Construction of a chimeric antigen receptor targeting the CD20 antigen and identification of the activity of genetically modified T cells.

Abstract

This invention provides a chimeric antigen receptor that targets CD20 and a method for producing the same. [Solution] The extracellular antigen-binding domain of the chimeric antigen receptor is a chimeric antigen receptor comprising a heavy chain variable region of an antibody comprising a specific amino acid sequence and a light chain variable region of an antibody comprising a specific amino acid sequence, and CAR-T cells (CAR-modified T cells) expressing this chimeric antigen receptor have the ability to kill tumor cells.

Description

[Technical Field]

[0001] The present invention relates to the sequence components of a chimeric antigen receptor targeting the CD20 antigen, and the modified T cells thereof. The present invention provides a method for producing (CART20) and its activity identification. Identify the structure of chimeric antigen receptors that treat tumors. [Background technology]

[0002] Hematological malignancies account for approximately 10% of all malignancies in humans, and 95% of hematological malignancies are caused by B phosphorus. It is derived from pacyl cells. Conventional chemotherapy and radiation therapy have been used to treat malignancies of the hematological system. It plays a vital role, and the therapeutic effect is remarkable in some patients, but the majority are difficult to cure. Effective treatment methods have long been the focus of research in this field.

[0003] Adoptive T-cell therapy has already shown strong therapeutic effects and promising results in the clinical treatment of malignant tumors. It shows the future. Inside, chimeric antigen receptors are independently deployed by several mechanisms. T cells modified by Chimeric Antigen Receptor (CAR) express CD19, which is associated with relapsed syndrome. He achieved unprecedented success in the treatment of refractory B-cell malignancies, particularly at the University of Pennsylvania School of Medicine. Therefore, CART19 was used to treat relapsed / refractory acute B-cell lymphoma (R / R B-ALL). In the clinical trial, 94% of patients experienced complete relief. The initial response rate of this clinical trial was high. However, nearly 40% of patients experienced relapses after complete relief within one month of treatment. Of the patients, as many as 60% showed immune evasion of CD19-negative tumor cells. Therefore, malignant recurrence To treat patients with lymphoma, C targets antigens other than CD19 that are associated with B-cell lymphoma. There is a strong need for screening of ART structures.

[0004] CD20 is a glycosylated protein and the first identified membrane marker for B cells, also known as B1. It is encoded by the MS4A gene. The CD20 molecule has four transmembrane hydrophobic regions, and its N Because both the terminal and C-terminus are located on one side of the cytoplasm, they form two loops extracellularly. These are called the large loop and the small loop, respectively. CD20 is specifically found in over 95% of normal and cancerous B cells. These cells are expressed in cells that are pre-B cells and in subsequent developmental stages, differentiating into plasma cells. CD20 is expressed continuously until [something happens]. Therefore, CD20 is ideal for immunotherapy of B-cell malignancies. It is the target.

[0005] Rituximab (MabThera®, Rituxan®) is the first to be approved by the US FDA and the European EMA. Therefore, first-generation CD20-targeted chimeras approved for the treatment of chronic lymphoma It is a noclonal antibody. Rituximab recognizes and binds to the large loop structure in the extracellular domain of CD20. Furthermore, the ADCC kills tumor cells through its toxic effect. However, rituximab monotherapy When used in Germany, its activity is limited and the duration of the reaction is short, but when used in combination with chemotherapy... This significantly enhances the effectiveness of chemotherapy. Rituximab is used to treat lymphoma, and half of the cases are treated with it. These patients have a complete (CR) or partial (PR) response.

[0006] Ofatumumab (Arzerra R ) is the first whole-human derived CD20 therapeutic antibody. Unlike rituximab, the epitope recognized by ofatumumab is the major CD20 group. - It includes a part of each of the large loop and the small loop. At the same time, the tumor killing means of ofatumumab is mainly through the complement-dependent pathway, and the next is the tumor killing effect depending on ADCC.

[0007] Obinutuzumab (Gazyvaro R , Gazyva R ) is a humanized type II anti-CD20 antibody whose affinity for FcγRIIIa is optimized by reducing fucosylation levels. Obinutuzumab recognizes and binds to the large loop portion of the CD20 extracellular molecule, mainly mediating the tumor killing effect by ADCC. At the same time, the binding of obinutuzumab to the CD20 molecule also has the effect of inducing apoptosis of tumor cells. Obinutuzumab is used in combination with bendamustine, a nitrogen mustard drug, in a phase III clinical trial for NHL that is ineffective in treatment with rituximab. In this trial, the combination of obinutuzumab and bendamustine had a progression-free survival period twice as long as that of bendamustine alone (the former was 29 months and the latter was 14 months). When obinutuzumab is used to treat CLL, the overall response rate (ORR, including CR and PR) reached 77.3%, while that of rituximab was 65.7%. Compared with therapeutic antibodies, cellular immunotherapy is a new tumor treatment method with significant therapeutic effects and a new treatment method for anti-cancer by autoimmunity. It is a method of culturing and amplifying immune cells collected from the patient's body

[0008] in vitro using biotechnology and biological agents and then returning them to the patient's body, aiming to treat tumors by activating and enhancing the body's autoimmune function. Those skilled in the art incorporate the development of new cellular immunotherapies, improve the effects of cellular immunotherapies, and their side effects are improved, and It reduces the effect. In recent years, many therapeutic antibodies like the ones mentioned above have been developed, but their clinical effects... None of the treatments have achieved the same level of therapeutic effect as CART19. Therefore, CD20 The development of CART therapies targeting [specific target cells] has enormous market value and social significance. [Overview of the Initiative]

[0009] Therapeutic antibodies targeting CD20 differ in their affinity and killing mechanism, and therefore we are different. A series of chimeric antigen receptors targeting CD20 are constructed using the DNA sequence of the antigen-binding region of an antibody. To build and identify the in vitro antitumor activity of genetically modified T cells of these chimeric antigen receptors and the differences between them. We conducted a comparison to determine the effectiveness of CAR-T therapy in the clinical treatment of CD20-positive leukemia and lymphoma. This provides new methods and formulations. The object of the present invention is to provide a CD20-targeting chimeric antigen receptor, a method for producing the same, and its use. That is what it is.

[0010] This invention relates to the construction of a structure for a chimeric antigen receptor that targets the CD20 antigen, and the construction of a structure that targets the CD20 antigen. This invention relates to a method for producing genetically modified T cells with chimeric antigen receptors and the identification of their activity.

