD38 can be applied to the preparation of drugs for malignant tumors. Attached Figure Description

[0021] Figure 1 This is a structural diagram of CD38-CAR, along with the names of each group and the structural diagrams of the leader, scfv, and co-stimulatory domains.

[0022] Figure 2 : Figure 2 A uses CD38 protein to detect the CAR-T cell positivity rate of CD38-CAR in each group (n=3) by flow cytometry; Figure 2 B is the ratio of the number of CD38-CAR cells amplified to the initial number of cells during a 5-day culture cycle, i.e., the amplification fold, which is determined by cell counting.

[0023] Figure 3 : Figure 3 A is a schematic diagram (n=3) of CD38+ target cells for detecting the specific cleavage of CD38 CART using the LDH method. Figure 3 B and 3C were detected using flow cytometry with the CBA method to assess the secretion of cytokines by T cells during the killing of target cells, namely IFN-γ and TNF (n=3).

[0024] Figure 4 : Figure 4 A was determined by examining cell viability on day 12 after trypan blue staining. Figure 4 B was performed on day 10 to detect the expression of CD38 on CD38-CAR-T cells. Anti-CD38 antibodies were used for the detection using flow cytometry. Figure 4 C. Large-scale amplification of A-DU-BB28 and A-DU-BB40 was performed, and the expression trend of CD38 protein was detected by flow cytometry at 14-day intervals. Detailed Implementation

[0025] To make the technical means, inventive features, objectives, and effects of the invention readily understandable, the invention is further illustrated below with reference to specific figures. However, the invention is not limited to the embodiments described below.

[0026] Example 1:

[0027] In the first step, in this embodiment, the CAR protein is guided by a signal peptide leader and contains an scfv single-chain antibody targeting the tumor antigen CD38, as well as a linker and transmembrane domain derived from CD8. The intracellular signaling domain consists of a co-stimulatory domain and a CD3-zeta signaling domain, as shown below. Figure 1 As shown in Table 1, we constructed different structural combinations based on the basic structure of CARs, including two different signal peptides (a guide peptide derived from CD8A and a guide peptide derived from FMC63-CD828Z, GenBank: MN702884.1), VL and VH regions derived from two CD38-targeting therapeutic antibodies DU and IS, and combinations of three intracellular co-stimulatory signals. These combinations resulted in 12 different CAR proteins, which were then transduced using lentiviral vectors encoding CARs to generate CAR-modified T cells (CART-38) that recognize CD38. The amino acid and nucleotide sequences of the 12 CAR proteins are shown in Table 1.

[0028]

[0029]

[0030] Table 1

[0031] like Figure 2 As shown in Figure A, flow cytometry was used to verify the CAR expression level on the surface of transduced T cells. The transduction efficiency of CAR genes in each group was relatively average, indicating that all 12 CAR structures could successfully package lentiviruses and express them on the surface of CAR-T cells. The A-IS-BB40 group had the highest transduction efficiency, but overall, the positive rate of all CAR-T cells exceeded 40%, indicating that the preparation of CAR-T cells was successful and there was no significant difference. Figure 2 As shown in Figure B, during the 5-day culture period, the amplification fold of each group was around 10-fold, with no significant difference.

[0032] like Figure 3 As shown in Figure A, K562 target cells stably expressing the CD38 antigen were constructed. After treatment with mitomycin C, the target cells were co-cultured with CAR-38 cells, with the CD38-based K562 cell line serving as a control. CAR-38 cells were co-incubated with both K562-CD38 cells and K562 cells. Both A-DU-BB28 and A-IS-BB28 showed high cytotoxic activity, with cytotoxic efficiencies reaching 60%, indicating that both specific antibody-derived scfvs can target the corresponding antigens and activate CAR-T cell cytotoxicity. Different structures resulted in different cytotoxic efficiencies, suggesting that cytotoxic ability is structure-dependent. BB28, with its co-stimulatory domain, exhibited stronger cytotoxic efficiency, which is more beneficial for the treatment of AML. Figure 3B and 3C represent the ability of CAR-T cells to release cytokines after receiving stimulation from target cells. It can be seen that they are basically consistent with the killing results. Both A-DU-BB28 and A-IS-BB28 have high cytokine release capabilities.

[0033] like Figure 4 As shown in Figure A, after culturing for 10 days, significant differences in cell growth status were observed among the groups. Cell viability testing revealed that the scfv group (IS group) had poorer cell condition. To explore the reasons for this, it was found that T cells express CD38 in their native state. Figure 4 As shown in Figure B, the expression of CD38 protein on CD38-CAR-T cells was detected on day 10. Surprisingly, CD38 expression in the DU group was lower than that in the IS group, demonstrating an unexpected inhibitory effect on CD38 expression. This may be because DU scfv binds more readily to CD38 protein intracellularly, resulting in a combined inhibitory effect. It was also observed that signal peptide A was more effective than signal peptide B, which is more conducive to cell proliferation and production. Furthermore, the reduction in CD38 levels increases the survival time of CD38-CAR-T cells in vivo, improving the therapeutic effect against AML. As shown in Figure C, we selected A-DU-BB28 and A-DU-BB40 for large-scale culture testing. The expression level of CD38 protein decreased with increasing amplification time, especially after day three, which is consistent with the expression cycle of CAR protein.

