Chimeric antigen receptors and uses thereof

Abstract

A chimeric antigen receptor is provided. The chimeric antigen receptor comprises an extracellular domain comprising a heavy chain variable region, a light chain variable region, and a CD8 hinge region of a single-chain antibody fragment; a transmembrane domain comprising an immune costimulatory molecule transmembrane domain; and an intracellular domain comprising an immune costimulatory molecule intracellular segment and a CD3 zeta chain. The chimeric antigen receptor can specifically recognize tumor cells expressing a specific antigen and achieve a specific killing effect against tumor cells expressing a highly specific antigen.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of Chinese Patent Application No. 201910741624.1, filed with the State Intellectual Property Office of China on August 12, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to the field of biomedicine, in particular, the present invention relates to a chimeric antigen receptor and uses thereof, more particularly, the present invention relates to a chimeric antigen receptor, a transgenic lymphocyte expressing said chimeric antigen receptor, a construct for preparing said transgenic lymphocyte, a lentivirus, a method, a therapeutic composition for treating cancer, and a method for improving the safety, efficacy or durability of lymphocyte therapy. [Background technology]

[0003] Tumor cells can escape from immune surveillance by inducing the expression of immunosuppressive receptors and stopping their immune response. Chimeric antigen receptor T cells (CAR-T cells) are T cells with chimeric proteins expressed on them, which are composed of antibody variable regions that can recognize certain tumor antigens linked with costimulatory structures and CD3 proteins; thus, CAR-T cells can specifically recognize and kill tumor target cells, and with the help of the cytotoxicity of T cells and the targeting effect of antibodies, can kill tumor cells more accurately and quickly, which has achieved remarkable results in hematological malignancies treatment and is a rapidly developing personalized tumor treatment program. However, there are some problems in the application of CAR-T. Improving and solving the existing problems in CAR-T treatment is the key to the development of CAR-T treatment in the future. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 5,449,752 [Patent Document 2] US Patent Application Publication No. 2002 / 0197266 [Patent Document 3] U.S. Patent No. 4,235,871 [Patent Document 4] U.S. Patent No. 4,501,728 [Patent Document 5] U.S. Patent No. 4,837,028 [Patent Document 6] U.S. Patent No. 5,019,369 [Patent Document 7] U.S. Patent No. 5,075,109 [Patent Document 8] U.S. Patent No. 4,452,775 [Patent Document 9] U.S. Patent No. 4,667,014 [Patent Document 10] U.S. Patent No. 4,748,034 [Patent Document 11] U.S. Patent No. 5,239,660 [Patent Document 12] U.S. Patent No. 3,832,253 [Patent Document 13] U.S. Patent No. 3,854,480 [Patent Document 14] U.S. Patent No. 5,087,616 [Patent Document 15] U.S. Patent No. 4,450,150 [Non-patent literature]

[0005] [Non-Patent Document 1] Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000 [Non-Patent Document 2] Peptide and Protein DrugAnalysis, Reid, R., Marcel Dekker Inc., 2000 [Non-licensed document 3] Epitope Mapping, Westwood, Oxford University Press, Oxford, United Kingdom, 2001

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0006] The present disclosure is based on the inventors' discovery and realization of the following facts and problems:

[0007] Currently, there are many problems in the treatment of chimeric antigen receptor gene-modified T cell therapy (CAR-T), such as high relapse rate, high cytokine inflammation, poor durability, and sometimes even neurotoxicity. [Means for solving the problem]

[0008] The object of the present invention is to solve one of the technical problems mentioned above. Based on the prior art of CAR-T, the inventors of the present application have designed a novel immune co-stimulatory molecule tandem structure of chimeric antigen receptor (CAR). CAR-T cells with this tandem structure CAR can effectively reduce the self-release of CAR-T cell inflammatory factors and stimulate central memory T cells (T CM It is surprising that CAR-T can increase the proportion of T cells that are activated by CAR-T antibodies, stimulate the immune response of normal T cells, and activate the function of normal T cells, which is expected to solve the existing problems in prior art CAR-T treatments.

[0009] In the first aspect of the present invention, the present invention provides a chimeric antigen receptor. According to an embodiment of the present invention, the chimeric antigen receptor comprises an extracellular domain comprising a heavy chain variable region, a light chain variable region, and a CD8 hinge region of a single chain antibody; a transmembrane domain comprising an immune costimulatory molecule transmembrane domain; and an intracellular domain comprising an immune costimulatory molecule intracellular segment and a CD3 zeta chain (CD3 zeta). According to an embodiment of the present invention, the chimeric antigen receptor can specifically recognize tumor cells expressing a specific antigen and achieve a specific killing effect against tumor cells highly expressing a specific antigen.

[0010] According to an embodiment of the present invention, the above chimeric antigen receptor further comprises at least one of the following additional technical features:

[0011] According to an embodiment of the invention, the immune costimulatory molecule transmembrane domain is a CD8 transmembrane domain; the immune costimulatory molecule intracellular segment is a 4-1BB intracellular segment and an ICOS or OX-40 intracellular segment.

[0012] According to an embodiment of the invention, the immune costimulatory molecule transmembrane domain is an ICOS transmembrane domain; the immune costimulatory molecule intracellular segment is a 4-1BB intracellular segment and an ICOS intracellular segment.

[0013] According to an embodiment of the invention, the immune costimulatory molecule transmembrane domain is an OX-40 transmembrane domain; the immune costimulatory molecule intracellular segment is a 4-1BB intracellular segment and an OX-40 intracellular segment.

[0014] Through experiments, the inventors found that when different immune costimulatory molecule transmembrane domains were combined with different immune costimulatory molecule intracellular segments, the expression titer of the chimeric antigen receptor, the killing effect of immune cells expressing the chimeric antigen receptor, and the release of cytokines were significantly affected.

[0015] According to an embodiment of the present invention, the immune costimulatory molecule transmembrane domain is a CD8 transmembrane domain, the immune costimulatory molecule intracellular segment is a 4-1BB intracellular segment and an ICOS intracellular segment, the N-terminus of the ICOS intracellular segment is linked to the C-terminus of the CD8 transmembrane domain, the C-terminus of the ICOS intracellular segment is linked to the N-terminus of the 4-1BB intracellular segment, and the C-terminus of the 4-1BB intracellular segment is linked to the N-terminus of the CD3ζ chain. The present inventors have found that when the immune costimulatory molecule transmembrane domain in the chimeric antigen receptor is linked to the intracellular segment in the above order, the chimeric antigen receptor has a high expression value in adenovirus, and the immune cells expressing the chimeric antigen receptor have a significant specific killing effect on tumor cells expressing CD19, and the non-specific killing effect and cellular inflammatory factor response are weak.

[0016] According to an embodiment of the present invention, the immune costimulatory molecule transmembrane domain is a CD8 transmembrane domain, the immune costimulatory molecule intracellular segment is a 4-1BB intracellular segment and an OX-40 intracellular segment, the N-terminus of the OX-40 intracellular segment is linked to the C-terminus of the CD8 transmembrane domain, the C-terminus of the OX-40 intracellular segment is linked to the N-terminus of the 4-1BB intracellular segment, and the C-terminus of the 4-1BB intracellular segment is linked to the N-terminus of the CD3ζ chain. The present inventors have found that when the immune costimulatory molecule transmembrane domain in the chimeric antigen receptor is linked to the intracellular segment in the above order, the release of cytokine INFγ is reduced, provided that the specific killing effect of immune cells expressing the chimeric antigen receptor on tumor cells expressing CD19 is equivalent to that of second generation CAR-T.

[0017] According to an embodiment of the present invention, the immune costimulatory molecule transmembrane domain is an ICOS transmembrane domain, the immune costimulatory molecule intracellular segment is a 4-1BB intracellular segment and an ICOS intracellular segment, the N-terminus of the 4-1BB intracellular segment is linked to the C-terminus of the ICOS transmembrane domain, the C-terminus of the 4-1BB intracellular segment is linked to the N-terminus of the ICOS intracellular segment, and the C-terminus of the ICOS intracellular segment is linked to the N-terminus of the CD3ζ chain. The present inventors have found that when the immune costimulatory molecule transmembrane domain in the chimeric antigen receptor is linked to the intracellular segment in the above order, the specific killing effect of the immune cells expressing the chimeric antigen receptor against the tumor cells expressing CD19 is significantly superior to that of the second generation CAR-T.

[0018] According to an embodiment of the present invention, the immune costimulatory molecule transmembrane domain connected to the immune costimulatory molecule intracellular segment has the amino acid sequence of any one of SEQ ID NOs: 1-6.