[0011] In a first aspect of the present invention, a chimeric antigen receptor (CAR) (sequence) is the chimeric antigen receptor The antigen-binding domain of the substance (i.e., scFv) is associated with the antibody shown in SEQ ID NO: 7, 9, or 33. Includes the heavy chain variable region and the light chain variable region of the antibody as shown in SEQ ID NO: 11, 13, or 35. It provides receptors. In another preferred example, the antigen-binding domain of the chimeric antigen receptor is given by the following formula It is represented as follows. V H -VL (However, V H is the variable region of the heavy chain of the antibody, V L is the variable region of the light chain of the antibody, and "-" is a linker peptide or a peptide bond.) In another preferred example, the amino acid sequence of the linker peptide is shown by SEQ ID NO: 15 . In another preferred example, the amino acid sequence of V H is shown by SEQ ID NO: 7, and the amino acid sequence of V L is shown by SEQ ID NO: 11. In another preferred example, the amino acid sequence of V H is shown by SEQ ID NO: 9, and the amino acid sequence of V L is shown by SEQ ID NO: 13. In another preferred example, the amino acid sequence of V H is shown by SEQ ID NO: 33, and the amino acid sequence of V L is shown by SEQ ID NO: 35. In another preferred example, 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 arbitrary leader sequence (Leader sequence, that is, a signal peptide sequence). H is a hinge region. TM is a transmembrane domain. CS is a co-stimulatory molecule derived from 4-1BB and / or CD28. CD3ζ is an intracellular signaling sequence derived from CD3ζ. V H , V L and "-" are as described above respectively.) In another preferred example, the sequence of L is shown by SEQ ID NO: 27. In another preferred example, the sequence of H is shown by SEQ ID NO: 17 or 19. In another preferred example, the sequence of TM is the transmembrane region of CD8a or the transmembrane region of CD28 The TM sequence is preferably represented by SEQ ID NO: 21 or 37.

[0012] In another preferred example, the CS structure is CD28-4-1BB, where CD28 is derived from CD28. It is a co-stimulatory molecule, and 4-1BB is a co-stimulatory molecule derived from 4-1BB. In another preferred example, the sequence of the co-stimulatory molecule derived from 4-1BB is shown in SEQ ID NO: 23. . In another preferred example, the sequence of the CD28-derived co-stimulatory molecule is shown in SEQ ID NO: 39. In another preferred example, the sequence of CD3ζ is shown in sequence number 25. In another preferred example, the sequence of the chimeric antigen receptor is sequence numbers 1, 3, 5, 29, 3 This is shown by 1.

[0013] In a second aspect of the present invention, the chimeric antigen receptor (CAR) described in the first aspect of the present invention is It provides nucleic acid molecules for use in this purpose. In another preferred example, the nucleic acid molecule comprises the hinge region selected from the following group. Includes nucleic acid sequences that encode: (a) Polynucleotides encoding the polypeptide shown in SEQ ID NO: 17 or 19; (b) Polynucleotides whose sequence is shown in SEQ ID NO: 18 or 20; (c) Homology of the nucleotide sequence with the sequence shown in SEQ ID NO: 18 or 20 is ≥90% (good) The poly(s) have a value of ≥95% and encode the amino acid sequence shown in SEQ ID NO: 17 or 19. nucleotide; (d) A polynucleotide complementary to any of the polynucleotides described in (a) to (c) .

[0014] In another preferred example, the nucleic acid molecule is selected from the following group to form a transmembrane of the CD8a. Includes nucleic acid sequences that code for the common region: (a) Polynucleotides encoding the polypeptide shown in Sequence ID No. 21; (b) Polynucleotide whose sequence is shown in Sequence ID No. 22; (c) Homology of the nucleotide sequence with the sequence shown in SEQ ID NO: 22 is ≥90% (preferably) A polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 21, with a tolerance of ≥95%; (d) A polynucleotide complementary to any of the polynucleotides described in (a) to (c) .

[0015] In another preferred example, the nucleic acid molecule is the 4-1BB(CD13) selected from the following group. 7) Includes a nucleic acid sequence encoding the intracellular signaling domain: (a) Polynucleotides encoding the polypeptide shown in Sequence ID No. 23; (b) Polynucleotide whose sequence is shown in SEQ ID NO: 24; (c) Homology of the nucleotide sequence with the sequence shown in SEQ ID NO: 24 is ≥90% (preferably) A polynucleotide that is ≥95% and encodes the amino acid sequence shown in SEQ ID NO: 23; (d) A polynucleotide complementary to any of the polynucleotides described in (a) to (c) .

[0016] In another preferred example, the nucleic acid molecule is a cell of CD28 selected from the following group. Includes nucleic acid sequences that encode the internal signaling domain: (a) Polynucleotides encoding the polypeptide shown in Sequence ID No. 39; (b) Polynucleotide whose sequence is shown in sequence number 40; (c) Homology of the nucleotide sequence with the sequence shown in SEQ ID NO: 40 is ≥90% (preferably) A polynucleotide that is ≥95% and encodes the amino acid sequence shown in SEQ ID NO: 39; (d) A polynucleotide complementary to any of the polynucleotides described in (a) to (c) .

[0017] In another preferred example, the nucleic acid molecule is a subset of the CD3ζ selected from the following group. Includes nucleic acid sequences encoding intracellular signaling domains: (a) Polynucleotides encoding the polypeptide shown in Sequence ID No. 25; (b) Polynucleotide whose sequence is shown in SEQ ID NO: 26; (c) Homology of the nucleotide sequence with the sequence shown in SEQ ID NO: 26 is ≥90% (preferably) A polynucleotide that has an amino acid sequence of ≥95% and is represented by SEQ ID NO: 25; (d) A polynucleotide complementary to any of the polynucleotides described in (a) to (c) .

[0018] In another preferred example, the nucleic acid molecule comprises a nucleic acid sequence selected from the following group. : (a) Polynucleotides encoding polypeptides represented by Sequence ID No. 1, 35, 29, or 31 Do; (b) Polynucleotides whose sequence is represented by sequence numbers 2, 4, 6, 30, or 32; (c) Homology of the nucleotide sequence with the sequence shown in SEQ ID NOs: 2, 4, 6, 30 or 32 ≥95% (preferably ≥98%) and the amino acid combination shown in SEQ ID NOs: 1, 35, 29, or 31 Polynucleotides that code for a sequence; (d) A polynucleotide complementary to any of the polynucleotides described in (a) to (c) . In another preferred example, the nucleic acid molecule is isolated.

[0019] In another preferred example, the nucleic acid molecule further comprises a leader sequence (leader sequence). It includes a polynucleotide encoding the quense (signal sequence), and the leader sequence The amino acid sequence is shown in Sequence ID No. 27, and preferably the leader sequence (signal sequence) is coded The polynucleotide to be used is shown in SEQ ID NO: 28. In another preferred example, the sequence of the nucleic acid molecule is shown in SEQ ID NOs: 2, 46, 30, or 32. It will be done.