[0034] The research approach of this embodiment is that the combination of A-DU-BB28 and A-DU-BB40 can inhibit the expression of CD38 protein in CD38-CAR-T cells, thereby improving the growth and maintenance of CD38 CAR-T cells, preventing autolysis of CD38 CAR-T cells, and thus enhancing the efficacy of CD38 CAR-T therapy for AML, making it more suitable for AML tumor treatment. Simultaneously, as third-generation CAR-T therapy products, A-DU-BB28 and A-DU-BB40's CD38 expression inhibition function does not affect CAR-T expression, maintaining a strong anti-tumor effect.

[0035] Example 2: Construction of CAR plasmid and packaging of lentivirus

[0036] The lentiviral backbone plasmid vector was pWPXL, derived from Eucardi; pPac-R, pPac-GP, and pEnv-G were all derived from Eucardi; DMEM medium was derived from Gibco; and 293T cells were derived from ATCC.

[0037] The 12 CAR structures obtained in Example 1 were constructed on the plasmid vector pWPXL.

[0038] The specific steps include: digesting the lentiviral backbone pWPXL with two restriction endonucleases, EcoRI and BamHI; recovering the 7000bp linear plasmid using an agarose gel DNA recovery kit after gel electrophoresis (120V, 20min); designing corresponding primers to synthesize the target gene, namely the anti-CD38-CAR plasmid gene, via polymerase chain reaction (PCR), which contains the leader membrane receptor signal peptide, scfv, CD8α chimeric receptor hinge region Hinge and CD8α chimeric receptor transmembrane region TM, co-stimulatory domain, and CD3ζ intracellular signal peptide in tandem; recovering the PCR product by agarose gel electrophoresis (120V, 20min) and purifying it using a purification kit; and then homologously recombinating the obtained vector linear plasmid and the target gene fragment using a seamless cloning kit to obtain the target plasmid containing the target gene.

[0039] Transfect 293T cells with an appropriate amount of the target gene plasmid as follows: Add 2500 μL CaCl2, three packaging plasmids (80 μg pPac-R, 100 μg pPac-GP, 60 μg pEnv-G), and 220 μg of the target plasmid CAR to a 15 mL centrifuge tube. Immediately add bacterial endotoxin test water to a final volume of 5000 μL, then add 5000 μL HBS. After adding all the ingredients, vortex for approximately 20 seconds. Take 293T cells, add 1 mL of the mixed transfection reagent, gently shake to mix thoroughly, and place in an incubator. After 6 hours of transfection, discard the culture supernatant and add 10 mL of 4% DMEM complete medium. After 24 hours of transfection, add another 10 mL of 4% DMEM complete medium. Viral supernatants were collected at 24, 48, and 72 hours of culture and stored at 4°C. After collection, the viral supernatants were filtered using a 0.45µm filter to remove cell debris. Polyethylene glycol 8000 (PEG8000) was added to concentrate the virus, and the mixture was incubated at 4°C for 12 hours. After centrifugation at 3000 rpm for 15 minutes, the supernatant was discarded, and an appropriate amount of sterile PBS was added to dissolve the viral pellet. The dissolved virus was aliquoted and stored at -80°C to obtain the recombinant lentiviral vector. This recombinant lentiviral vector contains the anti-CD38 CAR gene. The structure of the Anti-CD38-CAR plasmid gene is shown below. Figure 1 .

[0040] Example 3: Preparation of anti-CD38-CART

[0041] The lentiviral vectors obtained in Example 2 were transduced into primary T cells to prepare CAR-T cells, and the CAR positivity rate was detected by flow cytometry. The CAR positivity rate was defined as the percentage of cells that could bind to the CD38 antigen. The primary antibody for flow cytometry detection was Human CD38 Protein, His Tag, and the secondary antibody was anti-His fluorescence. The specific steps included: isolating peripheral blood mononuclear cells (PBMCs) using mononuclear cell separation medium; sorting lymphocytes with CD4 and CD8 separation magnetic beads; activating T cells with CD3 and CD28 antibodies; transducing T cells with lentivirus and incubating them in a 37°C, 5% CO2 incubator for 48 hours; changing the culture medium after 48 hours of transduction and counting and replenishing the medium every 1-2 days; detecting the CAR positivity rate by flow cytometry approximately 5 days after transduction, with the primary antibody being Human CD38 Protein, His Tag and the secondary antibody being anti-His fluorescence. The CAR positivity rate is as follows: Figure 2 As shown in Figure A.

[0042] Each day, CART cells were removed from the CO2 incubator and placed in a biosafety cabinet. After gently pipetting the cells in the mixing bottle with a disposable pipette, approximately 100 μL of the cell suspension was transferred to a 1.5 mL EP tube. Cell counts were performed using trypan blue staining, and the cell count and cell viability were calculated.