[0019] IYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 1)

[0020] IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 2)

[0021] IYIWAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 3)

[0022] FWLPIGCAAFVVVCILGCILICWLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 4)

[0023] VAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 5)

[0024] VAAILGLGLVLGLLGPLAILLALYLLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 6)

[0025] A chimeric antigen receptor having the amino acid sequence of SEQ ID NO:1 is referred to in this application as CAR3 (the structure from N-terminus to C-terminus may be represented as follows: scFv - CD8 hinge + CD8TM - ICOS - 4-1BB - CD3 zeta, where hinge is the hinge domain and TM is the transmembrane domain), a chimeric antigen receptor having the amino acid sequence of SEQ ID NO:2 is referred to in this application as CAR1 (the structure from N-terminus to C-terminus may be represented as follows: scFv - CD8 hinge + CD8TM - 4-1BB - ICOS - CD3 zeta), a chimeric antigen receptor having the amino acid sequence of SEQ ID NO:3 is referred to in this application as CAR6 (the structure from N-terminus to C-terminus may be represented as follows: scFv - CD8 hinge + CD8TM -OX40 - 4-1BB - CD3 zeta), and a chimeric antigen receptor having the amino acid sequence of SEQ ID NO:4 is referred to in this application as CAR4 (the structure from N-terminus to C-terminus may be represented as follows: scFv - CD8 hinge + The chimeric antigen receptor having the amino acid sequence of SEQ ID NO:5 was referred to in this application as CAR7 (the structure from N-terminus to C-terminus may be represented as follows: scFv - CD8 hinge + OX40TM - OX40 - 4-1BB - CD3 zeta), and the chimeric antigen receptor having the amino acid sequence of SEQ ID NO:6 was referred to in this application as CAR8 (the structure from N-terminus to C-terminus may be represented as follows: scFv - CD8 hinge + OX40TM - 4-1BB - OX40 - CD3 zeta).

[0026] According to an embodiment of the invention, the antigen comprises at least one selected from CD19, CD20, CD123, GPC3, MUC-1, GD2, BCMA, HER2, EGFR, VEGFR, cMet, MSLN, EGFRvIII, and claudin 18.2.

[0027] In a second aspect of the present invention, the present invention provides a construct. According to an embodiment of the present invention, the construct comprises a nucleic acid molecule encoding a chimeric antigen receptor of the present invention. The construct according to an embodiment of the present invention is introduced into a receptor cell, and said chimeric antigen receptor can be efficiently expressed in the receptor cell.

[0028] According to an embodiment of the invention, the above construct further comprises at least one of the following additional technical features: According to an embodiment of the invention, the construct further comprises a promoter, said promoter operably linked to the nucleic acid molecule; According to an embodiment of the present invention, the promoter comprises at least one selected from CMV, EF-1α, and RSV; According to an embodiment of the invention, the construct is a non-pathogenic virus; According to an embodiment of the invention, the virus is selected from a retrovirus, a lentivirus, and an adenovirus-associated virus.

[0029] In a third aspect of the present invention, the present invention provides a lentivirus. According to an embodiment of the present invention, the lentivirus carries a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 7 to 12. Expression of a chimeric antigen receptor in a receptor cell can be achieved by infecting a receptor cell according to an embodiment of the present invention, such as an activated T lymphocyte, with the lentivirus, and the cells expressing the chimeric antigen receptor, such as T lymphocytes, can achieve specific killing of tumor cells with high expression of a specific antigen, and furthermore, have weak non-specific killing and cellular inflammatory factor response, thereby overcoming the existing treatment problems of CAR-T cells.

[0030] ATCTACATCTGGGCGCCCTTGGCCGGGGACTTGTGGGTCCTTCTCCTGCTGTCACTGGTTATCACCCTTTACTGCTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGATACATGTTCATGAGAGCAGTGAACACAGCCAAAAATCTAGACTCACAGATGTGACCCTAAAAGCGGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCTTCATGCGCCCCGTGCAGACCACCACCAGGAGGAGGACGGCTGTAGCTGCCGCTTCCCCGGAGGAGGAGGGGCGGCTGCGAGCTG(sequence number 7)

[0031] ATCTACATCTGGGCGCCCTTGGCCGGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCTTCATGCGCCCCGTGCAGACCACCCAGGAGGAGGACGGCTGTAGCTGCGCTTCCCGAGGAGGAGGAGGCGGCTGCGAGCTGTGTGGCTTACACAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAATCTAGACTCACAGATGTGACCCTA(sequence number 8)

[0032] ATCTACATCTGGGCGCCCTTGGCCGGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCCGGCGACCAGCGCCTGCCCCCCGACGCCCACAAGCCCCCCGGCGGCGGCAGCTTCCGCCACCCCCATCCAGGAGGAGCAGGCCGACGCCCACAGCACCCTGGCCAAGAAGCTGCTGTACATCTTCAAGCAGCCTTCATGCGCCCCGTGCAGACCACCACCAGGAGGAGGACGGCTGTAGCTGCGCTTCCCCGAGGAGGAGGGGCGGCTGCGAGCTG(sequence number 9)

[0033] TTCTGGTTACCCATAGGATGTGCAGCCTTGTTGTAGTCTGCATTTTTGGGATGCATACTTATTTGTTGGCTTAAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCCAGCCCTTCATGCGCCCGTGCAGACCACCCAGGAGGAGGACGGCTGTAGCTGCGCTTCCCGAGGAGGAGGAGGCGGCTGCGAGCTGTGTGGCTTACAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGATACATGTTCATGAGAGCAGTGAACACAGCCAAAAATCTAGACTCACAGATGTGACCCTA(sequence number 10)

[0034] GTTGCCGCCATCCTGGGCCTGGGTCTGGGCTGCTGGGCCCCCTGGCCATCCTGGCCCCTGCTGTACCTGCTCCGGCGCGCACCAGCGCCCTGCCCCCGACGCCCACAAGCCCCCCGGCGGCGGCAGCTTCCGCCACAGCACCCTGGCCAAGAGCTGCTGTACATCTTCAAGCAGCCTTCATGCGCCCGTGCAGACCACCACCAGGAGGAGGACGGCTGTAGCTGCGCTTCCCCGAGGAGGAGGGGCGGCTGCGAGCTG(sequence number 11)

[0035] GTTGCCGCCATCCTGGGCCTGGGTGTGGGCTGGCTGCTGGGCCCCCTGGCCTCTGGCCATCCTGGCCCTGTCCTACCTGCTCAAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTTCATGCGCCCGTGCAGACCACCCAGGAGGAGGACGGCTGTAGCTGCGCTTCCCCGAGGAGGAGGCGGCTGCGAGCTGCGGGCGACCAGCGCCTCCCCCGACGCCCACAAGCCCCCCGGCGGCGGCAGCTTCCGCCACCCCCATCCAGGAGGAGCAGCCCTGGCCAAGATC(sequence number 12)

[0036] The nucleic acid having the nucleotide sequence of SEQ ID NO: 7 encodes the immune costimulatory molecule transmembrane domain and the immune costimulatory molecule intracellular segment of the chimeric antigen receptor CAR3, the nucleic acid having the nucleotide sequence of SEQ ID NO: 8 encodes the immune costimulatory molecule transmembrane domain and the immune costimulatory molecule intracellular segment of the chimeric antigen receptor CAR1, the nucleic acid having the nucleotide sequence of SEQ ID NO: 9 encodes the immune costimulatory molecule transmembrane domain and the immune costimulatory molecule intracellular segment of the chimeric antigen receptor CAR6, the nucleic acid having the nucleotide sequence of SEQ ID NO: 10 encodes the immune costimulatory molecule transmembrane domain and the immune costimulatory molecule intracellular segment of the chimeric antigen receptor CAR4, the nucleic acid having the nucleotide sequence of SEQ ID NO: 11 encodes the immune costimulatory molecule transmembrane domain and the immune costimulatory molecule intracellular segment of the chimeric antigen receptor CAR7, and the nucleic acid having the nucleotide sequence of SEQ ID NO: 12 encodes the immune costimulatory molecule transmembrane domain and the immune costimulatory molecule intracellular segment of the chimeric antigen receptor CAR8.

[0037] In a fourth aspect of the present invention, the present invention provides a transgenic lymphocyte. According to an embodiment of the present invention, the transgenic lymphocyte expresses a chimeric antigen receptor, or the transgenic lymphocyte is obtained by introducing said construct or said lentivirus into lymphocytes. The transgenic lymphocyte according to an embodiment of the present invention can specifically recognize and kill tumor cells with high expression of a specific antigen, and has a weak non-specific killing and cellular inflammatory factor response.

[0038] According to an embodiment of the invention, the transgenic lymphocyte further comprises at least one of the following additional technical features: According to an embodiment of the invention, the transgenic lymphocytes are CD3 + are T cells; According to an embodiment of the present invention, the transgenic lymphocytes are natural killer cells; According to an embodiment of the invention, the transgenic lymphocytes are natural killer T cells.

[0039] In a fifth aspect of the present invention, the present invention provides a method for preparing a transgenic lymphocyte as described above. According to an embodiment of the present invention, the method comprises the step of introducing the construct or the lentivirus into a lymphocyte.

[0040] In a sixth aspect of the present invention, the present invention provides a therapeutic composition for treating cancer. According to an embodiment of the present invention, the therapeutic composition comprises the above construct, the above lentivirus, and the above transgenic lymphocyte. The therapeutic composition according to an embodiment of the present invention produces a significant specific killing effect on tumor cells, and overcomes the problems of high recurrence rate, high cytokine inflammation, and poor persistence in the prior art CAR-T therapy.

[0041] According to an embodiment of the present invention, the aforementioned therapeutic composition further comprises at least one of the following additional technical features: According to an embodiment of the invention, the cancer comprises hematopoietic malignancies, gastrointestinal cancer, glioma, lung cancer, liver cancer, pancreatic cancer.

[0042] In a seventh aspect of the invention, the invention provides a method for improving the safety, efficacy or durability of lymphocyte therapy. According to an embodiment of the invention, the transgenic lymphocyte expresses a chimeric antigen receptor as described above.

[0043] In a seventh aspect of the invention, the invention provides a chimeric antigen receptor as described above for use in improving the safety, efficacy or durability of lymphocyte therapy. According to an embodiment of the invention, a transgenic lymphocyte expresses the chimeric antigen receptor as described above.