[0020] A third aspect of the present invention provides a vector comprising the nucleic acid molecule described in the second aspect of the present invention. do. In another preferred example, the vector is a lentiviral vector. A fourth aspect of the present invention includes, or contains, the vector described in the third aspect of the present invention. This provides a host cell in which an exogenous nucleic acid molecule described in the second aspect of the present invention is incorporated into the chromosome. do. In another preferred example, the cells are isolated cells, and / or the cells These are genetically modified cells. In another preferred example, the cells are mammalian cells. In another preferred example, the cell is a T cell.

[0021] In the fifth aspect of the present invention, a pharmaceutically acceptable carrier and the ki described in the first aspect of the present invention Mela antigen receptor, nucleic acid molecule according to the second aspect of the present invention, and the bean according to the third aspect of the present invention. The present invention provides a drug composition containing a cell or a cell as described in the fourth aspect of the present invention.

[0022] In the sixth aspect of the present invention, the chimeric antigen receptor described in the first aspect of the present invention, the present invention The nucleic acid molecule described in the second aspect, the vector described in the third aspect of the present invention, or the fourth aspect of the present invention The use of cells described in the aspect of a drug or preparation for treating a tumor or autoimmune disease. It provides use in the manufacture of [something]. In another preferred example, the aforementioned autoimmune disease is caused by the overexpression of B cells. It is a disease (for example, lupus erythematosus). In another preferred example, the tumor is a CD20-positive tumor.

[0023] A seventh aspect of the present invention is a method for treating a disease, wherein an appropriate amount of [unclear text] is applied to the subject requiring treatment. Chimeric antigen receptor as described in the first aspect of the invention, nucleic acid molecule as described in the second aspect of the invention, A vector as described in the third aspect of the invention, or a cell as described in the fourth aspect of the invention, or this The present invention provides a method comprising the step of administering a drug composition described in the fifth aspect of the invention. In another preferred example, the disease is a tumor.

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

[0025] A nucleic acid molecule described in the second aspect of the present invention or a vector described in the third aspect of the present invention is T The process includes obtaining the CAR-T cells by transduction into the cells,

[0026] Of course, within the scope of the present invention, each of the above technical features of the present invention and the following (for example) Each of the technical features specifically described in the examples can be combined with each other to create new or suitable technologies. It is understood that a treatment plan can be constructed. Due to space limitations, a detailed explanation will not be provided here. [Brief explanation of the drawing]

[0027] Description of the drawing [Figure 1] Figure 1 shows the structure of a chimeric antigen receptor targeting CD20. Each element of the designed CAR structure, as shown in the figure, includes a leader sequence, an antigen recognition sequence (ofatumumab, obinutuzumab, rituximab), a hinge region, a transmembrane region, a costimulatory signaling 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, while CAR-T20.19 and CAR-20.20 are forms of the L235E-N297Q mutation in the IgG4Hinge-CH2-CH3 linkage region of CAR-T20.14, and CAR-T20.20 is also a third-generation chimeric antigen receptor structure that simultaneously has costimulatory signaling molecular coding sequences for CD28 and 4-1BB. [Figure 2] Figure 2 shows the detection of the transduction efficiency of CD20-targeting chimeric antigen receptors into genetically modified T cells. The expression levels of proteins encoded by the CAR gene on the T cell membrane surface were identified in CAR-T20s cells cultured up to day 7 (A) and day 11 (B) using the Protein L method. [Figure 3] Figure 3 shows that 1 × 10⁵ cells each of NT, CART-20.13, CART-20.14, and CAR-T20.16 cells, cultured up to day 6, were taken and co-cultured for 18 hours in 200 μl of GT-551 medium in a 1:1 ratio with CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell line, respectively. The expression level of CD137 on the membrane surface of the T cells (A) and the secretion level of IFNγ in the co-culture supernatant (B) were then detected. [Figure 4]Figure 4 shows the detection of the level of apoptosis induction in tumor cells by CART-20. 1 × 10⁴ cells of CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines were taken and co-cultured for 4 hours with NT, CART-20.13, CART-20.14, and CAR-T20.16 cells, respectively, cultured in 200 μl of GT-551 medium in the ratios shown in the figure until day 11. The cells were then centrifuged to collect the precipitate, 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 the statistical analysis of Annexin V-positive cells in the corresponding co-culture samples. [Figure 5] Figure 5 shows the identification of the in vitro activation capabilities of hinge region mutation chimeric antigen receptors and third-generation chimeric antigen receptors constructed based on the sequence of the ofatumumab antibody. Using the Protein L method, the expression level of proteins encoded by the CAR gene on the membrane surface of T cells (A) was detected in CAR-T20.14, CAR-T20.19, and CAR-T20.20 cells cultured up to day 7. Then, 1 × 10⁵ cells each of NT, CART-20.14, CART-20.14, and CAR-T20.16 cells cultured up to day 7 were taken and co-cultured in 200 μl of GT-551 medium for 18 hours in a 1:1 ratio with K562, CD19 monopositive, CD20 monopositive, CD19 and CD20 bipositive K562 induced cell lines, and RAJI target cells, respectively. After co-culture, the expression level of CD137 on the membrane surface of T cells (B) and the secretion level 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. The results indicate that CAR-T20.19 can effectively suppress the proliferation of CD20-positive cells in the body.

[0028] Specific Embodiments The inventors have conducted extensive and in-depth research on chimeric antigen receptors targeting CD20. Although a method for producing and using the same was obtained, the extracellular antigen-binding domain of the chimeric antigen receptor was The heavy chain variable region of the antibody indicated by column number 1 and the light chain variable region of the antibody indicated by sequence number 2 Includes. Experimental results show that tumor cells with very high levels of the chimeric antigen receptor provided by the present invention This demonstrates its lethality against [the target].

[0029] Therapeutic antibodies targeting CD20 differ in terms of affinity, mechanism of killing, and so on. Because the transmembrane domain and intracellular domain significantly affect the activity of the chimeric antigen receptor, In the present, different transmembrane and By binding to intracellular regions, a series of chimeric antigen receptors targeting CD20 are constructed, and then... This method enables the expression of chimeric antigen receptors in primary T cells and allows for the detection of receptor expression intensity. To establish a method for the ability of these CAR-T cells to recognize the CD20 antigen in vitro and in vivo, and To identify the difference in activity between in vitro killing and in vitro elimination of malignant tumors with CD20 antigens, and for clinical applications... Novel and effective methods and formulations for the treatment of CD20-positive leukemia and lymphoma using CAR-T therapy. 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. It provides a receptor (CAR). The extracellular domain is a target-specific binding element (antigen-binding domain). It includes the main domain (also called the core). The intracellular domain consists of the co-stimulatory signaling region and the ζ chain region. It includes the co-stimulatory signaling region, which contains a portion of the intracellular domain of the co-stimulatory molecule. Aggressor molecules are cell surface molecules necessary for an effective response of lymphocytes to antigens, and they also act as antigen receptors. It is not that ligand.

