Construction of chimeric antigen receptor targeting CD20 antigen and activity identification of engineered t cells thereof

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 relates to sequence components of a chimeric antigen receptor that targets the CD20 antigen, and modified T cells thereof. The present invention provides a method for the preparation and activity identification of CD20-positive B-cell lymphoma. The present invention identifies chimeric antigen receptor structures that treat tumors. [Background technology]

[0002] Hematological malignancies account for approximately 10% of all malignant tumors in humans, 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 treating some patients, but the majority are difficult to cure. Effective treatment methods have been the focus of ongoing research in this field.

[0003] Adoptive T cell therapy has already demonstrated strong therapeutic effects and promising results in clinical treatment of malignant tumors. In this study, chimeric antigen receptors evolved independently by several mechanisms. T cells modified with chimeric antigen receptors (CARs) express CD19 and He has achieved unprecedented success in refractory B-cell malignancies, particularly at the University of Pennsylvania School of Medicine. CART19 was used to treat relapsed / refractory acute B-cell lymphoma (R / R B-ALL). 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, then relapsed again and again. Among patients with CD19-negative tumors, as many as 60% showed immune escape of CD19-negative tumor cells. CD40-targeting B-cell lymphoma-associated antigens other than CD19 to treat patients with lymphoma Screening of ART structures is urgently needed.

[0004] CD20 is a glycosylated protein that was the first identified membrane marker for B cells, designated B1 cells. The CD20 molecule has four transmembrane hydrophobic regions and its N Both the terminal and C-terminal ends 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 more than 95% of normal and malignant B cells. It is expressed in the cytoplasm of cells that are pre-B cell and subsequent developmental stages and differentiate into plasma cells. CD20 is expressed continuously until the tumor is induced. Therefore, CD20 is an ideal target for immunotherapy of B cell malignancies. It is a target.

[0005] Rituximab (MabTheraR, RituxanR) is the first to be approved by the U.S. FDA and the European EMA First generation CD20-targeted chimeric mouse to treat indolent lymphoma approved by the National Institute of Integrative Medicine Rituximab is a monoclonal antibody that recognizes and binds to the large loop structure of the CD20 extracellular domain. It kills tumor cells through ADCC. 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 had 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 located on the major loop of CD20. At the same time, the tumor-killing means of ofatumumab is This 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 affinity for FcγRIIIa by reducing the antibody level. Izamab recognizes and binds to the large loop of the CD20 extracellular molecule, and inhibits tumor proliferation mainly through ADCC. At the same time, the binding of obinutuzumab to the CD20 molecule mediates the killing effect of tumor cells, and at the same time, the binding of obinutuzumab to the CD20 molecule mediates the apoptosis of tumor cells. Obinutuzumab is effective in treating NHL that is refractory to rituximab. In a phase III clinical trial, In the study, the combination of obinutuzumab and bendamustine was more effective than bendamustine alone Progression-free survival was one-fold longer in the former (29 months vs. 14 months in the latter). When used to treat CLL, mab achieved an overall response rate (ORR, including CR and PR) of 77.3%, and was The rate for mab was 65.7%.

[0008] Compared with therapeutic antibodies, cellular immunotherapy has remarkable therapeutic effects and is a new approach to tumor treatment. This is a novel method of anti-cancer treatment through autoimmunity. This is a method in which immune cells collected from the body are cultured and expanded outside the body, and then returned 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 cell immunotherapy, improve the efficacy of cell immunotherapy, and In recent years, many therapeutic antibodies like those mentioned above have been developed, but their clinical applications remain unclear. None of the treatments have achieved the same therapeutic effect as CART19. The development of CART therapy targeting this gene has enormous commercial value and social significance. Summary of the Invention

[0009] Therapeutic antibodies targeting CD20 differ in their affinity and killing mechanisms, A series of chimeric antigen receptors targeting CD20 were constructed using DNA sequences from the antigen-binding regions of antibodies. We then identified and differentiated the in vitro antitumor activity of these chimeric antigen receptor-modified T cells. The efficacy of CAR-T in the treatment of CD20-positive leukemia and lymphoma in clinical trials was compared. The present invention provides new methods and formulations. The object of the present invention is to provide a chimeric antigen receptor targeting CD20 and a method for producing and using the same. To do so.

[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 identification of their activity.

