AU2020397271B2 - Chimeric antigen receptor T cell which can be efficiently prepared and safely used, preparation method therefor and use thereof - Google Patents

Abstract

Provided is a chimeric antigen receptor T cell which can be efficiently prepared and safely used, a preparation method therefor and the use thereof. The present invention also relates to a lentiviral recombinant vector which contains a CAR structure encoding sequence and a promoter MND which drives the expression of the CAR structure encoding sequence. The nucleotide sequence of the promoter MND is the positions 2838-3236 of sequence 1. In order to overcome the disadvantages of low transfection efficiency and a large number of cytokines being produced during current CAR-T treatment, a new chimeric antigen receptor is prepared by improving the initiation of the CAR structure by promoter, namely, using the MND promoter to initiate the CAR structure, and a T cell expressing the chimeric antigen receptor is prepared. The T cell has the property of having less of an impact on the expression of CAR molecules on the cell surface and has the advantage of alleviative cell killing effect when combined with a CD19 positive cell, which can greatly reduce the occurrence of cytokine storm and effectively improves safety.

Description

Description

Chimeric antigen receptor t cell which can be efficiently prepared and safely used, preparation method therefor and use thereof Technical Field The present invention relates to the field of biotechnology, in particular to a Chimeric antigen receptor t cell which can be efficiently prepared and safely used, preparation method therefor and use thereof

Background Art CAR-T therapy, known as Chimeric Antigen Receptor T-Cell Immunotherapy, is an innovative and promising form of precise targeted therapy for tumor immunotherapy, has achieved good results in the treatment of clinical tumors through optimization and modification in recent years, and has the potential to cure cancer precisely, rapidly, and efficiently. Despite the great success of CAR-T in clinical efficacy, there are still significant problems with its commercial deployment: firstly, it is expensive, for example, KymriahTM from Novartis is priced as high as $475, 000, and although the payment mode can be innovated, the cost for production and preparation remains high due to a failure to scale; there are many factors affecting the production cost, where low transfection efficiency is an important reason and still exists in the universal CAR-T, the improvement of the transfection efficiency can effectively promote the process development of clinical grade cell production, leading to obvious reduction of the preparation cost; secondly, some patients administered with CAR-T cell therapy suffer from severe or life-threatening side effects known as cytokine release syndrome (CRS) or cytokine storm, which is a systemic inflammatory response observed following the rapid release of large amounts of cytokines produced by infused activated T cells into the bloodstream, resulting in dangerous hypotension, high fever, and chills. Patients present with life-threatening condition in severe cases of CRS. Aiming at the problems of low transfection efficiency and high preparation cost of CAR-T, the current conventional solution is to adopt appropriate transfection reagents, maintain the optimal cell state or select the optimal transfection method; however, these changes still result in low transfection efficiency and high production costs. With regard to the occurrence of cytokine storm side effects side effects in patients administered with CAR-T cell therapy, current studies have found that the number of CAR molecules on CAR-T cells will seriously affect the binding efficiency and activation efficiency to target cells; the low expression efficiency of CAR molecules may result in the failure of effective activation of

CAR-T cells, thus affecting the therapeutic effect; however, excessive density of CAR molecules on the cell surface will cause an intense reaction during the course of treatment, leading to a production of a large amount of cytokines and an increase of the risk of cytokine storm. Therefore, it is an urgent problem to find an antigen receptor T cell which can be prepared with high efficiency and low cost and can reduce the cytokine storm while treating.

Summary of the Invention In an aspect, the present invention provides a nucleic acid molecule comprising a MND promoter driving the expression of a CAR structure and a CAR structure-encoding nucleic acid located downstream of the MND promoter; wherein the nucleic acid molecule comprises SEQ ID NO: 1. In another aspect, the present invention provides a recombinant vector comprising a nucleotide sequence of SEQ ID NO: 1. In certain embodiments, the recombinant vector is a lentiviral recombinant vector. In another aspect, the present invention provides a nucleic acid molecule comprising aMIND promoter driving the expression of a CAR structure and a CAR structure-encoding nucleic acid located downstream of the MND promoter. In certain embodiments, the nucleotide sequence of the promoter MND is at positions 2838-3236 of SEQ ID NO: 1. In certain embodiments, the CAR structure comprises, in order, an extracellular binding domain, one or more hinge domain or spacer domain, a transmembrane domain, one or more intracellular costimulatory signal transduction domains, and a primary signal transduction domain. In certain embodiments, the extracellular domain is selected from, but not limited to, at least one of an antibody or an antigen-binding fragment thereof, a tethered ligand, or an extracellular domain of a co-receptor to the following target antigens : folate receptor u, 5T4,UvP6 integrin, BCMA, B7 H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2(HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican 3(GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-Al+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-1lRa, IL-13Ru2, k, Lewis-Y, K, Mesothelin, Muc, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, and VEGFR2; the hinge domain or spacer domain is selected from, but not limited to, at least one of the transmembrane domains of CD8, CD4, CD45, PD1, and CD152; the transmembrane domain is selected from, but not limited to, at least one of the transmembrane domains of CD8, CD4, CD45, PD1, and CD152; the intracellular co-stimulatory signal transduction domain is selected from, but not limited to, at least one of CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD L2), CD274 (PD-LI), and CD278 (ICOS); the primary signaling domain is selected from, but not limited to, at least one of the primary signaling domain of CD3Q and FcRy.