[0031] Between the extracellular domain and transmembrane domain of CAR, or between the intracellular domain and transmembrane domain of CAR A linker may be introduced between the inlets. As used herein, the term "linker" "Typically, the transmembrane domain is the extracellular or intracellular domain of the polypeptide chain. A linker is any oligopeptide or polypeptide that performs the function of linking to a molecule. , 0 to 300 amino acids, preferably 2 to 100 amino acids, most preferably 3 to 50 amino acids It may contain anoacids.

[0032] In one preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention The compound contains an antigen-binding domain that targets CD20. The CAR of the present invention is expressed in T cells. In this case, the antigen can be recognized based on the specificity of antigen binding. When combined, it affects tumor cells, preventing them from growing and causing them to die, or in other ways. The patient's tumor burden is reduced or eliminated. The antigen-binding domain is a costimulatory component. It is preferable to fuse with one or more intracellular domains derived from the child and ζ chain. Preferably, the antigen-binding domain is the 4-1BB signaling domain and / or CD28 signaling domain. It fuses with the intracellular domain, which is a combination of the signaling domain and the CD3ζ signaling domain.

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

[0034] In one preferred embodiment of the present invention, a chimeric antigen receptor provided by the present invention The amino acid sequence of (CAR) is as follows: CAR-T20.13 (Sequence ID 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 36o 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 Note: In the original text, "36o" in line 16 should probably be "360". This has been corrected in the translation.RDPEMGGKPR RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT 660 YDALHMQALP PR 672

[0037] The DNA sequence encoding CAR-T20.14 (Sequence ID 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 (Sequence ID 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, more 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 (Sequence ID 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 anti The amino acid sequence of the proto-receptor (CAR) is as follows: CAR-T20.20 (Sequence ID 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 is a target-specific binding domain called an antigen-binding domain. The antigen-binding domain of the CAR of the present invention specifically targets CD and binds to the element. It is a typographical feature. In one preferred embodiment of the present invention, the antigen-binding domain is a heavy chain variable of the anti-CD antibody. Includes regions and light chain variable regions. In another preferred example, the amino acid sequence of the heavy chain variable region of the ofatumumab antibody The following applies: EVQLVESGGG LVQPGRSLRL SCAASGFTFN DYAMHWVRQA PGKGLEWVST ISWNSGSIGY 60 ADSVKGRFTI SRDNAKKSLY LQMNSLRAED TALYYCAKDI QYGNYYYGMD VWGQGTTVTV 120 SS 122 (Sequence ID 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 (Sequence ID 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 (Sequence ID 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] Furthermore, the amino acid sequence of the heavy chain variable region of the obinutuzumab antibody used in this invention is as follows: That's right. QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA PGQGLEWMGR IFPGDGDTDY 60 NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNV FDGYWLVYWG QGTLVTVSS 119 (array 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 (Sequence ID 34)

[0046] In another preferred example, the amino acid sequence of the light chain variable region of the ofatumumab antibody The following applies: 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 (Sequence ID 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 (Sequence ID 13) The DNA sequence encoding the single-stranded 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 (Sequence ID 14)

[0047] Furthermore, the amino acid sequence of the light chain variable region of the obinutuzumab antibody used in this invention is as follows: That's right. DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ LLIYQMSNLV 60 SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQNLELP YTFGGGTKVE IKRTV 115 (Sequence ID 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 (Sequence ID 36) In one preferred embodiment of the present invention, the mesh between the heavy chain variable region and the light chain variable region The no-acid linkage sequence is as follows: GGGGSGGGGS GGGGS 15 (Sequence No. 15) The DNA sequence that codes for it is as follows: GGTGGCGGTG GCTCGGGCGG TGGTGGGTCG GGTGGCGGCG GATCT 45 (Sequence No. 16)

[0048] Hinge region and transmembrane region Regarding the hinge region and transmembrane region (transmembrane domain), CARs merge with the extracellular domain of the CAR. It may be designed to include a combined transmembrane domain. In one embodiment, natural CA One of the domains in R and a related transmembrane domain are used. In some examples, By selecting a transmembrane domain or modifying it with amino acid substitutions, such domains can be created. By avoiding binding to the transmembrane domains of similar or different surface membrane proteins This minimizes interaction with other members of the receptor complex. In one preferred embodiment of the present invention, the hinge region has the following amino acid sequence (IgG4 Hin Includes the 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 (Sequence ID 17)

[0049] The DNA sequence that codes for 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 (Sequence ID 18) Alternatively, the hinge region may have the following amino acid sequence (IgG4 Hinge-CH2-CH3(L235E,N297Q) ) includes. 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 (Sequence ID 19)

[0050] The DNA sequence that codes for 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 ID 20).

[0051] In one preferred embodiment of the present invention, the amino acid distribution of the transmembrane region (CD8™) derived from CD8 The columns are as follows: IYIWAPLAGT CGVLLLSLVI TLYC 24 (Sequence ID 21) The DNA sequence that codes for it is as follows: ATCTACATCT GGGCGCCCTT GGCCGGGACT TGTGGGGTCC TTCTCCTGTC ACTGGTTATC 60 ACCCTTTACT GC 72 (Sequence ID 22) In one preferred embodiment of the present invention, the transmembrane region (CD28™) derived from CD28 is used The acid sequence is as follows: FWVLVVVGGV LACYSLLVTV AFIIFWV 27 (Sequence ID 37) The DNA sequence encoding the transmembrane region derived from CD28 (CD28™) is as follows: TTTTGGGTGC TGGTGGTGGT TGGTGGAGTC CTGGCTTGCT ATAGCTTGCT AGTAACAGTG 60 GCCTTTATTA TTTTCTGGGT G 81 (Sequence ID 38)

[0052] intracellular domain The intracellular domain in the CAR of the present invention is the signaling domain 4-1BB and CD3ζ Includes signal transduction domains. Preferably, the signaling domain at 4-1BB includes the following amino acid sequence. KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL 42 (Sequence ID 23) The DNA sequence that codes for it is as follows: AAACGGGCA GAAAGAAACT CCTGTATATA TTCAAACAAC CATTTATGAG ACCAGTACAA 60 ACTACTCAAG AGGAAGATGG CTGTAGCTGC CGATTTCCAG AAGAAGAAGA AGGAGGATGT 120 GAACTG 126 (Sequence ID 24) Preferably, the intracellular signaling domain derived from CD28 includes the following amino acid sequence. RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S 41 (Sequence ID 39) The DNA sequence that codes for it is as follows: AGGAGTAAGA GGAGCAGGCT CCTGCACAGT GACTACATGA ACATGACTCC CCGCCGCCCC 60 GGGCCCACCC GCAAGCATTA CCAGCCCTAT GCCCCACCAC GCGACTTCGC AGCCTATCGC 120 TCC 123 (Sequence ID 40)

[0053] Preferably, the intracellular signaling domain of CD3ζ contains the following amino acid sequence. RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPQ RRKNPQEGLY 60 NELQKDKMAE AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR 113 (Sequence ID 25) The DNA sequence that codes for 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 (Sequence ID 26)

[0054] vector Furthermore, the present invention provides a DNA construct that encodes the CAR sequence of the present invention. The nucleic acid sequence encoding the target molecule can be recombined using a known recombination method in this field, such as generating a gene. Screening libraries in cells exhibiting the gene, and libraries containing known genes. Obtaining the vector from a vector, or cells containing the gene and using standard techniques It can be obtained by directly isolating it from tissue. If necessary, the gene of interest can be obtained It can also be produced through synthesis.