[0011] In a first aspect of the present invention, there is provided a chimeric antigen receptor (CAR) (sequence), 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. Provide a receptor. In another preferred embodiment, the antigen-binding domain of the chimeric antigen receptor has the formula: It is expressed as: V H -VL (However, V H is the heavy chain variable region of an antibody, V L is the light chain variable region of the antibody, and "-" is the linking 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 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: L.V. H -V L -H-TM-CS-CD3ζ (however, L is an optional leader sequence (i.e., a 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 shown 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 includes the transmembrane domain of CD8a or the transmembrane domain of CD28. Preferably, the TM sequence is as set forth in SEQ ID NO: 21 or 37.

[0012] In another preferred embodiment, the CS structure is CD28-4-1BB, where CD28 is derived from CD28. , and 4-1BB is a costimulatory molecule derived from 4-1BB. In another preferred embodiment, the sequence of the costimulatory molecule derived from 4-1BB is shown in SEQ ID NO: 23. . In another preferred embodiment, the sequence of the CD28-derived costimulatory molecule is shown 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 provided. The present invention provides a nucleic acid molecule that encodes the nucleic acid. 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 a polypeptide set forth in SEQ ID NO: 17 or 19; (b) a polynucleotide having a sequence 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 set forth 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 molecule selected from the group consisting of: The nucleic acid sequence encoding the consensus region includes: (a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO:21; (b) a polynucleotide having the sequence set forth in SEQ ID NO:22; (c) the nucleotide sequence has a homology of ≥ 90% (preferably ≥ 22) with the sequence set forth in SEQ ID NO: ≧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 an intracellular signaling domain of: (a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO:23; (b) a polynucleotide having the sequence set forth in SEQ ID NO:24; (c) the nucleotide sequence has a homology of ≥ 90% (preferably ≥ 24) with the sequence set forth in SEQ ID NO: ≧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 selected from the group consisting of: The nucleic acid sequence encoding the internal signal domain includes: (a) a polynucleotide encoding the polypeptide set forth in SEQ ID NO:39; (b) a polynucleotide having the sequence set forth in SEQ ID NO:40; (c) the nucleotide sequence has a homology of ≥ 90% (preferably ≥ 40) 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 having the sequence set forth in SEQ ID NO:26; (c) the nucleotide sequence has a homology of ≥ 90% (preferably ≥ 26) with the sequence set forth in SEQ ID NO: ≧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; D; (b) a polynucleotide having a sequence 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. The gene encoding the leader sequence (a sequence, a signal sequence) is 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 invention, a vector is provided which comprises or is a vector according to the third aspect of the invention. or a host cell having integrated into its chromosome an exogenous nucleic acid molecule according to the second aspect of the invention. do. In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically modified cell. In another preferred embodiment, the cell is a mammalian cell. In another preferred embodiment, the cell is a T cell.

[0021] In a fifth aspect of the invention, a pharmaceutical composition is provided comprising a pharma- ceutically acceptable carrier and a compound 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; The present invention also provides a pharmaceutical composition comprising a vector or a cell according to the fourth aspect of the invention.

[0022] In a sixth aspect of the present invention, there is provided 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 use of the compound in the manufacture of In another preferred embodiment, the autoimmune disease is an autoimmune disease caused by overexpression of B cells. A 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 for treating a disease, comprising administering to a subject in need of treatment an appropriate amount of the compound. 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 the step of 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 transfection obtaining said CAR-T cells by transducing said CAR-T cells into a cell.

[0026] Of course, within the scope of the present invention, the above 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 the technical scheme can be constructed, but due to space limitations, it will not be described here one by one. [Brief description of the drawings]

