Construction of a chimeric antigen receptor targeting CD20 antigen and identification of its activity in genetically engineered T cells

Abstract

To provide a chimeric antigen receptor targeting CD20 and a preparation method thereof.SOLUTION: An extracellular antigen binding domain of a chimeric antigen receptor includes an antibody heavy chain variable region shown in a specific sequence and an antibody light chain variable region shown in a specific sequence, and is capable of killing tumor cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention provides sequence components of a chimeric antigen receptor that targets the CD20 antigen, and modified T cells thereof. The present invention provides a method for producing and identifying the activity of CD20-positive B-cell lymphoma cells (CART20). The present invention identifies chimeric antigen receptor structures that treat tumors. [Background technology]

[0002] Hematological malignancies account for approximately 10% of human malignant tumors, and 95% of hematological malignancies are B-lymphoma. Conventional chemotherapy and radiation therapy are important for the treatment of hematological malignancies. It plays an important role in the treatment of rheumatoid arthritis, and while some patients show remarkable therapeutic effects, the majority are difficult to cure. Effective treatments have been the focus of research in this field.

[0003] Adoptive T cell therapy has already demonstrated strong therapeutic efficacy and promising clinical applications in the treatment of malignant tumors. Among them, chimeric antigen receptors evolved independently by several mechanisms. T cells modified with chimeric antigen receptor (CAR) express CD19. He achieved unprecedented success in refractory B-cell malignancies, particularly at the University of Pennsylvania School of Medicine. CART19, which was developed by the In a clinical trial of this drug, 94% of patients achieved complete relief. However, nearly 40% of patients experienced complete relief within one month of treatment, followed by relapse and recurrence. Among these patients, as many as 60% showed immune escape of CD19-negative tumor cells, leading to malignant risk. C-cell lymphoma-associated antigens other than CD19 are targeted to treat patients with lymphoma. Screening of ART structures is highly desirable.

[0004] CD20 is a glycosylated protein and the first membrane marker identified for B cells, designated B1. The CD20 molecule has four transmembrane hydrophobic regions and its N Both the C-terminus and the C-terminus are located on one side of the cytoplasm, forming two loops outside the cell. These are called the large and small loops, respectively. CD20 specifically binds to more than 95% of normal and malignant B cells. It is expressed in the cytoplasm of cells, which are pre-B cells and later in development, and differentiate into plasma cells. Therefore, CD20 is an ideal target for immunotherapy of B-cell malignancies. It's a target.

[0005] Rituximab (MabThera®, Rituxan®) is the first to be approved by the US FDA and the European Medicines Agency First-generation CD20-targeted chimeric mouse approved for treating indolent lymphoma Rituximab is a monoclonal antibody that recognizes and binds to the large loop structure of the CD20 extracellular domain. However, rituximab alone does not When used alone, it has limited activity and short duration of response, but when combined with chemotherapy, Rituximab is used to treat lymphoma, and half of patients Patients have a complete (CR) or partial (PR) response.

[0006] Ofatumumab (Arzerra R ) is the first fully human CD20 therapeutic antibody. Unlike rituximab, the epitope recognized by ofatumumab is a major target of CD20. At the same time, the tumor-killing mechanism of ofatumumab is The tumor killing activity is primarily via a complement-dependent pathway, followed by ADCC-dependent tumor killing.

[0007] Obinutuzumab (Gazyvaro) R , Gazyva R ) is a humanized, fucosylated It is a type II anti-CD20 antibody with optimized FcγRIIIa affinity by reducing the antibody level. Izamab recognizes and binds to the large loop of the CD20 extracellular molecule, and inhibits tumor growth primarily through ADCC. The binding of obinutuzumab to CD20 mediates tumor cell killing, and simultaneously induces tumor cell apoptosis. Obinutuzumab is effective in treating NHL that is resistant to rituximab treatment. Phase III clinical trials using cyclosporine in combination with bendamustine, a nitrogen mustard drug. In the study, the combination of obinutuzumab and bendamustine was more effective than bendamustine alone Progression-free survival was approximately one-fold longer (29 months in the former and 14 months in the latter). When treating CLL, mab achieved an overall response rate (ORR, including CR and PR) of 77.3%, and The rate for mab was 65.7%.

[0008] Compared with therapeutic antibodies, cellular immunotherapy has significant therapeutic effects and is a new approach to tumor treatment. This is a novel method of anti-cancer treatment through autoimmunity. This method involves culturing and amplifying immune cells collected from the body outside the body and then returning them to the patient. The purpose of treating tumors is achieved by activating and enhancing the autoimmune function of the immune system. This will be incorporated into the development of new cellular immunotherapies, improving their efficacy and promoting their side effects. In recent years, many therapeutic antibodies such as those mentioned above have been developed, but their clinical applications have not been fully evaluated. None of the treatments have achieved the same therapeutic effect as CART19. The development of CART therapy targeting this has enormous commercial value and social significance. Summary of the Invention

[0009] Therapeutic antibodies targeting CD20 differ in affinity and killing mechanism, A series of chimeric antigen receptors targeting CD20 were constructed using the DNA sequence of the antigen-binding region of antibodies. We constructed chimeric antigen receptor-modified T cells and identified their in vitro antitumor activity. and clinical efficacy of CAR-T in the treatment of CD20-positive leukemia and lymphoma. and providing new methods and formulations. The object of the present invention is to provide a chimeric antigen receptor that targets CD20, and methods for producing and using the same. To do this.

[0010] The present invention relates to the construction of a chimeric antigen receptor targeting the CD20 antigen, The present invention relates to a method for producing genetically modified T cells with chimeric antigen receptors and to identifying their activity.

[0011] In a first aspect of the present invention, there is provided a chimeric antigen receptor (CAR) (sequence), wherein said chimeric antigen receptor The antigen-binding domain (i.e., scFv) of the receptor is that of the antibody shown in SEQ ID NO: 7, 9, or 33. The heavy chain variable region and the light chain variable region of the antibody set forth in SEQ ID NO: 11, 13, or 35. Provides receptors. In another preferred embodiment, the antigen-binding domain of the chimeric antigen receptor has the following formula: It is expressed as: V H -VL (However, V H is the heavy chain variable region of an antibody, and V L indicates the light chain variable region of the antibody, and "-" indicates the linked peptide. tide or peptide bond. In another preferred embodiment, the amino acid sequence of the connecting peptide is set forth in SEQ ID NO: 15. do. In another preferred embodiment, V H The amino acid sequence of is shown in SEQ ID NO: 7, and V L Net The amino acid sequence is shown in SEQ ID NO:11. In another preferred embodiment, V H The amino acid sequence of is shown in SEQ ID NO: 9, and V L Net The amino acid sequence is shown in SEQ ID NO:13. In another preferred embodiment, V H The amino acid sequence of is shown in SEQ ID NO: 33, and V L Net The amino acid sequence is shown in SEQ ID NO:35. In another preferred embodiment, the structure of the chimeric antigen receptor is represented by the following formula: LV H -V L -H-TM-CS-CD3ζ (however, L is an optional leader sequence (ie, signal peptide sequence). H is the hinge region. TM is the transmembrane domain. CS is a costimulatory molecule derived from 4-1BB and / or CD28. CD3ζ is an intracellular signaling sequence derived from CD3ζ. V H , V L and "-" are as above.) In another preferred embodiment, the sequence of L is set forth in SEQ ID NO:27. In another preferred embodiment, the sequence of H is set forth in SEQ ID NO: 17 or 19. In another preferred embodiment, the TM sequence is a transmembrane domain of CD8a or a transmembrane domain of CD28. Preferably, the TM sequence is set forth in SEQ ID NO: 21 or 37.

[0012] In another preferred embodiment, the CS structure is CD28-4-1BB, wherein CD28 is derived from CD28. 4-1BB is a costimulatory molecule derived from 4-1BB. In another preferred embodiment, the sequence of the 4-1BB-derived costimulatory molecule is set forth in SEQ ID NO: 23. . In another preferred embodiment, the sequence of the CD28-derived costimulatory molecule is set forth in SEQ ID NO:39. In another preferred embodiment, the sequence of CD3ζ is shown in SEQ ID NO:25. In another preferred embodiment, the sequence of the chimeric antigen receptor is SEQ ID NO: 1, 3, 5, 29, 3 It is indicated by 1.

[0013] In a second aspect of the present invention, a chimeric antigen receptor (CAR) according to the first aspect of the present invention is used as a covalent fusion protein. Nucleic acid molecules that encode the nucleic acid sequences are provided. In another preferred embodiment, the nucleic acid molecule comprises a hinge region selected from the group consisting of: The nucleic acid sequence encoding: (a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 17 or 19; (b) a polynucleotide whose sequence is set forth in SEQ ID NO: 18 or 20; (c) The nucleotide sequence has a homology of ≥ 90% (preferably ≥ 18 or ≥ 20) with the sequence set forth in SEQ ID NO: 18 or 20. Preferably, the polynucleotide has a repeat sequence of ≥ 95% and encodes the amino acid sequence shown in SEQ ID NO: 17 or 19. nucleotide; (d) a polynucleotide complementary to any one of the polynucleotides (a) to (c) .

[0014] In another preferred embodiment, the nucleic acid molecule is a transmembrane domain of the CD8a selected from the group consisting of: The nucleic acid sequence encoding the consensus region includes: (a) a polynucleotide encoding the polypeptide represented by SEQ ID NO: 21; (b) a polynucleotide whose sequence is set forth in SEQ ID NO: 22; (c) the nucleotide sequence has a homology of ≥ 90% (preferably ≥ 10%) with the sequence set forth in SEQ ID NO: 22 ≥ 95%) and encoding the amino acid sequence set forth in SEQ ID NO: 21; (d) a polynucleotide complementary to any one of the polynucleotides (a) to (c) .

[0015] In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of 4-1BB (CD13 7) A nucleic acid sequence encoding the intracellular signaling domain of: (a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 23; (b) a polynucleotide whose sequence is set forth in SEQ ID NO: 24; (c) the nucleotide sequence has a homology of ≥ 90% (preferably ≥ 10%) with the sequence set forth in SEQ ID NO: 24 ≥ 95%) and encoding the amino acid sequence set forth in SEQ ID NO: 23; (d) a polynucleotide complementary to any one of the polynucleotides (a) to (c) .

[0016] In another preferred embodiment, the nucleic acid molecule is a nucleic acid molecule encoding a CD28 cell selected from the group consisting of: The nucleic acid sequence encoding the inner signal domain includes: (a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 39; (b) a polynucleotide whose sequence is set forth in SEQ ID NO: 40; (c) the nucleotide sequence has a homology of ≥ 90% (preferably ≥ 10%) with the sequence set forth in SEQ ID NO: 40 ≥ 95%) and encoding the amino acid sequence set forth in SEQ ID NO: 39; (d) a polynucleotide complementary to any one of the polynucleotides (a) to (c) .

