CN111718957A

CN111718957A - Chimeric antigen receptor recombinant adeno-associated virus particle and application thereof

Info

Publication number: CN111718957A
Application number: CN201910224056.8A
Authority: CN
Inventors: 吴喜林; 吴稚伟; 瓦卡斯; 黄碧莲
Original assignee: Nanjing Anrui Biotechnology Co ltd
Current assignee: Y-CLONE MEDICAL SCIENCE Co.,Ltd.
Priority date: 2019-03-22
Filing date: 2019-03-22
Publication date: 2020-09-29
Also published as: WO2020192803A1

Abstract

The invention relates to a chimeric antigen receptor recombinant adeno-associated virus particle, which is a recombinant adeno-associated virus particle carrying a chimeric antigen receptor gene. The invention directly injects the recombinant adeno-associated virus particles loaded with the chimeric antigen receptor into a body, the chimeric antigen receptor recombinant adeno-associated virus particles directly infect T cells or NK cells in the body, and CAR cells are directly formed in the body, thereby realizing cell therapy of direct target cell killing.

Description

Chimeric antigen receptor recombinant adeno-associated virus particle and application thereof

Technical Field

The invention relates to the technical field of chimeric antigen receptors, in particular to a chimeric antigen receptor recombinant adeno-associated virus particle and application thereof.

Background

Chimeric Antigen Receptor (CAR) modified T cell (CAR-T) immunotherapy is a new type of cell therapy that has been improved to be used clinically in recent years. CAR-T cell immunotherapy T cells are isolated by collecting peripheral venous blood from a patient, extensively expanded under induction by various cytokines, and introduced with CAR molecules that target tumor antigens. The modification of the CAR molecule can ensure that the tumor killing effect of the T cell is not limited by a Major Histocompatibility Complex (MHC) when the T cell obtains the targeted killing capability, and finally the CAR-T cell is infused back into a patient body by means of intravenous injection or intradermal injection and the like to achieve the purpose of killing the tumor. The CAR-T cell has continuous amplification capacity and tumor targeted killing activity, and can ensure that the CAR-T cell can continuously and effectively kill tumor cells, so that patients can be cured. CAR-T cell therapy is currently of significant efficacy in the treatment of acute leukemias and non-hodgkin's lymphoma, and CAR-T cell immunotherapy is considered to be one of the most promising approaches to tumor therapy.

However, some patients die during the genetic modification and culture of T cells due to the disease progressing too rapidly, thereby losing the opportunity for CAR-T cell therapy; in addition, autologous T cell in-vitro expansion is utilized to prepare autologous CAR-T reinfusion for personalized treatment, so that the time and cost of CAR-T treatment are greatly increased, and the scale of CAR-T treatment is also limited. Therefore, there is an urgent need to develop universal CAR-T cells for clinical use or to obtain similar drugs that can be directly infused into the human body to achieve CAR-T cell therapy.

The CAR molecule needs to be transduced with a killer T cell by a viral vector, including retroviral systems, lentiviral systems, adenovirus, DNA plasmid, and RNA transfection systems. At present, the CAR-T cell can achieve stable integration in the treatment, has high transfection efficiency, and is widely used by retroviruses and lentiviruses. Retroviruses do not readily transfect resting T cells; lentiviruses can transfect resting T cells, but are costly, and both are genotoxic and risk of insertion carcinogenesis, so there is an urgent need for a safer and more effective viral vector to load CAR molecules. Adeno-associated virus (AAV) vectors are widely used for gene expression and in vivo expression of antibodies, and since AAV viral vectors exist in the cytoplasm in the form of episomes, there is no insertion mutation and they can be expressed for a long time.

Disclosure of Invention

The invention provides a chimeric antigen receptor recombinant adeno-associated virus particle and application thereof for solving the technical problems.

The technical scheme for solving the technical problems is as follows: a chimeric antigen receptor recombinant adeno-associated virus particle is a recombinant adeno-associated virus vector carrying a chimeric antigen receptor gene.

Further, the chimeric antigen receptor recombinant adeno-associated virus particle is obtained by packaging the chimeric antigen receptor gene through an adeno-associated virus packaging system.

Further, the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

Further, the extracellular domain is an antibody that recognizes the antigenic domain.

Further, the antibody recognizing the antigen domain is an scFV antibody or V_HH antibody.

Further, the scFV antibody is specific for CD4, CD19, CCR5 or CD 20.

The invention also provides the application of the chimeric antigen receptor recombinant adeno-associated virus particles in preparing a medicament for treating tumors.

Furthermore, the medicine is a targeted medicine applied to tumor immunotherapy.

The invention also provides a pharmaceutical composition, which comprises the chimeric antigen receptor recombinant adeno-associated virus particles and a pharmaceutically acceptable carrier or excipient.

Further, the dosage form of the pharmaceutical composition is injection.