[0044] In a seventh aspect of the invention, the invention provides the use of said chimeric antigen receptor in improving the safety, efficacy or durability of lymphocyte therapy. According to an embodiment of the invention, a transgenic lymphocyte expresses said chimeric antigen receptor.

[0045] The chimeric antigen receptors according to embodiments of the present invention, and lymphocytes expressing the chimeric antigen receptors, have the following advantages: 1. Effectively reduce CAR-T autoactivation and factor autorelease; 2. Central memory T cells of CAR-T cells (T CM ) and effector memory T cells (T EM ) increasing the proportion of T cell subsets, reducing the recurrence rate of cancer, and stimulating the immune activation and proliferation function of normal T cells. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1 shows the results of viral packaging of CARs with different structures and their positive expression rates according to an embodiment of the present invention. [Diagram 2] FIG. 1 shows the results of cytotoxicity of T cells carrying CARs with different structures according to embodiments of the present invention. [Diagram 3] FIG. 1 shows the results of INF-γ release from T cells carrying CARs with different structures according to embodiments of the present invention in a cytotoxicity experiment. [Figure 4] FIG. 1 shows the results of CAR positivity rates of T cells carrying CARs with different structures according to embodiments of the present invention after tumor cell stimulation. [Diagram 5] FIG. 1 shows the results of cytokine self-release of T cells carrying CARs with different structures according to embodiments of the invention when co-cultured with non-tumor cells. [Figure 6] FIG. 1 shows the effect of specific and non-specific killing of CAR-T3 after it is co-cultured with effector cells according to an embodiment of the present invention. [Figure 7] FIG. 1 shows the effect of CAR-T 3 according to an embodiment of the present invention on the CAR positive rate after co-culture with effector cells. [Figure 8] Figure 1 shows the results of cytokine release of anti-claudin 18.2 CAR-T with different structures according to an embodiment of the present invention when cultured alone or co-cultured with MGC-803-18.1 for 24 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Specific implementation The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the drawings. The following embodiments are illustrated by way of example with reference to the drawings, which are merely for the purpose of explaining the present invention, and are not intended to limit the scope of the present invention.

[0048] Detailed Description Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0049] The present invention relates to chimeric antigen receptors (CARs), which are molecules that combine antibody-based specificity for a desired antigen (e.g., a tumor antigen) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cellular immune activity.

[0050] T cells that express a CAR are referred to herein as CAR T cells or CAR-modified T cells.

[0051] In one embodiment, the CAR of the present invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and an intracellular domain.

[0052] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, and therefore, they are among the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0053] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences and RNAs that encode proteins may contain introns.

[0054] The term "operably linked" refers to the functional linkage of a control sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. By way of example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein coding regions, in the same reading frame.

[0055] The CARs (including functional parts and functional variants) disclosed in the embodiments of the present invention were obtained by methods known in the art. The CARs can be prepared by any suitable method of preparing proteins or polypeptides. Suitable methods for de novo synthesis of peptides and proteins are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, Reid, R., Marcel Dekker Inc., 2000; Epitope Mapping, Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; U.S. Patent No. 5,449,752. In addition, polypeptides and proteins can be produced recombinantly using standard recombinant methods and based on the nucleic acids described herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley&Sons, NY, 1994. In addition, some CARs of the present invention (including functional portions and functional variants) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, e.g., rats, humans, and the like. Isolation and purification methods are well known in the art. Optionally, the CARs described herein (including functional portions and functional variants) can be synthesized by commercial companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA).In this embodiment, the CAR of the present invention can be synthesized, recombinant, isolated, and / or purified.

[0056] Methods for testing antibodies for the ability to bind to any functional portion of the CAR of the invention are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, and U.S. Patent Application Publication No. 2002 / 0197266).

[0057] The present invention also includes within its scope functional variants of the CARs of the present invention described herein. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity with a parent CAR, which functional variant retains the biological activity of the CAR of which it is a variant. Functional variants include, for example, variants of the CARs described herein (parent CARs) that retain the ability to recognize target cells to a similar extent, to the same extent, or to a greater extent than the parent CAR. A functional variant can be, for example, at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99%, or more identical in amino acid sequence to the parent CAR, with reference to the parent CAR.

[0058] A functional variant may, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent CAR.

[0059] The amino acid substitutions in the CAR of the present invention are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. By way of example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid (e.g., Asp or Glu) in place of another acidic / negatively charged polar amino acid, an amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.) in place of another amino acid with a nonpolar side chain, a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) in place of another basic / positively charged polar amino acid, an uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.) in place of another amino acid with a beta-branched side chain (e.g., He, Thr, and Val), an amino acid with an aromatic side chain (e.g., His, Phe, Tip, and Tyr) in place of another amino acid with an aromatic side chain, etc.

[0060] The CARs of the present embodiments (including functional portions and functional variants of the present invention) can include synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, a-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, naphthylalanine, cyclohexylalanine, cyclohexylglycine, indomethacin, cyclohexylglycine ... phosphorus-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0061] An embodiment of the present invention also provides an antigen-binding portion of any of the antibodies described herein. The antigen-binding portion can be any portion having at least one antigen-binding site, such as Fab, F(ab')2, dsFv, sFv, diabody, and triabody. Single-chain variable fragment (sFv) antibody fragments, which are truncated Fab fragments containing the variable (V) domain of the antibody heavy chain linked to the variable (V) domain of the antibody light chain via a synthetic peptide, can be produced using routine recombinant DNA technology techniques. Similarly, disulfide-stabilized variable fragments (dsFv) can be prepared by recombinant DNA technology (see, e.g., Reiter et al., Protein Engineering, 7, 697-704 (1994)). However, the antibody fragments of the present invention are not limited to these exemplary types of antibody fragments.

[0062] Further provided by embodiments of the invention are nucleic acids comprising a nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof). The nucleic acids of the invention can comprise a nucleotide sequence encoding any of the extracellular domains, transmembrane domains, and / or intracellular domains described herein.

[0063] As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic, or obtained from a natural source (e.g., isolated and / or purified), and may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide linkages, such as phosphoramidate or phosphorothioate linkages instead of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, as discussed herein, in some cases, it may be appropriate for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions. In some embodiments, the nucleic acid may encode additional amino acid sequences that do not affect the function of the CAR, which may or may not be translated upon expression of the nucleic acid by a host cell.

[0064] The nucleic acid can comprise any isolated or purified nucleotide sequence encoding a CAR or any of its functional portions or variants. Alternatively, the nucleotide sequence can comprise a nucleotide sequence that is degenerate to any of the sequences or a combination of degenerate sequences.

[0065] Embodiments of the present invention also provide isolated or purified nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0066] Nucleotide sequences that hybridize under stringent conditions may hybridize under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of the nucleic acids described herein) in an amount detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish between polynucleotides with exact complementary sequences, or random sequences that happen to have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence, and polynucleotides that contain only a few scattered mismatches. Such small regions of complementarity are more easily melted than full-length complements of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such high stringency conditions tolerate few, if any, mismatches between the nucleotide sequence and the template or target strand and are particularly suitable for detecting the expression of any of the CARs of the present invention.

[0067] The present invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or more identical to any of the nucleic acids described herein, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical.

[0068] In an embodiment, the nucleic acid of the present invention can be incorporated into a recombinant expression vector or construct. In this regard, an embodiment of the present invention provides a recombinant expression vector or construct comprising any of the nucleic acids of the present invention. For purposes herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows a host cell to express an mRNA, protein, polypeptide, or peptide, when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and the vector contacts the cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell, the genetically modified oligonucleotide or polynucleotide construct causes the host cell to express the mRNA, protein, polypeptide, or peptide. The vector of the present invention is not naturally occurring in its entirety. However, portions of the vector may be naturally occurring. The recombinant expression vector of the present invention may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic, or partially obtained from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may contain naturally occurring or non-naturally occurring internucleotide bonds or both types of bonds. Preferably, the non-naturally occurring or altered nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0069] In one embodiment, the recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include vectors designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector can be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λGT10, λGTl1, λZapII (Stratagene), λEMBL4, and λNMI 149, can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEU-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector or a lentiviral vector. In some embodiments, the vector can be a transposon.

[0070] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981)). Transfection methods include calcium phosphate co-precipitation (see, e.g., Graham et al.), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22: 479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6: 742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)), and nucleic acid delivery using high-velocity biolistic projectiles (see, e.g., Klein et al., Nature, 327: 70-73 (1987)).

[0071] In one embodiment, the recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al. and Ausubel et al. The expression vector constructs, either circular or linear, can be prepared to contain a replication system that can function in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0072] Recombinant expression vectors are specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which they are to be introduced and may contain control sequences, such as transcription and translation initiation and termination codons, as appropriate, considering whether the vector is DNA- or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0073] The recombinant expression vector may contain one or more marker genes that allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, and the like. Suitable marker genes for the expression vectors of the present invention include, by way of example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0074] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding the CAR (including functional portions and functional variants thereof) or to a nucleotide sequence that is complementary to or hybridizes with the nucleotide sequence encoding the CAR. The selection of a promoter, for example, a strong, weak, inducible, tissue-specific, and development-specific promoter, is within the ability of one of ordinary skill in the art. Similarly, combining a nucleotide sequence with a promoter is also within the ability of one of ordinary skill in the art. The promoter may be a non-viral promoter or a viral promoter, for example, a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of the murine stem cell virus.

[0075] The recombinant expression vectors of the invention can be designed for either transient expression, stable expression, or both. Additionally, the recombinant expression vectors can be made for constitutive or inducible expression.

