CN120737215A

CN120737215A - Chimeric antigen receptor and application thereof in preparation of long-acting medicine for treating allergic diseases

Info

Publication number: CN120737215A
Application number: CN202510916080.3A
Authority: CN
Inventors: 赵勇; 李頔
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2025-07-03
Filing date: 2025-07-03
Publication date: 2025-10-03

Abstract

The application provides a chimeric antigen receptor, which comprises an antigen specific binding domain, a hinge, a transmembrane domain, a co-stimulus domain and a CD3 zeta signal transmission domain, wherein the antigen specific binding domain can specifically bind an antigen comprising immunoglobulin IgE, and the application of the chimeric antigen receptor in preparing a long-acting medicament for treating IgE-mediated allergic diseases. Compared with the prior art, the application combines the anti-IgE monoclonal antibody with the CAR-T technology, changes the generated IgE protein from targeting to targeting the B cell generating IgE, and has proved the killing effect of the IgE CAR-T on the mIgE ⁺ B cell in vitro and in vivo experiments, thereby solving the problems of short half-life period of the monoclonal antibody and high-frequency administration. In addition, the application also provides a scheme for in vivo experiments by using a humanized mouse model, and the treatment effect of the CAR-T is successfully verified.

Description

Chimeric antigen receptor and application thereof in preparation of long-acting medicine for treating allergic diseases

Technical Field

The application relates to the technical field of biological medicine, in particular to a chimeric antigen receptor and application thereof in preparing a long-acting medicine for treating allergic diseases.

Background

The prevalence of allergic diseases continues to rise worldwide, including food allergies, allergic asthma, atopic dermatitis, urticaria, allergic rhinitis, conjunctivitis, and chronic sinusitis, among others. Immunoglobulin IgE is a key molecule in allergic reactions. Various forms of allergic diseases associated with IgE affect about 30% of the world population.

When a patient is exposed to an allergen, B cells are stimulated to differentiate and IgE antibodies are produced by the combined action of dendritic cells, th2, tfh and other cells, and IL-4, IL-5, IL-13 and other cytokines. These IgE antibodies bind to mast cells and basophils and mediate their degranulation reactions to release inflammatory mediators, which in turn trigger a range of allergic symptoms. Therefore, igE becomes an important target for development of allergic disease drugs, and the anti-IgE monoclonal antibody Omalizumab is also a first target drug to block downstream allergic inflammatory reactions by neutralizing free IgE (IgE) and the like. Omalizumab was approved by the FDA for the treatment of severe allergic asthma in 2003, was approved for the treatment of urticaria in 2014, and has also been shown to have therapeutic effects on allergic diseases such as allergic rhinitis and food allergy in some studies. The Omalizumab treatment can effectively improve symptoms of allergic asthma and urticaria, and improve the life quality of patients. IgE is a membrane-bound form of IgE and is expressed on the surface of B cells and plasma cells, and it is generally believed that the free IgE secreted by the plasma cells and B cells of mIgE ⁺ is the major source of IgE in allergic reactions. However, omalizumab as an IgE neutralizing antibody has a main effect of neutralizing the produced free IgE with less effect on IgE production. Omalizumab is difficult to effectively maintain free IgE levels at low levels for long periods of time.

To date, igE-targeted allergic disease treatment regimens have been based on monoclonal antibodies. However, the half-life of the monoclonal antibody is short, the therapeutic effect depends on a certain use frequency, and great limitation is brought, so that more effective therapeutic means are still needed. Chimeric antigen receptor T cell (CAR-T) treatment is to modify human T cells in vitro by genetic engineering means to identify specific target antigens, amplify and reinject the human T cells into human bodies for treating diseases. CAR-T technology currently achieves a remarkable effect in the treatment of hematological malignancies, and the memory of T cells enables long-term inhibition of tumors. Thus, it is also possible to apply CAR-T to the treatment of allergy-related long-term chronic diseases.

Therefore, none of the current therapies targeting IgE achieve long-term inhibition of IgE levels in vivo, long-term relief of allergic symptoms.

Disclosure of Invention

In view of the above-mentioned technical limitations, the present application proposes chimeric antigen receptors and their use in the preparation of long-acting medicaments for the treatment of allergic diseases, which overcome the drawbacks and deficiencies mentioned in the background art.

In order to achieve the above purpose, the present application adopts the following technical scheme:

The application provides a chimeric antigen receptor, which comprises an antigen specific binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a CD3 zeta signaling domain, wherein the antigen specific binding domain can specifically bind an antigen comprising immunoglobulin IgE, and the antigen specific binding domain comprises a single-chain variable fragment scFv composed of an amino acid sequence shown as SEQ ID No.1 or a single-chain variable fragment scFv which has more than 80 percent of homology with the amino acid sequence shown as SEQ ID No.1 and has the same or similar functions.

Alternatively, the chimeric antigen receptor has the single-chain variable fragment scFv which has more than 80% of homology with the amino acid sequence shown in SEQ ID No.1 and has the same or similar functions, and the amino acid sequence of the single-chain variable fragment scFv is shown in SEQ ID No.2 or SEQ ID No. 3.

Alternatively, the chimeric antigen receptor described above, the costimulatory domain is selected from the group consisting of polypeptides of OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDS, ICAM-1, or a combination thereof.

Alternatively, the chimeric antigen receptor is characterized in that the hinge domain is selected from an amino acid sequence shown as SEQ ID No.5 or a hinge domain which has more than 80% homology with the amino acid sequence shown as SEQ ID No.5 and has the same or similar function, the transmembrane domain is selected from an amino acid sequence shown as SEQ ID No.6 or a transmembrane domain which has more than 80% homology with the amino acid sequence shown as SEQ ID No.6 and has the same or similar function, the CD3 zeta signal transduction domain is selected from an amino acid sequence shown as SEQ ID No.7 or a CD3 zeta signal transduction domain which has more than 80% homology with the amino acid sequence shown as SEQ ID No.7 and has the same or similar function, and the costimulatory domain is selected from an amino acid sequence shown as SEQ ID No.8 or SEQ ID No.9 or a costimulatory domain which has more than 80% homology with the amino acid sequence shown as SEQ ID No.8 or SEQ ID No.9 and has the same or similar function.

Alternatively, the chimeric antigen receptor described above, the hinge domain is encoded by the nucleic acid sequence shown in SEQ ID No.11, the transmembrane domain is encoded by the nucleic acid sequence shown in SEQ ID No.12, the CD3 zeta signaling domain is encoded by the nucleic acid sequence shown in SEQ ID No.13, and the costimulatory domain is encoded by the nucleic acid sequence shown in SEQ ID No.14 or SEQ ID No. 15.