The invention has the beneficial effects that: the invention utilizes the chimeric antigen receptor recombinant adeno-associated virus particles (AAV-CAR) obtained by loading the chimeric antigen receptor (CAR molecule) with the recombinant adeno-associated virus (AAV) to be directly injected into a body, the AAV-CAR directly infects T cells or NK cells in the body, and CAR cells are directly formed in the body, thereby realizing the cell therapy of direct target cell killing. AAV viruses (AAV-CD4-CAR, AAV-CCR5-CAR, AAV-CD19-CAR and AAV-CD20-CAR) loaded with different CAR molecules were successfully prepared in the present invention, and AAV-CAR was capable of directly infecting 293TT adherent cells and 293F suspension cells and displaying CAR molecules on the cell surface. In addition, AAV-CAR can directly infect PBMC cells and display CAR molecules on the surface of more than 40% of T cells to form CAR-T cells. The CAR-T cell can specifically kill target cells, and the killing efficiency can reach more than 40%; AAV viruses prepared to be loaded with the CAR molecule are injected directly into mice, and after several days, the detection shows that a large number of CAR-T cells are formed in the mice and the specific killing of target cells is shown. The AAV-CAR can be directly injected in vivo, a sequence of steps of blood collection, PBMC in vitro amplification, virus in vitro infected T cells, CAR-T self-feedback and other individualized treatments required by the traditional CAR-T cell treatment are directly skipped, only chimeric antigen receptor recombinant adeno-associated virus particles (AAV-CAR) are directly injected into a patient, AAV-CAR general batch cell treatment is realized, and the preparation time at the early stage of treatment, the treatment cost and the treatment efficiency are greatly saved.

Drawings

FIG. 1 is a Western-blotting result of cell lysates obtained after AAV-CD4-CAR in example 3 of the present invention has infected 293TT adherent cells and 293F suspension cells, respectively, wherein M is marker, lane 1 is cell lysate obtained after AAV-CD4-CAR infection 293F cells, lane 2 is cell lysate obtained after AAV-CD4-CAR infection 293F cells, and lane 3 is a control group;

FIG. 2 is a flow cytometer of example 3 of the present invention to identify cell surface CAR molecule expression after AAV-CD4-CAR infection of cells, wherein AAV-CD4-CAR transfection group with high dose is AAV-CD4-CAR (H), AAV-CD4-CAR transfection group with low dose is AAV-CD4-CAR (L), AAV-GFP transfection group is Mock;

FIG. 3 is CD3+ of AAV-CD4-CAR infected PBMC identified by flow cytometry in example 3 of the present inventionExpression of cell surface CAR molecules with Anti-F (ab')₂-FITC and Anti-CD4-Fc-FITC assay, wherein the high dose AAV-CD4-CAR transfection group is AAV-CD4-CAR (h), the low dose AAV-CD4-CAR transfection group is AAV-CD4-CAR (l), and the AAV-GFP transfection group is Mock;

FIG. 4 is a graph of specific killing of CD4+ T cells following direct infection of PBMCs with AAV-CD4-CAR by flow detection in example 4 of the present invention, wherein FIG. 4A is a schematic diagram of flow analysis and FIG. 4B is a proportion of CD4+ T cells following AAV-CD4-CAR treatment of PBMCs of different origins; FIG. 4C is a graph showing the proportion of CD8+ T cells normalized to the value of the control AAV-GFP treated group, after AAV-CD4-CAR treatment of PBMCs from different sources, set at 100%;

FIG. 5 is a schematic diagram of flow analysis of AAV-CD4-CAR flow-detection of tumor cell killing in example 4, FIG. 5A is a schematic diagram of flow analysis, FIG. 5B is a schematic diagram of the killing ratio of AAV-CD4-CAR directly infected with PBMC cells to MT-2 and Jurkat tumor cells, the non-specific killing ratio of Mock tumor cells is normalized to 0, and the AAV-CD 4-CAR-treated specific cell killing data is obtained by normalization of a reference control group;

FIG. 6 shows the identification of AAV-CD4-CAR virus-induced specific targeted killing in humanized mice of example 5 of the present invention, FIG. 6A shows the actual ratio of CD3+ CD4+ T cells in humanized mice before AV-CD4-CAR virus treatment, FIG. 6B shows the expression of CAR molecules specifically recognizing CD4 protein on the surface of human CD3+ T cells 2 weeks after AAV-CD4-CAR direct infection in humanized mice, FIG. 6C shows the ratio of CD3+ CD4+ T cells in humanized mice at different time points in flow assay, and FIG. 6D shows the monitoring of the body weight of humanized mice in different treatment groups.

Detailed Description

The principles and features of this invention are described in connection with the drawings and the detailed description of the invention, which are set forth below as examples to illustrate the invention and not to limit the scope of the invention.

In the previous work, the inventor finds that adeno-associated virus (AAV) can be expressed in vivo for at least more than 9 weeks, and in addition, the clinical trials of the AAV vectors show that 178 AAV vector-related clinical trials are available, so that no serious side effects are reported so far, and therefore, the AAV vectors are selected to load Chimeric Antigen Receptors (CAR) to obtain the chimeric antigen receptor recombinant adeno-associated virus particles (AAV-CAR).

