CN116789844A - Chimeric antigen receptor of targeted CD7 protein and application thereof - Google Patents

Abstract

The present invention provides a chimeric antigen receptor targeting CD7 protein comprising: an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence; the extracellular domain comprises an antigen recognition region, which is a nanobody targeting CD7 protein; the amino acid sequence of the nano antibody targeting the CD7 protein is shown as SEQ ID NO: 1-SEQ ID NO:3, respectively. The present invention provides chimeric antigen receptors that target CD7 proteins and are ubiquitinated deletion engineered. And the continuous proliferation capability test, the killing function test and the like under the in vitro continuous stimulation are carried out on the primary T cells of the human, and the tumor killing function test in the tumor-bearing mice is carried out, so that compared with the CAR sequences reported in the literature, the nano antibody CAR effect of the invention is more excellent.

Description

Chimeric antigen receptor of targeted CD7 protein and application thereof

Technical Field

The invention relates to the field of chimeric antigen receptors, in particular to a chimeric antigen receptor targeting CD7 protein and application thereof.

Background

The full name of CAR is chimeric antigen receptor (chimeric antigen receptor, CAR), whose composition mainly comprises: single chain antibodies (single chain antibody fragment, scFv) capable of recognizing and binding specific tumor antigens, the specificity of which can enable accurate recognition and "site-directed hits" of tumor cells; the intracellular domain of the zeta chain of the T Cell Receptor (TCR) can effectively activate T cells after the scFv structure recognizes tumor antigens, and the activated T cells can secrete a large amount of cytokines, so that the T cells can effectively kill the tumor cells while rapidly proliferate, and other immune cells are recruited by the secreted inflammatory factors, so that the killing and phagocytosis of the tumor cells are further enhanced; on the basis, the intracellular signal domains of 1 or 2T cell co-stimulatory molecules are coupled, so that the capacity of activating the T cells of the CAR can be enhanced, and the functions of the CAR-T cells can be improved.

T cells expressing a CAR targeting a specific tumor antigen are called CAR-T cells, and CAR-T therapy is cell adoptive immunotherapy that achieves accurate striking of tumor cells by reinfusion of CAR-T cells to tumor patients. In 2017, the U.S. FDA sequentially passes through two commercial CAR-T products targeting the CD19 antigen and is used for treating the recurrent refractory malignant B-cell leukemia and lymphoma, and the curative effect is remarkable. However, no established commercial CAR-T products against T cell tumors such as lymphomas and leukemias are currently available.

As a very invasive malignant blood tumor, T-cell acute lymphoblastic leukemia (T-ALL) has the characteristics of rapid development, high recurrence rate, low median survival rate and the like. Recurrent/refractory T cell lymphomas have limited effective treatment methods such as: allogeneic stem cell transplantation is difficult and high in cost; although the U.S. drug and food administration (FDA) has approved the chemotherapeutic drug nelarabine for the treatment of T-ALL where at least two regimens are ineffective or recurrent, nelarabine has not yet been marketed in China and has a complete remission rate for T-ALL of less than 30% according to the FDA published clinical study report. The difficulty in the treatment of T cell tumors, particularly relapsed/refractory T-ALL and T cell lymphomas, is seen as a great need to develop safer and more effective treatment protocols.

Disclosure of Invention

In view of the above-described drawbacks of the prior art, it is an object of the present invention to provide chimeric antigen receptors targeting CD7 proteins and uses thereof.

To achieve the above and other related objects, a first aspect of the present invention provides a chimeric antigen receptor targeting CD7 protein, comprising:

an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence;

the extracellular domain comprises an antigen recognition region, which is a nanobody targeting CD7 protein; the amino acid sequence of the nano antibody targeting the CD7 protein is shown as SEQ ID NO:1-SEQ ID NO:3, respectively. In a second aspect the invention provides a polynucleotide sequence selected from the group consisting of:

(1) Polynucleotide sequences encoding the aforementioned chimeric antigen receptor targeting CD7 protein; and/or

(2) The complement of the polynucleotide sequence of (1).

In a third aspect the present invention provides a nucleic acid construct comprising a polynucleotide sequence as hereinbefore described; preferably, the nucleic acid construct is a vector; more preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3'LTR, a 5' LTR and the polynucleotide sequences described above.

In a fourth aspect, the invention provides a lentivirus comprising the nucleic acid construct described above.

In a fifth aspect the invention provides a method of ex vivo activation of T cells, the method comprising the step of infecting the T cells with a lentivirus as described above.

In a sixth aspect, the invention provides a genetically modified T cell or a pharmaceutical composition comprising the genetically modified T cell, wherein the cell comprises the polynucleotide sequence as described above, or comprises the nucleic acid construct as described above, or is infected with the lentivirus as described above, or is prepared by the method as described above.

In a seventh aspect, the invention provides the use of a chimeric antigen receptor, polynucleotide sequence, nucleic acid construct or lentivirus as described above for the preparation of a T cell activator and/or for the preparation of a T cell degradation inhibitor.

In an eighth aspect the invention provides the use of a chimeric antigen receptor, polynucleotide sequence, nucleic acid construct, lentivirus, or genetically modified T cell as hereinbefore described for any one or more of the following: (1) preparing a tumor therapeutic drug; (2) preparing a formulation that increases the killing efficiency of the tumor; (3) preparing a preparation for maintaining proliferation capacity of T cells; (4) preparing a preparation for inhibiting tumor development; preferably, the tumour is selected from one or more of leukaemia or lymphoma; more preferably, the tumor is selected from the group consisting of T cell lymphoma, T cell type acute lymphoblastic leukemia, B cell type acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, and acute myelogenous leukemia.

As described above, the CD7 protein-targeting chimeric antigen receptor and the use thereof of the present invention have the following beneficial effects:

the present invention provides chimeric antigen receptors that target CD7 proteins and are ubiquitinated deletion engineered. And the continuous proliferation capability test, the killing function test and the like under the in vitro continuous stimulation are carried out on the primary T cells of the human, and the tumor killing function test in the tumor-bearing mice is carried out, so that compared with the CAR sequences reported in the literature, the nano antibody CAR effect of the invention is more excellent.

Drawings

FIG. 1 shows the specific recognition of human CD7 protein by positive monoclonal antibodies obtained by phage display library screening by ELISA (enzyme linked immunosorbent assay, ELISA) assay.

Figure 2 detection of CAR expression by flow cytometry of the level of self-activation in the quiescent state of CAR-T cells. The detection level of the coupling Myc tag at the CARN end indicates the membranous condition of the CAR and the T cell activation marker protein CD69 indicates the self-activation level of the CAR-T cell. (a. Partial nanobody CAR cell surface expression level; b. Partial nanobody CAR background self-activation level).

Figure 3 detects the expression of CAR by flow cytometry for the membranous condition and its background self-activation level in resting state and the level of cell depletion caused by this self-activation signal. The level of ICOS reflects the level of CAR-T cell activation at the T cell activation indicator protein CD69, and the level of CAR-T cell depletion at the T cell depletion indicator protein PD1, LAG3, TIM 3. (a. Flow detection of background self-activation level of CAR-T cells; b. Flow detection of depletion level of CAR-T cells in resting conditions).

Figure 4 detection of CAR activation levels by flow cytometry. (a. T cell lines expressing 3 nanobody CARs, CD69 expression levels after target cell stimulation; b. T cell lines expressing 3 nanobody CARs, CD69 expression levels after non-target cell stimulation).

FIG. 5 detection of the killing function of different nanobodies CAR and PA7-13 on CD7 positive target cells the killing capacity of CAR-T cells on target cells was reflected by the fluorescence intensity generated by the consumption of its substrate luciferin by firefly luciferase expressed by target cells.

FIG. 6 detection of killing function of primary T cells expressing E7-CAR on tumor target cells and non-target cells. After expressing the E7 nanobody CAR on human primary T cells after knocking out CD7 protein, the killing function against target cells as well as non-target cells was tested.

FIG. 7 is a graph of detection of cytokine secretion levels of E7 CAR-T cells under target cell stimulation by ELISA.

FIG. 8 is an in vitro assay for wild-type E7 CAR-T cells, and proliferation of ubiquitinated modified deleted E7 CAR-T cells under sustained stimulation of target cells. (detection of sustained proliferation ability of E7-CAR T cells after deletion of wild-type and ubiquitination under in vitro antigen stimulation).

Fig. 9 is a tumor killing experiment in a tumor-bearing mouse, and verifies that ubiquitination modified deleted E7 CAR-T cells can effectively kill tumor cells in the tumor-bearing mouse, and the survival time of the mouse is remarkably prolonged. ( a. Ubiquitination modification lacks the tumor killing function of E7 CAR-T cells in tumor-bearing mice; b. analysis of survival rate after cell therapy in tumor-bearing mice )

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.