[0076] Additionally, the recombinant expression vector can be made to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell that expresses the suicide gene. A suicide gene can be a gene that confers sensitivity to an agent, such as a drug, on the cell in which it is expressed, causing the cell to die when the cell is contacted or exposed to the agent. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0077] Included within the scope of the present invention are conjugates, such as bioconjugates, nucleic acids, recombinant expression vectors, host cells, host cell populations, or antibodies, or antigen-binding portions thereof, that comprise any of the CARs of the present invention (including any of the functional portions or variants thereof). Conjugates, as well as methods for synthesizing conjugates, are generally known in the art (see, e.g., Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005)).

[0078] An embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungal, or algal cell, or a prokaryotic cell, such as a bacterial or protozoan cell. The host cell can be a cultured cell, or a primary cell, i.e., a cell directly isolated from an organism, such as a human. The host cell can be an adherent cell, or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, by way of example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5α cell. For purposes of producing a recombinant CAR, the host cell can be a mammalian cell. The host cell can be a human cell. The host cell can be any cell type, originate from any type of tissue, and be at any stage of development, while the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell can be a T cell.

[0079] For purposes herein, a T cell can be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified. The T cell can be a human T cell. The T cell can be a T cell isolated from a human. The T cell can be any type of T cell and at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. The T cell can be a CD8+ T cell or a CD4+ T cell.

[0080] Also provided by embodiments of the invention are cell populations comprising at least one host cell as described herein. The cell population can be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the cell population can be a substantially homogeneous population, where the population primarily comprises host cells comprising (e.g., essentially consists of) a recombinant expression vector. The population can also be a clonal population of cells, where all cells of the population are clones of a single host cell comprising the recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the cell population is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0081] CARs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) (all of which collectively are referred to as the "CAR materials of the invention") can be isolated and / or purified. As used herein, the term "isolated" means removed from its natural environment. The terms "purified" or "isolated" do not require absolute purity or isolation; rather, it is intended as a relative term. Thus, for example, a purified (or isolated) host cell preparation is one in which the host cells are purer than the cells in their natural environment in the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity can be at least about 50%, can be greater than about 60%, can be greater than about 70%, can be greater than about 80%, or can be about 100%.

[0082] The CAR material of the present invention can be formulated into a composition, such as a pharmaceutical composition. In this regard, embodiments of the present invention provide a pharmaceutical composition comprising any of the CAR, functional portion, functional variant, nucleic acid, expression vector, host cell (including populations thereof), and antibody (including antigen-binding portion thereof), and a pharma- ceutically acceptable carrier. A pharmaceutical composition of the present invention containing any of the CAR materials of the present invention may comprise more than one CAR material of the present invention, e.g., a CAR and a nucleic acid, or two or more different CARs. Alternatively, a pharmaceutical composition may comprise the CAR material of the present invention in combination with other pharma- ceutical active agents or drugs, e.g., chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like. In a preferred embodiment, the pharmaceutical composition comprises the host cell of the present invention or a population thereof.

[0083] The CAR materials of the present invention can be provided in the form of a salt, e.g., a pharma- ceutically acceptable salt. Suitable pharma-ceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and organic acids, such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, e.g., p-toluenesulfonic acid.

[0084] For pharmaceutical compositions, the pharma- ceutically acceptable carrier can be any of those conventionally used, limited only by physicochemical considerations such as solubility and lack of reactivity with the active substance, and by the route of administration. The pharma-ceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. It is preferred that the pharma-ceutically acceptable carrier is chemically inert to the active substance and has no adverse side effects or toxicity under the conditions of use.

[0085] The choice of carrier is determined in part by the particular CAR material of the present invention, as well as by the particular method used to administer the CAR material of the present invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present invention. A preservative may be used. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. Optionally, a mixture of two or more preservatives may be used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0086] Suitable buffering agents may include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. Optionally, a mixture of two or more buffering agents may be used. The buffering agent or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition.

[0087] The concentration of the inventive CAR material in a pharmaceutical formulation can vary, for example, from less than about 1% by weight, typically about or at least about 10% by weight, and as much as about 20% to about 50% by weight or more, and can be selected primarily according to fluid volumes and viscosities in accordance with the particular mode of administration selected.

[0088] Methods for preparing administrable (e.g., parenterally administrable) compositions will be known or apparent to those skilled in the art and are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st Edition (May 1, 2005).

[0089] The following formulations for oral, aerosol, parenteral (e.g., subcutaneous, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, and intrathecal), and topical administration are merely illustrative and in no way limiting. More than one route can be used to administer the CAR materials of the invention, and in some cases, a particular route can provide a more rapid and effective response than another route.

[0090] Formulations suitable for oral administration may include or consist of: (a) a liquid solution, e.g., an effective amount of the CAR material of the present invention dissolved in a diluent such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, either as a solid or granules; (c) powders; (d) suspensions in a suitable liquid; and (e) suitable emulsions. Liquid formulations may contain diluents such as water and alcohol, e.g., ethanol, benzyl alcohol, and polyethylene alcohol, either with or without the addition of a pharma-ceutically acceptable surfactant. Capsule forms may be, for example, of the ordinary hard or soft gelatin type, containing surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablets may contain one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, gum acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffers, disintegrants, wetting agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms may contain the CAR material of the present invention in a flavoring, usually sucrose, and gum acacia or gum tragacanth, and pastilles contain the CAR material of the present invention in an inert base such as gelatin and glycerin, or sucrose and gum acacia, while emulsions, gels, and the like additionally contain excipients as known in the art.

[0091] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The CAR materials of the present invention can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids including water, saline, aqueous dextrose and related sugar solutions, alcohols, e.g., ethanol or hexadecyl alcohol, glycols, e.g., propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals, e.g., 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of a pharma- ceutical acceptable surface active agent, such as a soap or surfactant, a suspending agent, such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0092] The oil that can be used in parenteral formulations includes petroleum, animal, vegetable, or synthetic oils.Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petroleum, and mineral oil.Suitable fatty acids that can be used in parenteral formulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0093] Suitable fatty acid salts for use in parenteral formulations include fatty acid alkali metal, ammonium, and triethanolamine salts, and suitable surfactants include (a) cationic surfactants, such as dimethyldialkylammonium halides and alkylpyridinium halides, (b) anionic surfactants, such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates and sulfosuccinates, (c) nonionic surfactants, such as fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene-polypropylene copolymers, (d) amphoteric surfactants, such as alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0094] Parenteral formulations typically contain, for example, from about 0.5% to about 25% by weight of the CAR material of the invention in solution. Preservatives and buffers may be used, and such compositions may contain, for example, one or more non-ionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17 to minimize or eliminate irritation at the injection site. The amount of surfactant in such formulations typically ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide with propylene glycol. Parenteral formulations may be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, such as water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0095] Injectable formulations are according to the embodiments of the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986)).

[0096] Topical formulations, including those useful for transdermal drug delivery, are well known to those skilled in the art and are suitable in the context of the present invention for application to the skin. The CAR materials of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered by inhalation. These aerosol formulations can be placed into propellants that can accept pressurization, such as dichlorodifluoromethane, propane, nitrogen, and the like. They can also be formulated as pharmaceuticals for non-pressurized preparations, such as in a nebulizer or atomizer. Such spray formulations can also be used to spray mucous membranes.

[0097] An "effective amount" or "amount effective to treat" refers to a dose that is sufficient to prevent or treat cancer in an individual. Amounts effective for therapeutic or prophylactic use will depend, for example, on the stage and severity of the disease or disorder to be treated, the age, weight, and general health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the selected active agent, the method of administration, the timing and frequency of administration, the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular active agent, and the desired physiological effect. It will be understood by those skilled in the art that various diseases or disorders may require chronic treatment, possibly involving multiple administrations, with the CAR material of the invention at each or various rounds of administration. By way of example, and without intending to limit the invention, the dose of the CAR material of the invention may be about 0.001 to about 1000 mg per kg of body weight of the subject to be treated per day, about 0.01 mg to about 10 mg / kg body weight / day, about 0.01 mg to about 1 mg / kg body weight / day. When the CAR material of the present invention is a host cell, an exemplary dose of host cells can be a minimum of 1 million cells (1 mg cells / dose). When the CAR material of the present invention is a nucleic acid packaged in a virus, an exemplary dose of virus can be 1 ng / dose.

[0098] For purposes of the present invention, the amount or dose of the CAR material of the present invention administered should be sufficient to produce a therapeutic or prophylactic response in a subject or animal over a reasonable time frame. For example, a dose of the CAR material of the present invention should be sufficient to bind an antigen or detect, treat, or prevent a disease for about 2 hours or more, such as about 12 hours to about 24 hours or more, from the time of administration. In certain embodiments, the time may be even longer. The dose is determined by the potency of the particular inventive CAR material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0099] For purposes of the present invention, an assay can be used to determine the starting dose to be administered to a mammal, for example, comprising comparing the extent to which target cells are lysed and / or IFN-γ is secreted by T cells expressing a CAR of the present invention upon administration of a given dose of such T cells between a set of mammals, each of which is given a different dose of T cells. The extent to which target cells are lysed and / or IFN-γ is secreted upon administration of a particular dose can be assayed by methods known in the art.