2A

In certain embodiments, the extracellular binding domain (also known as an antigen-specific binding domain) comprises a light chain variable region of a CD19 antibody and a heavy chain variable region of a CD19 antibody; the hinge domain is a CD8 hinge domain; the transmembrane domain is a CD8 transmembrane domain; the intracellular costimulatory signal transduction domain is 4-1BB; and the primary signal transduction domain is CD3Q. In certain embodiments, the amino acid sequence of the light chain variable region of the CD19 antibody is at positions 23-129 of SEQ ID NO: 3 in the sequence listing; the amino acid sequence of the heavy chain variable region of the CD19 antibody is at positions 148-267 of SEQ ID NO: 3 in the sequence listing; the amino acid sequence of the CD8 hinge domain is at positions 268-307 of SEQ ID NO: 3 in the sequence listing; the amino acid sequence of the CD8 transmembrane domain is at positions 308-335 of SEQ ID NO: 3 in the sequence listing; the amino acid sequence of the intracellular co-stimulatory signal transduction domain 4-1BB is at positions 336-377 of SEQ ID NO: 3 in the sequence listing; the amino acid sequence of the primary signal transduction domain CD3Q is at positions 378-489 of SEQ ID NO: 3 in the sequence listing. In certain embodiments, the nucleotide sequence of the structural coding sequence of CAR is at positions 3251-4717 of SEQ ID NO: 1. In another aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule. In certain embodiments, the recombinant vector is a lentiviral recombinant vector. In an example of the present invention, the lentiviral recombination vector is a vector for homologous recombination of the DNA molecule at positions 2838-5863 of SEQ ID NO: 1 into a lentiviral expression vector to obtain the expression of the CAR structure; in an example of the present invention, the specific lentiviral expression vector used is a pDBR vector. The lentiviral recombinant vector in the present examples is the lentiviral recombinant plasmid pLV-M9BBz (M19BBz), with a nucleotide sequence of SEQ ID NO: 1. In another aspect, the present invention provides a recombinant microorganism packaged from the above-mentioned recombinant vector. In certain embodiments, the recombinant microorganism includes a recombinant virus. In certain embodiments, the recombinant viruses are those capable of expressing the CAR structure shown in positions 3251-4717 of SEQ ID NO: 1 and infecting immune cells. The viruses include lentiviruses, herpes viruses, macrophage viruses, Epstein-Barr viruses, hepatitis B viruses, hepatitis C viruses, or HIV viruses. In another aspect, recombinant cells containing the nucleic acid molecule, the recombinant vector or the recombinant microorganism as described above are also within the scope of the invention. In certain embodiments, the above-mentioned recombinant cells are immune cells expressing the above-mentioned lentiviral recombinant vector; the immune cells include immune effector cells; the immune cells include cytotoxic T lymphocytes, NKT cells, NK cells, helper T cells, or gamma delta T cells. In another aspect, the use of promoter MND for driving the expression of CAR structure in CAR-T cells is also within the scope of the present protection; the nucleotide sequence of the promoter MND is at positions 2838-3236 of SEQ ID NO: 1. In another aspect, the present invention provides a method of preparing CAR-T cells. In another aspect, the present invention provides a process comprising the steps of introducing above-mentioned nucleic acid molecule or the recombinant vector into T cells, or infecting T cells with above-mentioned recombinant microorganism, to express the above-mentioned CAR structure. In another aspect, the present invention provides a product for treating tumors or having a function of treating tumors with low cytokine storm comprising the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned recombinant microorganism or the above-mentioned recombinant cell. In certain embodiments, the product is a pharmaceutical composition, the active ingredient of the pharmaceutical composition is the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned recombinant microorganism or the above-mentioned recombinant cell. The present invention also provides the following use: the present invention provides the use of the MND promoter in driving the expression of the CAR structure, the nucleotide sequence of the MND promoter being at positions 2838-3236 of SEQ ID NO: 1; the present invention provides the use of the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned recombinant microorganism, or the above-mentioned recombinant cell in the preparation of a product for treating tumors; alternatively, the present invention provides the use of the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned recombinant microorganism, or the above-mentioned recombinant cell in the preparation of a product having the ability to treat tumors with low cytokine storm. The present invention also provides a method of preparing a CAR-T cell, comprising the steps of introducing the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector into T cells, or infecting T cells with the above-mentioned recombinant microorganism to express the above-mentioned CAR structure. The present invention also provides a method for reducing cytokine storm production in CAR-T cell therapy by using the promoter MND to drive the expression of CAR constructs in T cells; the nucleotide sequence of the promoter MND is at positions 2838-3236 of SEQ ID NO: 1. The above method comprises the steps of: 1) preparing the above-mentioned recombinant vector 2) introducing the recombinant vector into T cells to obtain CAR-T cells; and 3) reinfusing the CAR-T cells back into the body to reduce cytokine storm production in CAR-T cell therapy; or the above method comprises the steps of: 1) preparing the above-mentioned recombinant microorganism; 2) infecting T cells with the above-mentioned recombinant microorganism to obtain CAR-T cells; and 3) reinfusing the CAR-T cells back into the body to reduce cytokine storm production in CAR-T cell therapy. Furthermore, the T cells are autologous T cells. The present invention also provides a method of treating a hematological tumor comprising the steps of administering to a subject in need thereof T cells which drive the expression of the CAR structure with the MND promoter. The present invention also provides a method for treating tumors, including the steps of: 1) preparing the above-mentioned recombinant vector; 2) introducing the recombinant vector into T cells to obtain CAR-T cells; and 3) reinfusing the CAR-T cells back into the body for the treatment of tumors; or the above method comprises the steps of: 1) preparing the above-mentioned recombinant microorganism; 2) infecting T cells with the recombinant microorganism to obtain CAR-T cells; and 3) reinfusing the CAR-T cells back into the body for the treatment of tumors. The tumors of the present invention include solid tumors and hematological tumors. The hematological tumors include, but are not limited to, leukemia, lymphoma, and myeloma. Furthermore, the T cells are autologous T cells.

The invention also provides a use comprising any one of the uses of:

1) a use of theMND promoter in driving the expression of the CAR structure, the nucleotide

sequence of the MND promoter being at positions 2838-3236 of SEQ ID NO: 1;

2) a use of the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector,

5A the above-mentioned recombinant microorganism, and the above-mentioned recombinant cell in the preparation of a medicament for the treatment of tumors; and

3) a use of the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector,

the above-mentioned recombinant microorganism, and the above-mentioned recombinant cell in

the preparation of a medicament having a function for the treatment of tumors with low cytokine

storm.

In certain embodiments, the tumors comprise solid tumors and hematological tumors. In certain

embodiments, the hematological tumors comprise leukemia, lymphoma, and myeloma.

In various embodiments, the present invention provides genetically engineered immune effector cells comprising a vector designed to express a chimeric antigen receptor that targets cytotoxicity to tumor cells. These genetically engineered receptors referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for a target antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity. The term "chimeric" as used herein describes being composed of parts of different proteins or DNAs from different origins. Vectors contemplated herein comprise promoters such as MND, EFlu, CMV, and PGK, and a polynucleotide encoding a CAR, including colony stimulating factor signal peptides, FMC63 single chain antibody sequences, CD8 hinge and transmembrane sequences, 4-1BB costimulatory signal domain and CD3Q signal domain, and T2A and tEGFR. The CARs contemplated herein comprise an extracellular domain that binds to a specific target antigen (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain and an intracellular signaling domain. Engagement of the antigen binding domain of the CAR with its target antigen on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell. The main characteristic of CARs is their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific co-receptors.