[0017] In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of: The nucleic acid sequence encoding the intracellular signaling domain includes: (a) a polynucleotide encoding the polypeptide represented by SEQ ID NO: 25; (b) a polynucleotide whose sequence is set forth in SEQ ID NO: 26; (c) the nucleotide sequence has a homology of ≥ 90% (preferably ≥ 10%) with the sequence set forth in SEQ ID NO: 26 ≥ 95%) and encoding the amino acid sequence set forth in SEQ ID NO: 25; (d) a polynucleotide complementary to any one of the polynucleotides (a) to (c) .

[0018] In another preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of: : (a) a polynucleotide encoding a polypeptide set forth in SEQ ID NO: 1, 35, 29, or 31; Do; (b) a polynucleotide whose sequence is set forth in SEQ ID NO: 2, 4, 6, 30 or 32; (c) the nucleotide sequence has homology with the sequence set forth in SEQ ID NO: 2, 4, 6, 30 or 32 ≥ 95% (preferably ≥ 98%) and the amino acid sequence shown in SEQ ID NO: 1, 35, 29 or 31 a polynucleotide encoding the sequence; (d) a polynucleotide complementary to any one of the polynucleotides (a) to (c) . In another preferred embodiment, the nucleic acid molecule is isolated.

[0019] In another preferred embodiment, the nucleic acid molecule further comprises a leader sequence. a polynucleotide encoding a leader sequence (a signal sequence) The amino acid sequence is shown in SEQ ID NO: 27, and preferably contains the leader sequence (signal sequence). The corresponding polynucleotide is shown in SEQ ID NO:28. In another preferred embodiment, the sequence of the nucleic acid molecule is set forth in SEQ ID NO: 2, 46, 30 or 32. will be done.

[0020] In a third aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention. do. In another preferred embodiment, the vector is a lentiviral vector. In a fourth aspect of the present invention, a vector is provided which comprises or is a vector according to the third aspect of the present invention. or a host cell having an exogenous nucleic acid molecule according to the second aspect of the invention integrated into its chromosome. do. In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically modified cells. In another preferred embodiment, the cells are mammalian cells. In another preferred embodiment, the cell is a T cell.

[0021] In a fifth aspect of the present invention, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a compound according to the first aspect of the present invention. a nucleic acid molecule according to the second aspect of the invention; a vector according to the third aspect of the invention; The present invention also provides a pharmaceutical composition comprising a vector or a cell according to the fourth aspect of the present invention.

[0022] In a sixth aspect of the present invention, a chimeric antigen receptor according to the first aspect of the present invention, A nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a vector according to the fourth aspect of the invention. Use of the cells according to aspect 1 in a drug or preparation for treating a tumor or an autoimmune disease. The present invention provides a use of the compound in the manufacture of a In another preferred embodiment, the autoimmune disease is an autoimmune disease caused by overexpression of B cells. disease (e.g. lupus erythematosus). In another preferred embodiment, the tumor is a CD20-positive tumor.

[0023] In a seventh aspect of the present invention, there is provided a method of treating a disease, comprising administering to a subject in need of treatment an appropriate amount of the present invention. A chimeric antigen receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, A vector according to the third aspect of the invention, or a cell according to the fourth aspect of the invention, or A method is provided which comprises administering a pharmaceutical composition according to the fifth aspect of the invention. In another preferred embodiment, the disease is a tumor.

[0024] In an eighth aspect of the present invention, a CA expressing a chimeric antigen receptor according to the first aspect of the present invention is provided. A method for producing RT cells (CAR-modified T cells) is provided.

[0025] A nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention may be used in a T obtaining said CAR-T cells by transducing said CAR-T cells into cells;

[0026] Of course, within the scope of the present invention, the above-mentioned technical features of the present invention and the following (for example Each technical feature specifically described in the embodiments may be combined with each other to form a new or preferred technology. It is understood that a surgical plan can be constructed, but due to space limitations, it will not be explained here one by one. [Brief explanation of the drawings]

[0027] DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the structure of a chimeric antigen receptor (CAR) targeting CD20. Each element of the designed CAR structure is shown in the figure, including the leader sequence, antigen recognition sequence (ofatumumab, obinutuzumab, or rituximab), hinge region, transmembrane region, costimulatory signaling region, and CD3ζ signaling region. CAR-T20.14, CAR-T20.13, and CAR-T20.16 are CAR structures constructed based on the variable regions of the antibodies of ofatumumab, obinutuzumab, and rituximab, respectively. CAR-T20.19 and CAR-20.20 are CAR-T20.14 with the L235E-N297Q mutation in the IgG4 hinge-CH2-CH3 junction region. CAR-T20.20 is a third-generation chimeric antigen receptor (CAR) structure that also incorporates the coding sequences for the costimulatory signaling molecules CD28 and 4-1BB. [Figure 2] Figure 2 shows the transduction efficiency of gene-modified T cells with a chimeric antigen receptor targeting CD20. Protein L assay was used to determine the expression level of the protein encoded by the CAR gene on the T cell membrane surface in CAR-T20s cells cultured up to day 7 (A) and day 11 (B). [Figure 3] Figure 3 shows that 1x105 NT, CART-20.13, CART-20.14, and CAR-T20.16 cells were cultured up to day 6, and then co-cultured with the CD20-positive RAJI and RAMOS tumor cell lines, and the CD20-negative MOLT-4 tumor cell line, respectively, at a 1:1 ratio in 200 μl of GT-551 medium for 18 hours. The expression level of CD137 on the membrane surface of T cells (A) and the secretion level of IFNγ in the co-culture supernatant (B) were then detected. [Figure 4]Figure 4 shows the level of tumor cell apoptosis induced by CART-20. CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines (1 x 10 cells) were co-cultured for 4 hours with NT, CART-20.13, CART-20.14, and CAR-T20.16 cells, which had been cultured for up to day 11 at the ratios indicated in the figure, in 200 μl of GT-551 medium. The cells were then centrifuged to collect the cell pellet, washed twice with PBS, and stained with Annexin V-APC staining reagent at a 1:50 ratio in 100 μl of staining solution for 30 minutes. After washing once with PBS, the proportion of Annexin V-positive cells among CFSE-positive cells was analyzed by flow cytometry. The results shown in the figure represent statistical analysis results for the Annexin V-positive cells in the corresponding co-culture samples. [Figure 5] Figure 5 shows the in vitro activation ability of hinge region-mutated chimeric antigen receptors and third-generation chimeric antigen receptors constructed based on the sequence of the ofatumumab antibody. The protein L method was used to detect the expression level of the protein encoded by the CAR gene on the T cell surface in CAR-T20.14, CAR-T20.19, and CAR-T20.20 cells cultured up to day 7 (A). NT, CART-20.14, CART-20.14, and CAR-T20.16 cells cultured up to day 7 were selected (1x105 cells), and co-cultured with K562, CD19 monopositive, CD20 monopositive, CD19 and CD20 bipositive K562 stable cells, and RAJI target cells at a 1:1 ratio in 200μl of GT-551 medium for 18 hours. Then, the expression level of CD137 on the T cell surface (B) and the secretion level of IFNγ in the culture supernatant (C) were detected. [Figure 6] Figure 6 shows the results of detecting the ability of CAR-T20 cells to eliminate CD20-positive cells in the body. The results showed that CAR-T20.19 can effectively suppress the proliferation of CD20-positive cells in the body.

[0028] Specific Embodiments Through extensive and in-depth research, the present inventors have discovered chimeric antigen receptors and The extracellular antigen-binding domain of the chimeric antigen receptor is a sequence The heavy chain variable region of the antibody shown in SEQ ID NO: 1 and the light chain variable region of the antibody shown in SEQ ID NO: 2 Experimental results show that tumor cells with very high levels of chimeric antigen receptors provided by the present invention It can be seen that this weapon demonstrated its lethality against

[0029] Therapeutic antibodies targeting CD20 vary in affinity, killing mechanism, and Since the transmembrane domain and the intracellular domain significantly affect the activity of the chimeric antigen receptor, In this study, we used the amino acid sequences of the variable regions of several anti-CD20 antibodies to identify different transmembrane and A series of chimeric antigen receptors that target CD20 and are linked to an intracellular portion are constructed, and Achieving expression of such chimeric antigen receptors in primary T cells and detecting receptor expression intensity We established a method to investigate the ability of these CAR-T cells to recognize the CD20 antigen in vitro and in vivo, and their Identify the differences in ex vivo killing and in vivo elimination of malignant tumors bearing the CD20 antigen, and use them in clinical trials. Novel and effective methods and formulations for the treatment of CD20-positive leukemia and lymphoma with CAR-T in to provide.

[0030] Chimeric Antigen Receptor The present invention provides a chimeric antigen comprising an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain provides a target-specific binding element (antigen binding domain). The intracellular domain contains the co-stimulatory signaling region and the ζ chain region. The costimulatory signaling region comprises a portion of the intracellular domain of a costimulatory molecule. Stimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, and are antigen receptors or is not its ligand.

[0031] Between the extracellular and transmembrane domains of CAR, or between the intracellular and transmembrane domains of CAR A linker may be introduced between the two. As used herein, the term "linker" " refers to the extracellular or intracellular domain of a polypeptide chain. A linker is any oligopeptide or polypeptide that serves to link a 0 to 300 amino acids, preferably 2 to 100 amino acids, most preferably 3 to 50 amino acids It may also contain acetic acid.

[0032] In one preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention The CAR of the present invention comprises an antigen-binding domain that targets CD20. When it binds to a related antigen, it can recognize the antigen based on the specificity of antigen binding. When combined, they affect tumor cells, causing them to stop growing, die, or otherwise The antigen-binding domain is linked to a costimulatory molecule, and the patient's tumor burden is reduced or eliminated. Preferably, the fusion is with one or more intracellular domains derived from the ζ and ζ chains. Preferably, the antigen-binding domain comprises a 4-1BB signaling domain and / or a CD28 signaling domain. The transduction domain is fused to an intracellular domain combined with a CD3ζ signaling domain.

[0033] In one embodiment, the CD20-targeting CAR of the present invention is a specific signal transduction vector of the present invention. The transduction domain (the transmembrane domain of CD8, the intracellular signaling domains of CD137 and CD3ζ) is connected in series. Compared to other forms of CD20-targeting CARs, the signal The transduction domain improves the antitumor activity and persistence of CAR-T cells in vivo.