It is a second object of the present application to provide an isolated immune cell modified to express a chimeric antigen receptor comprising an antigen binding domain linked to a costimulatory domain and a CD3 zeta signaling domain, said antigen binding domain being capable of specifically binding to a single chain variable fragment scFv comprising immunoglobulin IgE, said single chain variable fragment scFv having the amino acid sequence shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3, preferably a T cell.

Alternatively, the isolated immune cell described above, the costimulatory domain is selected as a CD28 or 41BB polypeptide.

Alternatively, the chimeric antigen receptor expressed in the isolated immune cell is the chimeric antigen receptor described above.

Alternatively, the isolated immune cells described above are selected from the group consisting of CD4 ⁺ T cells, CD8 ⁺ T cells, or CD4 ⁺ and CD8 ⁺ T cells mixed in any ratio.

It is a third object of the present application to provide an expression vector encoding the chimeric antigen receptor as described above.

It is a fourth object of the present application to provide a host cell comprising the aforementioned expression vector.

A fifth object of the present application is to provide the use of the chimeric antigen receptor described above, the isolated immune cell described above, the expression vector described above and the host cell described above for the preparation of a long-acting medicament for the treatment of allergic diseases.

Alternatively, the above-mentioned use, the allergic disease is selected as any one or more of IgE-mediated allergic reaction type diseases, preferably allergic asthma, food allergy, urticaria.

A sixth object of the present application is to provide a long-acting pharmaceutical composition for the treatment of allergic diseases, which comprises the chimeric antigen receptor, the isolated immune cell, the expression vector and the host cell.

The core technology of the application is to combine an anti-IgE monoclonal antibody with a CAR-T technology, and use IgE monoclonal antibodies to form scFv extracellular domains of the CAR so as to target B cells or plasma cells expressing mIgE. A CAR lentiviral vector comprising IgE scFv fragments was constructed and lentiviruses were packaged by transfection 293T. T cells were isolated from human PBMC or CBMC, activated with CD3/CD28 Dynabeads, cultured and expanded using IL-2 supplemented media. Primary T cells are infected with IgE CAR lentiviruses to construct CAR-T cells. The expanded CAR-T cells kill mIgE ⁺ cells in vitro experiments, and mIgE ⁺ cells comprise a U266 cell line with low expression of mIgE and a Daudi-mIgE-mCherry cell line constructed by lentiviral transduction. Meanwhile, the application uses an HDM induced asthma model of a humanized mouse to verify the therapeutic effect of IgE CAR-T cells in vivo. In this asthma model, adoptive IgE CAR-T cells are also able to kill mIgE ⁺ B cells in the body, reduce IgE levels and maintain for long periods of time.

Compared with the prior art, the application has the following advantages:

firstly, the IgE monoclonal antibody is combined with the CAR-T technology, the generated IgE protein is targeted to be converted into the B cell for generating IgE, and the killing effect of the IgE CAR-T on the mIgE ⁺ B cell is proved in vitro and in vivo experiments. The problem that the half-life period of the monoclonal antibody is short and high-frequency administration is needed is solved.

Secondly, because the mIgE key sequences have large species differences in humans and mice, in vivo experiments using mouse animal models are difficult, and none of the prior art has demonstrated in vivo experimental data. The application provides a scheme for in vivo experiments by using a humanized mouse model, and successfully verifies the treatment effect of the CAR-T.

Drawings

FIG. 1 shows a schematic illustration of IgECAR-T construction, wherein,

FIG. 1A is a schematic diagram of plasmid construction (BB-1 and 28-1 are set CAR-1, BB-2 and 28-2 are set CAR-2, with the difference in antigen-specific binding domain), FIG. 1B is a flow-through statistics after staining CAR-T with protein L, comprising 3 donor-derived CAR-T, indicating membrane surface expression of the CAR, FIG. 1C is a statistical graph of the results of CD4 ⁺ and CD8 ⁺ cell grouping in the CAR-T, and FIG. 1D is a statistical graph of the naive T cell (naive), central memory T cell (effector) and effector T cell grouping in the CAR-T.

FIG. 2 is a graph showing the results of the construction of mIgE ⁺ target cells and the killing of mIgE ⁺ target cells by CAR-T,

FIG. 2A is a graph showing the statistics of the relative cell amounts of target cells after co-culturing CAR-T and U266 target cells, FIG. 2B is a schematic plasmid map for Daudi target cell construction, FIG. 2C is the expression of membrane surface IgE in U266 and Daudi target cells, FIG. 2D is a graph showing the statistics of the relative cell amounts of target cells after co-culturing CAR-T and Daudi-mIgE-mCherry, daudi-sIgE-mCherry, daudi-mCherry target cells, respectively (data show mean.+ -. SEM, subject to two-tailed T-test, p <0.05, p <0.01, p <0.001, nsp >0.05 are not significant, statistical tests are all labeled with this standard, and p-values are labeled as differences between groups at 10:1-effect target ratio and UTD group).

FIG. 3 shows the killing effect of the modified scFv part of the CAR-T, wherein FIG. 3A is a schematic diagram of CAR-T plasmid construction, BB-2 is original CAR-T, BB-2-m1 and m2 are modified mutant CAR-T, and FIG. 3B is a graph of statistical results of the detected relative cell amounts of target cells after co-culturing various CAR-T with Daudi-mCherry and Daudi-mIgE-mCherry target cells under the conditions of the effective target ratio of 1:1 and 10:1.

FIG. 4 shows a schematic representation of activation of individual CAR-T cells after co-culture with target cells, wherein,

FIG. 4A is a graph showing the ratio of CD107a, granzyme B, CD69, CD25, PD-1 expression and PD-1 ⁺TIM-3⁺ in CD8 ⁺ CAR-T when the CAR-T was co-cultured with Daudi-mIgE-mCherry, U266, daudi-sIgE-mCherry, daudi-mCherry, respectively, and the ratio of PD-1 to PD-3 when the CAR-T was cultured alone, FIG. 4B is a graph showing the ratio of TNF alpha and IFN gamma in the culture supernatant after the CAR-T was co-cultured with Daudi-mIgE-mCherry, U266, daudi-sIgE-mCherry, daudi-mCherry, respectively, and FIG. 4C is a graph showing the ratio of PD-1, LAG-3, TIM-3 expression of two CAR-T (CAR-1 and CAR-2) in depletion model.