[0027] Description of the drawings [Figure 1] FIG. 1 is a structural diagram of a chimeric antigen receptor targeting CD20. Each element of the designed CAR structure is shown in the figure, and the elements shown include a leader sequence, an antigen recognition sequence (ofatumumab, obinutuzumab, rituximab), a hinge region, a transmembrane region, a costimulatory factor signal region, and a CD3ζ signaling region. Here, CAR-T20.14, CAR-T20.13, and CAR-T20.16 are CAR structures constructed based on the variable regions of ofatumumab, obinutuzumab, and rituximab antibodies, respectively, and CAR-T20.19 and CAR-20.20 are in the form of L235E-N297Q mutation in the IgG4 Hinge-CH2-CH3 linkage region of CAR-T20.14, and CAR-T20.20 is a third-generation chimeric antigen receptor structure that also has the costimulatory signal molecule coding sequences of CD28 and 4-1BB at the same time. [Diagram 2] Figure 2: Detection of transduction efficiency of gene-modified T cells with chimeric antigen receptors targeting CD20. The protein L method was used to identify the expression levels of the protein encoded by the CAR gene on the membrane surface of T cells in CAR-T20s cells cultured up to day 7 (A) and day 11 (B). [Diagram 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 co-cultured with CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell line in 200μl of GT-551 medium at a 1:1 ratio for 18 hours, and then 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 detected. [Figure 4]Figure 4 shows the detection of the level of induction of apoptosis of tumor cells by CART-20. 1x104 tumor cell lines, CD20 negative (MOLT-4) or CD20 positive (RAJI, RAMOS), each labeled with CFSE, were taken and co-cultured for 4h with NT, CART-20.13, CART-20.14 and CAR-T20.16 cells, which were cultured in 200μl of GT-551 medium at the ratios shown in the figure until day 11, respectively, and then centrifuged to collect the cell pellet. The cells were washed twice with PBS, and then stained with Annexin V-APC staining reagent at a ratio of 1:50 in 100μl of staining solution for 30min, washed once with PBS, and the ratio of Annexin V positive cells in CFSE positive cells was analyzed by flow cytometry. The results shown in the figure show the statistical analysis results in the corresponding co-culture samples of Annexin V positive cells. [Diagram 5] FIG. 5: Identification of the in vitro activation ability of hinge region mutant 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 levels of the protein encoded by the CAR gene on the T cell membrane surface in CAR-T20.14, CAR-T20.19, and CAR-T20.20 cultured up to day 7 (A). NT, CART-20.14, CART-20.14, and CAR-T20.16 cells cultured up to day 7 were taken in 1x105 cells, and co-cultured with K562, CD19 monopositive, CD20 monopositive, CD19 and CD20 bipositive K562 stable transfectant cells, and RAJI target cells at a 1:1 ratio in 200μl of GT-551 medium for 18 hours, respectively, and then the expression levels of CD137 on the T cell membrane surface (B) and the secretion levels of IFNγ in the culture supernatant (C) were detected. [Figure 6] Figure 6 shows the detection results of the ability of CAR-T20 cells to eliminate CD20-positive cells in the body, and 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 a chimeric antigen receptor (CAGR) targeting CD20 and The extracellular antigen-binding domain of the chimeric antigen receptor is a sequence The heavy chain variable region of the antibody shown in sequence number 1 and the light chain variable region of the antibody shown in sequence number 2 Experimental results show that the chimeric antigen receptors provided by the present invention are highly expressed in tumor cells. It is clear that this weapon has demonstrated lethal power against

[0029] Therapeutic antibodies targeting CD20 vary in affinity, killing mechanism, etc. 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 multiple anti-CD20 antibodies to identify different transmembrane and A series of chimeric antigen receptors that are linked to intracellular segments and target CD20 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 To identify the differences in the activities of killing in vitro and eliminating malignant tumors bearing the CD20 antigen in vivo, and to develop clinical applications of Novel and effective methods and formulations for the treatment of CD20-positive leukemia and lymphoma with CAR-T in patients with to provide.

[0030] Chimeric Antigen Receptor The present invention relates to 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 a costimulatory signaling region and a 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 an effective response of lymphocytes to antigens. They are antigen receptors or is not the 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 insulators. As used herein, the term "linker" refers to " refers generally to the extracellular or intracellular domains of a polypeptide chain. A linker refers to any oligopeptide or polypeptide that serves to link a 0 to 300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids. The compound may contain an 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 the antibody 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. It is preferred to fuse the α- and β-chains to one or more intracellular domains derived from the α- and β-chains. Preferably, the antigen binding domain comprises a 4-1BB signaling domain and / or a CD28 signaling domain. The transduction domain is fused to the intracellular domain in combination with the CD3ζ signaling domain.