[0034] In one preferred embodiment of the present invention, the chimeric antigen receptor provided by the present invention The amino acid sequence of (CAR) is as follows: CAR-T20.13 (SEQ ID NO: 29) MALPVTALLL PLALLLHAAR PQVQLVQSGA EVKKPGSSVK VSCKASGYAF SYSWINWVRQ 60 APGQGLEWMG RIFPGDGDTD YNGKFKGRVT ITADKSTSTA YMELSSLRSE DTAVYYCARN 120 VFDGYWLVYW GQGTLVTVSS GGGGSGGGGS GGGGSDIVMT QTPLSLPVTP GEPASISCRS 180 SKSLLHSNGI TYLYWYLQKP GQSPQLLIYQ MSNLVSGVPD RFSGSGSGTD FTLKISRVEA 240 EDVGVYYCAQ NLELPYTFGG GTKVEIKRTV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK 300 DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV 360 LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPSQEEM TKNQVSLTCL 420 VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM 480 HEALHNHYTQ KSLSLSLGKI YIWAPLAGTC GVLLLSLVIT LYCKRGRKKL LYIFKQPFMR 540 PVQTTQEEDG CSCRFPEEEE GGCELRVKFS RSADAPAYKQ GQNQLYNELN LGRREEYDVL 600 DKRRGRDPEM GGKPRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG HDGLYQGLST 660 ATKDTYDALH MQALPPR 677

[0035] The DNA sequence encoding CAR-T20.13 (SEQ ID NO: 30) is as follows: atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcaggtgc aattggtgca gtctggcgct gaagttaaga agcctgggag ttcagtgaag 120 gtctcctgca aggcttccgg atacgccttc agctattctt ggatcaattg ggtgcggcag 180 gcgcctggac aagggctcga gtggatggga cggatctttc ccggcgatgg ggatactgac 240 tacaatggga aattcaaggg cagagtcaca attaccgccg acaaatccac tagcacagcc 300 tatatggagc tgagcagcct gagatctgag gacacggccg tgtattactg tgcaagaaat 360 gtctttgatg gttactggct tgtttactgg ggccagggaa ccctggtcac cgtctcctca 420 ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgatat cgtgatgacc 480 cagactccac tctccctgcc cgtcacccct ggagagcccg ccagcattag ctgcaggtct 540 agcaagagcc tcttgcacag caatggcatc acttatttgt attggtacct gcaaaagcca 600 gggcagtctc cacagctcct gatttatcaa atgtccaacc ttgtctctgg cgtccctgac 660 cggttctccg gctccgggtc aggcactgat ttcacactga aaatcagcag ggtggaggct 720 gaggatgttg gagtttatta ctgcgctcag aatctagaac ttccttacac cttcggcgga 780 gggaccaagg tggagatcaa acgtacggtg gagagcaagt acggaccgcc ctgcccccct 840 tgccctgccc ccgagttcct gggcggaccc agcgtgttcc tgttcccccc caagcccaag 900 gacaccctga tgatcagccg gacccccgag gtgacctgcg tggtggtgga cgtgagccag 960 gaagatccg aggtccagtt caattggtac gtggacggcg tggaagtgca caacgccaag 1020 accaagccca gagaggaaca gttcaacagc acctaccggg tggtgtctgt gctgaccgtg 1080 ctgcaccagg actggctgaa cggcaaagaa tacaagtgca aggtgtccaa caagggcctg 1140 cccagcagca tcgaaaagac catcagcaag gccaagggcc agcctcgcga gccccaggtg 1200 tacaccctgc ctccctccca ggagagatg accaagaacc aggtgtccct gacctgcctg 1260 gtgaagggct tctaccccag cgacatcgcc gtggagtggg agagcaacgg ccagcctgag 1320 aacaactaca agaccacccc tcccgtgctg gacagcgacg gcagcttctt cctgtacagc 1380 cggctgaccg tggacaagag ccggtggcag gaaggcaacg tctttagctg cagcgtgatg 1440 cacgaggccc tgcacaacca ctacacccag aagagcctga gcctgtccct gggcaagatc 1500 tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 1560 ctttactgca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1620 ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1680 ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1740 ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1800 gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1860 gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1920 atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1980 gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctag 2034

[0036] CAR-T20.14 (SEQ ID NO: 1) MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGRSLR LSCAASGFTF NDYAMHWVRQ 60 APGKGLEWVS TISWNSGSIG YADSVKGRFT ISRDNAKKSL YLQMNSLRAE DTALYYCAKD 120 IQYGNYYYGM DVWGQGTTVT VSSGGGGSGG GGSGGGGSEI VLTQSPATLS LSPGERATLS 180 CRASQSVSSY LAWYQQKPGQ APRLLIYDAS NRATGIPARF SGSGSGTDFT LTISSLEPED 240 FAVYYCQQRS NWPITFGQGT RLEIKESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI 300 SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW 360 LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY 420 PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH 480 NHYTQKSLSL SLGKIYIWAP LAGTCGVLLL SLVITLYCKR GRKKLLYIFK QPFMRPVQTT 540 QEEDGCSCRF PEEEEGGCEL RVKFSRSADA PAYKQGQNQL YNELNLGRRE EYDVLDKRRG 600 RDPEMGGKPR RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT 660 YDALHMQALP PR 672

[0037] The DNA sequence encoding CAR-T20.14 (SEQ ID NO: 2) is as follows: atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 cgggaagtgc agctggtgga gtctggggga ggcttggtac agcctggcag gtccctgaga 120 ctctcctgtg cagcctctgg attcaccttt aatgattatg ccatgcactg ggtccggcaa 180 gctccaggga agggcctgga gtgggtctca actattagtt ggaatagtgg ttccataggc 240 tatgcggact ctgtgaaggg ccgattcacc atctccagag acaacgccaa gaagtccctg 300 tatctgcaaa tgaacagtct gagagctgag gacacggcct tgtattactg tgcaaaagat 360 atacagtacg gcaactacta ctacggtatg gacgtctggg gccaagggac cacggtcacc 420 gtctcctcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgaaatt 480 gtgttgacac agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540 tgcagggcca gtcagagtgt tagcagctac ttagcctggt accaacagaa acctggccag 600 gctcccaggc tcctcatcta tgatgcatcc aacagggcca ctggcatccc agccaggttc 660 agtggcagtg ggtctgggac agacttcact ctcaccatca gcagcctaga gcctgaagat 720 tttgcagttt attactgtca gcagcgtagc aactggccga tcaccttcgg ccaagggaca 780 cgactggaga ttaaaagagag caagtacgga ccgccctgcc ccccttgccc tgccccccgag 840 ttcctgggcg gacccagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 900 agccggaccc ccgaggtgac ctgcgtggtg gtggacgtga gccaggaaga tcccgaggtc 960 cagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1020 gaacagttca acagcaccta ccgggtggtg tctgtgctga ccgtgctgca ccaggactgg 1080 ctgaacggca aagaatacaa gtgcaaggtg tccaacaagg gcctgcccag cagcatcgaa 1140 aagaccatca gcaaggccaa gggccagcct cgcgagcccc aggtgtacac cctgcctccc 1200 tcccaggaag agatgaccaa gaaccaggtg tccctgacct gcctggtgaa gggcttctac 1260 cccagcgaca tcgccgtgga gtgggagagc aacggccagc ctgagaacaa ctacaagacc 1320 acccctcccg tgctggacag cgacggcagc ttcttcctgt acagccggct gaccgtggac 1380 aagagccggt ggcaggaagg caacgtcttt agctgcagcg tgatgcacga ggccctgcac 1440 aaccactaca cccagaagag cctgagcctg tccctgggca agatctacat ctgggcgccc 1500 ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1560 1620 caagagaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1680 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1740 1800 cgggaccctg agatgggggg aaagccgaga aggagaacc ctcaggaagg cctgtacaat 1860 1920 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1980 tacgacgcccc ttcacatgca ggccctgccc cctcgctag 2019

[0038] CAR-T20.16 (SEQ ID NO: 3) MALPVTALLL PLALLLHAAR PQVQLQQPGA ELVKPGASVK MSCKASGYTF TSYNMHWVKQ 60 TPGRGLEWIG AIYPGNGDTS YNQKFKGKAT LTADKSSSTA YMQLSSLTSE DSAVYYCARS 120 TYYGGDWYFN VWGAGTTVTV SAGGGGSGGG GSGGGGSQIV LSQSPAILSA SPGEKVTMTC 180 RASSSVSYIH WFQQKPGSSP KPWIYATSNL ASGVPVRFSG SGSGTSYSLT ISRVEAEDAA 240 TYYCQQWTSN PPTFGGGTKL EIKESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLMISR 300 TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN 360 GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS 420 DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH 480 YTQKSLSLSL GKIYIWAPLA GTCGVLLLSL VITLYCKRGR KKLLYIFKQP FMRPVQTTQE 540 EDGCSCRFPE EEEGGCELRV KFSRSADAPA YKQGQNQLYN ELNLGRREEY DVLDKRRGRD 600 PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYD 660 ALHMQALPPR 670