[0100] In addition to the pharmaceutical compositions described above, the CAR material of the present invention can be formulated as an inclusion complex, such as a cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the CAR material of the present invention to a specific tissue. Liposomes can also serve to target the CAR material of the present invention to a specific tissue. Liposomes can also be used to increase the half-life of the CAR material of the present invention. Many methods are available for preparing liposomes, as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0101] Delivery systems useful in the context of embodiments of the present invention may include sustained release, delayed release, and extended release delivery systems, such that delivery of the compositions of the present invention occurs prior to sensitization of the site to be treated and has sufficient time to cause said sensitization. The compositions of the present invention may be used together with other therapeutic agents or treatments. Such systems may avoid repeated administration of the compositions of the present invention, thereby increasing convenience for subjects and physicians, and may be particularly suitable for certain composition embodiments of the present invention.

[0102] Many types of release delivery systems are available and known to those skilled in the art. These include polymer-based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Drug-containing microcapsules of the aforementioned polymers are described, for example, in U.S. Pat. No. 5,075,109. Delivery systems also include non-polymeric systems, including sterols such as cholesterol, cholesterol esters, and lipids, including fatty acids or neutral fats such as mono-, di-, and triglycerides; hydrogel release systems; sylastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosion systems in which the active composition is contained within a matrix, such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660, and (b) diffusion systems in which the active ingredient permeates through a polymer at a controlled rate, such as those described in U.S. Patent Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

[0103] Those skilled in the art will readily understand that the CAR material of the present invention can be modified in a variety of ways such that the therapeutic or prophylactic efficacy of the CAR material of the present invention is increased through modification. For example, the CAR material of the present invention can be conjugated to a targeting moiety either directly or indirectly through a linker. The practice of conjugating a compound, such as the CAR material of the present invention, to a targeting moiety is known in the art. For example, see Wadhwa et al., J. Drug Targeting 3: 111 (1995) and U.S. Patent No. 5,087,616.

[0104] Alternatively, the CAR material of the present invention can be modified into a depot form such that the manner in which the CAR material of the present invention is released into the body to which it is administered can be controlled with respect to time and location within the body (see, e.g., U.S. Patent No. 4,450,150).

[0105] A depot form of the CAR material of the invention can be an implantable composition comprising the CAR material of the invention and a porous or non-porous material, such as a polymer, in which the CAR material of the invention is encapsulated or diffused throughout the material and / or the non-porous material upon degradation. The depot is then implanted at a desired location within the body, and the CAR material of the invention is released from the implant at a predetermined rate.

[0106] When the CAR material of the present invention is administered with one or more additional therapeutic agents, the one or more additional therapeutic agents can be co-administered to the mammal. By "co-administering" is meant that the one or more additional therapeutic agents and the CAR material of the present invention are administered close enough in time that the CAR material of the present invention can enhance the effect of the one or more additional therapeutic agents, or vice versa. In this regard, the CAR material of the present invention can be administered first, and the one or more therapeutic agents can be administered second, or vice versa. Alternatively, the CAR material of the present invention and the one or more additional therapeutic agents can be administered simultaneously. For purposes of the methods of the present invention, when a host cell or cell population is administered, the cells can be cells that are allogeneic or autologous to the mammal.

[0107] It is contemplated that the pharmaceutical compositions, CARs, nucleic acids, recombinant expression vectors, host cells, or cell populations of the present invention can be used in methods of treating or preventing disease in a mammal. Without intending to be bound by any particular theory or mechanism, the CARs of the present invention have a biological activity, e.g., the ability to recognize an antigen, such that when the CAR is expressed by a cell, it can mediate an immune response against cells expressing the antigen for which the CAR is specific. In this regard, embodiments of the present invention provide a method of treating or preventing cancer in a mammal, comprising administering to a mammal a CAR, nucleic acid, recombinant expression vector, host cell, cell population, antibody and / or antigen-binding portion thereof, and / or pharmaceutical composition of the present invention in an amount effective to treat or prevent cancer in the mammal.

[0108]

[0013] An embodiment of the invention further comprises lymphodepleting the mammal prior to administering the CAR material of the invention. Examples of lymphodepletion include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body radiation, and the like.

[0109] For the purposes of the methods of the invention, when a host cell or cell population is administered, the cells may be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0110] The mammals referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and Lagomorpha, such as rabbits. Mammals may be from the order Carnivora, including Feline (cat) and Canine (dog). Mammals may be from the order Artiodactyla, including Bovine (cow) and Swine (pig), or Perssodactyla, including Equine (horse). Mammals may be from the order Primates, Ceboids or Simoids (monkeys), or Anthropoids (humans and apes). Preferably, the mammal is a human.

[0111] For the methods of the present invention, the cancer can be any cancer, including, but not limited to, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic bone marrow cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's disease, and / or other cancers. The present invention includes cancers such as lymphoma, hypopharyngeal cancer, renal cancer, pharyngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer and ureteral cancer. Preferably, the cancer is a hematological malignancy (e.g., a leukemia or lymphoma, including but not limited to Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphocytic carcinoma, acute myeloid leukemia, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma). Preferably, the cancer is characterized by expression of CD22.

[0112] As used herein, the terms "treat" and "prevent," as well as stem variations thereof, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art recognizes as having potential benefit or therapeutic effect. In this regard, the method of the present invention may provide any amount of any level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the method of the present invention may include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, that is being treated or prevented. Also, for purposes herein, "prevention" may include delaying the onset of the disease, or its symptoms or conditions.

[0113] Another embodiment of the invention provides the use of a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody or antigen-binding portion, or a pharmaceutical composition of the invention for the treatment or prevention of cancer in a mammal.

[0114] In addition, CAR function can be assessed by measuring cytotoxicity as described in Zhao et al., J. Immunol, 174: 4415-4423 (2005).

[0115] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0116] Viral packaging of chimeric antigen receptors 1. Synthesis of CARs with different structures. The structures of CARs with signal peptides are shown in Table 1 below.

[0117] [Table 1]

[0118] The amino acid sequence of each part of the CAR, including the signal peptide, and the nucleotide sequence of the corresponding encoding nucleic acid are as follows:

[0119] The nucleotide sequence of the signal peptide (GL): Atgggcgtcaaggtcctgttcgccctgatctgcatcgccgtcgccgaggcc

[0120] Signal peptide (GL) amino acid sequence: MGVKVLFALICIAVAEA

[0121] The nucleotide sequence of the CD8 hinge: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0122] CD8 hinge amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0123] Nucleotide sequence of CD8 transmembrane (TM): ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGG GTCCTTCTC CTGTCACTG GTTATCACC CTTTACTGC

[0124] CD8 transmembrane (TM) amino acid sequence: IYIWAPLAGTCGVLLLSLVITLYC

[0125] The nucleotide sequence of ICOS transmembrane (TM): ttctggttacccataggatgtgcagcctttgttgtagtctgcattttgggatgcatacttatttgttggctt

[0126] Amino acid sequence of ICOS transmembrane (TM): FWLPIGCAAFVVVCILGCILICWL

[0127] Nucleotide sequence of OX40 transmembrane (TM): gttgccgccatcctgggcctgggcctggtgctggggctgctgggccccctggccatcctgctggccctgtacctgctc

[0128] The amino acid sequence of OX40 transmembrane (TM): VAAILGLGLVLGLLGPLAILLALYLL

[0129] Nucleotide sequence of the 4-1BB immune costimulatory molecule intracellular segment: AAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACCACCCAGGAGGAGGACGGCTGTAGCTGCCGCTTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTG

[0130] Amino acid sequence of the 4-1BB immune co-stimulatory molecule intracellular segment: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0131] Nucleotide sequence of the ICOS immune co-stimulatory molecule intracellular segment: tgttggcttacaaaaaagaagtattcatccagtgtgcacgaccctaacggtgaatacatgttcatgagagcagtgaacacagccaaaaaatctagactcacagatgtgacccta

[0132] Amino acid sequence of ICOS immune co-stimulatory molecule intracellular segment: CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

[0133] Nucleotide sequence of the OX40 immune co-stimulatory molecule intracellular segment: CGGCGCGACCAGCGCCTGCCCCCCGACGCCCACAAGCCCCCCGGCGGCGGCAGCTTCCGCACCCCCATCCAGGAGGAGCAGGCCGACGCCCACAGCACCCTGGCCAAGATC

[0134] Amino acid sequence of the OX40 immune co-stimulatory molecule intracellular segment: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0135] The nucleotide sequence of CD3 zeta: CGCGTGAAATTTAGCCGCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGGGGCCGCGACCCCGAGATGGGCGGCAAGCCCCAGCGCCGCAAGAACCCCCAGG AGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAAGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGGGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC

[0136] The amino acid sequence of CD3 zeta: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0137] Taking the construction of CARs against CD19 or claudin 18.2 antigens as an example, the constructed nucleotide sequences of CAR1-10 and the expressed amino acid sequences of CAR1-10 with signal peptides are as follows:

[0138] Nucleotide sequence of anti-CD19 scFv: GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA

[0139] Amino acid sequence of anti-CD19 scFv: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGG SEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0140] Constructed nucleotide sequence of anti-CD19-CAR1:

[0141] Amino acid sequence of anti-CD19-CAR1 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCWLTKKKYSSSVHDPNGEYM FMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0142] Constructed nucleotide sequence of anti-CD19-CAR2:

[0143] Amino acid sequence of anti-CD19-CAR2 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKS RLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0144] Constructed nucleotide sequence of anti-CD19-CAR3:

[0145] Amino acid sequence of anti-CD19-CAR3 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0146] Constructed nucleotide sequence of anti-CD19-CAR4:

[0147] Amino acid sequence of anti-CD19-CAR4 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCWLTKKKYSSSVHDPNGEYM FMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0148] Constructed nucleotide sequence of anti-CD19-CAR5:

[0149] Amino acid sequence of anti-CD19-CAR5 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0150] Constructed nucleotide sequence of anti-CD19-CAR6:

[0151] Amino acid sequence of anti-CD19-CAR6 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0152] Constructed nucleotide sequence of anti-CD19-CAR7:

[0153] Amino acid sequence of anti-CD19-CAR7 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0154] Constructed nucleotide sequence of anti-CD19-CAR8:

[0155] Amino acid sequence of anti-CD19-CAR8 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVAAILGLGLVLGLLGPLAILLALYLLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRDQRLPPDAHKPPGGGSF RTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0156] Constructed nucleotide sequence of anti-CD19-CAR9:

[0157] Amino acid sequence of anti-CD19-CAR9 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRDQRLPPDAHKPPGGGSFR TPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0158] Constructed nucleotide sequence of anti-CD19-CAR10:

[0159] Amino acid sequence of anti-CD19-CAR10 with signal peptide: MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK LEITGGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0160] Nucleotide sequence of anti-claudin 18.2 scFv: GATATCGTGATGACACAGAGCCCTAGCTCTTTAACAGTGACCGCCGGCGAGAAGGTGACCATGAGCTGCAAGAGCAGCCAGTCTTTACTGAACTCCGGCAACCAGAAGAACTATTTAACTTGGTACCAGCAGAAGCCCGGCCAGCCCCCTAAGCTGCTCATCTACTGGGCCAGCACTCGTGAGAGCGGCGTGCCCGATAGATTCACTGGTTCCGGCAGCGGCACCGACTTTACTTTAACCATCAGCAGCGTGCAAGCCGAGGATTTAGCCGTGTACTACTGCCAGAACGACTATAGCTACCCCTTCACCTTCGGCTCCGGCACCAAGCTGGAGATTAAGGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTCAAGTTCAGCTGCAGCAACCCGGAGCCGAACTGGTGAGACCCGGAGCTAGCGTGAAGCTGTCTTGTAAGGCCTCCGGCTATACATTTACCAGCTACTGGATCAATTGGGTGAAGCAGAGACCCGGCCAAGGTTTAGAGTGGATCGGAAACATCTACCCCTCCGATAGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACACTCACAGTGGACAAAAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCCCACCTCCGAGGATAGCGCCGTGTACTATTGCACTCGTTCTTGGAGGGGCAATAGCTTCGACTATTGGGGCCAAGGTACCACCCTCACCGTGAGCAGC

[0161] Amino acid sequence of anti-claudin 18.2 scFv: DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKGGGGSGGGGS GGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS

[0162] Constructed nucleotide sequence of anti-claudin18.2-CAR1:

[0163] Amino acid sequence of anti-claudin18.2-CAR1 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVS STTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCWLTKKKYSSSVHDPNGEY MFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0164] Constructed nucleotide sequence of anti-claudin18.2-CAR2:

[0165] Amino acid sequence of anti-claudin18.2-CAR2 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYSYPFT FGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYC TRSWRGNSFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLCWLTKKKYSSSVHDPNGEYMFMRAVNTAKK SRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0166] Constructed nucleotide sequence of anti-claudin18.2-CAR3:

[0167] Amino acid sequence of anti-claudin18.2-CAR3 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVS STTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0168] Constructed nucleotide sequence of anti-claudin18.2-CAR4:

[0169] Amino acid sequence of anti-claudin18.2-CAR4 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVS STTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCWLTKKKYSSSVHDPNGEY MFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0170] Constructed nucleotide sequence of anti-claudin18.2-CAR5:

[0171] Amino acid sequence of anti-claudin18.2-CAR5 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVS STTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0172] Constructed nucleotide sequence of anti-claudin18.2-CAR6:

[0173] Amino acid sequence of anti-claudin18.2-CAR6 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVS STTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0174] Constructed nucleotide sequence of anti-claudin18.2-CAR7:

[0175] Amino acid sequence of anti-claudin18.2-CAR7 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0176] Constructed nucleotide sequence of anti-claudin18.2-CAR8:

[0177] Amino acid sequence of anti-claudin18.2-CAR8 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDVAAILGLGLVLGLLGPLAILLALYLLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRDQRLPPDAHKPPGGGS FRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0178] Constructed nucleotide sequence of anti-claudin18.2-CAR9:

[0179] Amino acid sequence of anti-claudin18.2-CAR9 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKG GGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVS STTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRDQRLPPDAHKPPGGGSF RTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0180] Constructed nucleotide sequence of anti-claudin18.2-CAR10:

[0181] Amino acid sequence of anti-claudin18.2-CAR10 with signal peptide: MGVKVLFALICIAVAEADIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTF GSGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTR SWRGNSFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0182] The designed nucleotide sequences encoding various CAR structures, synthesized by GENEWIZ, were cut with BamH I and Sal I, cloned into lentiviral vector LV-CAR (constructed by our company), transfected into stbl3 competent cells for amplification, and the plasmids were extracted as a standby. 2. Lentiviral packaging of anti-CD19 CAR or anti-claudin 18.2 CAR with different structures a. Transfect 293T cells at 3 x 10 into a 6-well plate containing 2 mL lentiviral packaging medium. 6 The cells were seeded at a density of 10 cells / well and incubated overnight in a 37° C., 5% CO 2 incubator. b. When the cell density reached approximately 95% as observed, lentiviral packaging was performed and the lentiviral packaging system was constructed according to the construction system listed in Table 2 below.

[0183] [Table 2]

[0184] The preformed Part A was transferred to Part B, and the mixture was mixed well and then allowed to stand at room temperature for 20 minutes. c. Remove 1mL medium per well of 6-well plate inoculated with 293T cells, and after removing the medium, add 500μL A+B mixture to each well, mix well, and then place the plate in a 37℃, 5% CO2 incubator for 6 hours, then replace the medium in the 6-well plate with lentivirus packaging medium, and place the 6-well plate in the incubator for another 52 hours, and collect the culture supernatant, concentrate 20 times (100μL / tube), and store at -80℃ as standby. EXAMPLES

[0185] 1. T cell activation and lentiviral infection 2mL of CD3 + The positive cells (viability 98.5%, live cells 5E+06) were added to a 15mL centrifuge tube containing 5mL AIM-V, the cells were collected by centrifugation at 500g for 5 minutes, the supernatant was discarded, the collected cells were resuspended in 10mL AIM-V medium (containing 20IU / mL IL-2), and the cell suspension was taken out to a 10cm Petri dish for culture. 180μL CD3 / 28 magnetic beads were washed by adding 4mL buffer, placed on a magnetic stand for 2 minutes, the supernatant was removed, and then resuspended in 180μL AIM-V medium (containing 20IU / mL IL-2). The magnetic bead suspension was added to the cell supernatant, they were mixed well, and placed in an incubator for culture. After 24 hours of culture, viral transfection was performed. 2. The above cell mixture was added to a 15mL centrifuge tube, the tube was placed on a magnetic stand for 2 minutes, and the cell-containing liquid of the cell mixture was pipetted into another 15mL clean centrifuge tube. 10μL of the cell-containing liquid was mixed with 10μL AO / PI staining solution, and the mixture was added to a cell counting chamber (viability 98.41%, live cell density 4.01E+06). 3mL of the cell-containing liquid was centrifuged at 500g for 5 minutes, and the supernatant was discarded. The remaining cells were diluted with 10 mL of PBS in 1.2mL AIM-V medium (containing 20IU / mL IL-2). 7 The cells were resuspended at a concentration of 10 cells / mL. 3. Viral infection: Activated T cells were infected in 24-well plates according to the conditions shown in Table 3 below. After mixing the cells and the virus, they were left at room temperature for 15 minutes and then placed in a cell incubator for culture. 24 hours after infection by centrifugation at 500g for 5 minutes, the cells were collected into a 1.5 mL centrifuge tube, and the collected cells were resuspended in 1 mL AIM-V medium (containing 20 IU / mL IL-2). The cells were passaged at a cell density of 0.5E+06. When the cell density of the cells reached 5E+06-1E+07, the cells were passaged. After continuous culture for 2 days, the CAR-T positive rate was detected by flow cytometry.

[0186] [Table 3]

[0187] 4. Collect different groups of CAR-T cells, each group containing approximately 10 6 cells, resulting in a total of 1 T cell negative control group and 10 experimental groups. 5. The cells were collected by centrifugation at 500g for 5 min and the supernatant was discarded; the cells were resuspended in 500μL PBS and the suspension was centrifuged at 500g for 5 min, the procedure was repeated twice and the supernatant was discarded. As a negative control, the cells were resuspended by adding 200μL PBS and stored at 4℃. 6. The experimental group was provided by a procedure including the steps of resuspending the cells in 500 μL PBS, adding 1 μL Protein L, and incubating in the dark at 4° C. for 30 minutes; then collecting the cells by centrifugation at 500 g at 4° C. for 5 minutes and discarding the supernatant; then resuspending the cells in 500 μL PBS, collecting the cells by centrifugation at 500 g at 4° C. for 5 minutes, discarding the supernatant, and repeating the steps three times; finally resuspending the cells in 200 μL PBS for flow cytometry.