[0055] Furthermore, the present invention provides a vector into which the DNA construct of the present invention is inserted. Retrovirus For example, lentivirus-derived vectors allow for long-term stable pairing of introduced genes. To enable implantation and proliferation in its daughter cells, long-term gene transfer is achieved. It is a suitable tool for this. Lentiviruses are introduced into cells that do not multiply, such as liver cells. Therefore, a vector for a carcinogenic retrovirus, such as mouse leukemia virus, can be used. It has superior advantages. It also has the advantage of low immunogenicity.

[0056] In other words, typically, nucleic acids that encode a CAR polypeptide or a portion thereof in a manipulable form are promoted By linking to a vector and incorporating the construct into an expression vector, CAR can be encoded. The vector enables the expression of natural or synthetic nucleic acids. The vector facilitates the replication and combination of eukaryotic cells. Suitable for cloning. Typical cloning vectors are used to regulate the expression of the target nucleic acid sequence. Includes usable transcription and translation terminators, start sequences, and promoters.

[0057] The expression construct of the present invention is delivered via a standard gene delivery protocol to nucleic acid immunity and gene It can also be used in therapy. Methods of gene delivery are known in this field. For example, Referencing U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, the full text is hereby taken by reference. In another embodiment, the present invention provides a gene therapy vector.

[0058] The nucleic acid can be cloned into various types of vectors. For example, the nucleic acid The vectors that can be cloned include plasmids, phages, phage derivatives, and animal viruses. This includes, but is not limited to, s and cosmids. A specific vector of interest is an expression vector. Includes vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0059] Furthermore, expression vectors can be delivered to cells in the form of viral vectors. The technology of viral vectors is well known in this field, and for example, Sambrook et al. (2001, Mole (Cyclical Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and It is described in other virology and molecular biology manuals. It can be used as a vector. The viruses involved include retroviruses, adenoviruses, adeno-associated viruses, and herpesviruses. This includes, but is not limited to, S and lentiviruses. Usually, there are few suitable vectors. Also, replication origins, promoter sequences, and convenient restriction enzyme digests that act in one type of organism. Includes site and one or more selectable markers (e.g., WO01 / 96584, WO01 / 290) (58 and U.S. Patent No. 6,326,193).

[0060] Many virus-based systems have already been developed for the introduction of genes into mammalian cells. It is used for, for example, retroviruses are useful for gene delivery systems. We provide a rat home. We insert genes selected using techniques known in this field into the vector. It can be packaged into retrovirus granules. This recombinant virus is They are then isolated and delivered to target cells inside or outside the body. Many retroviral cells Them is known in this field. In some embodiments, an adenovirus vector is used. Many adenovirus vectors are known in this field. In some embodiments, Therefore, lentiviral vectors are used.

[0061] Additional promoter elements, such as enhancers, regulate the frequency at which transcription is initiated. It is possible. Typically, these are located in the 30-110 bp region upstream of the starting point, but recently, Many promoters have been shown to include functional elements downstream of the starting point. The spacing between motor elements is such that the elements are inverted relative to each other. Alternatively, many are mobile so that the promoter's function can be maintained if it is moved. In the din kinase (TK) promoter, the spacing between promoter elements is active It can be increased up to 50 bp without decreasing. By the promoter, a single The elements act together or independently to initiate transcription.

[0062] An example of a suitable promoter is the pre-early cytomegalovirus (CMV) promoter sequence. The promoter sequence is any polynucleotide sequence that can be manipulated and ligated thereto. It is a powerful constitutive promoter sequence that can express at high levels. Another example of a motor is the extension factor 1α (EF-1α). However, other constitutive promo The ter sequence may also be used, Simian virus 40 (SV40) initial promoter, mouse mammary cancer Virus (MMTV), Human Immunodeficiency Virus (HIV) Long-chain Terminal Repeat (LTR) Promoter, Mo MuLV promoter, bird leukemia virus promoter, Epstein- Barr) virus pre-early promoter, Roussarcoma virus promoter, and human genetics This includes, but is not limited to, child promoters; human gene promoters include, for example, A cutin promoter, myosin promoter, heme promoter and creatine kinase promoter Examples include, but are not limited to, motors. Furthermore, the present invention is a constructive motor - Not limited to the use of -. Inducible promoters can also be considered as part of the present invention. The use of promoters provides a molecular switch, thereby creating a site where such expression is required. In addition, the expression of a polynucleotide sequence linked to an inducible promoter is initiated in a controllable manner. Alternatively, if expression is not necessary, expression can be terminated. (Inducible promoter) Examples include metallothionein promoters, glucocorticoid promoters, and progesterones. This includes, but is not limited to, tetracycline promoters and tetracycline promoters.

[0063] To evaluate the expression of a CAR polypeptide or a part thereof, an expression vector introduced into cells - may contain either or both of a selectable marker gene and a reporter gene so that expressing cells can be identified and selected from a group of cells transfected or infected with a viral vector. Also, the selectable marker can be carried on a single DNA fragment and used in the process of co-transfection. Appropriate regulatory sequences may be present in the regions adjacent to both the selectable marker gene and the reporter gene so that they can be expressed in a host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo. The reporter gene is used for the identification of cells that may have been transfected and for the evaluation of the functionality of regulatory sequences. Usually, the reporter gene encodes a polypeptide whose expression is not present in the recipient organism or tissue, or is expressed by the recipient organism or tissue and whose expression can be clearly demonstrated by a property that can be easily detected, such as enzyme activity When the DNA has already been introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time. Appropriate reporter genes include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, and green fluorescent protein (for example, Ui-Tei et al., 2000 FEBS

[0064] Letters 479:79-82). Appropriate expression systems are known and can be produced by known techniques or obtained commercially. Usually, the one showing the highest level of expression of the reporter gene Letters 479:79-82). Appropriate expression systems are known and can be produced by known techniques or obtained commercially. Usually, the one showing the highest level of expression of the reporter gene A construct having at least five flanking regions is identified as a promoter. The promoter region is linked to the reporter gene and regulates the promoter of the reagent. It can be used to evaluate the ability to activate transcription.