Figure 5 shows a graph comparing the killing capacity of CD4 ⁺ and CD8 ⁺ CAR-T against the results, wherein,

FIG. 5A is a graph showing the ratio of CD4 ⁺ to CD8 ⁺ cells in the sorted CAR-T, and FIG. 5B is a statistical plot of the relative cell amounts of target cells after co-culturing CD4 ⁺ and CD8 ⁺ CAR-T with Daudi-mIgE-mCherry target cells, respectively.

FIG. 6 shows a statistical plot of target cell relative cell mass after co-cultivation with or without addition of recombinant sIgE protein in the CAR-T and Daudi-mIgE-mCherry co-cultivation system, wherein the ratio of targets is 1:1, co-cultivation is carried out for 24 hours.

FIG. 7 shows the results of IgECAR-T treatment of humanized mouse asthma, wherein,

Fig. 7A and 7B are graphs showing the statistics of the ratio of CAR-T to human immune cells (hCD 45 ⁺) in the lungs and spleen of mice after the secondary challenge, fig. 7C and 7D are graphs showing the statistics of the ratio of mIgE ⁺ B cells to human immune cells in the lungs and spleen of mice after the secondary challenge (the significance of the figure is marked as a difference compared to the model group, the same applies below), fig. 7E is a measurement of the total IgE content in the serum of mice after the first challenge, fig. 7F is a measurement of the content of HDM-specific IgE protein in the serum of mice after the first challenge, fig. 7G is a measurement of the total IgE content in the serum of mice after the second challenge, fig. 7H is a measurement of the content of HDM-specific IgE protein in the serum of mice after the second challenge, and fig. 7I is a H & E staining of the lung section of mice after the second challenge (scale marked 100 μm).

Detailed Description

The present application will be described in further detail below in order to make the objects, technical solutions and advantages of the present application more apparent. It is to be understood that the description is only intended to illustrate the application and is not intended to limit the scope of the application.

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 application belongs, and the terms used herein in this description of the application are for the purpose of describing particular embodiments only and are not intended to be limiting of the application. Reagents and instruments used herein are commercially available, and reference to characterization means is made to the relevant description of the prior art and will not be repeated herein.

For a further understanding of the present application, the present application will be described in further detail with reference to the following preferred embodiments.

Example 1

The chimeric antigen receptor provided by the application comprises a leading chain domain, an antigen specific binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a CD3 zeta signaling domain, wherein the antigen specific binding domain can specifically bind an antigen comprising immunoglobulin IgE, and the antigen specific binding domain comprises a single-chain variable fragment scFv consisting of an amino acid sequence shown as SEQ ID No.1 or a single-chain variable fragment scFv which has more than 80% of homology with the amino acid sequence shown as SEQ ID No.1 and has the same or similar functions, and the amino acid sequence of the antigen specific binding domain is shown as SEQ ID No.2 or SEQ ID No. 3.

The co-stimulatory domain is selected from the group consisting of OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDs, ICAM-1, or a combination thereof.

The hinge domain is selected from an amino acid sequence shown as SEQ ID No.5 or a hinge domain which has more than 80% of homology with the amino acid sequence shown as SEQ ID No.5 and has the same or similar functions;

the transmembrane domain is selected from an amino acid sequence shown as SEQ ID No.6 or a transmembrane domain which has more than 80 percent of homology with the amino acid sequence shown as SEQ ID No.6 and has the same or similar functions;

The CD3 zeta signaling domain is selected as an amino acid sequence shown as SEQ ID No.7 or a CD3 zeta signaling domain which has more than 80 percent of homology with the amino acid sequence shown as SEQ ID No.7 and has the same or similar functions;

The costimulatory domain is selected from the amino acid sequences shown as SEQ ID No.8 or SEQ ID No.9, or the costimulatory domain which has more than 80% homology with the amino acid sequences shown as SEQ ID No.8 or SEQ ID No.9 and has the same or similar functions.

The homology may be 80%, 85%, 90%, 95% or 99%.

The hinge domain is encoded by the nucleic acid sequence shown in SEQ ID No. 11;

the transmembrane domain is encoded by the nucleic acid sequence shown in SEQ ID No. 12;

the CD3 zeta signaling domain is encoded by the nucleic acid sequence shown in SEQ ID No. 13;

the costimulatory domain is encoded by the nucleic acid sequence shown as SEQ ID No.14 or SEQ ID No. 15.

In addition to the sequences shown above, the chimeric antigen receptor of the application, the individual domains can be selected from the following sequences:

Upstream of the chimeric antigen receptor may or may not be provided a signal peptide selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof;

the hinge domain of the chimeric antigen receptor can also be selected from CD8, CD28, CD137, or a combination thereof;

The transmembrane domain of the chimeric antigen receptor may also be selected from CD3epsilon, CD4, CD8, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3, or a combination thereof;

The co-stimulatory domain of the chimeric antigen receptor may also be selected from OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDS, ICAM-1, or a combination thereof;

the chimeric antigen receptor 2A was selected as T2A, P2A, E2A, F a.

The application also provides isolated immune cells, i.e., T cells, modified to express the chimeric antigen receptor described above, selected from CD4 ⁺ T cells, CD8 ⁺ T cells, or a mixture of CD4 ⁺ and CD8 ⁺ T cells in any ratio.

The application also provides an expression vector which can code the chimeric antigen receptor.

The application also provides a host cell comprising the expression vector.

The application also provides chimeric antigen receptor, isolated T cells, expression vector and application of host cells in preparing long-acting medicine for treating allergic diseases.

The allergic disease is selected from one or more of IgE mediated allergic reaction diseases, preferably allergic asthma, food allergy, and urticaria.

The application also provides a long-acting pharmaceutical composition for treating allergic diseases, which comprises the chimeric antigen receptor, an isolated T cell, an expression vector and a host cell.

Example 2

1. Experimental materials and methods:

1) CAR-T construction:

First, an IgE-targeting CAR lentiviral plasmid is synthesized, the coding region of which comprises a signal peptide gene fragment, an anti-IgE single-chain antibody gene fragment, a transmembrane region gene fragment, an intracellular signal region gene fragment, including or not including a T2A linked EGFP tag gene fragment. CAR plasmids containing different single chain antibodies were constructed according to this protocol, as well as negative control plasmids that did not contain single chain antibodies. Each CAR lentivirus was individually packaged using 293T cells.