[0043] Example 4: In vitro functional verification of anti-CD38-CART

[0044] CD4 / CD8 magnetic beads, CD3 antibody, and CD28 antibody were all from Miltenyi Biotechnology; IL-2 was from Peprotech; A-IMV medium was from Gibco. The LDH killing kit was from CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, catalog number REF: G1782; A-IMV medium was from Gibco; K562 (CD38 negative) was from the Shanghai Cell Bank of the Chinese Academy of Sciences; and the CD38-overexpressing myeloid leukemia cell line K562-CD38 was from Shanghai Eucardi.

[0045] Twelve groups of effector cells were cultured separately; K562 and CD38-K562 target cells were cultured separately; effector cells and target cells were co-incubated in groups for cytotoxicity experiments, with an effector-to-target ratio of 5:1; after overnight incubation, the killing efficiency was measured using the lactate dehydrogenase (LDH) method, and the supernatant of effector cells and target cells was collected to detect cytokine levels.

[0046] The specific experimental steps are as follows: 1) Collecting target cell suspension: For adherent cells, discard the culture supernatant. Soak the bottom of each dish with 10 ml of physiological saline, then discard the saline. Add 1 ml of trypsin to each dish to digest the cells. The digestion time depends on the characteristics of the cell line. Observe under a microscope that the cells do not adhere, are suspended, and have a rounded shape. Add 3 ml of complete culture medium to stop digestion. Centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend in 5 ml of PBS. Centrifuge at 1500 rpm for 5 min, then discard the supernatant. 2) Plating target cells: Resuspend the target cells in 1 ml of AIM-V killing medium (AIM-V + 4% FBS). Count the cells using a hemocytometer. Add 1 x 10⁴ target cells to each well of a 96-well plate. Prepare the target cell suspension using killing medium according to the required cell volume. After preparation, plate the target cells in a 96-well cell culture plate, centrifuge at 250 g for 5 minutes, and incubate at 37℃ in a CO₂ incubator for 2 h to promote target cell adhesion. 3) Collect the effector cell suspension, mix the T cells thoroughly, and count them using a hemocytometer. Based on the required amount of effector cells for killing, take effector cells, centrifuge at 1500 rpm for 5 min, resuspend the effector cells in 1 mL of AIM-V killing medium (AIM-V + 4% FBS), and count them using trypan blue staining. 4) 1 x 10⁴ target cells have been added to each well of the 96-well plate. When the E:T (effector cells: target cells) ratio is 2.5:1, add 2.5 x 10⁴ effector cells per well; when E:T = 5:1, add 5 x 10⁴ effector cells / well; when E:T = 10:1, add 1 x 10⁵ effector cells / well, with a volume of 50 μL / well for each ratio. Prepare 1 x 10⁵ effector cells / well according to the calculated E:T = 10:1 ratio, and perform a 2-fold serial dilution using killing medium to prepare effector cell suspensions with E:T = 5:1 and E:T = 2.5:1 ratios. 5) After plating, seal the 96-well plate with sealing film and centrifuge at 250g for 5 minutes (3 increments, 1 decrement), then remove the sealing film and incubate at 37℃ for 24 hours. 6) Perform the assay according to the kit instructions, and finally measure the absorbance at 490nm using a microplate reader. 7) Cytotoxicity calculation formula: Cytotoxicity % = (Cytotoxicity in experimental group / Maximum cytotoxicity) x 100%.

[0047] Cytokine Detection Procedure: After co-incubating effector T cells and target cells, collect the supernatant in a 1.5 mL EP tube for cytokine detection. a. Sample Preparation: Centrifuge the cell suspension at 1500 rpm for 3 min, then collect the supernatant in a new 1.5 mL EP tube. b. Standard Preparation: Dilute the lyophilized standard powder with 2 mL of standard diluent and incubate at room temperature for 15 min. Then, perform 2-fold serial dilutions of the standard (10 dilutions from the highest concentration to the blank dilution). c. Add 50 μL of standard or sample to a new EP tube, followed by 50 μL of magnetic beads and 50 μL of detection antibody. Vortex mix thoroughly and incubate at room temperature for 3 h. d. Wash twice with Wash Buffer, discard the supernatant, resuspend in 200 μL Wash Buffer, and then detect the cytokine IFNγ / TNFα.

[0048] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A CD38-CAR-T, characterized in that, The amino acid sequence of the CD38-CAR is SEQ ID NO.

15.

2. The CD38-CAR-T as described in claim 1, characterized in that, The amino acid sequence is encoded by the following nucleotide sequence: SEQ ID NO.

3.

3. A CAR-T drug, characterized in that, The CD38-CAR-T of claim 1 and a pharmaceutically acceptable carrier.

4. A vector, characterized by, The nucleotide sequence of claim 2.

5. A host cell, characterized in that, The nucleotide sequence of claim 2 or the vector of claim 4.

6. The CD38-CAR-T of claim 1 for use in the preparation of a medicament for treating acute myeloid leukemia.

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