[0039] The DNA sequence encoding CAR-T20.16 (SEQ ID NO: 4) is as follows: ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG 60 CCGCAGGTGC AGTTGCAACA GCCTGGAGCT GAGTTGGTGA AGCCTGGTGC TTCTGTGAAG 120 ATGTCTTGTA AGGCTTCTGG ATACACATTC ACTTCTTACA ACATGCACTG GGTGAAGCAG 180 ACTCCTGGTA GGGGTTTGGA GTGGATCGGA GCTATCTACC CAGGAAACGG AGACACATCT 240 TACAACCAGA AGTTCAAGGG TAAGGCTACA TTGACTGCTG ACAAGTCTTC ATCTACTGCT 300 TACATGCAAT TGTCTTCTTT GACATCTGAG GACTCTGCAG TTTACTACTG CGCTAGGTCT 360 ACATACTACG GAGGTGACTG GTACTTCAAC GTGTGGGGAG CAGGTACCAC GGTCACTGTC 420 TCTGCAGGTG GAGGTGGATC TGGAGGAGGA GGATCTGGTG GAGGAGGTTC TCAAATTGTT 480 CTCTCCCAGT CTCCAGCAAT CCTGTCAGCT TCTCCTGGAG AGAAGGTGAC TATGACTTGC 540 AGGGCTTCTT CATCTGTTTC TTACATCCAC TGGTTCCAGC AGAAGCCTGG TTCTTCACCT 600 AAGCCTTGGA TCTACGCTAC ATCTAACTTG GCATCTGGAG TGCCTGTGAG GTTCTCTGGT 660 TCTGGTTCAG GTACTTCTTA CTCTTTGACA ATCTCTAGGG TGGAGGCTGA GGACGCTGCT 720 ACTTACTACT GCCAGCAGTG GACATCTAAC CCTCCAACAT TCGGAGGTGG TACTAAGTTG 780 GAGATCAAGG AGAGCAAGTA CGGACCGCCC TGCCCCCCTT GCCCTGCCCC CGAGTTCCTG 840 GGCGGACCCA GCGTGTTCCT GTTCCCCCCC AAGCCCAAGG ACACCCTGAT GATCAGCCGG 900 ACCCCCGAGG TGACCTGCGT GGTGGTGGAC GTGAGCCAGG AAGATCCCGA GGTCCAGTTC 960 AATTGGTACG TGGACGGCGT GGAAGTGCAC AACGCCAAGA CCAAGCCCAG AGAGGAACAG 1020 TTCAACAGCA CCTACCGGGT GGTGTCTGTG CTGACCGTGC TGCACCAGGA CTGGCTGAAC 1080 GGCAAAGAAT ACAAGTGCAA GGTGTCCAAC AAGGGCCTGC CCAGCAGCAT CGAAAAGACC 1140 ATCAGCAAGG CCAAGGGCCA GCCTCGCGAG CCCCAGGTGT ACACCCTGCC TCCCTCCCAG 1200 GAAGAGATGA CCAAGAACCA GGTGTCCCTG ACCTGCCTGG TGAAGGGCTT CTACCCCAGC 1260 GACATCGCCG TGGAGTGGGA GAGCAACGGC CAGCCTGAGA ACAACTACAA GACCACCCCT 1320 CCCGTGCTGG ACAGCGACGG CAGCTTCTTC CTGTACAGCC GGCTGACCGT GGACAAGAGC 1380 CGGTGGCAGG AAGGCAACGT CTTTAGCTGC AGCGTGATGC ACGAGGCCCT GCACAACCAC 1440 TACACCCAGA AGAGCCTGAG CCTGTCCCTG GGCAAGATCT ACATCTGGGC GCCCTTGGCC 1500 GGGACTTGTG GGGTCCTTCT CCTGTCACTG GTTATCACCC TTTACTGCAA ACGGGGCAGA 1560 AAGAAACTCC TGTATATATT CAAACAACCA TTTATGAGAC CAGTACAAAC TACTCAAGAG 1620 GAAGATGGCT GTAGCTGCCG ATTTCCAGAAA GAAGAAGAAG GAGGATGTGA ACTGAGAGTG 1680 AAGTTCAGCA GGAGCGCAGA CGCCCCCGCG TACAAGCAGG GCCAGAACCA GCTCTATAAC 1740 GAGCTCAATC TAGGACGAAG AGAGGAGTAC GATGTTTTGG ACAAGAGACG TGGCCGGGAC 1800 CCTGAGATGG GGGGAAAGCC GAGAAGGAAG AACCCTCAGG AAGGCCTGTA CAATGAACTG 1860 CAGAAAGATA AGATGGCGGA GGCCTACAGT GAGATTGGGA TGAAAGGCGA GCGCCGGAGG 1920 GGCAAGGGGC ACGATGGCCT TTACCAGGGT CTCAGTACAG CCACCAAGGA CACCTACGAC 1980 GCCCTTCACA TGCAGGCCCT GCCCCCTCGC TAG 2013

[0040] In another further preferred embodiment of the present invention, the chimera provided by the present invention The amino acid sequence of the antigen receptor (CAR) is as follows: CAR-T20.19 (SEQ ID NO: 5) MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGRSLR LSCAASGFTF NDYAMHWVRQ 60 APGKGLEWVS TISWNSGSIG YADSVKGRFT ISRDNAKKSL YLQMNSLRAE DTALYYCAKD 120 IQYGNYYYGM DVWGQGTTVT VSSGGGGSGG GGSGGGGSEI VLTQSPATLS LSPGERATLS 180 CRASQSVSSY LAWYQQKPGQ APRLLIYDAS NRATGIPARF SGSGSGTDFT LTISSLEPED 240 FAVYYCQQRS NWPITFGQGT RLEIKESKYG PPCPPCPAPE FEGGPSVFLF PPKPKDTLMI 300 SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFQSTYRVV SVLTVLHQDW 360 LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY 420 PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH 480 NHYTQKSLSL SLGKIYIWAP LAGTCGVLLL SLVITLYCKR GRKKLLYIFK QPFMRPVQTT 540 QEEDGCSCRF PEEEEGGCEL RVKFSRSADA PAYKQGQNQL YNELNLGRRE EYDVLDKRRG 600 RDPEMGGKPR RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT 660 YDALHMQALP PR 672

[0041] The DNA sequence encoding CAR-T20.19 (SEQ ID NO: 6) is as follows. atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggaagtgc agctggtgga gtctggggga ggcttggtac agcctggcag gtccctgaga 120 ctctcctgtg cagcctctgg attcaccttt aatgattatg ccatgcactg ggtccggcaa 180 gctccaggga agggcctgga gtgggtctca actattagtt ggaatagtgg ttccataggc 240 tatgcggact ctgtgaaggg ccgattcacc atctccagag acaacgccaa gaagtccctg 300 tatctgcaaa tgaacagtct gagagctgag gacacggcct tgtattactg tgcaaaagat 360 atacagtacg gcaactacta ctacggtatg gacgtctggg gccaagggac cacggtcacc 420 gtctcctcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgaaatt 480 gtgttgacac agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540 tgcagggcca gtcagagtgt tagcagctac ttagcctggt accaacagaa acctggccag 600 gctcccaggc tcctcatcta tgatgcatcc aacagggcca ctggcatccc agccaggttc 660 agtggcagtg ggtctgggac agacttcact ctcaccatca gcagcctaga gcctgaagat 720 tttgcagttt attactgtca gcagcgtagc aactggccga tcaccttcgg ccaagggaca 780 cgactggaga ttaaaagagag caagtacgga ccgccctgcc ccccttgccc tgccccccgag 840 ttcgagggcg gacccagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 900 agccggaccc ccgaggtgac ctgcgtggtg gtggacgtga gccaggaaga tcccgaggtc 960 cagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1020 gaacagttcc aaagcaccta ccgggtggtg tctgtgctga ccgtgctgca ccaggactgg 1080 ctgaacggca aagaatacaa gtgcaaggtg tccaacaagg gcctgcccag cagcatcgaa 1140 aagaccatca gcaaggccaa gggccagcct cgcgagcccc aggtgtacac cctgcctccc 1200 tcccaggaag agatgaccaa gaaccaggtg tccctgacct gcctggtgaa gggcttctac 1260 cccagcgaca tcgccgtgga gtgggagagc aacggccagc ctgagaacaa ctacaagacc 1320 acccctcccg tgctggacag cgacggcagc ttcttcctgt acagccggct gaccgtggac 1380 aagagccggt ggcaggaagg caacgtcttt agctgcagcg tgatgcacga ggccctgcac 1440 aaccactaca cccagaagag cctgagcctg tccctgggca agatctacat ctgggcgccc 1500 ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1560 ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1620 caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1680 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1740 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1800 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1860 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1920 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1980 tacgacgccc ttcacatgca ggccctgccc cctcgctag 2019

[0042] In another most preferred embodiment of the present invention, the chimeric antibodies provided by the present invention The amino acid sequence of the original receptor (CAR) is as follows: CAR-T20.20 (SEQ ID NO: 31) MALPVTALLL PLALLLHAAR PEVQLVESGG GLVQPGRSLR LSCAASGFTF NDYAMHWVRQ 60 APGKGLEWVS TISWNSGSIG YADSVKGRFT ISRDNAKKSL YLQMNSLRAE DTALYYCAKD 120 IQYGNYYYGM DVWGQGTTVT VSSGGGGSGG GGSGGGGSEI VLTQSPATLS LSPGERATLS 180 CRASQSVSSY LAWYQQKPGQ APRLLIYDAS NRATGIPARF SGSGSGTDFT LTISSLEPED 240 FAVYYCQQRS NWPITFGQGT RLEIKESKYG PPCPPCPAPE FEGGPSVFLF PPKPKDTLMI 300 SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFQSTYRVV SVLTVLHQDW 360 LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY 420 PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH 480 NHYTQKSLSL SLGKFWVLVV VGGVLACYSL LVTVAFIIFW VRSKRSRLLH SDYMNMTPRR 540 PGPTRKHYQP YAPPRDFAAY RSKRGRKKLL YIFKQPFMRP VQTTQEEDGC SCRFPEEEEG 600 GCELRVKFSR SADAPAYKQG QNQLYNELNL GRREEYDVLD KRRGRDPEMG GKPRRKNPQE 660 GLYNELQKDK MAEAYSEIGM KGERRRGKGH DGLYQGLSTA TKDTYDALHM QALPPR 716

[0043] The DNA sequence encoding CAR-T20.20 (SEQ ID NO: 32) is as follows: atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 cgggaagtgc agctggtgga gtctggggga ggcttggtac agcctggcag gtccctgaga 120 ctctcctgtg cagcctctgg attcaccttt aatgattatg ccatgcactg ggtccggcaa 180 gctccaggga agggcctgga gtgggtctca actattagtt ggaatagtgg ttccataggc 240 tatgcggact ctgtgaaggg ccgattcacc atctccagag acaacgccaa gaagtccctg 300 tatctgcaaa tgaacagtct gagagctgag gacacggcct tgtattactg tgcaaaagat 360 atacagtacg gcaactacta ctacggtatg gacgtctggg gccaagggac cacggtcacc 420 gtctcctcag gtggcggtgg ctcgggcggt ggtgggtcgg gtggcggcgg atctgaaatt 480 gtgttgacac agtctccagc caccctgtct ttgtctccag gggaaagagc caccctctcc 540 tgcagggcca gtcagagtgt tagcagctac ttagcctggt accaacagaa acctggccag 600 gctcccaggc tcctcatcta tgatgcatcc aacagggcca ctggcatccc agccaggttc 660 agtggcagtg ggtctgggac agacttcact ctcaccatca gcagcctaga gcctgaagat 720 tttgcagttt attactgtca gcagcgtagc aactggccga tcaccttcgg ccaagggaca 780 cgactggaga ttaaaagagag caagtacgga ccgccctgcc ccccttgccc tgccccccgag 840 ttcgagggcg gacccagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 900 agccggaccc ccgaggtgac ctgcgtggtg gtggacgtga gccaggaaga tcccgaggtc 960 cagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1020 gaacagttcc aaagcaccta ccgggtggtg tctgtgctga ccgtgctgca ccaggactgg 1080 ctgaacggca aagaatacaa gtgcaaggtg tccaacaagg gcctgcccag cagcatcgaa 1140 aagaccatca gcaaggccaa gggccagcct cgcgagcccc aggtgtacac cctgcctccc 1200 tcccaggaag agatgaccaa gaaccaggtg tccctgacct gcctggtgaa gggcttctac 1260 cccagcgaca tcgccgtgga gtgggagagc aacggccagc ctgagaacaa ctacaagacc 1320 accccctcccg tgctggacag cgacggcagc ttctctgt acagccggct gaccgtggac 1380 aagagccggt ggcaggaagg caacgtttt agctgcagcg tgatgcacga ggccctgcac 1440 aaccactaca cccagaagag cctgagcctg tccctgggca agttttggtt gctggtggtg 1500 gttggtggag tcctggctg ctatagcttg ctagtacag tggcctttat tattttctgg 1560 gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 1620 cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 1680 cgctccaaac ggggcagaaa gaactcctg tatatattca aaaaccatt tatgagacca 1740 gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaga agaagaagga 1800 ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1860 cagaaccagc tctataacga gctcaatcta ggacgaagg aggagtacga tgttttggac 1920 aagagacgtg gccgggaccc tgagatgggg ggaagccga gaggaagaaccctcaggaa 1980 ggcctgtaca atgactgca gaagataag atggcggagg cctacagtga gattgggatg 2040 aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 2100 accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 2151