[0065] Methods for introducing genes into cells and methods for expressing genes in cells are relevant in this field. This is known. In the content of the expression vector, the vector can be expressed in any way in this field This allows for easy introduction into host cells, such as those of mammals, bacteria, yeast, and insects. 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 and lipofibre. This includes injection, particle collision, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or foreign nucleic acids are well known in this field. For example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour) See (or Laboratory, New York). Which method is preferable for introducing polynucleotides into host cells? The method is calcium phosphate transduction.

[0067] Biological methods for introducing polynucleotides of interest into host cells include DNA and RNA vectors. This includes methods for using vectors. Viral vectors, especially retroviral vectors, are already It has become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors include lentiviruses, poxviruses, and herpes simplex virus. It may also be derived from adenoviruses or adeno-associated viruses, for example, US patents. See numbers 5,350,674 and 5,585,362.

[0068] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, for example, Oita Subcomplexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixtures This includes lipid-based systems such as composite micelles and liposomes. It also includes extracorporeal and intracorporeal delivery carriers (de An example of a colloidal system used as a livery vehicle is a liposome (for example, an artificial membrane (It is a vesicle.)

[0069] When using nonviral delivery systems, an exemplary delivery carrier is liposomes. It is conceivable to use this to introduce nucleic acids into host cells (ex vivo). (or in the body). Furthermore, the nucleic acid may be associated with lipids. Lipid-associated nucleic acids are liposolecules. It is encapsulated within the aqueous solution, dispersed within the lipid bilayer of the liposome, and the liposome and oligonucleotides The ocide attaches to the liposome via a linking molecule that connects to both ends, and is taken up by the liposome. It is then complexed with liposomes, dispersed in a lipid-containing solution, mixed with lipids, and combined with lipids. It is contained in lipids as turbidity, contained in micelles, complexed with micelles, or in other forms. It can bind to lipids in a specific state. The composition can bind to lipids, lipid / DNA, or lipid / expression molecules. The term "Kutar" is not limited to any specific structure in a solution. For example, the structure of a bilayer. They may exist as micelles or in a "disintegrated" structure. They disperse easily in solution. Furthermore, they may form aggregates of different sizes and shapes. Lipids are fatty substances, and even natural lipids. It may be a synthetic lipid. For example, the lipid is a lipid droplet that naturally occurs in the cytoplasm, as well as a long-chain aliphatic hydrocarbon and its derivatives, such as fatty acids, alcohols, amines, amino a compounds such as rchols and aldehydes. When using a non-viral delivery system, for example, gene editing techniques, such as CRISPR-Cas9, Z FN or TALEN to complete the present invention. In one preferred embodiment of the present invention, the vector is a lentiviral vector .

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

[0071] therapeutic use The present invention includes cells (for example, T cells) transfected with a lentiviral vector (LV) encoding the CAR of the present invention. The transfected T cells elicit a T cell response by the CAR . It can be rubbed.

[0072] Therefore, the present invention relates to the immune response by T cells against target cell groups or tissues in mammals. A method for stimulating the present invention, comprising the step of applying T cells expressing the present invention to a mammal. We will also provide the method.

[0073] In one embodiment, the present invention modifies the genes of T cells to express the CAR of the present invention. This includes cell therapy in which CAR-T cells are injected into the recipient who needs it. The cells being treated can kill the recipient's tumor cells. Unlike antibody therapy, CA RT cells can replicate in the body, resulting in long-term persistence in suppressing tumors.

[0074] In one embodiment, the CAR-T cells of the present invention undergo stable T cell proliferation in the body. This allows the effect to be sustained for an extended period of time. Furthermore, the immune response induced by CAR is used in adoptive immunotherapy. Even a portion of it is sufficient, and here, CAR-modified T cells have specific immunotherapy for the plateau-binding domain in CAR. It induces an epidemic response. For example, anti-CD20 CAR-T cells have a specific resistance to cells that express CD20. It triggers a specific immune response.

[0075] The data disclosed herein specifically pertain to anti-CD20scFv, hinge and transmembrane regions, and We have released a lentiviral vector containing 4-1BB and the CD3ζ signaling domain, but this The term "clarity" should be interpreted as including any number of variations in each component of the structure.

[0076] The indications for which it can be treated are CD20-positive tumors and diseases caused by excess B cells (e.g., autoimmune diseases). This includes diseases such as lupus erythematosus. CD20-positive tumors are CD20-positive non-solid tumors ( This includes blood tumors (such as leukemia and lymphoma) or solid tumors. The CAR of the present invention can cure The types of tumors or cancers treated include cancer, germ cell tumors and sarcomas, and some leukemias and lymphocytic malignancies. Benign tumors and malignant tumors, and malignant tumors include, for example, sarcomas, cancers and melanomas. However, it is not limited to these. It also includes adult tumors / cancers and childhood tumors / cancers.

[0077] Blood cancer is cancer of the blood or bone marrow. Examples of blood (or hematogenic) cancer include leukemia. In addition, acute leukemia (for example, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia) Diseases and myeloblastic, promyelocytic, myelomonocytic, mononuclear, and erythroleukemia, chronic leukemia (for example For example, chronic myelocytic (granulocytic) leukemia, chronic bone marrow leukemia and chronic lymphocytic leukemia), Hemocytosis, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and severe cases), multiple Myeloma, Waldenström macroglobulinemia, H chain disease, myelodysplastic syndrome This includes hair cell leukemia and spinal cord malformations.

[0078] Solid tumors typically do not include cysts or masses of fluid-filled tissue. Solid tumors can be benign or malignant. But that's fine. Different types of solid tumors are named according to the type of cells that form them. Examples of solid tumors include sarcomas, cancers, and lymphomas. Examples of solid tumors include sarcomas and cancers such as fibrosarcomas and mucosal tumors. This includes sarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer. The CAR-modified T cells of this invention are vaccines for in vitro immunization and / or in vivo therapy against mammals. It is also useful as such. Preferably, the mammal is human.

[0079] Regarding in vitro immunity, i) cell proliferation, ii) introduction of CAR-encoding nucleic acids into cells, and / or iii) at least one of the cell cryopreservations is performed outside the body before the cells are administered to mammals. It will be held at [location].

[0080] The in vitro protocol is publicly known in this field and will be discussed in more detail later. For example, cells isolated from mammals (preferably human) express the CAR disclosed herein. Gene modification is performed using a vector (i.e., in vitro transformation or transduction). CAR modification The cells can be administered to mammalian recipients and provide beneficial therapeutic effects. The recipient of the dairy animal can be a human, and the CAR-modified cells are the recipient's own cells. This is also fine. If necessary, cells can be allogeneic, syngeneic, or Different species are acceptable. Regarding in vitro immunity, other than cell-based vaccines, this invention provides in vitro immunity to patients. The present invention also provides compositions and methods for eliciting an immune response to an antigen.