Based on the excellent therapeutic effect of CAR-T cell therapy in B cell lymphomas, we hope to develop a safer and more effective CAR-T product against T cell tumors including T-ALL and lymphomas, bringing new therapeutic opportunities for a broad range of patients.

CD7 is a highly specific target on the surface of T cells and starts to be expressed early in T cell differentiation, and thus includes blast T cell tumors (T-ALL/lymphoblastic lymphoma), mature T cell tumors (peripheral T cell lymphoma, metaplastic cell lymphoma, etc.), NK/T cell tumors, acute Myeloid Leukemia (AML), etc. may express CD7. Thus, CD7 can be used as a tumor-associated target (tumor associated antigen, TAA) for hematological tumors, especially T-cell tumors, including T-cell lymphomas and leukemias [4-8]. However, the target point of killing the CAR-T cells by using the indicative protein co-expressed by the T cells has the following 2 main problems: the strong killing function of the 1, CAR-T cells can kill a great amount of normal T cells which also express CD7 protein while killing CD7 positive tumor cells, thereby bringing about serious immunodeficiency problems. However, studies have now shown that about 10% of normal T cells in humans are CD7 negative and have normal function, and that normal T cells that are not killed by CD 7-targeted CAR-T cells can function to some extent as cellular immunity. And it has been proposed that by constructing and reinfusion CD 7-deleted hematopoietic stem cells, they can be used for recovery of T and NK cells following CD 7-targeted CAR-T cell therapy. 2, car-T cells "stutter" to each other. Since CAR-T cells are also T cells and express CD7, they can be killed as target cells of other CAR-T cells, and thus "mutually kill" CAR-T cells, it is necessary to remove CD7 protein expressed on the surface of T cells in a certain manner (gene editing, retention of protein in cells, etc.) at the time of constructing CAR-T cells.

In conclusion, CD7 is a highly specific tumor-associated target for T cell tumors including lymphomas and leukemias, and the difficulty in the preparation and application processes of the CD7 can be overcome by the prior art, so that the CD7 is an ideal target for treating T cell lymphomas/leukemias by CAR-T cells.

According to the invention, a plurality of nano antibodies which specifically identify human CD7 proteins are obtained through screening by phage display screening technology, and are constructed into chimeric antigen receptors (chimeric antigen receptor, CARs); human junket T cell lines, each expressed in a knockout CD7, detect their specific recognition of antigen CD7 and their resultant level of CAR-T cell activation; and comparing the background expression level of each activation index of each CAR-T cell under the resting condition, and confirming the self-activation level of the background. And selecting a CAR structure sequence (E7) with good specificity, high stimulation response to CD7 antigen and low background self-activation level from the sequence, expressing the CAR structure sequence in human primary T cells, and performing in vitro cell level and function verification in tumor-bearing mice.

Combining with our existing patent (201910302508X), the sequence is subjected to ubiquitin deletion transformation, and continuous proliferation capability test, killing function test and the like under in vitro continuous stimulation are performed on human primary T cells, and tumor killing function test in tumor-bearing mice is performed, so that compared with CAR sequences reported in literature, the nano antibody CAR effect is more excellent.

The chimeric antigen receptor targeting CD7 protein comprises:

an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence;

the extracellular domain comprises an antigen recognition region, which is a nanobody targeting CD7 protein; the amino acid sequence of the nano antibody targeting the CD7 protein is shown as SEQ ID NO:1-SEQ ID NO:3 is shown in any one of the following figures;

D2(SEQ ID NO：1)：

EVQLVESGGGLVQPGGSLRLSCAASGRTFSPYVMGWFRQAPGKGRELVADISSSGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAKPWPPFGPYTYAYNYWGQGTQVTVSSGGS

D4(SEQ ID NO：2：

EVQLVESGGGLVQPGGSLRLSCAASGITFSTYNMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAASLYPDGYLSIYSYNYWGQGTQVTVSSGGS

E7(SEQ ID NO：3)：

EVQLVESGGGLVQPGGSLRLSCAASGRTFSTNRMGWFRQAPGKGRELVAAINSRDSRTWYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAGGEIYSLGDDWFGWGPYDYWGQGTQVTVSSGGS

the intracellular domain comprises a costimulatory signaling region and a CD3 zeta intracellular region which are connected in sequence to form a costimulatory signaling region-CD 3 zeta intracellular region;

in one embodiment, the costimulatory signaling region is selected from the group consisting of the intracellular region of CD27, CD28, CD134, 41BB or ICOS.

Preferably, the costimulatory signaling region is selected from the 41BB intracellular region.

In one embodiment, the amino acid sequence of the 41BB intracellular region is as set forth in SEQ ID NO:4 (wild type). The method comprises the following steps: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

In one embodiment, the amino acid sequence of the cd3ζ intracellular domain is set forth in SEQ ID NO:5 (wild type). The method comprises the following steps:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

in one embodiment, the costimulatory signaling region-CD 3 zeta intracellular region is a polypeptide formed by mutating a lysine in the wild-type costimulatory signaling region-CD 3 zeta intracellular region to arginine.

In one embodiment, the costimulatory signaling region-CD 3 zeta intracellular region has the amino acid sequence as set forth in SEQ ID NO:6 (41 BB-CD3 ζ wild type) or SEQ ID NO:7 (ubiquitination modified deletion type). The method comprises the following steps:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。(SEQ ID NO：6)

RRGRRRLLYIFRQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVRFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDRRRGRDPEMGGRPQRRRNPQEGLYNELQRDRMAEAYSEIGMRGERRRGRGHDGLYQGLSTATRDTYDALHMQALPPR。(SEQ ID NO：7)

further, the extracellular domain also includes a signal peptide and/or a hinge region. Forming a signal peptide-antigen recognition region-hinge region connected in sequence.

Preferably, the signal peptide is selected from the group consisting of CD8 a signal peptide and/or the hinge region is selected from the group consisting of the hinge region of CD8 a.

Optionally, the extracellular domain further comprises a tag. The tag may be a myc tag forming a signal peptide-tag-antigen recognition region-hinge region that are sequentially linked. The amino acid sequence of myc tag is shown in SEQ ID NO: shown at 8.

Specific: EQKLISEEDL. (SEQ ID NO: 8)

In one embodiment, the amino acid sequence of the CD8 a signal peptide is set forth in SEQ ID NO: shown at 9. Specific:

MALPVTALLLPLALLLHAARP。(SEQ ID NO：9)

in one embodiment, the hinge region of CD8 a has the amino acid sequence set forth in SEQ ID NO: shown at 10. Specific:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。(SEQ ID NO：10)

the transmembrane domain is selected from the transmembrane region of CD4, CD8 alpha, OX40 or H2-Kb. Preferably, the transmembrane region is selected from CD8 alpha.

In one embodiment, the amino acid sequence of the transmembrane region of CD8 a is set forth in SEQ ID NO: 11. Specific:

IYIWAPLAGTCGVLLLSLVIT。(SEQ ID NO：11)

In one embodiment, the chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID NO: 12-17. Specific:

MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASGRTFSPYVMGWFRQAPGKGRELVADISSSGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAKPWPPFGPYTYAYNYWGQGTQVTVSSGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：12，D2 CAR)

MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASGITFSTYNMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAASLYPDGYLSIYSYNYWGQGTQVTVSSGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：13，D4 CAR)

MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASGRTFSTNRMGWFRQAPGKGRELVAAINSRDSRTWYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAGGEIYSLGDDWFGWGPYDYWGQGTQVTVSSGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：14，E7 CAR)

MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASGRTFSTNRMGWFRQAPGKGRELVAAINSRDSRTWYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAGGEIYSLGDDWFGWGPYDYWGQGTQVTVSSGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRGRRRLLYIFRQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVRFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDRRRGRDPEMGGRPQRRRNPQEGLYNELQRDRMAEAYSEIGMRGERRRGRGHDGLYQGLSTATRDTYDALHMQALPPR (SEQ ID NO:15, E7 ubiquitination deleted CAR)

MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASGRTFSPYVMGWFRQAPGKGRELVADISSSGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAKPWPPFGPYTYAYNYWGQGTQVTVSSGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRGRRRLLYIFRQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVRFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDRRRGRDPEMGGRPQRRRNPQEGLYNELQRDRMAEAYSEIGMRGERRRGRGHDGLYQGLSTATRDTYDALHMQALPPR (SEQ ID NO:16, D2 ubiquitination deleted CAR)

MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASGITFSTYNMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAASLYPDGYLSIYSYNYWGQGTQVTVSSGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRRGRRRLLYIFRQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVRFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDRRRGRDPEMGGRPQRRRNPQEGLYNELQRDRMAEAYSEIGMRGERRRGRGHDGLYQGLSTATRDTYDALHMQALPPR (SEQ ID NO:17, D4 ubiquitination deleted CAR)

The above-described individual parts forming the chimeric antigen receptor of the invention may be directly linked to each other or may be linked by a linker sequence. The linker sequences may be linker sequences suitable for antibodies as known in the art, such as G and S containing linker sequences. Typically, a linker contains one or more motifs that repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are contiguous in the linker sequence with no amino acid residues inserted between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motif compositions. The length of the linker may be 3 to 25 amino acid residues, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a glycine linker sequence. The number of glycine in the linker sequence is not particularly limited, and is usually 2 to 20, for example 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc.