[0044] antigen-binding domain In one embodiment, the CAR of the present invention comprises a target-specific binding domain called an antigen-binding domain. The antigen-binding domain of the CAR of the present invention contains a specific binding element that targets CD4. It is a nt. In one preferred embodiment of the present invention, the antigen-binding domain is a heavy chain variable domain of an anti-CD antibody. The antibody comprises a light chain variable region and a light chain variable region. In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the ofatumumab antibody is as follows: EVQLVESGGG LVQPGRSLRL SCAASGFTFN DYAMHWVRQA PGKGLEWVST ISWNSGSIGY 60 ADSVKGRFTI SRDNAKKSLY LQMNSLRAED TALYYCAKDI QYGNYYYGMD VWGQGTTVTV 120 SS 122 (SEQ ID NO: 7) The DNA sequence encoding the heavy chain variable region of the ofatumumab antibody is as follows: GAAGTGCAGC TGGTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGCAGGTC CCTGAGACTC 60 TCCTGTGCAG CCTCTGGATT CACCTTTAAT GATTATGCCA TGCACTGGGT CCGGCAAGCT 120 CCAGGGAAGG GCCTGGAGTG GGTCTCAACT ATTAGTTGGA ATAGTGGTTC CATAGGCTAT 180 GCGGACTCTG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA GTCCCTGTAT 240 CTGCAAATGA ACAGTCTGAG AGCTGAGGAC ACGGCCTTGT ATTACTGTGC AAAAGATATA 300 CAGTACGGCA ACTACTACTA CGGTATGGAC GTCTGGGGCC AAGGGACCAC GGTCACCGTC 360 TCCTCA 366 (SEQ ID NO: 8) or, The amino acid sequence of the heavy chain variable region of the rituximab antibody is as follows: QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY 60 NQKFKGKATL TADKSSSTAY MQLSSLTSED SAVYYCARST YYGGDWYFNV WGAGTTVTVS 120 A 121 (SEQ ID NO: 9) The DNA sequence encoding the heavy chain variable region of the rituximab antibody is as follows: CAGGTGCAGT TGCAACAGCC TGGAGCTGAG TTGGTGAAGC CTGGTGCTTC TGTGAAGATG 60 TCTTGTAAGG CTTCTGGATA CACATTCACT TCTTACAACA TGCACTGGGT GAAGCAGACT 120 CCTGGTAGGG GTTTGGAGTG GATCGGAGCT ATCTACCCAG GAAACGGAGA CACATCTTAC 180 AACCAGAAGT TCAAGGGTAA GGCTACATTG ACTGCTGACA AGTCTTCATC TACTGCTTAC 240 ATGCAATTGT CTTCTTTGAC ATCTGAGGAC TCTGCAGTTT ACTACTGCGC TAGGTCTACA 300 TACTACGGAG GTGACTGGTA CTTCAACGTG TGGGGAGCAG GTACCACGGT CACTGTCTCT 360 GCA 363 (SEQ ID NO: 10)

[0045] The amino acid sequence of the heavy chain variable region of the obinutuzumab antibody used in the present invention is as follows: That's right. QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA PGQGLEWMGR IFPGDDGDTDY 60 NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNV FDGYWLVYWG QGTLVTVSS 119 (Sequence number No. 33) The DNA sequence encoding the heavy chain variable region of the obinutuzumab antibody is as follows: caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60 tcctgcaagg cttccggata cgccttcagc tattcttgga tcaattgggt gcggcaggcg 120 cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180 aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300 tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357 (SEQ ID NO: 34)

[0046] In another preferred embodiment, the amino acid sequence of the light chain variable region of the ofatumumab antibody is as follows: EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ GTRLEIK 107 (SEQ ID NO.11) The DNA sequence encoding the ofatumumab antibody is as follows: GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC 60 CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG CCTGGTACCA ACAGAAACCT 120 GGCCAGGCTC CCAGGCTCCT CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC 180 AGGTTCAGTG GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT 240 GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGCCGATCAC CTTCGGCCAA 300 GGGACACGAC TGGAGATTAA A 321 (SEQ ID NO: 12) Alternatively, the amino acid sequence of the light chain variable region of the rituximab antibody is as follows: . QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKPG SSPKPWIYAT SNLASGVPVR 60 FSGSGSGTSY SLTISRVEAE DAATYYCQQW TSNPPTFGGG TKLEIK 106 (SEQ ID NO: 13) The DNA sequence encoding the single-chain light chain variable region (VL) derived from the rituximab antibody is as follows: That's right. CAAATTGTTC TCTCCCAGTC TCCAGCAATC CTGTCAGCTT CTCCTGGAGA GAAGGTGACT 60 ATGACTTGCA GGGCTTCTTC ATCTGTTTCT TACATCCACT GGTTCCAGCA GAAGCCTGGT 120 TCTTCACCTA AGCCTTGGAT CTACGCTACA TCTAACTTGG CATCTGGAGT GCCTGTGAGG 180 TTCTCTGGTT CTGGTTCAGG TACTTCTTAC TCTTTGACAA TCTCTAGGGT GGAGGCTGAG 240 GACGCTGCTA CTTACTACTG CCAGCAGTGG ACATCTAACC CTCCAACATT CGGAGGTGGT 300 ACTAAGTTGG AGATCAAG 318 (SEQ ID NO: 14)

[0047] The amino acid sequence of the light chain variable region of the obinutuzumab antibody used in the present invention is as follows: That's right. DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ LLIYQMSNLV 60 SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQNLELP YTFGGGTKVE IKRTV 115 (SEQ ID NO: 35) The DNA sequence encoding the heavy chain variable region of the obinutuzumab antibody is as follows: gatatcgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gcccgccagc 60 attagctgca ggtctagcaa gagcctcttg cacagcaatg gcatcactta tttgtattgg 120 tacctgcaaa agccagggca gtctccacag ctcctgattt atcaaatgtc caaccttgtc 180 tctggcgtcc ctgaccggtt ctccggctcc gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggagtt tattactgcg ctcagaatct agaacttcct 300 tacaccttcg gcggagggac caaggtggag atcaaacgta cggtg 345 (SEQ ID NO: 36) In one preferred embodiment of the present invention, the amino acid sequence between the heavy chain variable region and the light chain variable region is The amino acid linkage sequence is as follows: GGGGSGGGGS GGGGS 15 (SEQ ID NO: 15) The DNA sequence encoding it is as follows: GGTGGCGGTG GCTCGGGCGG TGGTGGGTCG GGTGGCGGCG GATCT 45 (SEQ ID NO: 16)

[0048] Hinge and transmembrane regions The hinge region and transmembrane domain of CAR are fused to the extracellular domain of CAR. In one embodiment, the naturally occurring CA may be designed to contain a transmembrane domain. A transmembrane domain associated with one of the domains in R is used. Such domains can be obtained by selecting transmembrane domains or modifying them with amino acid substitutions. by avoiding binding of proteins to the transmembrane domains of similar or different surface membrane proteins. , minimizing interactions with other members of the receptor complex. In one preferred embodiment of the present invention, the hinge region has the following amino acid sequence: ge-CH2-CH3 hinge region). ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY 60 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 120 AKGQPREPQV YTLPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 180 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 229 (SEQ ID NO: 17)

[0049] The DNA sequence encoding it is as follows: GAGAGCAAGT ACGGACCGCC CTGCCCCCCT TGCCCTGCCC CCGAGTTCCT GGGCGGACCC 60 AGCGTGTTCC TGTTCCCCCC CAAGCCCAAG GACACCCTGA TGATCAGCCG GACCCCCGAG 120 GTGACCTGCG TGGTGGTGGA CGTGAGCCAG GAAGATCCCG AGGTCCAGTT CAATTGGTAC 180 GTGGACGGCG TGGAAGTGCA CAACGCCAAG ACCAAGCCCA GAGAGGAACA GTTCAACAGC 240 ACCTACCGGG TGGTGTCTGT GCTGACCGTG CTGCACCAGG ACTGGCTGAA CGGCAAAGAA 300 TACAAGTGCA AGGTGTCCAA CAAGGGCCTG CCCAGCAGCA TCGAAAAGAC CATCAGCAAG 360 GCCAAGGGCC AGCCTCGCGA GCCCCAGGTG TACACCCTGC CTCCCTCCCA GGAAGAGATG 420 ACCAAGAACC AGGTGTCCCT GACCTGCCTG GTGAAGGGCT TCTACCCCAG CGACATCGCC 480 GTGGAGTGGG AGAGCAACGG CCAGCCTGAG AACAACTACA AGACCACCCC TCCCGTGCTG 540 GACAGCGACG GCAGCTTCTT CCTGTACAGC CGGCTGACCG TGGACAAGAG CCGGTGGCAG 600 GAAGGCAACG TCTTTAGCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG 660 AAGAGCCTGA GCCTGTCCCT GGGCAAG 687 (SEQ ID NO: 18) Alternatively, the hinge region may have the following amino acid sequence: IgG4 Hinge-CH2-CH3(L235E,N297Q) ) is included. ESKYGPPCPP CPAPEFEGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY 60 VDGVEVHNAK TKPREEQFQS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 120 AKGQPREPQV YTLPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 180 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 229 (SEQ ID NO: 19)

[0050] The DNA sequence encoding it is as follows: GAGAGCAAGT ACGGACCGCC CTGCCCCCCT TGCCCTGCCC CCGAGTTCGA GGGCGGACCC 60 AGCGTGTTCC TGTTCCCCCC CAAGCCCAAG GACACCCTGA TGATCAGCCG GACCCCCGAG 120 GTGACCTGCG TGGTGGTGGA CGTGAGCCAG GAAGATCCCG AGGTCCAGTT CAATTGGTAC 180 GTGGACGGCG TGGAAGTGCA CAACGCCAAG ACCAAGCCCA GAGAGGAACA GTTCCAAAGC 240 ACCTACCGGG TGGTGTCTGT GCTGACCGTG CTGCACCAGG ACTGGCTGAA CGGCAAAGAA 300 TACAAGTGCA AGGTGTCCAA CAAGGGCCTG CCCAGCAGCA TCGAAAAGAC CATCAGCAAG 360 GCCAAGGGCC AGCCTCGCGA GCCCCAGGTG TACACCCTGC CTCCCTCCCA GGAAGAGATG 420 ACCAAGAACC AGGTGTCCCT GACCTGCCTG GTGAAGGGCT TCTACCCCAG CGACATCGCC 480 GTGGAGTGGG AGAGCAACGG CCAGCCTGAG AACAACTACA AGACCACCCC TCCCGTGCTG 540 GACAGCGACG GCAGCTTCTT CCTGTACAGC CGGCTGACCG TGGACAAGAG CCGGTGGCAG 600 GAAGGCAACG TCTTTAGCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG 660 AAGAGCCTGA GCCTGTCCCT GGGCAAG 687 (SEQ ID NO: 20).