The constructed CAR plasmids containing different single-chain antibodies or costimulatory domains (BB-2, 28-2 in FIG. 1 are different costimulatory domains; BB-2, BB-2-m1, BB-2-m2 in FIG. 3 are different single-chain antibodies) have the following structural profiles:

CD8 leader (leader domain, CD 8-SP) -single chain antibody gene fragment scFv-CD 8-range-CD 8-TM-4-1 BB or CD 28-CD 3 zeta-T2A EGFP;

the negative control CAR plasmid (BB-1 in FIG. 1) has the following structural profile:

CD8 leader-CD 8-range-CD 8-TM-4-1 BB or CD 28-CD 3 zeta-T2 AEGFP.

Wherein, each partial sequence is respectively:

(1) The sequence of the CD8 leader (CD 8 leader) is shown in SEQ ID No. 4:

MALPVTALLLPLALLLHAARP;

(2) When the single-chain antibody gene fragment scFv is selected to be of Omalizumab origin (BB-2 or 28-2 in FIG. 1), the amino acid sequence is shown in SEQ ID No. 1:

EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKR;

when the single-chain antibody gene fragment scFv is selected as a homologous sequence scFv-2-m1 (BB-2-m 1 in FIG. 3), the sequence is shown in SEQ ID No. 2:

EVQLVESGGGLVQPGGTLRLSCAVSGYSVTSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRISISRDDSKNTFYLQMNGLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQSHEDPYTFGQGTKVEIKR;

When the single-chain antibody gene fragment scFv is selected as the homologous sequence scFv-2-m2 (BB-2-m 2 in FIG. 3), the sequence is shown in SEQ ID No. 3:

EVQLVESGGGLVQPGGSVRLSCAVSGYSITSGYSWNWIRQAPGKALEWVASITYDGSTNYNPSVKGRITISKDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTISCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEVKR.

(3) The sequence of the CD8 hinge (CD 8-hinge) is shown in SEQ ID No. 5:

AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

(4) The sequence of the CD8 transmembrane region (CD 8-TM) is shown in SEQ ID No. 6:

IYIWAPLAGTCGVLLLSLVITLYC;

(5) The sequence of the CD3zeta signaling structure (CD 3 zeta) is shown in SEQ ID No. 7:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR;

(6) When the costimulatory structure is selected as 4-1BB, the sequence is shown as SEQ ID No. 8:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

when the co-stimulatory structure is selected as CD28, its sequence is shown in SEQ ID No. 9:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS;

(7) The sequence of the tracer structure (T2 AEGFP) is shown in SEQ ID No. 16:

EGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

The single chain antibody gene fragment scFv is linked to the CD8 leader via a short peptide SR and the single chain antibody gene fragment scFv is linked to the CD8 hinge domain via a short peptide PGAAA. The negative control CAR-1 plasmid does not contain scFv, and the CD8 leader is linked to the CD8 hinge structure via a linker peptide linker (SRLVLEHMHPGVAA).

The nucleic acid sequence of the amino acid sequence of the coding leader chain domain SEQ ID No.4 is shown as SEQ ID No.10, SEQ ID No.10:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg。

The nucleic acid sequence of the amino acid sequence of the coding hinge domain SEQ ID No.5 is shown as SEQ ID No. 11;

SEQ ID No.11:

gcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccc tgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat.

the nucleic acid sequence of the amino acid sequence of the coding transmembrane domain SEQ ID No.6 is shown as SEQ ID No. 12;

SEQ ID No.12:

atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc。

The nucleic acid sequence of the SEQ ID No.7 amino acid sequence of the encoding CD3 zeta signaling domain is shown as SEQ ID No. 13;

SEQ ID No.13:

agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc.

the nucleic acid sequence of the amino acid sequence of the coding co-stimulatory domain SEQ ID No.8 or SEQ ID No.9 is shown as SEQ ID No.14 or SEQ ID No. 15;

SEQ ID No.14:

aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagagg aagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg.

SEQ ID No.15:

cgaagcaagcggagccggctgctgcacagcgactacatgaacatgacccctagacggcccggaccaaccaga aagcactatcagccttacgctcctcctcgggacttcgccgcctatagatct.

The nucleotide sequence of the SEQ ID No.16 sequence of the coding tracer structure (T2 AEGFP) is shown as SEQ ID No. 23;

SEQ ID No.23:

gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa.

From the above, it can be seen that the specific structural sequences of CAR plasmids containing different single chain antibodies are as follows:

a negative control CAR plasmid (BB-1 in fig. 1) having the sequence shown in SEQ ID No. 17:

SEQ ID No.17:

MALPVTALLLPLALLLHAARPSRLVLEHMHPGVAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK;

A negative control CAR plasmid (28-1 in fig. 1) having the sequence as set forth in SEQ ID No. 18:

SEQ ID No.18

MALPVTALLLPLALLLHAARPSRLVLEHMHPGVAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

The constructed CAR plasmids (BB-2, 28-2 in FIG. 1 and BB-2-m2 in FIG. 3) containing different single-chain antibodies respectively have sequences shown as SEQ ID No.19 to SEQ ID No.22, and BB-2 (SEQ ID No. 19):

MALPVTALLLPLALLLHAARPSREVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRPGAAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK;

28-2(SEQ ID No.20):

MALPVTALLLPLALLLHAARPSREVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRPGAAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK;

BB-2-m1(SEQ ID No.21):

MALPVTALLLPLALLLHAARPSREVQLVESGGGLVQPGGTLRLSCAVSGYSVTSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRISISRDDSKNTFYLQMNGLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQSHEDPYTFGQGTKVEIKRPGAAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK;

BB-2-m2(SEQ ID No.22):

MALPVTALLLPLALLLHAARPSREVQLVESGGGLVQPGGSVRLSCAVSGYSITSGYSWNWIRQAPGKALEWVASITYDGSTNYNPSVKGRITISKDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTISCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTKVEVKRPGAAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

the nucleic acid sequence of the amino acid sequence of the negative control CAR plasmid (BB-1 in FIG. 1, SEQ ID No. 17) is shown as SEQ ID No. 24;

SEQ ID No.24:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtctagactagtcctcgagcatatgcaccccggggtggccgcagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa.

The nucleic acid sequence encoding the amino acid sequence of the negative control CAR plasmid (28-1 in FIG. 1, SEQ ID No. 18) is shown as SEQ ID No. 25;

SEQ ID No.25:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtctagactagtcctcgagCATATGcaccccggggTggccgcagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgccgaagcaagcggagccggctgctgcacagcgactacatgaacatgacccctagacggcccggaccaaccagaaagcactatcagccttacgctcctcctcgggacttcgccgcctatagatctagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa.