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

[0034] In one preferred embodiment of the present invention, the chimeric antigen receptor provided by the present invention is 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 ATTCCAGAA 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 antibody 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. This is a case in point. In one preferred embodiment of the 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 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 is correct. 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 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 is correct. 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 is correct. 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, an amino acid sequence between the heavy chain variable region and the light chain variable region is The amino acid linking 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 domains 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 that is linked to the CA. 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 the binding of phospholipase A 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 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 has the following amino acid sequence: IgG4 Hinge-CH2-CH3(L235E,N297Q) ). 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 (sequence number 20).

[0051] In one preferred embodiment of the present invention, the amino acid sequence of the transmembrane domain from CD8 (CD8TM) is 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: FWVLVVGGV LACYSLLVTV AFIIFWV 27 (SEQ ID NO: 37) The DNA sequence encoding the transmembrane domain derived from CD28 (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 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 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 the 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. Screening the library in cells expressing the gene of interest or by standard techniques to obtain cells and 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, lentivirus-derived vectors allow long-term, stable expression of introduced genes. This allows for long-term gene transfer by allowing for the transfer of genes into the host and their propagation in progeny cells. 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 low immunogenicity.

[0056] That is, a nucleic acid that operably encodes a CAR polypeptide or a portion thereof is typically The construct is then linked to a promoter and inserted into an expression vector to produce a CAR-encoding gene. The vectors provide for the expression of natural or synthetic nucleic acids that are capable of replication and synthesis in eukaryotic cells. 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 by standard gene delivery protocols, including nucleic acid immunization and gene delivery. 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, the nucleic acid 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 vectors include vectors for sequencing, replication, probe generation and sequencing.

[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. (Cold Spring Harbor Laboratory, New York) and It is described in other virology and molecular biology manuals. Viruses that cause infection include retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. Suitable vectors include, but are not limited to, vectors such as avian vectors and lentiviruses. Typically, suitable vectors include at least Replication origins, promoter sequences, and convenient restriction enzyme digests that function in only 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] A number of virus-based systems have been developed to transfer genes into mammalian cells. For example, retroviruses have been used as convenient platforms 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 vector 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. 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 initiation site, but recently It has become clear that many promoters 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 determines the activity of the The promoter can increase the length of the single The elements act jointly or independently to initiate transcription.

[0062] One 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 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 genomic DNA Human gene promoters include, but are not limited to, promoters of human genes, such as, for example, Cutin promoter, myosin promoter, heme promoter, and creatine kinase promoter In addition, the present invention relates to a constitutive promoter. 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 when the expression of the polynucleotide sequence is initiated, the polynucleotide sequence is operably linked to an inducible promoter. Alternatively, expression can be turned off when expression is not required. Examples of promoters include the metallothionein promoter, the glucocorticoid promoter, and the progesterone promoter. Examples of promoters include, but are not limited to, the cyclosporine promoter and the tetracycline promoter.

[0063] To assess the expression of the CAR polypeptide or a portion thereof, an expression vector introduced into the cell -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 are also expressed by a single DNA fragment. The selectable marker gene and Both the reporter gene and the promoter region must have appropriate regulatory sequences. Useful selectable markers include those that can be expressed in the host cell by , for example antibiotic resistance genes, such as neo.

[0064] Reporter genes allow identification of potentially transduced cells and confirmation of the functionality of regulatory sequences. Typically, reporter genes are present in the recipient organism or tissue. not be expressed by the recipient organism or tissue, and its expression is readily The polypeptide is encoded by a polypeptide that can be clearly demonstrated by a detectable property, for example an enzymatic activity. Once the DNA is introduced into the recipient cells, the reporter gene is Expression of the reporter gene is measured at appropriate times. Suitable reporter genes are 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 commercially available. These are commercially available products that usually show the highest levels of reporter gene expression. A construct having 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 agent. The method can be used to assess the ability of a target gene to activate transcription.

[0065] Methods for introducing genes into cells and expressing genes in cells are described in the art. In the context of expression vectors, the vectors can be synthesized by any method known in the art. They 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, lipofusion, and ELISA. injection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acid are known in the art. For example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, For example, see the ELISA Kit, ELISA Kit, or Laboratory, New York. The method is calcium phosphate transduction.