It will be appreciated that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed amino acid sequence, without affecting the activity of the sequence of interest. To construct fusion proteins, facilitate expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside of the host cell, or facilitate purification of recombinant proteins, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, for example, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-or carboxy-terminus of the fusion protein of the invention (i.e., the CAR) may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, poly-His, poly-Arg, strep-TagII, AU1, EE, T7,4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins.

The present invention provides a polynucleotide sequence selected from the group consisting of:

(1) Polynucleotide sequences encoding the chimeric antigen receptors described above; and/or

(2) The complement of the polynucleotide sequence of (1).

Preferably, the polynucleotide sequence is as set forth in SEQ ID NO: 18-23. Specific:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAACAGAAGCTGATCTCGGAGGAGGATCTGGAGGTGCAGCTGGTGGAGAGCGGTGGTGGTCTCGTGCAGCCCGGCGGTAGTCTGCGCCTCAGCTGTGCCGCCAGCGGTCGCACCTTTAGCCCATATGTGATGGGCTGGTTTCGCCAAGCCCCCGGCAAAGGTCGCGAACTGGTGGCCGATATTAGCAGCAGCGGTGGCAGCACCTACTACCCAGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACGCCAAGCGCATGGTGTATCTGCAGATGAACAGTCTGCGCGCCGAGGACACCGCCGTGTATTATTGTGCCGCCAAACCATGGCCACCATTTGGCCCATATACCTATGCCTACAATTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGCGGTGGTTCTACCACCACTCCCGCACCCCGCCCTCCTACTCCTGCCCCTACCATTGCtAGCCAACCGCTTAGTCTGAGACCTGAGGCCTGTAGGCCCGCTGCTGGTGGCGCTGTGCACACCCGAGGATTGGACTTCGCTTGCGACATCTACATCTGGGCACCTCTGGCTGGGACCTGCGGCGTGTTGTTGTTGAGCCTGGTGATTACGCTGTACTGTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC(SEQ ID NO：18，D2 CAR)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAACAGAAGCTGATCTCGGAGGAGGATCTGGAGGTGCAGCTGGTGGAGAGCGGTGGTGGTCTCGTGCAGCCCGGCGGTAGTCTGCGCCTCAGCTGTGCCGCCAGCGGTATCACCTTTAGCACCTATAACATGGGCTGGTTTCGCCAAGCCCCCGGCAAAGGTCGCGAACTGGTGGCCGCCATTTCCCGCACGGGCGGTAGCACCTACTACCCAGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACGCCAAGCGCATGGTGTATCTGCAGATGAACAGTCTGCGCGCCGAGGACACCGCCGTGTATTATTGTGCCGCCAGCCTGTATCCAGATGGCTATCTGAGCATCTATAGCTACAATTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGCGGTGGTTCTACCACCACTCCCGCACCCCGCCCTCCTACTCCTGCCCCTACCATTGCtAGCCAACCGCTTAGTCTGAGACCTGAGGCCTGTAGGCCCGCTGCTGGTGGCGCTGTGCACACCCGAGGATTGGACTTCGCTTGCGACATCTACATCTGGGCACCTCTGGCTGGGACCTGCGGCGTGTTGTTGTTGAGCCTGGTGATTACGCTGTACTGTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC(SEQ ID NO：19，D4 CAR)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAACAGAAGCTGATCTCGGAGGAGGATCTGGAGGTGCAGCTGGTGGAGAGCGGTGGTGGTCTCGTGCAGCCCGGCGGTAGTCTGCGCCTCAGCTGTGCCGCCAGCGGTCGCACCTTTAGCACCAATCGCATGGGCTGGTTTCGCCAAGCCCCCGGCAAAGGTCGCGAACTGGTGGCCGCCATTAACAGCCGCGATAGCCGCACCTGGTACCCAGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACGCCAAGCGCATGGTGTATCTGCAGATGAACAGTCTGCGCGCCGAGGACACCGCCGTGTATTATTGTGCCGCCGGCGGCGAAATCTATAGCCTGGGCGATGATTGGTTTGGCTGGGGCCCATACGATTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGCGGTGGTTCTACCACCACTCCCGCACCCCGCCCTCCTACTCCTGCCCCTACCATTGCtAGCCAACCGCTTAGTCTGAGACCTGAGGCCTGTAGGCCCGCTGCTGGTGGCGCTGTGCACACCCGAGGATTGGACTTCGCTTGCGACATCTACATCTGGGCACCTCTGGCTGGGACCTGCGGCGTGTTGTTGTTGAGCCTGGTGATTACGCTGTACTGTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC(SEQ ID NO：20，E7 CAR)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAACAGAAGCTGATCTCGGAGGAGGATCTGGAGGTGCAGCTGGTGGAGAGCGGTGGTGGTCTCGTGCAGCCCGGCGGTAGTCTGCGCCTCAGCTGTGCCGCCAGCGGTCGCACCTTTAGCACCAATCGCATGGGCTGGTTTCGCCAAGCCCCCGGCAAAGGTCGCGAACTGGTGGCCGCCATTAACAGCCGCGATAGCCGCACCTGGTACCCAGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACGCCAAGCGCATGGTGTATCTGCAGATGAACAGTCTGCGCGCCGAGGACACCGCCGTGTATTATTGTGCCGCCGGCGGCGAAATCTATAGCCTGGGCGATGATTGGTTTGGCTGGGGCCCATACGATTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGCGGTGGTTCTACCACCACTCCCGCACCCCGCCCTCCTACTCCTGCCCCTACCATTGCtAGCCAACCGCTTAGTCTGAGACCTGAGGCCTGTAGGCCCGCTGCTGGTGGCGCTGTGCACACCCGAGGATTGGACTTCGCTTGCGACATCTACATCTGGGCACCTCTGGCTGGGACCTGCGGCGTGTTGTTGTTGAGCCTGGTGATTACGCTGTACTGTAGACGGGGCAGACGCAGACTCCTGTATATATTCCGCCAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAGATTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACCGCAGACGTGGCCGGGACCCTGAGATGGGGGGAAGACCGCAGAGAAGGCGCAACCCTCAGGAAGGCCTGTACAATGAACTGCAGCGCGATAGAATGGCGGAGGCCTACAGTGAGATTGGGATGAGAGGCGAGCGCCGGAGGGGCAGAGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCCGCGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC (SEQ ID NO:21, E7 ubiquitination deleted CAR)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAACAGAAGCTGATCTCGGAGGAGGATCTGGAGGTGCAGCTGGTGGAGAGCGGTGGTGGTCTCGTGCAGCCCGGCGGTAGTCTGCGCCTCAGCTGTGCCGCCAGCGGTCGCACCTTTAGCCCATATGTGATGGGCTGGTTTCGCCAAGCCCCCGGCAAAGGTCGCGAACTGGTGGCCGATATTAGCAGCAGCGGTGGCAGCACCTACTACCCAGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACGCCAAGCGCATGGTGTATCTGCAGATGAACAGTCTGCGCGCCGAGGACACCGCCGTGTATTATTGTGCCGCCAAACCATGGCCACCATTTGGCCCATATACCTATGCCTACAATTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGCGGTGGTTCTACCACCACTCCCGCACCCCGCCCTCCTACTCCTGCCCCTACCATTGCtAGCCAACCGCTTAGTCTGAGACCTGAGGCCTGTAGGCCCGCTGCTGGTGGCGCTGTGCACACCCGAGGATTGGACTTCGCTTGCGACATCTACATCTGGGCACCTCTGGCTGGGACCTGCGGCGTGTTGTTGTTGAGCCTGGTGATTACGCTGTACTGTAGACGGGGCAGACGCAGACTCCTGTATATATTCCGCCAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAGATTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACCGCAGACGTGGCCGGGACCCTGAGATGGGGGGAAGACCGCAGAGAAGGCGCAACCCTCAGGAAGGCCTGTACAATGAACTGCAGCGCGATAGAATGGCGGAGGCCTACAGTGAGATTGGGATGAGAGGCGAGCGCCGGAGGGGCAGAGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCCGCGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC (SEQ ID NO:22, D2 ubiquitination deleted CAR)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAACAGAAGCTGATCTCGGAGGAGGATCTGGAGGTGCAGCTGGTGGAGAGCGGTGGTGGTCTCGTGCAGCCCGGCGGTAGTCTGCGCCTCAGCTGTGCCGCCAGCGGTATCACCTTTAGCACCTATAACATGGGCTGGTTTCGCCAAGCCCCCGGCAAAGGTCGCGAACTGGTGGCCGCCATTTCCCGCACGGGCGGTAGCACCTACTACCCAGATAGCGTGGAAGGCCGCTTCACCATCAGCCGCGATAACGCCAAGCGCATGGTGTATCTGCAGATGAACAGTCTGCGCGCCGAGGACACCGCCGTGTATTATTGTGCCGCCAGCCTGTATCCAGATGGCTATCTGAGCATCTATAGCTACAATTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGCGGTGGTTCTACCACCACTCCCGCACCCCGCCCTCCTACTCCTGCCCCTACCATTGCtAGCCAACCGCTTAGTCTGAGACCTGAGGCCTGTAGGCCCGCTGCTGGTGGCGCTGTGCACACCCGAGGATTGGACTTCGCTTGCGACATCTACATCTGGGCACCTCTGGCTGGGACCTGCGGCGTGTTGTTGTTGAGCCTGGTGATTACGCTGTACTGTAGACGGGGCAGACGCAGACTCCTGTATATATTCCGCCAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAGATTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACCGCAGACGTGGCCGGGACCCTGAGATGGGGGGAAGACCGCAGAGAAGGCGCAACCCTCAGGAAGGCCTGTACAATGAACTGCAGCGCGATAGAATGGCGGAGGCCTACAGTGAGATTGGGATGAGAGGCGAGCGCCGGAGGGGCAGAGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCCGCGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC (SEQ ID NO:23, D4 ubiquitination deleted CAR)