The nucleic acid sequence of the CAR plasmid (BB-2 in figure 1, SEQ ID No. 19) containing different single chain antibodies is shown in SEQ ID No. 26;

SEQ ID No.26:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtctagagaagtgcagctggtggagagcggaggaggactggtgcagcctggaggaagtctgagactgagctgcgccgtgagtgggtacagcatcacaagcgggtacagctggaactggatcagacaggctccaggcaagggactggagtgggtggctagtattacatatgacggctctaccaactacaaccccagcgtgaaaggcagaatcaccatcagcagggacgacagcaagaacaccttctacctgcagatgaactctctgcgggccgaggacaccgctgtgtattactgcgccagaggtagccactacttcggacattggcacttcgccgtgtggggccagggaacactggtgacagtgagcagcggcggaggaggatctggaggaggaggaagtggcggaggaggctctgatattcagctgacccagagccccagcagcctgtctgctagtgtgggagacagagtgaccatcacctgcagagccagccagagcgtggattatgatggagactcatacatgaactggtaccagcagaaacccggcaaggccccaaaactgctgatctacgctgctagctacctggaaagcggcgtgccaagcagattctcaggaagcggcagcggaaccgacttcaccctgactatcagcagcctgcagcccgaagacttcgccacatactactgccagcagtcccacgaagacccctacaccttcggccagggaaccaaagtggagatcaagagacccggggcggccgcagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa.

The nucleic acid sequence encoding the amino acid sequence of the CAR plasmid (28-2 in FIG. 1, SEQ ID No. 20) comprising the different single chain antibodies is shown as SEQ ID No. 27;

SEQ ID No.27:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtctagagaagtgcagctggtggagagcggaggaggactggtgcagcctggaggaagtctgagactgagctgcgccgtgagtgggtacagcatcacaagcgggtacagctggaactggatcagacaggctccaggcaagggactggagtgggtggctagtattacatatgacggctctaccaactacaaccccagcgtgaaaggcagaatcaccatcagcagggacgacagcaagaacaccttctacctgcagatgaactctctgcgggccgaggacaccgctgtgtattactgcgccagaggtagccactacttcggacattggcacttcgccgtgtggggccagggaacactggtgacagtgagcagcggcggaggaggatctggaggaggaggaagtggcggaggaggctctgatattcagctgacccagagccccagcagcctgtctgctagtgtgggagacagagtgaccatcacctgcagagccagccagagcgtggattatgatggagactcatacatgaactggtaccagcagaaacccggcaaggccccaaaactgctgatctacgctgctagctacctggaaagcggcgtgccaagcagattctcaggaagcggcagcggaaccgacttcaccctgactatcagcagcctgcagcccgaagacttcgccacatactactgccagcagtcccacgaagacccctacaccttcggccagggaaccaaagtggagatcaagagacccggggcggccgcagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgccgaagcaagcggagccggctgctgcacagcgactacatgaacatgacccctagacggcccggaccaaccagaaagcactatcagccttacgctcctcctcgggacttcgccgcctatagatctagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa.

The nucleic acid sequence of the CAR plasmid (BB-2-m 1 in FIG. 3, SEQ ID No. 21) containing different single chain antibodies is shown as SEQ ID No. 28;

SEQ ID No.28:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtctagagaagtgcagctggtggagagcggaggaggactggtgcagcctggaggaaccctgagactgagctgcgccgtgagtgggtacagcgtgacaagcgggtacagctggaactggatcagacaggctccaggcaagggactggagtgggtggctagtattacatatgacggctctaccaactacaaccccagcgtgaaaggcagaatcagcatcagcagggacgacagcaagaacaccttctacctgcagatgaacggtctgcgggccgaggacaccgctgtgtattactgcgccagaggtagccactacttcggacattggcacttcgccgtgtggggccagggaacactggtgacagtgagcagcggcggaggaggatctggaggaggaggaagtggcggaggaggctctgatattcagctgacccagagccccagcagcctgtctgctagtgtgggagacagagtgaccatcacctgcagagccagccagagcgtggattatgatggagactcatacatgaactggtaccagcagaaacccggcaaggccccaaaactgctgatctacgctgctagctacctggaaagcggcgtgccaagcagattctcaggaagcggcagcggaaccgacttcaccctgactatcagcggtctgcagcccgaagacttcgccacatactactgccagcagtcccacgaagacccctacaccttcggccagggaaccaaagtggagatcaagagacccggggcggccgcagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa.

the nucleic acid sequence encoding the amino acid sequence of the CAR plasmid (BB-2-m 2 in FIG. 3, SEQ ID No. 22) comprising the different single chain antibodies is shown in SEQ ID No. 29;

SEQ ID No.29:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtctagagaagtgcagctggtggagagcggaggaggactggtgcagcctggaggaagtgtgagactgagctgcgccgtgagtgggtacagcatcacaagcgggtacagctggaactggatcagacaggctccaggcaaggctctggagtgggtggctagtattacatatgacggctctaccaactacaaccccagcgtgaaaggcagaatcaccatcagcaaagacgacagcaagaacaccttctacctgcagatgaactctctgcgggccgaggacaccgctgtgtattactgcgccagaggtagccactacttcggacattggcacttcgccgtgtggggccagggaacactggtgacagtgagcagcggcggaggaggatctggaggaggaggaagtggcggaggaggctctgatattcagctgacccagagccccagcagcctgtctgctagtgtgggagacagagtgaccatcagctgcagagccagccagagcgtggattatgatggagactcatacatgaactggtaccagcagaaacccggcaaggccccaaaactgctgatctacgctgctagctacctggaaagcggcgtgccaagcagattctcaggaagcggcagcggaaccgacttcaccctgactatcagcagcctgcagcccgaagacttcgccacatactactgccagcagtcccacgaagacccctacaccttcggccagggaaccaaagtggaggtgaagagacccggggcggccgcagcgaagcccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa.

T cells were isolated from human mononuclear cells using a human T cell negative selection kit (Meitian and Twaiter) and cultured using X VIVO 15 medium (2.5% human AB serum, 1% penicillin-streptomycin, 100IU/mL IL ^-2 added). CD3/CD28 dynabeads (Invitrogen) was added to stimulate for 3 days during which T cells were individually infected with CAR lentiviruses to construct CAR-T. Positive rates were detected on days 7-10 of culture and sorted using EGFP fluorescent markers.