Example 1 Loading of CAR molecules with AAV viral vectors

1. pAAV-CAR plasmid construction

scFv (VL-VH) antibody sequences specific for CD19, CD4, CCR5 and CD20 and hinge, transmembrane and intracellular signaling domain sequences of third generation CARs were obtained by PubMed and HIV database website. Wherein

The amino acid sequence of the scFv (VL-VH) antibody of CD19 is shown in SEQ ID NO: 1: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFSSSWIGWVRQMPGKGLEWMGIIYPDDSDTRYSPSFQGQVTISADKSIRTAYLQWSSLKASDTAMYYCARHVTMIWGVIIDFWGQGTLVTVSS

Wherein the amino acids 1-107 of the sequence SEQ ID NO:1 are the light chain variable region, the amino acids 108-122 are the connecting peptide, and the amino acids 123-243 are the heavy chain variable region.

The amino acid sequence of the scFv (VL-VH) antibody of CD4 is shown in SEQ ID NO: 2:

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWANSTESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS

wherein the amino acids 1-112 of the sequence SEQ ID NO. 2 are the light chain variable region, the amino acids 113-127 are the connecting peptide, and the amino acids 128-249 are the heavy chain variable region.

The amino acid sequence of the scFv (VL-VH) antibody of CCR5 is shown in SEQ ID NO: 3:

ISCRSSQRLLSSYGHTYLHWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGYTFSNYWIGWVRQAPGKGLEWIGDIYPGGNYIRNNEKFKDKTTLSADTSKNTAYLQMNSLKTEDTAVYYCGSSFGSNYVFAWFTYWGQGTLVTVSS

wherein the amino acids 1-92 of the sequence SEQ ID NO. 3 are the light chain variable region, the amino acids 93-107 are the connecting peptide, and the amino acids 108-229 are the heavy chain variable region.

The amino acid sequence of the scFv (VL-VH) antibody of CD20 is shown in SEQ ID NO:4

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA

Wherein the amino acids 1-107 of the sequence SEQ ID NO. 4 are the light chain variable region, the amino acids 108-122 are the connecting peptide, and the amino acids 123-243 are the heavy chain variable region.

The choice of hinge region, transmembrane region and intracellular signaling domain of the CAR is a matter of routine choice in the art, and the structures of the CAR hinge region, transmembrane domain and intracellular signaling domain of this example are: CD28-4-1BB-CD3zeta, the nucleotide sequence of which is shown in SEQ ID NO: 5:

ATCGAGGTGATGTACCCCCCCCCCTACCTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTCATCATCTTCTGGGTGCGCAGCAAGCGCAGCCGCGGCGGCCACAGCGACTACATGAACATGACCCCCCGCCGCCCCGGCCCCACCCGCAAGCACTACCAGCCCTACGCCCCCCCCCGCGACTTCGCCGCCTACCGCAGCGGCGGTGGCGGCAGCAAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCGCTTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGGAGGCGGTGGCAGCCGCGTGAAGTTCAGCCGCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGCGACTACAAGATGACCACCATCAGC

nucleotide sequences encoding scFv (VL-VH) antibody of CD19 shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, scFv (VL-VH) antibody of CD4, scFv (VL-VH) antibody of CCR5 and scFv (VL-VH) antibody of CD20 were artificially synthesized, and the nucleotide sequences thereof were shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

SEQ ID NO:6

GCCATCCAGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCAGCGCCCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTTCAACAGCTACCCCTACACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGCTTCAGCAGCAGCTGGATCGGCTGGGTGCGCCAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTACCCCGACGACAGCGACACCCGCTACAGCCCCAGCTTCCAGGGCCAGGTGACCATCAGCGCCGACAAGAGCATCCGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGCGCCCGCCACGTGACCATGATCTGGGGCGTGATCATCGACTTCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC

SEQ ID NO:7

GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGTGACCATGAACTGCAAGAGCAGCCAGAGCCTGCTGTACAGCACCAACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAACAGCACCGAGAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTACTACAGCTACCGCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGTGGCGGAGGTTCTGGAGGAGGTGGAAGCGGAGGTGGCGGATCTCAGGTGCAGCTGCAGCAGAGCGGCCCCGAGGTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACGTGATCCACTGGGTGCGCCAGAAGCCCGGCCAGGGCCTGGACTGGATCGGCTACATCAACCCCTACAACGACGGCACCGACTACGACGAGAAGTTCAAGGGCAAGGCCACCCTGACCAGCGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGCGAGAAGGACAACTACGCCACCGGCGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC

SEQ ID NO:8

GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCCGCAGCAGCCAGCGCCTGCTGAGCAGCTACGGCCACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACGAGGTGAGCAACCGCTTCAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCAGCCAGAGCACCCACGTGCCCCTGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTACACCTTCAGCAACTACTGGATCGGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCGACATCTACCCCGGCGGCAACTACATCCGCAACAACGAGAAGTTCAAGGACAAGACCACCCTGAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCGGCAGCAGCTTCGGCAGCAACTACGTGTTCGCCTGGTTCACCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC

SEQ ID NO:9

CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATCCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACTTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGTGGTGGCGGAGGTTCTGGAGGAGGTGGAAGCGGAGGTGGCGGATCTCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCA

The scFv (VL-VH) antibody gene shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and the CD28-4-1BB-CD3zeta gene were enzymatically ligated to pAAV-MCS plasmid vector (Cell Biolabs, San Diego, USA), and finally a single-gene CAR molecule of scFv (VL-VH) -CD28-4-1BB-CD3zeta was loaded on the pAAV-MCS plasmid vector to construct pAAV-CAR (pAAV-CD 19-CAR, pAAV-CD4-CAR, and pAAV-CCR 5-and pAAV-CD20-CAR, respectively).

2. AAV-CAR viral packaging

AAV Helper-Free viral packaging system was purchased from Cell Biolabs, San Diego USA. The above pAAV-CAR was co-transfected with pHelper and pAAV-DJ plasmids using PEI transfection reagent at a mass ratio of 1:1:1 with AAV-293T cells. Supernatants were collected at 48, 72, 96 and 120 hours post transfection and concentrated with 5x PEG8000(sigma), and finally purified by cesium chloride density gradient centrifugation at 1.37g/mL to give AAV-CAR virions loaded with different CAR molecules, i.e., chimeric antigen receptor recombinant adeno-associated virions (AAV-CAR), including AAV-CD4-CAR, AAV-CD19-CAR, AAV-CCR5-CAR, and AAV-CD20-CAR, which were dissolved in PBS, identified and stored at-80 ℃ after packaging.

Example 2 AAV-CAR Virus Titer quantification

The copy number of the purified AAV-CAR was determined by Q-PCR, DNaseI was used to pre-digest the purified AAV-CAR, primers for CMV enhancer (AAV-Mono-CMV-F and AAV-Mono-CMV-R) were used to determine AAV-CAR virus copies, and other reagents were derived from SYBR Premix Ex Taq II (Takara) kit. Samples were tested in triplicate wells on a 7300 instrument. The real-time PCR was performed under the following cycle conditions of 2min at 50 ℃ for one cycle, 10min at 95 ℃ for one cycle, and 40 cycles of 15s at 95 ℃ and 60s at 60 ℃. The copy number of the virus is calculated by a standard curve made by the AAV-GFP plasmid, and finally the AAV-CAR virus with the virus size of 1x10^13gc/mL is obtained.

Wherein the sequence of AAV-Mono-CMV-F is shown in SEQ ID NO: 10:

CCATTGACGTCAATGGGTGGAGT

the sequence of AAV-Mono-CMV-R is shown in SEQ ID NO: 11:

GCCAAGTAGGAAAGTCCCATAAGG

EXAMPLE 3 AAV-CAR infected cells

AAV-CAR virus solutions with different concentrations are directly added into different cells (293TT cells, 293F cells and PBMC cells), and after 24h of culture, expression of CAR molecules is detected by Western-Blot, flow detection and the like.

In the case of AAV-CD4-CAR viral particles specific for CD4 protein infecting 293TT adherent cells and 293F suspension cells, the control group used AAV-GFP viral particles to infect 293TT cells, after infection for 24h, the cells were harvested and lysed, and the expression of CAR molecules in cell lysates was detected by Western-Blot using murine anti-human CD3zeta antibody, and the results are shown in FIG. 1, where the cell lysates of TT cells and 293F cells infected with AAV-CD4-CAR viral particles each had a specific band of 74kD size, and the control group did not have this band, indicating that AAV-CD4-CAR successfully infected 293TT cells and 293F cells, and expressed CD4-CAR molecules in large amounts in these two cells.

To further verify whether the CAR molecule was expressed on the cell surface after AAV-CD4-CAR infection of adherent and suspension cells, 293TT adherent and 293F suspension cells were infected with low dose AAV-CD4-CAR (L) (1x10^4gc/cell) and high dose AAV-CD4-CAR (H) (1x10^5gc/cell), respectively, and control group (Mock) used AAV-GFP, Anti-F (ab')₂The expression of CAR molecules on the cell surface after infection of 293TT adherent cells and 293F suspension cells was detected by flow cytometry using antibodies against FITC, and as a result, as shown in fig. 2, more than 20% of positive cells expressing CAR molecules were detected on the surface of 293TT cells and 293F cells infected with AAV-CD4-CAR (l); in the high dose AAV-CD4-CAR (H) -treated group, greater than 50% of 293TT and 293F cells expressed CAR molecules on the surface, indicating that AAV-CD4-CAR can directly infect adherent cells and suspension cells and display CAR molecules on the cell surface.