The polynucleotide sequences of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The invention also includes degenerate variants of the polynucleotide sequence encoding a fusion protein, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

The polynucleotide sequences described herein can generally be obtained using PCR amplification methods. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

The nucleic acid construct provided by the invention comprises the polynucleotide sequence.

The nucleic acid construct further comprises one or more regulatory sequences operably linked to the aforementioned polynucleotide sequence. The coding sequences of the CARs of the invention can be manipulated in a variety of ways to ensure expression of the protein. The nucleic acid construct may be manipulated according to the expression vector or requirements prior to insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequences may also be suitable leader sequences, untranslated regions of mRNA that are important for host cell translation. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

Preferably, the nucleic acid construct is a vector.

Expression of the polynucleotide sequence encoding the CAR is typically achieved by operably linking the polynucleotide sequence encoding the CAR to a promoter, and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

Polynucleotide sequences encoding the CARs of the invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector. The expression vector may be provided to the cell as a viral vector. Viral vector technology is well known in the art. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers.

More preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3'LTR, a 5' LTR and the aforementioned polynucleotide sequences.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the epstein barr virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the use of inducible promoters is also contemplated. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when expressed for a period of time and switching off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both of a selectable marker gene or reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or commercially available.

Methods for introducing genes into cells and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Chemical means for introducing the polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, lentiviruses provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into lentiviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

The invention provides a lentivirus comprising the nucleic acid construct described above. Preferably comprising said carrier. More preferably, the lentiviral vector.

The invention provides a method for ex vivo activation of T cells, comprising the step of infecting the T cells with a lentivirus as described above.

The CAR-T cells of the invention can undergo robust in vivo T cell expansion and last at high levels in blood and bone marrow for prolonged amounts of time and form specific memory T cells. Without wishing to be bound by any particular theory, the CAR-T cells of the invention can differentiate in vivo into a central memory-like state upon encountering and subsequently eliminating target cells expressing the surrogate antigen.

The invention also includes a class of cell therapies in which T cells are genetically modified to express a CAR as described herein, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to persistent tumor control.

The anti-tumor immune response elicited by the CAR-T cells can be an active or passive immune response. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-T cells induce an immune response specific for the antigen binding portion in the CAR.

The cancer that can be treated can be a non-solid tumor, such as a hematological tumor, e.g., leukemia and lymphoma. In particular, diseases treatable with the CAR, coding sequences thereof, nucleic acid constructs, expression vectors, viruses and CAR-T cells of the invention are preferably CD7 mediated diseases, in particular CD7 mediated hematological tumors.

In particular, herein, "CD7 mediated diseases" include, but are not limited to, leukemias and lymphomas, such as T cell lymphomas, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myelogenous leukemia.

The invention provides a genetically modified T cell or a pharmaceutical composition containing the genetically modified T cell, wherein the cell contains the polynucleotide sequence, or contains the nucleic acid construct, or is infected with the lentivirus, or is prepared by adopting the method.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as the relevant cytokine or cell population. Briefly, the pharmaceutical compositions of the invention may comprise a CAR-T cell as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.

The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease.

When referring to an "immunologically effective amount", "an" antitumor effective amount "and,In the case of "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be administered at 10 ⁴ To 10 ⁹ A dose of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Dosage of individual cells/kg body weight. T cell compositions may also be administered multiple times at these doses. Cells may be administered by using infusion techniques well known in immunotherapy. Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. The composition of T cells may be injected directly into the tumor, lymph node or site of infection.

In some embodiments of the invention, the CAR-T cells of the invention or compositions thereof can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressants. For example, treatment may be performed in combination with various radiotherapeutic agents, including: cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, fludarabine, rapamycin, mycophenolic acid, and the like. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablation therapy with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH.

Herein, "anti-tumor effect" refers to a biological effect that can be represented by a decrease in tumor volume, a decrease in tumor cell number, a decrease in metastasis number, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

"patient," "subject," "individual," and the like are used interchangeably herein to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

Use of the chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct or the lentivirus described above for the preparation of a T cell activator and/or for the preparation of a T cell degradation inhibitor.

Use of the chimeric antigen receptor, the polynucleotide sequence, the nucleic acid construct, the lentivirus, or the genetically modified T cell described above in any one or more of the following applications: (1) preparing a tumor therapeutic drug; (2) preparing a formulation that increases the killing efficiency of the tumor; (3) preparing a preparation for maintaining proliferation capacity of T cells; (4) preparing a preparation for inhibiting tumor development.

Preferably, the tumour is selected from one or more of leukaemia or lymphoma.

More preferably, the tumor is selected from the group consisting of T cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, and acute myelogenous leukemia.

Example 1: vector construction of CAR

The antigen specific antibody sequence of the target human CD7 protein CAR used in the invention is obtained by the following method: the extracellular end sequence of the recombinant human CD7 protein is taken AS an antigen, the recombinant human CD7 protein is fixed on a high adsorption pore plate, a nanoantibody phage display library (the nanoantibody phage display library is constructed in the laboratory, library design references such AS Zhong Z, yang Y, chen X, han Z, zhou J, li B, he X. Positive charge in the complementarity-determining regions of synthetic nanobody prevents aggregation. Biochemim Biophys Res Commun.2021 Oct 1;572:1-6.doi:10.1016/J. Bbic. 2021.07.054.Epub 2021 Jul 28.PMID:34332323. And McMahon C, baier AS, pascalutti R, wegreki, zheng S, ong JX, landson SC, hilger D, rasmassen SGF, ring AM, manglik A, AC. Yst surface display platform for rapid discovery of conformationally selective nanobioes. StrucMono biol 8.35:8:26/6:35, and the phage is subjected to amplification by the combination with the phage of the phage (26: 35:26: 35:35) of the amplification products of the recombinant human CD7 protein, and the phage (the phage were subjected to the amplification reaction product was constructed in the laboratory, and the phage were subjected to the amplification reaction product (the phage: 26: 35: 26: 35 to the phage: 26: 35). A total of 4 rounds of screening were performed, 156 positive clones were screened out, sequenced, and 14 different nanobody sequences were obtained after removal of the repeated sequences and constructed as CARs.

The extracellular segment structure of the CAR is formed by connecting a CD8 alpha signal peptide sequence, a myc tag sequence, a nano antibody sequence and a CD8 alpha hinge sequence in series; the transmembrane region sequence is the transmembrane region sequence of CD8 alpha; the intracellular segment structure is composed of human 41BB molecule intracellular segment sequence and human CD3 zeta intracellular segment sequence connected in series. The base sequences of all CARs in the invention are finally cloned into pHR-hEF1 alpha-IRES-EGFP vector by means of Gibson ligation.