2) Flow cytometry:

Cells were stained using antibodies PE/Cyanine7 anti-human CD45、Brilliant Violet 605anti-human CD3、PE/Cyanine5 anti-human CD4、Brilliant Violet 421anti-human CD8、APC anti-human CD69、Brilliant Violet anti-human CD25、Alexa Fluor 700anti-human PD-1、PE/Cyanine7 anti-human LAG-3、Brilliant Violet 750anti-human TIM-3、APC anti-human CD19、PE anti-human IgE、Brilliant Violet 510anti-human IgM、Brilliant Violet 421anti-human IgD、Brilliant Violet 605anti-human CD107a、PE/Cyanine7 anti-human Granzyme B、PE protein L、Brilliant Violet 711anti-human CD62L、PE/Cyanine7 anti-human CD45RA(, all available from BioLegend). The intracellular staining was performed using a cell fixation/permeabilization buffer (BD Bioscience) followed by antibody staining. Analysis was performed using a BD Fortessa flow analyzer, or sorting was performed using a BD Fusion flow sorter, and flow data analysis was performed using Flowjo software.

3) T cell killing efficiency detection:

CAR-T or UTD (UTD, cells refer to untransduced cells "Untransduced"; UTD cells refer to cells that have not been genetically modified, i.e., cells that have not been transduced with a particular gene), were co-cultured with target cells in an effective target ratio (E: T) by adding corresponding amounts of cells to U-bottom 96-well plates in RPMI 1640 (10% FBS and 1% penicillin-streptomycin). The single culture holes of the target cells are arranged, and the cell quantity is consistent with that of the co-culture holes. Daudi-mIgE-mCherry, daudi-sIgE-mCherry, daudi-mCherry target cells were all labeled with mCherry fluorescent, and U266 target cells were stained with CELLTRACKER CMTPX (Invitrogen) prior to co-culture. At the end of the co-culture, daudi wild-type cells were stained with CELLTRACKER CM-Dil (Daudi-Dil) as quantitative cells. Equal amounts of Daudi-Dil cells were added to each co-culture well or individual culture wells and the proportion of each population of cells within each well was measured by flow analyser. Target cell relative cell amount= (co-culture well target cell ratio/co-culture well Daudi-Dil cell ratio)/(single culture well target cell ratio/single culture well Daudi-Dil cell ratio).

4)ELISA:

Levels of tnfα and ifnγ in cell co-culture supernatants were detected using the Human tnfα Deluxe kit and the Human ifnγ Deluxe kit (BioLegend), respectively. The operation steps are carried out according to the instruction of the kit.

5) Humanized mouse asthma model:

Hu-HSC-NPG-GM3 humanized mice are used as experimental materials, and the mice are constructed by Venetherlands, and the humanized degree of the immune system is detected to be more than 40 percent. A humanized mouse asthma model was established by tracheal administration of HDM (GREER, XPB91D3A2.5) to the mice. Sensitization was performed by tracheal administration of 100 μg/40 μl HDM on day 0 and day 7, respectively. IgE CAR-T was constructed using T cells isolated from cord blood of the same donor as the humanized mice, and CAR-T adoptive was performed on day 12. HDM dosing challenge was performed again on day 14. HDM-specific IgE levels were analyzed by tail vein blood sampling on day 17. The relapse was again followed by 50. Mu.g/20. Mu.L of HDM dosing on days 32, 33, 34. Mice were sacrificed on day 36 and spleen, lung tissue and peripheral blood samples were collected for analysis.

2. Experimental results:

1) Construction of IgE CAR-T:

To examine whether IgE-targeted CAR-T can be used for the treatment of allergic diseases, the inventors first selected the scFv fragment composed of the IgE-targeted mab Omalizumab, constructed lentiviral plasmids as shown (fig. 1A), and transduced human PBMC-derived T cells by lentivirus. The current common costimulatory domain for the second generation of CAR-T is 4-1BB or CD28. Depending on the co-stimulatory domain, the completed CAR-T was designated BB-2 or 28-2, respectively, BB-1 and 28-1 being CAR-T controls that did not contain scFv.

By protein L staining and GFP positive analysis, CARs in BB-2 and 28-2 were both able to be expressed normally on cell membranes with positive rates above 60% (FIG. 1B). CAR-T cell typing as shown in figures (fig. 1C, fig. 1D), CD8 ⁺ T ratio was about 50%, effector T cell ratio was about 10% -20% with no significant difference between the two CAR-T, measured at 14-20 days of culture.

2) BB-2 and 28-2 both have specific killing effect on mIgE ⁺ target cells:

to examine the killing effect of IgE CAR-T on target cells in vitro, the inventors utilized the natural low-IgE expressing U266 cell line as target cells, and co-cultured IgE CAR-T with them in vitro to examine the killing effect. Experiments found that although BB-2 and 28-2 could not cause significant killing effect on U266 within 24 hours, killing was continued for 5 days with a lower effective target ratio of 1:1, U266 could be killed to below 60% after 5 days, and the killing capacity of 28-2 was slightly stronger than BB-2 (FIG. 2A). To further demonstrate the killing effect of BB-2 and 28-2 on mIgE ⁺ target cells, the inventors also constructed Daudi-mIgE-mCherry cells of human mIgE ⁺, and two strains of mIgE ^- cells, daudi-sIgE-mCherry expressing sIgE and a negative control Daudi-mCherry, respectively (FIGS. 2B, 2C). The target cell balance was examined by co-culturing IgE CAR-T with 3 target cells for 24 hours at different target ratios (FIG. 2D), and it was found that the target cell survival was significantly reduced and the killing effect increased with increasing target ratio after BB-2 and 28-2 were co-cultured with Daudi-mIgE-mCherry. BB-2 and 28-2 kill Daudi-mIgE-mCherry to less than 70% at an effective target ratio of 1:1, BB-2 and 28-2 kill the target cells to less than 30% at an effective target ratio of 10:1, and similar to the killing of U266, 28-2 has slightly stronger killing capacity than BB-2. BB-2 and 28-2 have no significant killing effect on Daudi-sIgE-mCherry and Daudi-mCherry compared to Daudi-mIgE-mCherry. The results indicate that BB-2 and 28-2 have significant specific killing capacity against mIgE ⁺ cells.