Brief Description of the Drawings FIG. 1 shows the construction of CAR vectors with different promoters EFlu and MND, (a) CAR vector map using EFlu promoter; (b) CAR vector map using MND promoter, (c) schematic diagram of the structure of CAR vector of different promoters EFla and MND; FIG. 2 shows the assay for transfection efficiency; FIG. 3 shows a streaming graph of the assay for transfection efficiency; FIG. 4 shows the difference in expression of CAR molecules in T cells transduced with E19BBz and M19BBz; FIG. 5 shows antigen-specific tumor clearance with T cells expressing CAR from different promoters and the amount of cytokines produced by CAR-T cells in corresponding experiments; FIG. 6 shows the effect of CAR-modified T cells under different promoters on tumor cell regression in tumor model mice after adoptive transfer; FIG. 7 shows the transfection efficiency, Relative MFI, T cell in-vitro culture amplification rate, and CAR-T in-vivo amplification rate of two CAR-T cells in clinical trials; FIG. 8 is a summary of patient responses following treatment in a clinical trial, (a) temperature changes of patient within 14 days after reinfusion; (b) the maximum value of fever temperature after treatment; (c) statistical CRS grading of patients after being treated; and FIG. 9 shows the long-term clinical results of patients treated with CAR-T cells produced from two promoters.

Detailed Description of the Invention The experimental methods applied in the following examples are conventional methods, unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available, unless otherwise specified. In the quantitative experiments of the following examples, triplicate experiments are set up, and the results are averaged. Example 1 Construction of Lentiviral Recombinant Plasmids 1. Lentiviral recombinant plasmid pLV-M19BBz (M19BBz) containing the MND promoter A lentiviral recombinant plasmid pLV-M19BBz (M19BBz) containing the MND promoter (as shown in FIG.lb and the lowerpanel of and FIG.lc) has anucleotide sequence of SEQ ID NO: 1 (whole gene synthesis), and can also be obtained by the homologous recombination of DNA molecule at positions 2838-5863 of SEQ ID NO: 1 into a pDBR vector (Calder6n-G6mez, E., et al., Reprogrammed quiescent B cells provide an effective cellular therapy against chronic experimental autoimmune encephalomyelitis. European Journal of Immunology, 2011. 41(6): p.

1696-1708.). Wherein in SEQ ID NO: 1, aMIND promoter is at positions 2838-3236, a nucleic acid encoding a colony stimulating factor signal peptide is at positions 3251-3316 (the amino acid sequence is at positions 1-22 of SEQ ID NO: 3), a nucleic acids encoding an extramembrane antigen binding region--a light chain variable region VL19 of a CD19 antibody is at positions 3317-3637 (the amino acid sequence is at positions 23-129 of SEQ ID NO: 3), Linker is at positions 3638-3691, and a nucleic acids encoding an extramembrane antigen binding region--a heavy chain variable region VH19 of a CD19 antibody is at positions 3692-4051 (the VH19 amino acid sequence is at positions 148-267 of SEQ ID NO: 3; in SEQ ID NO: 1, a nucleic acid encoding FMC63 single chain antibody is at positions 3317-4051 of SEQ ID NO: 1), a nucleic acid encoding CD8 hinge region is at positions 4052-4171 (the amino acid sequence is at positions 268-307 of SEQ ID NO: 3), a nucleic acid encoding CD8 transmembrane region is at positions 4172-4255 (the amino acid sequence is at positions 308-335 of SEQ ID NO: 3), a nucleic acid encoding intracellular costimulatory signal transduction domain 4-1BB is at positions 4256-4381 (the amino acid sequence is at positions 336-377 of SEQ ID NO: 3), and a nucleic acid encoding primary signal transduction domain CD3 ( is at positions 4382-4717 (the amino acid sequence is at positions 378-489 of SEQ ID NO: 3); a nucleic acid encoding self-cleavage sequence T2A is at positions 4718-4789 (the amino acid sequence is at positions 490-513 of SEQ ID NO: 3), a nucleic acid encoding tEGFR is at positions 4790-5863 (the amino acid sequence is at positions 514-870 of SEQ ID NO: 3), the CAR structure having a nucleotide sequence at positions 3251-4717 of SEQ ID NO: 1 is expressed in the lentiviral recombinant plasmid pLV-M19BBz (M19BBz) (at positions 1-489 of SEQ ID NO: 3). 2. Lentiviral recombinant plasmid pLV-E19BBz (E19BBz) containing the EF l promoter A lentiviral recombinant plasmid pLV-E19BBz (E19BBz) containing the EFlu promoter (as shown in FIG.la and the upper panel of and FIG.Ic), has a nucleotide sequence of SEQ ID NO: 2, and is obtained by replacing the MND promoter at positions 2838-3236 of SEQ ID NO: 1 with the EFla promoter at positions 2838-4131 of SEQ ID NO: 2, with the remaining nucleotide sequence unchanged. The recombinant vector was prepared according to the following method: a promoter sequence and a polynucleotide sequence of CAR were cloned respectively, then spliced by Overlap PCR with designed primers, and cloned into a pDBR vector (GenBank: FR822201.1) to construct pLV-E19BBz. The promoter EFlu of pLV-E19BBz (E19BBz) was replaced with MND by means of sequence synthesis and cloning, and the promoter was named pLV-M9BBz (M19BBz). Elements of each part of the vector were shown in FIG. 1. Example 2 Preparation of CAR T cells I. Preparation of CAR T cells 1. Package of lentivirus recombinant plasmids Lentiviral recombinant plasmids pLV-E19BBz (E19BBz) and pLV-M19BBz (M19BBz) were packaged separately to obtain corresponding lentiviral particles. The specific steps were as follows: (1) 293FT cells (Cyagen Biosciences (GuangZhou) Inc., HEKFT-30001) grown to 80%-90% in flasks (175) were removed from a 37°C, 5% CO2 cell incubator, then digested, washed, and collected, 4.5x106 cells and 9 mL of DMEM complete medium (purchased from Gibco, Cat. No. 11965-084) were added to each 10 cm cell culture dish, the mixture was shaken gently, and cultured in a 37°C, 5% CO2 incubator. On Day 2, the following reagents were added to each dish: 500 tl of buffer (purchased from Polyplus Transfection, Cat. No. B161116), 6 tg of lentiviral recombinant plasmid pLV-E19BBz (E19BBz) or pLV-M19BBz (M19BBz) prepared in Example 1, 3 tg of psPAX2 (purchased from Wuhan Miaoling Biotechnology Co., Ltd., Cat. No. P026), and 1.5 tg of pMD2.G (purchased from Guangzhou Geneseed Biotech Co., Ltd., Cat. No. 161220L08), the mixture was mixed homogeneously, then the packaging reagent (purchased from Polyplus Transfection, Cat. No. 114-15) was added to the system at 25 tl /10 cm culture dish, followed by mixing homogeneously, leaving standing at room temperature for 10 min to obtain a mixed solution. (3) 293FT cells used for virus packaging were taken out from the 37°C, 5% CO 2 cell incubator, and the mixed solution was evenly added to each culture dish, gently shaken well, and placed into the 37°C, 5% CO2 incubator for further culture. After incubation for 4 h, the culture medium was discarded, 5 ml of pre-heated PBS was added to wash the cells, then 9 ml of fresh pre-heated DMEM complete medium containing 10% (volume fraction) FBS was added, and the mixture was placed into the 37°C, 5% CO 2 incubator for culture. (4) After continuing to culture for 48-72 h, the culture supernatant was collected as virus stoste, which was filtered into a 50 ml centrifuge tube using a 0.45 tm filter, and high-speed centrifugation was performed at 18500g for 2 h at 4°C. The supernatant was discarded, DMEM complete medium (with a volume ratio of added medium to virus stoste of 1: 500) was added to the precipitate to re-suspend virus particles, resulting in virus concentrate. (5) The virus concentrate was aliquoted at 200 tl /tube and 10 tl was reserved for determination of virus titer. The aliquoted dispensed concentrated solution was stored in a -80°C refrigerator to obtain E19BBz virus and M19BBz virus. Virus titer was detected to be 2.6 X10 8/ml for E19BBz virus and 4.3 X 10 8/ml for M19BBz virus. In terms of detection, the E19BBz virus stoste and the M19BBz virus stoste obtained in (4) were quantified using the ELISA kit for p24 (TAKARA, 632200). The results were shown in FIG. 2b. It can be seen that the amount of p24 in the M19BBz virus stoste was greater than that in the E19BBz virus stoste. 2. Sorting of T cells