Example 2: human primary T cell culture, knockout of CD7 protein and lentiviral infection

Human primary T cells were all taken from healthy, informed volunteers. Primary T cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. Mu.g/ml streptomycin sulfate, 1mM sodium pyruvate, optional amino acids, 55. Mu.M 2-mercaptoethanol (all available from Gibco). To maintain proliferation of T cells, 200U/ml of hIL-2 (Sigma-Aldrich) was added to the medium.

Preparation of lentiviruses: will be 6.5X10 ⁵ The Lenti-X293T cells (TaKaRa# 632180) were resuspended in DMEM medium (Gibco#11995-065) containing 10% fetal bovine serum and seeded in 6-well cell culture plates (Corning#CLS 3516) for 24 hours. 650ng was reverse transcribed using a liposome transfection system (Mirus # 2300) Virus packaging plasmid Gel-pol and 400ng VSVG were mixed with 2ug of lentiviral plasmid to be packaged following the procedure described in the Liposome transfection protocol, and Lenti-X293T cells were added. Discarding the culture medium containing the liposome after 16-18 hours, and supplementing a proper amount of fresh culture medium; after 48 hours, the cell supernatant is collected, concentrated directly or by ultracentrifugation, and frozen in a refrigerator at-80 ℃ for later use.

Electrotransport RNP knockout CD7 protein: the gRNA for CD7 protein knockout was ordered by Nanjing Jinsri Co., ltd, and the sequence was: ggagcaggtgatgttgacgg (SEQ ID NO: 24), RNase-free water was diluted to 50. Mu.M. Cas9 protein was ordered by Beijing Yiqiao Shenzhou technologies Co., ltd (cat# 40572-A08B), and RNase-free water diluted to 30uM. The method comprises the steps of performing RNP electrotransformation on non-activated human Primary T cells by using a Lonza-AmaxaTM P2 Primary Cell 4D-NucleofectorTM X Kit S kit instruction, and knocking out CD7 protein.

Lentiviral infection of primary T cells: 96-well cell culture plates (100 ul/well) without TC treatment were coated with anti-human CD3 antibody (4 ug/ml) and anti-human CD28 antibody (4 ug/ml), and T cells were recovered 24 hours after activation of the electrotransformation; after 24 hours of incubation, the prepared retrovirus and polybrene with a final concentration of 4-8ug/ml are added for centrifugal infection (2000 rpm, centrifugation at 37℃for 1 hour, speed up 2, speed down 0); after 24 hours the virus-containing medium was discarded and replaced with fresh T cell complete medium. After 4-5 days of T cell stimulation, transferring to a culture plate without antibody coating for continuous culture, regulating the cell density to 0.8-1 x 10 x 6/ml by using a T cell complete culture medium, and supplementing fresh T cell complete culture medium every 2 days to maintain the cell density.

Example 3: flow cytometry analysis

Staining for cell surface markers: the antibodies were diluted in FACS buffer (phosphate buffer pbs+2% foetal calf serum) in the appropriate ratio and cells were resuspended in the appropriate amount of antibody dilution and incubated for 25 min in dark at 4 ℃; after washing the cells 3 times with FACS buffer, the cells were resuspended with FACS buffer containing DAPI at the appropriate concentration and detected on-line.

Data for flow cytometry were obtained by BD LSRFortessa machine (BD bioscience) and analyzed using FlowJo software (Tree Star). Antibody information used for flow cytometry is listed below.

Example 4: CAR-T in vitro killing function detection

Both CD7 positive and negative target cells express firefly luciferase genes. After counting target cells and CAR-T cells, respectively taking required amount of cells, centrifuging, discarding the original culture medium, and re-suspending the original culture medium to a corresponding volume by using a complete culture medium of T cells without IL-2; according to the effector cells shown: the ratio of target cells (E: T) is that different amounts of CAR-T cells and the same amount of target cells are added into a black 96-well cell culture plate, sample wells containing only the same amount of target cells are arranged, and the mixture is mixed uniformly and then subjected to static culture in a cell culture box for 16 hours. During detection, firefly luciferase substrate luciferin is added to a final concentration of 1.5mg/ml, and the fluorescence value in each well is rapidly read by an enzyme-labeled instrument. The ratio of target cells/non-target cells killed in each sample was calculated using the fluorescence reading of the sample well containing only the same amount of target cells as a baseline value (F0). The calculation formula is as follows:

Killing efficiency (%) = (fluorescence reference value F0-sample fluorescence value Fx)/F0 x 100%

Example 5: ELISA for detecting cytokine secretion level of CAR-T cells

After counting target cells and CAR-T cells, respectively taking required amount of cells, centrifuging, discarding the original culture medium, and re-suspending the original culture medium to a corresponding volume by using a complete culture medium of T cells without IL-2; according to the E:T ratio, a certain amount of CAR-T cells are incubated with different amounts of target cells, and the cells are mixed uniformly and then subjected to static culture in a cell culture box for 16 hours. The culture supernatant was collected, and the levels of cytokines such as IL-2, IFN-gamma, etc., in culture were measured according to ELISA kit instructions.

Example 6: detection of CAR-T cell proliferation capacity in vitro

E7-BBζ ^WT And E7-BBζ ^KR After CAR-T cell counting, mixing with irradiated target cell CD7 positive Jurkat cells 1:1 respectively, and re-suspending the cells with complete medium of T cells without IL-2 to a cell density of 1 x 10≡6/ml; viable cell counts were performed every 2 days to calculate cell proliferation and cell density was adjusted to 1 x 10≡6/ml with IL-2 free T cell complete medium; when the cell density obtained by counting living cells is less than or equal to 1 x 10≡6/ml, it indicates that the cells are no longer proliferating and the experiment is terminated.

Example 7: mouse tumor model and CAR-T cell function detection

In vivo subjects were 5 to 8 week old combined immunodeficiency (NSG) mice. To compare the in vivo anti-tumor effects of CAR-T, NSG mice were first vaccinated with 1 x 10 tail vein ⁶ CD7 positive Jurkat cells expressing firefly luciferase gene; after 4 days of in vivo growth of the target cells, NSG mice were given 2X 10 by tail vein injection ⁶ Treatment of individual CAR-T cells; the intensity of firefly luciferase carried by mouse tumor cells was detected weekly by a small animal in vivo imaging system, and the development of tumors in vivo was followed. Specific implementation operations of the living animal imaging system include: firefly luciferase substrate (D-fluorescein sodium salt) was administered to tumor-bearing mice by intraperitoneal injection, the substrate amount was administered in an amount of 0.15mg/g of mouse body weight; after 10 minutes, after the substrate had been fully circulated throughout the body of the mice, the mice were anesthetized with 2.5% -3.5% isoflurane gas and imaged. Bioluminescence imaging was performed by an IVIS spectral imaging system (Perkin Elmer) and fluorescence quantitative data was obtained by in vivo imaging software (Perkin Elmer). And the death time of the mice is recorded, or when the mice reach the animal experiment endpoint judgment standard due to tumor load, humane euthanasia treatment is given, the death time is recorded, and the survival rate of the mice is counted.

Example 8: analysis of experimental results

The nanobody phage of example 1 was screened using recombinant human CD7 protein (sine-biological, cat # 11028-H08H) purified in vitro as antigen, phage monoclonal was picked after 4 rounds of screening for ELISA detection, clones from which antigen human CD7 protein was recognized, but not the control protein Bovine Serum Albumin (BSA) were selected, and these clones were sequenced and aligned to remove the repetitive sequences. The ELISA detection results of the partial positive monoclonal are shown in FIG. 1.

Nanobodies specifically recognizing CD7 protein screened by ELISA detection were constructed as CARs, and after the CD 7-targeted CARs reported in the literature (No. PA7-13,) (Chen D, you F, xiang S, et al, chimeric antigen receptor T cells derived from CD nanobody.exhibit robust antitumor potential against CD7-positive magnetic antibodies.Am J Cancer Res.2021;11 (11): 5263-5281.Published 2021 Nov 15) MALPVTALLLPLALLLHAARPEQKLISEEDLMQVQLVESGGGLVQPGGSLRLSCAASGYPYSSYCMGWFRQAPGQGLEAVAAIDSDGRTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARFGPMGCVDLSTLSFGHWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 25) were expressed on the CD7 knocked-out human T cell line jurkat, the expression of the CARs was detected by flow cytometry after staining the cells with surface antigen, and the self-activation level in the resting state of the CARs was detected. The results are shown in figure 2, wherein the nano-antibodies are D2, D4 and E7, and the CAR has high expression level on cell membranes, low background self-activation level and meets the screening standard of people.