[0051] In one preferred embodiment of the present invention, the amino acid sequence of the transmembrane domain from CD8 (CD8TM) The columns are as follows: IYIWAPLAGT CGVLLLSLVI TLYC 24 (SEQ ID NO: 21) The DNA sequence encoding it is as follows: ATCTACATCT GGGCGCCCTT GGCCGGGACT TGTGGGGTCC TTCTCCTGTC ACTGGTTATC 60 ACCCTTTACT GC 72 (SEQ ID NO: 22) In one preferred embodiment of the present invention, the amino acid sequence of the transmembrane domain derived from CD28 (CD28TM) is The acid sequence is as follows: FWVLVVVGGV LACYSLLVTV AFIIFWV 27 (SEQ ID NO: 37) The DNA sequence encoding the CD28-derived transmembrane domain (CD28TM) is as follows: TTTTGGGTGC TGGTGGTGGT TGGTGGAGTC CTGGCTTGCT ATAGCTTGCT AGTAACAGTG 60 GCCTTTATTA TTTTCTGGGT G 81 (SEQ ID NO: 38)

[0052] Intracellular domain The intracellular domain in the CAR of the present invention is the signaling domain of 4-1BB and the signaling domain of CD3ζ. Contains a signaling domain. Preferably, the signaling domain of 4-1BB comprises the following amino acid sequence: KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL 42 (SEQ ID NO: 23) The DNA sequence encoding it is as follows: AAACGGGCA GAAAGAAACT CCTGTATATA TTCAAACAAC CATTTATGAG ACCAGTACAA 60 ACTACTCAAG AGGAAGATGG CTGTAGCTGC CGATTTCCAG AAGAAGAAGA AGGAGGATGT 120 GAACTG 126 (SEQ ID NO: 24) Preferably, the intracellular signaling domain from CD28 comprises the following amino acid sequence: RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S 41 (SEQ ID NO: 39) The DNA sequence encoding it is as follows: AGGAGTAAGA GGAGCAGGCT CCTGCACAGT GACTACATGA ACATGACTCC CCGCCGCCCC 60 GGGCCCACCC GCAAGCATTA CCAGCCCTAT GCCCCACCAC GCGACTTCGC AGCCTATCGC 120 TCC 123 (SEQ ID NO: 40)

[0053] Preferably, the intracellular signaling domain of CD3ζ comprises the following amino acid sequence: RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPQ RRKNPQEGLY 60 NELQKDKMAE AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR 113 (SEQ ID NO: 25) The DNA sequence encoding it is as follows: AGAGTGAAGT TCAGCAGGAG CGCAGACGCC CCCGCGTACA AGCAGGGCCA GAACCAGCTC 60 TATAACGAGC TCAATCTAGG ACGAAGAGAG GAGTACGATG TTTTGGACAA GAGACGTGGC 120 CGGGACCCTG AGATGGGGGG AAAGCCGAGA AGGAAGAACC CTCAGGAAGG CCTGTACAAT 180 GAACTGCAGA AAGATAAGAT GGCGGAGGCC TACAGTGAGA TTGGGATGAA AGGCGAGCGC 240 CGGAGGGGCA AGGGGCACGA TGGCCTTTAC CAGGGTCTCA GTACAGCCAC CAAGGACACC 300 TACGACGCCC TTCACATGCA GGCCCTGCCC CCTCGC 336 (SEQ ID NO: 26)

[0054] vector The present invention also provides a DNA construct encoding a CAR sequence of the present invention. Nucleic acid sequences encoding the desired molecules can be extracted using recombinant methods known in the art, e.g., gene expression. screening the library in cells expressing the gene of interest; or by standard techniques to obtain the vector from a human or animal source, or to obtain cells and If necessary, the gene of interest can be obtained by direct isolation from tissue. It can also be produced synthetically.

[0055] The present invention also provides a vector into which the DNA construct of the present invention is inserted. For example, lentiviral vectors allow long-term, stable expression of the introduced gene. This allows for long-term gene transfer, allowing for the transfer of genes to the offspring and their subsequent proliferation. Lentiviruses are suitable tools for transducing non-proliferating cells, such as hepatocytes. Therefore, oncogenic retroviruses, such as murine leukemia virus vectors, can be used. It also has the advantage of being less immunogenic.

[0056] That is, a nucleic acid that operably encodes a CAR polypeptide or a portion thereof is typically promoter. The construct is then inserted into an expression vector to produce a CAR-encoding gene. The vectors are capable of replication and assembly in eukaryotic cells and provide for the expression of natural or synthetic nucleic acids. A typical cloning vector is suitable for regulating the expression of a desired nucleic acid sequence. Usable transcription and translation terminators, initiation sequences and promoters are included.

[0057] The expression constructs of the present invention can be delivered to cells by standard gene delivery protocols, including nucleic acid immunization and gene transfer. Gene delivery methods are known in the art, for example, See U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, the entire contents of which are incorporated herein by reference. In another embodiment, the present invention provides a gene therapy vector.

[0058] The nucleic acid can be cloned into a variety of vectors. For example, Vectors that can be used for cloning include plasmids, phages, phage derivatives, and animal viruses. Vectors of particular interest include, but are not limited to, expression vectors and cosmids. These include vectors, replication vectors, probe generation vectors and sequencing vectors.

[0059] Additionally, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is known in the art and is described, for example, in Sambrook et al. (2001, Mol. cular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and It is described in other virology and molecular biology manuals. The viruses that cause this include retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. Suitable vectors include, but are not limited to, vectors containing at least Replication origins, promoter sequences, and convenient restriction enzyme digests that function in more than one organism and one or more selectable markers (see, e.g., WO01 / 96584, WO01 / 290 58 and U.S. Patent No. 6,326,193).

[0060] Many virus-based systems have already been developed to transfer genes into mammalian cells. For example, retroviruses have become a convenient platform for gene delivery systems. The selected gene is inserted into the vector by techniques known in the art. The recombinant virus can be introduced into the host and packaged into retroviral particles. The virus is then separated and delivered to target cells either inside or outside the body. Systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, For this purpose, lentiviral vectors are used.

[0061] Additional promoter elements, such as enhancers, regulate the frequency at which transcription is initiated. These are usually located in the region 30–110 bp upstream of the start site, but recently Many promoters have been shown to also contain functional elements downstream of the initiation site. The spacing between the motor elements is such that one element is inverted relative to another. Many of them are mobile so that the promoter function can be maintained if they are moved. In the tk promoter, the spacing between promoter elements is important for the activity of The promoter can increase the length by up to 50 bp without reducing the The elements act jointly or independently to initiate transcription.

[0062] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence may be any polynucleotide sequence operably linked to it. It is a strong constitutive promoter sequence that can express the appropriate promoter at a high level. Another example of a motor is elongation factor 1α (EF-1α). However, other constitutively promoters A target sequence may also be used, such as the Simian Virus 40 (SV40) early promoter, mouse mammary tumor promoter, Virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Mo MuLV promoter, avian leukosis virus promoter, Epstein-Barr Barr virus immediate early promoter, Rous sarcoma virus promoter, and human genetic Human gene promoters include, but are not limited to, promoters of the human genome, e.g., Cutin promoter, myosin promoter, heme promoter, and creatine kinase promoter Furthermore, the present invention also relates to constitutive promoters. The present invention is not limited to the use of inducible promoters. Inducible promoters are also contemplated as part of the present invention. The use of a promoter provides a molecular switch, whereby such expression can be controlled when required. In this case, the expression of a polynucleotide sequence operably linked to an inducible promoter is initiated, Alternatively, expression can be turned off when it is not needed. Examples of promoters include the metallothionein promoter, the glucocorticoid promoter, and the progesterone promoter. Examples of promoters include, but are not limited to, the cyclodextrin promoter and the tetracycline promoter.

[0063] Expression vectors introduced into cells to assess expression of CAR polypeptides or portions thereof. -Selectable marker genes or reporter genes, or both from a cell population transduced or infected with a viral vector by including Expressing cells can also be identified and selected. Selectable markers can also be expressed by a single DNA fragment. The cells can be placed on a strip and used in the co-transfection process. Both the reporter gene and the target gene must have appropriate regulatory sequences in their flanking regions. Useful selectable markers include those derived from the nucleotide sequence nucleotides ... , for example, antibiotic resistance genes, such as neo.

[0064] Reporter genes allow identification of potentially transduced cells and confirmation of functionality of regulatory sequences. Typically, the reporter gene is present in the recipient organism or tissue. not be expressed by the recipient organism or tissue, and its expression is readily The polypeptide may be encoded by a detectable property, such as an enzymatic activity. Once the DNA is introduced into the recipient cells, the reporter gene Expression of the gene is measured at appropriate times. Suitable reporter genes include luciferase, β- Galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase These include genes encoding ribosomal enzymes and green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are known and can be prepared by known techniques or obtained commercially. These are commercially available and typically show the highest levels of reporter gene expression. A construct with at least five flanking regions is identified as a promoter. Such a promoter region is linked to a reporter gene and regulates the promoter of the reagent. This can be used to assess the ability of a gene to activate transcription.

[0065] Methods for introducing genes into cells and expressing genes in cells are well known in the art. In the context of expression vectors, the vectors can be expressed by any method known in the art. can be easily introduced into host cells, such as mammalian, bacterial, yeast, and insect cells. For example, an expression vector can be introduced into a host cell by physical, chemical, or biological means. It is possible.

[0066] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipopolysaccharide precipitation, and lipopolysaccharide precipitation. injection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or foreign nucleic acids are known in the art. For example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor For a preferred method of introducing polynucleotides into host cells, see the ELISA Kit (Immuno- and Immuno-Rad Laboratories, New York). The method is calcium phosphate transduction.