(1) Fresh peripheral blood (donor informed consent) samples from healthy donors were transferred into centrifuge tubes, diluted with an equal volume of 0.9% normal saline, and mixed homogeneously; (2) 15 ml of lymphocyte isolation solution (Beijing DongFang HuaHui Biomedical Technology Co., Ltd., 25710) was placed into a new centrifuge tube, then 2 volumes (i.e., 30 ml) of the blood sample diluted in step (1) was slowly added to the upper layer of the lymphocyte separation solution, and centrifugation was performed, with the centrifugation parameters of 2000 rpm, 20 min, Ascend(7)/Descend(4), 25°C; (3) after centrifugation, there will be four layers of a diluted plasma layer, a monocyte layer, a lymphocyte separation solution layer, and an erythrocyte layer, the monocyte layer was transferred to a new centrifuge tube and washed with normal saline; (4) the monocytes obtained in step (3) were subjected to purification of CD3+ T cells with the kit (Miltenyi: 130-050-101), resulting in CD3+ T cells. 3. Preparation of chimeric antigen receptor T cells by transfecting target cells with lentiviral particles (5) x107 purified CD3+ T cells were plated into culture flasks, and 25 tl of CTS CD3/CD28 Dynabeads (Gibco: 40203D) was added to stimulate the activation and culture; (6)on the next day of culture, a certain amount of the E19BBz virus or M19BBz virus (with MOI of 0.25, 0.5, 1, 2, 4, and 8, respectively) prepared in step (5) in1 above-mentioned was added to a culture flask containing CD3+ T cells at room temperature, then the culture flasks were placed into a centrifuge for centrifuging under centrifuging parameters of 2000 rpm, 2 h, Ascend(4)/Descend(4), 35°C, to complete lentiviral transduction, thus obtaining CAR T cells containing M19BBz of different MOIs and CAR T cells containing E19BBz of different MOIs; (7) on the third day of culture, the cells in the culture flask were subjected to half cell culture media supplements (200 IU IL-2 in TexMACS (Miltenyi: 130-097-748 and 170-076-309) was added); (8) on the fifth day of culture, the expression of chimeric antigen receptor in T cells was detected by flow cytometry; (9) on the twelfth day of expanding culture, the transduction rate was detected by flow cytometry; (10) on the fourteenth day of expanding culture, the cells were collected to complete the preparation of chimeric antigen receptor T cells. II. Identification and detection of CAR T cells 1. Detection of transduction rate The cells cultured on the fifth day in the above-mentioned step (8) in I were subjected to flow cytometry to detect the transduction rate by CD4-FITC (Biolegend; 357405) and 7AAD

(Biolegend; 420403). Results for the transduction rate of different MOI values were shown in FIG. 2a, and it can be seen that T cell transduction rate of M19BBz was higher than that of E9BBz. As shown in FIG. 2c, the percentage of positive CAR+ cells (representing transduction rates) at different MOI values showed that equal amount of T cells were transduced with E19BBz virus and M19BBz virus at different MOI, respectively, and as the MOI was increased, there was a corresponding increase in the transduction rate for M19BBz, but not for E9BBz, indicating that M19BBz showed a higher T cell transduction rate than E9BBz. FIG. 3 is a representative graph with a MOI value of 0.25 and it can be seen that T cell transduction rate of M19BBz was higher than that of E9BBz. 2. Detection of CAR molecular expression The cells (MOI value of 0.5) cultured on the fifth day in the above-mentioned step (8) in I were subjected to flow cytometry to detect the expression of tEGFR by biotinylated Erbitux (anti-EGFR monoclonal antibody, Merck: Erbitux) with SA-PE (Biolegend: 405204) or SA-APC (Biolegend: 405207), and to detect the expression of CAR molecule using specific antibodies against the antigen CD19 Fc-FITC (ACRO: P15391-1) and FMC63 scFv (Bioswan: 019-01-647M). Untransduced CD3+ T cells were used as a control. As shown in FIG. 4a, the results showed that CAR can be expressed using different promoters; tEGFR was co-expressed with CAR, with a positively correlated expression efficiency. The flow cytometry results were further analyzed for mean fluorescence intensity (MFI) (Flow cytometry analysis software can generate MFI data directly). The results were shown in FIG. 4b, the expression of tEGFR (erb) was detected, the relative MFI value of tEGFR on the surface of CAR T cells expressing M19BBz (positive cell MFI/negative cell MFI) (12.19± 1.30) was slightly higher than that of CAR T cells expressing E19BBz (10.47± 1.16), with a difference between the two of about 16%. There was no significant difference between the two groups by detection of tEGFR. The relative MFI of CAR T cells expressing E19BBz and CAR T cells expressing M19BBz (4.37 ±0.59 vs 1.13 ±0.08) had a difference of -7 4 %, when the expression of the CAR molecules was

detected with CD19Fc; the relative MFI of E19BBz and M19BBz (2.26 ±0.37 vs 1.24 ±0.07) had a difference of -46% when the expression of the CAR molecules was detected with FMC63 scFv antibody (scFv), both of which showed a higher expression of CAR on the surface of E19BBz. Example 3 In vitro functional assay for chimeric antigen receptor T cells 1. Detection of killing and cytokine