[0188] result: 1. The positive rates of CARs with different structures are significantly different under the same amount of virus infection system. The second generation CAR structure (CAR10) is shorter than the CAR1-9 sequence and has a higher positive rate; 2. There is a large difference between the CAR positivity rates of the CAR1-9 structures, with the CAR3 (CAR positivity rate 41.8%) and CAR9 (CAR positivity rate 41.4%) structures having the highest positivity rates (CD19 CAR results are shown in Figure 1 ); 3. The same infection system yielded different CAR positive rate results, indicating that the CAR structure can affect the lentivirus packaging titer. In contrast, the lentivirus titers packaged in the CAR3 and CAR9 structures were the highest in CAR1-9. EXAMPLES

[0189] Detection of cytotoxicity of CD19 CAR-T with different structures 1. Cell culture Effector-target cells (NALM-6 / K562) were cultured in 1640 / IMDM+10%FBS medium in a 37°C, 5% carbon dioxide incubator. Suspension cells: The cells were observed once a day using an inverted microscope and the medium was changed every 2-4 days. The cells were passaged before the cell density in the flask reached 3E+06. 2. Treatment of effector cells (CD19 CAR-T with different structures) CAR-T cells with different structures were collected in a 15 mL centrifuge tube and centrifuged at 500 g for 5 min. The supernatant was discarded, and the cells were washed by adding 4 mL of 0.01 M PBS, centrifuged, the supernatant was discarded, and the cells were resuspended in approximately 10 7 The cells were resuspended in an appropriate amount of AIM-V medium to a cell density of 100 cells. The cell density and viability were determined. 3. Pretreatment of target cells 1) Target cell labeling: Target cells were harvested in logarithmic growth phase and washed once with 0.01M PBS. The target cell mixture was centrifuged at 500g for 5 minutes and the supernatant was discarded; then the target cells were resuspended in complete medium to obtain a suspension with a density of 1E+06 cells per mL. An appropriate amount of the suspension was taken, centrifuged, the supernatant was discarded, and CFSE solution (prepared from stock solution and pre-warmed PBS at a ratio of 1:500) was added and mixed well to maintain a final density of 1E+06 cells per mL. The cells were incubated in the dark at 37°C, 5% CO2 for 20 minutes. 2) 5 volumes of complete medium was added and incubated for 5 minutes. 3) The mixture was centrifuged at 500 g for 5 minutes, after which the supernatant was discarded. 4) The cells were resuspended in pre-warmed complete medium and allowed to sit at room temperature for 10 minutes before the next experiment. 4. Effector-target cell co-incubation 1) NALM6 + Effector cell population, K562 + The effector cell population was set up according to total effector cells: target cells = 10: 1. Target and effector cells were co-incubated for 4 hours, and cells were collected for flow cytometry.

[0190] result: 1. All ten designed CD19 CAR-T constructs produce specific killing effects on NALM6 (CD19 positive tumor cell line), but no obvious killing effects on the negative cell K562 (CD19 negative cell line) (results shown in Figure 2); 2. Compared with the currently used second-generation CAR-T (No. 10 CAR-T), the specific killing of No. 1, 3, and 4 in the designed No. 1-9 CAR-Ts was significantly stronger than that of the second-generation CAR-T (shown in Figure 2). 3. The specific killing of Nos. 2, 6, 7, 8, and 9 in the engineered Nos. 1–9 CAR-Ts is comparable to that of second-generation CAR-Ts (shown in Figure 2). 4. No. 5 in the designed No. 1–9 CAR-Ts is weaker than that in the second-generation CAR-Ts (shown in Figure 2 ). 5. The non-specific killing of No. 3 and No. 9 CAR-T is the weakest among the designed No. 1-9 CAR-T, which can reduce off-target and cellular inflammatory factor responses and improve the safety of CAR-T treatment in clinical application. EXAMPLES

[0191] Cytokine release assay of CAR-T with different structures in cytotoxicity experiments 1. After 24 hours of incubation in the cytotoxicity test, the supernatant was collected and diluted 5-fold for use; 2. The factor content in the samples was detected by using CBA Factor Detection Kit (Product No. 551809). 25μL diluted sample + 25μL mixed magnetic beads + 25μL PE detection reagent were added to each test well. After mixing well, it was incubated at room temperature for 3 hours. The mixture was washed 3 times with 200μL PBS and resuspended in 100μL PBS; 3. Flow cytometry and data analysis using FCAP software.

[0192] result: 1. CAR-T with different structures all produced obvious INF-γ release, with obvious differences between different structures, with No. 3 and No. 9 producing strong release (the results of CD19 CAR-T are shown in Figure 3 ); 2. The release of INF-γ correlates with the cytotoxicity of CAR-T to a certain extent. CAR-T with stronger cytotoxicity produces a higher release of INF-γ; Based on the above results of CAR+ positive rate, cytotoxicity experiment, and related factor release, No. 3 CAR-T (i.e., CAR-T3) performed best. EXAMPLES

[0193] Changes in phenotype and CAR positivity after co-incubation of CD19 CAR-T with different structures 1. 1mL each of CAR-T cells with different structures and CD3+ cells were collected by centrifugation at 500g for 5 minutes, the cells were resuspended in 500μL staining buffer, then transferred to a 96-well U-shaped plate, which was then centrifuged at 500g for 5 minutes to collect the cells. The collected cells were washed and resuspended three times according to the above conditions, and finally resuspended in 500μL staining buffer. 2. 1 μL of Protein L (FITC) was added to the corresponding detection wells and mixed well, then an equal volume of staining buffer was added to the corresponding negative control wells and reacted for 45 minutes at 4° C. After the reaction was completed, the cells were washed and resuspended according to step 1. 3. 5 μL each of anti-CD3 (APC-cy7), anti-CD4 (BV510), anti-CD8a (Percp-cy5.5), anti-HuCD45RA (BV421), and 20 μL of anti-CD197 (PE) were added to the resuspended cell detection wells, and an equal volume of staining buffer was added to the remaining wells and incubated at 4°C for 30 minutes. 4. After the reaction was completed, the cells were washed according to step 1 (adding 500 μL of staining buffer during washing) and the cells were collected. Finally, 200 μL of staining buffer was added to each well to resuspend the cells for use. 5. Preparation of color compensation samples: 200 μL of staining buffer was added to the corresponding 96-well U-shaped plate, 1 drop of BD comp-beads negative control (mix vigorously before use) and 1 drop of BD comp-beads anti-mouse Ig k pellet (mix vigorously before use) were added to each well, 5 μL of detection antibody (CD3 / CD4 / CD8 / CD45RA / CD4FITC) was added to the corresponding well; 1 drop of BD comp-beads negative control (mix vigorously before use) and 1 drop of BD comp-beads anti-rat Ig k pellet (mix vigorously before use) were added to another well, then 20 μL of anti-CD197 antibody was added, the plate was incubated at 4°C for 30 minutes, the pellet was washed and resuspended according to step 1 as waiting. 6. The flow cytometry results are shown in Figure 4, in which the abbreviations are TE (effector T cells), TN (native T cells), TCM (central memory T cells), and TEM (effector memory T cells, effector memory T cells).

[0194] result: 1. CAR-T 1, 2, and 3 all showed a significant increase in CAR+ positivity after stimulation with tumor cells (NALM-6), with no significant increase in other structures; when cultured alone or stimulated with unrelated tumor cells, CAR + There was no significant change in the positive rate, which may form a mechanism similar to immune response, with a higher contribution to CAR-T cytotoxicity; 2. After co-incubation of CAR-T 1, 2, or 3 with tumor cells, T CM For other CAR-Ts, T CM caused almost no change. 3. About CAR-T 3 CM increases, and T EM also showed a significant increase; 4. T after co-incubation of CAR-T 3 with tumor cells compared to other CAR-T CM and T EMAn increase in the ratio is beneficial for sustained CAR-T cytotoxicity; 5. ICOS structure as a costimulatory molecule is a T CM and T EM The ratio of can be increased. EXAMPLES

[0195] Detection of self-released factors of CD19 CAR-T 1. Supernatants after 24-hour culture of K562 and CAR-T were collected and diluted 5-fold for use. 2. The factor content in the samples was detected by using CBA Factor Detection Kit (Product No. 551809). 25μL diluted sample + 25μL mixed magnetic beads + 25μL PE detection reagent were added to each test well. After mixing well, the plate was incubated at room temperature for 3 hours. The mixture was washed once with 200μL PBS and resuspended in 100μL PBS. 3. Flow cytometry and data analysis using FCAP software.

[0196] The results are shown in Figure 5. When CAR-Ts were co-incubated with unrelated tumor cells, there were significant differences in cytokine release among the different CAR-Ts, with CAR-T 1 and 3 constructs producing lower INF-γ and IL10 release, and CAR-T 2, 9, 10 producing higher INF-γ and IL10 release, of which CAR-T 2 produced the highest release; With the exception of the CAR-T 2 construct, there is no obvious release of IL4; When CAR-T with different structures were co-incubated with K562 cells, the amount of released factors reflected the state of CAR-T autoactivation. The higher the factor release, the more the CAR-T structure was in an autoactivation state, which may lead to a higher apoptotic factor storm, and the continuous autoactivation state may lead to cell failure and reduce the persistence of CAR-T.