[0081] Typically, activated and proliferated cells, as described herein, occur in individuals without an immune response. It can be used to treat and prevent diseases. In particular, the CAR-modified T cells of the present invention It can be used to treat CCL. In some embodiments, the cells of the present invention This is used to treat patients who are at risk of developing CCL. Therefore, this invention is used to treat CCL A method for treating or preventing the disease, wherein a therapeutically effective amount of the CAR-modified T of the present invention is administered to a target in need. The present invention provides a method that includes the step of applying cells.

[0082] The CAR-modified T cells of the present invention can be used alone or as a diluent and / or other drug composition. Administer in combination with other components, such as IL-2, IL-17, or other cytokines or cell groups. In short, the drug composition of the present invention can be used to create biosynthetic drugs, such as monoclonal antibiotics. A body containing small molecule drugs, and one or more pharmaceutically or physiologically acceptable carriers, diluents. Alternatively, it may be combined with an excipient. Such a composition may be used with a buffer, such as neutral buffered saline. , sulfate buffered saline, etc., carbohydrates such as glucose, mannose, sucrose and dextrose Lan, mannitol, proteins, polypeptides or amino acids, such as glycine, acids Anti-oxidants, chelating agents, such as EDTA and glutathione, adjuvants (for example, hydroxide It may also contain aluminum and preservatives. The composition of the present invention is administered intravenously. It is preferable to prepare it as sea urchin.

[0083] The drug composition of the present invention is administered in a form suitable for a disease requiring treatment (or prevention). It is possible to administer the quantity and frequency of administration, depending on the patient's bed number and the type and severity of the patient's disease. The appropriate dosage is determined by factors such as the degree of the dose, but it is determined through clinical trials.

[0084] This is referred to as the "immunological effective dose," "antitumor effective dose," "tumor suppression effective dose," or "therapeutic dose." When providing information, the precise amount of the composition of the present invention to be administered should be determined based on the patient's (subject's) age, weight, and tumor size. The size of the lesion, the extent of infection or metastasis, and individual differences in disease symptoms are taken into consideration and determined by a physician. Typically, the drug compositions containing T cells described herein are 10 4 ~10 9 Dosage per individual cell / kg body weight, Preferably 10 5 ~10 6 Dosage per individual cell / kg body weight (all integer values ​​within these ranges) It can be administered (including). The T cell composition may be administered several times at these dosages. The cells are injected using injection techniques known in immunotherapy (e.g., Rosenberg et al., NewEng.J. of Med.319:167). It can be administered according to (6,1988). The optimal dosage and treatment plan for specific patients will be determined. Run can monitor the patient's disease findings and adjust treatment accordingly. This can be easily determined by engineers in the medical field.

[0085] The composition may be applied to the target by any means, including spraying, injection, oral administration, intravenous infusion, implantation, or transplantation. This may be carried out by convenient means. The compositions described herein are used subcutaneously, intradermally, intratumorally, intranodulately, It may be administered to the patient intraspinal, intramuscular, intravenous (IV) injection, or intraperitoneally. In terms of administration, the T cell composition of the present invention is administered to patients by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is administered by intravenous injection. Preferably, the T cell composition may be injected directly into the tumor, lymph node, or infected area. stomach.

[0086] In some embodiments of the present invention, the methods described herein or other methods known in the art may be used. Using cells activated and proliferated by a method that proliferates T cells to therapeutic levels, In conjunction with a specified number of related treatments (for example, before, simultaneously with, or after), the patient is administered The therapeutic means include antiviral therapy, cidofovir and interleukin-2, and bruises. Treatments using reagents such as cytidine (known as ARA-C), or for MS patients Treatment with natalizumab or treatment with efalizumab or PML in patients with psoriasis This includes, but is not limited to, other treatments for the patient. In further embodiments, The T cells of this invention are chemotherapy, radiation, immunosuppressants, such as cyclosporine A and azathioprine. Purines, methotrexate, mycophenolate mofetil, FK506, antibodies, or other immunosuppressants. It can be used in combination with disease treatment agents. In a further embodiment, the cell composition of the present invention is Bone marrow transplantation, chemotherapy, such as fludarabine, external phototherapy (XRT), cyclophosphamide It is administered to the patient in combination with sphamide (for example, before, simultaneously with, or after). For example, in one embodiment, the subject undergoes peripheral blood stem cell transplantation after high-dose chemotherapy. It is also possible. In some embodiments, after transplantation, the subject receives an injection of the proliferated immune cells of the present invention. In another embodiment, the proliferated cells are administered before or after surgery. It will be done.

[0087] Dosages of treatment exceeding those administered to the patient depend on the precise characteristics of the disease being treated and the treatment recipe. It varies depending on the individual. The dosage ratio for administration to humans is determined by the practices accepted in this field. It can be performed. Typically, 1 x 10 in one treatment or one treatment cycle. 6 pieces~1×10 10 Individual modified T cells of the present invention (e.g., CAR-T20 cells) are administered, for example, by means of intravenous infusion. Therefore, it can be administered to patients.

[0088] The advantages of this invention are as follows: (1) The chimeric antigen receptor of the present invention has an extracellular antigen-binding domain that is specific to anti-CD20 scFv, The specific anti-CD20 scFv CAR, which binds to a specific hinge region and intracellular domain, is very It exhibits powerful tumor cell-killing ability, yet has low cytotoxicity and few side effects. (2) The chimeric antigen receptor provided by the present invention is a lentwill carrying a CAR gene. After infecting T cells with CAR proteins, the goal is to achieve stable expression and membrane localization of CAR proteins. It is possible. (3) The CAR-modified T cells of the present invention have a long survival period in the body and a strong antitumor effect, and IgG4 Hing CARs with optimized e-CH2-CH3 linkage regions enable Fc receptor binding and subsequent ADCC activity (antibody-dependent). This can avoid cytotoxic effects.

[0089] Example 1: Construction of a lentiviral expression vector The coding plasmid was synthesized by Shanghai Boyi Biotechnology Co., Ltd., which also performed the synthesis and cloning of full-length DNA. We requested construction. In each coding plasmid, a different anti-CD20 scFv coding sequence was used. The cloning vector used was the pWPT lentiviral vector, and the cloning section The positions are BamH I and Sal I. The specific sequence structure is shown in Figure 1. The sequences of the amino acids and nucleotides are as shown above. In the following examples, CAR-T20.13, CAR-T20.14, CAR-T20.16, CA showed relatively good results. We will explain using R-T20.19 and CAR-T20.20 as examples.