CARs (SEQ ID NOS: 12-15) constructed by 3 nanobodies of D2, D4 and E7 and PA7-13 are respectively expressed on CD7 knocked-out human primary T cells, and after staining by cell surface antigens, the expression and coating conditions are detected by flow cytometry, and in a resting state, the background self-activation level and the cell depletion level caused by the self-activation signal are detected. As shown in fig. 3, the CAR-T cells are depleted in advance under the condition that the CAR-T cells are not stimulated by target cells, and the proliferation capacity of the depleted T cells is obviously weakened, and the killing capacity of tumor cells, the cytokine release level and the like are greatly reduced. In the above experiments, the 3 nanobody CARs screened and constructed have lower background self-activation level and lower induced cell depletion level, suggesting that the 3 nanobody CARs may have stronger sustained proliferation capability and tumor killing function.

After CAR constructed by 3 nanobodies of D2, D4 and E7 and PA7-13 are respectively expressed in a human T cell line jurkat for knocking out CD7, the activation level of the CAR constructed by 3 nanobodies is detected by flow cytometry after the CAR constructed by 3 nanobodies of D2, D4 and E7 and PA7-13 are respectively incubated with CD7 positive target cells and CD7 negative non-target cells for 16 hours by different effector-target cell ratios (E: T). As shown in fig. 4, each of the 3 different nanobody CARs constructed in the present invention can effectively recognize CD7 protein on target cells, thereby activating T cells, so that the T cell activation indicator protein CD69 significantly up-regulates expression, while hardly responding to CD7 protein negative target cell stimulation.

CARs constructed by 3 nanobodies of D2, D4 and E7 and PA7-13 are respectively expressed on CD7 knocked-out primary human T cells, and after the CARs and CD7 positive target cells are incubated for 16 hours in different E:T ratios, the firefly luciferase expressed by the target cells consumes substrate luciferin, and the generated fluorescence intensity reflects the killing capacity of the target cells. As can be seen from fig. 5, all 3 kinds of CAR-T cells constructed by the present invention can effectively recognize and kill tumor target cells, and the CAR constructed by the nanobody E7 is the CAR with the lowest background self-excitation level and the most remarkable response to human CD7 antigen, so the present invention will take the CAR constructed by E7 as an example, to further verify its function.

After expressing the E7 nanobody CAR on human primary T cells after knocking out CD7 protein, the killing function against target cells as well as non-target cells was tested. After CAR-T cells and control T cells were incubated with CD 7-positive target cells and CD 7-negative non-target cells, respectively, for 16 hours at different effector-target cell ratios (effector: target, E: T), substrate luciferin (luciferase) of firefly luciferase expressed by the target cells was administered, and the intensity of characteristic fluorescent signals generated by luciferin under the action of firefly luciferase was detected by an enzyme-labeling instrument, thereby reflecting the number of target cells and non-target cells that were present. As shown in fig. 7, the E7CAR can specifically recognize CD7 positive target cells and mediate CAR-T cell specific and efficient killing of tumor cells.

After CAR-T cells and control T cells were incubated with CD7 positive target cells, respectively, for 16 hours at different effector-target cell ratios (effector: target, E: T), culture supernatants were collected and secretion levels of effector cytokine IL-2 and IFN- γ in the culture supernatants were detected by ELISA. As shown in fig. 8, the E7 CAR-T cells can effectively respond to the stimulation of CD7 positive tumor target cells, secrete a large amount of effector cytokines related to T cell proliferation and function, thereby further stimulating a stronger immune response and playing a synergistic role in killing tumors.

The invention uses effector cells in a ratio of 1:1: target cell proportion, wild-type E7 CAR-T cells and ubiquitinated modified deleted E7 CAR-T cells (i.e., CAR modified T cells of SEQ ID NO:15 and CAR modified T cells of SEQ ID NO: 16) were given, a certain amount of irradiated target cells were stimulated, viable cell counts were performed every 2 days, cell proliferation was recorded and appropriate medium was supplemented, cell density was adjusted to 1X 10≡6/ml, and target cell stimulation was repeated every 6 days. The results of this experiment are shown in figure 8, and the ubiquitination modified deleted CAR-T cells, especially in the presence of target cells and in the presence of sustained stimulation, have a stronger and more significant sustained proliferation capacity, suggesting that they may have a stronger, more effective and more sustained tumor killing capacity.

Tumor-bearing mice are randomly divided into 2 groups, and normal T cells and E7 CAR of the equal amount of untransfected CAR are respectively given to the mice by tail vein injection ^KR (ubiquitin-modified deleted) -T cells the growth of tumor cells in mice was followed by a small animal in vivo imaging device (in vivo imaging system, IVIS). As shown in fig. 9, under the condition of reinfusion of equal amount of T cells, T cells not transfected with CAR have almost no effective killing function on tumors, while ubiquitination modified deleted E7 CAR-T cells can effectively control the growth of tumor target cells, and the survival rate of mice is remarkably improved.

While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Sequence listing

<110> Shanghai first people Hospital

<120> a chimeric antigen receptor targeting CD7 protein and uses thereof

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Pro Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Asp Ile Ser Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Lys Pro Trp Pro Pro Phe Gly Pro Tyr Thr Tyr Ala Tyr Asn

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser

115 120 125

<210> 2

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Tyr

20 25 30

Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ser Leu Tyr Pro Asp Gly Tyr Leu Ser Ile Tyr Ser Tyr Asn

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser

115 120 125

<210> 3

<211> 131

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Asn

20 25 30

Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val

35 40 45

Ala Ala Ile Asn Ser Arg Asp Ser Arg Thr Trp Tyr Pro Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Gly Glu Ile Tyr Ser Leu Gly Asp Asp Trp Phe Gly Trp

100 105 110

Gly Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

Gly Gly Ser

130

<210> 4

<211> 42

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 5

<211> 113

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 6

<211> 155

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

35 40 45

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

50 55 60

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

65 70 75 80

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn

85 90 95

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

100 105 110

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

115 120 125

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

130 135 140

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

145 150 155

<210> 7

<211> 155

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Arg Arg Gly Arg Arg Arg Leu Leu Tyr Ile Phe Arg Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Arg Phe Ser Arg

35 40 45

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

50 55 60

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Arg Arg

65 70 75 80

Arg Gly Arg Asp Pro Glu Met Gly Gly Arg Pro Gln Arg Arg Arg Asn

85 90 95

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Arg Asp Arg Met Ala Glu

100 105 110

Ala Tyr Ser Glu Ile Gly Met Arg Gly Glu Arg Arg Arg Gly Arg Gly

115 120 125

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Arg Asp Thr Tyr

130 135 140

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

145 150 155

<210> 8

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 9

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 10

<211> 45

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 11

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr

20

<210> 12

<211> 382

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu

20 25 30

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

35 40 45

Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Pro Tyr Val

50 55 60

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala

65 70 75 80

Asp Ile Ser Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Glu

85 90 95

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr Leu

100 105 110

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Ala Lys Pro Trp Pro Pro Phe Gly Pro Tyr Thr Tyr Ala Tyr Asn Tyr

130 135 140

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Thr Thr

145 150 155 160

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

165 170 175

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

180 185 190

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

195 200 205

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

210 215 220

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

225 230 235 240

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

260 265 270

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

275 280 285

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

290 295 300

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg

305 310 315 320

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

325 330 335

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

340 345 350

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

355 360 365

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

370 375 380

<210> 13

<211> 382

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu

20 25 30

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

35 40 45

Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Tyr Asn

50 55 60

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala

65 70 75 80

Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Glu

85 90 95

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr Leu

100 105 110

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Ala Ser Leu Tyr Pro Asp Gly Tyr Leu Ser Ile Tyr Ser Tyr Asn Tyr

130 135 140

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Thr Thr

145 150 155 160

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

165 170 175

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

180 185 190

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

195 200 205

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

210 215 220

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

225 230 235 240

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

260 265 270

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

275 280 285

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

290 295 300

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg

305 310 315 320

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

325 330 335

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

340 345 350

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

355 360 365

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

370 375 380

<210> 14

<211> 386

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu

20 25 30

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

35 40 45

Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Asn Arg

50 55 60

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala

65 70 75 80

Ala Ile Asn Ser Arg Asp Ser Arg Thr Trp Tyr Pro Asp Ser Val Glu

85 90 95

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr Leu

100 105 110

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Ala Gly Gly Glu Ile Tyr Ser Leu Gly Asp Asp Trp Phe Gly Trp Gly

130 135 140

Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly

145 150 155 160

Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

165 170 175

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

180 185 190

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

195 200 205

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

210 215 220

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

225 230 235 240

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

245 250 255

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

260 265 270

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

275 280 285

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

290 295 300

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

305 310 315 320

Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

325 330 335

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

340 345 350

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

355 360 365

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

370 375 380

Pro Arg

385

<210> 15

<211> 386

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu

20 25 30

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

35 40 45

Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Asn Arg

50 55 60

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala

65 70 75 80

Ala Ile Asn Ser Arg Asp Ser Arg Thr Trp Tyr Pro Asp Ser Val Glu

85 90 95

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr Leu

100 105 110

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Ala Gly Gly Glu Ile Tyr Ser Leu Gly Asp Asp Trp Phe Gly Trp Gly