[0067] Biological methods for introducing a polynucleotide of interest into a host cell 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. The virus may be derived from a virus such as adenovirus or adeno-associated virus. For example, U.S. Pat. See numbers 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 include 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 can be introduced into host cells using techniques such as in vitro, ex vivo, or in vivo. The nucleic acid may also be associated with a lipid. A nucleic acid associated with a lipid may be a liposome. The oligonucleotides are encapsulated in the aqueous interior of the liposome and are interspersed in the lipid bilayer of the liposome. The peptide is attached to the liposome via a linking molecule that connects both the peptides to the liposome, and is incorporated into the liposome. , complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, suspended They may be present in lipid suspensions, in micelles, complexed with micelles, or in other forms. The lipid, lipid / DNA or lipid / expression vector associated with the composition may be linked to the lipid. The vector is not limited to any particular structure in solution. For example, the structure of a bilayer They may exist as micelles or in a "collapsed" structure in solution. Lipids are fatty substances, and are also 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, etc. Includes compounds such as alcohols and aldehydes. When using a non-viral delivery system, 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 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, said signal peptide sequence comprises a sequence encoding said 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 the preparation of a CAR-transduced human CAR-transduced ... The transduced T cells induce a T cell response via the CAR. It can be rubbed.

[0072] Thus, the present invention provides a method for the preparation of ... A method for 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 the genetic modification of 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 that are delivered 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 expansion. The immune response induced by CAR can be sustained for extended periods of time, making it a promising tool for adoptive immunotherapy. The CAR-modified T cells may be part of a process to express an immune response specific to a antigen-binding domain in the CAR. For example, anti-CD20 CAR-T cells induce specific immune responses against cells expressing CD20. This triggers a specific immune response.

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

[0076] The indications that can be treated 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 For example, hematological tumors, such as 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 (such as acute lymphocytic leukemia, acute myeloid leukemia, and acute myeloid leukemia) leukemia and 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 macroglobulinemia, heavy chain disease, myelodysplastic syndrome , hairy cell leukemia and myelodysplasia.

[0078] Solid tumors do not usually contain a cyst or a mass of liquid tissue. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (for example, Solid tumors include sarcomas, carcinomas, and lymphomas. Examples of sarcomas and carcinomas include fibrosarcomas, myxomas, and 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 the cells, ii) introduction of a 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.

[0080] In vitro protocols are known in the art and are discussed more fully below. For example, cells can be isolated from a mammal (preferably a human) to express the CARs disclosed herein. Genetic modification (i.e., in vitro transformation or transduction) with a vector that encodes the 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 transgenic to the recipient. It may be of a different species. In addition to cell-based vaccines for ex vivo immunization, the present invention provides a method for in vivo immunization of patients. 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 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 method includes the step of applying the 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 Combined administration of ingredients such as IL-2, IL-17, and other cytokines or cells Briefly, the pharmaceutical compositions of the present invention can be used to produce biosynthetic drugs, such as monoclonal antibodies. and one or more pharma- ceutically 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 orchid, mannitol, proteins, polypeptides or amino acids, such as glycine, acid antioxidants, chelating agents such as EDTA and glutathione, adjuvants (e.g., hydroxypropyl The composition of the present invention may contain an antiseptic, an antibacterial agent, a sedative, an antiseptic, and an antiseptic agent. 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 dosage will depend on factors such as the severity of the condition, but appropriate dosages will be determined through clinical trials.

[0084] "Immunologically effective amount," "antitumor effective amount," "tumor suppression effective amount," or "therapeutic amount" When described above, the exact amount of the composition of the present invention to be administered depends 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 Dose 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 particular patient can be determined. Ran is able to monitor the patient's disease progression and adjust treatment accordingly. This can be easily determined by those skilled in the medical field.

[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, intranodal, or intravenously. 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 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 methods described herein or other methods known in the art are used. The cells are activated and expanded by a method for expanding T cells to therapeutic levels, administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any quantity of related therapeutic procedures The therapeutic means includes antiviral therapy, cidofovir and interleukin-2, azathioprine, Treatment with agents such as cytidine (known as ARA-C) or for MS patients Treatment with natalizumab for patients with psoriasis or treatment with efalizumab for patients with PML In a further embodiment, the present invention further comprises administering to the patient other therapeutic options, including, but not limited to, other therapeutic options for the patient. The T cells of the invention may be administered in combination with chemotherapy, radiation, immunosuppressants, 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 is treated with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the 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 recipe for the treatment. The dosage rates for administration to humans will vary according to accepted practices in the art. Typically, 1 × 10 6 pieces~1×10 10 The modified T cells of the 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 an intracellular domain to form a CAR. It exhibits extremely strong killing ability against tumor cells, and has low cytotoxicity and few side effects. (2) The chimeric antigen receptor provided by the present invention is a lentivirus carrying a CAR gene. To achieve stable expression and membrane localization of the CAR protein after infection of T cells with 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 immunoglobulin-specific. The CAR with an optimized e-CH2-CH3 linkage region is able to inhibit Fc receptor binding and subsequent ADCC (antibody-dependent This avoids the risk of cell-dependent cytotoxicity.