3) BB-2 variants have specific killing effects on mIgE ⁺ target cells:

the inventors further made mutations in OmalizumabscFv sequences, creating 2 variants of BB-2 CAR-T: BB-2-m1, BB-2-m2 (FIG. 3A). CAR-T was also co-cultured with Daudi-mIgE-mCherry and Daudi-mCherry at different target ratios for 24 hours and the target cell balance was detected (FIG. 3B). The results show that BB-2-m1, m2 has significantly higher killing effect on Daudi-mIgE-mCherry than Daudi-mCherry, and that the killing effect is stronger at 10:1 than at 1:1, whether at an effective target ratio of 1:1 or 10:1. Meanwhile, the killing capacity of BB-2-m1 and BB-2-m2 is basically equivalent to that of BB-2. The above results indicate that the variant CAR-T of BB-2 still has specific killing effect on the mIgE ⁺ target cells.

4) IgE CAR-T activates under target cell stimulation:

Flow staining analysis of CAR-T cells at the end of co-culture (fig. 4A) found that the proportion of CD107a, granzyme B, CD69, CD25, PD-1 positive cells in BB-2 and 28-2 was all increased under stimulation by Daudi-mIgE-mCherry, and varied more significantly at 28-2. Wherein CD107a and Granzyme B reflect the degranulation intensity and cytotoxicity, the positive proportion of BB-2 and 28-2 is obviously higher than that of UTD control group, the positive proportion of CD107a in BB-2 is about 15%, the positive proportion of Granzyme B in BB-2 is about 30% or more, and the positive proportion of Granzyme B in BB-2 is about 2%, and the positive proportion of BB-2 is as high as 20%. CD69 and CD25 reflect the level of cell activation, with positive ratios in BB-2 and 28-2 being significantly higher than in UTD groups, while 28-2 is also significantly higher than BB-2.PD-1 may also reflect to some extent the level of cellular activation, as 28-2 is also significantly higher than BB-2. Similarly, the levels of CD107a, granzyme B, CD25, PD-1 expression exhibited similar trends with U266 as the target cell. In contrast, BB-2 and 28-2 did not respond significantly to stimulation of both mIgE ^- cells. The supernatant of the co-culture system was taken for cytokine level detection (FIG. 4B), and the results show that BB-2 and 28-2 have significantly increased levels of secreted TNFα and IFNγ under the stimulation of Daudi-mIgE-mCherry cells, and have lower levels of increase under U266 cells, and have no obvious response to the stimulation of mIgE ^- cells. The above data indicate that BB-2 and 28-2 are specifically activated upon stimulation of the mIgE ⁺ target cells, with 28-2 activation levels being higher. Combining CAR-T co-culture killing and activation data, it is shown that BB-2 and 28-2 can specifically target mIgE ⁺ cells and exert killing effects, with 28-2 exhibiting higher activation levels and greater killing capacity.

It is generally believed that the CD28 co-stimulatory domain is more prone to the depletion of CAR-T than 4-1BB, and therefore the inventors also compared the depletion of CAR-T. At the end of 24 hour co-culture, the proportion of PD-1 ⁺TIM-3⁺ cells in CAR-T was examined and found that 28-2 was also significantly higher than BB-2, suggesting that CD28 caused a stronger depletion while eliciting a higher level of activation of CAR-T. To further demonstrate this, a depletion model was introduced in which CAR-T was subjected to repeated stimulation of Daudi-mIgE-mCherry target cells for a long period of time (10 days), at a low target ratio (1:10), and three depletion markers PD-1, LAG-3, TIM-3 expression were detected to show their depletion levels. The results of this experiment showed that, after stimulation, although BB-2 and 28-2 were similar in terms of PD-1 expression, the depletion markers LAG-3 and TIM-3 in 28-2 were both expressed higher than BB-2 (FIG. 4C), again demonstrating that CD28 aggravates 28-2 depletion.

The above data indicate that both BB-2 and 28-2 have killing ability against mIgE ⁺ cells in vitro, while 28-2 shows more severe activation and depletion.

5) Both CD4 ⁺ and CD8 ⁺ CAR-T have killing ability:

To compare the effect of CD4 ⁺ and CD8 ⁺ CAR-T, populations of CD4 ⁺ and CD8 ⁺ cells were sorted from CAR-T, respectively (fig. 5A), and tested for killing capacity by co-culturing with Daudi-mIgE-mCherry target cells for 24 hours. As shown (fig. 5B), both CD4 ⁺ and CD8 ⁺ CAR-T were able to kill Daudi-mIgE-mCherry target cells efficiently within 24 hours, with the degree of killing increasing with increasing target ratio. In comparison, CD8 ⁺ CAR-T is slightly more potent than CD4 ⁺ CAR-T, and all CD8 ⁺ CAR-T at 10:1 potency target ratio can kill target cells to below 20% while CD4 ⁺ CAR-T can also kill to below 40%. And 28-2 has a slightly stronger killing power than BB-2, both CD4 ⁺ and CD8 ⁺. Meanwhile, BB-1, 28-1 and UTD of the control group in CD8 ⁺ CAR-T showed lower non-specific killing relative to CD4 ⁺ CAR-T. Overall, both CD4 ⁺ and CD8 ⁺ CAR-T resulted in effective killing of target cells. It can thus be seen that the CAR-T products of the CD4 ⁺ and CD8 ⁺ cell populations, combined in any ratio, also have killing capacity against the mIgE ⁺ target cells.

6) Effect of sIgE on IgE CAR-T:

To simulate in vivo environment, the effect of the presence of free sIgE on killing IgE CAR-T was explored, and the addition of recombinant sIgE protein in the co-culture system of CAR-T and Daudi-mIgE-mCherry revealed that the extent of killing by BB-2 and 28-2 on Daudi-mIgE-mCherry was significantly reduced compared to the control group without sIgE, and BB-2 killing was reduced by nearly 60% and 28-2 killing by nearly 40% at an effective target ratio of 1:1 (FIG. 6), indicating that the killing effect would be interfered by free IgE. This is likely to be relevant to the use of Omalizumab-derived scFv capable of recognizing both sIgE and mIgE. Thus the use of CAR-T combinations of different scFv may have certain advantages.

7) Therapeutic effects of IgE CAR-T in humanized mouse asthma model:

To examine the therapeutic effect of IgE CAR-T on allergic diseases, the inventors established a humanized mouse asthma model. To better mimic the therapeutic effect of IgECAR-T in human patients, the model used Hu-HSC-NPG-GM3 humanized mice. The human immune system was reconstituted in mice by adoptively transferring hematopoietic stem cells (hCD 34 ⁺) derived from human cord blood to immunodeficient mice, and the degree of humanization was 40% or more in this experiment.