130 135 140

Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly

145 150 155 160

Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

165 170 175

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

180 185 190

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

195 200 205

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

210 215 220

Leu Val Ile Thr Leu Tyr Cys Arg Arg Gly Arg Arg Arg Leu Leu Tyr

225 230 235 240

Ile Phe Arg Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

245 250 255

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

260 265 270

Leu Arg Val Arg Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

275 280 285

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

290 295 300

Tyr Asp Val Leu Asp Arg Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

305 310 315 320

Arg Pro Gln Arg Arg Arg Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

325 330 335

Gln Arg Asp Arg Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Arg Gly

340 345 350

Glu Arg Arg Arg Gly Arg Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

355 360 365

Thr Ala Thr Arg Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

370 375 380

Pro Arg

385

<210> 16

<211> 382

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu

20 25 30

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

35 40 45

Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Pro Tyr Val

50 55 60

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala

65 70 75 80

Asp Ile Ser Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Glu

85 90 95

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr Leu

100 105 110

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Ala Lys Pro Trp Pro Pro Phe Gly Pro Tyr Thr Tyr Ala Tyr Asn Tyr

130 135 140

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Thr Thr

145 150 155 160

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

165 170 175

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

180 185 190

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

195 200 205

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

210 215 220

Leu Tyr Cys Arg Arg Gly Arg Arg Arg Leu Leu Tyr Ile Phe Arg Gln

225 230 235 240

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Arg

260 265 270

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

275 280 285

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

290 295 300

Asp Arg Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Arg Pro Gln Arg

305 310 315 320

Arg Arg Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Arg Asp Arg

325 330 335

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Arg Gly Glu Arg Arg Arg

340 345 350

Gly Arg Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Arg

355 360 365

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

370 375 380

<210> 17

<211> 382

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 17

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Glu

20 25 30

Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

35 40 45

Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Tyr Asn

50 55 60

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val Ala

65 70 75 80

Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Glu

85 90 95

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr Leu

100 105 110

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Ala Ser Leu Tyr Pro Asp Gly Tyr Leu Ser Ile Tyr Ser Tyr Asn Tyr

130 135 140

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Thr Thr

145 150 155 160

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

165 170 175

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

180 185 190

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

195 200 205

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

210 215 220

Leu Tyr Cys Arg Arg Gly Arg Arg Arg Leu Leu Tyr Ile Phe Arg Gln

225 230 235 240

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

245 250 255

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Arg

260 265 270

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

275 280 285

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

290 295 300

Asp Arg Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Arg Pro Gln Arg

305 310 315 320

Arg Arg Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Arg Asp Arg

325 330 335

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Arg Gly Glu Arg Arg Arg

340 345 350

Gly Arg Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Arg

355 360 365

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

370 375 380

<210> 18

<211> 1146

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaacaga agctgatctc ggaggaggat ctggaggtgc agctggtgga gagcggtggt 120

ggtctcgtgc agcccggcgg tagtctgcgc ctcagctgtg ccgccagcgg tcgcaccttt 180

agcccatatg tgatgggctg gtttcgccaa gcccccggca aaggtcgcga actggtggcc 240

gatattagca gcagcggtgg cagcacctac tacccagata gcgtggaagg ccgcttcacc 300

atcagccgcg ataacgccaa gcgcatggtg tatctgcaga tgaacagtct gcgcgccgag 360

gacaccgccg tgtattattg tgccgccaaa ccatggccac catttggccc atatacctat 420

gcctacaatt actggggcca aggcacccaa gtgaccgtga gcagcggtgg ttctaccacc 480

actcccgcac cccgccctcc tactcctgcc cctaccattg ctagccaacc gcttagtctg 540

agacctgagg cctgtaggcc cgctgctggt ggcgctgtgc acacccgagg attggacttc 600

gcttgcgaca tctacatctg ggcacctctg gctgggacct gcggcgtgtt gttgttgagc 660

ctggtgatta cgctgtactg taaacggggc agaaagaaac tcctgtatat attcaaacaa 720

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 780

gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 840

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 900

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgcagaga 960

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1020

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1080

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgcct 1140

cctcgc 1146

<210> 19

<211> 1146

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaacaga agctgatctc ggaggaggat ctggaggtgc agctggtgga gagcggtggt 120

ggtctcgtgc agcccggcgg tagtctgcgc ctcagctgtg ccgccagcgg tatcaccttt 180

agcacctata acatgggctg gtttcgccaa gcccccggca aaggtcgcga actggtggcc 240

gccatttccc gcacgggcgg tagcacctac tacccagata gcgtggaagg ccgcttcacc 300

atcagccgcg ataacgccaa gcgcatggtg tatctgcaga tgaacagtct gcgcgccgag 360

gacaccgccg tgtattattg tgccgccagc ctgtatccag atggctatct gagcatctat 420

agctacaatt actggggcca aggcacccaa gtgaccgtga gcagcggtgg ttctaccacc 480

actcccgcac cccgccctcc tactcctgcc cctaccattg ctagccaacc gcttagtctg 540

agacctgagg cctgtaggcc cgctgctggt ggcgctgtgc acacccgagg attggacttc 600

gcttgcgaca tctacatctg ggcacctctg gctgggacct gcggcgtgtt gttgttgagc 660

ctggtgatta cgctgtactg taaacggggc agaaagaaac tcctgtatat attcaaacaa 720

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 780

gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 840

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 900

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgcagaga 960

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1020

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1080

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgcct 1140

cctcgc 1146

<210> 20

<211> 1158

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaacaga agctgatctc ggaggaggat ctggaggtgc agctggtgga gagcggtggt 120

ggtctcgtgc agcccggcgg tagtctgcgc ctcagctgtg ccgccagcgg tcgcaccttt 180

agcaccaatc gcatgggctg gtttcgccaa gcccccggca aaggtcgcga actggtggcc 240

gccattaaca gccgcgatag ccgcacctgg tacccagata gcgtggaagg ccgcttcacc 300

atcagccgcg ataacgccaa gcgcatggtg tatctgcaga tgaacagtct gcgcgccgag 360

gacaccgccg tgtattattg tgccgccggc ggcgaaatct atagcctggg cgatgattgg 420

tttggctggg gcccatacga ttactggggc caaggcaccc aagtgaccgt gagcagcggt 480

ggttctacca ccactcccgc accccgccct cctactcctg cccctaccat tgctagccaa 540

ccgcttagtc tgagacctga ggcctgtagg cccgctgctg gtggcgctgt gcacacccga 600

ggattggact tcgcttgcga catctacatc tgggcacctc tggctgggac ctgcggcgtg 660

ttgttgttga gcctggtgat tacgctgtac tgtaaacggg gcagaaagaa actcctgtat 720

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 780

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 840

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 900

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 960

aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1020

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1080

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1140

caggccctgc ctcctcgc 1158

<210> 21

<211> 1158

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaacaga agctgatctc ggaggaggat ctggaggtgc agctggtgga gagcggtggt 120

ggtctcgtgc agcccggcgg tagtctgcgc ctcagctgtg ccgccagcgg tcgcaccttt 180

agcaccaatc gcatgggctg gtttcgccaa gcccccggca aaggtcgcga actggtggcc 240

gccattaaca gccgcgatag ccgcacctgg tacccagata gcgtggaagg ccgcttcacc 300

atcagccgcg ataacgccaa gcgcatggtg tatctgcaga tgaacagtct gcgcgccgag 360

gacaccgccg tgtattattg tgccgccggc ggcgaaatct atagcctggg cgatgattgg 420

tttggctggg gcccatacga ttactggggc caaggcaccc aagtgaccgt gagcagcggt 480

ggttctacca ccactcccgc accccgccct cctactcctg cccctaccat tgctagccaa 540

ccgcttagtc tgagacctga ggcctgtagg cccgctgctg gtggcgctgt gcacacccga 600

ggattggact tcgcttgcga catctacatc tgggcacctc tggctgggac ctgcggcgtg 660

ttgttgttga gcctggtgat tacgctgtac tgtagacggg gcagacgcag actcctgtat 720

atattccgcc aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 780

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgagatt cagcaggagc 840

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 900

cgaagagagg agtacgatgt tttggaccgc agacgtggcc gggaccctga gatgggggga 960

agaccgcaga gaaggcgcaa ccctcaggaa ggcctgtaca atgaactgca gcgcgataga 1020

atggcggagg cctacagtga gattgggatg agaggcgagc gccggagggg cagagggcac 1080

gatggccttt accagggtct cagtacagcc acccgcgaca cctacgacgc ccttcacatg 1140

caggccctgc ctcctcgc 1158

<210> 22

<211> 1146

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaacaga agctgatctc ggaggaggat ctggaggtgc agctggtgga gagcggtggt 120