[0089] Example 1 Construction of a lentiviral expression vector The coding plasmid was synthesized and cloned by Shanghai Boyi Biotechnology Co., Ltd. Each coding plasmid contained a different anti-CD20 scFv coding sequence. The cloning vector used was the pWPT lentiviral vector, and the cloning part The sites are BamH I and Sal I sites. The structure of the specific sequence is shown in Figure 1. The amino acid and nucleotide sequences are as shown 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 subjects 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 pretreated with CD3 monoclonal antibody at a final concentration of 5 μg / mL. Monoclonal antibody (OKT3) and Retronectin (purchased from TAKARA Co., Ltd.) 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 with saturated humidity and 5% CO2. Cultured. (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 10% CO2 per well. After incubating the cells for 12 hours 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 for the corresponding activity detection test. In the present invention, the manufacturing process of T cells modified with a CAR structure targeting the CD20 antigen is improved. Okay, so I cultured lymphocytes 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 0.5×10 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 levels of CAR20 protein on the membrane surface of T cells were analyzed. All of the designed CAR constructs, except for CAR-T20.13, used protein L to deliver chimeric antigens. It was shown that the receptors were localized at the membrane surface of the correspondingly modified T cells. .

[0092] Example 4 Detection of in vitro activation ability of CAR-T20s After co-culture with target cells using CART-20s cells cultured up to day 6 in Example 2, CD137 The upregulation level of IFNγ and the secretion level of IFNγ in the culture supernatant were detected. Culture CART-20 cells at 1 × 10 5 RAJI and RAMOS tumor cell lines, CD20 positive, respectively. and CD20-negative MOLT-4 tumor cell line, or 200 μl of GT-5 without tumor cell addition. After 18 h of co-culture in 51 medium at a 1:1 ratio, T cell membranes were analyzed by flow cytometry. The expression level of CD137 on the cell surface (Figure 3A) and the concentration of IFNγ in the culture supernatant were measured by ELISA. Secretion levels were detected (Figure 3B). The results in Figure 3 show that obinutuzumab-based CARs also significantly increased expression and membrane surface activity of the corresponding modified cells. The localization of obinutuzumab and 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 in the ratio shown in Fig. 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 binds obinutuzumab and Rituximab. Induce early apoptosis of CD20 target cells in vitro more effectively than simab-based CAR 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) Using CAR-T20s cells cultured up to day 7, produced by the method in Example 2 Under conditions where the transduction rates were almost the same (Figure 5A), 5 Take one cell K562, CD19 monopositive, CD20 monopositive, CD19 and CD20 bipositive K562 stable transfectants, 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 levels (Fig. 5B) and secreted levels of IFNγ in the culture supernatant (Fig. 5C). (2) From the results shown in Figure 5, when the infection efficiency was almost the same, the texture of the hinge region mutations was 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 IFNg-dependent activating ability in vitro was superior to that of the IFNg-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 caudal 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 injected into the tail vein of mice on Day 0. (5 × 10 6 On Day 0, Day 7, Day 14, and Day 21, the mice were imaged in vivo. The growth of tumor cells in the mouse body was observed, and changes in fluorescence intensity and the mouse body temperature were observed. The results were analyzed 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 embodiment of the present invention has been described in detail above, the present invention is not limited to the above-mentioned embodiment. The present invention is not limited to specific details, and various modifications and variations are possible 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 are within the scope of protection of the present invention.

[0097] In addition, the specific technical features described in the above specific embodiments may be arbitrarily used unless inconsistent. In order to avoid unnecessary duplication, the present invention provides The possible combinations are not described separately. In addition, different embodiments of the present invention may be arbitrarily combined, and the present invention is not limited to the above. Unless contrary to the purpose, the contents of the present invention are also deemed to be disclosed in the present invention.

Claims

[Claim 1] Chimeric antigen receptor (CAR).

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