[0197] Considering the overall cytotoxicity, factor release, phenotype change, and positive rate change, the CAR-T 3 structure is preferred. EXAMPLES

[0198] Cytotoxicity detection of anti-claudin 18.2 CAR-T (CAR-T 3) 1. Cell culture Validated target cells (MGC-803-18.2, i.e., self-assembled cell line with high expression of claudin 18.2 / MGC-803-18.1, i.e., self-assembled cell line with high expression of claudin 18.1) were cultured by using 1640 / IMDM+10%FBS medium in a 5% carbon dioxide incubator at 37°C. Adherent cells: The cells were observed once a day by using an inverted microscope and subcultured every 2 days. The cells were subcultured before the cell density in the flask reached 95% (covering the whole bottom by observing under a microscope). 2. Treatment of effector cells (anti-claudin 18.2 CAR-T) Anti-claudin 18.2 CAR-T cells were collected in a 15 mL centrifuge tube and centrifuged at 500 g for 5 min. The supernatant was discarded and the cells were washed by adding 4 mL of 0.01 M PBS, centrifuged, the supernatant was discarded, and the cells were resuspended in approximately 10 per mL. 7 The cells were resuspended in an appropriate amount of AIM-V medium to a cell density of 100 cells. The cell density and viability were determined. 3. Pretreatment of target cells 1) Target cell labeling: Target cells were harvested in logarithmic growth phase and washed once with 0.01M PBS. The target cell mixture was centrifuged at 500g for 5 min and the supernatant was discarded; then, target cells were labelled at 1 × 10 per mL. 6 The cells were resuspended in complete medium to obtain a suspension with a density of 1 × 10 cells per mL. An appropriate volume of the suspension was taken, centrifuged, the supernatant discarded, and CFSE solution (prepared from stock solution and pre-warmed PBS at a ratio of 1:500) was added, mixed well, and diluted to a density of 1 × 10 cells per mL. 6 The cells were incubated in the dark at 37° C., 5% CO2 for 20 minutes. 2) 5 volumes of complete medium was added and incubated for 5 minutes. 3) The mixture was centrifuged at 500 g for 5 minutes and the supernatant was discarded. 4) The cells were resuspended in pre-warmed complete medium and allowed to sit at room temperature for 10 minutes before the next experiment. 4. Effector-target cell co-incubation 1) Both MGC-803-18.1 effector cell group and MGC-803-18.2 effector cell group were set according to the ratio of total effector cells: target cells = 1: 1, 2: 1, 3: 1. Target cells and effector cells were co-incubated for 4 hours, and then cells were collected for flow cytometry.

[0199] The results are shown in Figure 6: Compared with the current second-generation CAR-T (CAR-T 10), the specific killing of CAR-T 3 is significantly stronger than that of the second-generation CAR-T; CAR-T 3 produces weaker non-specific killing, which can reduce off-target and cellular inflammatory factor responses and improve the safety of CAR-T treatment in clinical applications. EXAMPLES

[0200] Changes in phenotype and CAR positivity after co-incubation of anti-claudin 18.2 CAR-T with different structures 1. 1 mL each of anti-claudin 18.2 CAR-T cells and different target cells (treated in Example 7) was centrifuged at 500g for 5 minutes to collect the cells, the cells were resuspended in 500μL staining buffer, then transferred to a 96-well U-shaped plate, which was centrifuged at 500g for 5 minutes to collect the cells, the cells were washed and resuspended three times according to the above conditions, and finally resuspended in 500μL staining buffer. 2. 1 μL of Protein L (FITC) was added to the corresponding detection wells and mixed well, then an equal volume of staining buffer was added to the corresponding negative control wells and reacted for 45 minutes at 4° C. After the reaction was completed, the cells were washed and resuspended according to step 1. 3. 5 μL each of anti-CD3 (APC-cy7), anti-CD4 (BV510), anti-CD8a (Percp-cy5.5), anti-HuCD45RA (BV421), and 20 μL of anti-CD197 (PE) were added to the resuspended cell detection wells, and an equal volume of staining buffer was added to the remaining wells and incubated at 4°C for 30 minutes. 4. After the reaction was completed, the cells were washed according to step 1 (adding 500 μL of staining buffer during washing) and the cells were collected. Finally, 200 μL of staining buffer was added to each well to resuspend the cells for use. 5. Preparation of color compensation samples: 200 μL of staining buffer was added to the corresponding 96-well U-shaped plate, 1 drop of BD comp-beads negative control (mix vigorously before use) and 1 drop of BD comp-beads anti-mouse Ig k pellet (mix vigorously before use) were added to each well, 5 μL of detection antibody (CD3 / CD4 / CD8 / CD45RA / CD4FITC) was added to the corresponding well; 1 drop of BD comp-beads negative control (mix vigorously before use) and 1 drop of BD comp-beads anti-rat Ig k pellet (mix vigorously before use) were added to another well, then 20 μL of anti-CD197 antibody was added, the plate was incubated at 4°C for 30 minutes, the pellet was washed and resuspended according to step 1 as waiting. 6. The flow cytometry results are shown in FIG. 1) CAR-T 3 is a method to stimulate tumor cells (MGC-803-18.2) with CAR + showed a significant increase in positivity in CAR-positive cells, but not in other structures; when cultured alone or stimulated with unrelated tumor cells, + There was no significant change in the positive rate, and therefore it may form a mechanism similar to immune stimulation, with a higher contribution to CAR-T cytotoxicity; 2) After co-culture of CAR-T 3 with tumor cells, T CM For other CAR-Ts, T CM caused almost no change. 3) About CAR-T 3CM increases, and T EM also showed a significant increase; 4) Compared with other CAR-Ts, CAR-T 3 showed a significant improvement in T after co-culture with tumor cells. CM and T EM An increase in the ratio is beneficial for sustained CAR-T cytotoxicity; 5) The structure of ICOS as a costimulatory molecule is CM and T EM The ratio of can be increased. EXAMPLES

[0201] Detection of self-released factors of anti-claudin 18.2 CAR-T 1. The supernatants of anti-claudin 18.2 CAR-T monoculture and 24-hour coculture of MGC-803-18.1 and anti-claudin 18.2 CAR-T were collected and diluted 5-fold for use. 2. The factor content in the samples was detected by using CBA Factor Detection Kit (Product No. 551809). 25μL diluted sample + 25μL mixed magnetic beads + 25μL PE detection reagent were added to each test well. After mixing well, the plate was incubated at room temperature for 3 hours. The mixture was washed once with 200μL PBS and resuspended in 100μL PBS. 3. Flow cytometry and data analysis using FCAP software.

[0202] The results are shown in Figure 8. The CAR-T 3 construct results in less INF-γ and IL10 release.

[0203] Reference throughout this specification to "one embodiment," "one embodiment," "some embodiments," "examples," "particular examples," or "some examples" means that a particular feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the disclosure. A schematic illustration of an item above in this specification does not necessarily refer to that same embodiment. The appearance of "one embodiment," "one embodiment," "some embodiments," "examples," "particular examples," or "some examples" in various places throughout this specification does not necessarily refer to that same embodiment or example of the disclosure. Furthermore, particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0204] While illustrative embodiments have been shown and described, it will be understood by those skilled in the art that the above embodiments should not be construed as limiting the disclosure, and that changes, substitutions, and modifications may be made to the embodiments without departing from the spirit, principles, and scope of the disclosure.

Claims

1. an extracellular domain of a single-chain antibody fragment, comprising a heavy chain variable region, a light chain variable region, and a CD8 hinge region, wherein the single-chain antibody fragment specifically recognizes an antigen; a transmembrane domain, including an immune costimulatory molecule transmembrane domain; and Intracellular domain containing immune costimulatory molecule intracellular segment and CD3 zeta chain Including, the immune costimulatory molecule transmembrane domain is a CD8 transmembrane domain; the immune costimulatory molecule intracellular segment is a 4-1BB intracellular segment and an ICOS intracellular segment, the N-terminus of the ICOS intracellular segment is linked to the C-terminus of the CD8 transmembrane domain, the C-terminus of the ICOS intracellular segment is linked to the N-terminus of the 4-1BB intracellular segment, and the C-terminus of the 4-1BB intracellular segment is linked to the N-terminus of the CD3 zeta chain; the immune co-stimulatory molecule transmembrane domain connected to the immune co-stimulatory molecule intracellular segment has the amino acid sequence of SEQ ID NO:1; The antigen is a tumor antigen; Chimeric antigen receptor.

2. The chimeric antigen receptor described in claim 1, wherein the antigen includes at least one selected from CD19, CD20, CD123, GPC3, MUC-1, GD2, BCMA, HER2, EGFR, VEGFR, cMet, MSLN, EGFRvIII, and claudin 18.

2.

3. 3. A construct comprising a nucleic acid molecule encoding the chimeric antigen receptor of claim 1 or 2 and further comprising a promoter, wherein the promoter is operably linked to the nucleic acid molecule and comprises at least one selected from CMV, EF-1, and RSV.

4. 4. The construct of claim 3, which is a non-pathogenic virus, wherein the virus is selected from a retrovirus, a lentivirus, and an adenovirus-associated virus.

5. A transgenic lymphocyte which expresses a chimeric antigen receptor according to claim 1 or 2, or which has a construct according to claim 3 or 4.

6. A method for preparing a transgenic lymphocyte according to claim 5, comprising a step of introducing the construct according to claim 3 or 4 into a lymphocyte in vitro.

7. A therapeutic composition for treating cancer comprising the construct of claim 3 or 4, or the transgenic lymphocyte of claim 5, wherein the cancer comprises at least one selected from hematopoietic malignancies, gastrointestinal cancer, glioma, lung cancer, liver cancer, and pancreatic cancer.

8. 3. A chimeric antigen receptor expressed by a lymphocyte according to claim 1 or 2 for use in improving the safety, efficacy or durability of lymphocyte therapy.

Images