[0067] Biological methods for introducing polynucleotides of interest into host cells include DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have already It has become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors include lentiviruses, poxviruses, and herpes simplex viruses. They may be derived from viruses such as adenoviruses and adeno-associated viruses. For example, U.S. Pat. See Nos. 5,350,674 and 5,585,362.

[0068] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, e.g., polymer complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, These include lipid-based systems, including synthetic micelles, and liposomes. Exemplary colloidal systems used as livery vehicles are liposomes (e.g., artificial membranes). vesicles).

[0069] When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. It is contemplated that nucleic acids may be introduced into host cells using methods such as in vitro, ex vivo, or in vivo immunoassays. The nucleic acid may also be associated with a lipid. The nucleic acid associated with a lipid may be a liposome. The oligonucleotides are encapsulated in the aqueous interior of the liposome and interspersed in the lipid bilayer of the liposome. The linking molecule that connects both the nucleotides to the liposomes allows the nucleotides to be incorporated into the liposomes. , complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, suspended They may be contained in lipids as suspensions, contained in micelles, complexed with micelles, or in other forms. The lipid, lipid / DNA or lipid / expression vector associated with the composition may be bound to the lipid in the form The vector is not limited to any particular structure in solution. For example, a bilayer structure They may exist as micelles or in a "collapsed" structure in solution. Lipids are fatty substances, and can be found in natural lipids. The lipids may be synthetic. For example, the lipids may be the lipid droplets that occur naturally in the cytoplasm, as well as long-chain lipids. Aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino acids These include compounds such as alcohols and aldehydes. When using non-viral delivery systems, for example, gene editing techniques such as CRISPR-Cas9, Z The present invention is completed using FN and TALEN. In one preferred embodiment of the present invention, the vector is a lentiviral vector. do.

[0070] In one preferred embodiment of the present invention, the DNA construct further comprises a signal peptide. Preferably, the signal peptide sequence encodes the antigen-binding domain. Preferably, the signal peptide is a human-derived CD4+ signal peptide. 8a is a signal peptide. Preferably, the amino acid sequence of the signal peptide is as follows: The amino acid sequence of the CD8 leader sequence: MALPVTALLL PLALLLHAAR P 21 (SEQ ID NO: 27) DNA sequence encoding the CD8 leader sequence: ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG 60 CCG 63 (SEQ ID NO: 28)

[0071] therapeutic use The present invention relates to a method for producing a CAR-transduced human CAR-like cell line comprising: The transduced T cells induce a T cell response via the CAR. It can be rubbed.

[0072] Therefore, the present invention provides a method for the treatment of T cell-mediated immune responses against target cell populations or tissues in mammals. a method of stimulating a T cell that expresses a CAR of the present invention, comprising administering to a mammal a T cell that expresses a CAR of the present invention. A method is also provided.

[0073] In one embodiment, the present invention provides a method for genetically modifying T cells to express a CAR of the present invention. and the CAR-T cells are infused into a recipient in need thereof. The cells injected can kill tumor cells in the recipient. Unlike antibody therapy, CA RT cells can replicate in the body, resulting in long-term tumor suppression.

[0074] In one embodiment, the CAR-T cells of the present invention undergo stable in vivo T cell proliferation. Furthermore, the immune response induced by CARs can be used for adoptive immunotherapy. This can be part of a process in which CAR-modified T cells express immunity specific to the antigen-binding domain in the CAR. For example, anti-CD20 CAR-T cells induce specific immune responses against cells expressing CD20. triggering a specific immune response.

[0075] The data published herein specifically identify the anti-CD20 scFv, the hinge and transmembrane regions, and We have released a lentiviral vector containing the 4-1BB and CD3ζ signaling domains. The description should be construed to include any number of variations of each component part of the construct.

[0076] Therapeutic indications include CD20-positive tumors and diseases caused by excess B cells (e.g., autoimmune CD20-positive tumors include CD20-positive non-solid tumors (such as These include hematological tumors (e.g., leukemia and lymphoma) or solid tumors. The types of tumors or cancers treated include carcinomas, germ cell tumors, sarcomas, and some leukemias and lymphoid malignancies. benign and malignant tumors, including sarcomas, carcinomas and melanomas This includes, but is not limited to, adult tumors / cancers and childhood tumors / cancers.

[0077] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenic) cancers include leukemia, In addition, acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia) leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., For example, chronic myelocytic (granulocytic) leukemia, chronic bone marrow leukemia and chronic lymphocytic leukemia), Polycythemia, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and severe cases), polycythemia Myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome , hairy cell leukemia and myelodysplasia.

[0078] Solid tumors usually do not contain cysts or liquid-filled masses of tissue. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (for example, Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Examples of solid tumors include sarcomas and carcinomas, such as fibrosarcomas, myxomas, and lymphomas. Includes liquid sarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer. The CAR-modified T cells of the present invention can be used as a vaccine for ex vivo immunization and / or in vivo therapy in mammals. Preferably, the mammal is a human.

[0079] For ex vivo immunization, i) expansion of cells, ii) introduction of nucleic acid encoding a CAR into the cells, and / or or iii) cryopreservation of the cells in vitro prior to administering the cells to a mammal. It is held at.

[0080] In vitro protocols are known in the art and are discussed more fully below. For example, cells isolated from a mammal (preferably a human) expressing a CAR disclosed herein can be used. Genetic modification (i.e., in vitro transformation or transduction) with a vector that encodes CAR. The cells can be administered to a mammalian recipient to provide a beneficial effect of therapy. The mammalian recipient may be a human, and the CAR-modified cells may be the recipient's own cells. Optionally, the cells may be allogeneic, syngeneic, or It may be of a different species. In addition to cell-based vaccines for ex vivo immunization, the present invention also provides a method for administering the vaccine to a patient by in vivo immunization. Also provided are compositions and methods for eliciting an immune response to an antigen in a subject.

[0081] Typically, cells activated and expanded as described herein occur in individuals without an immune response. In particular, the CAR-modified T cells of the present invention can be used to treat and prevent diseases. can be used to treat CCL. In some embodiments, the cells of the present invention is used to treat patients at risk of developing CCL. The present invention provides a method for treating or preventing a CAR-modified T cell of the present invention, comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cell of the present invention. The present invention provides a method comprising the step of applying cells.

[0082] The CAR-modified T cells of the present invention may be administered alone or in a pharmaceutical composition containing diluents and / or other Applying ingredients such as IL-2, IL-17, or other cytokines or cell populations Briefly, the pharmaceutical compositions of the present invention can be used to treat biosynthetic drugs, such as monoclonal antibodies. a small molecule drug, and one or more pharmaceutically or physiologically acceptable carriers, diluents Such compositions may be combined with a buffer, such as neutral buffered saline. , sulfate buffered saline, etc., carbohydrates, such as glucose, mannose, sucrose, and dextrose orchids, mannitol, proteins, polypeptides or amino acids, such as glycine, acid antioxidants, chelating agents such as EDTA and glutathione, adjuvants (e.g., hydroxybenzoates), The compositions of the present invention may contain an antiseptic agent, such as a steroid agent, an antiseptic ... It is preferable to prepare it as follows.

[0083] The pharmaceutical composition of the present invention is administered in a form suitable for the disease to be treated (or prevented). The quantity and frequency of application will depend on the patient's bed and the type and severity of the patient's illness. The appropriate dosage will depend on factors such as the degree of toxicity, but the appropriate dosage will be determined through clinical trials.

[0084] "immunologically effective amount," "antitumor effective amount," "tumor suppression effective amount," or "therapeutic amount" When described, the exact amount of the composition of the present invention to be applied will depend on the age, weight, tumor size, and other factors of the patient (subject). The doctor will decide based on the size of the tumor, the degree of infection or metastasis, and individual differences in the disease. Typically, the pharmaceutical compositions comprising the T cells described herein are administered in a dose of 10 4 ~10 9 Dosage in cells / kg body weight, Preferably 10 5 ~10 6 Dose of cells / kg body weight (all integer values ​​within these ranges) The T cell composition may be administered at these doses several times. The cells can be injected using injection techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:167). 6, 1988). The optimal dosage and treatment plan for a specific patient can be determined. Ran can monitor the patient's disease symptoms and adjust treatment accordingly. This can be easily determined by a medical technician.

[0085] The composition may be administered to a subject by any convenient method, including spraying, injection, oral administration, infusion, implantation, or transplantation. The compositions described herein may be administered subcutaneously, intradermally, intratumorally, intranodally, or by any convenient means. It may be administered to a patient intraspinal, intramuscular, intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are administered by iv injection. Preferably, the T cell composition may be injected directly into a tumor, lymph node, or site of infection. stomach.

[0086] In some embodiments of the present invention, the method described herein or other methods known in the art are used. The cells activated and expanded by a method that expands T cells to therapeutic levels are used, and administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any number of related therapeutic procedures The therapeutic measures include antiviral therapy, cidofovir and interleukin-2, azathioprine, and the like. Treatment with agents such as cytidine (known as ARA-C) or for MS patients Treatment with natalizumab for psoriasis or efalizumab for patients with PML In a further embodiment, other treatments for the patient include, but are not limited to: The T cells of the present invention may be administered in combination with chemotherapy, radiation, immunosuppressive drugs such as cyclosporine A, azathioprine, Purines, methotrexate, mycophenolate mofetil, FK506, antibodies or other immunosuppressants In a further embodiment, the cell composition of the present invention can be used in combination with an immunotherapeutic agent. Bone marrow transplants, chemotherapy drugs such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, It is administered to a patient in combination with (e.g., before, simultaneously with, or after) sulfamide. For example, in one embodiment, the subject undergoes high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the present invention. In another embodiment, the expanded cells are administered pre- or post-surgery. will be done.

[0087] The dosage of the above treatment administered to the patient depends on the exact characteristics of the disease being treated and the treatment recipe. The dosage rate for administration to humans will vary according to accepted practice in the art. Typically, 1 x 10 6 pieces~1×10 10 The modified T cells of the present invention (e.g., CAR-T20 cells) can be administered, for example, by intravenous infusion. It can then be administered to the patient.

[0088] The advantages of the present invention are as follows: (1) The chimeric antigen receptor of the present invention has an extracellular antigen-binding domain that is a specific anti-CD20 scFv. The specific anti-CD20 scFv is bound to a specific hinge region and intracellular domain to form a CAR. It exhibits extremely potent killing ability against tumor cells, yet has low cytotoxicity and few side effects. (2) The chimeric antigen receptor provided by the present invention is a lentivirus carrying a CAR gene. After infecting T cells with the CAR gene, stable expression and membrane localization of the CAR protein was achieved. can be done. (3) The CAR-modified T cells of the present invention have a long survival time in the body, a strong antitumor effect, and are IgG4 H CARs with an optimized e-CH2-CH3 linkage domain are able to bind to Fc receptors and subsequently activate ADCC (antibody-dependent cell death). This can avoid the adverse effects of steroids on the immune system (e.g., immune-mediated cytotoxicity).