To further verify whether AAV-CD4-CAR can infect PBMC cells and display the CAR molecule on the surface of CD3+ T cells, resulting in CAR-T cells, PBMC cells were infected with low dose AAV-CD4-CAR (L) (1x10^4gc/cell) and high dose AAV-CD4-CAR (H) (1x10^5gc/cell), respectively, and control group (Mock) used AAV-GFP, Anti-F (ab')₂FITC antibody flow cytometric PBMC cell surface CAR molecule expression, results are shown in FIG. 3, low dose AAV-CD4-CAR (L) enables 28% of T cells to form CAR-T, and high dose AAV-CD4-CAR (H) infection of PBMC enables 49% of T cells to become CAR-T. To further verify that the CAR-T cells formed by AAV-CD4-CAR infected PBMCs recognized CD4 protein specifically, flow assays were performed using Anti-CD4-Fc-FITC, flow results are shown in fig. 3, and CD3+ T cells in PBMCs directly infected with AAV-CD4-CAR bound CD4 protein specifically; the positive rates of CD3+ T cells recognizing CD4 protein of the high and low dose AAV-CD4-CAR treated group were 75% and 31%, respectively, and the positive rate data are compared with Anti-F (ab')₂Detection of antibody by FITC approximateSimilarly. It was suggested that AAV-CD4-CAR directly infected PBMC were able to form high proportion of CAR-T cells, and that these CAR-T cells specifically recognized CD4 protein.

Example 4 AAV-CAR in vitro cell functional characterization

The successful construction of AAV-CAR cell killing experiments of CAR-T cells was performed according to the protocol of the radioisotopic-free-based CytoTox-Glo kit (Promega). The kit detects killed cells by detecting protease activity. The experimental procedure is briefly described as follows: AAV-CAR transduced PBMCs were diluted 2-fold in a gradient in 96-well plates with the highest cell number of 2.0x10 per well⁶The AAV-CAR PBMC cells: target cells were added at a ratio of 50:1 to the corresponding volume of target cells (target cells could be tumor cells expressing CD4, CD19, CD20 or CCR5, and could also be CD4+ T cells and CD19+ B cells). Cells at 37 ℃ 5% CO₂Incubating for 4 hours; CytoTox-Glo reagent was added to each well and after 15 minutes at room temperature, fluorescence was measured by the instrument; the reading of cell lysates containing target cells but not responding to CAR-T cells was taken as 100% lytic killing.

AAV-CD4-CAR was infected with PBMCs of different origins, 48 hours later, the ratio of CD3+ CD4+ T cells in the PBMCs was measured, and the flow-through results are schematically shown in FIG. 4A. CD3+ CD4+ CD8-T is a CD4+ T cell. Flow cytometry results for CD4+ T cells as shown in figure 4B, CD4+ T cells were significantly reduced in AAV-CD4-CAR treated PBMC cells compared to the negative control and were concentration gradient dependent, with the number of CD4+ T cells treated with high dose AAV-CD4-CAR being lower than the number of CD4+ T cells in the low dose AAV-CD4-CAR treated group; suggesting that the higher the AAV-CD4-CAR dose, the stronger the specific killing ability to CD4+ T; after 6 PBMC cells of different sources are directly infected by the AAV-CD4-CAR at high dose, the reduction rate of CD4+ T cells is about 50%, which indicates that the PBMC directly infected by the AAV-CD4-CAR can specifically target and kill CD4+ T cells in different individuals, and presents dose dependence, the flow cytometry result of the CD8+ T cells is shown in figure 4C, the CD8+ T cells in the PBMC of 6 different sources are not only reduced, but also the CD8+ T cells of individual PBMC cells have a rising trend, and the results indicate that the PBMC directly infected by the AAV-CD4-CAR can specifically kill the CD4+ T cells, but not nonspecifically kill the CD8+ T cells.

To further verify that AAV-CD4-CAR can specifically kill CD4+ tumor cells after directly infecting PBMC, different doses of AAV-CD4-CAR (high dose AAV-CD4-CAR group (H) and low dose AAV-CD4-CAR group (L)) were directly infected with PBMC cells, and AAV-GFP was used in the control group (Mock group), and CD4+ MT2 or CD4+ Jurkat tumor cells were added at a cell ratio of 1: 13 days later; specific dead MT-2 cells or Jurkat were detected by flow assay after 48 hours of co-incubation; flow results are shown in fig. 5, compared with a control group, the AAV-CD4-CAR directly infected PBMC can significantly kill CD4+ MT2 cells and Jurkat cells specifically, and the proportion of specific killing is 25% by subtracting non-specific killing, when the dosage of AAV-CD4-CAR infected PBMC is increased, the proportion of specific killing of MT2 cells and Jurkat cells is increased, the highest specific MT2 cell killing proportion reaches more than 40%, and the above results show that after AAV-CD4-CAR directly infects PBMC, CAR-T which specifically recognizes CD4 protein can be formed, so that CD4 tumor cells are specifically targeted and killed, and efficient specific targeted killing is achieved in a dosage-dependent manner.