A. Detection of specific killing efficiency M19BBz CAR T cells (MOI value of 0.25) and E19BBz CAR T cells (MOI value of 0.25) prepared as described in 3 of I in Example 2, were co-cultured with three different tumor target cells (NALM-6, Raji, 697 from Guangzhou Cellcook Biotech Co., Ltd.: B-cell acute lymphoblastic leukemia CC1928; ATCC: Burkitt's lymphoma cells CCL86; Shanghai Binsui Biotechnology Co., Ltd.: human pre-B cell leukemia cell line BSC-5209479641-01), respectively, the steps were as follows: Experimental group: target cells were stained with 1 mg/ml calcein (Calcein-AM, Invitrogen, C3099) in 200 ul of cell culture medium (RPMI 1640, Gibco, 22400-089 + 10% FBS ExCell Bio, FND500), 2X 10 4 target cells was added, and 2X 105 each type of CAR T cells was added, the mixture was incubated four hours at 3C, the released calcein in the supernatant was detected (by microplate reader Multiskan Sky, detection conditions: excitation wavelength of 490 nm and emission wavelength of 515 nm). Spontaneous release group: the target cells supernatant was directly detected after the same time to obtain a fluorescence value of spontaneous release group; Complete release group: the target cells were added with 100 tl of 2% Triton X-100 per well and the supernatants were assayed directly after the same time as the complete release group. Specific killing efficiency (%) = (fluorescence value of experimental group-fluorescence value of spontaneous release group)/(fluorescence value of complete release group-fluorescence value of spontaneous release group) x 100% As shown in FIG. 5a, the results indicated that there was no significant difference in killing ability between E19BBz CAR T cells and M19BBz CAR T cells in the two NAML-6 and 697 cell lines, while the killing ability of M19BBz CAR T cells was higher than that of El9BBz CAR T cells in the Raji cell line. In combination with the results of Example 2, it was shown that the number of CAR molecules on the surface of CAR-T cells of M19BBz CART cells was reduced, but the killing ability against tumor cells was not reduced. B. Detection of cytokine In 200 ul of cell culture medium (RPMI 1640, Gibco, 22400-089 + 10% FBS ExCell Bio, FND500), 2 X 104 target cells was added and 6 X 104 CART cells was added, the mixture was incubated 20 h at 37 °C, the supernatant was detected for cytokine using Multifactor Assay Kit (BioLegend LEGENDplex #740118). The results were shown in FIGs. 5b and 5c, it was found that the expression levels of cytokines TNF-u and IFN-y in M19BBz CAR T cells were significantly reduced compared to E19BBz CAR T cells, in combination with the results of FIG. 5a, it was demonstrated that M19BBz CAR T cells were safer and more effective in reducing cytokine storm than E19BBz CAR T cells without reducing tumor killing ability. 2. Detection of tumor killing effect in vivo by animal experiments NOD-SCID mice were used to establish a Raji-luciferase tumor model, which was constructed by infusing Raji human B cell lymphoma luc (luciferase)-labeled cells Raji-luciferase (provided by Yicon (Beijing) BioMedical Technology Inc.), and injecting the tumor cells in logarithmic growth phase into the blood vessels of mice via tail vein, the steps were as follows: The Raji-luciferase tumor model cells were prepared with PBS as a cell suspension at a concentration of Ix107/ml and inoculated into mice via tail vein, 100 tl/mouse; each mouse was inoculated with Ix106 cells, on the third day, mice were injected with luciferin, after anesthesia, fluorescence intensity was detected on the equipment (IVIS Lumina, Series III, PE), after detection, mice with consistent fluorescence intensity were selected and randomly divided into groups and administered (6 mice in each group). Control (no treatment): 200 tl of normal saline containing 2% (mass volume percentage content, unit: g/ml) human albumin (Hebei Daan Pharmaceutical Co., Ltd., GYZZ S200443042) was injected; M19BBz group (CART M19BBz): 200 l of a CAR T cells solution containing 5x106 M19BBz (MOI value of 0.25) (the total cell number was converted according to transfection efficiency, and the cells were resuspended in physiological saline containing 2% human albumin) was injected; E19BBz group (CART M19BBz): 200 l of a CAR T cells solution containing 5x106 E19BBz (MOI value of 0.25) (the total cell number was converted according to transfection efficiency, and the cells were resuspended in physiological saline containing 2% human albumin) was injected; the luminescence signal was measured every seven days and the survival of mice was observed. The results of the assay analysis were shown in FIG. 6, (a): tumor burden after E19BBz and M19BBz treatment compared to untreated group was detected in vivo imaging system; (b) (on the ordinate, mean light intensity represents tumor burden): FIG.6a was subjected to quantification and statistical analysis; the tumoricidal efficacy of M19BBz CAR T cells in mice was significantly better than that of E19BBz CAR T cells with statistical difference. 3. Experimental determination at the clinical level Through the results of clinical trials, the quality and safety of the product were statistically analyzed. Patients with refractory and relapsed acute B-cell leukemia, who have been screened by physicians and signed informed consent by the patient's representative, were enrolled in clinical trials, and leukocytes were collected in hospitals for cell production.

Table 1 Enrollment Details

E19BBz Serial Number Abbreviations for Names Gender Age (years)

X03 WMF Male 3

X06 MWX Male 3

X07 LLL Female 31

X08 XJX Female 16

X13 ZQ Male 13

X22 HSY Female 5

M19BBz Serial Number Abbreviations for Names Gender Age (years)

X01 DZ Male 22

X04 YS Female 29

X05 GLJ Male 27

X09 DSY Female 38

X10 ZQJ Male 20

X11 HBY Male 12

X12 WZH Female 12

X14 LHS Male 4 Leukocytes were treated according to the method in 3 of I in Example 1 to prepare a plurality of batches of M19BBz CAR T cells (a certain batch having an MOI value of 1) and E19BBz CAR T cells (a certain batch having an MOI value of 1) (each from different batch of cells), and the CAR-T cells were subjected to flow cytometry for measurement of transduction rate (by biotinylated Erbitux (anti-EGFR monoclonal antibody, Merck: Erbitux) with SA-PE (Biolegend: 405204) or SA-APC (Biolegend: 405207)) and MFI value, before reinfusion on the fourteenth day. The above cells were reinfused back into the patients, who remained in the hospital for observation until discharge. During hospitalization, the body temperature changes were monitored daily and the response was observed, and symptomatic treatment was given according to the corresponding conditions. Peripheral blood was detected at a fixed time, and the CAR-T cell expansion was calculated. An early assessment of effect was performed 14-28 days after the treatment. The results were shown in FIGs. 7a and 7b, it can be seen that the average transduction rate of E19BBz was lower than that of M19BBz. There was no significant difference in tEGFR relative fluorescence intensity between the two structures, indicating that the transduction rates of both viruses were consistent with tEGFR expression and previous in vitro functional results in clinical cell preparations. No statistically significant difference between E19BBz and M19BBz was shown by counting the number of cells harvested during in vitro culture, by counting the fold of culture expansion (FIG. 7c), and by counting the maximum number of cells expanded in vivo and the fold of the initial reinfusion dose (FIG. 7d). However, when observing the daily body temperature changes of subjects in the two groups, the average body temperature of M19BBz raised slowly (FIG. 8a), with a lower highest value of fever temperature (FIG. 8b), and a lower proportion of CRS above grade 2 (FIG. 8c). After long-term follow-up of patients, the overall survival of M19BBz was not significantly different from that of E19BBz (FIG. 9), representing that M19BBzCAR-T cells with a milder side effect were similar to E19BBz in terms of therapeutic effect. In summary, replacing the promoter changed the number of chimeric antigen receptors on the surface of CAR-T cells, and the safety of the product was further adjusted to reduce cytokine storm and provide a safer product.