[0090] Example 2: Production of CAR-T cells (1) Vessel blood was collected from healthy individuals, 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 cultured to the minimum size. Final concentration is 2 × 10 6 The solution was adjusted to achieve a cell / mL concentration. The cells were pre-treated to a final concentration of 5 μg / mL of CD3. Noclonal antibody (OKT3) and Retronectin with a final concentration of 10 μg / mL (purchased from TAKARA Corporation) The cells were inoculated into cell culture bottles coated with ). The culture medium was then mixed with recombinant hydroxypropyl alcohol to a final concentration of 1000 U / mL. Tointerleukin-2 (IL-2) was added and placed in an incubator at 37°C, saturated humidity, and 5% CO2. It was cultured. (3) On the second day, fresh culture medium, concentrated and purified CAR20s lentivirus solution, protamine Sulfate (12 ug / ml) and IL-2 with a final concentration of 1000 U / mL were added. The ink was prepared at 37°C with 5% CO2. After inoculating in a vibrator for 12 hours, discard the culture medium, add fresh medium, and set to 37°C and 5% CO2. The culture was continued in the incubator. (4) From day 6, CART20s cells were taken and the corresponding activity detection tests were performed. In this invention, the process for producing T cells modified with a CAR structure targeting the CD20 antigen is improved. Okay, lymphocytes were cultured in vitro in serum-free GT-551 medium supplemented with 2% human serum albumin. .

[0091] Example 3: Integration rate of CAR genes in the T cell genome and the genes encoded therein Detection of the expression level of proteins on the membrane surface Each is 0.5 × 10 6 In Example 2, the cells were cultured until day 7 (Figures 2A and 5A) and day 11 (Figure 2B). A sample of CART-20s cells was taken, stained with protein L, and measured using a flow cytometer. We then analyzed the expression level of the CAR20 protein on the membrane surface of T cells. From the results, in this study we found that The designed CAR structures, with the exception of CAR-T20.13, all use protein L to create chimeric antigens. The localization of the receptor to the membrane surface of correspondingly modified T cells was detected. .

[0092] Example 4: Detection of the in vitro activation ability of CAR-T20s After co-culturing target cells with CART-20s cells cultured up to day 6 in Example 2, CD137 Upregulation levels and IFNγ secretion levels in the culture supernatant were detected. These were monitored sequentially up to day 6. Cultured CART-20 cells 1 × 10 5 Individually isolated CD20-positive RAJI and RAMOS tumor cell lines, In addition, CD20-negative MOLT-4 tumor cell lineage, or 200 μl of GT-5 without the addition of tumor cells. After co-culturing in 51 medium at a 1:1 ratio for 18 hours, the membrane of the T cells was measured using a flow cytometer. The expression level of CD137 on the surface (Figure 3A) was determined by ELISA, and the IFNγ content in the culture supernatant was also determined. The secretion level was detected (Figure 3B). The results in Figure 3 show that obinutuzumab-based CAR also affects the expression of corresponding modified cells and membrane surface Localization was achieved, but the CAR structure based on the ofatumumab sequence was obinutuzumab and Superior in vitro activation capability and target antigen specificity compared to CARs constructed from rituximab. The conclusion was reached that it had been demonstrated.

[0093] Example 5: Detection of the activity of CAR-T20s cells in inducing early apoptosis in tumor cells In Example 2, CART-20.13, CART-20.14, and CAR-T20.16 cells cultured up to day 11 were used. , 1 × 10 in proportion to the ratio shown in Figure 4 4 Individual CFSE-labeled CD20-negative (MOLT-4) or CD20 Co-culture positive (RAJI,RAMOS) tumor cell lines in 200 μl of GT-551 medium for 4 hours, then centrifuge. After collecting the cell precipitate and washing the cells twice with PBS, stain them with Annexin V-APC staining reagent in a 1:50 ratio. After staining in 100 μl of staining solution for 30 minutes and washing once with PBS, the sample was subjected to flow cytometry. Therefore, we analyzed the proportion of Annexin V-positive cells among CFSE-positive cells. The results in Figure 4 show that the CAR structure based on the ofatumumab sequence is obinutuzumab and lituk Superior inducing early apoptosis of CD20 target cells in vitro compared to CARs constructed from simab. This demonstrates their ability to do so.

[0094] Example 6 Chimeric antigen receptors with hinge region mutations and third-generation chimeric antigen receptors Identification of extracorporeal activation ability (1) Using CAR-T20s cells cultured up to day 7, prepared by the method in Example 2 Using these methods, under conditions where the transduction rate was approximately the same (Figure 5A), each was 1 × 10 5 Take individual cells K562, CD19 monopositive, CD20 monopositive, CD19 and CD20 bipositive K562 stable transduced cell lines, and RAJI label After co-culturing target cells in a 1:1 ratio in 200 μl of GT-551 medium for 18 hours, CD137 was upregulated. The levels (Figure 5B) and IFNγ secretion levels in the culture supernatant (Figure 5C) were detected. (2) From the results shown in Figure 5, when the infection efficiency is nearly the same, the hinge region mutations The CAR-T20.19 antigen receptor has nearly equivalent in vitro activation capabilities to CAR-T20.14 (CD137 and IFNg). However, the third-generation CAR structure, CAR-T20.20, was superior to the second-generation CAR-T20.14 and CAR-T20.19. It was found that it showed superior in vitro activation ability compared to (CD137 and IFNg).

[0095] Example 7: Detection of the ability of CAR-T20 cells to eliminate CD20-positive cells in the body. (1) Raji-Luc cells expressing luciferase were injected into NCG mice via the tail vein (5 × 1 0 5 ( / animal). One week after vaccination, in vivo imaging revealed the proliferation of tumor cells in the body. The situation was observed and noted as Day 0. NT and CAR-T20.19 cells were collected from the tail vein of Day 0 mice. Injected (5 x 10 6 ( / animal). On Day 0, Day 7, Day 14, and Day 21, in vivo imaging was performed. The proliferation of tumor cells in the mouse body was observed, and changes in fluorescence intensity and the mouse body The analysis was based on changes in weight. (2) The results shown in Figure 6 indicate that CAR-T20.19 effectively promotes the proliferation of CD20-positive cells in the body. It can be seen that it can be suppressed.

[0096] Although preferred embodiments of the present invention have been described in detail above, the present invention is a tool used in the above embodiments. Not limited to physical details, but within the scope of the spirit of the present invention, various aspects of the technical solution of the present invention Various simple modifications can be made, and any of these simple modifications falls within the scope of protection of the present invention.

[0097] Furthermore, each specific technical feature described in the above specific embodiment is, to the extent that it does not contradict the other, The present invention can be combined in an appropriate form of intent, and to avoid unnecessary duplication, various Possible combinations will not be explained separately. Furthermore, different embodiments of the present invention can be arbitrarily combined, and the present invention is as described above. Unless otherwise stated, this shall be considered to be the same as the content disclosed in this invention.

Claims

[Claim 1] Chimeric antigen receptor (CAR).

Images