Example 5 evaluation of AAV-CCR for in vivo Effect

1. Construction of tumor model of humanized mouse

Cg-prkdcsccill 2rgtm1Wjl/szj (ncg) mice were purchased from university of tokyo model animals and, similar to NSG mice, were deficient in the IL2 receptor gene on a SCID mouse basis, resulting in the absence of mouse T cells, B cells and very few NK cells in vivo. 1.5x10⁷PBMC were injected intraperitoneally into NCG mice for 4-6 weeks; after three weeks, human T cells were collected and flow-tested, and the mice were judged to be successfully humanized by staining human CD45+, CD3+, CD4+, CD8+, and the proportion of human CD45 positive cells was 10% or more. Results all humanized mice contained greater than 10% human CD45+ cells, with actual ratios of human CD4+ T cells in mice as shown in figure 6A, with ratios of human CD4+ T cells as high as 20% or more, inoculated with tumor cells, and monitored for tumor size four weeks in succession.

2. Humanized mouse in vivo verification AAV-CD4-CAR therapeutic effect on tumor

AAV-CAR with different concentrations is injected into the humanized mouse body inoculated with the tumor in the abdominal cavity, and blood sampling detection is carried out at different time points; detecting the change trend of human CD4+ T cells by a flow cytometer; the body weight of the mice was also monitored.

Detecting the expression of the CD3+ T cell surface CAR molecule in the humanized mouse after 1, 2 and 4 weeks by directly injecting AAV-CD4-CAR virus into the humanized mouse through an abdominal cavity; flow results as shown in figure 6B, greater than 12% of the CD3+ T cells in the humanized mice of the AAV-CD4-CAR treated group expressed CAR molecules on the surface, while the proportion of CD3+ T cells expressing CAR molecules specifically recognizing CD4 protein in the humanized mice of the control AAV-CD20-CAR treated group was less than 2%. It can be seen that the direct injection of AAV-CD4-CAR virus can change more than 12% of CD3+ T cells in the humanized mice into CD4-CAR-T cells specifically recognizing CD4 protein, while the control AAV-CD20-CAR can not form CD4-CAR-T cells specifically recognizing CD4 protein in vivo.

To further validate whether CAR-T cells formed after direct infection of mice with AAV-CD4-CAR could specifically kill human CD4+ T cells in vivo, the human CD4+ T cell ratio was examined at different time points. Flow assay results as shown in fig. 6C, all mice in the AAV-CD4-CAR treated group showed a decrease in CD4+ T after 1 week of treatment, a decrease in human CD4+ T cells in mice of greater than 50% after 2 weeks, a further decrease in CD4+ T cells after 4 weeks, and no significant decrease in CD4+ T cells in the AAV-CD20-CAR control group, compared to the control group.

The results indicate that by directly injecting AAV-CD4-CAR virus into humanized mice, CAR-T cells which specifically recognize CD4 protein with high proportion can be directly formed, and the effect of highly efficient target killing of CD4+ target cells can be achieved. By monitoring the body weight changes at different time points after AAV-CAR treatment, the results are shown in FIG. 6D, and the body weight of mice directly infected with AAV-CD4-CAR virus did not significantly decrease, indicating that viral infection with AAV-CD4-CAR did not cause significant side effects on mice.

In conclusion, the invention successfully constructs a series of AAV-CAR viruses specifically targeting molecules such as CD4, CCR5, CD19 and CD 20. The experimental results show that AAV-CD4-CAR virus-infected cells can express CAR molecules specifically recognizing CD4 protein on the surface, and AAV-CD4-CAR virus can form high-proportion CAR-T cells after directly infecting PBMC, and can specifically target and kill CD4+ T cells and CD4+ tumor cells. In vivo experimental results further confirm that after 2 weeks of direct injection of AAV-CD4-CAR virus into humanized mice, more than 12% of T cells become CAR-T cells and can efficiently target and kill CD4+ target cells in vivo, the reduction ratio of CD4 target cells is more than 50%, and the reduction ratio is further reduced with time. The above results suggest that AAV-CAR viruses injected into vivo can directly infect PBMC cells in vivo and form CAR-T cells in vivo, which can achieve specific killing of target cells. Compared with the traditional CAR-T cell treatment, the method does not need complex processes such as blood collection, PBMC separation, T cell in vitro amplification, CAR-T in vitro preparation, body feedback and the like, and the CAR-T cell treatment can be realized only by directly injecting AAV-CAR virus into a body. Therefore, the AAV-CAR virus can be developed into a universal type efficient novel cell therapy. AAV-CAR virus can be widely applied to tumor cell therapy by targeted killing of tumor cells, and can achieve cell therapy aiming at virus infection by targeted killing of potentially or latently infected cells of the virus.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Nanjing Anrui Biotechnology Ltd

<120> chimeric antigen receptor recombinant adeno-associated virus particle and application thereof