Industrial Applications The experiments of the present invention demonstrate that in order to overcome the disadvantages of low transfection efficiency and large amount of cytokines produced during the current CAR T treatment, the present invention has achieved a significant increase in transfection efficiency, and reduced the production cost due to the ease of development of clinical grade cell production; by reducing the density of CAR molecules on the cell surface, the intensity of the reaction is reduced, thereby reducing the occurrence of CRS, making CAR-T treatment safer. The steps are as follows: a new chimeric antigen receptor is prepared by improving the initiation of the CAR structure by promoter, namely, using the MND promoter to initiate the CAR structure, and a T cell expressing the chimeric antigen receptor is prepared. The T cell has the property of having an impact on the less expression of CAR molecules on the cell surface and has the advantage of alleviating cell killing effect when combined with a CD19 positive cell, which can greatly reduce the occurrence of cytokine storm and effectively improve safety. Meanwhile, the chimeric antigen receptor T cell obtained by the preparation method has higher transduction efficiency and can be stably and durably expressed on the surface of the T cell, so that the chimeric antigen receptor T cell can play a role in killing target cells when being combined with antigens on the surface of the target cells, and the lasting expression can also ensure that the killing effect is durable, making it more clinically safe for the treatment of cancer. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge. It will be understood that the terms "comprise" and "include" and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied. It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.

SEQUENCE LISTING

<110>HEBEI SENLANG BIOTECHNOLOGY CO. LTD.

<120>Chimeric antigen receptor t cell which can be efficiently prepared and safely used, preparation method therefor and use thereof

<160>3

<170>PatentIn version 3.5

<210> 1 <211> 8953 <212> DNA <213> Artificial sequence

<400> 1 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 60 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaatccgcg cacatttccc 120 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc 180 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 240 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg 300 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg 360 ccagatatcg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc 420 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 480 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 540 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 600 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 660 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 720 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 780 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 840 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 900 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt 960 ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 1020 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg 1080 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga 1140 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct 1200 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact 1260 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag 1320 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg 1380 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt 1440 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct 1500 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac 1560 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat 1620 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc 1680 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta 1740 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga 1800 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc 1860 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata 1920 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc 1980 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag 2040 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg 2100 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg 2160 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa 2220 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt 2280 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata 2340 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt 2400 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac 2460 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta 2520 gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa 2580 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 2640 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg 2700 ggtttattac agggacagca gagatccagt ttggttagta ccgggcccgc tctagaggat 2760 ccggctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag 2820 aagtatgcaa agcatgcttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc 2880 tgtaggtttg gcaagctagg atcaaggtta ggaacagaga gacagcagaa tatgggccaa 2940 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gttggaacag 3000 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 3060 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt 3120 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt 3180 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccattaa 3240 ttaagccacc atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc 3300 ctttctgctg atccccgaca tccagatgac ccagaccacc tccagcctga gcgccagcct 3360 gggcgaccgg gtgaccatca gctgccgggc cagccaggac atcagcaagt acctgaactg 3420 gtatcagcag aagcccgacg gcaccgtcaa gctgctgatc taccacacca gccggctgca 3480 cagcggcgtg cccagccggt ttagcggcag cggctccggc accgactaca gcctgaccat 3540 ctccaacctg gaacaggaag atatcgccac ctacttttgc cagcagggca acacactgcc 3600 ctacaccttt ggcggcggaa caaagctgga aatcaccggc agcacctccg gcagcggcaa 3660 gcctggcagc ggcgagggca gcaccaaggg cgaggtgaag ctgcaggaaa gcggccctgg 3720 cctggtggcc cccagccaga gcctgagcgt gacctgcacc gtgagcggcg tgagcctgcc 3780 cgactacggc gtgagctgga tccggcagcc ccccaggaag ggcctggaat ggctgggcgt 3840 gatctggggc agcgagacca cctactacaa cagcgccctg aagagccggc tgaccatcat 3900 caaggacaac agcaagagcc aggtgttcct gaagatgaac agcctgcaga ccgacgacac 3960 cgccatctac tactgcgcca agcactacta ctacggcggc agctacgcca tggactactg 4020 gggccagggc accagcgtga ccgtgagcag cactacccca gcaccgcggc cacccacccc 4080 ggctcctacc atcgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc 4140 tggtggggcc gtgcataccc ggggtcttga cttcgcctgc gatatctaca tttgggcccc 4200 tctggctggt acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg 4260 cggtcggaag aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac 4320 tcaagaggag gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact 4380 gcgcgtgaaa ttcagccgca gcgcagatgc tccagcctac aagcaggggc agaaccagct 4440 ctacaacgaa ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg 4500 acgggaccca gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa 4560 cgagctccaa aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg 4620 cagaagaggc aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac 4680 ctatgacgct cttcacatgc aggccctgcc gcctcggctc gagggcggcg gagagggcag 4740 aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctagga tgcttctcct 4800 ggtgacaagc cttctgctct gtgagttacc acacccagca ttcctcctga tcccacgcaa 4860 agtgtgtaac ggaataggta ttggtgaatt taaagactca ctctccataa atgctacgaa 4920 tattaaacac ttcaaaaact gcacctccat cagtggcgat ctccacatcc tgccggtggc 4980 atttaggggt gactccttca cacatactcc tcctctggat ccacaggaac tggatattct 5040 gaaaaccgta aaggaaatca cagggttttt gctgattcag gcttggcctg aaaacaggac 5100 ggacctccat gcctttgaga acctagaaat catacgcggc aggaccaagc aacatggtca 5160 gttttctctt gcagtcgtca gcctgaacat aacatccttg ggattacgct ccctcaagga 5220 gataagtgat ggagatgtga taatttccgg aaacaaaaat ttgtgctatg caaatacaat 5280 aaactggaaa aaactgtttg ggacctccgg tcagaaaacc aaaattataa gcaacagagg 5340 tgaaaacagc tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg 5400 ctggggcccg gagcccaggg actgcgtctc ttgccggaat gtcagccgag gcagggaatg 5460 cgtggacaag tgcaaccttc tggagggtga gccaagggag tttgtggaga actctgagtg 5520 catacagtgc cacccagagt gcctgcctca ggccatgaac atcacctgca caggacgggg 5580 accagacaac tgtatccagt gtgcccacta cattgacggc ccccactgcg tcaagacctg 5640 cccggcagga gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca 5700 tgtgtgccac ctgtgccatc caaactgcac ctacggatgc actgggccag gtcttgaagg 5760 ctgtccaacg aatgggccta agatcccgtc catcgccact gggatggtgg gggccctcct 5820 cttgctgctg gtggtggccc tggggatcgg cctcttcatg tgaactagtg cggccgccgt 5880 ttaaacggcc ggccgcggtc tgtacaagta ggattcgtcg agggacctaa taacttcgta 5940 tagcatacat tatacgaagt tatacatgtt taagggttcc ggttccacta ggtacaattc 6000 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt 6060 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat 6120 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg 6180 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt 6240 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact 6300 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc 6360 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg 6420 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc 6480 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg 6540 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc 6600 tccccgcatc gataccgtcg acctcgatcg agacctagaa aaacatggag caatcacaag 6660 tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga 6720 ggtgggtttt ccagtcacac ctcatgtacc tttaagacca atgacttaca aggcagctgt 6780 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg 6840 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca 6900 gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct 6960 agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt 7020 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt 7080 tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc 7140 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 7200 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 7260 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagcat 7320 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 7380 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7440 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7500 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7560 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7620 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7680 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7740 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7800 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7860 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 7920 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 7980 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 8040 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 8100 aatctaaagt atatatgagt aaacttggtc tgacagctag aaaaactcat cgagcatcaa 8160 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt 8220 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg 8280 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat 8340 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag 8400 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc 8460 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg 8520 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc 8580 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt 8640 tttcccaggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt 8700 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac 8760 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc 8820 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc 8880 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg 8940 aatatggctc ata 8953