Asthma models were established on the basis of humanized mice. Following sensitization of mice with House Dust Mite (HDM), donor-derived BB-2 or UTD cells are adoptively followed by asthma challenge (first challenge) with HDM. All subsequent assays were directed to human proteins or cells. IgE levels in serum of mice after challenge were found to be elevated in both the total IgE levels and the HDM-specific IgE levels in the model group (model) and UTD adoptive group compared to the no-treatment group (NC), indicating successful model construction (fig. 7E, fig. 7F). Whereas the total IgE levels and HDM-specific IgE levels were significantly reduced in the BB-2 treated group compared to the model group (fig. 7E, fig. 7F).

Two weeks later mice were subjected to a second asthma challenge with HDM, followed by sacrifice for detection. The results of examination of immune cell composition in mouse tissues showed that BB-2 adoptive group was present in uniform and constant proportion of CAR-T cells in lung and spleen, accounting for about 2% -5% of all humanized immune cells (FIGS. 7A, 7B). The results of testing the proportion of mIgE ⁺ B cells showed that in the BB-2 treated group, the proportion of mIgE ⁺ B cells in total hCD45 ⁺ immune cells in the lung and spleen was significantly lower than in the NC and model groups (fig. 7C, 7D). Detection of total IgE levels and HDM-specific IgE levels in serum showed that the levels in both BB-2 treated groups remained significantly lower than in the model group (fig. 7G, fig. 7H). These data indicate that BB-2CAR-T is capable of long-term survival in vivo and effectively killing mIgE ⁺ B cells, reducing IgE production.

The H & E staining of the mouse lung sections after the second asthma challenge (fig. 7I) showed that the model group had an alveolar shape change compared to the untreated group, a thickening of the interval, a fibrotic character, and a large amount of inflammatory cell infiltration, whereas the degree of lung fibrosis was significantly improved in the BB-2 treated group compared to the model group, less inflammatory cell infiltration, demonstrating the therapeutic effect on asthma.

Taken together, the use of a humanized mouse asthma model demonstrates that BB-2CAR-T has a therapeutic effect on asthma, BB-2 treatment is capable of reducing its in vivo levels by killing mIgE ⁺ B cells, while also reducing the levels of total IgE and antigen-specific IgE in vivo, and also reducing the degree of pulmonary inflammatory cell infiltration and fibrosis, and more importantly, this effect continues from a first asthma challenge to a second asthma challenge, for a period of up to 24 days from CAR-T, whereas the half-life of typically monoclonal antibodies (e.g., omalizumab) in vivo is only 14 days, demonstrating the superiority of CAR-T therapy.

The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the application.

Claims

1. A chimeric antigen receptor is characterized by comprising an antigen specific binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a CD3 zeta signaling domain, wherein the antigen specific binding domain can specifically bind an antigen comprising immunoglobulin IgE, and the antigen specific binding domain comprises a single-chain variable fragment scFv consisting of an amino acid sequence shown as SEQ ID No.1 or comprises a single-chain variable fragment scFv which has more than 80 percent of homology with the amino acid sequence shown as SEQ ID No.1 and has the same or similar functions.

2. The chimeric antigen receptor according to claim 1, wherein the single chain variable fragment scFv having 80% or more homology with the amino acid sequence shown in SEQ ID No.1 and having the same or similar function has the amino acid sequence shown in SEQ ID No.2 or SEQ ID No. 3.

3. The chimeric antigen receptor according to claim 1, wherein the costimulatory domain is selected from the group consisting of OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDs, ICAM-1, and polypeptides in combination thereof.

4. The chimeric antigen receptor according to any one of claims 1 to 3, wherein the hinge domain is selected from the amino acid sequence shown in SEQ ID No.5 or a hinge domain having 80% or more homology to the amino acid sequence shown in SEQ ID No.5 and having the same or similar function, the transmembrane domain is selected from the amino acid sequence shown in SEQ ID No.6 or a transmembrane domain having 80% or more homology to the amino acid sequence shown in SEQ ID No.6 and having the same or similar function, and the CD3 zeta signaling domain is selected from the amino acid sequence shown in SEQ ID No.7 or a CD3 zeta signaling domain having 80% or more homology to the amino acid sequence shown in SEQ ID No.7 and having the same or similar function, and the co-stimulatory domain is selected from the amino acid sequence shown in SEQ ID No.8 or SEQ ID No.9 or a stimulatory domain having 80% or more homology to the amino acid sequence shown in SEQ ID No.8 or SEQ ID No.9 and having the same or similar function.

5. The chimeric antigen receptor according to claim 4, wherein the hinge domain is encoded by the nucleic acid sequence shown in SEQ ID No.11, the transmembrane domain is encoded by the nucleic acid sequence shown in SEQ ID No.12, the CD3 zeta signaling domain is encoded by the nucleic acid sequence shown in SEQ ID No.13, and the costimulatory domain is encoded by the nucleic acid sequence shown in SEQ ID No.14 or SEQ ID No. 15.

6. An isolated immune cell, wherein the isolated immune cell is modified to express a chimeric antigen receptor comprising an antigen binding domain linked to a costimulatory domain and a CD3 zeta signaling domain, the antigen binding domain being capable of specifically binding to a single chain variable fragment scFv comprising immunoglobulin IgE, the amino acid sequence of the single chain variable fragment scFv being shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3, the immune cell preferably being a T cell.

7. The isolated immune cell of claim 6, wherein the costimulatory domain is selected as a CD28 or 41BB polypeptide.

8. The isolated immune cell of claim 6 or 7, wherein the chimeric antigen receptor is selected as the chimeric antigen receptor of claim 4 or claim 5.

9. The isolated immune cell of any one of claims 6-8, wherein the immune cell is selected from the group consisting of a CD4 ⁺ T cell, a CD8 ⁺ T cell, and a CD4 ⁺ cell and a CD8 ⁺ T cell mixed in any ratio.

10. An expression vector encoding the chimeric antigen receptor according to any one of claims 1-5.

11. A host cell, characterized in that, the host cell comprising the expression vector of claim 10.

12. Use of the chimeric antigen receptor of any one of claims 1-5, the isolated immune cell of any one of claims 6-9, the expression vector of claim 10, and the host cell of claim 11 in the manufacture of a long-acting medicament for the treatment of allergic diseases.

13. The use according to claim 12, wherein the allergic disease is selected as any one or more of IgE-mediated allergic reaction type diseases, preferably allergic asthma, food allergy, urticaria.

14. A long-acting pharmaceutical composition for the treatment of allergic diseases, comprising the chimeric antigen receptor according to any one of claims 1 to 5, the isolated immune cell according to any one of claims 6 to 9, the expression vector according to claim 10 and the host cell according to claim 11.