<160>11

<170>SIPOSequenceListing 1.0

<210>1

<211>243

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly GlySer Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln

115 120 125

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

130 135 140

Lys Gly Ser Gly Tyr Ser Phe Ser Ser Ser Trp Ile Gly Trp Val Arg

145 150 155 160

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Asp

165 170 175

Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile

180 185 190

Ser Ala Asp Lys Ser Ile Arg Thr Ala Tyr Leu Gln Trp Ser Ser Leu

195 200 205

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg His Val Thr Met

210 215 220

Ile Trp Gly Val Ile Ile Asp Phe Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser

<210>2

<211>249

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Asn Ser Thr Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala Ser

130 135 140

Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val

145 150 155 160

Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile Gly

165 170 175

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe Lys

180 185 190

Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr Met

195 200 205

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser

245

<210>3

<211>229

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Ile Ser Cys Arg Ser Ser Gln Arg Leu Leu Ser Ser Tyr Gly His Thr

1 5 10 15

Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu

20 25 30

Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser

35 40 45

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu

50 55 60

Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser Thr His Val Pro

65 70 75 80

Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly

85 90 95

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

100 105 110

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

115 120 125

Cys Ala Ala Ser Gly Tyr Thr Phe Ser Asn Tyr Trp Ile Gly Trp Val

130 135 140

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Asp Ile Tyr Pro

145 150 155 160

Gly Gly Asn Tyr Ile Arg Asn Asn Glu Lys Phe Lys Asp Lys Thr Thr

165 170 175

Leu Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser

180 185 190

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Gly Ser Ser Phe Gly

195 200 205

Ser Asn Tyr Val Phe Ala Trp Phe Thr Tyr Trp Gly GlnGly Thr Leu

210 215 220

Val Thr Val Ser Ser

225

<210>4

<211>243

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

115 120 125

Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys

145 150 155 160

Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly

165 170 175

Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu

180 185 190

Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

195 200 205

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr

210 215 220

Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr

225 230 235 240

Val Ser Ala

<210>5

<211>837

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

atcgaggtga tgtacccccc cccctacctg gacaacgaga agagcaacgg caccatcatc 60

cacgtgaagg gcaagcacct gtgccccagc cccctgttcc ccggccccag caagcccttc 120

tgggtgctgg tggtggtggg cggcgtgctg gcctgctaca gcctgctggt gaccgtggcc 180

ttcatcatct tctgggtgcg cagcaagcgc agccgcggcg gccacagcga ctacatgaac 240

atgacccccc gccgccccgg ccccacccgc aagcactacc agccctacgc ccccccccgc 300

gacttcgccg cctaccgcag cggcggtggc ggcagcaagc gcggccgcaa gaagctgctg 360

tacatcttca agcagccctt catgcgcccc gtgcagacca cccaggagga ggacggctgc 420

agctgccgct tccccgagga ggaggagggc ggctgcgagc tgggaggcgg tggcagccgc 480

gtgaagttca gccgcagcgc cgacgccccc gcctaccagc agggccagaa ccagctgtac 540

aacgagctga acctgggccg ccgcgaggag tacgacgtgc tggacaagcg ccgcggccgc 600

gaccccgaga tgggcggcaa gccccgccgc aagaaccccc aggagggcct gtacaacgag 660

ctgcagaagg acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcgccgc 720

cgcggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggacacctac 780

gacgccctgc acatgcaggc cctgcccccc cgcgactaca agatgaccac catcagc 837

<210>6

<211>729

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

gccatccagc tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gggcatcagc agcgccctgg cctggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgac gccagcagcc tggagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag ttcaacagct acccctacac cttcggccag 300