<210> 2 <211> 9848 <212> DNA <213> Artificial sequence

<400> 2 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 60 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaatccgcg cacatttccc 120 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc 180 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt 240 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg 300 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg 360 ccagatatcg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc 420 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 480 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 540 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 600 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 660 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 720 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 780 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 840 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 900 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt 960 ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 1020 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg 1080 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga 1140 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct 1200 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact 1260 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag 1320 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg 1380 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt 1440 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct 1500 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac 1560 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat 1620 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc 1680 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta 1740 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga 1800 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc 1860 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata 1920 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc 1980 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag 2040 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg 2100 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg 2160 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa 2220 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt 2280 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata 2340 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt 2400 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac 2460 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta 2520 gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa 2580 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 2640 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg 2700 ggtttattac agggacagca gagatccagt ttggttagta ccgggcccgc tctagaggat 2760 ccggctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag 2820 aagtatgcaa agcatgcgtc gacgataagc tttgcaaaga tggataaagt tttaaacaga 2880 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg 2940 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt 3000 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt 3060 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc 3120 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg 3180 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac 3240 ctggctgcag tacgtgattc ttgatcccga gcttcggggt tggaagtggg tgggagagtt 3300 cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc 3360 gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata 3420 agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata 3480 gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg 3540 gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc 3600 caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg 3660 cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagtagcgt 3720 gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc 3780 gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag 3840 ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct 3900 cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc 3960 cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct 4020 tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc 4080 aaagtttttt tcttccattt caggtgtcgt gaggaatttc gacatttaaa tttaattaag 4140 ccaccatgct gctgctggtg accagcctgc tgctgtgcga gctgccccac cccgcctttc 4200 tgctgatccc cgacatccag atgacccaga ccacctccag cctgagcgcc agcctgggcg 4260 accgggtgac catcagctgc cgggccagcc aggacatcag caagtacctg aactggtatc 4320 agcagaagcc cgacggcacc gtcaagctgc tgatctacca caccagccgg ctgcacagcg 4380 gcgtgcccag ccggtttagc ggcagcggct ccggcaccga ctacagcctg accatctcca 4440 acctggaaca ggaagatatc gccacctact tttgccagca gggcaacaca ctgccctaca 4500 cctttggcgg cggaacaaag ctggaaatca ccggcagcac ctccggcagc ggcaagcctg 4560 gcagcggcga gggcagcacc aagggcgagg tgaagctgca ggaaagcggc cctggcctgg 4620 tggcccccag ccagagcctg agcgtgacct gcaccgtgag cggcgtgagc ctgcccgact 4680 acggcgtgag ctggatccgg cagcccccca ggaagggcct ggaatggctg ggcgtgatct 4740 ggggcagcga gaccacctac tacaacagcg ccctgaagag ccggctgacc atcatcaagg 4800 acaacagcaa gagccaggtg ttcctgaaga tgaacagcct gcagaccgac gacaccgcca 4860 tctactactg cgccaagcac tactactacg gcggcagcta cgccatggac tactggggcc 4920 agggcaccag cgtgaccgtg agcagcacta ccccagcacc gcggccaccc accccggctc 4980 ctaccatcgc ctcccagcct ctgtccctgc gtccggaggc atgtagaccc gcagctggtg 5040 gggccgtgca tacccggggt cttgacttcg cctgcgatat ctacatttgg gcccctctgg 5100 ctggtacttg cggggtcctg ctgctttcac tcgtgatcac tctttactgt aagcgcggtc 5160 ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag actactcaag 5220 aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc gaactgcgcg 5280 tgaaattcag ccgcagcgca gatgctccag cctacaagca ggggcagaac cagctctaca 5340 acgaactcaa tcttggtcgg agagaggagt acgacgtgct ggacaagcgg agaggacggg 5400 acccagaaat gggcgggaag ccgcgcagaa agaatcccca agagggcctg tacaacgagc 5460 tccaaaagga taagatggca gaagcctata gcgagattgg tatgaaaggg gaacgcagaa 5520 gaggcaaagg ccacgacgga ctgtaccagg gactcagcac cgccaccaag gacacctatg 5580 acgctcttca catgcaggcc ctgccgcctc ggctcgaggg cggcggagag ggcagaggaa 5640 gtcttctaac atgcggtgac gtggaggaga atcccggccc taggatgctt ctcctggtga 5700 caagccttct gctctgtgag ttaccacacc cagcattcct cctgatccca cgcaaagtgt 5760 gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct acgaatatta 5820 aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg gtggcattta 5880 ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat attctgaaaa 5940 ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac aggacggacc 6000 tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat ggtcagtttt 6060 ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc aaggagataa 6120 gtgatggaga tgtgataatt tccggaaaca aaaatttgtg ctatgcaaat acaataaact 6180 ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac agaggtgaaa 6240 acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag ggctgctggg 6300 gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg gaatgcgtgg 6360 acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct gagtgcatac 6420 agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga cggggaccag 6480 acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag acctgcccgg 6540 caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc ggccatgtgt 6600 gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt gaaggctgtc 6660 caacgaatgg gcctaagatc ccgtccatcg ccactgggat ggtgggggcc ctcctcttgc 6720 tgctggtggt ggccctgggg atcggcctct tcatgtgaac tagtgcggcc gccgtttaaa 6780 cggccggccg cggtctgtac aagtaggatt cgtcgaggga cctaataact tcgtatagca 6840 tacattatac gaagttatac atgtttaagg gttccggttc cactaggtac aattcgatat 6900 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 6960 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 7020 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct 7080 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 7140 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc 7200 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 7260 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt 7320 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 7380 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 7440 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc 7500 gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca 7560 atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg 7620 gttttccagt cacacctcat gtacctttaa gaccaatgac ttacaaggca gctgtagatc 7680 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac 7740 aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact 7800 acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac 7860 cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc 7920 ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca 7980 gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct 8040 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc 8100 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg 8160 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agcatgtgag 8220 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 8280 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 8340 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 8400 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 8460 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 8520 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 8580 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 8640 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 8700 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 8760 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 8820 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 8880 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 8940 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 9000 aaagtatata tgagtaaact tggtctgaca gctagaaaaa ctcatcgagc atcaaatgaa 9060 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta 9120 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg 9180 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt 9240 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat 9300 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg 9360 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc 9420 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg 9480 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc 9540 cagggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg 9600 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat 9660 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca 9720 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata 9780 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat 9840 ggctcata 9848