ggtctcgtgc agcccggcgg tagtctgcgc ctcagctgtg ccgccagcgg tcgcaccttt 180

agcccatatg tgatgggctg gtttcgccaa gcccccggca aaggtcgcga actggtggcc 240

gatattagca gcagcggtgg cagcacctac tacccagata gcgtggaagg ccgcttcacc 300

atcagccgcg ataacgccaa gcgcatggtg tatctgcaga tgaacagtct gcgcgccgag 360

gacaccgccg tgtattattg tgccgccaaa ccatggccac catttggccc atatacctat 420

gcctacaatt actggggcca aggcacccaa gtgaccgtga gcagcggtgg ttctaccacc 480

actcccgcac cccgccctcc tactcctgcc cctaccattg ctagccaacc gcttagtctg 540

agacctgagg cctgtaggcc cgctgctggt ggcgctgtgc acacccgagg attggacttc 600

gcttgcgaca tctacatctg ggcacctctg gctgggacct gcggcgtgtt gttgttgagc 660

ctggtgatta cgctgtactg tagacggggc agacgcagac tcctgtatat attccgccaa 720

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 780

gaagaagaag aaggaggatg tgaactgaga gtgagattca gcaggagcgc agacgccccc 840

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 900

tacgatgttt tggaccgcag acgtggccgg gaccctgaga tggggggaag accgcagaga 960

aggcgcaacc ctcaggaagg cctgtacaat gaactgcagc gcgatagaat ggcggaggcc 1020

tacagtgaga ttgggatgag aggcgagcgc cggaggggca gagggcacga tggcctttac 1080

cagggtctca gtacagccac ccgcgacacc tacgacgccc ttcacatgca ggccctgcct 1140

cctcgc 1146

<210> 23

<211> 1146

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaacaga agctgatctc ggaggaggat ctggaggtgc agctggtgga gagcggtggt 120

ggtctcgtgc agcccggcgg tagtctgcgc ctcagctgtg ccgccagcgg tatcaccttt 180

agcacctata acatgggctg gtttcgccaa gcccccggca aaggtcgcga actggtggcc 240

gccatttccc gcacgggcgg tagcacctac tacccagata gcgtggaagg ccgcttcacc 300

atcagccgcg ataacgccaa gcgcatggtg tatctgcaga tgaacagtct gcgcgccgag 360

gacaccgccg tgtattattg tgccgccagc ctgtatccag atggctatct gagcatctat 420

agctacaatt actggggcca aggcacccaa gtgaccgtga gcagcggtgg ttctaccacc 480

actcccgcac cccgccctcc tactcctgcc cctaccattg ctagccaacc gcttagtctg 540

agacctgagg cctgtaggcc cgctgctggt ggcgctgtgc acacccgagg attggacttc 600

gcttgcgaca tctacatctg ggcacctctg gctgggacct gcggcgtgtt gttgttgagc 660

ctggtgatta cgctgtactg tagacggggc agacgcagac tcctgtatat attccgccaa 720

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 780

gaagaagaag aaggaggatg tgaactgaga gtgagattca gcaggagcgc agacgccccc 840

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 900

tacgatgttt tggaccgcag acgtggccgg gaccctgaga tggggggaag accgcagaga 960

aggcgcaacc ctcaggaagg cctgtacaat gaactgcagc gcgatagaat ggcggaggcc 1020

tacagtgaga ttgggatgag aggcgagcgc cggaggggca gagggcacga tggcctttac 1080

cagggtctca gtacagccac ccgcgacacc tacgacgccc ttcacatgca ggccctgcct 1140

cctcgc 1146

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

ggagcaggtg atgttgacgg 20

<210> 25

<211> 381

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Met

20 25 30

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

35 40 45

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Pro Tyr Ser Ser Tyr

50 55 60

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val

65 70 75 80

Ala Ala Ile Asp Ser Asp Gly Arg Thr Arg Tyr Ala Asp Ser Val Lys

85 90 95

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

100 105 110

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

115 120 125

Ala Arg Phe Gly Pro Met Gly Cys Val Asp Leu Ser Thr Leu Ser Phe

130 135 140

Gly His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr

145 150 155 160

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

165 170 175

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

180 185 190

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

195 200 205

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

210 215 220

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

225 230 235 240

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

245 250 255

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

260 265 270

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

275 280 285

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

290 295 300

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg

305 310 315 320

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

325 330 335

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

340 345 350

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

355 360 365

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

370 375 380

Claims (16)

1. A chimeric antigen receptor that targets a CD7 protein, comprising:

an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence;

the extracellular domain comprises an antigen recognition region, which is a nanobody targeting CD7 protein; the amino acid sequence of the nano antibody targeting the CD7 protein is shown as SEQ ID NO:1-SEQ ID NO:3, respectively.

2. The chimeric antigen receptor targeted to a CD7 protein of claim 1, wherein the intracellular domain comprises a costimulatory signaling region and a cd3ζ intracellular region, which are sequentially connected, forming a costimulatory signaling region-cd3ζ intracellular region.

3. The CD7 protein-targeting chimeric antigen receptor according to claim 2, further comprising one or more of the following features:

a. the costimulatory signaling region is selected from the 41BB intracellular region;

b. the amino acid sequence of the CD3 zeta intracellular region is shown as SEQ ID NO: shown at 5.

4. A chimeric antigen receptor targeting a CD7 protein according to claim 3, wherein in feature a the amino acid sequence of the 41BB intracellular domain is set forth in SEQ ID NO: 4.

5. The chimeric antigen receptor targeted to CD7 protein according to claim 2, wherein the costimulatory signaling region-cd3ζ intracellular domain has the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO: shown at 7.

6. The CD7 protein-targeting chimeric antigen receptor according to claim 1, further comprising one or more of the following features:

a. the extracellular domain further comprises a signal peptide and/or a hinge region;

b. the transmembrane domain is selected from the transmembrane region of CD4, CD8 alpha, OX40 or H2-Kb.

7. The chimeric antigen receptor targeted to CD7 protein according to claim 6, wherein the signal peptide is selected from the group consisting of CD8 a signal peptide and/or the hinge region is selected from the group consisting of the hinge region of CD8 a.

8. The chimeric antigen receptor targeted to CD7 protein according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is as set forth in SEQ ID NO: 12-17.

9. A polynucleotide sequence selected from the group consisting of:

(1) A polynucleotide sequence encoding a CD7 protein-targeting chimeric antigen receptor according to any one of claims 1-8; and/or

(2) The complement of the polynucleotide sequence of (1).

10. The polynucleotide sequence of claim 9, wherein said polynucleotide sequence is set forth in SEQ ID NO: 18-23.

11. A nucleic acid construct comprising the polynucleotide sequence of any one of claims 9-10; preferably, the nucleic acid construct is a vector; more preferably, the nucleic acid construct is a lentiviral vector comprising a replication origin, a 3'LTR, a 5' LTR and a polynucleotide sequence according to any of claims 9 to 10.

12. A lentivirus comprising the nucleic acid construct of claim 11.

13. A method of ex vivo activating T cells comprising the step of infecting said T cells with the lentivirus of claim 12.

14. A genetically modified T cell or a pharmaceutical composition comprising the genetically modified T cell, wherein the cell comprises the polynucleotide sequence of any one of claims 9 to 10, or comprises the nucleic acid construct of claim 11, or is infected with the lentivirus of claim 12, or is prepared by the method of claim 13.

15. Use of the chimeric antigen receptor of any one of claims 1-8, the polynucleotide sequence of any one of claims 9-10, the nucleic acid construct of claim 11 or the lentivirus of claim 12 in the preparation of a T cell activator and/or in the preparation of a T cell degradation inhibitor.

16. Use of the chimeric antigen receptor of any one of claims 1-8, the polynucleotide sequence of any one of claims 9-10, the nucleic acid construct of claim 11, the lentivirus of claim 12, or the genetically modified T cell of claim 14 in any one or more of the following: (1) preparing a tumor therapeutic drug; (2) preparing a formulation that increases the killing efficiency of the tumor; (3) preparing a preparation for maintaining proliferation capacity of T cells; (4) preparing a preparation for inhibiting tumor development; preferably, the tumour is selected from one or more of leukaemia or lymphoma; more preferably, the tumor is selected from the group consisting of T cell lymphoma, T cell type acute lymphoblastic leukemia, B cell type acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, and acute myelogenous leukemia.