ggcaccaagc tggagatcaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 360

ggcagcgagg tgcagctggt gcagagcggc gccgaggtga agaagcccgg cgagagcctg 420

aagatcagct gcaagggcag cggctacagc ttcagcagca gctggatcgg ctgggtgcgc 480

cagatgcccg gcaagggcct ggagtggatg ggcatcatct accccgacga cagcgacacc 540

cgctacagcc ccagcttcca gggccaggtg accatcagcg ccgacaagag catccgcacc 600

gcctacctgc agtggagcag cctgaaggcc agcgacaccg ccatgtacta ctgcgcccgc 660

cacgtgacca tgatctgggg cgtgatcatc gacttctggg gccagggcac cctggtgacc 720

gtgagcagc 729

<210>7

<211>747

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gcgcgtgacc 60

atgaactgca agagcagcca gagcctgctg tacagcacca accagaagaa ctacctggcc 120

tggtaccagc agaagcccgg ccagagcccc aagctgctga tctactgggc caacagcacc 180

gagagcggcg tgcccgaccg cttcagcggc agcggcagcg gcaccgactt caccctgacc 240

atcagcagcg tgcaggccga ggacgtggcc gtgtactact gccagcagta ctacagctac 300

cgcaccttcg gcggcggcac caagctggag atcaagggtg gcggaggttc tggaggaggt 360

ggaagcggag gtggcggatc tcaggtgcag ctgcagcaga gcggccccga ggtggtgaag 420

cccggcgcca gcgtgaagat gagctgcaag gccagcggct acaccttcac cagctacgtg 480

atccactggg tgcgccagaa gcccggccag ggcctggact ggatcggcta catcaacccc 540

tacaacgacg gcaccgacta cgacgagaag ttcaagggca aggccaccct gaccagcgac 600

accagcacca gcaccgccta catggagctg agcagcctgc gcagcgagga caccgccgtg 660

tactactgcg cccgcgagaa ggacaactac gccaccggcg cctggttcgc ctactggggc 720

cagggcaccc tggtgaccgt gagcagc 747

<210>8

<211>747

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccccggcga gcccgccagc 60

atcagctgcc gcagcagcca gcgcctgctg agcagctacg gccacaccta cctgcactgg 120

tacctgcaga agcccggcca gagcccccag ctgctgatct acgaggtgag caaccgcttc 180

agcggcgtgc ccgaccgctt cagcggcagc ggcagcggca ccgacttcac cctgaagatc 240

agccgcgtgg aggccgagga cgtgggcgtg tactactgca gccagagcac ccacgtgccc 300

ctgaccttcg gccagggcac caaggtggag atcaagggcg gcggcggcag cggcggcggc 360

ggcagcggcg gcggcggcag cgaggtgcag ctggtggaga gcggcggcgg cctggtgaag 420

cccggcggca gcctgcgcct gagctgcgcc gccagcggct acaccttcag caactactgg 480

atcggctggg tgcgccaggc ccccggcaag ggcctggagt ggatcggcga catctacccc 540

ggcggcaact acatccgcaa caacgagaag ttcaaggaca agaccaccct gagcgccgac 600

accagcaaga acaccgccta cctgcagatg aacagcctga agaccgagga caccgccgtg 660

tactactgcg gcagcagctt cggcagcaac tacgtgttcg cctggttcac ctactggggc 720

cagggcaccc tggtgaccgt gagcagc 747

<210>9

<211>729

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

caaattgttc tctcccagtc tccagcaatc ctgtctgcat ctccagggga gaaggtcaca 60

atgacttgca gggccagctc aagtgtaagt tacatccact ggttccagca gaagccagga 120

tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt ccctgttcgc 180

ttcagtggca gtgggtctgg gacttcttac tctctcacaa tcagcagagt ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg actagtaacc cacccacgtt cggagggggg 300

accaagctgg aaatcaaacg tggtggcgga ggttctggag gaggtggaag cggaggtggc 360

ggatctcagg tacaactgca gcagcctggg gctgagctgg tgaagcctgg ggcctcagtg 420

aagatgtcct gcaaggcttc tggctacaca tttaccagtt acaatatgca ctgggtaaaa 480

cagacacctg gtcggggcct ggaatggatt ggagctattt atcccggaaa tggtgatact 540

tcctacaatc agaagttcaa aggcaaggcc acattgactg cagacaaatc ctccagcaca 600

gcctacatgc agctcagcag cctgacatct gaggactctg cggtctatta ctgtgcaaga 660

tcgacttact acggcggtga ctggtacttc aatgtctggg gcgcagggac cacggtcacc 720

gtctctgca 729

<210>10

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ccattgacgt caatgggtgg agt 23

<210>11

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

gccaagtagg aaagtcccat aagg 24

Claims

1. A chimeric antigen receptor recombinant adeno-associated virus particle is characterized in that the chimeric antigen receptor recombinant adeno-associated virus particle carries a chimeric antigen receptor gene.

2. The chimeric antigen receptor recombinant adeno-associated virus particle according to claim 1, wherein the chimeric antigen receptor gene is packaged by an adeno-associated virus packaging system.

3. The chimeric antigen receptor recombinant adeno-associated virus particle according to claim 1, wherein the chimeric antigen receptor gene encodes a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

4. The chimeric antigen receptor recombinant adeno-associated virus particle according to claim 3, wherein the extracellular domain is an antibody recognizing the antigen domain.

5. The chimeric antigen receptor recombinant adeno-associated virus particle according to claim 4, wherein the antibody recognizing the antigen domain is scFV antibody or V_HH antibody.

6. The chimeric antigen receptor recombinant adeno-associated virus particle according to claim 5, wherein the scFV antibody is specific for CD4, CD19, CCR5 or CD 20.

7. Use of the chimeric antigen receptor recombinant adeno-associated virus particle according to any one of claims 1 to 6 in the preparation of a medicament for the treatment of tumors.

8. The use according to claim 7, wherein the medicament is a targeted medicament for use in tumor immunotherapy.

9. A pharmaceutical composition comprising the chimeric antigen receptor recombinant adeno-associated virus particle of any one of claims 1 to 6, and a pharmaceutically acceptable carrier or excipient.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is in the form of an injection.