<210>3 <211> 870 <212> PRT <213> Artificial sequence

<400>3

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala 145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr 225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Pro Ala Pro 260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 275 280 285

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 290 295 300

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 305 310 315 320

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys 325 330 335

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 340 345 350

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 355 360 365

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 370 375 380

Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu 385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg Leu Glu Gly Gly Gly Glu Gly 485 490 495

Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 500 505 510

Arg Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His 515 520 525

Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile 530 535 540

Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His 545 550 555 560

Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val 565 570 575

Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln 580 585 590

Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu 595 600 605

Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn 610 615 620

Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu 625 630 635 640

Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys 645 650 655

Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys 660 665 670

Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln 675 680 685

Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr 690 695 700

Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro 705 710 715 720

Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu 725 730 735

Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val 740 745 750

Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala 755 760 765

Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys 770 775 780

Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly 785 790 795 800

Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly 805 810 815

His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly 820 825 830

Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile 835 840 845

Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu 850 855 860

Gly Ile Gly Leu Phe Met 865 870

Claims (16)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A nucleic acid molecule comprising a MND promoter driving the expression of a CAR structure

and a CAR structure-encoding nucleic acid located downstream of the MND promoter; wherein the

nucleic acid molecule comprises SEQ ID NO: 1.

2. A recombinant vector comprising a nucleotide sequence of SEQ ID NO: 1.

3. The recombinant vector according to claim 2, wherein the recombinant vector is a lentiviral

recombinant vector.

4. A recombinant microorganism packaged from the recombinant vector according to claim 2 or 3.

5. The recombinant microorganism according to claim 4, wherein the recombinant microorganism

is a recombinant lentivirus.

6. A recombinant cell comprising the nucleic acid molecule of claim 1, the recombinant vector of

claim 2 or 3, or the recombinant microorganism of claim 4 or 5.

7. The recombinant cell according to claim 6, wherein the recombinant cell is an immune cell.

8. The recombinant cell according to claim 7, wherein the immune cells include cytotoxic T

lymphocytes, NKT cells, NK cells, helper T cells, or gamma delta T cells.

9. A product for treating tumors or having a function for treating tumors with low cytokine storm

comprising the nucleic acid molecule of claim 1, the recombinant vector of claim 2 or 3, the

recombinant microorganism of claim 4 or 5, or the recombinant cell of claim 6 or 7.

10. The product according to claim 9, wherein the product is a pharmaceutical composition

comprising as an active ingredient the nucleic acid molecule of claim 1, the recombinant vector of

claim 2 or 3, the recombinant microorganism of claim 4 or 5, or the recombinant cell of claim 6 or

7.

11. A method, comprising any one of the methods of:

1) a method of preparing a CAR-T cell, comprising the steps of introducing the nucleic acid

molecule of claim 1 or the recombinant vector of claim 2 or 3 into T cells, or infecting T cells with

the recombinant microorganism of claim 4 or 5 to express the CAR structure of claim 1;

2) a method of reducing the cytokine storm production in CAR-T cell therapy by driving the

expression of the CAR structure of claim 1 in T cells with the MND promoter of claim 1; and

3) a method of treating a hematological tumor comprising the steps of administering to a subject in

need thereof T cells which drive the expression of the CAR structure of claim 1 with the MND promoter of claim 1.

12. The method according to claim 11, wherein the method 2) comprises the steps of:

a) preparing the recombinant vector of claim 2 or 3;

b) introducing the recombinant vector into T cells to obtain CAR-T cells; and

c) reinfusing the CAR-T cells back into the body to reduce cytokine storm production in CAR-T

cell therapy;

or the method 2) comprises the steps of:

d) preparing the recombinant microorganism of claim 4 or 5;

e) infecting T cells with the recombinant microorganism to obtain CAR-T cells; and

f) reinfusing the CAR-T cells back into the body to reduce cytokine storm production in CAR-T cell

therapy.

13. The method according to claim 11, wherein the method 3) comprises the steps of:

a) preparing the recombinant vector according to claim 2 or 3;

b) introducing the recombinant vector into T cells to obtain CAR-T cells; and

c) reinfusing the CAR-T cells back into the body for the treatment of tumors;

or the method 3) comprises the steps of:

d) preparing the recombinant microorganism according to claim 4 or 5;

e) infecting T cells with the recombinant microorganism to obtain CAR-T cells; and

f) reinfusing the CAR-T cells back into the body for the treatment of hematological tumors.

14. The method according to claim 11, wherein hematological tumors comprise leukemia,

lymphoma, and myeloma.

15. A use comprising any one of the uses of:

1) a use of the nucleic acid molecule of claim 1, the recombinant vector of claim 2 or 3, the

recombinant microorganism of claim 4 or 5, and the recombinant cell of claim 6 or 7 in the

preparation of a medicament for the treatment of hematological tumors; and

2) a use of the nucleic acid molecule of claim 1, the recombinant vector of claim 2 or 3, the

recombinant microorganism of claim 4 or 5, and the recombinant cell of claim 6 or 7 in the

preparation of a medicament having a function for the treatment of hematological tumors with low

cytokine storm.

16. The use according to claim 15, wherein hematological tumors comprise leukemia, lymphoma,

and myeloma.