[0089] Example 1 Construction of a lentiviral expression vector The coding plasmid was synthesized and cloned by Shanghai Boyi Biotechnology Co., Ltd. Each encoding plasmid contained a different anti-CD20 scFv coding sequence. The cloning vector used was the pWPT lentiviral vector, and the cloning section The sites are BamH I and Sal I sites. The specific sequence structure is shown in Figure 1. The amino acid and nucleotide sequences are as described above. In the following examples, CAR-T20.13, CAR-T20.14, CAR-T20.16, and CA We will use R-T20.19 and CAR-T20.20 as examples.

[0090] Example 2: Production of CAR-T cells (1) Venous blood was collected from healthy volunteers, and mononuclear cells (PBMCs) were isolated by density gradient centrifugation. (2) On day 0, PBMCs were cultured in GT-T551 cell medium containing 2% human serum albumin, and the cells were Final concentration is 2 x 10 6 The cells were pre-adjusted to a final concentration of 5 μg / mL of CD3 monoclonal antibody. Monoclonal antibody (OKT3) and Retronectin (purchased from TAKARA) at a final concentration of 10 μg / mL were used. The cells were inoculated into cell culture flasks coated with 1000 U / mL of recombinant human HIV-1. Add interleukin-2 (IL-2) and incubate at 37°C, saturated humidity, and 5% CO2. Cultivated. (3) On day 2, fresh culture medium, concentrated and purified CAR20s lentivirus solution, and protamine were added. The medium was incubated at 37°C, 5% CO2 with IL-2, sulfate (12 μg / ml), and IL-2 at a final concentration of 1000 U / mL. After 12 hours of infection in an incubator, the culture medium was discarded, fresh medium was added, and the cells were incubated at 37°C in 5% CO2. The culture was continued in an incubator. (4) From the sixth day, CART20s cells were harvested and the corresponding activity detection tests were carried out. In the present invention, we have improved the manufacturing process of T cells modified with a CAR structure that targets the CD20 antigen. Lymphocytes were cultured in vitro in GT-551 serum-free medium supplemented with 2% human serum albumin. .

[0091] Example 3: Integration rate of CAR gene in T cell genome and its encoded Detection of the expression level of proteins on the membrane surface 0.5 x 10 each 6 In Example 2, the cells were cultured for 7 days (FIGS. 2A and 5A) and 11 days (FIG. 2B). A sample of the CART-20s cells was taken, stained with protein L, and analyzed by flow cytometry. The expression level of CAR20 protein on the membrane surface of T cells was analyzed. All of the designed CAR structures, except for CAR-T20.13, used protein L to express chimeric antigens. It was shown that the receptor was detected at the membrane surface of the correspondingly modified T cells. .

[0092] Example 4: Detection of in vitro activation ability of CAR-T20s CART-20s cells cultured up to day 6 in Example 2 were used to co-culture with target cells, and then CD137 The upregulated levels of IFNγ and the secreted levels of IFNγ in the culture supernatant were detected. CART-20 cells were cultured at 1 × 10 5 RAJI and RAMOS tumor cell lines, CD20 positive, respectively. and 200 μl of GT-5 with or without the addition of CD20-negative MOLT-4 tumor cell line. After 18 hours of co-culture in 51 medium at a 1:1 ratio, T cell membrane activity was measured by flow cytometry. The expression level of CD137 on the surface of the cells was measured (Fig. 3A), and the level of IFNγ in the culture supernatant was measured by ELISA. Secretion levels were detected (Figure 3B). The results in Figure 3 show that obinutuzumab-based CARs also significantly increase the expression and membrane surface of the corresponding modified cells. The localization of obinutuzumab and CAR structures based on the ofatumumab sequence was achieved. Superior in vitro activation ability and target antigen specificity compared to rituximab-based CARs The conclusion was that it showed

[0093] Example 5: Detection of early apoptosis induction activity of tumor cells by CAR-T20s cells CART-20.13, CART-20.14, and CAR-T20.16 cells cultured up to day 11 in Example 2 were used. , 1 × 10 respectively at the ratios shown in Figure 4 4 CFSE-labeled CD20 negative (MOLT-4) or CD20 The cells were co-cultured with 200 μl of GT-551 medium for 4 hours and centrifuged. The cell pellet was collected, washed twice with PBS, and then stained with Annexin V-APC staining reagent at a ratio of 1:50. The cells were stained in 100 μl of staining solution for 30 min, washed once with PBS, and then analyzed by flow cytometry. Therefore, the ratio of Annexin V-positive cells among CFSE-positive cells was analyzed. As shown in Figure 4, the CAR structure based on the ofatumumab sequence is similar to that of obinutuzumab and Rituximab. Induce early apoptosis of CD20 target cells in vitro more effectively than CARs based on simamib It can be seen that they have demonstrated the ability to do so.

[0094] Example 6: Chimeric antigen receptors with hinge region mutations and third-generation chimeric antigen receptors Identification of in vitro activation capacity (1) CAR-T20s cells produced by the method in Example 2 and cultured for up to 7 days were used. Under conditions where the transduction rates were similar (Fig. 5A), 1 × 10 5 Take one cell K562, CD19 single-positive, CD20 single-positive, CD19 and CD20 double-positive K562 stable transfectant cells, and RAJI target cells Upregulation of CD137 after 18 h of co-culture with target cells at a 1:1 ratio in 200 μl of GT-551 medium The levels (Fig. 5B) and secreted levels of IFNγ in the culture supernatant (Fig. 5C) were detected. (2) From the results shown in Figure 5, when the infection efficiency is approximately the same, the texture of the hinge region mutations is The in vitro activation capacity of the LA antigen receptor CAR-T20.19 is similar to that of CAR-T20.14 (CD137 and IFNg). However, the third-generation CAR structure, CAR-T20.20, outperformed the second-generation CAR-T20.14 and CAR-T20.19. It can be seen that the IFN-g-dependent activating ability in vitro was superior to that of the IFN-g-dependent activating ability in vivo (CD137 and IFNg).

[0095] Example 7: Detection of the ability of CAR-T20 cells to eliminate CD20-positive cells in the body (1) Luciferase-expressing Raji-Luc cells were injected into NCG mice via the tail vein (5 × 1). 0 5 One week after inoculation, tumor cell proliferation in the body was confirmed by in vivo imaging. The condition was observed and recorded as Day 0. NT and CAR-T20.19 cells were administered into the tail vein of mice on Day 0. injected (5 × 10 6 On Day 0, Day 7, Day 14, and Day 21, in vivo imaging was performed. The growth of tumor cells in the mouse body was observed, and changes in fluorescence intensity and the mouse's body temperature were also observed. The analysis was based on changes in weight. (2) The results shown in Figure 6 indicate that CAR-T20.19 effectively inhibits the proliferation of CD20-positive cells in vivo. It is clear that it can be suppressed.

[0096] Although the preferred embodiments of the present invention have been described above in detail, the present invention is not limited to the above-described embodiments. The present invention is not limited to specific details, and various modifications may be made to the technical solution of the present invention within the scope of the technical spirit of the present invention. Various simple modifications can be made, and all of these simple modifications fall within the scope of protection of the present invention.

[0097] It should be noted that the specific technical features described in the above specific embodiments may be arbitrarily used unless they are inconsistent. In order to avoid unnecessary duplication, the present invention provides various The possible combinations are not explained separately. Furthermore, different embodiments of the present invention can be arbitrarily combined, and the spirit of the present invention is not limited to these. Unless contrary to the purpose, the contents of the present invention will be considered as the contents disclosed in the present invention.

Claims

1. A chimeric antigen receptor (CAR) comprising an anti-CD20 antigen-binding domain, wherein the anti-CD20 antigen-binding domain (i) a heavy chain variable region (V) having the amino acid sequence shown in SEQ ID NO: 7 Ｈ ), and (ii) a light chain variable region (V) having the amino acid sequence set forth in SEQ ID NO: 11 Ｌ ), V Ｈ is V Ｌ is located at the N-terminus of the anti-CD20 antigen-binding domain is a single-chain variable fragment (scFv) that specifically binds to CD20; The chimeric antigen receptor (iii) a signal peptide having the amino acid sequence shown in SEQ ID NO: 27; (iv) a hinge region having the amino acid sequence set forth in SEQ ID NO: 19; (v) a transmembrane domain having the amino acid sequence set forth in SEQ ID NO: 21; (vi) a costimulatory signaling region having the amino acid sequence set forth in SEQ ID NO: 23, and (vii) an intracellular signaling domain having the amino acid sequence set forth in SEQ ID NO: 25; further comprising: Chimeric antigen receptor.

2. From the N-terminus to the C-terminus, the signal peptide, V Ｈ , V Ｌ 2. The chimeric antigen receptor of claim 1, comprising the hinge region, the transmembrane domain, the costimulatory signaling region, and the intracellular signaling domain.

3. An immune cell comprising the chimeric antigen receptor of claim 1 or 2.

4. The immune cell of claim 3, which is a T cell.

5. A pharmaceutical composition comprising the immune cells of claim 3 or 4.

6. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1 or 2.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A pharmaceutical composition comprising the vector of claim 7.

9. An immune cell comprising the nucleic acid molecule of claim 6.

10. A pharmaceutical composition comprising the immune cells of claim 9.

11. Use of a chimeric antigen receptor according to claim 1 or 2, an immune cell according to claim 3, 4 or 9, a nucleic acid molecule according to claim 6, a vector according to claim 7, or a pharmaceutical composition according to claim 5 or 8 in the manufacture of a drug or formulation for treating a tumor or an autoimmune disease.

12. The use according to claim 11, wherein the tumor is a hematological tumor.

13. 13. The use according to claim 11 or 12, wherein the tumor is a B-cell malignancy.

14. 12. The use of claim 11, wherein the tumor is Hodgkin's disease, non-Hodgkin's lymphoma, leukemia, multiple myeloma (MM), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, B-cell lymphoma, or a combination thereof.

15. 12. The use of claim 11, wherein the immune cells are administered by infusion, injection, transfusion, implantation, and / or transplantation, or the immune cells are administered intravenously, subcutaneously, intranodally, intraspinally, intramuscularly, or intraperitoneally.

16. The use according to claim 11 , wherein the immune cells are administered by intravenous injection.

17. The use according to claim 11, wherein the autoimmune disease is lupus erythematosus.

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